Cell Culture Bioprocess Engineering

Cell Culture Bioprocess Engineering
Cell Culture
Bioprocess Engineering
Wei-Shou Hu
Department of Chemical Engineering and Material Science
University of Minnesota
Minneapolis, MN
With Contributions From:

Weichang Zhou
Gargi Seth
Sadettin Ozturk
Chun Zhang
Copyright © 2012 by Wei-Shou Hu
ISBN: 978-0-9856626-0-8
http://www.cellprocessbook.com/
Preface
For over two decades, we have assembled innovative guest lecturers to share
their research and best-practices at our annual cell culture bioprocessing short course
at the University of Minnesota. This course was created for industrial practitioners of
the production of biologics. This book is the culmination of two decades of accumulated
expertise, practical know-how and insight into future trends.
There have been many books and courses on cell culture technology covering
topics from a technical or business perspective. The goal of this course and this book is to
bring new knowledge from cutting-edge research into the very practical setting of today’s
industrial laboratories.
A second goal of this course is to prepare industrial practitioners and students from
different academic disciplines to collaborate in today’s cross-disciplinary teams. In the
course of delivering a molecule from a gene sequence in the laboratory to a product in the
manufacturing plant, scientists and engineers must quickly communicate, troubleshoot and
innovate. The fundamental knowledge for practicing industrial cell culture spans from cell
biology and physiology to process engineering principles in stoichiometry, reactor kinetics
and scale up. Thus, we have designed this course for students of diverse backgrounds.
The book is used in the classroom of our annual course. The layout of the book is
thus designed to facilitate the delivery of information. The left panels are graphs, tables,
diagrams, highlights of key points and space for note taking; while the right panels are
descriptive text.
This course has been given around the world: in Europe, East and South Asia, South
America and as an internal course at many corporations. Over three thousand industrial
biotechnologists have taken this course. With the technology of biologics production
spreading to wider regions of the world, this book will meet a timely need of many who
practice the technology but cannot attend the course in Minnesota. The book is published
in an electronic form to allow for more frequent future updates, and for easy distribution to
the parts of the world where the biologics manufacturing is quickly expanding.

Wei-Shou Hu
Acknowledgements
The authoring of this book has been influenced by many who have lectured in the
summer course at the University of Minnesota over the years. Foremost, thanks go to
Anthony J. Sinskey, Michael C. Flickinger, Donald McClure and Fredrick Srienc who started
the course with me originally. Konstantin Konstantinov, James Piret, James N. Thomas,
Randall Kaufman, Florian Wurm, John Aunins, Michael Betenbaugh, Sadettin Ozturk,
Matthew Croughan, Weichang Zhou, Chun Zhang and Gargi Seth all contributed to enrich
the course.
Many former and current members of my research laboratory at the University of
Minnesota contributed to the preparation of course materials. These include Derek Adams,
Marlene Castro, Bhanu Chandra Mulukutla, Anushree Chatterjee, Anna Europa, Patrick Fu,
Chetan Gadgil, Mugdha Gadgil, Anshu Gambhir, Patrick Hossler, Claire Hypolite, Nitya M.
Jacob, Kathryn Johnson, Anne Kantardjieff, Edmund Kao, Anurag Khetan, Rashmi Korke,
Huong Le, Jongchan Lee, Marcela de Leon Gatti, Sarika Mehra, Jason D. Owens, Yonsil Park,
Gargi Seth, Shikha Sharma, Kartik Subramanian, Siguang Sui, Katie Wlaschin, and Kathy
Wong. Gargi Seth, Sadettin Ozturk, Weichang Zhou and Chun Zhang, whose participation in
the course led to the development of new chapters, are noted as contributors.
This book, which began as a set of lecture notes, has gone through many years of
refinement in organization by many skillful hands. Kimberly Durand first took the notes to
digital form in a CD ROM. Ruth Patton, Radha Dalal, Katherine Matthews, Heather Wooten,
Kirsten Keefe, Jessica Raines-Jones, Kimberly Coffee and Kaitlyn Pladson continued to
shape it. At the long last, Erin Fenton and Jenna Novotny took it to current form. Kimberly
Durand also coordinated our final publication efforts.
This book is dedicated to the students, fellows and staff formerly and currently in
my laboratory at the University of Minnesota. It is through working with them that the
materials used in the book were distilled. It was also through their educating me with new
knowledge, new concepts, and new tools that this book took its shape. I must also thank my
dear friend and close colleague, Miranda Yap of Bioprocess Technology Institute, Singapore,
with whom I have had a wonderful and long collaboration.
Finally, I wish for my lovely family, Jenny, Kenny and my wife, Sheau-Ping to share
the joy of the book’s completion.
Wei-Shou Hu

Contents In Brief
Overview of Cell Culture Technology. . . . . . . . . . . . . . . . . . . . . . . 1
Cell Biology for Bioprocessing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Cell Physiology for Process Engineering. . . . . . . . . . . . . . . . . . . . . 57
Medium Design for Cell Culture Processing. . . . . . . . . . . . . . . . . . 97
Cell Line Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Stoichiometry and Kinetics of Cell Cultivation. . . . . . . . . . . . . . . . 147
Cell Culture Data Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Metabolic Flux Analysis in Cell Culture Systems . . . . . . . . . . . . . . 175
Cell Culture Bioreactors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Oxygen Transfer in Cell Culture Bioreactors . . . . . . . . . . . . . . . . . 213
Fedbatch Culture and Dynamic Nutrient Feeding. . . . . . . . . . . . . 233
Cell Retention and Perfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
Scaling Up and Scaling Down for Cell Culture Bioreactors. . . . . . 263
Cell Culture Genomics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
ACKNOWLEDGEMENTS | VII
Overview of Cell Culture
Technology
Cell Culture Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Cell Culture Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Virus Vaccines and Protein Therapeutics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Protein Molecule as Therapeutics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Industrial Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Biosimilars or Follow-on-Biologics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Manufacturing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Alternative Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Product Quality and Process Robustness. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Critical Feature of rDNA Proteins from Mammalian Cells . . . . . . . . . . . . . . . . . . 14
Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Cell Culture Engineering

In the past decade we have seen continuous
growth in mammalian cell culture bioprocessing,
driven primarily by the expansion of therapeutic
antibody production in the pharmaceutical
industry. The range and quantity of products
have both significantly increased over the past ten
years. Also fueling this growth are the increasing
numbers of therapeutic protein candidates in the
drug development pipeline that can potentially
render many more untreatable diseases treatable.
Recombinant therapeutic proteins have yielded major
advances in healthcare. Their societal impacts may
even rival those of antibiotics, whose discovery and
clinical applications transformed much of modern
medicine. Microbial fermentation technology
enabled pharmaceutical industry to make penicillin
widely available between 1950 and 1970. Today, we
see cell culture processing technology enabling this
new class of protein biologics to reach needy patients.
As with any product, manufacturers are under
continual pressure to produce more with less. In
OVERVIEW | 1
the case of cell culture bioprocess technology, we
see increasing demand for therapeutic proteins,
coupled with the strain of often prohibitively high
investment costs for new manufacturing facilities.
Thus, we must constantly re-evaluate, streamline
and refine, to increase production without the
luxury of totally new and improved facilities.
As we look to the future of cell culture processing, it
is useful to look back at the history and development
of penicillin. This specific case highlights lessons that
are almost universally relevant for the manufacturing
Fig. 1.1: Historical trend of penicillin titer and
of other products. Today’s innovations will all travel
value through some variation of these phases, from the
moment of discovery, expansion and distribution,
maturation and even demise of the product.
Pencillin is also representative of the many strides
made in the broader field of microbial natural
products that preceded today’s protein biologics.
Sir Alexander Fleming’s discovery of penicillin began
a new chapter in biotechnology. In the twenty-
five years following the first clinical applications
(pioneered by Edward Penley Abraham) both
the product titer and the production volume of
penicillin increased almost exponentially. This
rapid expansion in production quantities and
titer was then followed by a period of slower
but steady growth over the next fifty years.
The roughly three orders of magnitude increase
in production volume and product concentration
was the result of relentless effort on the part
of process scientists and engineers. These
engineers looked for hidden opportunities for
strain improvement, media development, and
much more. As a result, we have seen steady
productivity growth due to improvements in oxygen
transfer, heat transfer, and mixing characteristics.
Additional advances in on-line sensing, sterility
control, equipment reliability and process
control all contributed to technological success.
It should be noted that the success of process
technology also eventually drove down the
price. Penicillin G is no longer produced in the
United States; the cost of production is now
OVERVIEW OF CELL CULTURE TECHNOLOGY | 2

dramatically lower in other parts of the world.
Now, two decades after the first introduction
of therapeutic biologics, we have seen titers
in large manufacturing processes increase
from tens of milligrams per liter to more than
five grams per liter for many immunoglobulin
products today. Although little published
information is available, the production cost has
also decreased by at least an order of magnitude
since the beginning of cell culture products.
A graph of historical data for cell culture products
plotted one or two decades from now will likely
resemble that of penicillin. Cell culture production
today is likely around the transition from the
exponential growth stage to the steady and
slower growth period. However, it is important
to note that, in terms of both absolute quantity
of product produced and economic value, the
slower and steadier phase is as critical as the
early rapid growth stage for the product life cycle.
Even for penicillin, there were tremendous process
enhancements after the initial rapid growth phase.
Due to these improvements, major medicines
became affordable for the world’s population. The
next question for bioprocess scientists and engineers
is: How can cell culture processing accomplish what
the antibiotics industry has achieved for our society?
Bioprocess scientists and engineers possess
genomics and genome engineering tools that were
not previously available to antibiotic researchers or
even to the early innovators of cell culture processes.
These new genome-wide investigative and
engineering tools will greatly facilitate the designing
and engineering of cells with desired growth and
production characteristics. Process technologists
will need to harness the power of genomics and
genome engineering to enhance productivity and
process robustness. This will also facilitate the
expansion of biosimilars (i.e., “Follow-on” biologics)
and make many medicines available to needy
patients around the world at an affordable cost.
Much of the process technology employed in cell
culture biologics was developed for antibiotic

production. In transforming cell culture products
from laboratory discovery to clinical reality, many
innovations in the design and engineering of gene
constructs, cells, products, and processes have been
conceived and implemented. These technologies are
also likely to help new technologies move forward.
The next generation technology that will benefit
most from cell culture innovations is stem cell based
therapy. This technology is still in its infancy, but
its significant potential impact on our society will
compel cell culture technologists to push the evelope.
Cell Culture Products

Virus Vaccines and Protein Therapeutics Cell culture processes have been used to produce
viral vaccines for over half a century. Virus
production in animals or in tissues has been in
practice for over two centuries. The most notable
example is the pox vaccine from cow. Most of the
tissue-based production methods have since been
replaced by cell culture processes. A tissue system
that is still in use is the chick egg. This process is
begun by seeding a virus into 10-day-old embryos
in chicken eggs. A few days later, the replicated
virus is then isolated from infected embryos.
Early cell culture processes were an extension
of tissue culture, using primary cells explanted
from various tissues (such as chick embryos
and monkey kidneys) for the virus to infect
and replicate. The primary cells used in virus
production have mostly been replaced by cell
strains or even cell lines, which can be cultivated
over many generations to build up stocks (or a cell
bank) for routine use to ensure consistent quality.
Most viruses used as vaccines have been inactivated
by formalin treatment to render the virus incapable
of infection. However, the treated virus particles
retain a small degree of immunogenicity to elicit
the immune response in vaccine applications.
There are cases in which live attenuated viruses are
used. These attenuated viruses have been adapted,

Table 1. Principal Viral Vaccines Used in Prevention of often by the prolonged cultivation in a non-human
Human Virus Diseases host species so that the adapted strain is no longer
Condition of virulent to humans. These viruses are still capable
Disease Source of vaccine virus of replication, which significantly reduces the dose
Tissue culture (human Live required for immunization. However, they also carry
Poliomyelitis diploid cell line, monkey attenuated,
a very low, but non-zero, risk of reverting to their
kidney) inactivated
Tissue culture (chicken
wild type form and causing an infection in the patient.
Measles Live attenuated
embryo) Vaccine technology predated modern cell culture
Tissue culture (chicken for recombinant protein production by over
Mumps Live attenuated
embryo)
two decades. Although recombinant therapeutic
Tissue culture (duck
Rubella embryo, rabbit, or human Live attenuated proteins propelled the advances in cell culture
diploid) technology, proteins derived from tissues, and
Smallpox Lymph from calf or sheep
Live vaccinia even cell culture, were used for therapeutic
(vaccinia) (glycerolated, lyophilized)
purposes even before the arrival of recombinant
Smallpox Chorioallantois, tissue
(vaccinia) cultures (lyophilized)
Vaccinia DNA technology. These examples include
Tissue cultures and eggs
insulin, urokinase, Factor VIII, and interferon.
Yellow fever Live attenuated
(17D strain) The generation of recombinant DNA therapeutic
Highly purified subunit
proteins, such as human growth hormone and
forms of chicken embryo
Influenza allantoic fluid Inactivated insulin, were first produced in microorganisms.
(formalinized UV The next wave were human proteins, which
irradiated)
naturally circulate in human blood and require
Influenza Cell culture (MDCK, Vero) Attenuated post-translational modifications, such as complex
Rabies
Duck embryo or human
Inactivated disulfide-bond formation and glycosylation.
diploid cells
These proteins can not be replicated in microbial
Human diploid cell
Adenovirus Live attenuated systems. For the production of those proteins,
cultures
Mouse brain
mammalian cells must be, and were, employed.
Japanese B
(formalinized), cell Inactivated Initially, hybridoma cells were used. These are
encephalitis
culture
fusion products of the non-antibody-secreting,
Venezuelan
Guinea pig heart cell but continuously proliferating, myeloma cell
equine Live attenuated
culture
cephalomyelitis and the antibody-secreting, but non-dividing,
Eastern equine
Chicken embryo cell
Inactivated lymphocyte. This soon gave way to recombinant
culture
DNA technology. After the introduction of tissue
Chicken embryo cell
Western equine Inactivated plasminogen activation (tPA) by Genentech
culture
Russian spring -
in 1987, erythropoietin (EPO) and Factor VIII
Mouse brain also reached the market in following years.
summer Inactivated
(formalinized)
encephalitis
Antibody products and antibody-based fusion
proteins have since blossomed. They make up
the bulk of the protein drugs in clinical use.

Table 2. Therapeutic Protein Biologics Produced in
Non-Mammalian Host
Activity/Use
Granulocyte colony-
White blood cell growth for
stimulating factor
Neutropenia
(Neupogen)
Insulin (Humulin) Diabetes
α-Interferon (Intron-A) Anticancer, viral infections
Somatropin [human growth
Growth deficiencies
hormone] (Humatrope)
Somatropin [human growth
hormone] (Protopin/ Growth deficiencies
Nutropin)
Interleukin-2 (Proleukin) Kidney Cancer
Table 3. Non-Antibody Products Produced in Mammalian Cells

Trade name Type Therapeutic Use Manufacturer U.S. Host
approval
year
Aldurazyme Laronidase Mucopolysaccharid-eosis I Genzyme 2006 CHO
Cerezyme β-glucocerebrosidase Gaucher’s disease Genzyme 1994 CHO
Myozyme -galactosidase Pompe disease Genzyme 2006 CHO
Fabrazyme -galactosidase Fabry disease Genzyme 2003 CHO
Naglazyme N-acetylgalactosamie Mucopolysaccharideosis VI BioMarin Pharmaceutical 2005 CHO
4-sulfatase
Orencia Ig-CTLA4 fusion Rheumatoid arthritis Bristol-Myers Squibb 2005 CHO
Luveris Luteinizing hormone Infertility Serono 2004 CHO
Activase Tissue plasminogen Acute myocardial infraction Genentech 1987 CHO
activator
Epogen/ EPO Anemia Amgen/Ortho Biotech 1989 CHO
Procrit
Aranesp EPO (engineered) Anemia Amgen 2001 CHO
Pulmozyme Deoxyribonuclease I Cystic fibrosis Genentech 1993 CHO
Avonex Interferon-β Relapsing multiple sclerosis Biogen Idec 1996 CHO
Rebif Interferon-β Relapsing multiple sclerosis Serono 2002 CHO
Follistim/ Follicle stimulating Infertility Serono/NV Organon 1997 CHO
Gonal-F hormone
Benefix Factor IX Hemophillia A Wyeth 2000 CHO
Enbrel TNF receptor fusion Rheumatoid arthritis Amgen, Wyeth 1998 CHO
Tenecteplase Tissue plasminogen Myocardial infraction Genentech 2000 CHO
activator
(engineered)
ReFacto Factor VIII Hemophilia A Wyeth 2000 CHO
Advate Factor VIII Hemophilia A Baxter 2003 CHO
(engineered)

Table 4. Therapeutic Antibody Products
Trade name mAb type Therapeutic Use Manufacturer U.S. Host
approval
year
Orthoclone Muromomab CD3 Reversal of acute kidney Johnson & Johnson 1986 Hybridoma
OKT3 transplant rejection
ReoPro Anti-Abciximab Prevention of blood clots Centocor 1994 SP2/0
Rituxan Anti-CD20 mAb Non-Hodgkin’s lymphoma Genentech, Biogen 1997 CHO
IDEC
Zenapax Humanized, anti-α- Prevention of acute kidney Protein Design 1997 NS0
(Daclizumab) subunit T cell IL-2 transplant rejection Labs
receptor
Simulect Chimeric, anti-α- Prophylaxis of acute organ Novartis 1998
(Basiliximab) chain T cell IL-2 rejection in allogeneic renal
receptor transplantation
Synagis Humanized, anti-A Prophylaxis of lower- MedImmune 1998 CHO
(Palivizumab) antigen of RSV respiratory-tract disease
Remicade Anti-TNF- - mAb Active Crohn’s disease Centocor 1998 SP2/0
Herceptin Anti-HER2 mAb Metastatic breast cancer Genentech 1998 CHO
Mylotarg Anti-CD33 Acute myeloid leukemia Wyeth 2000 CHO
Campath Anti-CD52 mAb Chronic lymphocytic Millennium, 2001 CHO
leukemia Berlex, Genzyme
Zevalin Anti-CD20 murine Non-Hodgkins lymphoma Biogen IDEC 2002 CHO
mAb
Humira Anti-TNF- mAb Rheumatoid arthritis Abbott 2002 CHO
Xolair Humanized, Anti- Moderate/severe asthema Genentech 2003 CHO
IgE mAb
BEXXAR Anti- CD20 mAb Follicular non-Hodgkins GSK 2003 CHO
lymphoma
Raptiva Anti-CD11a mAb Chronic psoriasis Genentech 2003 CHO
Erbitux Chimeric antibody EGF receptor–expressing Imclone Systems, 2004 CHO
raised against metastatic colorectal cancer Bristol-Myers
human EGF Squibb, Merck
receptor
Avastin Anti-VEGF Metastatic colorectal cancer Genetech 2004 CHO
and lung cancer
Soliris Antibody binding Paroxysmal nocturnal Alexion 2007 NS0
to C5 hemoglobinuria
Vectibix Anti-EGFR mAb Metastatic colorectal cancer Amgen 2006 CHO
Protein Molecule as Therapeutics The early generation of protein therapeutics consisted

of all molecules native to humans. Many antibody
molecules retained part of the sequence of the
immunized species (e.g., mouse or rabbit), although
later generations of antibody molecules were all
humanized or were human antibodies. Some products
are engineered molecules with altered amino acid
sequences that enhance their drug characteristics.

Table 5. Industrial Cell Lines Subsequent products entail fusion proteins, in
Major Cell Strains and Lines for Human Biologics Production which domains (or fragments) of different human
Human Vaccines protein molecules are joined. A prominent example
Green monkey kidney cells (no is the fusion molecule of the Fc fragment of IgG and
Primary Cells longer used)
the TNFα binding fragment of the TNFα receptor.
Chicken embryo cells
This molecule was developed by then Immunex
Cell strains MRC5 (human lung fibroblast)
(now Amgen) for inhibition of TNFα to suppress its
Vero (monkey kidney epithelial inflammatory effect. Another class of product entails
Cell line
cell), MDCK
completely foreign proteins, such as recombinant
Recombinant Proteins
protein or designer proteins, which have enhanced
Species cell line derived from
potency for eliciting an immune response.
Human HEK 293, Per C6
Mouse C-127, NSO, hybridoma cells, SP2/0
Chinese Hamster CHO
Syrian hamster BHK
Table 6. Cell Lines Used in the Production of Veterinarian

Vaccines*
Vaccines Cell line
Bovine viral diarrhea virus MDBK
Bovine parainfluenza virus type 3 MDBK
Bovine rhinotracheitis virus MDBK
Bovine respiratory syncytial virus MDBK
Feline leukemia virus FL72
Feline panleukopenia virus CRFK
Feline chlamydia CRFK
Canine parvovirus CRFK
Canine distemper Vero
Canine adenovirus type 2 Vero
Ehrlichia canis DH82
Rabies BHK-21
Eastern equine encephalitis virus Vero
Western equine encephalitis virus Vero
Equine rotavirus MA104
Equine rhinopneumonitis virus type 1 and 4 Equine Dermal
Equine influenza virus MDCK
Foot and mouth disease virus BHK-21
Swine parvovirus ST, PK
Swine influenza virus MDCK
*This table was provided by Terry Ng, 2001. Organisms in
italics are intracellular parasitic bacteria.

Industrial Cell Lines For the production of traditional viral vaccines,
human diploid cell strains are the primary
production vehicle. Viral products differ from
protein products, in that the viral genome, along with
the entire virus particle, is injected into the patient
to elicit a response. Even though the virus particle is
inactivated by formalin or other treatment, there is
still a potential risk of recombination between the
virus genome and the host cell genome that may
result in the transmission of activated oncogenic
or foreign genetic elements to the patient.
Therefore, the vast majority of virus vaccines
are still produced in normal diploid human cells.
Vero and MDCK cells (along with chick embryos)
are notable exceptions of non-human continuous
cell lines used for human vaccine production.
For veterinary vaccines, the selection of host cells
is vastly wider. Both cell lines and tissue-derived
cell strains with limited life spans are widely used.
For the production of recombinant therapeutic
proteins, the cell lines that are primarily used are of
rodent origin and include mouse, chinese hamster,
and syrian hamster cells. Human cells are only
used for a handful of products. The vast majority is
produced using chinese hamster ovary (CHO) cells.
Biosimilars or Follow-on-Biologics Two decades after the introduction of mammalian

cell-based therapeutic proteins, many of those
medicines’ patents have expired. A number of
commercially successful therapeutic proteins will
go off patent between 2013 and 2017, including
the blockbuster drugs Remicade and Humira.
These prospects certainly have helped to draw
in investments to follow-on biologics. Generic
versions of those protein therapeutics have begun
to reach patients throughout the world. The terms
“biosimilar” or “follow-on biologic” refer to products
that are marketed after the expiration of patents. They
are expected to have similar properties to existing
biologic products. Sandoz was the first company
to launch a biosimilar-human growth hormone,

Omnitrope, in both Europe and the United States.
Follow-on biologics differ from traditional generic
Table 7. Approved Biosimilars in the EU
drugs, in that their biological activity, or the efficacy
Generic Product Launch
Name of their active ingredient, is not as easily defined as
Medice the traditional chemical and natural product drugs.
Recombinant
Arzneimittel Traditional drugs, like penicillin and statins, have very
human 2007
Putter clearly defined chemical structures that also confer
EPO-α
(Germany)
their biochemical activities. Protein therapeutics, on
Recombinant
Sandoz the other hand, cannot be entirely characterized by
Binocrit human 2007
(Austria)
EPO-α their chemical composition, or primary sequences.
Epoetin
Recombinant Hexal Therefore, their biological equivalency to their
human Biotech 2007 patented and branded counterparts cannot be
alfa Hexal
EPO-α (Germany)
established simply by structural similarity or identity.
Hospira
Retacrit 2007
Enterprises The status of molecular folding, glycan composition,
STADA etc. may affect their activity profoundly. The particular
Silapo Arzneimittel 2007
(Germany) host cell line that is used, as well as the production
Somatropin process, may affect subtle aspects of the protein’s
Sandoz
growth
(Austria)
2006 properties, thus posing a greater uncertainty
hormone about the “quality” of the product produced
Somatropin by manufacturers of those follow-ons. While a
Biopartners
Valtropin growth 2006
(Germany) biosimilar’s approval pathway has been established
hormone
in Europe, the U.S. has yet to lay down any guidelines.
Table 8. Marketed Biosimilars in India
Company Brand Name Biosimilar Launch
Ranbaxy Ceriton Epoetin 2003
Dr Reddy’s Grastim G-CSF 2001
Reditux MabThera 2007
Wockhardt Wosulin Insulin 2003
Wepox Epoetin 2001
Biovac-B Hepatitis B 2000
Biocon Insurgen Insulin 2004
BioMab-EGFR MabThera 2006
Recosulin Epoetin 2004
Intas Epofit, Erykine Epoetin 2005
Pharmaceuticals
Neukine G-CSF 2004
Shantha Shanpoietin Epoetin 2005
Biotechnics
Shanferon IFN α 2b 2002
Shankinase Strptokinase 2004
Shanvac B Hepatitis B 1997

Table 9. Marketed Biosimilars in China
Company Biosimilars
Dragon
Epoetin, filgrastim
Pharmaceuticals
Dongbao Insulin, G-CSF
Anhui Anke
HGH, interferon alpha
Biotechnology
Amyotop G-CSF, IL-11
G-CSF, PEG filgrastim,
GeneLeuk Biotech
interferon
HangzhouJiuyan
G-CSF, IL-11
Gene
Manufacturing Viral vaccines are administered to patients in

relatively minute quantities because a small amount
Table 10. Dose of Some Antibody Product of antigen proteins is sufficient to elicit immune
Approximate response. Cytokine, growth factors, or enzyme-
Disease Formulation types of proteins (such as EPO, human growth
Product Indication Company Configuration
hormone or tPA) are also given in small doses, in
7.5mg / 0.5ml;
Amevive Psoriasis Biogen terms of protein quantity. Depending on the market
15mg / 0.5ml
Enbrel RA Amgen 25mg size, the production facility of these products may
Breast be relatively small. The biological effect of antibody
Heceptin Genentech 440mg / 30cc
Cancer products is largely based on their binding to antigen;
Rheumatoid 40mg (1ml this event requires antibody and antigen molecules to
Humira Abbott
arthritis prefilled syringe)
be in some stoichiometric ratio to elicit downstream
Johnson &
Remicade
Crohn’s
Johnson, 100 mg / 20cc target killing or neutralization. Antibodies are
disease, RA
Centocor large molecules, as are many antibody-based
100mg / 10cc; fusion proteins. Thus, many antibody products are
Rituxan NHL Genetech/Idec
500mg / 50cc administered in relatively high doses. Thus, the
Respiratory product vessels, and the size of the manufacturing
Synagis syncytial MedImmune 100mg
virus plant for antibody products, tend to be larger.
Allergic Genetech/ The manufacturing process of protein therapeutics
Xolair 150mg / 5cc
Asthma Tanox/Novatis
is rather similar to that for traditional biochemical,
such as antibiotics and E. coli-based recombinant
proteins. A typical process entails a couple of seed
expansion reactor cultures before reaching the
production reactor. The process cycle tends to be
longer. Many cell culture manufacturing processes
are operated in fed-batch modes that last ten to
fifteen days. Some are operated as continuous
perfusion processes and last from two to six months.
The recovery process of cell culture products is
simpler than that for bacterial-based recombinant

Manufacturing Plants proteins. The vast majority of processes now employ
• Genentech’s Vacaville Facility, California a medium with a relatively low concentration of
• Started construction in 2004, started operation in proteins, to ease the purification operation. With
2009. Currently inoperative due to capacity reasons
• Investment: $800 million the high product concentrations in the range of
• Eight 25,000-liter bioreactor 5 – 10 g/L, the product molecule should be the
• Production of Herceptin, Avastin and Rituxan predominant protein in the medium at the end
• Bristol Myer Squibb, Devens, Masschusetts
• Started construction in 2007, validation in 2011 of cell culture process. The product isolation
• Investment: $750 million and purification process is substantially simpler
• Six 20,000- liter bioreactors, one purification strain than separating intracellular protein products.
• Production of Orencia and other biologics
• Biogen IDEC LSM Facility
• 245,000 ft2 production
• Multi-product facility
• Six 15,000L production reactor capacity
Fig. 2.2: Flow chart of a typical recombinant

antibody production process
Alternative Technologies
Other host cells used for biopharmaceutical production Mammalian cells, especially CHO and myeloma cells
include E.coli and Sacchromyces cerevisiae. Alternative such as NS0 and SP1/0, have been the workhorse
production systems include: for the production of protein therapeutics that
require post-translational modifications (e.g.,
• Insect cell culture
glycosylation, γ-carboxylation, etc). Although those
• Yeast ( Pichia ) post-translational modifications cannot be carried
• Transgenic animals out in bacterial systems (primarily E. coli), there
• Transgenic plants are a number of host systems that are capable of
performing N- and O-glycosylation and other post-
translational modifications. They have been explored
as the production vehicles of therapeutic proteins.

Insect Cell Culture Insect cells were explored as a production vehicle for
therapeutic proteins. The glycoforms of the proteins
Table 11. Insect Cell Culture produced in insect systems are rather different
Application Comments from those produced in mammals. Overall, such
Basic research Hundreds of genes have been expressed efforts have largely subsided. However, for other
using baculovirus.
applications, such as protein production for toxicity
Bioproduction Using baculovirus expression systems.
studies and for veterinary vaccine production, the
Gene therapy BV may be used as the gene-delivery vehicle. insect cell culture remains attractive because the
Bioreagent A number of bioreagent suppliers use BV to cultivation is relatively straightforward and the
production make target proteins, viral components and process development time can be relatively short.
other compounds for the research market.
Yeast The yeast in the genus Pichia is capable of

Table 12. synthesizing N-glycans that are not the mannose-
Product Company Use Status rich types produced in Saccharomyces. They
Medway Mitsubishi Blood On the have been used in the production of recombinant
(recombinant Tanabe Pharma expander market in proteins, including serum albumin. Advances
human serum Corporation, Japan have been made in ‘humanizing’ the glycosylation
albumin) Osaka, Japan
characteristics in Pichia systems for the production
Hepatitis B Shantha Hepatitis B On the
vaccine Biotechnics Ltd., market in of therapeutic proteins. Glycofi (Merck) has worked
India India towards a multistep genetic engineering process
Interferon- Shantha Hepatitis C/ On the where non-human glycosylation enzymes were first
alpha Biotechnics Ltd., Cancer market in eliminated and human glycosylation reactions were
India India
then introduced. A titer of ~ 1.4 g/L of recombinant
DX-88 Dyax Hereditary BLA
Corporation, angioedema submitted proteins has been reported. With further improved
Cambridge, (HAE), a secretion capacities and glycosylation patterns,
Mass. debilitating these engineered yeast strains may be capable of
condition
characterized producing proteins with consistent glycosylation
by acute patterns, or even with uniform glycans.
attacks of
inflammation.
Recombinant Biocon, India Diabetes, all On the
Human types market in
Insulin India
Recombinant Fibrogen Inc., Medical On the
collagen South San research market
Francisco reagents and
dermal filler
Botulism USAMRIID/ Botulism Phase I
vaccine DynPort vaccine (U.S.)
product
Anti- ThromboGenics Thrombosis Tx Phase II
thrombolytic Ltd.

Transgenic Animals Transgenic organisms for the production of
biotherapeutics have been in development for
two decades. These production systems require
a low initial capital investment and have a
relatively easy purification process for glycosylated
products. However, so far, the FDA has approved
only one product, ATryn, which is produced in
transgenic goat’s milk by GTC Biotherapeutics.
An advantage of transgenic animal production
is its high titer in milk, on the order of 2 – 10
g/L. However, over the years, the titer in cell
culture processes has also increased to 5 – 10
g/L range, thereby diminishing this particular
advantage of transgenic animal production.
Table 13. Transgenic Animal Products Approved or Under Development

Species Company Product Status Comments
Goat GTC Biotherapeutics, ATryn- recombinant human Approved Glycosylation patterns differ slightly ( involves
MA antithrombin-alpha N-glycolylneuramic acid- not seen in humans),
but was not a regulatory hurdle; Predicted
sales of $6-$10 million in 2009.
Goat PharmAthene, MD Protexia- recombinant human Development
butyrylcholinesterase (BChE)
Rabbit Pharming, Rhucin-Recombinant human Phase 3 trials For the treatment of hereditary angiodema.
Netherlands C1 esterase inhibitor
Chickens Origen Therapeutics, mAb Pilot Studies Functional Mabs produced at 3 mg/egg; some
(eggs) Medarex Inc., CA differences in glycosylation; Half life in mouse
serum half that of natural antibodies (reduced
from 200-100h)
Product Quality and Process Robustness
Critical Feature of rDNA Proteins from In spite of its dominance as the production vehicle
Mammalian Cells for therapeutic proteins, the mammalian cell
system does have some shortcomings in its process
• Folding and disulfide bond
characteristics. Compared to microbial systems,
• Glycosylation
mammalian cell systems have a slow growth rate
• N or O - glycosylation and a relatively low achievable cell concentration.
• Sulfation or phosphorylation of glycans The product titer is also substantially lower than
• Affect solubility, clearance and biological that of extracellular protein produced using fungal
activities systems. Finally, the optimal range of growth
• Other post-translational modifications environments for mammalian cells is much narrower
• Y-carbonxylation than the range for either plant or microbial systems.
• Lipidation After years of research effort, the low productivity
• Phosphorylation that used to be associated with the low cell and
product concentrations has largely been overcome.

Through cell adaptation and media development, the
Tissue Plasminogen Activator (tPA)1 complex nutritional requirements for mammalian
• Single polypeptide chain (70 kDa) or cell growth have been greatly simplified. Now,
proteolytically cleaved at ARG276.
the relatively low tolerance of mammalian cells
• Multiple N-linked carbohydrates: ASN117 (high
mannose), ASN184 (50% complex multiantenary, to their chemical and physical environment has
50% unoccupied), THR61 (O linked fucose). not prevented highly stressed conditions from
• Contains 35 cysteine residues, 17 pairs of disulfide being used in the final production stage. What has
bonds. CYS83 can form a disulfide with other free been lagging is our ability to control the quality,
thiols depending upon the growth medium and notably the glycosylation profile, of the product.
buffer composition.
• May form high molecular weight aggregate The mammalian cell system is chosen for protein
(complexes with protease inhibitors) and production, almost invariably for its capability of post-
proteolytically cleaved tPA. translational modifications on the product (such as
Erythropoietin the formation of multiple disulfide bonds of tPA and
• Contains 40% carbohydrate, only 2 disulfide the glycosylation of Factor VIII and EPO). Major post-
bonds.
translational modifications commonly seen in protein
• 3 N-linked ASN (24,38,83), 1 O linked (SER126) therapeutics, such as disulfide bond formation,
glycosylation sites.
N- and O-glycosylation, and phosphorylation,
• O-linked site not essential for in vitro or in vivo
activity. all involve extensive enzymatic reactions in the
endoplasmic reticulum or in the Golgi apparatus.
• Sialic acid residues (average 10 moles/mole
Epo) responsible for preserving pharmacokinetic The level of those enzymes, as well as the
behavior. Muteins lacking 2 or 3 N-linked sites are
poorly secreted. supply of precursors and co-factors, affects the
outcome of those reactions. The enzyme levels
• N linked glycosylation and sialylation is critical to
optimal secretion, structure, in vivo potency. vary with cell clone and growth stage, while the
supply of precursors and cofactors change with
the chemical environment. These variations
cause fluctuations in the glycans attached to N-
(asparagin) sites or to O- (serine or tyosine) sites.
For a given glycoprotein, regardless of whether it
is produced in culture or present in circulation,
the glycans attached to different molecules are not
identical. Rather, they are a mixture of different, but
related, forms. In fact, most glycoproteins in blood
circulation also have hetergeneous glycans. The
structure of glycan and the extent of glycosylation on
the protein molecule affect the blood circulation half-
life of the protein. In some cases, the glycan structure
even affects the protein’s biological functions. Thus,
confining glycan distribution to an acceptable range
is important for the quality control of the product.
The glycosylation pathway is long and complex,
and takes place in multiple compartments in
the cell. Producing a glycoprotein product with
a defined range of glycan structures throughout

Table 14. In Process Structural Alternations to Mammalian a product’s life cycle is still a challenge.
Protein Biologics
Glycosylation may affect the folding of the protein
Glycoform molecule, but it does not affect its structure. Other
Site occupancy post-translational modifications may affect protein
Altered sialic acid content Possibly caused structure. Failure to form a disulfide bond or the mis-
Uncapped galactosyl residue, High by stochasticity of pairing of a disulfide bond both give rise to an altered
mannose glycosylation process
protein structure or the improper cross-linking
Glycan distribution out of range (multimer formation) among different molecules.
Amino acid alterations in protein A lack of γ-carboxylation or phosphorylation
Error rate of amino acid also drastically changes a protein’s properties.
Mis-incorporation (codon
incorporation during
misreading) Errors in protein synthesis caused by amino acid mis-
translation (1/1000)
Deamidation (Asparagine)
incorporation have been reported. Many production
cell lines have multiple copies of the product gene;
Loss of terminal amino acid
• lysine in C-terminus Most likely occurred a non-silent mutation (i.e., a mutation causing a
of heavy chain IgG, in culture fluid, may change of an amino acid in the protein) in one of
enzymatic cleavage be affected by process
those genes will inevitably result in the presence
• cyclization of N terminus conditions, or even
glutamine product titer of some fraction of mutated protein molecules.
Glycation (addition of reducing An alteration of the amino acid structure may
sugar (glucose) to amino acids)
also result from chemical modifications after
May be caused by folding being secreted into the medium. After being
in ER or agglomeration
Protein aggregation secreted into culture medium, the product protein
in culture or in down
stream processing molecules are also subjected to modification by
enzymes released by cells, which are either actively
secreted or released from lysed cells. Extracellular
proteolytic cleavage can give rise to degradation of
Factor VIII, or can alter the ratio of single chain/
double chain molecules of tPA and Protein C. Also,
the sialic acid moiety in glycans may be cleaved
by sialidase released from lysed cells. Table 14
summarizes some more commonly-seen alterations
in protein molecules in cell culture processes.
In the past decade we have seen the productivity
of recombinant cells reaching or even exceeding
the production rate of professional secretors in our
body (such as liver cells or antibody- and insulin-
secreting cells). We have also seen the product titer
in the bioreactor approaching the concentrations
of antibody in ascites fluid. As the productivity and
product concentration of cell culture processes
approaching its “natural” biological counter
parts, we must also be cautious and ask ourselves
whther we are pushing cell’s protein folding
and processing machinery to operate at its limit.

The unprecedented high productivity is achieved by
operating the reactor at conditions that are neither
optimal for growth nor the natural homeostatic
state. Rather, they are often highly stressed to favor
producing the product of our design. In today’s
production cultures, both the intracellular and the
extracellular environments are extremely harsh.
While protein synthesis and secretion is boosted to
a nearly unprecedented level, the cellular machinery
for protein quality control may not be operated at the
same level of stringency as it is for optimal growth.
Process technologists must bear in mind that quality
consistency and process robustness must be the
highest priority when pushing productivity higher.
Concluding Remarks
Over half a century, cell culture processes have Cell culture processes now aim to provide optimal
evolved from tissue and small-scale cell culture for growth conditions for cell expansion, while often
vaccine production to large scale manufacturing employing highly-stressed conditions for the final
process for protein production. Therapeutic production stage. All must be accomplished without
proteins, especially antibody and antibody- compromising the quality of product produced.
based proteins, are the dominant products. The Achieving those aims through process innovation will
continuing pressure to meet increasing demand for be critical in the next phase of the technology, wherein
products has led to many process innovations and follow-ons or biosimilars will have an increasing
refinements over the past two decades. The cell presence. Cell culture engineering efforts in the
and product concentrations in today’s process are past quarter century have transformed bioprocess
nearly two orders of magnitude higher than they technology. The advances made in cell culture
were at the dawn of the recombinant protein era. technology will greatly facilitate the development
The success of this technology has also shifted the of the emerging stem cell and other cell therapy.
focus from production quantity to product quality.

Cell Biology for Bioprocessing
Cells: Source, Composition and Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Cell Source. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Cell Composition and Chemical Environment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Cell Membrane. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Cytoplasm and Organelles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Transport Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Major Mechanism of Transport. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Extracellular Matrices and Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Movement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Growth, Death and Senescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Cell Cycle and Growth Control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Senescence and Telomeres. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Cells: Source, Composition and Structure

Cell Source
The cells commonly used for the production of
Table 1. Cells Commonly Used in Bioprocessing biologics are derived from different tissues of
Tissue different species. Thus, they can vary widely at
Species Cell Type
Isolated the genomic level. Their differences are even
W1 - 38 Human Fibroblast Lung visible microscopically, with various numbers of
MRC - 5 Human Fibroblast Lung
FS - 4 Human Fibroblast Foreskin chromosomes. However, at a physiological and
HEK 293 Human Epithelial Kidney transcriptome level, cells from the same tissue of
Vero Monkey Epithelial Kidney
MDCK Dog Epithelial Kidney different species are strikingly similar. Their similarity
NS/SP2/0 Mouse Lymphoid Myeloma is much greater than different cell types from the same
CHO
Chinese
Epithelial Ovary
animal. For example, chicken embryo fibroblasts look
Hamster morphologically very similar to human fibroblasts
Syrian
BHK Epithelial Kidney from the lung or foreskin, while the epithelial MDCK
Hamster
cells look rather different from dog fibroblasts even
though they are both derived from the same species.
CELL BIOLOGY | 19 CELL ENGINEERING| 19

Even though there are about two hundred types of
cells in a vertebrate animal, most cells that are used
for the production of biologics are either epithelial
or fibroblast in nature. These two cell types are
• Among the ~200 different types of cells, fibroblasts, more amenable to isolation from tissues and to
epithelial cells and myeloma cells are most frequently in vitro culture, as demonstrated during the early
used cell types in biologics production explorations on tissue cell isolation more than half a
• Cells in culture bare closer physiological and century ago. NSO and CHO are the two prominent host
morphological characteristics of the tissue they were cell lines used for therapeutic recombinant protein
derived from than the species
production. They exhibit different behaviors and were
• Cells in vivo may be in a quiescent state or in a
proliferative state, but are all adapted to rapid derived from two different tissues and two different
proliferation in culture species. CHO cells were isolated from the ovary of
a Chinese hamster; NSO cells were isolated from a
mouse myeloma. Cells used for recombinant protein
production are primarily epithelial and lymphatic.
Both fibroblasts and epithelial cells are frequently
used for viral vaccine production. These cells differ in
both their functions and tissue locations. Epithelial
cells line the “boundary” of tissues, while fibroblasts
make up a larger part of the connective tissue.
Epithelial cells form tightly connected sheets, which
often get damaged, die, and are replenished by “new”
ones. Thus, many of them are constantly growing in
vivo. Conversely, fibroblasts are mostly quiescent.
They migrate into wounds and begin to grow only
when they are stimulated by various cues. Lymphatic
cells, especially the terminally-differentiated plasma
cells (from which myeloma cells are derived), are
needed to secrete antibodies against a particular
antigen, but only for a limited period of time after
the host’s exposure to the antigen. They undergo
apoptosis days after their differentiation into active
antibody-secreting cells, so that the host does not
continue to have unnecessary or maybe even harmful
antibody molecules in circulation. Such native
characteristics are often still evident in culture.
CELL BIOLOGY
20 | CELL | 20
BIOLOGY
Cell Composition and Chemical Most cells in culture have a diameter of about 12 – 18
Environment µm. Some types of stem cells are rather small and have
Table 2. Typical Composition of a Cell only a small amount of cytoplasm. In contrast, liver
Mammalian Cell E. coli cells (i.e., hepatocytes) in some species are rather large,
pg / Cell Range % %
with an average cellular diameter of 20 µm. A typical
Wet weight 3,000 3,000 ‑ 8,000
Dry weight 600 300 ‑ 1,200
cell has nearly 80% of its mass as water. Proteins make
Protein 250 200 ‑ 300 10‑20 15 up the next largest portion of cell mass, after water.
Carbohydrate 150 40 ‑ 200 1‑5 2
Lipid 120 100 ‑ 200 1‑2 2 Other than water and proteins, the other cellular
DNA 10 8 ‑ 17 0.3 1
RNA 25 20 ‑ 40 0.7 6 constituents are present in much smaller amounts
Water 80 ‑ 85 70
4 x 10‑9 and rarely exceed 10% of the total dry mass. Lipids
Volume
cm3 make up various membranes of the cell, including the
Diameter 18 μm 0.5-2 μm
cytoplasmic membrane and the membrane enclosing
all organelles. Lipids, thus, constitute a significant
portion (about 5-8% of total dry mass) of cell mass.
Table 3. Cellular and Extracellular Fluids Ion
Carbohydrates (such as glycogen) are used to store
Concentration
Plasma Intracellular osmolality energy in some cells. However, not all cells have a
(mmole / l) Interstitial (mmole / l) (mmole / l)
large amount of free carbohydrates. Carbohydrate
Na+ 140 14 molecules that serve as energy sources are quickly
K+ 4 4 140 metabolized to become intermediates in energy
Ca++ 1 1 10-4 metabolism. Most carbohydrate moieties that
Mg++ 0.8 0.7 20
remain in their carbohydrate forms exist as part of
Cl -
110 110 50
nucleotides or are conjugated to proteins or lipids.
The size of a haploid genome in a typical mammalian
cell is about 3 Gbp. That equates to about 5 pg of
DNA for a diploid cell. However, DNA is not the
most abundant nucleic acid in the cell. RNAs are
far more abundant than DNA in a cell and include
messenger RNA (mRNA), ribosomal RNA (rRNA),
• >10 fold concentation difference for K+ and Na+ across and others. Ribosomal RNA, which is a major
the plasma membrane constituent of the cell’s protein synthesis machinery,
• Opposite direction of concentration gradient for K+ and constitutes over 90% of all RNA in the cell.
Na+
Since water constitutes the largest fraction of all cell
• Extremely low concentration of Mg++ in intracellular materials, the chemical species that are present at a
fluid
high concentration in the cytosol are also major cellular
• Total osmolality ̴ 280 mOsm
constituents. Combined, all minerals contribute
a significant (~5%) proportion of the dry mass.
The concentrations of some ions are vastly different
inside the cell versus outside the cell. Maintaining these
concentration gradients is critical for cell functions.
The concentration ratio between intracellular and
extracellular K+ and Na+ is in the range of 15 to

30. Conversely, their direction of concentration
gradient is opposite: the concentration of K+ and
Na+ should be far higher inside and outside the cell,
respectively. The solutes in a solution exert osmotic
pressure, which is typically quantified by osmolality.
The osmolality of cellular fluid is about 280 mM (or
mOsm). A typical medium has its osmolality at the
same level as in cells, to avoid incurring osmotic stress.
Cell Membrane
Cultured mammalian cells have long been thought

Lipid Bilayer Composition as being extremely fragile to mechanical stresses
Phospholipids because their cellular materials are surrounded only
• Constitute the majority (35-70%) by cytoplasmic membrane; the only thing preventing
Glycolipids the cellular content from dissolving into the aqueous
• Neutral glycolipids (e.g. galactocerebroside) environment is that lipid bilayer. Yet in a modern
Gangliosides manufacturing plant, these tiny cells thrive in
• Have sialic acids bioreactors of tens of cubic meters in volume under
such highly turbulent conditions. The membrane
Four types of phospholipids surrounding a cell is not merely a double-layer of
• Three have glycerol as backbone,Phosphotidyl lipids, and the integrity of a cell is not merely dictated
ethanol amine, Phosphotidyl serine and by its membrane wrapping.
Phosphotidyl choline
The lipids which make up the lipid bilayer are
• Serine as backbone
amphipathic. They have a hydrophilic head group,
and a hydrophobic tail group made of fatty acids.
When suspended in an aqueous solution, amphipathic
molecules can form micelles. In such micelles, the
hydrophilic.
Fig.2.1: A phospholipid molecule with glycerol as backbone,

with an ethanol amine, a saturated and an unsaturatted
fatty acid.
CELL BIOLOGY
22 | CELL | 22
BIOLOGY
Lipid Bilayer
A lipid bilayer membrane behaves like a fluid. If the
lipid molecules in a specific location are labeled with
a fluorescent dye, the fluorescence disperses shortly
Characteristics of a Lipid Bilayer
thereafter due to molecular diffusion (instead of
• The lipid bilayer is a fluid staying in the same place as in a solid). The lateral
• As temperature decreases, the bilayer transitions diffusion coefficient of a phospholipid molecule in a
from a fluid state to a gel state bilayer membrane is about 10-8 cm2/s. A lipid molecule
• The degree of fatty acid “unsaturation” affects does not flip-flop (or change its side of a lipid bilayer)
the transition temperature of membrane from a fluid without the aid of membrane-bound phospholipid
state to a gel state
translocator. Gas species diffuse about equally fast in
• The magnitude of diffusion of various solutes in the a lipid bilayer as they do in water. Even large protein
cell membrane resembles that of a liquid
molecules diffuse in a lipid bilayer membrane.
Fatty acids make up the hydrophobic tail. At very
mild temperatures these acids undergo a phase
transition from a fluid to an ordered structure. Thus,
lipid bilayers also undergo phase transition to form a
“liquid crystal” at a relatively moderate temperature.
This tightly-packed, ordered structure acts as a very
good barrier to keep most molecules from freely
passing in or out of the cell. The permeability of
most biological molecules across a lipid bilayer
membrane is rather low. Even the smallest
nutrient, such as glucose and simple amino acids,
cannot pass by fast enough to support cell growth.
All major biological macromolecules (e.g., DNA,
proteins, and polysaccharides) are biopolymers
made of covalently-bonded monomers. A lipid
bilayer membrane is a not a polymer, rather, it is
an assembly of phospholipids. The non-covalent
nature of phospholipids within the cell membrane
allows it to be very dynamic: expanding, shrinking,
breaking, and fusing rapidly. The lipid bilayer also
Fig. 2.2: Lipid bilayer membrane at a crystaline state and envelops various organelles to compartmentalize
fluid state regions in the cell for specialized functions. Many of
those organelles are in a constant dynamic process
of membrane budding and fusion. For example,
in trafficking between organelles and in protein
secretion, the “cargo” is carried inside membrane
vesicles while transiting from one organelle to
another. This process occurs without the need to break
up and re-form a larger number of covalent bonds.
Three types of lipids make up a lipid bilayer
membranes in cells and organelles: phospholipids,

Characteristics: glycolipids, and gangliosides (phospholipids being
• One saturated, one cis-unsaturated (C14-C24) the most common). There are also different types
typically constitute the tail of the phospholipid. of phospholipids, with either glycerol or serine
as the backbone, with the former being the most
• Fatty Acids (the tail group) on the lipid affect the
packing of lipids in bilayer membrane. Saturated abundant type. Glycerol has three hydroxyl groups
fatty acids allow more dense packing; double attached to its three carbons and one of them has
bonds in unsaturated fatty acids creates kinks, a phosphate group, to which an ethanolamine or
reduce packing, increase fluidity. serine is attached. The phosphate moiety has a
• Cholesterol has a small head polar group linked to strong negative charge, thus making this end of
a rigid planar region of steroid rings followed by the molecule the highly hydrophilic head group.
a more flexible non-polar tail. They interact with The other two hydroxyl groups of glycerol are linked
phospholipids to stabilize the region closer to the to two fatty acids through an ester bond. Typically,
head group as well as to make the lipid bilayer
one of those two fatty acids is saturated and the other
less inclined to become crystalline. Overall, they
increase the membrane permeability to small is unsaturated, with a cis double bond in-between
compounds, and make the membrane less fluid. C14 and C24. The degree of unsaturation affects
the packing of the lipid bilayer. Saturated fatty acids
• Depending on temperature and the degree of
allow more dense packing, while the double bonds
hydration, lipid bilayer is in gel state or in liquid
crystalline state. The temperature of bilayer in the unsaturated fatty acids create kinks, which
phase transition from the crystalline lipid bilayers reduce packing and increase the membrane fluidity.
to fluid bilayers is affected by fatty acid and A lipid bilayer can be in a gel state or in a liquid
cholesterol composition. crystalline state depending on the temperature
and degree of hydration. A lipid bilayer’s phase
transition temperature is affected by its composition
Cholesterol in a Lipid Bilayer of fatty acid and cholesterol. As temperature
decreases, the lipid bilayer changes from a liquid-
crystalline state to crystalline (or gel) state. A
higher content of shorter, unsaturated fatty
acids increases the fluidity of the lipid bilayer
and decreases its phase transition temperature.
Another molecule playing a key role in the membrane
properties of animal cells is cholesterol. Cholesterol
has a small polar head group linked to a rigid planar
region of steroid rings that are further linked to a
more flexible non-polar tail. Cholesterol interacts
with phospholipids to stabilize the region closer to the
head group and to make the lipid bilayer less inclined
to become crystalline. Overall, cholesterol increases
the membrane permeability to small compounds
and makes the membrane less fluid. Cholesterol
Fig. 2.3: Schematic drawing of a cholesterol molecule content varies in different lipid bilayer membranes.
interacting with two phospholipid molecules in one leaflet of Its level in the cytoplasmic membrane is higher, but
a lipid bilayer
CELL BIOLOGY
24 | CELL | 24
BIOLOGY
Membrane Proteins in the membrane of many organelles it is very low.
A typical biological membrane has ~50% lipids and
~50% proteins, by mass. In terms of molecules,
however, the lipid:protein ratio is actually about
• A typical biological membrane has ~50% proteins by 50:1, since proteins have much higher molecular
mass; in terms of molecules, lipid:protein = 50:1 weights than lipids. The protein content of a
• Metabolically active mitochondrion has 75% protein in membrane is greatly affected by the tissue of origin
its membrane. and by the membrane’s function in the cell. The
• Na+/K+ ATPase acts as a pump, using ATP to pump 3Na+ mitochondrial membrane, through which many
out and 2K+ into the cell. molecules (e.g., amino acids, pyruvate, various ions
• The electric protential across the plasma membrane is and many other proteins) pass at a high flux, has a
about -80mV.
high protein content of about 75%, by mass. On the
other hand, the myelin membrane, which serves as
a protective sheath between the nerve cell and its
Table 4. Biochemical Composition of Hepatocyte Plasma surroundings, has a low protein content of about 25%.
Membrane
Protein/
Cholesterol/ Lipid bilayer membranes separate cellular
Total Total Lipid Cholesterol Phospholipids in
Phospholipid
Lipids Protein mass
molar ratio
in total lipids total lipids content from their surroundings and divide the
ratio
30-40%
50 -60% organelles from the cytosol. Not only do they
(by 1-2 0.4 - 0.8 12 - 20% 50 - 70%
(by mass)
mass) create a barrier for the physical retention of a
Adapted from The Liver: Biology & Pathology, 4th Ed., p. 78 (2001)
cell’s contents, but they create a rather different
chemical environment across membranes. For
example, cells maintain about an 80 mV electric
potential across the plasma membrane and about
140 mV across the mitochondrial membrane. The
ER membrane separates an oxidative environment
(inside the ER) from a reduced one (in the cytosol).
The maintenance of various chemical, electrical, and
redox potentials across a membrane is accomplished
by various membrane proteins. Rat small intestinal
enterocyte has about 150,000 Na+ pumps per cell,
which collectively allow each cell to transport about
4.5 billion Na+ ions out of the cell, each minute.
The sodium and potassium membrane gradients
generated by those pumps, as well as the electric
potential across cytoplasmic and mitochondrial
membranes, are fundamental to cellular bioenergetics.

Membrane Dynamics The cellular membrane is in a dynamic state;
membrane constituents are continuously being
added and removed. This is not only for membrane
Cellular membrane is in a dynamic state expansion and cell growth, but also for turnover and
contributed by: for vesicle trafficking. Like other cellular components,
• Lipid turn-over the turnover of the cellular membrane is necessary
• Inter-organelle shift of membrane vesicles
to replace lipid molecules that have been oxidized or
damaged, or to allow cells to change their membrane
• Secretion, endocytosis
composition to adapt to new environments.
The turnover rate of a cell membrane varies widely.
Homeostasis of cellular membranes Phospholipids are said to have a half-life of three
• Professional secretory cells in the body can add hours, while the half-life of cholesterol is about
0.5% per minute of their plasma membrane due
two hours. Cellular membrane proteins are also
to the fusion of secretory vesicles with the plasma
membrane; they must be recycled to maintain a turned over. Their half-life ranges from a couple of
balance minutes to a couple of days, whereas macrophage
• Phospholipids in the membrane are subject to turnover membrane proteins are turned extremely rapidly.
Inter-organelle trafficking and the secretion of proteins
into the extracellular environment also contribute to
a membrane’s dynamic state. Protein molecules that
are destined for export are carried from organelles to
the cytoplasmic membrane by vesicles. Upon reaching
the inner surface of the cytoplasmic membrane,
those vesicles fuse with the cytoplasmic membrane
and release their contents outside of the cell.
In the liver, each hepatocyte synthesizes ~120 x 103
albumin molecules per min (translating to about 15
pg/cell/day). All of those molecules are wrapped in
280 – 400 nm of vesicles and delivered to the basal
plasma membrane of the cell. The infusion of those
membrane vesicles would cause the membrane
surface to expand at a rate of 0.5%/min. However,
since hepatocytes are typically in a G0 state (i.e., not
dividing), the size of their cytoplasmic membrane
does not need to increase to accommodate cell growth.
Therefore, the lipid molecules that are added to the
cytoplasmic membrane must be recycled back into
the intracellular organelle (Golgi bodies) to maintain
the cytoplasmic membrane in a homeostatic state.
Similarly, cells active in endocytosis can internalize
up to 0.8%/min of a plasma membrane. The loss of
lipids from membrane caused by endocytosis must be
replenished to maintain the size of cell’s outer envelope.
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Cytoplasm and Organelles
The cytoplasm and nucleus are both enclosed by
cytoplasmic membrane in the cell. The cytoplasm
Total protein concentration in 150 g / L (~4 μM)
cytoplasm can be largely divided into two groups: various
Total protein concentration in 90 g / L (1.2 μM) organelles and the highly-viscous cytosol. The
plasma cytosol has a very high concentration of proteins (100
Albumin (MW 69,000) 45 g / L (0.65 μM) – 300 mg/mL). For comparison, the protein content
Globulins (MW 140,000) 25 g / L (0.18 μM) in blood plasma is only 90 mg/mL. The cytosol also
Fibrinogen (MW 400,00) 103 g / L (0.0075 μM) contains the inorganic solutes, building blocks, and
intermediates and metabolites of metabolic reactions.
The cytosol is not only full of soluble components.
It also contains large assemblies (or aggregates) of
particles. The ribosome is the main machinery for
making proteins; it is a complex particle consisting
of many ribosomal proteins and ribosomal RNAs
(rRNA). Each cell contains thousands of ribosomes
of ~30 nm in size. Many ribosomes are located on
the cytosolic surface of the endoplasmic membrane
and appear as a black spot, when viewed under an
electron microscope. Some enzymes also form large
complexes that can be seen under electron microscope,
such as pyruvate dehydrogenase complexes.
Also rich in the cytosol are the fiber-like structures of
• Cytoplasm is not a simple solution
the cytoskeleton. These large protein particles, enzyme
• Some protein complexes (like pyruvate dehydrogenase
and ribosomes) are aggregated complexes, cytoskeletal proteins, and organelles
make the cytoplasm of a cell very crowded and render
• Cytoskeletal network is interspersed in cytosol
its solution phase very dense in mass. Under light
microscopy, an animal cell appears to be primarily
cytoplasm, wrapped in a membrane, with a nucleus
sitting near the center spanning over half of the cell’s
diameter. Other than the nucleus, various organelles
include the mitochondria, the endoplasmic reticulum,
the Golgi apparatus, peroxisomes, endosomes,
etc., and are visible only by electron microscopy.

Nucleus
In bacterial cells, DNA molecules are roughly
endocytosis
rough
localized at the center of the cell. Both DNA and
endosome endoplasmic RNA synthesis occur in the nucleoid region that is
reticulum
golgi adjacent to the chromosome formed by the large DNA
apparatus
lysosome molecule. The nucleoid occupies a distinctive part
secretory of the cytoplasm and the DNA is tightly coiled and is
vessel
bound by many proteins. If completely extended, a
smooth DNA molecule of an E. coli cell is nearly 1-mm long.
endoplasmic
reticulum
In contrast, a eukaryotic cell’s genome is separated
into a number of DNA molecules, which each
form a chromosome. Then, the DNA molecules
nuclear chromatin
envelope are segregated into nuclear compartments. The
(nuclear plasma
membrane) membrane average genome of a mammalian cell is about three
nucleus
orders of magnitude larger than that of E. coli. If
nucleolus mitochondria
stretched, it extends to about 1-m in length. This
large amount of DNA is packed into a small space
Fig. 2.4: Organelles in an animal cell
by forming DNA-protein (histone) complexes.
DNA/RNA synthesis and ribosome assembly occur
in the nuclear compartment and are segregated
from the metabolic processes and protein synthesis
in the cytoplasm. Ribosomes are assembled in
nucleoli and are subsequently exported into the
cytoplasm to participate in protein synthesis. The
complex tasks of sorting out which segments of DNA,
or which genes, are to be transcribed into RNA at a
given moment occur in the nucleus. A large array
of transcription factors and other transcription
regulators are synthesized in the cytoplasm and then
imported into the nucleus where they bind to specific
genetic loci to perform their role in transcription.
Thus, there is a large volume of material trafficking
between the nucleus and the cytoplasm. Components
of the ribosome, nucleotides/deoxynucleotides,
nuclear structural proteins, and transcription
factors need to be imported into the nucleus. The
RNAs (mRNA, tRNA, and some non-coding RNA)
are exported into the cytosol for protein synthesis.
A double-layered membrane separates the
cytosol and the nucleoplasm. The nucleus and the
mitochondrion are two organelles in the cell that
have double membranes, instead of only a lipid-
bilayer membrane. Much of the trafficking occurs
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through nuclear pores on the surface of the nuclear
membrane (also known as nuclear envelope).
Mitochondria
The mitochondrian is the most common organelle
in a cell. With about 1,700 per cell, they make
Mitochondria are.... up 20% of the cell’s volume. Mitochondria are
• The most abundant organelle in a cell about the size of bacteria and are thought to
(about 1,700 per cell) have originated from bacteria-like structures
• Take up to 20% of cell volume that were acquired by primitive eukaryotes.
• In the catabolism of glucose to carbon Mitochondria serve as the cell’s power plants. The
dioxide, the oxygen atom in CO2 is most reactive reactions in the cell (e.g., oxidizing
contributed from water molecules. The
nutrients and generating energy through electron
oxygen reacts with H in NADH, FADH2, to
form water in mitochondria
transfer and oxidative phosphorylation) take
place in the mitochondria. Cells with different
• Active mitochondrion has a negative
energy needs have different numbers of these
140 mV electric potential across its inner
membrane, and 1.0 units of pH gradient power plants. In a high-energy demanding cell,
(inside mitochondria pH is higher [H+ there can be as many as 3,000 mitochondria.
concentration is lower] and pH is pumped The main ATP-generating process occurs via electron
against concentration gradient)
transfer, across the inner mitochondrial membrane.
• The membrane potential cannot be charged The total surface area of all mitochondrial inner
up too much. Therfore the homeostasis of
membranes in a cell is greater than that of the
mitochondria is critical.
cytoplasmic membrane. At the mitochondrial inner
• Cells meet long-term energetic needs by
membrane, reactive electrons in electron transfer
biogenesis of mitochondria.
react with oxygen to form H2O. Mitochondria are thus
rich in potentially damaging free radical species. By
confining these reactions to the mitochondria, the cell
can potentially reduce unintended cellular damage.
The mitochondrion resembles a bacterium, not
only in size but also by having its own genome
in the form of a circular DNA molecule. Each
mammalian mitochondrion contains one or
more mitochondrial genomes of about 18 kbp.
The control of mitochondrial DNA replication
is separate from the regulation of genomic DNA
replication. The biogenesis (i.e., the replication)
of mitochondria is independent of cell division.
An active respiring mitochondrion has a negative
140 mV electric potential and pH of 1.0 across its
inner membrane. The pH inside a mitochondrion is
higher, as the H+ ion concentration is lower inside,
so pH is pumped against the concentration gradient.
The pH gradient and the electric potential are critical

for cells. The electric potential and pH gradient are
created by pumping protons out of mitochondria.
This occurs while transferring electrons at a high
energetic state in NADH to a low energetic state
that can be received by oxygen to form water. In
other words, the chemical potential energy in the
high energetic electron is transformed and “stored”
in the electric potential and proton gradient.
The membrane potential cannot become too high; it
can burst the organelle. It is important to maintain
a homeostatic condition in mitochondria. When
the energetic need of a cell is high over a long
period, cells respond by increasing their number
of mitochondria. The flux of energy (primarily
pyruvate) into mitochondria is tightly controlled.
Mitochondria (along with other organelles) cannot
be generated merely from the genetic content in the
Fig. 2.5: Proton and electric potential (charge) gradient nucleus. A cell must have mitochondria at its origin in
across mitochondrial inner membrane. The direction of
fluxes of major species are indicated by and arrow. Note order to make more mitochondria as it proliferates.
NADH oxidation coupled electron transfer pumps protons The mitochondrial genome encodes a number of
against proton and charge gradient, while the movement mitochondrial proteins and RNA molecules, while other
of proton to drive ATP synthesis is in the direction of
proton and charge gradient. mitochondrial components are encoded by cellular
genomic DNA and imported into the mitochondria.
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Endoplasmic Reticulum The endoplasmic reticulum (ER) is largely classified
into the smooth ER and the rough ER, based on
morphology. The smooth ER is rich in enzymes
involved in chemical transformation reactions.
Smooth ER In liver cells, the smooth ER takes on the role of
detoxification; in the ovaries and testes, it makes
• Function varies with tissue, in liver cells it detoxifies; in
ovary and testes it makes hormones hormones. The rough ER is the site of folding and
processing of proteins destined for some organelles,
Rough ER
integral membranes, and secretion. It gains its name
• Proteins for some organelles, integral membrane through the attachment of a large number of ribosomes
proteins and secreted proteins are folded in ER
to its cytosolic domain surface, thus appearing to be
• Professional secretors in the body, such as pancreatic rough under the transmission electron microscope.
beta cell, hepatocyte and antibody secreting plasma
cell, all have abundant ER. As B cells differentiate to Professional secretors in the body have abundant
become plasma cell, ER and Golgi apparatus expand ER, such as the pancreatic beta cells that secrete
drastically, at least by 15 fold insulin and the antibody-secreting plasma cells.
Some characteristics of ER As B cells (non-antibody secreting) differentiate
• In hepatocyte, surface of ER is about 63,000 μm2 per to become plasma cells, the ER and Golgi
cell, or about 40 times of plasma membrane apparatus expand drastically, more than 15 fold.
• ER lumen very viscous, gel-like. Diffusion coefficient of Hepatocytes secrete many proteins, including albumin,
fluorescent probe is 9 - 18 times lower than in water
which are coagulation factors that constitute many of
• ER has much higher oxidative environment than the blood’s protein components. In the liver, some
cytoplasm, appropriate for disulfide bond formation hepatocytes specialize in protein secretion, while
• High free Ca2+ environment others play major roles in oxidative detoxification.
• Many proteins are present at very high concentrations These hepatocytes have distinctive ERs. Those
(PDI, GRP94, GRP74) involved in protein secretion have an abundance of
• A major site of protein folding, other post-translational rough ER, while those more specialized in xenobiotic
processing, Ca++ homeostasis, cholesterol synthesis metabolism have an abundance of smooth ER.
Protein processing occurs in ER ER is also a major site of protein post-translational
• Cleavage of signal peptide modifications, and is involved in Ca+2 homeostasis
• Addition of high mannose core oligosaccharide to Asn- and cholesterol synthesis. The ER lumen is rich in
x-Ser / Thr N-linked glycosylation site proteins that facilitate protein folding and catalyze
• Trimming of terminal glucose and mannose residues the formation of intermolecular disulfide bonds.
from initial glycan
• Fatty acid addition
• Disulfide bond formation

Golgi Apparatus and Protein Post- The classical view of the Golgi apparatus is as a
Translational Modification stack of flattened sacks. More recently, the Golgi
apparatus is viewed as a dynamic region where many
key reactions occur. Many proteins are modified
Protein Processing Occurs in Golgi in Golgi bodies after they are folded in the ER.
• Addition glycoform modification
The Golgi apparatus is loosely divided into four
• Sulfation of tyrosines or carbohydrates
compartments: cis, medial, trans, and the trans-
• glycosylation Golgi network (TGN). The protein cargo from the
• Peptide proteolytic cleavage ER is transferred to the cis Golgi and then to other
• Gamma-carboxylation of glutamic acid Golgi compartments through membrane vesicles.
• Beta-hydroxylation of aspartic acid The enzymes in the four Golgi compartments
are not identical. Thus, different reactions
may take place in different compartments.
Protein Secretion Through ER and Golgi

Apparatus For a high-producing industrial cell line, the secreted
recombinant product constitutes a very large fraction
of all of the total protein synthesized. Cells devote a
large portion of their protein processing capacity
Some Characteristics of Golgi to the secreted protein product. It is therefore
• Compartmentalized into functionally distinct regions: useful to review this process of protein secretion.
Golgi stack (consisting of cis, medial and trans
cisternae), and trans Golgi Network (TGN) In a professional secretor, approximately 30% of
• Proteins, lipids are sorted in Golgi for delivery to all cellular proteins are destined for organelles,
different cellular locations membranes, and secretion and are processed through
• From here proteins go to secretion (exocytosis) or the ER. Although some proteins are translocated
other organelles into the ER post-translationally, most (including
• During mitosis the Golgi apparatus breaks down and typical recombinant DNA protein molecules)
reassembles after mitosis
are translocated as nascent protein molecules.
• Different molecules of the same secretory protein
spend different amounts of time inside the cell, i.e. Proteins destined for secretion have a leader
there is a distribution of “holding time” in the cell sequence at the amino terminus that serves as
• Translation of a protein molecules takes only seconds, the signal peptide. After translation initiation, the
but the secretion process takes tens of minutes to signal peptide of the nascent protein is recognized
hours
by signal recognition particles (SRPs). This halts
translation and docks the nascent protein (which
has only the beginning segment of the entire
sequence) to a receptor on the ER membrane. It thus
prevents the protein molecule from being elongated
in the cytosol. The nascent polypeptide is then
transferred to a translocon on the ER membrane.
Subsequently, translation elongation resumes
and the elongating polypeptide passes through
the channel of the translocon into the ER lumen.
Folding of the polypeptide starts immediately upon
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translocation into the ER lumen. The signal peptide on
the elongating polypeptide in the ER lumen is cleaved
upon entry into the ER. The protein concentration in
the ER is estimated to be 100 mg/mL, a concentration
at which proteins would otherwise aggregate and fall
out of solution. A class of ER chaperones and other
proteins that facilitate protein folding act on the
nascent protein molecules to prevent aggregation
and assist in folding. Their actions require cellular
energy (ATP). An important member of the ER luminal
chaperones, BiP, is also a component of the translocon
complex. In addition to BiP (also known as GRP78)
major ER luminal chaperones include calnexin,
Fig. 2.6: Physiological changes incurred during the transition
from B cell to plasma cell calreticulin, and protein disulfide isomerase (PDI).
As will be discussed later, extensive glycolyation occurs
The Becoming of Plasma Cells -
A Hint to the Creation of a Super Secretor along the secretion process, starting in the ER and
• Overloading of secretory protein molecules induces continuing into Golgi bodies. In the ER, glycosylation
unfolded protein response (UPR), which triggers the also serves as an indicator of correct protein folding.
differentiation process of B cells to become non-
dividing plasma cells. Protein molecules that have completed the folding
process are exported from the ER by inclusion in
• B cells differentiate into plasma cells (or memory cells)
upon antigen stimulation, along with helper T cells, membrane vesicles. Vesicle fusion, fission, and
increasing their size significantly and their ER by at trafficking are the main forms of molecular transfer
least 15 fold (in 4 days). They also increase metabolic from the ER to different organelles. Secretory proteins
machinery significantly. in the vesicles are taken from the ER to the cis-
• Xbp1 codes for XBP-1 (55KDa). XBP-1 is post- Golgi, which, along with the trans-Golgi, comprises
transcriptionally modified upon UPR, and activates an array of tubules and vesicles on the opposite
transcription of many ER proteins. Increase in XBP-1
side of the medial-Golgi. The medial-Golgi, typically
coincides with an increase in antibody production (day
4 ), but lags in ER expansion. containing three to seven stacks of cisternae, is the
main site of glycan elongation for glycoproteins.
• ER appears to expand by increasing its abundance, not
merely selectively increasing some ER proteins. The There are two different views on how protein
expansion starts before mass antibody production. cargos are transported outward towards the TGN
ER is a very oxidized environment (unlike cytoplasm
and eventually to other organelles or secreted out
that is highly reduced). Disulfide bridges are formed
in ER and catalyzed by protein disulfide isomerase of the cell. The vesicle diffusion model hypothesis
(PDI). PDI and four oxidoreductase level increases states that the cargo from an earlier compartment
in ER as the B cells differentiate. Many of the redox is transported to the next compartment by the
balance enzymes in cytosol and mitochondria are also membrane vesicles. The cisternae maturation model
upregulated. There is also evidence to show that Golgi
views the cargo as stationary inside the stack,
increases along with ER.
once they enter the compartment. The cisternae
Note: the drastically increased antibody secretion is
(including the cargo) then moves outward, with its
through biogenesis of ER and other protein secretion
machinery, NOT merely by faster “throughput.” enzyme constituents changing along the way, and
the cargo protein molecules becoming “mature”.
Eventually, the cargo at TGN is transported through
the vesicles to its final destination, be it the plasma
membrane (for secretion) or to other organelles. As

the contents of the early compartment translocate
to the later compartment, they need to be recycled
after the cargo is delivered. Thus, there are vesicles
for retrograde transfer, in addition to anterograde
transfer. The ER, Golgi, lysosomes, and endosomes are
all part of the secretory network. They communicate
through the dynamic trafficking of membrane vesicles.
An estimated 100 to 200 glycosyltransferases,
transporters of various nucleotide-
sugars, are membrane proteins that
constitute the majority of Golgi enzymes.
After translation, it takes a finite amount of time to
Fig. 2.7: Secretion time of IgG heavy chain. Intracellular process protein molecules before they are excreted.
proteins were completely labeled with C14N15 - arginine, For an average protein of about 350 amino acids in
then switched to unlabeled medium. ~50% heavy chain is length, the translation takes only tens of seconds.
secreted in two hours.
However, the time required for synthesized
proteins to be secreted depends on the nature of
the protein, and can thus range from 30 minutes to
a few hours. For example, the α1-protease inhibitor
Table 5. Secretion time of liver proteins is among the fastest secreted proteins, with a half-
Half-life in ER Half-life in Golgi life of about 28 min. Transferrin, in contrast, takes
(min) (min)
around two hours to be secreted. Even for the
Transferrin 110 45
same protein, the secretion time is not uniform
Ceruloplasmin 80 30
for all molecules. Rather, we observe distribution
Anti-trypsin 30 - 40 10
between shorter and longer “holding times”.
IgG Total ~120
• Secretory proteins spend different amounts

of time in the ER and Golgi apparatus and in
different proportions
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Retrograde
transport
TGN
Trans Golgi
A
A
AA
AA
Anterograde
AA
AA
transport
A
AAAA
Medial Golgi
Endosome
Cis Golgi
Signal
peptide
Endocytosis
SRP
Bip
Translocon Endoplasmic
reticulum
SRP
AAAAA
Nucleus
Fig. 2.8: Synthesis and secretion of proteins to an extracellular

environment
Protein Secretion
• Nascent protein molecules destined for ER have a special ER signal sequence being synthesized in
organized polysome. They are recognized by SRP (signal recognition particle), a ribonucleoprotein.
• SRP binding transiently arrests elongation, directing the ribosome/nascent polypeptide complex (RNC)to
the receptor on ER membrane and transfer the growing polypeptide to translocon.
• SRP is released from the ribosome/nascent polypeptide complex.
• The nascent polypeptide begins to pass through translocon and elongate into ER lumen.
• Signal peptide on the elongating polypeptide is cleaved.
• Protein folding and post-translation modification begins as polypeptide continues to elongate.
• Major ER luminal chaperons: BiP, calnexin, calreticulin and PDI.
• Ribosome is released once the translation is complete.
• Folded protein (with inner core of glycan if it is a glycoprotein) concentrate at exit site of ER and is thought
to bud into vesicles and translocate to Golgi as pre-Golgi intermediates.
• Golgi apparatus is in a dynamic state. There is also retrograde transport (its own proteins need to be
recycled) and anterograde transport.
• After reaching trans Golgi network (TGN), secretory proteins are packaged into post Golgi vesicles and
move along cytoskeletal network through cytoplasm to fuse with plasma membrane and be secreted.
• Different molecules of the same secretory protein spend different amounts of time inside the cell, i.e.
there is a distribution of “holding time” in the cell.
• Translation of a protein molecules takes only seconds, but the secretion process takes tens of minutes to
hours.

