Process Intensification Yields Downstream Benefits: For Personal, Non-Commercial Use

Volume 32 Number 7
INTERNATIONAL
July 2019 The Science & Business of Biopharmaceuticals
www.biopharminternational.com
FOR PERSONAL, NON-COMMERCIAL USE
PROCESS INTENSIFICATION
YIELDS DOWNSTREAM
BENEFITS
MANUFACTURING PEER-REVIEWED RESIDUAL IMPURITIES

DESIGN CONSIDERATIONS CATALYTIC SITE ANALYSIS AND ANALYSIS OF
FOR A COMMERCIAL CHARACTERIZATION OF RESIDUAL IMPURITIES
CELL AND GENE A SOLVENT-TOLERANT IN CONTINUOUS
THERAPY FACILITY ALDO-KETO REDUCTASE MANUFACTURING
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Headaches go down.
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Table of Contents
BioPharm International integrates the science and business of biopharmaceutical research,

development, and manufacturing. We provide practical, peer-reviewed technical solutions
to enable biopharmaceutical professionals to perform their jobs more effectively.
COVER STORY
12 Catching Up Downstream
Although downstream efficiency still lags behind upstream,
engineering-driven innovation is breaking through boundaries.
Cover Design by Dan Ward
Images: Sebastian Kaulitzki - stock.adobe.com
FEATURES COLUMNS AND DEPARTMENTS
FROM THE EDITOR

PEER-REVIEWED FDA and USP take sides in debate
Catalytic Site Analysis on biologic drug standards.
UPSTREAM PROCESSING and Characterization of a Solvent- Rita Peters. . . . . . . . . . . . . . . . . . . . . . . . .5
Applying QbD to Tolerant Aldo-Keto Reductase
Upstream Processing Wei Jiang, Rui Pei, Weiliang Wu,
Susan Haigney PanPan Zhao, Libing Tian, REGULATORY BEAT
Using a QbD approach from early-stage and Shu-Feng Zhou New guidance documents
development through commercialization In this study, a novel aldo-keto clarify production standards
can ensure that upstream processes are reductase, AKR7-2-1, was cloned and and processes for developing
efficient and reliable. . . . . . . . . . . . . . . 17 purified, and its important conserved interchangeable biologic drugs.
sites were analyzed. . . . . . . . . . . . . . . . 34 Jill Wechsler . . . . . . . . . . . . . . . . . . . . . . .6

PERSPECTIVES ON OUTSOURCING
MANUFACTURING The increasing growth in the cell- and
QUALITY
Design Considerations gene-therapy markets is inspiring
for a Commercial Cell Removing Gaps in Data Integrity CDMOs to expand their services in
and Gene Therapy Facility Agnes Shanley this emerging biologic drug arena.
Feliza Mirasol FDA guidance is expected to improve Susan Haigney . . . . . . . . . . . . . . . . . . . . .8
The commercialization of cell and industry practices, but work is also
gene therapies has become a reality, needed to bridge disparate industry and
prompting deeper considerations of software engineering standards. . . . . . 41 EARLY DEVELOPMENT PIPELINE . . . .10
logistics, technology, and design for
manufacturing facilities. . . . . . . . . . . . . 23
AD INDEX . . . . . . . . . . . . . . . . . . . . . . . .49
OPERATIONS
RESIDUAL IMPURITIES Up-to-Date Systems ASK THE EXPERT
Analysis of Residual Impurities Streamline Operations Cultural and language discrepancies
in Continuous Manufacturing Amber Lowry during an audit can be resolved
Cynthia A. Challener Recently released equipment and products using what many call a “playbook,”
Real-time monitoring of product- and include microbioreactor systems, cell says Siegfried Schmitt, PhD,
process-related impurities remains a therapy automation software, and IIoT- vice-president, technical, Parexel
challenge. . . . . . . . . . . . . . . . . . . . . . . . 28 enabled flow sensors.. . . . . . . . . . . . . . . 48 Consulting. . . . . . . . . . . . . . . . . . . . 50
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4 BioPharm International July 2019 www.biopharminternational.com

From the Editor
A Biologics Partisan Divide
A
bipartisan effort by the Senate Health, Education, Labor and Pensions
(HELP) committee designed to lower healthcare and medication costs
has led to a contentious debate between FDA and standards-setting orga-
nization, the US Pharmacopeial Convention (USP). The committee voted 20–3
on June 26, 2019 to advance legislation with proposals that include changes to
patent policy, ending “surprises” in medical bills, increasing transparency in
healthcare, and reducing roadblocks that delay generic drugs and biosimilars
getting to market (1).
It is provisions in Section 207—designated as promoting biological drug
innovation—that are proving controversial, pitting FDA and USP in an
Rita Peters is the exchange of policy statements. According to HELP committee documents,
editorial director of Section 207 is intended to prevent delays in the licensure of biosimilar and
BioPharm International. interchangeable products by excluding biological products subject to regula-
tion under the Public Health Service Act from requirements to follow United
States Pharmacopeia compendial standards. A similar provision to make bio-
logics exempt from meeting USP standards was removed from the final 21st
FDA and USP Century Cures Act of 2016 (2).
“A biological product is so inherently complex and variable that the estab-
take sides in lished structure of the USP monograph standards process does not serve it well,
and in fact, can impede technological progress or innovation,” noted Steven
debate on biologic Kiozlowski, director of the Office of Biotechnology Products in FDA’s Office of
Pharmaceutical Quality in an interview published by FDA (3).
drug standards. Due to the inherent differences in biological products, the “sameness” stan-
dard used in USP monographs for chemical-based drugs cannot apply to bio-
logic drugs, FDA argues.

USP, backed by patient groups and pharmacists, argues that the elimination
of required standards would harm patients. In a letter to the committee, USP
noted the role standards play in providing quality benchmarks; supply chain
security; reliability and predictability for drug product development, manufac-
turing, and distribution; and promoting transparency and accountability that
leads to patient trust (4).
“Essential to the framework that safeguards the quality and safety of
medicines in the United States is the principle that public quality standards,
required under the law, establish and articulate quality expectations for medi-
cines, including biologics,” USP noted in the letter.
FDA has no legal authority to control the price of drugs. However, the
agency notes it is “committed to facilitating increased competition in the mar-
ket for prescription drugs through the approval of lower-cost, generic medi-
cines” and “makes sure that safe and effective drugs are available to improve
the health of consumers” (5). That’s a lot of priorities to balance in a high-
stakes, highly partisan environment.
References
1. S. 1895, US Senate, 116th Congress, 1st Session (Washington, DC).
2. J. Wechsler, “FDA Challenges USP Standards for Biologics,” www.PharmTech.com,
June 25, 2019.
3. FDA, CDER Conversation: Ensuring That Standardization Does Not Impede Biological
Product Innovation, www.fda.gov, June 12, 2019.
4. USP, Letter to Senate Committee on Health, Education, Labor and Pensions (HELP),
June 11, 2019.
5. FDA, Frequently Asked Questions about CDER, www.fda.gov/about-fda/center-drug-
evaluation-and-research/frequently-asked-questions-about-cder. X
July 2019 www.biopharminternational.com BioPharm International 5

Regulatory Beat
FDA Revamps Biosimilar

Quality Requirements
New guidance documents clarify production standards and
processes for developing interchangeable biologic drugs.
A
s part of its ongoing support for the investigational and commercial-scale lots, vali-
development and approval of competi- dation lots, and lots manufactured at difference
tive biotech therapies, FDA has updated scales. Additional sections clarify the submis-
and clarified standards and procedures for sion of reference standards and data on func-
ensuring the quality and similarity of bio- tional activity to demonstrate any structural
similars. A new draft guidance recommends a differences with a reference product. And FDA
process for developing comparative analytical provides the usual caveat that it will consider
assessment plans to demonstrate biosimilar- alternative approaches from sponsors in analyz-
ity, with added details on procedures for con- ing and presenting requested data.
ducting such assessments and for documenting
quality in a range of production lots to make SUPPORT FOR INTERCHANGEABLES
biosimilar development and production more FDA issued the revised quality assurance advi-
predictable and efficient (1). sory soon after publication of a final guidance
The new advisory replaces an earlier guidance on developing interchangeable biosimilars, a
on quality considerations for biosimilars issued much-anticipated and hotly debated option for
in 2015 and updates a 2017 draft guidance on manufacturers (2). That document updates a

statistical approaches, both withdrawn later by draft guidance from January 2017 that attracted
FDA due to industry concerns and objections. voluminous comment from multiple stakehold-
This revised document presents strategies for ers. The new version instructs manufacturers on
conducting comparative analytical studies to conducting switching studies, which FDA says it
assess biosimilarity to a reference product and usually will require to approve an interchange-
for presenting scientific and technical informa- able label. While some firms consider extensive
tion in the chemistry, manufacturing, and con- switching studies unnecessary, FDA justifies
trols (CMC) section of biosimilar applications. this approach as important for demonstrating
There’s more detail on how comparative ana- fully that safety and efficacy are not affected by
lytical assessments should cover key processes, changing treatment from brand to interchange-
including a product’s expression sys- able, a finding that is considered important
tem, manufacturing process, phys- to gain the trust of physicians and patients in
icochemical properties, functional pharmacy-level switching.
activities, target binding, impurities, Important for biosimilar makers, the new
reference product and reference stan- guidance supports the use of non-US-licensed
dards, finished drug product, and sta- comparator products to conduct tests and to
bility. produce the data needed to demonstrate inter-
VisionsofAmerica/Joe Sohm/Getty Images
To address concerns about lot-to- changeability. Even though FDA wants data
lot variability, FDA advises biosimilar from bridging studies to foreign-made reference
makers to include data from at least products, manufacturers still may benefit from
10 reference product lots acquired leeway to obtain samples of biologics that often
Jill Wechsler over several years to fully assess refer- are less costly outside the United States and can
is BioPharm International’s ence product drift. Similarly, spon- help reduce the cost and time for developing
Washington editor, sors should assess 6 –10 lots of the interchangeable therapies. Sponsors develop-
jillwechsler7@gmail.com. proposed biosimilar, including both ing interchangeable products may be eligible to
6 BioPharm International www.biopharminternational.com July 2019

Regulatory Beat
gain a year of market exclusivity current costly diabetes treatments interchangeable determination may
for being the first to develop such as multiple sponsors move into be affected by delivery of insulin
a copycat therapy. this field. These issues were dis- through a pump or over-the-coun-
The new guidance also appears cussed at an FDA public meeting ter device.
less didactic by dropping the fre- in May on the future of biosim- So far FDA has not approved any
quent use of terms such as “residual ilar insulins, just days after the biosimilars as interchangeable, but
uncertainty” and “fingerprint-like” appearance of the interchange- this is expected to change with
similarity between reference and able biologics guidance. As a rel- this added clarification of regula-
interchangeable products, which atively simple biotech product tory process and requirements. The
were common in the earlier advi- that has been available as a drug rising price of insulin therapies has
sories. FDA here aims to set gen- for 100 years (but ineligible for become one of the most conten-
eral standards and requirements for generic competition), manufactur- tious issues in the escalating drug
these therapies but notes, however, ers expect to be able to utilize a pricing debate, with Congressional
that the specific data and testing rather streamlined and straight- hearings generating multiple leg-
needed to document interchange- forward process for documenting islative proposals on this issue,
ability may vary with the struc- similarity and interchangeability many supporting the development
tural and functional complexity for these widely used therapies. of biosimilar and interchangeable
of the product and with clinical Firms planning to produce com- products.
experience with the reference prod- petitive insulin therapies propose
uct. Sponsors may seek to justify an that FDA require data from only REFERENCES
exemption from switching studies, one, small immunogenicity study, 1. FDA, Development of Therapeutic
and FDA says it will take a flexible as opposed to multiple switching Protein Biosimilars: Comparative Ana-
lytical Assessment and Other Quality-
approach. studies, and that analytic charac- Related Considerations, Guidance for
terization, bridging studies, and Industry (CDER, CBER, May 2019),
EYE ON INSULIN reliance on pharmacokinetic and www.fda.gov/regulatory-information/
In moving forward on standards pharmacodynamic data should sea rch-fda-g u ida nce-docu ments/
development-t herapeutic-protein-
for interchangeable products, FDA provide ample support for inter-
biosimilars-comparative-analytical-
sets the stage for the develop - changeable insulins. assessment-and-other-quality
ment and approval of competi- FDA says it plans additional guid- 2. FDA, Considerations in Demonstrat-

tive insulin products, which are ance on data needed on product ing Interchangeability With a Refer-
scheduled to transition to bio- container closure systems and ence Product, Guidance for Industry
(CDER, CBER, May 2019), www.fda.
logic status in March 2020. There delivery device constituent parts
gov/regulatory-information/search-fda-
is great anticipation that inter- to support the presentation of a guidance-documents/considerations-
changeable insulin therapies will proposed interchangeable product. demonstrating-interchangeability-ref-
provide important alternatives to Of interest is how a biosimilar or erence-product-guidance-industry ◆
Federal Court Decides US Stem Cell Clinics Adulterated and Misbranded Products
FDA announced on June 4, 2019 that a US District Judge their disregard of the law and prior FDA warnings. This
in the Southern District of Florida held that US Stem Cell decision today is a victory for the FDA’s work to stop
Clinic LLC, of Weston, Florida, and US Stem Cell Inc., of these bad actors and to protect patients,” said Peter
Sunrise, Florida, and their Chief Scientific Officer Kristin Marks, MD, PhD, director of the FDA’s Center for Biologics
Comella, PhD had adulterated and misbranded a stem cell Evaluation and Research, in a press release (1).
drug product made from a patient’s adipose tissue. The
ruling came after the US Department of Justice initiated Reference
action in May 2018 to seek a permanent injunction 1. FDA, “Federal Court Issues Decision Holding that US Stem Cell
Clinics and Owner Adulterated and Misbranded Stem Cell Products in
against the clinics after attempts by FDA to work with the
Violation of the Law,” Press Release, June 4, 2019.
company to become compliant with regulations failed.
“In the case against US Stem Cell Clinic, the clinic and
—The Editors of BioPharm International
its leadership have put patients at serious risk through

Perspectives on Outsourcing
Gene Therapies Propel Outsourcing Investment

The increasing growth in the cell- and gene-therapy markets is inspiring
CDMOs to expand their services in this emerging biologic drug arena.
G
ene therapies are becoming a fast- service its clients’ outsourcing needs in this area,
growing area in the biopharmaceutical Catalent acquired Paragon Bioservices, Inc., a
industry, creating a crowded pipeline of viral vector development and manufacturing
products in need of manufacturing. FDA expects company for gene therapies with expertise in
the agency will receive more than 200 investi- adeno-associated virus vectors, in May 2019 (2).
gational new drug applications for cell and gene Clark believes the acquisition will help
therapies by 2020, with the agency predicting Catalent accelerate its growth. “Paragon offers
that it will be approving 10–20 cell- and gene- its partners GMP-compliant manufacturing
therapy products each year by 2025 (1). capacity, scale-up expertise, and customized
Because gene therapies use the patient’s own downstream processing without the added time,
body to manufacture the active ingredient, costs, and risks of building a new viral facility,”
the treatment is specific to that patient. This says Clark. “The acquisition also brings comple-
tailoring can often be a more effective and mentary capabilities that will fundamentally
longer-lasting treatment, according to Chris enhance our biologics business and our end-
Murphy, general manager for viral vector ser- to-end integrated biopharmaceutical solutions
vices at Thermo Fisher Scientific. “Because of for customers. We have also gained the experi-

these developments, novel, life-changing thera- enced leadership and dedicated team at Paragon,
pies are being realized that may transform the who will continue to run the business going
lives of patients in need.” forward, bringing their expertise and capabili-
This growth is causing contract development ties to the Catalent family.”
and manufacturing organizations (CDMOs) to Thermo Fisher Scientific Inc. took a major
take a look at their portfolios as their clients step into the viral-vector manufacturing arena
require more and more biologics-related ser- with the announced purchase of Brammer Bio, a
vices. Catalent has been focused on the clinical leading viral vector CDMO, with 600 employees
pipeline of gene therapies and anticipating the and locations in Massachusetts and Florida. The
need for commercial manufacturing. “There is purchase agreement, announced in a March
an abundance of gene therapy product candi- 24, 2019 press statement, is for approximately
dates in the pipeline, and on the heels of market $1.7 billion in cash. Brammer Bio joins Patheon,
approvals of gene therapy products in 2017 and acquired in 2017, and Fisher Clinical Services,
the influx of venture capital funding for gene which have been combined to form the Thermo
therapy companies, FDA approved a second Fisher’s Pharma Services business (3).
gene therapy this year. We see this as a water- The acquisition enhances the company’s
shed moment, where gene therapies are gaining pharmaceutical and biotechnology customer
regulatory acceptance and setting a precedent value proposition by combining cell-culture
for other programs in the pipeline,” says Bernie media, single-use bioprocessing capabilities,
Clark, vice-president, marketing and strategy, analytical instruments, and related consum-
Don Farrall/Getty Images
Catalent Biologics & Specialty Drug Delivery. ables. “Brammer Bio is an exciting addition
According to Clark, outsourcing the manu- to our Pharma Services business. By sharing
facture of gene therapies requires relatively low our combined capabilities, expanding commer-
volumes to fulfill demand, making cial scale and broadening deep customer rela-
Susan Haigney it an attractive option. To better tionships, we will strengthen our position as a

