The Science & Business of Biopharmaceuticals
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Volume 28 Number 9
CMOs SPECIALIZE TO BUILD BETTER BIOLOGIC DRUGS
OUTSOURCING
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PROCESSING
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ES660010_BP0915_CVTP2_FP.pgs 08.20.2015 19:04 ADV blackyellowmagentacyan
The Science & Business of Biopharmaceuticals
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Volume 28 Number 9
CMOs speCialize tO build better biOlOgiC drugs
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ES668476_BP0915_CV2_FP.pgs 09.03.2015 02:31 ADV blackyellowmagentacyan
INTERNATIONAL
The Science & Business of Biopharmaceuticals
EDITORIALEditorial Director Rita Peters [email protected] Editor Agnes Shanley [email protected] Editor Susan Haigney [email protected] Editor Randi Hernandez [email protected] Science Editor Adeline Siew, PhD [email protected] Director Dan Ward [email protected] 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
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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 Principal Scientist, Analytical
Biochemistry, MedImmune, Inc.
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
EMD Millipore Corporation
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 Managing Director
Arlenda, USA
Paula Shadle Principal Consultant,
Shadle Consulting
Alexander F. Sito President,
BioValidation
Michiel E. Ultee Principal
Ulteemit BioConsulting
Thomas J. Vanden Boom Vice-President, Global Biologics R&D
Hospira, Inc.
Krish Venkat CSO
AnVen Research
Steven Walfish Principal Statistician
BD
Gary Walsh Professor
Department of Chemical and
Environmental Sciences and Materials
and Surface Science Institute
University of Limerick, Ireland
ES670766_BP0915_003.pgs 09.09.2015 22:54 ADV blackyellowmagentacyan
4 BioPharm International www.biopharminternational.com September 2015
Contents
INTERNATIONAL
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
BioPharm International ISSN 1542-166X (print); ISSN 1939-1862 (digital) is published monthly by UBM Life Sciences 131 W. First Street, Duluth, MN 55802-2065. Subscription rates: $76 for one year in the United States and Possessions; $103 for one year in Canada and Mexico; all other countries $146 for one year. Single copies (prepaid only): $8 in the United States; $10 all other countries. Back issues, if available: $21 in the United States, $26 all other countries. Add $6.75 per order for shipping and handling. Periodicals postage paid at Duluth, MN 55806, and additional mailing offices. Postmaster Please send address changes to BioPharm International, PO Box 6128, Duluth, MN 55806-6128, USA. PUBLICATIONS MAIL AGREEMENT NO. 40612608, Return Undeliverable Canadian Addresses to: IMEX Global Solutions, P. O. Box 25542, London, ON N6C 6B2, CANADA. Canadian GST number: R-124213133RT001. Printed in U.S.A.
BioPharm International is selectively abstracted or indexed in: • Biological Sciences Database (Cambridge Scientifc Abstracts) • Biotechnology and Bioengineering Database (Cambridge Scientifc Abstracts) • Biotechnology Citation Index (ISI/Thomson Scientifc) • Chemical Abstracts (CAS) • Science Citation Index Expanded (ISI/Thomson Scientifc) • Web of Science (ISI/Thomson Scientifc)
6 Guest Editorial Ensuring data integrity involves effort on an individual and global basis. Richard M. Johnson
8 Global News
10 Regulatory Beat Legislation to streamline drug development may get tangled up in user fee negotiations and drug pricing battles.Jill Wechsler
14 Perspectives on Outsourcing Suppliers indicate prices for single-use equipment are likely to increase. Eric Langer
18 Industry Insider FDA’s proposed guidance for quality metrics raises questions about quantifying the tangibles and intangibles of quality culture. Andrew Harrison and Susan Schniepp
52 Troubleshooting Three case studies illustrate some analytical methods important for stability testing. Stella-Christiana Chotou
55 Product Spotlight
56 New Technology Showcase
57 Ad Index
58 Vaccines Development Update
Contract Biomanufacturing Firms Become More SpecializedRandi Hernandez
Focusing on niche and specialty service
offerings gives contract biomanufacturing
organizations an opportunity to
differentiate in a crowded market. 22
High Titers and Perfusion Processes Challenge Cell Harvesting SystemsCynthia A. Challener
New single-use technologies and other
filtration systems are beginning to address
cost, throughput, and manufacturing
footprint demands. 28
Best Practices in Qualification of Single-Use SystemsWeibing Ding
The author discusses the current best
practices in technical qualification of
single-use systems. 32
Enhancing Protein Binding Studies with a Light-Scattering ToolkitDaniel Some
Light-scattering techniques are useful
for interaction studies when traditional
methods of analysis do not suffice. 40
Utilizing Run Rules for Effective Monitoring in ManufacturingAaron Spence
To enable efficient monitoring
systems, life-science companies
need to effectively apply run rules. 48
Volume 28 Number 9 September 2015
Dieter Spannknebel/Stocktrek Images/Getty Images; Dan Ward
ES670683_BP0915_004.pgs 09.09.2015 19:45 ADV blackyellowmagentacyan
www.gelifesciences.com/BioProcessGE, GE monogram, and Xcellerex are trademarks of General Electric Company. © 2014–2015 General Electric Company. First published Apr. 2014. GE Healthcare Bio-Sciences AB, Björkgatan 30, 751 84 Uppsala, Sweden.
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6 BioPharm International www.biopharminternational.com September 2015
Guest Editorial
Ensuring data
integrity involves
effort on
an individual
and global basis.
Data Integrity: Getting Back to Basics
I have been involved in the healthcare products industry for all of my profes-
sional life, more than 35 years in varied environments and operations in the
United States, Europe, Asia, Australia, and Latin America. During that time,
I have seen many changes in the manufacture and control of pharmaceutical
and biopharmaceutical products.
Some changes are inherent in the newer products that have been introduced,
but many of these changes have been technological: the advent of computer-
ized systems; instruments and sensors to monitor and control machine func-
tion; increasingly sophisticated software; and the ability to communicate data
via wire and wirelessly around the world.
One of the ways that we have applied these technologies is through the
generation of volumes of data that were previously either not accessible or
impractical to analyze. It is through the generation of data, and the review
of these data by knowledgeable persons in comparison to specifications and
standards, that we base our quality decisions. Even the least sophisticated
operation today employs electronic systems for many applications: inventory
control, analytical data capture and analysis, equipment control, and report-
ing. As these changes have occurred, there have been evolving changes in the
regulatory focus—from quality control and an emphasis on testing quality—to
quality assurance and process validation—to quality by design and lifecycle
management.
The human elementBut while electronic systems are becoming more widely used and generating
ever larger amounts of data, it is also true that all of our operations depend
on a network of people to perform a wide variety of functions, from material
manufacturing, transportation, finished-product manufacturing, testing, dis-
tribution, regulatory filings, and controlling records that involve a combina-
tion of both electronic and paper-based records.
The global elementIn addition, there is a growing globalization of the business and the depen-
dence on partners from outside the primary organization. Activities that used
to be done within a single organization are more likely today to include a net-
work of separate organizations that are interdependent on goods, services, and
data exchange. Many of the healthcare products used by patients around the
world involve manufacturers and suppliers from multiple continents who com-
municate in multiple languages.
The data elementA basic principle of assuring the quality of healthcare products is the review
of data. Industry experts review data from their partners; independent quality
groups review manufacturing and testing data; and regulators who are respon-
sible for monitoring the products for the public review all data. The accuracy,
trustworthiness, and the integrity of that data must not be in question, or all
of the checks and balances, control measures, and quality agreements will not
be effective. This is equally true of the traditional paper-based records and
electronic data.
The recent emphasis on data integrity is not new, but it has never been more
crucial. This principle should remind us of a basic tenet: All relationships are
based on trust and evidence. If the integrity of one’s data is questionable, the
loss of trust will have severe consequences. We should all be committed to
reinforcing the importance of data integrity. ◆
Richard M. Johnson is the president and CEO of the
Parenteral Drug Association.
ES668635_BP0915_006.pgs 09.03.2015 19:48 ADV blackyellowmagentacyan
ES670762_BP0915_007_FP.pgs 09.09.2015 21:33 ADV blackyellowmagentacyan
FDA Releases Guidance on
Biosimilar NomenclatureThe long-awaited draft guidance on biosimilar naming
has been posted by FDA, and within the document,
the agency suggests naming follow-on biologics by a
common nonproprietary name, followed by a suffix that
will identify the manufacturer of the particular biosimilar.
The naming scheme is consistent with the way the agency
labeled Zarxio (filgrastim-sndz), the first biosimilar
product to be approved by FDA. The naming convention
will be used for all future biosimilar products and will also
be applied retroactively, meaning the naming convention
will apply to biologic products (submitted for approval
through the 351[k] or 351[a] pathways) that are already on
the market.
The renaming of all biologic medications has a dual
purpose, says FDA: to facilitate the “routine” use of
suffixes for biologics, and to “avoid inaccurate perceptions
of the safety and efficacy of biological products based
on their licensure pathway.” In other words, FDA does
not want healthcare providers to judge a drug’s quality
based on its name. As such, FDA will use the same
nonproprietary name for biosimilars (i.e., the generic or
“core name”) plus a manufacturer-specific suffix; taken
together, the core name and suffix will produce a “proper
name”.
Although FDA did not provide a full rollout plan for
how all the existing biologics will be renamed, it did
say that it intends to “assign distinguishing suffixes
to a limited group of these products.” The originator
biologics that will get new designations first are those
reference products that will be susceptible to biosimilar
competition in the near future, and FDA is considering
a rulemaking action to implement the retrospective
naming of biologics. For future products, manufacturers
will propose suffixes for their products themselves in
application documents. It is still unclear if these suffixes
will be required to directly reference the manufacturer, as
in the case of Sandoz’s filgrastim-sndz, or if the suffix will
be chosen based on other parameters, but FDA said the
suffix should be unique, “devoid of meaning”, and should
not be promotional. For example, proposing adalimumab-
best as a proper name for Humira would likely be blocked
by the agency. The suffix also cannot be a commonly used
abbreviation in clinical practice, contain reference to a
drug core name, or be confusingly similar to the originator
product’s suffix (once assigned) or to a similar product’s
suffix.
Some biological products will be exempt from the
renaming initiative, says FDA; these include products
“for which a proper name is provided in the regulations”
and those medications that already have robust
pharmacovigilance programs in place. FDA attests that
current originator products will benefit from a suffix
title, as it will help with pharmacovigilance efforts. It’s
unclear how many originator biologics will actually be
renamed, as the directives in the FDA guidance are meant
as suggestions, not requirements, and it may be difficult
to assess which drugs already have “proper names” by FDA
standards and which ones do not.
Though FDA said the naming protocol is meant to
prevent inadvertent substitution among biologic products
that have not been determined to be interchangeable,
the agency did not say how it will label interchangeable
biologics and how these are to be differentiated among
the other products in the same class. All the agency
said was that it was “considering whether the suffix
should be unique or should be the same as the reference
product.” If an interchangeable product shared the
same suffix as the originator, however, the tracking of
a drug’s safety by its suffix would not be feasible, and
pharmacovigilance problems could persist. Determination
of interchangeability and the naming of interchangeable
products will be covered in a future guidance, and FDA
requested feedback on how to name interchangeable
products.
—Randi Hernandez
GSK to Divest Ofatumumab
to Novartis Pharma
GlaxoSmithKline plc announced on Aug. 21, 2015 an
agreement with Novartis Pharma AG, a subsidiary of
Novartis AG, to divest its rights in ofatumumab for
auto-immune indications, including multiple sclerosis
for up to $1 billion, plus royalties.
Earlier this year, Novartis Pharma acquired the
oncology indications for ofatumumab (Arzerra) as
part of a three-part transaction between GSK and
Novartis. After completion of the latest transaction,
Novartis Pharma will own rights to ofatumumab in
all indications.
The consideration payable by Novartis Pharma to
GSK comprises milestone payments of $300 million
payable at closing; $200 million payable subject to
the start of a phase III study in relapsing remitting
multiple sclerosis by Novartis; and further contingent
payments of up to $534 million payable on the
achievement of certain other development milestones.
Novartis Pharma will also pay royalties of up to
12% to GSK on any future net sales of ofatumumab in
auto-immune indications.
The transaction is expected to complete by the end
of 2015. Glo
be im
ag
e: C
ha
d B
ake
r/G
ett
y I
ma
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s; P
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to C
red
it:
8 BioPharm International www.biopharminternational.com September 2015
Global News
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10 BioPharm International www.biopharminternational.com September 2015
Regulatory Beat
Vis
ion
so
fAm
eri
ca
/Jo
e S
oh
m/G
ett
y Im
ag
es
The later months of 2015 wi l l be
important in determining the fate of
a number of programs slated to shape
drug development and industry health in
the years ahead. Legislation to advance bio-
pharmaceutical R&D faces hurdles on Capitol
Hill, despite overwhelming House approval
in July 2015. Important user fee negotiations
between FDA and biopharmaceutical compa-
nies have begun, but tight timeframes and
competing demands will make it difficult to
resolve issues quickly. A mounting backlash
against high price tags on important new
drugs, moreover, may bolster opposition to
initiatives designed to speed new therapies to
market.
Mov ing the 21st Cent ur y Cures Ac t
through Congress in 2015 will be tough, as
the Senate seeks changes and legislators con-
tend with the usual impasse over the federal
budget for fiscal year 2016, as well as debate
over the Iran nuclear arms pact and major
trade agreements. Next year will be even
worse, as the presidential election campaign
heats up, coinciding for the first time with
the process for re-authorizing user-
fee agreements. With a new admin-
istration and a new Congress in
January 2017, FDA fees and legisla-
tion need to be ready for review by
both outgoing and incoming offi-
cials by the middle of next year.
ProsPects for “cures”Despite broad enthusiasm for the
Cures legislation, the bi-partisan
support that moved it through the
House may fall apart in the com-
ing months. The measure nearly
faltered due to opposit ion from
consu mer advocate s c la i m i ng
it would bring more unsafe drugs to mar-
ket, and from fiscal conservatives opposed
to increased mandatory spending for the
National Institutes of Health (NIH). But
House Energ y & Commerce Committee
Chairman Fred Upton (R-Mich) and his allies
garnered support from hundreds of patient
groups, research organizations, medical soci-
eties, and biopharmaceutical companies, all
eager to increase basic research at NIH and
to provide FDA with added resources. These
and other stakeholders applauded provisions
to more fully incorporate patient experi-
ences in considering a drug’s benefits and
risks, expedite new drug approvals through
biomarker qualification, streamline clinical
trials, encourage sponsors to use data from
clinical experience, and provide incentives
for developing antibiotics and treatments for
rare diseases (1).
The House measure now faces revisions
in the Senate, where there is talk of devel-
oping a much narrower bill by fall. Some
Senate Republicans strongly oppose NIH bud-
get increases, which would erode Democratic
support. If the Senate does enact a slimmer
bill, it would face difficult conference com-
mittee negotiations with the House, and the
process could extend well into next year.
focus on feesConsequently, many provisions of the Cures
legislation may end up in a broad FDA bill
to reauthorize user fees for drugs, generic
drugs, medical devices, and biosimilars by
summer 2017. FDA launched negotiations
in June 2015 for revising the Generic Drug
User Fee Act (GDUFA II) and in July 2015 for
medical devices and for prescription drugs
(PDUFA VI). Monthly discussions with indus-
try will begin in September 2015, along with
Manufacturers Face Key Policy and Regulatory ChallengesLegislation to streamline drug development may get tangled up in user fee negotiations and drug pricing battles.
Jill Wechsler is BioPharm
International’s Washington editor,
chevy chase, MD, 301.656.4634,
ES670435_BP0915_010.pgs 09.09.2015 01:54 ADV blackyellowmagentacyan
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12 BioPharm International www.biopharminternational.com September 2015
regulatory Beatregulatory Beat
regular meetings for FDA to hear
the views of patients, consumers,
and health professionals.
The importance of user-fee
programs to FDA was evident at
the PDUFA meeting with both
act ing commissioner Stephen
Ost rof f and deput y commis -
sioner Rober t Cal i f f of fer ing
comments, along with Center for
Drug Evaluation and Research
(CDER) Director Janet Woodcock.
But fast agreements and leaner
authorizing measures won’t be
easy to achieve. Generic-drug
makers are unhappy with FDA’s
slow pace in meeting the goals
set by the init ial GDUFA pro-
gram, noting that timeframes for
reviewing abbreviated new drug
applications (ANDAs) actually
have gone up, and there has been
little gain in whittling down the
massive ANDA backlog. Slow FDA
approval of new generic drugs
only aggravates shortage situa-
tions and delays patient access
to important, affordable medi-
cines, industry reps observed at
the June 2015 meeting.
Generic-drug firms also com-
plained about r ising fees, par-
t icu la rly when FDA had not
spent a big chunk of the money
it already has collected. Small
companies want relief in facility
fees, especially companies seek-
ing their f irst approved prod-
uct. Manufacturers urged greater
t ransparency and better com-
munication on agency decisions,
notably about a stiffer “refuse to
accept” policy that permits the
agency to reject ANDAs lacking
key components.
At the PDUFA public meeting,
patient groups and professional
organizations offered a range
of initiatives meriting user fee
support: more biomarker vali-
dation, pediatric and neonatal
drug development, data transpar-
ency initiatives, and greater con-
sistency in review practices by
review divisions. A main theme
was expanding the use of “real-
world” evidence to accelerate
drug development. Greg Daniel
of the Brook ings Inst itut ion
highlighted strategies for tap-
ping clinical evidence to support
agency decisions and to docu-
ment product safety, while Allan
Coukell of the Pew Charitable
Trusts emphasized the value of
observational data from claims
databases.
FDA is expected to seek access
to some portion of drug applica-
tion fees during product devel-
opment, instead of waiting until
f iling. Biosimilar sponsors pay
a portion of the fee upfront to
support CDER’s time and invest-
ment in product development
meet ings, a process that has
expanded with the proliferation
of breakthrough drugs and other
cruc ia l medic ines. Current ly,
only biosimilar sponsors pay a
fee to FDA even if no applica-
tion is filed at the end, points
out Gillian Woollett of Avalere
Health, a model that FDA would
like to extend to other products.
Pricing PerilsUser-fee negotiations assume a
certain number of production
faci l it ies and applicat ions for
drugs and medical products each
year to generate expected fees.
But these project ions may be
tempered by a slowdown in bio-
pharma R&D and sales, as payers
and health organizations attack
companies for setting unaccept-
ably high prices, as seen with the
recent emergence of important
new treatments for hepatitis C.
Oncologists are livid over soar-
ing prices for new cancer drugs,
as seen in a July 2015 editorial
in the Mayo Clinic Proceedings
signed by 118 physicians that
urge action to halt the r ising
prices (2). The American Society
of Clinical Oncology has devel-
oped a Cancer Value Framework
to relate the value of a drug and
its price to clinical benefits and
toxicities, a strategy applauded
by pharmacy benefit managers.
FDA approval of new PCSK9
inhibitors to manage cholesterol,
which could be used by millions
of patients, has set off a cam-
paign by payers and insurers to
limit indications and prescrib-
ing. Praluent (alirocumab), devel-
oped by Sanofi and Regeneron
Pharmaceuticals, was launched
in Ju ly 2015 with a $14,600
list price per year—much more
than anticipated and way more
than currently avai lable cho-
lesterol meds, but with labeling
designed to limit use to seriously
ill patients. There is much hope
for new treatments to prevent
Alzheimer’s disease, but already
strong pushback against poten-
tially high costs.