Other Organelles In addition to the nucleus, the mitochondria,
the ER and the Golgi apparatus, a number of
other organelles are also in the cytoplasm.
A lysosome is an organelle with a low pH in its interior.
It is the site of degradation of ingested materials or
cellular materials that are no longer needed by the
Lysosome: cell. Most cellular materials have a useful life span,
• Low pH regardless of whether they are catalyzing chemical
• Site of degradation of cellular materials destined for reactions or playing structural or mechanical roles.
degradation and endocytosed material
Occasionally, a catalyzing enzyme can be improperly
• Part of secretory pathway
“locked up” in its transition state, resulting in an amino
Peroxisome: acid being modified to lose its catalytic capability.
• Site of fatty acid oxidation Even in the cellular environment, some amino acids
• Rich in oxidative enzymes in the protein may get oxidized. The accumulation
of such “damages” may render a protein non-
functional. Thus, most proteins have finite life span.
Endosome:
Proteins that need to be turned over are tagged by
• In endocytosis the invaginated plasma membrane
forms small organelles ubiquitin and sent to the proteosome for degradation.
• They move inward along the microtubule network Proteosomes are a complex of protealytic
enzymes that are capable of degrading proteins.
• There is extensive cargo distribution and sorting
• Some material sent to lysome Through a process known as endocytosis, eukaryotic
• Some recycle to plasma membrane cells can take up external particles by wrapping
the particles within cellular membranes and taking
them up as vesicles enclosed in lipid bilayers. This
process is not seen in prokaryotes. Some cells in
higher organisms are specialized “scavengers” that
engulf foreign particles or dead cells. Lysosomes are
the sites within those cells where engulfed particles
are degraded. Lysosomes contain a large number of
digestive enzymes. They have proton pumps in their
membrane to maintain a low interior pH (pH = 5.0).
In fat cells, the catabolism of lipid occurs in peroxisomes.
The reactions involved in such metabolism generate
large amounts of reactive oxygen and, thus, need to
be contained within these specialized organelles.
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Cytoskeleton As discussed earlier in this chapter, cells are not
merely a droplet-like structure with its constituents
enclosed by a lipid bilayer. They form various shapes
and sustain mechanical forces by transmitting and
responding to mechanical force stimuli within, and
also between, cells. A class of proteins makes up
the cytoskeleton, accounting for their shape and
their ability to transmit and exert force. The three
major components of cytoskeleton are microtubules,
actin fibers, and intermediate filaments.
Microtubules A microtubule is an assemblage of hollow tube

structures formed by polymerized α- and β-tubulin
• Long, hollow tubes of polymerized subunit tubulin
(MW 50 kD), about 25 nm diameter, more rigid than molecules. The polymerization and de-polymerization
actin filaments . occurs at the ends of the microtubules, allowing
• Typically long and straight, many have one end (-) them to extend and shrink their length rapidly.
attached to a single microtubule organizing center
(centrosome). The hollow organization enables cells to use
a smaller amount of material to give a longer
• Form and break down rapidly.
protrusion with a high structural rigidity, like the
• α- and β-tubuline (GTP) form heterodimers; the dimer
assembles in a head-to-tail fashion into chains called hollow legs of aluminum ladders sold in hardware
protofilaments, 13 of which make up the microtubule stores. If the same amount of material were made
wall. into solid legs, the legs would be rather thin and
• Microtubules also play a key role in intracellular would easily deform when subjected to stress.
organelles and small vesicle transport. Being tubes, microtubules are relatively straight
• For secretory protein, the movement of post- and do not bend in sharp angles or high curvatures.
Golgi vesicle to plasma membrane is mediated by
microtubules. Microtubules can be extended to protrude from some
region of the cell, and can rapidly shrink to retract a
part of the cell. They are also used as a “train track” in
the cytoplasm to transport “cargos”, such organelles
and membrane vesicles, to different parts of the cell.
When two ends of a microtubule are attached to
different objects, they can also pull them together
or push them apart. For example, during mitosis,
multiple microtubules work in coordination to
separate a pair of chromosomes, thus allowing
the daughter cells to each receive one copy.
Fig. 2.9: Structure of a microtubule molecule and its cellular

organization

Intermediate Filaments The main role of intermediate filaments is to transmit
• Play a structural role, stable, transmit mechanical force mechanical force, like the cable holding a suspended
• The subunit is not a globular protein, but fibril, bridge in place. Each fiber is made of subunit proteins
different from the tublin and actin oriented in the same direction. The tail end of a subunit
• 10 nm diameter, has a head, a tail and a α-helical rod; is locked into the head of the next subunit. Each
can be made of a wide variety of proteins in various intermediate filament fiber is made of multiple fibrils,
tissues. which are in turn made of a series of subunit proteins.
To increase the structural integrity, the tail-head “lock”
positions of different fibrils are at different locations in
the multi-fibril filament. These intermediate filament
fibers are flexible, capable of absorbing energy
exerted by external force and transmitting it to other
regions of the cell. In a tissue or in interconnected
cells in culture, intermediate filaments also help to
transmit forces between cells. Their deformable
nature allows them to act like a shock absorber
and to reduce the deformation of cells upon stress.
Fig. 2.10: Structure of an intermediate filament

molecule and its cellular organization
Actin Filaments Actins are two- or three-stranded filaments that

often form web-like bundles underneath the
plasma membrane. Like ropes that are woven into
a net, actin fibers are twisted strings of filaments.
This geometry enhances their structural integrity
while maintaining a high degree of flexibility.
The actin-rich region immediately underneath
the cell’s plasma membrane often has a high
concentration of actin, where it forms a gel-like
structure, called the cortex. They are like a mesh of
thin nets underneath the lipid bilayer membrane.
They act as the first absorber of external mechanical
perturbations and give the lipid bilayer local shape.
Cells extend their body and spread flat on a surface,
both in tissues and in culture. The edge of an adherent
cell has an irregular shape, much like a fried egg. In
the protruded regions, actin fibers localize in the
Fig. 2.11: Structure of an actin filament molecule and its
cellular organization lamellipodia and filopodia. In stationary cells, the
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• Two stranded filaments (F actin) of helical polymer of actin fibers form stress fibers throughout the cell,
actin (G actin)(50 kD) but in moving cells, these visible fiber structures
• 5-9 nm flexible structures, organized into linear tend to localize at the moving ends of the cell.
bundles, 2-D networks and 3-D gels
• Distributed all over the cell, but concentrated in the Actin filaments, along with the other two
cortex beneath the plasma membrane major components of the cytoskeleton,
• The subunit, G actin, is a globular protein require many other component proteins to be
• Projections from cells, like microvilli, lamellipodia, present for their polymerization, dissociation,
microspikes and filopodia are maintained by rigid cargo translocation, and other functions.
bundles of actin filaments
• In non-motile cells, actin filaments form bundles called
stress fibers, loose meshwork of filaments underlies
cell membrane.
• In actively moving cells, stress fibers disappear and
actin filaments concentrate at the leading edge.
• There are many actin-related-proteins which affect the
polymerization and motor functions of actin filaments
• Actin is involved in motor functions. Best example is
muscle contraction.
Transport Mechanisms
The lipid bilayer membrane separates cells from
their environment. It presents a barrier to keep
most compounds outside the cell and to prevent
those inside from leaking out. It has a very low
permeability for large molecules, like proteins
and polysaccharides. Even polar or charged
small molecules cannot pass through easily.
Among the nutrients and metabolites, only oxygen,
fatty acid, and ethanol pass through the membrane
Fig. 2.12: Order of magnitude estimation of the at a fast enough rate to meet growth requirements.
permeability of various molecules across lipid membrane Specialized transport mechanisms mediate the
movement of the vast majority of nutrients and the
excretion of metabolites. Cells have a large number
of transporters (sometimes called permeases)
that allow molecules to cross the cytoplasmic
membrane and membranes of various organelles.
Such transporter-mediated transport is used to
pass small molecular weight compounds (up to
about 1 kDa) across the cell membrane (e.g., sugar,
oligosaccharides, amino acids, oligopeptides,
nucleotides, cholesterol, ions, organic acids, etc.).
Macromolecules are transported across membranes
by membrane fusion (in the secretion process
through the ER and Golgi apparatus), pinocytosis,

or exocytosis. Specific receptors, such LDL and
transferrin receptors, may be involved in pinocytosis.
Major Mechanism of Transport

The cellular transport of solutes (as opposed to
macromolecules) is grossly divided into two categories:
transport along or against the concentration gradient
of the solute. The former is thermodynamically
favorable, whereas the latter (called active transport)
requires energy input to make the process possible.
The energy source of active transport can be
derived from coupling to a chemical reaction,
such as the hydrolysis of ATP. Active transport of
a species may also be coupled to the diffusion of
Three Classes of Transport Processes
another solute along its concentration gradient.
• Channel-mediated diffusion: Thus, the driving force to transfer the second
Molecules or ion specific; once channel is open, very
fast flux. solute along its gradient is used to “push” the first
• Facilitated difussion: solute to move up against concentration gradient.
Provides molecule specific opening in the membrane; Transport along the concentration gradient
barrier for molecular diffusion.
can be mediated by carrier (transporter)
• Active transport:
Moves molecules up against a concentration gradient. proteins or by channel proteins.
Requires ATP or ion gradients of ion (H+, Na+) as an Channel proteins open into a duct-like structure across
energy source to drive the transport.
the membrane that is specific for a specific solute,
such as water, Na+ or K+. Channel proteins exist either
in an open state or a closed state. Once the channel
is open, the transfer is very fast in the direction of
the concentration gradient. The flux is affected by the
number of channel protein molecules on the membrane
and by the time period that the channel is open.
Carrier-mediated transport is also called facilitated
diffusion. It entails, first, the diffusion of solute
from the high concentration side of the membrane
into the transporter, followed by the translocation
of the solute to the low concentration side of
the membrane. Once on the low concentration
side, the solute is free to diffuse away.
Under normal culture conditions, amino acids and
glucose are transported by facilitated diffusion. A
ubiquitous transporter for glucose is the glucose
transporter 1 (GLUT1). The rate of transport by a
transporter is dependent on the concentration of the
solute. More precisely, it depends on the concentration
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difference of the solute across the membrane.
Its mechanism is similar to a typical enzyme-
catalyzed conversion of a substrate to a product.
The dependence of transport rate to solute
concentration can be described by Michaelis-
Menton enzyme kinetics. At low concentrations,
the transport rate is in proportion to the solute
concentration, while at high concentrations, the rate
is constant as the transporter becomes saturated.
The half-saturation constant (km) for GLUT1 is about
0.1 mM. In the 0.01 – 0.1 mM range, the glucose
import rate of the cell increases with increasing
Fig. 2.13: Three types of transport processes across glucose concentration. In the range typically used in
the cell membrane
cell culture media (1 – 10 g/L, or 5.5 mM to 55 mM),
the rate is not affected by glucose concentration at all.
Transporters for facilitated diffusion and
active transport can also be categorized
according to the number of solutes each
carries and the direction of solute flow.
Uniporters transfer a single solute from a high
concentration side to a low concentration
side, for example the GLUT1 transporter for
glucose and the GLUT5 transporter for fructose.
General Types of Transporters Symporters and antiporters transfer two
• Uniporter: Transfers a single molecule (e.g. glucose, solutes, simultaneously. If the two solutes
fructose), usually uncharged.
move in the same direction, the transporter
• Bispecies-transporter (co-transporters): is called symporter. Conversely, antiporters
Requires stoichiometric exchange of two species
simultaneously; important in change balance. transfer two solutes in opposite directions.
• Symporter:Two species transported in the same Collectively, symporters and antiporters are called
direction. co-transporters. Co-transporters are often used to
• Antiporter:Two species transported in the transport charged organic molecules. Dissociable
opposite direction. solutes exit with a counter ion to maintain electric
charge neutrality. When a charged solute moves from
one side of the membrane to the other, the charge
neutrality must be maintained. Otherwise, the net
charge will accumulate across the membrane and
create impudence for further transfer of the solute.
For example, lactic acid exists as lactate in an
aqueous solution. If it is removed from the cell,
a negative charge will be moved along with it.
After a while, the cell membrane will be negatively
charged outside, creating a negative voltage. The
negatively charged outside will prevent further

excretion of the negatively charged lactate.
In order to prevent the buildup of a charge across
the membrane, charged solutes are transported by
co-transporters. Two mechanisms are commonly
seen: 1) co-transport with a counter ion (such as
H+ for lactate) by a symporter, and 2) co-transport
with an ion of the same positive or negative charge,
but in the opposite direction (such as Cl- for HCO3-).
In co-transporter mediated transfer, the transport rate
is not only affected by the concentration difference of
the solute, but also by the concentration gradient of
the co-transported ion. Thus, the transport of lactate
by the monocarboxylate transporter (MCT) is not only
affected by the concentration of lactate, but also by pH.
Co-transporters may also be involved in active
transport. One such case involves an ion species
Fig. 2.14: Three types of transporters categorized being transported along its concentration
by the solute being transported gradient. The tendency of the ion to “push”
across the transporter is used to “drive” the
transport of a solute against its gradient.
For example, the Na+/glucose transporter in the
epithelium of the intestine can take up glucose from
the digestive track, even when glucose is lower than
in the cell. This is accomplished by using the Na+/
glucose transporter to transport two Na+ atoms from
the lumen of the intestine into the cell, where the Na+
level is very low. As the sodium ion is transported, a
glucose molecule also binds to the transporter and is
transported simultaneously. The propensity of Na to
move across the membrane is so high that it can drive
glucose to move against a large concentration gradient.
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Transport of Nutrients
Major nutrients like glucose, other sugars, amino
acids and oligopeptides, are taken up by cells
through facilitated transport. The excretion of
lactate, ammonium, and some non-essential amino
acids are also transported by the same mechanism.
A large number of glucose and amino acid transporters
• There are 12 different facilitative glucose transporters are present in the mammalian genome. Different
(GluT) in animal cells. Different transporters are
glucose transporters are expressed in different tissues
expressed in different tissues.
for different cellular needs, but GLUT1 is present in all
• GluT 1: the major transporter in all cells
cells. GLUT4 is responsive to insulin and is expressed
• Amino acid transporters have overlapping amino acid
only in some tissues. Upon insulin stimulation, the
specificity. Some amino acids compete for the same
transporter. Many have alternative transporters. intracellular GLUT4 molecules are translocated
• MCTs transport lactate, pyruvate together with H+ to the plasma membrane to take up glucose.
The number of amino acid transporters in a
mammalian genome is also large. Some transport
entire classes of amino acids that share a
common property, such as the neutral amino acid
transporter for uncharged amino acids. Others are
specific for one or a small number of amino acids.
The monocarboxylic acid transporter (MCT),
the transporter for lactate, also transports
pyruvate. A number of MCTs are expressed
in different tissues. In a cell, different MCTs
are located at the cytoplasmic membrane and
others at the membranes of some organelles.
Another class of transporters for active transport is
the ATP-binding cassette (ABC) transporter, which
transports some hydrophobic compounds by utilizing
ATP. After prolonged exposure to methotrexate, some
cancer cells develop drug resistance by pumping
the chemical out of cells using ABC transporters.
Ion Transport
Bulk ion species (H+, Na+, K+, PO4-3, Cl-) are present
at very different concentrations across the cell
membrane. For instance, Na+ and K+ have opposite
directions in their concentration gradients across the
plasma membrane. The intracellular concentration
of K+ is 20 – 50 times higher inside than outside the
cell, while the Na+ concentration is 10 – 15 times
higher outside the cell versus inside the cell. Along
with the concentration gradients of major ions,

cells also maintain an electric potential gradient
of -80 mV across their plasma membrane. This
electric potential is fundamental to the transport
of many compounds across the membrane.
Because of this concentration gradient across the
membrane, sodium ions have a natural tendency to
flow into the cell, wherever they are allowed to pass.
The -80 mV membrane potential further enhances the
propensity of Na+ to influx, as the negative charge in
the inner surface of the membrane draws Na+ to move
across the membrane. The combined concentration
and electric potential gradients are used as a driving
force in the Na+-dependent glucose transporter,
which will transfer glucose from the lumen of
the digestive track into intestinal epithelial cells,
moving against a glucose concentration gradient.
The Na+/K+ ATPase transporter, which is present in
the cytoplasmic membranes of all animal cells, is
important for establishing sodium and potassium
gradients across the plasma membrane. ATPase
is an integral cell membrane protein that has
multiple subunits. It simultaneously transports two
K+ ions into the cell and three Na+ out of the cell.
Although the cytoplasmic membrane is relatively
impermeable to ions, it does allow a small but finite
diffusion of Na+ and K+ along their concentration
gradient (i.e., a net influx of Na+ and a net outflux of K+).
It should be noted that the membrane permeability
Fig. 2.15: Transporters involving the transfer of ions
for K+ is higher than Na+. Intracellular K+ also leaks
and ABC transporter
out through potassium channels. Overall, through
diffusion across the membrane and transport by
channel proteins, there is a net movement of ions
into the environment from the cytosol. This balance
is maintained by the action of the Na+/K+ ATPase.
Na+/K+ ATPase has three binding sites for Na+ and
two for K+. The Na+ binding sites on the cytosolic side
of the ATPase have a Km for Na+ in the range of sub-
millimolar concentrations. Because the cytosolic Na+
concentration is about 10 mM, virtually all three Na+
binding sites exposed to cytosol will be occupied.
Na+/K+ ATPase also has a binding site for ATP.
Hydrolysis of ATP results in the phosphorylation of
the protein subunit and release of ADP. This allows
two K+ ions to bind to the protein on the extracellular
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• The concentrations of other major ion species (H+, Na+, side, while simultaneously exposing the Na+ binding
K+, Ca2+, PO4-3, Cl-) across membrane are polarized. Na+ sites to the extra-cellular solution. At the external
and K+ have opposite direction in their concentration side of the enzyme, the Km of Na+ is at a higher
gradient across the plasma membrane, which is
value. As a result, Na+ is released to the outside
about ten fold difference. For Ca2+ the intracellular
concentration is so low that the gradient is extremely environment. After the release of Na+, the phosphate
steep. is released from the protein and K+ releases to the
• Na+-K+ ATPases play a major role in maintaining Na+ and intracellular environment. The Na+/K+ ATPase
K+ gradients. ATPase utilizes the hydrolysis of ATP as then resets, ready for another round of reactions.
the energy source.
The net result is that ATPase uses ATP to pump three
• Iron is extremely reactive and participates in many Na+ ions out and two K+ ions into the cell. With its
redox reactions. In biological systems, it exists as
3:2 stoichiometric ratio of sodium to potassium, a
“bound” form. In its free form, it catalyses the
formation of peroxide and peroxidizes unsaturated prolonged operation of ATPase without any balancing
fatty acids. In cell culture, it is supplied as transferrin- action will inevitably generate a large electric
bound or bound by other chelators and taken up via potential across the membrane. To counter this, cells
transferrin receptors. also have chloride ion pumps to pump negatively
• Another class of ions are transported into the cells via charged Cl- out of the cell. In some cells, Na+ and Cl-
binding to proteins and internalized through specific channel proteins also facilitate the maintenance of
transportors. One example is iron transport by the membrane electric potential in the correct range.
transferrin. Iron is extremely reactive, participates in
many redox reactions. In biological systems, it exists
as “bound” form. In its free form, it catalyses the
generation of peroxide and peroxidize unsaturated
fatty acids. In cell culture, it is supplied as transferrin
bound or bound by other chelators and taken up via
transferrin receptor.
Extracellular Matrices and Cell

Movement
ECM The vast majority of cells in the body are embedded
in tissues in an acelluar tissue structure. When
cultured in vitro to plastic or glass surfaces, they
secrete materials onto the surface after adhering.
Those excreted materials to which cells attach are
collectively called the extracellular matrix (ECM).
The ECM is made of proteins, proteoglycans,
and glucosaminoglycans. Different types of cells
excrete somewhat different ECM components.
Among the prominent members of ECM
proteins are collagens, laminins, and fibronectin.
The role of ECM is not merely to provide a surface
appropriate for cell adhesion. It is also important for
cell-surface signaling and growth control. Receptors
on the cell membrane establish cell adhesion
complexes with ECM components and a tension force
is then transmitted through cytoskeletal fibers and

ECM Proteins the cell’s internal signaling pathway to allow cell cycle
• Collagen to proceed. The cell adhesion receptors exhibit a wide
range of diversity. For example, integrin receptors
• Laminin
have a variety of α and β components and form a large
• Firbronectin number of combinations of integrin complexes that
Proteoglycan have different affinity for different ECM components.
As a result, different cell types often have different
• Chondroitin sulfate
ECM requirements for adhesion and growth.
Glucosaminoglycan
Many ECM components are highly negatively
• Heparin charged. This allows many protein growth
• Hyaluronic acid factors to be adsorbed to the ECM and released
• The extracellular matrix is rich in electrocharges, to surrounding cells, perhaps even serving as
allowing growth factors, cytokines etc. to be “stored” chemoattractants. Therefore, they also play a role in
inside them providing cues for cell migration and differentiation.
• The extracellular matrices are the substrate for
adhesion of many cell types, and provide cues for
growth, differentiation and development
Cell Movement The vast majority of cells are capable of

movement on surfaces. In general, cell
movement can be the result of an attraction to
chemicals, or unidirectional random movement.
Cell movement is a coordinated series not
unlike walking. It involves a restructuring of the
• The filopodia extend as a result of microtubules
cytoskeleton, a protrusion of the membrane, the
growing at the cell front, and establish a “grip” on the
establishment of surface adhesions on one side of
surface
the cell, and the detachment of the cell membrane
• The subsequent dissociation of cell-substrate from adhesion complexes in the rear end of the cell.
contact at the rear of the cell allows the cell’s center
The actin fibers and plasma membrane of moving
of mass to move forward
cells extend the cell to become more elongated in one
direction. The extended regions form lamellipodia,
which may also contain microspikes, or filopodia,
which are active even within a few minutes.
Cells moving in an “open” surface (i.e., not
crowded) move randomly. They exhibit locomotion
contact inhibition, meaning when two moving
cells encounter each other, both will move away
in opposite directions. Furthermore, the two
daughter cells of a dividing mother cell move away
from each other when cell division is complete.
Cell migration is a regulated by many factors,
including growth factors. For example, epithelial cells
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respond to hepatocyte growth factor by moving away
from each other and become more scattered, instead
of forming cell clusters typical of epithelial cells.
Cell migration is not intentionally controlled
or manipulated in cell bioprocessing. However,
in some cases, when seeding cells into three-
dimensional matrices for tissue engineering
Fig. 2.16: A cell moving on its substrate. Lamellipodia applications, efficient cell migration into the interior
extend in the leading edge on the right side. A few filopodia of matrices is important for subsequent growth.
are extruding out.
Growth, Death and Senescence

Cell Cycle and Growth Control
Positive and Negative Cues

Cell growth is the manifestation of a delicate
balance between positive and negative regulations
that respond to signals both outside and inside
the cell. Positive signals stimulate cell growth
Cell Cycle and proliferation and suppress the cell death
• G1, S, G2 and M constitute four consecutive phases mechanism, while negative signals suppress those
of cell cycle. S stands for DNA synthesis and M for events and promote cell death. External signals
mitosis. They are separated by two gaps. from the environment tell cells the availability or
• The duration of S and M phases is relatively constant. absence of nutrients necessary for DNA replication
Cells grow at different rates and spend differnt and biomass synthesis. External signals from other
amounts of time in G1 and G2. For mammalian cells,
the duration of S phase is 5-8 hours and that of M part of the body allow cells to coordinate their
phase is 1 hour. response to the need of the organism. The internal
• The progression from G1 to S, and from G2 to M phase signals modulate cellular programs to increase
is tightly controlled. Only when a cell is “ready”, will it cellular component content, to divide, or to die.
proceed to the next phase.
• Before M phase, chromosomes and all organelles, Eukaryotic cells progress through four stages in their
ribosomes and other cellular contents are all procession to grow in cell number: G1, S, G2, and M
duplicated from time 0 (immediately after cell phases. G1 and G2 refer to the gap phase. S and M
division).
phases derive their designation from DNA synthesis
and mitosis, respectively. The four stages constitute a
cell cycle and this cycle is repeated every time a single
cell becomes two daughter cells. Cells that are in a long
period of quiescence, such as terminally differentiated
cells, divert from G1 and enter G0 stage indefinitely.
Checkpoints are present between the different stages
of cell cycle control. After mitosis, cells increase in
size and mass. They only enter the S phase from G1 if
cellular conditions are right. Similarly, they only enter
mitosis from G2 if cellular components are ready.

Additionally, the decision to enter the S phase is
subjected to the regulation of external positive
mitogenic factors, such as insulin, insulin-like
growth factors, and fibroblast growth factors.
Anchorage-dependent cells also receive growth
stimuli by establishing contacts between the
surface receptors and the ECM, and thereby
maintaining tension in the cytoskeletal network.
Countering the actions of the mitogenic factors are
those factors that provide signals to cause growth
arrest. Cell-cell contact, for instance after reaching
a confluent state, causes growth to cease. Such
contact inhibition of growth has been noted for more
than five decades. Only recently have researchers
found that it is the interference of the adherent
junctions between cells that disrupts a cell’s
internal signaling networks to cause growth arrest.
Whether cells divide and grow or self-destruct is the
outcome of a balancing act of a network of external and
internal positive and negative factors. Loosening the
controls may lead to unscheduled proliferation and
Fig. 2.17: Phases of cell cycle and transformation of cells to their malignant derivatives.
approximate duration of each phase
Cyclins and CDKs The progression through each of the four phases
of the cell cycle (G1, S, G2 and M) is positively
regulated by cyclins and cyclin-dependent kinases
(CDKs) and negatively controlled by CDK inhibitors
(CDI), which deactivate cyclin-CDK complexes.
Each of these regulatory proteins displays
a characteristic periodic dynamic profile
throughout the cell cycle. Each protein’s profile
is the result of interactions of the other cell cycle
regulatory components with their expression,
activation, inactivation, or degradation
corresponding to a specific time frame.
An important cell-cycle checkpoint occurs along the
transition from the G1 to S phase. The pivotal player
in the G1/S phase transition is the CDK4/6-cyclin D
complex. The activated CDK4/6-cyclin D complex can
phosphorylate the regulatory protein retinoblastoma
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(pRB). pRB, in its unphosphorylated state, binds
to and inhibits the transcription factor E2F. Upon
phosphorylation, pRB dissociates from E2F, leading
to the activation of cyclin E transcription by E2F.
E2F activation positively regulates the transcription
Growth Control of genes involved in cell cycle progression.
• Growth, the increase in biomass and its contents (i.e. Inputs from growth factor signaling and cell
organelles and cytosol) as well as the increase in cell
number of almost all cells (except those which have adhesion-mediated signaling are prerequisites to the
been adapted in vitro) is regulated by growth factors, G1 phase. These two pathways are not independent
cytokines. of each other. On the contrary, they have rather
• The regulation balances positive factor (mitogenic) and extensive crosstalk. In normal untransformed
negative factors. which prevent cell death or apoptosis. cells, all the important growth factor signal
• The most common growth factors for cells of transduction cascades are regulated by integrin-
bioprocess interest are insulin or insulin-like growth
factors (IGF). mediated cell adhesion. As a result, adherent
cells rely on attachment to the ECM for growth.
• IGF and insulin have different receptors on cell surface.
Insulin regulates glucose metabolism and has a Except for vaccine production, where normal
mitogenic effect. IGF, which is used in much lower
concentrations and also has a mitogenic effect. diploid human fibroblasts are employed, virtually
all cells used for protein production are continuous
cell lines, including CHO, BHK, HEK 293, and
mouse myeloma cells, such as NS0 and Sp2/0. All
of these cell lines have lost their normal growth
control. Their cell cycle checkpoint controls have
been compromised and their entry into a quiescent
state in the absence of mitogen has been relaxed.

Fig. 2.18: Schematic representation of the interaction between cell cycle and apoptosis pathways. CDK Cell cycle-dependent
kinase, IRF-1 interferon-regulatory factor 1, pRb phosphorylated retinoblastoma, ERK extracellular signal regulated kinase, FADD
Fas associated death domain protein, FLIP FLICE-inhibitory protein, Cdc42 cell division cycle 41, EIF4E eukaryotic translation ini-
tiation factor 4E, Cyc Cyclin, XIAP cross-linkied inhibitor of apoptosis proteins, Apaf-1 apoptotic peptidase activating factor – 1,
BAX Bcl-2 associated X protein, BAK Bcl-2 homologous antagonist/killer, Ub ubiquitination, cyt C cytochrome C
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Apoptosis
• Cells, under some conditions, commit suicide, undergo Apoptosis is the process of regulated cell
programmed cell death; this is different from cell death death in response to developmental cues or
caused by injuries (necrosis). to accumulating non-lethal stresses, such as
• Necrosis may entail cell swelling, rupture and leakage nutrient depletion, growth factor deprivation,
of cellular materials. In vivo, necrosis may cause
inflammation of surrounding tissues after encountering virus infection, and/or metabolite accumulation.
cell debris. This process of programmed death is marked
• Apoptosis entails cell shrinkage, mitochondria breakage by specific cell morphological changes: DNA
and release of cytochrome C, DNA fragmentation, and
release of phosphatidylserine from phospholipids which condensation, chromatin shrinkage, and membrane
causes phagocytotic cells to engulf the cell fragments. bulging (also called blebbing). The final intracellular
• Apoptosis can be caused by the lack of positive signal/ event involves a series of cascades leading to cellular
growth factors (e.g. withdrawal of IGF) or the imposition destruction. These final acts of self-destruction
of negative signals. are similar in all apoptosis mechanisms; however,
• Two general pathways for apoptosis: one, induced by the initiating “signal” can differ. The two major
specific signals, occurs during cell development; another,
induced by a variety of stress conditions. apoptosis signaling pathways are the death
receptor pathway and the mitochondrial pathway.
Death Receptor Pathway

In many developmental events, individual cells
serve their function only for a finite period of time.
Beyond this period, their existence may interfere
with, or even imperil, the well being of the organism.
In those cases, cells are built to die after their
functional duration by endowing an individual
cell’s survival to depend on the presence of positive
factors or the absence of negative effectors.
In the event that a cell survival signal is absent
or a cell death signal is present, a cell undergoes
self-destruction and ceases to serve its function.
Developmentally related apoptosis is largely regulated
by death receptors on the cell surface. The death
receptor pathway is mediated by binding, or a lack of
binding, of ligands to death receptors. For example,
immature neurons die in large numbers during early
brain development because neuronal cells require
positive survival signals. The lack of such positive
survival signals leads to neurodegenerative disorders.
Ligand binding to death receptors initiates the
recruitment of an adaptor molecule, Fas-associated
death domain (FADD), to the cytoplasmic end
of the receptors. The presence of FADD causes
caspase 8 or 10 to associate with the receptor,
forming a death inducing signaling complex. The
caspase is then proteolytically activated, triggering

the activation of a series of downstream effector
caspases (3, 6 and 7). The activation of these effector
caspases leads to the final stages of cell destruction.
Mitochondrial Pathway In addition to its role in energy metabolism,

mitochondria also sequester pro-apoptotic proteins in
the space between their outer and inner membranes.
These pro-apoptotic factors are released in a controlled
manner in stressed cells to initiate apoptosis.
Mitochondrial Apoptotic Pathway
Mitochondrial cytochrome C, a major component
Non-Apoptotic State: of electron transfer, is also a signal for apoptosis.
The balance of anti-apoptotic and pro-apoptotic factors
As in the control of cell growth, the components of
• At non-apoptotic state anti-apoptotic factors
hold pro-apoptotic factors in check
the mitochondrial apoptosis pathway also involve
positive proapoptotic and negative anti-apoptotic
• Apoptogenic factors are held in inter membrane space
of mitochondria factors. The Bcl-2 family that consists of over 20
pro- or anti-apoptotic proteins is a major player
in the mitochondrial apoptosis pathway. The pro-
Apoptotic State:
apoptotic subfamily includes Bax, Bak, and Bok,
Upon insult of apoptotic inducing agents or environment which all contain BH1, 2, and 3 homology domains.
factors
• Anti-apoptotic factor(s) had conformational change Upon exposure to death signals, Bax undergoes
(exposing BH3) conformational changes and translocates to
• Membrane disruption releases cytochrome C the mitochondria, where it inserts into the
and other pro-apoptotic and apoptogenic factors outer mitochondrial membrane and forms
• The releases of procaspase3 form apoptosome channels. These channels allow the leakage of
with the adaptor (Apaf -1), becomes capase-3. cytochrome C and other pro-apoptotic molecules.
• Caspase-3 converts procaspase-9 to caspase-9
which becomes the executioner caspase and starts Cytochrome C proceeds to form a complex with
the apoptotic cellular event Apf-1, pro-caspase 9, and dATP, known collectively
as the apoptosome. In the apoptosome, the inactive
pro-caspase 9 is activated and the active enzyme
subsequently activates downstream caspases.
Two anti-apoptotic proteins, Bcl-2 and Bcl-xL,
counter the actions of the pro-apoptotic components.
Bcl-2 is localized on the mitochondrial membrane
and inhibits the release of pro-apoptotic molecules
from the mitochondria by maintaining membrane
integrity. Bcl-xL is localized in the cytoplasm and
binds to pro-apoptosis members of the Bcl-2 family.
The involvement of multiple protagonist and
antagonist factors ensures tight control of apoptotic
event. This scheme also provides an amplification
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of the desired signal. An excellent example of this
is the cascade of caspases. When cell destruction
is needed, the signal is greatly amplified through
a series of steps where one caspase activates
another caspase in an exponential fashion.
Fig. 2.19: Mitochondrial pathway mediated apoptosis
Senescence and Telomeres

The vast majority of animal cells isolated from tissues
require surface adhesion in order to multiply, since
they are anchorage-dependent cells. Cells are typically
isolated from tissues by an enzymatic dissociation of
the tissue. After dissociation and the removal of large
chunks of undissociated debris, cells are plated on a
compatible surface overlaid with media. Under the
microscope, cells in media suspension can be seen to
attach to the surface or out-grow from the remaining
tissue chunks. Subsequently, they extend their
body length, spread and begin to multiply. Those
cells derived from normal tissues generally possess
two sets of chromosomes and are diploid cells.
Fig. 2.20: “life” of normal cells from isolation to Eukaryotic cells enter an exponential growth phase,
senescence and occurrence of cell line similar to microbial cells. The growth rate slows as
they begin to cover the entire surface area to form a
“monolayer”. Upon reaching confluence, they stop
dividing. While the cell bodies of neighboring cells
may cross each other, their nuclei never overlap.
This is called contact inhibition of cell growth.
Cells can be dissociated from a surface after being
treated with trypsin (i.e., trypsinized) or with other
proteases. They can then be plated on a larger surface
area for continued growth. This process can then
be repeated to expand the population. Each round
of detachment and expansion is called a “passage”.
Non-immortalized cells cannot be grown in culture
indefinitely by simple repeated passage in culture.
Normal diploid cells from animals (except stem

cells) have a limited life span in culture. Fibroblasts
(a cell type from connective tissue) isolated from
a mouse embryo can be cultured in vitro for about
60 doublings. As that limit approaches, the cells
begin to fail to reach confluence. Eventually, high
passage cells will cease to grow. This is referred
to as having reached “crisis”. Such a limit in
tissue dissociation the proliferating potential is called “Hayflick’s
Plating in nutrient medium
phenomenon”. It is a common phenomenon for
all normal diploid cells obtained from vertebrates.
In a historical experiment carried out half a century
ago, the continued passaging of mouse fibroblast
cells beyond crisis gave rise to a small fraction of
Growth survivors. These cells eventually grew, expanded,
Replating to larger surface area
and could be cultured continuously in vitro without

Contact inhibition a limited life span. They were given the name 3T3
because the cells were passaged every three days by
expanding the surface area three times more. These
cells appear normal and are subjected to contact
Cell Dissociation
inhibition of growth under typical culture conditions.
However, although the parental mouse fibroblast
cells had diploid set of chromosomes before
reaching crisis, 3T3 cells have abnormal number
of chromosomes. Cells that succumb to Hayflick’s
constraint (e.g., those that are diploid and have a
Fig. 2.21: Anchorage dependency and contact
inhibition of cultured normal diploid cells limited life span) are called “cell strains.” The cells
that reestablish after crisis and can grow in culture
indefinitely, but are aneuploid (do not have a normal
set of chromosomes), are referred to as “cell lines.”
Cells derived from cancer also give rise to cell lines.
Cell lines can also be established by “immortalization”
through viral or oncogene transformation. These
transformed cells are also often aneuploid. They
are capable of growing beyond the monolayer, since
they are not subject to contact inhibition of growth.
Normal diploid cells, thus, appear to “count” their
number of doublings. They achieve this by using
their telomeres. Telomeres are special repetitive
sequences at the end of chromosomes. They are
not replicated by DNA polymerase during DNA
Fig. 22: Telomere at the end of a chromosome
replication, but are synthesized by telomerase. The
reaction is not precise for accurately reproducing
CELL BIOLOGY
54 | CELL | 54
BIOLOGY
the number of tandem repeats of the sequence, thus
there can be much variation in telomere length among
cells. As the number of passages increases, telomeres
decrease their length unless they are repaired by
telomerase. For instance, in stem cells, telomerase
activity is high to maintain the telomere length. Unlike
cell strains, stem cells do not exhibit senescence.
Concluding Remarks
In this long chapter we provided a condensed to be bound by the nature of cells; rather we should
overview of knowledge in cell biology that is employ means to “adapt” them to serve our goals
essential for biotechnologists to practice cell culture better. For example, most cells used for biologics
bioprocessess. The structure and make-up of production were anchorage-dependent originally
cells that gives them their functional versatility, and are now cultivated not only in suspension, but
also constrains their capability. In practicing also in highly turbulent flow conditions. Most of the
biotechnology, while exploiting their biological adaptation processes in the past two decades were
versatility, we also must understand cell’s structural conducted empirically. At a molecular level, what
and functional constraints and biological limits. In causes those cells to have the adapted behavior is
the meantime, we must also keep in mind that our poorly understood. By equipping ourselves with a
objectives are frequently different from scientists better knowledge of cell’s capability and limits, we will
studying the biology of the cell. To fully harness a be able to push the technological boundary further.
cell’s biological potential, we do not necessarily need

CELL BIOLOGY
56 | CELL | 56
BIOLOGY

Cell Physiology for Process

Engineering
Overview of Central Metabolism of Cultured Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Glucose and Energy Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Oxidation of Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Pentose Phosphate Pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Lactate Formation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Carbon Flow and Reaction Intermediates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
NADH Balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Role of Transport and Transporters in Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Glucose Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Lactate Transport. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Transport Across Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Regulation of Glucose Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Isozymes and Differential Allosteric Regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Signaling Pathways and Regulation by Growth Control . . . . . . . . . . . . . . . . . . . . . 77
Metabolic Homeostasis and Lactate Consumption. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Glutamine and Its Relation to Energy Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . 80
Amino Acid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Lipid Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Lipid Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Fatty Acid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Acetyl CoA shuttle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Cholesterol & Its Biosynthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Glycan Biosynthesis and Protein Glycosylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Importance of Glycan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Protein Folding and Glycosylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Glycan Extension in Golgi Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Glycan Types and Microheterogeneity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
Synthesis and Transport of Nucleotide Sugar Precursor. . . . . . . . . . . . . . . . . . . . . 93
Glycan Diversity Among Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
CELL PHYSIOLOGY FOR PROCESS ENGINEERING | 57

Overview of Central Metabolism of Cultured Cells
Glucose Oxidation Cells in culture take up sugar, amino acids, lipids,
C H O + 6O
6 12 6 2 6CO + 6H O
2 2
and nucleosides from their growth media. They
metabolize these components to derive energy and
Glucose Anaerobic Metabolism use them as building blocks to generate more cell
CH O
6 12 6 2CH6 $ CHOH $ COOH mass, create more cells, and produce products. The
processes of making more cell mass and protein
Glutamine Oxidation product are very energy intensive. Proteins constitute
CO (NH ) CH CH CH (NH ) COOH + 4.5O
2 2 2 2 2
over 50% of the dry mass in a typical cell; they are
2NH + 5CO + 2H O
3 2 2
essentially amino acids connected by peptide bonds.
The synthesis of each peptide bond costs at least 3
ATP, which is nearly 1/10 of the amount of energy that
can be obtained by oxidizing one glucose molecule.
One high-producing recombinant cell produces over
40 pg per day of IgG protein. Since an average cell
has about 400 pg of cell mass (or about 200 pg of
cellular proteins) it is easy to see that producing the
protein product is a major energetic load for cells.
A classical cell culture medium contains 1 – 5 g/L
of glucose, and somewhat lower levels of amino
acids (about 0.8 mM, or 1 g/L). The sum of the
nutrients, together, typically generates only about
1 – 3 x 109 cells/L, or approximately 0.1 – 0.3 g/L
of cell dry mass. The efficiency of producing cell
mass from glucose and other nutrients is rather low.
Glucose is the most important source of energy for
most cells. Even when another sugar such as galactose
or fructose is used as the sole carbohydrate source,
it still enters the metabolic pathway that has evolved
Fig. 3.1: Lactate and ammonium profiles in manufacturing runs for glucose (called glycolysis) to get catabolized.
with high product titers (blue) and low product titers (red).
Lactate profile correlates to productivity, but not ammonium. The complete oxidation of one glucose molecule
consumes six O2 and generates six H2O and six CO2. For
cells in culture, however, the majority of consumed
glucose is not completely oxidized; it is converted to
lactate and excreted. By converting to lactate instead
of completely oxidizing to CO2, much less energy is
derived from each mole of glucose. This is the root
cause of the low efficiency in conversion from glucose
to cell mass. For some cells, especially transformed
cell lines, each mole of consumed glucose produces
almost two moles of lactate, which is the theoretical
maximum of the glucose to lactate conversion.

Glucose, Glutamine Major Carbon Source This type of “wasteful” metabolism is common
• Both glucose and glutamine consumed in excess to to almost all vertebrate cells in culture. For
what are need to grow biomass bioprocessing, the accumulation of these byproducts
• Most glucose consumed, converts to lactate
• Excess glutamine consumption, results in ammonium inhibits cell growth and impedes productivity.
excretion Cells invariably produce lactate from glucose when
Lactate Acid and Fedbatch Culture Productivity growing rapidly. However, under some conditions,
• Lactate is produced in exponential growth phase such as the stationary phase of fed-batch culture,
• In stationary phase, some cultures continue to produce lactate may also be consumed. It is not unusual
lactate, others switch to lactate consumption
• Lactate consumption correlate to sustained higher
that under the same operating conditions, different
viability and high productivity culture runs have different metabolic outcomes. In
some runs, lactate production in the rapid growth
phase continues into the stationary phase. In
others, the transition from lactate production to
lactate consumption occurs in the stationary phase.
When production data from a manufacturing
plant were analyzed, it was found that the top
productivity runs switched from lactate production
to lactate consumption, while low productivity runs
remained in lactate production mode throughout the
culture. This is an indication that cell metabolism
plays a key role in determining productivity.

Glucose and Energy Metabolism
Glucose is mainly catabolized through three
pathways: glycolysis, the pentose phosphate
pathway (PPP), and the tricarboxylic acid cycle
Glucose Oxidation (TCA) cycle. In glycolysis, one mole of glucose is
converted to two moles of pyruvate. In this segment
• Main metabolic pathways in energy metabolism:
of catabolism, only a small fraction of the chemical
• Glycolysis
potential energy of glucose is converted to the
• TCA cycle (tricarboxylic acid cycle)(Kreb cycle)
“usable” form of chemical potential energy in the
• Pentose phosphate pathway (PPP) cell, i.e., ATP. Two moles of ATP are generated
per one mole of glucose. Pyruvate may enter the
TCA cycle for further oxidation, or it may become
a shunted product as lactic acid (at a neutral pH it
exists as lactate). Through the TCA cycle, the carbon
skeleton of glucose is finally broken down to CO2 and
H2O. The PPP is a shunt from glycolysis. It generates
five-carbon sugars for nucleoside synthesis
and supplies NADPH for many biosynthesis
reactions and to maintain a redox state in the cell.
In eukaryotic cells, glycolysis and PPP take
place in the cytosol, while the further oxidation
of pyruvate to CO2 occurs in the mitochondria.
It is in the mitochondria that the majority of
the chemical potential energy of glucose is
converted to ATP for use in cellular synthesis
and other energy-dependent cellular processes.
Oxidation of Glucose In glycolysis, glucose is broken down to two

pyruvates and generates two ATP and two
NADH. Pyruvate may then be further converted
to lactate as a final product, instead of entering
the TCA cycle. Thus, when glucose metabolism is
terminated at glycolysis, it produces two lactate
molecules and two ATP, but no additional NADH.
Although overall glycolysis generates high-energy
compounds (e.g., ATP and NADH), its first segment
actually consumes ATP. Two ATP are used to add a
phosphate group to each end of the glucose molecule.
The two phosphate groups pull their surrounding
electron clouds toward the two ends of the
molecule, thereby making the carbon-carbon bond

in the middle susceptible to enzymatic cleavage.

After cleavage, the six-carbon skeleton becomes
Glycolysis - Each Mole of Glucose (6 Carbons)
two three-carbon compounds: glyceraldehyde-3-
• Consumes two moles ATP (to activate to fructose, 1,
6-bisphosphate) phosphate (G3P) and dihydroxyacetone-phosphate
• Produces two moles NADH, 4 moles ATP (DHAP). These two compounds are interconvertable
• Net: through a reversible reaction. The continued
Becomes 2 Pyruvate reaction of glyceraldehyde-3-phosphate (G3P)
Produces 2ATP, 2NADH
effectively draws DHAP toward G3P and moves it
further downstream in glycolysis. The conversion
of two G3P to the end product of two pyruvates also
TCA cycle converts two NAD+ and four ADP to two NADH and
• Pyruvate enters mitochondrion four ATP. The net energetic consequence of the
• Pyruvate loses CO2, becomes acetyl CoA (2 carbons) conversion of glucose to two pyruvate in glycolysis
• Acetyl CoA enters TCA cycle,
is the generation of two ATP (because two ATP
• Becomes 2 CO2
• Produces NADH, FADH2, which stores energy are consumed to activate glucose) and two NADH.
• Never reacts with oxygen directly The further oxidation of pyruvate takes place in the
mitochondria, where it is first converted to acetyl
CoA, releasing one CO2 and generating one NADH.
Acetyl CoA is then fed into the TCA cycle where it is
NADH, FADH2 enter electron transfer pathway broken down to two CO2. The pathway is cyclic, with
passes its high energy electron down, also its proton four- to six-carbon skeletons cycling in a loop. At the
• As electron passes on energetic ladder, it pumps
beginning of the cycle, the four-carbon oxaloacetate
protons out of mitochondrion
• create a high pH inside mitochondrion (OAA) takes in acetyl CoA to become citrate. Citrate
• also a negative charge of ~120 mV across has three carboxylic acid groups; hence the name
mitochondrion inner membrane “tricarboxylic acid cycle”. The TCA cycle is also
• The electron and proton, at the bottom of energetic known as the citric acid cycle and the Krebs cycle.
ladder, react with oxygen to form water.
As noted before, molecular oxygen does not react
with the carbon compounds in the reactions from the
TCA cycle. CO2 is released, through decarboxylation
Oxidative Phosphorylation Pathway reactions, from the carbon skeleton without the
• The higher concentration of proton in cytosol and the participation of molecular oxygen. In two of the
negative charge inside mitochondrion drives proton to reactions catalyzed by pyruvate dehydrogenase
move into mitochondria
and α-ketoglutarate dehydrogenase, the energy
• Those protons moves into mitochondrion by
passing through ATP synthase; as they pass through from the breakup of the C-C bond is preserved in
ATP synthase, ADP is converted to ATP inside the high-energy compounds acyl CoA (acetyl CoA
mitochondrion and succinyl CoA, respectively) and NADH. In the
• There are a lot of fluxes across mitochondrial other case, one of the three carboxylic acid groups
membrane, inclusind
in citrate is released and one NADH is generated.
• going in: pyruvate, ADP, phosphate, H+,
• going out: ATP, CO2 If the carbon-carbon bond is broken by directly
reacting with oxygen, such as the case for
combustion, a very high temperature is necessary
to provide the activation energy. As we all know,
it takes a fire to burn wood. Furthermore, the

Figure 3.2. Major pathways in energy metabolism. Glucose and glutamine uptake, glycolysis, lactate excretion,
TCA cycle, and oxidative phosphorylation.
Symbols of metabolites in energy metabolism:
Glc: Glucose; g6p:Glucose 6-phosphate; f6p:Fructose 6-phosphate; f16bp (f16p2): Fructose 1,6-bisphosphate; f26bp (f26p2): Fructose
2,6-bisphosphate; gap: Glyceraldehyde 3-phosphate; Dhap: Dihydroxyacetone phosphate; 1,3bpg: 1,3-bisphosphoglycerate; 3pg:
3-phosphoglycerate; 2pg: 2-phosphoglycerate; pep: Phosphoenolpyruvate; pyr: Pyruvate; lac: Lactate; NADH: Nicotinamide adenine dinucleotide
(reduced); NAD: Nicotinamide adenine dinucleotide (oxidized); NADPH: Nicotinamide adenine dinucleotide phosphate (reduced); NAPD:
Nicotinamide adenine dinucleotide phosphate (oxidized); Gln: glutamine; Glu: glutamate; Asp: aspartate; ala: alanine; Mal: malate; αKG:
α-ketoglutarate; OAA: oxaloacetate; SucCoA: succinyl CoA; 6pg: 6-phosphogluconate; ru5p: ribulose 5-phosphate; r5p: ribose 5-phosphate; xyl5p:
xylulose 5-phosphate; e4p: erythrose 4-phosphate; s7p:sedoheptulose 7-phosphate
Symbols of enzymes and transporters in energy metabolism:

GLUT: Glucose transporter; HK: Hexokinase; GPI: Glucose phosphate isomerase; PFK: Phosphofructokinase; PFKFB: 6-phosphofructo-2-kinase/
fructose-2,6 bisphosphatase; ALDO: Aldolase; TPI; Triosephosphate isomerase; GAPD: Glyceraldehyde 3-phosphate dehydrogenase; PGK:
Phosphoglycerate kinase; PGM: Phosphoglycerate mutase; ENO: Enolase; PK: Pyruvate kinase; LDH: Lactate dehydrogenase; PYRH: Pyruvate
mitochondrial transporter; G6PD: Glucose 6-phosphate dehydrogenase; 6PGD: 6-Phosphogluconate dehydrogenase; RPE: Ribulose phosphate
epimerase; RPI: Ribose phosphate isomerase; TK: Transketolase
TA: Transaldolase; PRPPS: Phosphoribosylpyrophosphate synthetase; PYRH: Pyruvate mitochondrial transporter; MCT: monocarboxylate transporter

chemical potential energy would have been released

as heat. Cells utilize decarboxylation reactions
to form CO2 and to preserve energy in NADH.
Oxygen is then used to extract chemical potential
energy from NADH and FADH2, in order to
generate ATP that can be used in cellular work.
The participation of oxygen in the oxidation of
NADH/FADH2 generates the six O2 molecules
required to oxidize one glucose, as shown in the
stoichiometric equation of glucose oxidation.
Extraction of the chemical potential energy of
NADH and FADH2 takes place through an electron
Fig. 3.3: Structure of key compounds in glycolysis. transfer chain residing in the mitochondrial inner
membrane. The high-energy electrons of NADH
and FADH2 enters the electron transfer chain
down the energy ladder, mediated by electron
carriers such cytochrome C. The energy released
is then used to trigger a proton pump to drive
H+ out of the mitochondrial inner membrane. In
the last step of the electron transfer chain, the
electron reacts with oxygen and H+ to form water.
The export of H+ from the mitochondria creates a
single unit pH difference across the membrane, as
well as about -120 mV of electric potential. Because
of the higher pH (lower proton concentration) and
excess negative charge inside the mitochondrial
membrane, there is a propensity for the proton ions
outside the mitochondria to cross the mitochondrial
membrane. They enter the mitochondria through
an ATP synthase embedded in the inner membrane
of mitochondria. Through the process, the act of
a proton passing through ATP synthase brings an
ADP and a phosphate together to synthesize ATP.
Fig. 3.4: Structure of key compounds in TCA cycle The electron transfer and the generation of ATP are
and glutamine metabolism. often referred to as “oxidative phosphorylation”.
The amount of ATP generated per mole of glucose
varies somewhat among different species because
of their variable expression of ATP synthase.
However, in general, the number of ATP generated
per mole of glucose is about 30 – 32 for mammals.
The older literature tends to list the number as
36 moles of ATP / mole of glucose. Under some
physiological conditions, the electron transfer chain

and oxidative phosphorylation are uncoupled.
Instead of generating ATP, the energy from NADH
is released as heat to maintain body temperature.
The amount of energy, two ATP and two NADH (or
the equivalent of six ATP, since one NADH in the
cytosol can be roughly considered to be two ATP),
produced from splitting glucose into two moles of
pyruvate is only about 1/6 of what can be generated
from completely oxidizing glucose to CO2 and H2O. In
glucose oxidation, the majority of energy conversion in
glucose metabolism therefore occurs in the hundreds
of mitochondria in the cell and not in the cytosol.
Pentose Phosphate Pathway PPP is an important shunt from glycolysis that

supplies five carbon sugars and NADPH. PPP is
divided into two segments: an oxidative segment
and a monosaccharide transformation pool.
In the first segment, glucose-6-phosphate from
glycolysis is oxidized and then decarboxylated to
form the five-carbon ribulose-5-phosphate and two
PPP NADPH. The five-carbon sugar phosphate is used
• Two segments: in nucleotide (such as ATP and dATP) synthesis
• Oxidative: remove 1 CO2 from glucose-6- to supply the building units for RNA and DNA.
phosphate,
Cells use two different nicotinamide-adenine
• Generate 5 carbon sugar phosphate for
dinucleotides as reductive chemical potential
synthesis of nucleotides and other compounds
• produce 2 NADPH energy carriers: NADH and NADPH. NADH is used
• Molecular transformation to store chemical potential energy in glycolysis,
• Interconverts 5 carbon sugar phosphate to 3 the TCA cycle, and lipid catabolism. Eventually,
carbon and 6 carbon NADH is used to derive ATP in the mitochondria.
• To allow NADPH and 5 carbon sugar to be
NADPH, on the other hand, carries a chemical potential
produced at different ratios
• NADPH is important in biosynthesis and in that is used in biosynthetic reactions, such as in the
neutralizing ROS synthesis of lipids, nucleotides, etc. NADPH is also
used to reduce oxidized glutathione and to regenerate
it. The reduced form of glutathione is important in
maintaining the cell’s reductive environment and in
the suppression of reactive oxygen species (ROS).
The second segment of PPP is a molecular conversion
pool that allows a two-carbon aldehyde unit or
three-carbon keto units to be translocated among
a number of three-carbon to five-carbon aldoses.

This allows the interconversion of carbohydrate

molecules that are three to seven carbons in length.
This “mixing pool” enables five-carbon sugars
from the first segment of PPP to be connected to
glycolysis through six-carbon fructose-6-phosphate
or three-carbon glyceraldehyde-3-phosphate.
The first segment of PPP generates five-carbon
ribulose and NADPH at a molecular ratio of 1:2.
However, cells do not always need those two
compounds at a 1:2 proportion. The molecular
conversion in the second segment allows the net flux
from glycolysis to PPP to vary to meet the cellular
demand of ribose and NADPH at different proportions.
Lactate Formation Under anaerobic conditions, some bacteria and

yeast produce ethanol or lactate. In the absence of
Aerobic Glycolysis
oxygen, the electron transfer chain does not operate
• Cultured cells and cancer cells undergo glycolysis
and produce lactate even at high oxygen because no oxygen is available to receive the
concentration electron from NADH. When NAD is not regenerated
• This propensity toward lactate production is not by NADH oxidation, the TCA cycle ceases to
for lack of oxygen (anaerobic glycolysis) operate. The accumulated pyruvate from glycolysis
• At high glycolysis flux, not all NADH can be is then excreted as lactate or ethanol. (Note
oxidized by electron transfer in mitochondria that we denote NAD+ as NAD in text without the
• Lactate production serves to regenerate NAD superscript “+” associated with its positive charge).
• continuous supply of NAD is crucial for glycolysis Mammalian cells in culture take only a small portion
to proceed
of pyruvate generated in glycolysis into their
mitochondria, for further oxidation to CO2. They
appear to have a limited capacity to translocate
pyruvate into the mitochondria. The rest of glucose
is converted to lactate. This occurs in spite of the
presence of sufficient oxygen. The phenomenon is,
thus, different from anaerobic fermentation in bacteria
or yeast, and is referred to as “aerobic glycolysis”.
Not all cells in our body convert a large portion
of the glucose they take up into lactate. The vast
majority of cells in our body are in a quiescent (non-
proliferating) state. They consume less glucose than
proliferating cells. The contrast in cellular glucose
metabolism, known as the Warburg effect, was first
observed between normal tissues and cancer cells.
While normal cells have a lower glucose flux, cancer
and proliferating cells consume a larger amount of

glucose and convert much of the glucose to lactate.
Lactate synthesis is catalyzed by lactate
dehydrogenase. This reversible reaction
Energetic Yield of Aerobic Glycolysis takes one pyruvate and one NADH
1. Oxidation to become one lactate and one NAD.
Glu cos e + 2ADP + 2Pi + 2NAD+ 2Pyruvate In glycolysis, two ATP and two NADH are
+ 2ATP + 2NADH generated, along with two pyruvate. Continued
glucose metabolism through glycolysis requires
2. Reduction [oxidizing pyruvate to lactate (or ethanol as
in yeast)] continued supplies of both ADP and NAD as
reactants. ATP is used by cells to perform many
2Pyruvate + 2NADH 2Lactate + 2NAD + tasks, such as synthesis, maintaining osmotic
balance, etc. It is continually being consumed in
Net Reaction: various cellular reactions and converted back
Glu cos e + 2ADP + 2Pi 2 Lactate + 2ATP to ADP to resupply the reactant for glycolysis.
NADH is converted back to NAD through the electron
transfer chain in mitochondria. To be regenerated in
the electron transfer chain, cytosolic NADH must first
enter the mitochondria and the regenerated NAD
must be exported out of the mitochondria. A reaction
with lactate dehydrogenase allows NAD regeneration
from NADH to be carried out into the cytosol,
thereby enabling glycolysis to continue at a high flux.
Cells in culture convert about 90% of their glucose
to lactate. Most of the other 10% of glucose is
converted to CO2. At the completion of glycolysis,
two ATP are generated while about 30 ATP are
generated, following complete oxidation. The
90% of glucose converted to lactate generate
1.8 ATP (2 ATP x 0.9), while the other 10%
generate 3 ATP (30 ATP x 0.1). Aerobic glycolysis
of proliferating cells generates a significant
amount of total energy to allow for proliferation.

Carbon Flow and Reaction Among all of the pathways in the cellular metabolic
Intermediates reaction network, glycolysis has the highest flux
in terms of moles of substrate flowing through
it. For cells in culture, carbon flux (based on
the number of moles of carbon atoms, or the
Pyruvate is a controlling node number of carbons in the compound multiplied
• Generation rate of pyruvate is balanced by entry into by the number of moles of the compound) or
the mitochondrion and conversion to lactate molar flux (based on the number of moles of each
• Reduction of pyruvate to lactate recycles NAD+ for compound) of glycolysis is normally a few times
glycolysis to continue higher than that of the TCA cycle. PPP flux usually
• Lactate dehydrogenase (LDH) is reversible constitutes only about up to 5% of glucose intake.
• Transport of lactate by monocarboxylate transporter
which is coupled to proton gradient Glycolysis and the TCA cycle also supply precursors
to build cellular components. Culture media do not
necessarily supply cells with the right balance of
all of the components that they need to synthesize
cell mass. The three main pathways for energy
metabolism also serve as key distribution centers
for carbon skeletons needed for other cellular
functions. Glycolysis supplies glycerol phosphate,
which is for the synthesis of phospholipids.
Lactate dehydrogenase reaction
Pyruvate + NADH Lactate + NAD+ Glucose-6-phosphate and fructose-6-phosphate
are both a source of nucleotide sugars for glycan
synthesis, such as UDP-galactose, UDP-glucose, and
GTP-mannose. Except for liver cells (hepatocytes),
cells in culture have little gluconeogenesis activity;
that is, they cannot make hexose from lactate or
amino acids. So, even if cells can derive energy
from lactate and amino acids, they will still
need hexose to synthesize ribose and glycans.
Cells in culture take up a large quantity of amino

acids, especially glutamine. The intake of amino
acids exceeds what is needed to make cell mass and
product. The surplus of nitrogen is either excreted
as ammonia or transferred to pyruvate to form
alanine, and excreted. Since alanine is much less
growth inhibitory than ammonium, pyruvate has
some moderating effect on ammonium toxicity.

NADH Balance A total of 12 moles of reducing equivalent (10
NADH and 2 FADH2) are produced when 1
mole of glucose is oxidized to CO2. The 12 mole
• While glucose is oxidized in Glycolysis and TCA cycle, reducing equivalents consume 6 moles of O2 in
its carbons never react directly with O2 oxidative phosphorylation, consistent with the
• The energy is preserved to NAPH/FADH2, which is stoichiometry of glucose oxidation (1 glucose/6
then reacted with O2 in oxidative phosphorylation in O2). Among the 12 NADH/FADH2, 10 are produced
mitochondria to generate ATP in the mitochondria and the other 2 NADH are
• Altogether 12 reducing equivalents (NADH/FADH2) are produced in cytosolic glycolysis. The two reducing
generated to react with 6O2, generating 6H2O equivalents produced in the cytosol must then be
• 2 of the 12 reducing equivalents, two are generated in transported into the mitochondria where they drive
cytosol (in glycolysis) 10 in mitochondria the reaction that consumes the 6th molecule of O2.
NADH does not pass through the inner membrane

of mitochondria. Rather, it passes its reducing
potential through a carrier system called the malate-
aspartate shuttle. This system takes the reducing
equivalent into mitochondria through an exchange
of molecules between the mitochondria and the
cytosol. On the cytosolic side, NADH is oxidized
to NAD and transfers its reducing equivalent to
Glucose 6 Phosphate
malate by reducing oxaloacetate (OAA). Malate
NAD is then transported across the mitochondrial
membrane. Once inside the mitochondria, the
NAD
reducing equivalent in malate is transferred back
NAD NADH
to NADH by being oxidized to again become OAA.
Lactate Pyruvate
malate NAD
aspartate
shuttle
cytosol The net result of the transfer must be that only one
mitochondrion NADH in the cytosol can become one NADH in the
reducing oxidized
equivalent Pyruvate
mitochondria. To maintain that balance, cells employ
NADH NAD Equal moles of
Pyruvate and two transporters: one for transporting malate and
NADH reducing
equiavlent enters
the other for transporting aspartate (hence the
mitochondria name “malate-aspartate shuttle”). To maintain the
Election transfer
chain
NADH- Pyruvate Balance charge balance in the transport process, each of the
two transporters transfers a pair of compounds in
opposite directions (malate and α-ketoglutarate;
aspartate and glutamate). In this fashion, there
Fig. 3.5: NADH/NAD balance in cytoplasm. NADH generated
in glycolysis is recycled back to NAD via lactate production is no net change in total carbon or nitrogen on
and malate-aspartate shuttle to transfer the reducing either side of the mitochondrial membrane.
equivalent into mitochondria.
On each side of the membrane, the same glutamate-

OAA to α-ketoglutarate-aspartate amino-transfer
reaction occurs, but it is in the opposite direction.
The transfer of the reducing equivalent of NADH into

the mitochondria is therefore not only dependent

on the NADH concentration but also linked to amino
acid metabolism and the activities of the TCA cycle,
through the concentrations of associated carriers.
Glycolysis and the TCA cycle are thus connected by a

cytosolic balance of pyruvate and NADH. The flux of
both pyruvate and NADH are stochiometrically linked
in glycolysis, as well as through the LDH reaction. As
a result, the fluxes of pyruvate and NADH entering
the mitochondria are also stoichiometrically
related. Regardless of whether cells are at a
lactate-producing or -consuming state, the molar
ratio of pyruvate flux to NADH flux is always one.
Glyceraldehyde 1,2-Bisphospho Pyruvate
Glucose
3-Phosphate glycerate
NAD+ NADH
QAA NAD+
Lactate
Malate-Aspartate
Shuttle
Aspartate Glu αKG Malate
Mitochondria
Aspartate Glu αKG Malate
NAD+
Pyruvate
NADH
Oxidative Phosphorylation
OAA
ATP
Fig. 3.6: Detailed reactions of malate-aspartate shuttle.
• Each enzyme reaction/co-transportation is given in the same color

• Direction of reaction in NHDH reducing equivalent transport is indicated by an arrow
• Net transport of one reducing equivalent-1 NADH in cytosol, and +1 NADH in mitochondria
• Transport of 1 malate + 1 glutamate into mitochondria and 1 αKG + asparate in cytosol
Fig. 3.7: Transfer of NADH reducing equivalent from cytoplasm into mitochondria requires
continuous transport of four malate, aspartate, glutamate and α-ketoglutarate.

Fig. 3.8: Biochemical reactions in glycolysis, TCA cycle and Pentose phosphate pathway
Energetic Yield of Oxidation of Glucose

Cytosol
glu cos e 2pyruvate + 2NADH + 2ATP
Mitochondrion
2pyruvate 2acetylCOA + 2NADH + 2ATP + 2CO2
2acetylCoA 6NADH + 2FADH2 + 2GTP + 4CO2
• The overall energetic yield is ~30ATP, considering the cost of ATP transport out of mitochondria
• The NADH generated in cytosol (Glycolysis) is recycled back to NAD+, either through reduction of
pyruvate to lactate, or by NADH/NAD+ shuttle into mitochondria for oxidation.

Role of Transport and Transporters in Metabolism

Energy metabolism takes places in multiple
compartments, separated by lipid bilayer membranes.
First, glucose must cross the cytoplasmic membrane
to undergo glycolysis in the cytosol. The products
from glycolysis (pyruvate and NADH, in the form
of reducing equivalents) are transported into the
mitochondria. This exchange of molecules across the
membrane is mediated by membrane transporters.
Glucose Transporters Glucose transporters mediate the influx

of glucose across the cytosolic membrane.
Two Main Types of Glucose Transporters Generally speaking, there are two types
• GLUT transporters mediate facilitative diffusion across of glucose transporters: GLUT and SGLT.
plasma membrane.
The GLUT transporters are uniporters for
• SGLT, the sodium dependent glucose co-transporters
facilitated transport, allowing glucose to move
are expressed primarily in small intestinal absorptive
cells or renal proximal tubular cells. They use Na+-K+ along its concentration gradient. They have twelve
ATPase pump for active transport of glucose. transmembrane regions and intracellular carboxyl
and amino termini. According to common sequence
motifs, they are divided into three subclasses.
Glucose Transporter Isoform GLUT1 is ubiquitous, found in almost all cells. It can
transport glucose and galactose in a concentration-
• GLUT1 is highly expressed in all cells
dependent manner that is described by Michalis-
• Km is small for GLUT1. At culture glucose concentration
it operates at maximum rate. Menten kinetics. The Km for glucose is very low (1 –
2 mM). At the glucose concentration used in culture
medium, the flux of GLUT1 is at its maximum. In
some cells, GLUT1 is under the regulation of the
transcription factor HIF-1 (hypoxia inducible factor).
Under hypoxic conditions, the expression of GLUT1
is up regulated to increase the uptake rate of glucose.
The Km of GLUT1 for galactose is rather high.
When galactose is used as the only sugar, even
at a concentration of 25 mM, the uptake rate
is so low that only a little lactate is produced.
A few other notable GLUT transporters are: insulin
responsive GLUT4 and fructose transporting GLUT5.
In addition to GLUT1, cells in culture and in different
tissues may express other GLUT transporters at
Fig. 3.9: Michaelis-Menten kinetics plot for GLUT1 transporter
different proportions. The expression of different
transporters will give them different responses
to the concentration of glucose or other sugars.

The second type of glucose transporter, SGLT, is a
Table 1: Glucose Transporters co-transporter with Na+. It transports two sodium
Tissue ions and one glucose molecule into the cell. The
Affinity
Expression Na+ concentration is low intracellularly but is high
GLUT1 ubiquitous glucose, K m = 1 - 2 mM
in the medium and in body fluid. The large sodium
Liver, pancreas, glucose, K m = 16 - 20 mM
GLUT2 concentration difference and negative electric
intestine, kidney glucosamine K m = 0.8 mM
Class 1
GLUT3 brain, neurons glucose, K m = 0.8 mM potential across the cytoplasmic membrane gives
heart, muscle, rise to a high propensity of Na+ to enter the cell.
GLUT4 glucose, K m = 5 mM
adipose
GLUT5 intestine, testis fructose K m = 10 - 13 mM Thus, the chemical potential energy of the sodium
glucose, Km = 0.3 mM gradient and electric potential is used to drive the
GLUT7 intestine, testis
Class II fructose K m = 0.1 mM uptake of glucose against a concentration gradient.
GLUT9 kidney, liver fructose K m = ? mM
GLUT11 heart, muscle fructose K m = ? mM SGLT transport is abundant in intestinal epithelial
brain, spleen, cells and is responsible for moving glucose
GLUT6 glucose, K m = 5 mM
leukocytes
GLUT8 testis, brain, liver glucose, K m = 6 mM from the gut into the intestinal epithelial cells.
Class III GLUT10 liver, pancreas glucose, K m = 0.3 mM The glucose is then exported into the blood
heart, muscle,
GLUT12
prostate
not well known stream on the other side of the cellular barrier.
Lactate Transport Lactate and pyruvate are transported by

monocarboxylate transporters. These transporters
exist in two forms: one on the cytoplasmic membrane
and one on the mitochondrial inner membrane.
Lactate and pyruvate are both negatively charged.
Their movement across the cellular membrane will
cause a charge unbalance and create an electric
potential, unless measures are taken to counter
that imbalance. The monocarboxylate transporters
(MCT), which are responsible for their transport, are
a family of co-transporters that couple the transport
of lactate or pyruvate to the transport of a hydrogen
ion in the same direction to neutralize the charge
transfer. MCT is thus a symporter; its mechanism of
transport is facilitated diffusion.
Fig. 3.10: Monocarboxylate transporter for lactate Lactate transport is enhanced by a large difference
and pyruvate in lactate concentration between intracellular
and extracellular environments. pH also affect the
flux of lactate through MCT, however, whether the

effect is enhancing or retarding is dependent on the

direction of the proton gradient. MCT allows for
lactate transport in both directions, for excretion as
well as uptake. Keeping medium pH at a lower level
reduces lactate production during rapid growth
period, but enhanced lactate consumption in the
stationary phase.
Transport Across Mitochondria Cells in culture typically channel about 1/20 of the
carbons from their glucose intake to the TCA cycle
• The chemical potential energy generated by pyruvate
and oxidize them to CO2. The molar flux of pyruvate
oxidation/TCA cycle, i.e. NADH and FADH2, is not
necessarily all converted to ATP. The process can be into the mitochondria is thus about 1/10 of that of
decoupled to generate heat instead of ATP, as occurs in the glucose consumption rate. Each mole of pyruvate
hibernating mammals. entering the TCA cycle via acetyl CoA generates
• The mitochondrion is also the main site of molecular about 15 moles of ATP. These are exported to the
interconversion and degradation of amino acids and cytosol and require the import of equal moles of ADP
the main source of acetyl CoA. The excess glutamine and PO 3- for their synthesis. Each mole of pyruvate
4
consumed enters the TCA cycle through α-ketoglutarate.
generated from glycolysis or lactate consumption
For some cells, asparagine acts as a sink (in addition
to alanine), which is formed through oxaloacetate and is also accompanied by one mole of NADH, whose
aspartic acid. reducing equivalent is transferred into the
mitochondria through the malate-aspartate shuttle.
Additionally, cells in culture consume glutamine at
a high rate (approximately 1/5 to 1/10 of glucose
in molar ratio). Nearly half of the glutamine enters
the TCA via α-ketoglutarate. CO2 produced in the
TCA cycle is then exported out of the mitochondria.
Besides these major species, many other molecules
(including amino acids and nucleotides) are
transported into the mitochondria for DNA, RNA,
and protein synthesis. As will be described later, the
precursor for fatty acid and cholesterol synthesis,
acetyl CoA, is generated in the mitochondria, while
fatty acid and cholesterol synthesis occurs outside
the mitochondria. Acetyl CoA is very reactive
and does not get transported directly across the
inner membrane of the mitochondria. Rather, it
is transported out of the mitochondria as citrate.
After cleaving off acetyl CoA, the remaining four
carbons are returned to the mitochondria as
pyruvate or malate. Thus, the citrate and malate
flux across the mitochondrial membrane is also
substantial to sustain lipid and cholesterol synthesis.
The transport across the mitochondrial inner

membrane is dynamic and complex. Many
compounds crossing the membrane are
charged, yet their transport must not perturb
the proton and electric potential gradient. The
transport across the mitochondrial membrane
must be tightly regulated. Our understanding
of its regulation is still rather limited.
Regulation of Glucose Metabolism Under physiological conditions, the flux of glycolysis

and the TCA cycle is not controlled by one or a small
number of “rate-limiting” enzymes. Glucose flux is
the result of mutual constraints of many enzymes
in the pathway, through their feed-forward and
feedback inhibition and activation. A large number
of pathways are highly inter-connected and crosstalk
with each other through shared common substrates
or regulators. In mammals, different tissues serve
different metabolic roles to maintain the overall
homeostatic state of the organism. The partition of
metabolic roles is largely accomplished by giving
different tissues a different set of isozymes. Different
isozyme sets allow cells to respond to environmental
fluctuations or cellular cues differently.

Isozymes and Differential Allosteric Regulation

Different isoforms (isozymes) of glycolytic
enzymes catalyze the same reaction step, but have
very different kinetic properties and are often
• Cells express different isozymes in different tissues,
subjected to contrasting regulations. Isozymes
under different conditions of four glycolysis enzymes, hexokinase (HK),
• different isozymes have different kinetics and phosphofructokinase (PFK), pyruvate kinase (PK),
regulation and phosphofructokinase/fructose biphosphatase
(PFKFB), play key roles in controlling glycolysis
flux. Their different allosteric regulations give
them very distinct reaction characteristics.
The isoforms of those enzymes making up glycolysis
are different in proliferating and quiescent cells.
They are thought to be responsible for endowing
the high glycolysis flux and high lactate production
in proliferating and cancer cells and are thought
to be related to oncogenic transformation. They
also give tissues their metabolic capabilities.
PFK is pivotal in modulating the overall rate of
glycolysis and is a key node in energy metabolism.
Its activity is subjected to allosteric inhibition
by ATP and citrate, and is activated by AMP. PFK
has three isozymes: liver (PFKL), muscle (PFKM),
and platelet (PFKP). Among the three isoforms
of PFK, the muscle and the liver isozymes are
activated allosterically by fructose 1,6-bisphosphate
Fig. 3.11: Allosteric regulations in glycolysis. Note strong (F16BP). Muscle phosphofructokinase is inhibited
activation of glycolysis flux by F16BP and inhibition by
lactate. by lactate, a characteristic which may facilitate
reduced glycolysis flux at high lactate levels.
Fructose-2,6-phosphate is a shunted glycolytic
intermediate that plays a key regulatory role in rapidly
modulating the activity of PFK. All three PFK isozymes
are activated by fructose 2,6-bisphosphate (F26BP).
F26BP activates PFK1 by allosterically increasing its
affinity for F6P, even in presence of inhibitors such
as ATP, lactate, etc. The synthesis and degradation
of F26P is catalyzed by a bi-functional enzyme,
PFKFB (also known as PFK2); its kinase activity
catalyzes the synthesis of F26P and its phosphatase
activity catalyzes the hydrolysis of F26P to F6P.
PFKFB has four isozymes, each with different
kinase and phosphatase activities, allowing each to

respond differently to regulators. The brain isoform,
• F2,6P plays a key regulatory role PFKFB3, has the highest kinase to phosphatase
• Key regulatory enzyme of glycolysis activity and is expressed in several tumor cells. This
• PFK (PFK1) suggests that PFKFB3 can be accountable for the
• PFK2 glycolytic phenotype of reported cancerous cell lines
• PKM by allowing them to have high cellular F26P levels.
• Cancer cells (fast growing cells) and quiescent
cells have different isozymes The enzyme catalyzing the penultimate step of
glycolysis, pyruvate kinase, has three isozymes
in mammalian systems. The muscle isozyme is
expressed as either of the two splice variants, M1
or M2. The M1 isoform is mostly expressed in
adult tissues whereas the M2 isoform is expressed
exclusively in rapidly growing tissues, such as
fetal and tumor tissues, and also is thought to be
a critical player in the transformation leading to
cancer. In addition, the M2 isoform is known to be
under positive feed forward regulation by F16P.
Isozymes are often named after the tissue in which they
are the dominant isoform. However, it is important
to remember that the expression of isozymes is not
limited to one form in a cell type. Different isoforms
are often expressed in the same tissue or the same
cell. Different combinations of isozymes give rise
to different kinetics and regulatory behaviors
that may meet different physiological needs.
With the available genomic tools, we can easily
determine the relative expression of different
isoforms of the key enzymes of glycolysis, and further
evaluate how to influence cellular metabolism.

Signaling Pathways and Regulation by The regulation of cellular metabolism is tightly

Growth Control linked to the control of cell growth. The signaling
pathways that regulate growth rate (involving key
Signaling pathway/growth rate control of players like p53 and cMyc) also play regulatory roles
Glycolysis in regulating glucose metabolism. Transformation
• Insulin signaling that causes cells to switch from a quiescent state to
• positively regulating growth rate a proliferating state also triggers metabolic changes
• through AKT regulate glucose, amino acid metabolism to increase their glucose uptake and glycolysis flux.
• P53 (tumor suppressor) supresses glucose uptake and p53 is a major tumor suppressor that plays key
glycolysis roles in cell cycle arrest, senescence, and apoptosis.
• cMyc (proto-oncogene) stimulates glycolysis Its role in the regulation of glucose metabolism and
• Fast growing (tumor) cells have fast glycolysis (and oxidative phosphorylation was not well understood
lactate production) until only recently. It induces the overexpression of
TIGAR under mild oxidative stress conditions. TIGAR
contains a fructose-2,6-bisphosphatase catalytic
Regulation of Metabolism activity domain, which mediates the degradation of
Glucose fructose 2,6-phosphate, leading to a decrease in PFK1
p53 GLUT1,4 activity and the attenuation of the glycolytic flux.
Glucose
H2O
Glucose-6- NADPH p53 can also modulate glycolytic rate by regulating
TIGAR Phosphate
PPP
ROS
the activity of PGM, GLUT1, and GLUT4 transporters.
Fructose-2,6- Fructose-6-
Bisphosphate PFK2 Phosphate Furthermore it up-regulates mitochondrial
PFK1
Fructose-1,6- Transcriptional Inhibition oxidative phosphorylation by upregulating the
Transcriptional Activation
Bisphosphate
Allosteric Activation
expression of SCO2 (synthesis of cytochrome c
p53 PGM oxidase 2), which mediates the assembly and
Lactate Pyruvate
activity of the cytochrome c oxidase complex.
NADH
Pro-oncogenic genes may also provide the link
p53 SCO2 COX Mitochondria
ATP between increased glycolytic flux and oncogenic
Fig. 3.12: Tumor suppressor p53 negatively regulates transformation. Akt is a pro-oncogene which,
glycolysis flux.
when deregulated, affects many cellular functions
Akt and Myc Regulation of Metabolism including metabolism, cell proliferation, and protein
Glucose
synthesis. In tumor cells, constitutively activated
Akt
GLUT1
Glucose Activation by phosphorylation
AKT is sufficient to shift the cells from a primarily
HK
Glucose-6-
or localization
Transcriptional Activation
oxidative state to a primarily glycolytic state.
Phosphate
Allosteric Activation
Fructose-2,6- PFK2 Fructose-6- Myc is another proto-oncogene whose

Bisphosphate Phosphate
PFK1 pleiotropic regulatory roles include energy
Myc
Fructose-1,6-
Bisphosphate metabolism. Glycolytic enzymes have Myc
LDH
Lactate
canonical E-boxes in their promoter and are
Pyruvate
ASCT2
deregulated when Myc is overexpressed.
GLS SN2
Glutamate Glutamine Glutamine
Malate TCA (Extraacell
ular)
Mitochondria
Fig. 3.13: Signaling kinase, AKT and transcription factor, Myc,

positively regulate energy metabolism.

Metabolic Homeostasis and Lactate Consumption
Cells in culture, including those used in bioprocessing,
have all been selected for their capability to proliferate.
Their particular set of glycolytic isozymes directs
their metabolism to a high glucose flux and a high
rate of lactate production. However, even the exact
composition of isozymes is not monolithic among
different cells. Most cells express multiple isoforms
of the same enzyme in glycolysis and their proportion
is not identical. Consequently, even though the
• Metabolism is a balancing act of many constraining metabolism of all cultured cells share the common
reaction nodes trait of a high glucose flux and high lactate production,
• Glycolysis flux influenced by: their metabolic behaviors are not all identical.
• Regulation (feed back and growth) Lactate accumulation in culture inhibits cell growth
• Pyruvate/NADH flux into mitochondria
and hastens the decline of cell viability in the
stationary phase. A key to achieving a high cell
• LDH (NAD recycle rate)
concentration and high productivity is to direct cell
• Lactate export metabolism to minimize the accumulation of lactate.
• Glucose intake One may aim to alter the cells’ metabolism to reduce
or eliminate lactate production. However, it is
important to keep in mind that virtually all rapidly
proliferating cells produce lactate. Such a metabolic
state might be a default state that is “essential”
for cell growth and cannot be easily altered over a
long period without affecting growth behavior. It
is also important to keep in mind that through in
the course of culturing cells for manufacturing, the
duration of the actual production period is only
a very small fraction of the total process lifetime.
The long duration of the process time is mostly for
expanding cell numbers to reach a production scale.
Altering the metabolism from the cell’s “default”
state may have an unforeseen effect on cells.
Another approach for alleviating lactate inhibition
is to tamper with the cell metabolism only in the
production scale or in the final stages of production.
Increasing evidences show that the switch from
lactate production during the growth stage to
lactate consumption in late stages is correlated
with a high productivity. One may seek to alter
cell metabolism to change lactate consumption
in the last stage to be more reproducible.

The LDH reaction that catalyzes pyruvate to lactate

is reversible. The direction of the flux depends
on the concentrations of NAD, NADH, pyruvate,
and lactate. Those concentrations, in turn, are
related to fluxes of reactions that produce or
consume them, including pyruvate entry into
mitochondria and lactate export (or import).
Further observations related to lactate consumption:
Lactate consumption occurs in post-rapid stages of
growth, when the glucose consumption rate is low.
The pyruvate flux into the mitochondria is rather
limited. When cells are at a high glucose flux, the excess
pyruvate must be converted into lactate to regenerate
NAD+. Therefore, a high glucose flux state is always
linked to a lactate production state. Furthermore,
rapid proliferation is generally associated with
high glucose flux. Lactate consumption occurs
only when the growth rate is slow. Experimental
observation has demonstrated that the absence
Cycle of rapid growth and a low glucose consumption
are necessary conditions for lactate consumption.
Glucose is still being consumed while cells
consume lactate. While lactate is being taken
up by cells, its specific consumption rate is
relatively small. Lactate is converted to pyruvate,
and then it enters the mitochondria for further
oxidation and energy generation. Even though
energy is being derived through lactate oxidation,
cells still need many constituents derived from
glucose for glycan synthesis, including NADPH
(from PPP), glucosamine, and galactose. These
compounds cannot be derived from pyruvate in
most culture cells, since they lack key enzymes for
gluconeogenesis. They must be supplied through
glucose. The glucose consumption rate in the lactate
consumption stage is small, but it is never zero.
The conversion of lactate to pyruvate by LDH does
not occur in isolation. It is coupled to the reduction
of NAD to NADH (also catalyzed by LDH), and linked
Cycle to the reverse transport of lactate and H+ by MCT
from the medium to the cytosol. The propensity and
Fig. 3.14: Metabolic fluxes in lactate production and consump- rate of lactate consumption is affected by the pH
tion metabolism.
of the media. In addition to transporting pyruvate

formed from lactate into mitochondria, NADH
has to be transported from the malate-aspartate
shuttle into the mitochondria for oxidation.
Glutamine and Its Relation to Energy Metabolism

For most cultured cells, glutamine is the second
Glutamine highest consumed nutrient. Its molar consumption
rate is about 1/5 to 1/10 of that of glucose for most
• Consumed by growing cells at a high rate
cells. Glutamine is a major amino acid constituent
• Non-essential in vivo. Glutamine synthesase decreases of cellular proteins. It supplies amino groups for the
upon in vitro culture
synthesis of purine and pyrimidine bases, which are
• Supply TCA cycle intermediates the backbone of nucleic acids. However, the amount
• causes NH3 release of glutamine consumed by cells far exceeds what
• used in nucleotide and protein synthesis is needed for synthesizing cellular components.
Glutamine is not an essential amino acid for
mammals; it is only essential for cultured cells. Cells
in some tissues, like the liver, synthesize glutamine
by the incorporation of an ammonium into glutamate
at the expense of an ATP using glutamine synthase.
The transcript level of glutamine synthase in cultured
cells is low. It actually decreases by nearly two orders
of magnitude when liver cells are put into culture.
A large portion of glutamine is converted to glutamate
by glutaminase, and then to α-ketoglutarate before
entering the TCA cycle. Through α-ketoglutarate,
glutamine is a major contributor to central metabolic
flux. There is increasing evidence that glutamine
is needed to drive the TCA cycle for faster growth.
In the course of being converted to glutamate
and α-ketoglutarate, the nitrogen is released
as ammonium or transferred to an amino acid,
Fig. 3.15: Entry of glutamine into TCA cycle. Glutamine
is converted to glutamate by glutaminase. Glutamate is
such as alanine or asparagine, and excreted.
then convert to α-ketoglutarate by either transamination The ammonium that is released from glutamine
reaction (transfer its α-amino group to pyruvate or contributes to the waste metabolite accumulation.
oxaloacetate) or via oxidation by glutamate dehydrogenase
(converting NADP to NADPH and releasing ammonium).

