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BioPharmThe Science & Business of Biopharmaceuticals
INTERNATIONAL
www.biopharminternational.com
INTERNATIONAL
OPTIMIZING RESIN USE TO
ACHIEVE COST-EFFECTIVE
BIOPROCESSING
Bio
Ph
arm
Intern
atio
nal
MA
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arv
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March 2016
Volume 29 Number 3
UPSTREAM PROCESSING
BIOPHARMA TAKES
ON RAW MATERIAL
VARIABILITY
PEER-REVIEWED
A RISK-BASED GENETIC
CHARACTERIZATION
STRATEGY FOR RECOMBINANT
CHO CELL LINES
REGULATIONS
GENERIC-DRUG PRODUCTION
AND OVERSIGHT CHALLENGE
FDA AND MANUFACTURERS
COMPLEX SUPPLY, WITHOUT ALL THE CHAOS
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INTERNATIONAL
BioPharmThe Science & Business of Biopharmaceuticals
EDITORIAL
Editorial Director Rita Peters [email protected]
Senior Editor Agnes Shanley [email protected]
Managing Editor Susan Haigney [email protected]
Science Editor Randi Hernandez [email protected]
Science Editor Adeline Siew, PhD [email protected]
Community Manager Caroline Hroncich [email protected]
Art Director Dan Ward [email protected]
Contributing Editors Jill Wechsler, Jim Miller, Eric Langer, Anurag Rathore, Jerold Martin, Simon Chalk, and Cynthia A. Challener, PhD
Correspondent Sean Milmo (Europe, [email protected])
ADVERTISING
Publisher Mike Tracey [email protected]
West/Mid-West Sales Manager Steve Hermer [email protected]
East Coast Sales Manager Scott Vail [email protected]
European Sales Manager Chris Lawson [email protected]
European Sales Manager Wayne Blow [email protected]
C.A.S.T Data and List Information Ronda Hughes [email protected]
Reprints 877-652-5295 ext. 121/ [email protected] Outside US, UK, direct dial: 281-419-5725. Ext. 121
PRODUCTION
Production Manager Jesse Singer [email protected]
AUDIENCE DEVELOPMENT
Audience Development Rochelle Ballou [email protected]
© 2016 UBM. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical including by photocopy, recording, or information storage and retrieval without permission in writing from the publisher. Authorization to photocopy items for internal/educational or personal use, or the internal /educational or personal use of specific clients is granted by UBM for libraries and other users registered with the Copyright Clearance Center, 222 Rosewood Dr. Danvers, MA 01923, 978-750-8400 fax 978-646-8700 or visit http://www.copyright.com online. For uses beyond those listed above, please direct your written request to Permission Dept. fax 440-756-5255 or email: [email protected].
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EDITORIAL ADVISORY BOARDBioPharm International’s Editorial Advisory Board comprises distinguished
specialists involved in the biologic manufacture of therapeutic drugs,
diagnostics, and vaccines. Members serve as a sounding board for the
editors and advise them on biotechnology trends, identify potential
authors, and review manuscripts submitted for publication.
K. A. Ajit-Simh President, Shiba Associates
Rory Budihandojo Director, Quality and EHS Audit
Boehringer-Ingelheim
Edward G. Calamai Managing Partner
Pharmaceutical Manufacturing
and Compliance Associates, LLC
Suggy S. Chrai President and CEO
The Chrai Associates
Leonard J. Goren Global Leader, Human Identity
Division, GE Healthcare
Uwe Gottschalk Vice-President,
Chief Technology Officer,
Pharma/Biotech
Lonza AG
Fiona M. Greer Global Director,
BioPharma Services Development
SGS Life Science Services
Rajesh K. Gupta Vaccinnologist and Microbiologist
Jean F. Huxsoll Senior Director, Quality
Product Supply Biotech
Bayer Healthcare Pharmaceuticals
Denny Kraichely Associate Director
Johnson & Johnson
Stephan O. Krause Director of QA Technology
AstraZeneca Biologics
Steven S. Kuwahara Principal Consultant
GXP BioTechnology LLC
Eric S. Langer President and Managing Partner
BioPlan Associates, Inc.
Howard L. Levine President
BioProcess Technology Consultants
Herb Lutz Principal Consulting Engineer
Merck Millipore
Jerold Martin Independent Consultant
Hans-Peter Meyer Lecturer, University of Applied Sciences
and Arts Western Switzerland,
Institute of Life Technologies.
K. John Morrow President, Newport Biotech
David Radspinner Global Head of Sales—Bioproduction
Thermo Fisher Scientific
Tom Ransohoff Vice-President and Senior Consultant
BioProcess Technology Consultants
Anurag Rathore Biotech CMC Consultant
Faculty Member, Indian Institute of
Technology
Susan J. Schniepp Fellow
Regulatory Compliance Associates, Inc.
Tim Schofield Senior Fellow
MedImmune LLC
Paula Shadle Principal Consultant,
Shadle Consulting
Alexander F. Sito President,
BioValidation
Michiel E. Ultee Principal
Ulteemit BioConsulting
Thomas J. Vanden Boom VP, Biosimilars Pharmaceutical Sciences
Pfizer
Krish Venkat Managing Partner
Anven Research
Steven Walfish Principal Scientific Liaison
USP
Gary Walsh Professor
Department of Chemical and
Environmental Sciences and Materials
and Surface Science Institute
University of Limerick, Ireland
4 BioPharm International www.biopharminternational.com March 2016
Contents
BioPharmINTERNATIONAL
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.
COLUMNS AND DEPARTMENTS
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BioPharmThe Science & Business of Biopharmaceuticals
INTERNATIONAL
www.biopharminternational.com
INTERNATIONAL
OPTIMIZING RESIN USE TO
ACHIEVE COST-EFFECTIVE
BIOPROCESSING
March 2016
Volume 29 Number 3
UPSTREAM PROCESSING
BIOPHARMA TAKES
ON RAW MATERIAL
VARIABILITY
PEER-REVIEWED
A RISK-BASED GENETIC
CHARACTERIZATION
STRATEGY FOR RECOMBINANT
CHO CELL LINES
REGULATIONS
GENERIC-DRUG PRODUCTION
AND OVERSIGHT CHALLENGE
FDA AND MANUFACTURERS
Cover: BullStorm/Getty Images; Dan Ward
6 From the Editor
Thought leaders tackle drug shortages and biomanufacturing challenges. Rita Peters
8 US Regulatory Beat
Policy makers debate strategies for promoting access to less costly medicines. Jill Wechsler
10 Perspectives on Outsourcing
The outsourcing market starts 2016 with company expansions, acquisitions, and new offerings Susan Haigney
49 Ad Index
50 Ask the Expert
The auhors discuss how to create a robust CAPA system and how to identify root cause.Susan Schniepp
and Andrew Harrison
BIOPROCESSING
Achieving Cost-Effective
Bioprocesses
Randi HernandezExperts in the field share some best
practices for optimizing process
economics in bio-manufacturing. 14
UPSTREAM PROCESSING
Biopharma Takes On
Raw Material Variability
Cynthia A. ChallenerCollaborative efforts are
underway between suppliers
and drug manufacturers. 20
DOWNSTREAM PROCESSING
Adherent Cell Culture
in Biopharmaceutical
Applications: The Cell-
Detachment Challenge
Marcos Simon and Juan J. Giner-CasaresThe necessity to detach cells from a
culture substrate during cell harvesting
remains one of the most challenging
steps in a cell-culture process. 26
PEER-REVIEWED
A Risk-Based Genetic
Characterization Strategy
for Recombinant CHO Cell
Lines Used for Clinical and
Commercial Applications
Luhong He and Christopher FryeThe authors provide a comprehensive,
risk-based transgene characterization
strategy. 32
DATA INTEGRITY
How Important is Data
Integrity to Regulatory Bodies?
Bob McDowall and Joanne RatcliffData integrity is a widespread, global
problem that must be addressed. 42
COLD CHAIN
Cold Chain:
Going the Extra Mile
Agnes ShanleyReal-time GPS technology, better
IT connections, and more conservative,
controlled shipping temperatures
are improving the shipment of
sensitive pharmaceuticals. 45
Volume 29 Number 3 March 2016
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6 BioPharm International www.biopharminternational.com March 2016
From the Editor
Thought leaders
tackle drug
shortages and
biomanufacturing
challenges.
Keynote Series Addresses Crucial Industry Issues
At INTERPHEX 2016 at the Javits Center in New York, BioPharm
International and Pharmaceutical Technology will host a Keynote Series on
leading bio/pharma industry issues. Sessions will be presented on the
Innovation Stage in the exhibit hall. Admission is free to any attendee with an
exhibit hall pass. The following is a preview of the topics.
Overcoming Bottlenecks in Biopharmaceutical DevelopmentIn a panel discussion, industry experts will discuss how technology advances
are addressing challenges in biopharmaceutical development including qual-
ity control of raw materials, implementation of single-use technologies, process
monitoring, and downstream purification. The panelists are Marian L. McKee,
PhD, director, US development services, MilliporeSigma; Mike Goodwin, director
of R&D, SUT, Thermo Fisher Scientific; Alex Perieteanu, PhD, director, biophar-
maceutical services-Life Sciences, SGS Mississauga; Elizabeth Goodrich, director
of applications engineering, MilliporeSigma. (Tuesday, April 26, 10:15—11:45 AM)
Contract Services Market: 2016 UpdateHow will consolidation in the bio/pharmaceutical and contract services mar-
ket, a changing financial market, and an active political and regulatory year
shape the fortunes of the contract services market? In his annual presentation,
Jim Miller, founder and president, PharmSource Information Services will
offer his perspectives on the contract services landscape for the next few years.
(Wednesday, April 27, 10:30–11:30 AM)
Strategies and Innovations to Reduce Drug Shortages and Improve
Availability of Medicines Aging facilities and equipment, inadequate operator training, a lack of quality con-
trol, tighter regulatory enforcement, and business decisions to eliminate unprofit-
able product lines contribute to ongoing shortages of vital drug products. In this
session, industry thought leaders will identify triggers for drug shortages, methods
to avoid production line shutdowns and update facilities, and innovative industry
efforts to fulfill demand for needed therapies. (Wednesday, April 27, 1:30–3:15 PM)
An Interdisciplinary Approach to Address Drug Shortages. The effects
of drug shortages on patients, caregivers, hospitals, and medical professionals are
often not observed or understood by the bio/pharmaceutical manufacturing seg-
ment. This presentation will explore alternative, innovative, and cost-effective
ways to provide needed therapies to patients.
BARDA Innovation Initiatives in Medical Countermeasure
Manufacturing. This presentation will describe the Biomedical Advanced
Research and Development Authority’s (BARDA) program initiatives in manufac-
turing technologies for medical countermeasure advanced development, includ-
ing opportunities with the Centers for Innovation in Advanced Development &
Manufacturing and in continuous manufacturing.
Panel Discussion: Addressing Sterile Manufacturing Challenges Sterile injectables have been in extremely short supply, and industry efforts
have been focusing on root causes involving infrastructure, quality, and
efficiency. Experts involved in this work discuss recent initiatives, and offer
insights into what must be done to prevent injectables shortages in the future.
(Wednesday, April 27, 3:30–4:30 PM)
For more information about the Keynote Series, including sessions for
small-molecule manufacturing, visit: www.biopharminternational.com/bp/
Interphex2016. X
Rita Peters is the
editorial director of
BioPharm International.
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8 BioPharm International www.biopharminternational.com March 2016
Regulatory Beat
Vis
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It’s well known that generic drugs account
for 88% of prescription drug sales in the
United States and have saved billions for
patients and healthcare systems since Congress
enacted the Hatch-Waxman Act more than 30
years ago. That growth, though, has created
difficulties for FDA in processing the hundreds
of resulting abbreviated new drug applications
(ANDAs) and in inspecting an expanding num-
ber of generic-drug manufacturers and ingredi-
ent producers all over the world.
Concerns about ensuring the quality and
safety of medical products, moreover, have led
to the closure of outdated and noncompliant
facilities, contributing to shortages and price
spikes for certain widely used generics, particu-
larly sterile injectables. These developments have
raised questions about whether government reg-
ulatory policies limit competition in certain
drug classes and support monopoly pricing.
FEES ADD RESOURCESFDA’s ability to expeditiously approve new
generics was compromised by a budget squeeze
over many years. The Prescription Drug User
Fee program (PDUFA) of 1992 bolstered fund-
ing for new drug review by the Center for
Drug Evaluation and Research (CDER), but also
shifted resources away from generics.
ANDA approvals slowed to a crawl,
resulting in an enormous backlog of
pending applications.
Generic-drug makers finally agreed
to pay user fees in 2012 to strengthen
FDA regulation of generic-drug devel-
opment, review, and inspection. In its
first three years, the Generic Drug User
Fee program (GDUFA I) has generated
nearly $1 billion to support CDER’s
Office of Generic Drugs (OGD) and
certain operations of the new Office of
Pharmaceutical Quality (OPQ). FDA’s
field force also has increased inspections of over-
seas producers to help level the playing field
between US and foreign manufacturers.
As FDA and industry negotiate GDUFA
renewal in 2017, Congressional committees
and the broader healthcare community are
examining FDA policies and programs govern-
ing generics and the prescription drug market,
as seen at a January 2016 hearing before the
Senate Health, Education, Labor and Pensions
(HELP) Committee. Chairman Lamar Alexander
(R.Tenn.) cited concerns about “unnecessary
regulatory burdens” that can slow drug develop-
ment and the importance of a pharmaceutical
marketplace that “remains competitive” (1).
The panel also is developing a Senate version
of the “21st Century Cures” legislation, which
the House approved in July 2015. Instead of
combining multiple proposals into a compre-
hensive bill, Alexander and ranking Democrat
Patty Murray (D-Wash) are considering numer-
ous individual measures on FDA policies, disease
research, and expanded use of electronic data
technology to support broader research goals.
With deliberations running through April 2016,
though, there’s not much chance that Congress
will adopt any final “Cures” legislation this year,
but will wait until 2017 when action is required
to reauthorize FDA user fee programs.
NO MORE BACKLOGA main issue explored at the HELP hearing is
whether too-slow FDA approval of new generics
limits drug access and competition. Some legisla-
tors suggested that a pharma company would be
less likely to buy up a small drug firm with the
intent of boosting product prices if it knew that
FDA could quickly approve a new competing drug.
Despite complaints from generics makers about
still-delayed ANDA approvals, CDER director
Janet Woodcock made a strong case for agency
progress in addressing the backlog problem,
Generic-Drug Production and Oversight Challenge FDA and ManufacturersPolicy makers debate strategies for promoting access to less costly medicines.
Jill Wechsler is BioPharm
International’s Washington editor,
Chevy Chase, MD, 301.656.4634,
March 2016 www.biopharminternational.com BioPharm International 9
Regulatory Beat
speeding important new gener-
ics through the approval process
and expanding timely inspections
of manufacturing facilities. She
explained that generic-drug makers
submitted nearly 2500 applications
in 2013 and 2014, making it difficult
for OGD to process those documents
and to tackle long-pending submis-
sions, while also restructuring and
expanding its program (2).
Even so, in the past three years
CDER was able to “take action” on
approximately 85% of 4600 over-
due ANDAs and post-approval
supplements, Woodcock stated.
She promised that all the backlog
would be gone by 2017 and that
OGD would meet its goal for tak-
ing a “first action” within 10
months on ANDAs submitted this
year. No applications in the backlog
are first generics, she emphasized,
and OGD’s “express lane” policy
moves these products to the front
of the queue. She also highlighted
CDER efforts to promote advanced
manufacturing in the generic-drug
industry, as continuous, computer-
controlled production systems
would enable fast ramp-up of new
production.
Key to achieving these goals is an
FDA-industry effort to achieve more
first-cycle approvals. A “right-the-
first-time” policy permits rejection
of notably incomplete applications
when they first come in. CDER
also is issuing more guidance on
what data it wants from sponsors
and encouraging manufacturers
to conduct all necessary tests and
processes before sending in appli-
cations. A new pre-ANDA process
that addresses approval challenges
for particular drugs prior to applica-
tion submission may be included in
GDUFA II.
SUPPORTING COMPETITIONIn highlighting FDA efforts to
quickly approve first generics,
Woodcock acknowledged that mul-
tiple drugs per innovator may drive
down costs and facilitate patient
access to more affordable therapies.
Yet FDA does not approve a new drug
or generic in response to rising prices,
she noted, and does not have the
expertise to calculate what qualifies
as a “price hike:” would that involve
doubling a price from 10 cents to
20 cents, or possibly raising a list
price by more than 1000%?, she
queried, adding that a new report
from the US Department of Health
and Human Services (HHS) better
addresses generic-drug pricing (3).
The agency does keep a close eye
on sole-source products and those
with only one or two competitors,
as part of efforts to anticipate drug
shortages and supply disruptions.
Woodcock presented data indicating
that 99 innovator drugs have only
one generic competitor; 66 drugs
have two generics; and 623 drugs
have 3 or more generics. Of particu-
lar interest is the segment of 125
innovator drugs with no approved
generics (and no patent or exclusiv-
ity protections) (4).
These drugs may have limited
competition, Woodcock explained,
because they are orphans or spe-
cialized therapies that serve small
patient populations. Many topical
products, inhalants, and complex
substances also lack well-understood
methods for testing and document-
ing bioequivalence. To support the
development of generic versions of
such therapies, GDUFA provided
FDA with approximately $35 mil-
lion for research on new bioequiva-
lence test methods and guidances to
“open up previously blocked path-
ways” for new generics.
Woodcock acknowledged that, in
some cases, innovator firms take
steps to block and delay generic drug
entry. Generic-drug makers have
complained loudly about problems
in obtaining supplies for bioequiv-
alence testing of brand products
that are subject to Risk Evaluation
and Mitigation Strategies (REMS).
Woodcock said that FDA has advised
brand firms that REMS don’t war-
rant withholding drugs for research
purposes, and indicated that
Congressional action would help
address this problem more directly.
The Gener ic Pharmaceut ica l
Association also wants the legisla-
tors to repeal a recent budget provi-
sion that boosts Medicaid rebates
on generic drugs (5).
One strategy Woodcock strongly
opposed is to turn to drug com-
pounders to provide less costly
alternative medicines when gener-
ics fail to meet demand. She
emphasized that there are “very
great risks” in such proposals, cit-
ing two examples of compounded
drugs that sickened dozens of peo-
ple. Mass production of these drugs
without adherence to GMPs, she
warned, “could have put thousands
of people in the hospital.”