Perspectives on Outsourcing
trusted partner to our pharma and and manufacturing providers for built on Catalent’s track record in
biotech customers. We will accel- AAV vectors,” says Clark. biologic drugs development, which
erate advancements in gene and includes more than 115 global
cell therapy, meet the increasing EXPANDING OTHER SERVICES clinical trials and 11 commer-
demand of the customers we serve I n May 2 019, T he r mo Fi she r cially marketed monoclonal anti-
and bring life-changing medicines S c ie nt i f ic a n nou nc e d t hat it bodies using the company’s GPEx
to patients in need,” says Murphy. is investing more than $50 mil- cell line development technology,
lion into its global bioproduction and 20 approved products through
VIRAL VECTOR CHALLENGES capabilities. The expansion will fill/finish and commercial supply
Viral vectors are crucial to gene provide additional capacity for to global markets, the company
therapy because they deliver the manufacturing single-use biopro- reports. According to Clark, the
therapy into the patient. “Viruses cess container (BPC) systems. In company has multiple initiatives
are evolutionarily designed to enter Cramlington, United Kingdom, in the works to shorten timelines
mammalian cells and reproduce the company will expand assem- and increase efficiency (5).
themselves. Gene therapy har- bly capacity and add BPC systems “Time is often lost for sponsors
nesses this feature to transport the manufacturing. The proximity of on the path to clinic from contract
genetic material—or ‘active ingre- these capabilities to customers in negotiation, site inspections, hand-
dient’—to target tissue or cells. Europe will shorten lead times and offs, and poor communication
Viral vectors are engineered to be improve overall global efficiency, between multiple vendors,” com-
safe for humans and can target according to the company (4). mented Clark in a press statement
specific cells or tissues in our bod- In the United States, the com- announcing the service. “Through
ies to maximize the effect of the pany will expand cleanroom space our new OneBio Suite, Catalent is
treatment,” says Murphy. Thermo for BPC chamber and related assem- uniquely positioned to provide an
Fisher Scientific’s acquisition of bly production processes at its site integrated offering that can accel-
Brammer Bio’s viral vector process in Logan, UT, and further expand erate biologic development poten-
development and manufacturing capacity at its site in Millersburg, tially shaving weeks to months off
leverages capabilities in the com- PA. Construction is expected to be standard timelines and allowing
pany’s biologics business. completed by the end of 2020. our customers to get to clinic and

C l a rk a l s o e mph a si z e s t he “The demand for our bioproduc- market faster.”
i mp or t a nc e of v i r a l ve c tor s . tion products and services contin-
“During gene therapy biomanu- ues to outpace the market,” said REFERENCES
facturing, cells package the thera- C or y Stevenson, president of 1. FDA, “Statement from FDA Commissioner
peutic genetic material into viral Thermo Fisher Scientific’s biopro- Scott Gottlieb, M.D. and Peter Marks,
M.D., Ph.D., Director of the Center for
vesicles and secrete the vesicles duction business, in a company Biologics Evaluation and Research on
into the media to be purified, then press release. “These investments New Policies to Advance Development of
undergo formulation development will expand capabilities across our Safe and Effective Cell and Gene
Therapies,” FDA.gov, Jan. 15, 2019, www.
to be ultimately filled into a vial existing bioproduction network fda.gov/news-events/press-
or syringe,” says Clark. According while we look to extend our foot- announcements/statement-fda-
to Clark, however, few innova- print into new regions to meet commissioner-scott-gottlieb-md-and-peter-
marks-md-phd-director-center-biologics
tors have the capacity, expertise, increasing customer demand for 2. Catalent, “Catalent To Acquire Gene
or resources to manufacture viral our industry-leading single-use Therapy Leader Paragon Bioservices,
vectors. Catalent’s acquisition of technologies.” Inc. for $1.2 Billion,” Press Release,
April 15, 2019.
Paragon helps the company manu- To further develop its biolog- 3. Thermo Fisher Scientific, “Thermo
facture viral vectors for their cli- ics offerings, Catalent is launch- Fisher Scientific to Acquire Brammer
ents. “There are several types of ing OneBio Suite, a new offering Bio, a Leader in Viral Vector
Manufacturing,” Press Release, March
viral vectors that can be utilized for the integrated development, 24, 2019.
for gene therapy. Adeno-associated manufacturing, and clinical sup- 4. Thermo Fisher Scientific, “Thermo
Fisher Scientific to Invest $50 Million
virus (A AV) is the vector most ply of biologic drugs. The Suite
to Expand Bioproduction Capabilities,”
commonly used today in clinical is designed to address challenges Press Release, May 20, 2019.
trials and approved gene therapies, that arise when accelerating pro- 5. Catalent, “Catalent to Launch OneBio
Suite For Integrated Biologics
due to its safety and tissue-specific grams to clinic or market, while
Development, Manufacturing and
targeting abilities, and Paragon is also reducing complexity and risks Supply at BIO International 2019, Press
one of the leading development from projects. The OneBio Suite is Release, May 23, 2019. ◆

Early Development Pipeline
Source of Nucleic-Acid Asymmetry Tiny DNA Reader to Advance
May Advance Gene Therapy Anticancer Drug Development
New research from the Institute for Research in Scientists from Osaka University have developed a solution
Biomedicine (IRB Barcelona) published in the journal to the challenge of studying anticancer drugs incorporated
CHEM offers insight into the source of asymmetry into single strands of DNA (1).
between nucleic acid hybrids. This development may According to the university, despite constant effort from
prove to be an important contribution to improving gene researchers to develop new and improved therapies to
therapies (1). eliminate cancer cells—or at least halt their replication—a
The study, done in collaboration with the Centre for limited understanding of exactly how these drugs work
Genomic Regulation and the Institute for Advanced can sometimes make it difficult to advance treatments.
Chemistry of Catalonia, has analyzed the source and One such treatment, trifluridine, is an anticancer drug that
biological consequences of the asymmetry that occurs gets incorporated into DNA as it replicates. While similar
in RNA–DNA hybrids when the relation between purine to thymine, one of the four nucleotides that make up DNA,
(adenine and guanine) and pyrimidine (thymine and cytosine trifluridine cannot bind to thymine’s partner nucleotide,
or uracil) bases differs in RNA and DNA strands (2). adenine. This destabilizes the DNA molecule, resulting
According to the researchers, the results of the study in aberrant gene expression and, ultimately, cell death.
indicate that in contrast to the homoduplexes of DNA or However, exactly where trifluridine gets incorporated into
RNA, RNA–DNA hybrids show intrinsic asymmetry, which the DNA is unknown because it is not distinguished by
suggests that this property is important for biological traditional DNA sequencing methods, hampering efforts to
function and for biotechnological applications. When the fully understand and develop the technology.
DNA of the hybrids is rich in pyrimidine bases, the duplex In a study published in Scientific Reports, the university
is more stable and rigid than when the DNA chain is rich in researchers developed a DNA sequencing method that could
purine bases. This symmetry, according to the researchers, distinguish the drug molecules from normal nucleotides in
may lead to improvements in the efficiency of therapies short strands of DNA (2). Using microscopic probes, the team
based on hybrids, such as antisense therapy, which can, for passed an electrical current across a distance approximately
instance, control the regulation of genes that contribute to 65,000 times smaller than a grain of sand—a gap just wide
the progress of cancer progression and other diseases, as enough to fit a strand of DNA.
well as CRISPR-Cas9 gene-editing technology, which allows “Using this single-molecule quantum sequencing

a target gene to be cut and edited.
“Thanks to a combination of theoretical and
method, we successfully identified individual molecules in
the DNA based on differences in electrical conductance,”
experimental methods, we have been able to understand said lead author Takahito Ohshiro in a university press
the relationship between the sequence and stability release. “For the first time, we were able to directly detect
of DNA–RNA hybrids—structures that form in the cell anticancer drug molecules incorporated in the DNA.”
spontaneously and that have enormous therapeutic The researchers report that the conductance of
potential,” said Modesto Orozco, head of the Molecular trifluridine was lower than that of the four native
Modeling and Bioinformatics Laboratory at IRB Barcelona nucleotides, which also displayed divergent conductance
and senior professor at the University of Barcelona, in values, allowing it to easily be distinguished in the DNA
an institute press release. “Our results will allow further sequence. Based on these values, the team successfully
development of much more efficient methods to sequenced single DNA strands of up to 21 nucleotides,
block and edit genes and that can potentially become pinpointing the exact insertion sites of trifluridine.
therapeutic alternatives for diseases for which there are “Now that we have the ability to determine exactly
no effective treatments.” where the drug is incorporated, we can develop a better
understanding of the mechanism involved in DNA damage,”
References said senior author Masateru Taniguchi in the release. “We
1. Institute for Research in Biomedicine, “A Study Makes expect that this technology will aid in the rapid development
a Significant Contribution to Progress in Gene Therapy of new and more effective anticancer drugs.”
Efficiency by Identifying the Source of Asymmetry in RNA–
DNA Hybrids,” Press Release, June 13, 2019. References
2. M. Terrazas et. al, Chem, doi: 10.1016/j.chempr.2019.04.002 1. Osaka University, “Tiny DNA Reader to Advance Development
(April 25, 2019). of Anticancer Drugs,” Press Release, March 7, 2019.
2. T. Ohshiro et. al, Scientific Reports, doi: 10.1038/s41598-019-
40504-x (March 7, 2019). ◆

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and Canada.
Downstream Processing
Catching Up Downstream
Although downstream efficiency still lags behind upstream,
engineering-driven innovation is breaking through boundaries.
AGNES SHANLEY

T
he biopharmaceutical market is challenged by growth, Market pressures have driven improved process technologies,
restructuring, and time-to-market pressures. For con- which some observers refer to as “next-gen” bioprocessing, a cat-
tract development and manufacturing organizations egory that includes process intensification and the connection
(CDMOs), development timelines for standard vanilla of different unit operations, as well as continuous processing,
antibodies have been cut in half over the past few years, both up and downstream. “These improvements allow manu-
says Uwe Gottschalk, chief scientific officer at Lonza. facturing to run more efficiently and produce higher yields in
“That means going from DNA to clinical trials within one less time and space, reducing capital investment,” says Andrew
year,” he says, “and development times for all the individual Bulpin, head of process solutions at MilliporeSigma. “By 2020,
steps entailed, from cell-line construction to a new manu- we expect that approximately 20% of today’s molecular pipeline
facturing process, have all been shrinking dramatically.” will be manufactured using elements of next-generation biopro-
For downstream bioprocessing, the greatest test has cessing or continuous manufacturing,” he says.
been addressing ongoing productivity gains upstream. Downstream, these improvements include combined unit
“There is a gap, and it is widening, between what is coming operations, alternatives to chromatography, new process model-
out of the fermenters and the capabilities of downstream ing techniques that allow for the visualization of “the golden
processing systems,” says Gottschalk. While upstream batch,” and analytical approaches including greater use of process
processing is volume-driven, unit operations downstream analytical technologies (PAT). While the gap with upstream may
Sebastian Kaulitzki - Stock.Adobe.com
are mass driven, he says. One can make a kilogram of still be there, it is being addressed. This article highlights trends.
antibodies in a 1000-L bioreactor, or increase the output
to 10 kilograms by increasing expression levels and overall PROCESS INTENSIFICATION
yield, says Gottschalk. But to handle the larger amount Behind many of the new advances is process intensification,
downstream, at any capacity, will require chromatography which improves facility productivity by focusing on terms of
columns that are 10 times larger, or that are run in 10 kilograms of product manufactured per year, per square meter
cycles, he says. of facility footprint, says Peter Levison, executive director of

business development at Pall Corp. TRIUMPHS OF ENGINEERING membrane, monolith, or depth filter,” he
“Chromatography is a potential bottle- The move to continuous chromatog- explains.
neck downstream because sorbents have a raphy (i.e.,having multiple columns Although some research groups and
finite binding capacity, but process inten- running side by side) has also been a sig- companies, most notably Merck & Co.,
sification allows feed to be pre-concen- nificant advance, says Chatel. Univercells, have been exploring end-to-end continu-
trated prior to adsorption,” he says. “In for example, is developing continuous- ous biomanufacturing, more manufac-
addition, one can move from batch to process-based platforms and is work- turers are using continuous processing
continuous chromatography to maximize ing on virus manufacturing platforms strategically, both up and downstream.
the adsorptive capacity of the sorbent,” he in projects sponsored by the Gates Univercells has developed a continu-
says, noting the benefits of single-pass Foundation. “All are batch processes ous production system in which cells
tangential flow filtration (TFF). As higher because they depend on binary loops and are grown in batch mode but different
concentration formulations become the they need regeneration, but they are run process steps are run continuously. “We
rule, filtration systems will have to handle so that when one is purifying product, need to get away from the old approach,
concentrated and more viscous solutions the other column is being regenerated, in which you send all the contaminants
without damaging product, he says, and resulting in a continuous stream of prod- downstream,” says Chatel. As Gottschalk
virus and sterile filters are being developed uct output. Advances in engineering and says, continuous processing was always
to address these challenges. mathematics enabled this to happen, and used upstream for sensitive molecules
Bulpin also notes the importance have drastically reduced the amount of that could not endure conditions in the
of process intensification “Our work resin required,” he says. fermenter (e.g., enzymes such as Factor
with customers has shown that facility The result of all these improvements A for hemophilia treatments). Perfusion
throughput can increase by up to 75%, has given manufacturers access to a “scale- fermentation was essential in order
simply by converting one or two unit able toolbox” that can enable batch, con- to keep the molecule intact, but the
operations,” he says. tinuous, or hybrid process development approach has since been used with other
Alex Chatel, product manager at and scale up using stainless steel or single- more stable molecules such as antibodies,
Univercells, singles out TFF systems as use systems, says Levison. and is now moving into the broad bio-
a major process-intensification-based Examples he cites include, not only pharma world, he says.
advancement. TFF enables a large increase processes that enable continuous chro- Continuous processing is already a
in concentrations in a single pass, so that matography, but depth filters designed to reality for single-use systems, Levinson

feed streams needn’t be recycled many
times over through the membrane, he says.
clarify higher cell density fed batch cul-
ture; continuous diafiltration and contin-
says. “We can currently clarify cell cul-
ture media continuously using depth fil-
Bulpin recalls one case in which a manu- uous concentration; and the introduction tration; purify product using continuous
facturer installed a single-pass TFF device, of large-scale prepacked chromatography chromatography; carry out continuous
which now provides inline feed stream columns and new single-use chromatog- virus inactivation; and then complete
concentration and/or dilution, elimi- raphy skids to complement them. “With the ultrafiltration and diafiltration stages
nating the need for holding tanks and associated products such as sterile con- using continuous single-pass filtration
intermediate process steps. “With slight nectors, biocontainers, and single-use fil- technologies,” he says.
modifications to a couple of unit opera- ters, a closed, fully connected downstream Trends for the future relate more to
tions, companies can incorporate a more processing train can now be assembled, the modularity of the continuous down-
continuous approach while reducing their allowing for rapid scale-up and process stream platform, with connectivity
manufacturing footprint and scale,” he says. implementation,” says Levison. between each unit operation and inte-
Using process intensification, unit There has been a shift to single use grated PAT and automation and control
operations are being integrated so that, for downstream, Gottschalk says. In some platforms, Levison says. For a truly con-
example, cell removal and early clarifica- cases, single-use systems can run at tinuous upstream process, a perfusion
tion may be captured in one step, instead close to 100% variable cost eliminat- bioreactor could be used upstream, but
of the three or four that were previously ing the need to depreciate costs for the it would require continuous cell culture
required (i.e., typically centrifugation fol- facility and stainless steel equipment, fluid harvesting. Current cell-retention
lowed by depth filtration, sterile filtration he says. Gottschalk sees increased use technologies based on hollow fiber filtra-
and capturing column), says Gottschalk. of convective, instead of throughput- tion are prone to fouling, he says, so the
As a result, some downstream processes limited chromatography media, as a composition of the harvest is variable
have been reduced from 10 to three or key trend. “Filters are developed with and changes over time. One alternate
four steps, and, in the future, he believes, the ligands that would normally go on
two or three steps will be possible. chromatography resins, and used in a Contin. on page 16
www.biopharminternational.com July 2019 BioPharm International 13

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14 BioPharm International July 2019

