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PharmThe Science & Business of Biopharmaceuticals
INTERNATIONALINTERNATIONAL
Bio
Ph
arm
Intern
atio
nal
JAN
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016
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acto
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eria
lizatio
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9 N
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January 2016
Volume 29 Number 1
FRAMING BIOPHARMA
SUCCESS IN 2016
DOWNSTREAM
PROCESSING
GOING SMALL TO
ACHIEVE SUCCESS ON THE
COMMERCIAL SCALE
PEER-REVIEWED
PRECIPITATION AS
AN ALTERNATIVE TO
CHROMATOGRAPHY IN THE
INSULIN MANUFACTURING
PROCESS
REGULATIONS
POLITICS AND PRICING
WILL CHALLENGE
MANUFACTURERS IN 2016
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INTERNATIONAL
BioPharmThe Science & Business of Biopharmaceuticals
EDITORIALEditorial Director Rita Peters rpeters@advanstar.comSenior Editor Agnes Shanley ashanley@advanstar.comManaging Editor Susan Haigney shaigney@advanstar.comScience Editor Randi Hernandez rhernandez@advanstar.com Science Editor Adeline Siew, PhD asiew@advanstar.comCommunity Manager Caroline Hroncich chroncich@advanstar.comArt Director Dan Ward dward@media.advanstar.comContributing Editors Jill Wechsler, Jim Miller, Eric Langer, Anurag Rathore, Jerold Martin, Simon Chalk, and Cynthia A. Challener, PhD Correspondent Sean Milmo (Europe, smilmo@btconnect.com) ADVERTISING
Publisher Mike Tracey mtracey@advanstar.comWest/Mid-West Sales Manager Steve Hermer shermer@advanstar.comEast Coast Sales Manager Scott Vail svail@advanstar.comEuropean Sales Manager Chris Lawson clawson@advanstar.comEuropean Sales Manager Wayne Blow wblow@advanstar.comC.A.S.T Data and List Information Ronda Hughes rhughes@advanstar.comReprints 877-652-5295 ext. 121/ bkolb@wrightsmedia.com Outside US, UK, direct dial: 281-419-5725. Ext. 121 PRODUCTION Production Manager Jesse Singer jsinger@media.advanstar.com AUDIENCE DEVELOPmENT Audience Development Rochelle Ballou rballou@advanstar.com
UBm LIfE SCIENCES
Tom Ehardt, EVP & Senior Managing Director, Life Sciences Tom Mahon, Senior VP, Finance Georgiann DeCenzo, EVP & Managing Director, UBM Medica Mike Alic, EVP, Strategy & Business Development Dave Esola, VP & Managing Director, Pharm/Science Group Johanna Morse, VP & Managing Director, CBI/IVT Becky Turner Chapman, VP & Managing Director, Veterinary Group Joy Puzzo, VP, Marketing & Audience Development Francis Heid, VP, Media Operations Jamie Scott Durling, Director, Human Resources
UBm AmERICAS
Simon Foster, Chief Executive Officer Brian Field, Chief Operating Officer Michael Bernstein, Head of Legal
UBm PLC
Tim Cobbold, Chief Executive Officer Andrew Crow, Group Operations Director Marina Wyatt, Chief Financial Officer Dame Helen Alexander, Chairman
© 2016 Advanstar Communications Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical including by photocopy, recording, or information storage and retrieval without permission in writing from the publisher. Authorization to photocopy items for internal/educational or personal use, or the internal/educational or personal use of specific clients is granted by Advanstar Communications Inc. for libraries and other users registered with the Copyright Clearance Center, 222 Rosewood Dr. Danvers, MA 01923, 978-750-8400 fax 978-646-8700 or visit http://www.copyright.com online. For uses beyond those listed above, please direct your written request to Permission Dept. fax 440-756-5255 or email: mcannon@advanstar.com.
UBM Life Sciences provides certain customer contact data (such as customers’ names, addresses, phone numbers, and e-mail addresses) to third parties who wish to promote relevant products, services, and other opportunities that may be of interest to you. If you do not want UBM Life Sciences to make your contact information available to third parties for marketing purposes, simply call toll-free 866-529-2922 between the hours of 7:30 a.m. and 5 p.m. CST and a customer service representative will assist you in removing your name from UBM Life Sciences’ lists. Outside the U.S., please phone 218-740-6477.
BioPharm International does not verify any claims or other information appearing in any of the advertisements contained in the publication, and cannot take responsibility for any losses or other damages incurred by readers in reliance of such content.
BioPharm International welcomes unsolicited articles, manuscripts, photographs, illustrations, and other materials but cannot be held responsible for their safekeeping or return.
To subscribe, call toll-free 888-527-7008. Outside the U.S. call 218-740-6477.
EDITORIAL ADVISORY BOARDBioPharm International’s Editorial Advisory Board comprises distinguished specialists involved in the biologic manufacture of therapeutic drugs, diagnostics, and vaccines. Members serve as a sounding board for the editors and advise them on biotechnology trends, identify potential authors, and review manuscripts submitted for publication.
K. A. Ajit-Simh President, Shiba Associates
Rory Budihandojo Director, Quality and EHS Audit
Boehringer-Ingelheim
Edward G. Calamai Managing Partner
Pharmaceutical Manufacturing
and Compliance Associates, LLC
Suggy S. Chrai President and CEO
The Chrai Associates
Leonard J. Goren Global Leader, Human Identity
Division, GE Healthcare
Uwe Gottschalk Vice-President,
Chief Technology Officer,
Pharma/Biotech
Lonza AG
Fiona M. Greer Global Director,
BioPharma Services Development
SGS Life Science Services
Rajesh K. Gupta Vaccinnologist and Microbiologist
Jean F. Huxsoll Senior Director, Quality
Product Supply Biotech
Bayer Healthcare Pharmaceuticals
Denny Kraichely Associate Director
Johnson & Johnson
Stephan O. Krause Director of QA Technology
AstraZeneca Biologics
Steven S. Kuwahara Principal Consultant
GXP BioTechnology LLC
Eric S. Langer President and Managing Partner
BioPlan Associates, Inc.
Howard L. Levine President
BioProcess Technology Consultants
Herb Lutz Principal Consulting Engineer
Merck Millipore
Jerold Martin Independent Consultant
Hans-Peter Meyer Lecturer, University of Applied Sciences
and Arts Western Switzerland,
Institute of Life Technologies.
K. John Morrow President, Newport Biotech
David Radspinner Global Head of Sales—Bioproduction
Thermo Fisher Scientific
Tom Ransohoff Vice-President and Senior Consultant
BioProcess Technology Consultants
Anurag Rathore Biotech CMC Consultant
Faculty Member, Indian Institute of
Technology
Susan J. Schniepp Fellow
Regulatory Compliance Associates, Inc.
Tim Schofield Senior Fellow
MedImmune LLC
Paula Shadle Principal Consultant,
Shadle Consulting
Alexander F. Sito President,
BioValidation
Michiel E. Ultee Principal
Ulteemit BioConsulting
Thomas J. Vanden Boom VP, Biosimilars Pharmaceutical Sciences
Pfizer
Krish Venkat Managing Partner
Anven Research
Steven Walfish Principal Scientific Liaison
USP
Gary Walsh Professor
Department of Chemical and
Environmental Sciences and Materials
and Surface Science Institute
University of Limerick, Ireland
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4 BioPharm International www.biopharminternational.com January 2016
Contents
BioPharmINTERNATIONAL
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.
COLUMNS AND DEPARTMENTS
6 From the Editor Infrastructure and payer decisions will determine drug choices. Rita Peters
7 Investment Outlook
47 Troubleshooting Miniature bioreactors can predict the cell culture kinetics in scaled-up reactors. Mohd Helmi Sani and Frank Baganz
50 Analytical Best Practices This article defines the concept, justification, and method of removal of out-of-trend points in stability modelling and shelf-life prediction. Thomas A. Little
56 Product Spotlight
56 New Technology Showcase
57 Biologics News Pipeline
57 Ad Index
58 Ask the Expert Siegfried Schmitt, principal consultant, PAREXEL, discusses how to streamline the document management process during market expansion.
Cover: Nastco/Andrey Machikhin/Martin McCarthy/
Mina De La O/Getty Images; Dan Ward
BIG PICTURE FOR 2016
Framing Biopharma Success in 2016Rita C. Peters
Corporate restructurings, regulatory
initiatives, and biosimilars will shape
biopharma development in 2016. 8
mAbs to Watch in 2016Randi HernandezWhich monoclonal antibodies will
gain US regulatory approval in 2016? 12
Politics and Pricing Will Challenge Manufacturers in 2016Jill WechslerThe bio/pharmaceutical industry will face increased scrutiny of product quality and
cost drivers. 14
Outsourcing Outlook for 2016Susan HaigneyIndustry experts discuss what the
outsourcing market holds for 2016. 16
BIOREACTORS
Tools for Continuous Bioprocess DevelopmentRajeev J. Ram
Could perfusion microbioreactors bring
more agility to biomanufacturing? 18
DOWNSTREAM PROCESSING
Going Small to Achieve Success on the Commercial ScaleCynthia A. Challener
Scale-down modeling is instrumental in supporting the development of downstream
biopharma manufacturing processes. 26
PEER-REVIEWED
Precipitation as an Alternative to Chromatography in the Insulin Manufacturing ProcessMadhavan Buddha, Shailabh Rauniyar, Shabandri Qais, Dinesh Goudar, Sai Srikar Kandukuri, Siddharth Mahajan, Sinash Siddik, and Partha Hazra
The authors evaluated two precipitation strategies as alternatives to the conventionally
used chromatographic process. 30
SERIALIZATION
Serialization: Getting Past the Quick FixAgnes Shanley
Traceability and transparency will remain elusive if manufacturers continue to approach serialization projects on a
case-by-case basis. 36
HEALTH-BASED
EXPOSURE LIMITS
EMA Guideline on Setting Health-Based Exposure LimitsAndrew Teasdale, Bruce D. Naumann, Gretchen Allison, Wendy Luo, Courtney M. Callis, Bryan K. Shipp, Laura Rutter, and Christopher Seaman
The results of an industry workgroup’s examination of EMA’s guide on shared
facilities are presented. 41
Volume 29 Number 1 January 2016
fEATURES
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6 BioPharm International www.biopharminternational.com January 2016
From the Editor
Infrastructure and
payer decisions
will determine
drug choices in
emerging and
developed regions.
Emerging Nations Close the Medicine Use Gap
Biopharma industry headlines in 2015 reflected the positive (more drug
approvals and the first US-approved biosimilar); the negative (inflated
drug prices, Martin Shkreli, and tax inversion-driven mergers); and the
uncertain (multiple mega mergers and FDA initiatives).
In this issue, the editors look at key industry trends and expectations for the
year ahead, including these predictions for future global drug demand.
Improved patient access to chronic disease treatment and breakthrough
drug therapies will help reduce the “medicine use gap” between emerging and
developed markets, driving global spending on drugs to grow at a 4–7% com-
pound annual rate over the next five years. Regional economic conditions and
healthcare infrastructure, however, will dictate the types of drugs prescribed,
number of medicine doses, and ultimately, the volume of pharmaceutical sales.
By 2020, total patient spending on medicines will be $1.4 trillion, reports
the IMS Institute for Healthcare Informatics (1); however, the global spend-
ing increase from 2015 to 2020, estimated to be 29–32%, is below the 35%
increase of the prior five years.
Global medicine use will reach 4.5 trillion doses in 2020, the study predicts,
up 24% from 2015. Emerging markets will account for most of the increase,
led by India, China, Brazil, Indonesia, and Africa. Generic drugs, non-original
branded drugs, and over-the-counter products will account for 88% of total
medicine used in emerging markets, the report says. More than 90% of the
prescription drugs filled in the United States in 2020 will be generic drugs, up
from the present level of 88%.
Brand spending in developed markets is expected to reach up to $590 bil-
lion, a 34% increase in spending over 2015 on an invoice price basis, thanks to
new product launches, and price increases in the US—which may be offset by
discounts and rebates. Invoice price growth, which does not reflect discounts
and rebates received by payers, is expected to continue at historic levels during
the next five years; however, competition and payer resistance will keep net
price increases to 5–7% annually.
Spending growth will be curbed by patent expiries, which will result in $178
billion in reduced spending on branded products, including $41 billion on
biologics, as biosimilars become more widely adopted.
The rise of specialty medicinesGlobal spending on specialty medicines used to treat chronic, rare, or genetic
diseases is expected to reach 28% of the total spending by 2020. The report
estimates that more than 225 medicines will be introduced by 2020, with
one-third focused on treating cancer; other development areas are hepatitis C,
autoimmune disorders, heart disease, and rare diseases. Adoption of specialty
medicines will be most prevalent in established markets.
“During the next five years, we expect to see a surge of innovative medi-
cines emerging from R&D pipelines, as well as technology-enabled advances
that will deliver measurable improvements to health outcomes,” said Murray
Aitken, IMS Health senior vice-president and executive director of the IMS
Institute for Healthcare Informatics, in a press statement. “With unprec-
edented treatment options, greater availability of low-cost drugs and better
use of evidence to inform decision making, stakeholders around the world can
expect to get more ‘bang for their medicine buck’ in 2020 than ever before.”
Reference1. IMS Health, Global Medicines in Use in 2020 (Parsippany, NJ, November 2015).
Rita Peters is the editorial director of
BioPharm International.
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Investment Outlook
Ma
rk E
va
ns/E
+/G
ett
y Im
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es
Shire Acquires Baxalta in $32 Billion DealShire and Baxalta have announced a $32 billion agreement
under which Shire will combine with Baxalta, creating a
company with the top rare diseases platform in revenue and
pipeline depth, the companies reported in a Jan. 11, 2016 press
statement. The new company is projected to achieve annual
revenues of $20 billion by 2020.
Shire reports that the combined companies will have
products in growing franchises in hematology; immunology;
neuroscience; lysosomal storage diseases; gastrointestinal/
endocrine; hereditary angioedema; and oncology; as well as a
late-stage ophthalmics pipeline.
Baxalta shareholders will hold approximately 34%
ownership in the combined company. The parties expect the
transaction to close mid-2016.
Boehringer Ingelheim to Establish Biopharmaceutical Production Facility in ViennaBoehringer Ingelheim will make a significant investment in
biopharmaceutical production at its Vienna site. The company
will establish a new large-scale biopharmaceutical production
facility for active ingredients manufactured using cell cultures.
With the roughly half billion-euro investment, Boehringer
Ingelheim will also create more than 400 new jobs in the
Austrian capital.
“This is a decision for Europe as a pharma location,” said
Professor Andreas Barner, PhD, chairman of the board of
managing directors at Boehringer Ingelheim. “We took a close
look at various international options as part of the investment
decision, also considering the research environment at
potential sites. The clincher for Vienna was ultimately the
company’s desire to additionally secure the market supply
of biopharmaceutical products and to balance the risk by
establishing a further independent facility.”
In Vienna, the company has already produced
pharmaceutical active ingredients using microorganisms;
over the next few years, cell-culture technology will also be
transferred to the plant. According to the company, the new
production plant will go into operation by 2021. Boehringer
Ingelheim has already been operating two large-scale facilities
for the market launch and cell-culture-based manufacture of
biopharmaceuticals in Biberach, Germany.
GSK Acquires Bristol-Myers Squibb R&D HIV AssetsGlaxoSmithKline (GSK) announced its global HIV business,
ViiV Healthcare, has reached two separate agreements with
Bristol-Myers Squibb (BMS) to acquire its late-stage HIV R&D
assets and its portfolio of preclinical and discovery-stage HIV
research assets.
Under the terms agreed in the two transactions, ViiV
Healthcare will acquire late-stage assets. Including fostemsavir
(BMS-663068), an attachment inhibitor, currently in Phase
III development for heavily treatment-experienced patients.