The Institute for Clinical and
Economic Review (ICER) is devel-
oping an assessment of the value
of PCSK9 inhibitors as part of
its expanded program to pro-
vide independent reports on the
cost- and comparative-effective-
ness and budget impact of new
drugs where the value evidence
is controversial and where the
budget impact may be quite high,
as with treatments for cancer,
asthma, and diabetes. The esca-
lating focus on drug “value” will
be important to policies and pro-
grams for biopharma R&D, pro-
duction, and marketing.
references 1. Rules Committee Print 114-22 Text
Of H.R. 6, 21st Century Cures Act,
http://docs.house.gov/
billsthisweek/20150706/CPRT-114-
HPRT-RU00-HR6.pdf
2. Mayo Clinic, In Support of a Patient-
Driven Initiative and Petition to Lower
the High Price of Cancer Drugs, Mayo
Clinic Proceedings 90 (8) (August
2015), www.mayoclinicproceedings.
org/article/S0025-
6196%2815%2900430-9/fulltext ◆
ES670434_BP0915_012.pgs 09.09.2015 01:54 ADV blackyellowmagentacyan
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ES671382_BP0915_A13_FP.pgs 09.11.2015 21:08 ADV blackyellowmagentacyan
14 BioPharm International www.biopharminternational.com September 2015
Perspectives on Outsourcing
Do
n F
arr
all/G
ett
y Im
ag
es
Contract manufacturing organizations
(CMOs) have been enthusiastic adopters
of single-use technologies in recent years,
lauding their range of benefits and attributing
significant improvements to their use. In fact,
according to the latest industry study from
BioPlan Associates (1), there appears to be one
aspect of single-use application that CMOs feel
is inhibiting adoption: The cost.
As part of the BioPlan study, factors that
may restr ict the use of disposables were
examined and general agreement was found
among both biomanufacturers and CMOs
when segmenting these two groups. The top
two concerns for each group, for example,
were “breakage of bags and loss of produc-
tion” and “leachables and extractables”, with
the former the most widely held among bio-
manufacturers and the latter most common
among CMOs.
Other common pain points for both groups
include material incompatibility with process
fluids, single-source issues, and the high cost
of disposables, with each of these a factor in
restricting adoption of single-use devices for
between 55–60% of respondents. Meanwhile,
CMOs (62.5%) were far more likely than bio-
therapeutic developers (40.4%) to cite the
lack of clear regulatory guidance
on leachables and extractables
(L&E) as a hindrance.
When the more than 200 sur-
vey respondents were asked to
ident i f y the most impor tant
reason for not increasing use of
disposable technologies, a new
ranking of obstacles was found,
as differences between developers
and CMOs emerged (see Figure 1).
For biotherapeutic developers,
the most crucial reasons restrict-
ing greater use of disposables are:
• “Breakage of bags and loss of production
material is a concern” (19% of biothera-
peutic developers)
• “For regulatory reasons, we can’t change
our current systems” (15.5%).
For CMOs, however, the top reasons given
were:
• The high cost of disposables (consum-
ables), at 26.7% of respondents
• Leachables and extractables are a concern
(20%).
Cost obstacles for CMOs partly exist because
these service suppliers need to justify their cost
to their clients. CMOs also have significantly
more in-use experience with these devices, so
cost rises to the top as other factors decrease in
importance.
One area that CMOs indicate has not been
worked out is leachables and extractables. For
example, 13% of CMOs worry for the lack of
regulatory guidance on L&E—noting this as
their top obstacle. In combination with other
concerns about L&E, these could be viewed
as the leading technical challenge to CMO
adoption.
It is interesting that single-use equipment is
generally accepted as providing cost savings, yet
the single most-cited factor restricting further
use by CMOs is cost.
Given that CMOs expect to increase their
budgets by as much as 6% for new technolo-
gies to improve upstream and downstream effi-
ciency—with single-use equipment presumably
a part of this spending—budgetary constraints
are not holding back single-use adoption.
Is sIngle-use too expensIve?The relatively rapid growth in use of single-
use equipment for early-stage (and increas-
ingly commercial stage) manufacturing may
suggest that costs are considered reasonable.
Yet as in-use experience grows, it is expected
that the high cost of some of these con-
CMOs Concerned With Cost of Single-Use EquipmentSuppliers indicate prices for single-use equipment are likely to increase.
Eric Langer is president of
Bioplan Associates,
tel. 301.921.5979,
ES670466_BP0915_014.pgs 09.09.2015 02:06 ADV blackyellowmagentacyan
September 2015 www.biopharminternational.com BioPharm International 15
perspectives on outsourcing
sumables will create friction and
push-back. As industry adoption
increases, single-use products in
biopharmaceutical manufacturing
will likely see the same levels of
pricing pressure as other similar
equipment. Vendors can expect
to see increasing price push-back
from their customers, and poten-
tially greater levels of competition.
sAtIsfActIon wIth vendorsThe influence of pricing as a selec-
tion criterion is unsurprising, of
course. Cost factors are right up
there with product quality, on-
schedule delivery, and the provi-
sion of L&E data that regulators will
accept. However, for this industry,
and most others, cost factors are a
commonly complained about issue.
Frankly, it’s rare to find buyers that
are particularly satisfied with the
cost of anything they buy.
The dissatisfaction with the cost
of single-use devices among buy-
ers of single-use devices is more
likely the result of buyers not per-
ceiving the full value that these
technologies can provide. This per-
ception is partly because this is a
relatively new segment and there
is little economic data, or in-use
experience and cost justifications
that are readily available. As more
data are developed, it is likely this
dissatisfaction gap will decrease.
In addition, as more fully inte-
grated single-use systems facilities
are brought online, the value of a
totally disposable operation will
begin to be realized.
In examination of the gap
between the importance of vari-
ous factors—including pricing and
the satisfaction end-users have
expressed regarding their single-
use vendors— approximately one in
five respondents (21.6%) reported
being “satisfied” or “very satisfied”
with single-use device costs. By
comparison, close to three-quarters
reported being satisfied with the
quality of the products (see Figure 2).
In any case, satisfaction with
single-use pricing is unlikely to
change anytime soon. Separately,
the BioPlan study also surveyed 164
suppliers to the biopharmaceutical
industry on issues associated with
industry growth. Suppliers reported
having increased their pricing for
disposable, single-use devices by
an average of 3.1% in 2014, about
the same average price increase as
the year before. Suppliers, however,
projected an average 3.8% increase
in pricing for disposable, single-use
devices this year, outstripping pro-
jected increases for other bioman-
ufacturing-related areas including
services, consumables, and instru-
ments. These results suggest that the
overall market for single-use equip-
ment is trending towards higher
prices, most likely due to consistently
high demand.
does cost reAlly mAtter to cmos?Single-use equipment costs are
unlikely to decrease and, as noted,
complaints about product costs
may be simply lack of understand-
Figure 1: Selected “Most Important Reasons” for not increasing use of
disposables, biotherapeutic developer vs. CMOs.
26.7%
0.0%
6.7%
0.0%
20.0%
13.3%
13.3%
4.8%
7.1%
7.1%
10.7%
10.7%
15.5%
19.0%
High cost of disposables (consumables)
We do not want to become vendor-dependent (single-source issues)
Limited scalability over a broad range (such as 2 L to 2,000 L)
We have already invested in equipment for current system
Leachables and extractables are a concern
For regulatory reasons, we can't change our current systems
Breakage of bags and loss of production material is a concern
Biotherapeutic Developer CMO
Which is the MOST important reason for not increasing use of disposable technologies?
“Biotherapeutic Developers vs CMO”
Source: 12th Annual Report and Survey of Biopharmaceutical Manufacturing, April 2015, www.bioplanassociates.com/12th
Figure 2: Percentage point gap between importance of single-use systems (SUS)
product attributes and level of satisfaction, select responses.
46.00%
46.20%
52.80%
54.40%
58.40%
Performing leaches/extractables testing using my current systems
Deliver on schedule
Providing leachables andextractables data that regulators will accept
Cost of product
Willingness to accept and fx integrity (leakage) problems
Source: 12th Annual Report and Survey of Biopharmaceutical Manufacturing, April 2015, www.bioplanassociates.com/12th
SUS Vendor Service Delivery GapsRelative Gap Between
Satisfaction with Vendor Delivery & Attribute’s Importance
ES670465_BP0915_015.pgs 09.09.2015 02:06 ADV blackyellowmagentacyan
16 BioPharm International www.biopharminternational.com September 2015
perspectives on outsourcing
ing of the economics associated
with an effective application of the
technology. The market demand
continues, though, and CMOs give
a long list of reasons for increasing
use of disposables, from faster cam-
paign turnaround time (85.7%)
and reduced time to get the facility
up and running (75%) to decreased
risk of product cross-contamina-
tion (68.8%) and the elimination
of cleaning requirements (58.8%).
So given the benefits, does
the cost of these devices matter?
Indeed, while CMOs are more apt
than biotherapeutic developers
to find fault with single-use pric-
ing, they’re also more likely in
general to recognize the benefits
they afford.
While CMOs may ultimately
want the cost of single-use prod-
ucts to come down, high costs have
not stunted their adoption rates
to a considerable degree. Although
CMOs can pass the costs of such
single-use devices and operations
on to their clients, allowing CMOs
to reduce the potential need for
capital expenditures, there is an
additional factor noted that sug-
gests single-use systems device
costs are not fully relevant—almost
9 in 10 (86.4%) of CMOs surveyed
report at least “some” productivity
improvements due to the use of
disposable/single-use devices.
Thus, CMOs appear to be
addressing their most crucial trend
in global biomanufacturing—the
need to improve productivity and
efficiency, by adopting single-
use devices. So complaints about
costs may be a bit of disingenuous,
especially in relation to the pro-
ductivity benefits reportedly being
delivered.
reference 1. BioPlan Associates, 12th Annual
Report and Survey of
Biopharmaceutical Manufacturing
Capacity and Production (Rockville,
MD, April 2015), www.
bioplanassociates.com/12th. ◆
while cmos may ulti-
mately want the cost
of single-use products
to come down, high
costs have not stunted
their adoption rates to
a considerable degree.
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18 BioPharm International www.biopharminternational.com September 2015
Industry Insider
12
3re
nd
er/
E+
/Ge
tty Im
ag
es
The Metrics of Quality CultureFDA’s proposed guidance for quality metrics raises questions about quantifying the tangibles and intangibles of quality culture.
The long awaited, anxiously anticipated
FDA guidance on qual ity metr ics
was finally distributed for comment
on July 28, 2015. The official title of this
guidance for industry is Request for Quality
Met r ics, Guidance for Indust ry (1) and its
potential release has been looming on the
horizon since 2012.
The intent of FDA to establish quality
metrics first emerged in 2012 when Congress
passed the Food and Drug Administration
S a fe t y a nd I n novat ion Ac t ( F DA SI A)
enhancing FDA’s capability to proactively
react to, prevent, and alleviate drug short-
ages. Specifically, Title VII Section 705 of
the Act states FDA “shall inspect establish-
ments described in paragraph [1] that are
engaged in the manufacture, preparation,
propagation, compounding, or processing of
a drug or drugs (referred to in this subsec-
tion as ‘drug establishments’) in accordance
with a risk-based schedule established by
the Secretary.” Section 706 of the same act
allows FDA to request certain information
from companies in advance of or in lieu of
inspections by stating, “Any records or other
information that the Secretary may inspect
under this section from a person that owns
or operates an establishment that is engaged
in the manufacture, preparation, propaga-
tion, compounding, or processing of a drug
shall, upon the request of the Secretary, be
provided to the Secretary by such person, in
advance of or in lieu of an inspection …” (2).
In the Feb. 12, 2013 Federal Register Notice (3),
FDA asked the industry to “assist the Food
and Drug Administration in draft-
ing a strategic plan on drug short-
ages as required by the Food and
Drug Administration Safety and
Innovation Act …” This notice asked
a series of thought-provoking ques-
tions including “What metrics do manufac-
turers currently use to monitor production
quality?” and “How frequently would such
metrics need to be updated to be meaningful?”
After a few years of actively engaging and lis-
tening to industry in a variety of venues, this
new guideline has finally been released.
The metrics proposed in the guideline are
not new to industry. Many of them are cur-
rently being used by companies to internally
measure performance. In some cases, the speci-
fied metrics are also reported to the agency
via the annual report or are contained in the
annual product review. The bio/pharma indus-
try needs to review these metrics and ensure
they will provide meaningful data while avoid-
ing unintended consequences.
Defining quality cultureThe underlying and understated tenet used
to determine a company’s well-being is a
measure of their quality culture. The culture
of a company dictates the veracity of their
metrics. The best way to ensure the data
reported have merit is to assess the quality
culture of the submitting organization. It
is in this area that the new guidance lacks
clarity. The guidance leaves the opportunity
open to establish quality-culture metrics by
stating, “these metrics are not intended to
be an all-inclusive set of the quality metrics
that FDA could consider useful to assess a
product and manufacturer’s state of qual-
ity. For example, senior management com-
mitment to quality is an important factor
in evaluating the overall health of the PQS
[pharmaceutical quality system] and qual-
ity culture” (1) and “…the Agency is com-
mitted to a dialog with industry to consider
benchmarks and standards that could pro-
vide acceptable metr ics that specif ically
demonstrate senior management’s commit-
Andrew Harrison is chief regulatory
affairs officer and general
counsel, and Susan Schniepp is
distinguished fellow, both with
regulatory compliance associates.
ES669184_BP0915_018.pgs 09.04.2015 16:23 ADV blackyellowmagentacyan
That saved the life of my child.
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Make the connection with Clinigen CTS:
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ES668433_BP0915_019_FP.pgs 09.03.2015 02:28 ADV blackyellowmagentacyan
20 BioPharm International www.biopharminternational.com September 2015
industry insider
ment to a culture of quality …” (1).
This commitment to establishing
quality culture metrics is further
evidenced by the section in the
guideline titled “Optional Metrics
Related to Quality Culture and
Process Capability/Performance”.
In this section, FDA “acknowledges
the importance of quality culture
to the overall state of quality of the
product, process, and commitment
to quality” (1).
Metrics relateD to quality cultureFDA proposes three voluntary met-
rics to try to get at the elusive qual-
ity culture. The first optional metric
proposed is intended to measure
senior management engagement
by assessing whether the head of
the quality unit and the head of
the operations unit have signed
the annual product review (APR) or
product quality review (PQR). The
second optional metric proposed
is corrective action and preventive
action (CAPA) effectiveness. The
measurement for this metric is to
indicate the percentage of correc-
tive actions that required retrain-
ing of personnel, the assumption
being that the root cause of the orig-
inal deviation (real or due to insuf-
ficient analysis) was determined to
be insufficient or ineffectual train-
ing. The third proposed metric is
intended to measure a firm’s process
capabilities through a series of three
questions. The real question should
be if these three optional metrics,
taken together, shed any light on the
quality culture.
Management engagement
Achieving a quality culture requires
management and employees to
establish an environment where
responsibility, accountability, and
reliability are paramount, and to
understand the role each person
performs in delivering a high-qual-
ity product to the customer and
sustaining that performance on a
continual basis. Management must
educate employees and provide the
tools and environment where they
can perform their functions in an
atmosphere that encourages excel-
lence and continuous improvement.
Assigning the head of quality and
the head of operations the task of
signing the APR or the PQR does
not ensure management engage-
ment nor does it mean that the
quality culture is lacking. It is up
to an organization to establish the
appropriate level of responsibility
and signing authority for APR and
PQRs. It is up to senior manage-
ment to provide the people charged
with these activities the necessary
resources to complete the task in a
timely manner with the expectation
that they will be held accountable
for the contents.
Retraining personnel
The second optional quality cul-
ture metric is specific to CAPA. The
proposed metric is to report the
percentages of corrective actions
involving the retraining of person-
nel. Without context supporting
the retraining of personnel, this
metric does not offer insight into
the true culture of an organization.
It could be argued that any CAPA
that results in a reduction or elim-
ination of a recurring deviation
would require an element of train-
ing personnel. In fact, retraining of
personnel on the CAPA issue, how
it was solved, and how to imple-
ment the necessary change is evi-
dence of management engagement.
It should be expected that a major-
ity of CAPAs involve some retrain-
ing of personnel.
Critical quality attributes
The third quality-culture optional
metrics involves trying to use criti-
cal quality attributes (CQA) as a
key indicator of a quality culture.
Of the three optional metrics pro-
posed, this one does provide some
measurement of the existence of a
quality culture. On the surface, the
questions just seem to be a regurgita-
tion of information contained in the
APR or PQR. Upon closer evaluation,
however, it is clear that FDA is try-
ing to measure whether a company
drives for continuous improvement
through their review and assess-
ment of threshold levels established
with CQAs. Companies that have
established CQAs and linked them
to a requirement to issue a CAPA
when they exceed the established
threshold levels have demonstrated
a commitment to continuous
improvement. Continuous improve-
ment programs are, in fact, reliable
indicators of the presence of a qual-
ity culture.
Measuring intangiblesThe establishment of simple qual-
ity metrics that not only measure
the quality of the product but also
reflect the quality culture of an
organization is required to assist
FDA in establishing a risk-based
audit program. The problem is that
it is difficult to measure something
as intangible as culture with cold,
hard data. The remaining ques-
When choosing a
metric, it is important
that the architects
of the metric are
aware of unintended
consequences that
may inadvertently
drive negative
behavior.
ES669183_BP0915_020.pgs 09.04.2015 16:23 ADV blackyellowmagentacyan
September 2015 www.biopharminternational.com BioPharm International 21
industry insider
tion is: If taken together, are the
three proposed optional metrics
indicative of a quality culture? The
answer is, maybe.
Careful thought and consider-
ation should be exercised when
determining what to measure, how
often to measure, how to interpret
and communicate the data, and
what the expectation is for using
the data to drive positive change.
Management needs to be cognizant
of the fact that whatever metrics are
reported, they must be developed,
evolved, and adjusted over time to
maximize their impact on driving
positive change. When choosing
a metric, it is important that the
architects of the metric are aware of
unintended consequences that may
inadvertently drive negative behav-
ior. Management attempting to
incentivize achievement of the goal
such as offering a financial award
if the goal is achieved, may lead
to inappropriate behaviors that do
not address the real issue. In these
cases, it is generally not the met-
ric that will drive the behavior but
rather use of behavioral rewards.
Reward for achievement rather
than analysis of the real underlying
causes will not lead to sustainable
positive change. When managed
properly, metrics are an important
tool to help drive positive change
and quality process improvements.
Upon observation, an unhealthy
quality culture is easy to identify.
People in a poor culture do not
understand their job and its impor-
tance to the business. They often
appear stressed, and they hide their
mistakes or blame others for their
errors. There is no evidence of team-
work. People work in silos and rarely,
if ever, seek input or advice from
others. Metrics that could potentially
be used to measure a poor culture
include a large employee turnover,
an overabundance of deviations
attributed to human error, and
lack of pride in the performance of
employees’ jobs.
In contrast, a robust, healthy
quality culture can be evidenced by
alignment of goals between qual-
ity and operations, self-sustained
work teams that focus on continual
improvement, and employees who
incorporate quality into their jobs
on a daily basis. They are not afraid
to speak up and offer suggestions
for improvement to their colleagues.
People understand the importance
of their jobas and respect each other
and their management. This culture
welcomes inspections and views
these inspections as another tool
to use in their continual improve-
ment initiatives. Metrics that could
potentially be used to measure a
healthy quality culture include a
small employee turnover, deviations
that identify a root cause other than
human error, and pride in the per-
formance of their jobs.
conclusionWhen establishing a metrics pro-
gram, companies should evaluate
numerous data input points includ-
ing, but not limited to, product-qual-
ity attributes, manufacturing site
performance, people metrics, and
quality-system metrics. For product-
quality metrics, companies should
consider reporting on batch-specific
data such as trending drug product,
drug substance, and stability test
results against customer complaint
rates. Indirect product-quality met-
rics could include environmental
monitoring, water trend results, and
yield rates. When establishing site
metrics, the company could look at
inspection history including internal
audit findings and maintenance his-
tory such as equipment age versus
defect-failure rates. People metrics
should consider ongoing job-spe-
cific training and education, skills
and experience assessments, and
employee turnover rate by job func-
tion and site. Quality systems met-
rics might look at change control,
investigation root-cause trends, and
release-testing cycle times.
There is no set requirement on
which metrics a company should
track to measure their overall perfor-
mance. Each company should deter-
mine which metrics to track based
on their operations, number of facili-
ties they operate and where they are
located, what types of products they
manufacture, and what type of cul-
ture exists in their places of business.
The metrics chosen must be
meaningful and written to provide
a clear analysis of ongoing activities.