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Contin. from page 13 virus test, which cannot currently be out carrying out a detailed design of
brought online, but most other analyti- experiment). If the digital twin could
could be to use acoustic wave separa- cal tests can be run online,” he says. predict batch-to-batch variability and
tion (AWS), and platforms using AWS Bupin sees artificial intelligence (AI), process robustness then perhaps this
should be commercially available in the primarily machine learning, as driv- could reduce time to market of new
near future, he says. ing future improvements in PAT. “This medicines directly impacting on patient
approach should significantly improve health,” says Levison.
MODELING AND PAT quality and process robustness; combined
The biggest change in bioprocess auto- with near-real-time data acquisition, it BUFFER MANAGEMENT
mation has been the introduction of the will enable closed loop for the process One practical area where downstream
model-driven approach, says Bulpin, execution,” he says, improving commu- efficiencies are being improved is in
who explains that this approach allows nications and interaction between enter- buffer and waste management. “Single-
users to define process, interactions, and prise systems and manufacturing field use technologies offer benefits because
contexts in form of parameterized data. I/O (inputs/outputs), including sensors, they reduce cleaning and cleaning
This helps to reduce complexity, increase actuators, analysers and drives. validation requirements,” says Levison.
flexibility and reusability, and enable As more bioprocessing steps are “With the introduction of single-use
dynamic re-configuration of systems and automated, the approach is also proving mixers and single-use biocontainers
processes. Model-driven design can lead important for sampling, says Gottschalk. have come opportunities for storage of
to increased quality by increasing vis- Lonza is working on data mining and buffer concentrates with in-line dilution
ibility, providing higher abstraction layers new approaches using machine learning and/or conditioning to generate the
with less custom code, and empower- and other artificial intelligence concepts. buffer on demand at the point of use,”
ing the domain experts by focusing on “The goal is to use advanced concepts and he says. Fluid waste can be collected in
the process rather than underlying tech- multivariate data analysis to ensure that biocontainers which can then be asepti-
nology, Bulpin says. In the long term, we run only the ‘golden batches,’” he says. cally disconnected and disposed of, he
he expects it will lower the sensitivity Over the next few years, Levison adds. However, as facilities become
to change and help to bridge the gap expects new inline or at-line tools to smaller, buffers continue to pose com-
between business and technical domains. become available to monitor both criti- plex storage and logistical challenges
At the same time, analytical meth- cal quality attributes and critical process and can become a bottleneck for single

ods are improving, and use of PAT is
increasing. Chatel has seen advances in
parameters. “Whether this will involve
new sensing and detection technologies
use production, Levison says. One solu-
tion to increase facility buffer capacity
sensors for pH, dissolved oxygen, and or multi-attribute measurements (MAM) is through the use of concentrates or
traditional measurements, that have based on existing technologies, or indeed stock solutions, both enabled by single-
allowed companies to adopt monitor- a combination of both approaches, use mixers and single-use biocontainers.
ing and control practices for bioreactors remains to be determined. What is clear Managing buffers this way and delaying
and other steps such as chromatography. is that as we move towards advanced ana- the dilution of these concentrated solu-
“Future sensors will be able to detect lytics and PAT, more data will be gener- tions up to the point of use, allows sin-
metabolic concentrations in cell cultures ated more frequently so the demands gle-use facilities to increase their buffer
(e.g., measure titers at line and provide on the data storage, management and capacity and increase their productivity.
feedback control over feeding strategies) handling will increase and this all needs In the area of buffer management
to reduce burden of contaminants and to interact with the process control sys- and maintenance, MilliporeSigma
ultimately lead to more lean processes tem. AI and the Internet of Things will recently launched the BioContinuum
and reduce processing time,” he predicts. play a big part in these advances, which buffer delivery platform, Bulpin says,
Noting the increased use of Raman all fall under the umbrella of Industry which uses buffer concentrates and in-
spectroscopy-based methods, Gottschalk 4.0,” he says. line dilution to deliver buffer directly
says, “We can now monitor the most One of the benefits of Industry 4.0 into the system. The company expects
relevant critical parameters online or would be ability to use simulation (e.g., the platform to permit an 18% reduc-
at-line so that, after we have manufac- in the digital twin approach). “Provided tion in cleanroom area requirements in
tured the batch, we know whether it is that the digital twin can be shown to a 2,000-L bioreactor facility; reduction
acceptable and can be approved or not. be a true twin of the real process, then in buffer costs by up to 16%; and more
In biopharma, we have one or two criti- its use in method development and than 50% reduction in labor and capital
cal assays that need to be run at the end optimization may become a reality (e.g., costs for a facility that uses five 2,000-L
of production, such as the adventitious for determining process limits with- bioreactors, he says. ◆

Upstream Processing
Applying QbD to Upstream Processing

Using a QbD approach from early-stage development
through commercialization can ensure that upstream
processes are efficient and reliable.
FOR PERSONAL, NON-COMMERCIAL USE SUSAN HAIGNEY
T
he concept of quality by design (QbD) has been part exclusion, and such token or generally representative metab-
of the biopharmaceutical industry for more than a olites as glucose or lactate,” says Whitford.
decade. While similar approaches to QbD are used According to the International Consortium for
in the development and manufacturing of large-molecule Innovation and Quality in Pharmaceutical Development
and small-molecule drugs, the complex nature of large- (IQ Consortium), a collective of more than 35 biophar-
molecule drugs creates more factors that may affect process maceutical companies, there are also differences between
performances and the critical quality attributes (CQAs) of the use of QbD for large molecules versus small molecules
the drug. when considering antibody-drug conjugates (ADCs). ADCs
Because biomanufacturing involves living cells, there are are complex molecules composed of an antibody linked
more reactions happening in a bioreactor than usually hap- to a cytotoxic small-molecule drug. As noted by the IQ
pen in chemical reactions, according to Bill Whitford, stra- Consortium, companies that develop ADCs state that CPPs
tegic solutions leader, bioprocess, GE Healthcare. “While we and CQAs may also differ between the drug-linker (DL)
often understand nearly everything about a small-molecule component of the ADC and the related small-molecule
reactor development pathway, we have only a partial under- drug because the DL represents a small portion of the total
standing of all the interacting salvage, degradation, and mass of the ADC. Unlike small-molecule drugs, the dos-
biosynthetic pathways of mammalian cells. This includes ing frequency and scheme of the ADCs also may dilute the
science photo - Stock.Adobe.com
even many relevant metabolic pathways, inductions, flux, impurities in DL intermediates.

rates, and turnover. Finally, we often develop and control Another difference between small-molecule and large-
bioprocesses using very surrogate key quality indicator (KQI) molecule drugs, according to the IQ Consortium, is that
and critical process parameters (CPPs). Rather than measur- in early development, a platform process approach may be
ing precisely what we know (or suppose) to be relevant, we applied, which may accelerate timelines and reduce develop-
monitor such general parameters as pH, oxygen, trypan-blue ment burden.

Upstream Processing
QBD IN UPSTREAM Performing small-scale DoE by USING QBD TO DEVELOP

PROCESSING applying verified scale factors and CELL CULTURE PROCESSES
QbD is used in cell-culture expansion, using a risk-based approach are key Because cell culture has a direct
cell harvesting, and cell-culture media to implementing QbD in upstream impact on production efficiency and
development. However, according to processing, says Whitford. “The final product quality, using DoE can help
the IQ Consortium, many companies product will be a verified design space, determine the relationship between
find that clone selection and the devel- clearly communicated—being mindful cell-culture process parameters and
opment of the production-stage bio- of using accepted terms and defini- product quality, which then helps
reactor process benefit most from the tions—to regulators in the submission. determine the design of the operation
application of QbD due to the follow- Currently, upstream process develop- space for a process. DoE can also help
ing: “In clone selection, the final pro- ment focuses on digitally transformed determine the needed sensor technol-
ducer cell line is defined, which is then and intensified biomanufacturing,” ogies to establish robust closed-loop
used for the commercial process and Whitford states. model predictive controls, according
throughout the lifecycle of a product, to Whitford. “Unlike many other pro-
while the N-stage production bioreac- duction processes, biomanufacturing
Bioreactor process
tor is considered to be the most criti- has classically been hindered by a lack
cal step with respect to the CQAs and of real-time product attribute mea-
development has
essentially determines the final product surement. Even in nominally identi-
quality from an upstream perspective. cal bioprocesses, we can see diversity
been positively
In principle, all upstream process steps in product quality due to variability
can benefit from a QbD approach and in (e.g., such raw materials as cell
impacted by the
examples where companies apply those culture media). Using a QbD/PAT
also depend on the assessment of (busi- strategy directs the implementation
use of QbD and
ness) risks,” a representative of the IQ of advanced near-real time monitor-
Consortium says. Predefining developing through the application of newer,
empowered by
ment goals for titer ranges and product automated sampling techniques and
quality profiles, as well as assessing risk, multivariate data analysis. This sup-
also benefit from QbD. process analytical ports advanced in silico modeling that
FOR PERSONAL,technology.
NON-COMMERCIAL
The development of robust pro-
cesses that include good cell culture USE
can not only help control the process,
but predict product quantity and qual-
performance can be done through ity outcomes,” says Whitford.
design of experiments (DoE). In early Bioreactor process development has Implementing a feedback qual-
stage development, QbD can be used been positively impacted by the use of ity control strategy, as opposed to a
to identify a quality target product QbD and empowered by process ana- recipe-based control strategy, when
profile (QTPP), according to the IQ lytical technology (PAT), according to using QbD in developing cell culture
Consortium. Late-stage applications Whitford, by enabling continuous moni- processes may be the best approach,
include process characterization stud- toring of process parameters and product says Brandon Downey, principal engi-
ies to decide on one-factor-at-a-time characteristics throughout development neer, R&D at Lonza, because of the
(OFAT) experiments versus DoE- and manufacturing. “In the pursuit of challenges cell-culture processes pres-
based experiments and the defining process intensification, the influence of ent. “Many [cell culture] processes
and classifying of parameter ranges. altering process parameters on product have successfully used a recipe-based
“Currently, a claim of a design space characteristics can be rapidly assessed. control strategy, determined using a
is not pursued by most companies. This contributes to increasing an under- design space approach, to manufac-
However, achieving in-depth understanding of what types and ranges of ture quality product. Nevertheless, cell
standing is a goal (e.g., by advanced bioreactor parameters provide accept- culture processes pose some unique
data analysis approaches). Also, able product quality. This mapping of challenges when attempting to employ
detailed risk assessments can play an the relationship between bioreactor pro- a recipe-based control strategy,” says
important role in late-stage activities. cess characteristics and product CQAs Downey. And, the large number of
Commonly, the knowledge gained in will define a bioreactor design space, an raw materials used in cell culture can
process characterization studies (PCS) important element in both ensuring a make understanding the variation of
will be included in the control strategy,” robust process as well as current FDA each component difficult. “Although
the IQ Consortium states. expectations in filings,” says Whitford. fundamental understanding of how

Upstream Processing
variations in common raw material been around for decades, individual

attributes impact a cell culture process process optimization is beneficial to Made in
is increasing, this knowledge is still far individual cell strains, recombination Germany
from complete enough to encompass techniques, cloning approaches, and
the variation that has been observed culture systems. Continuous monitor-
in the cell culture media,” he states. ing is being enabled by PAT-derived
systems, says W hitford. In addi-
tion, highly accelerated bioprocess
Using a QbD development is supported by new
fully-automated, single-use multi-
approach to define parallel bioreactors. “Employing some
of the process monitoring and design
objectives for
FPLC &
approaches … in the context of high
throughput and automated systems for
establishing a new process development is now very popu-
manufacturing
lar. For example, some vendors offer as
many as 24 fully featured, single-use,
250 mL mini-bioreactors supporting
Prep LC
facility may be process development and optimization Flexible purification solutions
as well as selection or development in
beneficial. scale-down studies,” says Whitford.
Medium selection, however, varies
The behavior of the cull culture pro- based on practices and needs, accord-
cess may also be impacted by varia- ing to the IQ Consortium. “For exam- Modular system layout
tions in unmeasured impurities in raw ple, companies that employ a platform
materials, says Downey, making it dif- process approach, including a platform
ficult to determine the total impact cell culture media system, typically do
of raw material variation on the cell not require media selection for mol-

Matching detectors
culture process. “Finally, the cells in the
for your target
production cell culture step uniquely
ecules that fit the platform process.
However, for atypical modalities that
determine many of the structural do not fit the predefined selection cri-
attributes of biologic molecules which teria, media screening can be done to
could (and in some cases have been select the condition which yields attri-
clinically shown to) influence the func- butes that best fit the criteria,” says the
tion of the molecule.” IQ Consortium. Easy scale-up to 1 000 ml/min
QBD IN CELL LINE SELECTION QBD IN
When it comes to cell line selection, EQUIPMENT SELECTION
QbD can be used in the selection of a Using a QbD approach to define
final clone during early-stage develop- objectives for establishing a new Various columns
ment by using prior knowledge to pre- manufacturing facility may be ben-
for your application
define clone selection criteria including eficial. Risk assessments can be used to
cell-line stability, culture performance, evaluate different equipment against
and product quality attributes. “As the predefined objectives. The knowl-
Define your AZURA® system:
the selection process progresses, more edge gained through this process can
clone-specific knowledge is gained, show how different equipment may www.knauer.net
and the process continues until the impact the objectives, according to the
clone that best fulfills the selection cri- IQ Consortium.
teria is identified,” according to the IQ QbD can also be applied to the
Consortium. evaluation of equipment performance.
Whitford stresses that even though
many bioprocessing platforms have Contin. on page 22
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FUJIFILM Irvine Scientific
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Upstream Processing
Contin. from page 19 activity but its stability in storage,” is a limitation to how much quality
says Whitford. “Secondly, we don’t can be designed into the process dur-
The suitability of equipment can be yet fully understand the conditions ing early-stage development, and in
established through experimental of cell culture that will control those some cases, target quality characteris-
designs, says Whitford. “This is gen- molecular properties. Often, they are tics may not be clear. Some companies
erally accomplished through either discovered more empirically than by question what effort associated with
limited experimentation at scale, or pathway and structure understand- this should be taken early in develop-
through studies in scale-down models, ing. Finally, we are only beginning to ment. For example, early-stage CQAs
employing prior understanding of the implement comprehensive monitoring are more focused on the safety aspect of
scale-factors involved in extrapolat- of the bioreactor process, metabolites, the drugs and as clinical data and process
ing results to full-scale manufactur- and products that can provide robust experience are gathered over the course
ing. Premier single-use system vendors specific, relevant, and predictive pro- of the project, this informs the efficacy
have for years been supplying much cess parameter values and product aspect thus modifying the CQAs in late-
information to aid in such studies. quality assessment,” Whitford adds. stage development,” according to the
One example here is CFD data on IQ Consortium. They also warn that
single-use mixers and various scales, using in-licensing molecules may present
A robust cell culture
fill volumes, impeller speeds, and vis- unanticipated challenges. “This might,
cosity of diluents. We have seen such depending on the development stage of
platform process
techniques applied to the selection of the in-licensed molecule, shift focus from
single-use bioreactors and mixers, per- ‘design’ to comparability.”
can ensure that
fusion apparatus and even dynamically
feed-back controlled inline buffer con- CONCLUSION
ditioning systems.” CQA profiles are The implementation of QbD into the
When it comes to late-stage devel- bio/pharmaceutical industry aligns
consistent from
opment, the IQ Consortium agrees with regulatory and industry efforts to
that small-scale models (SSMs) are ensure the efficacy, safety, and quality
the early stages
essential to QbD when scale-down of medications. The complex nature of
models must be qualified to show biologics adds additional complexity to
of development
FOR PERSONAL, NON-COMMERCIAL
they represent large-scale operations.
“SSMs are used in PCS to understand USE
developing and manufacturing these
innovative therapies. Using a QbD
through
the process and determine parameter approach from early-stage develop-
ranges and classifications. Given that ment through commercialization can
commercialization. ensure that upstream processes are effi-
the PCS experiments are often large
and complex, it is important to bal- cient and reliable.
ance between representativeness and A robust cell culture platform pro- “As biologics increasingly take the
throughput of the SSMs,” according to cess can ensure that CQA profiles are form of novel modalities (e.g., not
the IQ Consortium. consistent from the early stages of mAbs), it will become increasingly
development through commercializa- important to be able to control the
CHALLENGES IN QBD tion, according to the IQ Consortium. quality of biologics so that the struc-
IN EARLY DEVELOPMENT It may be difficult, however, for compa- ture—functional relationships of
Designing quality into the biophar- nies without a platform process in place these new classes of compounds can
maceutical process early on can be to incorporate quality in early-stage be better understood in the clinic.
difficult because the nature of the development without performing addi- Furthermore, as medicines become
protein may not be fully understood, tional experimentation to determine the more personalized, the ability to tai-
says Whitford. There is also a range robustness of their process. This experi- lor the structural attributes of bio-
of variants, such as glycosylation in mentation can be done using a QbD logics molecules that are dictated by
monoclonal antibodies (mAbs), that approach and DoE methodology, says the process will become increasingly
occurs when biologics are gener- the IQ Consortium, when “molecules important. QbD will likely continue
ated, which can affect both biological cannot conform to the platform.” to play a foundational role in creat-
activity and/or the secondary or ter- This work, however, could have an ing the processes which can robustly
tiary structure of the molecule. “This impact on investigational new drug deliver these compounds,” concludes
can influence not only the entity’s application submission timelines. “There Downey.X