Amino Acid Metabolism

Mammals can synthesize only fewer than 10
of the 20 amino acids used in the translational
synthesis of proteins. 11 or 12 (depending on
the species) essential amino acids that they
cannot synthesize must be acquired through diet.
Cells in culture have more essential amino acids
requirements than the organism; a number of
non-essential amino acids become essential for
in vitro culture, including glutamine, tyrosine,
serine under some growth conditions, proline
Fig. 3.16: Major amino acid transporters for some cells. All essential amino acids must be
supplied in medium, while non-essential amino
• Amino acids are transported with overlapping
acids can be derived from other amino acids.
transporters.
• All IgG antibodies produced by mammalian cells Amino acids are taken up by cells through a large
are glycoproteins, with an N-linked oligosaccharide number of amino acid transporters. Most amino acid
attached to each heavy chain in the hinge region at transporters transfer a family of amino acids with
Asn-297 similar characteristics, such large neutral (uncharged
Amino Acid Degradation side chain) amino acids, cationic or anionic amino
• Amino acids are consumed at a rate of three to five acids. The rate of transfer for particular amino acids
times what is needed for making biomass and product. is thus not only dependent on the concentration
• Excess amino acid consumed must be excreted. difference of itself between intracellular
• The nitrogen (amino group) is removed from the carbon environment and extracellular medium, but also on
skeleton by transamination, oxidative or non-oxidative the competition with other amino acids for the same
deamination. The excess nitrogen is excreted as
ammonium ion or as excreted amino acids (e.g. alanine,
transporter. One amino acid may be taken up via more
proline, asparagine) than one transporter, albeit with different affinity.
• The carbon skeleton mostly enters the TCA cycle to be The amino acids taken up by cells are not likely
converted to excreted non-essential amino acids or to
be converted to pyruvate and lactate, or to contribute
to be in the “right” stoichiometric ratios to
to acetyl CoA in cytosol through citrate. meet their need. Excess amount of amino acids
• Large excess of glutamine consumed by cells in is converted to the non-essential amino acids
culture is converted to glutamate in the cytosol, which are not supplied in sufficient quantities,
or is transported into the mitochondria and then or is degraded. The interconversion and
converted to glutamate. Glutamate is then converted to
α-ketoglutarate and enters carbon metabolism. degradation of amino acids takes place through
• Glutamine (amide group) is used as an amino group
the intermediates of glycolysis and TCA cycle.
donor in adenosine (AMP), guanosine (GMP) and Before the carbon skeleton of an amino acid enters
Cytosine (CTP) biosynthesis.
carbon metabolism pathways its amino group or
• Aspartic acid and glycine are also used in nucleic acid
synthesis
other nitrogen containing functional groups is
first removed. Glutamine and glutamate become
• Methionine is methyl group donor. Tryptophan is used
in NAD synthesis α-ketoglutarate, asparagine and aspartate become
• Glutamate participates in a large number of reactions.
oxaloacetate; all are catabolized through the TCA cycle.
The flux of its synthesis or supply is expected to be high. Alanine becomes pyruvate, while leucine, isoleucine
and others enter carbon catabolic pathway through

acetyl-CoA. Although the degradation of amino
acids yields energy, it is not a major energy source.
The excess nitrogen from amino acid degradation
Alanine
Cysteine is excreted as ammonia or as the amino group of
Glycine
Serine non-essential amino acid, especially as alanine or
Threonine
Tryptophan proline. In some cell culture processes ammonium
Isoleucine
Pyruvate
Leucine accumulates to growth inhibitory levels. Although
Lysine
Threonine the adverse effect of ammonium on cell growth
CO2
Acetyl-CoA Acetoacetate is usually not as severe as lactate accumulation.
Isoleucine
Citrate
Asparagine Leucine
Aspartate Lysine
Oxaloacetate Threonine
Citric Isocitrate
acid
Aspartate cycle CO2
Phenylalanine Fumarate
Tyrosine α-Ketoglutarate
Succinyl-CoA Arginine
Isoleucine Glutamate
Methionine CO2 Glutamine
Valine Histidine
Proline
Fig. 3.17: Entry of amino acids into catabolism

Lipid Metabolism
Lipids play key roles in many physiological functions
critical to cellular properties of particular interest
Functions of Lipids to bioprocessing. In addition to forming the bilayer
• Contributes to the membrane fluidity membrane, lipids are also involved in cell signaling.
• Storage of precursors metabolized to second Lipid content in bilayer membrane affects membrane
messengers (diacylglycerol, inositol triphosphate) fluidity and permeability. Lipid bilayer membrane
• Lipids (such as, polyphosphoinositide) involved in partitions various organelles from the cytosol.
protein traffic and membrane fusion events Secreted recombinant proteins are processed first in
• Anionic lipids (such as, PS) involved in attachment of endoplasmic reticulum (ER) and the Golgi apparatus.
cytoskeletal proteins to membranes
They are excreted via membrane vesicles. For
• Cholesterol and sphingolipids form microdomains optimal protein secretion capacity, the membrane
or ‘rafts’: enriched in specific subsets of membrane
proteins homeostasis and biogenesis among organelles,
secretory vesicles and plasma membrane is critical.
Subcellular Localization of Lipid Metabolism in Not all lipid bilayer membranes are the same. The
Animal Cells lipid composition of ER, mitochondrial and plasma
Cytosol membrane differ from each other. The plasma
• NADHP synthesis (pentose phosphate pathway, malic membrane of hepatocytes is enriched in cholesterol;
enzyme) however, the amount of cholesterol in the rough
• Isoprenoid and early cholesterol synthesis and smooth inner ER is lower. There is very little
• Fatty acid synthesis cholesterol in the inner mitochondrial membrane.
Mitochondria:
• Fatty acid oxidation
• Acetyl-CoA synthesis
• Ketone body synthesis
• Fatty acid elongation
Endoplasmic Reticulum:
• Phospholipid synthesis
• Cholesterol synthesis (late stage)
• Fatty acid elongation
• Fatty acid desaturation
Peroxisome:
• Cholesterol precursors on synthesis
• Final steps of cholesterol synthesis also occurs in
peroxisome

Lipid Transport Although some cells can be cultured in lipid-free
medium, most cell culture media contain some
Lipid Transport Processes
fatty acids and lipids. Cells readily take up fatty
Intramembrane Transport:
Transbilayer movement of lipid molecule acids, phospholipids, and cholesterol from the
medium and incorporate them into cellular lipids.
• Transbilayer movement at eukaryotic ER: relatively non-
specific, ATP-independent process Lipids can be supplied as serum lipoproteins in the
• Three classes of transport: form of a complex with albumin or liposomes, or as
solubilized conjugates, such as sorbitol-fatty acid
• Aminophospholipid translocases (also called
flippases) esters. The cellular uptake of fatty acids is a passive,
• Bidirectional transporter or scramblases non-energy dependent process. After being taken
up by cells, fatty acids quickly become esters; the
• ATP Binding Cassette (ABC) pumps
intracellular levels of free fatty acids are quite low.
Intermembrane Transport:
Movement of lipid molecules from one distinct membrane Cholesterol is complexed to low density lipoprotein
domain to another (LDL) in the body and is taken up by cells through
Possible mechanisms: the LDL receptor. For cells in culture, cholesterol
• Monomer solubility and diffusion (for molecules such as is often supplied as a conjugate with serum
free fatty acids) albumin, or as complexes with cyclodextrin.
• Soluble carriers such as lipid transfer proteins
• Carrier vesicles
• Membrane apposition and transfer
• Membrane fusion

Fatty Acid Metabolism Most cells have the capability of synthesizing

various fatty acids. Under starvation conditions,
cells also perform β-oxidation to degrade fatty
acids into acetyl CoA in the mitochondria or
peroxisomes. Acetyl CoA then enters the TCA cycle
to generate energy. Typically, cells in culture do not
need to derive energy from fatty acid oxidation.
Fatty acids are synthesized from acetyl CoA in
the cytosol. The first step of fatty acid synthesis
involves adding a CO2 to acetyl CoA to form malonyl
CoA, which then reacts with acetyl CoA to become
a four-carbon fatty acyl CoA. It is noted that even
though CO2 is a catabolic product, it is also an
essential nutrient for biosynthesis. Fatty acid
synthesis, thus, involves the step-wise elongation
processes of using three-carbon malonyl CoA to
add a two-carbon unit to fatty acyl-CoA in each
cycle. NADPH is also consumed in this process.
There are a number of fatty acid synthetases that
can synthesize fatty acids to different lengths.
The fatty acid products from elongation
reactions are all saturated fatty acids. Double
bonds are then synthesized by unsaturation
reactions after saturated fatty acids are made.

Acetyl CoA shuttle Acetyl CoA is the building block of fatty acids.
It is generated primarily through the oxidative
Acetyl CoA shuttle decarboxylation of pyruvate in the mitochondria.
On mitochondria inner membrane However, fatty acid synthesis takes place in the
• Citrate transporter: transport citrate into cytosol cytosol. Acetyl CoA does not pass through the bilayer
• Malate, α-ketoglutarate transportor: transport into membrane. Rather, it is exported to the cytosol via
mitochondria
an indirect process. Citrate, formed by condensation
• Pyruvate transporter: transport into mitochondria of OAA and acetyl CoA in the TCA cycle, is then
In Cytosol transported into then cytosol, where it is split into
• Citrate lyase: citrate +CoA+ATPOAA+acetylcoA oxaloacetate and acetyl CoA. Oxaloacetate gets
• Malate dehydrogenase: OAA+NADH+Malate +NADH+ reduced to malate at the expense of one NADH. Malate
• Malic enzyme: malate+NADP Pyruvate +NADPH+CO2 is then converted to pyruvate, losing one carbon and
consuming one NADPH. Pyruvate then recycles into
In mitochondria matrix
• Pyruvate carboxylase: Pyruvate+CO2+ATPOAA+ADP the mitochondria. The process of making fatty acids
• Malate dehydrogenase: malate+NAD+OAA+NADH+ using acetyl CoA is thus energetically expensive.
• Citrate synthase: OAA+acetylCoACitrate +CoA
CO2
Pyr NAD Fatty acid
Malate NADH cholesterol
NADPH NADP synthesis
OAA acetyl CoA
ADP+Pi
Pyr CO2
Citrate ATP CoA
CoA acetyl CoA
OAA Citrate
Malate αKG Glu Glu Gln

Fum
Suc NHg NH3
Fig. 3.18: Citrate and acetyl CoA shuttle.
Cholesterol & Its Biosynthesis Cholesterol is a 27-carbon molecule. Mammals

require cholesterol as a constituent of membranes
and as a precursor for the synthesis of steroid
hormones, bile acids, and lipoproteins. Cholesterol
is relatively insoluble and resides exclusively
in various cell membranes. Its regulation is
particularly important since excess cholesterol
forms solid crystals, leading to cell death.
Proper cell function requires that cellular
Fig. 3.19: The structure of cholesterol membranes have the appropriate composition,

including cholesterol, in order to maintain bilayer

fluidity, impermeability and other characteristics
specific to different organelles. Cholesterol
constitutes ~10% of the dry weight of plasma
membranes, and plasma membrane cholesterol
accounts for 65% to 80% of total cellular cholesterol.
• Cholesterol metabolism is a tightly regulated pathway Cells in culture obtain cholesterol either by de
subjected to stoppage of cholesterol biosynthesis in the novo synthesis or through receptor-mediated
presence of excess cholesterol. uptake of exogenous low-density lipoproteins.
• Animal cells obtain cholesterol from both de novo Cholesterol is synthesized from acetyl CoA, which
biosynthesis and receptor mediated uptake.
is condensed by 3-hydroxy-3-methylglutaryl
(HMG)-CoA synthase (HMGCS) to form HMG-
CoA. HMG-CoA is converted to mevalonate by
HMG-CoA reductase (HMGCR). This enzyme
Cholesterol is the target of statins, the class of drugs that
• Component for membrane biogenesis and precursors suppress cholesterol synthesis in patients.
for the synthesis of steroid hormones, bile acids and
lipoproteins. Further synthesis of mevalonate to farnesyl-
• Cholesterol resides exclusively in cell membranes and diphosphate takes place in peroxisomes.
its regulation is particularly important since excess cho- Subsequent condensation of two molecules of
lesterol forms solid crystals leading to cell death. farnesyl diphosphate to form squalene, lanosterol,
• Cholesterol is ~10% of dry weight of plasma mem- lathosterol and finally cholesterol occur in the ER.
branes. Precise lipid composition of the plasma mem-
brane has been controversial since it is difficult to iso- Out of the 18 key enzymes taking part in
late high yield of pure samples from tissues or cultured cholesterol biosynthesis, 5 enzymes reside in the
cells.
peroxisome and 13 reside in the ER. HMGCS is
• Cells obtain cholesterol by de novo synthesis or through upstream of HMGCR and is found in the cytosol.
receptor-mediated uptake of plasma lipoproteins.
Thus, there are at least three different sub-cellular
compartments involved in cholesterol biosynthesis.
Although cholesterol in mammals is synthesized
primarily in the liver, most cells have the
capability of synthesizing cholesterol for their
own growth requirements. NS0 cells lack an
enzyme, 17-β-hydroxysteroid dehydrogenase,
which converts lanosterol to lathosterol. In
NS0 cells, 17-β-hydroxysteroid dehydrogenase
is silenced through methylation of a CpG island
upstream of its promoter, leading to the cell
line’s dependency on cholesterol for growth.

Fig. 3.20: Cholesterol biosynthesis takes place in mitochondrion, peroxisome
and endoplasmic reticulum.
Cholesterol Biosynthtic Pathway

• Cholesterol is synthesized from acetyl CoA, which is condensed by 3-hydroxy- 3-methylglutaryl (HMG)-CoA
synthase (HMGCS) to form HMG-CoA.
• HMGCS exists in a mitochondrial form in hepatic tissue and is present in cytosol as 53kD protein for other
tissues.
• HMG-CoA is converted to mevalonate by HMG-CoA reductase (HMGCR)
• HMGCR exists as a 97-kDa glycoprotein in the endoplasmic reticulum (ER) and is exemplified as the rate
determining the enzyme of the biosynthesis pathway.
• Mevalonate is further metabolized to farnesyl-diphosphate by a series of peroxisomal enzymes as shown in the
figure.
• First committed step to cholesterol byosynthesis is typified by condensation of two molecules of farnesyl
diphosphate to form squanlene by farnesyl diphosphate farnesyl transferase-1 (Fdft1), or (squalene synthase) a
47-kDa protein residing in the ER
• Squalene is converted to the first terol, lanosterol by the action of squalene epoxidase.
• Conversion of lanosterol to lathosterol involves a series of oxidations, reductions and demethylations. It is a 17

Glycan Biosynthesis and Protein Glycosylation

A vast majority of recombinant therapeutic proteins
are glycoproteins. The extent of glycosylation and
the structure of the glycans on those glycoproteins
may have a profound effect on their activities
N-linked Glycosylation
and circulatory half-life. Glycans are classified as
attachment of oligosaccharide to the protein through the O-linked or N-linked glycans. O-glycans attach to
amine group of an asparagine
the polypeptide through the -OH group of serine
or threonine. N-glycans link to protein through the
amide group of asparagine. For N-linked glycan,
O-linked glycosylation the asparagine is in an Asn-X-Thr/Ser recognition
attachment of oligosaccharide to the protein through the sequence, where X indicates no specificity.
hydroxyl group of a serine or threonine
N- and O-glycans attached to proteins are
All IgG antibodies produced by mammalian cells are structurally heterogeneous. The glycans attaching
glycoproteins, with an N-linked oligosaccharide attached to to the same attachment site of different glycoprotein
each heavy chain in the hinge region at Asn-297 molecules are often structurally different. Such
heterogeneity is called microheterogeneity.
Multiple glycosylation sites are often present on
a protein molecule. Not all glycan attachment
sites on a protein molecule may be occupied.
Different protein molecules may have different
combinations of occupied and free sites;
such difference in the occupancy on different
attachment sites is called macroheterogeneity.
Importance of Glycan Glycosylation starts while the protein is still being

translated and undergoing protein folding in the
Effect of Glycan ER. The presence of glycans affects the solubility,
• Facilitate protein folding in the ER aggregation, and the stability of the folding protein.
• Increase solubility
The glycan structure affects the half-life of blood
• Affect biological activities
circulation and immunogenicity of a therapeutic
• Fucose for ADCC activity
protein. The presence of carbohydrates delays
• Affect half-life in circulation and pharmacokinetics
clearance from blood, as demonstrated by a
Heterogeneity In Glycoforms comparison of glycosylated protein and its non-
• Macroheterogeneity: when multiple sites of glycosylated counter-part. Higher sialic acid content
glycosylation are present in a protein, the occupancy on increases the circulation half-life of erythropoietin
different sites differs on different molecules
(EPO). Under-sialylated glycoproteins are thought
• Microheterogeneity: the structure of the glycan
to be cleared by a higher liver uptake via the
occupying the same site differs among different
molecules hepatic asialoglycoprotein binding protein. It was
also postulated that the glycosylated recombinant
proteins are better trapped by the extra-cellular
matrix, thus having a longer bioavailability
in vivo than their unglycosylated variant.

Glycan on glycoprotein may also affect its biological
activities. Many therapeutic antibody IgG molecules
facilitate killing of target cells through antibody
dependent cellular cytotoxicity (ADCC). ADCC
activities of those antibodies have been reported
to be affected by its glycan structure. Molecules
without a fucose on its mannose core have a fifty
fold higher ACDD activity than those with a fucose.
Protein Folding and Glycosylation O-glycosylation initiates in either the ER or the Golgi.
Overall, our understanding of O-glycosylation is far
less than N-glycosylation. N-glycosylation is initiated
by the transfer of a preassembled oligosaccharide
(Glc3Man9GlcNAc2, an oligosaccharide of three
glucose, nine mannose, and two N-acetylglucosamine)
to an asparagine in the recognition sequence of
a nascent protein in the ER lumen. The addition
of glycan occurs during the process of protein
synthesis, prior to the completion of protein folding.
The first part of N-glycan synthesis involves

the assembly of a high mannose backbone on
a membrane anchored dolicol molecule, on the
outside surface of the ER. The glycan is linked to the
Fig. 3.21: Glycosylation of secretory proteins dolicol carrier through a pyrophosphate group in
an activated form. After the seven-sugar backbone
is formed (with five mannose and two N-acetyl
glucosamine), it flips over to the interior of the ER.
No transporters are needed for the transport of
backbone glycan; rather, a flippase catalyzes their
translocation into the ER lumen. Inside the ER,
the backbone acquires an additional four mannose
and three glucose to become a mature core.
The mature core is then transferred to a binding

site (Asn-X-Thr/Ser) on a nascent protein
molecule. However, not all glycan binding sites
may be occupied. This could be due to competition
between protein folding and core glycan transfer.
Hence, different permutations of site occupancies
in different protein molecules exist, giving rise to
Fig. 3.22: N-glycan processing of glycoproteins
in endoplasmic reticulum a macroheterogeneity of glycosylation patterns.
After being synthesized, chaperones surround

protein molecules to facilitate their folding process.
The three glucose molecules on the glycan core
serve as a quality control signal for the proper
folding of these glycoprotein molecules. Upon

completion of folding, the three glucose molecules

are removed from glycan, signaling the protein’s
readiness to be transitioned to the Golgi apparatus.
Proteins that are not folded properly, with glucose
still in their glycan, may be recycled through the
calnexin pathway for refolding. Some unfolded
proteins are sent to the proteosome for degradation.
The well-folded glycoprotein molecules are

enclosed in membrane vesicles of ER and then
bud from the ER and translocate to the Golgi
apparatus. Once there, they fuse with the Golgi
body membrane and the glycoprotein cargos are
released into the lumen of the Golgi apparatus.
Inside the Golgi, mannose is trimmed from

Glycan Extension in Golgi Apparatus
the N-glycan core, effectively reducing the
number of mannoses from nine to three, before
extension takes place. Three main carbohydrate
molecules constitute most of the extended glycan:
N-acetyl glucosamine, galactose, and sialic acid.
Different glycosyltransferases add a different
monosaccharaides to the growing core glycan on the
protein. The incoming monosaccharide provides
the activated carbonyl group and the receiving
carbohydrate moiety on the growing glycan on the
protein may have more than one hydroxyl group
available for extension. The glycosyltransferases
recognize different incoming monosaccharaides
and catalyze different glycosidic bond formations.
However, a number of glycosyltransferases do allow
for some flexibility in glycosidic bond formation.
Fig. 3.23: N-glycan extension in Golgi apparatus Intermediate glycans on the protein have more than
one sugar that can be extended and each sugar may
have more than one hydroxyl group that is available
for extension. Importantly, the extension reaction
does not take place on all of the available reaction sites.
Each growing glycan, thus, has multiple available
reaction paths for extension. The glycans can
grow into different numbers of branches, creating
structures such as biantennary and triantennary
glycans. In some cases, the reactions of adding
those sugars to different branches of the glycan
may occur in different sequential orders, but
lead to the same product. In other cases, the

addition of a particular glycosidic linkage may
hinder the reaction of the others; thus, the
reaction, itself, leads to different glycan structures.
Consequently, a very large number of glycan structures
can be formed in the N-glycosylation pathway;
however, the number of glycosyltransferases
for the extension reactions expressed in a cell
or tissue is relatively small (only a dozen or
so). However, diverse patterns of glycosylation
are often seen throughout many glycoproteins.
The web of glycan extension reactions forms
a complex network which, when drawn out
graphically, indeed resembles a network of
diverging and converging paths leading to a number
of different fully-extended N-glycan structures.
Glycan Types and Microheterogeneity N-glycan structures are generally classified into
three principal categories: high mannose, complex,
and hybrid. All of them share a common tri-
mannosyl (Man3GlcNAc2) core structure. The high
mannose glycans have five to nine mannose (Man5-
9
GlcNAc2) sugars. Those with two GlcNAc’s attached
to the tri-mannosyl core are called “complex”. As its
name implies, the hybrid types are a combination
of high mannose and complex glycans, and have at
least three mannose sugars but only one GlcNAc
on one nonreducing mannose. This diversity of
glycan sugar composition on each glycosylation
site is referred to as microheterogeneity.
N-glycan microheterogeneity arises through
alternative reaction paths of extension in the Golgi
apparatus, as described above. The Golgi apparatus
consists of stacks of membranous compartments
commonly grouped into cis, medial, trans, and
Fig. 3.24: Different types of N-glycans in glycoproteins trans-Golgi network (TGN) cisternae. These
cisternae are not biochemically homogeneous. As
the secretory glycoproteins traverse through these
Golgi compartments, the glycan extension reactions
are catalyzed by varying the composition of
glycosylation enzymes in each compartment. Adding
to this diversity, not all protein molecules spend an
equal length of time in different Golgi compartments;
some exit early while others linger. Some glycans

do not acquire a terminal sialic acid, while others

have multiple sialic acid molecules on them.
Synthesis and Transport of Nucleotide Glycosidic bond formation is mediated by nucleotide

Sugar Precursor sugars. The nucleotide (NTP) reacts with an
activated sugar (glucose-1-phosphate, or glalatose-
1-phosphate, N-acetyl-glucosamine-phosphate,
mannose-1-phosphate) to form an NDP-sugar. Uracil
is used for glucose- and galactose-based sugars
(e.g., UDP-glucose and UDP-galactose), guarnyl for
mannose and fucose, and cytidyl for sialic acid.
Mannose, galactose, and fructose are synthesized in
branches of the glycolysis pathway. All three sugars
are activated at their first carbon. Therefore, they
link to glycans through the formation of (1→n)
glycosidic bonds. For example, UDP-NAcGlc is added
to a growing core by forming an N-acetylglucosamine
β (1→n) mannose bond; there can be a number of
possible positions on mannose (e.g., 2, 3, 4, or 6).
For sialic acid, the second carbon is activated; thus
CMP-2-sialic acid will form a sialyl (2→n) bond
Fig. 3.25: Transport of nucleotide-sugar precursors
into organelles with galactose. The synthesis of all the precursor
sugars occurs in the cytosol, including a nine-
carbon sialic acid and N-acetyl neuraminic acid.
Similarly, all nucleotide sugars are formed in this
way, except for sialic acid. The activation of sialic
acid to CMP-sialic acid occurs in the nucleus.
The backbone of N-linked glycan is synthesized
on the cytosolic side of the ER membrane through
the membrane-anchored dolicol. The nucleotide
sugars used in the formation of the backbone, GDP-
mannose, UDP-N-acetyl glucosamine, and GDP-
glucose are synthesized in the cytosol and directly
react with dolicol or with the growing glycan
backbone. The assembled backbone is then “flipped”
into the ER and the subsequent reactions occur
inside the ER. Transporters for these nucleotide
sugars have been reported to be present in the ER.
The nucleotide sugars for the extension reactions
in the Golgi apparatus are also transported
through transporters. These include CMP-sialic
acid, GDP-fucose, UDP-N-acetyl glucosamine,

UDP-galactose, and the activated sulfate donor
(3’-phosphoadenosine, 5’-phosphosulfate). All of
• Glycan synthesis use nucleotide sugar as monomer unit these transporters are antiporters, meaning that
• Nucleotide sugar highly charged, require specific a stoichiometric exchange of nucleotide sugars
transporter into organelles with NMPs is responsible for the charge balance.
• Sialic acid synthesis occurs in cytosol, activation to CMP-
Sialic acid to places in the nucleus.
Fig. 3.26: Biosynthesis of precursors of glycans

Glycan Diversity Among Species Unless intended for vaccination, the

immunogenicity elicited by recombinant
proteins is a concern. An antibody elicited by
and against the protein therapeutic can result
in neutralization of the therapeutic protein and
Glycosylation
may result in an unintended drop in efficacy,
• Enzymes are somewhat different among species thus causing serious adverse clinical effects.
• Possible immunogeneity (e.g. high mannose glycan from
most yeast
The potential immunogenicity of recombinant
therapeutics may arise from the aglycosylated
protein core or from the glycan associated with
them. There are at least two mechanisms by which
glycans on a protein may affect the immunogenicity
of a human therapeutic: 1) by being a foreign
glycan structure, or 2) by shielding a segment of
the protein that is otherwise antibody inductive.
Different recombinant human therapeutic proteins
that are produced in different organisms are differently
glycosylated (such as those from CHO versus yeast)
or aglycosylated (such as from CHO versus E. coli).
Comparison of those proteins indicates that the
“shielding” effect of minimizing immunogenicity is
affected by the nature of the protein, as well as by
the source of the protein. The concern about the
immunogenicity of different glycoforms of the rDNA
proteins produced in insect cells and in transgenic
plants has hindered those technologies’ application
for rDNA therapeutic protein production.
Glycosylated proteins produced in CHO and
mouse myeloma cells are minimally immunogenic.
The glycosylation pathway is highly conserved
in mammals. Nevertheless, a divergence among
different species does occur. Host cells derived from
other species may possess a set of glycosylation
enzymes that are different from humans. Human
glycans have terminal N-acetylneuraminic
acid (NANA), whereas other mammals have
N-acetylglycolylneuraminic acid (NGNA). NGNA
is the hydrolytic product of CMP-sialic acid
hydroxylase, which is present in nearly all mammals
but absent in humans. Thus, glycoproteins produced
in CHO have some NGNA present among all sialylated
glycans. Similarly, glycoproteins expressed in CHO

cells have only terminal α(2,3)-linked sialic acids,
CHO Glycan in contrast to α(2,6) and α(2,3) seen in humans,
• Has NGNA instead of human NANA due to a varied composition of sialyltransferases.
• Has only α(2,6) sialic acid, human has both α(2,3) and, Such differences in glycan composition have posed
α(2,6). a concern; however, the antigenicity of recombinant
proteins directly caused by glycans is still scant.
Concluding Remarks
In this chapter, we presented a brief overview of to better understand cell metabolism and to possibly
broad areas of cellular metabolism. We explored the find different ways of manipulating cell metabolism
core of energy metabolism, the process of glucose to better redirect the process. In recent years, we
utilization through glycolysis, PPP and the TCA cycle, have developed a better understanding of the link
and how all of these affect cell growth behavior between glycolytic regulation and growth control.
productivity. Through interconnected pathways, We have also established better tools to probe the
the central corridor of energy metabolism also relationship between metabolic flux distribution
influences the synthesis, and even the glycosylation, and other aspects of physiology that influence both
of the product proteins. The excessive consumption productivity and product quality. With the benefit
of glucose and glutamine and the corresponding of global physiological perspectives, we continue to
accumulation of lactate and ammonium in culture gain a deeper understanding of metabolism. Global
all contribute to growth inhibition and low views at a systemic level will significantly enhance
productivity. Lactate consumption in the late stage our capacity to manipulate cell metabolism, and
culture has been positively associated with a high thus increase productivity and product quality.
productivity. There are, therefore, ample incentives

Medium Design for Cell
Culture Processing
Optimization of Cell Growth Environment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
A Guide for Medium Design—Body Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Types of Media and Classes of Medium Components. . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Types of Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Basic Components of Cell Culture Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Classes of Medium Components. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Components of Basal Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Water. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Sugars and Energy Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Vitamins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Nucleosides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Optimal Concentration of Organic Nutrients. . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Fatty Acids and Lipid Precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Non-Nutritional Medium Components. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
High Molecular Weight and Complex Supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Medium for the Industrial Production Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Optimization of Cell Growth Environment

Medium, like the food we eat, exerts a fundamental
Industrial Cell Culture Process influence on the well-being of cultured cells,
1. Cell expansion profoundly affecting their growth, metabolic
2. Production/differentiation
activities, and other biological capability. The
question of how best to devise culture medium
emerges whenever new in vitro cultivation-
• Cell expansion stages last much longer than production
based science or technology is on the horizon,
• Medium design for both stages as occurred three decades ago driven by the
revolution in recombinant mammalian cell based
biotechnology, and as is occurring now concomitant
with the emergence of stem cell science.
Cells are the heart of cell technology; however,
without proper medium, cell cultivation cannot
accomplish process goals. Most cell types share
common basic nutritional requirements although
their needs for growth factors and cytokines may
MEDIUM DESIGN | 97

differ. In the nearly five decades since scientists began
to isolate and cultivate cells, the focus of medium
• Classical Medium Design - Optimize Cell Growth design has been to optimize cell growth, maintain
• Optimization for production - squeeze cell’s last growth potential and sustain the differentiated
productivity out properties in cultures of differentiated cells.
• Resurgence of media research in stem cell culture
With the growing importance of biologics, the
• opportunities in growth factors, antogonists, and
signaling pathway manipulation
focus of medium design has been extended to
enhancing production characteristics, such as
productivity or product quality. However, even
with the focus shifting to production, optimizing
medium for cell growth remains important. In
fact, the time that cells spend in the production
stage in a manufacturing reactor is a comparatively
small portion of their life span. A cell spends the
majority of its lifespan in growth, yielding progeny
to generate a sufficiently large number of cells for
producing product. Providing cells with an optimal
medium during the expansion stage is critical.
Recent advances in stem cell science have spurred
renewed interest in elucidating the nutritional needs
of cells in culture. Although the fundamental aspects
of nutritional requirements of a stem cell are not
different from other cell types, their requirements
for growth factors, surface matrices, and other
microenvironmental factors makes medium design
for stem cells far more complex than that for any
cell lines used in protein biologics production.
Furthermore, stem cell applications require that
the stem cell progeny be directed to differentiate
to specific lineages, for which the growth factor
requirements pose an even greater challenge.
Regardless of traditional biologics-based cell
technology or stem cell bioprocessing, the culture
process will involve both cell expansion and product
formation or differentiation. Medium optimization
strategy for cell expansion and for production may be
rather different. For expansion, the long term healthy
state of a cell while proliferating must be safeguarded.
In contrast, for production of biologics, the cells are
approaching their final stage of utility, and after all
products have been released into the medium, cells
and product molecules must be separated. Hence,
even conditions that might ordinarily hamper
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growth or harm cells, such as reduced temperature
or increased osmolality, are sometimes used.
For stem cell processing, or for other cell therapy,
the distinction between cell expansion and produc-
tion is not as significant. Even though cells are no
longer being expanded during the differentiation
stage or other final stage of preparation for clini-
cal applications, cells must not to be subjected to
deleterious conditions. Since the final product is
cell mass itself, their survivability and functional
capability after the cell culture process is critical.
In the following sections we will focus on nutritional
needs for cell expansion first, as that is best known.
We will then discuss how “optimal” medium for pro-
duction compares with that required for growth.
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A Guide for Medium Design—Body To design an optimal medium for cell growth, it is
Fluids instructive to examine the chemical environment of
Table 1. Cellular Chemical Environment in vivo their natural niche. The ultimate objectives of medium
Approximate Concentrations in Cellular Environment design are cell expansion, differentiation, and
Interstitial Intracellular production, not necessarily to reproduce their niche.
(mM) (mM) Understanding their native chemical environment
Na+ 140 14 provides us with a starting point from which to
K+
4.0 140 devise an environment suited to process goals.
Ca 2+
1.2 0.01
The vast majority of cells in the body are not in direct
Mg 2+
0.7 20
CI-- 108 4
contact with blood, but are surrounded by interstitial
HCO3 -
28.3 10
fluid. The chemical composition of interstitial
HPO4 , H2PO43- 2-
2 11 fluid, especially the protein and hormone content,
SO43- 0.5 1 varies with tissues. However, the general chemical
Amino Acids 2 8 composition of small molecular weight solutes in
Lactate 1.2 1.5 interstitial fluid and in intracellular fluid is similar.
Glucose 56 The low molecular weight solute composition
Protein 02 4 of interstitial fluid bears a few important
Total Chemical Species characteristics. The total osmolarity is around 280-
(mmole/L) 301.8 302.2
300 mM (or mOsm). A couple of percentage points of
Corrected osmolar activity (mM) 281.3 281.3
error notwithstanding, the osmolarity can be taken
as the sum of molarity of all dissolved species in the
fluid. The largest contributor to the final osmolarity
is Na+, followed by Cl–. In addition to Na+, other
inorganic species are present; notable are K+, Mg2+,
and Ca2+. However, those positively charged ions are
all present at low concentrations. Since the net charge
in a solution must be neutral, the total molarity of
positively and negatively charge ions must be equal.
In general Cl– concentration is lower than Na+ because
bicarbonate (HCO3–) is also present at ~30 mM
contributing to the negative charge in the solution.
For many ion species the interstitial concentration and
intracellular concentrations are strikingly different.
Both Na+ and Cl– are present outside the cell at a ten-
fold higher concentration than inside the cell, as is
Ca2+. In contrast, K+, Mg2+ and PO43– concentrations
are much higher on the intracellular side.
Cells can tolerate deviations from “optimal”
conditions for some period of time. The estimated
range of non-lethal physiological concentrations of
key compounds vary considerably. Keep in mind
MEDIUM DESIGN | 100

that the non-lethal range for the human body can
Table 2. Non-Lethal Range of Medium Constituents be rather different from that for cultured cells.
Approximate Nonle- For example, cells in culture can tolerate low (80
Normal Range thal Limits mM) or high (140 mM) sodium concentrations, or
Oxygen 35 – 45 10 – 1,000 mm Hg
high osmolarity (400 mOsm), for a period of days,
Carbon dioxide 35 – 45 5 – 80 mm Hg
whereas these extremes would not be tolerated by
Sodium ion 138 – 146 115 – 175 mmol / L most aquatic animals for more than a few hours.
Potassium ion 3.8 – 5.0 1.5 – 9.0mmol / L
Calcium ion 1.0 – 1.4 0.5 – 2.0mmol / L
Chloride ion 103 – 112 70 – 130mmol / L
Glucose 75 – 95 20 – 1500 mg / dL
Body 98 – 98.8 65 – 110 (18.3 – 43.3)
temperature (37.0) F° (C°)
pH 7.3 – 7.5 6.9 – 8.0
Types of Media and Classes of Medium Components

Basal Medium A complete cell culture medium often has two
• Sugar major categories of components: basal medium
• Amino acids and growth supplements. The basal medium is the
• Fatty acids, lipid precursors nutrient mixture consisting of the small molecular
weight components including sugar, amino acids,
• Vitamins, nucleosides
vitamins, various salts, etc. The basal medium does
• Bulk salts, trace elements
not merely provide a nutritional source for deriving
• pH buffer
energy and making new cell mass and product,
it also provides balanced salt concentrations
Supplements and osmolarity to allow for cell growth.
• Serum, hydrolysates
However, most cells will not grow if provided
• Growth factors with basal medium alone, as basal medium
• Carrier proteins does not contain growth factors or other factors
necessary for “optimal” growth conditions.
Growth supplements that may be added to basal
medium include growth factors, phospholipids,
soy hydrolysate, serum, etc. These supplements
may promote cell growth by providing constituent
components for specific signaling pathways, or may
supply special nutritional needs (such as delivering
cholesterol), and may direct cellular differentiation
MEDIUM DESIGN | 101

or maintain cells at a particular differentiation state.
There are numerous formulations of basal
media that are made into powder, packaged
and sold for commercial or routine use. Sterile
filtration is the standard means of sterilizing
basal medium, but heat sterilization (as is
used for microbial media) is also possible.
Types of Medium Traditional cell culture medium contains up to

Complex Medium vs. Chemically Defined 15% animal serum in addition to basal medium.
Medium Serum is a highly complex fluid in terms of its
chemical composition. Such a medium, containing
a largely undefined chemical composition, is
called a complex medium. Many supplements
Chemically Defined Media
commonly used in industrial processes, e.g.,
• Progress in this area will likely be accelerated by:
plant hydrolysates, soy phospholipids, also
• an urgency to demonstrate control over all aspects fall into this category. Their use renders the
of production and downstream processing for
licensing by the FDA chemical composition of the medium undefined.
• the availability of recombinantly produced (in E. coli) A chemically defined medium contains only
tissue culture supplements (i.e. insulin, EGF) components whose chemical composition is
• genetic engineering of cells to produce their own known and characterized, and has all of its
growth factors
chemical species specified. It does not contain
• development of small, synthetic peptides that can any mixture of components with unknown or
mimic the action of the larger, naturally occurring
protein (i.e., RDG sequence) undefined composition. For example, “lipids” or
• acceptance of continuous bioreactor systems and
“phospholipids” are not well defined compounds,
the operation of these systems in a “maintenance” but are mixtures of a class of compounds and
mode are not chemically specified. A chemically
defined medium often contains growth factors,
cytokines, and carrier proteins. Thus, a chemically
defined medium is not necessarily protein-free.
A large number of industrial production processes

Serum-Free and Animal Component-Free
Medium of rDNA proteins has eliminated serum from the
medium in the past decade. The use of serum-
free and animal-component-free medium has
become the industrial manufacturing norm, with
the intent to minimize animal virus or prion
contamination. Recently, serum-free medium has
been increasingly used even during the cell line
development stage to eliminate exposure of cells
to serum and animal components throughout the
MEDIUM DESIGN | 102

entire cell banking and manufacturing process.
Serum-Free Media
• Serum-free medium consists of nutritionally Nonetheless, the use of serum-free medium is not
complete basal medium supplemented with an yet universally practiced. For example, bovine
empirically determined mixture of hormones, growth serum is still widely used in manufacturing viral
factors, attachment factors, attachment proteins and
binding proteins. vaccines. As well, it is often not an easy task to
eliminate animal serum from processes involving
• Many serum-free media contain a complex mixture
of undefined components, such as soy-meal the cultivation of primary differentiated cells.
hydrolysate, peptone or beef hydrolysate, or other
plant hydrolysate.
Protein-Free Medium By definition, protein-free medium contains no

protein. Most protein-free media are, as well,
chemically defined. However, a protein-free medium
may contain undefined lipids or fatty acids, and thus,
may not be chemically defined. Cells that grow well
in chemically defined medium are likely to be highly
adapted or transformed, which eliminates any
dependence on mitogenic molecules or lipid sources.
Basic Components of Cell Culture Medium

Classes of Medium Components Medium serves two important roles: to provide a
Stoichiometric vs. Habitation-Conducive chemical environment in which cells can grow, and
Basal Medium Components to supply the components cells need to generate
energy and convert to cell mass and products. Some
medium components are taken up by cells and
• Stoichiometric: glucose, amino acids, vitamins, “utilized” to make more cells and products; other
nucleotides, lipids, fatty acids, some growth factor, components provide the chemical environment
some salts but are not appreciably taken up by cells. The
• Habitation conducive: Bulk salts, such as sodium, majority of medium components, including glucose,
chloride, and some proteins, albumin, some amino acids, lipids, and vitamins, are consumed
growth factors. stoichiometrically. The more cells are made,
the more of those components are consumed.
In a most precise sense, nearly all medium
components are utilized to generate more biomass.
Even water is taken up by cells; as the cell volume
expands water must be taken up along with other
components that constitute cellular materials.
However, in practical terms, many medium
MEDIUM DESIGN | 103

components are consumed in such small quantities
that their consumption may not be measurable.
Stoichiometric medium components must be
supplied in sufficient quantities to reach the
cell, and must meet the product concentration
target of the production. To reach a higher
goal, more stoichiometric components must be
supplemented. For the unconsumed components
of the medium—components whose role is
only to provide a conducive environment—the
key issue is to maintain their concentration.
Examples of conducive, unconsumed medium
components include chloride ion, sodium ion, and
even phosphate ion. Under ordinary conditions, only
a negligible amount of these components are taken
up by cells. However, these normally unconsumed
salts may be taken up in substantial quantities
in fed-batch cultures where fortified feeding is
used to add more glucose, amino acids, and other
nutrients to grow cells to a higher concentration.
In that case, these will also need to be replenished.
Thus, unconsumed medium components
may become stoichiometric components
under high density cultivation conditions.
Example
• PO4-3 concentration in medium is typically 1 mM, while its cellular concentration is 11 mM. The
intracellular ATP/ADP concentration is about 2mM, which represents around 6mM of phosphate.
Total DNA and RNA contain another 35 mM equivalent of phosphate. A culture with cells of an
average volume of 2 x 10-12 L and at a cell concentration of 1010 cells / L take up about [(11+6+35)
mmole / L x 2 x 10-12 L / cell x 1010 cell / J= ~ 1 mmole / L of PO4-3.
• Cell growth will certainly cause PO4-3 concentration in the medium to decrease drastically at such a
cell concentration.
• For instance, phosphate and trace metals are consumed in very small amounts, although their
depletion may not lead to stoppage of growth immediately, they must still be supplied in
stoichiometric quantities.
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Stoichiometric vs. Catalytic Cell culture medium often contains macromolecules
Macromolecular Components such as insulin, fibroblast growth factor (FGF),
serum albumin, etc. These constituents serve a
variety of purposes; some are carrier proteins
• Some high M.W. medium components are carrying ligands into the cell, while others are growth
interalized and consumed factors that bind to receptors on the cell surface.
• Others are not internalized when exerting their
functions and are degraded only slowly
Molecules that are taken up by cells may need to
be replenished to maintain their concentration,
whereas molecules that transmit signals by
binding to cell surface receptors may not need to
be replenished as frequently during cultivation.
Many macromolecules are internalized, and
some are degraded (consumed), while others
are recycled. For example, the ferric ion carrier,
transferrin, binds to transferrin receptor and is
internalized. After being internalized, transferrin is
translocated to lysosome, and after releasing ferric
ion in the low pH environment of the lysosome,
transferrin is recycled to the extracellular medium.
As long as ferric ions are available (i.e., replenished),
transferrin can continue its role as ferric ion carrier.
Insulin, another commonly used growth factor,
binds to the insulin receptor and triggers a
signaling event that does not involve its own
internalization. However, when present at a high
concentration (a few microgram per ml), insulin is
rapidly internalized by hepatocytes and degraded.
Therefore, even though as a growth factor it is
not consumed, its concentration does decrease.
An example of a consumable macromolecule is low-
density lipoprotein (LDL). After an LDL particle
binds to an LDL receptor on the plasma membrane,
the receptor-ligand complex is internalized in a
clathrin-coated pit that pinches off intracellularly
to become a coated vesicle. The clathrin coat then
depolymerizes, resulting in an uncoated (smooth
surfaced) vesicle, often called an endosome. The
endosome then fuses with an uncoupling vesicle
that has an internal pH of about 5.0, which causes the
LDL particles to dissociate from the LDL receptors.
MEDIUM DESIGN | 105

The LDL receptors are then recycled back to the
plasma membrane. The vesicles containing the LDL
particles fuse with lysosomes, in which the cholesterol
esters are hydrolyzed to fatty acids and cholesterol.
Cholesterol is incorporated into cell membranes.
Components of Basal Medium

Water Mammalian cells are exceedingly sensitive to the
quality of water used for media preparation. City water,
• Types of Contaminants in City Water: the usual source of water for medium preparation
• Inorganics—heavy metals, iron, calcium, chlorine contains particulates, bacteria which are the source
• Organics—by-products of plant decay, detergents of endotoxin, trace organics, and various inorganic
• Bacteria—endotoxin or pyrogen ions including harmful heavy metals. Typical water
• Particles—colloids or particles preparation processes include deionization through
• A typical water preparation process involves filtration, ion exchange, microfiltration to remove particulates
Reverse Osmosis and bacteria, and finally reverse osmosis to reduce
• WFI used to be employed, now mostly pure water. conductivity (or increase resistance) to > 20 MΩcm.
In some cell therapy applications, because the
product (i.e., cells) is subjected to little purification
process before administration to the patient, cell
culture medium may be prepared using water
for injection (WFI) to avoid the entry of any
pyrogenic contaminants. WFI is prepared by low
evaporation rate distillation, minimizing the chance
of any water droplet in the evaporating stem from
carrying over any solute or particle from the water.
Sugars and Energy Source Glucose and glutamine are the primary nutrients
that supply a cell’s energy needs in culture. The
physiological concentration of glucose in blood
is 0.8 g / L. In culture, glucose is typically present
from 1 g / L (5.5 mM) to 5 g / L (27.5 mM). In the
production reactor, sometimes a high level of
glucose, as much as 15 g / L (82.5 mM), is used.
In this case, glucose is a large contributor to the
osmolarity of the medium, and adjustment of the
composition of the medium must be made (by
reducing sodium and chloride concentration) to
MEDIUM DESIGN | 106

maintain osmolarity in a growth permissible range.
Six-Carbon Sugars
All cultured cells express the GLUT1 transporter
• Glucose:1-5 g / L,
at a significant level, and take up glucose readily.
• Fructose, galactose, may also be used.
GLUT1 also transports galactose. Thus, galactose
• Galactose and glucose can both be transported by the
can be used as an alternative sugar to glucose. The
GLUT1 transporter, which is present in most cells.
galactose km for uptake is higher than for glucose. In
• Fructose is transported by a different transporter
(GLUT5); unless the transporter is expressed, the cell the concentration range used for glucose, galactose
may not be able to use fructose efficiently. is taken up by cells at a lower rate, resulting
• The alternative sugar is often taken up by cells at a in lower lactic acid production in the culture.
slower rate, which may reduce lactate production and
be good for cell maintenance. Fructose is also transported by the GLUT5
transporter. The Km for fructose transport by
• Pyruvate and ribose are sometimes supplied in small
quantities, insufficient to supply cell's energy needs. GLUT5 is also high. Thus, similar to galactose, the
uptake rate for fructose is lower than for glucose
unless a high concentration of fructose is used. At
a low uptake rate, the use of fructose also results in
lower lactic acid production compared to glucose.
Glutamine is an essential amino acid for cells in
Glutamine Serves Two Roles:
culture. Most cells in tissues express glutamine
• source of amino acid for protein synthase, and make glutamine from glutamic
• nucleoside synthesis, energy source. acid. In culture, they consume glutamine at
roughly 1/5 to 1/10 of the consumption rate of
glucose. Glutamine supplies the amino group
for nucleotide biosynthesis. It also supplies the
carbon backbone of TCA cycle intermediates by
• Glutamine is consumed in large quantities, converting to α-ketoglutarate. Through carbon
approximately 1/5 to 1/10 of glucose in molar amount. 13 tracer experiments, it has been shown that
• Glutamine is spontaneously degraded in aqueous glutamine contributes to lactate production
solution. during cellular energy metabolism in culture.
Glutamine spontaneously degrades in aqueous
solution, and releases ammonium. Consequently,
to avoid degradation, glutamine is typically
added to culture medium immediately prior to use.
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Amino Acids Amino acids are classified as essential or nonessential
based on nutritional studies using animals or tissue
Table 3. Essential and Non-Essential Amino culture cells. Cells lack the biosynthetic pathways for
Acids making essential amino acids, and rely on exogenous
Non-essential amino supply to meet growth needs. Some amino acids are
Essential amino acids†
acids essential only for cells in culture, but not for animals.
L-arginine* L-alanine
In animals, different tissues may cross feed each
L-cysteine* L-asparagine
other; amino acids synthesized in one tissue (e.g.,
L-histidine L-asperatic acid
liver) may be transported to cells in other tissues.
L-isoleucine L-glutamic acid
Some enzymes involved in amino acid biosynthesis
L-leucine L-glycine
are expressed at lower levels in cultured cells in vitro
L-lysine L-proline
L-methionine L-serine
than in cells from their tissue of origin. Glutamine
L-phenylalanine
is non-essential for animals; it is synthesized from
L-threonine glutamic acid through glutamine synthetase. The
L-tryptophan expression level of glutamine synthetase decreases
L-tyrosine* when cells are cultured in vitro. There is also a CpG
L-valine island in the promoter region of glutamine synthetase,
L-glutamine* which may be methylated to cause glutamine
*Essential for cells in culture, not for animals synthetase to be silenced in some cultured cells.
Upon cell isolation for in vitro culture, expression
of some amino acid synthesis enzymes is
suppressed, but expression of some amino
acid transporters is elevated, which allows for
a faster transfer rate to meet growth needs.
• Most culture media contain all twenty amino acids.
Cell culture media developed in the 1960s
• proline is required by mutant CHO cells; and 1970s contained at least the 14 essential
• serine is frequently required at clonal densities; amino acids. Those media were designed to be
• asparagine is required by certain malignant cells; used with serum supplementation, which also
supplies some amino acids. Media designed
• glycine sometimes needed in case of borderline folic
for serum-free culture include all amino acids.
acid deficiency or in the presence of folate analogues
(methotrexate and aminopterin) In medium preparation, amino acid mixtures are often
• Small peptides can serve the same function as amino prepared as concentrated stock solutions organized
acids--some of these are more stable (e.g., glycine- into groups: neutral, acidic, basic, etc. Although the
glutamine) or are transported more readily than their solubility characteristics of amino acids indicates
free amino acids counterparts. that solubilizing them is feasible, the kinetics of their
• Some non-essential amino acids are excreted into cul- dissolution is slow. Some stock solution preparation
ture medium; alanine is most commonly seen. Aspara- methods rely on pH adjustment using acid or base to
gine and proline may also accumulate in medium. increase the rate of dissolution. Those stock solutions
also introduce additional salts into the culture
medium. Thus, when using such stock solutions,
it is important to assess the osmolarity of culture.
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Vitamins The biological activities of vitamins vary. Even
though they are classified as a common class of
• Ascorbic acid may be beneficial for cells that synthesize
collagen. nutrient, their biological roles are diverse. They all
• Vitamin A can have a pronounced effect on growth and
share the common feature of being essential for the
differentiation of some cell types. vitality of humans, and are needed only in minute
• Vitamin K is required for gamma-carboxylation and quantities compared to glucose and amino acids.
correct processing of vitamin K dependent proteins. Some vitamins are cofactors involved in biochemical
• Vitamin E functions as an antioxidant. reactions, and are required by all cells. These include
• Vitamin D regulates Ca+2 and is regarded by many as a biotin, thiamine pyrophosphate (or its precursor),
hormone rather than a vitamin. Most toxic of all vita- riboflavin and cobalamin. Some vitamins such as
mins when present in excess. vitamin D, vitamin K, vitamin A, and vitamin C are
• Thiamine pyrophosphate catalyses the transfer of car- required by only certain differentiated cell types.
boxyl group, transketolase, transaldolase.
• Pyridoxal phosphate (pyridoxine vitamin B6) catalyses
transamination.
• Biotin is a carrier of activated CO2, and is involved in
pyruvate dehydrogenase, pyruvate carboxylase, and
fatty acid synthesis.
• Cobalmin (B12) is involved in free radical reactions of
intramolecular C-C bond rearrangement, methylation,
and conversion of ribonucleotides to deoxyribosenu-
cleotides.
Nucleosides Nucleosides are not included as essential components

of basal media when serum is supplemented.
Table 4. Nucleosides in Basal Medium Inclusion of small quantities of nucleosides is
RNA DNA common in serum-free medium. The small quantities
Adenosine Thymidine included reflect the low nucleoside content in
Cytidine 2’deoxyadenosine cell mass: nucleic acids (RNA and DNA together)
Guanosine 2’deoxycytidine constitute only about 5% of dry cell mass. A purine
Uridine 2’deoxyguanosine source (adenosine or hypoxanthine) together with
thymidine is beneficial when folic acid is in limited
supply (e.g., in the case of methotrexate selection).
Fatty Acids and Lipid Precursors Lipids constitute a significant portion of biomass of
mammalian cells. Lipid bilayer membrane forms
vesicles which play key roles in protein secretion
and virus replication. The composition of lipids in
membrane affects its fluidity. However, the effect
of lipid supply in medium is usually rather subtle.
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Unlike the supply of essential amino acids or sugar,
whose deficiency cause observable effects on cell
growth almost immediately (with only a delay of
one doubling time), the effect of supplementing
or removing lipid is not as easy to assess.
Mammalian cells can synthesize almost all
Fatty Acids lipids required for their growth, including fatty
acids, phospholipids and cholesterol except for
• Cells can synthesize fatty acid, but can’t introduce
double bond beyond C9. auxotrophic mutants. Cells can make fatty acids of
• Some cell lines benefit from cis-unsaturated all different carbon length in membrane, including
fatty acids, such as oleic acid, linoleic acid and most unsaturated fatty acids, which constitutes
arachidonic acid (a precursor for prostaglandin almost half of the fatty acids in phospholipid.
formation)
However, mammals do not introduce double bonds
Phospholipid Precursors beyond C9 into fatty acids. Linoleate (18:2 cis-∆9,
• Choline ∆12) and linolenate (18:3 cis-∆9, ∆12 ∆15) are thus
• Ethanolamine essential fatty acids. Hence complete medium
for serum-free culture usually contains oleic acid,
Inositol—precursor for phosphatidyl-inositol linoleic acid, sometimes also arachedonic acid.
biosynthesis However, the removal of essential fatty acids from
• Cholesterol—required by some cell lines (e.g. culture medium, does not cause immediate cessation
NS-0 myeloma) of growth, subtle changes in membrane properties
is often masked by its residual amount in the cell
and visible effect may take a long time to emerge.
Ideally supplementing cells with phospholipids is
a good practice. However, non-animal source of
phospholipids with reproducible quality is not easily
available. Often precursors of phospholipids (choline,
ethanolamine, inositol) are supplied. Cholesterol is
supplied to its auxotrophic mutants, such as NS0 cells.
Cholesterol and fatty acids have very low solubility.
Fatty acids are conjugated in serum albumin when
serum supplement is used. In albumin-free medium
fatty acids may mixed in with other hydrophobic
supplements. Cholesterol can be supplied as
derivatized ester with a organic acid attached to
its hydroxyl group to increase its solibility, or can
be supplied as cyclodextrin conjugated complex.
MEDIUM DESIGN | 110

Optimal Concentration of Organic
Nutrients Upon determining the essential and beneficial
components of medium, it is necessary to decide on
the optimal concentration of those nutrients. The
experimental determination of concentration range
is complicated by cell growth and consumption
that causes medium composition to change.
Furthermore, metabolites accumulate as cell grow,
making culture conditions change as time goes by.
This problem was resolved by using clonal growth
as originally demonstrated with ells of human,
chicken and other species.. Cells were seeded in
Optimal small petri dishes at very low density of about 10-
50 cells/cm2 as opposed to 104 cell/ cm2 At such
Suboptimal Inhibitory a low cell concentration, the amount of nutrients
consumed is negligible compared to the amount
present in the medium. It was further assumed
Growth Rate
Optimal range that nutrient utilization is completely independent;

typically spans
over 10 fold or thereby allowing the effect of one component to be
more
tested when all the other components are present
in their “optimal” concentrations determined by
the point of experimentation. After plating cells
are dispersed as single cells on the surface. A
Starvation couple weeks later, the extent of growth from all
the cells initially plated is estimated by the total
Nutrient Concentration
surface area covered by cells after cells on the dish
Fig. 4.1: Optimal range of nutrients for cell growth is stained with a dye. The optimal concentration
is one which gives largest cell growth area.
The optimal concentration for almost all
nutrients (amino acids and other small molecular
weight molecules) span over a wide range of
at least ten fold. One can thus conclude that
for the purpose of cell cultivation, most cells
have a wide range of optimal concentration
for most organic basal medium components.
Bulk Salts and Trace Elements

Mineral elements are also essential components of
cell mass. Phosphate constitutes part of nucleotides
and nucleic acids; magnesium is present in high
concentrations in the cell as it is conjugated to ATP,
which is present in the mM range; calcium, which is
MEDIUM DESIGN | 111

essential for signaling in some differentiated cells,
Table 5. Concentrations of bulk ions in basal medium
(mM) is present in high concentrations in endoplasmic
DMEM/ William’s DMEM RPMI F12 reticulum (ER) and in some organelles. Calcium
F12 (1:1) E and magnesium are also involved in cell-cell and
Na+ 150.31 143.71 155.12 137.74 144.03 cell-substrate adhesion. It must be noted that
K+
4.18 5.37 5.37 5.37 3.00 sodium and potassium, through their reverse
Mg2+ 0.71 0.81 0.81 0.41 0.60 abundance in cytosol and medium, balance
Ca 2+
1.05 1.80 1.80 0.42 0.30 membrane potential, which is fundamental to life.
Many trace metals play key biological roles. Ferrous
Cl- 126.66 125.33 118.48 108.03 134.83
PO43- 1.02 1.17 0.78 5.63 1.17
ion plays a key role in electron transfer complexes, as
HCO3 -
29.02 26.19 44.04 23.81 14.00
does copper ion. Zinc is present at high concentration
SO42- 0.41 0.81 0.81 0.41 0.00 in pancreas and is conjugated with insulin. Selenate
NO3 -
0.85 0 serves as an antioxidant. These elements are
Total 313 302 327 282 288 required by cells in minute quantities; however,
their long term deprivation is detrimental to cells.
Role of Bulk Ion
There is a wide range of concentrations of bulk ions
• Maintenance of membrane potential (Na+, K+)
that is conducive to cell growth. What is apparently
• Osmotic balance (Na+, Cl-, HCO3-etc.) contribute most most important for growth is the balance of
of the osmolality of fresh medium. Optimal range is 280 -
310 mOsm / kg. osmolarity. The most important contributors
to osmolarity are sodium ions, chloride ions,
• Biological roles:
and bicarbonate; while the concentrations of
Mg+ conjugate with ATP
these can be varied, the total osmolarity must
Ca2+, Mg2+ for cell adhesion
be maintained in the range of 270-330 mOsm.
PO43- for nucleotides
When bicarbonate is not used in culture medium its
• Signaling (Ca2+ )
contribution to osmolarity must be replaced by other
• Buffering (HCO-3, HPO3-4)
salts. It is common practice to add a mixture of NaCl
and KCl to maintain their molar ratio at about 30.
Table 6. Trace Elements in MCDB
104 (serum-free medium for hu- There is a wide range of conducible concentration for
man diploid cells) (mM) bulk ions. What is apparently most important is the
CuSO4·5H2O 1.0 x 10-6 balance of osmolarity during growth stage. The most
important contributors to osmolarity is sodium and
FeSO4·7H2O 5.0 x 10-3
chloride ions, the concentration of both can be varied,
MnSO4·5H2O 1.0 x 10-6 so is bicarbonate. However, the total osmolarity
(NH4)6Mo7O24·4H2O 1.0 x 10-6 must be maintain in the range of 270-330 mOsm.
NiCl2·6H2O 5.0 x 10-7
SeO2 3.0 x 10-5
Na2SiO3·9H2O 5.0 x 10-4
SnCl2·2H2O 5.0 x 10-7
NH4VO3 5.0 x 10-6
ZnSO4·7H2O 5.0 x 10-4
MEDIUM DESIGN | 112

When bicarbonate is not used in culture medium
Trace Elements
its contribution to osmolarity must be replaced by
• Those clearly required by cultured cells are: iron, man-
ganese, zinc, molybdenum, selenium, vanadium, copper. other salts. It is common practice to add a mixture
of NaCl and KCl to keep their molar ratio at about 30.
• Trace elements are ubiquitous contaminants of chemi-
cals and supplements used in preparation of medium.
• Some media contain rare trace elements such as rubid-
ium, cobalt, zirconium, germanium, molybdenum, nickel,
tin and chromium; may be needed for long-term growth in
protein-free medium.
• What are the roles of heavy metal ions? The tables
provide a reference of optimal starting
Non-Nutritional Medium Components
Some components of medium are additives that

make it operationally easier to grow cells. They can be
removed from medium without harmful effect, and
do not appear to be taken up by cells. However, their
presence in the medium, especially under process
conditions, can minimize operational deviations.
Sodium Bicarbonate Buffer Sodium bicarbonate provides a pH buffer in our body

fluid, and was used to buffer cell culture medium
in the early days of in vitro culture development.
However, the pKa of bicarbonate is 6.1 making it less
than ideal as a buffer for neutral pH. The buffering
capacity of bicarbonate derives from the equilibrium
with soluble carbon dioxide as shown in the inset. The
buffering action of bicarbonate requires the presence
of CO2. With a given bicarbonate concentration,
the pH is inversely proportional to the CO2 level;
thus, as gas phase CO2 goes up, pH goes down.
Typical cell culture medium contains 14-44 mM
NaHCO3, which at equilibrium requires 10% CO2 to
maintain pH at 7.4. As pH decreases due to lactate
production, the CO2 level in the gas phase can be
reduced, which takes the proton to the left hand
side of the equilibrium equation, to maintain pH.
Conversely, as cells begin to consume lactate in the
late stage of a fed-batch culture, the CO2 concentration
in the gas phase is increased to maintain pH.
MEDIUM DESIGN | 113

Bicarbonate Concentration in medium
44mM in DMEM, 14 mM in F12, 26 mM in circulating blood.
• It is necessary to use 5 – 10% CO2 in the incubation chambers; media that contain bicarbonate become
alkaline very rapidly due to loss of CO2 when removed from the incubator.
• The low pKa of bicarbonate (6.1) results in suboptimal buffering throughout the physiological pH range.
• NaHCO3 buffer requires appropriate CO2 concentrations in the gas phase. The reactions are:
CO2 dissolves in aqueous solutions.

The CO2 concentration in liquid is described by Henry’s Law.
H: Henry’s law constant

CO2 in an aqueous solution forms a bicarbonate ion.

The equilibrium is described as:
From the definition of pH and pKa
The pH of the solution is affected by PCO2 and HCO3-.

From the equation, one can plot the relationship among HCO3-, PCO2 and pH.
MEDIUM DESIGN | 114

How do bicarbonate and CO2 work together as a pH
buffer?
The equilibrium equation, with pKa = 6.36 can be used

to plot the relationship between pH and bicarbonate
concentration. The relationship is also dependent on
CO2(g). Two lines are shown for two different CO2(g)
concentrations. Each point on the line represents the
corresponding pH and carbonate concentration. To
buffer the medium at a given pH one has to select a
combination of bicarbonate and CO2(g) concentrations.
Figure 4.2: Relationship between sodium bicarbonate

concentration, atmospheric carbon dioxide and pH
Alternative Buffer Under some conditions, bicarbonate buffer is

insufficient for maintaining pH—for instance,
• Sodium beta-glycero-phosphate (20 mM) also when a CO2 incubator is not available. There are a
functions as a detoxifier of ferric chloride hydroxo number of buffers known to sustain cell growth.
compounds (i.e., Fe3+ chelator)
Some of these buffers have a pKa in the vicinity of
• Zwitterionic buffers: HEPES (N-2-
hydroxyethylpiperazine-N-2-ethane) is used 7.0, and are more suitable for sustaining neutral
between 10 – 50mM. pH than bicarbonate. Unfortunately, most of them
• Alternative buffers can be growth inhibitory at high have a lower maximum concentration that can
concentrations (>25 mM) be used without a negative effect on cell growth,
• Requires balance of osmolality (by adjusting bulk which limits the buffering capacity they can provide.
salt levels, e.g. if 2 mM is used and 40 mM of
NaHCO3 is eliminated, the osmolality must be It should be remembered that CO2 is a metabolite, a
balanced with NaCl / KCl. Need to maintain Na+ / K+ medium component for buffer, and is also a nutrient
ratio (~30:1). required for some biochemical reactions, such as
carboxylation reactions in fatty acid synthesis.
Normally, the role of CO2 as a nutrient is not
Table 7. Cell Culture Tested Biological Buffers apparent because, as cells respire, the CO2 produced
Working is sufficient to supply CO2 needed for carrying out
pKa value Anhydrous
Description Concentration carboxylation reactions in the cell. However, when
at 37° C Mol. Wt.
(mM)
cultivating cells in a bicarbonate-free medium at a
Glycine 1.0M 9.53 75.0 50 ‑ 200
low cell concentration, CO2 may become limiting for
Glycylglycine 7.95 132.1 10 ‑ 20
HEPES 7.31 238.3 10 ‑ 28
cell growth. If continuous air is continuous flown
MOPS 7.01 209.3 10 ‑ 20
over the culture and thus stripping of CO2 occurs,
Sodium CO2 produced by cells may be stripped away and
Bicarbonate 6.28 84.0 2 ‑ 26 may not be sufficient for supporting growth. Under
TRICINE 7.80 179.2 <50 conditions such as these, using an air supply with
a small supplement of CO2 (0.2-0.5%) will suffice.
MEDIUM DESIGN | 115

Antioxidants Reactive oxygen species (ROS) arising from
biochemical reactions cause transient presence of
superoxide radical (O2-) and hydrogen peroxide
Physiologically Relevant Antioxidants (H2O2). Their reactions with lipids, proteins, and DNA
• Vitamin E cause damage to cells and to media components.
• Taurine Physiologically, cells deal with ROS through
• Beta-carotene reducing reactions that use cellular reducing
“agents” such as glutathione. Conventionally, the
• Transferrin
issue is dealt with through medium design either
• Ceruloplasmin
by providing antioxidant compounds to cells to
• Selenium - Selenium-deficient cells are more facilitate their capacity to cope with ROS, or by
sensitive to oxygen toxicity (selenium is a cofactor
for glutathione peroxidase, an enzyme which supplying components to minimize oxidation
helps remove peroxidases from cells). of labile medium components in culture fluid.
• Catalase Except for mercaptoethanol, few other antioxidants
• Superoxide Dismutase are routinely used in cell culture. However, for
• Reduced glutathione the cultivation of differentiated cells or for the
• ß-mercaptoethanol cultivation of cells under stress conditions, some
additional antioxidants are included to prolong the
period before irreparable damage to cells occurs.
Mechanical Damage Protective Agents Some medium components are added with the
intention of modulating the physiochemical
environment of the cell. In addition to osmolarity,
viscosity was speculated to be important for
sustaining cell growth in culture in the early
Table 8. Synthetic Protective Agents Used in Cell
Culture days of serum free culture medium development.
Common name Chemical identity Most process cell culture media contain a
Block copolymer glycols protective agent, Pluronic F68, which was first
of poly(oxyethylene) and
employed in the 1960s in growing BHK cells
Pluronic F68 or F88 poly(oxypropylene) (M.W. ~8350)
Poly(oxyethylene) glycol (or polyeth- in a stirred tank bioreactor. Many Pluronic
PEG ylene glycol) (M.W. ~20,000) surfactants are available; all are block copolymers
PVA Polyvinyl alcohol (M.W. ~20,000) of polyoxyethylene and polyoxypropylene. The
Methylcelluloses (15 cps methocel) larger the POE (polyoxyethylene) group, the more
MC 0.1- 0.2%
CMC, Edifas B50 Sodium carboxymethylcellulose
hydrophilic the molecule is and the greater its
HES Hydroxyethyl starch
detergent-like activity and cell cushioning effects.
PVP Polyvinylpyrrolidone The larger the POP (polyoxypropylene) group, the
Dextran Dextran (M.W. ~78,5000, 20-60 g / L) greater the toxicity and the anti-foaming ability.
Presently, Pluronic F68, within a concentration
range of 0.01–0.1%, provides adequate cell
cushioning, but the degree of foaming is high.
Therefore, it is desirable to determine if a suitable
MEDIUM DESIGN | 116

replacement is available. The only Pluronics other
than F68 known to provide suitable growth/
productivity are F88 and F77. Both have somewhat
different micelle formation characteristics than F68.
Polyethylene glycol or carboxymethyl
cellulose have been used as protective
agents, but are seldom used now.
Antibiotics The use of antibiotics in cell culture is unevenly

Table 9. Antibiotics for Cell Culture practiced. For manufacturing processes, antibiotics
Antibiotic are rarely used, although in vaccine production,
Antibiotic Concentration
Spectrum the use of neomycin, polymyxin B, streptomycin,
Chlortetracycline Gram‑positive and and gentamicin are sometimes seen. For the
5 mg / L Gram‑negative cultivation of primary cells, which are not subjected
bacteria
to a long duration of quality control prior to
Gentamicin sulfate Gram‑positive,
Gram‑negative processing, the use of gentamycin reduces the rate
50 mg / L
bacteria and of contamination significantly. Some antibiotics
mycoplasma are only moderately more inhibitory to bacteria
Nystatin 50 mg / L (or 100
Fungi and yeasts than to cultured cells. Toxicity testing is necessary.
U / mL)
Penicillin G Gram‑positive
100 U / mL
bacteria
Spectinomycin Gram‑positive and
20 mg / L Gram‑negative
bacteria
High Molecular Weight and Complex Supplements

Some calls such as NS0, some hybridoma/
myeloma cells, and hepG2 cells are capable of
rapid growth using only basal medium without any
further supplement. However, such extraordinary
capability is the exception rather than the norm
of cells in culture. Most cells in culture require
supplementation of at least a number of growth
factors and carrier proteins. Many stem cells
and other normal diploid cell lines need high
concentrations of serum to grow. In fact, for isolation
of cells from tissue, serum is still commonly used, at
least in the early stage of cell cultivation. In discussing
the role of various supplements it is instructive
to start by examining the role of animal serum.
MEDIUM DESIGN | 117

Serum or Biological Fluids Serum is the blood fluid left behind after coagulation;
it is free of blood cells and most coagulation
proteins. Serum is an extremely complex mixture
that contains nutrient substances, metabolites,
Functions of serum in cell culture medium: hormones, plasma proteins, substances released
from damaged cells (e.g., hemoglobin and growth
• Protease inhibitors (alpha 2 macroglobin) neutralize
proteases used in trypsinization or produced by cells. factors from platelets), antibody molecules against
• Provides hormones and growth factors.
various antigens to which the animals have
been exposed, and may even harbor infectious
• Provides carrier proteins
agents such as viruses carried by the animal.
• for low molecular weight substances
(e.g.,transferrin) Fetal bovine serum (FBS) is the most widely used
• for nutrients which dissolve poorly (e.g., fatty serum in animal cell culture because it contains higher
acids, cholesterol, apolipoprotein) concentrations of growth stimulatory factors and
• Binds compounds which are toxic when present in lower concentrations of growth inhibitory factors.
excessive amounts, and releases slowly Other commonly used sera are human, bovine calf,
• Binds and/or neutralizes toxic substances (e.g. newborn bovine, donor bovine, and donor horse.
detergents).
Serum serves many different and important roles
in cell culture. In addition to providing nutrients
not sufficiently present in basal medium (e.g.,
cholesterol), serum provides factors for cell-
substrate attachment (e.g., vitronectin, fibronectin)
for adherent cells, modulates colloid osmolarity,
a physiological property of medium with respect
to viscosity. Serum contains protease inhibitors,
and neutralizes trypsin used in cell detachment
as well as other enzymes released by dead cells.
Serum contains the carrier proteins transferrin
and serum albumin. Carrier proteins function
to chaperone components that are in very low
concentration or are otherwise poorly soluble,
such as fatty acids (carried by albumin), or are
unstable such as ferric ion (carried by transferrin).
Serum is rich in “bulk” proteins (e.g., serum
albumin) that can prevent non-specific adsorption
of critical factors to culture vessels. Serum also
plays an important role as a scavenger. In the
course of cell cultivation, various contaminants
may arise from numerous sources. For example,
this could result from leaching of minute chemical
components from reactor parts, from the filter
used in medium preparation, or even from medium
MEDIUM DESIGN | 118

supplements. Some of these chemicals may be
Disadvantages of serum in cell culture medium
detrimental to cell growth or may have other
• potentially introduce animal viruses into cell culture negative effects on cells. In the presence of serum in
and other undesirable contaminants (e.g. adventitious
agents, antibiotics, proteases). the medium, those compounds may be sequestered
• Availability of high quality serum by adsorption to serum proteins before they can
act on cells, thus minimizing potential damage.
• high running costs and unnecessary capital outlay.
• Normally purchased in large lot sizes and costly Animal serum, however, has numerous disadvantages
storage. in addition to cost and the difficulty of controlling
• Serum lot testing tedious and costly. consistent quality. The most serious concern is the
• Increase complexity of downstream processing and possibility of contamination with animal viruses or
final product characterization. prions. The presence of serum in culture medium also
makes downstream processing more complicated,
and makes the task of final product characterization
more complex. Serum carries antibodies, some of
which may be against viruses that the animal donors
had been exposed to. For virus production processes,
if serum antibodies cross-react with the product
virus, the production will be drastically affected.
MEDIUM DESIGN | 119

Insulin and Insulin-like growth factor (IGF- Insulin plays a key role in regulating glucose uptake
1) by many cell types. In addition to modulating
glucose metabolism, insulin also exhibits mitogenic
effects, and stimulates cell growth through an
• Insulin stimulates glucose uptake by adipocyte overlapping pathway with IGF-1. IGF-1 has an
and other cells, also has a mitogenic effect at high acute effect on protein and carbohydrate anabolism
concentrations by increasing cellular uptake of amino acids and
• Insulin is used in culture at 1 - 10 µg /ml range. glucose, and by stimulating glycogen and protein
Blood insulin level is 4µU/ml or 1.3 µg / ml (1
µU=0.33 µg) ; IGF1 level is is 100 to 200 ng / mL. synthesis. IGF-1 also affects cell proliferation,
differentiation, and apoptosis. It is a potent mitogen
• Insulin and IGF have overlapping signaling
pathway through IR and 1GF1R acting to increase DNA synthesis and to stimulate
• IGF1 regulates cell growth, IGF2 invovles in the expression of cyclin D in a wide variety of cells.
development. IGF1 can replace insulin in cell Both insulin and IGF-1 bind to the insulin
culture at a much lower concentration
receptor (IR) and IGF receptor (IGF1R) but with
different affinities. After insulin binding, IR or
IGF1R is phosphorylated, leading to activation
of an insulin receptor substrate (IRS). There are
multiple isoforms of IRS that are distributed
differently in cells of different tissues. The signal
is then relayed to downstream signaling pathways.
The response of the cell to insulin and IGF is
dependent on the abundance level of the different
IRS isoforms. Most cells, including CHO cells, express
both IR and IGFR. However, NS0 cells express only
IGFR. Differential binding to IR and IGF1R, as well
as differential activation of various IRS isoforms
leads to different responses to insulin and IGF1.
Insulin is used in cell culture at concentrations
that are nearly a hundred-fold higher than found
in blood. At such high concentrations insulin can
trigger a mitogenic response. IGF has a much
stronger affinity for IGF1R and stimulates cell
growth at much lower concentrations than insulin.
MEDIUM DESIGN | 120

Industrial NS0 cells or other myeloma lines are
grown without insulin supplementation, and many
CHO cells have been adapted to grow without
insulin. It is likely that the signal transduction
pathways have been altered downstream
of IR or IGFR in those adapted cell lines.
Insulin was one of the first recombinant proteins to
be made available for therapeutic use. Recombinant
insulin has long been used in cell culture. IGF1 is
also used in cell culture, often in a commercial form.
in cell culture, often in the commercial form.
Transferrin Transferrin is the iron carrier glycoprotein in

mammals. Ferric ion is highly oxidative, and exists
• Typical concentration: 1 – 30 µg / mL (MW 80kd, 10 µg primarily bound to heme and other proteins with
/ mL=0.1 µM) iron centers. In circulation, ferric ion is bound to
• 80 kDa glycoprotein with homologous N-terminal and transferrin. Transferrin has a very high binding
C-terminal iron-binding domains. Binds to iron very constant for iron, but dissociates readily at low
strongly with a dissociation constant of approximately pH. Transferrin receptor-bound iron is taken up
1022M-1. by cells and translocated to lysosomes, where iron
• Low interspecific potency; for human and rodent cell is released for incorporation into cellular proteins.
lines can be replaced by other iron binding protein (i.e.
hemoglobin), For industrial processing, it is desirable that all
growth factors supplemented to culture medium be
• May be replaced by an iron-chelating agent, such as
citrate. derived from recombinant DNA expression rather
than having been isolated from human or animal
sources. Although recombinant insulin and other
growth factors have long been available, recombinant
transferrin became available only in recent years.
The specificity of transferrin binding to cross-
species transferrin receptor is not universally high.
Table 10. Some Iron Chelators as Transferrin The recombinant transferrin used commercially
Replacements
is primarily of human origin. The concentration
Ferric citrate 0.1 - 0.5 mM
Ferric iminodiacetic acid 0.001 µM
required for cells of different species may
Ferric ammonium citrate and differ and must be empirically determined.
Tropolone 2 - 10 µM
Transferrin can be replaced by iron chelating
agents, including citrate. Most chelating agents
have a much lower binding constant for iron than
does transferrin, and they are used in higher
concentrations than are needed for transferrin.
MEDIUM DESIGN | 121

