Cell and Tissue Reactor Engineering

© 2003 University of Minnesota

Overview of Cell Culture Technology

By Wei-Shou Hu
I. CELL CULTURE PRODUCTS
A. Conventional mammalian protein therapeutics
B. Principal vaccines used in prevention of human virus diseases
C. Leading biotech drugs (1997)
D. Major licensed recombinant therapeutic proteins expressed by mammalian cells
II. INDUSTRIAL CELL LINES
A. Major cell lines used for human biologics production
B. Cell lines used in the production of veterinarian vaccines*
III. CELL CULTURE PRODUCT MANUFACTURING
A. Recombinant antibody technology products
B. Expected titer
C. Genentech’s new Vacaville facility
D. Production Costs of Mammalian Cell Culture Proteins
E. Contract manufacturers and contract research organizations
IV. ALTERNATIVE TECHNOLOGIES
A. Competing Technologies
B. Insect cell culture
1. LEADING APPLICATIONS FOR BACULOVIRUS SYSTEMS
C. Transgenic Animals
1. ANIMAL SPECIES COMMONLY USED FOR EXPRESSION OF RECOMBINANT PROTEINS IN MILK
2. TRANSGENIC GOAT
3. AVIAN TRANSGENICS
D. Transgenic plants
V. IMPORTANCE OF PRODUCT INTEGRITY
A. Tissue Plasminogen Activator (tPA)
B. Erythropoietin
VI. TISSUE ENGINEERING AND CELL-BASED THERAPIES
I. CELL CULTURE PRODUCTS
A. Conventional mammalian protein therapeutics
Source Name Use
I. Animal tissue and fluid 1. Albumin Hypoproteinemia; plasma extender for shock, burns
2. Human IgG Augmentation of immune system capacity, especially during hepatitis or tetanus infection
3. Rh IgG
4. Equine antivenins (snake, house ant, black widow spider, etc.)
5. Human factor VIII
6. Human factor IX
7. Bovine thrombin Topical for control of oozing after injury or surgery
8. Bovine plasmin
9. Human Cohn fraction fibrinolysin
10. Hepatitis vaccine
11. Porcine lipase and amylase Pancreatitis; to increase absorption of dietary fats
12. Pepsin
13. Trypsin (and chymotrypsin) Cataract surgery; to reduce inflammation and edema after trauma
14. Insulin Control of glucose metabolism
15. Bovine DNAse
16. Glucagon To terminate severe hypoglycemia as a result of insulin overdose
17. Human growth hormone Induction of bone growth in hypopituitary dwarfism
18. Trypsin inhibitor (Trasylol)
19. Bovine collagen
20. Homones (FSH and LH) from pregnant woman Induction of ovulation
21. Chorionic gonadotropin Cyptorchidism in males; stimulation of spermatogenesis
22. Hyaluronidase (bovine testicular)

II. Conventional cell culture 1. Urokinase Dissolution of blood clots

2. Vaccines: DPT, MMR, polio, rabies, yellow fever, etc. Confer specific immunity
3. β-Interferon
B. Principal vaccines used in prevention of human virus diseases

Groupe Disease Source of vaccine Condition of virus Route of administration

I Poliomyelitis Tissue culture (hunian diploid cell line, monkey kidney) Live attenuated Oral
Killed Subcutaneous
d
Measles Tissue culture (chicken embryo) Live attenuated Subcutaneous
b
Mumps Tissue culture (chicken embryo) Live attenuated Subcutaneous
b,e
Rubella Tissue culture (duck embryo, rabbit, or human diploid) Live attenuated Subcutaneous
f
2 Smallpox Lymph from calf or sheep (glycerolated,lyophilized) Live vaccinia Intradermal: mutiple pressure, multiple puncture
Chorioallantois, tissue cultures (lyophilized)
Yellow fever Tissue cultures and eggs (17D strain) Live attenuated Subcutaneous or intradermal
Influenza Highly purified or subunit forms of chicken or embryo Killed Subcutaneous or intradermal
allantoic fluid (formalinized UV irradiated)
Rabies Duck embryo or human diploid cells Killed Stibcutaricous
g
Adenovirus Human diploid cell cultures Live attenuated Oral, by enteric coated capsule
h
Japanese B encephalitis Mouse brain (formalinized), cell culture Killed Subcutaneous
Venezuelan equineen Guinea pig heart cell culture Live attentuated Subcutaneous
cephalomyelitis
Eastern equine Chicken embryo cell culture Killed Subcutaneous
i
encephalomyclitis
Western equine Chicken embryo cell culture Killed Subcutaneous
h
encephalomyelitis
Russian spring -summer Mouse brain (formalinized) Killed Subcutaneous
h
encephalitis