Manufacturing
Design Considerations for

a Commercial Cell and
Gene Therapy Facility
FOR
ThePERSONAL,
commercialization ofNON-COMMERCIAL USE
cell and gene therapies has become
a reality, prompting deeper considerations of logistics,
technology, and design for manufacturing facilities.
FELIZA MIRASOL
W
ith the recent FDA approvals and commercializa- tions (individual processing steps) will be combined to build
tion of cell and gene therapies in the US market out a complete process.”
and a pipeline of cell and gene therapies progress- Further, the considerations for cell-therapy versus viral-vector
ing toward regulatory review, there is increased focus on manufacturing (for in-vivo gene therapy or as a starting mate-
establishing commercial manufacturing facilities for these rial for ex-vivo gene therapy) can be quite different from each
complex biotherapeutics. The workflows of a cell therapy or a other, adds Jonathan Fortin, global director of Engineering,
gene therapy differ from each other and from those of thera- Lonza. “Viral-vector manufacturing lends itself to a pre-defined
peutic antibodies, so the design and layout of the manufactur- manufacturing process flow and equipment design, which leads
ing facility must take these workflows into consideration. to greater uniformity in scale-dependent footprint for multiple
processes. Cell therapies, on the other hand, often have unique
WHAT TO CONSIDER processes that limit the level of flexibility possible in suite and
Gorodenkoff - Stock.Adobe.com
“There are multiple considerations to take into account, per- general equipment configurations,” Fortin explains.
haps most important, though, are capacity utilization at full He emphasizes that it is necessary to conduct a keen assess-
scale and space efficiency,” says Phil Vanek, general manager, ment of shared processing areas, taking into account multi-
Cell and Gene Strategy, GE Healthcare. “This means several product segregation needs. For example, closed processing steps
things, including how the materials and personnel will flow (e.g., pre-transfection bioreactor stages for viral vector) and
throughout the facility (unidirectional) and how unit opera- lower utilization activities (e.g., final filling followed by product

Manufacturing
changeover or media preparation) could bioreactor skids with large downstream need for scale-out rather than scale-
use shared spaces, driving up efficiency. In chromatography platforms, often segre- up in the case of autologous therapies
addition, material, personnel, and waste gated into two or more rooms. In mAb and the highly manual, and often open,
flows require special attention for the production, Chinese hamster ovary cells nature of processing requiring asep-
current design and in support of future are the workhorse of the industry, and tic processing operations, Ramaswamy
expansion needs. the process is mostly, if not completely, explains. “Facilities are commonly
“As a contract development and man- closed, notes Vanek. grade B cleanrooms with grade A bio-
ufacturing organization, building facili- Cell and gene therapies, on the safety cabinets (BSC) and incubator
ties to accommodate for multiple current other hand, cover a large number of cell banks. These requirements will see a
and future customers—and, therefore, types, are often patient-specific (autolo- dramatic change as closed automated
multiple known and unknown products, gous), and are dependent on equipment operations (e.g., Lonza Cocoon tech-
processes, and setups—and having a flex- made for other industries (e.g., blood nology for autologous or small-scale
ible floor plan are critical. We need to be processing or protein bioprocessing). cell and gene therapy and suspen-
able to accommodate for multiple prod- Next-generation technologies have to sion single-use bioreactors to scale up
ucts and different modalities in different consider the cell types and the batch larger-scale cell therapies) are adopted
phases, each with the ability to scale up approach (single per patient or scaled for these products.”
operations to commercial-scale manufac- up). Common to each, however, would Another important difference is
turing in one facility,” Fortin states. be design criteria to minimize risk in the high level of tracking and trac-
“For product-specific setup areas within manufacturing, including unidirectional ing, which is crucial in cell and gene
the facility, such as dedicated suites, the flow of personnel and materials in the therapies, especially autologous patient
key aspects come down to the nature and cleanrooms, proximity to a quality therapies, but not relevant to campaign-
the stage of the product: autologous versus control lab for in-process testing, and based batch mAb production. In addi-
allogeneic, clinical going towards com- wholly integrated quality management/ tion, given that autologous cell therapy
mercial versus commercial. We try to opti- electronic batch records to reduce oper- products often lead to a single dose for
mize the capital cost for our customers ations errors, states Vanek. patients with limited treatment options
and for us by maximizing the throughput “We have to split this discussion into and time, redundancy in manufacturing
and flexibility of the facility,” he adds. two parts for a successful comparison: capability to proactively mitigate against
In addition to current processing first, viral vectors for gene therapy and any production disruptions must also

needs, Vanek notes that a facility design
must be flexible enough to accommo-
second, cell and ex-vivo gene therapy,” be considered. “This is a stark differ-
remarks Senthil Ramaswamy, head ence from mAb or allogeneic products
date future changes to any processes. of R&D, Cell & Gene technologies, where a stock-up strategy can offer suf-
“Tomorrow’s processes may look very Lonza. Viral-vector production, he ficient mitigation. Necessary, geographi-
different from today’s, so anticipating notes, has a number of parallels with cal proximity to patients can also drive
what will be needed in terms of gases, mAb manufacturing, especially if multiple simultaneous manufacturing
power, and bench space should be con- using a suspension cell-culture process sites,” says Ramaswamy.
templated. And then, finally, a digital or an adherent format that has the “A common factor between viral vec-
solution encompassing electronic batch footprint of a bioreactor rather than tor and mAb production is that many
records, process monitoring, scheduling, dozens of small multilayer cell-cul- of the processing steps are/can be
and other key process attributes should ture vessels in incubators. The needed standardized and scaled. This makes
be designed-in early to add efficiency and infrastructure is also similar to mAb the design of a commercial facility
control to the process,” he states. manufacturing, taking into consider- for such products relatively straight-
ation the unique segregation needs for forward, even considering that there
DIFFERENT FROM MABS viral vectors when multiple products is no standard platform yet for viral
Considering the differences between are being manufactured in the same vectors,” adds Bertus Markies, head
monoclonal antibody (mAb) production facility. “The scales of processing at of Value Chain Management, Lonza.
and cell/gene therapy production is also the moment top out at about 2000 L “As stated by Senthil, the situation is
important when establishing a cell/gene of cell culture, which is in line with radically different for cell therapy prod-
therapy facility. Already well-established, smaller mAb manufacturing processes,” ucts. For both allogeneic and autolo-
mAb production is typically batch-ori- Ramaswamy states. gous products, the design of the facility
ented (in this way, making it similar On the other hand, cell and ex-vivo heavily depends on the process. The
to allogeneic cell therapy production) gene therapy currently have different technology in these fields is still far
and characterized by larger single-use facility needs for two primary reasons: from mature, and a wide variety is seen

Manufacturing
in what is used, ranging from differ- Figure 1. Simple schematic of a cell and gene therapy manufacturing facility
ences in equipment, media, and cell- layout. CAR-T is chimeric antigen receptor T cell. BSC is biosafety cabinet.
expansion to even small parts such
as tubing sets. In essence, these tech-
nologies have high potential once they
become industrialized.”
EQUIPMENT/TECHNOLOGY
Another important factor to consider
for a cell and/or gene therapy facility
is the equipment and setup. For mAb
bioprocessing and viral-vector manufac-
turing, the equipment needed is similar,
such as single-use bioreactors and stan-
dard harvest and downstream systems
for clarification, tangential flow filtration,
chromatography, and sterile filtration,
according to Ramaswamy.
Both mAb bioprocessing and viral-
vector manufacturing are usually divided
into upstream and downstream processes.
Upstream focuses on maximizing both ing systems, and controlled-rate freezing It is important to note, however, where
the number of cells per bioreactor unit and storage capabilities (see Figure 1). customization of single-use technology
volume (e.g., cells per liter) and then the Process-specific equipment are typically may be required. “Reliable sourcing and
productivity of the cell upon induction cell isolation, selection, and transfection quality standards are key considerations
of protein (mAb) or viral particle expres- systems,” Ramaswamy clarifies. when selecting single-use systems for
sion. “Getting these conditions just right Single-use systems are technologies cell and gene therapy because they are
requires balancing the cell line, expres- that are widely used in the bioprocess- often not designed with these therapies

sion system, media and growth factors,
and bioreactor conditions—such as stir
ing and blood processing industries
to protect product from contamina-
in mind,” points out Ramaswamy. For
example, for cell therapy, the cells are the
rate and gas transfer. For biologics and tion, and these systems are also appro- final product received by the patient and
viral vectors, the cells are a byproduct priately sized for most cell and gene are highly dependent upon the process
of production and, thus, often get lysed therapies, says Vanek. Single-use bio- and consumables used. Cell therapy pro-
and discarded in the downstream process, reactors that are either flask based duction does not have the numerous puri-
which is designed to optimize both the or rocking based are widely used, for fication steps and analytical tests that are
yield and the purity of the final product,” example, with stirred tank bioreactors standard to mAb production.
Vanek explains. being used in certain applications. To be able to scale up viral vector
In comparison, cell therapies are Cell collection and washing is mostly manufacturing, bioreactors—with 3D
different principally because the cell by centrifugation (either closed-system spacing rather than a cell-stack setup—
is not a byproduct, but the final prod- centrifugation or a special approach based are needed with automated downstream
uct. “Therefore, production strategy is on elutriation), states Vanek. “Simple processing and product fill. The use of
all about preserving the cell phenotype filtration is not commonly used; new bioreactors would also be needed for
across multiple production steps and platforms are popping up claiming to be allogeneic cell therapy production as well
scales, meaning that keeping the thera- gentler or simpler, but none have yet been as maximizing automation at commer-
peutic potency (defined individually for widely adopted,” he adds. “Scalability is cial scale, according to Ramaswamy. “As
every therapy) is paramount, as well as key, and few platforms if any can move we see more products entering com-
keeping the cells from becoming con- easily between small volumes (10s of mercialization, there is an urgent need
Figure courtesy of Lonza.
taminated by microbes,” states Vanek. mLs) to large volumes (100s of liters). to close and automate these process-
“For cell therapy today, it is often a mix What’s more, analytical platforms for in- ing steps leading to industrialization of
of standard and process-specific equip- process control and cell characterization cell therapy processes. This will lead to
ment. Standard equipment would be BSC, are lacking in the cell therapy industry greater standardization in the future,”
incubators, centrifuges, vial or bag-fill- but are being actively pursued.” he adds.◆

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Residual Impurities
Analysis of Residual Impurities

in Continuous Manufacturing
Real-time monitoring of product- and process-
related impurities remains a challenge.
FOR PERSONAL, NON-COMMERCIAL USE CYNTHIA A. CHALLENER
C
ontinuous bioprocessing provides numerous benefits, host cell or processing steps (host-cell proteins [HCP], host-
from high product quality and consistency to smaller cell DNA, residual Protein-A, etc.).
physical footprints, increased flexibility, and potentially “Technologies that utilize mass spectrometry (MS) capa-
lower capital and operating expenses. It also provides the bilities are, at this point, the seemingly best choice for resid-
greatest benefits if real-time monitoring of process parameters ual impurity detection, characterization, and/or quantitation
and product quality, including detection and quantitation of when coupled with liquid chromatography (LC) systems,”
residual impurities, is performed. While advances in analytical observes Amit Katiyar, director of analytical and formula-
methods and automation are occurring, practical implementation sciences for Thermo Fisher Scientific. Multi-attribute
tion of real-time monitoring of product- and process-related methods (MAM) capable of detecting, quantifying, and
impurities has yet to be achieved. characterizing multiple product quality attributes are cur-
rently being developed.
IDEAL SOLUTIONS? A triple-quadrupole system would be best suited for
“In an ideal world,” asserts Byron Kneller, senior director quantitative analysis of small molecules/peptides, with quad-
for analytical and formulation development with AGC rupole time-of-flight systems more appropriate for the
Biologics, “we would have simple, rapid, physicochemical characterization and semi-quantitative analysis of larger
anawat_s - Stock.Adobe.com
tests for residual impurities.” molecules, according to Charles Heise, senior staff scien-
Unfortunately, there are different types of residual impuri- tist for bioprocess strategy and development at Fujifilm
ties. They can be classified as product- and process-related, Diosynth Biotechnologies.
and as chemical impurities (typically small-molecule process
CYNTHIA A . CHALLENER, PhD, is a contributing editor to
additives such as isopropyl `-D-1-thiogalactopyranoside, BioPharm International.
antifoams, antibiotics, etc.) or biological impurities from the

Residual Impurities
An amalgamation of both technolo- chromatography in addition to immuno- from Fortebio, which relies on biolayer
gies for a single quantitative measure- assays such as ELISA and electrophore- interferometry. Similarly, he says that
ment covering a wide mass range would sis as the primary means for quantifying ELISAs for sensitive quantitation of
be an ultimate solution, he adds. A series product- and process-related impurities,” biological impurities that have slow
of standards could be used to generate notes Katiyar. response times (material quarantining
standard curves for target impurities and Often tests for process-related impuri- needed) could also be replaced with
the software needed for identification ties require specialized reagents such as equivalent ‘dip-and-read’ methods.
and quantification of peaks. Aspects of anti-HCP antibodies or natural products The new technologies (e.g., MAM,
high-throughput proteomics analysis like limulus amebocyte lysate and can be MS for HCP analysis, automation for
may also be applicable to the continuous more complex, adds Kneller. In addition, DNA, HCP, and Protein A residuals)
manufacturing environment. many impurities are heterogeneous (e.g., are just starting to be introduced in the
Other analytical techniques Heise HCP) and thus not amenable to detec- process development space, according
highlights include in-line spectroscopic tion and quantitation using simple tests. to Katiyar. “The transition from explor-
measurements such as Raman, Fourier- In practice for continuous processes, atory to fully validated methods will be
transform infrared (FTIR), ultraviolet/ in-line confounded (e.g., spectroscopy) slower for such methods in a cGMP
visible (UV/Vis), and refractive index or secondary parameter (e.g., off-gas, pH, environment where method performance,
methods that can give quantitative as well basic physical property) measurements equipment validation, and data-integ-
as qualitative, real-time analysis. “The par- are most user friendly, but accuracy of rity measures need to be at the highest
ticular technology used will depend on level. There will also be a significant cost
In practice, the
the impurity, its expected concentration aspect of not only reconfiguring current
range, and whether monitoring is per- laboratories to accommodate the equip-
approach used
formed during steady-state conditions,” ment, but also for training and/or hiring
he observes. New methods or advances new personnel with expertise in these
to detect process
in current technologies may also become new technologies,” he says.
available that allow in-line measurements
impurities may be
using nuclear magnetic resonance (NMR) SENSITIVITY AND
imaging, sensor arrays, or LC systems. MATRIX CHALLENGES
determined by
The final option, Heise says, is to do At the process level, identifying the resid-

no process monitoring and rely solely
on the quality-by-design data generated USE
ual impurities present and the appropri-
available instruments, ate analytical assays, with the required
during process development to define detection limits and sensitivity ranges,
assays, time, and
the operating ranges and edges of failure. to monitor them across the process is
critical, stresses Dan Pettit, senior staff
PRACTICAL REALITIES staff capabilities. scientist for analytical development at
“In practice, the choice of method may Fujifilm Diosynth Biotechnologies. At
be constrained by the availability of the residual impurity detection is sacrificed the operations level, he notes that iden-
instrumentation, turn-around time, ana- for real-time control, as a result of the tifying suitably equipped labs to develop
lyst training, and potential interference heterogeneous nature of the signal, or and validate methods for GMP analysis
from the complex matrices that may be to allow for detection of a secondary is vital because of the heterogeneity of
involved in the downstream purifica- response, according to Heise. process impurities and the consequent
tion,” comments Ian Parsons, director of He also notes that off-line LC–MS, analytical issues they pose.
analytical development for biologics at where mass windows can be limited by Heterogeneous process-related
Charles River Laboratories. His com- buffer solution and scanning rates, or impurities, specifically HCPs, are often
pany has focused primarily on the use LC–evaporative light scattering detec- quantified in early-phase projects using
of high-performance LC (HPLC) and tion (ELSD), is typically used for small generic kits that may result in key impu-
the enzyme-linked immunosorbent assay molecules, but these are not sensitive rities going undetected during purifi-
(ELISA), with MS and gas chromatog- enough and can take too long to run for cation process development. “These
raphy (GC) also used frequently. large-molecule manufacture. impurities have the potential to cause
“The pharmaceutical industry is still These approaches could potentially problems at production scale,” Kneller
heavily utilizing traditional separa- be substituted for sensor technologies says. Current ELISA techniques lack the
tion techniques such as size-exclu- with equivalent modalities, accord- ability to identify specific immunogenic
sion, ion-exchange, and reversed-phase ing to Heise, such as the Octet system proteins, thereby making the process