Serum albumin and other carrier proteins The most abundant protein in serum is albumin.
Albumin is a versatile molecule and is a carrier for
Serum Albumin
• used at 0.1 – 5 mg / mL many compounds that may have low solubility in
aqueous solution. Most notably, albumin is a carrier
• High interspecific potency
for fatty acids, which can be toxic when present
• Fatty acid composition and content depend on method
of preparation and species. in free form at high concentrations. Albumin also
binds bilirubin, heavy metal ions, and other agents
• Most defined medium use fatty acid-free albumin
coupled to specific fatty acids, particularly oleic acid or that may harm cells. Albumin is probably the
linoleic acid. most important protein that mediates scavenger
functions of serum in cell culture medium.
Recombinant forms of human albumin are also
Table 11. Transport and Carrier Proteins
available. However, unlike other recombinant
Transport Source Structure Effects
proteins proteins, which are easily characterized for use in
Supplies free fatty a chemically defined medium, the diverse binding
Serum albumin Plasma
1-chain acids capacity of albumin makes its complete chemical
(MW=68000) Detoxifyer contains
trace elements
properties harder to define. Not all albumin
1-chain Supplies iron preparations are the same—albumin from different
Transferrin Plasma
(MW=77000) detoxifyer preparations may be bound to different amounts
High density
Particle Accepts and and varieties of fatty acids or other compounds.
(multiple transports
lipoprotein Plasma In addition to transferrin and serum albumin,
protein cholesterol and
(HDL)
subunit) cholesterol esters serum also contains other carrier proteins as
Low density Transports shown in the table. Most are rarely used in culture.
Particle (Apo
lipoprotein Plasma cholesterol and
B)
(LDL) cholesterol esters
Trenscobalamin Plasma Binds vitamin B12
1-chain
Ceruloplasmin Plasma (MW= Binds copper
135000)
4 subunits
Red
Hemoglobin (MW Transports O2
cells
~65000)
Cell Adhesion Molecules Anchorage dependent cells are employed in the

vaccine industry and in emerging stem cell and
cell therapy-based technologies. In this section we
will address adhesion of cells only to conventional
stationary surface cultivation, not microcarriers.
Although stainless steel or chemically modified
polymer surfaces are still used, the vast majority
of cells are grown on conventional plastic or glass
surfaces. Many suspension-adapted cells revert to
attachment when grown on adhesive (positively
MEDIUM DESIGN | 122

charged) surfaces. Adhesion can be enhanced
in the presence of serum or by coating surfaces
with adhesion molecules. For the cultivation of
industrial cell lines, there is, in general, no need
for coating the surface with adhesion molecules.
Adhesion molecules are used to promote
adhesion of various stem cells, differentiated
cells, or some highly transformed cells that do
not attach well to tissue culture flask surfaces.
Commonly used adhesion molecules, as shown
in the table, include biological molecules
(fibronectin, laminin), ECM extract (matrigel,
• Cells clumping and cells sticking to vessel surfaces can
which is rich in laminin), and synthetic molecules
be partially corrected by adding to medium:
(poly-L-lysine or RGD peptide (arg-gly-asp).
• Pluronic F68 (0.01 – 0.1%)
• Heparin (10–100 ug / mL) These adhesion molecules may be used to
revert suspension cells to an adherent state for
cell cloning. Under adherent conditions, cells
form colonies on the surface of culture dishes
and can be easily isolated using a cloning ring.
In some cases, one might want to prevent cell
adhesion to a surface. Inclusion of heparin, heparin
sulfate, or Pluronic in the medium, along with use of
a non-adhesive surface can minimize cell adhesion.
Table 12. Adhesion Molecules Used for Cell Culture

Adhesion proteins Source Effects
Fibronectin Plasma, cell lines Promotes attachment growth of mesenchymelly derived cells
Promotes attachment and growth of ectodermally and endo-
Laminin Extracellular matrix
dermally derived cells
Skin, extracellular Promotes attachment and growth either directly or through
Collagens (I-IV)
matrix, placenta the binding of other adhesion proteins
Vitronectin Plasma Promotes attachment and growth of a variety of cell types
Fetuin FBS Promotes attachment of cells to glass and plastic
Promotes attachment of many cell types (even in the presence
Poly-d-lysine Synthetic Polymer
of serum)
MEDIUM DESIGN | 123

Protein Hydrolysates Hydrolysates or extracts from animal or plant tissues
were commonly used in cell culture processes to
reduce the dependence on serum. A beef extract,
Ex-Cyte, was an excellent source of phospholipids;
however, the use of animal extracts has been largely
• Soybean hydrolysate and peptone are commonly discontinued, and has been replaced by hydrolysates
used
from soy, rice, and other plants derived by enzymatic
• Peptones derived from acid or enzyme hydrolysates
of casein, gelatin, meat, soy, egg and lactalbumin or acid hydrolysis. Lot to lot variation among such
have been used as supplements in cell culture extracts is huge. In a transcriptome analysis of CHO
• Contain a mixture of amino acids, small peptides, cell samples grown under different reactors, pH,
inorganic ions, carbohydrates and vitamins and other conditions, it was found that the specific
• Possible roles of hydrolysate lot of hydrolysate overrode other experimental
• Source of amino acids in the form of variables in sample clustering. In a data mining
oligopeptides experiment encompassing data from more than 100
• Some oligopeptides may mimic analogues of manufacturing runs, it was found that hydrolysate
signaling molecules by having non-specific lot had a strong correlation with productivity.
binding various cell surface receptors
• Such effects may be growth-promoting or The roles of hydrolysates are not completely
apoptosis-retarding understood. Hydrolysates are complex mixtures
of amino acids, peptides, derivatized peptides,
carbohydrates, and some lipids. They provide some
nutrients and minerals and may also act as scavengers
through undefined molecular interactions with
possible contaminants. Hydrolysates may have some
growth-stimulating or anti-apoptotic activities.
Given that synthetic peptides have been found to
interfere with signaling pathways by binding to
signaling intermediates, the possibility cannot be
excluded that some hydrolysates have similar effects.
MEDIUM DESIGN | 124

Lipid Supplements Lipids are sometimes added to serum-free culture
medium, although they must be used with caution.
Most lipids in circulation are associated with
carriers. Too much free lipid in culture medium is
not desirable. Phosphatidyl choline, phosphatidyl
ethanolamine, and/or sphingomyelin may be added
to cell culture medium in the form of liposomes
or may be dissolved in DMSO. Reconstituted lipid
supplements containing a defined composition of
phospholipids are commercially available for use in
chemically defined medium.
Medium for the Industrial Production Culture

Medium design for industrial cell culture
processes encompasses two aspects, cell
expansion and production. For cell expansion
the focus is on optimal growth and sustained
viability; for production, the objective is rapid
growth to production cell density, sustained
viability, and productivity at high cell density. For
the production of non-cell products including
recombinant proteins and viruses, the viability of
the culture at the end of production may not need
to be high; thus an extreme composition that favors
production at the expense of viability may be used.
In the past decade it has become a common practice
to use high osmolarity in the final stage of production
culture. Glucose concentrations as high as 15 g/L
(83 mM) may be used at the initiation of production
culture. At such a high level, glucose contributes
significantly to overall osmolarity; thus, the
concentration of NaCl is often reduced to compensate
for the additional glucose. High osmolarity
affects the growth rate of most cells, although a
high glucose concentration does not appear to
affect growth of some CHO and myeloma cells. As
discussed in the section on Fed-Batch cultures, most
industrial production is initiated at about 70% of
reactor volume, and concentrated nutrient mixtures
of amino acids, glucose, etc. are added during
cultivation. The concentrated nutrient mixtures
usually carry extra salts used to dissolve amino
MEDIUM DESIGN | 125

acids, which additionally increase the osmolarity.
As a consequence, it is not unusual to reach an
osmolarity of 400 mOsm by the end of production.
In recent decades, the concept of industrial medium
design has undergone a major shift. The focus on
providing cells with optimal growth medium is now
restricted to applications involving cell line isolation,
banking, and expansion. A new focal point is the
design of medium for production cultures. In some
cases, even in the expansion of the seed train toward
a production scale, the medium used has been altered
from traditional “optimal” formulations; for example,
sometimes high glucose concentrations are used. It
has not been shown that such conditions provide
any advantage for cell expansion, but new points of
view have replaced the notion that “optimal” growth
conditions are likely to be found by recreating
the native niche of a particular tissue of origin.
For industrial processes, the purpose of medium
design is not to provide conditions that cells like, but
to devise conditions that can be imposed on cells to
maximize their productivity. Significant advances
toward that objective have been accomplished.
MEDIUM DESIGN | 126

Methods and Strategies in Cell Line Development
Cell Line Development
With Contributions From Gargi Seth
Host Cells and Recombinant Protein Production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Transient Transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Stable Cell Line Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
What Makes a Hyper-Producing Cell Line? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Basic Steps for Generating a High-Producing Cell Line . . . . . . . . . . . . . . . . . . . 131
Basic Elements on a DNA Plasmid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Cell Adaptation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Stability of Selected Clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Automation and High Throughput Technology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
Host Cells and Recombinant Protein Production

Animal cells are powerful vehicles for the production
of therapeutic recombinant proteins. Therapeutic
proteins, along with viral vaccines and cell therapy
products, are generally called biologics to differentiate
from small-molecule drugs. The process of
transforming the host cell line is critical to producing
cells of high productivity. Cells must be screened for
those that contain a superior level of production
and growth characteristics. This chapter discusses
the process of generating cell lines suitable for the
large-scale production of recombinant proteins.
Industrial expression of recombinant proteins is
generally categorized into two types: transient or
stable. Transient expression is frequently used for
the generation of small quantities of protein (up
to gram quantities), for protein characterization
METHODS AND STRATEGIES IN CELL LINE DEVELOPMENT | 127

or animal testing. This process does not generate
Transient Expression:
a clonally derived cell line; it temporarily
• Not suitable for long term production or commercial
manufacturing transduces an existing cell line (e.g. HEK293 or
COS) with plasmids encoding the product protein.
• Useful for rapid production of small quantities of
research materials: Conversely, stable cell lines are generated for the
• Drug candidate identification and in-vivo industrial production of therapeutic proteins. These
evaluation
cell lines must be capable of producing product of
• Assay development (e.g. binding assays) the same quality in different batches, and in different
• Structural studies locations, throughout the years. Once a production
• Toxicology studies line is transduced and selected, cells are expanded to
establish a master cell bank, which is stockpiled. Cells
from a mater cell bank are then expanded further
to create a working cell bank from which frozen
vials of cells are taken for use in the production.
The banked cells, typically stored in liquid nitrogen,
are used for manufacturing purposes throughout
the production cycle. In generating the production
line, both the choice of the host cell and the choice
of the vector carrying the product’s gene are pivotal.
Transient Transfection
The overexpression of exogenous gene(s) in target
cells is an important technique used to understand
the functional significance of an unknown gene.
Often, the desired effects can be observed with a
transient expression of the protein, so in these cases,
Transient Expression Protein Production stable transduction is often un-warranted and un-
• Transgene in plasmid DNA or virus (advenovirus or necessary. Transient expression is also frequently
vaccinia virus) is introduced into the cell, the nucleus used to produce and isolate protein products encoded
where it exists as an extrachromosomal unit. by a transgene. The aim, in this instance, is to simply
• Cells are heavily loaded with vectors to increase the obtain a sufficient quantity of the product protein
production in a short amount of time. The scale of production
• The heterologous DNA is not integrated into the host in these cases is often small, but could also be fairly
genome.
large (e.g., up to tens of liters of reactor volume).
• plasmids generally do not replicate
For protein production through transient
• viral vector may be maintained inside the cell as an
autonomous replicon expression, the host cells are loaded with a very
large amount of exogenous DNA, such as a plasmid.
In transduction, only a small fraction of exogenous
DNA is actually taken up by cells and even smaller
fractions actually enter the nucleus where the
transgene gets transcribed. Thus, to see high levels
of exogenous protein expression, researchers
employ the use of a strong promoter to drive gene

transcription and transduce large quantities of
starting material. However, if too successful, the
overexpression of some genes can be toxic to cells.
Host Cells Most Frequently Used for Transient When transiently expressed, the exogenous vector
Expression: does not get integrated into the genome. After
• HEK-293 (human embryonic kidney fibroblasts) transfection, they exist as extra-chromosomal
• COS (from green monkey kidney cells) elements. Most plasmid vectors, except for some
• BHK cells episomal vectors, do not self-replicate and are
• CHO cells (to a lesser extent) gradually lost in about a week’s time. Even some
viral vectors, such as adenoviral vectors, do not
persist over a long time and are largely lost in a
month. This is fitting for the overproduction of
protein, where the aim is to generate a short and
intense, rather than a long and sustained, burst of
protein production. Thus, in transient situations, the
percentage of cells actually taking up and expressing
exogenous DNA (i.e., the transfection efficiency)
Production Life of the Transient Expression System is can significantly affect the overall expression level.
Usually Limited by:
HEK-293 (293; human) and COS (African green
• Loss of DNA from the cell with time
monkey) cells are frequently used for the transient
• Deleterious effect of foreign DNA on cell viability
expression of proteins, but product quality
• Typical titers ranges from >1 to 100mg / l in 5 - 10 days parameters, such as glycoform profile, may differ for
(~ 0.1-1 pg / cell / day)
materials derived from different species (e.g., human
versus Chinese hamster). However, CHO cells are
unique. Transient material produced in CHO cells is
more representative of the material produced from
stable CHO cell lines, so there has been an increasing
use of CHO cells for stable expression. Unfortunately,
the transient transfection productivity in CHO is
relatively low compared to that of 293 host cells.
Stable Cell Line Development

What Makes a Hyper-Producing Cell Line?
Two types of cells are most commonly used as
host cells for the generation of stable production
cell lines: CHO cells and myeloma cells. Virtually
all therapeutic proteins made with these cell lines
are secreted out of the cell and can be harvested
from their culture fluid. CHO cells, which do not
secrete any protein in an appreciable amount in
their native state, must be made to develop the
capability of protein secretion while becoming
producers. In contrast, myeloma cells have well-

developed protein secretion machinery, left over
from their original purpose of secreting antibodies.
Many scientists have studied the changes that
occur during the maturation of B-cell to plasma
cell, both at the proteome and transcriptome
levels. The cellular alterations appear to
include elevated energy metabolism, higher
Host Cell Protein protein secretion and glycosylation capacity,
Secretion as well as an increased redox balance, to
CHO DG44 serum dependent, adherent, DHFR-/- nil counter the effects of reactive oxygen species.
CHO DXB11 serum dependent, adherent, DHFR +/-
nil
CHO K1 serum dependent, adherent, proline nil B-cells and plasma cells have only one functional
dependent immunoglobulin gene in their genome. In the
SP2/0 serum dependent, suspension plasma developmental maturation process, one of
cell
the two gene copies in the diploid alleles is
Myeloma serum dependent, suspension, plasma
NS0 cholesterol-dependent cell inactivated, thus preventing the production of
two immunoglobulin molecules in a single cell
(i.e. “allele exclusion”). Even a single copy of
a transgene is sufficient for a cell to become a
super secretor, like the plasma cell. Therefore, to
transform a myeloma cell into a high producer of
protein, one needs only to introduce a single copy
of the product gene into the appropriate locus of
the genome. The pre-existing cellular machinery
Typical Characteristics of Producing Lines is already tuned to secrete a high level of protein.
• Serum independent CHO cells, contrary to myeloma cells, must have
• Suspension growth modifications to enhance their protein secretion
• Highly secretory, associate physiological change machinery and to acquire characteristics of high-
• Energetic producing cells. In addition to bolstering the
• Secretory pathway protein secretion machinery, a high-performing
cell line should also have superior growth and
• ROS-redox balance
metabolic characteristics. Manufacturing conditions
• Sustained growth in stationary phase
differ profoundly from laboratory cultivation.
• Ready switch to lactate consumption
For example, the prolonged stationary phase in
• Glycosylation capacity a fedbatch culture, in the presence of lactic acid,
allows for a longer period of protein production
at a stage when the cell concentration is high, thus
leading to a maximal product titer. The important
question, in this case, is how to identify candidate
cell lines that harbor those desirable traits.
In the past decade, numerous transcriptome
and proteome studies have been conducted to
examine the traits leading to hyper-productivity.
Biotechnologists now realize that there is

‘Best’ Producer probably no single master regulator that can be
Cumulative effect of
combination of “turned on” to make a CHO or NS0 cell a hyper-
different changes
may not be the
same, some may not
producer. Hyper-productivity is the culmination
be additive
Potentially a of multiple changes in multiple cellular pathways,

large
Different changes
combination such as metabolism, secretion, redox balance, and
may give rise to
may lead to the
same incremental
improvement of cell
similar
productivity.
growth/death control. The acquisition of hyper-
characteristics
necessary for high productivity is more likely to involve diminutive
productivity
gene expression changes on a vast scale, rather than
larger alterations in only a few master switches.
Energy Secretion Redox Growth/Death
Metabolism Control
Fig. 5.1: Hyperproductivity of recombinant protein in producing
cells contributed by multiple cellular functions. Many alternative
combinations of superior traits may lead to high-productivity.
Basic Steps for Generating a High-

Producing Cell Line
A few basic steps are generally followed to make
a host cell become a high producer of the desired
product. First, a transgene coding for the product
protein is typically introduced to the host cell using
a plasmid. In addition to the transgene, the plasmid
carries a gene that confers a selectable trait, such
as antibiotic resistance, so that after transfection,
a selective pressure can be applied to enrich for
those cells which have internalized the plasmid. The
Steps in Cell Line Creation
plasmid does not replicate in mammalian cells and
• Transfection
would otherwise be gradually lost as cells multiply.
• Selection
By applying selective pressure over an extended
• Amplification period, all of the cells that are selected for would
• Single cell cloning have the plasmid integrated into the chromosome.
• Screening
After a stable cell population is obtained,
• Adaption preliminary in vitro screening is then performed
to screen for clones with the highest production
levels. This is often fulfilled by assaying the product
concentration in the supernatant of a 96-well plate.
Another common practice is to grow cell clones
as small colonies on soft agar and then perform
immunoassays in situ to identify those with a larger
immunoprecipitation zone around the colony.
The next step, the amplification of transgene copy
number, is practiced in some instances (such as
when CHO cells are used), but not all (such as when
myeloma cells are used). To do this, the stable cells
are subjected to a high concentration of an inhibitor

to the amplification marker, which the cells require
for survival. This high concentration of inhibitor
kills the vast majority of cells, except those that have
multiple copies of the marker and resistance genes.
As the amplification marker multiplies, the adjacent
integrated transgene(s) are also co-replicated.
The number of copies may increase dramatically
in different regions of the genome, thereby giving
rise to high levels of transcription and translation.
Through this process, in some high-producing cells,
the transcript level of recombinant IgG heavy chain
becomes the most highly expressed transcript in the
cell. An excessive expression of protein can overwhelm
cell’s protein folding capacity and lead to an unfolded
protein response in the endoplasmic reticulum
and, thereby, induce apoptosis. Consequently, cells
which have not developed appropriate machinery
to handle the increased production may not survive.
After amplification, the selected cells not only have
multiple copies of the transgene and a high level
expression of resistance and product genes, but they
also have developed the secretory capacity to allow
for enhanced protein secretion. These clones have a
high propensity to become high producers, but not all
of them do. Hyper-productivity also requires many
other traits, such as the capability to quickly grow to
a high cell density and the capability to sustain a high
viability over a long duration in the stationary phase.
Fig. 5.2: Typical steps in introducing a transgene Subsequently, single-cell cloning is performed
for generating high producing cell lines for
on those surviving cells, typically by sorting
manufacturing.
single cells into culture wells by flow cytometry.
Cloning can also be performed by dispensing cells
(approximately 0.2 cell per well) into multi-welled
culture plates, so that the probability of having
more than one cell in each well is very low. Thus,
all cells that arise from a given well all originated
from the same cell. Single cell cloning is necessary
because a pool of stable cells would have vastly
different genetic backgrounds, perhaps in the loci
of exogenous gene integration, and potentially have
different mutations caused by those integration
and other unknown aberrations. Such a mixture of
cells of genetic heterogeneity is called a “cell pool”.

After single cell cloning, the productivity of each clone
is then assessed and those with high productivity are
isolated. The selected clones are further expanded
for stock preparation, growth characterization and
product quality assessment. In some cases, the
producing cells are then adapted to growth conditions
more amenable to manufacturing conditions.
Single cell cloning is considered critical for
establishing a production cell line, as the cells arising
from a single cell are genetically homogenous.
It is practiced regardless of whether there is an
amplification step or not. Although the host cells used
for establishing production lines are all aneuploid
and can be prone to further genome reorganization
and epigenetic reprograming, a culture of cloned
cells are much more homogenous than cell pools.
From a single cell at the beginning of single cell
clone to the end of a production run, the production
cell may have gone through more than 60 doublings.
If that is extended to the entire production lifetime,
the number of cell doublings may be greater than 80
doublings. It is important to minimize the outgrowth
of mutated cells in the original pool, during the
course of cell expansion, by single cell cloning
during the generation of production cell lines.
Methods of Gene Transfer

A number of different methods are commonly used
DNA-Calcium Phosphate Co-Precipitation to introduce expression vectors into host cells.
DNA and calcium chloride is added dropwise into a HEPES The choice of method is dependent on cell type,
buffer with sodium phosphate (1 mM). A fine precipitate the available quantity of cells and plasmids, and
forms in 5-30 min, and is added directly to the cells (~2-
the experience of the lab practicing it. Although
40μg/106).
different methods depend on different mechanisms
Electroporation of plasmid uptake by cells, all methods require the
Expose cells to a high-voltage electropulse in the presence cytoplasmic membrane to first become permeable
of DNA solution. This introduces pores in the plasma to plasmids. Plasmid delivery by calcium phosphate
membrane, allowing entry of DNA. Duration of pulse and precipitation, cationic polymers, and liposomes
strength of electric field varies with cell type. relies on direct interactions of the particles, or
Lipofection/Lipid Mediated Gene Transfer lipid vesicles containing plasmid DNA, with the
A mixture of DNA with amphipathic compound (DOTMA, cellular membrane through an endocytosis-
DOPE, etc), that simultaneously interacts with DNA and like of mechanism. In electroporation and
hydrophobic portions of the membrane, allowing passage microinjection, physical force is used to introduce
of DNA into the cell. openings in the cell membrane for DNA entry.
The “Methods of Gene Transfer” table lists

DNA-Calcium Phosphate Co-Precipitation commonly used methods of DNA introduction. All
DNA and calcium chloride is added dropwise into a HEPES methods use a very high plasmid-to-cell ratio, but
buffer with sodium phosphate (1 mM). A fine precipitate only a moderate DNA concentration. While up to
forms in 5 - 30 min, and is added directly to the cells (~2- a thousand copies of the plasmid can enter cells,
40μg/106). only a small proportion actually translocate to
Electroporation the nucleus and are transcribed to allow for the
expression of selectable marker resistance gene.
Expose cells to a high-voltage electropulse in the presence
of DNA solution. This introduces pores in the plasma
membrane, allowing entry of DNA. Duration of pulse and
strength of electric field varies with cell type.
Table 2. Estimates of Number of DNA Molecules per Cell for

Commonly Used Non-Viral Gene Transfer Methods.
Method DNA Cell Number
concentration concentration of DNA
(μg/mL)/pM (106 cells/mL) molecules
per cell
Calcium 50 / 15 5 1.8*106
phosphate
DEAE dextran 10 / 3 5 3.6*105
Lipofection 40 / 12 5 1.5*106
Electroporation 40 / 12 10 7.3*105
Basic Elements on a DNA Plasmid

Plasmids facilitate the introduction of specific
genes into mammalian cells. They also contain
elements that enhance the transcription and
translation of the product gene. Both viral and
bacterial plasmid vectors are commonly used for
introducing transgenes into mammalian cells for
research. However, the prevailing vehicle used for
introducing product gene for recombinant protein
production is a plasmid vector rather than a viral
vector. Although viral vectors are used often in gene
therapy, they are rarely employed for establishing
production cell lines for therapeutic proteins.
Fig. 5.3: A typical vector for introducing transgene into The expression of the product gene may be
host cells for recombinant protein production. driven by a constitutive, inducible, or conditional
promoter. Conditional promoters, driven by specific
endogenous factors or events, are frequently used
in the research of differentiation and development.
It allows a reporter gene or selectable gene to be
expressed after a particular differentiation event. It
enables the selection or sorting of differentiated cells

in a population. For example, the albumin promoter
is expressed and activated specifically in liver cells.
When driving the expression of green fluorescent
Elements in a Vector
protein (GFP), cells of the liver lineage become green.
• Promoter
• Coding sequence (often with introns) of gene of For protein production, the vast majority of
interest vectors employ a constitutive, and very strong,
• poly A signal
• Selectable marker promoter. Traditional viral promoters, such
• Other elements of plasmid (cloning site, origin of as SV40 late promoter or the CMV promoter,
replication) are frequently used. In the past few years,
constitutive promoters, such as the promoters
of elongation factor 1 (EF-1) and glyceraldehyde
dehydrogenase (GAPDH), from CHO, have been
isolated and used in product gene expression.
Target Gene
• Positional cloning: screen library for chromosomal In addition to a strong promoter, enhancer
markers known to flank the gene of interest, and then elements can also be included in the intron of
“walk”, testing individual genes identified in the region.
the transgene construct, to ensure a high level of
• PCR amplify
transcription. It is rather common to see that at
least one intron is included in the product gene
construct. Furthermore, the DNA sequence of
Promoter the product gene is often “codon optimized” to
• Constitutive match the efficiency of translation machinery
• Conditional of the host cell. Through codon optimization,
• Inducible one can replace the codon for a rare tRNA with
• Native dynamic regulated
a more abundant tRNA, to avoid the translation
rate being limited by the supply of the rare tRNA.
In addition to the promoter, enhancer, and
the product gene, the vector must also has
a selectable marker, and if amplification is
to be involved, the amplification marker.
In the course of introducing DNA into host cells,

only a fraction of them actually express the
plasmid. Although the number of plasmids entering
each cell is likely to be very high (probably in the
order of hundreds to thousands of plasmids),
the probability of their entry into the nucleus
and subsequent integration onto the genome is
very low. These plasmids are not capable of self-
replication; they only replicate after integrating
into the host chromosome. Free plasmids in the
cell are, thus, gradually degraded or otherwise lost.
After transfection a very large fraction cell
population are untransfected. To identify and select

those that have the transgene, a selectable marker is
included in the plasmid. Cells that have at least one
copy of plasmid integrated into the chromosome
and express the selectable marker gene will
survive in the presence of selectable marker.
Selectable markers are classified into two
categories: dominant and recessive. A recessive
marker resides in cells with a particular genetic
Recessive Selection background and causes a growth deficiency.
• DHFR (dihydrofolate reductase) Then, the introduction of a compensatory gene
• On DHFR deficient background leads to overcoming the deficiency. For example,
• TK (thymidine kinase)
• On TK deficient background a cell line without a functional dihydrofolate
Dominant Selection reductase (DHFR) gene requires the additional
• Antibiotic resistance supplementation of thymidine and glycine in the
• Neomycin culture medium for cell growth. The introduction
• Hygromycin of a functional DHFR enables it to grow without
• MDR (multi-drug resistance)
the supplements. Therefore, after the transfection
• DHFR
of plasmid containing DHFR, only the transfectants
will grow in the absence of thymidine/glycine.
Similarly thymidine kinase (TK)-defective mutants
require thymidine to be included to the culture
Reporter Gene medium. The introduction of a functional TK gene
• gfp family allows for cell growth in the absence of thymidine.
• β-galactosidase
Conversely, the presence of a dominant selective
• luciferase
• secreted alkaline phosphatase agent is lethal to the cell. By introducing the selectable
marker gene to the cell, the cell is endowed with a
resistance to the selective agent. The most frequently
used selectable markers, and their mode of action in
mammalian cells, is listed in the adjacent table. Each
resistance gene encodes an enzyme, which modifies
the selective chemical agent to destroy its activity.
The phosphorylation and acetylation reactions
employed for inactivating antibiotics require
intracellular reactants (ATP, acetyl group donor).
Those enzymes are, thus, only effective in destroying
the selective agents intra-cellularly. The hydrolysis
enzyme, on the other hand, may be active even when
released into medium after cell lysis. In any case,
in the selection process, the concentration of the
selective chemical agent decreases with time and the
rate of decrease is dependent on the concentration
of transfected cells. Thus, the optimal concentration
for selection for each agent is not only dependent

on cell line but also on its concentration. Clonal
selection and population selection may have rather
different optimal concentration of selective agent.
Another class of selective agents interferes
with the uptake of a toxic selective agent. The
multidrug resistance gene (MDR) confers cells
with resistance by increasing their ability to
pump toxic substances, such as colchicine, out
of cells. Its overexpression allows for selection
from a background of cells not expressing MDR.
Table 2. Commonly Used Drugs for Selection of Stably Transfected Mammalian Cells
Antibiotic Family Mode of Action Resistance gene Mode of Gene Drug
resistance size (bp) concentration
range
(μg / mL)
Geneticin Aminoglycoside Block protein synthesis Neomycin Phosphorylation 795 100 - 800
(G418) by inhibiting elongation phosphotransferase of Geneticin
(npt)
Hygromycin B Aminocyclitol Inhibit protein Hygromycin Phosphorylation 1011 10 - 400
synthesis by disrupting phosphotransferase of Hygromycin B
translocation (hpt)
and promoting
mistranslation
Puromycin Aminonucleoside Block protein synthesis Puromycin Acetylation of 603 0.5 - 10
by causing pre-mature N-acetyltransferase Puromycin
chain termination (pac)
Blasticidin S Peptidylnucleoside Inhibit protein Blasticidin S Deamination of 396 1 - 10
synthesis by interfering deaminase (bsr) Blasticidin S
with peptide bond
formation
Zeocin Bleomycin Intercalate into and Bleomycin Bind 375 0.1 - 50
(Glucopeoptide) cleave DNA resistance protein stoichiometrically
(ble) and prevent
Zeocin from
binding DNA

Amplification Two systems are commonly used for gene
amplification in mammalian cells: DHFR and
glutamine synthetase (GS). The most commonly
used gene amplification system is based on the DHFR
gene, whose chemical antagonist, methotrexate
(MTX) can be used to drive gene amplification. DHFR
is an enzyme which catalyzes the conversion of folate
to tetrahydrofolate, a compound required for the
biosynthesis of glycine, thymidine monophosphate
and purine. Methotrexate, a folate analogue, binds
• The strategy is to use a mutated form of the enzyme and inhibits DHFR, thereby leading to cell death in
that has a lower catalytic activity or to use an enzyme the absence of thymidine and purine in the medium.
inhibitor
When cells are selected for growth in methotrexate,
• DHFR with methotrexate (MTX)
the surviving population contains increased levels
• DHFR:
of DHFR which results from an amplification
dihydrofolate+NADPH → tetrahydrofolate+NADP
of the DHFR gene. This is also accompanied by
• GS with methionine sulphoximine (MSX) amplification of 10 – 10,000 kilobases of DNA
• The metabolic enzyme (amplifiable marker) needs to surrounding the site of integration. Therefore,
be amplified in order to supply sufficient reaction rate by introducing a gene of interest (i.e. protein
for cell survival
product gene) alongside the DHFR gene, co-
• Glutamine Synthetase: amplification of the product gene can be achieved.
Glutamate + ATP + NH3 → Glutamine + ADP + Pἱ
The amplification process can be perform as
a single step of MTX exposure over one to two
weeks, or in multiple step-wise increases in
methotrexate concentration. As MTX concentration
is increased, surviving cells with higher degrees
of DHFR gene amplification are obtained.
Highly methotrexate resistant cells may contain
several thousand copies of the DHFR gene.
DHFR based amplification is more efficient in a
DHFR defective genetic background. Otherwise,
endogenous DHFR may get amplified without
concurrent amplification of the product gene.
Chinese hamster ovary cells deficient in DHFR
were isolated after ethyl methanesulfonate- and
UV irradiation-induced mutagenesis. These DHFR-
deficient cells require the addition of thymidine,
glycine, and hypoxanthine to the media. These cells
do not grow in the absence of added nucleosides
unless they acquire a functional DHFR gene.
The glutamine synthetase (GS) selection system is
based on the biosynthetic pathway of glutamine

from the substrates glutamate and ammonia.
Most mammalian cell lines require glutamine
supplementation in their culture media to grow
since their endogenous GS activity is low. The
promoter region of GS in CHO cells is rich in
CpG and evidences indicate that GS in CHO is
silenced possibly by methylation of cytidine.
The GS expression vector contains a glutamine
synthetase gene along with the gene of interest,
allowing for selection by growth in glutamine-
free cell culture media. The GS gene is usually
driven by a weaker promoter, typically the
SV40 promoter. With a high concentration of
the glutamine synthetase inhibitor, methionine
DHFR Amplification sulphoximine (MSX), it is possible to select
• More efficient in DHFR- background for transfectants with gene amplification.
• CHO DXB11; one DHFR is deleted, the other has Several variations on the systems described above
missense mutation have been developed for increasing achievable
• CHO DG44: both DHFR deleted expression levels. DHFR is used in conjunction
• With sufficiently high levels of MTX, amplification can
with an impaired neomycin resistance gene. After
be carried out in DHFR+ background transgene induction and under G418 selection only
cells with the vector integrated in a transcriptionally
active region will express neomycin resistant
transcripts at high enough level to survive. Since
the locus of integration is transcriptionally
active, the high expressing clones isolated after
amplification have only a few integrated gene copies.
After amplification, lasting for a week to two weeks
typically, the concentration of antagonist selective
chemical agent is reduced. With a lower level of
selective pressure the number of copies of transgenes
may also decrease and resulting in a decrease in its
transcript level and productivity. The propensity for
losing transgenes is probably affected by the loci of
integration on the chromosome, with those near the
distal end of the chromosome arm being more prone
to dislodge from genome. Usually a clone becomes
more stable after the initial drop of copy number
and product titer upon the reduction of selection
pressure. In most cases the remaining transgenes are
stable in subsequent cell cultivation. Nevertheless a
low level of selection pressure is often maintained
to suppress any possible deleterious mutants which
may have a lower copy number and faster growth rate.

Classical DHFR Amplifiable Vector
There have been a number of variations of this method.

Some methods use another resistance marker, such
hygromycin or neomycin resistance, for the first selection
of cells co-transfected with DHFR and the gene of interest,
followed by amplification by selection of amplified
cells in the presence of high concentration of MTX.
The DHFR/MTX system has been widely used for the

generation of antibody-producing cells. The example
Fig. 5.4: A vector for DHFR based amplification of transgene. shown uses a two-plasmid system. DHFR resides on the
Classical DHFR transfections employ a plasmid in plasmid containing the gene encoding the light chain. Note
which DHFR is tethered downstream to the gene of that instead of cDNA, the gene of interest is interspersed
interest driven by a promoter (e.g., SV40 enhancer/ between two intron sequences. The heavy chain gene,
promoter). The plasmid is used to transfect CHO cells on another plasmid, has Neo as the drug resistance
(such DXB11) deficient in DHFR. The transfected cells are marker. The two plasmids are co-transfected into CHO
selected in nucleotide-free medium, in the presence of cells, often at a stoichiometric ratio that slightly favors
methotrexate (MTX). Subsequently, MTX concentrations the heavy chain plasmid. The transfected cells (which
are increased to enrich for cells that have multiple co-express DHFR/light chain plasmid and the heavy
copies of DHFR and, concomitantly, the gene of interest. chain plasmid) are then enriched using MTX treatment.
Fig. 5.5: A gene construct for introducing heavy chain

and light chain molecules of immunoglobulin gene.

Glutamine Synthetase (GS) as Selectable Amplifiable Marker
Glutamine synthetase (GS) is selectable marker in
most mammalian cells, as they require glutamine
for growth in culture. It is used frequently in my-
eloma and CHO cells. The transfectants are selected
by growth in a glutamine-free medium. Vector am-
plification can subsequently be achieved by using
the inhibitor of GS, methionine sulphoximine (MSX).
Fig. 5.6: A vector with GS selectable marker.
Directing Integration to a Transcriptionally Active Region
The locus of transgene integration influences the expression level, due to a position
effect on gene expression. There are strategies to select clones whose transgenes
are more likely to have integrated into transcriptionally active region (hot spots).
An impaired neomycin phosphotransferase, which confers neomycin G418 resistance, has

been used successfully for hot spot integration. In the impaired enzyme, the translation
initiation site has been mutated to reduce its translation initiation efficiency; thus more
transcripts are needed to synthesize the same levels of proteins that confer G418 resistance.
Moreover, an artificial intron is introduced to decrease the level functionally active Neo transcripts, by increasing the
frequency of unsuccessful splicing. As a result, only those clones which have the Neo inserted into a transcriptionally
active region of the chromosome will have a high enough expression level to overcome drug selection.
Most of the selected resistance clones have only one copy of the transgene. The selected clones can be further
amplified using the traditional DHFR system. With the increased probability of transcription, fewer clones will
need to be screened to obtain high producers, and most obtained clones have low levels of DHFR amplification.
Furthermore enhancement is obtained using a single promoter (CMV) for heterologous protein and selectable
(amplifiable) marker, while having an IRES between the heterologous gene and selectable marker gene.

Cell Adaptation In a typical cell line development process, a large
number of product-secreting clones are selected
and subjected to further screening of their growth
characteristics, product titer, and product quality,
• From transfection to working bank takes about 4 - 6 glycosylation patterns, and other post-translational
months. modifications. In some cases, the cells do not grow
well in the culture environment used in production
(e.g., high agitation rates or altered medium
composition). They often have to be “adapted” to
new culture conditions by long-term cultivation
with a gradual change of environmental factors. Over
time, the cells gradually develop the ability to grow
under the new chemical and physical environment.
The presence or absence of a physical surface
• adaptation to suspension growth
for cell attachment is probably the most drastic
Host
cell
• anti-apoptotic gene
• glycosylation modulation
difference in cultivation conditions. Most normal
Target
gene
diploid cells used for virus production are strictly
anchorage dependent. Some cells, like myelomas
Transfection and hybridomas, are suspension cells that can
be readily grown in a mixing vessel. Many cell
Cell pool, lines commonly used for recombinant protein
selection,
cell clone production, including CHO, BHK, and HEK293
cells, are derived from adherent cells. Although,
Selection of
higher and not being strictly anchorage dependent, they often
stable producer
prefer adherent growth given a compatible surface.
Initial drug testing
(chemistry, biological,
Adaptation to
suspension
Initial process When cultivated in suspension, the unadapted
development
and animal) growth cells either fail to grow or form large aggregates
Adaptation to
with extensive cell-cell contacts and intercellular
serum-free/
Animal-component-free
adhesion. They can be adapted to grow in suspension
growths
by being cultured in shaker flasks or spinner flasks.
Working cell banking
The initial growth rate is slow, and dispersing agents
like heparin sulfate, reduced serum (if any), or
Process development & Scale-up calcium incorporation may be necessary to prevent
• feed batch/perfusion
• metabolic shift adhesion to the wall of the culture vessel in the early
Fig. 5.7: Typical steps in cell line development. stage of adaptation. Gradually, the growth rate is
increased and the cells eventually adapt and appear
indistinguishable from regular suspension cells.
The adaptation of cells to a new nutrient environment
is also commonly practiced in cell line development.
For example, the requirement of complex lipid
additives and growth factors may be reduced or even
eliminated through adaptation, although sometimes
these adapted cells exhibit lower productivity.

Fig. 5.8: A timeline for generating an industrial producing cell line.
Stability of Selected Clones

Mutations and epigenetic alterations occur in cultured
cells at relatively high frequencies. Some of these
events are affected by culture conditions. For example,
many types of stem cells are prone to differentiation,
• From thawing (2x108 cell) to production (20m3) needs even by changes in cell density, oxygen tension,
at least 16 doublings. growth factor concentration, and cell aggregation
• Stability is tested over 40 doublings state, depending on the particular cell type.
Cells used for human vaccine production are
mostly diploid and are considered to be stable
under established cultured conditions. These cells
• Normal diploid cells used in vaccine production are do not exhibit any visible phenotypic alterations
“stable” within the accepted population doublings or macroscopic chromosomal abnormalities
• Continuous cell lines have variable karyotypes in within the accepted range of doubling, or until
culture they reach senescence. The stability of these
cells is not a general concern in bioprocessing.
In contrast, the extensively selected, hyper-producing
recombinant cell harboring transfected, and often
amplified, transgenes have a higher propensity to lose
their high productivity. To begin with, the aneuploid
host cells used to generate those hyper-producing
cells are less stable than their diploid counterparts.
In addition to having abnormal chromosomal counts,
many of the chromosomes also have macroscopic
structural aberrations. Such chromosomal
alterations accumulate over time, generating cells of
divergent karyotypes from the same parental line.
Divergent karyotype and chromosomal abnormality

is, thus, an inherent nature of producing cells derived
from continuous cell lines. Even though production
lines may not be stable (in relation to ploidy), they
Cell Line Stability Issues must maintain their production characteristics
• Productivity decrease over time related to growth, productivity, and product quality
over the number of population doublings required
• Product quality changes over time
for creating sufficient cell banks. They must also
• Karyotype changes over time
be hardy throughout the thawing process, as well.
• Microscopic chromosomal aberration occurs over time
Assuming that a product life time of 10,000 runs
• The last two issues may not be critical for protein
biologics, but are of concern for cell therapy at 10,000 liter scale, with a final cell concentration
applications of 1010 cells/L, the selected cell clone will have to
double nearly 80 times. This number of doublings
greatly exceeds that required of a fertilized
egg to grow into a hamster, a mouse, or even a
human adult. In that long duration of time, the
occurrence of mutations, epigenetic changes,
Possible Causes of Instability in Recombinant Protein and chromosomal rearrangement in some cells
Productivity of the population is unavoidable and probably
• Mutation, especially in intergenic region in pivotal cannot be eliminated with today’s technology.
product gene loci
In discussing cell line stability, we decided to
• a producing cell may contain many copies of
focus on property changes that affect productivity
product genes, but maybe only a dominating few
contribute to most product transcript and product quality. The critical component for
• Epigenetic silencing of pivotal genes, at DNA level or at sustaining productivity over time is to prevent
histone reprogramming level any cell with a lower productivity from overtaking
• Loss of copies of product gene due to deletion or the population, or to prevent a very high rate
chromosomal rearrangement of productivity loss in the majority population.
A gross and rapid change of productivity in a large
fraction of cells may occur upon the removal or
reduction of selective pressure after transgene
amplification. This problem is alleviated by
employing a lower degree of amplification and by
establishing the clone only after the copy number of
transgene has stabilized. For long-term stability, one
could carry out serial cultures for 40 to 60 doublings
and examine the productivity, as well as the transgene
copy number. From a thawed liquid-nitrogen frozen
cell bag of 1010 cells to a production reactor, cells
may undergo 15 doublings, so a 40-doubling test
of stability will certainly give sufficient margin.
The stability of product quality is more difficult to
assess. Mutations in genes affecting product quality,
such glycosyl-transferases, may occur and lead
to a subpopulation of cells that produce inferior

product. Mutations causing protein sequence
• Production cell lines are tested for their ability to
alteration may occur in one or more copies of
retain the product gene in the genome and produces
the product product genes in all, or a subpopulation of, cells.
• The focus is production stability, not cell/genome Since not all copies of product genes in the cells
stability are transcribed and translated at equal efficiency,
not all mutations of product genes in a production
cell population will be manifested to the same
degree. With deep sequencing technology, one may
be able to detect such mutations, even at minute
level, in the consequent producing cell population.
Automation and High Throughput Technology

Developing a cell line for production purpose is
a very labor-intensive process. To increase the
probability of obtaining a very high producer, a
large number of cells need to be isolated at every
step, which involves productivity variation from
successive rounds of selection and amplification.
In the past decades, lab automation and high-
throughput technology have become an integral part
of bioprocess development. Liquid and cell handling,
PerkenElmer - Basic Model both cell pool and cell clones, quantification of
product titer, data acquisition, data processing and
analysis, and archiving have all becoming automated.
Many of the automated systems are based on culture
plates, or wells, and resemble other liquid handling
systems for high throughput chemical screening.
The difference is that a incubation system, with
temperature and atmospheric control for gas mixture
and humidity, is necessary. Robotic arms are often
used to move plates onto the working “stage” and
allow multiple manipulations to be performed on
multiple plates without human interference. In many
cases, the system is installed inside a clean room or
clean hood to minimize microbial contamination.
Tecan -- more automated with multi-plate capabilities The culture handling system is usually integrated
and computer interface with an assay system to assess product titer and cell
growth. Multi-step assays and screening protocols
Fig. 5.9: Examples of high throughput cell clone
screening system. can be performed by transferring culture fluid
into automated assay systems. The results can be
directly integrated into culture handling systems
to further expand wells or plates selected for

• Basic liquid handling manipulations further investigation. An integrated microscopic
• Distribution of liquid to 96 - 384 well plates imaging system can provide the added capability
• Sampling/removal of liquid of determining anything from clonal colony
• Transfer from plate to plate growth to colony morphology to ensuring that
cells picked from each well are single-celled clones.
• Cherry picking – transfer from well to well
Another type of automated system integrates cell
cloning with product titer assessment by performing
More complex models are capable of: cell screening on agar plates. In this case, the
• Cherry picking secreted product molecules (mostly antibodies) are
entrapped in agar that contains antibodies against
• Multiple plate handing (i.e. movement of plates
from a “hotel” to the pipetting stage) the product. A halo ring of immunoprecipitation
• Multiple “steps” performed sequentially (e.g. DNA zone is formed around the colony. The size of the
preparation protocols) halo ring reflects the amount of product secreted.
• Other “add-ons” like PCR machines, incubators, Image analysis is then used to extract the data
spectrophotometers, etc for selecting high producing clones to pick.
Concluding Remarks
Under best culture conditions, a hyperproducing of stability is still labor intensive. The use of host
industrial cell line derived from CHO or myeloma cells cell lines, which have been adapted or modified to
can secrete 50-100 pg protein/cell-day, a level which harbor all desirable growth characteristics, have
rivals professional secretors in vivo. Such remarkable greatly reduced the need of adaptation. There is
cell lines are created through the combination an increasing movement toward miniaturizing
of optimized genetic constructs, selection, cell culture while still simulating large-scale
amplification, cell clone screening, and the insight reactors, although this progress is still limited.
of picking the “right” clones. Although the specific
The advance in genomics has brought about a
productivity of the producing cells has increased
fundamental change in the way we can study the
by about one order of magnitude, the methodology
process of cell line development and brightened
of generating high producing cells has remained
the prospects that we can gain mechanistic insight
largely the same in the past three decades. The entire
into hyper-productivity. This knowledge may allow
process is still empirical and very labor intensive.
us to quickly select the “right” clone by examining
High-throughput cell handling and screening the transcriptome or genome of the candidate cells.
systems allow for the screening of a large number of With more tools for genome engineering becoming
potential high producing clones in the early stages available, it may also become feasible to impart on
of cell line development. However, subsequent steps the cells favorable genome-wide modifications.
of adaptation, growth characterization, and testing

Stoichiometry and Kinetics of
Cell Cultivation
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Cell Mass and Composition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
Cell Mass and Size. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
Material Balance on Cell Growth. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Variation in Cell Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Amino Acid Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Intracellular Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Growth of Mammalian Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
Quantitative Description of Cell Growth & Product Formation . . . . . . . . . . . . . . . . . . 156
Stoichiometric Ratio and Yield Coefficient. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Integral Cell Concentration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Kinetic Model of Cell Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
A Model Describing Growth and Production. . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
Monod Model and its Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
Environment, Kinetics and Stoichiometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Introduction
Cultured cells take up nutrients to generate energy
and to make more cell mass and products. For
manufacturing, it is important to supply a sufficient
quantity of nutrients. These nutrients allow cells
to grow and produce product while minimizing
the formation of waste product. To make the
manufacturing process efficient one must also
produce the targeted product quantity within a
given period. Therefore, one must not only know
how much nutrients to supply, but often also how
fast to deliver in order to sustain the production
environment. We use stoichiometric principles to
determine how to supply the correct quantity of
nutrients; and use kinetic principles to guide the
process along a desired path. Awareness of these
concepts is key to an overall understanding of how to
culture cells. This chapter discusses stoichiometry,
STOICHIOMETRY AND KINETICS | 147

kinetics of cell growth, and also gives the typical
values of stoichiometric and kinetic parameters
commonly encountered in cell culture processes.
Cell Mass and Composition

Cell Mass and Size Both microbial and mammalian cells vary widely in
size. In general, the dry biomass of bacteria, yeast,
Table 1. Typical Dry Weight of Cells and animal cells is in the order of 10-12, 10-11, and 5
x 10-10 g per cell, respectively. The most abundant
Bacterial 10-12 g / cell chemical species in a cell is water, accounting
Yeast 10-11 g / cell for 90% of the volume of plant cells, 80 – 85% of
Average Animal Cell 3-6 x10-10 g / cell
animal cells, and 70% of bacterial cells. Since cell
cultivation is carried out in aqueous environments
and the amount of water taken up by cells during
Table 2. Average Composition of an Animal Cell growth is extremely difficult to assess, the material
balance on cell culture is typically performed only
Pg/Cell Range Percentage % of Dry
Biomass on “dry” matter, excluding water. Our discussion on
Wet weight 3500 3000 - 8000 stoichiometry will be largely based on dry biomass
Dry weight 600 300 - 1200 of cell number, as commonly practiced in cell culture.
Protein 250 200 - 300 10 - 15 ~50 - 70
Carbohydrate 150 40 - 200 ~1 - 5 ~5
Macroscopically, cells are made of a few classes
Lipid 120 100 - 200 ~1 - 2 ~5 of macromolecules (protein, DNA, and RNA) or
DNA 10 8 - 17 ~0.3 ~2 macromolecular assemblies (primarily lipid bilayer
RNA 25 20 - 40 ~0.7 ~4 membranes). These organic matters constitute
Water 55 - 80 the vast majority of the dry mass in a cell. Protein
Volume 4x10-9cm3 molecules constitute the largest portion among
them, providing the machinery for DNA, RNA, and
protein synthesis. Protein molecules also serve as
the structural components of the cell and execute
all of the catalytic, transport, and communication
functions. The lipid content of an animal cell is
greater than in a bacterium. The abundance of
organelles contributes to their higher content and
their lipid bilayer membrane in an animal cell.
Intracellular carbohydrates exist as oligosaccharides
on many proteins and lipids. Carbohydrate also
exists as ribose in DNA, RNA, and nucleotides (e.g.,
ATP, GTP, etc). Only a small fraction exists in a free
(or phosphorylated) form. The cellular content
of carbohydrate is harder to estimate, because
it usually exists as a part of other molecules.

Material Balance on Cell Growth
The principle of material balance holds true in cell
culture. The total mass of inputs and outputs, and
the amount accumulated in a system, is always
Growth of Biomass Involves: in balance. The two most common, and most
• Consumption of nutrients
abundant, nutrients in cell culture are glucose and
glutamine (although some cultures do not require
• Production of new biomass
glutamine). They serve both as constituents of
• Excretion of products
cell mass and as sources of energy. Other common
inputs to cell culture processes include lipids,
Glucose lipid precursors, vitamins, and salts; these will
Amino acids be discussed in the Medium Design chapter, as
Other Biomass these nutrients supply key cellular constituents,
macronutrients Product
(lipid, but contribute less to generating energy.
Carbon dioxide
nucleotides, → CELLS → Water
etc.) To generate energy, glucose is converted to lactate,
Lactate, NH3
Micro-organic Excreted amino CO2, and H2O through glycolysis, the TCA cycle,
nutrients
(vitamins)
acids and the pentose phosphate pathway. Glutamine is
Bulk salts deaminated, releasing NH3, before its carbon skeleton
Trace minerals is used for energy metabolism. Consequently, the
Oxygen
accumulation of metabolites, including lactate, NH3,
CO2, and H2O, is commonly seen in cell culture. In
many cases, the amino group from the metabolized
glutamine and other amino acids are exported as non-
essential amino acids (such as alanine, asparagine,
and proline), in addition to being excreted as NH3.
Energy metabolism satisfies the energetic needs of
Example of the “equation” for
making biomass, through the biosynthesis of DNA,
hybridoma growth:
RNA, protein, and organelles. Other important
energy-intensive aspects of cellular events are
C6H12O6 (glucose) + 0.15C6.14H12.36N1.50O2.08
the uptake of nutrients and the sustained balance
(weighted average of amino acids) of cellular osmosis and membrane potential. As
+ 0.34C5H10N2O2 (glutamine) + 1.39O2 will be discussed later, the cell’s cytoplasmic
→2.37CH1.97N0.26O0.49 (cell mass) + 0.0058CH1.83N0.14O2.06 and mitochondrial membranes have a negative
(antibody) + 1.53CO2 + 1.28H2O + 1.44C3H6O3 electric potential that must be maintained, at
(lactate) + 0.16NH3 + 0.13C4H7NO2 (alanine) the expense of energy, to sustain cell viability
The process of growing cells and producing
a product can be formulated into an “overall
Such a formula is used when one performs metabolic
biomass synthesis” equation. This equation can be
flux analysis.
viewed as, essentially, the “apparent” composite
of all reactions involved in generating energy and
synthesizing biomass. At the center of the reaction
will be biomass, so a formula for the cell mass

can then be established, based on its elemental
composition. Usually, we use a formula that neglects
all elements except for carbon (C), hydrogen (H),
nitrogen (N), and oxygen (O). This formula is only
useful for describing the ratio of those elements.
One can arbitrarily assign the stoichiometric
numbers to give them different “formula weights”.
In the example shown, the stoichiometric
number of carbon is chosen to be 1. Others may
prefer to assign the formula mass to be 100.
The inputs in a cell growth process include
all nutrients consumed by cells to proliferate.
Since only C, H, N, and O are considered in the
stoichiometric equation, we consider only glucose,
glutamine, and other amino acids. Other minute
media components containing C and N (such as
vitamins or nucleotides) are neglected. Instead
of writing down all amino acids separately, one
may also use a weighted average, according to
the stoichiometric ratio of their consumption.
The outputs include “new” cell mass that has been
generated as the result of nutrient consumption,
as well as the metabolites and product that have
been excreted. One can write a molecular formula
describing the formation of the protein product, by
knowing its composition. Typically, the metabolites
excreted also include lactate and ammonia,
as well as some non-essential amino acids.
Variation in Cell Volume The volume of a typical animal cell is a few pico
Table 3. Size of Animal Cells liters (about 1,000 times larger than bacteria). The
average cellular diameter ranges from 10 to 20 µm.
Cell Type Volume (μm3) Diameter (μm)
Hybridomas
Even for the same cell line, one can detect great size
12 - 20
variations over a range, since cells immediately,
Endothelial Cells 1400 - 2500 17
Trypsinized and reattached
before and after mitosis are about two times
2000
before spreading occurs different in their size. At a given time in a growing
Chinese Hamster Ovary
1200 - 1800 ~14
culture, cells are in different stages of the cell cycle
cells (suspension)
and their size distribution is somewhat larger than
Chinese Hamster Ovary
Cells (anchored)
1300 - 1800 two fold. For aneuploid cells, the distribution of
Human foreskin fibroblasts size is typically greater than normal diploid cells.
7000
(FS-4)
The distribution of cell size changes with the
growth stage. Rapidly growing and quiescent
cells may have different sizes. Furthermore, in

a culture, cells that lose viability often become
• Cell Volume exhibits a distribution at any point in
visibly smaller, as measured by flow cytometry.
time
• Dead cells are often smaller Cell size varies amongst different cell types. Many
• Varying with culture stage types of stem cells are fairly small. Their nucleus
spans more than 70% of the cell diameter and
their cytoplasm is relatively small. Liver cells and
antibody-secreting plasma cells are at the other end
of the cell size spectrum, and contain a significant
amount of cytoplasm for protein secretion.
It is instructive to remember that the volume of
a sphere (which is a reasonable approximation
of a cell) is proportional to its diameter raised
to the third power. Therefore, cells that are
twice as large in diameter are eight times
larger in cell volume. Although cell number is
traditionally used for the quantification of cell
concentration, it may not sufficiently capture
the difference when comparing different
processes, in which cell sizes are very different.
Fig. 6.1: Cell size change during a batch

culture

Amino Acid Composition Microbial and plant cells often grow on simple
carbon sources supplemented with an inorganic
Table 4. Amino Acid Composition of Cells and IgG nitrogen source, such as ammonium or urea. The
Cell Composition Standard IgG cells convert inorganic nitrogen to all 20 natural
Mean deviation composition amino acids that are used to make proteins. Animal
ALA 9.03 0.32 5.31 cells lack the capability to make 11 to 12 of those
ARG 4.74 0.32 2.43 20 natural amino acids. These essential amino
ASN 3.49 acids must be supplied for animal cell culture,
10.08 0.59
ASP 3.95 to enable them to grow and make products.
CYS 0.26 0.04 2.43 Thus, knowing the amino acid composition of
GLN 5.01 cells, and of the protein product, is important.
12.62 0.63
GLU 5.16
The protein content and composition of cells change
GLY 9.14 0.57 6.98
under different growth conditions; however, they are
HIS 2.22 0.07 1.67
seldom measured. Nevertheless, literature values of
ILE 5.73 0.35 2.43
some cells are available, as well as the amino acid
LEU 9.00 0.68 6.83
composition product, IgG. Given target levels of
LYS 6.85 0.49 6.98
biomass and product to be produced, a stoichiometric
MET 2.27 0.14 1.37
amount of all essential amino acids must be supplied.
PHE 3.73 0.31 3.49
PRO 5.51 0.58 7.13 In addition to essential amino acids, which must
SER 6.19 0.16 12.90 be provided, non-essential amino acids are usually
THR 5.42 0.22 7.74 also supplied. However, these can be derived
TYR 2.73 0.14 4.10 by metabolic transformation from other amino
VAL 6.54 0.27 9.10 acids and can be considered “substitutable”.

Intracellular Fluid Water constitutes 70 – 80% of total cell volume.
The soluble components in water also make up
Table 5. Intracellular Concentrations of Amino Acids
a large fraction of the biomass. The intracellular
mM mM fluid contains electrolytes, carbohydrates,
Ala 0.2 - 2.0 Lys 0.1 - 0.6 amino acids, metabolism reaction intermediates,
Ang <.05 Met 0.01 nucleotides (ATP, ADP, etc.), and many other
Asp 0.4 - 0.8 Ornithine 0.120 components. Most amino acids are present at
Asn 0.4 - 0.8 Phe 0.3 - 0.5 the 0.05 – 0.5 mM range in the intracellular fluid.
Asp 0.306 Proline 0.137
The vast majority of intracellular amino acids
reside in cellular proteins and only a tiny fraction
Citrulline 0.036 Ser 0.149
exists as free amino acids in intracellular fluid.
Glu 0.3 - 12 Thr 0.1 - 4
Gln 0.05 - 4 Tyrosine 0.059
Typical concentrations of other major soluble
components in intracellular fluids are listed in
His <0.05 - 0.09 Valine 0.171
Tables 5 and 6, along with the typical extracellular
Ile 0.3 - 0.5
environment conditions, in vivo. Potassium,
Leu 0.1 - 0.4 magnesium, and phosphate are present at high
concentrations. A large fraction of Mg2 + is associated
Table 6. Approximate Concentrations in Cellular with ATP, which is typically present at the 1 – 3 mM
Environment range. In addition to free phosphate, phosphate is
Interstitial Intracellular also present in phosphorylated compounds (DNA,
(mM) (mM) RNA, nucleotides, phosphorylated sugars, lipids, etc).
Na+ 140 6 - 14
K+
4.0 100 - 140 Many inorganics, including phosphate, potassium,
Ca2+ 1.2 0.01 and magnesium, are present at much higher
Mg 2+
0.7 3 - 20 concentrations intracellularly, compared to the
Cl -
108 4 extracellular fluid or culture medium. As cells grow,
HCO3 -
28.3 10 they take up nutrients at large enough quantities
HPO42- H2PO4- 2 11 that they accumulate in the intracelluar fluid. It
SO42- 0.5 1 is important to ensure those nutrients are also
Phosphocreatine supplied in sufficient quantities. By knowing their
Camosine 14 intracellular content, the stoichiometric amount
Amino acids 2 8 required to produce the biomass can be estimated.
Creatine 0.2 9
In addition to the major inorganic species (K+,
Lactate 1.2 1.5
Na+, phosphate, Mg2+, and Cl-), many minute
Adenosine triphosphate 1.5
inorganic elements are also constituents of cellular
Glucose 56 0.05
components, including iron, copper, selenium, zinc,
Protein 0.2 3-4
cobalt, etc. Many primarily exist as a prosthetic
group of proteins. These elements must also be
supplied in enough quantities to generate biomass.
Unfortunately, the cellular content of those elements
are seldom reported and may vary widely among
different cell types or even under different culture
conditions. For example, the zinc content is much
higher in pancreatic cells than in other cells. Similarly,

iron is rich in muscle and red blood cells. Without
quantitative data on the cellular contents of those
elements, one can only resort to titration experiments
under defined culture conditions to see whether the
supply of those elements is limiting the cell growth.
Growth of Mammalian Cells

A cell culture process can be described by its cell
growth curve, nutrient consumption curves, and
product concentration profile. Cell growth, in
general, is divided into different growth stages: lag
phase, exponential growth phase, stationary phase,
and death (or the decline phase). The exponential
growth phase is characterized by a linear increase
in cell concentration on a semi-logarithmic plot over
time and is easy to determine. In many cases, the
extent of cell expansion in a culture is small (only 3 –
5 fold increase in cell concentration). Also, the time
point for the transition from lag phase to exponential
phase and from the exponential phase to stationary
phase may not be clearly depicted in a growth curve.
Some cultures may experience a slow, or no-growth,
period ranging anywhere from a few hours to a few
Fig. 6.2: Growth phases in a batch culture
days. This may be caused by using the inoculum
from cultures that had already reached a stationary
or decline phase, or by inoculating cells into vastly
different media or culture conditions. Low inoculum
Table 7. Doubling Time of Some Culture Cells
cell concentrations may also cause poor initial
Cell type/contitions Doubling Time growth, due to insufficient conditioning factors in the
Mouse embryonic stem cell ~12 hr culture medium. In general, anchorage-dependent
Human Diploid Fibroblast (10% FBS) 24 - 40 hr cells should be inoculated at a minimum of 105
Human Diploid Fibroblast (2% FBS) 40 - 60 hr
cells/mL or 104 cells/cm2; while suspension cell
cultures are generally started at about 105 cells/mL.
CHO K1 (5% FBS or in rich medium) 16 - 24 hr
NSO cell 16 - 24 hr The exponential phase is marked by a constant cell
growth rate, or doubling time. Doubling times of
different cells span over a wide range, even under
favorable culture conditions. For instance, while
mouse embryonic stem cells divide every 11 – 12
hours, cells commonly used in bioproduct production
double every 15 – 30 hours, depending on medium
composition. Some human fibroblastic cells take
nearly two days to double their number, even under
optimal conditions with a high serum concentration.

After a period of rapid cell growth in the exponential
phase, the cell growth rate reduces and the culture
enters the stationary phase. Many reasons may
cause this transition, including the exhaustion or
suboptimal supplementation of key nutrients in
the culture, and/or the accumulation of growth-
inhibitory metabolites. A flat cell growth curve in
the stationary phase may indicate that a culture
that has ceased to proliferate. It may also reflect
a balance between cell growth and death. In the
latter case, the growth curve is characterized
by a constant viable cell concentration, along
with an increasing concentration of dead cells.
In the decline, or death phase, viable and total cell
concentrations decline due to an exhaustion of key
nutrients, the accumulation of metabolites to an
inhibitory level, or adverse culture conditions (such
as high osmolality). The growth behavior at a late
stage of culture often varies, depending on whether
cells are adherent or in suspension. Many anchorage-
dependent cells grow substantially slower once
cell density on the surface approaches confluence.
They can also sustain a confluent cell density, over
a period of days, without entering the death phase.
Conversely, cells grown in suspension, especially
those with an inherently strong receptor-mediated
apoptotic mechanism, often enter the death phase
soon after the viable cell concentration peaks.
In a production process, the rate of product
formation is at its highest when cell concentration is
maximal. A good production practice is to reach the
target cell concentration quickly, while sustaining
the culture as long as possible at that point.

Quantitative Description of Cell Growth & Product Formation
A cell culture process can be characterized by its
Specific Rates cell growth, nutrient consumption, and product
accumulation profiles. To quantitatively describe cell
Growth rate (G) (change in cell concentration per unit culture kinetics, three classes of quantities are used:
time, number of cells/L-hr; or gm of cells/L-hr)
concentrations of components (e.g., cells, nutrients,
metabolites, and product), activity parameters (e.g.,
G = dx (Eq. 1)
dt specific rates of growth, nutrient consumption,
x: viable cell concentration and product formation), and stoichiometric ratios.
After obtaining the concentration profiles of key
Specific Growth Rate (μ) (number of cells/cell-hr or
gm cell/gm cell-hr) process variables, the next step is to calculate
dx = nx the rate of change of cell, nutrient, and product
dt concentrations. These are referred to as volumetric
(Eq. 2)
n = 1 dx rates because they are normalized to culture
x dt
Doubling Time volume. A quickly-changing culture has a high
volumetric rate, which may be the result of having
ln x2 = n (t2 - t1) more cells, or by having cells that are more active.
x1
(Eq. 3)
Usually, cell volume constitutes only a very small
td = ln 2 = 0.693 fraction of the volume in a suspension cell culture.
n n
Specific Nutrient Consumption Rate (gm nutrient/gm The volumetric rate is essentially based on the liquid
cell-hr) volume. In some cases, such as with high-density
qs = - 1 ds
(Eq. 4) solid microcarrier culture, the volume occupied by
x dt solid beads is large; thus liquid volume and culture
s: substrate (nutrient)
volume are not equal. Usually the concentrations of
concentration
nutrients, metabolites, and product are measured
Specific Product Formation Rate based on liquid volume, not on culture or reactor
volume. In such cases, the “volume” used for
dp (Eq. 5)
qp = 1 different kinetic parameters must be clearly stated.
x dt
Another set of descriptors, called the specific
p: product concentration
rate, is normalized to a per cell basis. These
activity parameters describe how active each
unit of the cell is for activities like making new
biomass (specific growth rate), consuming
glucose (specific glucose consumption rate), or
producing lactate (specific lactate production rate).
Cell growth is autocatalytic, meaning the rate is
dependent on the cell concentration. Because
cell concentration may be measured in different
ways, such cell mass, cell number, or even cellular
DNA or protein, the specific growth rate may
also be based on different cell measurements.

Different measurements may give somewhat
A Model Considering Cell Death
different specific growth rates, because cell size,
dxv = nx - dx (Eq. 6) cell mass, and cellular content are not necessarily
dt
v v
proportional to each other at all culture stages.
It is important to note the difference between
dxd = dx (Eq. 7)
dt
v volumetric cell growth rate (the number of
cells/L/hr) and the specific cell growth rate (μ,
xt = xv + xd (Eq. 8) number of cells/cell/hr or hr-1). The two are
related quantities; one describes how fast the
culture is increasing in cell concentration, the
qs = - 1 ds (Eq. 9)
xv dt other describes how active cells are proliferating.
The cell doubling time is easily obtained from
n = 1 dxt (Eq. 10) experimentally-determined growth curves. By
xv dt
separating the variables x and t into two sides of the
d = 1 dxd differential equation and integrating with respect
xv dt (Eq. 11)
to time and cell concentration, respectively, one
With Cell Lysis obtains the relationship between cell concentration
and time, given a constant specific growth rate (Eq.
dxv = nx - dxv - m x (Eq. 12) 3). The doubling time is the time period it takes to
v v v
dt develop a two-fold increase in cell concentration.
dxd = dx - m x (Eq. 13)
In the case that non-viable cells are present in a
v v d
dt
significant portion of the cell concentration, a specific
λv: Dead Cell Lysis Rate death rate, α, can be incorporated into the equation
for cell balance, with xd representing the dead cell
concentration (Eqs. 6, 7). In some rare cases, cell
lysis occurs in culture. Lysed cells are not observable
by cell counting. To describe the growth kinetics of
such a culture, one can also include a cell lysis term.
The specific nutrient consumption rate is obtained by
dividing the volumetric nutrient consumption rate
(ds/dt) by the cell concentration. The specific product
formation rate is defined, similarly. In all cases, the
cell concentration used to calculate the specific rate
is the viable cell concentration. In other words, one
assumes that dead cells are metabolically inactive.
Experimentally, the specific rates can be calculated
from the changes in concentration over time.
These specific rates are important for describing
the dynamics of various activities in cultures.

Stoichiometric Ratio and Yield In evaluating a process, we need to assess the
Coefficient efficiency of material conversion; that is, how
much of a raw material is actually converted to
the product or to cells. If we know a theoretical
maximum of the conversion efficiency, or know
Yield of Biomass on Substrate its historical value, we can then assess how much
the current process can be further optimized.
Y = Dx , dx
x
(Eq. 14)
Ds ds
s
A yield coefficient is the ratio of the quantity
Yield of Product on Substrate of the product or cell produced, to that of the
raw materials used. A yield coefficient for a
Dp dp (Eq. 15) cell can be based on different input materials,
Y =
p ,
s Ds ds
e.g., glucose, ammonium, or other nutrients.
Stoichiometric ratio of lactate to glucose
In microbial processes, yield coefficient is
a = DL , dL (Eq. 16) frequently used to evaluate material utilization
DG dG efficiency. The yield coefficient is given a symbol
Stoichiometric ratio of product to substrate Yx/s or Yp/s, for cell mass or product, respectively,
and is based on the consumption of a particular
(Eq. 17)
substrate. These variables are key indicators
of process efficiency, because substrate cost is
often a major portion of the total product cost.
The efficiency of substrate utilization is essential.
Yield coefficient is rarely used in animal cell culture
Description of the “State” of the Cultures processes. Cell concentration in a typical animal
• Physiological state cell culture process is rather low and is seldom
measured in mass. Furthermore, bulk materials
• There is no unique or universal definition for
that are used by cells, i.e., glucose and amino acids,
physiological state. In general, the kinetic
parameters described above are sufficient to are not the major contributors to the cost of goods.
describe the cell’s physiological state The stoichiometric ratio of various nutrients and
metabolic products is more frequently used in
• Metabolic state cell culture processes. Under different metabolic
• The stoichiometric ratios can be used to de- conditions or in different growth stages, cells utilize
scribe metabolic state various nutrients differently and change the amount
of different metabolites produced. The stoichiometric
ratios of various nutrients and metabolites are
indicative of such alterations in metabolism.
For example, the stoichiometric ratio of lactate
to glucose, e.g., the ratio of the amount of lactate
produced to that of glucose consumed, is a
strong indication of energy metabolism being
at a highly glycolytic or highly oxidative state. If
most glucose is channeled through glycolysis to

lactate, the ratio is close to two moles of lactate
per mole of glucose. Conversely, if most glucose
is directed toward the TCA cycle for aerobic
oxidation, the ratio will be close to zero, while the
stoichiometric ratio of oxygen to glucose will be
closer to six moles of oxygen per mole of glucose.
The stoichiometric ratio and yield coefficient,
based on a given pair of compounds, can be
expressed in different units, e.g., mol/mol or g/g.
Integral Cell Concentration Product accumulation rate in a culture can be

described by multiplying the specific product
formation rate by cell concentration (Eq. 18).
Integrate over the culture period from t0 to
tf, to obtain the product concentration at tf .
If qp is constant, one can take it out of integral.
One can see that the final product concentration is
proportional to the integral of cell concentration.
In a plot of cell concentration (x) vs. time, the
integral is the area under the curve of the x curve.
This is often called the integral cell concentration.
With the assumption that qp is constant, integral
cell concentration is thus proportional to
product concentration, and can be used as a first
approximation estimate of product concentration.
Integral Cell Number is the Area Under the Growth Curve
(Eq. 18)
(Eq. 19)

Example of Experimental Data Processing and Plotting of a Fedbatch Culture
Typical process data include those from on-line and off-line measurements. On-line data are often continuously
recorded except that some control actions (e.g. turning on base pump or oxygen valve) may be discrete in time.
Off-line measurements are invariable only on discrete time points. The measured data should be plotted to discern
inconsistency and outliers. Then the calculated (or derived) data are also displayed. The example data shows that the
oxygen uptake rate (OUR) peaks slightly before cell concentration, since OUR is a more sensitive indicator of cell’s
activity. O2 flow rate often reflects OUR. We also see that even though specific rates may change over time, some
stoichiometric ratios remain constant.
Fig. 6.4: Plots of process data of a typical cell culture

Example of Stoichiometric Ratios as Indicators of Metabolic States
Myeloma cells were grown in batch or fedbatch cultures. In the fedbatch cultures, glucose and glutamine
were at lower concentrations, initially. Once the concentration reached the set point, concentrated
glucose or glucose/glutamine was fed continuously via computer control to maintain the concentration(s)
around the set point. The final product concentration, the amount of glucose, glutamine, and oxygen
consumed, and the total amount of lactate and ammonium produced were used to calculate the
stoichiometric ratios. The duration of fedbatch culture was substantially longer than the batch culture.
It is notable that more glucose, glutamine, and oxygen were consumed in the fedbatch culture, because the
culture lasted longer and the cell concentration was higher. However, from the stoichiometric ratio, one can
see that the lactate to glucose ratio decreased from the initial value, similar to the batch culture, indicating a
metabolic change by limiting glucose at a low level. This also indicates that by controlling glutamine, one can
affect lactate production. This metabolic change is confirmed by the oxygen/glucose ratio. The lower lactate/
glucose ratio was accompanied by a higher oxygen/glucose ratio, suggesting that more glucose was channeled
into the TCA cycle for oxidation. Glutamine control also resulted in a lower production of ammonium.
• Stoichiometric ratios change under different • A combination of stoichiometric ratios can reflect
metabolism the metabolic states of cells.
• In a fedbatch culture with glucose or glucoflutamine
level control, the stoichiometric ratio change can
reflect metabolic shift
Table 8. Typical stoichiometric ratios of hybridoma cells under different metabolism in batch and fed-batch cul-
tures
Batch Fedbatch with Fedbatch with Glucose
Glucose Control Glutamine Control
Growth Data Initial glucose conc. (mM) 17 1.4 1.4
Initial glutamine conc. (mM) 4 0.3 0.3
Glucose conc./set point (mM) 0.55 0.55
Glutamine conc./set point (mM) 4 ~0.5 0.2
Maximal viable cell conc. (10 cells / mL)
6
2.4 12 10.5
Antibody conc. (mg / L) 8 60 200
Consumption/ Glucose consumption 12 46.5 27
Production
Glutamine consumption 4 17 10.1
(mmole/mmole)
Oxygen consumption 24 143 122
Lactate production 3 37.4 17
Ammonium production 3 9.7 4.5
Stoichiometric Lactate/glucose 1.5 1.60 → 0.16 0.90 → 0.05
Ratio (mmole/
Oxygen/Glucose 1.85 1.9 → 6.0 2.7 → 4.7
mmole)
Ammonium/Glutamine 0.75 0.5 0.31 → 0.10
Alanine/Glutamine 0.35 0.34 → 1.35 0.08 → 0.42
Osmolality (mosm / kg) ~350 410 400

Kinetic Model of Cell Growth
Utility of Mathematical Models A mathematical model can be
used for different purposes:
1. Summarizing experimental data
2. Probing concepts and testing hypothesis (1) To summarize a large volume of experimental
data. Data, or plots of data, are extremely difficult to
3. Predicting and optimizing processes
describe in words. If data are fit to a mathematical
model, regardless of whether the model is empirical
or mechanistic, the behavior of the data can then be
recreated, given the model and value of the parameters.
(2) To explore concepts and test hypotheses. When
we study a physical system and its behavior (such as
growing cells consuming glucose), we first develop
a verbal description of the system and the behavior.
We may then propose a hypothesis, also in verbal
form. The verbal description can be translated
into a mathematic form. The mathematical
model can then be used to explore the system’s
possible behavior under different conditions.
(3) To predict the behavior of the systems, given a model
with sufficient complexity. The model can be used to
predict regions of parameter space that have not been
experimentally tested, previously. It can also be used
to optimize or control the dynamics of the system.
With an increasing emphasis on the notion
of Quality by Design, the application of the
mathematical model will gain importance. Proper
applications of well-posed models will help
explore system behaviors and define optimal
operating regions, in a potentially vast design space.
A Model Describing Growth and If we consider only a cell, a nutrient (e.g., glucose)
Production and a product (e.g., a recombinant antibody) as the
three most important variables in a culture system,
the mathematical model will consist of three material
balance equations for the concentrations of the three
species. The change of cell concentration will be due
to growth, which is described by the multiplication
of specific growth rate and cell concentration.
Similarly, the rate of change of substrate and product
concentrations is described by multiplication of

Balance Equations for a Batch Culture Growth Kinetics the respective specific rate and cell concentration.
Cell balance These three equations describe only the balance
(Eq. 20) of those three species. To describe the “dynamics”
of the system, we will need to have description of
Glucose balance how the parameter (or the specific rate) changes for
important variable(s). For anchorage-dependent
(Eq. 21)
cells, the growth rate is dependent on the extent of cell
“confluence” on the surface. Models for anchorage-
Product balance
dependent cells, thus, attempt to describe the
(Eq. 18)
dependence of the specific growth rate on cell density.
Lactate balance For process design or optimization purposes,
(Eq. 22) one first needs to identify the variable that most
affects the process outcome and then develop a
Models describing growth rate or production rate relationship between specific growth rate and this
dependence on environmental factors
variable. For example, if the glucose concentration
Cell density dependence is an important variable, then one can develop a
model describing the relationship between growth
(Eq. 23)
rate and glucose concentration. Then, as the glucose
Nutrient dependence concentration changes, so does the specific growth
rate. On the other hand, if the most important factor
(Eq. 24) affecting the cell is lactate concentration, then one
Inhibitor dependence will need a balanced equation for the production
of lactate and a model relating cell growth (µ), and
(Eq. 25) possibly also specific glucose consumption and
product formation rates, to lactate concentration.
Once a model is available, one would still need
experimental data to identify the parameter values
What is needed to develop a mathematical in the equations. Then, with appropriate initial
description of a cell culture processs?
conditions (i.e., the initial concentrations of cell
• A description for cell growth (and death), product mass, glucose, lactate, and product), the system
formation, nutrient utilization
behavior can be explored by simulation of the model.
• Growth model - dependence of specific consumption
rate on the “controlling variable” (growth rate, nutrient A number of mathematical models have been
concentration, etc.) tested for describing animal cell growth in culture.
• Product formation - dependence of specific production Most are based on Monod-type models that were
rate on “controlling variable” traditionally used to describe microbial growth.
• Experimental data - the model will have some Some employed more complicated structured or
parameters (such as half saturation constants). The segregated models. These models are empirical
experimental data are used to determine the value of in nature. However, using these models to
those parameters predict growth conditions outside the range of
• Material balance equations for “state variables” (the conditions under which the kinetic parameters
concentrations of cell, nutrients, product, inhibitors, are obtained may not give reliable prediction.
etc.)