It is important for operations and
quality to agree on the metrics and
how to report them to management
to avoid overreaction to the data. It
is not sufficient to simply report the
data. The interpretation of the data
is of crucial importance because it
may include a root-cause analysis of
its own.
references 1. FDA, Request for Quality Metrics
Guidance for Industry (Rockville, MD,
July 2015).
2. The Food Drug Administration Safety
and Innovation Act, Pub. L. 112-144,
126 Stat. 993 (2012).
3. FDA, “Food and Drug Administration
Drug Shortages Task Force and
Strategic Plan; Request for Comments”
Federal Register Feb. 12, 2013) online,
www.gpo.gov/fdsys/pkg/FR-2013-02-
12/html/2013-03198.htm, accessed
Aug. 17, 2015. ◆
When managed
properly, metrics
are an important
tool to help drive
positive change
and quality process
improvements.
ES669185_BP0915_021.pgs 09.04.2015 16:23 ADV blackyellowmagentacyan
22 BioPharm International www.biopharminternational.com September 2015
Die
ter S
pannkneb
el/S
tocktr
ek
Imag
es/
Gett
y Im
ag
es;
Dan W
ard
Assessing the capabilities of
a contract manufacturing
o r g a n i z a t io n (C M O) i s
a col laborat ive proces s ,
involving numerous communications
be t ween t he b iopha r maceut ica l
company and the CMO. Choosing a
manufacturing partner solely based
on scale, on prior experience with
the CMO client, or on the supposed
reputat ion of the CMO may have
been fruitful strategies in the past—
but as the requirements for purity,
quality, and comparability become
more precise, the select ion of an
appropriate business partner for the
manufacture of a sensitive biologic
product can sometimes prove to be
as challenging as the bioprocessing
techniques themselves.
Whi le the demand for outsourc-
ing se r v ices i s s tead i ly inc reas -
ing, say Eric Langer and Ron Rader
of BioPlan Associates, sizing up the
various partner options and capa-
bilit ies among CMOs and contract
development and manufac tur ing
organizations (CDMOs) can be diffi-
cult. Experts within the outsourcing
industry are unclear about the total
number of CMOs/CDMOs. Even gaug-
ing the percentage of CMOS/CDMOs
focused specifically on biomanufac-
turing is a challenge. In June 2014,
15 CMOs/CDMOs united to become
part of the Pharma & Biopharma
Outsourcing Association (PBOA)—a
contract biomanufacturing firms become more specialized
Randi Hernandez
Focusing on niche and specialty
service offerings gives contract
biomanufacturing organizations an
opportunity to differentiate in a crowded market.
contract biomanufacturing
ES670654_BP0915_022.pgs 09.09.2015 19:00 ADV blackyellowmagentacyan
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24 BioPharm International www.biopharminternational.com September 2015
nonprofit trade association for
CMOs/CDMOs representing the
interests and concerns of the
industry on matters affecting
the development and/or manu-
facture of pharmaceutical prod-
ucts—but the organizations that
have joined the trade group (now
at 18 members) encompass out-
sourcing companies from both
pharma and biopharma. Led by
journalist-turned-advocate, Gil
Roth, who is president, the trade
group covers issues such as the
assignment of Gener ic Dr ug
User Fee Amendment (GDUFA)
fees for facility inspections, and
the quality agreements between
CMOs and their clients.
C M O s h a v e d i f f e r e n t
approaches to quality agreements,
and the PBOA hopes that the
FDA’s draft guidance on the topic
will help lead to harmonization
or a proliferation of best practices
in that area. In an adjacent area,
the association is working with
FDA to help clarify the agency’s
recent draft guidance, Request for
Quality Metrics (1). Roth spoke at
an FDA public meeting on the
draft guidance, where he pushed
for clarity on the role of CMOs in
providing quality metrics to help
assure product safety, without cre-
ating a huge compliance burden.
The PBOA is also concerned that
certain metrics could favor in-house
manufacturing over outsourcing,
and Roth hopes PBOA can ensure
that the CMO perspective is repre-
sented when FDA ultimately imple-
ments the quality metrics (2).
Langer and Rader est imate
that there are as many as 80
biologics CMOs, and approxi-
mate ly 20 of these ac t ua l ly
make biologics. The three com-
panies in the top tier—Lonza,
Boehringer Ingelheim (BI), and
Patheon—make up the major-
it y of the biolog ics market ,
accord ing to BioPlan. Other
industry insiders argue, how-
ever, that while Lonza, BI, and
Patheon are among the biggest
players in the f ield, Celltr ion
and Samsung have signif icant
capacity in mammalian cell cul-
ture—and Sandoz is likely the
biggest CMO for microbial bio-
logics. Two CMO players that
fo l low t he a fo r e me nt ione d
companies in terms of business
activities with biologics include
Fujifilm Diosynth Technologies
and CMC Biologics, followed by
approximately 10 other com-
panies that make up the rest at
clinical scale. Jon S. Gingrich,
manager of business develop-
ment at Avid Bioservices, says
that Avid’s planned commercial
facility expansion will “triple its
present commercial capacity.”
The remaining 60-plus biolog-
ics CMOs are “specialty” CMOs,
according to Langer and Rader,
many of which are local businesses.
In short, approximately 15–20% of
the biologics CMOs actually man-
ufacture drugs, but five times as
many CMO facilities provide ser-
vices that support the production
of large-molecule medications.
“The market for biolog ica l
CDMO services—both in drug
substance and drug product—
is certainly robust,” observes
Roth. Areas such as biosimilars
and antibody-drug conjugates
(ADCs) present huge opportuni-
ties for drug makers and CDMOs
alike, he notes, adding that these
fields require significant levels
of expert ise and capital—and
players lacking in these attr i-
butes wil l be kept out of the
game. “The biologic space isn’t
for the fainthearted, and there
are a range of CDMO models,”
says Roth. “This demonstrates
that there isn’t one clear ‘plan of
attack’ for services and technol-
ogy solutions providers.”
The successful CMOs are those
that have continuously diversi-
fied their service offerings, note
Langer and Rader, and those
that have specialized in a par-
ticular niche area in the process
stream outside of the basic ser-
vice offerings of typical CMOs.
Some examples of this variety—
outside of the typical CMO ser-
vice offerings, such as process
development—include exper-
tise in bioconjugation and ADCs
(SAFC, Cata lent, Lonza, and
Goodwin Biotechnology); Baxter
Biopharma Solutions’ proficiency
in formulation development and
lyophilization cycle development
for biopharmaceuticals; Althea’s
ability to create stable crystal-
line formulations for therapeu-
tic products in suspension and
its expertise in protein expres-
sion using a non-endotoxic bac-
terial platform; KBI Biopharma’s
expanded microbial development
contract biomanufacturing
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September 2015 www.biopharminternational.com BioPharm International 25
and manufacturing services via
its acquisition of a Merck facil-
ity; Fujifilm Diosynth’s aptitude
in diverse gene therapy; WuXi
AppTec’s mammalian cell cul-
ture capabilities through the use
of disposable bioreactors and
its dedication to enhancing the
manufacture of cell therapeu-
t ics; Lonza’s exper ience with
viral gene and virally modified
cell products; and MicroProtein
Technologies’ proficiency in a
fu l ly automated, plate -based
prote i n produc t ion met hod
using Escher ichia coli . Langer
calls MicroProtein Technologies’
recombinant protein platform
“quite revolutionary”; the com-
pany touts itself as the “first com-
pany to produce recombinant
biologics on an industrial scale
without employing the conven-
tional fermentation process” (3).
Expectations of a CMO
handling unstable products
As it relates to the handling of
products made in living cells,
“a good CMO will be able to
support both development and
manufacturing programs with a
raft of orthogonal techniques to
analyze both in-process samples
and final purified material,” says
Daniel Smith, chief scientif ic
officer, Cobra Biologics. CMOs
i n t he b iolog ic space must
comprehend the factors affecting
product stabi l ity (parameters
such as temperature, pH, and
buf fer concentrat ion, among
others); have a good grasp on
p ote nt i a l p ro duc t s t r e s sor s
(shear force and pump type);
have informat ion on proven
hold t imes for intermediates
and final products; have a solid
s c ient i f ic u nde r s t a nd i ng of
protein degradation pathways;
a n d a c c e s s t o e x c e l l e n t
charac ter i zat ion methods to
me a su re pu r i t y a nd de te c t
aggregation.
Bioassays are still on top
Contract service providers are
expected to benef it f rom the
surge of interest in biosimilar
development, especially when
it comes to product character-
izat ion assays used to assess
comparability between a refer-
ence product and a biosimilar.
The demand for accurate and
sens it ive b ios im i la r compa-
rabil ity tools has prompted a
renewed interest in the use of
orthogonal techniques to char-
acterize elusive structural dif-
ferences between molecules (4).
Disciplined formulation prac-
tices for biosimilars are of para-
mount importance, says Wendy
Saffell-Clemmer, research and
development director at Baxter
BioPharma Solutions. With inno-
vator medications, the process
defines the product—whereas for
biosimilars, the process must be
designed to deliver the targeted
product, she adds. Thus, prod-
uct characterization through the
use of orthogonal bioanalytical
test methods can help provide
insight on whether the develop-
ment process has been appropri-
ately designed.
“FDA’s tota l ity of ev idence
approach has analytical char-
acterization at its foundation,
which will l ikely result in an
increase in analyt ical serv ice
requests during cell-line selec-
t ion, fermentat ion, and puri-
f icat ion deve lopment ,” says
Saffell-Clemmer. A recent survey
from BioPlan Associates found
that biomanufacturers rely pri-
marily on CMOs for analytical
test ing and for bioanaly t ica l
methods that measure the purity
or biological activity of a drug
product (5). Approximately 86%
of biologics manufacturers use
CMOs for analytical testing ser-
vices; a slightly lower number
compared to 89% in 2014 (6).
The other four activities mak-
ing the top five most commonly
outsourced serv ices in 2015,
according to the BioPlan sur-
vey, are the same as they were
in 2014: plant ma intenance
services, f ill/finish operations,
validation services, and toxic-
ity testing. Plant maintenance
services jumped from the fifth
most com mon ly out sou rced
activity in 2014 to the second
most commonly outsourced ser-
vice in 2015, perhaps indicating
that biopharmaceutical compa-
nies’ aging facilities may drive
businesses to outsource to CMOs
in lieu of investing in new equip-
ment for their own facilities. The
increased use of automation and
disposable technologies in asep-
tic processes are also expected to
drive business to CMOs, as many
CMOs have already invested in
the technology to do this work,
such as the purchase of large-
scale isolators for the preparation
of potent compounds (7).
An uptick in service requests
related to the development of
biosimilars has been observed
by many of the CMOs that spoke
to BioPharm International. These
services are primarily related to
analytical testing, with a special
interest in the understanding of
product glycosylation patterns,
notes Smith. Gingrich confirms
that his company has seen a
signif icant increase in glyco-
prof i l ing requests; this assay
is currently one of the most-
requested types at Avid.
O t he r a na ly t ic a l s e r v ice s
expected to gain popularity as
more biosimilar candidates flood
the pipeline include formula-
tion screenings, high-throughput
screenings, and quality and com-
parability studies, according to
Sébastien Ribault, PhD, director
of Provantage Biodevelopment/
e nd - to - e nd s e r v ice s , Me rc k
contract biomanufacturing
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26 BioPharm International www.biopharminternational.com September 2015
contract biomanufacturing
BioDevelopment, Millipore S.A.S.,
France. To f ind the opt imal
clone, comparative analytics for
biosimilars should ideally begin
as early in the process as cell-line
development, suggests Marion
Schrader, PhD, senior director of
marketing at Rentschler. Despite
the increased interest in biosimi-
lars, Schrader ment ions that
Rentschler expects “to see some
consolidation in the area, as not
all of the current clinical projects
will have commercial success.”
Other commonly outsourced
activities include: API manufac-
ture, cell-line development, cell
banking (master cell banks and
working cell banks), process devel-
opment, formulation develop-
ment, conjugation development,
media development, and routine
production processes. Activities
that present unique facility con-
cerns—such as the manufacture
of ADCs, cell and gene therapies,
compendial and stability test-
ing, or viral vaccines and vec-
tors—are usually outsourced as
well. Firelli Alonso-Caplen, PhD,
senior director of biotherapeutics
and vaccines outsourcing at Pfizer
Biotech, notes that keeping more
complex projects in house, how-
ever, helps her company in par-
ticular retain internal full-time
employees. She says these chal-
lenging projects “become motiva-
tional drivers for more innovation
for our own scientists.”
T he r e w a s no c on s e n s u s
among industry experts as to
whether viral clearance studies
are usually outsourced or not.
According to Gingr ich, most
CMOs do not have experience
working with viral banks and
many of them do not want to
have any viral material onsite.
On the other hand, P f i zer ’s
Alonso-Caplen says that v iral
vaccines and vectors pose a risk
to in-house biopharmaceutical
manufactur ing faci l it ies, and
such act iv it ies are, therefore,
likely be outsourced to CMOs.
While many activities are out-
sourced in a piecemeal fashion,
and the need for certain services
can vary widely depending on
a biopharmaceutical sponsor’s
existing core capabilities, there
is always the possibility that a
product sponsor could come to a
CMO with a completely defined
process and ask the CMO to
solely assume a production role.
The reason for this production-
driven request could be because
the pharma c l ient has over-
whelmed its in-house capacity,
or because it wants the CMO to
manufacture “a secondary supply
of clinical or commercial prod-
ucts for [the purpose of] risk mit-
igation,” explains Smith.
When outsourcers outsource
A CMO may decline a project
if the client requests fall out-
side of the scope of its capabili-
t ies. When a CMO suspects it
cannot complete certain tasks
as requested, it is important for
that company to “be upfront and
transparent with the client so
that [the client is] able to pursue
discussions with other CMOs,”
notes Jennifer Cannon, PhD,
senior director of commercial
strategy at Ajinomoto Althea.
Out-of-scope requests are one
of the “great chal lenges that
sharing project or partnership details
CMOs and their pharma clients are generally not allowed to disclose details of their partnerships, although the decision to disclose
information varies by company. Some see the value in public disclosures, says Jennifer Cannon, PhD, senior director of commercial
strategy at Ajinomoto Althea, whereas others want to keep their business dealings private. Daniel Smith, chief scientific officer,
Cobra Biologics, says pharma’s reticence to disclose partner/manufacturing details is often due to the competition to be first-to-
market with a product. Jon S. Gingrich, manager of business development at Avid Bioservices, says that its contracts in highly
competitive areas, such as biosimilars, explicitly state that information on the molecules being developed remain confidential. In
the quest to produce biosimilars of comparable quality and efficacy to innovator products, biosimilar manufacturers may comb
through patent documents to gain insight about compound formulation and process platforms used by branded drug makers. This
is why details surrounding the partnerships between CMOs and branded biologic manufacturers may be another information entry
point for biosimilars to gain a competitive advantage for comparability, and perhaps eventually, interchangeability.
Some clients give permission to serve as references, or opt to have CMOs make joint presentations with clients at
conferences, says Marion Schrader, PhD, senior director of marketing at Rentschler. Despite what each company decides to
do regarding information sharing, Schrader points out that for approved drugs, the name of a CMO that has worked with a
pharmaceutical client can be found in drug-approval documentation.
According to Cynthia Wooge, global strategic marketing at SAFC, companies rarely disclose their supplier relationships
until after the drug is approved and a commercial supply agreement is established. There is a growing trend, however, for
companies to disclose supplier relationships earlier in development, but Wooge says “a significant majority continue to hold
information fast until disclosure is necessary or beneficial later in development.” –Randi Hernandez
ES670655_BP0915_026.pgs 09.09.2015 19:00 ADV blackyellowmagentacyan
September 2015 www.biopharminternational.com BioPharm International 27
can continually test the CDMO/
deve lopment compa ny re la -
tionship,” says Cynthia Wooge,
global st rateg ic market ing at
SAFC. Specifically, the analyti-
cal tasks required for a project
create the greatest chance for
out-of-scope work requests, she
says. “Upfront diligence in defin-
ing the scope, deliverables, and
requirements of a program pro-
vides the best opportunity to
limit out-of-scope occurrences.”
A n out sou rc i ng f i r m c a n
also expand its capabilities by
partnering with a second CMO
for activities such as PEGylation
or antibody conjugation. Cobra
Biologics has such a partnership
with Quiapeg to complement its
service offerings. Other options
inc lude ask ing the c l ient to
perform product-specif ic tasks
it se l f , says Johannes Re ite r,
head of biotech cooperat ions
at S a ndoz , or bu i ld i ng t he
technology at the CMO level,
“but this evaluation is done on
a case-by-case basis only when it
makes sense,” adds Reiter.
According to Gingrich, every
CMO has a list of core capabili-
ties and a running list of out-
sourced service providers they
rely on. “The goal is to match
what can be done in-house and
what needs to be outsourced,”
Gingrich asserts. To establish
a successful partnership with
a client, it’s best for a CMO to
conduct a thorough technical
evaluation of a client request
for proposal (RFP), notes Saffell-
Clemmer, and possibly meet
with the client after review of an
RFP to clarify any outstanding
concerns. Wooge concurs that
managing customer expectations
is crucial: “CDMOs are wise to
apply significant rigor in assem-
bl ing detai led proposals that
clearly articulate the assump-
tions and expected work identi-
fied in the RFP process.”
If a selected CMO cannot per-
form all of the duties required
for the manufacture of a bio-
logic from start to finish, three
to four points of contact with
di f ferent CMOs can be typi-
cal. Smith emphasizes that if a
product were to be developed
from cell-line selection through
to distr ibution to clinical tr i-
als, seven to eight CMO part-
ners could even be probable. For
example, multiple points of con-
tact could conceivably be used
for the following steps: a primary
CMO to handle linker-payload
production, a second CMO to
handle conjugation of the drug
substance, a third to handle the
formulation and aseptic fill/fin-
ish of the drug product, and a
f inal CMO for the purpose of
release and stability testing. This
supply chain design adds a sig-
nificant amount of complexity,
says Alonso-Caplen, more room
for error, and “higher external-
ization risks, especially if [the]
CMOs are global ly situated.”
When it comes to the coordina-
tion of multiple service provid-
ers, Gingrich says that the cGMP
CMO is usually responsible for
the management of all of the
other outsourced activities.
In the c ircumstance that a
customer outsources a drug sub-
stance from a different location
than a drug product, a client
can, and typically will, ask a
CMO to perform a more rigor-
ous API testing and release pro-
gram than it normally would for
a drug substance manufactured
by the customer itself or by a
domestic partner, notes Saffell-
Clemmer.
In the case of biosimilar com-
pa rab i l i t y te s t i ng , mu lt ip le
samples of a drug product are
sourced from various regions or
production sites—and it’s up to
the primary CMO to determine
the degree of similarity between
the originator product and the
biosimilar, notes Gingrich.
1. FDA, Draft Guidance for Industry,
Request for Quality Metrics, (Rockville,
MD, July 2015).
2. The editors of Pharmaceutical
Technology, “A Voice of Their Own,”
Outsourcing Resources, supplement to
Pharm. Technol. 39, pp. s24–30 (2015).
3. MicroProtein Technologies, Inc.,
company homepage, www.mptbiotechs.
com, accessed July 24, 2015.
4. The editors of BioPharm International,
“Biopharma Advances Demand
Specialized Expertise,” Outsourcing
Resources eBook, BioPharm Int., pp.
12–19 (June 2015).
5. BioPlan Associates, Inc., 12th Annual
Report and Survey of Biopharmaceutical
Manufacturing Capacity and Production,
E.S. Langer, Ed. (Apr. 2015).
6. BioPlan Associates, Inc., 11th Annual
Report and Survey of Biopharmaceutical
Manufacturing Capacity and Production,
E.S. Langer, Ed. (Apr. 2014).
7. E.S. Langer, “Fill/Finish Trends,”
Outsourcing Resources eBook,
BioPharm Int., pp. 20–25 (June
2015). ◆
contract biomanufacturing
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28 BioPharm International www.biopharminternational.com September 2015
SC
IEP
RO
/Gett
y Im
ag
es
Cell harvesting is a crucial step
in biopharmaceutical manufac-
turing that can have significant
impacts on product quality and
the design of remaining downstream
processes. Growing titers and viable cell
densities, due to improved media and
cell lines, and growing use of perfusion
cell culture are making this downstream
purification step more challenging.