Fostemsavir has received a Breakthrough Therapy Designation
from FDA, and the company expects to file for regulatory
approval in 2018. Another Late-stage asset is a maturation
inhibitor (BMS-955176), currently in Phase IIb development for
both treatment-naive and treatment experienced patients. A
back-up maturation inhibitor candidate (BMS-986173) is also
included in the purchase.
Assets in preclinical and discovery phases of development
include a novel biologic (BMS-986197) with a triple mechanism
of action, a further maturation inhibitor, an allosteric integrase
inhibitor, and a capsid inhibitor. A number of BMS drug
discovery employees will also be offered the opportunity to
transfer to ViiV Healthcare.
According to GSK, the late-stage asset purchase comprises
an upfront payment of $317 million, followed by development
and first commercial-sale milestones of up to $518 million,
and tiered royalties on sales. The purchase of preclinical
and discovery-stage research assets comprises an upfront
payment of $33 million, followed by development and first
commercial-sales milestones of up to $587 million, and further
consideration contingent on future sales performance. The
two transactions are anticipated to complete independently
during the first half of 2016, subject to necessary approvals,
anti-trust, and regulatory clearances.
Takeda Acquires US Biologics Manufacturing FacilityTakeda Pharmaceutical Company Limited announced the
acquisition of a Brooklyn Park, Minnesota-based biologics
manufacturing facility from Baxalta Inc. Takeda is a Japan-
based pharmaceutical company focused on research and
development. It is the largest pharmaceutical company in
Japan.
Takeda intends to use the facility primarily for the
manufacture of Entyvio (vedolizumab) and other biologic
products. FDA approved Entyvio in May 2014 for the
treatment of ulcerative colitis and moderate-to-severe
Crohn’s disease. Terms of the transaction were not
disclosed.
January 2016 www.biopharminternational.com BioPharm International 7
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8 BioPharm International www.biopharminternational.com January 2016
Nastc
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art
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Dan W
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The US biopharmaceutical indus-
try turned a new page in 2015
when FDA approved Zarxio (fil-
grastim-sndz), the first biosimi-
lar approved for use in US markets, in
March 2015. Biotechnology stocks con-
tinued to outperform the Standards
& Poor’s 500 Index and NASDAQ
Composite Index. Venture capital invest-
ment and initial public offerings were
also near record levels (1). If the intro-
duction of generic alternatives is a sign
of maturity in the biologic-based drug
segment, the biopharma companies
may want to examine lessons learned by
small-molecule based pharma companies
as the market transitions.
In the past 20 years, biopharma
companies enjoyed success as block-
buster drugs dominated the market,
made adjustments as the patent cliff
approached, and in some cases, aban-
doned exist ing R&D programs to
seek alternate, more profitable routes.
Counterfeit drugs and drug shortages
disrupted the delivery of safe, effective
therapies to patients. Oversized, ineffi-
cient research, development, and manu-
facturing engines were downsized, or
transferred to outsourcing providers.
In 2015, FDA’s Center for Drug
Eva luat ion and Research (CDER)
approved 45 new molecular entity
and new biologic l icense applica-
tion approvals (2), the second highest
annual total. The Center for Biologics
Eva luat ion and Research (CBER)
approved more than a dozen new bio-
logics, vaccines, blood products, and
diagnostics. These numbers, and types
Framing Biopharma Success in 2016
Rita C. Peters
The Big Picture for 2016
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10 BioPharm International www.biopharminternational.com January 2016
of drugs approved, indicate that
the drug-development market may
be headed in a new direction, rais-
ing questions about the ability of
the current R&D and manufactur-
ing infrastructure to adapt to the
changing market. While approv-
als are up, FDA cautions that the
number of new drug applica-
tions has been flat, indicating the
potential of a slowdown ahead.
BREAKTHROUGHS, BIOSIMILARS, AND BOTTLENECKSMany of the new molecular enti-
ties—41% of the approvals in
2014 and 47% of the approvals in
2015—were for rare diseases that
target smaller patient populations
and, therefore, require smaller
quantities of drug doses required
compared to blockbuster drugs.
This shift in production require-
ments—combined with lower
profit margins for former block-
buster brand drugs now prescribed
as lower-cost generic drugs—has
implications for drug development
and manufacturing processes.
A market research study esti-
mates that the global biosimilars/
follow-on-biologics market will be
$26.5 billion by 2020, up from
$2.55 billion in 2014. Although
the compound annual growth rate
is expected to be of 49.5% from
2015 to 2020, the growth is slower
than anticipated due to a lack of
regulatory guidelines in China
and the United States and a lack of
economies of scale for small-scale
manufacturers (3).
FDA’s expedited pathways for
drug approval have proved suc-
cessful; 60% of the 2015 approvals
were designated in one or more of
the expedited pathways (fast track,
breakthrough, priority review, and
accelerated approval). While this
faster route to market of needed
therapies is good news, FDA’s
inspection process, and the inabil-
ity of drug manufacturers to accel-
erate production timeframes, are
potential roadblocks. John Jenkins,
director of CDER’s Office on New
Drugs, reported at the FDA/CMS
Summit in December 2015 that
manufacturing and inspections—
not clinical development—are often
the “rate-limited steps” for expe-
dited approval.
INVESTING IN INFRASTRUCTUREAttractive credit terms in recent
years prompted some biopharma
manufacturers to invest in facili-
ties and equipment. Encouraging
compa n ie s to ma ke cap it a l
improvements to avoid future
quality and manufacturing prob-
lems, however, can be a tough
financial and regulatory argu-
ment. In a 2015 survey of 100
executives from top biopharma
companies (4), less than half
said they planned to increase
investments in late-stage R&D.
Instead, marketing/distribution
(79%), drug discovery/early-stage
R&D (70%), clinical trials (60%),
technology acquisit ion (59%),
and patenting (50%) were higher
investment priorities.
To encourage manufacturers to
adopt new production technolo-
gies, FDA issued a draft guidance
document (5) in December 2015
that provides a framework for drug
manufacturers and FDA to discuss
manufacturing design and devel-
opment issues early in the process.
The goal is to educate FDA staff
about the new technologies in
advance of chemistry, manufac-
turing, and controls (CMC) sub-
mission, thereby expediting the
approval process.
The Big Picture for 2016
The view from the biopharma trenches
The big decisions about company acquisitions, mergers, and business development
may be made in the boardrooms of bio/pharma companies, but the frontline
professionals—those involved in formulation, development, and manufacturing
functions—can offer interesting observations about the impact those business
strategies have on the day-to-day tasks of bringing a drug to market.
More than 450 biopharma professionals from around the world participated in the
2015 BioPharm International annual employment survey (1). Respondents expressed
opinions about job security (74.6% feel secure in their current position), seeking new
opportunities (59.2% would like to leave their job), and increasing dissatisfaction
with salary levels. The respondents also expressed opinions about trends in the
industry, how changes impact their daily work, and future business prospects.
Almost 40% of those responded said business at their company increased in
2015, similar to data reported in the previous two years; 27.5% reported downsizing.
One-quarter of the respondents reported that their company had been through a
merger or acquisition in the past two years, down slightly from the 2014 survey.
Overall, respondents expressed a positive outlook for the bio/pharmaceutical
industry for the next year; however, fewer respondents said business would improve
(55.4% in 2015 compared with 62.2% in 2014). When looking at a longer-term horizon
(five years), nearly two-thirds of respondents (65.4%) predicted that business will
improve; however, 13.5% expect business to improve overseas, but not domestically.
More than half of the respondents (52%) predicted that their company’s business will
improve in 2016; more than one-third (34%) expected no significant change.
Reference
1. 2015 BioPharm International Employment Survey.
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January 2016 www.biopharminternational.com BioPharm International 11
QUALITY WARNING SIGNSFDA plans to use quality metrics
to develop risk-based inspection
scheduling of drug manufactur-
ers; to improve its ability to predict
and mitigate drug shortages; and
“encourage the pharmaceutical
industry to implement state-of-the-
art, innovative quality manage-
ment systems for pharmaceutical
manufacturing” (6). Industry feed-
back to the d ra f t g u idance
reflected concerns about how FDA
will use the reported data to guide
further regulatory action, what
players in the supply chain were
responsible for reporting to FDA,
how the data would be interpreted
to assess overall product quality,
and factors used to determine the
success of a quality program. A key
issue was the perceived burden and
additional resources needed to per-
form quality testing.
THE MERGERS, ACQUISITIONS AND COLLABORATIONS PICTUREIn 2015, biopharma companies
and contact service organizations
actively repositioned their business
profiles though mergers, acquisi-
tions, and collaboration agree-
ments, as well as divestitures of
specific product lines or business
units. Senior executives of large
biotechnology and pharmaceutical
companies surveyed by ReedSmith
(3) plan to stay on the investment
track; 94% plan to initiate an acqui-
sition during the next 12 months;
more than one-third plan to
divest assets. The search for exter-
nal opportunities was a priority;
more than half of the respondents
said they are planning peer-to-peer
research partnerships; 85% report
that they plan to hire a contract
research organization.
More respondents (74%) said
major pharmaceutical produc-
ers would target companies with
strong drug discovery or early-stage
R&D potential versus companies
involved in late-stage R&D (69%).
RIGHT-SIZING R&D EFFORTS FOR IMPROVED RETURNSFor the past six years, Deloitte
and GlobalData analysts exam-
ined R&D results for the top 12
publicly held, research-based life-
science companies (measured by
R&D spending in 2008–2009); in
2015, four mid- to large-cap com-
panies were added to the study.
Analysis of the pharma industry’s
performance in generating return
on investment in new drug devel-
opment revealed a continuing pat-
tern of declining returns, from
10.1% in 2010 to 4.2% in 2015.
While the cost to develop an asset
increased 33%, from $1.188 billion
in 2010 to $1.576 billion in 2015,
the average peak sales per asset
dropped 50%, from $816 million
in 2010 to $416 million in 2015.
The numbers, the report authors
say, “do not add up for life-sci-
ences R&D to generate an appro-
priate return” (7).
There was some good news; the
mid- to large-cap companies out-
performed the top 12 publicly held
companies, indicating that different
R&D business models could pro-
duce better results.
Despite the decline in return on
investment, the companies in the
study continued to increase invest-
ment in R&D as a proportion of
cash generated, from 25.5% in
2004 to 29.4% in 2014. Investors,
however, seek returns. Companies
are more likely to return cash to
shareholders via dividends and
share buybacks than they are to
invest in acquisitions, product
licenses, and internal R&D, the
study concludes.
At larger R&D companies, legacy
infrastructure, which may be dif-
ficult to improve, and excess over-
head are major contributors to R&D
costs. The smaller companies in the
study did not have the large infra-
structure, but risk becoming too
complex and lose their R&D pro-
ductivity advantage as they grow.
Both groups must challenge addi-
tional investments at each phase
and evaluate returns for each asset,
the study reports.
The authors suggest that focusing
R&D efforts on stable therapy areas
and specialty therapeutics will add
to scientific, regulatory, and com-
mercial value propositions. Agility,
flexibility, and a focus on science
will allow external sources of inno-
vation to be optimized. Reducing
development complexity by stream-
lining functions and addressing
unproductive infrastructure can
improve returns.
FRAMING THE 2016 AGENDAEarly indications are for mergers
and acquisitions activity to con-
tinue in 2016, and prospects for
new drug approvals also are strong.
FDA has a busy agenda of legislative
approvals ahead, complicated by
an election year. Among these dis-
tractions, the industry must drive
ahead to seek shareholder value and
affordable, effective patient thera-
pies on the same route.
REFERENCES 1. Z. Tracer, Health Care in 2016: Eight
Charts You Need to Follow the Sector,
online, www.bloomberg.com/news/
articles/2016-01-08/the-eight-charts-
you-need-to-understand-health-care-
in-2016, accessed Jan. 10, 2016.
2. FDA, Novel New Drugs Summary
2015, online, www.fda.gov/Drugs/
DevelopmentApprovalProcess/
DrugInnovation/ucm474696.
htm, accessed, Jan. 5, 2016.
3. R. Deshmukh, World Biosimilars/Follow-
on-Biologics Market - Opportunities
and Forecasts, 2014–2020 (2015),
www.alliedmarketresearch.com/
pharma/global-biosimilars-market
4. ReedSmith, Life Lines: Life Sciences
M&A and the Rise of Personalized
Medicine (London, 2015).
5. FDA, Advancement of Emerging
Technology Applications to Modernize
the Pharmaceutical Manufacturing Base
Guidance for Industry, Draft Guidance
(Rockville, MD, December 2015).
6. FDA, Request for Quality Metrics
Guidance for Industry, Draft Guidance
(Rockville, MD, July 2015).
7. Deloitte Centre for Health
Solutions, Measuring the Return
from Pharmaceutical Innovation
2015 (London, 2015). ◆
The Big Picture for 2016
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12 BioPharm International www.biopharminternational.com January 2016
Of the 45 new molecular enti-
ties and new therapeutic bio-
logical products approved by
FDA in 2015, nine products
were monoclonal antibodies (mAbs).
And there may be many more of these
types of proteins on the horizon—as
many as 14 are projected to hit the mar-
ket in 2016, according to data compiled
by EvaluatePharma, in collaboration with
BioPharm International (*).
MABS IN DEVELOPMENTIxekizumab—Eli Lilly’s ixekizumab is an
interleukin-17 drug that works by target-
ing the IL-17 ligand. This mAb is being
developed for the treatment of psoriasis
and psoriatic arthritis, and may directly
compete with Novartis’ Cosentyx
(secuk inumab) and Ast raZeneca/
Valeant’s brodalumab. Brodalumab,
however, will work a bit differently—it
is an antibody that binds to the IL-17
receptor, whereas secukinumab and
ixekizumab focus on the IL-17 ligand.
In clinical trials, ixekizumab demon-
strated superiority over Enbrel (etaner-
cept) for clearing skin plaques.
Brodalumab—Valeant and AstraZeneca’s
IL-17 therapy, brodalumab got some bad
press in May 2015 when Amgen dropped
out of a co-development project with
AstraZeneca for the drug after clinical-trial
results suggested that the drug was associ-
ated with suicidal ideation in patients. In
September 2015, however, Valeant stepped
in and agreed to be responsible for the
commercialization and development of
the project. Brodalumab was shown in
clinical trials to clear the scaly skin patches
associated with psoriasis more efficiently
than Johnson & Johnson’s (J&J) Stelara
(ustekinumab).
Obiltoxaximab—Elusys Therapeutics’
mAb, obiltoxaximab, which will go by the
trade name Anthim, differs significantly
from the rest of the mAb pack. The inves-
tigational agent for the treatment of inha-
lational anthrax infection is supported by
the Biomedical Advanced Research and
Development Authority (BARDA). Even
before the product’s approval by FDA,
BARDA granted Elusys its first delivery
procurement order for Anthim so that the
agency could add Anthim to its Strategic
National Stockpile (SNS) as a countermea-
sure against a potential bioterrorist attack.
Elusys has already received more than
$220 million in grants from government
agencies to develop the mAb. The drug,
meant to be used as both a treatment after
anthrax exposure and as an anthrax pro-
phylactic, is being investigated as an intra-
venous treatment as well as an emergency
injectable.
Atezolizumab—This PD-L1 checkpoint
inhibitor is being investigated for the treat-
ment of various solid tumors, including
indications for non-small cell lung can-
cer (NSCLC), bladder cancer, renal cell
carcinoma, and breast cancer. In clinical
trials, treatment with atezolizumab cor-
responded to an average 7.7 month-exten-
sion of life when compared with those
who received docetaxel chemotherapy;
this represents a statistically significant
survival benefit. If approved, atezolizumab
will likely compete with Merck’s Keytruda
(pembrolizumab), Bristol-Myers Squibb’s
Opdivo (nivolumab), and AstraZeneca’s
durvalumab, which is being investigated in
Phase II and III trials.