Monod Model and its Derivatives
A Monod model employs two parameters, μmax
Modod Model
and ks, to define the relationship between the
nmax s (Eq. 26)
n= specific growth rate, μ, and the limiting substrate
Ks + s
concentration, s. The specific growth rate is affected
not only by substrate concentration, but is also a
function of pH, temperature, the status of other
nutrients or growth supplements (e.g., serum), and
the presence of waste products. In applying a Monod
model, one assumes only substrate s is limiting and
that all factors are supplied in enough quantities.
The term-limiting substrates have been used
in two different contexts: 1) a stoichiometric-
limiting nutrient refers to the nutrient that
Fig. 6.5: Growth rate dependence on rate limiting
is first depleted in a batch culture and whose
substrate based on Monod model
depletion causes the cessation of growth; or 2)
If s » Ks, then μ → μmax a rate-limiting nutrient is the nutrient whose
concentration restricts the growth rate of cells.
• Two parameters, (μmax and ks), define the relationship
between μ and limiting substrate concentration s. The equation gives a saturation type of kinetic
• μ is also a function of pH, temperature, nutritional behavior; meaning μ increases with increasing
status, (i.e. serum), waste products; it may also depend substrate concentration, until it reaches a maximal
upon cell density for anchorage-dependent cells which
are subject to contact inhibition. value. At low concentrations, increasing the rate
limiting nutrient concentration increases the specific
Notes
growth rate, linearly. At very high concentrations,
• These models are all empirical models that can be used the specific growth rate is constant at μmax.
for first order approximation of cell growth
• Under most process conditions growth is not limited by A Monod model can be incorporated into the
nutrient availability balance equations describing the batch culture
• For stem cells and primary cells, growth factors have growth kinetics to provide a relationship between
more profound effects than nutrients the equations for cell growth and substrate. Indeed,
the resulting equation can be used to describe
batch growth when glucose is used as the limiting
substrate. Starting at a high concentration of
substrate, cells grow at μmax; as the substrate
concentration decreases so is the growth rate. The
simulated growth curve will show the entry into
stationary phase as the substrate decreases to a level
where μ is substantially reduced and, eventually,
growth ceases when the substrate is depleted.
However, animal cells require multiple nutrients for
proliferation. In addition to glucose, glutamine is
also a major nutrient. Many amino acids and other

nutrients are also required, although often most
are provided in excess and are not limiting. Monod
models are modified to describe the dependence of
Multiplicative Saturation Kinetics Model growth rate on multiple nutrients. Many include
multiplicative terms to incorporate multiple
s2 : s2 (Eq. 27) substrate utilization and product inhibition.
n = nmax :
K1 + s1 K2 + s2
In the multiplicative model for cell growth, two
(Eq. 28) substrates, S1 and S2, are considered to be growth
rate limiting. Each substrate has its corresponding
half saturation constant for saturation kinetics. In
most common applications, the two substrates are
glucose and glutamine. The model can be extended
to consider metabolite inhibition. For example, in the
case where A and B are inhibitory metabolites. Most
models consider lactate and ammonia as inhibitors.
Environment, Kinetics and Stoichiometry

We have discussed balance equations for cells,
substrates, products, and the model relating
growth rate to limiting substrate. Together, they
can be used to describe cell growth in culture. For
simple microbial systems, this is often sufficient.
However, for animal cells, the specific substrate
consumption and production rates are profoundly
affected by their environment. One, thus, also
seeks to describe the relationship between qx,
Specific Product Formation Dependence on Growth qp, and key variables affecting their behavior.
q=
p αµ + β (Eq. 29) The kinetics of product formation in microbial
systems are frequently categorized, according to
their relationships to growth rate. qp is considered to
be influenced by two factors: a “growth-associated”
term, α, which describes dependence on specific
growth rate; and a “non-growth associated” term,
β. Depending on the relative magnitude of α and
β, the production can be growth associated, non-
growth associated, or mixed growth associated.
Such classifications of production kinetics are
useful for microbial fermentation of amino acids,
organic acids, and antibiotics. However, their
applicability to animal systems are limited. In
general, the specific production rates of animal cell
culture products are less sensitive to growth rate.
For some recombinant proteins, the productivity

is somewhat higher in the stationary phase of
fedbatch cultures. At that stage, many factors,
including osmolality, lactate, and CO2 concentration,
all have deviated from optimal growth conditions.
It is possible that some of those factors exerted a
stronger effect on productivity than growth rate.
Another complication in applying a Monod-type of
model is that cell growth is rarely limited by substrate
concentration in cell culture bioprocess. Cells enter
the stationary phase often due to the accumulation
of metabolites (e.g., lactate and ammonium) or the
accumulation of salts (Na), due to base addition or CO2.
Lactate and ammonium are produced from glucose
and amino acid (primarily glutamine) metabolism,
while Na accumulation arises from base addition
to neutralize lactate and maintain pH. CO2 comes
from both cell metabolism and pH control actions.
Concluding Remarks
Stoichiometric and kinetic relationships among glucose consumption and high lactate production
process variables are important in describing to low glucose and lactate consumption is the
different cultures. From an experimental perspective, key to a high product accumulation. The simple
these parameters and variables provide a basis models described in this chapter can be made
for quantitatively comparing different processes. more effective models by including a mechanistic
They also allow the simulation of a culture’s kinetic description of how a cell’s metabolic shift occurs in
behavior under different growth conditions. A high response to environmental factors. Incorporation
productivity process often includes a cell growth of a mechanistic model for describing cell culture
phase and a prolonged stationary phase, in which bioprocesses will enhance our physiological
cells are kept at a high concentration and high understanding of cell metabolism and increase
productivity state. Growing evidences suggest the utility of models for optimizing the process.
that a switch of a cell’s metabolism from high

Cell Culture Data Analysis
Process Data Analysis and PAT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

Data Processing Pipeline. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
Standardized Templates for Data Logging and Processing . . . . . . . . . . . . . . . . . 168
Cell Culture Data Processing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
A Typical Spreadsheet for Analysis of Cell Culture Data. . . . . . . . . . . . . . . . . . . 170
Data Visualization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
Mapping Data to Pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Process Data Analysis and PAT

Process analytical technology (PAT) is gaining
greater attention for its potential role in enhancing
bioprocess robustness. PAT encompasses: 1)
the acquisition of data pertaining to process and
product attributes, related to both productivity
and product quality, through on-line and off-
line measurements; 2) the analysis of process
data in each run; 3) developing and employing
mathematical models to relate process variables
to process outcome for a better control; and 4) the
data mining and recognition of hidden patterns
of behavior in historical data for further process
enhancement. Every element of PAT has been
used in process development for decades. The new
emphasis is on integrating each individual element
to create a more comprehensive understanding of
the process and to generate useful information.
Modern production plants are thoroughly
electronically monitored and controlled. They
CELL CULTURE DATA ANALYSIS | 167

employ instrumentation in the reactor for
process monitoring, as well as to measure raw
material and product quality. A significant
challenge in the application of PAT is the
accumulation of the vast amount of data, which
can potentially impede process understanding.
The key to successfully implementing PAT
is, thus, to produce a better pipeline for data
processing and to have a better appreciation
of methodologies for bioprocess data analysis.
Data Processing Pipeline

Standardized Templates for Data The importance of having a standardized data format
Logging and Processing for data collection and archiving cannot be overstated.
After collection, data is processed to remove faulty
values (such as data resulting from wrong entry
Use Standardized Template in Data Processing or sensor failure). The raw data obtained from
• Speed up routine data analysis different sources, such as from different analytical
• Automatically perform calculations instruments, may have different units or be formatted
• Automatically generate standardized plots differently and are homogenized. The data are then
• Automate data regression and calculation of
estimated values. further used to calculate derived variables and
are plotted for visualization. Finally, the data are
• Ensure consistency and accuracy archived and sometimes employed in data mining,
• Uniform terminology, units, graphs
• Built-in formulas minimizes mistakes in calculations for generating more process insights. Throughout
• Up-to-date set of key plots eases communication this transformation, the data are transferred
and data interpretation within teams. between many different software platforms.
• Facilitate data archiving Many of those data processing steps are often
• Develop a format which allows easy transfer repeated over and over; not only in manufacturing
of data to other programs for analysis and
visualization settings but also in research environments. They
• Incorporate other important parameters, such are often performed by different workers at
as passage number, medium composition, and different times and at different manufacturing
operating information.
sites. Thus, setting a standardized data format
• Additional Consideration of Spreadsheet and uniform data processing protocol is critical
Templates to increase efficiency and minimize frustration.
• Metabolic flux analysis, visualization process
modeling or modeling. What exactly should be standardized? Highest on
• Data archiving: the list are the terminology, nomenclature, units
• Searching
• Experimental / run details of quantities, and data format. The same object
• Standardized upload / download platforms should be named the same way, consistently across
all data sets, and should use the same symbol, units,

and equal numbers of significant figures in values.
When treating data, the formula must be uniformly
applied to different data sets. However, inadvertent
mistakes may occur through incidents, such
as errors from formula entry. To facilitate the
workflow, the use of a uniform data format and the
minimization of inadvertent errors are advisable to
set up data entry and process templates for various
programs. The same templates should be used by
all colleagues performing similar experiments.
By setting up a template, various tasks can be
automated. For example, upon the entry of
experimental data into the spreadsheet template,
the related specific rate and stoichiometric
ratios can be automatically calculated, and their
profiles over time can be plotted instantaneously.
With templates for different software programs
in place, even data transfer from one software
program to another can be automated.
Cell Culture Data Processing The first level of bioprocess data is raw data that
Three Types of Data has been acquired from various measurements,
including the concentrations of cells, nutrients
• Measurement Data
and metabolites, pH, and oxygen. Subsequently,
• Cell concentration stoichiometric ratios and specific rates are calculated
• Nutrient and metabolite concentrations over a time course to discern the trend of metabolic
• Process parameters: pH, DO, temperature, etc. and productivity changes in the entire culture. The
• Calculated Integrated Data stoichiometric ratio can then be determined from
the amount of nutrients consumed over a time
• Cumulative nutrient consumption and metabolite
production period, and is an integral quantity in nature. Specific
• Integral viable cell concentration (IVCC) rate is the rate of change of the quantity, further
divided by cell concentration. It is determined from
• Calculated Differentiated Data
the slope of the curve of the quantity produced
• Specific rates or consumed over time. Specific rates are,
• Stoichiometric ratios therefore, quantities determined by differentiation.
Except in simple batch cultures, the concentration
Two Types of Calculations profiles of nutrients and products are often not
• In-process calculations monotonic functions. Even culture volume may not
• Process monitoring be constant. Feed medium is added occasionally,
• Troubleshooting and diagnosis resulting in step changes in nutrient and metabolite
• Post-process calculations levels. Typical culture profiles, thus, often entail
a step increase in nutrient levels after feeding
• Data smoothing for analysis
and a step decrease in metabolite levels, due

to a dilution caused by the volume of the feed.
The first step in data analysis is the compilation of
cumulative data pertaining to nutrient consumption
and metabolite production/accumulation.
Instead of using concentration profiles to perform
calculations, the cumulative amounts of nutrients
consumed and the amount of product produced
are calculated. The cumulative curves are mostly
monotonic, except for those few nutrients or
metabolites that are consumed at one time and
produced at another. A concentration profile with
many step changes cannot be easily subjected to
regression. Cumulative curves have no step changes,
thus allowing a regression to be performed on the
entire profile. The regression equation is then
used to calculate the slope for the determination
of stoichiometric ratios and specific rates.
Cell culture processes often extend over a long
duration, from 5 to 15 days up to a couple
of months. Data processing starts while the
process is still in progress and continues after
its completion. In-process data analysis allows
for potential outliers and faulty conditions to be
detected and corrected. Post-process analysis often
involves additional chemical analyses to provide
more process insight. A template spreadsheet
can be used for the first stage of data analysis.
A Typical Spreadsheet for Analysis of A spreadsheet template shall contain columns of

Cell Culture Data raw data entry followed by columns of cumulative
data for all measurement data for nutrients which
are consumed and products. Any time point of row
that incurs feed addition will incur a total mass
balance that takes volume change into consideration.
A simple way to account for volume change is
to perform calculations in total mass balance
(concentration of nutrient: x volume) instead of
on concentrations alone. In this case the amount
consumed between two time points is simply
StVt- St+1Vt+1)=St cumulative consumption is then
the sum of St over time. Forgetting to account
for volume change in material balance is a

Cumulative Data common mistake in fedbatch culture data analysis.
t
IVCt = # \ $ V $ dt . IVC
t-1
The calculation of cumulative data can be automated
+ \t $ vt - \t - 1 $ vt - 1
0 once the measurement data are entered. The next
t k set of columns are specific rates. Specific rates are
#
Si,t = qi,t $ x $ V $ dt = Vt0 $ si,t0 - Vt $ si,t + Vfk $ sbest
fk /calculated for cumulative data by regression.
0
0
The regression of cumulative data can be automated.
Si,t: Cumulative amount (mMole or g) of nutrient i Usually a third order polynomial fitting works well
consumed or produced at time t
V: Volume of culture
for most data. However, inspection is necessary
si: Concentration of component i in the culture broth to ensure a good fit. The measurement data,
Vf: Volume of feed medium added cumulative consumption/production and specific
sf: Concentration of component i in the feed medium
k: Total number of feed medium additions up until time t
rate data shall be all automatically for visualization.
IVC: Integral Viable Cell number Upon the calculation of cumulative data
Stoichiometric Ratio stoichiometric ratios are also automatically plotted.
This allows for detection of metabolic changes in
Si,t - Si,t T
2 1
=c S i
m culture. If a stoichiometric ratio deviates significantly
S j,t - S j,t TS j t
from historical data, it may also serve as a diagnosis
2 1
ai,j: Stoichiometric ratio for nutrient i with respect to nutrient alert for checking pasable process abnormality.
j at time t
An add-on to the spreadsheet template is an algorithm
for metabolic flux analysis. If the measurements
include all the major carbon compounds, glucose,
lactate, glutamine and other amino acids (if not all, the
majority), ammonium, then material balance can be
performed on the nitrogen balance. Carbon balance
will require the measurement of CO2 produced in
metabolism and is not easily done without isotope
labeling. If oxygen consumption data is available,
one can assume that R.Q. being 1.0 and set CO2
production to be same as oxygen consumption.
From the extent of carbon, nitrogen balance one
can assess the reliability of some stoichiometric
ratio data. If the carbon and nitrogen is reasonably
closed the data can then be further subjected to
metabolic flux analysis.MFA algorithm is typically
Fig. 7.1: Plots of cumulative consumption data in MatLab or other mathematical solvers. The Excel
template can build in an exportable table for ready
transfer of the specific rate data to those programs.
Specific Rates
• Two-point specific growth rate calculations and
specific nutrient calculation for fedbatch culture
1 dS 1 S - S1
qs = $ . $ 2
x $ V dt x2 $ V2 x1 $ V1 t2 - t1
+
2
• Slope calculation from curve of regression data

Data Visualization Visualizing the data is critical for developing a
deeper understanding of the effect of various
Multidimensional/Interactive Data
parameters on process performance. Each cell
Exploration
culture run usually entails many measurements
over multiple time points. Instead of browsing
• Process data are intrinsically multidimensional and
should be examined in multiple dimensions (e.g. time data through tables, we plotted each quantity
course and stoichiometric ratios) to provide different as a concentration profile over time. We also
insights
plotted data of one variable against another
• Data from multiple cultures can be consolidated and variable, to specifically examine the ratio of
examined for trends
specific rates or the stoichiometric ratios.
• Software for visualization interactive for multiple
dimensional viewing analysis – E.g.: Spotfire As data accumulate over time, it is even more
DecisionSite.
important to plot data of multiple runs together,
so that different runs under the same or different
experimental conditions can be compared
easily. In such analyses, mathematical and
statistical tools are important; however, the
importance of data visualization in the analysis
of multiple runs cannot be overemphasized.
When working with a large set of data from
multiple runs, visualization software is very useful.
Quick access to data, the rearrangement of data
into different combinations of dimensions, or the
filtering of data by different process performance
or other criteria can greatly facilitate deeper
insight. In the plot shown, lactate concentration
profiles from over 250 runs are colored by the
final product titer. One can see that high-titer runs
mostly consume lactate in the late stage of culture,
while the low-titer runs produce lactate. The trend
is easily seen when all data are plotted together.
We then plot the specific rates of glucose
consumption and lactate production at different
time points, for all runs. It can be seen that lactate
consumption (negative values) occurs only when
glucose consumption is also low. One can further
see that even when the cells are consuming lactate,
Fig. 7.2: Plots of archived historical data for discerning the glucose consumption rate is still significant.
process patterns
With the aid of a visualization tool, data
from all of these runs can be easily plotted in
different ways. To take advantage of the data
trove from large number of runs, a means
for quick visualization is very important.

Mapping Data to Pathways
Pathway related data is another type that its also exhibit three different glucose consumption
analysis can greatly benefit from a visualization rates. By plotting fluxes on to a metabolic map,
tool. An example is the fluxes through different it can be seen that high glucose consumption
reaction steps in various pathways obtained from rate leads to high lactate production, high
metabolic flux analysis (MFA). Another is the gene glutamine consumption and high TCA cycle flux.
expression transcript level data from microarray Such visualization will become almost essential
analysis. Presentation of the flux data on a when dealing with very complex pathways
pathway map makes it easy to compare metabolic like glycan biosynthesis on proteins passing
change over time or under different conditions. through Golgi apparatus before being secreted.
An example below shows flux distribution in
cells grown under three different conditions that
Metabolic Flux Analysis/Pathway Mapping
Metabolic flux analysis

uses culture data to
estimate fluxes of
intracellular species
through metabolic
pathways. It is useful to
transfer these numbers
onto a map of central
metabolism to visualize
the differences among
calculated values.
Mapping of data into

formats that incorporate
physiological information
facilitates interpretation
of complex datasets.
This example compares

fluxes of lactate
production state and
lactate consumption
state.
Fig. 7.3: Metabolic chart for visualizing metabolic fluxes

Concluding Remarks
Data analysis holds the key to understand and sample data to demonstrate the calculation and
improve the process. A large amount of software plotting. They can be used as a starting point for
for data processing, analysis and visualization is modification to meet individual needs. The use of
available. Setting up a consistent and efficient template will also make it easier to compile and
way of processing and plotting the data can analyze historical data accumulated over time. A
be hugely beneficial. A set of templates are good practice of data processing and analysis is
included in the template folder, along with some fundamental to process analytical technology.
Visualization of Glycan Profiles

• The Pathways leading to N-glycans form a
complex network
• A small number of enzymes are involved to
form a large number of glycans
• Most enzymes are used multiple times
• Most glycans, intermediate glycans and late
glycans may all appear in the final product
• Visualizing the distribution of glycans can
help deduce the plausible paths taken to
from each species
Comparison of Intracellar and

Culture Medium N-Glycan
Fig. 7.4: Visualization of glycosylation fluxes

Metabolic Flux Analysis
in Cell Culture Systems
Overall Material Balance for Reaction Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Chemical Reaction Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
MFA on a Cellular System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Utility and Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
General Approach. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Overall Material Balance for Reaction Systems
In cultivating cells, it is sometimes necessary to

know where and through which pathways the
absorbed nutrients have traveled. This allows
one to manipulate the distribution of nutrients
and to optimize the process. One useful tool
for performing such tasks is material balance
analysis. When such balance is used to analyze the
distribution of materials in biochemical systems
(either on whole cells or on specific pathways),
it is called metabolic flux analysis (MFA).
Metabolic reactions are chemical reactions.
They are governed by stoichiometric principles.
However, there are special characteristics in cellular
systems that set them apart from chemical reaction
systems. In a test tube or in a chemical reactor,
the reactions (i.e., the conversion of reactants to
products) occur directly in the fluid phase. For cells
in culture or in tissue, the reactants (i.e., nutrients)
are delivered to individual cells. Products are also
METABOLIC FLUX ANALYSIS| 175

formed in and excreted by individual cells into
the fluid phase. The reactions take place not in
a continuous phase, but in many discrete cells.
By applying MFA to cells in culture, one could
theoretically combine all of the cells’ mass into one
entity and could consider all cells as one biotic phase
and the fluid as one abiotic phase. All of the nutrients
taken up by cells (not just the nutrients added to the
culture) and products excreted into the fluid are
considered to be inputs and outputs, respectively.
Sometimes, MFA is applied to a pathway or a group
of pathways. In this case, the balance is applied to a
system, which does not have a physical boundary, like
a cell or even a group of cells. The pathway of interest
(for example, glycolysis or mitochondrial reactions)
occurs in many different locations within the cell
mass. And yet, we would treat all reactions of interest
in ALL cells as a system and perform a balance on it.
Another unique characteristic of the cell system is
that cells are growing and expanding in biomass, as
well as in cell volume. The output of cellular reactions,
thus, includes cell biomass. Furthermore, because
the volume of cells is expanding, it has a dilution
effect on the concentration of cellular materials.
In this chapter, we will first use a familiar
chemical reaction system to emphasize the
notion that the fundamental concept of MFA
is material balance. This will then be followed
by a discussion of the basic steps in MFA.
Chemical Reaction Systems

Consider a chemical reaction system, in which
multiple reactions are taking place simultaneously
and we want to determine the extent, or the flux, of each
reaction. We can solve this problem by performing
measurements on the concentrations of compounds
in the inflow and outflow streams of the system
and their relative flow rates. Then, we can apply
material balance on the stoichiometric equations
for all reactions known to occur in the system.
In the example shown below, we aim to determine
METABOLIC FLUX ANALYSIS | 176

Material Balance on Reaction Systems
Example: Incomplete combustion of CH4 to CO2, CO and H2O in a batch reactor. Two reactions can possibly
occur. We don’t directly measure the extent of each reaction, but we can measure the inputs and outputs at
the beginning and the end of reaction, and see how much has been consumed (i.e. the amount of reactants
left at the end subtracted from the amount put in initially), and how much has been produced. From these
balances we can calculate the flux of each reaction.
Known Reactions Output
CH 4 + O2 → CH 4 + 2O2 ξ
→ CO2 + 2 H 2O→ CO + CO + H O
2 2
1−ξ
2CH 4 + 3O2 → 2CO + 4 H 2O
If all the inputs and outputs (i.e., the amount of CH4, O2 consumed and that of CO2, CO, H2O produced) are
completely balanced, the fraction of CH4 going to each reaction can be determined. On the other hand, if the
material balance is not closed, there will be uncertainty about the solution, and the distribution of materials
can only be estimated.
Case I. 4 moles of CH4 and 7 moles of O2 are combusted to produce 2 moles each of CO2 and CO and 8
moles of H2O.
In this case, all three elements involved—C, H and O—are completely balanced. The only
unknown ξ can be easily calculated using a balance on any element, e.g., use C balance:
 mole C  ξ = 2 mole CO2 mole C
 4 mole CH 4 ×1  ×1
 mole CH 4  mole CO 2
ξ = 0.5
In Case I, in principal, one does not need to know the quantities of all inputs and outputs to find the solution.
From three elemental balances (C, H, O), we can have three linear equations. As long as there are only three
unknowns, we can also solve for ξ. For example, even if the quantities of CO and H2O are not measured, the
solution will be the same.However, in the real world, there is always a high degree of uncertainty about the
accuracy of measurement; the overall balance is always an important check of the validity of the results.
Case II: 4 moles of CH4 and 7 moles of O2 are combusted to form 1.5 moles of CO2 and 1.6 moles of
CO2. The amount of H2O produced is not measured. Determine the fraction of methane completely
combusted.
In this case, the input and output materials are not balanced. There are a total of 4 moles of C combusted
but only 3.1 moles are accounted for in the products. This presents a problem regardless of whether H2O is
measured.
The cause of input and output materials not being balanced is not always known. It may be due to measurement
error, or possibly other reactions have occurred but are not accounted for are not accounted for. In this case,
we can only get a plausible answer of how much CH4 goes to complete and incomplete combustion. If we
know the extent of various measurement errors and the amount of H2O, we can get a more reliable estimate.

the fluxes of methane for two reactions, which occur
simultaneously in the furnace, using measurements
of compounds in the inlet and outlet. The
concentration differences between inlet and outlet
of the furnace give the value of net consumption and
net production. They are the inputs and outputs into
the reaction system. In case 1, the carbon, hydrogen
and oxygen entering the system are all accounted
for in the products. One can easily determine how
methane distributes itself between the two reactions.
On the other hand, as in many cases, the material
balance from the measurement is not closed. This
happens frequently because of measurement
errors. Sometimes, it can be caused by compounds
in inputs or outputs that are not accounted for. For
instance, there might have been some unknown
reaction that occurred, leading to the emergence
of products that are not measured and included
in the output. Under such a situation, uncertainty
on the outcome of material balance inevitably
occurs. One then has to rely on the knowledge
of the reaction system to make appropriate
assumptions, or to perform further measurements.
A Systematic Way to Solve Material We will use the methane combustion in a furnace
Balance Problems as an example to illustrate how to set up equations
for MFA. Although the problem is so simple
that it can be solved using a piece of paper and a
pencil, the approach shown is more systematic
and will be suitable for calculating fluxes for a
large reaction system, such cellular metabolism.
Setting Up Material Balance Equations We will perform material balance on the materials
consumed and produced, in order to determine the
fluxes for the reactions involved. First, we define
the system, including its inputs and outputs (i.e., net
consumption and production), as well as the chemical
(or metabolic) reactions involved. In the example
shown, two reactions are listed, for which the chemical
formula of the compounds and the stoichiometric
coefficients of the reactions are all known.
The rate of consumption and production is given

the symbol “q”. In MFA, q’s are always the specific
Step 1.
rates. Consumption is designated as a negative
Write material balance equations for every species
value and production as a positive value. We
express the flux of each reaction as “J”. By denoting
the flux of reaction 1 as J1, the fluxes of methane,
oxygen, carbon monoxide, and water reacted in
reaction 1 are -2J1, -3J1, +2J1 and 4J1 respectively.
Their fluxes are related by the stoichiometric
coefficients (-2, -3, 2, and 4) of the reaction.
Next, we set up a material balance equation for
each species, which is present in the system.
Each compound involved in the system is given
a differential equation describing the change of
its concentration in the reactor as the balance of
its consumption, production, and other input and
output, if any. The balance equation for CH4, thus,
reflects that the rate of change of CH4 is the sum
of its input (qcH4) and its reaction fluxes (-2J1).
Quasi-Steady State
The equations set up above are differential equations.
Step 2.
For a short duration of time, the concentration of
Pseudo-Steady State Assumption
all species in the reactor (or in the cell) may not be
changing very rapidly. In that case, the left-hand
side of the differential equations can be assumed
to be negligible (e.g., the system is assumed to be
at a pseudo-steady state). With this assumption,
the left-hand side of all differential equations
becomes zero. The equations, thus, become a
simple system of linear algebraic equations.
Stoichiometric Matrix, Flux Vectors and To solve a large set of equations, it is more efficient
Solution to perform matrix operations. In these cases,
the matrix is called the stoichiometric matrix.
The stoichiometric matrix is the collection of
stoichiometric coefficients organized by reactions in
one dimension, and by species involved in reactions
in the other dimension. In the example shown,
there are two vectors for J1 and J2, respectively.
The two columns in the matrix correspond to the
stoichiometric coefficients of reactions 1 and 2. Each
row in the matrix represents the stoichiometric

coefficients in the balance of a compound in the
system. For example, the balance of CH4 is the net of
-2J1 (+0J2) and the output from the furnace -qCH4. If a
Step 3. compound exists only in the reactor but is not part
Write the equations in the matrix form of the input or output, then its value in the vector
on the right hand side is zero. In applying MFA to
cell systems, many reaction intermediates exist
only intra-cellularly (such as glucose-6-phosphate
and fructose-6-phosphate), so their q is zero.
Under most conditions, the fluxes are unknown. The
inputs, outputs, and q’s can usually be measured.
In setting up flux analyses, one typically considers
only the branching points (called nodes) of the
Matrix Form
reactions, in which one reactant is split into two or
Specific rates vector r: more reactions, or two reactions lead to a common
• Positive : production product. In a linear pathway, the material flow
• Negative : uptake is easily related by a stoichiometric relationship
along the path. For example, the flux of glycolysis
Stochiometric Matrix A: is a linear pathway, but only if the small diversion
• Each column is a reaction
of glyceraldehyde-3-phosphate to glycerol-3-
• Each row is a species
phosphate for lipid biosynthesis is ignored. At steady
• Solve simultaneous equations AX=Q state, hexokinase flux is half of that of pyruvate
• Over specified system: Least-squares method kinase flux and, on a molar basis, is defined by the
1:2 proportion of their stoichiometric coefficients.
There is no need to set up balances for reactions
along the pathway between the two reactions.
Next, the equations that are not independent are
removed from the system of equations. The resulting
system of equations may be overspecified (in the
case of having more independent equations than
unknowns), underspecified (in the case of having
more unknowns than independent equations), or
have a unique solution (in the case of having the same
number of independent equations as unknowns). For
underspecified systems, one seeks to perform more
measurements. For overspecified systems, one has
to use his/her knowledge to remove a less significant
reaction or find a set of solutions that gives rise to
minimal errors. In general, for biological metabolic
reactions, the system is always overspecified.

MFA on a Cellular System
Utility and Limitations Metabolic flux analysis is the application of material

balances on metabolic systems. The principle and
approach are not different from the example shown
for a chemical reaction system. When MFA is
applied to a cellular system, usually the inputs and
outputs into cells are measured and known. One
needs to enlist all of the reactions involved and set
up material balance equations for all intracellular
components that are considered, and then proceed
to solving the resulting system of equations. In
most cases, the effect of cell expansion and the
effect of dilution due to volume enlargement are
ignored. This is regarded as acceptable because
the timeframe considered in the analysis is usually
much shorter than the doubling time of cells.
MFA provides an additional dimension to analyze
experimental data. Instead of merely examining
various specific rates, MFA provides a bird eye’s
view of the distribution of nutrients to different
metabolic pathways. This insight is especially
useful when comparing different metabolic
behaviors under different culture conditions
or growth stages. By combining MFA with
transcriptome data, which provides a global view of
the changes of transcripts in cells, one can possibly
gain much more insight into the dynamics of cell
metabolism, which is not easily seen otherwise.
However, like material balance in chemical
reactions systems, the accuracy of MFA is limited by
the knowledge of reactions involved in the system
and the accounting of material balance. Further
complicating the analysis is the fact that cellular
metabolism is compartmentalized in organelles
and cytosol. This poses additional constraints
on the balancing of many molecules playing
key roles in inter-organelle communications.
Experimentally, a complete accounting of materials
in a cell culture system is challenging. Material
balance in a reaction system should be balanced on
every elemental species, or at least for the major ones

(C, N, H and O). The consumption of major nutrients,
including glucose and all amino acids, can be easily
accomplished. Oxygen consumption can also be
measured. The products, including lactate, some
non-essential amino acids, and the protein product,
can also be quantified. However, a major metabolic
product, carbon dioxide, is not easily measured.
A Generalized Approach to MFA
A typical cell culture medium contains a rather
high concentration of bicarbonate, as pH buffer,
Measure Specific Rates making it very difficult to quantify CO2 produced by
cells, unless isotope is used. One way to estimate
carbon dioxide production takes into account the
Check Carbon/Nitrogen Balance fact that the respiratory quotient of most cells
under typical culture conditions is about 1.0. By
Stoichiometric Matrix measuring oxygen consumption rate and assuming
RQ is 1.0, one can obtain the CO2 production rate
and use the value for closing the carbon balance.
Calculate Fluxes J
Nitrogen balance can be obtained by measuring
the consumption of amino acids and accounting
for the production of cellular protein and product.
Visualize Fluxes on Metabolic Map
However, with measurement errors and uncertainty
in estimating biomass composition, it is common to
see the total nitrogen in inputs and outputs differ by
Examine Calculated Specific Rates more than 10%. Still, this is often better than the
extent of closure one often obtains for carbon balance.
Some cell culture media contain complex
Examine Calculated Carbon/ components, such as serum or plant hydrolysate.
Nitrogen Balance Those components provide peptides, lipids,
and fatty acids for growth. The extent of their
Fig. 8.1: A flow chart for metabolic flux analysis consumption is hardly measured, making it
even more difficult to close the material balance.
MFA can give much insight into the metabolic
characteristics of cell culture processes, but
one should be extra diligent in the execution of
experiments and collection of measurements,
to ensure the results of MFA are reliable.

General Approach A typical mammalian cell expresses about 15,000
Selecting Reactions for Analysis genes at a given time. Only a fraction of the genes
are actually involved in material flow (or fluxes);
others are involved in cellular structure or in signal
flow. MFA primarily concerns the flow of materials.
For all practical purposes, we can account for
only carbon- and nitrogen-containing species.
The flow of ionic species (e.g., sodium, potassium,
phosphate, etc) are not considered in MFA.
Even considering only the flow of carbon- and
nitrogen-containing compounds, the number of
reactions is over a thousand. However, only a
portion of these reactions has a significant flux.
Overall, the glycolysis pathway has the highest
flux for cultured cells. There are only a hundred,
or so, reactions whose flux is >5% of that of
glycolysis. The vast majority of the reactions have
a flux in the order of 1% of glycolysis, or lower.
Considering the errors in carbon and nitrogen
balances, MFA is best applied to analyze the flux of
major arteries in carbon flow. It does not provide
accurate estimates for minor reactions, such as
glycan distribution in the product protein. To
apply MFA for the reactions with minor fluxes, an
accurate quantification of the specific formation
rate of key reaction intermediates is necessary.
The first step in applying MFA to a cell system
Fig. 8.2: Simplification of metabolic pathways for metabolic flux
analysis. is to reduce whole cell reaction networks to a
manageable and meaningful subset of pathways. In
general, those pathways include glucose and amino
Applying MFA to entire cell metabolism
acid metabolism, and the biosynthesis of building
• a vastly large number of reactions involving material
blocks for biomass and product formation. In the
flux
course of simplifying the reaction network, different
• most of them have very small fluxxes, much smaller
than errors in closing balance of materials assumptions are made and often a different set of
reactions are included in the analysis. The selection
• select the reactions and pathways which are relevant
and sufficiently large of pathways will impact the flux distribution.
• to zoom in on pathways with very small fluxxes, use a
root type tracer to examine fluxxes of local reactions

Compartmentalization Cells are compartmentalized to segregate various
reactions; genome replication and RNA synthesis
occurs in the nucleus and glycolysis takes place in
• Carbohydrate metabolism takes place in cytosol and cytosol. Among the major metabolic reactions, TCA
mitochondria cycle, oxidative phosphorylation, and fatty acid
• lipid metabolism involves even more compartments oxidation occur in the mitochondria. Across the
• intercompartmental traffic of pyruvate and malate- boundary of cytosol and mitochondria, pyruvate,
aspartate shuttle also involve amino acids and TCA glutamine, and components of the malate-aspartate
cycle intermediates shuttle pass at high fluxes. The flux of the malate-
• flux balance must be met for the intercompartmental aspartate shuttle transports the reducing equivalent
traffic
of NADH into the mitochondria, at the same level as
pyruvate. The flux of the citrate shuttle generates
acetyl CoA in the cytosol and is typically lower, but
under some growth conditions it can be rather large.
These shuttles and transfers across the
mitochondrial membrane pose further constraints
on material flow. Imposing those constrains on MFA
is important for obtaining a good estimate of fluxes
of energy metabolism. The regulation of fluxes
across the cytosol and the mitochondria may play a
role in shift of metabolism. Therefore, building a flux
analysis model based on two compartments of the
cytosol and mitochondrion is a worthwhile effort.
Biomass Equations With the exception of cell mass, all outputs of a

cell system have a defined chemical composition.
Their production rates are measured and readily
• biomass equation is very difficult to construct and is used in MFA. In contrast, the cell concentration
subject to errors
is often measured by the cell number, and
• under fast growing and lactate production conditions, not biomass. Furthermore, the composition
biomass formation constitutes only a small fraction of of the cell mass is seldom characterized.
total carbon
Under most culture conditions, the vast majority of
carbon taken up by cells as nutrients is converted to
lactate and carbon dioxide. Only a small fraction is
actually incorporated into new cell mass. Because
this amount is such a small fraction of the total
carbon consumed, the error in the estimation of
biomass does not significantly affect the MFA results.
However, under an oxidative metabolic state
(characterized by reduced specific glucose
consumption and very little lactate production or
consumption), the amount of carbon and nitrogen
channeled to biomass becomes a significant

portion of the overall material flow. Since the
Biomass Equation Derived components of the biomass are drawn from
from Cellular Components
fluxes leading to building blocks, the composition
Proteins of cell mass affects the results of flux analysis.
Glucose
DNA/RNA BIOMASS
Amino acids
Lipids/Carbohydrates, etc. Despite their potential influence on the estimation
of fluxes, the stoichiometric equations of biomass
Macromolecule pg per cell Elemental composition of cell
formation are not often addressed. Based on
Protein 300
DNA/RNA 15/30
C N H O elemental analysis of C, N, O, and H of a mouse
Lipids/Carbohydrates
Total dry weight
55
400
1 0.2605 1.975 0.489 hybridoma cell line, a general compositional
formula was given as: CH1.975N0.2605O0.489 . The
Fig. 8.3: Determining elemental composition for biomass general range of cellular composition of lipids,
stoichiometric formula proteins, nucleic acids, and polysaccharides is also
available. The amino acid composition of cellular
proteins has been reported for a number of cell
lines. Overall, the available data is very limited.
One should be aware of this potential error when
applying the literature to the biomass equation.
Solution and Analysis A simplified cellular reaction system may consider

only the metabolism of glucose and amino
acids, while simultaneously lumping lipid and
nucleotide synthesis into a biomass formation
equation. The resulting reaction network consists
of about fifty fluxes involving a similar number of
compounds. Such a system can be solved using
software such as Mathmatica and MatLab, using
the least square method to minimize residue.
The solution gives a set of values to unknown fluxes,
as well as to the specific rates whose measured
values are already known. One should compare
the values given by the solution and the measured
values. It is also prudent to check material balance
based on the solution values. If major deviations
are seen, the results need to be reevaluated.
The results of MFA involve tens of variables
depicting the fluxes in the reaction network.
Such data are difficult to comprehend, without a
proper graphic presentation. To gain insight, it is
helpful to link the results of MFA to a metabolic
map for visualization. Such visual images greatly
enhance our ability to discern shifts in metabolism.

An Example For an example of MFA, a MatLab algorithm is
presented and its solution is included. The reactions
considered in the reaction network are listed in
the accompanying table. These include reactions
for glycolysis, TCA cycle, amino acid degradation
pathways, biomass, and antibody synthesis. The
metabolic network is compartmentalized into the
mitochondria and the cytosol. Reactions in the
malate-aspartate shuttle (also known as the NADH
shuttle) are included to account for the transfer
of the reduction potential of NADH generated in
cytosol into the mitochondria, thereby regenerating
the levels of cytosolic NAD. Further, three
enzymes, including mitochondrial malic enzyme,
cytosolic malic enzyme, and pyruvate carboxylase,
are also included in the reaction network. The
respiratory quotient is assumed to be 1.0; in other
words, the carbon dioxide production rate is
assumed to be the same as the oxygen uptake rate.
The specific rates determined for two metabolic
states of NS0 cells: one produces lactate in the
exponential growth phase and the other consumes
lactate in late growth stage. These rates are used to
solve the fluxes in each situation. Upon the solution,
the results are displayed in a metabolic chart.
The contrast of the two metabolic states is seen,
not only in the specific rates of glucose and
glutamine, but also in the internal distribution.

Table 1. List of Energy Catabolism Reactions for MFA
# Reaction Compartment Pathway
1 GLCc + 2NADc → 2PYRc + 2NADHc + H2O Cytosol Glycolysis
2 PYRc + NADHc → LACc + NADc Cytosol Lactate Dehydrogenase Reaction
3 PYRm + NADm → AcCoAm + NADHm + CO2 Mitochondria Krebs Cycle
4 OAAm + AcCoAm → CITm Mitochondria Krebs Cycle
5 CITm + NADm + H2O → AKGm + NADHm + CO2 Mitochondria Krebs Cycle
6 AKGm + NADm → SUCCoAm + NADHm + CO2 Mitochondria Krebs Cycle
7 SUCCoAm + H2O → FUMm Mitochondria Krebs Cycle
8 FUMm + H2O → MALm Mitochondria Krebs Cycle
9 MALm + NADm → OAAm + NADHm Mitochondria Krebs Cycle
10 GLNc → GLUc + NH3 Cytosol Glutaminolysis
11 GLUm → AKGm + NH3 Mitochondria Glutaminolysis
12 PYRc + GLUc → ALAc + AKGc Cytosol Alanine Synthesis
13 SERc → PYRc + NH3 Cytosol Amino Acid Degradation
14 GLYc → SERc Cytosol Amino Acid Degradation
15 CYSm → PYRm + NH3 Mitochondria Amino Acid Degradation
16 ASNc → ASPc + NH3 Cytosol Amino Acid Degradation
17 HISm → GLUm + NH3 Mitochondria Amino Acid Degradation
18 ARGm + AKGm → 2GLUm Mitochondria Amino Acid Degradation
19 PROm → GLUm Mitochondria Amino Acid Degradation
20 ILEm + AKGm → SucCoAm + AcCoAm + GLUm Mitochondria Amino Acid Degradation
21 VALm + AKGm → GLUm + CO2 + SucCoAm Mitochondria Amino Acid Degradation
22 METm → SucCoAm Mitochondria Amino Acid Degradation
23 THRm → SucCoAm + NH3 Mitochondria Amino Acid Degradation
24 PHEm → TYRm Mitochondria Amino Acid Degradation
25 TYRm + AKGm → GLUm + FUMm + 2AcCoAm Mitochondria Amino Acid Degradation
26 LYSm + 2AKG → 2GLUm + 2 CO2 + 2AcCoAm Mitochondria Amino Acid Degradation
27 LEUm + AKGm → GLUm + 3AcCoAm Mitochondria Amino Acid Degradation
28 0.0104GLNc + 0.0110ALAc + 0.0050ARGm + 0.0072ASNc + 0.0082ASPc Cytosol/Mitochondria Antibody Synthesis
+ 0.005CYSm + 0.0107GLUc + 0.0145GLYc + 0.0035HISm + 0.0050ILEm +
0.0142LEUm + 0.0145LYSm + 0.0028METm + 0.0072PHEm + 0.0148PROm +
0.0267SERc + 0.0160THRm + 0.0085TYRm + 0.0189VALm → CH1.539N0.2645O0.314
29 0.208GLCc + 0.0377GLNc + 0.0133ALAc + 0.0070ARGm + 0.0ASNc + 0.0261ASPc Cytosol/Mitochondria Biomass Synthesis
+ 0.0004CYSm + 0.0006GLUc + 0.0165GLYc + 0.0033HISm + 0.0084ILEm +
0.0133LEUm + 0.0101LYSm + 0.0033METm + 0.005PHEm + 0.0081PROm +
0.0099SERc + 0.0080THRm + 0.0040TYRm + 0.0096VALm → CH1.975N0.2605O0.489
30 CITm + MALc → CITc + MALm Cytosol/Mitochondria Fatty Acid Synthesis
31 CITc → AcCoAc + OAAc Cytosol Fatty Acid Synthesis
32 MALc → MALm Cytosol/Mitochondria Glutaminolysis
33 GLUc → GLUm Cytosol/Mitochondria Glutaminolysis
34 OAAm + GLUm → AKGm + ASPm Mitochondria Malate Aspartate Shuttle
35 OAAc + NADHc → MALc +NADc Cytosol Malate Aspartate Shuttle
36 AKGc + ASPc → OAAc + GLUc Cytosol Malate Aspartate Shuttle
37 ASPm + GLUc → ASPc + GLUm Cytosol/Mitochondria Malate Aspartate Shuttle
38 MALc + AKGm → MALm + AKGc Cytosol/Mitochondria Malate Aspartate Shuttle
39 MALm + NADm → PYRm + CO2 + NADHm Mitochondria Malate Decarboxylation (Malic Enzyme)
40 MALc → PYRc + CO2 Cytosol Malate Decarboxylation (Malic Enzyme)
41 2NADHm + O2 → 2NADm Mitochondria Oxidative Phosphorylation
42 2FADH2 + O2 → 2FAD Mitochondria Oxidative Phosphorylation
43 PYRm + CO2 → OAAm Mitochondria Pyuvate Caboxylation (Pyruvate Carbox-
ylase)

Table 2. Abbreviation of Intermediates of Reaction Modes
Abbreviation Name
AB Antibody
AcCoA Acetyl Coenzyme A
AKG A-Ketoglutarate
ALA Alanine
ARG Arginine
ASN Asparagine
ASP Aspartate
BIOMASS Biomass
CYS Cysteine
CO2 Carbon Dioxide
FUM Fumarate
GLC Glucose
GLN Glutamine
GLU Glutamate
GLY Glycine
HIS Histidine
ILE Isoleucine
LAC Lactate
LEU Leucine
LYS Lysine
MAL Malate
MET Methionine
NH3 Ammonia
OAA Oxaloacetate
PHE Phenylalanine
PRO Proline
PYR Pyruvate
SER Serine
SucCoA Succinate Coenzyme A
THR Theonine
TYR Tyrosine
VAL Valine
O2 Oxygen
NADH Nicotinamide Adenine Dinucleotide
(Reduced)
NAD Nicotinamide Adenine Dinucleotide
(Oxidized)
FADH2 Flavin Adenine Dinucleotide (Reduced)
FAD Flavin Adenine Dinucleotide (Oxidized)
Subscript Compartment
c Cytosol
m Mitochondria

Concluding Remarks
MFA is an important analytic technique of
quantitative physiology. It can provide insight into
process optimization and metabolic engineering.
A flux balance can be written for each metabolite,
within a cellular or metabolic system, to yield the
dynamic mass balance equations that interconnect
various metabolites. With the knowledge of
stoichiometry and steady state assumption, one can
obtain the flux estimate for individual reactions.
MFA is a powerful tool, but it is invariably
based on a simplified reaction network. The
assumptions made in simplifying the reaction
network will affect the results of the analysis. It
is best used for a first approximation to obtain
some insights for further exploration of ideas.

Cell Culture Bioreactors
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
Basic Types of Bioreactors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Stirred Tank (Well Mixed) Vs. Tubular Reactor (Plug Flow) . . . . . . . . . . . . . . . 193
CSTR and PFR with Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
Implication When Growth or Reaction Occurs in the Reactor . . . . . . . . . . . . . 195
Heterogeneous Reactor- High Solid Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Operating Mode of Bioreactors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Batch and Continuous Processes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Batch Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Fedbatch Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
Tissue Culture and Disposable Cell Culture Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Disposable Culture Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Cell Support Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
Suspension Culture vs. Adherent Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
Microcarriers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Cell Culture Bioreactors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .206
Simple Stirred Tank Bioreactor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Airlift Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Fluidized Bed Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Membrane Bioreactor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
Multiple Membrane Plate Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
Other Bioreactor Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Microsphere Induced Cell Aggregates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Agarose Cell Immobilization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Microencapsulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Membrane Stirred Tank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Spin Filter Stirred Tank. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Vibromixer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
CELL CULTURE BIOREACTORS | 191

Introduction
In the past decade, industrial mammalian cell
culture has become the production vehicle of choice
in the fast growing field of medical biologics. These
products now extend from therapeutic proteins
and viral vaccines to cells and viruses for cellular
and gene therapy. Before the arrival of recombinant
DNA technology, industrial mammalian cell
culture was used only in the production of viral
vaccines, and was confined to relatively small
scale operations, involving the culture of adherent
cells in tissue culture flasks. The arrival of rDNA
technology led to significant changes. Now, the host
mammalian cells are invariably continuous cell
lines, and the scale of operations has skyrocketed.
The transition from cultivation of adherent cells in
flasks to a truly industrial scale has transformed
bioprocess. The most dramatic changes have been
in the process, rather than in the basic design
of the reactor. In the 1980s and early 1990s,
research and commercial development focused
on exploring possibilities for new bioreactors.
Researchers pushed to overcome a number
of real and perceived hurdles, e.g., the notion
that mechanical agitation and air sparging had
detrimental effects on mammalian cells, the fact
that the culture systems supported only low cell
numbers and resultant low product concentrations,
and that the accumulation of metabolites caused
growth inhibition limiting potential production.
What was unexpected was the extraordinary
capability of cells to adapt to suspension
growth, and to adapt to serum-free, and
even protein-free, conditions. Over time, the
bioreactors used for industrial cell culture
have evolved into a few basic configurations.
This chapter will discuss the fundamentals of
bioreactors and highlight specific bioreactors
developed in the 1980s. These examples are
useful for their conceptual novelty and help to
illustrate the limitations on their use as industrial
bioreactors. Although these reactors are not
commonly used in current processes, they might

find specialized applications in other areas, such
as tissue engineering, or gene and cellular therapy.
Basic Types of Bioreactors
Bioreactors can be generally categorized according
to their mixing characteristics. The two extremes
Tubular Bioreactor of mixing are, at one pole complete, instantaneous
Fig. 9.1: Schematic of a tubular reactor mixing and, at the opposite pole, a solid-like
complete absence of mixing. The differences are
best illustrated using a continuous flow reactor,
although many bioreactors are used in batch fashion.
The two extreme mixing patterns characterize
two types of idealized continuous reactors:
well-mixed stirred tank and plug-flow (tubular)
reactor. In an ideal well-mixed bioreactor, the
mixing is assumed to be intensive such that the
fluid is homogeneous throughout the reactor.
When a new solution is added to the reactor, the
solute is instantaneously, uniformly distributed.
Stirred Tank Bioreactor
Fig. 9.2: Schematic of a well-mixed stirred tank reactor.
A tubular reactor is at the other extreme of an
idealized bioreactor; it has absolutely no mixing.
Idealized Bioreactor It is also called a plug-flow reactor, or piston-flow
• Plug flow reactor (no mixing) bioreactor. As its name implies, a solution entering
• Well mixed reactor (instantaneously perfectly mixed) the bioreactor will move downstream like a wall or
a plug, and steadily forward until it reaches the exit.
Stirred Tank (Well Mixed) Vs. Tubular The distinction between a well-mixed continuous
Reactor (Plug Flow) stirred tank reactor (CSTR) and plug flow reactor
(PFR) is best illustrated by a comparison of their
behavior after a step change in feed concentration.
F F Consider a continuous reactor that has an inlet
x
C stream (feed) and an outlet stream that are equal in
volumetric flow rate; thus, the volume of the reactor
is constant. Initially both the fluid in the reactor and
the feed stream are colorless. At time=0, the feed
Co
V stream is switched to a fluid containing a red dye
at a concentration of C0. We then observe how the
concentration of the dye has changed at the outlet.
In the case that the reactor is well mixed (i.e., a
Continuous Stirred Tank Bioreactor CSTR) as soon as the feed stream is switched,
the color is distributed uniformly in the reactor.
Fig. 9.3: A continuous flow stirred tank bioreactor with a Because the dye is uniformly distributed, regardless
switch for tracer injection.

of where the effluent stream is drawn from, it will
also acquire the red dye at the same concentration
as in the reactor. Of course, for the purpose of our
discussion, we neglect the time delay caused by
flow in the pipe lead into and out of the reactor.
As more colored feed comes into the reactor, the
concentration of the color seen in the outlet will
gradually increase. The balance equation for the
concentration of the dye that gives the color is
shown, where F is the flow rate, V the reactor volume,
Fig. 9.4: The concentration profile of the tracer after C the dye concentration, and subscript o denotes
injection into a CSTR
the feed. Note that in this case, the concentration
in the outlet flow is the same as in the reactor,
because of the assumption of being well mixed.
By setting initial condition C=0, one can solve the
equation, and plot the profile of the normalized
Fig. 9.5: A plug-flow reactor with a switch for tracer injection. concentration of the dye (C/Ci) over time. V/F has units
of time and represents the holding time (θ), the time
it takes one reactor volume of feed to pass through
the reactor. One can see that after a holding time, the
concentration of the dye in the reactor is 0.63 of that
in the feed. It takes three holding times (3θ) for the
concentration to approach (~98%) that in the feed.
In a PFR, the red colored dye moves downstream
like a sharp band, since there is no backmixing
or diffusion to blur the sharp boundary between
the color and colorless streams. After switching
Fig. 9.6: Concentration of tracer detected at the end of a PFR the feed stream to the red color, it takes one
after tracer injection.
holding time for the red color to appear at the
exit. When the color appears at the exit, its
concentration is identical to the feed concentration.
Initial condition: t = 0, C = 0
CSTR and PFR with Reaction The discussion above considers only mixing
behavior in the reactor, essentially just mixing
The same reaction carried out in PFR or CSTR gives tanks. In a bioreactor, consumption (of nutrients)
different kinetic behavior: and generation (product formation and growth)
• In PRF, as the feed moves downstream, the take place; these processes (reaction) will cause
reactant(s) is consumed and the product(s) is the concentration profile to differ from that in a
produced. Different locations in the reactor have
different concentrations. simple mixing tank. For CSTR, the assumption that
• In CSTR, the concentration of reactant(s) and the concentration at the outlet is identical to that
product(s) is homogeneous. The concentration at in the reactor holds. The effect of the reaction is
the exit is the same as in the fluid in the reactor. dealt with by adding a term to the material balance
equation to account for the reaction. One equation

is written for each of the reactants and products.
The equation describing the overall balance of the
dye in a PFR differs from that for a CSTR. In a PFR, the
concentration is no longer constant throughout the
reactor, but is dependent on the position within the
reactor. Because the concentration of both reactants
and products changes along the flow direction in the
reactor, the size (i.e., the length) of the reactor, or the
holding time in the reactor, affects their values. To
account for the effect of position, we do a material
balance on the dye over a very small section along the
reactor. The equation has both time and position as
independent variables. Once a steady state is reached,
the dependence of time can be eliminated, and the
equation becomes an ordinary differential equation
with the position as the only independent variable.
It can be seen that in the presence of reaction,
the concentration of the reactant decreases as
the feed stream moves downstream, while the
product concentration increases. For bioprocess
applications using a PFR, the nutrient level
will decrease while metabolite concentration
increases along the bioreactor. At some position,
the nutrients will be depleted, and a reactor
longer than that length becomes unproductive.
Implication When Growth or Reaction Plug-flow bioreactors are intrinsically more difficult
Occurs in the Reactor to scale up than mixing vessels, as the concentration
gradient of essential nutrients, oxygen in particular,
will inevitably become limiting in the downstream
region of the reactor. One way to overcome the
limitation is to increase the nutrient supply rate
by using a higher concentration in the feed, or by
operating at a higher flow rate. There are, however,
practical limits on both nutrient concentration and
flow rate. For example, nutrient concentration is
limited by its solubility, and a high flow rate will
require a higher capacity pump to overcome a higher
pressure drop across the reactor. These kinds of
limitations are especially true for growth of aerobic
organisms, because oxygen solubility in water is
very low and is quickly depleted unless supplied
continuously. Thus, the size and scalability of a PFR

reactor is rather limited. In a CSTR model, all cells
in the reactor encounter the same environment. The
nutrients that feed into the reactor are distributed
uniformly, albeit at abundant or suboptimal levels.
Heterogeneous Reactor- High Solid A bioreactor typically encompasses three phases:

Content a liquid phase, a solid phase (cells can be regarded
as “solid”), and a gas phase (bubbles for aeration).
Often, cell mass represents a very small fraction
of the culture volume; thus, the content of a cell
culture bioreactor is often treated as homogeneous,
as if having a single liquid phase, and cells, like
nutrients, are treated as part of the liquid phase;
no special consideration of the volume taken up by
Fig. 9.7: Schematic of a heterogeneous reactor cells in establishing material balance equations .
However, in microbial fermentation and in plant
cell culture, cells may make up a large fraction
(up to 50%) of total reactor volume. The reactor
volume, thus, is partitioned into cell volume and
culture medium volume. The cell and nutrient
concentrations calculated from medium volume
or total reactor volume have different values.
microcarrier Some so called heterogeneous cell culture

bioreactors contain a significant volume fraction
of solid phase such as microcarrier beads. In a
microcarrier bioreactor for example, microcarrier
beads often constitute 10-30% of the culture
volume. In this circumstance, even cell concentration
Heterogenous Stirred Tank Bioreactor needs to be well defined. For example, it must be
specified whether 107 cells per milliliter is with
Fig. 9.8: A reactor supporting cell growth on microcarriers is a
heterogeneous reactor respect to total culture volume or to liquid volume.
Operating Mode of Bioreactors

Batch and Continuous Processes
The operation of a bioreactor is generally classified as
batch or continuous mode. In a continuous process,
the feed is continuously being introduced to the
reactor, and product stream is continuously being
withdrawn from the reactor. In a batch process, the
medium, including all nutrients (except oxygen), is
added at the beginning, and no other nutrient addition
occurs until the end of cultivation. Continuous
culture is not commonly practiced in the cultivation

of mammalian cells unless in conjunction with cell
recycle. This topic will be discussed in a later chapter.
Batch cultures are often limited by the fact that
in many cases the product concentration is
not sufficiently high. To achieve a high product
concentration it is necessary to have a high cell
concentration; for that the nutrient concentration
in the medium must also be high. In many cases
high concentrations of nutrients are not attainable
in batch culture because the initial concentration
at the start of culture is limited by the solubility of
some nutrients, or by the concentration at which
cells can be grown without growth inhibition. It
may be necessary to add additional substrate or
product precursor to sustained continued growth
to reach higher cell and product concentrations. In
other cases, such as intermittent fed-batch cultures,
instead of terminating a batch process at the end of
culture, a portion of the culture is kept, and fresh
medium is added to start the process again.
Batch Cultures Batch processes are simple and are widely used.
In fact, batch process culture is used much more
frequently than the fed-batch process. Before
reaching production scale, cell expansion is carried
out in a series of batch cultures with increasing
reactor volumes. This series of batch cultures
for cell expansion is often referred to as seed
train. During expansion in batch cultivations, the
culture is discontinued in the reactor and cells
are transferred to the next larger reactor while
still rapidly growing. If a culture is allowed to run
its course, cell growth will be inhibited by the
accumulation of metabolites, and the resulting
Fig. 9.9: Kinetics of growth and product formation in a batch
product concentration will be low. In general, batch
culture
processes do not result in high productivity. Batch
cultures are used in viral vaccine production, or,
if used in recombinant protein manufacturing,
are largely limited to the cell expansion stage.

Fedbatch Cultures Fed-batch processes do not differ significantly
from batch cultures. The simplest form of fed-
Intermittent Harvest
batch culture involves intermittent harvest. At
a late exponential growth stage of the culture, a
portion of the cells and product are harvested, and
the culture is replenished with fresh medium. This
avoids metabolite inhibition of cell growth and
replenishes nutrients for continued cell growth.
This process may be repeated several times.
This simple strategy is commonplace for the
production of viral vaccines produced by persistent
infection, as it allows for an extended production
period. It is also used in roller bottle processes
with adherent cells. By intermittent harvesting,
Fig. 9.10: Kinetics of growth and product formation in a fed- turn-around time involved in cleaning up and
batch culture with intermittent harvest restarting the process is saved. Furthermore, after
replenishing the medium, the culture reaches
peak cell concentration faster than if the culture
is started de novo with fresh inoculum, which
usually is at a significantly lower cell concentration.
Fedbatch
For production of recombinant proteins and
antibodies, a more traditional fed-batch process
is typically used. A fed-batch culture is started
in a volume much lower than the full capacity of
the bioreactor (approximately 40% to 50% of
the maximum volume, or at a level sufficient to
allow the impeller to be submerged). Nutrients,
usually in a more concentrated form than in basal
medium, are added during the cultivation to allow
cell and product concentrations to reach much
Fig. 9.11: Kinetics of growth and product formation in a fed-
batch culture with increasing culture volume
higher levels than can be attained in batch culture.

Tissue Culture and Disposable Cell Culture Systems
In this section we will describe reactors used for
large-scale cultivation of mammalian cells. Arguably,
the oldest bioreactors are animals themselves or
their tissues. The application of virus vaccines dates
back two hundred years, when Edward Jenner used
cow pox to inoculate humans for protection against
small pox. Many viral vaccines developed in the
first half of the 20th century employed animal or
animal tissues as production vehicles. Use of animal
tissues (e.g., chicken eggs, rodent brains) for virus
production is still practiced today for some viruses,
although cell culture is considered the norm.
Disposable Culture Systems Many different types of disposable, single-use

plastic bioreactors including T-flasks, roller
Single Use Bioreactor bottles, and multiple flat panels are commonly
used for production of recombinant proteins and
• Simple, low-capital investment, fast to implement,
ease of equipment validation
viral vaccines, using both attachment-dependent
and suspension cells. The plastic surface may be
• A variety of systems with vastly different mixing
mechanisms treated to facilitate attachment of adherent cells.
• Less well-characterized in fluid dynamics, mass
Suspension cells can be readily grown in a stirred
transfer vessel. However, there is an increasing trend toward
• Suitable for seed culture, scales up to hundreds of the use of disposable (single-use) bioreactors at
liters small and moderate scales. Factors such as ease of
• Possible faster turn around time operation, shorter time to scale up, lower capital
• Lacking the strength of steel; some operations investment, and reduction of process validation
routinely done on steel vessels, such as pressurized burden have contributed to their wide adoption.
liquid transfer, cannot be performed on single use
vessels
Roller Bottles Roller bottles are cylindrical screw-capped bottles
with a total volume ranging from 1 to 1.5 liters, and
are suitable for a culture volume of 0.1 to 0.3 liters.
Stacks of bottles are placed on a rack and rotated at
1 to 4 rpm. For small-scale operations, roller bottles
provide many advantages for the cultivation of
adherent cells. The system is relatively inexpensive
to set up, and allows for rapid adjustment of
production throughput in response to changing
needs. Furthermore, complete replacement of
medium, e.g., from growth medium to product
medium, is rather straightforward. Roller bottles

are particularly useful in the case that the serum-
containing medium needs to be replaced by a serum-
free or protein-free medium for the production
phase. The transparent glass or plastic wall allows
visual or microscopic examination of the culture
status. Microbial contaminated bottles can be readily
identified and discarded before mixing with others.
For large-scale production of biologics, however,
there are numerous drawbacks to roller bottles.
On-line environmental monitoring and control is
virtually impossible or at least impractical. Aseptic
bottle handling for inoculation, protease-treatment
for cell detachment and expansion, medium
exchange, and product harvest are labor intensive
and must be performed by a skilled technicians
to ensure a low failure rate. A batch of a size
suitable for manufacturing purposes may require
hundreds or even thousands of bottles, and the
large number of manual steps involved dramatically
increases the risk of microbial contamination.
Despite these significant drawbacks, roller bottles
are widely used in the production of recombinant
proteins and viral vaccines. Often, they are used
primarily because the product involved has already
been approved by regulatory agencies and a process
change would incur significant costs for regulatory
approval and could even result in product
comparability issues. In some cases, roller bottles
Fig. 9.12: A roller bottle and roller bottles on racks for cell were selected because the process was easy to
culture implement and the production capacity needed was
manageable. However, when faced with the need to
increase production, e.g., due to market expansion,
scale-up of a roller bottle process is challenging.
Notable examples of industrial roller bottle processes
include the production of erythropoietin (EPO)
using recombinant Chinese Hamster Ovary cells, the
production of live attenuated chickenpox (varicella)
vaccine, and herpes zoster (shingles) vaccine using
the adherent secondary human lung fibroblast cell
strain, MRC-5. In some manufacturing facilities a fully
automatic, robotic roller bottle handling system is
available. However, a robotic system cannot handle
a large number of bottles in parallel. Handling

a large number of roller bottles is a prolonged
process. For example, in a process entailing virus
inoculation or a culture condition switch to induce
product formation, inoculation and induction
time can vary very widely from the processing of
the first bottle to the processing of the last bottle.
Multiple Plate System Nunc Cell Factories® (NCFs) are widely used in
laboratories and in industrial production of viral
vectors, vaccines, and recombinant proteins.
Although designed to culture adherent cells,
NCFs can also be used for suspension cells. A 40-
tray NCF has a capacity of 25,280 cm2 and ~8 L
liquid volume. For large-scale manufacturing, a
mechanical handling system can be used. One
notable example is the production of multivalent
Rotavirus vaccine using Vero cells, a continuous
African green monkey kidney cell line. The vaccine
is a live, attenuated virus vaccine that contains
five different human-bovine virus reassortants,
each produced from a different process.
Another multiple plate system (CellCube Module)
entails nine-inch square polystyrene plates stacked
vertically with 1 mm spacing in a resin case. The
space between stacks is completely filled with
Fig. 9.13: A commercial multiple plate system for cell culture medium. Continuous medium circulation is used
for oxygen and nutrient supply. CellCube has
been used in the cultivation of MRC-5 cells for the
production of attenuated hepatitis A virus (HAV)
and for the production of VAQTA, an inactivated
HAV vaccine. These disposable bioreactors have
also been somewhat popular for intermediate levels
of production of cells and gene therapy vectors.
Bags, Cylinders on Shakers, Movers Blood bags have long been used for the cultivation
of cells for cell therapy. Small bags are placed in
incubators for temperature control. For larger single
use bag systems, mechanical mechanisms provide
mixing and oxygen transfer. Bags are especially
suitable for cell expansion before reaching final
production scale up. WaveTM consists of pre-
sterilized, disposable Cellbags and a special rocking
platform. The rocking motion of the platform

induces waves in the culture fluid to provide mixing
and oxygen/gas transfer.
In manufacturing settings, validating the equipment
for restart of a process contributes a significant
part of the overall operating costs. A more extreme
case of single use alternatives to a conventional
fermentor is a retrofit product to mimic an existing
stainless steel bioreactor vessel. It consists of a
reusable stainless steel outer support container and
a single use cell chamber with a working volume of
up to 1000 liters, which can be integrated into the
existing bioreactor control system. These modest
scale, single use reactors are not intended to replace
a conventional stirred tank made of stainless steel
Fig. 9.14: A single-use bag cell cultivation system
with fixed piping, auxiliary equipment, etc. Because
of its single use construction, its operation is limited.
For example, a single use fermentor cannot be
pressurized to quickly transfer its contents to other
process units, or even while receiving inoculum.
Cell Support Systems

Suspension Culture vs. Adherent Many cell lines used in the production of
Cultures vaccines and biologics grow in suspension cells.
Disposable systems are invariably limited in
their scale of operation. For processes whose
production scale is relatively small, a disposable
system provides some advantages. For protein
therapeutics needed in large quantities, the use
of disposable systems may be limited to inoculum
preparation. Large scale operations, thus, continue
to employ fermentors or other bioreactors.
The majority of cells used for protein production via
rDNA are suspension cells, which, as suggested by
the name, have no need for attachment to a surface
and are simply suspended in the medium in a
bioreactor. For growth of adherent cells, cell supports
are needed to provide adherent surface. The cell
support is placed in a bioreactor which provides a
mechanism to supply nutrients and oxygen to cells. In
some cases, cell support is also used for suspension
cells, allowing cells to be kept in the reactor while
the medium is being perfused or exchanged.

Microcarriers Most normal diploid cell strains or primary cells are
anchorage-dependent. For large scale operations in a
stirred tank bioreactor, microcarriers are used to provide
adherent surfaces. Conventional solid or microporous
microcarriers are suitable for normal diploid cells
which require attachment and spreading. Macroporous
microcarriers also allow cells to grow within the interior,
and are used for a wide variety of continuous cell lines.
Solid and Microporous Microcarriers The use of microcarriers for cell culture was first
demonstrated by Van Wezel (1967). The basic
concept is to allow cells to attach to the surface
of small suspended beads so that conventional
stirred tank bioreactors can be used for cell
cultivation. To facilitate suspension of these cell-
laden microcarriers, their diameter and density are
usually in the range of 100–300 μm and 1.02–1.05
g/cm3, respectively. This diameter range provides
good growth surface area per reactor volume. Even
at a moderate microcarrier concentration occupying
only 8–15% of the culture volume, a significantly
larger surface area per reactor volume can be
Human fibroplast FS4 on cytodex 1 microcarriers achieved than that possible with roller bottles.
Most anchorage-dependent cells do not develop their
normal morphology, and some do not multiply well
on surfaces with an excessively high curvature. These
cells do not grow well on small microcarriers. On the
other hand, some cells, especially transformed cells,
multiply well even on small microcarriers. When
these cells are grown on very small microcarriers,
they agglomerate to form aggregates and continue
CHO on cytodex 3 microcarriers to grow to high density. The small microcarriers,
usually with a diameter of about 50 μm, serve as
Fig. 9.15: Cell cultivated on microcarriers nuclei for the initiation of aggregate formation.
Microcarriers can be made of many different
materials including dextran, gelatin, polystyrene,
glass, and cellulose, not all of which are commercially
available. In general, microcarriers have a wettable
surface, and are sometimes coated with collagen or
other adhesion molecules to enhance cell adhesion
and spreading. The most widely used microcarriers,
which are based on dextran, are derivatized

with charged molecules or denatured collagen.
An advantage of microcarrier culture is the ease
Table 1. Desired Characteristics of Microcarriers of separating cells from culture medium. Many
Only slightly higher than microcarrier cultures require medium exchanges
Density ~1.02 g / cm3 water for easy suspension
and setting during cultivation to remove lactate, ammonia,
and other metabolites and to replenish nutrients.
Should be the smallest
This can be accomplished by simply allowing cell-
Diameter 150 - 200 μm possible and yet allow for
cell spreading laden microcarriers to settle so that the spent
medium can be withdrawn and replenished.
Prefer solid, for use as
From solid to In large-scale operations, continuous or semi-
Porosity inoculum bead to bead
nearly 90%
transfer continuous perfusion is more frequently used.
ECM coating,
This can be accomplished by withdrawing medium
Surface slightly Positive charge enhances through a coarse screen that retains microcarriers
Properties positively initial attachment in the reactor but allows medium to pass.
charged
A wide variety of cell types have been grown on
microcarriers including fibroblasts, epithelial cells,
hepatocytes, neuroblastoma cells, and endothelial
cells from various species. Overall, microcarrier
culture is a convenient laboratory and research tool,
and has the advantage of being amenable to large-scale
production if a large quantity of product is needed.
Macroporous Microcarriers Macroporous microcarriers contain large internal
pores. The void space inside allows cells to be
cultivated on internal and external surfaces. Cells
in the interior are less susceptible to mechanical
damage caused by agitation and gas sparging. On the
downside, cells in the interior of microcarriers are
more likely to be subject to oxygen limitation due to
the long diffusional distance, especially since most
macroporous microcarriers have a larger diameter
(500 μm–2 mm) than standard microcarriers.
Fig. 9.16: Scanning electron micrograph of a collagen based
macroporous microcarrier bead Macroporous microcarriers are made of various
materials including gelatin, collagen, and plastic.
Many cell lines have been successfully grown on
macroporous microcarriers including Vero, HepG2,
CHO, and 293 cells. The final cell concentration
achieved tends to be higher than that obtained
with an equivalent volumetric concentration
of conventional microcarriers. In some cases,
Cofocal microscopic optical section of
however, the growth kinetics are slower because the
HepG2 cells in collagen macroporous
microcarriers
penetration of cells into the interior may be slowed
Fig. 9.17: Cofocal optical section of cells grown in a or even retarded by the restrictive opening of the
macroporous microcarrier pores.

Table 2. Listing of Microcarriers
Different Types of Microcarriers
Dextran Cross-linked dextran matrix with various surface modifications
(GE Healthcare)
Cytodex 1 Positively charged DEAE group
Cytodex 3 Denatured type I collagen coating
Polystyrene Cross-linked polystyrene core material with a wide variety of
(SoloHill Engineering) surface modifications
Plastic No surface modification, or positively charged
ProNectin F Recombinant fibronectin coating
FACT III Type I porcine collagen coating
Glass High silica glass coating
Hillex Surface modified with cationic trimethyl ammonium
Cellulose Microgranular DEAE-cellulose microcarrier
DE-52 Anion exchange capacity of 1 meq / g dry materials
DE-53 Anion exchange capacity of 2 meq / g dry materials
Alginate Global Eukaryotic Microcarrier (GEM), composed of alginate
(Hamilton) matrix with modification of surface
Glass (Biosilon) Hollow glass
Macroporous Microcarriers
Gelatin CultiSpher-G and CultiSpher-S, cross-linked gelatin matrix with
(Percell Biolytica) pore size 10-20 µm, CultiSpher-S has a higher thermal and
mechanical stability due to a different cross-linking procedure
Cellulose Cytopore, cross-linked cellulose matrix with a pore size
(GE Healthcare) averaging 30 µm. Its surface is modified by the introduction of
DEAE group
Collagen Cellagen, prepared from bovine corium insoluble collagen by
(MP Biomedicals) pepsin treatment
Polyethylene Cytoline, composed of high-density polyethylene weighted with
(GE Healthcare) silica
Polypropylene Fibra-Cel disk, composed of polyester mesh with polypropylene
(New Brunswick) support

Cell Aggregates Some transformed cells can grow as aggregates
when cultivated in shaker flasks or in stirred
vessels. Different methods have been used to induce
aggregate formation. Aggregation can be promoted
by manipulating the calcium concentration in
conjunction with the agitation rate. Aggregate
cultures have advantages similar to microcarrier
cultures. They can be cultivated in conventional
stirred tank reactors with environmental
Fig. 9.18: Confocal section of HEK293 cell aggregates.
Green: viable cells, red: dead cell nuclei control. They can be allowed to settle relatively
rapidly by stopping agitation, permitting
easy medium replenishment or perfusion.

Simple Stirred Tank Bioreactor Stirred tanks, or conventional fermentors, have been
widely used for culturing suspension cells since the
1960s. By using microcarriers, adherent cells can also
be cultured in a stirred tank. The basic configuration
of stirred tank bioreactors for mammalian cell
culture is similar to that of microbial fermentors.
A major difference is that the aspect ratio (ratio of
height to diameter) is usually smaller in mammalian
cell culture bioreactors. The power input per
unit volume of bioreactor is also substantially
lower in mammalian cell culture bioreactors.
While the Rushton type impeller is the norm in
microbial fermentors, mammalian cell culture
fermentors mostly employ axial flow type impellers.
The difference reflects the different purposes of
agitation in microbial fermentation and in cell
culture. In microbial fermentation, agitation is
needed at a higher power input to disperse air
bubbles and to increase oxygen transfer efficiency,
whereas in mammalian cell culture, the primary
purpose of agitation is to maintain relatively
uniform suspension of cells or microcarriers.
In general, the mixing time in a mammalian cell
culture bioreactor is substantially longer than
that in a microbial fermentor of similar scale. The
oxygen transfer capacity in a cell culture bioreactor
is also substantially lower than that in a microbial

fermentor. However, the typical oxygen demand in a
mammalian cell culture is 10 to 50 times lower than
that in microbial fermentation.
Airlift Bioreactor A variant of the bubble column reactor with internal

circulation loops is used for the cultivation of
mammalian, insect, and plant cells. In these airlift
reactors, internal liquid circulation is achieved
by sparging through the internal draft tube. The
fluid in the draft tube has a lower effective density
than the bubble-free section, which, along with
the upward momentum generated by air flow,
induces liquid circulation. Medium flows upward
through the sparged section (riser) and downward
in the bubble-free section (downcomer). This
method of generating circulation has a low energy
requirement compared with stirred-tank reactors.
Airlift bioreactors for cell cultivation are considered to
be low-shear devices because there is no mechanical
Air agitation. They have been used successfully
Fig. 9.19: Schematic of an air-lift bioreactor with suspension cultures of BHK 21, human
lymphoblastoid, CHO, hybridomas, and insect cells.
Fluidized Bed Bioreactor Fluidized bed reactors have long been used in
chemical catalysis. The fluid stream (often gas
in catalysis) enters through a flow distributor
at the bottom at high velocity to suspend the
solid catalyst particles. The reactants enter the
catalyst and products diffuse out into the fluid
to be carried out through the top of the reactor
where a separator prevents any particles from
being blown out. The main advantage of a fluidized
bed is the high heat transfer efficiency between
the high velocity fluid and the catalyst surface.
When applied to cells or microcarriers directly, the
density difference between solid phase and liquid
phase is small, making particle retention difficult.
In the 1980’s, collagen macroporous beads were
used in commercial fluidized beds offered by Verax.
The carriers were weighted by inclusion of iron
particles to increase the density difference between
fluid and carrier so that the particle could be
retained in the reactor at the flow rates required for
supplying sufficient oxygen to support cell growth.

Membrane Bioreactor
Hollow Fiber Bioreactor The use of hollow fiber reactors for cultivation of
mammalian cells dates back to the early 1970s. A
hollow fiber system can be used for both anchorage-
dependent and suspension cells. It consists of a
bundle of capillary fibers sealed inside a cylindrical
tube. The basic configuration is rather similar to
the hollow fiber cartridge used in kidney dialysis.
The hollow-fiber entails a porous polymeric
layer that provides mechanical support covered
by a thin membrane which provides selectivity
based on the size of molecules. In most cases, an
ultrafiltration membrane is used. The molecular
weight cut-off (MWCO) of the membrane differs
according to the specific application, ranging from
a few thousand to a hundred thousand daltons.
The culture media is pumped through the fiber
Fig. 9.20: Schematic of a hollow-fiber bioreactor lumen, and cells grow either in the extracapillary
space, or on the shell side. Supply of low-molecular
weight nutrients to the cells and the removal of waste
products occur by diffusive transport across the
membrane between the lumen and the shell spaces.
The ultrafiltration membrane prevents free diffusion
of secreted product molecules from passing through
the membrane and allows them to accumulate in
the extracapillary space to a high concentration.
Although the use of microfiltration hollow fiber
membranes for cell culture is infrequent, it does
appear in various research applications for studying
metabolism and for producing small quantities of
materials for research or diagnostic applications.
Multiple Membrane Plate Bioreactor
Scaling up of a hollow-fiber system is limited by
the ability to extend the axial length of the fiber
due to oxygen transfer limitation. Use of a very
high flow rate to supply more oxygen is limited
by the capacity of the pump and the mechanical
strength of the membrane. Expanding the cartridge
diameter to increase the capacity eventually faces
the problem of uneven flow distribution among the
fibers. In principle, one can mix two different fibers
to supply oxygen and medium separately. However,
mixing different hollow fibers in a cartridge poses

a major challenge in manufacturing. An alternative
configuration using multiple flat membranes has
been attempted. However, this seemingly versatile
reactor also suffers from practical manufacturing
complexity, and is not used in large scale operations.
Other Bioreactor Systems

Microsphere Induced Cell Aggregates Some cells do not form aggregates readily. When
suspended in culture the rate of aggregate formation
is slow and cells lose viability over time. One way to
induce aggregate formation is to add microspheres
to cell a suspension to promote agglomeration to the
microspheres. These agglomerated microspheres
become aggregates as cells grow. If the aggregate
diameters become too large, necrotic centers
(a) CHO cells attaching to micro- can formed as a result of transport limitations.
spheres and agglomeration of Many cell lines including BHK, CHO, 293, and
beads. swine testicular cells have been cultivated as
(b) Sections of HEK 293 cells aggregates with sizes ranging between 90 and
forming microspheres induced 400 μm without the formation of necrotic centers.
aggregates.
(c) SEM micrograph of HEK 293

cells on microspheres
Figure 9.21: Microspheres induced cell aggregates
Agarose Cell Immobilization Agarose entrapment of cells is usually accomplished

by passing a cell/agarose suspension through a
small orifice into an air parallel jet stream. The
droplets of agarose containing entrapped cells
are collected in a chilled oil phase to allow the
agarose to gel. Alternatively, the cell-agarose
suspension may be allowed to drop onto the center
of a fast rotating disk. The centrifugal spinning
force causes droplets to form and be dispensed
outside the disk. Agarose beads tend to be rather
large, commonly, hundreds of micrometers in
diameter. The large size of agarose beads limits
oxygen transfer at high cell concentrations. In
addition, agarose beads lack sufficient mechanical
strength to sustain mechanical optimization even in
a moderately small scale (tens of liters) bioreactor.

Microencapsulation Another method of cell entrapment entails entrapping
cells in a polymeric matrix to form spheres followed
by coating with a polymeric film to control diffusivity
of molecules. The idea is to allow fast nutrient
diffusion while retaining the product molecules.
One of the polymers most often used for cell
entrapment is calcium alginate. Cells are suspended
in sodium alginate and added dropwise into
a calcium chloride solution, which allows for
gelation. The alginate beads may be coated with
polylysine for increased mechanical and chemical
stability. The alginate gel inside the polylysine
coated bead can be liquefied through treatment
with a calcium chelator such as EDTA or citrate.
Large-scale application using this
microencapsulation technique is not easy.
However, the microcapsule provides a
Fig. 9.22: Formation of microencapsulated cell beads means of immunoisolation of transplanted
cells or tissues, and could be suitable for
some tissue engineering applications.
Membrane Stirred Tank The membrane stirred tank was developed by

Professor Jürgen Lehmann in the 1980s. This
bioreactor uses long microporous polypropylene
tubing wrapped around rotating rods. By adjusting
the air pressure in the polypropylene tubing, the
micropore expands to allow gas to be in direct
contact with medium, thereby providing bubble-free
aeration. The rotation of the tubing also provides
gentle agitation to microcarriers or suspended cells.
Fig. 9.23: A membrane stirred tank bioreactor
Spin Filter Stirred Tank The centerpiece of a rotating wire cage bioreactor
is the wire cage, which is often mounted on the
agitation shaft. The bottom of the cage is solid,
while the side is made up of wire mesh with
openings ranging from 25 to 60 μm. The average
diameter of cells is approximately 10 to 15 μm.
Typically, fresh medium is added continuously
outside the draft tube and the culture fluid is

withdrawn from inside the cage at the same rate.
Under certain operating conditions, the cell
concentration inside the wire cage is lower than that
outside the cage, thus achieving cell retention in the
bioreactor. Under optimal conditions, the device
does not act as a filter, and no cake is formed. The
retention of the cells does not appear to be due to
centrifugal force exerted by the rotating motion of the
cage, because the terminal velocity of the cells due to
centrifugal force is two to three orders of magnitude
lower than that of the fluid velocity across the screen
due to perfusion. The electrostatic effect is also
unlikely to be responsible, since the ionic strength of
the culture fluid is relatively high, and the thickness
of the Debye layer is only in the order of 1 nm.
The rotating wire cage bioreactor has also been used
in aggregate and microcarrier cultures. In these
cases, the mechanism of cell retention is relatively
straight forward as the particle is much larger than
the openings on the cage.
The mechanism of retaining cells in suspension by
this device is still not clear. It is plausible that the
retention is caused by a fluid mechanical effect,
maybe one similar to the behavior of particles of
low Reynolds number near the wall in a Poiseuille
flow or laminar boundary layer flow along a flat
Fig. 9.24: A spinner filter stirred tank bioreactor plate. Lacking a mechanistic understanding of cell
retention has made scale up of this device difficult.
Many recently installed spin filters are operated
at very high rotation rates. At such a high sped of
rotation rate centrifugal force plays a key role in cell
separation by dispelling cells away from the surface
of the rotating cage. Cell separation in these reactors
is certainly different from the low speed wire cage
reactors described in this section.

Vibromixer A vibromixer uses a perforated disk as the mixing
mechanism rather than a conventional impeller. The
disk vibrates in a vertical direction (perpendicular
to the plane of the disc) at high frequency causing
liquid to circulate through the perforated holes
and provide mixing. The vibromixer was used in
the 1960’s for the cultivation of suspension cells
and virus production. Its use in cell cultivation has
diminished in recent decades. However, it is still used
in some cases to keep concentrated microcarriers
in suspension for cell detachment during the
Fig. 9.25: A vibro-mixer based bioreactor trypsinization step. The fluid mixing provides gentle
shear to detach trypsinized cells from microcarriers.
high frequency
vibration
liquid moved by the

vibrating plate as going
through the holes
Fig. 9.26: Movement of culture liquid around the vibromix-
ing plate
Concluding Remarks
Mammalian cells are widely used in the production As mammalian cell culture processes are increasingly
of viral vaccines and therapeutic proteins. Most of being used in manufacturing, one also witnesses an
those processes employ cells growing in suspension; increasing adoption of disposable (or single-use)
however, some applications, particularly in vaccine cell culture devices. Traditional roller bottles are still
production, use adherent cells. Stirred tank often seen. Additionally, multiple flat plates or parallel
bioreactors are most widely used for mammalian cell trays, along with bag type cell growth chambers and
culture processes. When a stirred tank bioreactor even plastic stirred tanks, are used in moderate scale
is used for growing adherent cells, a supporting production and in the seed culture propagation for
surface for cell attachment is provided through the large scale bioreactors. These disposable devices
use of microcarriers or macroporous microcarriers. offer simplicity in operations, and ease process
Alternatively, adherent cells may be grown as validation in the production of biopharmaceuticals.
aggregates in suspension. Fed-batch culture is Some innovative bioreactors developed nearly two
commonly practiced since it facilitates reaching decades ago are now finding new applications in
high cell density and high product concentrations. tissue engineering and cell therapy. As these new
When a stirred tank bioreactor is operated in technologies advance and the demand of those
a continuous mode, it is a common practice to specialized cells grows we will continue to see
employ a cell retention device for perfusion in innovative developments in bioreactor technology.
order to increase cell and product concentrations.