Residual Impurities
development aspect less strategic in and leachables like Protein A are sensi- monitoring during continuous opera-
design, agrees Michael Farris, scientific tive to extreme pH, high salt concentration must be resolved,” he adds. “Here
manager of analytical and formulation tions, and certain detergents. The typical again, sensor technology could be the
sciences with Thermo Fisher Scientific. means for overcoming the signal sup- way to go (e.g., online Octet dip-and-
“Building toxicology and immunogenic- pression or interference associated with read-type systems); however, the tech-
ity databases for various HCPs and other their presence is to dilute the sample, nology is not sufficiently developed yet.
process impurities would help fine-tune which acts to decrease the sensitivity The final technology offerings need to
process development and, in itself, pro- of the method to maintain acceptable be diverse, robust, accurate, reliable, and
vide a deeper insight into process perfor- precision and accuracy,” Farris explains. rapid,” he concludes.
mance and robustness,” he asserts. As a consequence, sensitivity constraints For off-line analysis of residuals from
Identifying/developing suitably imposed by matrix/API interference a continuous process, Parsons notes that
sensitive methods is also challenging, must be a point of consideration when methods for aliquot/sample withdrawal
according to Pettit. “Typically, ppm or demonstrating that a method is fit for are needed. In addition, the sampling
ppb levels of analyte in a complex mix- use for process characterization and/or frequency should be sufficient to pro-
ture must be detected, and therefore, process validation activities. vide a statistical sampling of the mate-
isolation/derivatization of the analyte For products with small total batch rial flowing through the process in order
may be required. These activities tend volumes, the quantities of material to account for any transient variation
to be prohibitively expensive to out- required for analytical method devel- that may occur while avoiding significant
source,” he observes. opment and sample analysis can also depletion of the process flow.
In addition, isolation of impurities present a challenge, Parsons notes. “If The analytical testing strategy associ-
prior to quantitation may introduce arti- method sensitivity is also an issue, an ated with continuous processes can, how-
facts or cause the generation of various approach can be taken to spike an ali- ever, be more demanding in terms of the
altered states during the isolation pro- quot of upstream material with a higher number of sampling time points if in-
cess. For example, physical manipula- known concentration of analyte and then line or on-line monitoring is not possible,
tion of samples prior to the detection demonstrate fold-removal of the ana- according to Farris. “Process analytical
of multiple analytes may alter the HCP lyte across the downstream purification technologies have generally been geared
antigen profile and relative abundance step(s),” he comments. more toward product quality, cell-culture
of the analytes themselves, according to viability, and growth than active moni-

CONSTRAINTS IN
Farris. Additional studies to understand
CONTINUOUS BIOPROCESSING
the stability of isolated product(s) mayUSE toring of process impurities. If a deeper
understanding of any particular impurity
therefore also be needed. For continuous processes, the turn- is required, more complicated assays will
Other process impurities such as Long around time of typical assays is a likely be required,” he observes.
R3 IGF-1, which is a component of some key issue, according to Kneller. “The
cell-culture processes, have a tendency to challenge is to increase our test- RESIDUALS ANALYSIS
adhere to certain plastics, thereby making throughput, decrease our reliance WHEN MOVING FROM
ing accurate quantitation more difficult, on heterogeneous reagents, and to find BATCH TO CONTINUOUS
Farris adds. Sampling instructions must ways to either simplify the method types When moving from batch to continu-
be clearly defined so as to not artificially used or engineer robust, easy-to-use sys- ous processing, it is likely that the same
deplete the analyte during sampling and/ tems for more complicated analyses (e.g., analytical methods for residual impuri-
or storage prior to analysis. mass spectrometry),” he says. ties can be used, but potentially in higher
Parsons agrees that the main chal- Real-time monitoring of representa- throughput versions. Offline analysis
lenges are focused around developing tive material for modeling the residence using LC, GC, ELISA, and MS should
methods of sufficient sensitivity that also time distribution in continuous pro- be possible as long as an appropriate
minimize product and matrix-related cesses is also required for process con- sampling approach can be implemented,
interference, and thereby accomplish trol, according to Pettit. “The range of Parsons observes.
sufficient recovery of the analytes. In appropriate analytical sensors for con- The throughput capability and time-
numerous instances, Farris points out tinuous monitoring of product specific to-result for the analytical method
that the quantitation and/or character- critical quality attributes and impurities employed are also important aspects if
ization of impurities is hindered by the is limited. Additionally, sensor drift over at some point the impurity is deemed
API and/or the matrix composition. the operating time, any on-going sensor process critical, according to Darshini
“Immunoassays, such as ELISA, used calibration needs, and system suitabil- Shah, senior scientist and group lead
for quantitation of host-cell proteins ity testing for quality critical attribute for downstream process development at

Residual Impurities
Thermo Fisher Scientific. Automation sensitive quantitation of the process tools capable of monitoring residual
of methods (e.g., liquid-handling or use residuals. Nevertheless, if a continuous impurities for either quantitation or
of systems such as the Octet for HCP process can be shown (perhaps by an characterization. “Offline analysis is the
analysis) can provide higher throughput online monitoring method) to be robust, predominant means of analysis and the
both in terms of overall speed and ana- then reduced sampling of the continu- only on-line/in-line tools are related to
lyst effort, according to Kneller. ous process for quantitation of residuals product quality aspects,” he says.
The analytical target profile must also may be justifiable,” he notes. Current solutions exist around auto-
be evaluated to ensure methods are capa- In fact, because continuous processes mated sampling for at-line analytics,
ble of monitoring fluctuating analyte are expected to operate under steady- where process changes occur over a longer
levels throughout continuous process- state conditions, control through predic- time span than the analysis time, such
ing. “Retention rates or biomass removal tive modeling or by exception should as in the case of mammalian bioreactor
may result in periods of correspondingly be possible, Heise adds. “Multi-variant metabolite analysis, according to Pettit.
lower impurity levels, and the analytical analysis during development may iden- On-line process analytical techniques
method must be sensitive enough and tify simple in-line monitoring techniques such as near-infrared spectroscopy (NIR)
support a large enough dynamic range that can control the process to ensure or mid-infrared spectroscopy (mid-IR)
to be able to actively monitor the process residual impurities do not pass through and UV may provide some general infor-
range without the need to constantly the whole process. However, the dynam- mation on clearance of residuals but
repeat analyses to better target the oper- ics of the process will be different at the are unlikely to be specific or sensitive
ating range of the assay,” Shah explains. outset until the steady state is reached. A enough to enable detailed quantitation of
In addition, the typical approach in testing strategy defining the frequency of analytes, according to Parsons.
batch processing involves analysis of
When moving from batch to continuous
a small discrete sample number, with
reference standards bracketing the
processing, similar offline analytical
samples and system suitability tests per-
formed prior to analysis, according to
methods may be used. Significant
Heise. Rotating through multiple identi-
cal analyzers would allow continuous
development is needed, however, before
monitoring, but the solution would be

costly. “Validation of both system suit-
on-line/in-line
ability tests and the analytical methodsanalysis of residual USE
for continuous control/monitoring will
impurities can be widely implemented.
need additional work to demonstrate
that results do not require bracketing
with reference standards and that the
detection methods do not drift or get measurement pre- and post-steady state Parsons does note that low-field
poisoned over time when operating for will be required to identify when process NMR is a potentially promising tech-
90+ days,” he says. Failure mode mitiga- equilibrium has been reached and when nique for on-line analysis of process
tion strategies will also need to be devel- it begins to fail,” he observes. residuals. “The system is simple to oper-
oped, for example with respect to system “Ideally,” Heise continues, “we ate, potentially applicable to both batch
suitability failure. don’t really want to be testing any- and continuous processes, and could be
In addition, Parsons points out that thing real-time and on-line. Instead, performed by stop-flow analysis with
for in-line and on-line analysis, the we want to have confidence that the direct connection of the flow path to
inherent requirements of sensitivity and process will deliver consistently over a the downstream purification process,”
specificity for analysis of process residu- defined time scale.” he says. Attractive features of NMR in
als suggests that many of the current this regard are its inherently quantita-
and potential online methods would POTENTIAL FOR tive response factor and its relatively
have limitations and may perhaps be ON-LINE/IN-LINE ANALYSIS high resolving power, and hence higher
able to best provide supportive general OF RESIDUAL IMPURITIES specificity than a technique such as
information, rather than specific quan- Significant development is still needed UV, while drawbacks include barriers
titation of residuals. “Offline analysis before on-line/in-line analysis of residual to implementation of a conceptually
using traditional validateable techniques impurities will be widely implemented. complex analytical technique and limi-
might still be required for specific and Shah is not aware of any on-line/in-line tations on sensitivity. ◆

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Peer-Reviewed
Catalytic Site Analysis

and Characterization
of a Solvent-Tolerant
Aldo-Keto Reductase
WEI JIANG, RUI PEI, WEILIANG WU, PANPAN ZHAO, LIBING TIAN, AND SHU-FENG ZHOU
ABSTRACT
Chiral alcohols are important intermediates of various drugs. Compared
with traditional chemical methods, the biocatalytic methods used for the
synthesis of chiral alcohols exhibits many advantages, such as mild conditions
and high enantioselectivity. Aldo-keto reductases are regarded as promising
enzymes that can be potentially applied in the biocatalytic synthesis of
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chiral alcohols. In this study, a novel aldo-keto reductase, AKR7-2-1, was

cloned and purified, and its important conserved sites were analyzed.
In addition, this study analyzed the catalytic potential of AKR7-2-1. The
optimum reaction conditions were studied; AKR7-2-1 showed excellent
thermal stability and pH stability even when the temperature reached 80 °C
or pH reached 9.0. Furthermore, AKR7-2-1 has strong enzymatic activity
when 11 ketone-containing compounds are used as substrates, indicating
the broad substrate spectrum of the enzyme. Most importantly, AKR7-
2-1 has superior organic solvent tolerance even in an organic solvent of
30% volume per volume (V/V) or 10 hours in a 10% V/V organic solvent,
FOR PERSONAL, NON-COMMERCIAL USE where 60% enzyme activity was retained. It is worth mentioning that AKR7-
2-1 can catalyze the reduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-
propanamine to (S)-N,N-dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine,
an intermediate of the antidepressant drug duloxetine. All this shows that
AKR7-2-1 has broad application prospects in the field of biomedicine.
C
hiral alcohols are a class of cofactor (NAD(P)H)-dependent enzymes
*
Wei Jiang , wjiang@hqu. compounds having a hydroxyl belonging to the oxidoreductase family (4).
edu.cn, Rui Pei, Weiliang group attached to a chiral car- AKRs are widely found in nature, such
Wu, PanPan Zhao, Libing bon atom. T he compounds as bacteria (5), plants (6), and animal tis-
Tian, and Shu-Feng Zhou*, are structurally stable and are important sues (7). AKRs show great application
szhou@hqu.edu.cn, are all intermediates for the synthesis of chiral prospects in the field of biomedicine due
at the College of Chemical drugs, perfume f lavors, and agricultural to their ability to catalyze the reacton of
Engineering, Huaqiao chemicals (1, 2). Compared with tradi- prochiral ketones to form chiral alcohols.
University, Fujian, China. Wei tional chemical methods, biochemical syn- AKR, for example, can be used for the
Jiang and Rui Pei contributed thesis provides a promising strategy for asymmetric reduction of ethyl 4-chloro-
equally to this work. the production of chiral alcohols with the 3-oxobutanoate (COBE) to ethyl (R)-4-
advantages of high efficiency and enanti- ch loro-3-hyd rox ybutanoate (CHBE),
*
To whom correspondence oselectivity and mild reaction conditions. known as an important intermediate in
should be addressed. Enzymes and microorganisms are used to organic synthesis, which can be applied
catalyze the production of various enantio- in synthesis of L-carnitine, HMGCOA
PEER-REVIEWED merically pure alcohols (3). reductase inhibitor (8, 9). Catalytic reduc-
Article submitted: Feb. 2, 2019 The aldo-keto reductase (AKR) is a tion of ethyl 2-oxo-4-phenylbutanoate
Article accepted: April 4, 2019 class of nicotinamide adenine dinucleotide with Bacillus subtilis-derived AKR is an

Peer-Reviewed
important way to obtain ethyl (R)-2- substrate prof ile of AKR7-2-1 and engineering, and the recombinant
hydroxy-4-phenylbutanoate, which strong organic solvent tolerance indi- vector pET-28a-akr7-2-1 was trans-
can be used as an intermediate for cate the great potentials in the synthe- formed into E. coli BL21 (DE3)
anti-hypertension drugs (10). Only a sis of high-value chiral alcohols. cel ls for heterologous ex pres-
limited number of AKRs have been sion. The AKR7-2-1 was heterolo-
obtained and applied to the synthe- MATERIALS AND METHODS gously expressed, and 200 mL of LB
sis of chiral alcohols, however. Five Strains, vectors, chemicals medium was added with kanamycin
AKRs were cloned for highly stereose- Escherichia coli (E. coli) DH5α and at a concentration of 100 μg/mL. The
lective reduction of bulky ketones by BL21 (DE3) were cultured in Luria- temperature was set to 37 °C and the
C. Liang et al. through genetic mining Bertani (LB) medium. They were used cells incubated at 200 rpm. E. coli
from microorganisms such as Candida for cloning and heterologous expres- BL21(DE3) was cultured for three to
albicans (CaCR), Saccharomyces cere- sion. Plasmid pET-28a was used in four hours until the optical density
visiae (ScCR), Kluyveromyces marxia- this study. All enzymes used in this (OD) value reached 0.6–0.8. After
nus (KmCR), and Candida parapsilosis work were from TaKaRa Co., Ltd. that, the temperature was changed
(CPR-C1, CPR-C2) (11). X. Luo et al. (Dalian, China). Plasmid Mini Kit I to 18 °C, and isopropyl β-D-1-
cloned an AKR from Kluyveromyces (200) and Gel Extraction Kit (200) thiogalactopyranoside (IPTG) was
lactis XP1461, which can be used for were purchased from OMEGA Co. added to the culture medium with a
the synthesis of chiral alcohols (12). (United States). Nicotinamide adenine final concentration of 0.1 mM. The
Y.H. Ma et al. cloned a stong heat- dinucleotide phosphate (NADPH) was E. coli BL21(DE3) cells were further
resistant AKR from thermophilic purchased from Sigma-Aldrich Co. cultured for 14 hours. Afterwards, E.
bacteria, Tm1743 (13). C. Ning et (Shanghai, China). All other chemi- coli BL21(DE3) cells were collected
al. cloned three kinds of AKRs from cals were purchased from Aladdin by centrifugation and washed three
Lodderomyces elongisporus—LEAKR (Germany). All chemicals used were times with phosphate buffered saline
48, LEAKR 49, and LEAKR 50— chromatographically pure or analytical (PBS). A sonicator was then used
which can be applied to synthesize grades and therefore required no fur- to lyse the cells, and the broken cell
ethyl 4-chloroacetoacetate (14). In ther purification in use. debris were centrifuged at 12,000
addition, AKR from S. cerevisiae, rpm for 30 minutes. The supernatant
YOL151W, can be used for asymmet- Amino acid sequence was collected and stored at 4 °C for
ric synthesis of (S)-3-chloro-1-phenyl- analysis and 3D modeling further use. (The previous experi-