Reslizumab—Teva is gunning for FDA
approval in 2016 for its biologic resli-
zumab (trade name Cinquil), which tar-
gets IL-5. Although the company will first
seek an approval for the medication for
the treatment of eosinophilic asthma,
it is also researching the drug’s efficacy
in the treatment of eosinophilic esopha-
gitis. Reslizumab is in the same class as
AstraZeneca’s Nucala (mepolizumab).
According to Teva, a decision from FDA on
reslizumab is expected by March 2016.
Dupilumab—Sanofi and Regeneron’s
dupilumab is currently in late-stage devel-
opment for asthma and eczema/atopic der-
matitis. The drug is promising because of
mAbs to Watch in 2016Randi Hernandez
The Big Picture for 2016: Emerging Therapeutics
*Editor’s note: The forecasted
approval dates are drawn directly
from company-disclosed information
(e.g., press releases, company presen-
tations, etc.). As such, there may be
products that are filed or in Phase III
that are not included in these reports,
due to the lack of information regard-
ing approval/launch dates from
either of these sources. Similarly,
the original data were requested
by and provided to BioPharm
International by EvaluatePharma
in August 2015; therefore, some
of the original submissions have
been excluded and/or modified by
BioPharm International to reflect
regulatory changes. As examples,
although daratumumab, elotu-
zumab, and idarucizumab were orig-
inally included in EvaluatePharma’s
list as mAbs that would gain regula-
tory approval in 2016, these three
molecules actually gained US FDA
approval in 2015.
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January 2016 www.biopharminternational.com BioPharm International 13
its ability to inhibit the biological
effects of both IL-4 and IL-13. It will
compete with AstraZeneca’s Nucala
(mepolizumab), which was approved
by FDA in November 2015, although
Nucala works by another mechanism
(through IL-5). Teva’s IL-5 inhibitor
Cinquil (reslizumab, currently in
Phase III trials), Roche’s IL-13 inhibi-
tor lebrikizumab (in Phase II trials),
and AstraZeneca’s IL-13 inhibitor
tralokinumab (in Phase III trials for
asthma) may also compete for market
share in this crowded uncontrolled
asthma mAb market.
Farletuzumab—Farletuzumab is
an investigational humanized IgG1
antibody targeting tumor cell surface
protein folate receptor alpha (FRA),
which is typically overexpressed
in epithelial tissue-derived can-
cers. Farletuzumab works by block-
ing the function of FRA. The mAb,
which is a radiotherapeutic that is
bound to a cytotoxic radioisotope,
received Orphan Drug designation in
the United States, European Union,
and in Switzerland for its potential
to treat platinum-sensitive ovarian
cancer. The investigational agent is
being developed by Eisai subsidiary
Morphotek and the Targeted Alpha
Therapy Group (TAT Group) at the
University of Gothenburg in Sweden.
Ibalizumab—The medication ibali-
zumab is believed to be a promising
treatment for HIV, as it represents
a treatment alternative for patients
who have become resistant to tradi-
tional antiretroviral therapies (ARTs).
Known as a viral-entry inhibitor, the
anti-CD4 human mAb of murine ori-
gin works by blocking HIV-1 entry in
vitro and has demonstrated in clini-
cal trials to reduce HIV viral load.
EvaluatePharma cites Roche,
Biogen, and The Aaron Diamond
AIDS Research Center as co-develop-
ers of the drug.
Inotuzumab ozogamicin—Pfizer’s
drug candidate for the treatment
of acute lymphoblastic leukemia
(ALL) is the only antibody-drug
conjugate on the potential approval
list for 2016. The drug received a
Breakthrough Therapy designation
from FDA in October 2015 based on
the results of the drug’s performance
in clinical trials compared with
long-term chemotherapy for patients
with relapsed or refractory CD22-
positive ALL. Current treatments
for ALL include Gleevec (imatinib)
and Sprycel (dasatinib), but these
are both examples of treatments for
patients whose leukemia cells have
the Philadelphia chromosome.
Ocrelizumab—Roche’s ocrel i-
zumab is a mAb being developed
to treat relapsing multiple sclerosis
(MS) and primary progressive mul-
tiple sclerosis (PPMS). In clinical tri-
als, ocrelizumab was shown to be
superior to interferon beta-1a, a
widely used treatment for relapsing
forms of the disease. There are cur-
rently no approved treatments for
the progressive form of the disease.
Ocrelizumab is designed to selec-
tively target CD20-positive B cells
that are thought to contribute to
myelin and axonal damage. Roche’s
Chief Medical Officer and Head of
Global Product Development Sandra
Horning, MD, stated in a press release
that ocrelizumab is the “first investi-
gational medicine to slow a clinically
meaningful and statistically signifi-
cant effect on the progression of dis-
ease in primary-progressive MS.”
Sarilumab—This IL-6-receptor
inhibitor from Sanofi and Regeneron
works by blocking the binding of
IL-6 to its receptor and interrupting
the cytokine-mediated inflamma-
tory signaling cascade. The drug is
being tested in clinical trials as a first-
and second-line therapy in combi-
nation with methotrexate or other
disease-modifying antirheumatic
drugs (DMARDs) and also as a mono-
therapy. Sarilumab is likely to com-
pete with first-line therapies such as
Bristol-Myers Squibb’s Orencia (abata-
cept), Genentech’s IL-6 inhibitor
Actemra (tocilizumab), and AbbVie’s
tumor necrosis factor (TNF)-blocker
Humira (adalimumab). The company
said it will have clinical trial results
comparing sarilumab’s efficacy to
adalimumab by 2016.
Tildrakizumab—This mAb from
Merck is in Phase III clinical trials
for the treatment of plaque psoria-
sis. Potential competitors that tar-
get IL-23 include J&J’s Stelara
(ustekinumab) and Janssen’s gusel-
kumab, which is currently in Phase
II trials.
Tremelimumab—Tremelimumab
is an immune checkpoint inhibi-
tor that prevents the blocking of the
action of cytotoxic T-lymphocytes,
allowing the immune system to more
effectively destroy cancer cells. The
fully humanized mAb binds to the
protein CTLA-4 on the surface of
activated T-lymphocytes, turning off
inhibitory immune signals.
In the ARCTIC, MYSTIC, and
NEPTUNE trials, MedImmune is
testing the drug’s efficacy in com-
bination with investigational agent
durvalumab for the first-line treat-
ment of metastatic non-small cell
lung cancer and head and neck can-
cers. FDA has also granted Fast Track
designation to tremelimumab as a
monotherapy for the treatment of
malignant mesothelioma, an aggres-
sive, rare form of cancer that affects
the lining of the lungs and abdomen.
Daclizumab—Daclizumab was orig-
inally approved by FDA in 1997 to
prevent organ rejection after trans-
plantation. The mAb’s new indica-
tion for multiple sclerosis would
be billed under the trade name
Zinbryta. According to co-developers
Biogen and AbbVie, it demonstrated
“superior outcomes” in clinical tri-
als when compared with Biogen’s
Avonex (interferon beta-1a).
Unlike investigational agent
ocrelizumab, which binds to CD20,
daclizumab binds to IL-2 subunit
CD25, modulating the activity of
IL-2 against abnormally activated
T-cells that attack myelin. Zinbryta
is believed to modulate the function
on IL-2 without disrupting general
immune system responses. ◆
The Big Picture for 2016: Emerging Therapeutics
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14 BioPharm International www.biopharminternational.com January 2016
The escalating attack on rising
expenditures for prescription
drugs and biologics is prompting
more proposals from Congress
and political candidates to strengthen
oversight of biopharmaceutical devel-
opment, manufacturing, and market-
ing. While Democrats have been most
vocal in challenging industry practices,
Republicans have joined the campaign,
citing the need to manage medical costs
as part of proposals for revising the
Affordable Care Act. All parties acknowl-
edge the importance of supporting bio-
medical and genomic discoveries able
to cure lethal conditions such as cancer
and hepatitis, but also want to ensure
that innovative therapies are available to
patients who stand to benefit.
The debate thus is moving beyond
industry “bad actors” and drug reim-
bursement issues to encompass a broad
range of policies affecting pharma opera-
tions. The Justice Department, for exam-
ple, is assessing competitive issues related
to pricing strategies of products marketed
by Eli Lilly, Merck, Mylan, and others.
Pharma mergers and acquisitions will face
even more scrutiny of potential impact
on competition and pricing, as well as
jobs, operations, and R&D. The Trans-
Pacific Partnership agreement and other
trade pacts may lose support among those
who consider these deals overly protective
of pharma intellectual property rights at
home and abroad.
The spreading epidemic of opioid
abuse and deaths from overdoses also will
increase scrutiny of manufacturer R&D
and marketing activities. State and local
governments are filing lawsuits that tar-
get industry practices seen to encourage
excessive opioid prescribing and dispens-
ing. FDA is encouraging development
of abuse-resistant formulations and new
easy-to-use opioid antidotes as it seeks to
provide patient access to needed painkill-
ers less likely to cause harm.
CONGRESSIONAL CONCERNSOne result of the anti-pharma rhetoric
has been to delay approval of the 21st
Century Cures legislation, as questions
multiply about the value of developing
new medicines that may be too costly
for many individuals to access. Such con-
cerns slowed efforts in the Senate to craft
a companion “Cures” bill and may dim
prospects for provisions in the House-
approved measure that provide added
incentives for drug research and testing.
Congressional committees will exam-
ine drug access issues more closely in
early 2016, as Republican leaders in the
House and Senate respond to calls for
reform, as well as pressure from De
mocrats to tackle rising costs. The
House Oversight & Government Reform
Committee has opened an investigation
into prescription drug pricing and expects
further hearings this year. The Senate
Special Committee on Aging set the pace
with a hearing in December 2015 focused
on price increases by Valeant and Turing
Pharmaceuticals. And the House Judiciary
Committee will continue to explore drug
pricing as part of its examination of fac-
tors affecting competition in the US
healthcare system, as seen at a November
2015 hearing on the role of pharmacy
benefit managers.
The Obama administration will fur-
ther examine strategies for manag-
ing outlays on drugs by public health
programs, as discussed at a high-profile
pharmaceutical forum in November
2015. A wide range of experts addressed
whether value-based purchasing and
other approaches can improve patient
access to medicines while maintaining
Politics and Pricing Will Challenge Manufacturers in 2016
Jill Wechsler
Jill Wechsler is BioPharm International’s
Washington editor, Chevy Chase, MD,
301.656.4634, jwechsler@advanstar.com.
The Big Picture for 2016: Regulations
ES722544_BP0116_014.pgs 01.15.2016 01:15 ADV blackyellowmagentacyan
January 2016 www.biopharminternational.com BioPharm International 15
incentives for biomedical innova-
tion. The White House has backed
calls for Medicare to have more lee-
way to negotiate drug prices, and
states are looking more closely at
options for revising Medicaid drug
reimbursement policy.
REGULATORY PRESSURESThese developments have entan-
gled FDA in cost and competition
issues much more than it desires.
At the November 2015 Senate hear-
ing to weigh the confirmation of
Robert Califf as the next FDA com-
missioner, Committee members
repeatedly pressed the nominee on
how the regulatory process could
improve access to medicines. Califf’s
main response was to promise to do
“a good job” to move new generic
drugs through the approval process
and to eliminate the generic-drug
application backlog. Similarly, FDA
approval of more biosimilars would
enhance patient access to less expen-
sive biotech therapies, a process
that would be facilitated by agency
clarification of controversial issues
related to biosimilar development,
such as product naming and inter-
changeability.
Califf also acknowledged the need
to streamline regulation of combi-
nation products, a topic of contin-
ued importance to biopharma and
medical-device manufacturers. And
he mentioned FDA efforts to set
standards for legitimate drug com-
pounding, which could produce
alternatives to costly medicines, as
proposed for a compounded version
of Turing’s costly antiparasitic treat-
ment. FDA aims to do more to track
and prevent drug shortages, with an
eye to avoiding monopoly situations
leading to price increases, as seen in
allegations involving manufacturers
of saline solutions and other com-
mon generic injectables.
Some of these issues may be
addressed through FDA initiatives to
improve drug and biotech product
quality. The agency hopes to final-
ize a program for collecting quality
metrics data that can help dem-
onstrate the state of operations at
pharma manufacturing facilities
and refine its risk-based method
for scheduling site inspections.
The larger benefit will be to reduce
drug recalls and quality-related
shortages and to reward manufac-
turers able to document high-per-
formance systems.
FDA also is stepping up scrutiny
of the accuracy of clinical and man-
ufacturing information submitted
to FDA by manufacturers in appli-
cations and reports, as part of its
increased focus on data integrity.
The agency wants to ensure that
information filed in all agency sub-
missions is complete and truthful,
particularly in numerous documents
related to production quality and
adherence to GMPs. FDA regards
data integrity as basic to any qual-
ity system, commented Doug
Stearns, director of the Office of
Enforcement and Import Operations
in FDA’s Office of Regulatory Affairs
(ORA), at the November 2015 FDA
Inspections Summit sponsored by
FDA News. False or altered data often
are hard to fix, particularly cases
involving overseas operations and
where errors arise more from neg-
ligence or carelessness than from
criminal activity. The agency still
will take enforcement action against
fraud, Stearns commented, but
noted that FDA seeks to rely on com-
panies to have “appropriate quality
systems” to address such problems
when they emerge.
In addition to ensuring indus-
try compliance with standards and
rules, FDA’s larger goal is to support
development and appropriate testing
of innovative therapies. The agency
is working on policies to advance
research on cellular and gene thera-
pies, which involve complex process-
ing and quality control issues.
In vitro diagnostics are in the
news, as drug and medical-device
makers look for clearer FDA stan-
dards for co-development and
appropriate use of these products.
The agency also is working with
genomic testing operators to develop
rational approaches for enhanc-
ing consumer access to personal
genomic information.
Many of these issues will be
on the table as FDA and industry
negotiate the next version of the
Prescription Drug User Fee Act
(PDUFA VI); the program has to be
reauthorized in 2017 to keep indus-
try payments flowing to support
FDA oversight of drugs and biologics,
as well as fees for medical devices,
generic drugs, and biosimilars. All
stakeholders look to continue ini-
tiatives for integrating patient per-
spectives into drug development and
regulatory decision-making, while
also enhancing FDA information
technology and its workforce and
ensuring the long-term stability of
fee programs. But hopes for quickly
enacting a “clean” user-fee reautho-
rization bill will continue to fade as
the political rhetoric heats up. ◆
The Big Picture for 2016: Regulations
Regulatory Topics to
Watch in 2016
In 2016, the US presidency, one-third
of the Senate seats, and all seats
in the House of Representatives
are up for election. In this active
political environment, the following
crucial issues affecting the bio/
pharma industry will be part of the
national discussion:
• Intellectual property protection
and competition
• Drug pricing and affordability
• International trade and regulations
• New FDA commissioner
• Alleviating drug shortages
• Improving product quality
• Data integrity oversight
• Development of innovative therapies
• Prescription Drug User Fee
Act reauthorization
ES722543_BP0116_015.pgs 01.15.2016 01:15 ADV blackyellowmagentacyan
16 BioPharm International www.biopharminternational.com January 2016
To find out what the outsourcing
industry may look like in 2016,
BioPharm International spoke with
Elliott Berger, vice-president of
global marketing and strategy at Catalent
Pharma Solutions; Saharsh Rao Davuluri,
president of contract research, Neuland
Labs; Michael Lehmann, president, global
PDS and executive vice-president global
sales & marketing, Patheon; Paul Dupont,
vice-president marketing and business
development, Ropack Pharma Solutions;
Mark Rogers, senior vice-president, Life
Science Services, SGS North America; and
Peter Soelkner, managing director Vetter
Pharma International GmbH.