Oxygen Transfer in
Oxygen Transfer Through Gas-Liquid Interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Oxygen and Carbon Dioxide Concentration in Medium . . . . . . . . . . . . . . . . . . 217
Oxygen Consumption and CO2 Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Method of Supplying Oxygen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
Gas Sparging in Cell Culture Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Bubble Size and Sparger Orifice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
Experimental Measurement of KLa and OUR . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
Effect of Hydrostatic Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Damage to Cells by Gas Sparging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
Mass Transfer Resistance in Cell Immobilization Reactor . . . . . . . . . . . . . . . . . . . . . . . 229
Oxygen Transfer in Plug-Flow Bioreactors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Oxygen Transfer Through Gas-Liquid Interface

In cell cultivation, oxygen is transferred from air
through the gas phase (via either gas bubbles or
the gas space in the flask) and the liquid phase
interface into the medium where it is consumed by
cells. Simultaneously, CO2 produced by the cells is
excreted into the medium and then diffuses into the
gas phase. Transfer of oxygen or CO2 molecules from
one phase to the other occurs only when oxygen
or CO2 in the two phases is out of equilibrium. If
oxygen in the two phases are in equilibrium (i.e.,
the medium is already saturated with oxygen),
there will not be any net transfer in either direction.
If a liquid deprived of any gas molecule is in contact
with air composed of 79% N2 and 21% O2, both N2
and O2 will diffuse into the liquid and eventually
Fig. 10.1: Concentrations and partial pressure of oxygen across
reach equilibrium between the two phases. (Of note,
an air bubble in culture fluid because N2 has a very low solubility and is hardly
OXYGEN TRANSFER IN CELL CULTURE BIOREACTORS | 213

consumed by cells, it will not be further considered
in this chapter.) At equilibrium, if the oxygen content
Description of Dissolved Oxygen Concentration in the gas phase is increased to 40%, then oxygen
• Solubility in water at 37°C with air of 1 atm, 21% O2 in the liquid phase will no longer be saturated
• 0.2 mmole / L and, gradually, oxygen will diffuse into the liquid
• 0.64 mg / L phase until the dissolved oxygen concentration
• Since air in equilibrium contains 159.6 mm Hg or 0.21 becomes equilibrated in the gas phase. Conversely
atm, sometimes solubility is expressed also in mm Hg, if the oxygen level in the gas phase is decreased to
especially for blood oxygen level 10%, then oxygen will escape from the liquid into
the gas phase until a new equilibrium is reached.
Thus, in order for mass to transfer between the two
To transfer oxygen or CO2 between gas and liquid phases phases, a deviation from equilibrium is essential. Such
through interface a deviation is often referred to as the “driving force
• The two phases must not be in equilibrium for mass transfer”. Quantifying the driving force, or
the degree of deviation from equilibrium is the first
step toward calculating the rate of mass transfer.
Oxygen level in the gas phase is usually described
as mole percentage or mole fraction (yO2). It can
also be expressed as partial pressure (PO2), or
the mole fraction of oxygen multiplied by total
pressure. If the ambient pressure is 1 atm (or
760 mm Hg) and the oxygen mole fraction is
0.21 (yO2 = 0.21), then the partial pressure of
oxygen in the air is 0.21 atm (or 159.6 mm Hg).
The oxygen concentration in a liquid phase (or
dissolved oxygen concentration) is often expressed
as mmole/L. When pure water is saturated with
oxygen under a pressure of 1 atm and 21% O2 at 37°C,
its oxygen concentration is 2.2 mmole/L. Because
this concentration is the equilibrium value with an
oxygen partial pressure of 159.6 mm Hg, sometimes
it is expressed as 159.6 mm Hg. Expressing dissolved
oxygen concentration in terms of its gas phase partial
Fig. 10.2: Driving force for oxygen transfer across air-liquid pressure is common in the medical profession.
interface Since oxygen is a critical nutrient in the living
world, one needs to be familiar with the multiple
descriptors used to describe its concentration.
For example, water which is at 50% of saturation
with air at 1 atm and at 37°C has a dissolved oxygen
concentrationof0.11mmole/L, or 79.8mmHg.Ifwater
containing 0.11 mmole / L oxygen is in equilibrium
with a N2/O2 mixture, then that mixture would
have 79.8 mm Hg [or 10.5% (mol/mol)] of oxygen.

If water containing 0.11 mmol/L of oxygen is
put in contact with air (with 21% O2 at 1 atm at
37°C), it is undersaturated and is 0.11 mmol/L
away from saturation. In other words, the water
could hold 0.11 mmol more oxygen per liter.
One could also say the water is 79.8 (159.6 -
79.8) mm Hg below oxygen saturation. Such
concentration difference from saturation is the
driving force for oxygen transfer. One can then
quantify the driving force using different units;
describing it in terms of liquid phase concentration
(mM), or gas phase concentration (mm Hg).
A concentration profile of oxygen across an interface
of gas phase and liquid phase is depicted in the
figure. In the bulk liquid (i.e., the liquid at some
distance away from the interface), the concentration
of oxygen is C. In the bulk gas phase, the oxygen
fraction is yO2, and its partial pressure is PO2.
To dissect the mechanism of oxygen transfer, we
model the interface as having a boundary layer or
an imaginary film separating the two sides. Let us
denote the oxygen concentration in the liquid as C* if
the system is at an equilibrium with gas phase (whose
oxygen partial pressure is PO2), and denote the gas
phase concentration as P*O2 when it is in equilibrium
with liquid phase (which is at a concentration of
C). The liquid phase system is therefore C*-C away
from equilibrium. In other words, the driving
force for the liquid phase to reach equilibrium is
C*-C. Recall that we can also use gas phase units
to describe the liquid phase concentration, so the
driving force can also be described as PO2-P*O2. The
two different descriptions of the same driving force
are related by a constant as will be seen again later.
Again the driving force across the boundary of the
two phases can be expressed in either gas phase
concentration or liquid phase concentration. Thus,
when the driving force is not zero, oxygen will diffuse
across the boundary layer to move the system toward
equilibrium. The higher the concentration gradient
across the interface is (i.e., a stronger driving
force), the faster the oxygen transfer rate will be.
The rate of oxygen transfer is dependent on three

factors: 1) the magnitude of the difference between
Oxygen Transfer Rate (OTR) = KLa • (C*-C) =KLa ΔC C* and CL, 2) the area that is available for oxygen
[mmole / l • hr] = [cm / hr] • [1 / cm] • [mmole / L] transfer, and 3) the mass transfer coefficient.
C*: solubility of oxygen, The second factor, available surface area, is typically
ΔC: oxygen concentration gradient (i.e., the driving force) expressed as interfacial area per volume of liquid.
It, thus, has a unit of inverse of length (L-1, like cm-
1). Think of water evaporation, or mass transfer
of water vapor, from a liquid water surface into
Three Factors Affecting OTR
dry air. Therefore, if a given amount of water were
• Mass transfer coefficient (KL)
contained in a cup, it would take longer for it to
• Specific transfer area (i.e., the interfacial area) evaporate than if that same amount of water was
• Driving force (i.e., the gradient across interface) spread out in a flat dish because the surface area
is much larger in a dish. Another example of using
a large surface to increase oxygen transfer is our
lungs, which, when fully expanded, can yield an area
of nearly 150 m2. This is why using a lung to transfer
To Improve Oxygen Transfer
oxygen is far more effective than using our skin.
• Increase KL (make the interface more “turbulent”)
• Increase a (use smaller bubbles for sparging, or use The third factor affecting the rate of oxygen
silicone tubing) transference is the mass transfer coefficient.
• Increase ΔC (use oxygen enriched air, or don’t maintain This is a measurement of the resistance for mass
dissolved oxygen (C) at an unnecessarily high level.) transfer at the interface. It is affected by the
specific molecules that are being transferred
and the physical and chemical properties of the
liquid. The mass transfer coefficient (KL) has
the same units as velocity (L/t, like cm/sec).
One can imagine the boundary layers in the interface
as a stagnant film surrounded by a bulk liquid,
which is under some mechanism of localized mixing.
The oxygen molecules have to diffuse across the
boundary layer to reach other phases. If the system
is under vigorous mixing, the boundary layer would
be thinner and the transfer would be faster. Again
considering water evaporation in a dish, if that
dish were being stirred and the air is being blown
across the dish, the mass transfer coefficient at
the interface would be higher and the evaporation
would be faster than when the dish is unperturbed.

Oxygen and Carbon Dioxide
Concentration in Medium
Calculation From Henry’s Law Because the rate of oxygen transference between a
gas phase and a liquid phase is dependent on how
far it is from equilibrium, it is important to first
determine its saturation level, i.e., concentration
at equilibrium, to accurately estimate the oxygen
Henry’s Law transfer rate. Oxygen is sparingly soluble in water.
PA
xA =
H
For such gas-liquid pairs, the solubility of a gas
solute in the liquid phase is described by Henry’s
xA: mole solute A / mole
solution Law. Henry’s Law states that, at equilibrium, the
PA: partial pressure of A concentration of a gas in the liquid phase (i.e., its
CA: mole A / L solubility) is proportional to the pressure of the gas.
H: Henry’s law constant
The concentration in each phase is described in
different ways. The most commonly-used variations
Table 1. Henry’s Law Constants for O2 are partial pressure for gas phase (which is the
(atm/ mole O2/mole H2O)
mole fraction of the gas solute multiplied by the
T, ˚C 5 20 25 30 35 37 total pressure) and the mole fraction for the liquid
10-4 x H 2.91 4.01 4.38 4.75 5.07 5.18 phase. For process calculations, one may find it
convenient to express liquid phase concentration
in mmole/L. In the medical profession, the
Example: at 37° C, in 1 atm air (PO2 = 0.21 atm),
O2 concentration in H2O is:
gas phase composition is often expressed in
C* = (0.21atm) / (5.18x104atm / moleO2 / (Eq. 1) mm Hg, with 760 mm Hg = 1 atm. The value of
moleH2O) x (55.5 mole H2O / L H2O) Henry’s law constant for a gas-liquid pair is, thus,
= 0.22 mmole O2 / L H2O dependent on how the concentration is expressed.
• In ambient air, (oxygen partial pressure = 0.21 Henry’s law applies well to an ideal solution. Despite
atm), C* ~ 0.22 mmole / L in H20 at 37 °C = 160 all of its components, cell culture medium is still close
mmHg = 5.8 mg / L (5.8 ppm) to an ideal solution so the saturation concentration
calculated using an aqueous solution applies to
cell culture media. However, cell culture media is
frequently used under a gas phase containing 1 - 10%
CO2. Also, in a bioreactor, the air pressure is usually
• Oxygen solubility is not affected by other greater than 1 atm. Thus, oxygen solubility needs to
dissolved species in medium. Its solubility is be corrected for the differences in pressure and CO2.
virtually identical to PBS and water.

The solubility of CO2 can similarly be calculated
using Henry’s law. However, in an aqueous
solution, CO2 associates with water molecules
and then dissociates into HCO3- and H+. The value
calculated using Henry’s law is the sum of CO2(aq)
and H2CO3 in pure water. Thus, the solubility of
CO2 will be affected by the pH of the solution.
• Solubility in medium should be adjusted by CO2 used

in gas phase (i.e., the fraction of O2 in gas phase is
usually not 0.21).
6CO2 (g) @ = H $ 6CO2 (aq) @

(Eq. 2)
PCO2
CCO2 =
H (Eq. 3)
Table 2. Henry’s Law Constants for CO2

(atm / mole CO2 / mole H2O)
T, °C 0 5 10 15 20 25 30 35 40
10-3 x H 0.728 0.876 1.04 1.22 1.42 1.64 1.80 2.09 2.33
Example: Using 10% CO2 in air, CO2 concentration at

37°C is
Solubility of CO2 affected by pH

CO2 (g) ? CO2 (aq) (Eq. 4)
CO2 (aq) + H2 O ? H2 CO3 (aq)

H2 CO3 ? HCO3- + H+

Oxygen Consumption and CO2 The objective in oxygen transfer is to supply oxygen
Production at a rate that is enough to maintain the dissolved
oxygen at a set level (or optimal level) for cell
growth and production. For most mammalian cells,
Optimal Oxygen Concentration a 30% saturation with ambient air is sufficient for
• Most cells grow well at a dissolved oxygen level of 30% optimal growth. Given that the saturation level
of saturation with air at 1 atm of ambient air is about 0.2 mmole/L, the
optimal value for cell growth is about 0.06 mmole/L.
The amount of oxygen cells consume per culture
Oxygen Demand by Cells
• On the average q02 ≈ 1.0 x 10-10 mmole / cell-hr volume per unit of time is called the oxygen uptake
rate (OUR). As you may recall from the kinetics
chapter, OUR is dependent on the specific oxygen
CO2 Production by Cells
consumption rate (qO2) and the cell concentration.
• Respiratory quotient of cells is about 1.0, i.e., 1 mole
CO2 is produced for every mole of O2 consumed, or qCO2 Most oxygen taken up by cells is used to oxidize
≈ 1.0 x 10-10 mmole / cell-hr carbon sources. The most important carbon
• A high CO2 concentration is inhibitory sources for mammalian cells in culture are glucose
• Inhibitory level usually starts at 15% to 20% CO2 and glutamine. The oxidation reaction of both
compounds yields CO2 at a stoichiometric ratio
Stripping of CO2 by Aeration of 1.0 and 0.9, respectively. This CO2 to O2 ratio
• CO2 is required for cell growth (e.g. in fatty acid is called the respiratory quotient (RQ). The CO2
synthesis), in addition to providing pH buffer in vivo. produced by cells will need to be removed from
• Cells can grow well even in sodium-bicarbonate-free the culture to avoid excessive accumulation, which
medium, especially in a stationary culture, because would cause a drop in pH or an inhibition of growth.
CO2 produced by cells accumulates in medium to
condition their growth. In cell culture operation, aeration accomplishes two
• When cell concentration is low in a sodium- goals: supply of oxygen and stripping carbon dioxide
bicarbonate-free medium, excessive aeration may strip out of culture medium. CO2, inhibitory at high
off CO2 to inhibit cell growth, unless some CO2 (~0.5 –
1%) is in the gas. concentrations, is also an essential component of the
medium. A certain level of gaseous CO2 is necessary
to maintain the pH in a neutral range. CO2 is also
used in a number of biosynthetic reactions, notably
fatty acid synthesis. If CO2 is completely stripped
from the medium, cell growth may be inhibited.

Method of Supplying Oxygen To supply oxygen, one can introduce it through
a gas phase in contact with culture medium. In a
small-scale reactor or flask, a gas head space is
Method of Supplying Oxygen often sufficient in supplying oxygen. In a large-scale
• Surface aeration reactor, air or oxygen-enriched air is introduced
• Silicon tubing/membrane as gas bubbles from the bottom. In some cases,
• Sparging direct bubbling of gas phase into culture medium
is to be avoided to minimize foaming or other
Balance of Oxygen in a Reactor
adverse effects. Under such conditions, the gas
dC phase may be introduced through a membrane
V
= OTR ⋅ V − OUR ⋅ V
dt with high oxygen permeability, such as silicone or
= K L a ⋅V ⋅ (C * − C2 ) − qO 2 ⋅ x ⋅ V Teflon membranes. In other cases, the gas phase
OTR: Oxygen Transfer Rate
is not introduced into the reactor but rather the
OUR: Oxygen Uptake Rate medium is pumped out and passed through an
V: Culture volume oxygenator. The oxygen-enriched medium is then
recirculated back to the reactor. A hollow fiber
bioreactor is typically operated in this fashion.
To control oxygen transfer, one can manipulate any
of the three factors affecting oxygen transfer. One
way is to increase the driving force by enriching the
KL is about 3 cm / hr – 6 cm / hr for surface aeration air with more oxygen. If surface aeration is used,
in cell culture vessel one can install a surface aerator to increase the mass
transfer coefficient on the liquid surface. If tubing or
Example: What cell concentration can be reached in
a 400 ml spinner flask (diameter 8 cm)? a membrane is used, one can use a larger membrane
Estimate: KL = 5 cm / hr or more tubing to increase the surface area.
Surface area A= (4 cm)2 • π = 50.2 cm2
a = 50.2 cm2 / 500 cm3 = 0.1 cm-1 (a is surface area per
In small-scale reactor operations, direct sparging
culture volume) (i.e., bubbling air) is more prone to problems. The
Assume D.O. is almost zero at maximum cell agitation rate in a cell culture reactor is comparatively
concentration (maximum driving force) low compared to a microbial reactor. A microbial
ΔC = 0.18 mmole / l
qO2 = 1.0 x 10-10 mmole / cell • hr
fermenter employs turbine impellers at a very high
qO2 • x = OTR = 5 cm / hr • 0.1 / cm • 0.18 mmole / l = agitation rate to make gas bubbles entrenched in
0.09 mmole / ℓ-hr the liquid. The bubbles take a torturous path to rise
x = 0.09 mmole / l-hr ÷ 1 x 10-10 mmole / cell • hr and eventually burst from the liquid surface. The
= 9 x 108 / l = 9 x 105 / ml
holding time of a bubble in the liquid is relatively
long. In comparison, in a cell culture bioreactor, the
bubbles rise in a relatively straight, vertical direction
and emerge from the liquid phase quickly. Therefore,
the efficiency of oxygen transfer for a given air flow
rate in a bioreactor is low. But, as the scale increases
in cell culture, the holding time also increases with
the length of the traveling path of the bubbles,
thus increasing the efficiency of oxygen transfer.

Gas Sparging in Cell Culture Bioreactor
In a cell culture reactor, the most practical means of
supplying oxygen is by blowing air into the reactor
through a sparger. If one were to supply oxygen
through the medium flow, the medium would have
to be recirculated many times per hour because
it can only carry a small amount of oxygen. Thus,
air sparging is commonly used in bioreactors.
Direct sparging in microcarrier culture poses a
number of challenges. Cells attached to microcarriers
render the microcarrier prone to stick to air bubbles.
Fig. 10.3: Supply of oxygen through liquid circulation As the bubbles rise to the liquid surface, carriers
may also rise as well. The associated fluid shear
caused by bubble rising may also cause cell damage.
v dc = F (Cin - C) - qo xv If a foam layer is allowed to accumulate, one may
dt 2
see microcarriers accumulate on top of the foam.

The use of antifoam may alleviate the problem,
or one may use a screen/sieve separator to allow
bubbles to be sparged in a microcarrier free zone.
Example:
Medium flow rate required to supply oxygen without To enhance the oxygen transfer rate in a bioreactor,
gas phase aeration:- one may make changes to manipulate each of
the three factors affecting oxygen transfer rate
qO $ x = 5 # 10- 10 mmole cell $ hr $ 2 # 10 9 cell l
2 (OTR): KL, a, and ΔC. Increasing the airflow rate
= 1 mmole l $ hr will increase the interfacial area, a. Increasing
assume Cin = 0.2 mmole l (saturated by air) oxygen concentration increases the driving force,
F V = 5hr- 1 ` need to recirculate medium ΔC. Increasing the agitation power input increases
5 times every hour KL and enhances bubble breakup to increase the
interfacial area, a. Smaller bubbles give a larger
Sparging interfacial area at a given air flow rate. In microbial
fermentation, a high agitation power input is used
• Short bubble residence time in small bioreactors
to break up air bubbles. The agitation power input
• Increasing the agitation rate increases oxygen transfer
through better surface aeration and longer bubble for mammalian cell culture is relatively low. The
residence time. range of airflow rate used in cell culture is also
• Direct sparging in microcarrier culture is possible but low, compared to microbial culture. A high air
more difficult sparge rate causes excessive foam formation, but an
• Can sparge in the "microcarrier free" zone (use a wire extensive use of antifoam agent causes cell damage.
cage) Therefore, enriching the oxygen content in the air
supply and using smaller bubbles are commonly
practiced, especially in small-scale cultures.

Bubble Size and Sparger Orifice Dispersions of gases in liquids are widely employed
in fermentation processes. The interfacial area
Effect of Orifice Diameter on Bubble Size can be very large: 3-mm spherical bubbles with a
1/ 3 gas holdup of 26% (i.e., a liter culture containing
 6d s 
d p =  0  26% volume of gas bubbles and 74% medium)
 ∆ρg  provide 52cm2/cm3 — a very large value.
d0= orifice diameter Bubbles behave very much like oil drops, but their
s = surface tension
buoyancy and rising velocity are greater. Mass
Δ ρ = difference in density between
transfer within the bubble is rapid, because the
liquid and gas
molecular diffusivity in gases is large. Normally
the resistance to mass transfer on the liquid
side of the interface is the dominant resistance.
In the case that a single bubble is slowly released
from a submerged orifice or small capillary, if the
gas flow is very slow, the bubble is released when
the buoyant force just overcomes the surface
tension, at which time the bubble diameter is
governed by the balance between buoyancy
and surface tension exerted on the bubble.
The force balance between buoyancy and surface
tension indicates a modest decrease of diameter,
dp, with an increase in the orifice diameter,
do. Inertial effects become dominant at larger
flow rates employed in industrial multi-orifice
spargers, thus rendering the initial bubble
size almost independent of the orifice size.
Using orifice size to control bubbles is thus not very
effective. In small-scale bioreactors, one may employ
spargers with a fine opening to generate very small
bubbles. The fine bubbles agglomerate as they rise
to the liquid surface and coalesce into large bubbles.
Thus, in large-scale reactors (on the order of
hundreds of liters), the effectiveness of fine bubbles
in an increasing interfacial area begins to diminish.
Bubble size may also affect bubble rising velocity
and the mass transfer coefficient. In principle,
larger bubbles have a higher terminal rising
velocity and a higher KL if bubbles are considered
to be rigid spheres. However, as bubble diameter
increases, the bubble tends to deform and become
flatter on top, thus decreasing the rise velocity.

Experimentally, it has been observed that both
rising velocity and KL only increase with bubble
diameter when the bubble size is small (<3
mm). Beyond that, they are relatively invariant.
Overall, manipulating bubble size to be very fine
is beneficial in small reactors as it increases the
gas transfer area for both oxygen and carbon
dioxide. This is important because the impeller
and agitation rate used is not effective for
breaking up gas bubbles. On a larger scale, though,
controlling bubble size becomes difficult because
of the high gas flow rate and bubble coalescence.

Experimental Measurement of KLa and Mass transfer coefficient can be determined
OUR experimentally. Overall, KL is relatively insensitive
to bubble size, except for very small bubbles and
dC = KL a (C* - C) under simple flow patterns. The interfacial area, a,
dt is difficult to estimate or to measure in bioreactors.
As a result, most experimental measurements of
mass transfer characteristics lump KL and a together
Initial Condition and report the value as “KLa”. KLa is sometimes
t=0, C=CO
called the volumetric mass transfer coefficient.
After Integration To measure KLa, one can aerate a reactor that initially
ln (C* - C) = KL a $ t has a very low oxygen concentration. The dissolved
Plot oxygen concentration will steadily increase until
ln(C*— C) vs. t reaching saturation. A semilog plot of C*-C vs. time
will give the slope as KLa. One can also perform the
experiment by stripping oxygen from medium that
was, initially, at a higher dissolved oxygen level. In
this case, the slope in the semilog plot will be -KLa.
In most operations, the aeration rate will change
over time. One can develop a correlation between
KLa and airflow rate for use during cultivation.
Fig. 10.4: Measurement of KLa by degassing, or stripping of

oxygen from the liquid phase

OUR or the specific oxygen consumption rate
can be measured by filling up a container with
Measure OUR in laboratory a cell suspension without leaving room for a
gas phase. Since there is no oxygen supply, the
change in dissolved oxygen level will be only
from cell consumption. The slope of the linearly
decreasing dissolved oxygen level will yield the
oxygen uptake rate. Then, the specific oxygen
consumption rate can be obtained by dividing OUR
by the cell concentration in the cell suspension.
Figure 10.5: Measurement of oxygen uptake rate
Effect of Hydrostatic Pressure In very large bioreactors, circulating cells will

occasionally reach a region where the hydrostatic
Effect of Hydrostatic Pressure pressure is high. This is particularly a concern
• 10 m liquid height gives a 1 atm hydrostatic pressure at for air-lift bioreactors, in which the aspect
the bottom of tank ratio (height over width) is much larger than
• Up to almost 9 atm has no adverse effect on hybridoma stirred-tank reactors. Experimentally, it has
cell growth and specific glucose consumption. been shown that cells grow normally at up to
nine atm and exhibit little discernible metabolic
differences, compared to normal conditions.

Damage to Cells by Gas Sparging
In an aerated mixing vessel, forces exerted on
cells may cause cellular deformation in several
ways. Forces normal to the cell surface may
squeeze the cell, cells may impinge on moving
mechanical objects, and/or cells may be subjected
to a fluid shear field, such that one side of the cell
is subjected to a fast flower than the other side.
Damage to cells by gas sparging
Despite the stress, cells have a substantial
• Cells and cell-laden microcarriers adhere to gas
capability to change their shape. The shape change
bubbles
involves reorganizing the cellular cytoskeleton
• Energy dissipation in gas sparging
and redistributing the lipid bilayer membrane
• It is generally thought that bubble breakup at (both the cytoplasmic and organelles). If the rate
liquid surface dissipates most energy and is most
of deformation is fast and the extent is extensive,
damaging to cells
the cytoskeletal network and the membrane
• Protective agents are added to medium in sparged
integrity will be compromised; therefore leading
(see media section)
to stressed conditions, or even cell death.
Vigorous sparging of air bubbles through
the culture can also cause cell damage. The
mechanism likely to cause most severe damage
is the fluid shear field caused by bubbles.
Several possible events may dissipate the
high intensity of mechanical energy when
bubbles traverse through cell suspension:
1. Release of the bubble from the orifice of the
sparger. As the bubble escapes from orifice, a
volume suddenly becomes void, causing the
cell suspension to rush in to fill the void space.
A velocity gradient, and thus a shear field, will
inevitably occur under such flow conditions.
2. A rising bubble will cause velocity gradient
around itself and thereby change the liquid
velocity in proximity to the surface of the
bubble. Conversely, far from the bubble, the
liquid velocity is the same as bulk velocity.
Fig. 10.6: Deformation of a cell by compressing force 3. Similarly, bubbles coalesce or break-
up, also causing liquid velocity gradient.
4. Bubbles bursting from the liquid surface cause the
volume originally occupied by gas to be rapidly
replaced by liquid, thereby causing a shear field.

It is generally recognized that the last event
generates the greatest shear stress. As the bubbles
burst off the liquid surface, the high liquid velocity
at the interface with the air decreases to the bulk
liquid velocity over a very small distance (of about a
couple of hundred micron). The momentum of the
Cell
liquid movement caused by the bursting bubbles
causes the liquid to rise above the liquid level
fast surface. As a result, the liquid moving downward
moving to fill the empty space left by the bubble reverses
object
its direction to rise upward. In the upward moving
Fig. 10.7:
Liquid Liquid velocityondifference
Velocity difference two sides of on
a celltwo sides
causes shearof a cell causes
stress
sheer stress
liquid cone, the velocity is highest in the center and
lowest on exterior. Also in the process of bubble
bursting, there is a rapid change of the gradient
direction when the liquid overshoots the surface
and then drops down after reaching a maximum
height. The damaging effect on cells entrapped
in the bubble-bursting zone can be tremendous.
Fig. 10.8: Possible ways of damaging cells by sparging in a

bioreactor

As visualized through high-speed video
photography, a bubble is often surrounded by
a layer of cells, possibly due to hydrophobic
interactions between the cell and bubble surfaces.
Once bubbles begin to rise, the liquid stream forces
cells to the tail, or the wake, of the moving bubble.
As the bubble bursts from the liquid surface, cells
can be severely damaged, if not outright killed.
Block copolymer pluronic F68 reduces the number
of cells adhering to the rising bubble through its
surface tension modulation effect. Pluronic F68 thus
“protects” cells from the damaging effect of sparging.
Fig. 10.9: Conceptual depiction of fluid movement surrounding

the point at which a bubble emerges from the liquid surface.
Fig. 10.10: Accumulation of cells to a bubble surface due to

hydrophobic interactions and accumulation to the tail end while the
bubble rises.

Mass Transfer Resistance in Cell Immobilization Reactor
Cells are sometimes cultivated inside particles
or in a stagnant liquid phase. Notable examples
are macroporous microcarrier and hollow fiber
systems. In these systems, oxygen not only needs
to be supplied continuously to the culture medium,
but it also needs to diffuse to the position where
oxygen is supplied to the interior, where cells reside.
As oxygen diffuses, it is also consumed by cells.
An oxygen concentration gradient is, thus, created
between the position of oxygen delivery and the
deepest point of cells where oxygen must reach.
The rate of concentration decrease is dependent
on how fast the cells consume oxygen and the
geometry of the particle. Farther into the interior,
oxygen may become depleted or become too low
to support a high rate of growth or productivity.
Figure 10.11: Concentration gradient of oxygen in a cell To avoid such oxygen limitations, one might resort
aggregate or an immobilized cell matrix particle
to using smaller particles or to maintain a higher
oxygen concentration in the culture medium. The
extent of oxygen transfer limitation can be estimated
by comparing the mass diffusion and consumption
rates. The theoretical calculation is presented
as plots of two variables, often referred to as
effectiveness factor and Thiele’s modulus, which are
discussed in standard chemical reactor textbooks.
Oxygen Transfer in Plug-Flow Bioreactors

Some bioreactors used in cell cultivation employ
a cell compartment that lacks direct contact with
• Oxygen transfer limitation occurs in both axial and the gas phase. Hollow fiber bioreactors and some
radial directions recirculatory flat bed bioreactors, especially in
• Periodically reversing the direction of flow should tissue engineering applications, fall into this
help category. In such a system, oxygen is supplied
by recirculating the medium. Often, an external
oxygenator, such as a hollow fiber membrane device
or a mixing vessel, is used to replenish oxygen in the
medium before it is recirculated to the cell chamber.
In such cases, the oxygen concentration gradient
exists in two dimensions. As the medium flows
downstream in the cell chamber, oxygen is diffused

through the membrane into the cell compartment.
Oxygen concentration, thus, decreases along
the axial direction. In the radical direction,
oxygen diffuses toward the cell chamber; along
the path the oxygen concentration decreases.
In a hollow fiber bioreactor, oxygen in the
recirculating medium diffuses from the bulk liquid
to the wall of the fiber, then through the fiber
and into the cell chamber. Typically, the diffusion
resistance is highest across the wall. Outside, the
wall diffusion occurs simultaneously with oxygen
consumption by cells. At a high cell concentration,
Fig. 10.12: Oxygen concentration gradient in the axial direction
the drop in oxygen in the cell chamber is steep
of fluid flow in a plug flow reactor so, often, a very high concentration of oxygen is
maintained in the medium to ensure the center
of the cell chambers receive enough oxygen. To
improve oxygen transfer, one may increase the flow
rate of medium recirculation, introduce a gas phase
compartment (and have hollow fibers for air flow),
or reverse the flow direction periodically. In any case
the longitudinal distance that can be used before
oxygen transfer becomes limiting is restricted.
Fig. 10.13: Photograph of a hollow fiber bioreactor
Fig. 10.14: Oxygen gradient in the radial direction of a fiber in a

hollow fiber bioreactor

Concluding Remarks
The solubility of oxygen is very low in cell culture supplying oxygen is by bubbling air through the
medium. It must be supplied continuously to sustain bioreactor. Although air bubbles cause damage to
cellular demand from a gas phase. The transfer rate cells, the problem can be overcome. However, the
of oxygen through the liquid-gas interface is affected air supply rate per reactor volume tends to decrease
by mass transfer coefficient, interfacial area and as scale increases. Therefore oxygen transfer and
driving force, or the deviation from equilibrium. All carbon dioxide removal are among the key issues that
three factors can be manipulated to enhance oxygen should be addressed in scaling-up or scaling-down.
transfer. The most direct and effective method of This will be further discussed in the scale-up chapter.

Fedbatch Culture
and Dynamic Nutrient Feeding
With contributions from Weichang Zhou
Fedbatch Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

Types of Fedbatch Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
Intermittent Harvest and Feed. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
Extended Fedbatch Culture: Fortified Feed and Addition. . . . . . . . . . . . . . . . . . 235
Fedbatch With Metabolic State Manipulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
Designing Feed Medium for Fedbatch Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
Feed Medium Design for Consumed Nutrients: Stoichiometric Principle. . . . 238
Feed Medium Design for Unconsumed Components. . . . . . . . . . . . . . . . . . . . . 240
Control Strategies for Fedbatch Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Control Objective and Criteria: Productivity and Product Quality. . . . . . . . . . 241
Feeding Strategies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
Feeding by Direct Measurement of Nutrient Consumption. . . . . . . . . . . . . . . . 243
Proportional Feeding With Base Addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
Proportional Feeding With Turbidity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
Proportional Feeding With Oxygen Uptake Rate (OUR) . . . . . . . . . . . . . . . . . . 245
Delivery of Feed Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Online Estimation of Stoichiometric Feeding. . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
Fedbatch Culture
In the first decade of the 21st century, we witnessed
Batch Culture a rapid expansion of therapeutic proteins in
• Limited nutrient concentration in medium clinical applications. We also saw productivity
• Solubility of amino acids increase rapidly through intense process
• High osmolarity at high nutrient levels development. During this time, fedbatch culture
processes have emerged as the predominant
• Growth inhibition by high nutrient levels
mode for producing recombinant proteins.
• Feed nutrients throughout the cultivation extending
the growth and production period In a batch process, the constraint of osmolarity
• Higher cell and product concentration limits the amount of nutrients that can be added
initially. This low-nutrient level prevents the culture
from attaining high cell and product concentrations.
In fedbatch cultures, medium is added during
cultivation to prevent nutrient depletion, thus
prolonging the growth phase and ultimately
increasing cell and product concentrations.
FED-BATCH | 233
Types of Fedbatch Culture
Fedbatch processes are widely used in multi-
Fedbatch Culture purpose, multi-product facilities because of their
simplicity, scalability, and flexibility. A variety of
• Predominant mode of production rDNA proteins
fedbatch operations, ranging from very simple
• Intermittent harvest used in inoculum to highly complex and automated, are seen in
preparation, also in virus production
current production facilities. Compared to a
typical batch operation, the operation time of all
types of fedbatch cultures are extended, resulting
in higher total cell and product concentrations.
Intermittent Harvest and Feed The “intermittent harvest and feed” fedbatch
process does not deviate significantly from a batch
culture. The culture is initiated using a medium and
culture conditions, very similar to that used in batch
culture. Cells are then allowed to grow exponentially,
with no additional manipulation, until cells reach
the late growth phase. A portion of the cells and
product are then harvested and fresh medium is
added to replenish the culture volume. This process
is repeated several times. This simple strategy is
commonly used for the production of viruses which
cause persistent infection in cells, as it allows for an
Fig. 11.1: Time profile of a fedbatch culture with intermittent
harvest extended production period. It is also used in roller
bottle processes with adherent cells. In some cases
this type of fedbatch culture is also used to supply
inoculum for imitating a reactor production chain.
FED BATCH | 234

Extended Fedbatch Culture: Fortified In “fortified feed and addition” type of fedbatch
Feed and Addition culture, the period of rapid cell growth is prolonged
by adding concentrated feed media (usually a 10-
15 times more concentrated than basal medium,
often without salts or with 1x concentrations of
feeding concentrated
feed salts). The concentrated medium is added either
nutrients continuously (as shown) or intermittently to supply
additional nutrients, thereby further increasing
cell concentrations and lengthening the production
initial phase. To accommodate the extra addition of media,
final
a fedbatch culture is initiated at a volume less than
Fig. 11.2: Depiction of a fedbatch culture with incremental
the full capacity of the bioreactor. This initial volume,
feeding
typically enough to allow the impeller to be submerged,
is kept at a low level to allow for the addition of
Extended Fedbatch Culture
extra media throughout the expansion phase.
The addition of extra nutrients results in a higher
cell concentration, prolongs the production
period, and, ultimately, results in a substantially
higher product titer. In recent years, it has become
common practice to decrease the operating
temperature after a period of rapid growth. The
temperature shift reduces the growth rate and
Fig. 11.3: Time profile of a extended fedbatch culture with nutrient consumption rates, further prolonging the
increasing culture volume
production period and increasing the product titer.
Typical Fedbatch Culture for rDNA Protein Production

• 7 - 15 days
• 8x106-2x107 cells / mL
FED BATCH | 235

Fedbatch With Metabolic State In batch cultures and most fedbatch processes,
Manipulation lactate, ammonium, and other metabolites eventually
accumulate in the culture supernatant over time.
These metabolites, along with high osmolarity
and reactive oxygen species, are growth inhibitory
and contribute to the eventual loss of viability and
productivity of the culture. The effects of lactate and
ammonia on cultured cells are complex. Detectable
changes in growth, productivity, and metabolism
have all been documented. Additionally, metabolite
accumulation has been found to affect product quality.
By limiting the availability of glucose and glutamine
Figure 11.4: Growth profile of a fedbatch culture with
using controlled feeding strategies, cell metabolism
controlled glucose level to induce metabolic shift can be directed to a more efficient state, characterized
by the reduced production of lactate. Such a change
in cell metabolism is often referred to as metabolic
shift. By extending the methodology to limit the
consumption of both glucose and glutamine, both
lactate and ammonium accumulation can be reduced.
In the example of fedbatch culture shown, glucose
concentration was kept at a low level by continuous
feeding of a concentrated glucose solution. In this
scenario, lactate was still produced and, after a period,
lactate production ceased, thus indicating a metabolic
shift. The shift can also be seen from the decrease
in the stoichiometric ratios of lactate production
to glucose consumption during the cultivation.
Fig. 11.5: Stoichiometric ratio change during a fedbatch culture
with metabolic shift A metabolic shift alters the stoichiometric
ratio of lactate to glucose throughout the
entire culture period. Even after averaging
Table 1 Characteristic Stoichiometric Ratios of Key
Nutrients for Cell Growing in Different Metabolic States the initial culture period (which includes no
stoichiometric Without Metabolic Lactate metabolic shift), less lactate and ammonium
ratio (mmole/ Metabolic Shift consuming are produced for a given nutrient consumed.
mmole) Shift cells
Cells can also consume lactate under slow growth and
lactate/glucose 1.4 - 2.2 0.05 - 0.5 0.4 - 1.0
high lactate and low pH conditions. Recently, it has been
ammonia/ 0.5 - 1.3 0.1 - 0.3 -
glutamine shown that after the rapid-growth stage in fedbatch
alanine/ 0.2 - 1.3 0.01 - 1.3 - cultures, cells can switch to lactate consumption
glutamine from lactate production. The consumption of
oxygen/glucose 1.0 1.0 - 2.0 - lactate alleviates growth inhibition and gives rise
FED BATCH | 236

to a higher product concentration, compared to the
cultures that remain at a lactate production state.
Designing Feed Medium for Fedbatch Cultures

Medium Components The vast majority of fedbatch culture employs
• Consumed by cell (almost all organics, growth factors) fortified nutrient mixtures to prolong growth and
production phases. Critical to a successful fedbatch
• Concentration decreases unless being replenished
process is the design of feed media. A well-designed
• Not consumed
feed medium should enable cell growth and
• Or consumption rate too small to cause product formation, without depleting any medium
concentration change in medium
component or allowing the build-up of excessive
Fedbatch Culture nutrients or metabolites. Furthermore, adding feed
• Volume can be expaniding due to feeding media to the culture should not introduce factors that
adversely affect cell growth or product formation.
• Volume expansion may cause dilution
The chemical species in media fall into two
general categories: 1) those whose consumption
rates are significant and measurable and 2) those
whose concentrations greatly exceed the amount
consumed by cells during the cultivation period.
Chemical species that are consumed should be
replenished by the feeding medium to maintain
their concentration in an optimal range. Conversely,
nutrients that are hardly consumed may not be
added; however, because the culture volume
increases substantially in a fedbatch culture,
even a nutrient that is not consumed by cells
may be diluted due to culture volume expansion.
Consequently, feeding of nutrients not consumed by
cells is necessary to sustain growth and production.
FED BATCH | 237

Feed Medium Design for Consumed During the growth phase, the goals of feeding
Nutrients: Stoichiometric Principle are largely to replenish the nutrients which have
been consumed and to ensure that nutrient
levels stay within an optimal range for optimal
• Glucose, amino acids can be added proportional to growth or for optimal product formation.
their stoichiometric ratio during fedbatch culture
Under balanced growth conditions, the specific
• Determine the stoichiometric ratio of medium consumption rates of various nutrients are relatively
components
constant and, correspondingly, the consumption
• Choose one reference component (e.g. glucose, of nutrients or production of metabolites relative
lactate, glutamine, oxygen), feed all other
to one another are proportional. These rate
nutrients by stoichiometric ratio
proportionalities are called stoichiometric ratios.
• Stoichiometric ratio may change over time of
A well-formulated feed media is designed to add
cultivation
nutrients at appropriate stoichiometric ratios
to match their consumption rates. This strategy
should allow all nutrients to be replenished at
each particular cell line’s rate of consumption.
Stoichiometric ratios can be calculated using
historical culture data obtained from the cell line
of interest, as discussed in the stoichiometry and
kinetics chapter. Ideally, the data should be obtained
from cultures under relevant cultivation conditions.
Typically, one medium component is chosen as a
reference nutrient. The consumption rates of other
nutrients are related to the reference nutrient,
using stoichiometric ratios. Common choices for
reference nutrients are glucose, glutamine, oxygen,
and lactate, as they are consumed or produced
in larger quantities among all nutrients and
metabolites and are relatively easy to measure.
The idea of stoichiometric feeding is that if all
nutrients are consumed in relative proportions,
once the amount of the reference nutrient
consumed is known, one can calculate the amount
consumed of all the other nutrients using the
known stoichiometric ratios. The stoichiometric
ratio can thus be used as a basis for designing feed
media. Using this strategy, one would determine
the consumption of the reference nutrient and
then feed the culture with the feed medium to
appropriately replenish lost nutrients, according
to their calculated stoichiometric consumption.
Stoichiometric ratios are not necessarily constant.
FED BATCH | 238

Cells may not necessarily consume all nutrients
in the same proportion in different culture
conditions or stages. The feed media composition
may need to be adjusted, according to known
changes of stoichiometric ratios under different
culture conditions or in different growth stages.
Feed medium design, using this method, is best
approached as an iterative process, where a design
is tested, analyzed, and refined until an optimal
medium emerges. Most basal media are designed
with an excess supply of amino acids, especially
glutamine. In addition to being used for protein
synthesis, glutamine supplies precursors for
nucleotide synthesis and replenishes intermediates
for the tricarboxylic acid cycle. The consumption of
glutamine and other amino acids can often be reduced
without affecting growth and product formation.
Fig. 10.6: Use of cumulative nutrient consumption data to The reduced consumption of these nutrients also
calculate stoichiometric ratio decreases ammonium production. In the feed
medium optimization process, the concentration
Table 2. Amino Acid Specific Consumption Rate and
Composition in Cell Biomass and IgG of some amino acids may also be reduced if excess
Normalized Specific Rate (relative Normalized ammonium accumulation becomes growth inhibitory.
to lysine at low ΔL/ΔG state) Composition
The first round of iteration usually employs data
High ΔL/ Intermediate Low ΔL/ Cell IgG
ΔG state State ΔG state from batch culture growth, in which amino acids
ALA -11.50 -0.93 0.08 1.32 0.76 are supplied in excess. The stoichiometric ratios
ARG 1.25 1.25 1.25 0.69 0.35 determined from batch culture are then used to
ASN 0.23 0.25 0.03 0.00 0.50 prepare the initial version of feed medium. Often,
ASP 0.05 0.23 0.10 1.47 0.57 the concentration of nutrients in the feed medium
CYS 0.00 0.00 0.00 0.04 0.35 is about 10- to 15-times that in basal medium,
GLN 16.25 9.00 4.50 0.00 0.72 in order to reduce the required feed volume.
GLU 0.50 0.25 0.13 1.84 0.74
During the optimization runs, the concentration of
GLY 0.35 1.28 0.10 1.33 1.00
the reference nutrient (usually glucose, glutamine,
HIS -0.75 0.75 0.50 0.32 0.24
or oxygen), which can be measured on-line or off-
ILE -0.75 1.25 1.00 0.84 0.35
line rapidly, is measured periodically to determine
LEU 0.75 1.75 1.25 1.31 0.98
the consumption rate. The amount of feed medium
LYS 1.50 2.25 1.00 1.00 1.00
needed to replenish the reference nutrient to the
MET 0.78 0.58 0.10 0.33 0.20
original level or a set point is then calculated and
PHE -0.40 0.38 0.45 0.54 0.50
added. If the stoichiometric ratios are estimated
PRO 0.00 0.00 0.00 0.80 1.02
SER 1.08 0.90 0.45 0.90 1.85
correctly, the level of nutrients should remain within
THR 2.00 1.50 0.75 0.79 1.11
the set range. If the stoichiometric ratio for a nutrient
TYR -0.50 0.25 0.50 0.40 0.59 is not accurate, its concentration will then increase
VAL -1.25 0.75 1.00 0.95 1.30 or decrease. After the optimization run, a new set of
ΔL/ΔG: stoichiometric ratio of lactate to glucose
stoichiometric ratios is then determined and is used
FED BATCH | 239

for the design of the next feed medium. Using these
methods, a very effective medium is typically achieved
with fewer than three rounds of optimization.
Feed Medium Design for Unconsumed Some medium components are consumed in very
Components small quantities and their concentrations remain
virtually unchanged in culture. This includes many
inorganic ions, such as sodium, calcium, and sulfate.
From a stoichiometric point of view, the feed
medium does not need to include these nutrients.
As feed is added, however, the volume of the culture
increases and causes the medium components to be
diluted. Thus, these components are included at low
levels in feed medium (typically 1X concentration
or less). In some cases, inorganic salts, such as NaCl,
are completely eliminated from the feed medium
• For Mg+2 and PO4-3, intracellular concentration >> to reduce any changes in culture osmolarity.
medium concentration
Some ions, such as magnesium (complexed with ATP),
• At high cell concentration, cell consumption can phosphate (as free phosphate or in nucleotides),
cause depletion in medium
and potassium, are present at a much higher
• Need to supplement about 1x in feed medium concentrations inside the cell than in the medium.
At high cell concentrations, the amount of these
ions taken up by the cells may become significant.
Therefore, it is often necessary to compensate their
consumption by supplying them in the feed medium.
In addition to basal medium components, protein
hydrolysates, serum, insulin, transferrin, vitamins,
and lipid additives are also used in culture. These
additives supply minute nutrients, which are
consumed or become inactive with time. However,
the concentrations of these medium components
are not usually measured and their consumption
rate is mostly unknown. In the absence of a reliable
measurement consumption rate for those additives,
one has to rely on an order-of-magnitude estimate
of the upper and lower limits of their consumption
rate. The feeding rate of those additives is then
chosen to maintain their concentrations above
a minimum threshold, while below a maximum
tolerable limit which is experimentally determined.
FED BATCH | 240

Control Strategies for Fedbatch Cultures
How the feed medium is delivered to a culture may
affect the performance of a fedbatch process. On-
line measurements may be employed to determine
the consumption of the reference nutrient in real
Developing Fedbatch Strategy time, then used to drive a fully-automated system
Define objective of feeding. Possibilities include: to feed nutrients continuously. At the other ex-
treme, one can use off-line monitoring of nutrient
• Sustain nutrient level
level and manually feed the nutrient periodically.
• Manipulate growth rate in a range
Three elements should be considered in devel-
Define what criteria to manipulate
oping a feeding scheme: 1) the control objective
• e.g., what level of nutrient to control?
and the control criterion (level of different nu-
Mode of feeding trients to be maintained), 2) the mode of feed-
• Continuous vs. intermittent ing (continuous or intermittent feeding), and
How to deliver the feed medium 3) a control strategy for determining the tim-
ing and amount of feed medium to be delivered.
• By direct measurement of nutrient or infer from
indirect measurement The simplest strategy is to allow nutrient levels
to vary, within a wide range, by adding large
quantities of feed media at widely-separated time
intervals (e.g., once or twice per day) based on
off-line measurements or historical data. Such
a periodic feeding scheme is very simple and is
usually sufficient to avoid depletion or overfeeding
of nutrients. For more specialized cases, especially
those aiming to manipulate cell metabolism, a more
frequent measurement of parameters, along with
well-controlled feeding schemes, are necessary.
Control Objective and Criteria: A common objective of nutrient feeding is to increase

Productivity and Product Quality product titer by increasing cell concentration.
In recent years, the objective has been gradually
• Control objective extends beyond acheiving high extended to assuring product quality and consistency.
productivity to high product quality and consistency Culture conditions may affect glycoform or protein
• Require an understanding of relationship between folding. The depletion of a particular amino acid
manipulated variables and control objective may cause an amino acid to be mis-incorporated
into the protein product. Culture conditions
may also affect the frequency of deamination or
glycation of the protein product after it is secreted
into the medium. Furthermore, extensive cell death
may cause product degradation or desialylation.
The control objective of feeding may be to reduce
glycation by setting a control criterion to keep
FED BATCH | 241

Possible Control Objective and Control Criteria glucose concentrations below a certain level, or
to minimize amino acid mis-incorporation by
Control Objective
maintaining amino acids above a certain level.
• Reducing desialylation by reducing cell death and
sialydase release Another method of control is to minimize protein
quality deterioration by maintaining a long,
• Reducing glycation
slow growth phase and avoiding a death phase.
Control Criteria After defining the control objective, the next step is to
identify the process variables that cause the process
• Control osmolarity below set point
to deviate from the control objective. Furthermore,
• Control glucose level below set point
a relationship between the key process variable
and the control objective, such as inducing lactate
consumption, should be available so that the process
variable can be controlled within an optimal range.
For mammalian cell culture, although the control
objectives can be defined, the factors that affect
the path to the objective are not easily identified.
Lacking knowledge of the relationships between
manipulated variables and the control objective
makes the optimization of control criteria difficult.
In the past few years, it has become empirically evident
that maintaining a moderately high osmolarity in a
late stage of culture can increase the productivity of
recombinant proteins. A high osmolality increases
productivity, perhaps, by enhancing the expression
of stress responsive proteins, which facilitate
protein folding, although the exact mechanisms
are not known. By keeping the osmolality only at a
moderately high level to avoid causing a rapid decline
of cell viability the culture can be sustained over a
longer period of high specific productivity, leading
to high final product concentration. This practice
is commonly used in industrial manufacturing.
An increasingly common practice is to employ
atypical culture conditions, which may not be
optimal for long-term maintenance or expansion
of cells in the final production reactor. These
conditions may include very high glucose
concentrations (15 g / L or 83 mM), low pH (6.9 vs.
7.2 for optimal growth), and/or low temperature
(33/34 oC vs. 37 oC). These conditions have been
found to affect the duration of culture and the
overall cell metabolism, resulting in an increased
product titer. A high concentration of glucose
FED BATCH | 242

also increases osmolality. This is compensated by
reducing the salt concentrations to keep osmolality
around 300 mOsm during the cell growth stage.
Feeding Strategies
Feeding by Direct Measurement of Direct measurement of nutrient concentrations
Nutrient Consumption is the most straightforward way to determine the
amount, rate, and timing of feed to be added. Based
on current concentrations of nutrients, one can
determine how much medium should be added to
sustain the nutrient level for a given period of time.
In-Time Measurement for Control The concentrations of some nutrients can
• On-line or off-line be determined on-line, although off-line
Example nutrient measurement is more common.
• On-line sampling coupled Glucose and glutamine are the two nutrients
• HPLC for amino acids most commonly measured and used as
• Enzyme assays for glucose, glutamine, controls because their concentrations can be
lactate determined rather rapidly in laboratories.
Direct measurement of these compounds on-line
Different Way of Coupling Feeding to On-Line can be implemented using an auto-sampling device,
Measurement
in series with commercially-available immobilized
enzyme/membrane-based measurement devices.
• Single stream with fixed stoichiometric
ratio HPLC can also be implemented as an on-line
• Multiple streams ratios among different approach for the measurement of glucose and amino
nutrients can be adjusted acids. This technique requires a series of processing
steps for sample preparation before injection into
the HPLC. A significant lag time between sampling
and delivering control action is unavoidable.
However, since the doubling time of mammalian
cells in culture in generally exceeding fifteen hours,
even an hour lag time in HPLC measurement of
amino acids is acceptable. Industrially HPLC on-line
analysis has been implemented at pilot plant scales.
FED BATCH | 243

Proportional Feeding With Base A widely-used control strategy in bioprocessing is to
Addition proportionally add medium according to the amount
of base added to the culture for controlling pH. This
provides the option of continuous on-line feeding
Rationale without an on-line nutrient measurement device.
• In lactate production state L / G is relatively constant In most cultures, a large fraction of the glucose
• Base added to neutralize pH at 1 mole base / mole consumed is converted to lactic acid. To
lactate maintain culture pH, one mole of base is added
• Use stoichiometric ratios to lactate to feed to neutralize each mole of lactic acid produced.
• Easy to implement, but its sensitivity is limited by
If the stoichiometric ratios between lactic acid
buffer in medium
production, glucose consumption, and other
nutrients are relatively constant, the rate of
lactic acid production can be used to estimate the
consumption rates of glucose and other nutrients.
Barring the effects of pH buffers (such as sodium
bicarbonate or HEPES), the base addition rate is
indicative of lactic acid production. This method
is simple and easy to implement. However, it is
highly sensitive to CO2 level in the gas phase and
sodium bicarbonate concentration in the medium.
Proportional feeding according to base addition
is not well-suited for processes requiring a highly
accurate control of feed rate. Medium buffer
capacity can cause delays in base addition and
decrease the overall sensitivity of the method.
Proportional Feeding With Turbidity The use of an online laser turbidity probe can provide
accurate estimations of cell concentration in culture.
Simple proportional feeding with turbidity works
• Turbidity is a good indicator of total cell density well during the exponential growth phase, when
(but not viability)
viability is high and the growth rate is relatively
• Feed nutrients proportional to cell density constant. However, near the end of the exponential
• Reliable during growth stage growth phase, when viability drops, an assessment
• Capacitance probe can measure viability of viability or metabolic activity must be used to
adjust feed rates to avoid over-feeding. An alternative
measurement to turbidity is measurement of
capacitance, which reflects viable cell volume, i.e.,
viable cell concentrations and cell sizes. This may
be used for better control of nutrient feeding rate.
FED BATCH | 244

Proportional Feeding With Oxygen Among different measurements of nutrient
Uptake Rate (OUR) consumption, the oxygen uptake rate (OUR) is most
accurate for assessing cellular metabolic activities.
OUR measurements, unlike pH, are not masked by
buffers in the medium. A small amount of oxygen
consumed in culture, even in the range of 10 µM,
• Most sensitive among all on-line measurement of can be accurately determined using a dissolved
metabolic activity
oxygen sensor. For other nutrients which are often
• Require developing a computer algorithm
present at millimolal or hundreds of micromollal
• Need to develop stoichiometric ratios to oxygen
levels, such sensitivity of measurement cannot be
easily achieved especially for on-line measurement.
With its high sensitivity, small changes of OUR
can be confidently detected on-line and in real
time, thereby providing an immediate indication
of changes in metabolic rate. On-line oxygen
consumption data can then be used to determine
nutrient demand, using established stoichiometric
ratios and control continuous feeding.
Delivery of Feed Medium

After determining the amount of feed media to be
Parameters in Feeding added, one needs to decide on the mode of medium
• Initial culture volume vs. feeding volume delivery (e.g., the frequency and amount of feed to be
• Feed concentration delivered at each feeding). The method of feed media
• High concentration desired, but stability/ delivery is constrained by equipment and also by the
precipatation concern
composition of the feed medium. The feed medium
• High salt content from solubilization
usually consists of a solution of concentrated
• Single feed vs. multiple feed solution amino acids and other organic nutrients, which,
• Need adjustable stoichiometric ratio when kept over a long time, tend to precipitate.
• Continuous vs. step-wise feeding
The main consideration in determining the proper
• Step-wise feeding
feeding frequency is the acceptable range of nutrient
• Frequency concentration. The concentration of nutrients will
• Equal volume each time or proportional to fluctuate between a high level at the time of feeding
cell density
and the low point immediately before the next
• Operational simplicity vs. controllability
feeding. More frequent feeding reduces the deviation
from the set point. On-line feeding, by coupling to
base addition, turbidity, or to OUR measurement,
is easily implemented by computer control and
is almost continuous. When feeding is coupled
to less frequent off-line measurements, medium
is typically added manually, a few times a day.
FED BATCH | 245

Processes employing continuous feeding to
control nutrient levels in a small range will
require substantially more effort than intermittent
feeding strategies. While allowing more control
over environmental conditions, the superiority of
continuous feeding in terms of extending culture
lifespan and increasing productivity has not been
clearly documented, except in the case where a
metabolic shift is the desired outcome. In fact, with
simple off-line monitoring, robust, intermittent
feeding strategies is standard industrial practice.
Online Estimation of Stoichiometric A challenging issue of stoichiometric feeding is the

Feeding adjustment of the feeding rate, or feed composition,
in response to metabolic changes throughout a
Detecting Stoichiometric Ratio Change During culture [12]. This is particularly critical when
the Culture fedbatch cultures are used to elicit a metabolic shift.
• Monitor glucose, lactate, glutamine, OUR Changes in metabolism over the course of a
• Monitor ∆L, ∆G, ∆G in, ∆OUR and check their ratios culture are commonly seen, as evidenced by the
over time nonlinear relationships between specific nutrient
consumption rates. Many such changes bear
little consequence on cell growth or productivity,
but in some cases the effect is profound.
For simplicity, major changes in feed composition
or feed rate are only made when profound changes
in metabolism are observed. For example, in a
late stage of growth, cells may cease to produce
lactate and consume glucose at a slower rate.
Excessive glucose feeding may, then, reverse the
metabolism in the favor of lactate production.
Major changes in the metabolic rates of glucose,
lactate, and glutamine can be detected by
monitoring their stoichiometric ratios throughout
the course of a culture. On-line measurement of
nutrient levels would allow for timely detection
of changes in stoichiometric ratios; however,
its widespread implementation in industrial
processes will require further development
of reliable, automated sampling methods.
Without on-line, direct measurements of glucose
and lactate to detect changes in stoichiometric
FED BATCH | 246

ratios, one may resort to indirect estimation. For
example, changes in OUR and the base addition
rate may be an indication of a changing metabolic
state, as the ratio of OUR to lactate consumption
changes significantly when cells switch from a
lactate production state to a consumption state.
Concluding Remarks
Fedbatch culture is the prevailing mode of cell productivity, high cell concentration, and high
culture in the final production of manufacturing product accumulation. The design of feed medium
recombinant proteins. It is also commonly used and the selection of the feeding control strategy are
for extending the period of cell expansion for critical to the successful implementation of fedbatch
pre-production culture. In the latter, the culture culture. By applying stoichiometric principles to
conditions and feeding strategy are designed feed medium design and by using a well-designed
for optimal cell growth, whereas in the former, feeding strategy, an optimal fedbatch culture
the conditions are often designed to elicit a high process can be implemented with relative ease.
FED BATCH | 247

FED BATCH | 248
Cell Retention and Perfusion
With contributions from
Sadettin Ozturk, Chun Zhang, and Weichang Zhou
Practice of Perfusion Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

Analysis of Perfusion Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Material Balance on Perfusion Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Effect of Recycling Factor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
Methods of Cell Retention. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
Sedimentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
Incline Settling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
Acoustic Resonance Enhanced Settling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Spin Filter Separation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
Microfiltration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
Alternating Tangential Filtration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
Practice of Perfusion Culture

Marketed cell culture products Many sectors of the chemical process industry
using perfusion bioreactors underwent major transformation during the
Product Company
second half of the 20th century. One of the most
significant changes is the switch from batch
• RecombinantTM, Antihemophilic
Factor (recombinant), (Factor VIII)
Baxter processes to continuous processes. By minimizing
• Kogenate-FS (Factor VIII) Bayer equipment turnover time and process start-up
• Aldurazyme time, a continuous process can be sustained at
BioMarin a high rate of productivity over a long time and
• Naglazyme
• ReoPro (IgG Fab Fragment) achieve a higher throughput than a batch process.
Centocor
• Remicade (IgG1
(J&J) Increasing adoption of the continuous process
• Simponi (IgG1)
• Xigris (Protein C) Eli Lilly method also sparked much research into
• Cerezyme bioprocessing. Many enzymatic biocatalyst
Genzyme
• Fabrazyme processes have long been operated continuously,
(Sanofi)
• Myozyme/Lumizyme similar to most waste treatment or biodegradation
Serono processes. Contrary to the practices of the
• Gonal-F (follicle stimulating hormone)
(EMD)
chemical process industry, however, continuous
• Vpriv (velaglucerase alfa)
Shire process only became more common in the later
• Replagal (agalsidase alfa)
Wyeth years of microbial and cell culture bioprocessing.
• ReFacto (Factor VIII)
(Pfizer) A vast majority of biochemical processes involving
microbial or animal cell cultivation are batch
CELL RETENTION AND PERFUSION | 249

processes for many reasons. First, unlike catalysts
and reactants do not change their behavior over
time in chemical reactors (although catalysts may
gradually lose their activity), microbes and cells
Advantages may mutate, evolve, and change the make-up of
Better Product Quality
their population and production capacity. Second,
• Better controlled culture environment (nutrients
& byproducts) the risks of microbial contamination and equipment
failure make long operations undesirable, especially
• Pseudo steady state operation (ease of control)
in manufacturing, for which process robustness is
• Shorter residence time
of paramount concern. This is especially true when
• Higher cell viabilities & lower concentration of producing high value pharmaceuticals. Finally,
impurities
the current product capture and purification
• Critical for unstable molecules
operations are all designed for batch mode. Even
if the production is operated in a continuous
More Economical mode, the process is not designed to realize
• Higher cell concentrations & higher all of the advantages of a continuous process.
productivities
A continuous culture is constrained by the maximal
• Smaller bioreactor size
flow rate at which it can operate. One cannot operate
• More flexible
at a flow rate that is faster than cells’ growth rate;
• Faster start up in process development otherwise cells are washed out and incapable of
replenishing the culture content. This is particularly
Disadvantages daunting for processes that must be performed at a
Longer Cycle Time flow rate higher than the cell growth rate. In some
• Longer process development & validation time cases, cells produce growth inhibitors that must be
• Higher contamination risk continuously removed by media replenishment. In
other cases, cells must be grown at a low growth rate
• Higher equipment failure risk
to achieve a high productivity, resulting in the media
• Potential regulatory/licensing issues
being removed at a faster rate than the growth rate.
To overcome this shortcoming, a cell recycling
system can be added to continuous culture. By
recovering cells from the effluent flow and returning
them to the reactor, one can operate a continuous
culture beyond its natural limitation of the dilution
rate (flow rate divided by the bioreactor volume).
With a higher cell concentration in the reactor, the
overall throughput of the reactor is then also higher.
When cell culture was first adopted as the production
vehicle for biopharmaceuticals, continuous
operation was explored as an industrial process.
Researchers realized the growth of mammalian cells
in batch culture is impeded by lactate and ammonium
accumulation. A continuous process, thus, alleviates
growth inhibition by removing metabolites through

a continuous flow of medium. However, the high cost
of medium, especially serum, and the low product
concentration made continuous culture impractical.
To enable continuous operation, cells are retained
in the reactor while the media flow flushes
out metabolites. This can be accomplished by
immobilizing cells on some solid particles to
prevent them from being flushed out by media
flow, or by separating cells out from the effluent
stream and recycling them back to the reactor.
Thus, the general idea is continuous culture with
cell retention. Theese processes are often called
perfusion, which is reminiscent of the procedure
of flowing fluid through an organ or tissue.
A number of biotherapeutic proteins are produced
by perfusion processes. Some recombinant
antibodies could easily be produced with a fedbatch
process instead. It should be noted that the selection
of process mode is often based on the availability
of in-house expertise and many other factors.
However, in the cases of labile products that may
be degraded or otherwise inactivated over time, a
perfusion culture alleviates the loss of productivity
that cannot be easily overcome in a batch culture.
For products that accumulate only at very low
concentrations, a perfusion process may also present
a competitive advantage over a batch or fedbatch
Analysis of Perfusion Culture

Material Balance on Perfusion Culture One can analyze a perfusion culture system by
performing material balances on the reactor system.
The flow rates (F) of fresh medium entering and
exiting the system are the same, to keep the volume
in the reactor constant. The fresh medium stream is
free of cells. We also assume that the reactor is well
mixed, so that the nutrient (substrate) concentration
in the reactor is the same that it is in the effluent
stream. A cell separator is used to process the reactor
effluent stream to return a concentrated cell stream
with a given cell concentration, cx, back to the reactor.
Note that the effluent stream from the reactor has a
Fig. 12.1: Schematic of a continuous culture with cell recycle
higher flow rate than the fresh feed, with a flow rate
of (1+α)F instead of F, to balance the recycle stream

For the balance equation on biomass for the bioreactor is at the αF flow rate. The symbols used are the same
dx
V = α Fcx − (1 + α ) Fx + μ xV as those in the stoichiometry and kinetics chapter.
dt
Balance on the cell recycle system gives: Material balance can be performed on both the
(1 + α ) Fx = αFcx + Fx2 reactor and the cell recovery/recycling device for
both cells and substrate. An important parameter
(1 + α − α c) Fx =Fx2 affecting the performance of the system is the
ratio of cell concentration in the purge stream, x2,
x2
=1 + α − α c to that in the reactor, x. This ratio is affected by
x
The balance on the substrate on the bioreactor the recycling factor, α, and the cell concentration
ds μx
factor, c by x2/x = 1+α-αc. Note the ratio should
V = Fs0 − V − F (1 + α ) s + α Fs be always smaller than 1, so that there is a
dt Y
concentration effect by the cell recovery device.
Defining F/V=D. Applying steady state conditions:
An important conclusion from the steady state
0 α Dcx − (1 + α ) Dx + μ x
= D = Dilution rate
analysis is that with cell recycling, the dilution rate,
μ= D(1 + α − α c) which is defined as the flow rate divided by the
practically c>1, so D>μ
μx reactor volume, is larger than the specific growth
0=Fs0 − V − Fs
Yx s rate; whereas without cell recycling (i.e., α=0), the
μx
0= D( s0 − s) −
Yx s dilution rate is always the same as the specific growth
rate. As seen in the figure about cell recycling, the cell
concentration in the reactor is higher than without
cell recycling and the reactor can be operated at a
dilution rate higher than the growth rate. Therefore,
for cells with a doubling time of one day, the maximum
flow rate that can be used without cell recycling
would be one volume a day. With a perfusion
culture, even a few volumes a day may be operated
depending on the cell retention factor (c). A higher
retention factor permits a higher cell concentration.
Depending on the retention device used, the
efficiency of retention may vary; for instance, it
may decrease rapidly at the high dilution rate,
Fig. 12.2: Comparison of cell concentration profile at causing the cell concentration to decrease rapidly.
different dilution rates with and without cell recycle
The analysis described above is for a bioreactor
Cell recycle (or retention) can accomplish: with an external cell recovery device. The same
principle applies to a system with an internal
• A higher cell density in reactors
device. The consequence of employing an internal
• A higher dilution rate than maximum specific device is the same: the dilution rate can be higher
growth rate than otherwise. It allows a fast nutrient flow rate
to be used to reduce metabolite concentrations,
while keeping cells in the reactor and allowing
cell concentrations to become maximal.

Effect of Recycling Factor In a perfusion system operated at steady state, the
amount of cells purged (Fx2) from the cell recovery
device equals the amount of cells produced through
reproduction (µxV). To keep a high viability and
steady state, a small amount of cells are purged,.
The system is operated at a dilution rate, D.
For a given dilution rate, one may employ a different
recovery device with a different cell retention
efficiency. Using the equation as an illustration,
given the same dilution rate and specific growth rate,
one may choose different combinations of α and c. If
one chooses a highly efficient cell concentrator with
a large c, then α that is used would be smaller, and
vice versa.
This concept illustrates that at a given dilution rate,
when using a highly efficient cell separator (such
as a centrifuge that creates a concentrated recycle
stream), a low recycling rate (αF) can be employed.
Fig. 12.3: A high cell concentration factor allows for a low
recycle ratio while achieving the same enhancing effect of cell
Conversely, when using an inefficient cell separator
cycle that gives a low degree of cell concentration, then a
large recycling rate needs to be used; in other words,
cells will need to be pumped out of the reactor and
pass through the cell separator to recycle more
frequently.
In large-scale operations, the cell stream could
potentially stay outside the reactor for a long time,
so oxygen starvation is a concern. Consequently, the
fluid stream out of the reactor is often chilled, first,
to reduce oxygen consumption. With a device that
gives a low c value, cells will need to be subjected
to the extra environmental perturbation of being
chilled and pumped more often. This factor should
be considered in selecting a cell recovery method.

Methods of Cell Retention
Selecting the method of cell separation very much
depends upon the way cells grow. The larger the
particles are, the easier they are separated. Many
processes employ microcarriers with particle
diameters ranging from 0.2 mm to 2 mm. These cell-
laden microcarriers can be easily separated from
media stream by sedimentation. In some cases,
cells are grown as large clumps, or aggregates, of 1
– 2 mm in size, also making sedimentation readily
applicable. Other considerations in selecting a cell
separator are the required throughput purge rate, the
concentration factor (c), and the scale of operation.
Because of the limit of the concentration factor
(c) of a cell separator, even though the purge
rate (F) may not be large, a large recycling factor
(α) may be necessary. Note that the capacity of
the separator required is not only based on the
dilution rate (D, or F/V), but also on the flow rate
out of the reactor ((1+α)F). If a device with a small
concentration factor, c, is used, the capacity of the
separator will have to be substantially larger than
what is needed to process the purge stream alone.
Sedimentation The simplest cell separator is perhaps a settling

cyclone. The fed stream from the bioreactor enters
Conical Settler into the settler in its midsection, where the stream
• Selective removal of dead cells splits into flows in two directions. The upward
• Low separation efficiency stream, drawn by a pump for a purge stream,
• Cell settling velocity ~ 2-10 cm / h encounters a large cross sectional area and, thus, the
• Good for larger cells vertical velocity is much smaller than the cell’s setting
velocity, so the cells move downward. The downward
• Long residence time outside of the bioreactor
stream, on the other hand, faces a decreasing cross
sectional area and, thus, increases its vertical
velocity as it carries cells downward. A transient
zone separates the two well-developed upward and
downward flow regions. In this transition zone, cells
are separated and carried downward into the reactor.
Such a settler is a convenient device for laboratory
operations. As the reactor scale increases, the flow
rate also needs to increase proportionally, but the
cross sectional area of the settler increases only
Fig. 12.4: A settling cone for cell retention

with 2/3 power of the settler volume. The efficiency
of cell separation decreases rapidly as the scale
increases, making it ill suited for larger operations.
Incline Settling In a simple settling tank, the direction of fluid flow

and cell settling are along the same axis (both
Operating parameters vertical). A sufficiently long transient zone is
• Cell size and concentration necessary to separate the cell and cell-free streams.
• Perfusion rate To enhance the separation efficiency, the
• Settling area settler is often inclined so that an angle exists
• Length of the plates between the fluid flow direction and the particle
Inclined Settling
settling direction. A particle is considered
“collected” once it settles on a settler’s surface,
• While a particle is moving upward with flow, it also
settles toward the bottom plate since the fluid velocity on the surface is zero.
• It is “collected” upon hitting the bottom In an inclined settler, the feed cell stream enters
• Eventually, the particle rich zone has a higher fluid at the bottom and moves upward. Inside the
density and begins to move downward settler, cells begin to “settle” down vertically
• The particle-rich stream is recycled to the bioreactor due to gravity. If a cell particle hits the surface
at the lower plate, it is “collected” and does
not get carried out by the effluent stream.
purge stream
Eventually, the cells settled on the bottom plate form
a layer of fluid with a higher density than the stream
above. This heavy stream then moves downward,
Particle carrying the cells along. At the steady state, there
Setting Particle are three streams in the system: the feed stream,
Recovery
the effluent stream (carrying unsettled cells),
m
ea
and the concentrated cell stream at the bottom.

str
ll
ce
ed
rat
In industrial design, multiple inclined plates

t
ce
n
co
are used in a single settler. In such designs, the

feed stream and the returning cell stream do not
feed from reactor recycle to reactor
cross each other by partitioning their flow path
Fig. 12.5: Cell separation in an inclined settler for cell
retention in different zones. In some cases, mechanical
vibration is applied to the plates to prevent settled
cells from sticking to the surface and being lysed.
The residence time in the settler has to be at least
as long as the particle settling time. With a high cell
concentration in the stream, oxygen starvation is a
major concern, as it may induce apoptosis and cell
lysis. Therefore, the stream passing through the settler
is often chilled to reduce the cell’s metabolic rate.

Centrifugation Centrifugation is a standard unit operation in many
downstream recovery processes. In the early days of
Centrifugation Technology
perfusion process development, it was among the first
• Excellent separation efficiency to be exploited for cell retention. As early as the mid
• High perfusion capacity 1980s, the Japanese company Teijin had employed
• Little clogging centrifuges for perfusion culture of hybridoma cells
used to produce antibodies for cancer imaging.
• Easy scale-up
• Vulnerable to mechanical failure in long term However, most centrifuges are not designed for
continuous operation long term and aseptic operations. Early use of the
centrifuge for perfusion was only intermittent and was
for batch harvest and cell biomass recycling, as well
Three different designs:
as for the replenishment of fresh medium. A number
• Westfalia Disc type of autoclavable or stem-sterilizable centrifuges
• Continuous cell recycle back to fermenter are now available. These and the disposable bag-
• Centritech Lab based centrifuge are all capable of processing
• Disposable separation unit up to hundreds of liters of medium a day, and are
suitable for continuous use in perfusion culture.
The disc-type centrifuge is analogous to a multiple
parallel plate settler, except that the parallel plates
are rotating and generate a centrifugal field for
cell settling. The disposable bag system employs
three tubes: a feed tube, an outflow tube for the
heavy (cell-rich) stream, and an outflow tube for
the light stream. The unique design of an inverted
question mark allows the three tubes to rotate,
along with the centrifuge, without being twisted.
Fig. 12.6: The cell loading and cell ejecting regions of a
disc centrifuge for cell recycle

concentrated
cell stream
cell
dilute stream
feed
cell movement
Fig. 12.7: A disposable bag based centrifuge (Centritech) for

cell recycle
Acoustic Resonance Enhanced Settling This device uses acoustic energy to enhance
cell agglomeration. As the cell stream from the
Mechanical/Acoustic Trapping reactor passes through the acoustic chamber,
• Enhanced sedimentation (Cell aggregation) cells are induced to agglomerate. This gives
rise to a faster settling velocity. With increased
• Slightly favorable for viable cell retention
settling velocity, sedimentation is easily
• Heat generation may create temperature gradient
accomplished without resorting to multiple plates.
• Low separation efficiency
As cell concentration or operating conditions (i.e.,
• Moderate capacity (200L/day per unit) flow rate and temperature) change, the energy
Operating parameters and residence needed for agglomeration may also
• Cell density, perfusion rate, power input

change. Sensors for detecting cell agglomeration
“light” single cell stream
moves upward will help stabilize the operation of the device.
“heavy” aggregate
stream moves
downward
reflector transducer
agglomeration
zone
feed
recycle stream cell aggregates
Fig. 12.9. Picture of an acoustic cell retention

Fig. 12.8: An acoustic cell agglomeration apparatus
device for cell retention
Spin Filter Separation The term “spin filter” is used to refer to two different
designs that may have rather different mechanisms
of operation. Both employ high porosity filter with
relatively large openings (~20 - 100 µm) installed
on the wall of a rotating cage. Both are submerged in
culture fluid. A pump draws medium from inside the
cage as the purge stream. The culture fluid passes
through the filter to enter the cage, then is withdrawn
by the pump to become the effluent stream exiting
the reactor. The flow into the cage has a lower
cell concentration than the bulk culture fluid, thus
achieving the overall retention of cells in the reactor.
Fig. 12.10: A spinning filter (or rotating cage) for
cell retention
Rotational Cage A rotating cage rotates along the center shaft of the
impeller agitator at a low speed. The centrifugal
Internal and External
field is typically insufficient to push away cells along
• Remove dead cells/debris
the outside wall of the cage. Yet, a boundary layer
Operating Parameters Affecting Performance of liquid around the cage probably exists, in which
• Screen pore size (1-120 μm)
• Perfusion rate the cell concentration is lower than in the bulk. As
• Rotation speed a result, the fluid drawn across the filter is lower
• Screen surface area than in the bulk. Furthermore, there is little filter
• Draft tube cake formation. The screen on the cage is, thus,
• Screen materials:
not exactly a filter. Nevertheless, the system has
• Stainless steel, DNA & RNA deposit,
• Teflon, Polyamide 66, polyethylene, better been employed in scales of up to hundreds of liters.