REFERENCES 1. US Senate Committee on Health,
Education, Labor & Pensions, “Alexander: Despite Extra $1 Billion to Speed Generic Drug Approvals, FDA Process Still Too Slow,” Press Release, Jan. 28, 2016, www.help.senate.gov/chair/newsroom/press/alexander-despite-extra-1-billion-to-speed-generic-drug-approvals-fda-process-still-too-slow., accessed Feb. 2, 2016.
2. Implementation of the Generic Drug User Fee Amendments of 2012 (GDUFA), Testimony of Janet Woodcock, MD, Before the Committee on Health, Education, Labor and Pensions, Jan. 28, 2016, www.help.senate.gov/imo/media/doc/Woodcock5.pdf, accessed Feb. 2, 2016.
3. Office of the Assistant Secretary for Planning and Evaluation, Understanding
Recent Trends in Generic Drugs, Jan. 27, 2016, https://aspe.hhs.gov/pdf-report/understanding-recent-trends-generic-drug-prices, accessed Feb. 2, 2016.
4. FDA, Slides from FDA GDUFA presentation before the Senate Health, Education, Labor and Pensions Committee, January 2016, www.biopharminternational.com/generic-drug-production-and-oversight-challenge-fda-and-manufacturers-0.
5. GPhA, “GPhA to Congress: Embrace Five Opportunities for More Generic Drug Savings,” Statement by Chip Davis, President and CEO, GPhA, Feb. 1, 2016, www.gphaonline.org/gpha-media/press/gpha-to-congress-embrace-five-opportunities-for-more-generic-drug-savings, accessed Feb. 2, 2016. ◆
10 BioPharm International www.biopharminternational.com March 2016
Perspectives on Outsourcing
Do
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A survey of drug manufacturers reveals
that cost considerations, as well as
mergers and acquisitions, will con-
tinue to drive the use of outsourced services
(1). Distribution activities was the top function
biopharmaceutical companies will outsource,
followed by packaging and labeling, and drug
product manufacturing.
For the broader pharmaceutical market, the
top five activities that drug companies out-
source are drug product manufacturing,
packaging and labeling, distribution, small-mol-
ecule manufacturing, and holding and storage,
according to Kate Hammeke of ISR Reports (1).
Service providers are expanding their ser-
vices and capabilities to keep up with the
high demand. The following are some exam-
ples of the growth the outsourcing industry is
experiencing.
COMPANY DEVELOPMENTS, EXPANSIONS, AND ACQUISITIONSThe first few months of 2016 has seen an array
of investments, expansions, and acquisitions
in the pharmaceutical outsourcing market.
Outsourcing companies appear to be looking
to the early-phase development and clinical
trial markets to increase their portfolios.
Austrianova completed a new facility, add-
ing GMP cell-banking and fill-and-finish
services for cell therapy products to its encap-
sulation services and technology, the company
announced in a Jan. 25, 2016 press release (2).
Austrianova offers the production of master
cell and working cell banks (MCB and WCB)
at the scale required for Phase I and II stage
clinical trials using its isolator-based produc-
tion facility. The company can also fill bulk
cell product into syringes or vials in its GMP
facility. This new cell banking and
filling service is called GMP4Cells.
MCBs and WCBs are required for
all cell therapy products like stem-
cell therapies and biologics produced from
cells such as vaccines, antibodies, and recom-
binant proteins.
LabConnect, a Seattle-based provider of lab-
oratory services to biopharmaceutical, medical
device, and contract research firms, has built a
new 5000-sq-ft biorepository in Johnson City,
TN, that includes space for ambient, refriger-
ated, cold (-20 °C), and ultra-low temperature
(-70 to -80 °C) storage as well as liquid nitrogen
vapor phase storage (-190 °C) (3).
The facility includes storage capacity for
more than eight million samples, validated
and mapped backup freezers and generators,
redundant HVAC systems, building and biore-
pository security systems, and a temperature
monitoring system for freezers and refrigera-
tors with a 21 Code of Federal Regulations Part 11
compliant audit trail. LabConnect also tracks
sample locations and consolidates data within
a centralized database.
Catalent Pharma Solutions announced on
Feb. 2, 2016 (4) an investment of $4.6 million
to expand its Singapore clinical supply facility
by building GMP space for secondary packag-
ing. The investment doubles the ambient stor-
age space and quadruples cold-storage capacity,
the company reports.
The site provides clinical supply services
including project and supply-chain manage-
ment, comparator sourcing, clinical label print-
ing, secondary packaging, clinical storage,
import/export management, importer of record
service, and returns and destruction manage-
ment services. It has served as a regional hub
for studies in Australia, Singapore, Korea, Hong
Kong, and other countries in Southeast Asia.
SGS, a bio/pharmaceutical analytical and
bioanalytical contract solutions provider,
announced on Jan. 19, 2016 that after the acqui-
sition of Quality Compliance Laboratories in
December 2015, its global network now offers
chemistry and microbiology testing services
Biopharma Outsourcing Market Expands The biopharmaceutical outsourcing market starts 2016 with company expansions, acquisitions, and new offerings.
Susan Haigney is managing editor of
BioPharm International.
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Perspectives on Outsourcing
for cosmeceuticals, natural health
products, and medical marijuana
from facilities in Canada (5).
New capabilities within the
two facilities include an induc-
tively coupled plasma-mass spec-
trometer and inductively coupled
plasma-optical emission spec-
trometer for elemental impurities
analysis, automated tablet disso-
lution apparatuses, flow-through
USP dissolution apparatus, and
Vitek for bacterial identification.
Vet te r a nnounced on Ja n.
28, 2016 that the company’s
Schuetzenstrasse multi-functional
building in Ravensburg, Germany,
has been completed on schedule
and departments crucial to its
operation have started to move in
(6). The $32 million (€29 million)
investment is part of a $331 mil-
lion (€300 million) total invest-
ment strategy announced by the
company in September 2015.
The continued demand by large
and small customers for enhanced
drug development services, as well
as the need for ever-more future-
oriented sophisticated IT systems
to protect their data, created the
need for the new facility, accord-
ing to the company.
The 91,500-sq-ft, six-story build-
ing contains non-cGMP labora-
tories for development support,
laboratory space for microbiologi-
cal analysis, office workplaces for
Vetter Development Service and
IT, and a data processing center
with enhanced security systems,
including a safety cell that protects
technology and data from external
physical hazards in the event of an
emergency.
Novasep is bui lding a new
synthesis laboratory and adding
kilogram-scale production at its
existing US facility in Boothwyn,
PA (7). This extension will allow
Novasep to offer both chemistry
and purification services and to
produce the initial kilogram-scale
batches of synthetic molecules
that are needed for biological test-
ing and preclinical trials.
The new laboratory, equipped
with reactors up to 50 L in size,
will start operation in May 2016.
It will offer cryogenic capacities
and standard chemistry, as well as
preparative purification chroma-
tography processes.
PBOA EXPANDS MEMBERSHIPT he Pha r ma & B iopha r ma
Outsourcing Association (PBOA),
founded in 2014, has been
advocating for the pharma out-
sourcing industry as the global mar-
ket changes and expands. “We’re
focused on working on the reautho-
rization of the Generic Drug User
Fee Amendment (GDUFA), while
keeping an eye on FDA’s Quality
Metrics initiative, and helping make
sure that CMO/CDMOs [contract
manufacturing organizations/con-
tract development and manufactur-
ing organizations] are prepared for
track-and-track/serialization regula-
tions as they roll out in the United
States and the European Union
in the next few years,” says PBOA
President Gil Roth.
I n Febr u a r y 2 016 , PB OA
expanded its membership (8). IDT
Biologika and Ei SolutionWorks
joined the PBOA as general mem-
bers; 3M Drug Delivery Systems
(DDS) joined as a sustaining mem-
ber. Diego Romeu, manufacturing
and supply chain director at 3M
DDS, was also voted to a three-year
term on the board of trustees, along
with Rajan Puri, director of business
development at Therapure, and Lee
Karras, CEO of Halo Pharmaceutical.
“As we continue our mission to
represent the CMO/CDMO indus-
try before FDA, Congress, and other
stakeholders, it’s critical that we
increase our membership and pro-
vide a true voice for our industry,”
said Roth. “We’ve been successful in
bringing the CMO/CDMO perspec-
tive to issues such as GDUFA, quality
metrics, and serialization, and we’re
delighted to bring in new member
companies and add fresh points of
view to our Board of Trustees.”
REFERENCES 1. A. Shanley, “Surveys Examine
Outsourcing Trend,” Pharmaceutical
Technology, Supplement: Partnerships
in Outsourcing, 40 (13), pg 32-33,
www.pharmtech.com/surveys-
examine-outsourcing-trend
2. BioPharm Editors, “Austrianova Offers
GMP Cell Banking and Fill/Finish
Services,” BioPharmInternational.com,
www.biopharminternational.com/
austrianova-offers-gmp-cell-banking-
and-fillfinish-services-0
3. LabConnect, “LabConnect Builds New
Biorepository, Expands Services,
Offers Absolute Sample Protection,”
Press Release, Feb. 3, 2016, www.
labconnectllc.com/Documents/
New%20Biorepository%20PR%20
22016%20-%20New%20
Biorepository%20Expands%20
Services%20Offers%20Absolute%20
Sample%20Protection.pdf, accessed
Feb. 16, 2016.
4. Catalent, “Catalent Invests $4.6M to
Further Expand Asia-Pacific Clinical
Trials Hub in Singapore,” Press
Release, Feb. 2, 2016, www.catalent.
com/index.php/news-events/news/
Catalent-Invests-4.6M-To-Further-
Expand-Asia-Pacific-Clinical-Trials-Hub-
In-Singapore, accessed Feb. 16, 2016.
5. SGS, “SGS Announces Expansion and
Integration of Chemistry &
Microbiology Testing Offer Following
Canadian Acquisition,”Press Release,
Jan. 19, 2016, www.sgs.com/en/
news/2016/01/sgs-announces-
expansion-and-integration-of-
chemistry-and-microbiology-testing,
accessed Feb. 16, 2016.
6. Vetter, “Vetter Announces Completion
of Multi-Functional Building for
Development Service and State-of-the-
Art IT,” Press Release, Jan. 28, 2016,
www.vetter-pharma.com/en/
newsroom/vetter-news/news-l-vetter-
announces-completion-of-multi-
functional-building-for-development-
service-and-state-of-the-art-it,
accessed Feb. 16, 2016. 7. Novasep, “Novasep Adds Synthesis
and Kilo Lab Extensions at US Facility,” Press Release, www.novasep.com/home/about-novasep/media-events/press-release/novasep-adds-synthesis-and-kilo-lab-extensions-to-us-facility.html, accessed Feb. 16, 2016.
8. PBOA, “PBOA Welcomes New Members and Trustees,” Press Release, Feb. 10, 2016, www.pharma-bio.org/news/pboa-welcomes-new-members-and-trustees/, accessed Feb. 16, 2013. ◆
For US inquiries please contact [email protected] Q For Asia Pacifi c inquiries, please contact
infoAsiaPacifi [email protected] Q For EU and other international inquiries, please contact [email protected]
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Although many of the newest
methods to optimize process
economics focus squarely on
the implementation of single-
use systems and the realization of contin-
uous processing, there are other methods
of cost cutting that could help a biopro-
cess become more sustainable. Operators
should look to methods to optimize the
use of solvents, get the most from their
resins, explore hybrid approaches and
miniature bioreactors for improved pro-
cess understanding, and investigate alter-
natives to mammalian vectors to improve
cost calculations.
OPTIMIZATION OF MEDIA VOLUMESResin prices can range from low single-
digit thousands of dollars per liter for ion
exchangers to tens of thousands of dol-
lars per liter for affinity resins, estimates
Kevin Isett, CEO and founder of Avitide,
which makes high- affinity resins. The
costs associated with chromatography
resins can significantly contribute to
overall manufacturing costs, especially
if two or three chromatographic steps
are required in a bioprocess, according
to Alex Xenopoulos, principal research
scientist at Mil l iporeSigma. Isett
explains that resin manufacturers his-
torically demanded a “premium” price
for resins that could withstand rigor-
ous cleaning and sanitation conditions
and facilitated a better unit economy
by allowing manufacturers to use less
expensive sanitization buffers. According
to Cobra Biologics’ Technical Director
Tony Hitchcock, cost has to be delicately
balanced with overall purity achieved,
Achieving Cost-Effective Bioprocesses
Randi Hernandez
Experts in the field share
some best practices for optimizing
process economics
in bio-manufacturing.
Bioprocessing
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binding capacities, and the num-
ber of cycles that can be performed
with the resin.
One strategy to lower the over-
all cost of bioprocessing is to mini-
mize resin-related costs. “For a
large commercial process produc-
ing one ton of antibody per year,
chromatographic resins contribute
about 10% of the downstream cost,
while filters contribute up to 30%,
because of the single-use nature
of depth, virus, and sterile filters,”
notes Xenopoulos. “For a smaller,
single-use process producing 50 kg
of antibody per year, the percentage
of cost contributed by chromato-
graphic resins goes down to a few
percentage points, because capital
and labor now constitute a larger
fraction of the total cost.”
Rathore et al. estimate that in a
typical monoclonal antibody (mAb)
platform process, 60% of down-
stream costs come from chroma-
tography (1); the cost of a typical
Protein A resin is 50% higher than
the cost of traditional chromato-
graphic media. Although Gjoka et
al. agree that economic optimiza-
tion of chromatography is critical,
these authors estimate that Protein
A resin is closer to “an order of mag-
nitude more expensive than other
non- affinity sorbents (including
most ion exchangers)” (2).
The switch to single-use systems
has been highly cited as a way to
reduce costs in biomanufacturing.
Although Xenopoulos mentions
that the use of disposables generally
increases the consumable compo-
nent of the cost of bioprocessing,
single-use chromatography devices,
such as prepacked columns and
membrane adsorbers, could offer
overall “cost advantages because of
the elimination of column pack-
ing and media washing and stor-
age steps.” Hitchcock points out
that at smaller processing volumes,
single-use products can reduce the
amount of liquids that are typically
used in cleaning operations.
Resin reuse
Cycling of resins can occur, and
resin reuse typically lowers the
cost burden of a process. A resin
can be used for up to 200–300
cycles, and smaller columns are
typically used to facilitate resin
reuse. Validating even more reuses
of resins can reduce costs further,
says Xenopoulos, although he says
that the benefit above 50 reuses is
quite small and the cost of valida-
tion should be taken into account.
Not all resins have the same num-
ber of reuses, adds Isett: “Ligand sta-
bility, resin matrix, and chemistries
employed to immobilize the ligands
are key determinants to the reusabil-
ity of any bioprocess resin.”
Reusing resin, although poten-
tially cost-effective, increases
total purification time, decreasing
throughput (1). Long-term use of a
resin has also been associated with
a resin’s decreased efficiency in
terms of product recovery as a result
of resin fouling, ligand degradation,
or reduction in pore surface area.
Using a simple depth filter before
loading, notes Xenopoulos, can
reduce the incidence of resin foul-
ing and maximize the number of
potential resin reuses.
Improving resin selectivity
Improving resin selectivity could
potentially help operators eliminate
a downstream chromatographic
step entirely, says Xenopoulos, or at
the least, reduce the load on down-
stream steps. It’s a double-edged
sword, however: although selecting
a resin with a high binding capac-
ity can limit the amount of resin
used, “such high-performance res-
ins usually have a higher per-liter
cost,” asserts Xenopoulos. Newer
separation technologies may play
a major role in improving process
economics, notes Isett. “Identifying
highly selective, product-specific
resins can enable reduced purifica-
tion unit operations, afford efficient
step-elution schemes, and permit
flow-through polish applications,
which will ultimately have the larg-
est beneficial impact on resin usage
and media/buffer volume costs in
batch and continuous downstream
processing.”
Finally, other small changes in
a bioprocess (e.g., proper selec-
tion of wash and elution buffers to
maximize product recovery, opti-
mizing solution conditions such
as dilution or pH changes, using
f low-through conditions, and
potentially, overloading a column
beyond nominal binding capacity)
can reduce resin volume and could
possibly improve the performance
of some of the chromatographic
steps, says Xenopoulos.
Additionally, some manufactur-
ers use in-line mixing and dilution
of buffer components, which could
help reduce the amount of media
used in downstream processing,
notes Benoit Mothes, scientific and
innovation downstream processing
head at Sanofi in France. Mothes says
that regarding buffers, adoption of
“in-line dilution and in-line concen-
tration will be the next improvement
to reduce the amount of media in
downstream processing.”
Continuous operations
Harnessing continuous downstream
operations has been cited as a good
way to both improve the utiliza-
tion of chromatography resins and
decrease the demand on filters (3,
4). Multi-column systems, in par-
ticular, allow resins to enjoy the lon-
gest lifespan (3). Adds Xenopoulos,
“Continuous, multi-column chroma-
tography has certainly been shown
to reduce resin volume used by sig-
nificant amounts, up to 80%. The
benefits depend on factors such
as product titer, batch time, and
cycling schedule and are higher
when the resin is reused multiple
times during cycling.”
Klutz et al. found that in upstream
operations for the manufacture of
mAbs, however, continuous pro-
Bioprocessing
March 2016 www.biopharminternational.com BioPharm International 17
Although not all products normally produced in mammalian
cells can be produced in microbial vectors—as the
glycosylation step necessary for antibody-dependent
cell-mediated cytotoxicity does not occur in microbial
cells—fragments of monoclonal antibodies (mAbs) can
successfully be produced in microbial vectors, such as
Saccharomyces cerevisiae, Pichia pastoris, and Escherichia
coli (1). Although certain post-translational modifications
such as glycosylation don’t currently occur in microbial
models, a few glycoengineering experiments with the genes
in P. pastoris vectors have focused on the humanization of
the N-glycosylation pathway (2, 3). If successful, this gene-
editing venture would allow for the production of full-length,
glycosylated mAbs in P. pastoris (2).
The use of microbial vectors offers manufacturers a way
to avoid costly perfusion operations and hard-to-model
mammalian cell metabolism conditions (2). Mammalian cells
are also highly susceptible to shear stress and are typically
associated with low product yields, risk of viral contamination,
and a requirement for animal-based serum (2).