1-propanol (15). The new AKR from The National Center for Biotechnology mental steps were all performed on
L. elongisporus, NRRL YB-4239, can
Information (NCBI) basic local align- ice at all times). AKR7-2-9 was then
be used to synthesize ethyl (R)-4-
ment search tool (BLAST) is commonly purified using an AKTA Prime sys-
chloro-3-hydroxybutanoate (16). So used in the analysis of bioinformatics (18). tem equipped with a 10-mL Ni-IDA
far, chiral compounds have been The amino acid sequence of AKR7-2-1 column (GE Healthcare, US). The
among the top 10 drugs in history, was blasted in NCBI, and nine sequences purification results were subsequently
and it is expected that by 2020 chi- from other sources having similarity to detected using a 12% sodium dodecyl
ral compounds will still dominate the the sequence of AKR7-1-2 were selected sulfate polyacrylamide gel electropho-
best-selling drug list (17). Although for multiple sequence alignment analysis. resis (SDS–PAGE) gel. Finally, the
the demand for chiral drugs in the The AKR7-2-1 was constructed in a fully protein concentration of AKR7-2-1
medical field is extremely large, there automated protein structure homology- was measured using a protein assay kit
are still few studies on biocatalytic modeling server (SWISS-MODEL) (Bradford Protein Assay Kit, Sangon
synthesis of chiral alcohols, which commonly used in the construction of Biotech [Shanghai] Co., Ltd.).
cannot meet the demand for chiral three-dimensional models of proteins (19).
drugs in the medical field. Therefore, Enzyme activity assays
it is necessary to continue to excavate Cloning, expression, and The total volume of the reaction
new types of AKRs to meet the syn- purification of AKR7-2-1 system was 220 μl, which contained
thetic needs of chiral drugs. The gene of AKR7-2-1 was ampli- 10 μl of methyl pyruvate, 170 μl of
In this study, a novel AKR (AKR7- f ied by bacterial liquid polymerase PBS, 10 μl of NADPH, and 30 μl
2-1) was cloned from Bacillus mega- chain reaction (PCR), and the primer of enzyme AKR 7-2-1. One unit
terium. The catalytic performance of used in this process was synthesized of AKR 3-2-9 was def ined as the
AKR7-2-1 was subsequently investi- by Bioengineering Biotechnolog y amount of enzyme that catalyzed
gated. The three-dimensional struc- (Shanghai) Co., Ltd. The DNA the oxidation of 1 μmol NADPH
ture and important sites of AKR7-2-1 fragment of AKR7-2-1 was cloned per min. The rates of reduction were
were also analyzed. The extensive into the pET28a vector by genetic assayed at 37 °C by measuring the

Peer-Reviewed
Figure 1. Homologous comparison of AKR7-2-1 with the other nine amino used. The maximum enzyme activ-
acid sequences. Conserved sites are marked in red with a blue box labeled ity was defined as 100%. Meanwhile,
as a conserved region rich in glycine and a catalytically active tetrad the pH stability of AKR7-2-1 was
(DX4YX34KX40H) labeled as a black triangle. investigated by incubating it for 24
hours in the buffer with different
pH ranges as described previously.
After the incubation was completed,
t he residua l enz y me ac t iv it y of
AKR7-2-1 was determined accord-
ing to a sta nda rd mea su rement
met hod. T he ma x imum enz y me
activity was defined as 100%.
Organic solvent tolerance

of the AKR7-2-1 enzyme
Si x industria l ly common organic
solvents—inc lud ing met ha nol,
ethanol, isopropanol, ethyl acetate,
acetonitrile, and dimethyl sulfox-
ide ( DMSO) — were selec ted to
study the organic solvent tolerance
of AKR7-2-1. The concentration of
organic solvents ranged from 10%
to 30% V/V. The residual enzyme
activity of AKR7-2-1 under differ-
change in absorbance of NADPH Effect of temperature and pH ent conditions was then determined
at 340 nm (ε= 6.22 mM −1 cm −1). Methyl pyruvate was used as a sub- according to standard methods. In
Apparent kcat and Michaelis-Menten strate to study the effect of temper- addition, the organic solvent sta-
constant of AKR7-2-1 (Km) were ature and pH on the reaction. To bility was also studied. The above

calculated by using the Lineweaver- study the optimum temperature of si x org a n ic solvent s were s epa-
Burk double reciprocal plot. AKR7-2-1, a gradient was set at a rately added to the reaction system.
2-°C inter val bet ween 30 °C and The concentration of organic sol-
CHARACTERIZATION OF 50 °C. The reaction system without vent was set at 10% V/V. AKR7-2-1
THE AKR 7-2-1 ENZYME coenzyme NAPDH was incubated was placed in the system (without
Substrate specificity of AKR7-2-1 at the corresponding temperature NAPDH) in a 4 °C refrigerator for
The substrate specificity of AKR7-2-1 for five minutes. After the comple- 10 hours. Every two hours, NAPDH
was investigated under the standard tion of the incubation, NA PDH was added to the reaction system
assay conditons by using 11 ketone- was added to rapidly measure the and its residual activity was quickly
containing compounds as substrates. residual activity of AKR7-2-1 using determined by standard methods.
The total volume of the reaction sys- a microplate reader. To determine The maximum enzyme activity was
tem was 220μl, in which the substrate, the temperature stability of AKR7- defined as 100%.
PBS buffer, cofactor NADPH, and 2-1, a gradient was set every 10 °C
AKR7-2-1 were set to 10 μL, 170 μL, from 30 °C to 80 °C. The AKR7-2-1 RESULT AND DISCUSSION
10 μL, and 30 μL, respectively. The was allowed to stand at the corre- Gene cloning and sequencing
maximum activity of AKR3-2-9 was sponding temperature for one hour. analysis of AKR3-2-9
defined as 100%. In this experiment, After one hour, the residual activity In the present study, the cloned DNA
11 substrates were used: 3-methylcyclo- of AKR7-2-1 was determined under fragment of AKR7-2-1 was 963 bp
All figures courtesy of the authors.
hexanone, methyl pyruvate, phenoxy- standard methods. in leng t h. T he DNA sequence

acetone, ethyl levulinate, 2-octanone, To s t u d y t h e o p t i m u m p H was translated into an amino acid
acetyl, acetone, 5-methyl-2-hexanone, o f A K R 7-2 -1 , C H 3 C O O H - sequence with a length of 320 bp,
4-methyl-2-pentanone, phenylmeth- CH3COONa buffer w ith pH of which was then subjected to BLAST
ylketone, N-Boc-3-piperidone, and N, 4 . 0 – 6 . 0 , Na H 2 P O 4 -Na 2 H P O 4 in NCBI. Nine sequences with dif-
N-Dimethyl-3-keto-3-(2-thienyl)-1- buffer with pH of 6.0–8.0, and Tris- ferent similarities were selected,
propanamine (DKTP). HCl buffer with pH of 8.0–9.0 were and multiple sequence alignment

Peer-Reviewed
analysis was performed. The result Figure 2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis
is shown in Figure 1. AKR7-2-1 is of expression products. Lane M = marker; Lane 1 = not induced with isopropyl
97% simila r to a ldo/ keto reduc- β-D-1-thiogalactopyranoside (IPTG); Lane 2 = crude enzyme solution after IPTG
t a s e f r o m B a c i l l u s m e ga te r i u m induction; Lane 3 = purified AKR3-2-9.
(NO.:AUO10603.1), 96% similarity
to aldo/keto reductase from Bacillus
aryabhattai (NO.:WP_088048797.1),
76% similarity to aldo/keto reduc-
t a s e f r o m F i c t i b a c i l l u s e n c l e n-
sis (NO.:W P_ 061968434.1), 73%
similarit y to the aldo/keto reduc-
tase from Ktedonobacterales bacte-
rium Uno11 ( NO.:GCE2128 0.1),
72% similarity to aldo/keto reduc-
tase from Anaerobacillus isosaccha-
rinicu s ( NO.:W P_ 0713171 2 3.1),
68% simila rit y to the a ldo/ keto
reductase from Youngiibacter fragi-
lis (NO.:W P_ 023384671.1), 64%
similarit y to aldo/keto reductase
from Sporolactobacillus laevolacti-
cus (NO.:W P_ 023510697.1), 63%
similarit y to aldo/keto reductase
from Sporolactobacillus pectinivorans
(WP_100488283.1), 58% similarity to
aldo/keto reductase from Paenibacillus a vital role in the spatial conformation reduction of DKTP to DHTP is a key
cellulosilyticus (NO.:WP_110042924.1), of proteins. step in the synthesis of the antidepres-
57% similarity to aldo/keto reduc- sant duloxetine (26, 27). These results
t a s e f r o m P a e ni ba c i l l u s l a u t u s Expression and purification indicate that AKR7-2-1 has a good

(NO.:WP_096776379.1). In addition, of AKR7-2-1 application prospect in the field of
in the N-terminal and middle (blue
After induction with 0.1 mM IPTG, biomedicine because of its broad sub-
box, see Figure 1) of the amino acid
the recombinant vector pET-28a- strate spectrum.
sequence of AKR7-2-1, a glycine-rich akr7-2-1 was successfully heterologously
sequence was present, indicating that expressed in E. coli BL21 (DE3). The Effect of temperature and pH
this motif is highly conserved. The sonicated crude enzyme solution was Temperature and pH are important
amino acid sequence of AKR7-2-1 purified. The SDS–PAGE results can factors inf luencing enzyme activity,
also has four highly conserved cata- be seen in Figure 2. and their effects on AKR7-2-1 were
lytically active sites located at Asp48, investigated in this study (see Figure
Tyr53, Lys88, and His129 (20). So Substrate specificity 4). Figure 4A shows that the optimum
far, the specific mechanism of action To detect the substrate specificity of temperature for AKR7-2-1 is 37 °C, at
for catalytically active tetrads is not AKR7-2-1, 11 substrates with ketone which point enzyme activity was the
well understood. A limited number groups were selected in this study, and highest. Enzyme activity was shown
of studies have shown that Lys (21) the results shown in Figure 3 demon- to increase at 30 °C and then decrease
is stable in binding to the coenzyme strate the different degrees of cata- as the temperature approached 50
NAD(P)H by enhancing the stabil- lytic activity on these substrates by °C. Further, enzyme activity slowly
ity of the charge interaction and par- AKR7-2-1. Among them, AKR7-2-1 decreased as the temperature exceeded
ticipating in the catalytic reaction; Tyr exhibited the highest catalytic activ- 37 °C, but still retained 70% or more
(22, 23) contains a phenolic hydroxyl ity when methyl pyruvate, which is an relative activity even when it reached
group, which binds to the substrate important raw material in pharmaceu- 50 °C. Previous studies by other
and participates in the catalytic reac- tical production, is used as a substrate researchers had reported an optimum
tion; His (24) is a proton donor in the (26). When acetylacetone and DKTP temperature of 30 °C for an aldo-keto
catalytic process and also relevant to were used as substrates, the catalytic reductase cloned from Kluyveromyces
the reduction reaction in combination activity of AKR7-2-1 is also relatively lactis XP1461 with enzyme activity
with the substrate; and Asp (25) plays high. It is worth mentioning that the decreasing sharply when the tempera-

Peer-Reviewed
Figure 3. Substrate spectrum of AKR7-2-1. The number 1 represents ture exceeded 35 °C. The reductase in
3-methylcyclohexanone; the number 2 represents methyl pyruvate; the number those previous studies kept less than
3 represents phenoxyacetone; the number 4 represents ethyl levulinate; the 20% activity when the temperature was
number 5 represents 2-octanone; the number 6 represents acetylacetone; the set to 50 °C (12). Compared with the
number 7 represents 5-methyl-2-hexanone; the number 8 represents 5-methyl-2- aldo-keto reductase in previous studies,
hexanone, 4-methyl-2-pentanone; the number 9 represents phenylmethylketone; AKR7-2-1 was well resistant to high
the number 10 represents N-Boc-3-piperidone; and the number 11 represents temperature. The thermal stability of
N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine (DKTP). This experiment was AKR7-2-1 was investigated by incu-
repeated three times, and the error bars represent the standard error of the bating the enzyme at a temperature
mean. The maximum enzyme activity of AKR7-2-1 was defined as 100%. range of 30 °C to 80 °C (Figure 4B).
It was shown that relative enzyme
activity decreased with the incremen-
tal increase in temperature; however,
more than 70% activity remained
when the temperature reached 80 °C,
showing superior thermal stability. In
comparison, an aldo-keto reductase
cloned from Lodderomyces elongispo-
rus NRRL YB-4239 by Q. Wang et
al. completely lost activity when incu-
bated for 30 minutes at 45 °C (28).
As can be seen in Figure 4C , the
optimum pH of AKR7-2-1 is 6.0.
When the pH is lower than 6.0, the
enzyme activity of AKR7-2-1 is dras-
tically decreased, with activity fall-
ing to 20% when pH is reduced to
5.0. Interestingly, as the pH dropped
Figure 4. Effect of temperature and pH on AKR7-2-1. (A) Optimum temperature
from 5.0 to 4.0, it decelerated the
of AKR7-2-1 (B) Temperature stability of AKR7-2-1. (C) Optimum pH of AKR7-2-1

reduction of enzyme acitivity. On the
50 mM acetate buffer (•), 50 mM potassium phosphate (■) and 50 mM Tris-
HCl (▲) were used. (D) pH stability of AKR3-2-9. All of the above experiments
other hand, the activity of AKR7-2-1
evaluated residual enzyme activity under standard assay conditions. The decreased only mildly when the pH
maximum enzyme activity was defined as 100%. All experiments were was above 6.0, maintaining 60% activ-
performed in triplicate, and the error bars represent the standard error of the ity even when the pH reached 9.0. In
mean. addition, there is an interesting phe-
nomenon that even under the same
pH conditions, the enzyme activity
of AKR7-2-1 in the phosphate buffer
was higher than in the acetate buffer
or the Tris-HCl buffer. This is simi-
lar to the results studied by Y. Wang
et al. (29). AKR7-2-1 exhibited the
best stability in a buffer with pH of
6.0. as shown in Figure 4D. Its activity
decreased to less than 20% when incu-
bated in buffer at a pH below 5.0 for
24 hours. The relative enzyme activity
also decreased at a pH ranging from
6.0 to 9.0, although it still retained
40% activity when the pH reached
9.0, which indicates an ability to resist
extreme pH. In a similar study by Q.
Wang et al. (28), the enzyme retained
less than 30% of activity even when it

Peer-Reviewed
was only incubated in a buffer of pH Figure 5. (A) Detection of the resistance of AKR7-2-1 to organic solvents. The six
8.0 for 60 minutes. organic solvents were methanol, ethanol, isopropanol, ethyl acetate, acetonitrile,
and d imethyl sulfoxide (DMSO). Each organic solvent was set to a concentration
Kinetic analysis gradient of 10% V/V to 30% v/v. (B) Organic solvent stability of AKR7-2-1.
In this study, the kinetic analysis of puri- Incubate for 10 hours in six organic solvents at 10% V/V and test once every two
fied AKR7-2-1 was also performed. The hours. The enzyme activity of AKR3-2-9 was measured using standard methods.
results of various kinetic parameters of The experiment was performed in triplicate and the error bars represent the
AKR7-2-1 show that Vmax, Km, Kcat, standard error of the mean.
and Kcat/Km of the enzyme was 110.0
uM/min, 4.380 uM, 57.29 min-1, and
13.04 min-1uM-1, respectively. These
kinetic parameter data indicate the strong
binding ability to the substrate (methyl
pyruvate). In addition, the Kcat/Km
of AKR7-2-1 is 13.04 min-1uM-1 (218
s-1mM-1), demonstrating an extremely
high catalytic efficiency compared to
other reported Kcat/Km of AKRs. For
example, the Kcat/Km of kmAKR-
W297 is 60.97 s-1mM-1 (29), Tm1743 is
54.6 s-1mM-1 (13), and CgKR1-F92L is
33.2 s-1mM-1 (30).
Detection of AKR3-2-9-tolerant
organic solvent properties
Enzyme catalytic systems containing
organic solvents have many advantages
in industrial production. Many natural
enzymes, however, are poorly resistant

to organic solvents (31). As shown in
Figure 5A , methanol, ethanol, isopro-
panol, ethyl acetate, acetonitrile, and
DMSO were used to study the organic
solvent tolerance of AKR7-2-1, which
was able to survive to 10% V/V of
organic solvents with more than 80%
residual activity. When the concentra-
tion of the organic solvent in the enzy- in methanol, ethanol, and acetonitrile MODEL, AKR7-2-1, and the Crystal
matic reaction increased from 10% V/V under the same reaction conditions. An structure of the E. coli Tas protein
to 30% V/V, the enzyme activity of aldo-keto reductase cloned by X. Luo et (SMTL ID: 1lqa.1) were shown to be
AKR7-2-1 began to gradually decrease al. from Kluyveromyces lactis XP1461 structurally similar (Figure 6A). This
but still retained more than 60% activity. was only able to tolerate up to 5% V/V protein is an NADP(H)-dependent
Furthermore, the organic solvent of organic solvents such as isopropanol aldo-keto reductase (32). The amino
stability of AKR7-2-1 was also studied and ethyl acetate (12). In addition, an acid sequence of AKR7-2-1 is com-
by incubating the enzyme in methanol, aldo-keto reductase (Tm1743) cloned pared with the amino acid sequence
ethanol, isopropanol, ethyl acetate, ace- from Thermotoga maritima has been of 1lqa.1 as shown in Figure 6B. The
tonitrile, or DMSO with the concen- reported to withstand up to 10% V/V of homology modeling results of AKR7-
tration of 10% V/V for 10 hours, and organic solvent (13). Compared with the 2-1 illustrated that it may have a
the enzyme activity of AKR7-2 was aldo-keto reductase reported, AKR7-2-1 binding site for NADPH coenzyme
detected every two hours. As shown showed superior organic tolerance. with a high conservative. As shown
by Figure 5B, AKR7-2-1 retained more in Figure 6B, the amino acid positions
than 50% enzyme activity in DMSO Molecular modeling marked with white letters on a red
after 10 hours of incubation, while A three-dimensional model of AKR7- background are possible binding sites
it retained approximately 40% activity 2-1 that was constructed using SWISS- for NADPH and AKR7-2-1.