In addition, manufacturers desire to
achieve more efficient, cost-effective pro-
cessing with a smaller footprint. “The
biopharmaceutical market is now fac-
ing increasing demand to be quicker
to market, with lower production costs
and a smaller industrial footprint while
improving productivity,” says Alain
Lamproye, president of the biopharma
business unit of Novasep. New filtration
and separation technologies, including
single-use systems for larger scale har-
vesting operations, are helping manufac-
turers meet these needs.
The move To single useCurrent interest in monoclonal antibody
(mAb) therapeutic candidates produced in
CHO cell expression systems is dominat-
ing development pipelines at major drug
producers, according to Timothy D. Hill,
director of upstream process development
for FujiFilm Diosynth Biotechnologies
USA. “One major shift in mAb production
is the replacement of stainless-steel reac-
tors with single-use bioreactors in order to
streamline operations, decrease change-
over times, eliminate cleaning validation,
and enable rapid capacity expansion as
product demand rises,” he notes.
high Titers and Perfusion Processes Challenge Cell harvesting systems
Cynthia A. Challener
New single-use technologies
and other filtration systems are beginning
to address cost, throughput, and
manufacturing footprint
demands.
Cynthia A. Challener, PhD,
is a contributing editor to
BioPharm International.
Downstream Processing
ES668644_BP0915_028.pgs 09.03.2015 19:48 ADV blackyellowmagentacyan
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• Continual troubleshooting and status updates during
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ES668438_BP0915_029_FP.pgs 09.03.2015 02:28 ADV blackyellowmagentacyan
30 BioPharm International www.biopharminternational.com September 2015
In addition to lowering upfront
capita l costs and of fer ing a
higher level of flexibility, single-
use technologies for cell harvest-
ing have also addressed one of the
major issues concerning this pro-
cess step—the risk of cross con-
tamination, according to Frank
Meyeroltmanns, head of product
management for purification tech-
nologies, Sartorius Stedim Biotech.
Single-use technologies also pro-
vide cost-effective drug develop-
ment options for products under
investigation. “Cell removal using
conventional centrifugation drives
up cost due to the high capital out-
lays required for the equipment and
operating costs—which are primar-
ily incurred due to required main-
tenance and cleaning-in-place—are
avoided with single-use technolo-
gies,” Meyeroltmanns adds.
imPaCT of PerfusionContinuous-processing technolo-
gies for both cell culture and down-
stream purification steps are some
of most advanced latest develop-
ments in terms of outcomes, accord-
ing to Lamproye. “In many cases,
continuous processes enable high
productivity while keeping produc-
tion costs low. These types of pro-
cesses also have smaller industrial
footprints and constant productiv-
ity and benefit from on-line control
that automatically adjusts processing
parameters to maintain optimal set-
tings,” he observes.
Improvements in both fed-batch
and perfusion processing have con-
tributed to increasing viable cell
densities and titers and are driv-
ing suppliers to provide improved
clarification technologies so that
recoveries are in line with the
higher outputs of the upstream
process, according to Joe Codamo,
senior project manager for biologics
at Patheon. He adds that continu-
ous processes—both upstream and
downstream—are also attractive as
an approach for reducing unit oper-
ations in order to lower manufac-
turing costs, shorten timelines, and
minimize product losses.
High CHO cell-density cultures
achieved using perfusion bioreac-
tor technology are, in fact, becom-
ing more standard to minimize
the upstream production scale and
reduce the footprint of reactors in the
manufacturing facility, thus lowering
overall capital investment and oper-
ating costs. “Typical peak CHO cell
densities have been reported publi-
cally by manufacturers experienced
with perfusion culture to range from
30–50x106 viable cells/mL culture
medium, with some reports of peak
cell densities over 100x106 viable
cells/mL in experimental bench scale
systems. Furthermore, mAb titers are
reported to exceed 10g/L in produc-
tion cycles of 2–3 weeks,” Hill says.
Thoughts about harvest technol-
ogy have also shifted in response to
adoption of perfusion cell culture
in the industry, according to Hill.
“The goal of harvest has broadened
to include cell and product retention
during production, as well as separa-
tion of product from cells during the
end of production,” he observes.
Challenge of higher TiTersCurrent single-use depth filters need
to handle ever-increasing cell con-
centrations yet deliver continuously
higher yields, but they often suffer
from blockage at lower loading capac-
ities, particularly when such volumes
have high biomass concentrations.
Major process-relevant parameters
for cell cultivation with respect to
clarification are particle-size distribu-
tion and particle quantity, accord-
ing to Meyeroltmanns. “For typical
cell-culture applications, particle sizes
vary between 0.1 µm and 25 µm,
whereas the quantity of total bio-
mass-relevant particles ranges from
10 million/mL to more than 100 mil-
lion/mL. These levels present a chal-
lenge for conventional clarification
techniques, and often multi-step fil-
tration regimes are required that use
up large numbers of filter units and
entail extensive case-based adapta-
tions,” he explains.
Centrifuges are predominantly
used to harvest cells from process vol-
umes above 500 L to reduce cost and
waste, according to Meyeroltmanns.
“Large-scale unit operations up to
2000 L require both scale-up and
scale-down concepts, as well as linear
scalability,” he asserts.
Hill does note that 3M has made
significant improvements to its line
of commercially available tradi-
tional depth filters for cell removal,
and Codamo adds that advances in
depth-filter media have also contrib-
uted to significant improvements
in filter capacity, flux, and product
recoveries. Hill agrees, however, that
depth filtration becomes less cost
effective compared with centrifuga-
tion at CHO cell densities of approxi-
mately 30x106 cells/mL or greater.
new large-sCale, single-use filTraTion TeChnologySartorius Stedim Biotechnologies
has adapted “body-feed filtration,”
(BFF) technology from the blood and
plasma fractionation industry for cell
harvesting to enable large-scale, sin-
gle-use filtration. The new Sartoclear
Dynamics system, which was intro-
duced earlier in 2015, is designed to
replace both centrifuges and depth
filters with a single-step process that
achieves cell clarification of up to
2000-L batch volumes.
“The main principle of BFF tech-
nology involves the addition of a fil-
ter aid. This pharmaceutical-grade
diatomite, a fine diatomaceous earth
(DE) is highly porous, increasing the
permeability of the filter cake that
builds up as clarification progresses
in the filtration system connected
downstream. As a result, the DE pre-
vents filters from becoming blocked,
providing a double advantage of sig-
nificant time savings and high flow
rates,” says Meyeroltmanns. He also
notes that special single-use bags
containing ultrapure DE are also
Downstream Processing
ES668650_BP0915_030.pgs 09.03.2015 19:49 ADV blackyellowmagentacyan
September 2015 www.biopharminternational.com BioPharm International 31
available for attachment using new,
patented quick connectors and dust-
free DE transfer directly into the cell-
culture fluid in a bioreactor.
“We believe that as a linearly
scalable filtration technology, BFF
closes the biggest gaps by enabling
single-use cell harvesting of fluids
from 2000 L standard single-use bio-
reactors and eliminating the need
for centrifugation technology for
removal of cells from such volumes.
This technology also provides a high
level of flexibility while helping to
significantly reduce upfront capital
investments,” Meyeroltmanns states.
TeChnologies for Cell harvesTing from Perfusion ProCessesPerfusion technology presents the
challenge of retaining cells and prod-
uct within the bioreactor until the
end of the production run, when
the product is separated from the
cells. Hollow-fiber filters initially
developed for the growth of adher-
ent cells have more recently been
used as physical barriers for cell
and product retention, while allow-
ing the passage of fresh and spent
media for exchange in the bioreactor,
according to Hill. He also notes that
single-path tangential flow through
hollow-fiber filters has been replaced
by alternating path flow (ATF, alter-
nating tangential flow) to prevent
cell attachment and filter fouling. By
selecting the appropriate filter pore
size, the process can be highly selec-
tive for operation in the retention or
permeate modes.
The combination of clarification
and product capture into one unit
operation also reduces timelines
and improves product recoveries,
according to Codamo. He points
to Patheon’s expanded-bed adsorp-
tion chromatography technology
as an example.
meeTing fuTure neeDsGoing forward, market needs will
be the main drivers of further cell-
harvesting technology develop-
ment, and particularly the need to
improve the production economics
for biological products, according to
Lamproye. As the biologics indus-
try continues to grow and com-
petition within the industry also
increases with the rise of biosimilars,
Codamo agrees that advances in
cell-harvesting technology will lead
to improved processing efficiencies,
reduced costs, and greater flexibility
for use with all of the mammalian
cell types used in the industry and
the various recombinant proteins
currently in the pipeline.
With respect to the technol-
ogy itself, Meyeroltmanns expects
growing mid-term and long-term
adoption of both large-scale unit
operations and single-use equipment
that provides continuous process-
ing capabilities for CMO environ-
ments and multi-product facilities.
Automation will also be of increasing
importance for biologics production,
according to Hill.
“Particular challenges include the
need to continually improve depth
filter efficiency in single-use applica-
tions to deal with ever-evolving cell
densities and productivities, and the
need to reduce process timelines fur-
ther, especially for products suscepti-
ble to degradation during cell harvest
Downstream Processing
Harvesting adherent cells for therapeutic applications
Cell-based therapies are still in the early stages of development, and one key
unanswered question relates to harvesting of the cells at commercial scales.
“Harvesting of adherent therapeutic cells is very different from the harvesting of
suspension cells that need to be separated from the desired product. In the former
case, the cell viability and activity of the surface proteins must be retained during
the harvesting process in order to preserve the therapeutic properties of the cells,”
according to Alun J. Fowler, commercial marketing manager for EMEA vaccines
and biologics in the Laboratory Products Group of Thermo Fisher Scientific.
“Currently there is no platform technology available for the harvesting of
therapeutic cells,” he notes. “One factor is the variety of cell-culture techniques
being used; most have very different culture conditions and harvesting
requirements, which makes the development of a platform technology quite
challenging,” says Fowler. Some cells (e.g., stem cells) are grown as sheets
while others are grown on microcarriers, and yet others are intended as
scaffold systems for regenerative medicine. Harvesting of therapeutic cells on
a large scale is also difficult because many of these techniques are not easy to
implement for large quantities of cells, according to Fowler. One possible solution
is massive parallel cell culture as a scale-up approach.
Alternatives to actual harvesting are also being explored, such as detachment
technologies. In one example (the UpCell Surface), adherence to a surface is
switched on and off with changes in temperature, thus allowing the grown cells
to detach as sheets without the need to use enzymes that could affect their
properties. It is possible that harvesting cells as sheets rather than individually
may be cheaper and easier at larger scale.
A second alternative involves the use of biodegradable scaffolds for growth of
therapeutic cells. The plant-based material can be digested by cellulase without
impacting the viability and make-up of the product cells, according to Fowler. He
also notes that enzymatic detachment from microcarriers is being investigated,
but this method requires careful selection of enzymes that will not affect the
crucial aspects of the cell structure.
Contin. on page 39
ES668647_BP0915_031.pgs 09.03.2015 19:48 ADV blackyellowmagentacyan
32 BioPharm International www.biopharminternational.com September 2015
Single-use technology has been
around for approximately two
decades if disposable capsule
filters are taken into consider-
ation. Aseptic connector devices, mixer
bags, storage bags, and bioreactors fol-
lowed. Currently, single-use systems
(SUS) a lso include disconnectors,
disposable filling needles, and sen-
sors. The technology is still evolving,
and the industry can expect further
advances in the future.
The dynamic nature of SUS provides
excitement for professionals from both
suppliers and end users, who predomi-
nantly believe that SUS is a key technology
for the future of biomanufacturing because
of the following major advantages:
• Flexibility
• Less up-front capital investment
• No batch-to-batch cross contamination
• No re-use cleaning validation
• Less work on scale up
• Shortened time-to-patient.
When end users actually implement
SUS in cGMP biomanufacturing processes,
however, there are still many challenges
including the following:
• Lack of thorough understanding of new
materials (e.g., compatibility with pro-
cessing fluids/extractables)
• Lack of deep knowledge of interaction
between SUS and biologics
• Lack of good practices to prevent leak-
age of SUS (manufacturing/ shipping/
handling)
• Limited interchangeability of compo-
nents (e.g., connectors)
• Limited industrial and regulatory guid-
ance on qualification of SUS.
These challenges have prompted both
suppliers and end users to work collabora-
tively to adequately qualify SUS prior to its
implementation.
SUS ProceSS DeveloPmentSingle-use systems are being used in
almost all stages of biomanufacturing pro-
cesses, including upstream, downstream,
and fill/finish operations. Figure 1 depicts
a typical monoclonal antibody (mAb) drug
manufacturing process. Single-use bioreac-
tors are widely used in cell line expansion,
and the use at production scale is increas-
ing, notably in contract manufacturing.
Single-use buffer filtration system and stor-
age bag application is common in down-
stream processes. Final drug-product sterile
filtration and transfer using SUS has also
being increasingly applied to fill/finish
operations.
The implementation of SUS usu-
ally starts with development of the user
requirement specification (URS). Typically,
the development engineers at biophar-
maceutical companies then work closely
with the design engineers and application
engineers from suppliers to select compo-
nents and standard assemblies as available,
or if not, generate suitable drawings, which
will then be subjected to internal approval
within the end-user company. There are
many factors to be evaluated, including
technical assessment, supply chain, busi-
ness aspects, and supplier quality. PDA
Technical Report No . 66, Application of
Single-Use Systems in Pharmaceutical
Manufacturing, has been developed to
provide comprehensive, high-level guid-
ance about qualification of SUS (1). This
paper focuses on technical assessment in
more detail.
technical conSiDerationS: chemical comPatibilityUnlike traditional stainless-steel equip-
ment, SUS is generally made from poly-
mers including plastics and elastomers.
These polymers are obtained through
best Practices in Qualification of Single-Use Systems
Weibing Ding
The author discusses the current best
practices in technical qualification of single-use
systems.
Weibing Ding, PhD, is principal
scientist Process Development, amgen
inc., thousand oaks, ca 91320
Single-Use Systems
ES669074_BP0915_032.pgs 09.04.2015 01:57 ADV blackyellowmagentacyan
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34 BioPharm International www.biopharminternational.com September 2015
polymerization of organic mono-
mers in the presence of catalysts. As
a result, they are susceptible to attack
by organic solvents as well as acids
and bases; some, at high concentra-
tions, can even dissolve polymers.
Because biomanufacturing may
involve processing of fluids contain-
ing these chemical compounds, the
chemical compatibility of single-
use components with process fluids
under process conditions should be
the first factor to be evaluated.
Table I lists the common polymers
used in the major single-use com-
ponents. Table II provides the gen-
eral chemical incompatibility of
these polymers with common flu-
ids. Because chemical compatibility
is determined by the concentration
of contacting fluid, the nature of the
polymer, the contact time, and tem-
perature, Table II can only be used
as a general guideline. When one
intends to use SUS with these fluids,
a pre-use qualification test should be
performed to validate that the pro-
cess conditions are not incompatible
with the material in question (2–4).
The consequences of incompatibil-
ity would be leaks from the closed
system (e.g., tubing, connector, chro-
matography column, and biobag),
inaccurate readings from sensors,
failed filtration (filter), and potential
failures in process performance and
product quality.
In addition, pretreatment methods
such as steam sterilization, e-beam
irradiation, gamma irradiation,
vaporized hydrogen peroxide, and
ethylene oxide could have an effect
on the properties of the polymers.
Therefore, the specific process param-
eters of these pre-treatments should
not exceed the supplier’s product
claim, which should be proven by
the supplier’s validation study.
technical conSiDerationS: FitneSS For PUrPoSeSUS used for each unit operation
should be fit for purpose. For exam-
ple, a single-use bioreactor should be
sterile and not adversely affect cell
growth due to certain leachables. It
should provide sufficient mixing and
have acceptable gas exchange, and
should not leak during operation.
For buffer filtration and storage, the
SUS should be sterile, not present any
concerns for extractables/leachables,
and not leak. For the SUS in fill and
finish, it should be sterile, not present
any concerns for extractables/leach-
ables, not leak, and in addition, not
raise any concerns for particulates
and endotoxins.
The questions are: How can we
ensure these requirements? Which
part is the responsibility of the sup-
plier? Which part is the responsibility
of the end user?
In general, process validation
remains the responsibility of the
biopharmaceutical manufacturer.
Supplier data should be appropri-
ately used. Leveraging supplier data
requires the end user to understand
how the supplier data were devel-
oped and assess their suitability to
the processes.
regUlatory reQUirementS For finished pharmaceuticals, FDA
has issued regulations explain-
ing cGMPs in 21 Code of Federal
Regulations (CFR) 210 and 211 (5, 6).
For APIs or drug substances, how-
ever, the agency has not issued reg-
ulations. Nevertheless, the agency
issues guidance documents to articu-
late its current practice on related
topics. Although there is no specific
regulation dedicated to SUS, FDA’s
Q7A Good Manufacturing Practice
Guidance for Active Pharmaceutical
Ingredients (7), which is based on
ICH Q7 (8), is a guidance document
Single-Use Systems
SU component Polymers as materials of construction
FiltersPolyvinylidenefuroride, polyethersulfone, polyamide, polyethylene terephthalate, polypropylene,
polysulfone, polyetherimide
Connectors/disconnectors Polypropylene
Aseptic connectors/disconnectors Polyvinylidenefuroride, polycarbonate, polysulfone, silicone
Biobags Low density polyethylene, ethylene vinyl acetate copolymer
Chromatography columns Polymethylmethacrylate
Tubing Silicone, thermoplastic elastomers
Sensors Silicone, polycarbonate, polysulfone
Filling needles Polyetheretherketone
Table I: Common polymers used in the major single-use components and in contact with process fuids.
ES669075_BP0915_034.pgs 09.04.2015 01:57 ADV blackyellowmagentacyan
September 2015 www.biopharminternational.com BioPharm International 35
that should be studied, understood,
and followed. Components and sys-
tems used for single-use technologies
should be assessed for incoming test-
ing. Key factors listed in the guidance
document (7) include:
“A supplier’s certificate of analysis
can be used in place of performing
other tests, provided that the manu-
facturer has a system in place to eval-
uate suppliers.
“Suppl ier approva l should
include an evaluation that provides
adequate evidence (e.g., past qual-
ity history) that the manufacturer
can consistently provide material
meeting specifications.
“P roce s s i ng a id s , ha z a rd -
ous or highly toxic raw materi-
als, other special materials, or
materials transferred to another
unit within the company’s con-
trol do not need to be tested if
the manufacturer’s certificate of
analysis is obtained, showing that
these raw materials conform to
established specifications. Visual
examination of containers, labels,
and recording of batch numbers
should help in establishing the
identity of these materials. The
lack of on-site testing for these
materials should be justified and
documented.
“Equipment should be constructed
so that surfaces that contact raw
materials, intermediates, or APIs do
not alter the quality of the intermedi-
ates and APIs beyond the official or
other established specifications.”
Failure to follow the guidance
could result in receiving an inspec-
tional observation from regulatory
authorities (9).