Microbial models, on the other hand, have historically
been associated with low cultivation costs (2), low cell-
culture media costs, and high yields. Rathore points out that
although some microbial vectors (e.g., E. coli) contain some
endogenous proteases that can cause protein degradation,
the use of protease-free strains and secretion of proteins
in the periplasm—where there are fewer proteases—have
been methods used to account for potential degradation
issues. In contrast with more traditional microbial models
that resulted in inclusion bodies—which produced significant
host-cell impurities—newer microbial fermentations
produce “purer product compared with mammalian cell
culture,” says Alex Xenopoulos, principal research scientist at
MilliporeSigma. Still, he says, secreted systems “suffer from
low productivity compared with inclusion body systems.” If
the biological product is not secreted, says Kevin Isett, CEO
and founder of Avitide, then “the feed streams can contain
considerable higher host-cell protein levels when compared
to mammalian or insect feed streams,” which can put a
“significant strain on downstream purification operations,
particularly the capture step.”
Yeast vectors are beneficial in that they have high yields
and are capable of post-translational modifications. There
are many ways to optimize microbial vectors through
glycoengineering so that they produce proteins that function
properly, according to experts in the field (1, 2).
Despite the fact that microbial models offer some
exciting production alternatives that could slash costs,
Xenopoulos believes that the ability for mammalian cells to
produce proteins with complex secondary structures, their
continuously increasing titers, and the fact that they are
already well understood outweighs all of the benefits of
microbial models. Ultimately, concludes Xenopoulos, “the
selection of a production system really depends on the
structure of the desired protein, with cost being secondary
considerations.” Summarizes Cobra Biologics’ Technical
Director Tony Hitchcock, “ The perceived advantage of
microbial systems are around costs of fermentation media
and processing times, and this has to be balanced out
against more complex and capitally intensive recovery
operations.”
References1. A. Rathore and J. Batra, BioPharm Int. 29 (2),
pp. 18–23(February 2016).2. O. Spadiut et al., Trends Biotechnol. 32 (1),
pp. 54–60(January 2014).3. J.L. Corchero et al., Biotechnol. Adv. 31, pp. 140–153 (2013).
– Randi Hernandez
Bioprocessing
Microbial Vectors for the Production of mAbs
cesses use more fermentation
media for perfusion than did fed-
batch operations (84 pounds per
grams mAb vs. 59 pounds per
grams mAb, respectively), making it
more expensive to use continuous
processes upstream. Downstream
continuous operations, neverthe-
less, were more efficient from a cost
perspective—which the authors
attributed to better utilization of
chromatography resins in down-
stream operations. Plus, using fed-
batch operations upstream instead
of continuous perfusion did not
affect overall yield, according to
Klutz and his colleagues: “In all
cases, fed-batch fermentation is
more cost efficient than the perfu-
sion fermentation at the same level
of cell-specific productivity.” Klutz
et al. concluded that a hybrid
approach—consisting of fed-batch
operations upstream and continuous
chromatography downstream—was
the most cost-effective model, corre-
sponding to a 15% reduction in cost
of goods (CoGs) (3).
As a whole, it appears a hybrid
approach uses the least amount of
media. Although hybrid methods
“lack the elegance of completely con-
tinuous templates,” according to
Xenopoulos, they do address cost
pressures related to high cell-culture
media usage.
18 BioPharm International www.biopharminternational.com March 2016
Bioprocessing
Although the aforementioned
study by Klutz el al. did not find
an increase in productivity asso-
ciated with continuous perfusion,
other experts in the field say that
the productivity increase out-
weighs the amount of media con-
sumption in continuous upstream
operations. “Continuous process-
ing provides clear advantages in
terms of space-time-yields,” says
Christel Fenge, vice-president of
marketing and product manage-
ment, fermentat ion technol-
ogy, Sartorius Stedim Biotech.
In other words, “the amount of
material produced per unit time
and volume is higher compared
with conventional fed-batch pro-
cesses.” Xenopoulos concurs that
while continuous perfusion uses
more media—which he estimates
can total 1–10 bioreactor volumes
per day—he says the increased
cost is “somewhat mitigated” by
the increased productivity, as mea-
sured by the cost per gram of prod-
uct. Scott Waniger, vice-president
of bioservices at the Cell Culture
Company, also asserts that gener-
ating greater production “makes
up for the additional raw material
consumption of relatively inexpen-
sive liquid tissue culture media.”
To control overall costs, “there
is also an effort to reduce the cost
of the media itself, with develop-
ment efforts directly targeted to
perfusion cell culture media,” notes
Xenopoulos. “Biomanufacturers
usually work with media vendors,
but sometimes invest in internal
development of media, showing
the importance of this aspect.” In
an attempt to reduce cell-culture
media volume used, some compa-
nies opt to use richer media, which
Xenopoulos says can unfortunately
also increase media cost per liter.
An additional strategy to optimize
media consumption, says Waniger,
is to perform an offline analysis
of spent media to characterize
the consumption and production
of media elements. “The resulting
information allows for identifica-
tion of the components that are
limited, and have been consumed
by the cell line,” Waniger articu-
lates. “With this knowledge, the
operator can add concentrated
amounts of nutrients as a supple-
ment to replace any specific ones
that have been exhausted.”
Buffer recycling
Jungbauer and Walch write that
buffer recycling positively impacts
process economics (5). The reuse
of buffers could decrease waste
streams and save money that
goes into wastewater treatment
efforts. The authors suggest the
use of multi-column separations,
integrated continuous counter-
current chromatography, and
countercurrent tangential f low
chromatography as effective ways
in which buffers can be recycled.
They note that continuous pro-
cesses and the introduction of filtra-
tion into chromatography systems
would allow for a substantial reduc-
tion in solvent consumption.
Unlike resins and other solvents,
expert consensus is that buffers
don’t significantly contribute to
overall biopharmaceutical manu-
facturing costs. “Cost savings of
recycling would most probably
be countered by the cost of the
recycling equipment and by the
need to validate and test the recy-
cled buffers,” notes Xenopoulos.
“Buffer recycling could address
environmental concerns of dis-
posal, especially if a particularly
exotic buffer is used.” Hitchcock
adds that the operation and vali-
dation challenges associated with
buffer recycling may not make it
“ worthwhile for a majority of pro-
duction processes.”
Rather than focus on salvag-
ing buffers, Sanofi’s Accelerated
Seamless Antibody Purification
(ASAP) platform focuses on avoid-
ing the use of nonvaluable buffers
as a technique to reduce down-
stream purification costs. “Most
of purification processes include
a minimum of three chromato-
graphic steps made in a sequence
of distinct unit operations,” says
Mothes, who runs the ASAP pro-
gram in France. He says that unit
operations cannot normally be
operated in a continuous mode, “as
adjustment of pH, molarity, and
protein concentration are neces-
sary between each chromato-
graphic or filtration step.” The
ASAP platform, however, elimi-
nates the need to perform what
he calls these “non-added-value
unit operations” and shortens pro-
cess cycle time to less than three
hours. Use of the system facilitates
a reduction in the buffer volumes
that are necessary for processing,
notes Mothes. While a typical
mAb purification relies on a total
of nine buffers, the ASAP model
requires only four. Mothes adds
that Sanofi’s process will “provide
an entirely purified mAb in a few
hours while reducing the volume
of resin used” and will enable
the reduction of buffer volumes
because of the platform’s small col-
umns and singular process skid.
OTHER FACTORS THAT INFLUENCE COSTAdditional costs related to biophar-
maceutical manufacturing can be
tied to events that occur after an
actual product is manufactured.
These factors can relate to drug
safety, speed to clinic, and time to
market, notes Fenge. Hitchcock esti-
mates that 80% or more of costs
are locked up in the manufactur-
ing design of a product; therefore,
“understanding the implications of
choices within the development
phase” and manufacturing pro-
cesses is key. He adds, “Once these
choices have been made, there is
often only a limited amount of
cost reductions that can really be
achieved.”
March 2016 www.biopharminternational.com BioPharm International 19
Bioprocessing
Miniature Bioreactors for Improved Process Understanding
Better process understanding has the potential to reduce
cost, and this is increasingly being achieved through the
use of miniature and microbioreactor systems. Christel
Fenge, vice-president of marketing and product management,
fermentation technology, Sartorius Stedim Biotech says
the miniature systems and their corresponding single-use
vessels “more precisely mimic larger-scale bioreactors” in
terms of stirring, gas, and pH control. Citing work by Lewis et
al. (1), Fenge comments that “these systems more accurately
predict cell-line productivity at larger scale, ensuring that
high-productivity cell lines (that also work at scale) are
selected at an early stage.” Multiparallel systems that
incorporate automated feeding and sampling have also driven
process efficiencies and allowed for speedier clone selection
and initial parameter selection, Fenge adds. “In combination,
these micro- and mini-bioreactor systems provide efficiency
gains as a result of automated high-throughput cell line,
media, and process development that can enable a faster
throughput of pipeline projects and allow larger experiment
designs, leading to a wider process understanding.”
Process efficiencies equal cost savings, says Cobra
Biologics’ Technical Director Tony Hitchcock, as reduced
development times, improved productivity, and enhanced
characterization time allow products to be brought to
clinical stages and the market more quickly. Scott Waniger,
vice-president of bioservices at the Cell Culture Company
stresses that cost-savings also come in the form of a
decrease in validation efforts. “As long as the miniature
bioreactors are scalable, the savings obtained from reduced
validation as the process scales up are drastically high,”
Waniger notes. “Using a system in which the cell-occupied
space does not change in form, fit, and function—and can
be scaled up with the insertion of more cell spaces in a
parallel manner—helps generate data that can be directly
extrapolated from small to large scale and prevents costly
and time-consuming revalidation at each step.”
Reference1. G. Lewis et al., Bioprocess J. 9 (1), pp. 22–25 (2010).
– Randi Hernandez
Fenge mentions that incorpo-
rating a fully integrated upstream
platform early—combined with a
similar downstream strategy—can
help reduce development-related
costs. “By selecting tools that are
efficient in their own right but that
have also been designed to work
together can significantly reduce
the resources and time needed to
develop processes and can ensure
low cost of goods in manufactur-
ing with reliable high productiv-
ity and consistent product quality.
For example, by deciding to use an
expression platform with a track
record of high-productivity cell
lines that has a proven performance
from small- to large-scale bioreac-
tors, the risk of delays and high
costs associated with determining
optimum process parameters and
control strategies are considerably
reduced, and a rapid path into [the]
clinic is provided.”
Costs re lated to outsourc-
ing must also be managed, and
Waniger suggests taking extra mea-
sures to ensure that the technol-
ogy transfer, method performance,
and manufacturing process are all
addressed in the original request
for proposal (RFP). “CDMOs [con-
tract and development manu-
factur ing organizat ions] may
unintentionally add costs to proj-
ects where the RFP does not fully
describe the needs of the manu-
facturing process,” Waniger says.
“Ultimately, these costs are passed
on to the patient.” Waniger also
suggests conduct ing compre-
hensive stability studies on the
final product, which he says will
maximize product shelf life and
reduce the frequency of batch pro-
duction.
“The pace of discovering novel
drugs and engineering highly
p ro duc t ive b iopha r mace ut i -
cal production systems, in large
par t, has outpaced the eng i-
neer ing of high-per formance
separation ligands and resins,”
concludes Isett, who bel ieves
that the industry should put an
increased focus on optimizing
downstream operations to make
biopharmaceutical manufactur-
ing more cost-effective overall.
“This is particularly true for vac-
cine and biosimiliar companies,
where sensitivities to manufac-
turing costs/pressures are more
pronounced.”
REFERENCES 1. A. Rathore et al., BioPharm Int. 28 (3),
pp. 28–33 (March 2015).
2. X. Gjoka et al., J. of Chrom. A 1416, pp.
38–46 (Oct. 16, 2015).
3. S. Klutz et al., Chem. Eng. Sci. 141, pp.
63–74 (2016).
4. A. Xenopoulos, J. Biotechnol. 213, pp.
42–53 (2015).
5. A. Jungbauer and N. Walch, Curr. Opin.
Chem. Eng. 10, pp. 1–7 (2015). ◆
20 BioPharm International www.biopharminternational.com March 2016
Ted H
oro
witz/F
use/G
ett
y Im
ag
es
Raw material variability can
have impacts across the board,
causing inconsistent processes
and process yields, produc-
tivity/efficiency issues, and problems
with quality compliance. Any one of
these problems can ultimately impact
drug efficacy and patient safety. While
increased outsourcing of raw material
manufacturing to emerging regions can
contribute to greater issues regarding
raw material consistency and trace-
ability, overall raw material quality
and reliability has increased in recent
years. At the same time, the sensitiv-
ity of analytical instruments and the
awareness of the impact of raw material
variability on biologic drugs have also
increased, leading to a need for further
definition. Raw material suppliers and
biopharmaceutical manufacturers both
have roles to play in addressing this
issue. The key to success will be open
and transparent communication.
MANIFOLD AND SIGNIFICANT IMPACTSVariability in raw materials from media
to packaging can have an impact on the
characteristics and quality of drug prod-
ucts that potentially impact safety and
efficacy. More specifically, lot-to-lot vari-
ability of raw materials may impact drug
product critical quality attributes (CQAs)
such as identity, purity, quality, and sta-
bility and cause lot-to-lot variability of
the drug product during its lifespan,
according to Nataliya Afonina, presi-
dent of AN Biologics Consulting. “Such
variability may ultimately lead to out-
Biopharma Takes On Raw Material Variability
Cynthia A. Challener
Collaborative efforts are underway between suppliers
and drug manufacturers.
Cynthia A. Challener, PhD,
is a contributing editor to
BioPharm International.
Upstream Processing
PharmaGrade™ RAW MATERIALS FOR COMMERCIAL USE
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PharmaGrade is a trademark of Sigma-Aldrich Co. LLC. Sigma-Aldrich Corp. is a subsidiary of Merck KGaA, Darmstadt, Germany.
CONFIDENCE
SUPPLY
SAFC’s PharmaGrade Raw Materials are designed to meet customer needs and regulatory guidances
for use in commercial biopharma processing. Each product has full supply chain transparency and easy
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22 BioPharm International www.biopharminternational.com March 2016
of-specification (OOS) results for
drug products and, in addition to
impacting patient safety, affect the
drug product clinical or commer-
cial supply chain and regulatory
submissions, which are all signifi-
cant consequences,” she asserts.
“The impact of raw material
variability can indeed be mani-
fold and very significant,” agrees
Raphael Gübeli, technical product
manager for liquid formulation at
Merck KGaA. Some effects, such
as reduced overall yield of the bio-
pharmaceutical manufacturing
process, mainly have a negative
economic impact. Unexpected
raw material impurities can, in
some cases, chemically alter the
drug substance or remain in the
final formulated drug product at
elevated levels, potentially affect-
ing efficacy and safety. “If not
detected during final release test-
ing by the manufacturer, there
could be harmful consequences for
the patient,” Gübeli says.
In some cases, minute quantities
of an impurity can have a measur-
able effect. “For example, increas-
ingly more information is being
gained on the effect of variable
elemental metal impurities on cell
health and the yield of cell-culture
processes. As more knowledge is
gained, it has become clear that
even at extremely low levels, a pro-
cess can still be impacted by the
presence of metal or other impuri-
ties,” observes Gary Perkins, head
of process solutions customer rela-
tions in the quality services group
of MilliporeSigma, the life-science
business of Merck KGaA.
Consequently, all raw materials
used to manufacture the API, plus
any excipients and other materials
used during formulation, must be
fully characterized and monitored
for changes in quality/properties.
Packaging materials must also be
monitored, as unexpected leach-
ables or other impurities can also
have an effect on product quality.
BETTER QUALITY, BUT GREATER SENSITIVITYConcerns raised about raw mate-
rial variability are not necessarily
due to a decline in raw material
quality, say the experts inter-
viewed for this article. There have
been some issues with increasing
raw material variability due to out-
sourcing of raw material manu-
facture to China, India, and other
developing countries and the use
of repackagers, which can reduce
transparency in the supply chain,
according to Afonina. She notes
that audits of even reputable ven-
dors must encompass the complete
chain of suppliers and repackagers.
In general, raw material vari-
ability hasn’t changed in recent
years, but analytical capabili-
ties have improved significantly,
increasing the ability of both sup-
pliers and drug manufacturers to
analyze, characterize, and under-
stand that variability, according
to Perkins. These improved capa-
bilities are a double-edged sword.
“We encounter more variability as
our ability to measure and analyze
it is enhanced. In fact, variabil-
ity that was previously invisible
can now be identified, pushing
manufacturers to further stream-
line processes to mitigate potential
impacts of even the slightest varia-
tions,” Perkins explains. He adds
that as detection limits continue
to reach ever lower levels, the
challenge becomes standardiza-
tion, as it often does in the phar-
maceutical industry.
It is also important to note,
according to Gübeli, that expec-
tations for raw material consis-
tency and control of processes
and final product quality have
increased dramatically, and thus
the variability has been reduced
in many cases. Parag Kolhe, group
leader and senior principal sci-
entist with Pfizer Biotherapeutic
Pharmaceutical Sciences, agrees
that raw-material suppliers are pro-
viding consistently better quality
materials. He provides one example
in the primary packaging space;
prefilled-syringe manufacturers
have acknowledged the sensitivity
of biotech products toward silicone
oil and tungsten and improved
their processes so that their pri-
mary packaging component specs
are more tightly controlled.
Raw materials such as formula-
tion excipients, however, which are
present in drug products in high
amounts, have received much less
attention than active biopharma-
ceutical ingredients, according to
Gübeli. “Many parameters defining
the quality aspects of excipients
are not yet routinely monitored or
included as part of supplier certifi-
cates of analysis, and thus are not
strictly under control,” he states.
MANY SOURCES OF VARIABILITYExcipients are just one of many
potential sources of raw material
variability that can impact bio-
pharmaceutical manufacturing. In
general, variability can arise from
inefficient/ineffective raw material
manufacturing controls and resid-
ual impurity controls in starting
materials. They can come from the
basic starting materials (e.g., nat-
ural sugars and other plant-based
compounds) used to manufacture
biopharmaceutical raw materials or
Upstream Processing
In general,
raw material variability
hasn’t changed in
recent years, but
analytical capabilities
have improved
significantly.
March 2016 www.biopharminternational.com BioPharm International 23
Upstream Processing
be an artifact of the manufacturing process itself (e.g.,
impurities in recycled solvents, chemicals released into
the raw material process fluid from the equipment, etc.).
The most common types of raw material variability
can be placed into three general categories, accord-
ing to Gübeli. The first group includes trace impuri-
ties that alter the biopharmaceutical API, either by
directly modifying it or by catalyzing its modification.