MARKET CONSOLIDATIONBioPharm: The contract services and man-
ufacturing market has experienced con-
solidation in the past year. What are the
implications of consolidation for drug
owners in terms of services available and
the cost of those services?
Berger (Catalent): Ongoing consolida-
tion enables major pharmaceutical com-
panies to optimize their supply networks
and source a greater range of services and
technologies from fewer partners. With a
more rationalized approach to sourcing, it
is easier for buyers to work with long-term
strategic partners to create joint quality
and operational scorecards against which
to measure suppliers, and this gives flex-
ibility to monitor and improve global sup-
ply chains.
Consolidation of allied services into
larger organizations creates partners that
are, on the whole, more flexible to clientsÕ
demands and have an attitude of expan-
sion and investment in new technologies,
global capacity, and world-class quality
systems to match the future demands of
sponsors. This may include co-creating
large custom manufacturing suites that
can accommodate new launches or tech
transfers with specific complex require-
ments from pharmaceutical clients who
look to free-up internal capacity; or the
placement of products which have been
launched or acquired into the companyÕs
portfolio and where no in-house capacity
currently exists.
As ever in the pharma industry, new
medicines are becoming increasingly more
complex, and the ability of contract service
companies to create customized solutions
is key to ongoing success. Products are
becoming more ÔglobalÕ, and the ability of
service providers to offer a global supply,
with the oversight of multiple regulatory
agencies, has never been more prescient.
Lehmann (Patheon): ItÕs true that the
contract development and manufactur-
ing industry has experienced significant
consolidation in recent years. The good
news is that there are solid benefits for
pharmaceutical companies that come from
outsourcing partners who have achieved
strategic consolidations. The increased
bandwidth, capacity, and capabilities,
together with an expanded regional and/or
global presence, are valuable to biopharma
companies. Improved economies of scale
and a simplified supply chain are also
important benefits.
Soelkner (Vetter): Consolidation has a
variety of implications for drug owners. It
reduces the number of different supplier
options for individual services. Also drug
owners want to simplify their service pro-
vider network and reduce the number of
providers, essentially creating a one-stop-
shop-approach whenever possible. As a
consequence, they expect their partners to
be strategic, not tactical. In practice, this
means that drug companies would rather
look at outsourcing opportunities for a
pipeline of several products, not just one
drug. This includes looking for attractive
cost models, which the provider can offer
to successfully contribute to developing,
Outsourcing Outlook for 2016
Susan Haigney
The Big Picture for 2016: Outsourcing
The Market Consolidates for Better Service
ES722541_BP0116_016.pgs 01.15.2016 01:15 ADV blackyellowmagentacyan
January 2016 www.biopharminternational.com BioPharm International 17
commercializing, and supplying this
drug pipeline.
Davuluri (Neuland Labs): As a result
of consolidation, vertically integrated
[contract manufacturing organiza-
tions] CMOs offer a Ôone-stopÕ solu-
tion to drug owners. Although this
idea has been promoted for quite
some time, consolidation could
potentially increase this focus. These
integrated CMOs are pitching their
services rather carefully to drug own-
ers. As a pure play API CMO, we tend
to focus on the advantages of being
API specialists with a lot of depth
in handling API-specific [chemistry,
manufacturing, and controls] CMC
issues. Integrated CMOs are also
separating their proposals for APIs
and drug products in order to avoid
coming across as a company who
insists that they do everything (or
nothing) and demonstrate skills that
are comparable to a standalone spe-
cialist who focuses purely on APIs
or finished products. Integrated
CMOs offer a potential cost savings
that drug owners can realize if they
choose the one-stop solution.
Dupont (Ropack): As consolidation
continues, benefits of long-term part-
nership between sponsors and [con-
tract research organizations] CROs/
CMOs become more apparent. Drug
development is becoming more com-
plex, and more venture capital fund-
ing and Big Pharma acquisition of
smaller and mid-size pharmaceuti-
cal companies continues to fuel new
product development. Both sponsors
and venture capitalists require that
candidates identify the drug candi-
dateÕs value point as quickly as pos-
sible. Yes, consolidation effectively
reduces the individual options avail-
able to sponsors. However, consoli-
dation provides sponsors with more
single-source options [and] helps
CROs and CMOs keep up with tech-
nology and develop more compre-
hensive service offerings.
Rogers (SGS): Like M&A activities in
any market, the consequential reduc-
tion in competition is not generally
an advantage to the consumer, and
the contract services and manu-
facturing market is no different.
However, such consolidation may
also make it easier for the drug own-
ers to find a Ôone-stop shopÕ, which
alleviates the need for the coordi-
nation and management of several
different providers but may also
increase the compliance and opera-
tional risks associated with a single-
source supplier.
MORE CONSOLIDATION AND OTHER CHANGES TO COMEBioPharm: Do you expect more con-
solidation or other changes in 2016?
Soelkner (Vetter): We expect con-
solidation to continue in 2016 and
beyond. Regularly, there is news
within the industry regarding a
merger or acquisition between large
and small companies, or at times
the consolidation of two equal large-
market participants. We believe this
will continue. But we do not see large
changes that can disrupt the indus-
try, but rather, the continuation of
existing challenges such as the ever-
increasing complexity of develop-
ment projects, greater expectations
for a high degree of flexibility at the
service providers pertaining to tim-
ing and batch size, as well as ever-
increasing regulatory demands.
Lehmann (Patheon): We know that
our clients are looking for partners
who can work differently with them
to deliver in a rapidly changing phar-
maceutical landscape .... The expand-
ing demand for sterile development
and manufacturing, biologics capac-
ity, and solutions for poorly soluble
compounds drive some of the con-
solidation efforts in the industry.
Berger (Catalent): On a global level,
the pharma industry is still very frag-
mented and continues to consolidate,
so we would expect the pharma ser-
vices sector to do the same. There
is increased demand for specialized
outsourced services, including global
solutions as well as customized and
full service options. At Catalent,
we have seen a marked increase in
the demand for complex products
requiring drug delivery expertise as
well as specialty handling services
including scheduled storage, cold
chain logistics and handling of DEA-
licensed drugs and potent and cyto-
toxic compounds.
Davuluri (Neuland Labs): API CMOs
will look at further modernizing
technology. New drug applications
are now required to include [qual-
ity-by-design] QbD data for any pro-
cesses where there could be control
or quality issues. Techniques such as
QbD can be effectively implemented
into development only if certain
infrastructure for lab scale, pilot, ana-
lytical, safety, and computer software
is in place. In addition, CMOs will
have to look at using modern tech-
niques in R&D and manufacturing
such as usage of parallel chemistry,
new chromatographic technologies,
flow chemistry, etc. This requires an
investment not just in infrastruc-
ture, but also in technologists with
the appropriate training and, most
importantly, a commitment from
management to make these technol-
ogies commercially viable long term.
Dupont (Ropack): Consolidation
within the drug-development mar-
ket continues to heat up. Ideally,
drug owners prefer to develop their
candidates under one roof and ben-
efit from a continuum of quality
throughout the development pro-
cess. This places greater pressure on
CMOs to offer additional services
supported by capital investments in
infrastructure, in equipment, and
in the recruitment of qualified per-
sonnel, while maintaining costing
models acceptable to sponsors and
clients. I believe that for these rea-
sons, CROs and CMOs will require
additional resources and revenue
to support the growing demand for
drug development and clinical mate-
rials. Meeting demand for compre-
hensive development capabilities can
be attained in the near-term only
through the M&A process. ◆
The Big Picture for 2016: Outsourcing
ES722542_BP0116_017.pgs 01.15.2016 01:15 ADV blackyellowmagentacyan
18 BioPharm International www.biopharminternational.com January 2016
Ole
g P
rikho
dko
/Gett
y Im
ag
es
As biopharmaceutical man-
ufactur ing matures, it i s
natural to consider whether
concepts and trends used in
other manufacturing sectors, such as
automotive and electronics, could be
applied to bioprocessing.
In semiconductor manufacturing,
for example, a thorough understand-
ing of process variation allows com-
panies to manufacture circuits with
billions of transistors at high yields.
These variations are translated into a
set of design rules, which help ensure
that designs will be manufactured suc-
cessfully and meet safety and other
regulatory requirements.
The ability to codify manufacturing
into robust design rules has had a tre-
mendous impact on the semiconduc-
tor industry, and enabled it to move
from a simple vertically integrated
model where manufacturers designed
and manufactured product, to a model
in which a few global suppliers out-
source all manufacturing.
Would this structure be desirable
for biopharmaceutical manufactur-
ing? Although their respective regu-
latory environments are radical ly
different, the two industries share
some common elements: use of con-
tract manufacturers, for instance,
and a manufacturer focus on discov-
ery and validation. A key difference is
that biopharmaceuticals are manufac-
tured using living organisms, which
have yet to be fully characterized,
designed, and produced with preci-
sion genetic modifications. Therefore,
Tools for Continuous Bioprocess Development
Rajeev J. Ram
Could perfusion microbioreactors
bring more agility to
biomanufacturing?
Rajeev J. Ram is professor of
electrical engineering at MIT.
Bioreactors
ES721315_BP0116_018.pgs 01.13.2016 16:14 ADV blackyellowmagentacyan
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20 BioPharm International www.biopharminternational.com January 2016
the equivalent design rules and
computer-a ided design tools
that are used in the electron-
ics industry cannot be applied
and the manufacturing process
must be developed, empirically,
for each new product. Bioprocess
development requires interaction
between manufacturer and cre-
ator because biopharmaceutical
product characteristics and qual-
ity are affected by the manufac-
turing process.
R e l i a b l e a n d s t a n d a r d -
ized scale-down models would
allow large-scale manufacturing
and product development to be
decoupled. If the large-scale bio-
reactor is well characterized, a
small-scale system can be used to
replicate all of its operating con-
ditions and potential variations.
Proper scale down requires good
simulation of large-scale bioreac-
tor conditions. The small-scale
reactor needn’t have the same
shape or geometry.
The industry needs technolo-
gies that can enable upstream
processes to move quickly and
seamlessly between partners,
whether internal or external.
Currently, cultural challenges
and established ways of working
pose challenges in moving to this
model. For example:
• Upstream process performance
(as measured by doubling time,
viability, and product quality)
is sensitive to a large number
of parameters (e.g., pH, gas con-
centration, shear, nutrient feed
rates, temperature, etc.).
• Bioreactors themselves, which
control this large number of
parameters, are large, complex
to operate, and have not been
standardized.
• Standardization is hampered
by use of various platforms and
lack of design rules.
For instance, in cell culture,
the typical biopharmaceutical
company uses at least a dozen
different platforms, including
shallow-well microtiter plates;
deep-well microtiter plates; shake
f lasks; benchtop bioreac tors
of various impeller and sparger
designs with various culture vol-
umes and larger bioreactors of var-
ious impeller and sparger designs
with various culture volumes
ranging from 100 L–15,000 L.
Generally, production teams
choose the platform that affords
them the greatest control of
the various process parameters.
Without design rules for pro-
cesses, this inevitably results
in choosing platforms that are
“familiar,” even if they might
not be the best choice for the
given process. In addition, dis-
covery teams tend to select plat-
forms that are easiest to use and
that offer the possibility of high-
throughput development.
As a result, biopharmaceutical
process development and scale
up are rarely linear. Fundamental
understanding of the cellular and
biochemical processes involved
is often sacrificed because it isn’t
always clear whether or not the
metabolism of the production
cells remains the same across var-
ious platforms.
In extreme cases, product qual-
ity can be affected. Consider the
case of Myozyme (alglucosidase
alfa), a lysosomal glycogen-spe-
cific enzyme indicated for use in
patients with Pompe disease. In
2008, FDA rejected Genzyme’s
application to produce Myozyme
in a 2000-liter-scale facility under
the same approval authorization
given for its 160-liter-scale plant.
The FDA ruling stated that the
glycosylat ion of the products
manufactured at each scale dif-
fered, and, thus, the 2000-liter
product required a new biologic
license application (1).
Why Is proCess Transfer harD?Scale up and scale down are well-
known barriers to biologics produc-
tion. Because volume and surface
area scale differently with length,
even in similar benchtop bioreac-
tors, the physical and chemical
environment seen by the cells will
be different from what is seen on
the industrial scale. The physical
and chemical environment of the
cells can strongly affect the cells’
physiology and productivity and,
hence, must remain constant or
above critical values during scaling.
First, the gas transfer rate of O2
and CO2 must be suffciently high
so that the dissolved oxygen (DO)
level remains above the oxygen
uptake rate of the cells, and waste
gases such as carbon dioxide are
effciently removed.
Bioreactors
Process Understanding
In the near future, biopharmaceutical companies will be looking into building
cellular function models that will help them understand the effects of feed rate,
and physical and chemical stresses on the productivity and growth of their
cell lines. Having predictive models of the impact of manufacturing conditions
on industrially relevant cell lines would greatly accelerate upstream process
optimization. Often, the overexpression of a recombinant protein is rate-limited
by an enzyme whose kinetics are not well understood. Understanding the rate-
limiting steps affecting the productivity of the cells will greatly reduce the effort
needed to find the optimal processing conditions of the recombinant cell line.
The large data sets required to form a complete cellular function model require
a high-throughput platform that can run at a lower cost than bench-scale
bioreactors but with the same set of instrumentations. —Rajeev J. Ram
ES721892_BP0116_020.pgs 01.13.2016 23:10 ADV blackyellowmagentacyan
January 2016 www.biopharminternational.com BioPharm International 21
AL
L F
IGU
RE
S A
RE
CO
UR
TE
SY
OF
TH
E A
UT
HO
R.
Secondly, the maximum shear
rate seen by the cells must remain
the same or below the critical value
that affects productivity during
translation. This is especially impor-
tant for mammalian cells such as
Chinese hamster ovary (CHO) cells
due to their shear sensitivity.
The circulation time is also
an important parameter, since it
affects the frequency at which
the cells see the high shear. The
repeated deformat ion of the
endoplasmic reticulum can be
detrimental to protein glycosyl-
ation. Bioreactors with differ-
ent chamber volumes will have
different circulation times, and
hence, some benchtop bioreac-
tors are equipped with a circula-
tion line that allows the physical
env i ronment of the ce l l s to
mimic the circulation time seen
in large industrial-scale bioreac-
tors. When designing scale-down
models of bioreactors, the energy
dissipation rate has to be main-
tained constant so that the trans-
fer of internal energy to the cell
remains constant.
Decades of research have estab-
lished these guidelines for pro-
cess transfer from one platform
to another. While not complete,
they provide a set of recommen-
dations that are grounded in an
understanding of cellular pro-
cesses and biochemistry. Many
important principles of process
transfer are rarely followed, how-
ever, for the following reasons:
• Microtiter plates and shake
f lasks are incapable of repro-
ducing more than one (gas
transfer) of these parameters
(e.g., shear, circulation time,
and energy dissipation rate)
relative to a production biore-
actor.
• Most companies strive to keep
only a subset of these param-
eters constant as they scale
upstream processes. A presenta-
tion by Biogen illustrated using
either the kLa or the power dis-
sipation per unit volume as
scale-down parameters (2).
• Maintaining geometric similar-
ity over-constrains the problem
so that all of the relevant param-
eters cannot be held constant
across scale. A paper from ETH
Zurich (funded by Novartis)
demonstrated that geometric
similarity fundamentally does
not allow for scale invariance
(from 3 L to 15,000 L) of kLa,
shear stress, and mixing time
simultaneously (3).