The rotating cage is notoriously difficult to scale up,
Centrifugal Cage
as its operating mechanism is not well understood.
dilute purge stream
Later modifications of spin filters increased its
rotation rate up to hundreds of rpm, allowing it
recycle stream to operate like a centrifugal filter. The centrifugal
force is sufficient to push the cells away from the
surface of the filter, thus drawing liquid through
with a lower cell concentration than in the bulk.
spinning
Using such a device, the operation is less prone
to variation, due to changes in fluid dynamics.
stationary
It gives the freedom of operating in different
regions in the reactor. In some variations, it is
installed outside of the reactor and used as an
external cell retention device. However, the device
differs from the traditional centrifugal filter used
in the recovery process in chemical industry
in an important way in that no filter cake is
formed. In fact, if cell cake is formed on the
Fig. 12.11: A Centrifugal filter for cell retention screen wall of the cage, cell death is likely to
occur in the cake and leads to process failure
Microfiltration Microfiltration uses membranes of different

External Loop for Cell/Harvest Separation configurations, including parallel plates and hollow
• High shear, tangential flow
fiber devices, and has a pore size of around 4 µm.
• At high transmembrane pressure, cell deformation
occurs Microfiltration was among the first techniques
• Fouling caused by high molecular weight DNA, protein, used for cell retention. Its widespread use has been
lipids, anti-foam occurs after days of operation impeded by membrane fouling, which is especially
• Difficult to scale-down severe when a high concentration of proteins or
• The degree of concentration in a single pass is
complex medium is used in the culture. With the
relatively small
use of low protein medium in the past decade,
the problem of protein fouling has lessened,
Cross Filtration Model but clogging by dead cells remains problematic.
feed
return to
reactor
cell-free permeate
cell
membrane
very high flux may
cause cell damage
Fig. 12.12: Microfiltration membrane for cell retention

Alternating Tangential Filtration Different configurations of microfiltration
devices all use tangential flow, meaning the
feed stream flows in a direction parallel to the
membrane, while the filtrate flows across the
membrane. The more recent use of a pulsatile flow
system, in which a diaphragm is used to rapidly
reverse the flow direction, has reduced fouling.
return to reactor from reactor
product harvest product harvest

stream stream
diaphragm in
closed position
diaphragm in
open position
air
air
Rapid pulsatile flow in reverse
directions minimizes fouling.
Fig. 12.13: An alternating tangential flow hollow fiber device for cell retention

Concluding Remarks
Perfusion culture operation was explored very early volumes of inoculum that are used to scale up. The
on in cell culture development for obvious reasons: carrying over of a large amount of spent medium
(1) the low throughput from batch operation can from seed culture is undesirable; evidence seems
be enhanced by switching to continuous operation, to suggest that a high metabolite concentration at
(2) a simple continuous culture would have too inoculation can negatively affect a cell’s metabolic
low of a cell concentration to make the process characteristics in the main culture. Thus, using
economical, and (3) a dilution rate faster than the the cell recovery devices for preparing inoculum,
cell growth rate is needed to purge the metabolites especially to increase the initial cell density,
accumulated in culture. Its widespread adoption could potentially enhance process performance.
was inhibited, however, by concerns about
Another area that may change in the near future is
regulatory requirements and the lack of a clear
the increased use of fortified medium in perfusion
definition of BATCH for product manufacturing.
culture, as we have seen in fedbatch cultures.
Researchers were also concerned about the lack of
Using fortified medium (instead of medium with
a scalable cell recovery device for long operations.
a standard composition) can reduce the flow rate
These hindering factors have largely been overcome.
and increase cell and product concentrations.
Although cell line stability for sustained production
of a product is still a concern, evidences have Given the intrinsic advantages of continuous
shown that, with proper control of cell age and cell operations and the advances in cell retention
stock, stability may not be an overriding concern. technology, we may begin to see more widespread
application of perfusion culture in the coming
As cell retention technologies become mature, one
years. It has considerable potential to increase
may also see this application go beyond perfusion
the capacity of high throughput processes, reduce
culture. The same devices can be used to concentrate
reactor sizes, and possibly minimize product quality
cells and remove metabolites, as well as to quickly
fluctuations through steady state operations.
replace the fluid phase for cell freezing operations.
Current inoculation operations are limited by the

Scaling Up and Scaling Down
for Cell Culture Bioreactors
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Mechanical Agitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Mechanism of Agitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Power Consumption and Mixing Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
Power Consumption of Agitated Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
Other Dimensionless Numbers for Stirred Tank Reactors . . . . . . . . . . . . . . . . . 268
Effect of Scale on Physical Behavior of Bioreactors . . . . . . . . . . . . . . . . . . . . . . . 269
Mixing Time. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Nutrient Starvation Time. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Mixing Time. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
Nutrient Enrichment Zone, Mixing Time vs. Starvation Time . . . . . . . . . . . . . 272
Mixing Time Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Scaling Up and Mechanical Forces on Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
Scaling Up and Oxygen Transfer and Carbon Dioxide Removal. . . . . . . . . . . . . . . . . . 275
Material Balance on Oxygen in Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
Aeration Rate and Superficial Gas Velocity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
Gas Flow Rate in Scaling Up: A Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
Effect of Scaling Up on CO2 Removal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Introduction
Translation of the process scale is one of the most
difficult issues in bioprocessing, and it is probably one
of the least systematically studied subjects in the field.
Few engineers are involved in designing large-scale
equipment using small-scale experimental data, but
many will be developing processes in laboratories
and at pilot plant scales for eventual implementation
in a production scale. Others may be involved
in troubleshooting investigations for production
plants using laboratory equipment. Therefore,
an understanding of the factors affected by scale-
translation is important in carrying out those studies.
SCALING UP AND SCALING DOWN ON CELL CULTURE BIOREACTORS | 263

Translation of Scale A reactor may be scaled up geometrically similarly
Objective or non-similarly. Geometrical similarity refers
• Prediction of process performance to maintaining the ratio of the main geometrical
• Specify operating conditions from one scale to lengths, such as height over diameter, as well
another as the relative size of the internal parts (e.g.,
Major Effect of Scale impeller, flow diverter, etc). In this chapter, our
discussion will focus on geometrically similar cases,
• Oxygen (Gas) transfer which are conceptually easier to grasp, although
this is not always the best approach in scaling.
• Heat transfer
In scaling up geometrically similarly, all length
• Shear force
dimensions of the rector are scaled proportionally.
• Compression force Consequently, the surface area will increase with
the length dimension to the second power, while
• CO2 removal the volume will increase to the third power of the
length. As a result of scaling up, the scale-related
surface area per unit volume of equipment will
decrease. In microbial fermentation, the decrease in
surface area to volume ratio causes the impediment
for removal of heat generated from metabolism
and mechanical agitation. In mammalian cell
processing, the metabolic heat generated is less
a concern. However, the process may still be
sensitive to other variables related to scale change.
As the scale of an equipment changes, the physical
and mechanical parameters may not be maintained
constant. Frequently, it will not be possible to keep
all key operating parameters constant between
different scales. This may lead to changes in
the chemical environment and, ultimately, cell
physiology and productivity. The objective of
scaling-up and scaling-down is, therefore, not to
strive to keep scale-related parameters at constant,
but to define the operating range of scale-sensitive
physical and mechanical parameters so that
the cellular physiological state and productivity
can be maintained within an acceptable range.
At times, this may require an adjustment of
Fig. 13.1: Schematic of a large
scale cell culture fermenter the chemical environment at different scales.
SCALING
SCALING UP
UP AND
AND SCALING
SCALING DOWN
DOWN ON
ON CELL
CELL CULTURE
CULTURE BIOREACTORS
BIOREACTORS | 264
Mechanical Agitation
Purpose of Agitation Mechanical agitation in a stirred bioreactor keeps
• Gas-liquid mass transfer cells in suspension, provides mixing to create
a more homogeneous chemical environment,
• The higher shear field near the impeller tip
produces small bubbles, thereby increasing and creates a flow pattern that increases the
gas-liquid interfacial area (provided that bubble retention time of gas bubbles in the culture fluid
coalescence is not correspondingly increased) to enhances oxygen transfer. In the cases that
• Suspension of solid (e.g. microcarriers, soymeal) or cells are grown as aggregates, agitation also helps
dispersion of liquid
reduce the formation of oversized particles.
• Liquid-liquid, liquid-solid mass transfer (e.g.
hydrocarbon culture, quick mixing of pH neutralizing In microbial fermentation, oxygen demand is rather
base) high (often exceeding 150 mmole / L-hr). To increase
• Minimization of pellets or aggregates the efficiency of the oxygen supply, extensive
• Pellets are cell aggregates or mycelial agitation is used to break up air bubbles. In many
microorganisms (streptomyces, molds) fermentations of mycelial mold or actinomycete,
• Mixing, especially for viscous fluid (e.g. xanthan gum) extensive agitation is used to overcome the
• Fermentation, broth of mold culture high viscosity of culture fluid and to reduce the
mycelial pellet size to enhance oxygen transfer.
In cell culture processes, the oxygen demand is
nearly two orders of magnitude lower than that
in microbial fermentation. Adequate oxygen
supply can usually be accomplished by much less
intensive agitation, which is also sufficient for
providing sufficient mixing and suspension of cells.
Mechanism of Agitation The flow patterns generated by different impellers

in a stirred tank are generally classified as one of two
types: axial flow or radial flow. An axial flow pattern
refers to primarily upward or downward flow due to
Table 1. Characteristics of Impellers the pumping action of the impeller. In a radial flow
Characteristics Propeller Disk Turbine pattern, the liquid moves primarily outward toward
the wall of the vessel. In cell culture processing,
Flow direction Axial Radial
impellers generating axial flow are used because
Gassing Less suitable Highly suitable
the shear fields generated by axial flow patterns are
Dispersing Less suitable Highly suitable lower than those generated with radial flow patterns.
Suspending Highly suitable Less suitable The Rushton disk turbines, as are often used with
Blending Highly suitable Suitable multiple installations in large reactors, are the
predominant type used in microbial fermentation.
In this design, the sparger is placed directly
underneath the disk turbine. Gas bubbles from the
sparger rise to hit the disk and are directed outward.

The blades, rotating at a fast speed, then break the
bubbles up. In the immediate surrounding region of
the blade, a very high-energy dissipation is predicted
by computer simulations, which would contribute
to bubble break up, but also create a high shear
zone which can potentially cause damage to cells.
The propeller or pitch blade type impellers, on the
other hand, focus on moving the liquid to create
the lift for mixing and suspending solids. The
impeller diameter to tank diameter ratio should
Fig. 13.2: Fluid flow patterns in a stirred tank reactor: axial flow be higher for microcarrier culture. While the
type vs. radial flow type
“propeller three blades” is used extensively in
microbial fermentation to enhance oxygen transfer,
the “axial flow three blades” provides less shear
stress and a more uniform velocity in the entire
discharged area than the “propeller three blades.”
Flat Blade (Rushton Turbin)
Axial Flow Blade
Propeller Three Blade

Fig. 13.3: Three types of impellers commonly
seen in stirred tank reactors
Power Consumption and Mixing Characteristics

Power Consumption of Agitated In designing equipment and analyzing physical
Bioreactors systems, of which the scale spans over a wide
N PO range, one often needs to develop a correlation
between different design variables. When data
collected from different scales and under different
conditions are plotted together, they inevitably
H
Fig. 13.4: Notation of an impeller give rise to different correlations. Each of those
V
based mixing reactor. H: liquid
height, V: liquid volume, N: impel-
correlations is good for that particular scale or a
Di ler rotation rate, Po: agitation range of scales. They are, thus, of limited value.
power, Di: impeller diameter, DT:
DT
tank diameter.

In order to find a correlation that is applicable to
different scales over a wide range of operating
variables, the experimental data are often plotted in
“dimensionless variables” to develop correlations
among the experimental data. These dimensionless
variables are a combination of experimental variables.
In such combinations, the units of dimensions from
individual variables cancel each other out with the
idea being that a correlation between dimensionless
variables is not sensitive to scale. The dimensionless
correlations obtained from experiments performed
on different scales should hold on any scale,
including those that have not been investigated.
Various relationships expressed in dimensionless
numbers are fundamental to fluid mechanics, mass
Fig. 13.5: Relationship between impeller Power number and
impeller Reynolds number for different types of impellers transfer, heat transfer, etc. The correlation between
friction factor (f) and Reynolds number (Re) is used
universally in the design of fluid flow in pipes. The
plot of friction factor and Re show two regions: at
low Re, f decreases linearly until Re = ~2000, where
there is a short break, then it continues at a relatively
constant value at a high Re region. The first region
is recognized as the laminar (or viscous) flow region
and the constant tail is the turbulent flow region.
2 A similar plot has been generated for power
Impeller Reynolds Number (ReI): NDi t
n consumption in a stirred tank reactor. The Reynolds
number is now denoted as ReI (Impeller Reynolds
Power Number (Np): 3Po 5 number) to indicate that it is based on the length
N Di t
(diameter) of the impeller. The dimensionless
Po = Impeller power (ungassed power, Po indicated number for power consumption by impeller is the
indicated ungassed) power number, Np. Correlations between Np and ReI
N = Impeller speed have been generated for various types of impellers.
Di = Impeller diameter They all exhibit a similar behavior to the f vs. Re
ρ = Fluid specific gravity plot for fluid flow in a pipe. In all these Np vs. ReI
μ = Fluid viscosity plots, a rapid decrease with increasing impeller
Reynolds number is followed by a constant value
• For all practical purposes a bioreactor is always
operated in the turbulent region
region; the decreasing region and the constant
region represent the two correlations for viscous
In turbulent regions: (Np) is constant, independent of flow and turbulent flow regimes respectively. In
(ReI) the turbulent regime, the power number is constant
Po = K (Eq. 1) over a wide range of impeller Renolds number, but
the value changes with different types of impellers.
3 5
tN Di

In bioreactor operations the flow is always in the
turbulent regime. Viscous flow is encountered in
a stirred tank only when a very viscous fluid, like
glycerol, is used. Therefore the power number for
impellers is constant for the same type of impeller. In
other words, the impeller power divided by N3Di5 (N
is agitation rate, Di is impeller diameter) is constant.
Other Dimensionless Numbers for Three other dimensionless numbers are frequently
Stirred Tank Reactors used to predict the performance of a stirred tank
when the scale changes. They deal with three
important aspects of bioreactor operations:
Three other dimensionless numbers, in addition to velocity, volumetric flow rate, and mixing time.
power number, are used:
The maximum velocity in a mixing tank occurs at the
• Dimensionless velocity, v / ND
tip of the impeller. This velocity can be represented
• Dimensionless pumping number, Qp / ND3
by the multiplicative product of the rotation speed
• Dimensionless blending (mixing time), ϴN of the impeller times its diameter, NDi. (Note: in
this chapter, we will ignore π in the discussion of
v: tip velocity
Qp: liquid pumping perimeter, area of circle, etc. The constant value π
θ : mixing time is cancelled out when comparing different scales.)
The amount of fluid that the impeller can move
(called “pumping”) is directly dependent on its
rotating velocity and the area of the impeller blades.
• At turbulent regions, all those numbers are Because we are considering scale translation under
relatively constant. geometrically similar conditions, we can use the
length of impeller, instead of the impeller blade, to
represent the length scale. The pumping, then, is
the projected area (D2) of the impeller multiplied
by the velocity of its rotation (ND), which gives ND3.
For mixing time, the representative time
scale (called “characteristic time”) in a mixing
tank is the inverse of rotation speed (1/N).
The dimensionless numbers for the three properties
can by obtained by taking the representative velocity
(v), liquid volumetric flow rate (Qp), and mixing time
(Ө), and divide by their respective characteristic
counterpart (e.g., ND, ND3, and 1/N). The plots of
dimensionless velocity, pumping, and mixing time
against ReI all show profiles similar to the power
number plot, with two distinct flow regimes: laminar
(viscous) flow and turbulent flow. The values at
high ReI turbulent regimes are relatively constant.

Effect of Scale on Physical Behavior of Using the correlations based on the dimensionless
Bioreactors numbers, one can explore the effect of a changing
scale on different variables. We assume that
Scale Translation Approaches
the equipment in different scales will remain
• Constant K a
L geometrically similar. In that case, the effect of
• Constant impeller tip speed (ND )
i different reactor sizes can be compared using
• Constant power per unit volume (Po/V)
characteristic length D (the tank diameter). If the
• Constant mixing time
tank diameter increases by 10 fold, all the other
reactor parts (tank height, impeller diameter, etc.)
will increase by the same proportion of 10 fold.
In scaling up different processes, one needs to
keep the most important variable(s) constant or
within an acceptable range. The commonly used
criteria for scaling up are (1) a constant KLa, so that
Table 2. Examples of Scaling Up by Keeping mass transfer can be maintained, (2) a constant
Different Parameters Constant.
The reactor is scaled up 15.6 times by volume while impeller tip speed, to sustain a critical value of
keeping geometric similarity. high shear velocity to break up agglomerating
particles or pellets of mycelial cells, (3) a constant
Property Pilot Scale power input per volume (usually for less power
Plant scale, 2500 l
160 l intensive processes such as crystallization,
blending), and (4) a constant mixing time.
P 1.0 15.6 98.0 6.2
P/volume 1.0 1.0 6.2 0.4 Consider the case of scaling up by maintaining power
input per reactor volume constant. Recall that the
N 1.0 0.54 1.0 0.4
power number is constant in a turbulent region and
D 1.0 2.5 2.5 2.5 the power input (PO) is proportional to N3D5. The
Qp
reactor volume is described by πHD2. Because of
1.0 8.5 15.6 6.2 geometrical similarity, we can represent H by D and
(pumping)
ignore the constant π that does not contribute to
Qp/volume 1.0 0.54 1.0 0.4
scale comparison. The reactor volume (V) is thus
NDi
1.0 1.35 2.5 1.0 represented by D3. In keeping PO/V constant, N3D2
(tip speed)
is also constant in different scales. In scaling up
as D increases, the rotation speed must decrease
by 1/D2/3. It is inevitable that larger reactors will
need to be operated at lower rotation speeds.
By similar algebraic manipulation one can also see
that scaling up by a constant power per volume (PO/
N3D2) constant will lead to increasing the amount f
total pumping (ND3) with the scale. However, pumping
per volume will decrease as the scale increases. For
mixing time, the trend is an increase with scale.
The table compares the effects of scaling up

by setting different parameters at a constant
value. Cases considered include constant power
per volume, constant agitation rate, constant
pumping rate, and constant tip speed. Often,
one chooses not to keep a single value constant,
and not to scale entirely geometrically similarly.
Scaling Up Geometrically Similarly By Keeping Power Per Unit Volume Constant
PO/N3DI5ρ is constant in turbulent region. Density of water, ρ, is constant. Thus,

PO = KN3DI5 (Eq. 1)
The volume of reactor can be expressed as characteristic length raised to the third power,
V = πHDt2 = cDi3 (Eq. 2)
The power per unit volume is described as
Po/V = K N3DI5/ cDI3 = K’N3DI2 = constant
This leads to the conclusion that when power per unit volume is kept constant, N3DI2 is also constant.
N3DI2 = constant (Eq. 3)
Effect on Agitation Rate Effect on Liquid Pumping

Comparing scale 1 and scale 2
N13DI12 = N23DI22 The capacity of liquid pumping can be described
N1/N2 = (DI2/ DI1 )2/3 (Eq. 4)
by the impeller tip speed, NDi, by the area that it
The agitation rate N decreases with increasing scale. moves against the liquid, Di2.
When the diameter increases eight times, the agitation
rate is ¼ in the larger scale. Under the condition of constant power per volume,
N13Di12 = N23Di22. Multiply both sides by diameter to
Effect on Impeller Tip Speed
the seventh power.
Tip speed is described by N multiplied by Di. from Eq. 3 (Eq. 7)
N13DI1 3/ DI1 = N23DI23/ DI2 N13Di19 / Di17 = N23Di29 / Di27

N1Di13 / N2Di23 = (Di1 / Di2)7/3
N13DI13/ N23DI2 3 = DI1/ DI2
Liquid pumping capacity increases with scale. By
N1DI1 / N2DI2 = (DI1/ DI2)1/3 (Eq. 5) dividing both sides by characteristic length raised
Tip speed increases with increasing scale, but only at to the third power, we can obtain the pumping
1/3 power of the length of scale. capacity on a per volume basis
NDi3 / Di3 = pumping per volume = Qp/V (Eq. 8)
Effect on Mixing Time
(Qp1/V1) / (Qp2/V2) = (Di1 / Di2)-2/3 = (Di2 / Di1)2/3
The decreased pumping per volume also causes
an increasing in mixing time when scale increases.
The pumping capacity per volume actually
The dimensionless mixing time is ΘN. Its value
decreases with increasing scale.
is relatively constant in the high Re number
turbulent region.
Θ1N1 = Θ2N2
From Eq. 4
(Eq. 6)
Θ1 / Θ2 = N2 / N1 = (Di1 / Di2)2/3

Mixing Time
The nutrients that are added at the beginning of
the culture will eventually become well mixed
in the culture fluid. (Note: This may not be true
for some microbial fermentation in which some
nutrients are supplied in a solid form and dissolve
gradually). Mixing problems may arise for those
components that are added continuously or
intermittently during the cultivation. If the nutrient
is added in a fixed position(s), it may not get carried
to other locations in the reactor fast enough to
meet the cells’ metabolic needs. In some cases,
the additive needs to be supplemented at a high
enough concentration that has an adverse effect
to the culture, and must be dispersed quickly. In
these cases, adequate mixing must be provided.
Nutrient Starvation Time In cell culture processes, oxygen is almost always the
first nutrient to be depleted. Due to its low solubility
Because of its solubility, oxygen is the first nutrient species
in medium, it must be continuously supplied.
to be completely consumed
In comparison, the concentration of glucose
maintained in the medium is usually a couple orders
of magnitude higher than oxygen. Their ratio of
Table 3. Comparison of Oxygen and Glucose Saturation molar specific consumption rates ranges from about
Time in a Typical Culture (For 1010) 1.0 (when most glucose is converted to lactate) to
Oxygen Glucose close to 6.0 (when most glucose is converted to
0.1 mM CO2). For oxygen, at a high cell concentration, the
C in Culture (50% saturation with
air space)
1 g / L (55 mM) depletion time can be as short as a few minutes,
whereas the depletion time for glucose is orders
Specific 0.15 – 1.0 X 10-10 mmole of magnitudes longer. Therefore, in reactor
1 x 10-10 mmole /
consumption / cell-hour
rate
cell-hr scaling, special attention is always paid to oxygen.
Volumetric
1 mmole / L-hr 0.15 – 1 mmole / L-hr
consumption
Time to
0.1 hr (6 min) 12 hr
depletion

Mixing Time To measure the mixing time, one can inject a
Mixing Time Measurement dye into the reactor under operating conditions
• Measurement and then use a sensor placed in a fixed location
to record the dye concentration over time. The
• At t = 0 add tracer
concentration will fluctuate over a large range
• Measure terminal mixing time, defined as the
point when an arbitrary chosen uniformity, 90%, initially, but will eventually reach a steady value.
is reached The time needed for the concentration to reach
a range of steady value (such as 90% of its final
homogenous concentration) is considered the
“mixing time.” If one plots the concentration
deviation from its final steady value, ΔC, the time
profile can be approximated by first order kinetics.
Fig. 13.6: Measurement of averaged mixing time in a stirred

tank
Nutrient Enrichment Zone, Mixing Time In a stirred tank reactor, a nutrient is added at a fixed
vs. Starvation Time position. Consider a fluid element carrying cells. When
it passes by this position, it acquires the nutrient and
then moves away to circulate around the reactor. On
average, the same fluid would return to this feeding
zone after a duration of one characteristic mixing
time. It is important that the amount of nutrient
that the fluid acquires at the nutrient enrichment
zone is sufficient to sustain the metabolic needs of
the cells before it returns to the zone. Therefore,
the mixing time needs to be shorter than the
starvation time. The starvation time is dependent
on cell concentration and the consumption rate.
If the mixing time is longer than the starvation
time, a discernible concentration difference of the
nutrient would appear in different locations in
the reactor. Pockets of low concentrations would
emerge and their locations may change over time,
as the fluid flow pattern and cell concentrations are
not constant. A sensor that is at a fixed position
may, thus, not reveal the presence of such pockets.

Mixing Time Distribution
If we follow a particular fluid element in a
reactor, it will come to the nutrient enrichment
zone, go away, and come back repetitively. The
time interval between its return to the nutrient
enrichment zone, however, will not be uniform.
The mixing time described above is an average mixing
time. But the mixing time that is physiologically
important (e.g., the return time to the nutrient
Fig. 13.7: Measurement of circulation time distribution enrichment zone) is not a single value of the
in a stirred tank average mixing time, but is distributed over a range.
Mixing Time Distribution Measurement Imagine that we use a ball that emits a radio signal.
The ball has the same density as the fluid and is
• Add radio emitter to the reactor i, a sensor picks up
the signal when the emitter circulates around and carried around by the fluid motion. A sensor at a
passes by fixed position in the reactor would pick up the
• Measure circulation time for each encounter and signal when the ball is close and record the time
plot the frequency distribution of the circulation interval between consecutive returns. This time
time
interval of return will distribute over a range as
• Determine the mean and median circulation time the ball sometimes returns shortly after it moves
and the standard deviation σ
away, while at other times it roams around the
• One can plot distribution of circulation time as
a population density function. The portion of reactor for a while before returning to the sensor.
circulation whose time of circulation lies between The histogram of the time-interval distribution can
t and t + Δt is the area under the curbe between t
and t + Δt. be converted to a mixing time distribution function.
In general, the distribution follows a logarithmic
normal distribution. The mean or median of mixing
time is a descriptor of mixing characteristics, but it
does not present the entire picture of mixing. Given the
same median or mean mixing time, two reactors may
still have a very different mixing time distribution.
Imagine that the location of the sensor is also
the position of nutrient feeding. Those cells
circulating with the particular fluid element
receive nutrient only when the fluid returns to
that position. If the circulation time is longer that a
critical time, then the nutrient level seen by those
cells may fall below the critical value. A wide
distribution of mixing time can be a concern.
Even though the frequency of exceedingly long
circulation time is low, nutrient starvation may
occur in those rare occasions and trigger apoptosis
or cause other irreparable damage to cells.
In reactor operation, the fraction of mixing

time that is longer than the critical time should
be minimized by either improving mixing or
Population Density Distribution f(t) by setting a limit on the cell concentration.
Probability of Starvation
τ
0 1 2
Critical Circulation Time
Circulation Time
Fig. 13.8: Mixing time distribution and critical mixing
time for nutrient depletion
Mixing Time Distribution

• The mixing time in a tank is not uniform. If the aver-
age mixing time is 6 minutes, many fluid elements will
have a mixing time shorter than 6 minutes, and others
will have longer times.
• If the critical mixing time is 6 minutes, the average
mixing time should be shorter than that.
Scaling Up and Mechanical Forces on Cells

In a turbulent flow, the fluid’s kinetic energy is
transferred by swirling pockets of fluid, called “eddies”.
Turbulent regimes are comprised of eddies of
different sizes, characterized by velocity fluctuations.
The hydrodynamic forces experienced by the cells
may arise from fluid-cell and cell-mechanical
parts interactions. Cells, being neutrally buoyant
particles, follow the motion of the relatively larger
eddies. In general, the direct impact of cells
on the impeller is minimal and cells generally
flow by impeller blades without suffering much
direct mechanical impact. However, these large
eddies cause the formation of cascades of smaller
eddies, which may impart damage to cells by
dissipating all of their energy on the cell surface.
The size of the eddy relative to the size of the cell
is thought to be an important factor that damages
cells. Smaller eddies in the size range of cell surface
motif, i.e., much smaller than cell diameter, are
considered to be more damaging than eddies that
are larger than cells. Studies examining cell death

• Microeddies cause cell damage
caused by turbulent flow often assume that the
cell death rate is proportional to the Kolmogorov
eddy concentration, and cell damage occurs when
the eddy size is smaller than a critical eddy size.
As the scale increases, the eddy length increases.
Thus, strictly from the viewpoint of the average eddy
size, the effect of turbulence on cell damage will
not become more severe in scaling up. One should
be cautioned that the eddy size is not uniform, but
distributed over a range. As the scale increases, the
distribution function of the eddy size also varies.
Further, the discussion here does not take into
account the effect of gas mixing. The phenomenon
of mechanical stress caused by combined agitation
Fig. 13.9: Eddies surrounding a cell suspending in a stirred
and aeration is rather complex in scaling up.
tank
• Eddy size increases with scale if the power per unit
volume decreases with scale
Scaling Up and Oxygen Transfer and Carbon Dioxide Removal

When scaling up a process, we aim to produce cells
and product in quantities proportional to the scale.
To meet that goal, we normally provide all nutrients
in a proportional amount, to meet the increased
metabolic needs of cells. For liquid nutrients,
increasing the nutrient provision in proportional to
cellular needs on a large scale is easily met. However,
for oxygen and CO2, which are supplied and removed
through gas aeration, scaling up presents a challenge
because of the constraints of physical factors.

Material Balance on Oxygen in Supplying oxygen to the growing cells in the
Bioreactor bioreactor involves blowing air through the culture
fluid, and transferring oxygen from the gas bubbles
into the culture fluid. The amount of oxygen carried
• The dynamics of the dissolved oxygen out from the reactor is less than that supplied
concentration are described by the balance
into the reactor. This difference is the amount
between OTR and OUR at a quasi-steady state.
transferred into the liquid phase. Material balances
on oxygen can be performed on the gas entering
and leaving the reactor (the gas phase balance),
(Eq. 9) as well as on oxygen being transferred from gas
At steady state, OTR=OUR
bubbles to liquid (the liquid phase balance). The
oxygen transfer rates calculated from the gas phase
balance and from the liquid phase balance are equal.
(Eq. 10)
Oxygen Balance on Gas Phase Gas phase balance is performed by taking the
difference between the oxygen input rate at the
inlet and the oxygen output rate at the outlet.
• On the gas side, the oxygen transferred from the That difference is the amount of oxygen that has
gas side to liquid side is reflected in the difference been transferred into the liquid. While oxygen is
of oxygen concentration between gas inlet and gas transferred into the liquid phase, CO2 (produced by
outlet. cells in the medium) and water vapor are stripped
out of the culture broth, thus the volume flow rate
in the outlet may differ from the inlet. Assuming
ideal gas behavior (PV = nRT), the molar flow rate of
the oxygen at the inlet and the outlet is the total air
(Eq. 11) flow rate (PQ/RT) multiplied by the molar fractions
of oxygen at the inlet and the outlet YO and YO
2,in 2,out
respectively. The oxygen transferred into the

liquid is simply the difference between the molar
flow rates of oxygen in the inlet and the outlet.

Oxygen Balance on Liquid Phase On the liquid side, the oxygen transfer rate (OTR)
is described by the overall mass transfer coefficient
(KLa) and the driving force (C*-C). For small
reactors, we assume that liquid is well mixed.
On the liquid side OTR and OUR is described by Eq. 11 This assumes that the concentration measured at
since the rate of change of dissolved oxygen is small. In cell
culture process, the air flow rate can be considered to be the outlet is the same as the concentration in the
the same at the inlet and outlet. Overall, the relationship is reactor. C* at the air outlet should be used for the
described as: driving force calculation. For a large reactor, one
assumes that the gas phase behaves like a tubular
reactor (plug flow), and a logarithmic mean of
(Eq. 12) the driving force is used. We assume that a quasi-
steady state, i.e., the change in dissolved oxygen
(Note: P/RT converts volume flow rate Q to molar flow rate
(dC/dt), is very slow compared to the oxygen
using ideal gas law. P is the pressure of gas phase)
consumption and rate of transfer. Thus, the oxygen
uptake rate (OUR) can be approximated by the OTR.
C* is the dissolved oxygen concentratoin in equilibrium

with the gas, which may differ in different parts of the
bioreactor.
For small scale bioreactors, one can assume both liquid

phase and gas phase are well mixed. The gas phase in the
reactor is thus the same as that in the exit gas stream.
Thus, C* is related to the oxygen concentration at the ex-
haust gas by Henry’s law constant:
(Eq. 13)
For large scale bioreactors, the inlet and outlet oxygen con-
centrations may be very different. One uses the logrithmic
mean driving force described below:
(Eq. 14)

Aeration Rate and Superficial Gas Now we examine the gas phase balance. In scaling
Velocity up, if we keep Q/V constant, we will be able to
maintain the same oxygen level at the inlet and
outlet (Yin, Yout) and sustain the same oxygen transfer
rate (OTR). However, when scaling up, Q/V is likely
to decrease. If OUR is to be sustained, then Yout
has to be smaller to maintain the material balance.
Then, consider the liquid phase balance. OTR is KLa
multiplied by (C*-C). Because oxygen level at outlet,
D = tank diameter
Q = aeration rate Yout, is lower, C* is also lower. How can OTR be kept
vl = culture volume in at the same level as in the small scale? One possibility
reactor is to increase KLa while keeping C at the same level
Vs = gas superficial velocity
A = cross-sectional area of as in the small scale (thus allowing (C*-C) to be
the tank smaller). The will require an increase in agitation
H = heights of culture power. The other possibility is to increase (C*-C) to
volume
the same level as in the small scale by allowing C to
be maintained at a lower level, if the reduced C has no
adverse effect on culture performance. Alternatively
one can use oxygen enriched air to increase C* to
maintain (C*-C) at the same level as in the small
scale. However, this will lead to increased level of CO2
Superficial gas velocity = gas flow rate/reactor cross accumulation as will be discussed in the next section.
sectional area
Therefore, overall oxygen transfer becomes a
vs = Q/A = Q/(πDt2) (Eq. 15) challenge in scaling up because the aeration rate
The reactor volume increases with length scale raised to
cannot be increased proportionally with the scale.
the third power, while the cross sectional area increases
only with the second power.

V1 / V2 = D13/ D23 A1 / A2 = D12/ D22
When scaling up one may choose to increase the air

flow rate proportional to the increasing reactor volume,

Q1 /Q2 = V1 / V2
One can see that

vs1 / vs2 = D1/ D2 (Eq. 16)
Superficial gas velocity will increase linearly with in-

creasing scale.

Now we examine the gas phase balance. In scaling
Aeration Rate and Oxygen Transfer
up, if we keep Q/V constant and keep KLa constant
Driving Force
in the liquid phase, we will be able to maintain
the same oxygen level at the inlet and outlet (Yin,
Yout) and sustain the same oxygen transfer rate
(OTR). However, when scaling up, Q/V will actually
decrease so if OUR is to be sustained, then Yout
has to be smaller to maintain the material balance.
Then, consider the liquid phase balance. We have
kept KLa constant in this analysis. To keep OTR
also constant, the driving force must be sustained.
However, the driving force is the logarithmic
mean of the oxygen concentration at the inlet and
the outlet. With a lower level of Yout, the driving
force for oxygen transfer will actually be lower.
Therefore, overall oxygen transfer becomes a
challenge in scaling up because the aeration rate
cannot be increased proportionally with the scale.
When scaling up, we aim to maintain OUR and OTR at the same level.
Considering mass balance in the gas phase:
From Eq. 12:
Given that Q2/V2 is smaller, and Yin (oxygen concentration in the inlet air) is the same, Yout,2 in the
large scale will be smaller
From Eq. 13 and Eq. 14, since Yout,2 is smaller, so is C*.
Considering the liquid phase, OUR and OTR will be maintain at the same level in the two scales:

(KLa)1(C*1 – C1) = (KLa)2(C*2 – C2)
This can be accomplished by
• Allowing C*2 to be lower while maintain C2 = C1. KLa2 in the large scale must increase.
Keep KLa2 at the same level as in small scale, then the concentration driving force of oxygen must
be increased to the level of the small scale by
• Allowing C2 to decrease
• Increasing C*2 by using enriched oxygen

Gas Flow Rate in Scaling Up: A The physical constraint of a reduced cross-sectional
Summary area to a reactor volume ratio with increasing
scale poses a challenge in oxygen transfer. In
scaling up, the airflow rate may not be increased
proportionally to the culture volume because an
overly high superficial air velocity is problematic.
In microbial fermentation, a high airflow rate
eventually causes “flooding” (i.e., the impeller is
swamped by gas bubbles) and loses its capacity for
pumping liquid. For cell culture, the aeration rate
used is substantially lower than the flooding aeration
rate. However, potentially a different problem may
arise. Antifoam agents are not used as extensively
in cell culture as in microbial fermentation because
of potential damage to cells. At a high superficial
Table 4. Effect of Scale on Oxygen Transfer velocity control of foaming may become problematic.
Constant When scaling up, aeration rate is not increased
Reference Constant
Superficial
Scale Air Flow
velocity proportionally to the culture volume. Because less air
Scale (volume) 1 1,000 1,000 is given to the same volume of culture, more oxygen
Cross Sectional has to be taken out from the gas phase to meet the
1 100 100
Area oxygen demand. This causes the oxygen level in the
Air Flow Rate 1 1,000 100 gas phase to be lower. As a result the driving force
Superficial Air
1 10 1 for oxygen transfer is also lower in the large scale.
Velocity
O2 Consumption/
In scaling up it is a common practice to take
1 1,000 1,000
CO2 Production a middle ground. The airflow rate per reactor
(yin –yout) has volume is decreased somewhat, but the superficial
to be very
Q(yin –yout) 1 1 large, i.e. yout is velocity is allowed to increase (albeit less than
small proportionally to the culture volume) to minimize
Need to
the loss of the oxygen transfer driving force. In
May reach some cases, the air is enriched with oxygen, while
Comments increase KLa or
flooding
power input in other cases, the agitation rate is increased when
oxygen falls below a set point during the cultivation.
If air supply increases proportionally with scale, foaming
can become serious and flooding may occur.

The respiratory quotient for most cells, using
Effect of Scaling Up on CO2 Removal
glucose and glutamine as the main source of
energy, is very close to 1.0. Thus, every mole of
oxygen consumed by the cells generates about one
• The mass transfer coefficients for oxygen and carbon
mole of CO2. A very active culture with a high cell
dioxide are about the same.
concentration can produce more than 100 mmole/L
• R.Q. (moles of CO2 produced/ moles O2 consumed)
for mammalian cells is very close to 1.0. So, oxygen of CO2 per day. In comparison, a cell culture medium
uptake rate (OUR) and carbon dioxide evolution rate has about 20 - 40 mM of sodium bicarbonate.
(CER) are about equal. The amount of CO2 produced by cells, thus, far
• The toxic level of CO2 is around 15–20% (110 mm Hg – exceeds that which is added as buffer to the media.
150 mm Hg).
Many cells, such as hepatocytes, are rather tolerant
• The carbon dioxide removed from the reactor
(at steady state) is the difference between its to CO2 but others are more sensitive. The growth
concentrations in the inlet and outlet gas: of most cells may begin to be affected at a CO2
concentration of 15% (114 mm Hg) so continuous
removal of CO2 from the culture is important.
(Eq. 17) When scaling up, the airflow rate per reactor
• CER and CCO2, in are the same in reactors of different volume may not increase proportionally. As a
scales consequence, less air is used to strip CO2 from an
• If cell concentration and metabolic activity remain equal volume of culture media. If the metabolic
constant.
activity of the culture remains the same, the same
• Q / V is smaller in large scale. amount of CO2 produced by the cells is now being
• Inevitably CCO2, out will have to be higher on a large removed using less air. The CO2 level will then be
scale.
higher in the air exiting from the large-scale reactor.
• The driving force for CO2 removal decreases with
increasing scale. The rate of CO2 stripping is dependent on the
concentration difference of CO2 in the liquid and the
gas phase. A higher concentration in the gas phase
diminishes the stripping efficiency. The increased
CO2 concentration in the exit air, thus, causes
further accumulation of CO2 in the liquid phase.
To compensate for the reduced O2 driving force
from scaling up, one can use O2-enriched air.
However, such a measure cannot compensate
for the diminished stripping efficiency of CO2.
Therefore CO2 accumulation patterns in large scale
and small scale bioreactors can be rather different.

• To achieve the same molar transfer rates for oxygen The overall mass transfer coefficient for CO2 is slightly
and carbon dioxide (although in opposite directions), lower than that of oxygen because of its larger molecular
the driving force for carbon dioxide has to be around
100 mm Hg, a level similar to that for oxygen transfer.
weight. However, the difference, approximately the
square root of their molar weight ratio, is very small.
• The upper bound of CL for CO2 should be below the
inhibitory level of 110-150 mm Hg. If the driving force One can consider the KLa to be about the same.
is 100 mm Hg, then C* will have to be 10-50 mm Hg.
That is 1.5-6.5% CO2 in air.
Unlike O2, the solubility of CO2 in aqueous solution is
very high. At the gas bubble interface, O2 in the liquid
• To strip off carbon dioxide a sufficiently fast air flow
rate must be used to ensure the CO2 concentration in phase can be assumed to be in equilibrium with the
the gas phase is low enough to provide a large driving gas phase, so (C*-C) is a good estimate of the driving
force. force. The same assumption is not always valid for CO2.
CO2 in the medium exists as CO2, HCO3, and CO3-2. At
(Eq. 18)
the interface, CO2 crosses the film and is transferred
out of solution, but HCO3- does not. So, HCO3-must
dissociate to CO2 before being transferred to the
gas phase. The kinetics of HCO3-, the dominant
form of CO2 in aqueous solution, to dissociate
to CO2 is slow. Because of the slow kinetics, the
actual driving force is smaller than what can be
estimated from the total CO2 (g) concentration.

Chemical Environment in Scale In scaling up or scaling down, the chemical
Translation environment may also be affected. For cell culture
processes, many of those changes are caused by
different CO2 accumulations at different scales. CO2is
a major contributor of the pH buffer in a cellular
environment. CO2 produced by the cells is excreted
to maintain a physiological range of intracellular pH.
Carbon dioxide is transported through the plasma
membranes as CO2 and HCO3-. CO2 can diffuse
through cellular membranes, while HCO3 transport
is mediated by transporters. A symporter co-
transports HCO3- and H+, while another antiporter
co-transports Cl- and HCO3- in the opposite direction.
As CO2 builds up in the culture fluid, a higher Cl-
gradient is needed to “drive” HCO3- out, otherwise
the intracellular HCO3- level will be higher. Because
the airflow rate per reaction volume is not kept
constant in scaling up, the CO2 level in the culture
will be higher on a larger scale. This will affect
the intracellular CO2/HCO3- levels. However,
experimental evaluation of the effect of scale on
cellular level of CO2 and intracellular pH is still lacking.
Fig. 13.12: Schematic of the removal of carbon dioxide produced
by cells
• CO2 produced by cells can diffuse through the cell

membrane
• Most CO2 becomes HCO3- and is excreted through
transporters
• Accumulation of CO2 in medium may affect
intracellular pH

Concluding Remarks
In scale translation, the relationship of geometry- CO2 stripping, to vary at different scales. Difference
related physical parameters are not kept constant, in pH control actions may further change the
because the volume and the surface area of the chemical environment of the culture. Given that the
reactor change in different proportions relative physical and chemical parameters related to scaling
to length. Therefore, one has to define the critical up cannot easily be manipulated or controlled, one
range of various scale-sensitive variables and may resort to selecting cells which are less sensitive
choose scaling up criteria to ensure the operation to those parameters. Understanding scale-sensitive
is within the optimal region. In most cases, one parameters and a sound knowledge of estimating
chooses not to scale up completely geometrically the range of those critical parameters will greatly
similarly. Most large-scale reactors have a larger facilitate the scale translation of cell culture
height to diameter ratio than the smaller scale ones. processes.
Nevertheless, the physical constraints on scaling up In process development involving scale translation,
are the same regardless of whether one scales up one should aim to reproduce or to predict the
geometrical similarly or not. In scaling up, the gas conditions of physical constraints, as well as the
flow rate is also likely to change in its proportion to resulting chemical environment. It may not be
the reactor volume. This causes the mass transfer possible to replicate all physical and chemical
characteristics to be different for different scales. parameters on drastically different scales.
While the dissolved oxygen can be controlled at the Ultimately, one should identify and aim to control
same level, the CO2 concentrations profiles are likely critical physical parameters, to minimize variations
different for reactors of different scales. Differences in the chemical environment.
in CO2,concentration in the reactor will cause pH
control actions, including base and CO2,addition and

Cell Culture Genomics
Gene and Genome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

What is a Gene?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Organization of Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
Packaging DNA into Chromatin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
Epigenome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
Molecular Mechanisms that Mediate Epigenetic Regulation . . . . . . . . . . . . . . . 291
Genome Scale Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
Transcriptome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
Proteome Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Sequencing Technologies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
Sanger Method and Sequencing Technology Evolution . . . . . . . . . . . . . . . . . . . 302
High Throughput NextGen Sequencing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
Gene Expression Exploration in Cell Culture Processing . . . . . . . . . . . . . . . . . . . . . . . . 306
Gene and Genome
What is a Gene? A gene is a sequence of DNA that encodes for an RNA

and protein product. Up until a quarter century ago
the prevailing notion was that one linear sequence
of DNA directly encodes for one gene. After the
discovery of alternative splicing, however, our
understanding quickly changed. Recent findings
have highlighted a relatively large number of
alternatively splicing genes in the mouse genome.
A large number of these genes are translated into
Mouse Genome Encodes
different protein sequences. Our knowledge of the
• ~30,000 genes coding for proteins
relationship between a gene and its expression
• ~1,600 genes coding for RNA product is evolving. For instance, we now know
• 800 tRNA genes that two genes may reside on opposite strands of
• 350 tRNA genes the same segment of DNA (and will be transcribed
• ~450 other ncRNA (noncoding RNA genes) in opposite directions), or they may reside in
the same strand of DNA and are overlapping..
A gene may encode for two types of end products: 1)
CELL CULTURE GENOMICS | 285

a protein, or 2) a non-protein-coding RNA (ncRNA).
Table 1. Distribution of Mouse Genes In its entirety, the mouse genome encodes for a
total of about 37,000 genes. Among them, just over
Protein coding ~30,000 30,000 genes code for proteins, 4,000 code for so-
genes
called pseudogenes (genes whose identity can be
Pseudogenes ~4,000 traced to an ancestor but are no longer translated
RNA coding genes ~1,000 into a functional protein), and the remaining
1,600 code for RNAs (including 800 rRNAs, 350
rRNA ~800
tRNAs, and other non-coding RNA, or ncRNA).
tRNA ~350
The average mammalian gene spans a region
snRNA ~150 encompassing approximately 23 to 28 thousand
miRNA ~300 base pairs (kbp) in length. Typically, the promoter
region is located upstream of the transcribed region
rcRNA ~450
(about 1 kbp or longer, sometimes as long as 10
kbp) and the transcribed region is used to generate
Gene Structure in Eukaryotes a primary transcript, or pre-mRNA. The primary
• Exons make up the mRNA; intervening sequences transcript starts with a 5’ end untranslated region
called introns are also present
(5’ UTR) and ends with a 3’ UTR. It also contains
• Splicing of pre-mRNA entails removal of intronic a number of protein coding exons and introns.
regions, addition of polyA tail and 5’ cap
After the introns are spliced out, the mRNA, or
• Alternative splicing is common
mature transcript, remains and includes: 1) the
5’UTR, 2) the exons, 3) the 3’UTR, and 4) a newly-
added polyA tail. The mRNA is ultimately exported
out of the nucleus for translation in the cytoplasm.
Many primary transcripts undergo alternative
splicing. In these cases, portions of exons are
stitched together under different conditions (e.g., in
different tissues, at different times, or occurring at
different frequencies) to give different mature mRNA
species. The stitching point of two consecutive
exons may not be the same under alternative
splicing. In this case, splicing may result in a shift
of the reading frame, which would affect the exons
downstream of the splice junction and perhaps
give rise to completely different protein sequences.
The Functional Annotation of the Mammalian
genome (FANTOM) consortium has generated the
most complete mouse gene sequence database to
date. It has uncovered a large number of protein
coding genes with alternative splicing. These
events have been found to produce very different
protein sequences, a large number of polymerase
II transcribed ncRNAs (with polyA tail), and

antisense RNAs that may have regulatory roles.
The prevalence of so many classes of variants poses
ambiguity in the definition of a “gene”. For instance,
if one were to count each independent gene
product as a different “gene”, the total number of
genes could be estimated to be upwards of 50,000.
Organization of Eukaryotic Genes
~1 kbp 23-28kbp
AUG UGA
intergenic region Promotor Exon1 Intron1 Exon2 Intron2 Exons intergenic region
5’ UTR Transcription 3’ UTR
AUG UGA
primary transcript
Alternative splicing
Splicing
5’ UTR polyadenylation 3’ UTR AUG UGA AAAAAAAAA
AUG
AUG UGA
UGA AAAAAAAAAAAA start stop
Export to cytosol
AUG UGA AAAAAAAAAAAA

start stop
Translation
protein 1 protein 2
Fig. 14.1: Expression of an eukaryotic gene. Alternative splicing into two different proteins are shown.
Organization of Genome The number of genes in each organism varies greatly,

from 700 genes in a simple parasitic mycoplasma
to over 30,000 in mammals. Those studying the
Composition of a Typical Mammalian Genome
“minimum set of genes” that are required for life have
• Coding Sequences ~ 1-2%
derived a gene set ranging from 270 to 350 genes.
• Intronic Sequences ~ 20-25%
• Repetitive Sequences ~45-55% As the number of genes increases with increasing
• Other intergenic sequences complexity of organism, the additional genes
and gene products acquired tend to affect a cell’s
interaction with its environment (e.g., transporters,
signaling molecules, and their receptors). In
other words, data suggest that the increased
complexity of higher organisms requires the
formation of a more sophisticated communication,
both at the cellular and organism level.
With an increasing number of genes, one also sees
an increasing size of the genome. By convention, the
size of a genome is quantified using the number of
bases in a haploid genome. A bacterium’s genome

is about 3 Mbp to 8 Mbp, while a fungus’s genome
Eukaryotic Genome is Organized into
Chromosomes is a bit less than one order of magnitude larger
• Human: 22 pairs; x,y than that of bacteria, from 12Mbp to 30Mbp.
• Mouse: 19 pairs; x,y In contrast, mammals have about a genome size
• Rat: 20; x,y of 2-3 Gbp, nearly 1,000 fold higher than bacteria.
• Chinese hamster: 10 pairs; x, y While the mammalian genome is nearly 1,000-fold
larger, it contains only 10 times more genes. A typical
• Mouse chromosome size: 61 Mbp (Chr 19)to 197
Mbp (Chr. 1) protein-coding gene in bacteria is about 1 kbp, or
330 amino acids, while an equivalent sequence in
mammals is about 1.3 kbp. Including introns and
UTRs, a mammalian gene in total is 23-28 kbp,
Repetitive Sequence which is substantially larger than a bacterial gene.
• Short RNA-derived interspersed elements (SINES, The gene-encoding regions, including introns,
90-300 bp)
account for only about 25% of the mammalian
• Long interspersed nucleotide element (LINES,>500 genome. The rest of the genome consists of other
bp)
intergenic sequences, including promoters,
• Retrovirus like elements with long terminal repeats
(LTR)
regulatory elements, and regions not yet explored
by scientific inquiries. Additionally, nearly 50%
• DNA transposons
of a mammalian genome consists of repetitive
• Widely distributed simple sequence repeats:
sequences. That number is even higher in some
• Direct repetitions of short k-mers such as (A)n, plants, giving them a genome size even larger than
(CA)n or (CGG)n
mammals. The repetitive sequences reside not only
• Segmental duplications, consisting of blocks of
10–300bp of the genome
in intergenic regions, but also in introns, UTRs and
upstream or regions adjacent to genes. Repetitive
• Blocks of tandem repeated sequences, such as at
centromeres, telomeres, the short arms of acro- sequences fall into different categories; some are
centric chromosomes and ribosomal gene clusters, short, while others are long, up to 500 bp. Some
these also include satellites and microsatellites. are the result of transposons or the remnants
of retrovirus infection throughout evolution.
The presence of repetitive sequences presents a
barrier to the quick and accurate assembly of DNA
sequencing reads. In DNA sequencing, the DNA
molecule is first fragmented and then the independent
fragments are sequenced. The assembly algorithm
searches for the overlapping regions and attempts
to stitch them together into longer contiguous
sequences (or contigs). Therefore, a fragment
that has a repetitive end can often be assigned to
multiple loci as it is not a unique sequence. One
solution to this is to sequence very long fragments
of DNA, over the stretches of repetitive sequences.

Table 2. Genome Size of Representative Organisms
Common Name Taxonomy Species Genome Size Estimated Number of Genes
Mycoplasma TENERICUTES Mycoplasma pneumoniae FH 8.11E+05 670

Bacteria PROTEOBACTERIA Escherichia coli DH10B 4.69E+06 4,271
Bacteria FIRMICUTES Bacillus subtillis subtillis 168 4.21E+06 4,354
Yeast FUNGI-ASCOMYCOTA Saccharomyces cerevisiae 1.21E+07 6,273
S288C
Slime Mold PROTISTS-MYCETOZOA Dictyostelium discoideum AX4 3.40E+07 13,362
Roundworm NEMATODES Caenorhabditis elegans 1.00E+08 20,935
Fruit Fly ARTHROPODA Drosophila melanogaster 1.37E+08 21,116
Chicken CHORDATA-BIRDS Gallus gallus 1.00E+09 17,935
Frog CHORDATA-AMPHIBIA Xenopus tropicalis 1.70E+09 20,500
Human CHORDATA-PRIMATES Homo sapiens 3.17E+09 53,894
Mouse CHORDATA-MAMMALS Mus musculus C57BL/6J 2.72E+09 37,261
Rat CHORDATA-MAMMALS Rattus norvegicus BN/SsNHs- 2.70E+09 35,427
dMCW
Dog CHORDATA-MAMMALS Canis lupus familiaris 2.40E+09 24,661
Chinese Hamster CHORDATA-MAMMALS Cricetulus griseus 2.70E+09 32,476

Packaging DNA into Chromatin In bacteria, the genome is typically arranged into a
single, large chromosome. Most chromosomes are
circular, although linear chromosomes are also seen.
Conversely, in eukaryotes, the genome is segmented
DNA molecule into different chromosomes. Chromosomes are not
merely DNA molecules wrapped loosely together.
A typical E. coli cell (2 µm x 1 µm in size) has a
packed with genome of about 1.5 mm in length. If all 46 diploid
histone proteins chromosomes of the human genome were strung
to form together, they would form a ~2 m x 2 nM string. The
chromatin chromosome is, thus, not merely a string of DNA
chromatin packed haphazardly into the nucleus. It requires
forms fiber-like extensive manipulation and the work of specialized
structure machinery to allow it to be packed into a dense volume,
while still remaining accessible for transcription.
may condense tightly Each chromosome is a molecule of double-stranded

or packed loosely in DNA. Packaging of DNA occurs at multiple levels.
different regions of At the local level, small regions of a DNA molecule
chromosomes form a complex with DNA binding proteins,
chiefly histones, to form a “beads-on-a-string”-
like structure. That form is further condensed
Fig. 14.2: Packing of a segment of DNA into highly condensed into packed beads, called nucleosomes. In further
region and relatively open region condensation of the structure, some regions are
more open and accessible to transcription (this is
called euchromatin), while other regions are more
densely packed, and are called heterochromatin.
• Chromatin: complex of DNA and protein in which
genetic material is packaged within the cell
• Histones: principal protein components of chromatin
• Nucleosome: fundamental sub-unit of chromosome
which consists of 165 bp of DNA wrapped around an
octamer of core histones

Epigenome
The term ‘epigenetics’ was introduced in the 1940’s to Although the genome of each cell of a higher mammal
describe “the interactions of genes with their environment, may encode more than 30,000 genes, at a given time
which bring the phenotype into being.”
it may actively transcribe only a fraction of these. A
• ‘Heritable changes in gene expression not encoded in typical CHO cell in culture, for instance, expresses only
the DNA’
about 16,000 genes. Some genes are ubiquitously
• Essential for genome packaging and fundamental to
development expressed in all tissues; others are tissue- or time-
dependent. At the transcriptional level, a large
• Epigenetic alterations influenced by the environment;
for example, identical twins can be susceptible to array of transcription factors are responsible for
different diseases regulating the expression of genes according to the
• Epigenomics: Representing the totality of epigenetic tissue type, timing by developmental stage, or by
marks in given cell type event (such as stress or exposure to some signaling
molecule). At a higher level, transcription is also
regulated by the accessibility of the gene loci, which
is further controlled through epigenetic events.
Epigenetic regulation does not result in a mutation, as

Molecular Mechanisms that Mediate
no change occurs to the underlying DNA sequence. It
Epigenetic Regulation
does, however, cause a heritable change in the cellular
phenotype. Unlike a mutation, which originates in
a single genomic locus of a single cell, epigenetic
changes can occur and affect gene expression
on a global level. For instance, when stem cells
differentiate, or when fibroblasts are transformed
into iPS (induced pluripotent stem) cells, global
epigenetic changes occur on the chromosomes
to affect the reprogramming of genetic circuits.
In cell culture, cells are often “adapted” to new
culture conditions, such as differing growth factors or
adjusting to growth in suspension. In such processes,
the entire population of cells shift their phenotype.
Such processes are less likely to be mutation events
and are more likely to involve epigenetic regulation.
In the generation of producing cells, the host cell
transforms from a non-secretor to a professional
secretor in a short time, accompanied by a vast change
of cellular properties. Although it is possible that
mutations may be responsible for some of the changes,
it is very likely that epigenetic events are the major

drivers to in the dramatic shift to high productivity.
• Chromatin modification The major mechanisms of epigenetic modification
• Covalent modifications of histones involve DNA modifications and histone
• Histone variants modifications. Of the DNA modifications, the
• Nucleosome remodeling methylation of Carbon 5 of cytosine is one of the
• DNA Methylation most common. Once methylation occurs, the mark
• Non-coding RNAs is highly stable and can be passed on to daughter
cells. Note that this change does not constitute a
change or mutation in the DNA sequence. Much of
the DNA methylation occurs on cytosines that reside
in CpG dinucleotides (its complementary strand, 3’-
GpC -5’, is also methylated). Regions of the genome
Table 3. Types of Histone Modifications
with a high GC content, where the CpG sequence
Chromatin Residues Functions Regulated is very frequent, are often called “CpG islands”.
Modifications Modified Such regions, when upstream of a promoter, can
Acetylation Lysine Transcription, play key roles in the regulation of gene expression.
Repair, Replication,
Condensation Methylation of a CpG islands primarily leads to
Methylation Lysine Transcription, Repair gene silencing, as has been shown in the cholesterol
Methylation Arginine Transcription dependence of NS0 cells. For instance, Hsd17b7, a
Phosphorylation Serine, Transcription, Repair, key gene in cholesterol synthesis, is silenced by CpG
Threonine Condensation
methylation in NS0 cells. Accordingly, demethylation
Ubiquitination Lysine Transcription
treatment of NS0 cells led to the rapid emergence of
cholesterol-independent cells. Methylation is also
likely to be involved in the glutamine dependence
of CHO cells, due to methylation of a CpG island
upstream of the glutamine synthetase gene.
Of note, methylation does not only occur in CpG
islands; nearly a quarter of methylation seen in
embryonic stem cells is not in a CG context. This
non-CG methylation, however, is more transient
and mostly disappears after differentiation.
At the histone level, acetylation, methylation,
phosphorylation, and ubiquitination may occur
on different amino acid residues of histone
proteins. Each histone protein has multiple
sites that may be modified, resulting in a large
combination of possible histone modifications,
each affecting the packing of DNA and the
accessibility of genes in the region. Both histone
and DNA chemical modifications require specific
enzyme-mediated reactions. Their maintenance
and removal also requires specific enzymes.

Knowing the mechanisms of epigenetic regulation,
it is possible to intervene using chemical inhibitors
of the necessary enzymatic reactions. For example,
5’-Azacytidine is used to facilitate demethylation.
TrichostabinA and sodium butyrate are also used to
interfere with histone modifications. The alternation
of the epigenetic status can also be induced by the
introduction of exogenous genes, as in the process of
reprogramming induced in the derivation of iPS cells.
reprogramming induced in the derivation of iPS cells.
Genome Scale Analysis

Transcriptome At a given time a typical mammalian cell transcribes
about 15,000 genes into RNA. The vast majority
of those transcripts are present at only very low
Mouse Genome Encodes levels, with some even as low as a few copies in
• ~15,000 genes are expressed in a given cell each cell, with another small fraction expressed at
• Highly abundant genes generally don’t change intermediate levels. Only a very small number of
transcript level over a wide range genes are expressed at extremely high levels. Genes in
• Rare genes can be very dynamic the last class, the so-called “abundant genes”, encode
• 1,000 fold change in transcript level in 30 min proteins such as ribosomes, GAPDH (3-phospho-
is common in bacteria. A similar change in glyceraldehyde dehydrogenase), and actin.
differentiating stem cells usually takes days.
In some recombinant cells, the product gene, which
is highly amplified, also falls into this category.
Because the sheer number of transcripts for each
abundant gene is very high, the total mass of those
RNAs can constitute up to ~10% of all mRNA.
Such genes usually do not undergo a very large
degree of change in their expression level. For
Table 4. mRNA in a Typical Somatic Human Cell instance, one rarely sees even two-fold changes in
Number of Species % of mRNA by the transcript level of most abundant genes under
mass
different culture conditions. However, keep in mind
Superprevalent 10 - 15 10 - 20
(Abundant)
that, because they are so abundant, even a 10%
change in the level of a gene in this category is much
Intermediate 1,000 - 2,000 40 - 50
greater than even a 10-fold change in a rare gene.
Complex (rare) 15,000 - 20,000 40 - 45
The genes that most frequently undergo very
large changes in expression are the rare genes.
These rare genes often encode for products
that are gene regulators or other products that
are powerful even at minute levels. For this
reason, they are kept at very low expression
levels and are not expressed when not needed.

Expressed Sequence Tags (ESTs) The transcript levels of many genes are relatively stable
at different times and under different conditions,
• Transcripts from cells of tissues are isolated, reverse
while some are relatively dynamic. Overall, the rate
transcribed to cDNA and cloned into E. Coli to
construct an EST library of change of gene expression in mammalian cells is
rather slow compared to bacteria. We see over three
• Clones of E. Coli are sequenced to give rise to fragment
orders of magnitude decrease in transcript levels
or the entire length of transcript
within half an hour in bacteria; however, even under
• Sequences are assembled and annotated
stem cell differentiation conditions, a similar level of
• The data gives transcriptome profiles of (abundance change in mammalian cells usually occurs over days.
level) of transcripts of different genes under different
conditions To explore the dynamics of gene expression in
different tissues and in different diseases or
• The sequence data are used to design microarray and
differentiation stages, transcripts were isolated
assemble the genome
from those tissues and directly sequences. Those
transcripts are typically called expressed sequence
tags (ESTs). The collection of those ESTs form the core
of the database of various genes in different species.
Capturing the dynamics of transcripts at a global level,
i.e., on a genome scale, has become readily available in
the past decade through the use of DNA microarrays
and, more recently, through deep sequencing.
Transcriptome profiling through arrays and
sequencing remains the cellular analytical tools that
are truly global and capable of genome-wide survey.

DNA Microarrays Two prevailing forms of microarrays are currently
utilized: cDNA microarrays (commonly used for
complex mammalian genomes) and oligonucleotide-
based microarrays. The difference between these
methods lies in their type of probes. In the case of
DNA microarrays for microbial species the primers
are designed to specifically amplify gene fragments
from genomic DNA that will then serve as probes.
These probes are designed specifically for unique
segments of gene sequences. The probes spotted on a
cDNA microarray for mammals, on the other hand, are
amplified from cloned cDNAs using universal vector
primers. Usually designing probes based on specific
gene sequences is too costly with the large number
of genes involved. They usually lack the specificity to
Fig. 14.3: Classical DNA microarray prepared from cDNA clones differentiate many isoforms or alternatively-spliced
of an EST library and the use of it as a two-dye microarray variants. Oligonucleotide-based microarrays,
in contrast, utilize much shorter (20 - 80 bp),
specifically designed, then chemically-synthesized
probes. Multiple probes covering different regions
of each gene are often used to increase the specificity.
With the decreasing cost of oligoDNA microarrays
Table 5. Available Microarray Technologies and direct sequencing, cDNA microarrays
Affymetrix Agilent Nimblegen are being phased out. cDNA arrays rely on
Manufacturing Photolithographic Non- Maskless using two fluorescence channels for relative
technology manufacturing contact synthesis
inkjet using digital measurement renders them inconvenient for
printing micromirro comparison of a large number of samples.
device
Probe Length 25 - mer 60 - mer 60 - mer Long oligo microarrays are typically comprised
Feature size 5 - 18 μm 65 μm 16 μm
Multiplexing No 2-, 4-, and 2- and 4 - plex
of 50 - 70 bp probes synthesized onto a glass
8-plex slide. Affymetrix arrays are made by the direct
synthesis of eleven sets of short 25-mer probes
onto the chip through photolithography-based
technology. Typically, multiple probes are employed
for a given gene or contig over a region of a few
hundred base pairs of each target transcript.
The photolithographic in situ synthesis technique
requires the construction of masks for each layer
of nucleotides added to the probes. The process
is extremely costly. In contrast, using a digital
micromirror, NimbleGen technology directs light to
tiny spots to allow chemical reactions to occur only in
the lighted spots without using masks, thus drastically

reducing the manufacturing cost of making the array.
Both Agilent and NimbleGen allow multiplexing (i.e.,
multiple independent samples can be hybridized
light source to separate arrays on a single slide). Both of these
array formats also support a dual mode system
that provides the option of using the routine two-
mask (each layer color experimental design (Cy3 / Cy5 based)
four masks for A,
T, G, C) or one-color (Cy3 only) on a single platform.
When the DNA array is used for two-channel
comparison, usually a common reference is used.
array “layer by layer” synthesis (total ~25 Such common references are usually acquired
layer for 25-mers of DNA) by mixing mRNA samples from different tissues
Fig. 14.4: Mask-based layer-by-layer in-situ photosynthesis under different culture conditions to ensure
of oligonucleotide on array surface (Affymetrix system). that the vast majority of transcripts are present.
When the DNA array is used for two-channel
comparison, usually a common reference is used.
Such common references are usually acquired
by mixing mRNA samples from different tissues
under different culture conditions to ensure
digital mirror that the vast majority of transcripts are present.
light
source
“layer by
layer”
no mask needed synthesis
~60-mers of
DNA
Fig. 14.5: Digital mirror-based photosynthesis of oligo-

nucleotides on array surface (NimbleGen system).
RNA-seq
With the ability to generate tens of gigabases
• Direct counting of abundance level of reads for in a single run, high throughput sequencing
each gene, normalized to gene length technologies are becoming an affordable and
• Very wide dynamic range of detection powerful tool for transcriptome profiling.
• Quantification not affected by low sensitivity or
saturation as in fluorescence detection A first step in transcript profiling is the removal of
rRNA by oligo(dT) capture of mRNAs, which targets
• Does not require sequence information for
their polyA. Subsequent reverse transcription
for cDNA synthesis is performed either by polyA-
based priming or by using random primers.
RNA-seq methods take mRNA samples, shears them

into fragments, and reverse transcribes them into
cDNAs, which are then sequenced using one of the
high throughput sequencing technologies. The
resulting output is a large number of sequences on
the order of 50 - 150 bp. Each string of sequence
is called a “read”. Depending on the depth of
sequencing and the abundance level of the nucleic
acid fragment, a segment of nucleic acid may be
sequenced only a couple times or up to million times.
RNA-seq is not 3’ end biased; the abundance level of
a transcript is represented by the number of times
that segments of the transcript have been sequenced.
The more abundant and the longer the transcript is,
the more frequently its sequence reads will appear.
Using this method, one can sequence as deeply
as is necessary to detect almost all transcripts in
the cells, even those that are rare. In microarray
analysis rare transcripts usually do not yield enough
signal to give confidence to results. Furthermore,
very high abundant genes are usually detected in
the non-linear, near-saturation range of signal,
thus lacking good sensitivity for quantification.
Such problems are not present when RNA-seq
is used, as it gives a much wider dynamic range.
Another major advantage of RNA-seq is that
it requires no prior EST database or genome
sequences of the species to be probed, whereas for
DNA array probe design, at least the sequences of
the genes to be probed must be available. For the
species whose genome sequence or EST database
is available, the reads from sequencing are mapped
to the exon sequences for enumeration of hit reads
and for normalization to sequence length. Even if
no genome sequence or EST database is available,
the reads can be still assembled into contigs. In
most such cases, the contig annotation can be
obtained from a related species, and the reads can
be mapped back to each contig and then counted
to give an estimate of transcript abundance.

Table 6. Commonly Used DNA Microarray Formats
Microarray Probe sequence Common Uses Sample Numbers Hybridization Quantification
Type generation method Method
cDNA Universal primer Commonly used Normally Typically mRNAs Measure the ratio
amplified EST probes for mammalian EST two channel are isolated and of intensity of the
library based array comparisons (can reverse transcribed same transcript from
go up to four) to cDNA which is different samples.
Sequence specific primer Generally used for
the labeled with a Comparison of
amplified probes microbial species
fluorescent dye multiple specimens
whose genome has
from different
been sequenced
samples must
use ratio of ratios
unless a common
reference for all
samples is used.
oligoDNA Synthetic specific Cross hybridization Two Channel
Can use a pooled
50-60mer, can have among different
RNA as a common
multiple probes per gene closely related
reference to facilitate
sequences can be
the comparison of
minimized
multiple samples.
Direct comparison
of levels of different
transcript in the
same specimen is
difficult.
Single Channel
Photolithographically Interrogate Single Channel mRNA is reverse The intensity gives

synthesized 25mers, multiple segments transcribed, then an estimate of
multiple probe set per of about 500 bp transcribed into the abundance of
sequence region using both cRNA, which is transcript for each
“perfect match” then fragmented gene. Data can be
and “mismatch” and biotin labeled used for both intra-
probes to compute before hybridizing array comparison
the signal to the probes on (different genes in
the array. the same specimen)
as well as inter-array
comparison (ratio of
expression level as in
cDNA array).
RNA-Seq Direct sequencing. Use for reaching Single sample Short reads Direct counting of
Coverage depends on depths sufficient per channel, are sufficient sequence reads per
sequencing depth. For to detect rare or multiplexing for sequenced gene, normalized to
CHO, 20GB gives good genes. Discerning barcoded sample genome. If gene length.
coverage of most genes heterogeneity assembly is
in transcript or required, long
genome. Also for reads are
transcriptome preferred.
profiling of
unsequenced
genome

Proteome Profiling Proteome profiling is another means of surveying
global changes in gene expression. It is a powerful
complement to microarray transcriptome profiling,
serving to examine gene function directly at the protein
level. Proteomic analysis allows for the investigation
of the primary amino acid sequence, protein-protein
interactions, and post-translational modifications
on a large scale. In proteomics, mass spectrometry
has established itself as an indispensable tool.
2D Gel Electrophoresis 2D-gel electrophoresis allows for the simultaneous

analysis of a many protein molecules. In this
method, a complex protein sample is resolved in
two dimensions according to charge (isoelectric
point) and mass on a polyacrylamide gel, followed
by staining. The stained protein spots are then
characterized using sophisticated image analysis
tools (such as PDQuest). The difference in
staining intensity of observed spots allows for
relative quantification between protein samples.
This method, however, is not suitable for some
proteins. For instance, proteins present at
lower levels are not easily detected. Also, some
proteins co-migrate and cannot be resolved.
Figure 14.6: Typical flow of 2-D electrophoresis-based protemics.
Finally, proteins with charges outside of the
• 2D gel electrophoresis isoelectric range of the gel or highly hydrophobic
• First Dimension: Isoelectric focusing (IEF), proteins are also unable to be resolved in the gel.
separation by charge. IPG (immobilized pH gradient) Once a protein spot of interest is identified, the
strips; usually pH 4 to 7
spot is excised and purified for further analysis,
• Second Dimension: SDS-PAGE, separation by such as direct sequencing or mass spectrometry
molecular weight
for protein identification. Electrospray ionization
• Staining types: Coomassie Blue (sensitivity in the ug (ESI) and matrix-assisted laser desorption/
range); silver staining (sensitivity in the ng range);
ionization (MALDI) are two techniques commonly
SYPRO RUBY(sensitivity in the pg range; fluorescent
dye; most suitable for quantification) used to volatize and ionize the proteins or
peptides for mass spectrometric analysis.
• Image Analysis: identification of differentially
expressed spots by eye or with the aid of specific
software packages (PDQuest)
• MALDI-TOF and ESI
• Differentially expressed spots are extracted from 2D
gel, and subjected to proteolytic digest, and peptide
finger print analysis using MALDI-TOF

2D LC In applying two-dimensional liquid chromatography
(LC) for analysis of protein mixtures, a proteolysis of
the protein (typically using trypsin) is first performed.
Shotgun LC Based Methods This process reduces proteins to oligopeptides of
SILAC mostly 15 - 30 amino acids long, which facilitates
separation by liquid chromatography. In the first
• Use non-radioactive isotope for labeling cultured
samples dimension, the peptides are separated into fractions.
Each of the selected fractions is then injected into
• Mix samples for isolation or enrichment of some
cellular fractions
the second LC for further separation before injection
into the mass spectrometer (MS) for detection.
• Sample can be analyzed by PAGE or 2D LC
LC-LC/MS methodologies have the advantage
• The fractions can be analyzed in mass-spec for
identification of being capable of analyzing complex protein
mixtures; however there was previously no way to
iTRAQ
quantify different expression levels among samples
• Use isobaric tag for different samples
until recently, due to the application of stable-isotope
2D LC labeling to LC-LC/MS. This method makes use of the
• Protein mixture is subjected to proteolytic digestion fact that pairs of chemically-identical analytes with
peptides are amenable to LC separation to reduce different isotope compositions can be differentiated
complexity in a mass spectrometer by their difference in mass-to-
• Column types: First dimension often exchange, second charge ratio. The ratio of signal intensities for the pair
dimension reverse phase accurately indicates an abundance ratio for the two
• Electrospray injection into mass spectrometer analytes. Two commonly used labeling techniques
• For identification of molecules, not quantification are SILAC (Stable Isotope Labeling by Amino Acid
in Cell Culture), and iTRAQ (Isobaric Tagging
for Relative and Absolute Protein Quantitation).
Although all proteins in a sample are, technically,
included in the analysis using non-gel-based
techniques, these methods are still not a true global
surveying tool. In actuality, only a few hundred to a
few thousand proteins are identified in the analysis
of a sample due to limitations in the resolving
power of liquid chromatography. The capture
of a peak from a sample is stochastic, and highly
abundant proteins are detected over those present
at lower levels. The isolation and identification
of low abundance proteins can be enhanced by
repetitively analyzing the same fractions in the mass
spectrometer and by excluding peptides that have
already been identified in the previous analysis. This
is very expensive and tedious so exhaustive surveys
Fig. 14.7: iTRAQ labeling of proteolytic peptides for 2-D liquid of the proteome space are not commonly practiced.
chromatography.

ITRAQ Chemical isotope labeling of proteins in vitro
(after protein isolation) using isobaric Tags for
Relative and Absolute Quantification (iTRAQ) is a
highly versatile and widely-used method. iTRAQ
tags have a reporter group, a balancer group, and
a peptide reactive group that tags the N-terminus
of every peptide. One of the unique features of
iTRAQ is that up to eight samples can be labeled
with different tags and analyzed simultaneously,
while other methods can only label two samples.
The combined balancer and reporter groups have
an identical mass of 145 for all four labeling pairs of
balancers (mass 28 to 31) and reporters (mass 114-
117). The MS spectra for each of the four labeled
samples look identical. The reporter-balancer
Fig. 14.8: Isotopic labeling of peptides.
fragment stays intact, giving rise to the same
m/z ratio. This allows for protein identification
using the combined signal of the four samples.
Upon MS/MS fragmentation, the bond between
the reporter and balancer group is broken. The
reporter groups then appear as peaks in the low
mass region, and quantification of the peak area for
each reporter group gives the relative abundance
for a peptide between the labeled samples.
In vivo labeling methods, such as Stable Isotope La-

SILAC
beling with Amino acids in Cell culture (SILAC), use
deuterated leucine, or other isotope labeled amino
acids, to differentially label one of the protein sam-
ples by replacing an amino acid in the cell culture me-
dium, thereby allowing the isotope to be incorporat-
ed into the cellular proteins. The combined labeled
and unlabeled samples are then analyzed by LC-LC/
MS, generating two spectra for each peptide frag-
ment, one shifted precisely by the mass of the deu-
terated amino acid. Differences in peak height pro-
vide the means of quantification between samples.
This method allows two samples to be mixed prior
Fig. 14.9: Stable isotope labeling of amino acids in cell culture to protein isolation, thereby eliminating systematic
(SILAC) for proteomics.
errors due to protein isolation efficiencies. It is
well suited for use when subcellular enrichment
protocols are used (as in the case of organelle
fractionation) prior to LC-LC/MS analysis. One

notable limitation of this method is that the labeling
time required is relatively long, to allow for sufficient
incorporation of the isotope into cellular proteins.
Sequencing Technologies
Sanger Method and Sequencing DNA sequencing technology has undergone
Technology Evolution revolutionary changes in the past few years.
Traditional Sanger sequencing has dominated
the field for nearly three decades, and it is still
the prevailing method used for sequencing small
regions of DNA. For large-scale, or genome-wide
sequencing, a number of high throughput methods
have rapidly changed the scope of sequencing.
They are now used for genome sequencing,
transcriptome profiling, transcription initiation
Table 7. Summary of Sequencing Methodologies site surveys, transcription factor binding site
Technology Read Characteristics Applications profiling, and epigenetic alteration studies.
Sanger Sequencing 500-1000 bp 384 Gold standard all
(ABI) reads/run purpose sequencing Sanger sequencing gives relatively long reads,
Roche Sequencer
FLX/454
400 bp/read
1-7 Gbp/run
Re-sequencing,
expression profiling,
while newer methods give shorter reads. Some
SNP/methylation of the reads are too short for efficient assembly
Illumina/Solexa 36-150 bp/read Re-sequencing,
20 Gbp/run expression profiling,
and are used primarily for “resequencing”, i.e., for
SNP/methylation sequencing the genome of individuals of a species
SOLiD (ABI) 30-50 bp/read Re-sequencing,
20 Gbp/run expression profiling,
whose genome sequence is already available. Other
SNP/methylation newer methods, such as 454 and Illumina, produce
Helicos 35 bp/read Re-sequencing,
20 Gbp/run expression profiling, reads that are at least long enough for assembly.
SNP/methylation
All of the new methods and the Sanger method share
the same “reading” scheme. Each time a nucleotide
is incorporated into an elongating DNA molecule a
• Target DNA template is used for DNA synthesis signal is emitted. Two approaches are adopted to
• Fluorescently labeled nucleotide analogues detect the emitted signal, (a) amplifying the signal
(dideoxynucleotides) is added to the synthesis by having many DNA molecules emitting the same
reaction; wherever an analogue is incoporated into the signal, (b) using very sensitive detection methods to
elongating DNA, the reaction terminates detect even the signal emitted from a sigle molecule.
• Each of the four dideoxynucleotide chain terminators
is labelled with a different florescent dye. In Sanger sequencing, each target DNA molecule is
first cloned into E. coli so that a sufficient amount
• The newly synthesized and labeled DNA fragments
are separated by size (with a resolution of just one of pure DNA molecules can be obtained by growing
nucleotide) the E. coli clone. Those DNA molecules are then used
as templates for DNA synthesis. By using altered
nucleotide analogues that cannot be used in DNA
synthesis, the elongation stops randomly as soon as
an analogue (instead of the genuine nucleotide) is
incorporated. Given a proper titration of the ratio

of analogues to normal nucleotides, there will be a
DNA molecule terminated at every base of the DNA
template. After synthesis, the mixture is elongated
and the terminated molecules are separated by
chromatographic separation at a single base
resolution. Since each analogue is labeled with a
fluorescent color, the colorimetric chromatogram
is finally used to read off the sequence.
This clone handling is very expensive and
time consuming. Furthermore, single-base
resolution can be accomplished only up to at
most 1.2 kbp, even with capillary electrophoresis.
Fig. 14.10: Conventional Sanger sequencing of DNA.
Sanger Sequencing
• Enrich target DNA fragments by cloning into a E. coli
plasmid
- Specific primers at two ends of target DNA
Collect plasmids, use specific primer to start
DNA synthesis from one end
• Add nucleotide analog, ddNTPs in addition to dNTPs at
low frequency in the reaction mixture
• Incorporation of a ddNTP terminates DNA elongation.
• Probabilistically a small fraction of elongating DNA
is terminated at every base, resulting a collection of
synthesized DNA fragments of different length
• Separate those DNA molecules using capillary
electrophoresis
• Each of the four ddNTPs fluoresce at a different
wavelength. As each DNA fragments comes out of
electrophoresis, the fluorescence is “read” to give the
identity of the base

The most fundamental change from the Sanger
High Throughput NextGen Sequencing method to newer methods is the omission of E. coli
clones. Two approaches were taken to bypass cloning.
Next Generation Sequencing Technologies In the first approach, localized PCR amplification is
performed: a single DNA fragment is amplified in
• Massively Parallel Local Amplification
a droplet or in a locus on a surface. This allows a
• ‘Sequencing-by-Synthesis’ large number of identical DNA fragments to be used
to generate signals for detection as they incorporate
nucleotides when used as templates for synthesis.
In the second approach, single molecule detection is
achieved. As long as each molecule is separated from
the others, the signal from nucleotide incorporation
into an elongating DNA can be detected. The
increased sensitivity eliminates the need for cloning.
The current prevailing methods, sometimes
referred to as “next generation sequencing
technologies”, fragment the DNA molecules
randomly without resorting to cloning. A key
feature of these methods is the massive and parallel
amplification of each DNA fragment individually,
thus creating up to millions of clusters, or
colonies, of DNA molecules of the same sequence.
With the 454 technology, each fragment is
immobilized on a bead contained in an oil /
water emulsion droplet, thus forcing all of
the PCR products of that particular fragment
to also be immobilized on the same bead.
On the other hand, the Illumina Solexa sequencing
technology immobilizes the fragment on a
solid surface and confines the PCR products of
the fragment in the locale, thereby forming a
cluster of identical sequences on the surface.
Fig. 14.11: Two strategies of localized PCR amplification of DNA
molecules for enhanced signal detection. These ingenious approaches allow a large number
of identical sequences to be isolated so that the
light emitted from reactions in these clusters
can be detected using a single sensor, such as a
CCD camera. This contrasts with the traditional
Sanger method, which requires a sensor at
the end of each electrophoretic capillary, thus
drastically reducing the cost of sequencing.
Both methods then employ base-by-base synthesis.
The 454 technology works by sequential addition of

one of the four nucleotides and photons are emitted
454 Sequencing Technology upon the incorporation of a base. The Illumina
• Immobilize one DNA fragment per bead Solexa methodology employs fluorescently-labeled
nucleotides that are also reversible terminators.
• Suspend each bead in an emulsion droplet
One base is incorporated and interrogated at
• PCR amplify DNA in each bead a time, since further elongation of the chain is
• Place each bead in one well prevented. When all clusters are scanned at the
• Perform synchronized synthesis by adding one end of a cycle and the base has been determined
nucleotide at a time for each colony, the fluorophores are cleaved
• Each nucleotide incorporation emits a photon off and terminating bases are activated, thereby
• Homo-oligomer emits stronger light intensity (works allowing another round of nucleotide incorporation.
only for short homo-oligomer sequences) The drawback of the current generation of these
• Can sequence up to 1000 bp in length new sequencing technologies is their reliance on the
complete synchronization of serial reactions. The
Illumina Sequencing Technology
reads obtained are relatively short, compared to those
from Sanger sequencing. However, this drawback
• Immobilize DNA fragments on surface with adaptor
is more than compensated by their improved
• Localize PCR reaction to create clusters of DNA capability of massively paralleled “reading” of
fragments with complementary strands
millions of sequences at high speeds and at relatively
• Perform synchronized DNA synthesis from one strand, low costs. Their tremendous parallel sequencing
then on the other strand
abilities allow for up to a million fragments of a very
• Upon the extension of one base, the reaction is abundant mRNA species (such as the transcript for
terminated because the functional group for further
the recombinant protein product) to be “read” in a
extension of the added nucleotide is protected; this
prevents incorporation of more than one nucleotide single run, while only single reads can be obtained
when homo-oligomer sequences are encountered for thousands of rare genes. Such a dynamic range
in sequencing outputs has made them great tools
• Can sequence up to over 100 bp; very high throughput
for assessing transcriptome-wide abundance levels.