Examples include peroxides, aldehydes, reducing sug-
ars, and catalytically active metal ions. The second
category comprises trace impurities that are them-
selves toxic to humans, such as lead and aluminum.
The third class consists of microorganism contami-
nants (and their associated endotoxins) that lead to
variabilities in the bioburden of raw materials and can
cause severe immunological responses in patients.
As mentioned previously, raw material variability
often occurs due to inadequate control of raw mate-
rial manufacturing processes and/or analytical release
testing by suppliers, and is most common at small
outsourcing companies, according to Afonina. Specific
problems to watch for include contamination with
antibiotics or other foreign raw materials due to a lack
of appropriate segregation of processing/handling facil-
ities, equipment, or control of processes used for the
manufacture of raw materials; poor GMP and analyti-
cal practices resulting in the approval of out-of-specifi-
cation material; switching of suppliers for a given raw
material; and deficiencies in the auditing of raw mate-
rials suppliers by biopharmaceutical manufacturers.
SUPPLIER RESPONSIBILITIESVendors are responsible for controlling/minimizing
raw material variability, according to Kolhe. They must
control their own manufacturing processes and audit
manufacturers of any raw materials they purchase. If
manufacture of these materials is outsourced, they need
to audit all facilities in the supply chain. If changes are
made to manufacturing processes, comparability stud-
ies must be performed and the customer notified. “All
these actions are important for all raw materials, but
specifically for animal- and plant-derived materials for
which properties are difficult to control,” says Afonina.
“Overall,” she adds, “strong risk-based management
and quality systems should be in place.”
Even repackagers must have the ability to trace spe-
cific raw material lots back to any changes that were
made at their suppliers. “If a client asks a supplier for
more information in order to investigate batch-to-
batch variability in raw material quality, the supplier
should be able to provide supply-chain documenta-
tion, including origins and analysis. If the answers
are not there, more analysis should be done on the
material,” Perkins says. All these key points should
be covered in the quality agreement between the raw
material vendor and the drug product manufacturer,
according to Afonina.
To best aid manufacturers as they deal with raw
material challenges, suppliers can, in addition to
meeting specifications of pharmacopoeia mono-
graphs, provide in-depth raw material characteriza-
tion data including customer-specific parameters and
historical data that can be used for the prediction of
future batch-to-batch consistency and information on
appropriate handling and storage conditions, accord-
ing to Gübeli. Trust can also be built by assuring
independent supplier auditing and certification, such
as is offered through the EXCiPACT voluntary interna-
tional certification scheme for excipients.
Most manufacturers select raw materials that are
excipient-grade or of similar quality due to the fact that
the raw material or its impurities have the potential to
be transferred to the final formulated biopharmaceuti-
cal product. For raw materials classified as excipients
(e.g., according to US or European pharmacopoeia), the
most important types of variability are part of the phar-
macopoeia monographs and are controlled and speci-
fied in the certificate of analysis (CoA) by the supplier,
according to Gübeli. Unfortunately, he also notes that
the pharmacopoeia monographs generally lag behind
24 BioPharm International www.biopharminternational.com March 2016
state-of-the-art knowledge about
raw material variability.
COMMUNICATION IS KEYThere is, however, a shared respon-
sibility between the vendor and
the client. “Clients need to be
aware of their needs with respect
to factors that are critical to the
performance of their processes and
make sure that suppliers under-
stand these factors,” Perkins states.
Biopharmaceutical companies also
need to understand if the typi-
cal variability in a raw material
is acceptable for a given process/
product or if more controls are
needed, according to Kolhe.
In general, it’s the responsibility
of the manufacturer of a biophar-
maceutical to select the appropri-
ate raw material that fulfills the
requirements for the production
of a safe and high-quality bio-
pharmaceutical under GMP. “Each
biopharmaceutical and associated
manufacturing process is unique
and influenced by many raw-mate-
rial associated factors to a different
extent. These phenomena are only
known by the manufacturers of
the biopharmaceuticals. Therefore,
we encourage manufacturers to
cooperate with us as suppliers and
exchange information on critical
raw material parameters. Only then
can we as a supplier assure consis-
tency of these particular param-
eters by introducing them into
custom CoAs,” Gübeli comments.
Biopharmaceutical manufactur-
ers need to implement raw-material
management strategies, including
processes/systems for the release
of raw materials for use based on
specifications, quality agreements,
routine audits, and raw-material
change communications, according
to Kolhe. Drug product manufac-
turers should also have segregated
areas for receiving/handling raw
materials to avoid the potential for
contamination from other areas in
of the manufacturing plant and
have a system in place to moni-
tor trends in the analytical data,
according to Afonina.
Many drug companies are tak-
ing risk-based and science-based
approaches to managing raw mate-
rial variability. Such approaches
require significant product under-
standing regarding the impact of
raw-material attribute variability.
“Formal risk assessment exercises
are conducted to evaluate the poten-
tial risks associated with raw mate-
rial availability with respect to final
product quality, safety, and efficacy.
The obtained results are then used to
understand the risks and determine
any actions needed in terms of raw-
material quality attributes,” Kolhe
explains. Integration of control strat-
egies for final drug substances and
drug products with raw-material
control measures also help ensure
consistent product quality.
“One of the issues here is that
there is no standard or specific guid-
ance on the management of raw
material variability,” says Perkins.
“In addition, the patchwork of reg-
ulatory guidance documents that
are applicable make references
to science-based and risk-based
approaches to the minimization
of raw material variability, but
most are open to interpretation.”
MilliporeSigma and other compa-
nies, as participants in initiatives
of associations like the BioPhorum
Operations Group, are opening up
lines of communication, even across
competitors. “A consistent indus-
try approach to variability analy-
sis will enable greater supply chain
transparency, standardized process
control, and key materials charac-
terization,” Perkins asserts. “The
general idea is that manufacturers
would be accountable for a set of
clear material definitions, so cus-
tomers know that they are choosing
the right raw materials,” he adds.
Transparency between a buyer
and supplier is key so that man-
ufacturers can understand and
address variability issues with the
potential to affect product quality
and patient safety. “If the buyer
provides information on the end
use of a material, the supplier
can then determine what infor-
mation they need to know about
where the material is coming from
(if repackaging) or about key raw
materials needed for its manu-
facture, as well as what analyses
may be necessary,” states Perkins.
Suppliers can in turn assist bio-
pharmaceutical customers with
risk assessment processes by pro-
viding streamlined documentation
involving detailed raw material
processing and characterization
information, according to Gübeli.
He adds that drug manufacturers
will have the greatest success in
obtaining raw materials that meet
their detailed demands if they
begin interacting with suppliers
early in the development process.
MilliporeSigma is also work-
ing with customers and indus-
try groups to develop an eData
exchange format. The system will
enable secure electronic sharing of
comprehensive raw material pro-
duction and test data. Not only
are the data priceless; multi-variant
analysis techniques provide a bet-
ter understanding of variability
and its impact on final drug prod-
ucts, according to Perkins. X
Upstream Processing
Transparency
between a buyer
and supplier is key so
that manufacturers
can understand and
address variability
issues.
April 26-28, 2016Javits Center | New York City
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26 BioPharm International www.biopharminternational.com March 2016
And
rew
Bro
okes/G
ett
y Im
ag
es
Cell culture is widely employed
in biomedical applications and
has numerous applications,
spanning from diagnosis, ther-
apy, and the production of biological
drugs. Cells used for biopharmaceuti-
cal applications are mainly of animal
origin and can be cultured in suspen-
sion or adherent cell cultures. A unique
characteristic of adherent cells is their
dependence on an anchorage surface
on which to attach to exert their nor-
mal metabolic activity and prolifer-
ate. While cell attachment may prove
advantageous for some purposes, such
as supernatant harvesting, cell detach-
ment may become an enormous chal-
lenge when cells are to be harvested.
Cell detachment is probably one of the
most relevant processes hindering a
faster development for cell-based bio-
technology. In this article, the authors
focus on different strategies available for
animal adherent cell detachment.
Animal adherent cell cultures are
derived either from tissue explants
or from cell suspensions. In a stan-
dard culture process, once cells have
attached to the culture support, they
undergo a lag phase and then start
Adherent Cell Culture in Biopharmaceutical Applications: The Cell-Detachment Challenge
Marcos Simon and
Juan J. Giner-Casares
The necessity to detach cells from a culture
substrate during cell harvesting
remains one of the most challenging
steps in a cell-culture
process.
Marcos Simon, PhD, is founder of The
Bolt-on Bioreactor project and Technology
Transfer Manager at CIC biomaGUNE.
Juan J. Giner-Casares, PhD, has
been a postdoctoral researcher in
the BioNanoPlasmonics Lab led by
Luis Liz-Marzán at CIC biomaGUNE.
Downstream Processing
March 2016 www.biopharminternational.com BioPharm International 27
growing exponentially at a high
metabolic activity, until conflu-
ency is reached. Then, growth
stops and the culture reaches a
stationary phase. Most often, cells
are harvested when population
density suppresses growth. The
on-demand release of cultured
cells, however, would be greatly
beneficial for certain biomedical
applications in which a certain
development step of the living
cells is pursued, rather than sim-
ply the pursuit of a large num-
ber of collected cells. This release
should be efficient and triggered
by a simple stimulus.
CHOICE OF CELL-DETACHMENT METHOD: FACTORS TO CONSIDERCell behavior and the degree of
adhesion of cells to the culture sup-
port are greatly affected by several
culture parameters. However, adhe-
sion degree is not the only factor
to be considered when choosing
the most convenient cell detaching
method within a given cell-culture
process. The authors consider dif-
ferent factors and requirements that
influence the choice of the right
cell-detachment method.
Degree of cell adhesion
Vigorous cell-culture processes,
such as culture on microcarriers,
require strong cell adhesion that
needs be matched by the harshness
of the detaching step. On the other
hand, cell culture on t-flasks is
more suitable for cells that establish
weak interactions with the support.
Further use of detached cells
When subsequent reattachment
is expected from detached cells
(e.g., in a subpassage step during
cell expansion or for therapeutic
cells production), cell viability
and membrane component integ-
rity are more important than in
the case of cell harvesting for fur-
ther preparation of cell extracts.
Similarly, when a cell sheet is to
be obtained, cell-to-cell interac-
tions must remain intact, but not
when cell suspension is the target
product. In addition to physical
integrity of the cells, an optimal
functionality/metabolic state of
such cells is also desirable.
Process compatibility
Some chemicals necessary for par-
ticular cell-detachment procedures
may interfere with subsequent
downstream processing steps that
are unaffected by other alternative
detachment methods. Therefore,
detachment based on physical
processes is certainly preferable
to chemical treatments, and help
mitigate concerns related to the
impact of process additives on a
final product.
Culture support
Cell-detaching methods where
direct access to the support is nec-
essary, such as cell scraping, are
not compatible with cell-culture
supports such as microcarriers or
hollow fibers.
Process scale
Manual cell-detachment tech-
niques such as cell scraping may
be adequate at laboratory scale but
unfeasible at industrial scale due to
the laboriousness of the method.
Reusability of
culture substrates
Some re spons ive subst rate s
designed to promote cell detach-
ment under the effect of a particu-
lar stimulus undergo an irreversible
structural modification that pre-
vents repeated cell culture on the
same substrate.
Regulatory constraints
Reg u lator y rest r ic t ions may
exclude some cell-detachment
methods from consideration in
particular applications or require
extensive validation before autho-
rization of use is granted.
Spatial resolution
Applications such as co-culture of
different cells in a given geometri-
cal pattern or single-cell harvesting
require the ability to detach cells
only from selected areas without
affecting cells growing in other
areas of the support.
Temporal resolution
Some stimuli used to promote cell
detachment are deleterious for
cells. In these cases, it is important
to control the temporal resolution
of the cell-detachment technique.
Compatibility with
sterilization methods
In case sterilization of the cell culture
device is necessary prior to use, care
should be taken to ensure that the
sterilization method does not affect
the characteristics of the cell detach-
ment technique (e.g., when using
supports with modified surfaces).
Shelf-life
Cell-culture devices are often pro-
vided as ready-to-use devices that
are stored for a long period of time
before use. Some components of
chemical and biological origin
contained in sophisticated cell-
detachment systems are unstable
and have a short shelf-life.
Production costs
Expensive cell-detachment systems
negatively affect the overall cell-
culture process cost and determine
the applicability of the technique.
ALTERNATIVE CELL-DETACHMENT METHODSFor many years, treatment with the
protease enzyme trypsin has been
the standard method for cell detach-
ment. This method, widely named
trypsinization, is based on the addi-
tion of an active concentration of
the enzyme to the cell culture and
subsequent digestion of cell-mem-
brane proteins that establish the
interaction with the support surface.
Downstream Processing
28 BioPharm International www.biopharminternational.com March 2016
Trypsinization variants have been
developed along the years to try and
circumvent the drawbacks associ-
ated to the deleterious effect of this
enzyme on the cells and other limi-
tations derived from the chemical
nature of the stimulus employed to
disrupt the interaction of the cells
with the support.
The successful development of
biopharmaceuticals produced from
animal cell culture and other appli-
cations of adherent cells in the bio-
pharmaceutical industry has spurred
research on alternative cell-detach-
ment methods that address deficien-
cies of the trypsinization technique
and help realize the huge potential
of adherent cells to enable therapeu-
tic and diagnostic solutions.
Cell attachment requires the inter-
action of cell membranes with the
culture support, and in order to dis-
tort this interaction, different cell-
detachment methods use different
types of stimuli to target either the
cells, the membrane support, or both.
Besides, the effect of different stim-
uli on the target may have a differ-
ent degree of reversibility, having an
impact on the applicability of the dif-
ferent cell-detachment alternatives.
The following is a brief description
of these alternative cell detachment
methods, currently on different
degrees of development, with poten-
tial impact in biopharmaceutical
applications. This information is
summarized in Table I.
In cell scraping, a rubber or plastic
spatula is used to physically remove
the cells from the culture support.
This manual method based on a
mechanical stimulus is limited to
devices with a smooth culture sur-
face accessible to the laboratory
technician and has important auto-
mation limitations. The method is
quick and easy when performed in
a reduced number of devices but dis-
ruptive to the cells and may result in
significant cell death. The absence
of chemical reagents, surface mod-
ifications, or complex equipment,
however, makes this technique an
attractive alternative for laboratory
scale cell detachment from t-flask-
like culture devices.
An alternative to cell scraping for
use in microcarrier-based cell cul-
ture is the combination of vigorous
shaking with a mild trypsinization
treatment (1). This cell-detachment
technique combines two types of
stimuli: mechanical and chemical.
The method also has the potential
for scalability. Careful analysis of
the combined effect of both stim-
uli on the cells and on the subse-
quent processing of the product,
however, is necessary. Apart from
concerns raised by the use of proteo-
lytic enzymes, this method is easy to
implement in large-scale microcar-
rier cell culture. A variation of this
method exploits the strong shear
forces in microfluidic systems (2).
In such a method, cells are cultured
on the internal walls of channels in
microfluidic chips. When a strong
liquid flow is passed through the
microfluidic channels, the cells are
subjected to a high shear force that
provokes an efficient detachment.
This method is considered a harsh
detachment procedure, however,
often resulting in significant damage
and even death of the cells.
Enzymatic treatment with alterna-
tive proteolytic enzymes such as col-
lagenase or Pronase, a commercially
available mixture of several nonspe-
cific endo- and exoproteases pro-
duced by Streptomyces griseus, are
alternatives to the traditional tryp-
sinization method. These enzymes
digest proteins exposed in the cell
surface to distort the interaction of
the cells with the culture support.
Enzymatic activity on the cell sur-
face can be deleterious to the cells
and irreversible damage such as the
apoptotic effect induced by the tryp-
sin treatment (3) can be expected
from other proteases. This method
has poor temporal resolution, lead-
ing to extended activity of the
enzyme on the cells and subsequent
cell damage. Strong regulatory con-
siderations are also associated with
the use of enzymes in biopharma-
ceutical applications. On the other
hand, this method provides an effi-
cient way to disrupt cell-to-cell inter-
actions, a useful feature of interest
for harvesting cell suspension rather
than cell sheets.
Non-enzymatic chemical treat-
ment is also an option. Enzymatic
treatment is usually combined with
Ethylenediaminetetraacetic acid
(EDTA), a chelating agent for diva-
lent cations that helps trypsin do
its task and inhibits the interaction
of some of the proteins involved in
cell-to-cell interactions and cell-to-
support interaction. EDTA is also
applied alone when cells are loosely
attached to the culture support. The
citric saline method (4) has also been
reported as a very gentle treatment
for cell detachment. Non-enzymatic
chemical treatment provides a gen-
tle cell-detachment method. Besides
poor spatial and temporal resolu-
tion, these methods suffer from
poor performance in terms of the
ratio between required amount
of chemical and cell detachment.
Therefore, applicability of this tech-
nique is restricted to a limited num-
ber of cell lines.
Thermoresponsive substrates
have been used to promote the
detachment of adhered cells. In this
method, the cell-culture support
is coated with a thermoresponsive
polymer such as poly(N-isopro-
pylacrylamide) (pNIPAAm) fol-
low i n g d i f f e r e nt c he m ic a l
strategies. Cells are cultured on the
pNIPAAm-coated support, and cell
detachment is achieved by drop-
ping the temperature of the culture
below the lower critical solution
temperature (LCST) of pNIPAAm
(32 °C). Below LCST, pNIPAAm
undergoes a phase transition from
a shrunken state to a swollen state
that induces cell detachment due
to changes in the hydrophobicity of
the polymer. This strategy, initially
Downstream Processing
March 2016 www.biopharminternational.com BioPharm International 29
Downstream Processing
Cell-detachment technique
Stimulus Major advantages Major drawbacks Comments
Cell scraping Mechanical scratch of cells from surface
High temporal resolution, simplicity, low cost
Cell damage, poor scalability, highly invasive
Simple method with poor applicability beyond research
Vigorous shaking Shear forces on cells combined with proteolytic digestion of adhering proteins on cells
Scalable, low to medium cost
Cell damage, impact on downstream processing
Applicable to microcarrier cell culture to diminish deleterious effect of enzymatic treatment
Enzymatic treatment Proteolytic digestion of adhering proteins on cells
Well established, tunnable to adhesion strength, can be used on different types of supports
Cell damage, impact on downstream processing, regulatory concerns
Trypsinization and its variants are well established, straightforward methods. Cell-to-cell interactions are affected.