In response to the increasing
need for parallelization and minia-
turization of controlled and moni-
tored bioreactors, commercial and
academic research groups have
developed microbioreactors with
working volume below 1 L to:
• De l ive r h i gh - t h roughput ,
easy-to-use platforms that can
replace the microtiter plates
and shake flasks used by dis-
covery teams
• Achieve the same gas-transfer
rates, shear, circulation, and
energy dissipation of 15,000-L
product ion reactors in the
high-throughput platform
• P rov ide backward compat-
ibility to all existing in-line
and final assays/instruments
performed by process develop-
ment teams.
sTaTe of The arT for sCale-DoWn Approaches to miniaturization
vary greatly, due to the many
different technologies currently
b e i ng deve lop e d . Howeve r,
advances in microbioreactors are
occurring, as the following exam-
ples demonstrate:
• pH and DO monitor ing are
increasingly being integrated
at microscale (4,5), as dispos-
able and non-invasive optical
pH and DO sensors are vali-
dated against the electrochemi-
cal probes used by large-scale
bioreactors.
• Optical DO sensors have been
implemented in shake flasks
as a first step to allowing the
monitoring of conditions in
these containers (6, 7).
• pH and feed control are being
implemented in shake flasks
with integrated syringe pumps
for l iqu id i nje c t ions a nd
pH probes (8), as shown by
Weuster-Botz et al.
Bioreactors
Figure 1: The fgure depicts the schematic for a disposable 1-mL polycarbonate
microbioreactor. The disposable device supports four input feed streams as well as a
perfusion output and waste output.
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22 BioPharm International www.biopharminternational.com January 2016
Bioreactors
• Equipment is being minia -
tur ized for control l ing DO
and pH (9, 10) by integrat-
ing optical pH and DO sen-
sors with 24-well plates and
sparging oxygen and carbon
d iox ide th rough a per me-
able membrane at the bottom
of the well, as seen with Pall
Corporation’s Micro-24 (M24)
reactor. The reactor has been
demonst rated in microbia l
cultivations (10), and has also
been shown to work for chi-
nese hamster ovary (CHO) cell
cultures as well (11).
• Microf luidics is being used
in well plates to improve the
mic rot ite r plate format in
appl icat ions such as m2p -
labs’ BioLector Pro. This tool
e nable s automate d l iqu id
inject ions to be per formed
using pneumatic valves in the
microfluidic section under the
well plate.
• Microbioreactors are also being
designed with optical density
(OD), conductiv ity, and pH
sensors (12). SimCell’s solution
for high-throughput minia-
turization is to design 700-μL
microbioreactors in the form
of cassettes agitated by a rota-
tor. The SimCell model uses
a robotic arm to transfer the
cassette from the rotator to a
sensing platform to measure
pH, DO, and OD. These online
measu rement s a re supple -
mented by sample removal for
off ine measurements of glu-
cose, viability, titer, and prod-
uct quality (13). The device
can use up to 324 cultures
simultaneously, allowing it to
be used for process optimiza-
t ion, screening, and media
optimization (14, 15).
• Mo s t r e c e nt l y, S a r t o r i u s
Sted im’s TAP ambr 15 and
ambr 250 have been gaining
tract ion for scale down. In
addition to optical pH and DO
sensors, the system combines
miniatur i zed st i r red tanks
with a pipett ing robot and
automated feed pumps, greatly
improving automation of the
fermentation process. By uti-
lizing a biosafety cabinet, the
system ensures that sterility is
maintained during automated
pipetting operations required
for feeding, inoculation, and
sampling. Both microbial and
mammalian processes have
been studied in the systems
and have shown reasonable
correlation with bench-scale
stirred tanks (16, 17). These
studies have also shown how
certain parameters, such as
agitation and power dissipa-
t ion, do not sca le l inearly
with bioreactor size, especially
when the st ir red-tank form
factor is maintained (18).
Nearly a l l of these micro -
bioreactors are capable of both
batch and fed-batch operation.
For the needs of conventional
biomanufacturing, these culti-
vat ion modes a re su f f ic ient .
However, cont inuous culture
operation, in all its forms—che-
mostat, turbidostat, and perfu-
sion— can suppor t metabol ic
flux analysis for increased pro-
cess understanding (see sidebar).
Continuous perfusion holds the
promise of improved product
quality and consistency because
of steady-state operat ion and
because the secreted molecule
does not have to be held until
the end for harvest.
Rea l i z ing such cont inuous
perfusion operation can be chal-
lenging for microbioreactors,
however. It requires long-term
cultures, and places greater stress
on sterile interfaces for the con-
tinuous in-flow and out-flow of
medium and cells.
For continuous perfusion pro-
cesses to be eff icient, the cell
density must be high, up to 100
million cells/mL, to convert the
nutr ient-r ich media into pro-
tein product, ef f ic iently (19).
Such high cell densities can be
demanding for gas t ranspor t
and fluid addition. In addition,
removal with cell retention is a
challenge. In fact, operation in
continuous perfusion culture mode
has not been reported for any of
the microbioreactors systems
described in previous passages.
sCale-DoWn for ConTInuous BIoproCessIngFor continuous perfusion bio-
processing, a lack of scale-down
technology is a major barrier to
entry (20). Because typical per-
fusion process experiments con-
sume 1–3 working volumes per
day and can run for 20–60 days,
culture media costs alone con-
tribute to prohibitively expen-
sive experimental campaigns. For
example, perfusion process devel-
opment experiments (21) using
a 4-L working-volume Wave bag
and alternating tangential f low
(ATF) or tangential flow filtration
(TFF) cell-retention filter, con-
Figure 2: An image of the instrument
that provides the fuidic interfaces and
the optical interfaces for the gas, pH,
and, dissolved oxygen (DO) samples.
ES721313_BP0116_022.pgs 01.13.2016 16:14 ADV blackyellowmagentacyan
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24 BioPharm International www.biopharminternational.com January 2016
sumed between 55 L and 717 L,
with a median of 105 L. With a
typical medium cost of approxi-
mately $50/L, media costs alone
contribute to $5,000 per data
point. The high cost associated
with running perfusion experi-
ments is mainly due to the work-
ing volumes available for current
perfusion technology.
Hollow-fiber bioreactors are
currently used for small-scale
perfusion processing. These con-
sist of semipermeable tubing that
allows nutrients to diffuse to cul-
tured cells and removes waste
products. FiberCell Systems, for
example, provides disposable per-
fusion cartridges with volumes
as low as 2.5 mL. Typical usage
of the FiberCell system involves
seeding cells in the space out-
side of the fibers and pumping
fresh media through the fibers to
maintain a proper environment
for cell growth (22). Since cells
are typically seeded in the hous-
ing of the FiberCell system, it is
difficult to measure parameters of
the culture environment such as
dissolved oxygen and lack of mix-
ing makes cell sampling difficult.
External hollow-f iber systems
such as the ATF and TFF couple
hollow-fiber filters to a conven-
tional bioreactor. These systems
circulate cells between the con-
vent ional bioreactor and the
hollow fibers to allow for more
environmental control represen-
tative of large-scale production.
The tangential flow geometry of
these filters, in particular, the
alternating tangential flow of ATF
systems, helps to prevent fouling
of the hollow fiber filters and pro-
long filter life in high cell den-
sity applications. However, these
systems are currently unable to
operate at scales as small as the
FiberCell System. For example,
both Pall Corporation’s iCELLis,
used for adherent perfusion cul-
ture, and Repligen Corporation’s
ATF, used for suspension culture,
have minimum working volumes
of 1 L. Therefore, there is a real
need for small-volume perfusion
bioreactors capable of supporting
the high cell densities desired for
continuous perfusion processes.
T he p r i m a r y t e c h no lo g y
hurdle for existing microbiore-
actors is the development of
h igh- t h roughput mea ns for
continuous f luid addition and
harvesting with cell retention
capable of supplying up to 100
working volumes over 30 days.
This is diff icult for well-plate
technologies due to the close
proximity of growth chambers,
which constrains external fluid
connections. In addit ion, for
robotic pipetting architectures,
fluid removal with cell retention
cannot be accomplished with
standard pipette tips.
Microf luidic technology has
been applied to continuous cul-
ture over the past decade. This
technology has been success-
ful primarily for the culture of
attachment-based cells and tis-
sue culture (23–25). The large
surface area to volume ratio of
microf luidic devices facilitates
efficient diffusion of nutrients
while providing mechanical sup-
port for the cells. Unlike hollow-
fiber-type perfusion bioreactors,
microfluidics allows for the inte-
gration of various sensors and
also facilitates the precise control
of environment. There are a few
early examples of continuous sus-
pension culture using microflu-
idics (26, 27); these examples do
not incorporate control of pH and
dissolved gases, which is typically
required in a bioreactor.
A few commercial microf lu-
idic perfusion systems without
active mixing are currently avail-
able. The Mil l ipore Cel lASIC
traps cells in a closed chamber
and uses microfluidic channels
smaller than the size of the cell
to deliver media and reagents.
The small 2.8-mm diameter cul-
ture chambers can be observed
using microscopy and can be
used for screening and gene-
expression experiments (28).
neW aDvanCeMenTs In The fIelD of MICroBIoreaCTorsRecently, a microfluidics-based
bioreactor system was introduced
by Pharyx—a recent start-up out
of MIT—for continuous and per-
fusion processes (Figures 1 and 2).
This microbioreactor’s smaller
1–2 mL volume enables long-
term continuous experiments
with neglibile media usage. In
addition, all pumps and control
valves are integrated on-chip as
disposable elements, greatly sim-
plifying setup and operation.
Mixing is performed by moving
silicone diaphragms to push the
fluid between mixing chambers.
The relatively large surface area
of the fluid also allows for bub-
ble-free gas transfer through the
silicone diaphragms, with kLas
greater than 30 h^-1. Bubble-free
gas transfer enables online opti-
cal density measurements as well,
which provides new functionality,
such as automated cell bleeding
and on-line cell density control.
The form factor of the microbiore-
actor also enables integration of a
perfusion filter membrane directly
inside the growth chamber, where
constant mixing of the culture
provides passive filter cleaning,
prolonging the lifetime of the per-
fusion filter.
The original experiments with
a similar microfluidic bioreactor
focused on the demonstration of
various cell-culture modes and
on the development of metabolic
models (29). Due to the ability
to switch between multiple input
streams and accurately measure
the optical density, pH, and oxy-
gen, a variety of functions were
Bioreactors
ES721317_BP0116_024.pgs 01.13.2016 16:15 ADV blackyellowmagentacyan
January 2016 www.biopharminternational.com BioPharm International 25
Bioreactors
possible. Multiple experiments in
chemostat and turbidostat modes
with dif ferent media compo-
sitions were demonstrated in a
single device. These experiments
proved that modulation of input
sources was possible, high-per-
formance liquid chromatography
(HPLC) sample collection times
could be fast enough to look at
dynamics, and control of oxygen
during continuous culture could
be implemented. Additionally,
operation for three weeks without
evaporation was demonstrated,
all while maintaining sterility.
Modrirez et al. have used micro-
fluidic perfusion devices to study
therapeutic protein production
using Pichia pastoris (30). A perfu-
sion filter (polyethersulfone) with
a 1-cm diameter was incorporated
underneath the growth chamber
to allow for fluid flow-through
while maintaining all of the cells
inside the growth chamber and
enabling the switching of induc-
tion media. The ratio of the filter
surface area to bioreactor volume
was 0.758 – a factor of three greater
than in previously reported surface
area measurements in high-per-
formance, bench-scale perfusion
bioreactors (21). A combination of
perfusion flow and cell density con-
trol enabled process optimization of
various parameters, such as inducer
concentration and perfusion flow
rate. Cultures were also shown to be
stable, producing consistent levels
of both hGH and interferon alfa 2b
for more than 10 days.
In-line cell density and precise
flow-rate control enabled Han et
al. to study genetic switching of
Saccharomyces cerevisiae during
exponential growth in a steady-
state turbidostat (31). Cells were
genetically modified to produce
different recombinant proteins
in response to different chemi-
cal inducers and it was shown
that by testing the synthetic cir-
cuits in turbidostatic steady state,
circuit induction increased over
8x and the standard deviation
of activation decreased by more
than 50% when compared to
growth in test tubes.
W h i le cont i nuou s m ic ro -
bioreactors are early in their
development and commercial
deployment, the preliminary data
with microbial cell lines demon-
strate the capabilities for high
gas-transfer rates—supporting the
high cell densities required for
scale-down models of continuous
biomanufacturing, the ability to
maintain sterile conditions with
continuous in-flow and out-flow,
and the ability to support high
perfusion rates over a wide range
of operating conditions.
A mature perfusion microbio-
reactor platform has the poten-
tial to fill a crucial need in the
evolv i ng l a nd scape of b io -
manufacturing. As with earlier
microbioreactors for batch bio-
processing, perfusion microbio-
reactors could lower the cost for
bioprocess development. More
importantly, such devices can
fulfill multiplexing performance
and ease-of-use demands in early-
stage development and discov-
ery. Providing development and
discovery teams for robust scale-
down models in biomanufactur-
ing has the potential to help the
industry advance tremendously.
referenCes 1. G. Mack, Nature Biotech.
26, pp. 592 (2008).
2. V. Janakiraman et al., “Application
of Multivariate Analysis Tools
and Design of Experiments (DOE)
to Model the Design Space for
Characterization of a Mammalian Cell
Culture Process,” presentation from
the AIChE Annual Meeting (2012).
3. M. Soos et al., “Characterizing
Heterogeneity of Environmental
Conditions in Various Bioreactor
Scales Used for Cell Cultivation,”
presentation at the AIChE
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4. H.R. Kermis et al., Biotech. Prog.
18 (5), pp.1047–1053 (2002).
5. M.A. Hanson et al., Biotech. 97
(4), pp. 833–841 (2007).
6. A. Gupta and G. Rao, Biotech.
Bioeng. 84 (3), pp. 351–358 (2003).
7. C. Wittmann et al., Biotech. Letters
25 (5), pp. 377–80 (2003).
8. D. Weuster-Botz, J. Altenbach-
Rehm, and M. Arnold, Biochem.
Eng. J. 7 (2), pp. 163–170 (2001).
9. A. Chen et al., Biotech. Bioeng.
102 (1), pp. 148–160 (2009).
10. K. Isett et al., Biotech. 98 (5),
pp. 1017–1028 (2007).
11. S.R.C. Warr, Anim. Cell Biotech.
(1104), pp. 149–165 (2013).
12. A. Buchenauer et al., Biosensors
Bioelectronics 24 (5), pp.
1411–1416 (2009).
13. R. Legmann et al., Biotech. Bioeng.
104 (6), pp. 1107–1120 (2009).
14. A.P. Russo et al., “Multi-Parameter
Process Optimization Using
the SimCell System,” in the
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Meeting of the European Society
for Animal Cell Technology, N.
Jenkins, N. Barron, and P. Alves,
Eds. (Springer, Dublin, Ireland,
5th ed., 2009), pp. 515–518.
15. Z. Xiao et al., Methods Mol. Biol.
(1104), pp. 117–137 (2014).
16. V. Janakiraman et al.,
Biotechnol. Prog. doi: 10.1002/
btpr.2162 (2015).
17. M. Tai et al., Biotechol. Prog.
(5), pp. 1388–1395 (2015).
18. A.W. Nienow et al., Biochem. Eng.
J. (76), pp. 25–36 (2013).
19. M.S. Croughan, K.B. Konstantinov,
and C. Cooney, Biotech. Bioeng.
112 (4), pp. 648–651 (2015).
20. E.S., Langer and R.A.
Rader, BioProcess. J. (13),
pp. 43–49 (2014).
21. M.F. Clincke et al., Biotechnol.
Prog. 29 (3), pp. 754–767.
22. W.G. Whitford and J.J.S. Cadwell,
Bioproc. Int., (10), pp. 54–63 (2009).
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Bioeng. 78, pp. 257–269 (2002).
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Microdev. 6, pp. 279–287 (2004).