a
For diseases in group I immunization is recommended for the general public; for those in group II recommended only under certain conditions (epidemics, exposure, travel,
military).
b
Available also as combined vaccines.
c
Killed measles vaccine was available for a short period. However, a serious delayed hypersensitivity reaction often occurs
when children who have received primary immunization witli killed measles vaccine are later exposed to live Wastes virus. Because of this complication, killed measles vaccine
is no longer recommended.
d
With less attenuated strains, immune globulin USP is given in another linib at the time of vaccination.
e
Neither monovalent rubella vaccine nor combination vaccines incorporating r-ubella should be administered to a postpubertal susceptible woman unless she is not pregnant and
understands that it is imperative not to become pregnant for at least 3 months after vaccination. (The time immediately postpartum has been suggested as a safe period for
vaccination.) f Since smallpox virus seems to have been totally eradicated fruni the world, vaccination is no longer recommended. However, stocks of vaccine are held in depots,
to be made available if cases should reappear.
g
Recently licensed but recommended only for military populations in which epidemic respiratory disease caused by adenovirus is a frequent occurrence.
h
Not available in the United States except for the armed forces or for investigative purposes.
iAvailable for use in domestic animals (front the U.S. Department of Agriculture) and for investigative purposes.
C. Leading biotech drugs (1997)

1997 worldwide
Product Biological basis Activity/Use Disease sales ($ millions)

Epogen Erythropoietin Red blood cell growth Anemia 1,161

Neupogen Colony-stimulating factor White blood cell growth Neutropenia 1,056
Procrit Erythropoietin Red blood cell growth Anemia 1,000*
Humulin Insulin Diabetes Diabetes 936
Intron-A α-Interferon Anticancer, infections Cancers, hepatitis 598

Engerix-B Hepatitis B Vaccine Hepatitis B 584

Cerezyme Glucocerebrosidase Genetic deficiency Gaucher’s disease 333
Activase Tissue plasminogen Heart attack/stroke Heart attack/ 261
activator embolism, stroke
Humatrope Somatropin Growth deficiencies Growth deficiency/ 260*
renal insufficiency
ReoPro PGIIb/IIIa antibody Prevents blood clots Blood clots in 254
angioplasty

Avonex Interferon beta-1a Multiple sclerosis Multiple sclerosis 240

Protopin/Nutropin Somatrem/somatropin Growth deficiencies Growth deficiency 224
Pulmozyme Dornase alpha Cystic fibrosis Cystic fibrosis 92
Proleukin Interleukin-2 Cancer Kidney cancer 71
Leukine Colony-stimulating factor White blood cell growth 53
Factor VIII Hemaphilia 250
Anti-CD3 MAL Transplant rejection 80

Note: Figures unable for Roferon α-Interferon, Recombinvax hepatitis B vaccine, and antihemophilia blood clotting factors such as Kogenate and
Recombinate. * C&EN estimates. Source: Company reports
 D. Major licensed recombinant therapeutic proteins expressed by mammalian cells

Tissue plasminogen activator (TPA) CHO Heart attack/embolism, stroke (1987)

Erythropoietin (EPO) CHO Anemia (1989)
Human growth hormone C127 Growth deficiency, renal insufficiency (1989)
Granulocyte colony stimulating factor CHO Neutropenia (1991)
Factor VIII CHO/BHK-21 Hemophilia A (1992 & 1993)
Deoxyribonuclease I (DNase) CHO Cystic fibrosis (1993)
Glucocerebrosidase CHO Gaucher’s disease (1994)
Interferon-β CHO Multiple sclerosis (1996)
Factor IX CHO Hemophilia B (1997)
Monoclonal Antibody (MAb) GPIIb/IIIa CHO Blood clots in angioplasty (1994)
Anti-IL 2 receptor α chain (CD25) NS0 Renal transplantation (1997 & 1998)
Anti-CD20 CHO Non-Hodgkin’s Lymphoma (1997)
Anti-RSV NS0 Respiratory syncytial virus (1998)
Tumor necrosis factor α CHO Active Crohn’s (inflammatory bowel) disease (1998)
Anti-HER2 CHO Metastatic breast cancer (1998)
Tumor necrosis factor receptor CHO Rheumatoid arthritis (1998)
1999 total sales: > 5 billion U.S. dollars Weichang Zhou,  2000 Merck & Co., Inc. All Rights Reserved

II. INDUSTRIAL CELL LINES

A. Major cell lines used for human biologics production

•Chinese Hamster Ovary Cells

–tPA, EPO, Antibodies, Factor VIII
•Baby Hamster Kidney Cells
–CD molecules, Factor VIII
•Human 293 Cells, PER C6, Mouse C127
–Protein C, viral vectors for gene therapy
•Hybridomas, myelomas, SPO, NSO
•Vero
–Viral vaccines
•Normal diploid cells for human vaccines
–MRC-5
B. 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.