Non-enzymatic treatment
Molecular conformational changes on adhering molecules on the cells
Mild effect on cell integrity Applicable only to loosely attached cells
Alternative to enzymatic treatment with reduced impact on cell viability, but poor detachment efficency
Thermoresponsive substrates
Temperature change on the substrate
Noninvasive, automation potential can be used on different types of supports
Potential issues with leachables and extractables, cost of surface modifications
Technique with high potential for automation that can be adapted to most culture surfaces
pH-responsive substrates
pH change in substrate surface
Automation potential can be used on different types of supports
Very sensitive to process parameters, requires surface modification of the substrates
Future potential for this technique depends on availability of compatible pH-sensitive polymers
Electro-responsive substrates
Electric discharge on substrate
High temporal and spatial resolution, noninvasive, automation potential
Very complex surface modifications required, limited to small areas, not valid for microcarrier culture
Sophisticated technique with poor potential application beyond research
Photo-responsive substrates
Light-induced molecular modifications on substrate surface
Noninvasive, automation potential, scalable
Regulatory concerns due to required surface modifications, complex chemistry
Highly applicable technique once substrate modification issues are solved
Plasmonic substrates
Light-induced temperature change in localized nanoparticles on substrate surface
Noninvasive, automation potential, scalable, simple surface modification
Requires substrate modifications
Besides attractive features of photo-responsive substrates, easy surface modification allows for use on different types of substrates
Magnetic detachment
Magnetic field pull-on cells Allows for precise re-location of detached cells
Chemical modification of cell surface is required prior to application, regulatory concerns, poor scalability
Interesting technique for research applications
Shock-waves Shear forces on cells High spatial and temporal resolution
Cell damage, complex implementation, only for localized cell detachment
For use in localised cell detachment but complex compared to alternative techniques
Freeze-thaw Freezing cells Noninvasive, easily implemented
Cell damage This method is of application on processes where cell damage is of no concern. Scalable only when culture is performed on microcarriers following a supernatant separation step
Table I: Cell-detachment methods with potential impact on biopharmaceutical applications.
30 BioPharm International www.biopharminternational.com March 2016
applied to the recovery of cell sheets
from t-flask type culture devices (5),
has been extended successfully to
microcarrier-based cell cultures (3).
A key issue in this approach is raised
by regulatory concerns associated
with the use of current thermo-
responsive polymers (6). Chemical
synthesis of such polymer brushes
onto the surfaces of cell-culture sup-
ports requires a number of steps and
reagents that might hinder their
applicability. Otherwise, these sys-
tems are interesting due to their
potential for automation, scalability,
and noninvasive cell detachment.
The use of pH-responsive poly-
mers grafted on cell-culture sup-
ports has allowed researchers to
promote cell detachment by low-
ering environmental pH from 7.4
to 6.5 (7). Cell detachment can
occur following structural changes
that take place in the pH respon-
sive polymers upon pH decrease.
Changes in the polymer charge
f rom posit ive to negative in
response to a pH increase have been
used to detach cells from chitosan-
coated supports (8). This method
could potentially be employed to
detach cells from microcarriers or
from t-flask-like cell culture devices,
but similar to thermoresponsive
substrates, treatment with chemi-
cals, and enzymatic treatment,
requires the homogeneous modifi-
cation of the overall culture envi-
ronment conditions, which could
result in poor temporal resolution.
Sophisticated gold-coated elec-
tro-responsive substrates have been
employed to hydrolyze—under a
transient electrical potential—the
ether bond that retained the cell-
adhesive ligands to which cells are
attached (9). In this example, the
complex structure of the system is
a drawback for large-scale and mass
production. Besides, the reaction is
irreversible, because the cell-adhe-
sive ligand molecules remain bound
to the cells after the ether bond is
hydrolyzed. Moreover, the applied
electric potential might induce paral-
lel electrochemical reactions. Despite
the aforementioned drawbacks, the
potential automation of the system
may result in future efforts to further
develop electro-responsive systems.
Photo-responsive substrates based
on different designs and with dif-
ferent features have been studied.
Some of these systems are based
on changes on the hydrophilicity
of the substrate by light illumina-
tion (10). Some are based on revers-
ible structural changes in molecules
grafted on the surface of the sup-
port (11), and others use the irrevers-
ible cleavage of a photolabile linker
to release a cell-adhesive molecule
bound to the substrate (12). In all
cases, the high spatial and tempo-
ral resolution of light sources is an
important advantage of these cell-
detachment systems. Some of these
systems induce an irreversible modi-
fication on the substrate or a surface
modification on the cell membrane.
Complex modifications of the sub-
strate are necessary to sensitize cul-
ture supports and prepare them to
respond to light. Otherwise, use of
light as stimulus for cell detach-
ment is one of the most promising
alternatives for automated, nonin-
vasive cell detachment for biophar-
maceutical applications, including
large-scale production of biophar-
maceuticals using different types of
culture supports.
Downstream Processing
Figure 1: Cell detachment from plasmonic substrates. Transmitted light images of
HeLa cells grown on plasmonic substrates before (A) and after (B) irradiation with
a near-infrared laser at 980 nm. Power density of 340 mW/cm2 during 20 min.
(A)
(B)
FIG
UR
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Downstream Processing
Plasmonic substrates incorporate a
particular type of light-induced cell-
detachment properties. In this type
of substrate, a transient hyperther-
mic effect results from the interac-
tion of light of a given wavelength
with nanoparticles integrated in the
support. To date, available systems
were based on the use of near-ultra-
violet light, resulting in an aggressive
treatment for the growing cells and
in loss of spatial and temporal resolu-
tion due to the formation of reactive
oxygen species (ROS) that diffuse in
the culture medium (13). Efforts in
this direction have led to the devel-
opment of a plasmonic substrate that
promotes cell detachment upon a
short illumination with near-infra-
red (NIR) light (14). This technique
is based on plasmonic substrates pro-
duced by embedding gold nanoparti-
cles on the surface of the cell-culture
support. The controlled size and
geometry of the nanoparticles, as
well as their close contact, leads to
an intense plasmonic phenomena
upon interaction with NIR light—
better suited for cell treatment than
ultraviolet light—widely known to
induce mutagenic alterations in cel-
lular DNA. Besides, the system com-
bines the attractive properties of
light as a controlled stimulus with
a simple and robust support readily
transferable to different cell culture
devices. Most advantageously, NIR
light can be applied from outside the
cell-culture device, thus providing a
noninvasive cell-detachment system.
The effect of NIR light on cells grow-
ing on plasmonic substrates is shown
in Figure 1.
Cells labeled with magnetic
nanoparticles or liposomes have
been used to demonstrate the via-
bility of magnetic systems for cell
detachment. In these systems, cells
lightly adhered on the culture sup-
port are treated with positively
charged liposomes that readily bind
to the negatively charged cell mem-
branes (15). Then, a magnetic field
is applied to detach the cells from
the culture surface. The good spatial
and temporal resolution associated
to the localized application of the
magnetic field and the possibility to
deliver detached cells to designated
locations are of potential interest.
However, the complexity associated
to this method and other consider-
ations, such as the specific features
required for the cell membrane to
interact with the liposomes, preclude
its application beyond dedicated
research experiments.
Shock waves originated from a
piezoceramic source adapted from a
commercial lithotripter have been
used to detach adherent cells (16).
In this work, the ability of surviving
detached cells to reattach and propa-
gate was not assessed. However, cells
detached by laser-induced shock-
waves have been shown to adhere
again (17). This technique has good
spatial and temporal resolution.
There are few advantages to this com-
plex and shear-intensive technique,
however, when applied to the culture
of adherent cells for biopharmaceuti-
cal applications.
For particular applications that are
more related to research and produc-
tion of cell fragments, freeze/thaw is
a traditional method for cell detach-
ment, where cell viability is not of
major concern (18). This method has
been proposed for cell harvesting
from microcarriers (19), especially for
harvesting protease-sensitive biologi-
cal materials, and it is often used to
obtain cell fragments. Freeze/thaw
has a detrimental effect on cell via-
bility and integrity and a poor tem-
poral resolution. On the other hand,
regulatory concerns related to this
technique are scarce.
CONCLUSIONSome of the discussed methods for
cell detachment are already avail-
able commercially, while others are
in different stages of development.
In coming years, the authors expect
to see commercial variants of these
methods implemented in existing
devices for cell culture, as is the case
of the ongoing efforts of the authors
to incorporate plasmonic cell-
detachment features into the Bolt-on
bioreactor (20). Refinement of the
methods that have great potential
for enabling efficient cell detach-
ment is also necessary, as many of
the newer techniques are still too
complex and/or sophisticated to be
widely used by the cautious biophar-
maceutical industry.
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3907–3910 (2011) doi: 10.1002/
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795–798 (2004).
18. N. Nishishita et al., Am. J. Stem Cells 4 (1),
pp. 38–49 (2015).
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Press, April 25, 2011).
20. The Bolt-on Bioreactor Project. www.
boltonbioreactor.com. ◆
32 BioPharm International www.biopharminternational.com March 2016
ABSTRACT
The Chinese hamster ovary (CHO) cell lines used for the production of recombinant
therapeutic proteins are immortalized cells with a relatively high degree of genetic
plasticity. Given this inherent genetic flux, recombinant genes (or transgenes,
also referred to as the expression construct) expressed in CHO cells—used for
therapeutic bioproduct development—can be subject to genetic alteration that
may potentially impact the integrity and/or stability of those transgenes and, in
turn, impact drug substance production. Provided is a comprehensive, risk-based
transgene characterization strategy; its implementation is based on chemistry,
manufacturing, and control (CMC) development phases to ensure that the
integrity and stability of the transgene is maintained for clinical and commercial
CHO production cell lines. Early-phase assessment includes characterization
of the expression plasmid prior to cell-line generation (transfection); evaluation
of transcript integrity of those transgenes expressed transiently and stably in
CHO cells after transfection but prior to single-cell cloning of the candidate
production cell lines using single-cell sorting (or alternative methods); and
profiling of transgene copy number in cell-line populations across cell generations
spanning the manufacturing window. Mid-phase assessment includes further
characterization of the integrity and stability of the integrated transgenes using
the defined commercial cell-culture processes. Finally, the presented strategy
includes the late-phase characterization of the expression construct using cells
at the limit for in vitro cell age harvested from the commercial cell-culture
process to support the marketing authorization applications. Together, the
presented strategy is integrated with other existing drug substance analytical
control and product characterization strategies to ensure the integrity and
consistency of the drug substance used for clinical and commercial applications.
A Risk-Based Genetic Characterization Strategy for Recombinant CHO Cell Lines Used for Clinical and Commercial Applications
Luhong He and Christopher Frye
Luhong He, PhD, is senior research
scientist, and Christopher Frye is research
advisor, both in the department of Bioprocess
Research and Development at
Eli Lilly and Company.
Email: [email protected]
PEER-REVIEWED
Article submitted: Nov. 30, 2015.
Article accepted: Dec. 17, 2015.
Peer-ReviewedA
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March 2016 www.biopharminternational.com BioPharm International 33
Over the past several decades,
numerous recombinant proteins
have been approved as therapeu-
tic drugs by regulatory authorities,
and many more are currently undergoing
clinical development (1). Chinese hamster
ovary (CHO)–derived cell lines have become
the predominant host for the manufactur-
ing of glycosylated therapeutic proteins.
During this period of explosion in the appli-
cation of CHO expression systems, consid-
erable efforts have been made to improve
recombinant protein production to meet
the demands of high quantity and consis-
tent quality of biopharmaceutical products.
Those efforts can be categorized into two
fundamental areas: improving therapeutic
protein developability by protein engineer-
ing based on structure-activity relationship
(SAR) studies, and improving therapeutic
protein production systems with a focus on
host-cell engineering, cell-culture medium,
and process development.
While the aforementioned efforts suc-
cessfully introduced clinical candidates
with more desirable therapeutic traits (e.g.,
enhanced activity, chemical and physical sta-
bility) and boosted the productivity of CHO
cell lines from < 100 mg/L to > 10g/L, unin-
tended consequences have resulted from the
extensively-engineered transgenes expressed
in CHO hosts, which inherently possess a
relatively high degree of genetic instability.
The authors’ internal development experi-
ence, which is consistent with published
literature, indicates that transgenes inte-
grated into the CHO genome can, in some
cases, result in unintended protein species
caused by a number of potential mecha-
nisms including transgene RNA (transcript)
aberrant splicing (2), genetic mutation (3, 4),
and amino acid misincorporation (5–7) dur-
ing cell-culture processes. If not removed by
the purification process, these unintended
byproducts are typically considered prod-
uct-related impurities (PRI), as defined by
International Council for Harmonisation of
Technical Requirements for Pharmaceuticals
for Human Use (ICH) Q6B (8), and could
have potential implications on the safety and
efficacy of the intended products. In addi-
tion, the integrated transgenes may be par-
tially or completely lost or silenced (through
epigenetic mechanisms) during the CHO
cell-culture process (9–13), which may poten-
tially lead to a lack of robustness of the drug
substance manufacturing process.
ICH Q5B (14) recommends that, “segments
of the expression construct should be ana-
lyzed using nucleic acid techniques in con-
junction with other tests performed on the
purified recombinant protein for assuring the
quality and consistency of the final product.”
This manuscript presents a comprehensive
genetic characterization strategy developed
and implemented based on bioproduct chem-
istry, manufacturing, and control (CMC)
development phases to ensure that the integ-
rity and stability of the transgenes is main-
tained during the cell-culture process and in
the cells at the limit for in vitro cell age. It is
crucial to note that the genetic characteriza-
tion strategy presented here is not an isolated
practice but rather an important part of a
holistic, integrated strategy assembled dur-
ing bioproduct development, including an
appropriate analytical control strategy cou-
pled with extensive product characterization.
In addition to meeting the regulatory
requirements that ensure the safety and
efficacy of biopharmaceuticals for clinical
and commercial applications, the strategy
presented here also provides an approach
that addresses business considerations. CMC
development is a complex, expensive, and
resource-intensive process. There are differ-
ent strategies to addressing business needs.
At one extreme would be initiation of early
clinical development using drug substance
from a manufacturing process that would
have to change significantly for commercial-
ization. This approach will require additional
effort to demonstrate comparability between
the early-phase and commercial product,
which potentially creates a risk of the need
for additional clinical trials. An alternative
approach is to develop a “commercializable”
process early and leverage that process for all
clinical development. This approach has the
potential advantage of lowering risk to prod-
uct comparability assessment, but requires
additional early process development invest-
ment. The strategy presented in this paper
is based on the latter approach, where the
process development objective is to develop
a commercial production cell line for first
human dose (FHD) or first-in-human (FIH)
application, and leveraging a “commercial-
Peer-Reviewed
34 BioPharm International www.biopharminternational.com March 2016
izable” platform for cell culture and down-
stream process development. At the heart of
this strategy is the view that the production
cell line is the foundation of any bioprocess,
and thus, appropriate genetic characteriza-
tion of the production cell line is absolutely
crucial to the success of process development.
PHASE–APPROPRIATE STRATEGY FOR CHARACTERIZATION OF TRANSGENES EXPRESSED IN CHO CELLSThe CMC development phases discussed in
this manuscript refer to the three phases com-
monly associated with clinical and commer-
cial development. The early phase begins from
generation of the clonally derived production
cell line and ends with the release of the for-
mulated drug product for FHD/FIH. The mid-
phase involves activities including product
resupply for ongoing clinical trials (first effi-
cacy dose [FED]) and development of the com-
mercial manufacturing process. Late-phase
development starts from the manufacture of
product for the first registration dose (FRD)
using commercial manufacturing processes
and ends with process validation and sub-
mission of the product licensing applications.
Table I summarizes the CMC development
phase–appropriate activities for the character-
ization of the transgenes encoding the thera-
peutic proteins expressed in CHO cells.
For clarity, two terms, genetic suitability
and genetic stability, are used to describe the
characterization of the transgenes expressed
in CHO cells. The genetic suitability of a pro-
duction cell line refers to the acceptability
or appropriateness of the cell line for clini-
cal drug substance manufacturing. In con-
trast to genetic suitability, more traditional
genetic stability studies refer to the charac-
terization of the production cell lines used for
commercial drug substance manufacturing
followed the ICH Q5B guidance. In conjunc-
tion with protein characterization and quality
assessment, the genetic stability studies using
nucleic acid techniques examine the integ-
rity and stability of the expression construct,
which could potentially impact protein integ-
rity and process consistency. The genetic sta-
bility studies are performed in two phases: the
mid-phase study, referred to as the “genetic
stability risk assessment,” which evaluates the
integrity and stability of the expression con-
struct under defined commercial cell-culture
process, and the late-phase study, referred to
as the “expression construct characterization,”
which generates the genetic characterization
data package (based on the commercial cell-
culture process) that forms part of the market-
ing authorization application (MAA).
TRANSGENE CHARACTERIZATION ACTIVITIES DURING EARLY-PHASE DEVELOPMENT As shown in Table I, four genetic characteriza-
tion activities are implemented during early-
phase development to minimize the risk of
choosing inappropriate production cell lines.
Those activities are summarized in the fol-
lowing passages.
EVALUATION OF THE EXPRESSION PLASMID USED FOR TRANSFECTIONThe cloning of genes coding for therapeutic
proteins within an expression plasmid back-
bone is usually a straightforward procedure.
It is a common practice and a recommenda-
tion of ICH Q5B to confirm the nucleotide
sequence of the coding region of the gene(s)
of interest and associated flanking regions
that are inserted into the plasmid backbone.
After the sequence of the expression plasmid
is confirmed, the expression plasmid DNA
is usually isolated at larger scale to prepare
sufficient plasmid DNA to enable cell-line
generation. From time to time, researchers
encounter plasmid instability, such as loss
of plasmid or changes in plasmid structure
during large-scale bacterial cultivation (15).
Through the evaluation of expression plas-
mid batches prior to transfection, the authors
identified plasmid structural changes and
point mutations that resulted from a larger-
scale preparation, even though the expression
plasmid was previously confirmed from a
small-scale preparation (mini-prep). Because
the issues were discovered and corrected prior
to transfection, they did not cause significant
delays in the generation of the production cell
line.