SUPPlier PracticeSThe basic document generated by
the supplier and shared with the
end user is the validation guide for
a component or a SUS. At a mini-
mum, the following topics should
be evaluated; for some components,
other specific tests may also need to
be performed:
• Materials of construction (Animal-
derived ingredients free or meeting
the EMA410.01 rev.03)
• Particulates/Endotoxin (United
S t a t e s Pha r macope ia [USP ]
<788>/USP <85>)
• Extractables (USP <661> and
/ or other more robust test
designs [10])
Single-Use Systems
Polymers Incompatible chemical compounds
Polycarbonate High pH fuids, strong acids, chlorinated hydrocarbons, and hot water with constant exposure
Polysulfone Dimethylsulfoxide, dimethylacetamide, chlorinated hydrocarbons, and acetone
PolyethersulfoneDimethylsulfoxide, dimethylacetamide, chlorinated hydrocarbons, acetone, and polyethylene
glycol at high temperature
Polyvinylidenefuroride Dimethylformamide, diethylacetamide, and acetone
Polyamide Low pH fuids
Polyethylene terephthalate High pH fuids
Polyetherimide Methylethylketone and chlorinated hydrocarbons
Low density polyethyleneChlorinated hydrocarbons and some types of detergents/disinfectants that induce stress
cracking
Ethylene vinyl acetate copolymer Concentrated mineral acids, ketones, and chlorinated hydrocarbons
Polymethylmethacrylate Strong acids and alkalis, ketones and chlorinated hydrocarbons
Silicone Concentrated acids and alkalis, methylene chloride, and methylethylketone
Thermoplastic elastomers Chlorinated hydrocarbons
Polyetheretherketone Concentrated mineral acids and halogenated hydrocarbons
*Note: Aromatic and aliphatic hydrocarbons are not considered here because they are rarely used in bioprocessing. The table provides
general guidance, and the actual evaluation should be based on end user’s process conditions
Table II: General chemical incompatibility of these polymers with common fuids in bioprocessing* (see references 2–4).
ES669073_BP0915_035.pgs 09.04.2015 01:57 ADV blackyellowmagentacyan
36 BioPharm International www.biopharminternational.com September 2015
Single-Use Systems
• Biocompatibility (International
Organization for Standardization
(ISO) 10993-4)
• Cytotoxicity/Bioreactivity (USP
<87>/USP<88>)
• Sterility (USP <71>)
• Transportation validation (ISTA-2A
or American Society for Testing
and Materials [ASTM] D7386-12)
• Shelf life (at least two years post
gamma irradiation)
• Sterilization (ISO11137)
• Integrity or gross leak
• Functional tests.
Functional tests are specific to
each SU component. For bioreactor
bags, film gas barrier tests and film
mechanical strength tests should
Chemical compatibility
Supplier End user
Based on the intended use of the single-use component,
perform chemical compatibility tests and publish the results in
the validation guide. When designing the tests, consider using
worst-case application conditions (e.g., time, temperature, and
pre-sterilization conditions) as well as typical process fuids
(chemical constituents and pH) (12–15).
Evaluate the generic data from supplier. Assess the applicability
of the data to the process. If there is a gap, work out a mitigation
plan, which includes either choosing another component that
is compatible or performing additional testing to confrm the
compatibility within process conditions.
Gross leak
Supplier End user
Perform gross leak test prior to packaging at component
level and system level. Then perform shipping validation
for packaged system after gamma irradiation. For aseptic
connectors, side load testing should be performed to provide
useful information for end user’s actual operation to prevent
potential leakage.
Evaluate supplier approach and data. In some cases, shipping
validation using the actual shipping lane is also warranted.
Perform risk assessment, and perform post-installation pre-use
gross leak test, if deemed necessary based on risk assessment.
During the production, closely monitor if there is leak. After the
production, inspect thoroughly.
Extractables
Supplier End user
Perform extractables study using a recommended standardized
extractables protocol (10) that includes selection of test articles,
test solvents, surface area to volume ratio, pre-sterilization
conditions, test conditions, and analytical methods. Evaluate
effect of extractables on cell growth for bioreactors. When there
is change in materials of construction and/or manufacturing
process, perform extractables assessment.
Evaluate supplier approach and data against the recommended
standardized extractables protocol. Perform a science-based
risk assessment, and perform additional extractables testing,
if there are gaps. Perform toxicity-based safety evaluation as
part of extractables/leachables evaluation. For bioreactors,
perform cell culture growth test, if needed. For drug substance
containers and SUS in fll/fnish, product stability study should
be performed to ensure extractables/leachables do not
negatively interact with drug product.
Particulates and endotoxin
Supplier End user
Perform quarterly testing on master system that includes
representative components. For fll and fnish applications,
perform additional test per lot.
Evaluate supplier approach and data. Perform science-based
risk assessment, and perform additional test, if there are gaps.
Change management
Supplier End user
After the launch of SU components with comprehensive
validation guide, when there is any change in materials of
constructions, manufacturing processes and sites, and
test methods, supplier needs to perform science-based
assessment, qualify the change, and provide change notifcation
as well as the qualifcation documentation to end user.
Ensure that a change notifcation agreement is in place with
suppliers (including sub-suppliers) and that such an agreement
provides suffcient time to assess the effects of any changes
on SUS functionality. Evaluate and assess supplier’s change
notifcation and associated change qualifcation documentation.
If the supplier’s assessment is not suffcient, the end user may
have to perform independent testing.
Table III: Best practices in technical qualifcation of single-use systems (SUS).
ES669070_BP0915_036.pgs 09.04.2015 01:57 ADV blackyellowmagentacyan
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ES668437_BP0915_037_FP.pgs 09.03.2015 02:28 ADV blackyellowmagentacyan
38 BioPharm International www.biopharminternational.com September 2015
be performed. Carbon dioxide, oxy-
gen, and water vapor transmission
studies can follow ASTM F2476,
ASTM D3985/ASTM F1307, and
ASTM F1249/ISO 15106-3, respec-
tively. Mechanic strength tests
include tensile strength (ASTM
D882/ISO 527), tear strength
(ASTM D1004), seal strength
(ASTM F2097/ISO 15747), brittle-
ness (ASTM D1790), and dynamic
mechanical properties (ASTM
D5026). In addition, drop tests on
bags are performed according to
ASTM D4169-05. For SU assemblies,
the joints between tubing and
barbs should be validated to pre-
vent potential leakage.
In addition to the validation guide,
suppliers may have additional sup-
porting documents, including but
not limited to detailed extractables
report, certificate of quality, and cer-
tificate of gamma doses.
enD USer PracticeSAside f rom qual i fy ing a sup-
plier through a quality audit, a
technical due diligence visit is
necessary to understand the sup-
plier’s manufacturing process,
sources of variation, and their
control. The goal is to ensure
that the supplier can provide SUS
or components with high qual-
ity consistently, which do not
negatively impact the end user’s
biomanufacturing process and/or
drug product quality and safety.
To achieve this goal, technical
transparency is essential. During
the v isit, the technical team
members from both companies
should review test designs, con-
firm test methods/results, review
test repor ts, and understand
the test results. When needed,
sub -suppl iers should a lso be
involved.
The supplier’s release criteria
and end-user’s receiving accep-
tance criteria should be aligned
with each other. These include,
but are not limited to, packag-
ing integrity, appearance/identity,
contamination, and extractables
assessment. The alignment of cri-
teria should include any variabil-
ity in appearance, such as minor
blemishes on the exterior of SUS
arising from transport and han-
dling, which do not impair the
function of the assembly. When
developing specifications, func-
tional specifications should be
taken into account to allow SUS
or component interchangeability
wherever feasible.
The next step is to perform a gap
analysis in the context of the specific
application. The major points to con-
sider are as follows:
• Is the extractables study relevant
and report useful?
• Are the particulate/endotoxin tests
adequate?
• Is the irradiation process validated
and results certified?
• Is cell culture growth in the biore-
actor acceptable?
• Is a gross leak test carried out prior
to irradiation?
• Is shipping validation performed
after irradiation?
• Does the use of the SUS affect
product stability?
Based on the gap analysis, addi-
tional qualification tests/assessments
may be warranted.
beSt PracticeS To ensure successful biomanufac-
turing, the SUS must not leak and
must not adversely affect process
performance and product quality
during production. To ensure the
drug product quality and patient
safety, the SUS shall not be reac-
tive, additive, or absorptive so
as to alter the safety, identity,
strength, quality, or purity of the
drug product (11). The technical
Single-Use Systems
Figure 1: Typical mAb manufacturing process.
DS
DP
Vialthaw
Drug
substance
container
Various
column
purifcation
Inoculum
Formulation Final fll & fnishSterile
fltration
Bioburden
reduction
fltration
storage
UF/DF Protein A
Expansion Harvest RecoveryProduction
Sterilefltration
Viralfltration
ViralInactivation
AL
L F
IGU
RE
S A
RE
CO
UR
TE
SY
OF
TH
E A
UT
HO
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ES669071_BP0915_038.pgs 09.04.2015 01:57 ADV blackyellowmagentacyan
September 2015 www.biopharminternational.com BioPharm International 39
Single-Use Systems
due diligence should be carried
out prior to the implementation
of SUS. Table III provides a partial
list of best practices to consider
in the technical qualification of
SUS used in biomanufacturing
(12–15).
conclUSionThe partnership between end
user and supplier is essential to
successfully implement SUS in
manufacturing drugs to achieve
success in implementation and
cont inued operat ion, and to
ensure the quality and safety of
the drug products. The supplier
needs to be fully transparent
about materials of construction,
supply chain, manufactur ing
process, sources and control of
variability, impact of changes,
and qual i f icat ion/va l idat ion.
The end user needs to perform
quality audits and technical due
diligence visits to ensure com-
pliance as well as a deep under-
standing of all these aspects, and
then carry out science-based risk
assessment considering the prox-
imity of SUS to the final drug
product container closure system
by applying a phase-appropriate
approach. Any gaps found should
be bridged or filled in to ensure
the successful implementation of
SUS in biomanufacturing.
acknowleDgmentSThe author greatly appreciates
Duncan Low’s, Executive Director,
Process Development, Amgen,
guidance and constructive review
of the manuscript. In addition,
the author sincerely thanks the
collaboration from Amgen’s Raw
Materials & Devices, Supplier
Qual ity Management, Global
Strategic Sourcing, and Process
Development, as well as major sup-
pliers for SUS.
reFerenceS1. PDA, Technical Report No.66, Application
of Single-Use Systems in Pharmaceutical
Manufacturing (PDA, 2014).
2. H. Domininghaus, Plastics for Engineers,
Materials, Preperties, Applications
(Hanser Publishers, 1993).
3. Plastics Design Library, Chemical
Resistance, Second Edition (William
Andrew Inc., April 1996).
4. P. A. Schweitzer, Corrosion Resistance
Tables, Third Edition, Revised and
Expanded (Marcel Dekker, Inc., 1991).
5. FDA, Part 210 Current Good
Manufacturing Practice in Manufacturing,
Processing, Packing, or Holding of
Drugs; General, www.accessdata.
fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?CFRPart=210
6. FDA, Part 211 Current Good
Manufacturing Practice for Finished
Pharmaceuticals, www.accessdata.
fda.gov/scripts/cdrh/cfdocs/cfcfr/
cfrsearch.cfm?cfrpart=211
7. FDA, Guidance for Industry, Q7A Good
Manufacturing Practice Guidance for
Active Pharmaceutical Ingredients,
www.fda.gov/downloads/Drugs/
uidanceComplianceRegulatoryInformation/
Guidances/UCM073497.pdf
8. ICH, Harmonised Tripartite Guideline, Q7
Good Manufacturing Practice Guide for
Active Pharmaceutical Ingredients, www.
ich.org/fileadmin/Public_Web_Site/
ICH_Products/Guidelines/Quality/
Q7/Step4/Q7_Guideline.pdf
9. FDA, Form 483, 2013, www.fda.gov
/downloads/AboutFDA/CentersOffices/
fficeofGlobalRegulatoryOperationsand
Policy/ORA/ORAElectronicReadingRoom/
UCM377864.pdf
10. W. Ding et al., Pharmaceutical Engineering
(November/December 2014), pp74-85
11. FDA, CGMP for Finished Pharmaceuticals;
21 CFR 211.65(a), www.accessdata.
fda.gov/scripts/cdrh/cfdocs/cfcfr/
CFRSearch.cfm?fr=211.65
12. V. Vinci and S Parekh, Handbook
of Industrial Cell Culture:
Mammalian, Microbial and Plant
Cells (Humana Press, 2003).
13. P. Culter, Protein Purification Protocols,
Second Edition (Humana Press, 2004).
14. F. Jameel and S. Hershenson,
Formulation and Process Development
Strategies for Manufacturing
Biopharmaceuticals (Wiley, 2010).
15. L. Ducry, Antibody-Drug Conjugates
(Humana Press, 2013). ◆
and in the presence of host-cell
impurities,” Codamo observes. He
does note, however, that these issues
have been addressed to some extent
by newer technologies, but further
improvements will be needed. One
example is the development of sin-
gle-use technologies that are better
suited for low-temperature harvesting
and thus would support improved
product quality.
For cell harvesting with perfu-
sion processes, Hill believes that
while ATFs currently provide a good
level of automation, the equipment
is still cumbersome to setup, ster-
ilize, and operate. Cell fouling of
hollow-fiber filters has been the
most frequent problem at large scale
(2000 L). “The concept of separation
of product from cells in a continuous
or semi-continuous manner is neces-
sary on the front end of manufac-
turing to provide a constant process
flow through purification,” he also
notes. Presterilized single-use filter
units would also greatly improve
robustness and build efficiency for
setup and operation of ATF systems,
according to Hill.
He adds that technologies are
also needed to increase the speed
of product separation from cells
through hollow f iber f i lters,
because currently this process is
much slower than continuous cen-
trifugation equipment (up to 24 hrs
for ATF compared with less than 2
hours for continuous centrifuga-
tion at the 2000-L scale). Repligen
Corp., which manufactures ATF
systems, expects to launch a single-
use hollow fiber filter within the
next 12 months, according to Hill,
but he is not currently aware of
any technology under development
that would improve the harvest
time of ATF systems. ◆
Downstream Processing—Contin. from page 31
ES669069_BP0915_039.pgs 09.04.2015 01:57 ADV blackyellowmagentacyan
40 BioPharm International www.biopharminternational.com September 2015
Biophysical binding studies
ut i l i z ing sur face plasmon
resonance, biolayer interfer-
ometry, isothermal titration
calorimetry, or related techniques are
central to the selection and optimi-
zation of biotherapeutic candidates
based on proteins, immunoglobulin
G (IgG), or advanced formats, includ-
ing bispecifics and fusion proteins.
Though these screening and char-
acter ization technologies are well
established, in a variety of circum-
stances, binding measurements may
be ambiguous or even fail to provide
useful data. This article discusses a
suite of complementary techniques,
all based on light scattering, that are
useful in troubleshooting many of the
underlying characterization issues.
These techniques can help investiga-
tors assess solution quality prior to
running binding experiments, qual-
ify aggregation behavior of analytes,
and character ize complex interac-
tions that may not be amenable to
standard characterization method-
ology. Judicious use of a biophysical
light-scattering toolkit is essential for
robust and reliable interaction studies.
Common problems in interaCtion measurementsExperienced practitioners of biophysi-
cal techniquesÑdesigned to measure
biomolecular interactions, such as
surface plasmon resonance (SPR), bio-
layer interferometry (BLI), isothermal
titration calorimetry (ITC), and oth-
ersÑwill recognize the following not-
uncommon scenarios:
• Poor fits of the signals to theoretical
binding curves
• Ambiguous determination of the
appropriate association model
• Erratic or irreproducible data
• Results that vary with immobiliza-
tion chemistry or chip coating
• Resu lt s that depend on which
binding partner is immobilized or
titrated
• Results that vary greatly from lot to
lot of reagent
• Fouling of microfluidic channels.
The causes of these dif f icult ies
are var ied, so it is convenient to
divide the most common into two
categor ie s : qua l i t y cont rol a nd
method limitations.
Quality control issues typical ly
arise from suboptimal sample prepa-
ration, purification, or formulation.
Manifestations may include aggre-
gates, particulates, other impurities,
concentration-dependent self-associ-
ation, or undesirable analyte surface
adhesion (e.g., stickiness). While sam-
ple quality control problems tend to
be more prevalent during early phases
of expression, purification, and pro-
cess development of a biomolecule,
established processes and commer-
cially available reagents are by no
means immune to such excursions
from the normal standard of quality.
M e t h o d l i m i t a t i o n s i n c l u d e
those imposed by su r face -based
methodologies and those imposed by
indirect interaction reporter signals.
The former are generally related to
l igand immobil izat ion, while the
latter is common to many types of
solut ion-based techniques. Every
experimental technique is subject
to specific pros and cons, and often,
the application of several techniques
is necessary to complete the picture
of an interaction or to cross-validate
results. This is particularly true when
the complexes that form go beyond
standard homo/heterodimer or trimer
stoichiometries.
enhancing protein binding studies with a light-scattering toolkit
Daniel Some
Light-scattering techniques
are useful for interaction
studies when traditional
methods of analysis do not suffice.
Daniel Some, PhD, is principal scientist
and director of marketing at Wyatt
technology Corp., [email protected].
protein Characterization
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September 2015 www.biopharminternational.com BioPharm International 41
AL
L F
IGU
RE
S A
RE
CO
UR
TE
SY
OF
TH
E A
UT
HO
R.
light sCattering: the solution for CharaCterizing solutionsMa ny of t hese t rouble some
phenomena may be avoided
or overcome by applying one
or more characterization tech-
niques based on light scatter-
ing. Described in the following
examples are the light scatter-
ing tools most complementary
to biophysical interaction analy-
sis: size-exclusion chromatogra-
phy–multi-angle light scattering
(SEC–MALS), dynamic light scat-
tering (DLS), composition-gradi-
ent–multi-angle light scattering
(CG–MALS), and composition-
gradient– dynamic l ight scat-
tering (CG–DLS). Each of these
tools consists of a sample prep-
arat ion and del ivery method
combined with one of the two
primary flavors of light scatter-
ing for solution character iza-
tion: multi-angle light scattering
(MALS) and dynamic light scat-
tering (DLS).
malsMALS is a first-principles tech-
n ique fo r de te r m i n i ng t he
molecular weight and size of
macromolecules and nanopar-
ticles in solution/suspension (1).
A beam of light impinges on the
solution. While most of the light
traverses the solution volume
unimpeded, a f ract ion of the
photons meet solute molecules
and scatter in a l l d irect ions.
The magnitude R of scattered
intensity relative to the incident
intensity (excess Rayleigh ratio)
is directly related to the molecu-
lar weight of the solute M, the
solute’s mass/volume concentra-
tion c, and the relative differ-
ence in refractive index of the
solute and solvent dn/dc. There
is also an angular dependence to
the scattered intensity when the
particles are larger than approxi-
mately 25 nm in diameter, but
because most proteins and other
biomolecules involved in inter-
act ion studies do not exceed
this size limit, we will hereafter
ignore the angular dependence.
The fundamental thermody-
namic relation, which may be
considered the “ideal gas law”
for light scattering—representing
the limit of a dilute system of
point-like particles—is expressed
as R=K·M·c∙(dn ⁄dc)2 where K is a
system constant. By measuring
the scattered intensity and the
concentration, MALS provides
a first-principles determination
of molar mass. When more than
one species of solute is present,
MALS measures the weight-aver-
age molar mass Mw=∑iMici/∑ici .
DlsDLS is a f irst-pr inciples tech-
n i q u e f o r m e a s u r i n g t h e
diffusion coefficient of macro-
molecules and nanopar t ic les
in solution/suspension and for
est imat ing par t ic le sizes and
size distr ibutions (2). As in a
MALS measurement, in DLS, a
laser beam impinges on the solu-
tion and scatters from particles.
Unlike in MALS, a DLS measure-
ment does not consist of simply
measuring the average scattered
intensity, but in determining its
rate of f luctuation. These f luc-
tuations arise from the scatter-
ers’ Brownian motion, the rapid
jittering of particles buffeted by
solvent molecules. Because the
rate of fluctuation is correlated
to the particle’s diffusion rate,
diffusion coefficient(s) may be
determined by analyzing the
DLS fluctuations. A measure of
particle size known as the hydro-
dynamic radius rh is calculated
from the Stokes–Einstein equa-
tion rh=(kBT ) ⁄(6πηDt).
When more than one popula-
tion of particle size is present
in the solut ion, DLS analysis
protein Characterization
Figure 1: Size-exclusion chromatogram of bovine serum albumin (BSA) and
hemoglobin (Hb). Points indicate molar masses measured by on-line multi-angle
light scattering (MALS) (DAWN, Wyatt Technology) at each elution time. The
identifcation of the early peak as BSA dimer is confrmed by its molar mass
relative to the monomer. The late elution time of Hb would erroneously indicate a
much lower molecular weight than BSA. This pitfall is avoided by use of MALS,
which also indicates the decreasing molar mass across the Hb peak’s trailing
shoulder; this is a result of dynamic dimer-tetramer dissociation in the Hb complex.