Gene Expression Exploration in Cell Culture Processing
Transcriptome analysis is a powerful tool for
studying cell culture processes, as it is extremely
revealing for both subtle and not-so-subtle
changes that can occur in many situations (e.g.,
cell cultivation, the variation of cell behavior over
time, and cell line development). It has been used
to compare cell performance in different reactor
scales, cells of varying productivities, and cells
growing under different media composition, and
to probe cell changes after long-perfusion culture.
In a striking example, a cell line was subjected to
studies under differing culture conditions. The
variables included temperature, pH, and reactor
size. There was also an uncontrolled variable. Two
different lots of the same hydrolysate were used in
each run, as is customary (when one lot of material
is exhausted, another lot is used). The time-course
transcriptome data from all runs were collected and
subjected to clustering. All the runs with the same lot
of hydrolysate were found to be clustered together,
thus overriding the other variables of the experiment.
Such revelation is possible because of the large
number of genes probed. Many subtle changes which,
Fig. 14.12: Comparative transcriptome dynamics between individually, may not be used to draw a confident
(a) mouse hybridoma cells treated with butyrate and (b)
differentiating mouse stem cells (MAPC). MAPC cells were
conclusion, can collectively point to a trend or
grown on liver lineage differentiation medium for 6 days. correlation that is not otherwise easily discernible.
Mouse hybridoma cells were treated with 1mM butyrate for 27
h. Both MAPC and hybridoma cell samples were referenced to There has been some effort in comparing cells of
a corresponding, untreated time sample. The transcript levels varying levels of productivity. It has now been
were probed with the Affymetrix MOE430A array. realized that the “hyperproductivity trait” is a
For each dataset, average intensities are plotted along the y-axis,
and the log2 of the expression ratios are plotted on the x-axis. complex phenomenon that is not simply a result of
Each marker represents a gene on the array. The vertical lines turning on a small number of master genes. Rather,
mark the bounds of a two-fold expression change. Markers the generation of high producers is likely to involve
lying outside of these lines are more than two-fold up or down
regulated between the two samples compared. In comparing colossal changes in gene expression that occur in a
Figures 2a and b, the number of genes that are more than two wide range of genes, but each only to a modest extent.
fold differentially expressed is substantially higher for the stem cell
differentiation study than for the butyrate treated hybridoma cells. A most important feature of either sequencing- or
microarray-based transcriptome analysis is its global
coverage of gene expression. This type of analysis
sometimes reveals unnoticed cellular processes that
may play a key role in some physiological events.
For example, it was found that the endocytosis and
secretory vesicle retrograde transport pathways

are upregulated in high recombinant protein-
producing cells. This led to the realization that
high producers have an increased capability
to recycle components of membrane vesicles
that carry proteins destined for secretion from
the Golgi apparatus to the cytosolic membrane.
One of the important features of transcript
profiling in cell culture processes is the small
number of genes differentially expressed and
the lower magnitude in the observed fold-
changes, as compared to those observed in other
biological processes. For microbial cells, it is not
unusual to observe more than 20% of the genes
as differentially expressed by more than two-fold
within ten minutes of changing culture conditions.
For cultured mammalian cells, it is rare to see over
two-fold expression changes occurring in more
than a small percentage of the genes surveyed.
It is important to realize that the cells we deal with
are cultivated in artificial conditions, not in their
native niche. For an organism the development
of a fertilized egg to an adult body incurs many
major events and gene expression changes to
guide those events. Once the cell is guided to its
destined developmental status, the perturbations
Fig. 14.13: Intracellular processes differentially expressed of other environmental factors are relatively
between high and low producing NS0 cell lines. (a) Each node minor in magnitude by comparison. The various
depicts an intracellular process with a large number of differ-
entially expressed genes. (b) Schematic of the steps involved in
events that cells in culture may encounter are
vesicle-mediated transport (nodes 4, 5, and 6). only relatively small perturbations compared to
developmental or differentiation events that their
genome has been evolved to accommodate. It is
not surprising that colossal big changes in gene
expression are rarely seen in cell culture bioprocess
but are frequently seen in stem cell differentiation.
The small change in gene expression, as compared
to other organisms and to the in vivo processes,
requires one to be more versatile and more
skillful in data analysis. However, the power of
global transcriptome analysis will fundamentally
change our practice in cell culture processing.
nor perturbations in comparison to development.
The small degree of gene expression changes, as

compared to other organisms and to the in vivo pro-
cesses, requires one to be more versatile and more
skillful in data analysis. However, the power of glob-
al transcriptome analysis will fundamentally change
our practice in cell culture processing.
Concluding Remarks
In the past few years we have seen dramatic advances way we deal with bioprocess challenges. The
in genome science and technology. The availability application of “-omics” technology in cell culture
and affordability of sequencing technology has processing is still largely limited to process analysis.
changed sequencing effort from species sequencing One can foresee that, in a not too distant future,
(i.e., focusing on obtaining a “representative genome “-omics” technology will be increasingly used for
sequence”), to individual sequencing (i.e., aimed to the generation of producing organisms, as well
acquire genome sequence of an individual human, as in the design of biological processes. In some
organism or cell line). The depth of information ways, genomics science may also bring about
we are acquiring from genome, transcriptome, transformative changes in cell culture bioprocessing.
proteome and epigenome, is transforming the

Index Alginate microcarriers, 205t
Allosteric regulation, differential, of glucose metabolism, 75–76,
75f
Note: Page references followed by f or t indicate material in
Alternating tangential filtration, 260, 260f
figures or tables, respectively.
Alternative splicing, 285–286
Amino acid(s)
A
alterations, in bioprocessing, 16–17, 16t
ABC transporter, 43, 44f, 84
analysis of, 299–302
Abraham, Edward Penley, 2
in cellular growth, 150, 151, 151t
Abundant genes, 293, 293t
degradation of, 81–82
Acetylation of histones, 292–293, 292t
essential, 81, 108, 108t, 151
Acetyl CoA
in fedbatch culture, 238–240, 239f, 239t, 246–247
in cholesterol synthesis, 87
in intracellular fluid, 153, 153t
in lipid metabolism, 86, 86f
in medium, 81, 106–108
metabolism of, 81
metabolism of, 58, 81–82, 81f, 82f, 186
in tricarboxylic acid cycle, 61, 73
non-essential, 81, 108, 108t, 151
Acetyl CoA shuttle, 86, 86f
stock solutions of, 108
Acoustic resonance enhanced settling, 257–258, 258f
transport of, 40–41, 43, 81
Actin fibers, 37, 38–39, 38f, 46
Aminophospholipid translocases, 84
Active transport, 40–42, 41f
Ammonia, in cultured cell metabolism, 67, 236
Activity parameters, 156, 160f
Amphipathic lipids, 22–23
Adaptation, in stable cell lines, 131, 142, 142f, 143f
Amplification
ADCC. See Antibody dependent cellular cytotoxicity
localized, in DNA sequencing, 304, 304f
Adenosine, in medium, 109, 109t
for stable cell lines, 131–132, 138–141, 140f
Adenosine triphosphate (ATP)
Amplification maker, 135
in active transport, 40
Anaerobic glucose metabolism, 58, 65
consumption in glucose metabolism, 60–61
Aneuploid cells, 54, 133, 143, 150
mitochondrial production of, 29
Animal(s)
production in glucose metabolism, 60–64, 66, 70
as production vehicles, 199
Adenosine triphosphate synthase (ATP synthase), 61, 63
transgenic, 12, 14, 14t
Adherent cultures, vs. suspension cultures, 202
vaccines for, 8t, 9
Adhesion, cellular, 45–46, 53, 142
Animal component-free serum, 102–103
microcarriers for, 203–204, 204t, 205t
Animal serum, in medium, 102, 117–119
vs. suspension, 202
Anterograde transport, 34, 35, 35f
Aeration. See also Oxygen transfer, in bioreactors
Antibiotic(s)
stripping of carbon dioxide by, 219
declining price of, 2–3
surface, for oxygen supply, 220
development of and advances in, 2–4
Aerobic glycolysis, 65–66
in medium, 117, 117t
Affym etrix microarrays, 295–296, 295t
as selection markers, 137t
Agarose cell immobilization, 209
Antibody dependent cellular cytotoxicity (ADCC), 90
Aggregation, 206, 206f, 209, 209f, 212
Antibody products, 5–8, 7t
Agilent microarrays, 295t, 296
manufacturing of, 11–12
Agitation, in bioreactors, 265–266
quantity for administration, 11, 11t
impellers for, 206, 265–268, 265t, 266f
stoichiometric ratio in, 11
mechanism of, 265–266
Antibody synthesis, analysis of, 186
purpose of, 265
Antioxidants, in medium, 116
Airlift bioreactor, 207, 207f, 225
Antiporters, 41, 42f
Akt, in regulation of glucose metabolism, 77, 77f
Apf-1, 52
Alanine
Apoptosis, 51–53
in medium, 108
cell cycle and, 50f
metabolism of, 81–82
death receptor pathway in, 51–52
Albumin
mitochondrial pathway of, 52–53, 53f
production of, 13
morphological changes in, 51
recombinant, 122
vs. necrosis, 51
secretion of, 31
Apoptosome, 52
serum, in medium, 118, 122
Ascorbic acid (vitamin C), in medium, 109
INDEX | 309
Asparagine impellers for, 265–268, 265t, 266f
in medium, 108 mechanism of, 265–266
metabolism of, 81 purpose of, 265
Aspartate, metabolism of, 81–82 basic types of, 193–196
ATP. See Adenosine triphosphate batch processes in, 196–198, 197f
ATP-binding cassette (ABC) transporter, 43, 44f, 84 cell culture, 206–212
Attenuated vaccines, 4–5 cell retention in, 249–261. See also Perfusion culture
Autocatalytic growth, 156 cell support systems in, 202–206
Automation, for cell line production, 145–146, 145f continuous processes in, 196–197
Axial flow impellers, 206, 265–266, 265t, 266f disposable or single-use systems in, 199–202
Azacytidine, 293 fedbatch cultures in, 198, 198f, 212
mixing characteristics of, 193, 266–270
B mixing time in, 206, 268–274
Baby hamster kidney cells. See BHK cells operating mode of, 196–198
Bacterial genome, 287–288, 289t, 290 oxygen balance in, 220, 276–280
Bag-based centrifuge, 256 oxygen supply for, 220–228
Bag culture systems, 201–202, 202f by medium recirculation, 221f
Bak protein, 52 by silicon tubing/membrane, 220
Balance equations, 162–165, 175–180. See also Material balance by sparging, 220–228
Basal medium, 101 by surface aeration, 220
components of, 106–117 oxygen transfer in, 206–207, 213–231
optimal concentration of organic nutrients in, 110, 110f driving force for, 214–216, 220, 279–280
Base addition, proportional feeding with, 244 enhancing or improving, 216, 220, 221
Base-by-base synthesis, 304–305, 304f experimental measurement of, 224–225, 224f, 225f
Batch culture (processes), 233. See also Fedbatch culture hydrostatic pressure and, 225
in bioreactors, 196–198, 197f in immobilization reactor, 229, 229f
vs. continuous processes, 249–251 mass transfer coefficient in, 216
Bax protein, 52 objective of, 219
B cells, transition to plasma cells, 33, 33f, 130 in plug-flow reactors, 229–230, 230f
Bcl-2 proteins, 52 rate of, 215–216, 220
Bcl-xL protein, 52 scaling up and, 275–280, 280f, 280t
Beef extract, in medium, 124 surface/interfacial area and, 216
Beta-carotene, in medium, 116 through gas-liquid interface, 212–219, 213f, 214f
Beta cells, pancreatic, 31 phases in, 196
BHK cells, 8t, 19t power consumption in, 266–270, 266f
adhesion of, 142 reactions and reaction kinetics in, 194–196
aggregates of, 209 scale translation for, 263–284
as continuous cell line, 49 and carbon dioxide removal, 275, 281–283
for transient expression, 129 and chemical environment, 283, 283f
veterinarian vaccines from, 8t constant parameters in, 269–270, 269t
Bicarbonate buffer, 113–114, 115f, 115t dimensionless variables in, 267–270
Biogen, IDEC ISM facility, 12 and driving force, 279–280
Biological fluids, in medium, 118 effect of scale on physical behavior, 269–270
Biologics, 127. See also specific types geometric nonsimilarity in, 264
Biomass, 147–148, 148t geometric similarity in, 264
growth of. See also Growth major effects of scale, 264
material balance on, 149–154, 149f and mechanical forces on cells, 274–275, 275f
as objective of medium, 97–99, 125 mixing time in, 271–274, 271t
overall synthesis equation for, 149–150 nutrient starvation time in, 271–274, 271t
intracellular fluid in, 153–154 objective of, 264
medium to generate, 103–104 and oxygen transfer, 275–280, 280f, 280t
metabolic flux analysis of, 184–185, 185f physical and mechanical parameters of, 264
as output of reaction, 176 Reynolds number in, 267–268, 267f
on substrate, yield of, 158 and superficial gas velocity, 278, 278f
Bioreactors, 191–212. See also specific types tracer concentration response in, 193–195, 194f
agitation in, 265–266 velocity of, 268, 278, 278f
INDEX | 310
volumetric flow rate in, 268–270 cDNA microarrays, 295, 295f, 298t
Biosimilars, 3, 9–10 Cell(s)
approval in Europe, 10, 10t chemical environment of, 21–22, 21t, 97–101
marketed in China, 11t composition of, 21–22, 21t, 148, 148t
marketed in India, 10t in culture. See Cultured cells
uncertainty about quality of, 10 death of, 51–53
Biotin, in medium, 109 apoptosis vs. necrosis, 51
BiP chaperone protein, 33, 35 cell cycle and, 50f
Bispecies-transporters, 41–42 consideration in growth rate, 157
Blasticidin S, as selection marker, 137t death receptor pathway in, 51–52
Blood bags, 201–202 from injury (necrosis), 51
Bok protein, 52 mitochondrial pathway of, 52–53, 53f
Bristol Myer Squibb, Devens (Massachusetts) facility, 12 morphological changes in, 51
Bubble size, in gas sparging, 222–223 diameter of, 21
Buffer systems, in medium, 113–115, 115f, 115t mass of. See Biomass
Bulk ions, in medium, 111–112, 111t movement of, 46–47
nutritional requirements of, 97–99. See also Medium
C senescence of, 53–55
Calcium size of, 21, 148, 148t
intracellular and extracellular concentrations of, 100, 100t sources of, 19–21, 19t
in medium, 111, 111t volume of, variation in, 150–151, 150t, 151f
Calcium phosphate precipitation, 133–134, 134t Cell adhesion, 45–46, 53, 122–123, 142
Calculated differentiated data, 169 microcarriers for, 203–204, 204t, 205t
Calculated integrated data, 169 vs. suspension, 202
Carbohydrates, as cellular component, 21, 148 Cell adhesion molecules, in medium, 122–123, 123t
Carbon Cell aggregates, 206, 206f, 209, 209f, 212
in amino acid metabolism, 81–82 CellCube Module, 201
in cellular growth, 149–150 Cell culture engineering. See also specific processes and
flow or flux of, 67 materials
in glucose metabolism, 59, 61–67 advances and growth in, 1–4
in glutamine metabolism, 59 process robustness in, 14–17
metabolic flux analysis of, 182f, 183 Cell culture products, 4–12. See also specific products
in pentose phosphate pathway, 64–65 alternative technologies for, 12–14
Carbon dioxide biosimilar or follow-up biologics, 3, 9–10, 10t, 11t
in buffer system, 113–114 in-process structural alterations to, 15–17, 16t
cellular tolerance of, 281 manufacturing of, 11–12
concentration in medium, 217–218 from perfusion bioreactors, 249t
in glucose metabolism, 60, 61, 66 quality of, 14–17
production of, 219 Cell cycle, 47–49, 48f
removal in bioreactors, 219 and apoptosis, 50f
scaling up and, 275, 281–283 checkpoints in, 47–49
transfer (diffusion) of, 212–219 cyclins and CDKs in, 48–49
g-Carboxylation, 12, 14, 16 positive and negative cues in, 47–48
Carrier-mediated diffusion, 40–42, 41f Cell expansion. See also Growth; Growth control
Carrier proteins medium for, 97–99, 125
in medium, 117, 118, 122 Cell lines
transport by, 122, 122t adaptation of cells in, 131, 142, 142f, 143f
Caspases, in apoptosis, 51–53 amplification for, 131–132, 138–141, 140f, 141f
Catabolism automation and high throughput technology for, 145–146,
of glucose, 60 145f
of lipids, 36 basic steps for generating, 131–133, 132f
in metabolic flux analysis, 188t continuous, 49, 143–144
Catalase, in medium, 116 development of, 127–146
Catalytic macromolecular components, of medium, 105–106 gene expression in, 306–308, 306f, 307f
CDK4/6-cyclin D complex, 48–49 genomics and, 146, 306–308, 306f, 307f
CDK inhibitors (CDI), 48–49 host cells for, 127–129
INDEX | 311
hyper-producing, 129–133, 130t, 131f recombinant proteins from, 8t
immortalization of, 54 for stable expression, 129–130, 130t, 146
industrial, 8t, 9 therapeutic antibody products from, 7t
screening for, 131 for transient expression, 129
selection of cells for, 131–132 volume of, 150t
single-cell cloning for, 131–133 Chloride ions
sources of, 19–20, 19t intracellular and extracellular concentrations of, 100, 100t
stability of clones selected for, 143–145 in medium, 104
stability of product quality in, 144–145 transport of, 43–45
stable, 127, 129–146 Chlortetracycline, in medium, 117
transfection for, 131–137 CHO. See Chinese hamster ovary cells
veterinary, 8t, 9 Cholesterol
vs. cell strains, 53f, 54 biosynthesis of, 86–88, 88f
Cell membrane, 22–27 function of, 83, 86–87
composition of, 22–25 in lipid bilayer, 24–25, 24f, 83, 87
dynamic nature of, 23, 26 structure of, 85, 85f
homeostasis of, 26 turnover rate of, 26
lipid bilayer of, 22–27, 83, 87 Choline, as backbone of phospholipid, 22
potentials across, 25–26, 25t Chondroitin sulfate, in extracellular matrix, 46
protein content of, 25 Chromatin
transport across, 23, 26–27, 39–47 DNA packaging into, 290, 290f
turnover rate of, 26 modifications of, 292–293, 292t
Cell pool, 132–133 Chromium, in medium, 112
Cell recycling, 250–251 Chromosome(s)
Cell retention abnormalities, in cell line, 143–144
methods of, 249–261 in cultured cells, 54–55
in perfusion culture, 249–261 in mammalian genome, 288
Cell separation methods, 254–261 Cis Golgi, 32, 33
Cell signaling, 45–46 Cisternae maturation model, 33
Cell strain, 53f, 54 Citric acid cycle. See Tricarboxylic acid cycle
Cell support systems, 202–206 City water, contaminants in, 106
Cellulose, for microcarriers, 203–204, 205t Clonal growth, 110
Centrifugal cage, 259, 259f Clones
Centrifugation, 256, 256f, 257f in Sanger sequencing, 302–303
Centritech Lab centrifuge, 256 selected for cell lines, 131–132, 143–145
Ceruloplasmin, in medium, 116 Cloning, single-cell, 131–133
Channel-mediated diffusion, 40–42 CMV promoter, 135
Chaperone proteins, 33, 35, 90 cMyc, in regulation of glucose metabolism, 77, 77f
Checkpoints, in cell cycle, 47–49 Cobalamin, in medium, 109
Chemical environment Cobalt
of bioreactors, scale translation and, 283, 283f in intracellular fluid, 153
of cells, 21–22, 21t, 97–101 in medium, 112
Chemically defined medium, 102, 103 Codon optimiziation, 135
Chemical reaction systems Collagen
cellular system vs., 175–176 in extracellular matrix, 45–46
material balance for, 175–180 in medium, 123t
Chick egg, 4, 9, 19 for microcarriers, 204, 204f, 205t
China, biosimilars marketed in, 11t Combustion, 61–63, 176–178
Chinese hamster ovary (CHO) cells, 8t, 9, 19t, 20 Compartmentalization, 184
adhesion of, 142 Complex glycans, 92, 92f
aggregates of, 209, 209f Complex medium, 102
as continuous cell line, 49 Complex supplements, in medium, 117–125
doubling time of, 154t Concentration factor, in perfusion culture, 254
genome of, 288, 289t Concentration gradient
on microcarrier, 203f, 204 across cell membrane, 21–22, 21t, 40–42, 43–45
non-antibody products from, 6t across mitochondria, 29–30, 30f
INDEX | 312
Conditional promoters, 134–135 Data analysis, 167–174
Conical settler, 254–255, 254f Data processing, 167–174
Constitutive promoters, 134–135 cell culture, 169–171
Contact inhibition, 46, 48, 53, 54f fedbatch culture, 160, 160f
Continuous cell lines, 49 mapping data to pathways, 173, 173f. See also Metabolic flux
Continuous processes, 249–251 analysis
advantages of, 250 pipeline for, 168–173
in bioreactors, 196–197 spreadsheets for, 168, 170–171
with cell retention, 251. See also Perfusion culture standardized templates for, 168–169
disadvantages of, 250 Data visualization, 172, 172f, 174f
economics of, 250 Death, cellular
flow rate vs. growth rate in, 250 death of, 51–53
Continuous stirred tank reactor (CSTR), 193–196, 193f, 212 apoptosis vs. necrosis, 51
reaction and reaction kinetics in, 194–196 cell cycle and, 50f
tracer concentration response in, 193–195, 194f consideration in growth rate, 157
Copper death receptor pathway in, 51–52
in intracellular fluid, 153 from injury (necrosis), 51
in medium, 112 mitochondrial pathway of, 52–53, 53f
COS cells, for transient expression, 129 morphological changes in, 51
Co-transporters, 41–42, 42f Death phase, of cell growth, 154–155, 154f
Cow pox vaccine, 4, 199 Death rate, specific, 157
CpG islands, 292 Death receptor pathway, 51–52
CRFK cells, 8t Decline phase, of cell growth, 154–155, 154f
Crisis, 54 Delivery of feed medium, 245–247
Cross filtration model, 259, 259f Deoxyribonucleic acid. See DNA
CSTR. See Continuous stirred tank reactor Derived parameters, 160, 160f
Cultured cells Dextran
crisis of, 54–55 in medium, 116t
epigenetic changes in, 291–293 for microcarriers, 203–204, 205t
growth of. See Growth DH82 cells, 8t
Hayflick’s phenomenon and, 54–55 DHFR amplification system, 138–141, 140f
life span of, 54–55 Differential allosteric regulation, of glucose metabolism, 75–76,
medium for. See Medium 75f
metabolism of. See also Metabolism Differentiated data, calculated, 169
glucose, 58–70 Diffusion
overview of, 58–59 in bioreactors, 212–219, 213f, 214f
passages of, 53–55 carrier-mediated, 40–42, 41f
stoichiometry and kinetics of, 147–166 channel-mediated, 40–42
Cumulative data, 171 facilitated, 40–42
Cyclins, 48–49 Dihydroxyacetone-phosphate (DHAP), 61
Cylinders on shakers, 201–202 Dilution rate, in perfusion culture, 254, 254f
Cytidine, in medium, 109t Dimensionless variables, in scale translation, 267–270
Cytochrome C Diploid cells, 53–55, 143, 150
in apoptosis, 52 Diploid chromosomes, 290
in electron transfer chain, 63 Direct DNA sequencing, 295
Cytokine(s) Direct measurement, for fedbatch culture, 243
in medium, 97–98 Disc centrifuge, 256, 256f
quantity for administration, 11 Disposable bag-based centrifuge, 256
Cytoplasm, 27–36 Disposable cell culture systems, 199–202
Cytoskeleton, 27, 37–39 Dissociation, of cells from surface, 53–54, 54f
Cytosol, 27–29 Disulfide-bond formation, in protein therapeutics, 5, 14
acetyl CoA shuttle in, 86 DNA
lipid metabolism in, 83, 85, 86, 87 as cellular component, 21
location in cell, 28
D methylation of, 292–293
Data, types of, 169 mitochondrial, 29–30
INDEX | 313
modifications of, 291–293 Epithelial cells
packaging into chromatin, 290, 290f movement of, 46–47
synthesis of, 28, 47 use in bioprocessing, 19–20, 19t
DNA–calcium phosphate Co–precipitation, 133–134, 134t EPO. See Erythropoietin
DNA microarrays, 294–297, 298t ER. See Endoplasmic reticulum
cDNA, 295, 295f, 298t Erythropoietin (EPO)
layer-by-layer synthesis in, 295, 295t, 296f development of, 5
oligonucleotide-based, 295–296, 298t glycan and, 89
for pathway-related data, 173 product quality and process robustness, 15
RNA-seq, 296–297, 298t quantity for administration, 11
for two-channel comparison, 296 Escherichia coli
DNA sequencing, 288–289, 302–305 genome of, 28, 290
in cell culture processing, 306–308, 306f as host system, 12
direct, 295 in Sanger sequencing, 302–303
evolution of technologies for, 302–303 ESI. See Electrospray ionization
next generation technologies for, 304–305 Essential amino acids, 81, 108, 108t
Sanger method of, 302–303, 302t, 303f ESTs. See Expressed sequence tags
Dog (MDCK) cells, 8t, 9, 19–20, 19t Ethanol, transport of, 39
Dominant marker, 136 Euchromatin, 290
Doubling time, 154–157, 154t Eukaryotes
Driving force, for oxygen transfer, 214–216, 220 chromosomes of, 290, 290f
scaling up and, 277, 279–280 gene structure in, 286
Dry mass, of cells, 148, 148t genome of, 287–288, 289t
European Union, biosimilar approval in, 10, 10t
E Ex-Cyte, 124
E. coli. See Escherichia coli Exocytosis, 39–40
E2F transcription factor, 49 Exons, 286
ECM. See Extracellular matrix Exponential phase, of cell growth, 151f, 154–155, 154f
Eddies, in bioreactors, 274–275, 275f Expressed sequence tags (ESTs), 294
EF-1. See Elongation factor 1 Extended fedbatch culture, 235, 235f
Electron transfer chain, 61, 63–64, 66 Extracellular fluid, 100–101, 100t
Electroporation, 133–134, 134t Extracellular ion concentration, 21–22, 21t, 100–101, 100t
Electrospray ionization (ESI), 299 Extracellular matrix (ECM), 45–46
Elongation factor 1 (EF-1), 135 composition of, 45–46
Endocytosis, 26–27, 36 functions of, 45–46
Endoplasmic reticulum, 27, 31 Extracellular matrix extract, in medium, 123
expansion of, 33
glycan biosynthesis in, 93 F
glycosylation in, 90 Facilitated diffusion, 40–42
lipid metabolism in, 83, 88f F actin, 39
protein secretion through, 31–35, 35f, 83 Factor VIII
rough, 31 in-process structural alterations to, 16
smooth, 31 recombinant, 5
Endosomes, 27 tissue-derived, 5
Endothelial cells, volume of, 150t FADD. See Fas-associated death domain
Enzyme(s) FADH2, from glucose metabolism, 63, 68
in glucose metabolism regulation, 74–76, 75f FANTOM. See Functional Annotation of the Mammalian genome
Michaelis–Menton kinetics of, 41 Fas-associated death domain (FADD), 51–52
Epigenetics Fatty acids
in cell culture, 291–293 in lipid bilayer, 23–24
definition of, 291 in medium, 84
inhibition of, 293 metabolism of, 85–86
mechanisms of, 292–293 oxidation of, 36
molecular mechanisms mediating, 291–293 saturated, 85
Epigenome, 291–293 transport of, 39, 84, 122
Epigenomics, definition of, 291 FBS. See Fetal bovine serum
INDEX | 314
Fedbatch culture, 233–247 Folding, of proteins, 32–33, 89–91
in bioreactors, 198, 198f, 212 Follow-up biologics, 3, 9–10
control objective and criteria in, 241–243 approval in Europe, 10, 10t
control strategies for, 241–243 uncertainty about quality of, 10
data processing and plotting of, 160, 160f Formalin, viral inactivation with, 4, 9
delivery of feed medium in, 245–247 Fortified feed and addition culture, 235, 235f
feeding parameters in, 245 454 sequencing technology, 304–305
feeding strategy for, 241–245 Friction factor, in scale translation, 267
direct measurement of nutrient consumption, 243 Fructose
proportional with base addition, 244 in glycan biosynthesis, 93–94
proportional with oxygen uptake rate, 245 in medium, 108
proportional with turbidity, 244 transport of, 41, 71
fortified feed and addition, 235, 235f Fructose 1,6-bisphosphate (F16BP), 75, 75f
habitation-conducive components of, 104 Fructose 2,6-bisphosphate (F26BP), 77
for industrial production, 125 Fructose-6-phosphate, 67
intermittent harvest and feed, 198, 198f, 234, 234f FS-4 cells, 19t
lactate and production in, 59 Functional Annotation of the Mammalian genome (FANTOM),
medium for, 233, 237–240 286–287
for consumed nutrients, 238–240, 239f, 239t Fungus, genome of, 288
for unconsumed components, 240 Fusion proteins, 8
with metabolic state manipulation, 236–237, 236f, 236t
metabolic stress in, 161, 161t G
on-line estimation of stoichiometric feeding in, 246–247 G1 phase of cell cycle, 47–48, 48f
productivity and product quality of, 241–243 G2 phase of cell cycle, 47–48, 48f
for stable cell lines, 130 G actin, 39
stoichiometric ratio in, 160f, 236–240, 236f, 236t, 239f, 239t, Galactose
246–247 in glycan biosynthesis, 93–94
types of, 234–237 in medium, 108
Fermentation technology, 1 transport of, 71
Fermentors, 206–207 Gangliosides, in lipid bilayer, 22, 24
large-scale, 264f GAPDH. See Glyceraldehyde dehydrogenase
Ferrous ion, in medium, 111 Gap phase of cell cycle, 47–48, 48f
Fetal bovine serum (FBS), 118 Gas-liquid interface, oxygen transfer through, 212–219, 213f,
Fetuin, in medium, 123t 214f
Fibroblast(s) Gas phase, in bioreactor, 196, 276, 278–279
doubling time of, 154, 154t Gas sparging, 220–229
human vs. chicken embryo, 19 damage to cells by, 226–228, 226f, 227f, 228f
life span in culture, 54 direct, 221
on microcarrier, 203f orifice and bubble size in, 222–223
use in bioprocessing, 19–20, 19t Gelatin, for microcarriers, 203–204, 205t
vs. epithelial cells, 20 Gene(s), 285–287
Fibroblast growth factor, 48 abundant, 293, 293t
Fibronectin alternative splicing of, 285–286
in extracellular matrix, 45 coding for non-protein RNA, 285–286
in medium, 123, 123t coding for proteins, 285–286, 288
Filopodia, 38–39, 46 coding for RNA, 285–286
Filtration, 259–260 definition of, 285
FL72 cells, 8t environment and, 291–293
Fleming, Sir Alexander, 2 minimum set of, 287
Flippases, 84, 90 number of, 287, 289t
Flooding, in bioreactors, 280 rare, 293–294, 293t
Fluid structure in eukaryotes, 286
extracellular, 100–101, 100t Gene expression, 285
intracellular, 153–154, 153t in cell culture processing, 306–308, 306f, 307f
Fluidized bed bioreactor, 207 epigenetic regulation of, 291–293
Flux vectors, 179–180 proteome profiling of, 299–302, 299f
INDEX | 315
transciptome analysis of, 293–297 insulin in, 120
Genentech, Vacaville (California) facility, 12 lactate consumption in, 59, 78–80, 79f
Geneticin, as selection marker, 137t lactate conversion in, 58–59, 60, 65–66, 67
Gene transfer lipid metabolism and, 86
amplification in, 131–132, 138–141, 140f, 141f metabolic flux analysis of, 186
cell adaptation to, 142, 142f, 143f NADH balance in, 68–69, 69f
direction to transcriptionally active region, 141 reaction intermediates in, 67
methods of, 133–134, 134t regulation of, 74–78
promoters for, 134–135 differential allosteric, 75–76, 75f
selectable marker in, 135–137, 137t growth control and, 77, 77f
for stable cell line, 131–145 isozymes in, 74–76, 75f
for transient expression, 128–129 signaling pathways and, 77, 77f
Genome(s) transport in, 71–74
and complexity of organism, 287 Warburg effect in, 65–66
environment and, 291–293 yield of, 60–61, 70
eukaryotic, 28 Glucose oxidation, 58–64, 70f
mammalian, 287–289 Glutamate, metabolism of, 81–82
mitochondrial, 29–30 Glutamine
mouse, 285–287, 288, 289t, 293 in cellular growth, 150
organization of, 287–289, 287f in medium, 106–108, 149
prokaryotic, 28, 287–288 metabolism of, 58, 59, 67, 81–82
repetitive sequences in, 287–288 in regulation of glucose metabolism, 80, 80f
species comparison of, 287–288, 289t Glutamine oxidation, 58
Genome engineering, 3 Glutamine synthetase (GS)
Genome scale analysis, 293–297 for amplification, 138–141, 141f
Genomics, 3, 285–308 for glutamine synthesis, 108
cell line, 146, 306–308, 306f, 307f Glutathione
protein (proteome profiling), 299–302 reduced, in medium, 116
transcriptome, 293–297 reduction of, 64
Gentamicin, in medium, 117, 117t GLUT transporter(s), 71, 72t
Geometric-nonsimilar scale translation, 264 GLUT1 transporter, 40–41, 43, 71, 72t, 107
Geometric-similar scale translation, 264 GLUT2 transporter, 72t
Germanium, in medium, 112 GLUT3 transporter, 72t
Glass, as microcarrier, 203–204, 205t GLUT4 transporter, 43, 71, 72t, 77
Gluconeogenesis, 67 GLUT5 transporter, 41, 71, 72t, 107
Glucosaminoglycans, in extracellular matrix, 45–46 GLUT6 transporter, 72t
Glucose GLUT7 transporter, 72t
in cellular growth, 149–150 GLUT8 transporter, 72t
in fedbatch culture, 236–237, 238–240, 239f, 246–247 GLUT9 transporter, 72t
lactate ratio to, 158–159, 236–237, 236f, 236t GLUT10 transporter, 72t
in medium, 106–107, 149 GLUT11 transporter, 72t
metabolism of. See Glucose metabolism GLUT12 transporter, 72t
specific consumption rate of, 156 Glycan(s)
transport of, 39–43, 42f, 71–72, 71f, 72t biosynthesis of, 67, 89–96
Glucose-6-phosphate, 64, 67 diversity among species, 95–96
Glucose metabolism, 58–77, 62f, 70f effect/importance of, 89–90
aerobic, 65–66 extension in Golgi apparatus, 91–92, 91f, 93–94
amino acid metabolism and, 81–82, 82f and immunogenicity, 95–96
anaerobic, 58, 65 macroheterogeneity of, 89
ATP consumption in, 60–61 microheterogeneity of, 89, 92–93, 92f
ATP production in, 60–64, 66, 70 N-linked, 89–92, 90f, 91f, 174, 174f
carbon flow or flux in, 67 nucleotide sugar precursors of, 93–94, 93f, 94f
carbon production in, 59, 61–67 O-linked, 89
in culture, 149 types of, 92–93, 92f
in electron transfer chain, 61, 63–64, 66 visualization of data on, 174, 174f
glutamine and, 80, 80f Glyceraldehyde dehydrogenase (GAPDH), 135
INDEX | 316
Glycerol, as backbone of phospholipid, 22, 22f, 24 Growth curve, 154–155, 154f
Glycine Growth factors
as buffer in medium, 115t in cell cycle, 48, 49
in medium, 108 in cell migration, 46–47
Glycoforms, heterogeneity in, 89 in medium, 97–98, 117, 118
Glycolipids, in lipid bilayer, 22, 24 Growth hormone. See Human growth hormone
Glycolysis, 58–70 Growth rate, 53–55, 53f, 156–157
aerobic, 65–66 GS. See Glutamine synthetase
carbon flow and reaction intermediates in, 67 GTP-mannose, 67
in culture, 149 Guanosine, in medium, 109t
lactate consumption in, 78–80
metabolic flux analysis of, 186 H
pentose phosphate pathway as shunt from, 60, 64–65 Habitation-conducive components, of medium, 103–104
regulation of, 74–78 Hamster cells, 8t, 9, 19t, 20. See also BHK cells; Chinese hamster
transport and, 71–74 ovary cells
yield of, 60–61, 70 Hayflick’s phenomenon, 54
Glycosylation, 5, 12–14, 89–96, 90f Heavy metal ions, in medium, 112
diversity among species, 95–96 HEK 293 cells, 8t, 19t
multiple sites of, 89 adhesion of, 142
N-linked, 89–92, 90f, 91f aggregates of, 209, 209f
O-linked, 89, 90 for transient expression, 129
in secretion process, 33 Helicos sequencing technology, 302t
visualization of data on, 174, 174f Henry’s law, 217–218
Glycosyltransferases, 34 Heparin
Glycylglycine, as buffer in medium, 115t in extracellular matrix, 46
Glyeraldehyde-3-phosphate (G3P), 61 in medium, 123
Golgi apparatus, 27, 32–34 Hepatocyte(s)
classical view of, 32 cell membrane of, 25, 25t
compartments of, 32 endoplasmic reticulum of, 31
dynamic nature of, 32 size of, 21, 151
glycan extension in, 91–92, 91f, 93–94 Hepatocyte growth factor, 46–47
glycosylation in, 90 HEPES buffer, 115, 115t
protein secretion through, 32–35, 35f, 83 HepG2 cells, on microcarrier, 204, 204f
transport across, 33–34 Heterochromatin, 290
Green monkey kidney cells, 8t, 9, 19t, 129, 201 Heterogeneous bioreactor, 196, 196f
Growth Hexokinase (HK), in regulation of glucose metabolism, 75
autocatalytic, 156 hGH. See Human growth hormone
balance equations for, 162–165 High-mannose glycans, 92, 92f
cell death consideration in, 157 High-molecular-weight supplements, in medium, 117–125
doubling time of, 154–157, 154t High-performance liquid chromatography (HPLC), for fed-batch
kinetic model of, 162–165 culture, 243
mammalian cell, 154–155, 154f, 154t High throughput technology
material balance on, 149–154, 149f, 162–165 for cell line production, 145–146
medium for. See Medium for DNA microarrays, 296–297
multiplicative model of, 165 for DNA sequencing, 302–305
overall synthesis equation for, 149–150 for proteome profiling, 299–302, 299f
phases of, 151f, 154–155, 154f Histone, 28, 290, 290f
quantitative description of, 156–161 Histone modification, 292–293
Growth control HMG-CoA, 87–88, 88f
apoptosis in, 51–53 HMG-CoA reductase (HMGCR), 87–88, 88f
cell cycle and, 47–49 HMG-CoA synthase (HMGCS), 87–88, 88f
contact inhibition in, 46, 48, 53, 54f Holding time, in secretion, 32, 34
Hayflick’s phenomenon and, 54 Hollow fiber bioreactor, 208, 208f, 229
loss, in continuous cell lines, 49 Homeostasis, of cell membranes, 26
in regulation of glucose metabolism, 77, 77f Hormone(s)
telomeres and, 54–55, 54f in interstitial fluid, 100
INDEX | 317
in serum, 118 scale translation for, 263–284. See also Scale translation;
Host systems, 5–6, 12–14, 127–129. See also specific systems Scaling up
Hot spot integration, 141 In-process calculations, 169
HPLC, for fedbatch culture, 243 Insect cell culture, 12–13, 13t
Human growth hormone (hGH) Insulin
biosimilar, 9–10 in cell cycle, 48, 49
quantity for administration, 11 in glucose metabolism, 120
Humira, expiration of patent, 9 in glucose transport, 43, 71
Hyaluronic acid, in extracellular matrix, 46 in medium, 120–121, 240
Hybrid glycans, 92 mitogenic response to, 120
Hybridoma, 5 recombinant, 121
adhesion of, 142 as tissue-derived protein therapeutic, 5
gene expression in, 306, 306f Insulin-like growth factor(s), 48
growth of, 149 Insulin-like growth factor-1, in medium, 120–121
stoichiometric ratio and metabolic stress in, 161, 161t Insulin-like growth factor-2, in medium, 120
therapeutic antibody products from, 7t Integral cell concentration, 159, 159f
volume of, 150t Integrated data, calculated, 169
Hydrogen Interactive data exploration, 172
in cellular growth, 149–150 Interferon(s), as tissue-derived protein therapeutic, 5
transport of, 43–45 Intermediate(s), of reaction modes, abbreviation of, 189t
Hydrogen peroxide, 116 Intermediate filaments, 37, 38, 38f
Hydrolysates, in medium, 124, 240 Intermittent harvest and feed, 198, 198f, 234, 234f
Hydrostatic pressure, and oxygen transfer, 225 Interstitial fluid, 100–101, 100t
Hydroxylethyl starch (HES), in medium, 116t In-time measurement, for fedbatch culture, 243
Hygromycin B, as selection marker, 137t Intracellular fluid, 153–154, 153t
Hyper-producing cell line, 129–133. See also Stable cell lines Introns, 286, 287, 288
basic steps for generating, 131–133, 132f Ion(s)
characteristics of, 129–131, 130t, 131f bulk, in medium, 111–112, 111t
gene expression in, 306–308 intracellular vs. extracellular concentration of, 21–22, 21t,
100–101, 100t
I transport of, 43–45
IEF. See Isoelectric focusing (IEF) Ion channels, as transport mechanism, 40–42, 41f
IGF. See Insulin-like growth factor(s) Iron
IgG. See Immunoglobuin G free vs. bound form of, 45
Illumina sequencing technology, 302t, 304–305 in intracellular fluid, 153–154
Immobilization reactors in medium, 111, 112
agarose, 209 reactivity of, 45
oxygen transfer in, 229, 229f transport of, 45, 122t. See also Transferrin
Immortalization, 54 Iron chelators, 121, 121t
Immunogenicity, glycans and, 95–96 Isobaric Tagging for Relative and Absolute Protein Quantitation
Immunoglobuin G (IgG) (iTRAQ), 300–301, 300f, 301f
in cellular growth, 151, 151t Isoelectric focusing (IEF), 299
Fc fragment, in fusion protein, 8 Isoleucine, metabolism of, 81–82
secretion time of, 34f, 34t Isozymes, in glucose metabolism regulation, 74–76, 75f
Impellers, in bioreactors, 206, 265–268, 265t, 266f iTRAQ, 300–301, 300f, 301f
amount of fluid moved by (pumping), 268–270
constant tip speed, for scale translation, 269–270, 269t J
velocity of, 268 Jenner, Edward, 199
Inactivated vaccines, 4, 9
Incline settling, 255, 255f K
India, biosimilars marketed in, 10t Karyotype, variable, in cell lines, 143–144
Inducible promoters, 134 a-Ketoglutarate
Industrial cell lines, 8t, 9 in amino acid metabolism, 81
Industrial production in glucose metabolism, 61, 68, 80
bioreactors for, 192–193. See also Bioreactors in medium, 108
medium for, 125–126 Kinetics
INDEX | 318
in bioreactors, 194–196 Live attenuated vaccines, 4–5
of cell cultivation, 147–166 Liver, endoplasmic reticulum of, 31
model of growth, 162–165 Localized amplification, in DNA sequencing, 304, 304f
KL. See Mass transfer coefficient Logging data. See also Data processing
Krebs cycle. See Tricarboxylic acid cycle standardized templates for, 168–169
Low-density lipoprotein (LDL)
L in medium, 105–106
Lactate transport of, 84
accumulation in culture, 78, 236 Lymphoid cells, use in bioprocessing, 19–20, 19t
in fedbatch culture, 236–237, 246–247 Lysis, 157
metabolism of, 58–59, 65–66, 78–80 Lysosome, 36
consumption in glucose metabolism, 59, 78–80, 79f
correlation with productivity, 58f, 59 M
production in glucose metabolism, 58–59, 60, 65–66, 67 MA104 cells, 8t
specific production rate of, 156 Macroheterogeneity, of glcyans, 89
transport of, 41–42, 42f, 43, 72–73, 72f Macromolecules
Lactate dehydrogenase, 66, 67, 79 catalytic, in medium, 105–106
Lactate-to-glucose ratio, 158–159, 236–237, 236f, 236t transport of, 39–40
Lag phase, of cell growth, 154, 154f Macroporous microcarriers, 203, 204, 204f, 205t, 229
Lamellipodia, 38–39, 46 Madin-Darby bovine kidney (MDBK) cells, 8t
Laminins Madin-Darby canine kidney (MDCK) cells, 8t, 9, 19t
in extracellular matrix, 45 Magnesium ions
in medium, 123, 123t in fedbatch culture, 240
Large scale, translation for. See Scale translation; Scaling up intracellular and extracellular concentrations of, 21, 100,
Large-scale fermenter, 264f 100t, 153, 153t
LDL. See Low-density lipoprotein in medium, 111, 111t
Lehmann, Jürgen, 210 Malate-aspartate shuttle, 68–69, 186
Length, in scale translation, 264 MALDI. See Matrix-assisted laser desorption ionization
Leucine, metabolism of, 81–82 Mammalian cells. See also specific cells
Lipid(s) bioreactors for, 192–193. See also Bioreactors
amphipathic, 22–23 critical feature of rDNA proteins from, 14
catabolism of, 36 fragility of cells in, 22
in cell membrane, 22–27 genome of, 28
as cellular component, 21 growth of, 154–155, 154f, 154t
functions of, 83 limitations of, 14
in medium, 84, 125, 240 product quality and process robustness in, 14–17
metabolism of, 83–88 products from, 5–6, 6t
acetyl CoA shuttle in, 86, 86f transgenic animals in, 12, 14, 14t
subcellular localization of, 83 Mammalian genome, 287–288, 289t
transport of, 84 Manganese, in medium, 112
Lipid bilayer, 22–27, 83, 87 Mannose, in glycan biosynthesis, 90–94
characteristics of, 23 Manufacturing plants, 12
composition of, 22–25 Manufacturing processes, 11–12, 12f. See also specific processes
dynamic nature of, 23, 26 and products
function of, 25 Mass. See Biomass
permeability of, 23, 39 Mass spectrometry, in proteome profiling, 299–302
phase transition of, 23, 23f, 24 Mass transfer coefficient (KL)
transport across, 23, 26–27, 39–47 for carbon dioxide, 282
turnover rate of, 26 for oxygen, 216
Lipofection/lipid-mediated gene transfer, 133–134, 134t constant, in scale translation, 269–270, 269t
Lipoprotein(s) experimental measurement of, 224–225, 224f
in medium, 105–106 scaling up and, 277
transport of, 84 sparger orifice and bubble size and, 222–223
Liquid chromatography, 2D, 300–302 Material balance
Liquid chromatography/mass spectroscopy, 300 on cell growth, 149–154, 149f, 162–165
Liquid phase, in bioreactor, 196, 277–279 in fedbatch culture, 236–237, 236f, 236t, 238–240, 239f, 239t,
INDEX | 319
246–247 for production, 97–99, 125–126
on oxygen, in bioreactors, 220, 276–280 protective agents in, 116–117, 116t
on perfusion culture, 251–253, 252f, 253f protein-free, 103
for reaction systems, 175–180. See also Metabolic flux protein hydrolysates in, 124, 240
analysis serum albumin in, 118, 122
setting up equations, 178–179 serum-free, 102–103, 108, 109
systematic way to solve problems, 178 serum in, 102, 117–119, 240
Mathematical model for stem cells, 97–99, 117
of growth, 162–165 sugars and energy source in, 106–107
information needed for developing, 163 tolerance of deviation from optimum, 100–101, 101t
Monod and Monod derivatives, 163–165, 164f trace elements in, 111–112, 112t
purposes of, 162 transferrin in, 105, 116, 118, 121, 240
Matrigel, in medium, 123 types of, 101–103
Matrix-assisted laser desorption ionization (MALDI), 299 vitamins in, 109, 240
Matrix operations, 179–180 water in, 106
MDBK cells, 8t Membrane, cell. See Cell membrane
MDCK cells, 8t, 9, 19–20, 19t Membrane bioreactor, 208–209
MDR. See Multidrug resistance gene Membrane fusion, 39–40
Measurement data, 169 Membrane potentials, 25–26, 25t, 29–30, 30f, 43–45
Mechanical/acoustic trapping, 257–258, 258f Membrane stirred tank, 210, 210f
Mechanical damage protective agents, in medium, 116–117, Mercaptoethanol, in medium, 116
116t Messenger RNA (mRNA), 21, 286
Medial Golgi, 32, 33, 35f abundant, intermediate, and rare, 293–294, 293t
Medium in RNA-seq, 296–297
amino acids in, 81, 106–108, 238–240, 246–247 Metabolic flux analysis (MFA), 173, 173f, 175–187
animal component-free, 102–103 abbreviation of intermediates in, 189t
antibiotics in, 117, 117t biomass equations in, 184–185, 185f
basal, 101, 106–117 catabolism reactions for, 188t
buffer systems in, 113–115, 115f, 115t on cellular system, 181–186
bulk ions in, 111–112, 111t compartmentalization in, 184
carrier proteins in, 117, 118, 122 example of, 186
cell adhesion molecules in, 122–123, 123t general approach to, 182f, 183–185
for cell expansion, 97–99, 125 selecting reactions for, 183, 183f
chemically defined, 102, 103 solution and analysis in, 185
classical, composition of, 58 utility and limitations of, 181–182
complex vs. chemically defined, 102 Metabolic state, of culture, 158, 236–237, 236f, 236t
components of Metabolic stress, stoichiometric ratio as indicator of, 161, 161t
basic, 103–117 Metabolism. See also specific types
classes of, 103–106 amino acid, 58, 81–82, 81f, 82f
non-nutritional, 113 central, in cultured cells, 58–59
stochiometric vs. catalytic macromolecular, 105–106 glucose, 58–70
stochiometric vs. habitation-conducive, 103–104 lactate, 58–59, 65–66, 78–80
design for, 97–126 lipid, 83–88
for fedbatch culture, 233, 237–240 transport and transporters in, 71–74
for consumed nutrients, 238–240, 239f, 239t Methane combustion, 176–178
for unconsumed components, 240 Methionine sulphoximine (MSX), in glutathione synthetase
fundamental influence of, 97 amplification, 138–141
high-molecular-weight and complex supplements in, 117–125 Methotrexate (MTX), in DHFR amplification, 138–141, 140f
for industrial production, 125–126 Methylation
insulin and insulin-like growth factors in, 120–121, 240 DNA, 292–293
lipids in, 84, 125, 240 histone, 292
nucleosides in, 109 Methylcelluloses (MC), in medium, 116t
objectives of, 98 MFA. See Metabolic flux analysis
optimal concentration of organic nutrients in, 110, 110f Micelles, 22–23
optimization of cell growth environment in, 97–101 Michaelis–Menton enzyme kinetics, 41
osmolarity of, 111–112 Microarray analysis. See DNA microarrays
INDEX | 320
Microcarriers, 203–204, 205t Myelin membrane, protein content of, 25
characteristics of, 204t Myeloma cells, 20. See also NSO cells
macroporous, 203, 204, 204f, 205t, 229 as continuous cell line, 49
microporous, 203–204, 203f, 205t for stable expression, 129–130, 130t, 146
solid, 203–204, 203f stoichiometric ratio and metabolic stress in, 161, 161t
Microencapsulation, 210, 210f
Microfiltration, 259–260, 260f N
Microheterogeneity, of glcyans, 89, 92–93, 92f NADH
Microporous microcarriers, 203–204, 203f, 205t balance of, 68–69, 69f
Microsphere-induced cell aggregates, 209, 209f carrier system for, 68–69
Microtubules, 37, 37f, 46 in electron transfer chain, 63–64, 66
Minerals, in medium, 111–112 in glycolysis, 60–64, 66, 68–69, 69f
Minimum set of genes, 287 in lactate metabolism, 79–80
Mitochondria, 29–30 in lipid metabolism, 86
acetyl CoA shuttle in, 86 in tricarboxylic acid cycle, 61–63, 68–69, 69f
as cell’s power plant, 29 NADPH
genome of, 29–30 in lipid metabolism, 85
lipid metabolism in, 83, 85, 86, 88f in pentose phosphate pathway, 64–65
membrane potential of, 29–30, 30f Necrosis, 51
pH of, 29–30 Neomycin
protein content of, 25 in gene transfer, 139–140
size of, 29 in medium, 117
transport across, 73–74 Next generation sequencing technologies, 304–305
Mitochondrial apoptotic pathway, 52–53, 53f Nickel, in medium, 112
Mitochondrial DNA, 29–30 NimbleGen microarrays, 295–296, 295t
Mitogenic factors, 48, 49 Nitrogen
Mitosis, 47–48, 48f in cellular growth, 150
Mixing time, in bioreactors, 206, 268–274 in cultured cell metabolism, 67
distribution of, 273–274, 273f, 274f metabolic flux analysis of, 182f, 183
measurement of, 272, 272f N-linked glycosylation, 89–92, 90f, 91f, 174, 174f
in scale translation, 269–274 Non-essential amino acids, 81, 108, 108t
vs. starvation time, 272 Non-nutritional components, of medium, 113
Molybdenum, in medium, 112 NSO cells, 8t, 19t, 20
Monkey cells, 4, 8t, 9, 19t, 129, 201 as continuous cell line, 49
Monocarboxylate transporter (MCT), 42, 43, 72–73, 72f doubling time of, 154t
Monod-derivative models, 164–165 gene expression in, 307f
Monod models, 164–165, 164f for stable expression, 130t
Monolayer, 53 therapeutic antibody products from, 7t
MOPS buffer, 115t Nuclear envelope, 29
Mouse cells, 8t, 9, 19t, 49. See also NSO cells; SP2/0 cells Nuclear membrane, 29
embryonic stem, doubling time of, 154t Nuclear pores, 29
gene expression in, 306, 306f Nucleoid, 28
Mouse genome, 285–287, 288, 289t, 293 Nucleoside(s), in medium, 109
Movement, cellular, 46–47 Nucleosomes, 290
M phase of cell cycle, 47–48, 48f Nucleotides, precursors of glycans, 93–94, 93f, 94f
MRC-5 cells, 19t Nucleus, 27–29, 28f
MRCS cells, 8t Nunc Cell Factories, 201
mRNA. See Messenger RNA Nutrient consumption curve, 154–155
MSX, in glutathione synthetase amplification, 138–141 Nutrient consumption rate, specific, 156–157
MTX, in DHFR amplification, 138–141, 140f Nutrients, transport of, 43
Multidimensional data exploration, 172 Nutrient starving time, 271–272, 271t
Multidrug resistance (MDR) gene, 137 Nystatin, in medium, 117t
Multiple membrane plate bioreactor, 208–209
Multiple plate system, 199, 201, 201f, 212 O
Multiplicative model, of growth, 165 OAA. See Oxaloacetate
Myc, in regulation of glucose metabolism, 77, 77f Off-line data, 160, 160f
INDEX | 321
Oligonucleotide-based microarrays (oligoDNA), 295–296, 298t P
Oligopeptides, transport of, 43 p53 tumor suppressor, in regulation of glucose metabolism, 77
O-linked glycosylation, 89 Pancreas, beta cells of, 31
Omnitrope, 9–10 Passage, 53–55
On-line data, 160, 160f PAT. See Process analytical technology
On-line measurement, for fedbatch culture, 243 Patents, expiration of, 9–10
Organelle(s), 23–24, 27–36, 28f. See also specific organelles Pathway-related data, 173, 173f. See also Metabolic flux analysis
Organic nutrients, in medium, optimal concentration of, 110, PCR. See Polymerase chain reaction
110f PDI. See Protein disulfide isomerase
Osmolarity PDQuest, 299
of interstitial fluid, 100 Penicillin(s)
of medium, 111–112, 125 declining price of, 2–3
OTR. See Oxygen transfer rate development and production of, 2–4, 2f
OUR. See Oxygen uptake rate discovery of, 2
Oxaloacetate (OAA), in glucose metabolism, 61, 68 Penicillin G
Oxidative phosphorylation pathway, 61 in medium, 117t
Oxygen production outside U.S., 2–3
cellular demand for, 219 Pentose phosphate pathway (PPP), 60, 62f, 64–65
concentration in medium, 217–218 carbon flow or flux in, 67
consumption of, 219 in culture, 149
dissolved concentration of, 214 molecular transformation in, 64–65
optimal concentration of, 219 oxidative segment of, 64
transport of, 39 Peptides, in medium, 108, 123t
Oxygen balance, in reactor, 220, 276–280 Peptone, in medium, 124
on gas phase, 276, 278–279 Perfusion culture, 249–261
on liquid phase, 277–279 analysis of, 251–254
Oxygen starvation time, 271–272, 271t cell culture products from, 249t
Oxygen supply, for bioreactors, 220–228 cell retention in, 251, 254–261
by medium recirculation, 221f external vs. internal recovery device in, 252
by silicon tubing/membrane, 220 material balance on, 251–253, 252f, 253f
by sparging, 220–228 recycling factor in, 254, 254f
damage to cells by, 226–228, 226f, 227f, 228f Permeability, of lipid bilayer, 23, 39
direct, 221 Permeases, 39
orifice and bubble size in, 222–223 Peroxisomes, 27, 36
by surface aeration, 220 lipid metabolism in, 83, 85, 87, 88f
Oxygen transfer, in bioreactors, 206–207, 213–231 PFK. See Phosphofructokinase
driving force for, 214–216, 220, 277, 279–280 PFKFB. See Phosphofructokinase/fructose biphosphate
enhancing or improving, 216, 220, 221 PFR. See Plug-flow reactor
experimental measurement of, 224–225, 224f, 225f pH
hydrostatic pressure and, 225 of bioreactors, scale translation and, 283, 283f
in immobilization reactor, 229, 229f of fedbatch culture, 244
mass transfer coefficient in, 216 of medium, 113–115
objective of, 219 of mitochondria, 29–30
in plug-flow reactors, 229–230, 230f Phenotype, epigentic changes in, 291–293
rate of, 215–216, 220 Phosphate
scaling up and, 275–280, 280f, 280t in fedbatch culture, 240
surface/interfacial area and, 216 intracellular and extracellular concentrations of, 100, 100t,
through gas-liquid interface, 212–219, 213f, 214f 153, 153t
Oxygen transfer rate (OTR), 215–216, 220 in medium, 104, 111, 111t
balance with oxygen uptake rate, 220, 276–280 transport of, 43–45
scaling up and, 276–280 Phosphatidyl choline, in medium, 125
Oxygen uptake rate (OUR), 160, 160f, 219, 220, 224–225, 225f Phosphatidyl ethanolamine, in medium, 125
balance with oxygen transfer rate, 220, 276–280 Phosphofructokinase (PFK), in regulation of glucose metabolism,
in fedbatch culture, 245–247 75, 75f
scaling up and, 276–280 Phosphofructokinase/fructose biphosphate (PFKFB), in
regulation of glucose metabolism, 75–76, 75f
INDEX | 322
Phospholipids Process analytical technology (PAT), 167–168
in cell membrane, 22–27 Product accumulation rate, 156, 159
turnover rate of, 26 Product concentration profile, 154–155
types of, 22, 22f Product formation. See also specific products
Phosphorylation in fedbatch culture, 241–243
of histones, 292–293, 292t kinetic model of, 162–165
in protein therapeutics, 14, 16 lactate metabolism and, 58f, 59
Photolithographic synthesis, in microarray analysis, 295, 295t, quantitative description of, 156–161
296f specific rate of, 156–157, 159, 165–166
Physiological state, of culture, 158 Programmed cell death. See Apoptosis
Pichia (yeast) cell culture, 12–13, 13t Proline, in medium, 108
Pinocytosis, 39–40 Promoter
Piston-flow reactor. See Plug-flow reactor for gene transfer, 134–135
PK. See Pyruvate kinase in mammalian genes, 286, 288
PK cells, 8t for transient expression, 128–129
Plants, transgenic, 12 Pro-oncogenic genes, in regulation of glucose metabolism, 77,
Plasma cells, 20, 33, 33f, 130, 151 77f
Plasma membrane. See Cell membrane Proportional feeding
Plasmid(s). See also Gene transfer with base addition, 244
basic elements on, 134–137, 134f with oxygen uptake rate, 245
free, 135 with turbidity, 244
method of delivery, 133–134, 134t Protease inhibitors, in serum, 118
selectable marker for, 135–137, 137t Proteases, for dissociation, 53
for stable cell line, 131–137 Proteasome, 36
for transient expression, 128–129 Protective agents, in medium, 116–117, 116t
Plastic, for microcarriers, 204, 205t Protein(s)
Plug-flow reactor (PFR), 193–196, 193f carrier
oxygen transfer in, 229–230, 230f in medium, 117, 118, 122
reaction and reaction kinetics in, 194–195 transport by, 122, 122t
tracer concentration response in, 193–195, 194f in cell membrane, 25
Pluronic F68, in medium, 116–117, 116t, 123 in cellular growth, 151
Pluronic F77, in medium, 117 in cytoplasm, 27, 27t
Pluronic F88, in medium, 116t, 117 in cytoskeleton, 37
PolyA tail, 286 in extracellular matrix, 45–46
Poly-d-lysine, in medium, 123t folding of, 32–33, 89–91
Polyethylene glycol (PEG), in medium, 116t gene expression in, 299–302
Poly-L-lysine, in medium, 123 genes coding for, 285–286, 288
Polymerase chain reaction (PCR), 304 in interstitial fluid, 100
Polymyxin B, in medium, 117 secretion of
Polypropylene microcarriers, 205t and cell membrane, 26
Polystyrene, for microcarriers, 203–204, 205t in endoplasmic reticulum, 31–35, 35f, 83
Polyvinyl alcohol (PVA), in medium, 116t in Golgi apparatus, 32–35, 35f, 83
Polyvinylpyrrolidone (PVP), in medium, 116t time of, 32, 34, 34f, 34t, 35, 35f
Post-process calculations, 169 Protein C, in-process structural alterations to, 16
Post-translational modification Protein disulfide isomerase (PDI), 33
analysis of, 299 Protein-free medium, 103
in endoplasmic reticulum, 31 Protein hydrolysates, in medium, 124, 240
in protein therapeutics, 5, 12–17 Protein molecules, as therapeutics, 7–8. See also Protein
Potassium chloride, in medium, 111–112 therapeutics
Potassium ions Protein therapeutics, 4–8
intracellular and extracellular concentrations of, 21–22, 100– alternative technologies for, 12–14
101, 100t, 153, 153t biosimilar or follow-up biologics, 9–10
in medium, 111–112, 111t g-carboxylation in, 12, 14, 16
transport of, 43–45 disulfide-bond formation in, 5, 14
PPP. See Pentose phosphate pathway fedbatch culture for, 233, 235f. See also Fedbatch culture
pRB. See Retinoblastoma protein from fusion proteins, 8
INDEX | 323
glycosylation in, 5, 12–16, 89–96, 90f material balance for, 175–180
growth and advances in, 1 Reactive oxygen species (ROS)
host cells for, 127–129 glutathione and, 64, 116
immunogenicity of, 95–96 in medium, 116
industrial cell lines for, 8t, 9 Reactor state parameters, 160f
in-process structural alterations to, 15–17, 16t Read, in DNA sequencing, 297
instability in production of, 144 Recessive marker, 136
from mammalian cells, 6t Recombinant technology, 5, 192. See also specific applications
manufacturing of, 11–12 and products
from non-mammalian host, 6t Recovery process, 11–12
phosphorylation in, 14, 16 Recycling factor, in perfusion culture, 249–261, 254, 254f
post-translational modification of, 5, 12–17 Remicade, expiration of patent, 9
product quality and process robustness for, 14–17 Repetitive sequences, in genome, 287–288
quantity for administration, 11 Reporter gene, 136
recombinant technology for, 5 Respiratory quotient (RQ), 219, 281
stable cell lines for, 127, 129–146 Retention, cell
stoichiometric ratio in, 11 methods of, 254–261
tissue-derived, 5 in perfusion culture, 249–261
transient expression of, 127–129 Retinoblastoma protein (pRB), 48–49
Proteoglycans, in extracellular matrix, 45–46 Retrograde transport, 34, 35, 35f
Proteome profiling, 299–302, 299f Reynolds number, 267–268, 267f
iTRAQ labeling in, 300–301, 300f, 301f RGD peptide, in medium, 123t
SILAC labeling in, 300–302, 301f Riboflavin, in medium, 109
2D liquid chromatography in, 300–302 Ribonucleic acid. See RNA
Proton pumps, 36 Ribose
Pseudogenes, 286 in medium, 107
Pulsatile flow, in microfiltration, 260, 260f synthesis of, 67
Pumping, in bioreactor, 268–270 Ribosomal RNA (rRNA), 21, 27
Purines, in medium, 109, 109t removal, in microarray analysis, 296–297
Puromycin, as selection marker, 137t Ribosomes, 27, 31
Pyridoxine, in medium, 109 Rice hydrolysate, 124
Pyruvate RNA
in amino acid metabolism, 81 abundant, intermediate, and rare, 293–294, 293t
as controlling node, 67 as cellular component, 21
in glycolysis, 60, 61, 66, 67, 70 genes coding for, 285–286
lactate conversion to, 79–80 location in cell, 28
in lipid metabolism, 86 non-protein coding, 285–286
in medium, 107 synthesis of, 28
NADH balance and, 68–69, 69f RNA-seq, 296–297, 298t
transport of, 43, 72–73, 72f Robustness, of cell culture processes, 14–17
in tricarboxylic acid cycle, 60, 61 Roche sequencer, 302t
Pyruvate kinase (PK), in regulation of glucose metabolism, 75–76 Roller bottles, 199–201, 200f, 212
ROS. See Reactive oxygen species
Q Rotational cage, 258, 258f
Quality, of cell culture products, 14–17 Rough endoplasmic reticulum, 31
Quality by Design, 162 RQ. See Respiratory quotient
Quantitative description, of growth, 156–161 rRNA. See Ribosomal RNA
Quasi-steady state, 179 Rubidium, in medium, 112
Rushton impellers, 206, 265–266, 265t, 266f
R
Radial flow impellers, 206, 265–266, 265t, 266f S
Rare genes, 293–294, 293t Sacchromyces cerevisiae systems, 12
Rate-limiting enzymes, 74 Saturated fatty acids, 85
Rate-limiting nutrient, 165 Scale translation
Reaction systems and carbon dioxide removal, 275, 281–283
cellular vs. chemical, 175–176 and chemical environment, 283, 283f
INDEX | 324
constant parameters in, 269–270, 269t Sodium chloride, in medium, 111–112
dimensionless variables in, 267–270 Sodium/glucose transporter, 42
and driving force, 279–280 Sodium ions, 21–22, 21t
effect of scale on physical behavior, 269–270 intracellular and extracellular concentrations of, 21–22, 21t,
geometric nonsimilarity in, 264 100–101, 101t, 153, 153t
geometric similarity in, 264 in medium, 104, 111–112, 111t
major effects of scale, 264 transport of, 43–45
and mechanical forces on cells, 274–275, 275f Sodium/potassium ATPase transporter, 44–45, 44f
mixing time in, 271–274, 271t Software, for data visualization, 172
nutrient starvation time in, 271–274, 271t Solid microcarriers, 203–204, 203f
objective of, 264 Solid phase, in bioreactor, 196
and oxygen transfer, 275–280, 280f, 280t SOLiD sequencing technology, 302t
physical and mechanical parameters of, 264 Solutes, cellular transport of, 40
Reynolds number in, 267–268, 267f Soybean hydrolysate, 124
and superficial gas velocity, 278, 278f SP2/0 cells, 19t, 49
Scaling down, 263–264. See also Scale translation for stable expression, 130t
Scaling up therapeutic antibody products from, 7t
and carbon dioxide removal, 275, 281–283 Sparging, 220–228
and driving force, 279–280 damage to cells by, 226–228, 226f, 227f, 228f
and mechanical forces on cells, 274–275, 275f direct, 221
and oxygen transfer, 275–280, 280f, 280t orifice and bubble size in, 222–223
and superficial gas velocity, 278, 278f Specific death rate, 157
Secretion time, 32, 34, 34f, 34t, 35, 35f Specific growth rate, 156–157
Sedimentation, 254–255, 254f Specific nutrient consumption rate, 156–157
Selectable marker, in gene transfer, 135–137, 137t Specific product formation rate, 156–157, 159, 165–166
Selenate, in medium, 111 Specific rate, 156–157, 169
Selenium Spectinomycin, in medium, 117t
in intracellular fluid, 153 S phase of cell cycle, 47–48, 48f
in medium, 112, 116 Sphingomyelin, in medium, 125
Senescence, 53–55 Spin filter separation, 258, 258f
Separation methods, 254–261 Spin filter stirred tank, 210–211, 211f
Sequencing, DNA. See DNA sequencing Spreadsheets, 168, 170–171
Serine SRPs. See Signal recognition particles
as backbone of phospholipid, 22, 24 Stable cell lines, 127, 129–146
in medium, 108 adaptation of cells in, 131, 142, 142f, 143f
Serum amplification for, 131–132, 138–141, 141f
disadvantages of use, 119 automation and high throughput technology for, 145–146,
in medium, 102, 117–119, 240 145f
Serum-free medium, 102–103, 108, 109 basic steps for generating, 131–133, 132f
Settling cyclone, 254–255, 254f gene expression in, 306–308
SGLT transporters, 71–72 genomics and, 146
Shotgun liquid chromatography, 300 screening for, 131
Sialic acid, in glycan biosynthesis, 93–94 selection of cells for, 131–132
Signaling, cellular, 45–46 single-cell cloning for, 131–132
Signaling pathways, in regulation of glucose metabolism, 77, 77f stability of clones selected for, 143–145
Signal recognition particles (SRPs), 32–33, 35, 35f stability of product quality in, 144–145
SILAC, 300–302, 301f transfection for, 131
Silicon tubing/membrane, for oxygen supply, 220 Stable Isotope Labeling by Amino Acid in Cell Culture (SILAC),
Simple stirred tank bioreactor, 206–207 300–302, 301f
Single-cell cloning, 131–133 Standardized templates, for data logging and processing, 168–
Single molecule detection, in DNA sequencing, 304 169
Single-use bioreactor, 199 Starvation time, in bioreactors, 271–272, 271t
Smooth endoplasmic reticulum, 31 State of cultures, 158
Sodium beta-glycero-phosphate buffer, 115 Stationary phase, of cell growth, 151f, 154–155, 154f
Sodium bicarbonate buffer, 113–114, 115f, 115t ST cells, 8t
Sodium butyrate, 293 Stem cell(s), 4
INDEX | 325
differentiation in culture, 143 veterinarian vaccines from, 8t
medium for, 97–99, 117
mouse embryonic, doubling time of, 154t T
size of, 21, 151 Tangential flow, in microfiltration, 260, 260f
Stirred tank bioreactor, 193, 193f Taurine, in medium, 116
agitation in, 265–266 TCA. See Tricarboxylic acid cycle
mechanism of, 265–266 Telomerase, 54
purpose of, 265 Telomeres, 54–55, 54f
continuous, 193–196, 193f, 212 Temperature, and lipid bilayer, 24
reaction and reaction kinetics in, 194–196 T-flasks, 199
tracer concentration response in, 193–195, 194f Thiamine pyrophosphate, in medium, 109
heterogeneous, 196, 196f 3T3 cells, 54
impellers in, 206, 265–268, 265t, 266f Thymidine, in medium, 109, 109t
membrane, 210, 210f TIGAR, 77
mixing time in, 206, 268–274 Tin, in medium, 112
power consumption in, 266–270, 266f Tissue culture systems, 199–202
simple, 206–207 Tissue plasminogen activator (tPA)
spin filter, 210–211, 211f development of, 5
velocity of, 268 in-process structural alterations to, 16
volumetric flow rate in, 268–270 product quality and process robustness, 15–16
well-mixed, 193–196 quantity for administration, 11
Stock solutions, amino acid, 108 Titers, increases in, 2–3, 2f
Stoichiometric components, of medium TNFa. See Tumor necrosis factor a (TNFa)
vs. catalytic macromolecular components, 105–106 tPA. See Tissue plasminogen activator
vs. habitation-conducive components, 103–104 Trace elements, in medium, 111–112, 112t
Stoichiometric-limiting nutrient, 165 Transcription, 28
Stoichiometric matrix, 179–180 Transcription factors, 28, 291
Stoichiometric ratio, 11, 156, 158–159 Transcriptome analysis, 293–297
in data processing, 169, 171, 171f in cell culture processing, 306–308, 306f, 307f
in fedbatch culture, 160f, 236–240, 236f, 236t, 239f, 239t, DNA microarrays for, 295–297, 295t, 298t
246–247 Transfection. See also Gene transfer
as indicator of metabolic stress, 161, 161t for stable cell line, 131–137
of lactate to glucose, 158, 236–237, 236f, 236t Transferrin
of product to substrate, 158 iron chelators as alternative to, 121, 121t
Stoichiometry, of cell cultivation, 147–166 iron transport by, 45, 122t
Streptomycin, in medium, 117 in medium, 105, 116, 118, 121, 240
Stress, metabolic, stoichiometric ratio as indicator of, 161, 161t recombinant, 121
Substrate secretion time of, 34, 34t
stoichiometric ratio of product to, 158 Transgenic animals, 12, 14, 14t
yield of biomass on, 158 Transgenic plants, 12
yield of product on, 158 Trans-Golgi network (TGN), 32, 33, 35, 35f
Sugars, in medium, 106–107 Transient expression, 127–129
Superoxide dismutase, in medium, 116 host cells for, 129
Superoxide radical, 116 production life of system, 129
Support systems, cellular, 202–206 Translation of scale. See Scale translation; Scaling down; Scaling
Surface aeration, 220 up
Surface area, and scale translation, 264 Transport
Surfactants, in medium, 116–117 classes of processes, 40
Suspension culture, 202, 212 mechanisms of, 39–47. See also specific mechanisms
SV40 late promoter, 135 in metabolism, 71–74
Symporters, 41–42, 42f nutrient, 43
Syrian hamster cells (BHK), 8t, 9, 19t Transporters, 40–42. See also specific types
adhesion of, 142 Tricarboxylic acid cycle (TCA), 60–64, 62f
aggregates of, 209 amino acid metabolism and, 81–82, 82f
as continuous cell line, 49 carbon flow or flux in, 67
for transient expression, 129 in culture, 149
INDEX | 326
glutamine and, 80, 80f Vitamin D, in medium, 109
lipid metabolism and, 86 Vitamin E, in medium, 109, 116
NADH balance in, 68–69, 69f Vitamin K, in medium, 109
regulation of, 74–78 Vitronectin, in medium, 123t
transport and, 71–74 Volume of cells, variation in, 150–151, 150t, 151f
TrichostabinA, 293 Volumetric flow rate, in bioreactor, 268–270
TRICINE buffer, 115t Volumetric rates, 156–157
Trypsin
for dissociation, 53, 212 W
for proteolysis, 300 W1-38 cells, 19t
Tubular bioreactors, 193–196, 193f Warburg effect, 65–66
oxygen transfer in, 229–230, 230f Water
reaction and reaction kinetics in, 194–196 as cellular component, 21, 148, 153
tracer concentration response in, 193–195, 194f in medium, 106
Tumor necrosis factor a (TNFa), in fusion protein, 8 Water for injection (WFI), 106
Turbidity, proportional feeding with, 244 WaveTM, 201
2D liquid chromatography, 300–302 Well-mixed stirred tank reactor, 193–196, 193f
Westfalia disc centrifuge, 256
U WFI. See Water for injection
Ubiquitin, 36
Ubiquitination of histones, 292–293, 292t X
UDP-galactose, 67 XBP-1, 33
UDP-glucose, 67
Unfolded protein response (UPR), 33 Y
Uniporters, 41, 42f, 71 Yeast (Pichia) cell culture, 12–13, 13t
Untranslated regions (UTRs), 286, 288 Yield coefficient, 158–159
UPR. See Unfolded protein response
Uridine, in medium, 109t Z
Urokinase, as tissue-derived protein therapeutic, 5 Zeocin, as selection marker, 137t
UTRs. See Untranslated regions Zinc
in intracellular fluid, 153
V in medium, 111, 112
Vaccine(s) Zirconium, in medium, 112
veterinary, 8t, 9 Zwitterionic buffers, 115
viral. See Viral vaccines
Vanadium, in medium, 112
Vector. See Gene transfer
Velocity, in bioreactors, 268, 278, 278f
Vero cells, 8t, 9, 19t, 201
Vesicle diffusion model, 33
Veterinary vaccines, 8t, 9
Vibromixer, 212, 212f
Viral vaccines, 4–5
cell sources for, 20
inactivated, 4, 9
industrial cell lines for, 8t, 9
live attenuated, 4–5
manufacturing of, 11–12
principal, in prevention of human disease, 5t
quantity for administration, 11
Viscosity, of medium, 116–117
Vitamin(s), in medium, 109, 240
Vitamin A, in medium, 109
Vitamin B6, in medium, 109
Vitamin B12, in medium, 109
Vitamin C, in medium, 109
INDEX | 327

Cell Culture Bioprocess Engineering

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Cell Culture Bioprocess Engineering

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Cell Culture Bioprocess Engineering

With Contributions From:

Overview of Cell Culture Technology. . . . . . . . . . . . . . . . . . . . . . . 1

Cell Biology for Bioprocessing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Cell Physiology for Process Engineering. . . . . . . . . . . . . . . . . . . . . 57

Medium Design for Cell Culture Processing. . . . . . . . . . . . . . . . . . 97

Cell Line Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Stoichiometry and Kinetics of Cell Cultivation. . . . . . . . . . . . . . . . 147

Cell Culture Data Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

Metabolic Flux Analysis in Cell Culture Systems . . . . . . . . . . . . . . 175

Cell Culture Bioreactors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Oxygen Transfer in Cell Culture Bioreactors . . . . . . . . . . . . . . . . . 213

Fedbatch Culture and Dynamic Nutrient Feeding. . . . . . . . . . . . . 233

Cell Retention and Perfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

Scaling Up and Scaling Down for Cell Culture Bioreactors. . . . . . 263

Cell Culture Genomics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

Cell Culture Engineering

OVERVIEW OF CELL CULTURE TECHNOLOGY | 2

OVERVIEW OF CELL CULTURE TECHNOLOGY | 3

Cell Culture Products

OVERVIEW OF CELL CULTURE TECHNOLOGY | 4

OVERVIEW OF CELL CULTURE TECHNOLOGY | 5

Table 3. Non-Antibody Products Produced in Mammalian Cells

OVERVIEW OF CELL CULTURE TECHNOLOGY | 6

Protein Molecule as Therapeutics The early generation of protein therapeutics consisted

OVERVIEW OF CELL CULTURE TECHNOLOGY | 7

Table 6. Cell Lines Used in the Production of Veterinarian

OVERVIEW OF CELL CULTURE TECHNOLOGY | 8

Biosimilars or Follow-on-Biologics Two decades after the introduction of mammalian

OVERVIEW OF CELL CULTURE TECHNOLOGY | 9

OVERVIEW OF CELL CULTURE TECHNOLOGY | 10

Manufacturing Viral vaccines are administered to patients in

OVERVIEW OF CELL CULTURE TECHNOLOGY | 11

Fig. 2.2: Flow chart of a typical recombinant

OVERVIEW OF CELL CULTURE TECHNOLOGY | 12

Yeast The yeast in the genus Pichia is capable of

OVERVIEW OF CELL CULTURE TECHNOLOGY | 13

Table 13. Transgenic Animal Products Approved or Under Development

Product Quality and Process Robustness

OVERVIEW OF CELL CULTURE TECHNOLOGY | 14

OVERVIEW OF CELL CULTURE TECHNOLOGY | 15

OVERVIEW OF CELL CULTURE TECHNOLOGY | 16

OVERVIEW OF CELL CULTURE TECHNOLOGY | 17

Cells: Source, Composition and Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Cells: Source, Composition and Structure

CELL BIOLOGY | 19 CELL ENGINEERING| 19

CELL BIOLOGY | 21 CELL ENGINEERING| 21

Cultured mammalian cells have long been thought

Fig.2.1: A phospholipid molecule with glycerol as backbone,

CELL BIOLOGY | 23 CELL ENGINEERING| 23

CELL BIOLOGY | 25 CELL ENGINEERING| 25

CELL BIOLOGY | 27 CELL ENGINEERING| 27

CELL BIOLOGY | 29 CELL ENGINEERING| 29

CELL BIOLOGY | 31 CELL ENGINEERING| 31

Protein Secretion Through ER and Golgi

CELL BIOLOGY | 33 CELL ENGINEERING| 33

• Secretory proteins spend different amounts

Fig. 2.8: Synthesis and secretion of proteins to an extracellular

CELL BIOLOGY | 35 CELL ENGINEERING| 35

Microtubules A microtubule is an assemblage of hollow tube

Fig. 2.9: Structure of a microtubule molecule and its cellular

CELL BIOLOGY | 37 CELL ENGINEERING| 37

Fig. 2.10: Structure of an intermediate filament

Actin Filaments Actins are two- or three-stranded filaments that