III. CELL CULTURE PRODUCT MANUFACTURING
A. Recombinant antibody technology products

Company Product Application

Abgenix ABX-IL8 (fully human MAB Treatment of psoriasis
specific for interleukin 8)
Antisoma plc Theragym (radiolabelled Treatment of ovarian cancer
MAB)
Cambridge Antibody Fully human MAB against Inflammation
Technology TGF-β
Knoll/BASF Pharma Fully human MAB against Treatment of rheumatoid
TNF-α arthritis
Knoll/BASF Pharma & Fully human MAB against IL- Inflammatory and
Wyeth-Ayerst/GI 12 autoimmune diseases
Genentech Murine humanized MAB anti- Treatment of asthma
IgE
Herceptin (humanized anti- Treatment of advanced
HER2 MAB) breast cancer
Medarex, Inc. Bispecific antibody (CD64 Prostate cancer
and CD89)
Centocor Reopro (anti-integrin receptor
chimeric MAB)

B. Expected titer

•Most newer processes are fed-batch cultures

•Small number of perfusion cultures, productivity
advantage is small
•Batch 5-7 day, 50-250 mg/L; 800 mg/L (?)
•Fed-batch 7-12 day, 300-600 mg/L
•High end titer, 1-3 g/L over two weeks
C. Genentech’s new Vacaville facility

•Started construction in 1995, FDA licensure 2000

•Cost: $250 M (too low?)
•Eight 12,000-liter fermentors, three seed trains, one recovery train
•500 employees
•1,000 kg capacity
•Produce Herceptin (for breast cancer) and Xolair (for allergic asthma)

•There are a few other large-scale antibody production plants around the world
•Some estimate there is a need for 16 equivalent facilities

D. Production Costs of Mammalian Cell Culture Proteins (bulk product)

Contract production
$2000/g-$5000/g

Target cost of goods

$250/g-$100/g

Dose
a few mg/dose EPO
30 mg/dose tPA
50 mg-1 g/dose antibodies
 E. Contract manufacturers and contract research organizations
•Boehringer-Ingleheim
•Lonza
•Diosynth
•BioInvent Prodution AB (Lund, Sweden)
•Heme Biotech (Copenhagen, Denmark)
•Bio Gaia Fermentation AB (Lund, Sweden)
•CAMR (Center for Applied Microbiology & Research) (Salisbury, UK)
•Collaborative BioAlliance (Stony Brook, NY),
•Smith-Kline BeechamNY)

IV. ALTERNATIVE TECHNOLOGIES

A. Competing Technologies
• Insect cell culture
• Recombinant E. coli
• Transgenic animals
• Transgenic plants

B. Insect cell culture

1. LEADING APPLICATIONS FOR BACULOVIRUS SYSTEMS

Application Comments
Basic research Hundreds of genes have been expressed using baculovirus.
Bioproduction Possibly 25 or more compounds in clinical trials are being produced using baculovirus expression
systems.
Gene therapy A growing number of cell types have been shown to work with BV as the gene-delivery vehicle.
Display systems Demonstrations of the ability to produce large numbers of proteins suggests BV display systems could
lead to combinatorial proteomics.
Bioreagent A number of bioreagent suppliers use BV to make target proteins, viral components and other
production compounds for the research market.
Artificial The large BV genomes suggest use as artificial chromosomes. So far, one patent has been awarded
chromosomes for this application.
Biopesticides There has been an enduring interest in using BV for biopesticides; many attempts have failed. One of
the latest is to use a gene for a scorpion toxin against targeted insects.
C. Transgenic Animals
Genzyme Transgenics Corp (Framingham, MA) Goat
PPL Therapeutics (Roslin, UK) Sheep
Pharming Group, N.V. (Leiden, Netherlands) Rabbit
Geneworks (Ann Arbor, MI) Chicken

1. ANIMAL SPECIES COMMONLY USED FOR EXPRESSION OF RECOMBINANT PROTEINS IN MILK

Species Reproductive Age Average # of Average Yield per Natural
(months) Offspring Lactation (l)
Mouse 1 10 0.0015
Rabbit 6 8 1.5
Pig 8 9 120
Sheep 8 2 300-400
Cattle 15 1 10,000

2. TRANSGENIC GOAT
•α-1-proteinase inhibitor, MAb, tPA have been expressed
•16 mg/ml milk protein
•Relatively easy purification process
•Glycosylated
•Low initial capital investment
•No product has been approved by FDA

3. AVIAN TRANSGENICS
Geneworks
–Cost $0.05/egg @ 30 ml egg white, 2.5g albumin
–250 eggs/year/hen
•Method
–Gene transfection into embryo
–Transformed males used for breeding,
•2,000 inseminations/month/rooster
 D. Transgenic plants
•Advantages
–Cost of goods
–Large quantity
–Low capital investment (in corn seed 8% of soluble protein, 0.5-1 kg/acre)
–Stable product in situ–No prion
•Disadvantages
–Presence of plant glycan
–Good chemical characteristics, but low in vitro activity in cell-based assays

V. IMPORTANCE OF PRODUCT INTEGRITY

A. Tissue Plasminogen Activator (tPA)1

• Single polypeptide chain (70 kDa) or proteolytically cleaved at ARG276.