200,000
160,000
120,000
80,000
40,000
32.0 36.0 40.0 44.0
Mo
lar
ma
ss (
g/m
ol)
Time (min)
BSA monomer
(66 kDa)
BSA dimer
(133 kDa)
Hemoglobin
(52-62 kDa)
ES670477_BP0915_041.pgs 09.09.2015 02:10 ADV blackyellowmagentacyan
42 BioPharm International www.biopharminternational.com September 2015
may ref lect their distribution.
Depending on the range of sizes,
the d ist r ibut ion i s t y pica l ly
described in one of two ways:
• Polydispersity: If the size range
is l imited around a central
value, DLS reports the aver-
age size and the polydispersity
index, which represents the
width of the distribution.
• Multi-modal distributions: If
size populations are separated
by a factor of 3–5x, a distribu-
tion exhibiting distinct peaks
around each size range may be
calculated. The polydispersity
index of each peak may also be
assessed.
SEC –MALS uses SEC coupled
w ith MALS to determine an
accurate distribution of solution
molecular weights of a sample
from first principles. SEC sep-
arates molecules according to
hydrodynamic size, and in stan-
dard SEC analysis, the reten-
tion time is related to molecular
weight via a series of calibration
standards. Hydrodynamic size
may or may not be re lated
directly to molecular weight; the
specific size/weight relationship
depends on a molecule’s con-
formation. Additionally, mole-
cules may interact non-ideally
with column-packing materials,
or columns may age, leading to
skewed retention times. Hence
standard SEC calibration using
globular, hydrophilic proteins
often does not ref lect the true
molecular weight of the elut-
ing sample, as shown in Figure
1. SEC–MALS overcomes these
limitations by determining inde-
pendently the molar mass of
each elution volume, regardless
of elution time (3). While there
are a variety of important uses
of SEC–MALS for protein charac-
terization, including analysis of
the solution molecular weights
and conjugation state of glyco-
proteins and membrane proteins,
the appl icat ions most useful
in the context of biomolecular
interactions are:
• Determination of a protein’s
oligomeric state in solution
• Assessment of aggregates
• Testing for self-association
• Preliminary analysis of hetero-
complex stoichiometry, espe-
cially when binding affinity is
high enough to survive dilu-
tion in the SEC column.
CG –MALS couples a MALS
detector with a composition gra-
dient (CG) system, which auto-
matically prepares a series of
concentrations or compositions
and injects them sequentially
to the MALS and concentration
detectors, with no accompanying
separation step (4, 5). Each com-
position is analyzed by MALS to
determine molecular weight (Mw),
which increases with the forma-
tion of complexes in a direct and
intuitive fashion with respect
to the nature of the complexes
formed. Hence, dimer formation
results in complexes that scatter
twice as much as the two individ-
ual monomers combined; trimer
formation results in complexes
that scatter three times as much
as the three individual monomers.
A complete CG–MALS analy-
sis determines equilibrium dis-
sociation constants Kd as well as
absolute molecular stoichiome-
tries—the number of molecules
of each type in the complex,
rather than the ratio of moles
of each type that react to form
the complex. Hence, CG–MALS
excels at analyzing self-associa-
tion to determine the oligomer(s)
formed, as well as complicated
interactions involving multiple
complexes, simultaneous self-and
hetero-association, and coopera-
tive interactions—all in solution
without labeling or immobiliza-
tion. As a general rule of thumb,
CG–MALS is appropriate when
the molar mass of the complex
is at least 10% greater than the
largest constituent monomer’s
molar mass.
Batch DLS simply refers to DLS
measurements in a cuvette or
other vessel with no separation.
protein Characterization
Figure 2: High-throughput–dynamic light scattering (HT–DLS) analysis
of proteins samples in situ in a 96-well plate (DynaPro Plate Reader, Wyatt
Technology). The analysis is color-coded to highlight different levels of purity,
indicating which solutions will provide high-confdence results and which are not
suitable for further use.
Monodisperse / low aggregation
Polydisperse / moderate aggregation
Signifcant aggregate content
ES670480_BP0915_042.pgs 09.09.2015 02:10 ADV blackyellowmagentacyan
Register for free at
http://www.biopharminternational.com/bp/Purifcation
Monoclonal Antibody Purification by Protein A Affinity and Hydroxyapatite Mixed Mode Multi-Column Continuous Chromatography
LIVE WEBCAST: Wednesday, October 7 2015 at 2:00-3:00pm EDT
EVENT OVERVIEW:
Increasing manufacturing capacity and decreasing cost per
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the drawback of higher levels of impurities, a burden that can
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Multi-column continuous chromatography (MCC), a form of
simulated moving-bed chromatography (SMBC), is a scalable
technology previously demonstrated to improve productivity
and lower the cost of Protein A afnity chromatography versus
the standard single-column batch process.
This presentation will explain how implementing a continuous
downstream process, through the use of MCC as a cost-reduc-
ing capture step and hydroxyapatite for aggregate removal and
concurrent depletion of impurities following Protein A purifca-
tion of mAbs, can increase efciency and reduce the costs of
mAb production.
Who Should Attend:
n Chromatographers, method developers, and process engineers who are involved with the development of downstream purifcation processes that include Protein A capture and aggregate removal steps.
For questions contact Kristen Moore at [email protected]
Key Learning Objectives:
n Discover how multi-column continuous chromatography difers from traditional chromatographic methods.
n Understand how high-capacity protein A resin and hydroxyapatite, in conjunction with multi-column continuous chromatography, can be an efective means of increasing process efciency and reducing production costs.
n Learn how to select the appropriate conditions to make the most efective use of high-capacity Protein A and hydroxyapatite resins in a multi-column continuous chromatography mAb separation.
Presenter
ANTHONY GRABSKI, Ph.D
Director of Research and
Development
Semba Biosciences Inc.
Moderator
RITA PETERS
Editorial Director
BioPharm International
Sponsored by Presented by
ES668454_BP0915_043_FP.pgs 09.03.2015 02:29 ADV blackyellowmagentacyan
44 BioPharm International www.biopharminternational.com September 2015
protein Characterization
High-throughput–dynamic light
scatter ing (HT–DLS) performs
standard DLS analysis in high
throughput format, in situ, in
standard microwell plates, mak-
ing possible analyses that oth-
erwise would be too onerous to
carry out on a regular basis. Figure
2 illustrates a common solution
quality visualization scheme.
CG –DLS is simi lar in con-
cept to CG–MALS, but the aver-
age size (rather than the average
molar mass, as in CG–MALS) is
measured as a function of con-
centrat ion and/or concentra-
tion to assess interactions (6).
While the range of Kd that may
be quantified via CG–DLS is sev-
eral orders of magnitude less that
CG–MALS, and analysis of sizes
is not as rigorous as analysis of
masses to calculate interaction
parameters, CG–DLS does offer a
few advantages, including much
lower s a mple consu mpt ion,
simple operation in a microw-
ell-plate format, and the ability
to readily carry out temperature
ramps to determine entropy and
enthalpy of the interaction via
van’t Hoff analysis. Perhaps one
of the most useful aspects of
CG–DLS is its ability to quickly
screen hundreds of binding part-
ners, in solution, with a simple
determination or validation of
complex stoichiometry.
Quality Control With light sCatteringClean protein solut ions with
minimal aggregates, particulates,
or other impurities are crucial to
obtaining accurate and repeat-
able binding data. A publication
(7) outlined how poor solution
quality may impact SPR or BLI,
with phenomena that include
noisy signals, unusual/poorly fit-
ting binding curves, low active
concentrations, and even fouling
of microf luidic channels. The
presence of two analytes (e.g.,
monomer and dimer) that bind
to the immobilized ligand, for
example, will lead to a binding
curve that includes two “on”
rates and therefore w i l l not
be well represented by a sin-
gle exponential fit. In another
example, an aggregate contain-
ing multiple binding sites will
often exhibit avidity, or anom-
alously enhanced binding and
slow “off” rates not governed by
the usual fit that assumes a sin-
gle binding site.
Other interaction analysis tech-
niques are no less susceptible to
skewed and erroneous results
caused by poor solution quality.
All solutions (including pure buf-
fer) should be prescreened to test
for aggregation, particulates, etc.
The quickest and easiest means
for assessing solution quality is
batch DLS, which can be per-
formed with as little as 1 µL of
sample (4–20 µL is more typical)
and just a few seconds of mea-
surement time (10–30 seconds
is typical). Batch DLS provides
a low-resolution size distr ibu-
tion covering 0.2–2500 nm in rh,
highlighting the presence of large
particles or aggregates as well as
of low oligomers.
In a qua l it y- d r iven work-
flow, all solutions would be pre-
screened by batch DLS. Solutions
with large particulates would be
filtered or centrifuged to remove
the pa r t ic les , and the solu-
tion rechecked. Buffers should
be cleaned up to see no appre-
ciable particles above ˜0.3 nm.
For protein solutions, if no large
Figure 3: Size-exclusion chromatography–multi-angle light scattering (SEC–
MALS) data indicating dynamic equilibrium between monomers and oligomers, via
the concentration dependence of molar mass (DAWN, Wyatt Technology). Three
injections with different starting concentrations and concentration variation across
each peak all point to rapid, reversible self-association.
140
120
100
80
60
200 μg
cmax = 0.10 mg/mL
100 μgcmax = 0.05 mg/mL
50 μgcmax = 0.02 mg/mL
17 18 19Time (min)
Mo
lar
ma
ss (
kD
a)
20
light-scattering
analyses are
generally
straightforward,
intuitive, and
informative.
ES670482_BP0915_044.pgs 09.09.2015 02:10 ADV blackyellowmagentacyan
September 2015 www.biopharminternational.com BioPharm International 45
particles (i.e., more than 3–5
times the size of the monomer)
remain, then the polydispersity
of the protein peak should be
checked. Because it is possible
to f i lter protein solut ions to
0.02 µm using syringe-tip filters
(Whatman), most larger oligo-
mers may be removed as well.
When large numbers of solu-
t ions a re to be prescreened,
HT–DLS offers a ready solution.
Because it uses standard microw-
ell plates and measures in situ
in the plate with no liquid han-
dling, HT–DLS is ideal for inte-
grating with interaction analysis
technologies that sample from
these plates (SPR, ITC) or make
the measurements directly in the
plates (BLI).
The final quality check of a
protein solution consists of a
SEC–MALS measurement to ver-
ify the oligomeric state of the
protein and determine size and
quant it ies of low ol igomers.
More than one interaction mea-
surement has been led astray by
assuming that the protein was
monomeric in solution because a
single peak appeared in the SEC
run, or because a denaturing gel
assay indicated the monomeric
molecular weight. SEC–MALS
prov ides con f idence i n t he
reagents to be used in the bind-
ing measurement, and the work-
flow should only proceed if an
investigator is satisfied with the
SEC–MALS results.
overComing methoD limitations With light sCatteringSurface-based techniques such as
SPR and BLI necessarily rely on
assumptions regarding the expo-
sure of relevant epitopes to the
solution, which may not hold
in certain circumstances. More
common, though, are confus-
ing phenomena such as avidity
(the anomalously strong bind-
ing to immobilized ligands of
analytes presenting two binding
sites), surface-chemistry-depen-
dent results (of ten related to
the charge of the immobiliza-
tion layer such as dextran), and
complex multi-valent or coop-
erative interactions that cannot
occur when one binding partner
is immobilized.
While the limitations of sur-
face interaction techniques are
genera l ly overcome by solu-
tion-based techniques such as
ITC, f luorescence anisotropy,
or mic rosca le ther mophore -
sis (MST), most of these solu-
tion-based assays are subject to
their own limitations. A com-
mon drawback is the need for
fluorescent labeling, potentially
modifying the interaction. In
addit ion, most solution-based
measurements involve an indi-
rec t repor te r s ig na l t hat i s
assumed to represent the effect
of a binding interaction, but can-
not be unequivocally assigned to
the formation of a specific bio-
molecular complex. For exam-
ple, ITC measures the release or
uptake of heat; while there is a
good probability that this ther-
mal signal is the result of asso-
ciation or dissociation, ITC does
not offer direct proof of com-
plex formation or dissociation
and cannot indicate unambigu-
ously which complex(es) form,
especially when characterizing
self-association. In instances of
purely entropic binding, no ther-
mal signal is available to report
the interaction.
Analy t ica l u lt racent r i f uga -
tion—sedimentation equilibrium
is quite useful for analyzing a
variety of interactions. Its pri-
mary limitation is the long time
required to equilibrate, during
which it is possible that sensitive
proteins degrade.
Use of SEC–MALS, CG–MALS,
and CG –DLS helps overcome
protein Characterization
Figure 4: A complete analysis of antibody-antigen self- and hetero-association
by means of composition-gradient–multi-angle light scattering (CG–MALS)
(Calypso, Wyatt Technology). The left and right sections correspond to single-
species, stepwise concentration gradients that determine the molecular weight
and self-association properties of each protein. The center section corresponds
to a stepwise cross-over gradient that gradually reduces the fraction of antibody
and increases the fraction of thrombin, covering the complete range of hetero-
association stoichiometries. The data indicate expected excess scattering over the
signal if no interaction occurs, pointing to increasing molar mass due to complex
formation. The data are analyzed to determine the molecular composition of the
complex and its binding affinity, even if self-association is present.
Antibodygradient
4
3.5
3
2.5
2
1.5
1
0.5
0Lig
ht
sca
tte
rin
g s
ign
al
(kD
a*
g/L
)
0 20 40 60Time (min)
Actual LS signal LS signal if no interaction
80 100 120 140
Thrombingradient
Light scattering
Hetero-association gradient
ES670479_BP0915_045.pgs 09.09.2015 02:10 ADV blackyellowmagentacyan
46 BioPharm International www.biopharminternational.com September 2015
protein Characterization
most of aforementioned l imi-
tations and are excellent com-
plements to other interaction
analysis technologies. In some
cases, CG–MALS may be the only
technique capable of fully teas-
ing out a complex interaction
with relative ease.
For self-association, it is often
simplest to begin with SEC–MALS.
Plots of molecular weight that vary
along the peak in a concentration-
dependent manner and that vary
similarly with the concentration
of the injected aliquot are good
indicators of a self-associating pro-
tein, as shown in Figure 3. Under
certain assumptions, it is even
possible to determine Kd of simple
oligomerization such as dimer or
trimer formation (8). Alternatively,
other techniques may suggest that
self-association occurs. In a robust
approach, the workflow would
continue from an initial self-asso-
ciation indication to a CG–MALS
analysis, which would accurately
determine the oligomers that
form—even if multiple oligomeric
states are involved—and the bind-
ing affinity for each oligomer.
For hetero-associat ion, too,
SEC–MALS may be an excel-
lent initial indicator of complex
stoichiometry. A series of pre-
incubated aliquots prepared at dif-
ferent stoichiometric ratios may be
tested, with analysis consisting of
determining the molar mass of the
eluting complexes and unbound
monomers (2). For example, if the
complex is 1:1, and the aliquot is
prepared at a 1:1 ratio, then little
to no unbound monomer should
remain, but if the complex is
2:1, then there will be an excess
of B and a significant amount of
unbound B should elute in a sepa-
rate peak from the complex. While
surface-based techniques are quite
limited in this respect, other solu-
tion-based techniques also give
good initial indications of hetero-
association stoichiometry.
A robust workf low designed
for fully characterizing a het-
ero-association should proceed
to a CG–MALS analysis such as
that of Figure 4. CG–MALS will
validate the true molecular stoi-
chiometry of the complex and
determine Kd by fitting the data
of Mw vs. composition under the
correct association model.
A variety of advanced interac-
tions are not addressed well by
most techniques. These include
simultaneous self- and hetero-
association, interactions between
t wo mult i -va lent molecu les ,
higher-order self-assembly, and
cooperative effects that lead to
formation of higher-order com-
plexes. Because CG –MALS is
a f i r s t-pr i nc iple s , solut ion-
based technique that provides a
direct reporter signal (molecular
weight), it is well suited for tack-
ling such challenging interactions
and providing a comprehensive
analysis. In parallel, SEC–MALS
may provide an initial indica-
tion of what forms are present in
solution, guiding the CG–MALS
method design.
CG–DLS may be used in a simi-
lar manner as CG–MALS, with a
lower range of measurement and
less robust analysis albeit much
lower sample consumption. CG–
DLS becomes limited in terms of
analysis when the order of the
interaction becomes high, since the
relationship between stoichiometry
and rh may become ambiguous.
summaryInteractions between molecules are
complicated. Careful characteriza-
tion of reagents, as well as a criti-
cal examination of binding studies,
is necessary to guarantee accurate
and repeatable results, regardless of
the techniques applied. Many of the
challenges related to both reagent
characterization and validation
of interaction measurements may
be addressed successfully through
a suite of analytical tools involv-
ing light scattering. Light-scattering
analyses are generally straightfor-
ward, intuitive, and informative.
They solve analytical questions with
minimal time and effort, enhance
productivity, and minimize ambi-
guities in interaction studies.
referenCes 1. P.J. Wyatt, Analytica Chimica Acta
272, pp. 1–40 (1993). 2. I. Teraoka, Polymer Solutions: An
Introduction to Physical Properties, (John Wiley & Sons, Inc. ISBN 0-471-38929-3, New York, NY, 2002).
3. J. Wen, T. Arakawa, and J.S. Philo, Analytical Biochemistry 240, pp. 155–166 (1996).
4. D. Some and S. Kenrick, “Characterization of Protein-Protein Interactions via Static and Dynamic Light Scattering” in Protein
Interactions, J. Cai and R. Wang Eds. (InTech, 2012), pp. 401–426.
5. D. Some, Biophysical Reviews 5 (2), pp. 147–158 (2013).
6. A. D. Hanlon, M. I. Larkin, and R. M. Reddick, Biophysical Journal 98 (2), pp. 297–304 (2010).
7. D. Some, “Protein Quality Control in SPR and BLI High-Throughput Screening Studies,” www.wyatt.com/files/literature/white-papers/protein-quality-control-high-throughput-screening-studies.pdf, accessed Aug. 14, 2015.
8. S. Das et al., J. Bacteriol. 190, pp. 7302–7307 (2008). ◆
Clean protein
solutions with
minimal aggregates,
particulates, or
other impurities are
crucial to obtaining
accurate and
repeatable binding
data.
ES670481_BP0915_046.pgs 09.09.2015 02:10 ADV blackyellowmagentacyan
eve
ryth
ingpossib
le/G
etty Im
ages
SPECIAL SESSION
CPhI Pharma Insights Briefing
The Outlook for the Bio/Pharmaceutical Contract Manufacturing Industry
Presented by Jim Miller, President, PharmSource
Wednesday, October 14 from 12:00 – 12:45 PM
While contract manufacturing organizations (CMOs) consolidate their positions in traditional markets and
technologies, they are not addressing some of the most important trends in pharmaceutical manufacturing.
Learn about these trends, as well as the status of the CMO industry, opportunities it offers to clients, and the
challenges it must face to remain relevant in the future.
Located in The Pharma Forum, outside of Halls 5 & 6.
GEt tHE LAtESt
COVERAGE OF:• ProcessDevelopment
• ManufacturingTrends
• Formulation
• AnalyticalTechnology
• RegulatoryCompliance
• QualityAssurance
• APIsynthesis
• Packaging
• Outsourcing
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SPECIAL EDUCATIONAL PROGRAMMING
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LEADING PHARMACEUtICAL DEVELOPMENt &
MANUFACtURING PUBLICAtION IN EUROPE.
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Come See US at Booth 1a3
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ES668455_BP0915_047_FP.pgs 09.03.2015 02:29 ADV blackyellowmagentacyan
48 BioPharm International www.biopharminternational.com September 2015
So
rend
ls/G
ett
y Im
ag
es
To maintain a state of control
and comply with regulatory
authorities, many pharma-
ceutical, biotech, and med-
ical-device companies have adopted
continued process verification (CPV)
initiatives for manufacturing pro-
cesses (1). By adopting a proactive
approach to monitoring, life-science
manufacturing companies can iden-
tify changes in their manufacturing
processing prior to a quality event
such as a batch failure. Control charts
are the predominant monitoring tool
used to monitor parameter data across
batches with control limits defined
for the normal manufacturing pro-
cess. Control chart run rules generate
signals when non-random processes
occur and flag potential process issues.