Peer-Reviewed
Figure 6. (A) The 3D model of AKR7-2-1 and (B) alignment of the sequence of 6. I. Gavidia, P. Pérezbermúdez, and
H.U. Seitz, European Journal of
AKR7-2-1 with the sequence of 1lqa.1. The amino acid positions marked with Biochemistry 269 (12) 2842–2850 (2010).
white letters on a red background are possible binding sites for nicotinamide 7. E. Kozma et al., Journal of Biological
adenine dinucleotide phosphate and AKR7-2-1. Chemistry 277 (18) 16285–16293 (2002).
8. J. Keju et al., Preparative Biochemistry
& Biotechnology 35 (3) 203–215 (2005).
9. M. Kataoka et al., Applied
Microbiology & Biotechnology 51 (4)
486–90 (1999).
10. Y. Ni et al., Journal of Biotechnology
168 (4), 493–498 (2013).
11. L. Chen et al., Bioresources &
Bioprocessing 5 (1) 33 (2018).
12. X. Luo, Y.J. Wang, and Y.G. Zheng,
Enzyme & Microbial Technology 77,
68–77 (2015).
13. Y. H. Ma et al., Biotechnology Letters
35 (5) 757–762 (2013).
14. C. Ning, E. Su, and D. Wei, Archives
of Biochemistry & Biophysics 564,
219–228 (2014).
15. C.Y. Hee et al., Applied Microbiology &
Biotechnology 87 (1) 185–193 (2010).
16. Q. Wang et al., Journal of Industrial
Microbiology & Biotechnology 41 (11)
1–8 (2014).
17. R. Kratzer, J.M. Woodley, and B.
Nidetzky, Biotechnology Advances 33
(8) 1641–1652 (2015).
18. T. Madden, “The BLAST Sequence
Analysis Tool,” in The NCBI Handbook,
Jo McEntyre and Jim Ostell, Eds.
(National Center for Biotechnology
Information, Bethesda, MD, 2002).
19. M. Biasini et al., Nucleic Acids
Research 42 (W1) W252–W258 (2014).

20. E.M. Ellis, Fems Microbiology Letters
216 (2) 123–131 (2002).
CONCLUSION AUTHOR CONTRIBUTIONS 21. K. Regina and N. Bernd, Biochemical
Journal 389 (2) 507–15 (2005).
In this study, a novel aldo-keto reductase Rui Pei did experiments, Weiliang 22. B.P. Schlegel, J.M. Jez, and T.M.
AKR7-2-1 with conservative site was Wu, PanPan Zhao, and Libing Tian Penning, Biochemistry 37 (10) 3538–
excavated. The broad substrate spectrum provided help for the experiment. Pei 3548 (1998).
showed that the enzyme was especially and Wei Jiang wrote and modified the 23. O.A. Barski et al., Biochemistry 34 (35)
11264–11275 (1995).
able to catalyze DKTP to DHTP, an article, and Jiang and Shu-Feng Zhou 24. K.M. Bohren et al., Biochemistry 33 (8),
important precursor of the antidepressant designed and supervised the work. 2021–2032 (1994).
drug duloxetine. In addition, the enzyme 25. X. Liu et al., Protein Science 23 (11)
was shown to have superior thermal sta- CONFLICT OF INTEREST 1540–1549 (2015).
26. P. Soni and U.C. Banerjee, Applied
bility, pH stability, and excellent toler- The authors state that they have no Microbiology & Biotechnology 67 (6)
ance to a wide range of organic solvents. competing interests. 771-777 (2005).
These characteristics make AKR7-2-1 a 27. M.P. Knadler et al., Clinical
promising enzyme to be applied in the REFERENCES Pharmacokinetics 50 (5) 281–294 (2011).
1. W. Kroutil et al., Current Opinion in 28. Q. Wang et al., Journal of Industrial
medicine field. Microbiology & Biotechnology 41 (11)
Chemical Biology 8 (2) 120–126 (2004).
2. X. Wu and J. Xiao, Chemical 1–8 (2014).
ACKNOWLEDGMENTS Communications 38 (24) 2449–2466 29. Y.J. Wang et al., Enzyme & Microbial
The work was supported by the Natural (2007). Technology 107, 32–40 (2017).
3. Y. Ni and J.H. Xu, Biotechnology 30. G.W. Zheng et al., ACS Catalysis 7 (10)
Science Foundation of China (No. Advances 30 (6) 1279–1288 (2012). 7174–7181 (2017).
21808073), the high-level personnel 4. J.M. Jez and T.M. Penning, Chemico- 31. N. Doukyu and H. Ogino, Seikagaku
activation fee of Huaqiao University Biological Interactions 130 (1–3) 499– the Journal of Japanese Biochemical
525 (2001). Society 48 (3) 270–282 (2009).
( No. 6 0 0 0 0 5-Z17 Y0 0 7 2), a n d
5. D.L. Eric, R.A. Elling, and D.K. Wilson, 32. G. Obmolova et al., Proteins: Structure,
Quanzhou City Science & Technology Biochemical Journal 400 (1) 105–114 Function, and Bioinformatics 53 (2),
Program of China (No. 2018C008). (2006). 323–325 (2003).X

Quality
Removing Gaps in Data Integrity

FDA guidance is expected to improve industry practices, but work
is also needed to bridge disparate industry and
software engineering standards.
FOR PERSONAL, NON-COMMERCIAL USE AGNES SHANLEY
O
ver the past few years, after some notable failures at administrative role for managing data, separate from func-
a number of companies (1), global regulators have tions that are generating and recording that data. FDA also
been paying much closer attention to how pharma- introduced the ALCOA acronym to emphasize the fact that
ceutical manufacturers safeguard the integrity of their data, data must be “attributable, legible, contemporaneous, original
to prevent accidental manipulation as well as fraud. Data (or a true copy), and accurate.”
integrity is the foundation of the current good manufactur- In December 2018, FDA updated this guidance (4). “The
ing practices (cGMPs). According to FDA, between January new guidance serves to clarify data integrity requirements
2015 and May 15, 2016, 21 out of the 28 warning letters that have consistently challenged organizations. It firms up
issued to pharmaceutical manufacturers centered around expectations around data/metadata and the data lifecycle
problems with data integrity (2). within a risk-based structure of systems and design controls,”
As Orlando López, Kansas-based senior site automation says Kir Henrici, CEO of the Henrici Group, a consultant
specialist for a Big Pharma company, has noted (3), con- for the Parenteral Drug Association (PDA), which pub-
nections between different data points (i.e., initial data cap- lished best practices on laboratory data integrity in 2018 and
ture, metadata, and records) cannot be weakened or broken plans to publish manufacturing data integrity guidance by
because they provide the only objective evidence that opera- the end of 2019.
tions meet regulatory requirements and are being managed FDA has emphasized the need for careful risk manage-
responsibly. These intact connections are also needed for ment, the importance of audit trails and access controls,
process validation and continuous process verification. and the need to save all records that could be relevant to
FDA’s 2016 draft guidance clarified a number of cGMP compliance. Its latest data integrity guidance also
best practices, such as the need for paper and electronic
recordkeeping to use the same basic practices, and for an Contin. on page 44

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Quality
Contin. from page 41 which data you will focus on, you will be
in a much better position to demonstrate
ALCOA addresses
underscores the importance of senior full cGMP controls to the authorities,”
management support and involvement says López. “If you have done both the
the ‘why’ but not
in data integrity efforts. “Leadership risk assessment and the process mapping
and culture are key, and, if underdevel- correctly, there should be no doubt that
the ‘how’ of data
oped, are the starting points for com- you are implementing controls to the
pliance risks and gaps,” notes Henrici. correct records,” he says. Decisionmaking
integrity, leaving
Training and ongoing communica- processes must be documented, says
tion with senior management are crucial Henrici—not only reported data but any
questions for
to establishing and maintaining this data that support quality decisions—and
support, says López. Expressing poten- they need to be justified, retrievable, and
IT professionals
tial losses or liabilities in dollars and reviewable, she says. Maintaining a clear
cents terms can be especially effective, audit trail and using the best approaches
and engineers
he says, noting a 2017 hacking inci- to computer system validation will be
dent at Merck & Co., which cost $105 crucial and will require new training and
implementing new
million to fix (5) and might have been staffing practices, says Henrici. “Next-
prevented if investment had been made generation quality assurance will consist
platforms.
in data security. He also recommends of multiple disciplines or working teams engineers and IT professionals imple-
keeping senior management aware of to keep pace with data integrity require- menting systems.
regulatory citations in a way that allows ments in the era of smart manufacturing, Currently, there are a number of
them to connect data issues to lost big data management, and artificial intel- different data integrity guidelines. In
batches, product recalls, incorrect labels, ligence,” she says. pharma, these include regional ones
or formulation changes. “Senior manag- as well as those developed by pro-
ers needs to hear about money. It gets GETTING TO A DEEPER LEVEL fessional societies such as PDA and
their attention immediately,” he says. In a sense, however, data integrity the European Compliance Academy
is only the first step in a continuum. (ECA). In addition, the software
RISK-BASED APPROACH “Pharmaceutical industry guidelines industry has codes of its own, as does

It is also important to take a systematic,
risk-based approach to data integrity pro-
often refer to data reliability, which
is a step beyond data integrity,” says
the manufacturing industry in gen-
eral. The focus of each of the relevant
grams, says Henrici. Some organizations López. “But, ultimately, the goal for pharma guidelines is different, López
may roll out data integrity programs in our industry is data quality, which says, and the definition of data integ-
fragments, or as a reaction to a specific is a step beyond reliability. We can- rity in software engineering standards
event, but then allow efforts to trail off, not reach that point with the guide- (i.e., as mainly about data security
she says. “Pharmaceutical companies lines that we have now,” he says. “And and not changing the records), differs
need to structure a systematic, risk-based cGMP requirements alone will not somewhat from its definition in the
approach that integrates the quality man- suffice.” (6) Another challenge is pharmaceutical industry. The result can
agement system (QMS), which assures the fact that existing pharmaceutical be confusing to IT professionals, at
the efficacy and sustainability of the entire industry data guidelines don’t go deep least initially, and take time to sort out.
data integrity initiative.” enough, to the engineers and IT pro- “I would love to see integra-
Process mapping is especially fessionals who are implementing the tion between pharma standards and
important in ensuring that the systems, says López. “ALCOA may the software engineering standards,
right documentation is easily acces- give them the ‘why,’ but it doesn’t give because currently the industry is not
sible to staff, but also to regulators. them the ‘how,’” he says. speaking the same language as soft-
“Companies should track their ‘qual- Annex 11, first published in 1986, ware development academics. For
ity decisions’ to identify where non- addresses data integrity in a more example, for the pharma industry, vali-
cGMP records (e.g., emails) will be explicit way than the US 21 Code of dation is a process associated with the
required to demonstrate cGMP com- Federal Regulations (CFR) Part 11, he system lifecycle (SLC), which con-
pliance,” says Henrici. notes. López recalls one Big Pharma tinues from the start of a project until
“You need to understand each spe- company that correlated Annex 11 decommissioning. For software devel-
cific process. Then, if you do a good risk with Part 11 computer system require- opment, validation is just the testing
assessment, to record which systems and ments, which made things easier for phase of the SLC. So when you hire a

Quality
new graduate, he or she won’t under- for a “Data Integrity 4.0” strategy, a sets, and intended use, and simply apply
stand the difference,” says López. framework for matching IT and data what we have learned during the past
Without understanding the pharma integrity rules. She also noted that 30 years to the new technologies,” adds
validation concept and the relation- FDA approved 12 artificial intelligence López (9).
ship between software engineering and algorithms in 2018. The industry has come a long way
software quality, people in pharma’s If the industry is to reap the ben- in improving data integrity since the
IT and engineering departments will efits of using algorithms and artificial Barr decision in 2005 (1), a land-
have different understandings and the intelligence, she said, companies will mark case that resulted in mandates
resulting programs will be incomplete, need to create multifunctional data for recordkeeping and the investiga-
he says. López recalls seeing such a governance teams to bring different tion of out-of-specification conditions.
situation when asked to consult for perspectives to this effort, and facilitate New guidance and better integration
one mid-sized pharmaceutical com- communication between industry and of existing best practices—not only
pany several years ago. Data security regulators, and data will need to be safe, those designed for pharma’s end users,
had been made part of the system’s secure, and relevant (8). but for implementation specialists in
user requirements, but, as one looked IT and engineering—promise to push
at the different processes throughout Common standards pharmaceutical manufacturers beyond
the product lifecycle, there was one data integrity in the future.
point where the computer system was and definitions
missing provisions for security, so that REFERENCES
anyone could make changes to the data. will be the key to 1. J. Gallant, “Data Integrity: Detecting
and Mitigating Risk,” presented at the
“There was testing, but no transparency, Life Sciences Trainers and Educators
and in the design document, there had moving the industry Network annual conference, 2017,
been no steps taken to ensure security https://cdn.ymaws.com/www.l-ten.
throughout the lifecycle,” he says. beyond data org/resource/resmgr/2017_Annual_
Conference/Data_Integrity.pdf.
Common standards and defini- 2. S. Barkow, “Current Expectations and
tions will be the key to moving the integrity to data Guidance Including Data Integrity
and Compliance With cGMPs,”
industry beyond data integrity to presented at the International Society
data quality, López says, explaining quality. for Pharmaceutical Engineers (ISPE)

that the origin of the International Data Integrity workshop, June 5, 2016,
Bethesda, MD, April 2016. https://www.
Society for Pharmaceutical Engineers’ In the end, the required knowl- fda.gov/media/99556/download.
(ISPE’s) GAMP 5 guidance (7) is the edge is already there for implementing 3. O. López, Pharmaceutical
International Standards Organization’s new IT approaches, says López, who Technology 43(6) 2019, p. 32.
4. FDA, Data Integrity and Compliance
(ISO’s) 9000-3, which is also the foun- developed an analytics and business with cGMP, Q&A–Guidance for
dation of the pharmaceutical industry’s intelligence platform at a Big Pharma Industry (FDA, 2018), https://www.
guidelines. “Why are we speaking dif- company several years ago, before the fda.gov/media/97005/download.
5. C. Souza, Pharmaceutical Executive 38(12),
ferent languages?” he asks. “We need term “Big Data” had even become a December 10, 2018, www.pharmexec.
to synchronize all the different stan- buzz phrase. He advises breaking the com/lessons-pharma-merck-cyber-attack.
dards (e.g., ISO, ISPE, The Institute problem down to its simplest level: 6. O. Lopez, “Comparison of Health
Authorities’ Data Integrity Expectations,”
of Electrical and Electronics Engineers the data wave and input/output (I/O). presented at the 4th Annual Data
[IEEE]), and pharma’s so that we are “With a Big Data project, instead of Integrity Validation Conference,
speaking one common language and one, you might have 20 data waves Boston, MA, August 2018.
7. C. Plagiannos, “What is GAMP 5 and
so that we will understand each other,” and I/Os. The pharmaceutical industry How Do I Use It Effectively, montrium.
says López. Without that common has been working on all the technolo- com, November 30,2015. https://blog.
understanding, gaps will persist, some gies that industries are exploring (e.g., montrium.com/experts/what-is-gamp5-
and-how-do-i-use-it-effectively.
worse than others, he says. wireless, industrial internet of things, 8. A. Shanley, “PDA Strengthens its Global
The pharmaceutical industr y ’s and Big Data). In the end, each type Presence,” pharmtech.com, April 15,
approach to data integrity and quality involves software and hardware, input 2019, http://www.pharmtech.com/
pda-strengthens-its-global-presence.
will need to evolve as the industry and output, and there are certain tools 9. O. López, “Electronic Records Integrity
moves to a Pharma 4.0 model and best suited for each situation,” he says. in Data Warehouse and Business
adopts data analytics and artificial “On the fundamental level, we need to Intelligence” in Data Integrity in
Pharmaceutical and Medical Devices
intelligence. At INTERPHEX 2019 understand the relationship between Regulation Operations (CRC Press, Boca
in April, Henrici spoke of the need the system, process mapping, data wave Raton, FL, 1st ed., 2017), pp. 341–351. ◆

SPECIAL SPONSORED SECTION
Quantifying Viruses?
A Q&A Here’s What Matters Most
ith viruses playing a greater role in everything from vaccine development to gene
W therapy, ensuring their rapid, accurate, and biologically relevant enumeration isn’t
just an analytical “plus”; it’s the first step in the development of biological tools that
support human health.
The catch? Traditional virus-quantification methods haven’t always cleared the high bar
that contemporary needs set. That began to change a few years back when a startup in
Antje Schickert , PhD
Product Manager, Virus Analytics Boulder, CO, designed a new platform for rapidly enumerating total virus particles using reagent
Sartorius Stedim Biotech technologies that are both biologically relevant and biologically specific.
Seeing the potential in this emerging technology—called the Virus Counter platform—
Sartorius Stedim Biotech acquired it in 2016 and made it even better, engineering it into a
robust commercial platform. We sat down with Antje Schickert, product manager for virus
analytics at Sartorius Stedim Biotech, to learn how the Virus Counter makes even the trickiest
virus enumerations…well, count.
BioPharm International: Which industries Enumeration and a very good understanding

have a need for virus quantification? of the composition of these products are part
Schickert: Virus enumeration plays a critical of that in-depth characterization.