25. M. Reichen et al., PLoS ONE
7 (12):e52246 (2012).
26. F.K. Balagadde et al., Science
309, pp. 137–140 (2005).
27. References 5–9 as originally
cited in reference 28.
28. P. Lee, T. Gaige, and P. Hung,
Methods Cell Biol. (102),
pp. 77–103 (2011).
29. K.S. Lee et al., Lab on a Chip
(11), pp. 1730–1739 (2011).
30. N.J. Mozdzierz at al., Lab on a Chip
(15), pp. 2918–2922 (2015).
31. N. Han et al., “Microfluidics for
Control in Synthetic Biology,”
presentation at the 18th
International Conference on
Miniaturized Systems for Chemistry
and Life Sciences, MicroTAS
2014, pp. 312–314 (2014). ◆
ES721314_BP0116_025.pgs 01.13.2016 16:15 ADV blackyellowmagentacyan
26 BioPharm International www.biopharminternational.com January 2016
mst
ay/
Gett
y Im
ag
es
Biopharmaceutical manufactur-
ing involves a series of complex
unit operations linked together
to provide high-purity, biologic
actives with specified physicochemi-
cal and pharmacokinetic properties.
The development of commercial-scale
processes that consistent ly pro -
vide high-quality product that meets
those specifications requires extensive
examination of numerous process vari-
ables and potential process variations.
Conducting the required number of
studies on the commercial scale is not
practical; scale-down models are there-
fore employed to determine optimum
conditions for downstream separation
processes, including chromatography
and viral clearance steps. Qualification
that such models provide results truly
representative of commercial-scale pro-
cesses becomes necessary as molecules
move closer to commercialization.
whaT counTs as a scale-Down moDel?A scale-down model is a bench-scale
process designed to predict the results
that will be obtained when the process
is run at commercial scale. Unlike ini-
tial processes that are used during the
discovery and early development phases
to produce material for characteriza-
tion, efficacy, safety, and other studies,
scale-down models are intended to aid
in optimization and characterization of
the process that will be implemented
for manufacture of large quantities of
a biologic once the product has been
approved.
going small to achieve success on the commercial scale
Cynthia A. Challener
Scale-down modeling is
instrumental in supporting the development
of downstream biopharma
manufacturing processes.
Cynthia A. Challener, PhD, is
a contributing editor to
BioPharm International.
Downstream processing
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January 2016 www.biopharminternational.com BioPharm International 27
While process simulations are an
ideal surrogate, given the complex-
ity and inaccuracy of many theo-
retical models, there often remains
no substitute for a physical scaled-
down model, according to Adam
J. Meizinger, a process engi-
neer in Genzyme’s Purification,
Manufac tur ing Sc ience, and
Technology Laboratory.
Scale-down models are used in
the development of nearly every
unit operation in the overall bio-
pharmaceutical manufacturing
process, including both upstream
and downstream steps, and sepa-
ration and filtrations steps, such
as chromatography and viral fil-
tration processes. A scaled-down
chromatography process, for
instance, is designed considering
the residence time, flow rate, and
column bed height of the large-
scale process. Additional param-
eters that are considered include
buffer preparation and feed stream.
“The ideal model behaves identi-
cally to the scaled-up process such
that the results obtained in the lab
exactly predict the results that will
be obtained in the manufacturing
plant,” says Paul Jorjorian, director
of global technology transfer for
Patheon. Because they are mod-
els, however, such a situation does
not occur. The goal, therefore, is to
mimic the commercial-scale pro-
cess as closely as possible.
The impacT of QbD anD DoeThe qual ity-by-design (QbD)
approach to process development
requires characterization of the
impacts of different process param-
eters on product critical quality
attributes (CQAs), which in turn
necessitates considerable experi-
mentation, according to Meizinger.
“While the consideration of up-
front risk assessments, access to
general scientific understanding,
and the use of design-of-exper-
iment (DoE) methodology mini-
mizes the required number of runs
and maximizes the informational
output, it is impractical to conduct
such characterization experiments
at full scale. Qualified scaled-
down models are instrumental
for conducting such characteriza-
tion work, as well as for viral clear-
ance studies, continuous process
improvement, and for conducting
impromptu investigations in sup-
port of large-scale operations, such
as when deviations occur,” he adds.
Dealing wiTh imperfecTionWhile ideal scaled-down models
correlate exactly with their large-
scale counterpart processes, gener-
ally a perfect match is not achieved
between the bench and commer-
cial scales, and therefore, model
processes are not perfectly scalable.
The causes, according to Jorjorian,
may not relate directly to the pro-
cess, but can be attributed to small,
nuanced differences that still have
an impact on the results at differ-
ent scales. “The issues are often
unavoidable and thus must be
addressed by developing correction
factors and/or transfer functions
that enable the accurate prediction
of the behavior of manufacturing
processes,” explains Jorjorian. “It is
important, however, to minimize
the need for correction factors and
transfer functions, and for those
that are required, provide thor-
ough justification and clearly dem-
onstrate their suitability,” he adds.
Another aspect of scale-down
models that can create issues when
predicting large-scale behavior is
the fact that models are qualified
at the center point of the process
design space. “This approach can
lead to discrepancies in predicted
and actual results when the model
is applied to the entire design
space,” Jorjorian observes. He adds
that timing and planning of mod-
eling runs is also important and
can negatively influence results if
not done effectively. For example,
it is important to use the same feed
used for production runs in scale-
down modeling runs; therefore,
modeling runs must be scheduled
when the feed is available. In many
cases, that isn’t possible, and the
feed may need to be frozen, which
introduces a new variable that can
impact the reliability of the model.
Specifically for chromatography
operations, some key consider-
ations in developing scaled-down
models are minimizing the dif-
ferences in the extra-column
volume, which varies consider-
ably with scale, contending with
increasing wall effects inherent in
small-diameter chromatography
columns, and accounting for dif-
ferences in detector-specific path
lengths and the associated non-lin-
earity of detector output, according
to Meizinger. He also notes that
some steps (e.g., gas overlays for
intermediate holds) may be chal-
lenging to implement as part of
sca led-down model develop -
ment, and mixing dynamics and
heat transfer often remain differ-
Downstream processing
while process
simulations are an
ideal surrogate, given
the complexity and
inaccuracy of many
theoretical models,
there often remains
no substitute for a
physical scaled-down
model.
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28 BioPharm International www.biopharminternational.com January 2016
ent between scales. “The impact
of these differences must be care-
fully assessed for their resulting
impact on scaled-down model per-
formance,” he says.
Scale-down modeling of mem-
brane chromatography processes
can also be difficult, according to
Jorjorian. The reason often relates
to the designs used for small- and
large-scale membranes; they are
often different, with large-scale
membranes having added flutes or
pleats or other features that affect
their performance such that it is
difficult to develop bench-scale
processes that can reliably predict
production-scale outcomes.
QualificaTion aT laTer sTagesThere is nothing new about the
use of bench-scale process mod-
els during the development of
biopharmaceutical manufactur-
ing processes. What has become
an important trend in process
development is the need to dem-
onstrate that scaled-down models
truly represent their large-scale
counterparts. “It has only recently
become standard practice to dem-
onstrate that scaled-down models
provide results that are statisti-
cally equivalent to those obtained
from commercial-scale versions
of the same processes,” states
Meizinger.
Often during earlier stages of
development, even through Phase
I clinical trials, scale-down models
of downstream biopharmaceuti-
cal processes are generally not for-
mal, qualified models. “For these
models, the basis for use of each
model and the rationale for why
it is expected to be representa-
tive of the process at large scale
are documented,” notes Jorjorian.
As a molecule progresses toward
commercialization, however, for-
mal, qualified models are required.
The ability of the model to pre-
dict results at large-scale must be
demonstrated by performing at
least triplicate runs. The use of any
correction factors and/or transfer
functions must also be justified
and their application demonstrated
to provide robust results.
Qualification of most models
for chromatographic separations
is fairly straightforward, accord-
ing to Jorjorian. “There is a lot of
documentation and analyses to
complete, so in essence, qualifica-
tion of scale-down models is often
an exercise in coordination, rather
than presenting a substantial
technical challenge,” he observes.
Meizinger agrees that the use of
statistical equivalence for test-
ing quality attributes and process
performance indicators between
scales is generally not an issue.
“Demonstration of both individual
unit and linked unit output (recov-
ery, product quality attributes,
product purity, chromatographic
profiles/transition analysis, etc.)
comparability between small and
large scales remains both practical
and accepted,” he states.
moving To high-ThroughpuT moDelsQualification of scale-down mod-
els can be challenging, however,
when very small column sizes
are involved. There is significant
interest in using high-throughput
approaches for scale-down mod-
eling to speed up development
times. There are, however, techni-
cal issues that arise due to the dif-
ferent behaviors observed in very
large and very small columns,
such as the wall effects mentioned
previously.
“It is definitely more challeng-
ing to establish robust scale-down
models using high-throughput
methodologies because typically
transfer functions and correction
factors are required. As a result,
it is more difficult to adequately
demonstrate the scalability of
the models to the level required
for qua l i f icat ion,” Jor jor ian
says. He adds that there is a lot
of effort being devoted to over-
coming these issues to enable
high-throughput modeling of
downstream bioprocesses.
A few groups are also investi-
gating the application of first
principles calculations to the scale-
down modeling of both upstream
and downstream unit operations,
and particularly for chromatog-
raphy, for which there is signifi-
cant understanding of separation
behaviors and existing mathemati-
cal models. “This approach to
scale-down modeling is in the ear-
liest phases and largely limited to
large pharmaceutical companies
and university research groups
with the resources and computing
power required,” Jorjorian says.
exisTing Technology is highly valuable“Despite the limitations of exist-
ing scale-down modeling meth-
ods, the use of sca led-down
models is recommended wherever
possible, including for process
characterization, viral clearance
studies, continuous improvement,
manufacturing-related investiga-
tions, evaluation of changes in
raw materials, and so on, bar-
ring only validation efforts and
clinical trial/commercial manu-
facturing requirements for per-
formance of large-scale runs,”
asserts Meizinger. “Today, scaled-
down models serve as efficient
and cost-effective representations
of full-scale processes. While the
inability to demonstrate equiva-
lence between scaled-down and
large-scale operations is often
inevitable for some metrics, fur-
ther understanding of these scale-
related differences, particularly
through the development/refine-
ment of associated engineering
models, promises to continuously
improve the accuracy and util-
ity of scaled-down models,” he
states. ◆
Downstream processing
ES721336_BP0116_028.pgs 01.13.2016 16:29 ADV blackyellowmagentacyan
How will you respond when the FDA asks:“Are you efectively monitoring your contract lab?”
Thursday, February 11, 20168 a.m. PST | 10 a.m. CST | 11 a.m. EST
Attend our
www.EurofinsLancasterLabs.com/Webinars
Presented by
EVENT OVERVIEW
In response to FDA draft guidance regarding the Quality
Metrics Program issued in July, 2015, the bio/pharma-
ceutical industry faces increasingly stringent regulatory
requirements in order to safeguard product quality and
patient safety. Moreover, suppliers and contractors are
now being viewed as an extension of the pharmaceutical
and biopharmaceutical manufacturer’s own facility rather
than an independent resource, forging a unique, strategic
relationship between vendors and sponsors.
While critical to meeting the recent regulations set forth
by FDA, the process of maintaining control over suppliers
requires effective organization, risk management, imple-
mentation of a strong quality culture, and quality over-
sight, which can lead to an extensive amount of time and
resources if not managed appropriately.
Join us for this informative webinar to learn how to foster
your relationships with contract partners to develop a qual-
ity surveillance program that will meet FDA scrutiny.
KEY TOPICS INCLUDE
• Critical aspects of an effective quality agreement
• Essential elements of a comprehensive quality process
• Best practices for a successful audit process
• Strategies for obtaining qualitative and quantitative
metrics reporting
WHO SHOULD ATTEND
Scientists, Managers, Directors and Quality Assurance/
Control Validation personnel in a bio/pharmaceutical
manufacturing company who are responsible for managing
contract testing services.
PRESENTERS
Arthur PezzicaSenior Director,
Quality Compliance
Eurofins Lancaster Laboratories
For questions, contact Kristen Moore
at kmoore@advanstar.com
Hosted by
Brittany CloudSenior Specialist,
Quality Compliance
Eurofins Lancaster Laboratories
Susan SchnieppFellow,
Regulatory Compliance
Associates
ES720989_BP0116_029_FP.pgs 01.13.2016 03:04 ADV blackyellowmagentacyan
30 BioPharm International www.biopharminternational.com January 2016
Mo d e r n d o w n s t r e a m
process ing of b iolog-
ics has elucidated sev-
e ra l spec i f ic prote i n
precipitation techniques that could
considerably reduce the number of
purification steps within a process.
Some of the specific precipitants (2)
used for these techniques include
polyelectrolytes, af f inity l igands,
metal ions, and protein-binding dyes.
The authors evaluated two pre-
c ipitat ion st rateg ies— one using
polyelectrolyte polyvinyl sulfonic
acid (PVS) and the other using zinc
chloride—as alternatives to the con-
ventionally used chromatographic
process. The authors also compared
the benefits of these strategies with
ion-exchange chromatography as
they relate to product purity, reduc-
tion of process-related impurities,
and cost of operation.
materiaLs and metHodsCell-free supernatant used for pre-
cipitation trials was generated in the
laboratory by a fermentation process
using a P. pastoris strain engineered
to secrete a human insulin precursor.
ABSTRACTRecombinant human insulin production using Pichia pastoris typically involves the expression of an insulin precursor as a single chain that is subsequently secreted into fermentation media. The first step in the
downstream process after cell separation typically involves capture by ion-exchange chromatography. In the capture step, the protein is concentrated by ion-exchange chromatography, which also results in the removal of a
significant proportion of the process-related impurities, such as pigments and host-cell proteins (HCPs) (1). The capture step requires an armamentarium of materials, including chromatographic resin media, various buffers, and
highly skilled personnel to handle the process. Although chromatography is a widely used step in this process, there exists a strong impetus to evaluate alternative process technologies from the perspective of process economics.
Precipitation as an alternative to Chromatography
in the insulin manufacturing Process
Madhavan Buddha, Shailabh Rauniyar, Shabandri Qais, Dinesh Goudar, Sai Srikar Kandukuri, Siddharth Mahajan, Sinash Siddik, and Partha Hazra
Madhavan Buddha, is senior scientific
manager; Shailabh Rauniyar is senior scientist;
Shabandri Qais is principal scientist; Dinesh Goudar is principal scientist; Sai Srikar Kandukuri is senior scientist; Siddharth
Mahajan is principal scientist; Sinash Siddik is senior scientist; and Partha Hazra is chief
scientific manager; all of whom are in the
research and development department at
Biocon research Limited in Bangalore, india.
PEER-REVIEWED
article submitted: may 29, 2015.
article accepted: aug. 19, 2015.
MA
RT
IN M
CC
AR
TH
Y/G
ET
TY
IM
AG
ES
Peer-reviewed
ES722494_BP0116_030.pgs 01.15.2016 01:07 ADV blackyellowmagentacyan
January 2016 www.biopharminternational.com BioPharm International 31
Polyvinyl sulfonic acid (PVS), zinc chlo-
ride, phenol, sodium hydroxide, ortho-
phosphoric acid used for the precipitation
trials were procured from MilliporeSigma.
The standard Pierce BCA Protein Assay
Kit (Thermo Fisher Scientific) was used
to analyze the total protein content in
the samples. Sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS–
PAGE) was performed using Thermo Fisher
Scientific’s Novex NuPAGE 12% Bis–Tris
precast gel (1mm*10 well).
A Synergy HT microplate reader (BioTek)
was used to measure the optical density
of samples. Agilent’s 1260 Infinity HPLC–
Chip/MS was used to check the purity
profile and specific protein content of the
samples.