EVALUATION OF INTEGRITY OF THE TRANSGENE MRNA EXPRESSED IN CHO CELLSRNA splicing is a natural process in
mammalian cells that removes introns and
joins exons in a primary transcript to create
mature messenger RNA (mRNA) for transla-
tion. For biopharmaceutical development, the
Peer-Reviewed
March 2016 www.biopharminternational.com BioPharm International 35
recombinant genes coding for the mature
mRNA transcripts (i.e., cDNA versions) are com-
monly used for the production of drug sub-
stance to avoid complexity of RNA splicing in
the production host. However, with the imple-
mentation of SAR studies and advancements
in DNA manipulation techniques, more and
more recombinant genes undergo extensive
and complex genetic manipulation to improve
the encoded candidate’s therapeutic traits.
Although the protein engineering process has
been successful in that regard, the full impact
of some nucleotide sequence modifications is
difficult to predict. It has been observed that
several engineered antibody genes expressed in
CHO cells produced unintended transcripts—
resulting from aberrant RNA splicing at cryptic
splicing sites—in addition to the expected full-
length transcript (2, 16). Aberrant mRNA splic-
ing can lead to unexpected low-level expression
of the recombinant transgenes (2) and/or give
rise to truncated product-related impurities
(16). In one case, the authors experienced an
antibody-producing cell line, which had cryp-
tic aberrant mRNA splicing that gave rise to a
truncated light chain (LC) product. Although
the truncated LC was effectively removed
through the downstream purification process,
the overall purification yield was significantly
reduced (to approximately 10%). A new pro-
duction cell line was subsequently generated
for Phase II/III and commercial applications.
The aberrant splicing was eliminated in the
new cell line by site-specific mutagenesis at the
cryptic splicing sites.
To mitigate the risk of unintended splicing,
the authors identified and eliminated cryptic
aberrant splicing proactively during clinical
candidate selection to avoid potential delays
to the clinical development or the need to
switch cell lines for commercial applications.
The authors’ experiences and the literature
indicated that several publicly available
splice-site-recognition programs were unable
Peer-Reviewed
CMC development phase
Timing Characterization activity
Early (gene to FHD)
Prior to transfection
Evaluation of the integrity of the expression plasmid used for transfection to ensure the DNA used has the expected gene coding sequence. Assays include restriction mapping and DNA sequencing of the expression cassette.
Prior to single-cell cloning
Evaluation of integrity of the transgene mRNA expressed in CHO cells to ensure engineered genes do not give rise to unintended aberrant mRNA splicing or other modifications. Assays include RT-PCR and DNA sequencing.
Prior to production cell-line selection
Genetic suitability evaluation of transgenes in the clonally derived cell lines to eliminate cell lines that display significant changes in their transgene population profiles due to cell aging. Assays include single-cell qPCR.
Prior to drug product release for clinical applications
Confirmation of nucleotide sequence of transgenes in MCB to ensure the MCB carries expected transgene sequence. Assays include RT-PCR and DNA sequencing.
Mid (FHD to FRD)Prior to commercial cell-culture process determination
Genetic stability risk assessment of the chosen production cell line under defined cell-culture process at various cell ages from bench and pilot scales to ensure the cell line and cell-culture conditions are acceptable for commercial development. Assays include coding sequence confirmation, restriction endonuclease mapping for integration pattern analysis and transgene copy number determination.
Late (FRD to MAA)Prior to commercial process validation at manufacturing sites
Expression construct characterization in MCB and EOPC at the limit for in vitro cell age from fully representative commercial manufacturing process for MAA. The assays developed during genetic stability risk assessment will be applied.
Table I: Phase-appropriate characterization activities for therapeutic transgenes expressed in
Chinese hamster ovary (CHO) cells. FHD=first human dose, FRD=first registration dose,
PCR=polymerase chain reaction, qPCR=quantitative polymerase chain reaction, MCB=master cell
bank, RT–PCR=reverse transcription polymerase chain reaction, EOPC=end-of-production cells,
MAA=marketing authorization application.
36 BioPharm International www.biopharminternational.com March 2016
to identify the aberrant splicing sites used
in the CHO cell environment (2). The most
effective methods to identify the presence
of aberrant splicing events are reverse
transcription polymercase chain reaction
(RT–PCR) and Northern blot. The authors’
data indicate that RT–PCR is a more sensitive
method to detect low level of aberrant splic-
ing compared to Northern blot (unpublished
data). The potential bias due to PCR primer-
binding capability can be overcome by
applying multiple pairs of primers.
RT–PCR-based methods have been devel-
oped and implemented to screen all new clini-
cal candidates for the aberrant splicing events
in both transiently and stably transfected
CHO cells before they are chosen to gener-
ate production cell lines supporting clinical
development. This early screening approach
has identified cryptic aberrant splice sites in
multiple therapeutic candidates including
monoclonal antibodies, bispecific antibod-
ies, and fusion proteins, therefore preventing
significant investment in the development
of certain candidate-encoding genes (unpub-
lished data).
EVALUATION OF TRANSGENE DISTRIBUTION PROFILES IN CHO CELL LINESThe inherent adaptive ability of CHO
cells and their capacity for the expression
and secretion of recombinant proteins
have been the most important factors
that have enabled the adoption of CHO
cells as the industry’s predominant host
for development and manufactur ing
of therapeutic proteins (17). However, as
immortalized cells, CHO cell lines exhibit
a high level of genetic and phenotypic
diversity and instability (17). In spite of
repeated rounds of single-cell cloning by
limiting dilution or florescence-activated
cel l sor t ing (FACS), c lonal ly-der ived
CHO cell lines have often been observed
to diverge, becoming a heterogeneous
populat ion over long per iods of sub-
culturing (17–22). Extensive efforts have
been devoted to the screening process
Peer-Reviewed
Figure 1: Transgene copy number distribution profiles of cell lines expressing recombinant protein at
generations of 0, 30, 45, and 60. Generation 0 (G0) represents the generation of a master cell bank, G30
represents the generation of cells harvested from a 5000-L bioreactor, and G60 represents the limit for
in vitro cell age designed for a commercial manufacturing process. The distribution profiles shown in the
scatterplot and histograms (1A: cell line 3E4; 1B: cell line 7H2) were generated by Oneway platform of JMP
software (SAS Institute, Inc, Cary, NC) as outlined in (12). The cycle threshold (Ct) values of transgene were
generated by a single-cell quantitative polymerase chain reaction (qPCR) assay. The number of tested
single cells (number), mean and standard deviation (std dev) of the Ct values are shown below the plots.
1A: Cell line 3E4
40
35
30
25
20
3E4-G
0
3E4-G
30
3E4-G
45
3E4-G
60
3E4-6
0
3E4-G
30
3E4-G
45
3E4-G
60
Ct
Sample Name
Means and std DeviationsMean Std DevNumberLevel
3E4-G0
3E4-G30
3E4-G45
3E4-G60
46474848
30.430.330.730.6
0.60.60.70.6
1B: Cell line 7H2
40
35
30
25
20
Ct
7H
2_G
O
7H2_GO7
H2
_G
3O
7H2_G3O
7H
2_G
45
7H2_G45
7H
2_G
60
7H2_G60
7H
2_G
0
7H
2_G
30
7H
2_G
45
7H
2_G
60
Sample Name
Means and Std Deviations
Level Number Mean Std Dev
47484546
29.030.631.230.8
2.11.51.00.8
FIG
UR
E 1
IS
CO
UR
TE
SY
OF
TH
E A
UT
HO
RS
.
March 2016 www.biopharminternational.com BioPharm International 37
to identify desired characteristics and
suitability of recombinant CHO cell lines.
Historically, suitability has been evalu-
ated utilizing phenotypic measures (pro-
ductivity) assessing cell-line productivity
across a generational span encompassing
the manufacturing window. Given the vari-
ability of the phenotypic assessment, the
current and preferred measure of suitability
is to assess the genetic profiles of the produc-
tion cell-line population, thus obtaining a
measure of the genetic consistency of the cell
line across generations. The profiles provide
an indication of significant loss of the trans-
gene (% negative cells) or significant levels
of genetic heterogeneity within the cell-line
population (e.g., broad transgene copy dis-
tribution or standard deviation) across gen-
erations (12). Cell lines displaying genetic
heterogeneity may have a higher risk of not
meeting commercial manufacturing require-
ments and, therefore, should be eliminated
during production cell-line screening. The
methodology used for testing transgene het-
erogeneity was initially developed and imple-
mented to evaluate the genetic suitability of
clonally-derived CHO cell lines expressing
IgG1 and IgG4 monoclonal antibodies. It
has now been adapted to permit the evalu-
ation of candidate cell lines expressing Fab,
Fc fusion proteins, proteins (with or without
glycosylation), as well as bispecific or bifunc-
tional molecules. Undesired cell lines—mea-
sured by three parameters including the
percentage of negative cells, standard devia-
tion of cycle threshold (Ct) value (a measure
of population heterogeneity), and mean Ct
changes during aging (a measure of popula-
tion drift) (12) —were identified regardless
of the type of transgenes being used. In the
authors’ experience, approximately 20% of
clonally-derived candidate production cell
lines analyzed showed significant transgene
population heterogeneity over generations.
As examples, Figure 1 shows the transgene
distribution profiles of two cell lines express-
ing identical recombinant therapeutic pro-
teins obtained from the same transfection.
The cell line in Figure 1A (designated 3E4)
represents a relatively homogenous trans-
gene population across the generational span
needed for the manufacturing process, while
the cell line in Figure 1B (designated 7H2)
displayed a significant population shift when
the cells were aged, as indicated by the Ct
mean change of >1 and overall large stan-
dard deviations across generations.
CONFIRMATION OF NUCLEOTIDE SEQUENCE OF TRANSGENES IN THE MASTER CELL BANKAlthough it has been a common practice to
verify the nucleotide sequence of transgenes
encoding the therapeutic proteins in mas-
ter cell banks (MCB) for the MAA as rec-
ommended by ICH Q5B guidance, it was
only recently recommended by the European
Medicines Agency (EMA) that the sequence
of the coding region should be confirmed
prior to the initiation of clinical trials (23).
For recombinant CHO cell lines, trans-
genes are integrated into CHO chromosome.
The most common technique to verify the
nucleic acid sequence encoding the product
is sequencing of coding regions amplified by
the polymerase chain reaction (PCR) from
pooled cDNA isolated from the production
cell line. The nucleic acid sequence of the
predominant transgene transcripts should be
identical—within the limits of detection of
the methodology—to the expected sequence
encoding for the protein.
In summary, the early-phase characteriza-
tion activities focus on evaluating and mini-
mizing the potential risks associated with a
transgene’s integrity and consistency when
expressed in CHO cells. By implementing
these activities, manufacturers can ensure that
potential issues are identified during cell-line
generation and are prevented from posing
risk to the development of the manufacturing
process. It should be noted that the transgene
expression system used for the generation
of a production cell line may also impact its
integrity and stability. Two of the most com-
mon expression systems leveraged for the pro-
duction of therapeutic proteins in CHO cells
are the dihydrofolate reductase (DHFR)-based
methotrexate (MTX) selection system and
the glutamine synthetase (GS)-based methi-
onine sulfoximine (MSX) selection system.
Observations have been reported of genomic
DNA mutations in the cell lines utilizing the
DHFR/MTX system (24), and the mutation
rates measured by 6-thioguanine (6-TG) assay
positively correlated with the MTX concen-
trations used to select the recombinant cell
lines (3). Because multiple rounds of ampli-
fication are often applied using the DHFR/
Peer-Reviewed
38 BioPharm International www.biopharminternational.com March 2016
Peer-Reviewed
MTX system—which can result in as much as
a 1000-fold increase in transgene copy num-
ber (25)—more detailed DNA and amino acid
sequence analyses may be necessary to ensure
consistent product quality (3). In contrast, the
GS–MSX expression system typically does not
require multiple rounds of amplification and,
thus, usually results in relatively low trans-
gene copy numbers. The authors’ internal data
indicate that the average transgene copy num-
ber is approximately 5 in 62 clonally-derived
production cell lines generated leveraging the
Lonza GS expression system. The relatively
low transgene copy number may reduce the
risk of DNA alterations, although it does not
eliminate the possibility of modification (26).
TRANSGENE GENETIC STABILITY RISK ASSESSMENT DURING MID-PHASE DEVELOPMENTAlthough significant efforts are devoted to the
identification of production cell lines with
the fewest potential risks for commercializa-
tion, many of these studies are performed
using cells grown in shake flasks or small
bench-scale bioreactors under nonoptimized
cell culture conditions. Therefore, it is cru-
cial to further evaluate the integrity and sta-
bility of the transgenes using commercial
cell-culture conditions during the mid-phase
development. This approach can enable cor-
rective actions to be taken to avoid costly
changes later. It has been reported that unex-
pected genetic alterations were identified in
the MCB, the manufacturing working cell
bank (MWCB), the end-of-production cell
bank (EPCB), and in production cells (27).
Deta i ls of this eva luat ion, termed
the “genetic stability risk assessment” can
be found in Table I. Mid-phase assessment
focuses on evaluating the impact of cell age
and cell-culture process using DNA and RNA
isolated from the MCB or premaster research
cell bank (pmRCB), end-of-production cells
(EOPC) from a typical defined cell-culture
process, and EOPC inoculated from the pro-
posed limit for in vitro cell age. It includes
assessment of integrity of predominant cod-
ing transcripts, consistency of integration
patterns, and average transgene copy
numbers for the aforementioned samples.
The established assessment methods will be
applied to characterize the expression con-
struct needed for future MAA. The tested
limit for in vitro cell age will be used to pro-
pose the commercial manufacturing operat-
ing space for marketing applications.
CHARACTERIZATION OF THE EXPRESSION CONSTRUCT FOR MAAThe ultimate goal of genetic characterization
of the production cell line is to demonstrate
the integrity and stability of the expres-
sion construct carrying the transgenes. This
includes demonstrating that these transgene-
carrying constructs are stably maintained from
the starting cell banks (MCB and/or WCB) to
the EOPC at the limit for the in vitro cell age
inoculated from a WCB and harvested from
the commercial drug substance manufacturing
process. This characterization—which con-
firms that the commercial cell culture process
does not lead to unintended changes in the
transgenes—is performed not only to meet
regulatory expectations for the MAA, but also
to provide assurance of safety and manufactur-
ing consistency when coupled together with
product characterization and an analytical
control strategy. The characterization, focused
on the integrity and consistency of the expres-
sion construct, consists of three aspects:
t� 7FSJGJDBUJPO�PG�QSPUFJO�DPEJOH�TFRVFODF�JO�
production cells through the end of pro-
duction. This is commonly accomplished
by nucleotide sequence analysis of the
transgene-specific PCR products amplified
from pooled cDNA.
t� "TTFTTNFOU�PG� JOUFHSBUJPO�QBUUFSOT �XIJDI�
provides insight into potential insertions
and/or deletions of the expression con-
struct. The most common methodology
for this assessment is restriction endonu-
clease mapping analysis by Southern blot.
t� %FUFSNJOBUJPO�PG� BWFSBHF� USBOTHFOF� DPQZ�
number. The current common method is
quantitative PCR (qPCR). Transgene-specific
qPCR assays and a normalizer qPCR assay
targeting a host genome region are usually
applied. The characterization, focused on
the integrity and consistency of the expres-
sion construct, consists of three aspects,
which are methods established during the
mid-phase “genetic risk assessment”.
The in vitro cell age is defined by ICH
Q5B as “measure of time between thaw of
the MCB vial(s) to harvest of the produc-
tion vessel measured by elapsed chronologi-
cal time in culture, by population doubling
March 2016 www.biopharminternational.com BioPharm International 39
Peer-Reviewed
(PD) of the cells, or by passage level of the
cells when sub-cultivated by a defined pro-
cedure for dilution of the culture” (14). In
experience with the authors’ CHO cell lines,
a typical cell age from a 5000-L production
bioreactor is found to be approximately 30
PD including the cell age of a WCB, seed
train, and bioreactor expansion and growth
in the production vessel. Extra cell culture
passages in the cell expansion stages are
added to generate the cells at the limit for in
vitro cell age to increase flexibility of manu-
facturing operation. The typical limit for in
vitro cell age is in total approximately 45–60
PD, depending on the growth rate of each
individual cell line and the specific cell-cul-
ture expansion process. The mid-phase risk
assessment enables the design of limit for in
vitro cell age for the commercial process.
The impact of a change in cell-culture pro-
cess after the commercial process has been
defined should be evaluated to determine if
re-testing of the cells at the limit of in vitro
cell age is necessary. Changes in cell-culture
process scale and/or manufacturing site may
not require re-testing of cells at the limit for
in vitro cell age, provided the available data
meet the predetermined acceptance criteria
and were obtained using cells with sufficient
in vitro cell age to cover the additional cell gen-
erations resulting from the increased scale and
cell-culture performance. Changes in media
components and growth conditions often
result in changes in cell culture profiles, and
one should consider re-testing the integrity
and stability of the expression construct at the
limit for in vitro cell age in those situations.
Although a two-tier cell-banking system
(MCB and WCB) is commonly established
for the commercial manufacturing of drug
substance, it is considered that the character-
ization of the expression construct for each
WCB is unnecessary if the following criteria
are met:
t� 5IF�.$#�IBT�CFFO�GVMMZ�DIBSBDUFSJ[FE�BOE�
the expression construct is confirmed to
be stable. If the characterization cannot be
carried out on the MCB, it should be car-
ried out on each WCB.
t� 5IF�DFMM�BHF�PG�UIF�DVSSFOU�BOE�UIF�GVUVSF�
WCB, as well as the EOPC derived from
the future WCB, will be controlled within
the previously approved limit for in vitro
cell age.
By implementing a development-phase
appropriate genetic characterization strat-
egy, manufacturers can be assured that the
integrity and stability of transgenes in all
clonally derived CHO cell lines intended
for drug substance commercial manufactur-
ing meet regulatory requirements for MAA.
Harnessing this strategy has facilitated the
early identification of unsuitable/unstable cell
lines, thus enabling effective investments of
time and resources only on those cell lines
that are appropriate candidates. This strategy
also meets business needs consistent with
aggressive development timelines and the
industry-wide movement toward more effi-
cient practices, from therapeutic candidate
selection to product launch.