• Roller bottle produced material predominately in the two chain form, approximately 25% more
active that the single chain form.
• Multiple N-linked carbohydrates - ASN117 (high mannose), ASN184 (50% complex multiantenary,
50% unoccupied), THR61 (O-linked fucose).
• Substitution of TYR103 by ASN changes glycosylation at ASN117 (from high mannose to
complex).
• Contains 35 cysteine residues, 17 pairs of disulfide bonds. CYS83 can form a disulfide with other
free thiols depending upon the growth medium and buffer composition.
• The presence of serum results in formation of high molecular weight aggregated (complexes with
protease inhibitors) and proteolytically cleaved tPA.

1
Lubiniecki, A.S. and J.H. Lupkert. 1994. Purified protein products of rDNA technology expressed in animal cell culture. Biologicals 22: 161-169.
 • To avoid this, the production process uses serum-free media.

B. Erythropoietin

• Contains 40% carbohydrate, only 2 disulfide bonds.

• 3, N-linked ASN (24,38,83), 1 O-linked (SER126) glycosylation sites. Glycosylation at these sites
may be responsible for resistance to denaturing conditions.
• O-linked site not essential for in vitro of in vivo activity.
• Sialic acid residues (average 10 moles/mole Epo) responsible for preserving pharmacokinetic
behavior. Muteins lacking 2 or 3 N-linked sites are poorly secreted.
• N-linked glycosylation and sialylation is critical to optimal secretion, structure, in vivo potency.

VI. TISSUE ENGINEERING AND CELL-BASED THERAPIES

Current Products and Pipeline, 1999
Tissue Company Product Name Status Technology

Skin Organogenesis, Inc., Apligraf Approved by FDA in May, 1998; First human Human dermal fibroblasts seeded onto
Canton, MA living TE skin product bilayered collagen matrix, to treat venous
ulcers

Advanced Tissue Sciences, Inc., La TransCyte Approved by FDA in March 1997; First human Human dermal fibroblasts seeded onto
Jolla, CA non-living TE skin product synthetic polymer scaffold, to treat full- and
partial-thickness burns

Dermagraft Clinical Trials Human dermal fibroblasts seeded onto

bioresorbable polymer scaffold, to treat
diabetic foot ulcers

Ortec International, Composite Cultured Clinical Trials Human epidermal and dermal cells seeded
New York, NY Skin onto bovine collagen matrix

Cartilage Genzyme Tissue Repair, Inc., Carticel Approved by FDA in 1995: First cell-based Allogeneic cells cultured in vitro and
Cambridge, MA cartilage product injected into knee defect site, to treat
articular surface defects

Liver Circe Biomedical, ELAD Clinical Trials Extracorporeal, immunosiolated liver-assist

Lexington, MA device as bridge to transplantation

Hepatix ELAD Clinical Trials Extracorporeal, immunosiolated liver-assist

device as bridge to transplantation

Algenix, Inc. (University of ELAD Clinical Trials Extracorporeal, immunosiolated liver-assist

Minnesota) device as bridge to transplantation

Spinal Cord CytoTherapeutics, -- Clinical Trials, efficacy phase suspended in Encapsulated bovine adrenal cells secrete
Nerves Lincoln, RI June, 1999 analgesics for severe pain control

Blood Osiris Therapeutics, Allogen Clinical Trials Infused allogeneic human mesenchymal
Baltimore, MD stem cells for cell-based therapy of cancer
patients undergoing chemotherapy

Dendreon, Inc., APC8015 Clinical Trials Immunotherapy treatment for prostate

Seattle, WA cancer patiens by using dentritic cells
Competing Technology: Biomaterials and Biomolecules-based therapies, Stem Cell technology

• e.g. Bone: Creative Biomolecules (OP-1 Implant)

• e.g. Skin: Integra LifeSciences (INTEGRA Artificial Skin); LifeCell (AlloDerm)
• e.g. Spinal Cord Nerves: Guilford Pharmaceuticals (neuroimmunophilins); Regeneron (brain derived
neurotrophin factor and neurotrophin-3)

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