HPLC anaLytiCaL metHodA high-performance liquid chromatogra-
phy (HPLC) method—operable at alkaline
pH—was developed to analyze post-pre-
cipitation samples, because the polyvinyl
sulfonate/insulin complex precipitated at
acidic pH. A Zorbax C18 column from
Agilent (4.6*150mm) was used for purifi-
cation. Buffer A consisted of 100mM tris
and 15mM magnesium chloride and buffer
B consisted of 100% acetonitrile (HPLC
grade). An in-house qualified insulin stan-
dard with concentration of 4 mg/mL was
used to calculate the product content in
post-precipitation samples.
All precipitation samples were analyzed
using a 30-minute reverse-phase HPLC
method, and the HPLC column was main-
tained at 25°C. Elution was done with a
gradient formed by mixing buffers A and
B as follows: 85–60% B (0–15 minutes);
60–20% B (15–20 minutes); 20% B (18–
23 minutes); 20–85 % B (23–25 minutes);
85% B (25–30 minutes). The flow rate was
maintained at 1 mL/min, and the column
effluent was monitored at 215 nm.
BCa assay for Protein estimationThe authors followed the standard proto-
col described for the Pierce BCA Protein
Assay Kit (3) to establish a protein standard
curve. The protein samples were diluted to
approximately 1.5 g/L before performing
the assay. The BCA reagent (4), mixed with
the protein sample (total volume being
200 µl), was pipetted into a 96-well cell-
culture plate and absorbance was recorded
at 562 nm using a Synergy HT Multi-
Detection Microplate Reader from BioTek.
sds–PaGeThe authors followed the standard proto-
col for the NuPAGE kit (5). MES running
buffer (2-(N-morpholino)ethanesulfonic
acid), de-staining solution, sample buf-
fer (5X), staining dye were prepared as per
the procedures mentioned in the proto-
col. The samples were diluted to 1 g/L of
product concentration. Five volumes of
sample were mixed with one volume of
sample buffer, and the mixture was incu-
bated at 85 °C for 15 minutes. Next, 25 µl
of the sample was loaded into respective
wells of the precast gel. The gel was run
at 175V for 60 minutes. Post-completion,
the gel was rinsed in destaining buffer for
10 minutes followed by staining for three
hours. The gel was destained for nine
hours and examined for protein bands.
exPerimentaL ProCedure PVS-based precipitation
PVS concentrat ions of 0.10%, 0.25%,
0.50%, and 1% v/v were screened at pH
2.5, 3.5, and 4.5 to determine the opti-
mal conditions for precipitation. PVS was
added to clarified cell-free supernatant
under mixing and was allowed to mix
for approximately 8–10 minutes. The pH
was adjusted using concentrated ortho-
phosphoric acid. The mixture was further
mixed for 15 minutes and centrifuged at
7000 g for 25 minutes. The centrifuged
supernatant was analyzed for product
loss. The entire process was carried out
at approximately 20 –25 °C. The pellet
obtained was dissolved in 1M tris buffer
and checked for various process attributes
such as product purity, specific protein
content, and pigment content.
Zinc-based precipitation
Phenol concentration was screened at con-
stant zinc chloride and pH to determine
the optimal concentration required for
the precipitation process. The clarif ied
cell-free supernatant was treated with con-
centrations of 0.125%, 0.5%, and 0.75%
v/v of 100% phenol and allowed to mix for AL
L F
IGU
RE
S A
RE
CO
UR
TE
SY
OF
TH
E A
UT
HO
RS
.
Peer-reviewed
ES722492_BP0116_031.pgs 01.15.2016 01:07 ADV blackyellowmagentacyan
32 BioPharm International www.biopharminternational.com January 2016
10 minutes. This was followed by addition
of 5% v/v of 4% w/v zinc chloride solu-
tion and mixed for 5–7 minutes. The pH
of the precipitation mixture was adjusted
to 4.5 by slow addition of 1N hydrochloric
acid. Post-pH adjustment, the precipitation
mixture was allowed to settle at 2–8 °C for
10–12 hours. The supernatant was decanted,
and the remaining slurry was centrifuged
at 6000 g for 25 minutes at 23+2 °C. The
obtained pellet was dissolved in 1M tris
to measure various process attributes such
as product purity, specific protein content,
and pigment content.
resuLts and disCussion The development of a precipitation pro-
cess for a protein to maximize yield and
product purity requires the screening
and optimization of various factors, such
as the nature and concentration of the
precipitant, the pH, the temperature, the
ionic strength, and the dielectric constant
of the medium. The precipitation of an
insulin precursor in the cell-free superna-
tant (CFS) stage strongly depends on the
buffer composition and various other fac-
tors that are present in the supernatant.
Polyelectrolyte-mediated precipitation
Polyelectrolytes are essentially charged
polymers that can either be anionic or
cationic in nature. Polyelectrolytes dis-
sociate in aqueous medium, which leads
to the formation of protein polyelectro-
lyte complexes (6). In addition to electro-
static forces, complex formation is also
influenced by hydrogen bonds and hydro-
phobic forces. This phenomenon—polyelec-
trolyte-mediated precipitation—has been
used in the protein purification process.
The earlier work by McDonald et al. on
antibody precipitation (7) demonstrated
the successful replacement of the chro-
matography step with polyelectrolyte pre-
cipitation. Such precipitation is able to
remove host-cell proteins, DNA, and other
impurities, and results in a product with
a purity that matches that of a product
obtained with the chromatographic pro-
cess. The precipitation of antibodies has
no influence on the activities of the anti-
bodies, which enables the iterative use of
the antibodies in additional protein puri-
fication processes. These antibody results
led the authors to evaluate a similar pre-
cipitation strategy for the purification pro-
cess of a human insulin precursor.
Various factors affecting polyelectro-
lyte precipitation of an insulin precursor
include: conductivity of the medium, pH
of the medium, and polyelectrolyte con-
centration. In the case of an insulin pre-
cursor, preliminary experiments revealed
that precipitation did not occur above the
conductivity of 25 mS/cm. However, there
exists a f lexibility of using higher con-
centration of polyelectrolytes to counter
the effects of higher conductivity of the
medium. The effect of pH (Figure 1) can
be explained by the charge neutralization
of the proteins as the pH increases toward
the isoelectric point (pI). Based on the
results (see Figure 1), PVS concentration
of 0.5% v/v and pH range of 2.5–3.5 were
found to be optimal for protein recovery
by precipitation. At higher concentrations
of polyelectrolytes, the electrostatic repul-
sion effect between the excess of free poly-
electrolyte and polyelectrolyte bound to
the protein increases the solubility of the
insulin precursor.
Zinc phenol-mediated precipitation
One of the fundamental methods of pre-
Peer-reviewed
Figure 1: Effect of concentration of polyvinyl sulfonic acid (PVS)
and pH on product recovery.
100
% R
eco
very
90
80
70
60
31.47 33.06
9.59
0.1% PVS 0.25% PVS
pH 2.5 pH 3.5 pH 4.5
0.5% PVS 1% PVS
68.23
75.6
93.7990.35 92.08
89
4.554.542.7
50
40
30
20
10
0
ES722496_BP0116_032.pgs 01.15.2016 01:07 ADV blackyellowmagentacyan
January 2016 www.biopharminternational.com BioPharm International 33
cipitating insulin precursor molecules
involves the use of metal ions. Zn+2 is the
most preferred metal ion because it is spe-
cific to insulin-like molecules (8). Zinc
induces the hexamerization of an insulin
precursor (9) and thus, stabilizes the mol-
ecule. Precipitation using zinc ions was
also evaluated as a substitute for the chro-
matography step. Hexamerization of insu-
lin precursor requires 0.3 moles of Zn+2
for every mole of insulin precursor. In
the current study, 5% v/v of 4% zinc chlo-
ride solution is added for precipitation,
which is in excess of molar requirement
for hexamerization of an insulin precur-
sor. In the authors’ unpublished study, it
was found that a molar excess of insulin
precursor is required to ensure complete
precipitation of an insulin precursor.
In the case of zinc phenol-mediated pre-
cipitation (precipitant concentration being
mainly phenol), the pH of the medium
and the protein concentrat ion before
addition of precipitants play a key role in
recovery of insulin precursor. Based on
the results (see Table I), 0.5% v/v of phenol
was found to be optimal for precipitation
of an insulin precursor. A higher-than-opti-
mal amount of phenol has shown to incur
higher product losses due to an increase in
solubility of the insulin precursor.
Product quality
assessment after precipitation
The product quality obtained through the
precipitation method was comparable to
the quality of a product purified through
traditional chromatography routes.
Certain parameters were monitored
to assess the quality of medications pro-
duced using precipitation methods com-
pared with products obtained through
chromatography. Host-cell proteins (HCPs),
host-cell pigments, and product purity
(as measured by HPLC) were studied. The
SDS–PAGE (see Figure 2) results show a
comparable band pattern for the chroma-
tography route and the polyelectrolyte
precipitation technique. The optical den-
sities at 600 nm, 450 nm, and 652 nm
and the specif ic protein contents were
recorded for the three strategies (the two
precipitation strategies in addition to tra-
ditional chromatography, as compared
in Table II). Reductions in the color of the
solution at a constant product concen-
tration indicate the removal of different
pigments. The optical densities and spe-
cific protein contents of the CFS at various
stages are tabulated in Table II. Reduction in
optical density is indicative of the removal
of the host-cell pigments by the respective
strategies. Specific protein is a measure of
the amount of insulin precursor relative to
the total protein content in the sample.
The product purities of the three strate-
Peer-reviewed
Phenol (%v/v) Zinc (%v/v) Recovery (%)
0.125 5 43.8
0.5 5 83.4
0.75 5 50.9
v/v: volume by volume
Table I: Effect of phenol concentration on
recovery of an insulin precursor with a constant
zinc concentration.
Figure 2: Sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) for precipitation feed and precipitate
samples.
Lane 1: Cell-free supernatant (CFS)
Lane 2: Clarifed CFS
Lane 3: Polyelectrolyte precipitate re-dissolved
Lane 4: Zinc-based precipitate re-dissolved
Lane 5: Ion-exchange elution pool
Lane 6: Human insulin drug substance re-dissolved
Lane 7: Protein ladder
1 2 3 4 5 6 7
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34 BioPharm International www.biopharminternational.com January 2016
Attributes
Optical density Specific protein based
on BCA Assay (%)
HPLC product
purity (%)450nm 600nm 652nm
Cell-free supernatant (insulin precursor, before clarification) (n=3)
0.30±0.05* 0.083±0.01 0.073±0.01 27.00±1.00 84.16±1
Polyelectrolyte precipitate redissolved (strategy 2) (n=3) 0.073±0.01 0.049±0.01 0.048±0.01 68.00±1.00 92.18±0.5
Zinc-based pellet redissolved (strategy 3) (n=3) 0.072±0.01 0.043±0.01 0.04±0.01 68.58±1.00 89.30±0.5
Cation-exchange (CIEX) elution pool (strategy 1) (n=3) 0.048±0.01 0.041±0.01 0.04±0.01 86.90±1.00 89.72±0.5
*The values represent mean ± standard deviation.
PVS precipitation Zinc phenol-precipitation Ion-exchange chromatography
Raw material Quantity required (g or mL)
Price($)
Raw materialQuantity required (g or mL)
Price ($)
Raw material Quantity
required (g or ml)
Price($)
1Polyvinyl sulfonic acid (PVS)
10 mL 2.80 Zinc chloride 4 g 0.34Ion-exchange
resin*166 mL 7.51
2Orthophosphoric acid
1 mL 0.02 Phenol 10 mL 0.62Sodium chloride
55 g 1.24
1N hydrochloric
acid 1 mL 0.01
Glacial acetic acid
4.5 mL 0.13
Sodium hydroxide
27 g 1.16
Total 2.82 0.97 10.04
*Resin reusability is considered in all cost calculations.
Table II: Optical densities and specific protein contents at various stages of the precipitation process. HPLC=high-
performance liquid chromatography.
Table III: Major raw material cost comparison of polyvinyl sulfonic acid (PVS)-based precipitation and zinc phenol-
mediated with ion-exchange chromatography to process 10 grams of insulin precursor.
gies measured by HPLC were comparable
(see Table II). Considering the purity of the
CFS under study, all the strategies gave a
significant increase in overall purity. This
could be connected to the increase in spe-
cific protein content as well after employ-
ing various strategies. Enrichment of the
specific protein content was accomplished
by precipitation strategies, although chro-
matography is a superior technique.
Removal of pigments prior to subse-
quent purification steps helps to improve
purification efforts. Whether these pig-
ments are associated with the insulin
precursor is still in question, but there
are instances where these pigments have
affected the chromatographic purification
of proteins (10). In a separate unrelated
study, these pigments—which are under-
stood to be alcohol-oxidase crystalloids—
were shown to interact with hydrophobic
proteins (such as human growth hormone),
resulting in altered charge and polarity
characteristics of the molecule (11). These
pigments considerably affect overall pro-
cess economics, leading to a lower bind-
ing capacity of target proteins, reduced
lifespan of the chromatographic media,
reduced yields, and lower product purity.
Cost considerations of precipitation
Use of a precipitation strategy is econom-
ical compared with the use of an ion-
exchange chromatography method. The
costs of the major raw materials used to
process 10 g of an insulin precursor using
PVS-mediated precipitation, zinc phenol-
mediated precipitation, and ion-exchange
chromatography are compared in Table III.
Even though ion-exchange resins can be
reused for 80–100 cycles, the cost of pro-
cessing by ion-exchange chromatography
Peer-reviewed
ES722616_BP0116_034.pgs 01.15.2016 02:09 ADV blackyellowmagentacyan
January 2016 www.biopharminternational.com BioPharm International 35
is 3.5 times higher than it is with the PVS-
based precipitation process and 10 times
higher than it is in zinc phenol-mediated
precipitation. Hence, precipitation pro-
vides a significant cost advantage in terms
of raw materials.
ConCLusion Precipitation offers several advantages
over chromatography. Precipitat ion is
cheaper, facilitates high-throughput pro-
cessing, and leads to higher concentration
of proteins. Chromatographic processes
are limited by the binding capacity of
the chromatographic media, a higher
cost of goods, and a larger volume of elu-
tion pools. In the present context, con-
sidering the example of enzyme reaction
after recovery of an insulin precursor
through various strategies, the authors
conclude that use of a precipitation step
offers the flexibility of working at higher
concentrations and at the buffer com-
positions suited for an enzyme reaction
that is associated with higher yield and
purity. Chromatography limits the choice
of eluents, which may be composed of
chemicals that could affect subsequent
processing steps.
The current study did not evaluate a
hybrid approach, which would include the
use of chromatography and precipitation
for purification. A hybrid approach that
employed a precipitation step, however,
may be a desirable approach in the future
for successive chromatography steps.
P ur i f icat ion of biolog ics t y pica l ly
involves several chromatography steps.
Thus, precipitation strategies discussed
here reduce the number of chromatog-
raphy steps required in a purif ication
process by serving to replace capture chro-
matography. A hybrid strategy could also
offer significant cost advantages by reduc-
ing the number of chromatographic steps
involved in the purification process.
In terms of infrastructure requirement,
precipitation involves the use of stirred
tanks for mixing and the solid-liquid sepa-
ration by either using centrifugation or fil-
tration. In contrast, the chromatographic
processes require a complex infrastructure
of chromatographic systems integrated to
buffer tanks, which significantly limits
the throughput of the process.
reCommendationsResults obtained in the current study are
promising in terms of cost reduction of
capture step of purification process with-
out compromising on the quality and func-
tionality of the product. Purification by
precipitation offers the opportunity for cost
reduction in capture steps without compro-
mising the quality and functionality of the
product. Therefore, precipitation is a promis-
ing method that could serve as an alternative
to expensive chromatography techniques.
referenCes 1. R.L. Fahrner et al., Biotech. Gen. Eng.