POTENTIAL APPLICATIONS OF NEW TECHNOLOGIES FOR THE CHARACTERIZATION OF TRANSGENESAs the biotechnology industry continues to
mature, so does the ability to understand the
processes used to manufacture biopharma-
ceutical products. Part of this understand-
ing involves the recognition and ability to
characterize production cell lines as popu-
lations of cells exhibiting various levels of
genetic and phenotypic heterogeneity. The
described strategy includes characterization
of production cell-line populations in addi-
tion to more traditional genetic characteriza-
tion methodologies. Although this strategy
has historically served well in accomplish-
ing its intended purpose, it is also recognized
that there is a need to continue to monitor
and assess the potential value of new nucleic
acid-based technologies, such as next-genera-
tion sequencing (NGS), to potentially further
enhance the capability for genetic character-
ization (28). NGS can be utilized as a com-
plementary/orthogonal analysis tool for the
investigation of bioproduct-related impuri-
ties. NGS can also provide a means of better
understanding impurities if they are related
to genomic mutations or nutrient limitations
in a cell-culture process. New approaches and
technologies are becoming available and hold
promise for permitting better characterization
of cell lines and cell-culture processes, which
could lead to improved insight into how to
develop manufacturing processes more holis-
tically. Implemented together, the described
phase-appropriate genetic characterization
40 BioPharm International www.biopharminternational.com March 2016
Peer-Reviewed
strategy, orthogonal product characterization,
and appropriate product control strategies will
ensure the safety and efficacy of clinically vali-
dated therapeutic proteins.
CONCLUSIONPotential risks related to the integrity
and stability of recombinant transgenes
expressed in CHO cells are inevitable during
biopharmaceutical development. These risks
include unintended aberrant mRNA splicing
in the coding regions due to the bioengi-
neering process and potential instability of
the integrated recombinant genes as a result
of the global genetic instability inherent
in CHO cells. The genetic characterization
strategy described here can play a crucial
role in assessing and managing these risks.
The authors’ implementation of this strategy
has prevented problematic expression con-
struct/cell lines from being developed inap-
propriately, thereby enabling more efficient
and effective process development programs.
The data presented here demonstrate that
the CHO production cell lines developed by
the authors have relatively low integrated
transgene copy numbers, and those genes
are stably maintained from their respective
master cell banks to the end of production
cells at the limit for in vitro cell age used for
drug substance manufacturing. To ensure
the most effective genetic characterization
strategy, new technologies will continue to
be evaluated for potential future applications.
ACKNOWLEDGMENTThe authors would like to thank Christal
Winterrowd and Monica Myers for provid-
ing copy number analysis and Dennis Gately
and Matthew Hilton for providing technical
input to the characterization strategy. We’d
also like to thank Matthew Hilton, Michael
De Felippis, Tongtong Wang, and Anli
Ouyang for critical review of the manuscript
and helpful discussions. In addition, the
authors wish to recognize and thank Lonza
Biologics (Slough, UK) for the licensing of
and permission to modify the GS-CHO
expression system.
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12. L. He et al., Biotechnol. Bioeng. 109 (7),
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15. F.A. Al-Allaf et al., 3 Biotech. 3 (1), pp. 61–70
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16. J.M. Reichert, N.M. Jacob, and A. Amanullah,
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23. EMA/CHMP/BWP/534898/2008 Committee
for Medicinal Products for Human Use
(CHMP), Guideline on the Requirements for
Quality Documentation Concerning Biological
Investigational Medicinal Products in Clinical Trials
(March 15, 2012).
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42 BioPharm International www.biopharminternational.com March 2016
Data integrity is a major regula-
tory topic in GMP-regulated
laboratories. The problem is
widespread, as cases of non-
compliance have also been observed
in laboratories regulated by good labo-
ratory practice (GLP) and good clini-
cal practice (GCP), not just GMP.
Non-compliances from the European
Medicines Agency (EMA) or warning
letters from FDA show examples from
companies in the United States, Canada,
the United Kingdom, and Italy, as well
as China and India. So, it is not just a
problem for Asia—it is a global issue.
The non-compliances are not con-
fined to falsification and fraud. In fact,
in the majority of labs, the main data
integrity issues concern poor data man-
agement, which account for 95% of
non-compliance cases; only 5% are a
result of falsification or fraud. Problems
arise from basic errors such as rely-
ing on paper as raw data and failing to
protect electronic records, rather than
deliberate manipulation of data. It is
the latter, however, that gets the major
headlines. In July 2014, FDA issued a
stern warning that data integrity was a
key focus of its enforcement efforts (1).
REGULATORY GUIDANCE ON DATA INTEGRITYIn January 2015, the UK Medicines
and Healthcare products Regulatory
Agency (MHRA) went further and issued
Guidance for Industry on Data Integrity
(revised in March 2015) (2). This guid-
ance consists of descriptions, defini-
tions, and expectations based around
those definitions, which include raw
data, original records, file structures,
and audit trail. The regulatory expecta-
tions presented in this guidance are use-
ful. However, the definitions are simply
presented as a shopping list; a diagram
explaining and linking key definitions
would be more beneficial.
The guidance presents data integrity
as the extent to which all data are “com-
plete, consistent, and accurate” through-
out the data lifecycle, which covers the
period from data acquisition through to
interpretation, reporting, and archiving
and then destruction after the record
retention period. There are similarities
in the US regulations; a 20-year-old FDA
definition of data integrity describes the
degree to which a collection of data is
complete, consistent, and accurate (3).
In European GMP regulations, doc-
umentation constitutes a key part of
quality assurance and, therefore, com-
pliance with GMP (4). An organization
must have good documentation, follow
standard operating procedures (SOPs),
and demonstrate compliance with the
applicable regulations. Several require-
ments focus on data integrity for com-
puterized systems (5). If critical data
are entered into a data system, a second
check is required, which can either be
a second person or can be automated
using the computer system itself. In
the US, the regulations for laboratory
records focus on the concept of com-
plete data (6).
A review of data integrity warning
letters reveals several citations for fail-
ure to have complete data including a
failure to have a complete procedure (7),
a failure to fully document the work
that is carried out (8), or being selective
in reporting data (e.g., using test sam-
How Important is Data Integrity to
Regulatory Bodies? Bob McDowall and
Joanne Ratcliff
Data integrity is a widespread,
global problem that must be addressed.
Dr. Bob McDowall is director of
R D McDowall Ltd., rdmcdowall@
btconnect.com. Dr. Joanne
Ratcliff is communications
project manager at Mettler Toledo
GmbH, [email protected].
Data Integrity
March 2016 www.biopharminternational.com BioPharm International 43
ples to “check” if an instrument is
working correctly) (9). Complete
data includes the actual observa-
tion, which can be visual or by
computerized system. Contextual
metadata, which puts the data or
result in context, is also required.
Examples of metadata include
operator ID, units of measurement,
sample information, identity,
batch number, instrument suit-
ability, and readiness. Audit trail
events are also part of the meta-
data. If a hybrid system or a fully
electronic system is used, FDA
and European regulatory agencies
require companies to review and
evaluate audit trail events to see if
data have been manipulated with-
out authorization.
FDA guidance comprises a list
of commonly asked questions
and answers that are crucial for
maintaining data integrity (10).
Detailed explanations are given
for:
t� 8IZ� QBQFS � SFDPSET � G SPN � B�
hybrid system should not be
defined as raw data—instead it
should be the underlying elec-
tronic records; Although the
reasoning focuses on chroma-
tography, it is applicable to any
computerized system.
t� 8IZ� TIBSJOH�PG�VTFS� JEFOUJUJFT�
in a computerized system is not
allowed—as it makes it impossi-
ble to attribute the work carried
out to a single individual.
t� 8IZ� BDUVBM� TBNQMFT� TIPVME�
not be used as system suitabil-
ity tests to see whether a batch
passes or not. FDA warning let-
ters reveal many citations for
this transgression.
DATA INTEGRITY CRITERIAThe criteria for data integrity are
defined by the acronyms ALCOA
and ALCOA+. ALCOA, which stands
for attributable, legible, contempo-
raneous, original, and accurate, was
developed by an FDA GMP inspec-
tor in the late 1980s. ALCOA has
been used in many areas that are
regulated both by FDA and other
regulatory agencies worldwide. In
2010, EMA published a paper on
electronic source data for clini-
cal records, in which another four
requirements relating to elec-
tronic data were added: complete,
consistent, enduring, and avail-
able (11). The Good Automated
Manufacturing Practice (GAMP)
Data Integrity Special Interest Group
(SIG) now refers to ALCOA+, which
includes the nine attributes or crite-
ria for data integrity (see Table I).
INSPECTION TRENDS In the past, an inspector would
review piles of paper; to view
a computer system, he would be
shown print-outs of the screen.
Now, the focus is on the comput-
erized systems and the electronic
records within; the paper output
is secondary. Inspectors will focus
on the electronic records, looking
at how they were generated and
manipulated within the applica-
tion. In the light of these changes,
consideration needs to be given to
the person who will manage the
system during an inspection. How
is the system configured to pro-
tect the electronic records? Are
electronic signatures being used in
the application, ensuring that the
configuration of the application is
documented and reflects the set-
tings within the software?
Annex 11 now requires audit
trail entries to be reviewed, and
FDA considers these entries as part
of the complete data. Many find-
ings of non-compliance during
inspection have been discovered
by looking at audit trail entries,
therefore, an approach to review-
ing audit trail events is needed.
Citations have noted when audit
trails were been turned off, the
audit trail had not been reviewed,
or user identities were shared,
which is not allowed under the
regulations. Additional citations
can be found in the September
2014 issue of LCGC (12).
THE TEN COMPLIANCE COMMANDMENTSThe 10 compliance command-
ments for computerized laboratory
systems described in Table II should
be considered (12).
Data Integrity
Table I: Good Automated Manufacturing Practice (GAMP) criteria for data integrity—ALCOA+.
ALCOA Term
Criteria Definition
A Attributable Who performed the action and when? If a record is changed, who did it and why? Link to the source data.
L Legible Data must be recorded permanently in a durable medium and be readable.
C Contemporaneous The data should be recorded at the time the work is performed and date/time stamps should follow in order.
O Original The information must be the original record or a certified true copy.
A Accurate No errors or editing performed without documented amendments.
+ Complete All data including any test, repeat, or reanalysis performed on the sample.
+ Consistent Consistent generation of records and application of date time stamps in the expected sequence.
+ Enduring Data should be recorded on controlled worksheets, in laboratory notebooks or in validated electronic systems.
+ Available Data needs to be available and accessible for review, audit, or inspection over the lifetime of the record.
44 BioPharm International www.biopharminternational.com March 2016
WHERE SHOULD THE ELECTRONIC DATA TRANSFER BEGIN?Let’s consider preparation of stan-
dards by way of example. In an
analytical lab, standards are usu-
ally prepared using an analyti-
cal balance. The actual weight
is typically printed on a strip
printer and pasted into a lab jour-
nal. Afterwards, the standards are
widely used for analytical meth-
ods, such as high-performance liq-
uid chromatography (HPLC), gas
chromatography (GC), titration, etc.
Data management and documenta-
tion for analytical instruments are
usually managed by dedicated soft-
ware or a laboratory information
management system (LIMS). This
procedure is currently allowed by
regulatory bodies, which state that
print-outs representing original
data for simple devices (such as bal-
ances) are acceptable (2) but is not
the case for more complex devices.
Nevertheless, it is important that
reference standards are accurate
and traceable because they repre-
sent the starting point of many
BOBMZTFT��8BSOJOH�MFUUFST�IBWF�DJUFE�
“no details available on the prep-
aration of standards or solutions,
especially of analytical reference
standards” (13, 14). Independent of
process, it is important to ensure
that all the data are available.
This then begs the question:
8IFSF�TIPVME�UIF�FMFDUSPOJD�USBOT-
fer begin? In the process described
above, there is a gap in the data
transfer between the “simple
instrument” (the balance) and the
“complex instrument” (e.g., the
HPLC). Clearly, this is not recom-
mended because it introduces an
additional level of risk; it is obvi-
ous why no gaps should occur.
Capturing the data at the point
of origin and transferring the data
electronically throughout the
whole workflow is a much lower
risk approach and reduces the risk
of errors during the early stages of
a process, giving additional confi-
dence in the compliance of a work-
flow or laboratory.
WHAT DATA SHOULD BE TRANSFERRED?T h e n e x t q u e s t i o n t h e n
CFDPNFT�� ù8IBU�EBUB�OFFE� UP�CF�
transferred? It is essential to associ-
ate results with metadata to build
context around the values col-
lected. Although integrating even
a simple instrument can be tricky,
the advantages of an integrated
TPMVUJPO� BSF�PCWJPVT��8IFO� FMFD-
tronic data transfer starts from
the beginning of the process, each
piece of information needs to be
input only once for it to be avail-
able throughout the whole system.
This allows seamless movement of
data and other information from
the start of a process to the end,
without the need for manual effort,
such as manual transcription, creat-
ing an efficient work environment.
Data Integrity
Table II: The 10 compliance commandments.
Commandment Comment
1 Management is responsible. Management must take the lead in making certain that the integrity of data in the lab is managed and maintained.
2 Use a networked system, ideally with a database.
Stand-alone systems should not be used in a regulated environment.
3 Document the system configuration and manage all changes to it.
You must document and ensure the configuration protects the records.
4 Work electronically and use electronic signatures.
Try to work electronically wherever possible. The advantage is that the data are maintained within the system. Don’t use a hybrid because you have two incompatible formats (worst possible situation).
5 Allocate each user a unique identity and use adequate password strength.
Ensure that you have unique user identities, so that you can attribute the work to a single individual.
6 Separate roles to avoid conflict of interest. Separate the roles within any computerized system. Typically “Admin” should only be accessible by IT or a small group of people, not standard laboratory workers. Exception if there are only 1–2 users (in which case it is necessary to share the roles).
7 Define methods that can and cannot be adjusted.
Consider which methods within a system can actually be changed and which cannot.
8 Have a standard operating procedure for data manipulation.
For chromatography data systems, a standard operating procedure (SOP) for both automatic and manual integration is necessary.
9 Ensure staff are trained and competent. The need for an SOP is clear, but do people understand it and are they competent to use it? Ensure the people are trained, both in data integrity and the instrumental techniques they are using.
10 Carry out effective self-inspections or internal audits.
Make certain that internal audits don’t just focus on paper. Instead, they should go deeper and look at things within the computerized system.
Contin. on page 48
March 2016 www.biopharminternational.com BioPharm International 45
Mura
t S
en/g
ett
y im
ag
es
Shipping temperature-sensitive
pharmaceuticals, such as biologi-
cal products, has never been easy.
Today, changing global require-
ments and a more complex supply chain
are driving more conservative approaches
to temperature control. For products such
as cell therapies and tissue culture, cryo-
genic shipping is now preferred. Other
biopharmaceuticals are being shipped at
lower tempertures, and even small-mol-
ecule-based pharmaceuticals that might
have been shipped at ambient tempera-
tures a few years ago, must now be kept at
controlled temperatures.
Transport has become much more
than simply moving product from one
place to another. It’s now being seen
as a “mobile form of storage,” says
Volker Kirschner, director of tempera-
ture control solutions for World Courier
Management Co, part of Amerisource
Bergen. This is especially true in Europe,
he says, where the European Union (EU)
began strengthening its good distribution
practice (GDP) guidelines three years ago.
Nothing can be taken for granted.
“From political unrest to pandemic
concerns, the possibility of changes in
shipping patterns and logistics strategies
is quite real,” he says, and developing
a global transport strategy has become
more complex.
Specialists in cold-chain shipment
are responding to regulatory challenges
and uncertainty by investing in new
technology and IT, and strengthening
partnerships with specialty logistics
providers. For their pharmaceutical and
biopharm customers, they caution, care-
ful risk-assessment is the only way to
avoid costly product shipment problems.
Cold Chain: Going the Extra Mile
Agnes Shanley
Real-time GPS technology,
better IT connections,
and more conservative,
controlled shipping
temperatures are improving the shipment
of sensitive pharmaceuticals.
Cold Chain
46 BioPharm International www.biopharminternational.com March 2016
Many are adding services and prod-
ucts to help make this task easier.
THE RISE OF GOOD DISTRIBUTION PRACTICES The most pronounced recent
change in the cold-chain market
has been more complex global
regulations, from the EU’s GDP
guidelines for pharmaceuticals to
the Drug Supply Chain Security
Act in the United States. “It can be
difficult for companies, especially
smaller ones, to know where to
begin, and how to revamp compli-
ance processes to stay ahead,” says
Wanis Kabbaj, director of global
healthcare strategy for UPS.
Additional requirements (e.g.,
for controlled room temperature)
have increased compliance chal-
lenges. Many drugs that were once
shipped at ambient emperatures
must now be shipped at 15 °F to
25 °C, increasing costs at a time
when most pharmaceutical com-
panies are trying to reduce logis-
tic and supply chain operations
budgets, says Ariette Van Strien,
chief commercial officer at Marken
Global Life Science Supply Chain
Solutions, which specializes in cold
chain and logistics.
Compliance has become espe-
cially challenging for clinical trial
supply shipments, she says, noting
that suppliers must scrutinize their
global network, enhance tracking
capabilities, and improve quality
standards.
Of utmost importance, says
Kirschner, are quality management
systems (QMS) and documentation;
personnel and training; risk man-
agement, especially regarding stan-
dard operating procedures (SOPs);
facilities and storage requirements;
and transport. “Quality depart-
ments within manufacturers’ sup-
ply-chain operations are gaining
more and more influence,” he says,
noting that regulatory agencies
expect pharmaceutical manufac-
turers to take a risk-based approach
and enforce audits and quality
agreements.
MANAGING RISKThe most c r it ica l aspec t to
maintaining stability and integ-
rity is choosing the most appropri-
ate temperature conditions for the
product and, consequently, the best
packaging for maintaining those
conditions, says Mark Sawicki,
chief commercial officer at the cold
chain specialist, Cryoport.
A perfect example, Sawicki says, is
the distribution of critical biomarker
samples. Under currently accepted
practice, these samples are shipped
on dry ice. However, he notes, this
shipping method has been con-
nected with a 15–30% product com-
promise or failure rate. As a result,
more companies are opting to use
cryogenic temperatures to ship bio-
markers, to help eliminate potential
risks associated with transit times
and temperature excursions.
PCI Pharma Services, a diversi-
fied contract services firm that offers
cold-chain expertise, has seen a dra-
matic increase in demand for storing
product at ultra-low temperatures,
from -40°C down to -196°C, and has
adapted its products and services to
focus on supporting these temper-
atures for storage, packaging, and
shipments, says Samantha James,
associate director of clinical services.
Pharmaceutical manufactur-
ers need to conduct more critical,
comprehensive shipping studies
on all materials transported in
support of a clinical program,
says Sawicki, not only looking at
sample failure but the impact of
the selected transport medium on
assay or product performance.