The maximum partial pressure of
CO2, for example, may have an unde-
sired upward trend due to a machine
calibration issue, and a signal would
prompt a process engineer to investi-
gate before drug potency was affected.
Valid signals can reduce costs, improve
process understanding, and enhance
operational reliability (2).
Despite the well-defined benefits of
run rules, many life-science companies
misuse them due to lack of guidelines
and/or statistical expertise regarding
statistical process control, which may
lead to incorrect signals (i.e., the inabil-
ity to differentiate between signals and
random noise) and, ultimately, failure
of the monitoring system. Essentially,
the goal when using run rules is to
ensure that valid signals are generated,
Utilizing Run Rules for Effective Monitoring in Manufacturing
Aaron Spence
To enable efficient
monitoring systems, life-
science companies
need to effectively
apply run rules.
Aaron Spence is manager of
analytic consulting, BIOVIA.
Process Controls
ES668637_BP0915_048.pgs 09.03.2015 19:48 ADV blackyellowmagentacyan
September 2015 www.biopharminternational.com BioPharm International 49
correct signals are not overlooked,
and false signals are not created.
This article discusses the life-
science manufacturing industry’s
current use of monitoring tech-
niques and provides guidance
on how to improve the value
obtained from monitoring pro-
grams and run-rule signals.
CURREnt IndUstRy PRACtICEsLife-science manufacturing compa-
nies with mature CPV initiatives
apply run rules to critical/key pro-
cess parameters and quality attri-
butes for trending purposes (4).
Despite small variation among
companies, run rules are typically
applied to parameters based on
risk assessments (e.g., process fail-
ure mode and effects analysis, esti-
mated process capabilities, etc.).
Two common issues life-science
companies often face in the early
stages of adopting a CPV initia-
tive are over-alerting (e.g., generat-
ing false, non-value-added signals)
or under-alerting (e.g., overlook-
ing valid signals). Over-alerting
is often the result of monitoring
every parameter with every run
rule. Conversely, under-alerting
can occur when companies only
evaluate if critical release param-
eters fall within specification lim-
its and adequate monitoring is not
performed. The objective of run
rules in a mature CPV program is
to generate valid signals that pro-
vide useful information for engi-
neers and process experts.
One of the principal benefits
companies receive from using run
rules is the enablement of a mon-
itor ing-by-exception solution.
Instead of manually reviewing
control charts on a regular basis,
selected software programs can
automatically alert users to run-
rule signals (i.e., when a run rule
is violated). Then, resources may
be allocated appropriately to eval-
uate parameters with signal viola-
tions. A monitoring-by-exception
solution that can scale up to mul-
tiple sites and products is essential
to a CPV model.
Regardless of their specific prac-
tices, companies should clearly
document the proper use of run
rules in their monitoring proce-
dures to avoid significant regulatory
risk, and responsible individuals
should be assigned to create and
maintain these crucial documents.
Implementation will also likely
require ongoing evaluations and
support from the company.
GEttInG thE RIGht sIGnAlsGenerating valid run-rule signals
provides a variety of benefits,
primarily through cost reduc-
t ions and deployment of an
early-warning system to prevent
product quality issues. Obtaining
valid signals to drive these busi-
ness benefits, however, is depen-
de nt on f ive mat he mat ic a l
assumptions: parameters must
be baselined from an in-control
process, baselined on an accept-
able sample size, detailed regard-
ing data prec ision, normal ly
distributed, and independent.
Receiving correct value-added
signals requires the use of his-
torical data to establish base-
line control limits for ongoing
monitor ing. Many companies
Process Controls
Table I: Monitoring assumptions checklist.
AssumptionAssessed?
Typical assessments Suggested correction(s)
In-control process Process knowledge, outlier identification Remove special cause for baseline
Sample size ≥ 25 or 30 batches availableObtain additional batches, limit run rules, k-factor corrections (3)
Data precision ≥ 5 different parameter values (excluding outliers)Improve data collection procedures, limit run rules, revise data agreements with contract manufacturing organizations
Normality Shapiro-Wilk, Anderson Darling, Q-Q plotTransformations, non-parametric limits, rational subgrouping
IndependenceDubin-Watson, Ljung Box Q, autocorrelation function/partial autocorrelation function (ACF/PACF) plots
Long-term standard deviation for limits, regression methods (e.g., autoregressive integrated moving average [ARIMA], generalized least squares [GLS]), rational subgrouping
ES668636_BP0915_049.pgs 09.03.2015 19:48 ADV blackyellowmagentacyan
50 BioPharm International www.biopharminternational.com September 2015
overlook the criticality of base-
lining parameters, which results
in inf lated control l imits and
missed valid signals. To ensure
the process is in control, special
cause variation needs to be iden-
tified and removed during the
baseline process. Special cause
variation results from variability
caused by events outside of the
normal manufacturing process,
such as operator error, power
failure, etc. Eliminating special
cause var iat ion provides l im-
its that more accurately reflect
nominal manufactur ing pro -
cesses. Baselining should occur
after a known process change
occurs (e.g., equipment change,
supplier change, etc.) or on a
regular basis (e.g., annually or
semi-annually) depending on the
availability of staff, the number
of batches produced in a year,
and other parameters. When
one is baselining parameters, at
least 25–30 batches should be
available to generate targets that
accurately reflect the manufac-
turing process. The monitoring
system should allow engineers to
differentiate valid signals from
random process noise, as well.
To produce valid signals that
generate value, an organization
needs to measure and record pro-
cess variability accurately with
sufficient data precision. This is
especially important when the
decimal precision for param-
eters results in a small number
of unique values—an issue that
often plagues the life-sciences
manufac tur ing indust ry. For
example, if pH is reported at a
single decimal precision and val-
ues are always 6.7, 6.8, or 6.9,
run rules cannot be appropri-
ately applied and false signals
may be generated or valid signals
may be overlooked.
For an organization to effec-
tively use control charts and run
rules, parameters need to fol-
low a normal distribution. The
assumption of a normal distribu-
tion relies on the fact that only
1 out of 370 observations will
fall beyond control limits just
by chance (5). Abnormal data
Process Controls
Table II: Common run rules used in the manufacturing industry. Control limits = 3 standard deviations (SD) from mean; warning
limits = 2 SD from mean; inner limits = 1 SD from mean.
Signal Signal typeWestern
electric ruleNelson
ruleFollow-up
1 point outside of control limits
Single event Rule 1 Rule 1Examine other issues or events (e.g., operator error, machine calibration) or any other abnormal observations from other parameters within the batch.
2 out of 3 points outside of warning limits
Shift
Rule 2 Rule 5
Investigate process or supplier changes; utilize group difference tests (e.g., t-tests, analysis of variance models [ANOVAs]) for categorical process variables such as machines, cleanrooms, etc.; conduct correlations with parameter that displays shift.
4 out of 5 points outside of inner limits
Rule 3 Rule 6
9 points on the same side of the central line
Rule 4 Rule 2
6 points in a row increasing/decreasing
Decreasing variability N/A Rule 3Investigate correlations with the parameter that displays the trend; determine if machine maintenance or calibrations are required.
14 points in a row alternating in direction (increasing/decreasing)
Increasing variability N/A Rule 4Investigate correlations with parameter that displays oscillation; conduct group difference tests if multiple populations are represented.
15 points in a row all within the inner limits
In-control process N/A Rule 7Investigate correlations with parameter that displays decreased variability; examine process improvements.
8 points in a row, none of which are within the inner limits
In-control process N/A Rule 8
Investigate seasonality effects or non-random cycles (e.g., changing suppliers, using different machines, etc.); conduct group difference tests; conduct correlations with parameter that displays cycle.
ES668649_BP0915_050.pgs 09.03.2015 19:49 ADV blackyellowmagentacyan
September 2015 www.biopharminternational.com BioPharm International 51
Contin. on page 57
Process Controls
results in an inflated likelihood
that data will fall beyond control
limits (e.g., a one out of 40 chance)
(6). Non-normal distributions are
highly prevalent in life-science
manufacturing processes, and
they are often expected when
processes have lower or upper
bounds (e.g., yield, hold time,
etc.). Not correct ing for this
assumption violation, however,
may lead to false signals, which
have the effect of devaluing the
significance of a violated con-
trol limit. Over-alerting by gen-
erating false signals can result in
employees simply ignoring the
monitoring system.
Non-independent parameters
have narrower control l imits
and are more likely to generate
false signals, resulting in wasted
resources and an inef f ic ient
monitoring system. The assump-
tion of independence assumes
that prev ious batches do not
affect subsequent batches, and
non-independence often indi-
cates there is a pattern in the
data. It is crucial that CPV and
monitoring plans provide direc-
t ions on how to test assump-
tions and correct for assumption
violations so that monitoring-
by-exception solutions generate
appropriate signals.
To achieve the most va lue
from a monitoring system and
generate valid signals, one needs
to continuously evaluate and
correct the assumptions underly-
ing a system. Without these cor-
rections, false signals will waste
company resources and valid sig-
nals may be overlooked. Table I
provides assessments and suggested
actions for the aforementioned
assumptions.
PROPER REsPOnsE tO sIGnAlsLi fe - sc ience companies w ith
mature CPV models derive value
from run-rule signals by imple-
menting appropriate follow-up
practices. When a monitoring
system is configured correctly,
i ncor porat i ng a l l t he s teps
defined in Table I, signals should
be valid and indicate that the
process has changed in some
way. Thus, when a run-rule vio-
lation occurs, it is important to
appropriately follow up on the
signal. Conversely, life-science
manufacturing companies with
immature CPV initiatives spend a
lot of non-value added time chas-
ing false signals, or they revert to
the other extreme of not follow-
ing up on signals at all. Limited
resources and statistical expertise
can make following up on viola-
tions a struggle for many life-
science manufacturers.
It is c ruc ia l to understand
that run-rule signals should be
t reated d i f ferent ly than out-
of-specification quality events.
Quality events require imme-
diate cor rec t ive and preven-
tive actions and investigations,
whereas not every run-rule sig-
Figure 1: Examples of signals denoting process events/changes.
Single event
Nelson rule 1
Cyclical pattern
Nelson rule 2,5,6,8
Oscillation
Nelson rule 4
Increasing variability
Nelson rule 8
Shift
Nelson rule 2,5,6
Trend
Ind
ivid
ua
ls -
Tre
nd
Ind
ivid
ua
ls -
Sh
ift
Ind
ivid
ua
ls -
Sin
gle
eve
nt
Ind
ivid
ua
ls -
Cycl
ica
l p
att
ern
Ind
ivid
ua
ls -
Osc
illa
tio
n
Ind
ivid
ua
ls -
In
cre
asi
ng
va
ria
bil
ity
Batch ID Batch ID Batch ID
Batch ID Batch ID Batch ID
4
3
2
1
0
-1
-2
-3
-4 F8
10
39
F8
10
37
F8
10
35
F8
10
33
F8
10
31
F8
10
29
F8
10
27
F8
10
25
F8
10
23
F8
10
21
F8
10
19
F8
10
17
F8
10
15
F8
10
13
F8
10
11
F8
10
09
F8
10
07
F8
10
05
F8
10
03
F8
10
01
4
3
2
1
0
-1
-2
-3
-4 F8
10
39
F8
10
37
F8
10
35
F8
10
33
F8
10
31
F8
10
29
F8
10
27
F8
10
25
F8
10
23
F8
10
21
F8
10
19
F8
10
17
F8
10
15
F8
10
13
F8
10
11
F8
10
09
F8
10
07
F8
10
05
F8
10
03
F8
10
01
4
3
2
1
0
-1
-2
-3
-4 F8
10
39
F8
10
37
F8
10
35
F8
10
33
F8
10
31
F8
10
29
F8
10
27
F8
10
25
F8
10
23
F8
10
21
F8
10
19
F8
10
17
F8
10
15
F8
10
13
F8
10
11
F8
10
09
F8
10
07
F8
10
05
F8
10
03
F8
10
01
4
3
2
1
0
-1
-2
-3
-4 F8
10
39
F8
10
37
F8
10
35
F8
10
33
F8
10
31
F8
10
29
F8
10
27
F8
10
25
F8
10
23
F8
10
21
F8
10
19
F8
10
17
F8
10
15
F8
10
13
F8
10
11
F8
10
09
F8
10
07
F8
10
05
F8
10
03
F8
10
01
4
3
2
1
0
-1
-2
-3
-4 F8
10
39
F8
10
37
F8
10
35
F8
10
33
F8
10
31
F8
10
29
F8
10
27
F8
10
25
F8
10
23
F8
10
21
F8
10
19
F8
10
17
F8
10
15
F8
10
13
F8
10
11
F8
10
09
F8
10
07
F8
10
05
F8
10
03
F8
10
01
4
3
2
1
0
-1
-2
-3
-4 F8
10
39
F8
10
37
F8
10
35
F8
10
33
F8
10
31
F8
10
29
F8
10
27
F8
10
25
F8
10
23
F8
10
21
F8
10
19
F8
10
17
F8
10
15
F8
10
13
F8
10
11
F8
10
09
F8
10
07
F8
10
05
F8
10
03
F8
10
01
Nelson rule 3
AL
L F
IGU
RE
S A
RE
CO
UR
TE
SY
OF
TH
E A
UT
HO
R
ES668648_BP0915_051.pgs 09.03.2015 19:49 ADV blackyellowmagentacyan
52 BioPharm International www.biopharminternational.com September 2015
Sve
ta D
em
ido
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ett
y Im
ag
es
Troubleshooting
Stability testing of biopharmaceuticals
must be performed according to regula-
tory requirements for drug development
programs to establish the re-test/expiry date
for drugs. During such studies, however, it is
not uncommon that atypical or unexpected
data may arise, and given the crucial need
for these studies, effective and rapid response
is necessary to prevent impact on drug-devel-
opment timelines. The cause of any issue
must be identified, and any newly observed
impurities/degradants must be characterized
and assessed for any risk to safety, quality,
and efficacy.
This article presents three common scenarios
observed during stability testing of biologics and
the use of orthogonal analysis, stability experi-
ence, and troubleshooting measures to identify
causes, assess risk, and define subsequent steps in
the development of a biopharmaceutical.
Protein Product and acidic sPeciesIn the first study, Molecule X was a lyophilized
formulation of a protein, a sterile drug product
contained in a vial with an intended long-term
storage condition of +5 °C ± 3 °C. The material
had been tested by gel isoelectric focusing in the
early phase of drug development. The method
that was used was standard and had not been
further reviewed or optimized during the pro-
gram. The isoelectric focusing (IEF) gel profiles
showed unclear banding with poor resolution.
To prov ide g reater understanding of
charged-based variants of the molecule, a
method with greater sensitivity was devel-
oped using imaged capillary isoelectric focus-
ing (cIEF). As expected, cIEF indicated that
the poorly resolved bands on the
IEF gel actually represented four
charged-based variants. These con-
sisted of the main product isoform
that focused at the expected isoelectric point
of the product (i.e., at pI [isoelectric point] 6.8),
two acidic isoforms, and one basic isoform
(Figure 1). This test was validated and incorpo-
rated into the release and stability programs
for the product. During routine testing at the
three-month time point, however, an atypi-
cal isoform was observed that had not been
detected during previous testing procedures or
during method development/validation. The
data indicated that the change in cIEF profile
was accompanied by a change in the ELISA
results for the molecule, thus showing that the
atypical peak impacted product activity.
To further characterize this atypical peak,
the sample was tested against a frozen retained
sample from T=0, by electrospray mass spec-
trometry quadrupole-time-of-flight (ESI MS
Q-ToF) analysis. The data confirmed that the
T=3 sample had an increased level of deamida-
tion of approximately 15% (Figure 2). In addi-
tion, MS/MS sequencing confirmed that the site
of deamidation was located at an asparagine
residue that was located in the complementar-
ity-determining region (CDR) of the molecule.
These data correlated with tertiary structural
changes identified by near-ultraviolet circular
dichroism (near-UV CD), where changes in the
spectral profile were observed. Supporting data
from Fourier transform infrared (FTIR) analysis
showed that secondary structural changes in
the reduction of β-sheets had occurred.
The investigation therefore showed that the
molecule had an amino acid residue that had
an increased propensity to deamidate in the
active domain. Unfortunately, this was not
identified in early development, as the original
method had insufficient resolution to distin-
guish between charged variants.
Based on the results, the product sequence was
reviewed and modified to remove the susceptible
Stability Testing in BiopharmaThree case studies illustrate some analytical methods important for stability testing.
Stella-Christiana Chotou is team
leader of stability services at sGs Life
science services.
ES670448_BP0915_052.pgs 09.09.2015 01:56 ADV blackyellowmagentacyan
September 2015 www.biopharminternational.com BioPharm International 53
residue. The development program
was significantly delayed as various
additional supporting studies were
required. This delay could have been
prevented if an optimized method
had been developed upfront in the
early development phase.
A forced degradation study dur-
ing early development would con-
firm key degradation pathways,
and determine whether such a
method is stability-indicating for
the drug molecule or not. These
degradation data should also be
used in the validation of the cho-
sen method for charge-based vari-
ant analysis of the product.
MonocLonaL igG druG substance and ParticuLates In a second study, Molecule Y was a
recombinant protein drug substance
in a liquid formulation. The drug
product itself was produced follow-
ing the vialing and lyophilization of
the drug substance. The drug sub-
stance had an intended long-term
storage condition of +5 °C ± 3 °C,
and data had been used to establish
a two-year shelf life.
During ongoing and established
stability studies, activity testing
of the drug substance unexpect-
edly failed at T=6 months. This
result did not correlate with his-
torical data or any of the other
data available. During the investi-
gation, it was noted that all testing
for the sample was performed at
site A with the exception of activ-
ity testing, which was performed
on a small aliquot of the sample
at site B. In this case, the shipping
procedure for the sample used in
activity testing differed from the
one normally used. The courier
had intended to ship the sample
to the testing lab directly, as had
been done previously; however,
this time the courier had made an
error and shipped the sample to
an incorrect location. The sample
then had to be transported to the
correct location. The temperature
monitoring data indicated that the
cold chain had been adequately
maintained throughout the ship-
ment. The only item of note was
that because of the need to cor-
rect the delivery of the sample,
the product had been subjected to
additional eight hours of transport
by truck, which had not occurred
in previous shipments.
troubleshooting
Figure 1: Examples of a) an imaged capillary isoelectric focusing (cIEF) electropherogram at T=0 months, and b) of a cIEF
electropherogram at T=3 months.
A0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
-0.054.0
Abso
rbance
-4.22
-9.45
4.5 5.0 5.5 6.0 6.5 7.0
pl
7.5 8.0 8.5 9.0 9.5 10.0
Abso
rbance
-4.22
-9.45
pl
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
-0.054.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
B
AL
L F
IGU
RE
S A
RE
CO
UR
TE
SY
OF
TH
E A
UT
HO
R.
Figure 2: Electrospray mass spectrometry quadrupole-time-of-fight (ESI MS
Q-ToF) data demonstrating deamidation in the complementarity-determining
region (CDR) domain at T=3 months.
T=3
T=0
This species is doubly charged. Therefore, an m/z increase of 0.5 of a doubly charged signal corresponds to a mass increase of 1 Da.
This is consistent with deamidation.
100
100
893
894.03894.75895.64 896.67 897.60
898.15899.91
900.43
900.92
901.43
901.93
900.43
900.94
901.43
901.93
910.93
902.42
902.62 904.71904.98 906.03907.93
908.41909.44
909.90 911.91
894 895 896 897 898 899 900 901 902 904903 905 906 907 908 909 910 911
893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911m/z
m/z0
0
%%
ES670445_BP0915_053.pgs 09.09.2015 01:55 ADV blackyellowmagentacyan
54 BioPharm International www.biopharminternational.com September 2015
troubleshooting
Based on this, a small-scale agi-
tation study was performed in
which the product was subjected
to agitation at 30 rpm over 0, 4,
8, 16 and 24 hours. Samples were
tested using low-volume methods
to assess levels of visible particles
by visual appearance, sub-visible
particles by light obscuration and
smaller size particles by size exclu-
sion chromatography (SEC) and
dynamic light scattering (DLS).