role in vaccine development and production,
viral vector manufacturing, and many other
In the past, the availability of quick and
reliable quantification methods often failed to
processes where the viruses are often the effectively provide these important insights.
final product. The increasing demand for viral
vectors, requires more efficient manufacturing BioPharm International: How did these
processes to keep pace with the needed industries traditionally enumerate
quantities of viruses. viruses?
Optimization of those manufacturing Schickert: Traditional methods of virus enu-
processes commands a very detailed under- meration include plaque titer assays or TCID50.
standing of how the concentration, and also These are often referred to as functional assays,
the quality, of the product—which, in this case, but they present a drawback in the delay they
is a virus—is influenced by different param- impose because of the labor-intensive nature
eters such as different growth conditions or of the techniques. Results often aren’t avail-
purification steps. able for days or weeks because we have
In applications such as gene therapy, to wait until the viruses replicate in the cell
viruses are often directly administered to to actually quantify them. That can slow or
patients as a therapeutic, and in this case, it’s pause manufacturing processes, or provide
really vital to characterize these viral therapies results that might not be as relevant as when
in detail to ensure the safety of the patient. the sample was taken.
SPECIAL SPONSORED SECTION QUANTIFYING VIRUSES? HERE’S WHAT MATTERS MOST
Many of these traditional methods are also inherently vari- fluorophores that are directly associated with the viruses as
able, and that can decrease confidence in the results that they they pass the laser. The Virus Counter instrument then detects
deliver. So, these methods really cannot keep pace with the the emitted light and uses it, together with the sample flow rate,
modern requirements of cell and gene therapies. to calculate total particle concentration in the virus sample.
BioPharm International: What are some of the more BioPharm International: What is the significance of
modern approaches that are used to prevent the delays counting total virus particles in a direct manner?
caused by traditional assays? Schickert: Virus samples can be incredibly heterogeneous
Schickert: To address the delays that functional assays mixes of infectious viruses, nonfunctional particles, and unas-
cause, methods such as PCR and ELISA were more recently sociated nucleic acids and proteins that weren’t assembled
introduced into the virus quantification field. These methods into virus particles.
are more rapid than more traditional functional assays, but Diverse virus quantification methods actually quantify dif-
they often rely upon measurements of viral components only ferent sub-fractions of the virus sample. For example, plaque
such as viral proteins or nucleic acids; then, they calculate the assays only enumerate functional particles. Depending on the
titer from these measurements. That means that results can virus type and the manufacturing process, however, functional
potentially be biased, as they’re derived values rather than particles can actually be a very small fraction of total particles
values based upon direct measurements of intact viruses. within the sample. ELISA assays usually quantify viral proteins
in the sample, and then use that data to calculate a virus titer.
BioPharm International: How does the Virus Counter PCR quantifies virus-specific nucleic acid sequences in the
address these gaps in the field of virus quantification? sample and derives a titer value from that measurement.
Schickert: The Virus Counter platform was designed to mea- So, the heterogeneity of the sample and the nature of these
sure virus titers directly and with high speed and precision. quantification assays are responsible for the diverse results
The readout is biologically relevant because we look at total that we obtain with different quantification methods when we
viral particles that are directly measured using specific binding measure the same virus sample.

reagents. That can have a significant impact on, for example,
patient safety.
The Virus Counter directly quantifies all intact virus particles
by measuring a fluorescent signal—so the readout here is
Sartorius offers different reagents to address diverse total particles, and it’s really critical for the in-depth under-
customer needs. We have a more universal quantification standing of the virus sample composition and for the safety
reagent that has very broad utility and can be used with a of the patient.
large variety of different viruses, but we also offer an antibody-
based reagent line that allows virus detection with high affinity BioPharm International: Where do you see the advan-
and specificity. tages of the Virus Counter technology over existing
We also emphasize the ease of use of our instrument. From enumeration assays?
system operation to data analysis, everything is software Schickert: The Virus Counter platform enables users to
assisted, and that means we limit the risk of any kind of user measure virus samples in near real time and with very high
error or variability. precision, and that makes Virus Counter results actionable.
The technology empowers our users to increase viral vector
BioPharm International: Can you describe how the Virus yields by comparing, for example, different growth conditions
Counter platform works? and recovery during process development. Users can also
Schickert: The Virus Counter platform was purpose-built to track virus titers throughout their process and determine
enumerate viruses. As mentioned, the platform includes an ideal conditions and even detect possible challenges early
instrument, software, and the reagents we’re using to detect on in their process. Additionally, the Virus Counter platform
viruses. Viruses are directly labeled with either fluorescent increases safety and efficiency by facilitating the in-depth
dyes or antibody-based reagents. Inside the instrument, the characterization of virus samples and increases the under-
virus sample is guided in a fluidic stream and focused to a standing of the total particle concentration in the final product.
small core of moving fluid. A laser is then used to excite the
Operations
Up-to-Date Systems
Streamline Operations
Recently released equipment and products include microbioreactor
systems, cell therapy automation software, and IIoT-enabled flow sensors.
FOR PERSONAL, NON-COMMERCIAL USE AMBER LOWRY
N
ew and updated technologies have been released over system’s new functionality, election of clones, media, and
the past few months for a range of bioprocessing feeds can be performed under perfusion mimic conditions
tasks to spark innovation and support efficiency. The to bleed large volumes of culture and quickly remove spent
following are a sampling of such products. media from the microbioreactors. The company states that
a new flexible workstation layout and an expanded tip bin
CELL CULTURE MICROBIOREACTOR SYSTEM capacity provide greater operator walk-away time.
A new generation of the ambr 15 cell culture automated New culture passage steps and rapid vessel drain function-
microbioreactor system from Sartorius Stedim Biotech ality allow for the adaptation of cell lines to different media
(SSB) offers increased flexibility and expanded capability for for media screening studies to be performed in the microbio-
clone selection, media and feed optimization, and early pro- reactors. New media mixing steps automate the creation of
cess development work (1). media blends, eliminating the need to pre-mix. Clone stabil-
The Generation 2 system replicates laboratory-scale bio- ity studies can also be set up with serial passages performed
reactor performance at the 10–15 mL microscale and con- and fully automated in the microbioreactors. Rapid vessel
trols up to 48 single-use bioreactor cultures in parallel. The drain functionality for automated cell passaging and media
updated design incorporates new features to improve process exchanges in the microbioreactors supports cell and gene
flexibility, expand the system’s functionality, and allow more therapy applications. New culture station design provides
applications to be investigated. lower stirrer speed control suitable for more sensitive cell lines.
New features include a one-year license of SSB’s clone The system also provides a robust screening platform
selection software, which provides simplified, streamlined for development of cell and gene therapy processes, includ-
multivariate data analysis for faster, more consistent cell-line ing HEK293 for viral vector production, T-cells, induced
screening and ranking, according to the company. Using the pluripotent stem cells, and other immune-derived cell lines.

Operations
AUTOMATION SOFTWARE According to WMF TG, the gas- sensor provides local indication of all
FOR CELL THERAPY kets use new materials—polytetra- relevant operating and diagnostic data.
The Chronicle web application, the fluoroethylene and a synthetic rubber The sensor is available as a normally
next generation of GE Healthcare’s and fluoropolymer elastomer (Viton, open and normally closed version.
my.Cryochain software, now supports Chemours)—to provide a high level of Analog outputs can be switched with a
the complete cell therapy workflow chemical and steam resistance. Each of signal from 4 to 20 mA. According to
(2). Chronicle automation software the high-purity gaskets have been engi- the company, these signals can be inter-
is a GMP-compliant, fit-for-purpose neered to deliver improved sealing per- preted directly by many controllers.
digital solution for the optimization of formances under clamping compression. The sensor’s IO-Link capability and
complex cell therapy process develop- The BioPure range offers lot traceability the Ethernet interface enable users to
ment and manufacturing. on every component, silicone products communicate with existing controllers.
Chronicle automation is suited to manufactured and packed in an ISO Communications protocols integrated
increase efficiency while meeting reg- 14644-1 Class 7 cleanroom, compliancy into the device include Open Platform
ulatory compliance using real-time with FDA regulations CFR 21 177.2600, Communications Unified Architecture
supply chain tracking, hardware per- dedicated validation and qualification and Message Queuing Telemetry
formance monitoring, SMS or email data to help achieve cGMP require- Transport. A web-based dashboard
alarms, and comprehensive electronic ments, and USP Class VI compliance shows real-time data for the users.
batch records. Software capabilities and animal-derived component free.
include a unified digital space that REFERENCES
monitors all facility manufacturing IIOT-ENABLED FLOW SENSOR 1. Sartorius Stedim Biotech, “Sartorius
Stedim Biotech Launches New ambr
operations and supply chain logistics The Aventics AF2 flow sensor from 15 Cell Culture Microbioreactor
with real-time data acquisition and Emerson continuously monitors air con- System,” Press Release, May 2, 2019.
notifications. According to the company, sumption, which allows for rapid leak- 2. GE Healthcare, “GE Healthcare
Announces Commercial Launch of
electronic batch records increase pro- age intervention in applications such as Chronicle Automation Software for Cell
ductivity, reinforce GMP compliance, packaging (4). The sensor continuously Therapy,” Press Release, May 15, 2019.
and improve the security of patients’ monitors air consumption in pneumatic 3. Watson-Marlow Fluid Technology
Group, “Watson-Marlow Fluid
samples through increased traceability. systems, enabling compliance with the Technology Group Expands
Built by cell therapy platform solu- energy management standard DIN BioPure Gasket Range,” Press

tion engineers, the software supports ISO 50001. The device is one of vari- Release, May 2, 2019.
4. Emerson, “IIoT-Enabled Flow Sensor
electronic standard operating proce- ous Industrial Internet of Things (IIoT) Monitors Air Loss in Pneumatic
dures, which are suitable for specific components offered by the company for Systems to Optimize Energy Usage,”
processes to manage deviations, pro- networked plants in factory automation. Press Release, April 2, 2019. ◆
mote adherence to protocol, and “The Aventics AF2 series flow sensor
provide guidance to ensure sensitive is ideally suited to those companies aim-
patient cells are handled appropriately.
Additionally, the software integrates
ing to control and optimize their energy
usage,” said Andreas Kliewe, expert for
Ad Index
with the full range of GE instruments energy management with Emerson’s
as well as many third-party instruments machine automation business, in an Company Page
and has been independently audited April 2, 2019 press release. “The device BIO RAD LABORATORIES 51
against GAMP5, 21 Code of Federal records the flow rate and compressed air
CATALENT PHARMA SOLUTIONS 52
Regulations Part 11, and EU Annex 11. consumption in a system, sending a sig-
nal to the controller when pre-set levels EPPENDORF 14–15
EXPANDED GASKET RANGE are exceeded. This helps avoid excessive
IRVINE SCIENTIFIC 20–21
Watson-Marlow Fluid Technology energy loss and enables rapid interven-
Group ( WMF TG) expanded its tion in the event of an issue.” KNAUER GMBH 19
BioPure gasket range for high purity fluid The company states that the device’s LABVANTAGE SOLUTIONS, INC 26–27
management (3). The high-purity gaskets modular construction makes it suitable
cover a range of applications and support for installation where space is limited. MASTER CONTROL SYSTEMS 2
leak resistant connectivity within phar- The sensor is delivered precalibrated MILLIPORE SIGMA 11
maceutical and biotechnology produc- with its filter, which can be configured
PDA 32–33
tion processes, reducing validation risks in in any maintenance unit. An organic
contamination-free applications. light-emitting diode display on the SARTORIUS STEDIM BIOTECH 42–43, 46–47

Ask the Expert
Playbooks Are Not Just Child’s Play

Cultural and language discrepancies during an audit can be
Siegfried
Siegf
Si friiedd Schmitt,
Schhmiitt
itt PhD,
PhD
resolved using what many call a “playbook,” says Siegfried vice-president, technical
Schmitt, PhD, vice-president, technical, Parexel Consulting. at PAREXEL Consulting
Q: We are preparing for our first inspec-

tion by an overseas regulatory agency.
This will be a challenge from several perspec-
Without a playbook, the answer may go like this:
‘In step 5 after vessel 234 on the second floor,
remember you saw this reactor on the tour of the
tives: language, culture, and presentation style. facility. We have installed a 0.2-micron filter …’
We believe we have covered the first two issues With a playbook, the answer would be more
through highly experienced translators and cul- similar to this: ‘We have a bioburden control
tural training sessions with coaches. Our concern strategy that takes into account environment,
is about how to present and respond in an appro- process equipment, material handling, product
priate manner. Can you give any advice? properties, and regulatory expectations. For the
A: Hosting a foreign agency for the first

time is always challenging, but as with all
inspections, nothing beats preparation, practice,
five steps produced in our facility, we have pre-
pared a strategy paper, which has subsequently
been verified during the validation studies and
practice, and more practice. Even seasoned per- ongoing monitoring.’
sonnel can become flustered, nervous, or even Of course, should the inspector then want
unsure how to respond to unfamiliar questions to know more details, this is when all the evi-
or requests, especially when asked in a foreign dence can be presented. Again, it may be useful
tongue. One tool proven to be particularly useful to have a playbook ready for this, especially if
in such situations is a playbook. Playbooks may many activities should be organized in a logical

be known by other terms, but essentially these sequence and timeline.
are aide-memoires that function as references for Playbooks have several benefits, as they help
company representatives who need to present those responding in an inspection to give clear
or answer questions from inspectors or auditors. answers. They also help prepare the site person-
The playbook may include prepared answers to nel to find answers to difficult questions (e.g.,
anticipated questions the auditor may ask and/or why certain deviation investigations are overdue,
detailed information about process steps. or why a particular change hasn’t been docu-
How detailed should these playbooks be and mented as per requirements). They also help
in what format? Here is what has stood the test summarize convoluted event histories or complex
of time: situations. Often, one has to overcome obstacles
• Always start with a succinct, logical, and to reach a successful outcome, and playbooks
convincing few lines of text and/or graphics. help to focus on the positives, rather than to
• If necessary, have a second playbook with overemphasize the difficulties experienced.
more detailed, supporting information. Then why should one have these playbooks
• Have a third party (i.e., someone unfamil- reviewed by someone from the outside? Well,
iar) review your playbook and/or provide the inspector will be exactly such a person (i.e.,
feedback. an outsider who isn’t necessarily familiar with
The first point is the most important. As we the terminology, the facility, or the processes
all are experts in what we do, we can talk in used at the site). Therefore, by having an out-
great detail about it. But inspectors do not have side third party review the playbook, you essen-
Fanatic Studio/Getty Images
unlimited time. They want to receive answers tially perform a dry run of the inspection.
that provide them with clear and easy-to-com- In summary, these playbooks are a useful tool
prehend information. This is best illustrated with in any inspection or audit, whether foreign or
an example. The inspector may ask: ‘What is local, but writing these in a clear and concise
your bioburden control strategy for this product?’ manner is anything but child’s play. ◆

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Volume 32 Number 7

July 2019 The Science & Business of Biopharmaceuticals

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MANUFACTURING PEER-REVIEWED RESIDUAL IMPURITIES

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Explore how quality changes everything at www.mastercontrol.com

The Science & Business of Biopharmaceuticals

EDITORIAL EDITORIAL ADVISORY BOARD

European Senior Sales Executive

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BioPharm International integrates the science and business of biopharmaceutical research,

FEATURES COLUMNS AND DEPARTMENTS

FROM THE EDITOR

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A Biologics Partisan Divide

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FDA Revamps Biosimilar

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Gene Therapies Propel Outsourcing Investment

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That's why the entire range of

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MilliporeSigma, the vibrant M and Supelco are

© 2019 Merck KGaA, Darmstadt, Germany and/or its

The Life Science Business

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Applying QbD to Upstream Processing

FOR PERSONAL, NON-COMMERCIAL USE SUSAN HAIGNEY

even many relevant metabolic pathways, inductions, flux, impurities in DL intermediates.

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QBD IN UPSTREAM Performing small-scale DoE by USING QBD TO DEVELOP

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variations in common raw material been around for decades, individual

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FUJIFILM Irvine Scientific

With more than four decades of experience,

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Let our culture work for you.

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Design Considerations for

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• /I]RSXIWTIEOIVWMRGPYHMRKPeter Marks, • *(%'IRXIVERH'SQTPMERGI9THEXIW