“We consult with every client
to select the best system and tem-
perature-control solution, whether
dry ice, liquid nitrogen, or phase-
change materials (PCMs) to ship
pharmaceutical products, diagnos-
tic specimens, biotherapeutics, and
tissue samples,” says Van Strien.
Generally, the most crucial fac-
tors to consider include therapeutic
indication, route, mode of trans-
portation, and product sensitivity,
she says. “Because a pharmaceuti-
cal’s stability is not always known
in early trial phases, additional
caution may be necessary, and pro-
tection for any changes in environ-
mental conditions.”
Van Strien advocates developing
a thorough risk assessment to help
ensure product stability from ori-
gin to destination, including stud-
ies of the following:
t� 1BDLBHJOH�
t� 1BZMPBE�DPOGJHVSBUJPO
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strategy.
For clinical products that are
only available in small samples,
incorrectly handled shipping and
transportation can put millions or
even billions of revenue dollars at
risk by compromising clinical stud-
ies, potentially delaying product
commercialization, notes Kirschner.
When minimizing risk, it is impor-
tant for manufacturers to consider
the package type and choice of tem-
perature-control mechanisms, envi-
ronmental concerns, value of the
ingredients in the product, temper-
ature requirements and variables,
and overall shipping costs.
Suppliers need to master non-
technical issues as well, including
local politics and security issues,
especially when conducting clini-
cal trials in new geographic mar-
kets. Proactive planning is required,
Kirschner says, to help protect the
integrity of products and, ulti-
mately, the safety of patients who
use those pharmaceuticals.
Generally, he notes, up-to-date
methods should be used to assess
risk. For example, he says, failure
mode and effects analysis (FMEA)
can be conducted, and risk prior-
ity numbers (RPNs) assigned, based
Cold Chain
March 2016 www.biopharminternational.com BioPharm International 47
on the probabilities and severities
of certain adverse events occur-
ring during transport. These RPNs
would help determine whether mit-
igation is required, and what type
would work best for the specific
product, market, and application.
Temperature is not the only
variable to consider. Many phar-
maceuticals and biopharma man-
ufacturers require a great deal of
stability protection from temper-
ature, shock, vibration, humid-
ity, and light, explains Kabbaj.
“Healthcare logistics has little
room for error, so specialized pack-
aging, advanced temperature and
location monitoring leveraging
global control towers, and in-tran-
sit intervention are all critical.”
In add it ion, Kabbaj says ,
extended time in transit when
product is being transported inland
may expose the packaging to tem-
perature fluctuations that increase
risks. Ocean transportation risk fac-
tors include container placement
on the vessel, sunlight exposure,
container insulation, and dwell
time on the dock—all introducing
additional packaging stressors.
He suggests that pharmaceuti-
cal manufacturing professionals
take the following steps to ensure
that they are covering all the bases
required for risk mitigation:
t� Test packaging, including seals,
for endurance, and put prototypes
t hrough compression, impac t ,
vibration, temperature, humidity,
and shock tests. Package optimi-
zation should include validation
through design qualification,
operational qualification, and
per for mance qua l i f icat ion
protocols.
t� Optimize packaging and transpor-
tation together. Do not develop
a cold-chain solution sequen-
tially with assumptions based
on transportation, and then
the transportation solution that
comes with pack-out expiry.
Pairing a lighter and cheaper
cold-chain packaging with expe-
dited air transportation, for
example, may prove to be more
effective than opting for a slower
mode of transportation (e.g.,
ground shipping) with complex,
heavy, and bulky packaging.
t� Innovate continually to create
bet ter ef f iciencies. Opt imize
the shipping carton, mini-
miz ing unused space, and
select more precise packaging
configurations.
“Pharmaceutical manufactur-
ers that do not continually review,
evaluate, and update packaging
best practices leave themselves
open to product risk and cost inef-
ficiencies,” Kabbaj says. “Leave
room for new ideas, material inno-
vations, and new packaging manu-
facturers by engaging cold chain
partners in a collaborative mode.”
TECHNOLOGIES ENABLE FASTER RESPONSECold-chain service providers have
been developing new technologies
to help pharmaceutical companies
better manage risk and respond to
problems. “New requirements are
driving developments in packag-
ing and monitoring technologies,”
says Kirschner. As an example, he
points to newer semi-active pack-
aging technologies that allow for
longer durations of temperature
control with lighter materials that
have less environmental impact.
In addition, he says, passive pack-
aging solutions have been devel-
oped that can provide proven
temperature control over long dis-
tances. Independent testing has
shown these solutions to perform
five to seven times more efficiently
than semi-active solutions, he says.
Kirschner also points to new phase-
change materials (PCMs), comprised
of paraffin or salt-based solutions,
that allow for more precise tem-
perature control to maintain prod-
uct stability over long distances or
through extreme climates.
Many packaging manufactur-
ers are now developing their own
vacuum-insulated panel (VIP)
containers, combined with PCM
solutions, for easier handling and
storage. Additionally, he says, on
the monitoring side, developments
in global positioning software
(GPS) and tracking equipment
now include automatic start-up
and shutdown mechanisms that
can provide a real-time view into
a shipment’s status. Many tech-
nology providers have invested in
real-time, GPS-enabled data log-
gers. Combined with improved
data access, through customer
portals and better IT, the technol-
ogy offers immediate insights into
bottlenecks and delays.
LOGGING MORE THAN JUST TEMPERATURE“These data loggers provide the
ability to track both condition and
chain-of-custody, in real time,”
says Sawicki. They can track, not
just temperature, but also monitor
the impact of external influences
on container integrity including
orientation and package damage,
he adds, and can also tell whether
the container has been opened.
Examples of new cold-chain
data loggers include Sensitech’s
TempTale and Nexleaf’s ColdTrace
systems. Contract service suppli-
ers are also offering enhanced IT
connections. PCI Pharma Services,
for instance, has developed a por-
tal to allow clients to access real-
time data, 24/7. “They can monitor
stock levels, and view and trace
all live shipments associated with
their project, with direct links to
courier tracking,” says James.
Last year, Marken introduced
Sentry, a GPS-enabled sensor plat-
form, designed specifically for
pharma with real-time track and
trace capability through custom-
designed software that monitors,
records, and reports on location,
temperature, motion, shock, expo-
Cold Chain
48 BioPharm International www.biopharminternational.com March 2016
Cold Chain
sure to light (i.e., when the box
opens), atmospheric pressure, and
remaining battery life. The device
transmits in real time and commu-
nicates through customized cloud-
based software that connects with
Marken’s Maestro IT system, says
Van Strien.
Today, major cold-chain trends
include increased use of temper-
ature-managed shipping, and an
interest in evaluating transport
methods and equipment critically,
to maximize sample integrity, says
Sawicki. There is also an increased
emphasis on protecting the prod-
ucts of cell, gene, and immuno-
therapy, says Van Strien, and the
complex supply chain logistics that
these products require.
CRYOGENIC SHIPMENT FOR CELL THERAPIESA growing number of biopharm
companies must now ship cell- and
gene-based therapies, says Kabbaj.
Such products require cryogenic
storage at temperatures -238 °F
and below. In the past, shippers
relied on dry ice (which performs
best at approximately -108.4 °C),
he explains, but dry ice emits car-
bon gas, which can damage pro-
teins in biologic shipments, and
might be harmful to handlers and
to the environment. “Not only is
dry ice shipping subject to a wide
range of international regulations,
it simply does not provide a cold
enough temperature for many new
specialty therapies” he says.
Yet another important trend,
says Van Str ien, is increased
demand of remote shipping, for
example, of clinical trial dosages
to patients’ homes. “We will con-
tinue to develop further services
to allow patients to participate
in clinical trials from home, or
remote sites of their choosing,”
she says.
Marken has also introduced an
enhanced online booking app
for investigator sites that is pre-
programmed by study. It has also
integrated enhanced scanning
technologies throughout its global
network. “More mobile technol-
ogy will help treat patients at
locations of their choosing,” says
Van Strien. ◆
This transfer of data is achiev-
able using a laboratory execu-
tion system (LES), such as LabX,
which enables a variety of instru-
ments to be directly connected
( ba lances , t it rator s , densit y
meters, refractometers, thermal
analysis instruments, and pH
meters). Useful features such as
SOP-user guidance on the instru-
ment terminal, automatic results
capture in a database, and real-
time data access support trace-
ability of data and compliance
with the GAMP data integrity cri-
teria in ALCOA+.
As regulators continue to tighten
their inspection approaches, it is
crucial for managers and scientists
in regulated GXP laboratories to
understand these criteria for data
integrity and to assess and improve
laboratory data management
processes to ensure compliance
with current regulations. Only
after all these points have been
addressed can data integrity truly
be achieved.
REFERENCES 1. C. Rosa, “Current Regulatory/Inspection
Issues Related to Supply Chain,” Food and Drug Law Institute (FDLI), Conference Understanding cGMPS–What Attorneys Need to Know, Washington DC, July 15, 2014, www.fdli.org/docs/cgmps/carmelo-rosa.pdf?sfvrsn=0
2. MHRA, Guidance for Industry on Data
Integrity (MHRA, March 2015), www.gov.uk/government/uploads/system/uploads/attachment_data/file/412735/Data_integrity_definitions_and_guidance_v2.pdf
3. FDA, “Glossary of Computer System Software Development Terminology,” 1995, www.fda.gov/iceci/inspections/inspectionguides/ucm074875.htm
4. European Commission, EudraLex, The
Rules Governing Medicinal Products in
the European Union, Volume 4, Good
Manufacturing Practice, Chapter 4 Documentation (June 2011), http://ec.europa.eu/health/files/eudralex/vol-4/chapter4_01-2011_en.pdf
5. European Commission, EudraLex, The
Rules Governing Medicinal Products in the
European Union, Volume 4, Good
Manufacturing Practice, Annex 11 Computerised Systems (January 2011), http://ec.europa.eu/health/files/eudralex/vol-4/annex11_01-2011_en.pdf
6. US Electronic Code of Federal
Regulations, 21 CFR 211.194(a). 7. FDA, FDA Warning Letter to Trifarma
S.p.A, July 2014, www.fda.gov/ICECI/enforcementactions/warningletters/ 2014/ucm404316.htm
8. FDA, FDA Warning Letter to Ipca Laboratories Ltd., January 2016, www.fda.gov/ICECI/enforcementactions/warningletters/ucm484910.htm
9. FDA, FDA Warning Letter to Micro Laboratories Ltd., January 2015, www.fda.gov/iceci/enforcementactions/warningletters/2015/ucm431456.htm
10. FDA, Questions and Answers on Current
Good Manufacturing Practices, Good
Guidance Practices, Level 2 Guidance–Records and Reports, www.fda.gov/Drugs/ Guidance ComplianceRegulatoryInformation/Guidances/ ucm124787.htm
11. EMA, GCP Inspectors Working Group publication, Reflection paper on expectations for electronic source data and data transcribed to electronic data collection tools in clinical trials (London, June 2010), www.ema.europa.eu/docs/en_GB/document_library/Regulatory_and_procedural_guideline/2010/08/WC500095754.pdf
12. R.D. McDowall, LCGC 27 (9) (September 2014), www.chromatographyonline.com/role-chromatography-data-systems-fraud-and-falsification
13. FDA, FDA Warning Letter to RPG Life Sciences Ltd., May 2013, www.fda.gov/ICECI/EnforcementActions/WarningLetters/2013/ucm355294.htm
14. FDA, FDA Warning Letter to Wockhardt Ltd., July 2013, www.fda.gov/ICECI/EnforcementActions/WarningLetters/ 2013/ucm361928.htm ◆
Data Integrity—Contin. from page 44
March 2016 www.biopharminternational.com BioPharm International 49
Ask the Expert
retrieved and sent for analysis. The
green gel was identified as contain-
ing copper. The company was still
convinced that the contamination
was due to poor quality control on
the part of the vial manufacturer
and continued operations and work-
ing with the vial manufacturer to
determine the source of the copper.
To help facilitate the investigation,
the firm hired a consultant to go to
the glass manufacturer and reaudit
their facility. The auditor could not
identify the source of the copper at
the vial manufacturer and recom-
mended that the company reevalu-
ate their operations for the presence
of copper. The company continued
to manufacture product but agreed
to reevaluate the facility for potential
sources of copper.
This time the facilities and main-
tenance personnel opened up the
line as they would during a routine
maintenance shut down. When they
opened the depyrogenation tunnel,
they discovered the presence of cop-
per on the top of the HEPA filters as
well as a coating of green on the side
doors to the tunnel. Manufacturing
was finally halted on the line until
the in-depth evaluation could be
performed.
The root cause of the problem was
determined to be a faulty cooling
valve in the depyrogenation tunnel,
which was identified as a potential
root cause but ultimately not pur-
sued because it was at the bottom of
the probability list and rated as pos-
sible but highly unlikely. The tunnel
in question was 20 years old and was
bulit with copper piping above the
line and the HEPA filters.
Although the line was routinely
maintained and checked, there was
no alarm associated with the cool-
ing valves to indicate a failure. The
failing valve caused liquid to con-
dense on the copper lines and drip
onto the HEPA filters. As more and
more liquid collected on the fil-
ters, the stress caused the filters to
breach, relieving the pressure. This
breaching happened on a predict-
able seven-day cycle. Ultimately, the
incident involved the investigation
of lots manufactured on the line
over a sixth-month period, which
was the last time the cooling valve
was inspected and determined to be
functioning to standards. Once the
problem was properly identified, the
effective corrective action could be
taken; however, by that time, 28
lots of product manufactured for
several different clients during the
two-month period were rejected
because of the presence of green
vials intermittantly detected dur-
ing the inspection process. If the
site had conducted a thorough
investigation and cleared their
equipment, personnel, etc., by pur-
suing the identified root causes
before jumping to the conclusion
the vial manufacturer was at fault,
they could have stopped manufac-
turing and corrected the problem
before the loss of 28 lots of product.
CONCLUSIONThe bottom line is there are many
perspectives on what constitutes
a good CAPA system, but the real-
ity is the quality and thoroughness
of the investigations ultimately
drive the effectiveness of the CAPA.
When conducting the investiga-
tion, it is important not to jump
to conclusions on what caused the
non-conformance. The investiga-
tion should use root-cause analy-
sis tools and should address why
potential areas are either elim-
inated as the root cause or are a
potential cause of the non-con-
formance. If you can conduct a
complete investigation, you will
ultimately have a robust CAPA
program. ◆
Your opinion matters.
Have a common regulatory or compliance question? Send it to [email protected] and it may appear in a future column.
The key to a robust
CAPA system lies in
the thoroughness
and quality of the
investigation.
Ad Index
Company Page
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EMD MILLIPORE 5
EUROFINS LANCASTER LABORATORIES 11
INTERPHEX 25
SAFC BIOSCIENCES SIGMA ALDRICH 2, 21
TOSOH BIOSCIENCE 15
VETTER PHARMA-FERTIGUNG GMBH 13
WATERS CORP 51
WOODLEY EQUIPMENT CO 23
WUXI APP TEC 52
Ask The Expert—Contin. from page 50
50 BioPharm International www.biopharminternational.com March 2016
Fa
na
tic S
tud
io/G
ett
y I
ma
ge
s
Susan Schniepp and Andrew Harrison, both of Regulatory
Compliance Associates, discuss how to create a
robust CAPA system and how to identify root cause.
Q: I work for a contract manufacturer,
and I think we have a fairly robust cor-
rective actions and preventive actions (CAPA)
system. Sometimes we close our CAPAs before
we have completed our effectiveness check due
to the timeline needed to implement and mea-
sure the effectiveness of the preventive action.
Occasionally, we fail our effectiveness checks,
requiring us to reopen our CAPA. This creates a
lot of concern during audits. Is there something
I can do to prevent this from happening?
A: The key to a robust CAPA system lies
in the thoroughness and quality of the
investigation. Based on the limited informa-
tion, it sounds like you are having trouble rec-
ognizing the root cause(s) identified during the
investigation. The investigation process should
make use of root-cause analysis tools designed
to examine the impact of the equipment, pro-
cess, people, materials, environment, and man-
agement on the identified non-conformance.
The investigation process should review each
possible root cause; the investigators should
either eliminate it or it should become part of
the corrective action. As the elimination pro-
cess progresses, the investigation will naturally
and logically hone in on the root cause(s) of
the non-conformance. Any potential root cause
that can’t be eliminated needs to be remedi-
ated. Most companies make the mistake of
stopping too soon and not pursuing all the
possible root causes that can’t be eliminated in
the investigation stage, which leaves them in
the situation you have described.
ROOT CAUSE CASE STUDYIt might help if we look at a situation where
a probable root cause was identified but not
pursued as part of the preventive action. The
incident involved the detection of a discolored
vial during inspection of lyophilized vials. The
vials inspectors discovered several green-hued
vials before the packaging phase of the opera-
tion. What seemed to be a simple issue isolated
to one batch grew and ultimately affected more
than 28 batches produced over a two-month
period. Several green-hued vials were discov-
ered during inspection of a lyophilized batch
of product that was produced on a 20-year-old
automated line.
The inspectors that discoved the vials imme-
diately informed the quality department, and
an investigation was opened. The lot was put
on quality assurance (QA) hold, and the vials
were sent out for analysis. Manufacturing on
the line was continued while the investigation
was being performed. The results of the analysis
indicated the green color in the vial was due to
the presence of copper. The firm was unable to
determine the source of copper in their opera-
tion and concluded the copper was most prob-
ably due to contamination of the vial at the vial
manufacturer and the investigation was closed.
Seven days after the detection of the first
green vials during the manufacturing of a
different lyophilized product, the same issue
occurred. The original investigation was re-
opened and a for-cause audit was performed at
the vial manufacturer. The results of the for-
cause audit were inconclusive with no defini-
tive source of the copper identified. During
the investigation into the vial manufacturer,
the company continued to manufacturer other
products and implemented a 100% inspection
of incoming vials before use.
Seven days later, a line operator noticed a vial
exiting the depyrogenation tunnel that con-
tained a green, gel-like blob in it. The manu-
facturing run was stopped, and the vial was
Creating Robust CAPA Systems
Susan Schniepp is distinguished fellow, and Andrew
Harrison is chief regulatory affairs officer and general
counsel, both of Regulatory Compliance Associates.
Ask the Expert
Contin. on page 49
Combine our core organizational knowledge with an unmatched breadth of
capabilities, powerful technologies and robust workflows, and it’s easy to see
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