DLS was used to assess the lev-
els of higher-molecular-weight
(HMW) species at particle sizes
from 10nm to 1μm. The results
showed significant levels of higher
MW species in the later time
points compared with the con-
trol sample at T=0 hours, which
was demonstrated in a series of
correlograms (Figure 3). The con-
trol sample showed a steep sig-
moidal correlogram as a result of
the correlogram function, indi-
cating small monomeric-type spe-
cies, whereas the degraded sample
showed a broad, slow decay, indi-
cating the formation of multiple,
large, slowly diffusing, high-MW
species. It was also observed in
the sample wells that no parti-
cles were evident in the T=0 hours
well, but it was obvious that there
were particles in the T=16 hours
sample well. These particles were
identified as consisting of dimers,
trimers, and multimers of the
drug molecule, and it was con-
cluded that the material was sensi-
tive to agitation. This meant that
shipment as a liquid was not a
suitable procedure to use for this
drug product.
Following confirmation that the
product was stable after a freeze/
thaw test procedure, the ship-
ment conditions for the material
were selected as frozen. This issue
could have been identified during
early development had an agita-
tion assessment been performed. It
is recommended that an agitation
assessment be performed as part of
an early-phase forced-degradation
study or that it is incorporated into
the first stability study.
HorMone druG Product teMPerature excursionIn a third study, Molecule Z, a hor-
mone drug product in a liquid
formulation, had an intended long-
term storage condition of +5 °C ±
3 °C. The product was in ongoing
Phase II clinical trials, and during
shipment of a batch for use in the
next stage of clinical trials, the tem-
perature that the sample was being
held at dropped to 1 °C, outside of
the required range.
During shipment, customs had
retained the product at the air-
port to review paperwork. Such a
delay is not uncommon; however,
during this period the container
was held outside in cold weather
conditions, and the temperature
in the container dropped to 1 °C
for up to 5 hours (Figure 4). The
cold-chain data for this product
indicated it was not stable upon
freeze/thaw at temperatures rang-
ing from -20 °C to ambient; how-
ever, no data were available at 1 °C.
The regulators were informed and
Figure 3: Comparison of dynamic light scattering (DLS) correlograms at T=0
hours and T=16 hours.
Figure 4: Temperature data during shipment showing the excursion to 1 °C.
1.18
1.16
1.14
1.12
1.10
1.08
1.06
1.04
1.02
1.00
0.98
0.960.10
Inte
nsi
ty A
uto
corr
ect
ion
1.00 10.0 100.0 1.0E+3
Time (μs)
1.0E+4 1.0E+5 1.0E+6 1.0E+7
T=0 T=16
Temp tale data9
8
7
6
5
4
3
2
1
0
Temperature
lower limit
upper limit
Time
Tem
pe
ratu
re (
oC
)
06:0
0:00
07:0
0:00
08:0
0:00
09:0
0:00
10:0
0:00
11:0
0:00
12:0
0:00
13:0
0:00
14:0
0:00
15:0
0:00
16:0
0:00
17:0
0:00
18:0
0:00
19:0
0:00
20:0
0:00
21:0
0:00
22:0
0:00
23:0
0:00
ES670447_BP0915_054.pgs 09.09.2015 01:55 ADV blackyellowmagentacyan
September 2015 www.biopharminternational.com BioPharm International 55
troubleshooting
the clinical trial was put on hold
until the quality of the material
was reviewed.
An urgent excursion study
on the material was performed,
with a representat ive sample
being taken from the batch and
held in a programmed thermal
cycling chamber at +5 °C for 4
hours, followed by 1 °C for 5, 10
and 15 hours. Stability tests were
performed on an unexposed con-
trol sample and on the exposed
samples, with the resulting data
confirming that the product qual-
ity was unaffected following this
level of exposure. The data were
used to provide scientifically sound
justification that product quality
had not been affected and the clini-
cal trials were continued.
Excursion studies for new prod-
ucts are not mandatory. Studies
that include mapping of the
intended shipment routes, how-
ever, can be used to perform
risk analyses and identify any
high-risk excursions in advance.
Conducting a small-scale study to
identify the impact of most likely
shipping events upfront allows
drug developers to be proactive,
rather than reactive, when an
unexpected temperature excur-
sion occurs.
concLusionStability studies are an integral
part of drug development with
str ingent timelines for analyt-
ical testing. However, it is not
uncommon that at y pica l or
unexpected stability data may
arise that cause severe impact
on the drug development pro-
gram. It is crucial, therefore, for
drug developers to take rapid
and effective action that mini-
mizes the risk to their timelines
and satisfies regulatory authori-
ties. As discussed in this article,
it is recommended that potential
risks to the biopharmaceutical
due to temperature excursions
or agitat ion dur ing shipment
are ident i f ied in early devel-
opment rather than late-phase
stages. Furthermore, it is vital
to develop appropriate stability-
indicating analytical methods to
be able to identify degradants
that present potential r isk to
patient safety. ◆
PRODUCT SPOTLIGHT
Biocontainer Assemblies
Provide Repeatable PerformanceMeissner’s FlexGro single-
use biocontainer assemblies,
designed for use with
rocker-style bioreactors,
feature the TepoFlex
polyethylene (PE) multi-
layer film, are available in
up to 50 L and are delivered
presterilized for immediate
use. The TepoFlex film
offers a clean film platform,
devoid of slip agents,
which provides robust
and repeatable performance for cell-cultivation
processes, the company reports. FlexGro
biocontainer assemblies can be customized to
specific end-user requirements via various sampling
and process connection options, or companies
can choose from standard assembly designs.
Meissner Filtration Products
www.meissner.com
Serialization Software Helps
Companies Stay CompliantAdents’ Pharma Suite serialization
software, developed in compliance
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following GAMP5 guidelines, features
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generation of unique barcodes and
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management for origin identification
(bundle, pallet, etc.), and distribution
of various types of serialized code.
The software is compatible with both information
systems (e.g., enterprise resource planning, manufacturing
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address both current and emerging regulations.
The software includes the Adents Pharma Exchange
gateway to transit serialization data, the Adents Pharma
Supervisor to manage and distribute serialized codes,
and the Adents Pilot to direct printing and vision systems
and manage communication with line equipment.
Adents
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ES670446_BP0915_055.pgs 09.09.2015 01:55 ADV blackyellowmagentacyan
56 BioPharm International www.biopharminternational.com September 2015
New Technology Showcase
Pa
ge h
ea
de
r im
ag
e: A
rth
ur
S. A
ub
ry/G
ett
y I
ma
ge
s
Protein A resin
Protein A resins constitute a substantial cost
in state-of-the-art mAb purification
processes. Factors such as operating cycles,
capacity, and mAb titer can have an impact on
total costs associated with mAb purification.
High capacity TOYOPEARL AF-rProtein A HC-650F resin from Tosoh Bioscience
LLC has a binding capacity of >70 g/L, generating increased product
throughput, reduced operating costs and increased manufacturing
productivity. Tosoh Bioscience, tel: 484.805.1265, chris.manzari@tosoh.
com, www.tosoh.com
BioFlo® 320 From ePPendorF:
the smArter solution
The BioFlo 320 is the next generation in
bioprocess control. Designed as a universal
platform, it combines a new industrial
design, flexibility between
interchangeable autoclavable and single-use vessels, and universal gas
control strategy for both microbial and cell culture applications.
The BioFlo 320 has the right combination of features to get the job done.
Eppendorf AG, tel: +49 40.53801.0, [email protected],
www.eppendorf.com/contact
nuviA cPrime hydroPhoBic
cAtion exchAnge mediA
The Nuvia™ cPrime™
chromatography media are a new
addition to Bio-Rad’s family of
mixed-mode purification products.
The media are designed for process-
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selectivity allows method developers to use hydrophobic and cation
exchange interaction modes to achieve effective purification.
Bio-Rad Laboratories, Inc., www.bio-rad.com/nuvia
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Introducing the next generation in chemically-defined CHO fed-batch media. This contemporary media and feed platform was developed across a wide range of CHO cell lines commonly
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euroFins lAncAster lABs
As a member of Eurofins’ BioPharma
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Lancaster Laboratories supports all functional areas of bio/pharmaceutical
manufacturing, including method development, microbiology, process
validation, and quality control throughout all stages of the drug
development process. Eurofins Lancaster Labs, tel. 717.656.2300,
www.EurofinsLancasterLabs.com
sievers 500 rl
online toc AnAlyzer
The Sievers 500 RL Online Total Organic
Carbon (TOC) Analyzer is designed to
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If you need to monitor organics, choose
the most reliable technology that is also the simplest to install,
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GE Analytical Instruments, 6060 Spine Road, Boulder CO 80301,
tel. 303.444.2009, [email protected], www.geinstruments.com/500
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online viABle cell
density monitoring
Hamilton’s Incyte, viable cell density sensor, enables measurement of viable cells without influence from changes in the media, microcarriers, dead cells, or debris. Designed
for use in mammalian cell culture, yeast and bacterial fermentation, its 12 mm diameter, PG13.5 thread and 120 thru 425 mm lengths fit all reactor sizes. Either 2 or 4 sensors connect to the Arc View Controller, which displays, records, and exports measurement data in 4-20 mA, OPC or Modbus formats. Hamilton Company, tel: 800.648.5950, [email protected], www.hamiltoncompany.com/sensors
wuxi APPtec Begins extensive
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by Q2 2016. WuXi AppTec, [email protected], www.
wuxiapptec.com/biologics
ES670674_BP0915_056.pgs 09.09.2015 19:12 ADV blackyellowmagentacyan
September 2015 www.biopharminternational.com BioPharm International 57
Process Controls—Contin. from page 51
nal should be investigated. If a
parameter has been relatively
stable with infrequent and ines-
sential signals for example, it is
acceptable to take no immedi-
ate action. However, it is impor-
tant to track parameter signals
over time so that multiple viola-
tions or problematic patterns are
not overlooked. If a signal does
require follow-up, it is crucial
to identify the type of violation
and fol low-up with appropr i-
ate investigative techniques to
detect a root cause. Although a
multitude of process changes can
occur, run rules are designed to
detect some of the more com-
mon s ig na l s : s ing le event s ,
sh i f t s , t r end s , o sc i l l at ions ,
increasing/decreasing variability,
and cycles (see Figure 1).
Identifying the type of signal
and appropriate follow-up tech-
niques will bring significant value
to a business by reducing resource
and time requirements. Each sig-
nal indicates that a different pro-
cess change/event occurred, and
investigations should be tailored
based on the signal (see Table II).
Once a signal has been detected
and a decision has been made to
pursue it, typical follow-up steps
include:
• Confirm the value is correct
and not a data entry error.
• Determine if there is a readily
apparent root cause or if other
issues occurred in the process.
• Discuss the signalwith subject
matter experts.
• Examinethelottraceabilityand
review parameters related to the
parameter of interest.
• Escalatetoothergroupsforfur-
ther statistical investigation.
While run ru les prov ide a
useful tool for detecting some
non-random processes, not all
patterns can always be flagged.
Additionally, signals can be over-
looked for parameters that are
being trended with few run rules.
Thus, it is v ital to review all
parameters regularly (e.g., annu-
ally) to ensure that trends are
not missed. Individuals respon-
sible for conf ig ur ing, main-
taining, and reporting on the
monitor ing system should be
clearly identif ied in company
documents. Properly responding
to signals will result in more effi-
cient use of resources and time.
Additionally, investigating sig-
nals can enhance process under-
standing, which should improve
process monitoring and opera-
tional reliability.
COnClUsIOnProper CPV monitoring is a cru-
cial business value investment
for a company. A monitoring-by-
exception system that generates
valid signals can reduce resource
requirements, proactively iden-
t i f y issues pr ior to a qual ity
event, and create a regulatory
compliant environment. Today’s
life-science industry can improve
productivity and compliance by
developing more mature CPV ini-
tiatives and adopting practices
to ensure that they get the right
signals. Furthermore, performing
appropriate follow-up on valid
signals is vital to an effective and
continuously improving monitor-
ing solution.
REfEREnCEs1. FDA, Guidance for Industry: Process
Validation: General Principles
and Practices (Rockville,
MD, January 2011).
2. BioPhorum Operations Group,
Continued Process Verification:
An Industry Position Paper with
Example Plan, BPOG–Biophorum
Operations Group (2014).
3. R.J. Seely, L. Munyakazi, and
J. Haury, BioPharm Int. 14
(10), pp. 28–34 (2001).
4. J. M. Juran, Juran on Quality by Design:
The New Steps for Planning Quality
into Goods and Services (Simon and
Schuster, New York, NY, 1992).
5. D. Montgomery, Introduction to
Statistical Quality Control (John
Wiley, Hoboken, NJ, 5th ed., 2005).
6. S. Steiner, B. Abraham, and J.
MacKay, Understanding Process
Capability Indices (University of
Waterloo, Ontario, 1997).◆
Process Controls
Ad Index Company Page
BIO RAd lABORAtORIEs Cover tip
EMERGEnt BIOsOlUtIOns 17
ClInIGEn 19
CPhI 33, 37
EPPEndORf nORth AMERICA 11
EMd MIllIPORE 13
EUROfIns lAnCAstER lABORAtORIEs 29
GE AnAlytICAl InstRUMEnts 60
GE hEAlthCARE lIfE sCIEnCEs 5
hAMIltOn CO 9
IntERPhEX 23
sAfC BIOsCIEnCEs sIGMA AldRICh 59
sGs lIfE sCIEnCE sERVICEs 7
tOsOh BIOsCIEnCE 2, 43
ES668645_BP0915_057.pgs 09.03.2015 19:48 ADV blackyellowmagentacyan
EMA Releases Positive Opinion
for GSK’s Malaria VaccineThe Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA) has announced a positive scientific opinion for GlaxoSmithKline’s (GSK’s) malaria candidate vaccine, Mosquirix (RTS,S), for use outside of the European Union (EU) in children aged 6 weeks to 17 months.
While other vaccines tackle viruses or bacteria, RTS,S has been designed to prevent malaria caused by the Plasmodium falciparum parasite, which is prevalent in sub-Saharan Africa (SSA) and also protects against hepatitis B. In 2013, there were an estimated 584,000 deaths from malaria with approximately 90% of these occurring in SSA, and 83% in children under the age of five in SSA, the World Health Organization (WHO) reports.
RTS,S aims to trigger the body’s immune system to defend against the Plasmodium falciparum malaria parasite when it first enters the human host’s bloodstream and/or when the parasite infects liver cells. It is designed to prevent the parasite from infecting, maturing, and multiplying in the liver, after which time the parasite would re-enter the bloodstream and infect red blood cells, leading to disease symptoms, GSK reports in a statement.
Limited effectivenessData from a clinical trial conducted in seven African countries showed that Mosquirix provides modest protection against Plasmodium falciparum malaria in children in the 12 months following vaccination. The vaccine was effective at preventing a first or only clinical malaria episode in 56% of children aged between 5–17 months and in 31% of children aged 6–12 weeks. The efficacy of the vaccine decreased after one year. The safety profile of the vaccine was considered acceptable, EMA reports.
CHMP concluded that despite its limited efficacy, the benefits of Mosquirix outweigh the risks in both age groups studied and the benefits of vaccination may be particularly important among children in high-transmission areas in which mortality is very high.
Because the studies showed that Mosquirix does not offer complete protection, and the protection it provides decreases in the longer term, EMA states that established protective measures, such as insecticide-treated bed nets, should continue to be used in addition to the vaccine.
Next steps in developmentThe vaccine was submitted to EMA under a regulatory procedure (Article 58) that allows EMA to assess the quality, safety, and efficacy of a medicine or vaccine and its benefit-risk balance, although it will not be marketed in the EU. EMA reports that the CHMP worked closely with WHO and regulatory authorities from the relevant countries and applied the same rigorous standards as for medicines to be marketed within the EU.
The EMA approval was a pre-requisite for a WHO policy recommendation for the use of Mosquirix in vaccination
programs. WHO will consider additional factors not addressed by EMA including feasibility of implementation, affordability, cost-effectiveness, and the public health value of the vaccine in relation to other available malaria control measures and vaccines. WHO will provide its recommendations on use of Mosquirix by November 2015. Regulators in the countries where the vaccine will be used will decide on licensing in their jurisdictions.
RTS,S, which was developed in partnership with the PATH Malaria Vaccine Initiative (MVI), is the first candidate vaccine for the prevention of malaria to reach this milestone, GSK reports. RTS,S was created in 1987 by scientists working at GSK laboratories. Early clinical development was done in collaboration with the Walter Reed Army Institute for Research. In January 2001, GSK and PATH, with grant monies from the Bill & Melinda Gates Foundation to PATH, entered into a public-private partnership to develop an RTS,S-based vaccine for infants and young children living in malaria-endemic regions in sub-Saharan Africa.
GSK reports the company has invested more than $365 million to date and expects to invest a further $200 to $250 million until development is completed. Between 2001 and the end of 2014, the MVI, supported by grants from the Bill & Melinda Gates Foundation, invested more than $200 million to advance the RTS,S project.
In a press statement, GSK noted that the company has committed to a not-for-profit price for RTS,S; if approved, the price of RTS,S would cover the cost of manufacturing the vaccine with a return of approximately 5% that will be reinvested in research and development for second-generation malaria vaccines, or vaccines against other neglected tropical diseases.
CMC Biologics to Manufacture
mAbs for the Treatment of MalariaCMC Biologics will manufacture monoclonal antibodies (mAbs) and provide process development services for the PATH Malaria Vaccine Initiative (MVI), CMC announced on Aug. 12, 2015. The company will use its proprietary production cell line for the project, and will test MVI’s investigational mAb for its ability to target circumsporozoite protein (CSP) and protect against infection by Plasmodium falciparum, the parasite that causes malaria.
“We will deliver cGMP material for MVI’s preclinical and Phase I/II clinical studies in a remarkable 12 months—the fastest development timeline in the industry from DNA to delivery,” Gustavo Mahler, PhD, global chief operations officer at CMC Biologics said in a press release.
Based on the success of the project with MVI, the group may hire CMC to investigate additional mAbs that bind to alternate epitopes. “Assuming this initial study is successful, we will evaluate other antibodies targeting novel vaccine targets for their capacity to protect humans from infection and therefore, inform future vaccine development efforts,” confirmed Ashley J. Birkett, PhD, director at MVI.
58 BioPharm International www.biopharminternational.com September 2015
Vaccines Development Update
Ho
u Y
uxu
an
/Ge
tty I
ma
ge
s
ES669072_BP0915_058.pgs 09.04.2015 01:57 ADV blackyellowmagentacyan
Introducing a next generation, chemically-deÀned CHO fed-batch media platform from SAFC. Developed
across a range of widely used industrial CHO cell lines, this newest portfolio media delivers exceptional
titers and economic efÀciencies. Adaptation is simple. Celebrate performance and accelerate your
bio-development process.
Celebrate! Request your complimentary sample at
sigma-aldrich.com/CHOperformance
Cat. Nos: 14366C, 24366C, 24367C, 24368C
EX-CELL® ADVANCED™ CHO FED-BATCH SYSTEM
CELL CULTURE
PERFORMANCE
©2015 Sigma-Aldrich Co. LLC. All rights reserved. Sigma-Aldrich, SAFC, and EX-CELL are trademarks of Sigma-Aldrich Co. LLC, registered in the US and other countries.
NEXT GENERATION MEDIA PLATFORM
ES668428_BP0915_CV3_FP.pgs 09.03.2015 02:27 ADV blackyellowmagentacyan
www.geinstruments.com/M9
Your workday can be a chaotic place, tools that boost
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Enjoy peace of mind using the perfect tool for
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ES668453_BP0915_CV4_FP.pgs 09.03.2015 02:28 ADV blackyellowmagentacyan
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