Rev. 18 (1), pp. 301–327 (2001).
2. M.Q. Niederauer and C.E. Glatz, “Selective
Precipitation,” in Bioseparation Advances
in Biochemical Engineering/Biotechnology,
G.T. Tsao, Ed. (Springer-Verlag, Berlin,
1st ed., 1992), 47, pp. 159–188.
3. T. Adilakshami and R.O. Laine, J. Biol.
Chem. 277, pp. 4147–4151 (2002).
4. P.K. Smith et al., Anal. Biochem. 150,
pp. 76–85 (1985).
5. I.M. Szalo et al., Clin. Diagn. Lab.
Immunol. 11 (3), pp. 532–537 (2004).
6. M. Braia et al., J. Chrom. 873
(2), pp. 139–143 (2008).
7. P. McDonald et al., Biotech. Bioeng.
102, pp. 1141–1151 (2009).
8. M. Sahyun, J. Biol. Chem. 138,
pp. 487–490 (1941).
9. G.D. Smith et al., Proc. Natl. Acad. Sci.
USA 81, pp. 7093–7097 (1984).
10. S.A. Minyasab et al., “A method of purifying
human growth hormone and purified growth
hormone thereof,” US Patent Application
WO2010134084 A1, Nov. 2010.
11. L.M. Damasceno et al., Protein Expr.
Purif. 37 (1), pp. 18–26 (2004). ◆
Peer-reviewed
ES722497_BP0116_035.pgs 01.15.2016 01:08 ADV blackyellowmagentacyan
36 BioPharm International www.biopharminternational.com January 2016
M_a_y_
a/E
+/G
ett
y Im
ag
es
As traffic in counterfeit and adul-
terated pharmaceuticals grows
(see Sidebar), the idea of a fully
traceable pharmaceutical supply
chain is taking shape. The concept has
changed drastically since the past decade,
when the first radio-frequency identifica-
tion (RFID)-based serialization systems
were piloted, and Florida set the first pedi-
gree requirements.
Today, 2-D matrix barcoding is the
technology of choice, and 40 countries
around the world have set requirements
for lot- and unit sales-level serialization
and traceability, each of which offers
specific challenges (Figure 1).
In the United States, the Drug
Supply Chain Security Act set a dead-
line of January 2015 for lot-level
traceability and verification. Unit seri-
alization requirements will take effect
in November 2017, and 2023 will be the
deadline for having serialization-based
track-and-trace systems in place. In the
European Union, the Falsified Medicines
Act has set a deadline of the end of 2018
for unit serialization and end-to-end ver-
ification and authentication.
Pharmaceutical serialization, aggre-
gation, and traceability pose huge
technical challenges, requiring the
easy transfer of, and access to, massive
amounts of data. These data will need
to flow from manufacturers—some of
whom have merged with or acquired
other companies—but also from their
contract manufacturing and packaging
partners. Data will also have to connect
to wholesale distributors, dispensers,
pharmacies, and clinics.
Serialization: Getting Past the Quick Fix
Agnes Shanley
Traceability and transparency
will remain elusive if
manufacturers continue to approach
serialization projects on a case-by-case basis.
Serialization
ES722125_BP0116_036.pgs 01.14.2016 17:40 ADV blackyellowmagentacyan
January 2016 www.biopharminternational.com BioPharm International 37
Over the past few years, the
pharmaceutical industry has made
progress in developing serializa-
tion strategies and investing in
the information technology (IT)
required. In UPS’ 2015 Pain in
the Chain Survey (1), 67% of 421
respondents viewed their invest-
ment in serialization IT as success-
ful in improving product security
(Figure 2).
However, challenges remain.
Even at the manufacturing level,
where IT connections will be
required between manufactur-
ers and contract manufacturing
and packaging organizations,
companies are at different levels
of achievement. Generally, Big
Pharma companies were ready for
serialization before the deadlines,
notes Darryl Brown, vice-president
of global strategic marketing at
Systech International. For smaller
firms and contract manufactur-
ing organizations (CMOs), readi-
ness varies. “Some companies have
robust programs in place, but oth-
ers have been working tactically, to
meet the bar set in different parts
of the world,” says Shabbir Dahod,
CEO of TraceLink, which offers a
cloud-based IT solution that won
a CPhI Award in 2015 for innova-
tion in supply chain and logistics
management.
“With less than two years to
meet the US deadline, both manu-
facturers and CMOs realize that
they have to move fast,” says
Steve Peterson, project manager at
CRB Consulting in St. Louis, Mo.
However, he says, over the past six
months, lead time for line-level
serialization equipment has gone
from 12–14 weeks to 10–16 weeks.
IF CMOS haven’t Started yet, they May already be tOO lateGenerally, it can take the smallest
pharmaceutical CMOs from one
to two years to develop a strategy
and foundation for serialization,
medium-sized CMOs from two
to three years, and large CMOs
more than three years, Vivian
McCain, vice-president of third-
party operations, Americas, at
Teva Pharmaceuticals, and pro-
gram owner, CMO Serialization
Program, told attendees at the
First Serialization Roundtable for
CMOs, held in Philadelphia, PA
on October 8, 2015 (2). For mid-
to-large CMOs that haven’t started
laying the groundwork, it may
already be too late, McCain said.
There are many complex ques-
tions to decide, he explained,
such as who should “own” label-
ing artwork issues or IT connec-
tivity projects, or how quality
assurance and exception manage-
ment should be handled.
Teva Pharmaceuticals began
developing its internal serialization
strategy four years ago, focusing on
its global network of 20 sites. This
year, the company began expand-
ing the program, funding a CMO
Serialization
A new standard aims to improve connectivity
The global market in counterfeit pharmaceuticals has been seeing double-
digit growth, according to Charlie Gifford, executive director of the Open
Serialization Communications Standard (Open-SCS) working group. Open-
SCS, launched in 2015 by the Open Platform Communication Foundation,
involves the participation of pharma companies that include Abbott, Teva
Pharmaceutical Industries Ltd., and Mylan Pharmaceutical, and vendors
including SAP AG, OptelVision Inc., Systech International, Werum IT Solutions
GmbH, and Antares Vision Srl. Its mission is a standard that would help
promote open communications and nonproprietary data interfaces.
The explosive growth in demand for generic drugs, and the fact that contract
manufacturing organizations now manufacture these drugs, has resulted in
huge opportunities for counterfeiters, Gifford says. “This issue of counterfeiting
is really a World Health Organization-type problem, and we’re seeing nothing
short of a pandemic,” he notes, “but it is only being addressed by countries and
individual manufacturers.”
The problem is that regulators in different countries, typically, don’t know
anything about manufacturing or supply-chain systems, or aggregating
information, Gifford says. In addition, he says, most regulatory agencies
are not actively participating in standards efforts, often out of concern for
the appearance of potential conflicts of interest. As a result, serialization
requirements can be difficult to meet, given typical plant floor realities.
Global Standards One (GS-1) has standardized communications protocols,
and current serialization data standards include its Electronic Product Code
Information Services (EPCIS) with pieces of various manufacturing process
control standards such as ISA 95, S88, and Make 2Pack, says Gifford.
Open-SCS would develop a comprehensive standard that would help
streamline and simplify data integration, says Gifford, reducing costs. The
group studied offerings from 10 leading packaging serialization equipment
vendors and found that architectures and data syntax varied greatly, potentially
increaasing the cost of serialization and traceability. Gifford notes two
challenges: vendor’s preference for proprietary systems and companies’
reluctance to subsidize employees’ participation in standards development.
Nevertheless, the group plans to launch new phases of the project this year.
For more information, visit www.opcfoundation.org/open-scs-working-group/
ES722129_BP0116_037.pgs 01.14.2016 17:40 ADV blackyellowmagentacyan
38 BioPharm International www.biopharminternational.com January 2016
support program to help oversee
partner serialization efforts in its
external network of more than 700
CMOs. The program focuses on
issues that include IT connectiv-
ity, quality assurance, tools, and
applications. A CMO serialization-
management dashboard allows the
company to track each CMO’s seri-
alization readiness and progress
and assess the risk of noncompli-
ance. Teva is developing risk pro-
files for each partner to assess the
potential business impact of serial-
ization noncompliance.
To ensure flexibility, it’s impor-
tant to have an IT solution that’s
loosely coupled to the enterprise
system, says TraceLink’s Dahod.
However, Dahod sees non-standard
data exchange and integration
approaches as one of the big-
gest potential obstacles that the
industry faces in meeting upcom-
ing serialization and traceability
regulations.
Global Standards One (GS-1),
the nonprofit organization that
maintains standards for supply
chains across different industries,
and its Electronic Product Code
Information Services (EPCIS) stan-
dard have played a key role in help-
ing drive a common data exchange
framework between packaging
sites, enterprise serialization sys-
tems, and network trade partners,
he says.
He notes the potential of newer
standards as well. “The Open
Ser ia l izat ion Communicat ion
Standard (Open-SCS) Working
Group could extend these data
exchange harmonization efforts
even deeper in the serialization
process,” he says. “Particularly
between the packaging line and
packaging site level, where the
sheer diversity of line-management
systems and equipment manu-
facturers have made readiness for
serialization a great challenge.”
Jean-Pierre Allard, global serializa-
tion program manager for Optel
Vision, has called it “the standard
that CMOs need” (3).
Foundations for the Open-
SCS Working Group were laid
in February 2015, and executive
director Charlie Gifford sees a real
need for this work. “With trace-
ability, ultimately, we need to val-
idate the bottle that reaches the
patient,” Gifford says, “and the
current process—which includes
serializing at the bottle level,
aggregating and serializing in all
forms, and then de-aggregating to
the pharmacy level—is still vulner-
able to criminal interference.”
The idea behind Open-SCS is to
focus on the three or four data and
communication layers (line and
equipment, distribution centers
and warehouses, plant and ware-
house operations management)
between the plant and the corpo-
rate enterprise resource planning
(ERP) system. For serialization,
such a standard would help phar-
maceutical companies and CMOs
improve the aggregation of seri-
alization master data between
plants, and optimize plant-floor
strategies depending on whether
their manufacturing departments
assign a serialization number, or
whether a government regulatory
agency is providing numbers to
apply to product.
Given the number and variety
of regulations, and differences
between different vendors’ prod-
ucts, the challenges are great. “If
you walk into any CMO’s packag-
ing line today, every single piece of
equipment there is from a different
vendor. Manufacturing Execution
System (MES) and ERP vendors sell
proprietary systems. By the time
you get up to the top level to cre-
ate a report, it’s a mapping night-
mare,” Gifford says. As he explains,
line-equipment vendors tend to
promote proprietary solutions; at
Serialization
Figure 1. Different global requirements for serialization challenge pharma companies today. Respondents to UPS’ 2015 Pain
in the Chain survey (1) singled out the most challenging national regulations, as shown here (1).
International regulations that cause the most pain (global)
European Union GoodDistribution Practice
39%
16%
19%
7%
European Union Medical DevicesDirectives and Requirements
Brazil Serialization China Medical DeviceGood Supply Practice
Korea Medical Device andin-vitro Diagnostics Practice
U.S. Drug Supply ChainSecurity Act
Q. When thinking about regulatory compliance issues, what are the international regulations that cause you most pain?
39%
16%
24%
16%
All
fig
ure
s co
urt
esy
of U
PS
ES722127_BP0116_038.pgs 01.14.2016 17:40 ADV blackyellowmagentacyan
ON-DEMAND WEBCAST Originally aired December 8, 2015
Register for free at
www.biopharminternational.com/bp/UNit
EVENT OVERVIEW:
Formulation scientists are expected to do a lot more with less—
more constructs, higher concentrations, less volume. Novel
antibody structures and ADCs present a diferent challenge for
development. Platform formulations that were used for monoclonal
antibodies don’t always work and degradation can show up from a
number of biophysical and chemical pathways. Developing assays
that can characterize and predict the manufacturability, developa-
bility, and long-term stability of these complex biologics isn’t easy.
Obtaining information about their stability, whether it be thermally
driven structural changes, aggregation kinetics, viscosity, and more
can help make sense of what’s going to happen to the protein. In
this webinar, case studies from literature will be presented and a
powerful tool—the Unit—which can perform all of these measure-
ments with a lot less sample, will be presented and discussed.
Key Learning Objectives
n Learn the benefts of measuring Tm
and Tagg
simultaneously
n Learn how small sample sizes enable faster screening of
biologics
n Learn why full spectrum fuorescence and static light scattering
are critical for understanding protein unfolding and aggregation
PRESENTERS:
DAN LUND, PhD
Field Applications Scientist
Unchained Labs
MODERATOR:
SARA BARSCHDORF
Multimedia EditorBioPharm International
Who Should Attend
n Pre-formulation and formulation
scientists
n Biologic characterization scientists
n Structural biologists
Presented by
Way more biologic stability, a lot less protein
For questions contact Sara Barschdorf at sbarschdorf@advanstar.com
Hosted by
ES720981_BP0116_039_FP.pgs 01.13.2016 03:03 ADV blackyellowmagentacyan
40 BioPharm International www.biopharminternational.com January 2016
Serialization
the MES level, they are looking at
the batch record, and, at the ERP
level, the batch record is different
from the master batch record. Also,
not all countries are standardized
on GS-1, so five different countries
may have five different require-
ments for reporting out of the
plant, Gifford says.
EPCIS addresses some, but not
all of these issues, so many proj-
ects become one-offs using custom
solutions. “This type of work does
not scale. What works in one plant
may not be able to accommodate
change, and work in five plants
around the world five years from
now,” says Gifford.
Another major problem with
one-offs is lack of transparency,
says Systech’s Brown. “Temporary
solutions will get you through the
checkbox for serialization, but if
there’s a problem down the line,
there will be no way to diagnose
it and retain the data, so it will
then be very difficult in situations
where, say, you need to recall mil-
lions of dollars worth of product.”
Supply Chain Wizard’s manag-
ing partner Burak Tiftikci sug-
gests that manufacturers “design
for tomorrow.” As he noted dur-
ing the October CMO conference,
“You can always dumb down the
richness of data to meet a specific
requirement” (4). He noted, how-
ever, that it can be “exponentially
more challenging” to work the
other way around.
A prerequisite to successful
serialization is having a strong
IT system that can handle serial
number mapping. Peterson adds,
“you need to be able to handle
high volumes of data, and to have
a disaster recovery and backup sit-
uation in place.”
A l l of the major ERP ven-
dors have introduced platforms
designed to facilitate serializa-
tion, although consultants note
that some may still be unproven.
In September 2015, SAP intro-
duced Adva nced Trac k a nd
Trace for Pharma (ATTP), which
replaces its prev ious Auto-ID
Infrastructure (AII) and object
event repository (OER) programs
as a way to generate serial num-
bers, send codes to packaging
lines, and then track them. That
same month, TraceLink intro-
duced an SAP Migration Kit to
allow users of SAP serialization
products to take that functional-
ity into the cloud.
POInt-OF-uSe COnneCtIOnS ChallenGe PharMaWell beyond the manufacturing
and CMO portion of the supply
chain, a major challenge is work-
ing with pharmacies, clinics, and
hospitals at the point of service,
Gifford says. “Today, between
20–50% of the cost of serialization
is due to paper pushing,” he says.
“We’re going into a rudimentary
IT world and telling them to put in
yet more systems.”
Concerns that can play out at
the point of service were recently
seen in Brazil, whose regula-
tory agency, ANVISA, postponed
a December 2015 deadline for
its traceability pilot phase. The
requirements cal led for drug
license holders to report to the
government, and pharmacies
feared that access to patient data
might give suppliers too much
information about stock levels and
trends, and allow them to control
pricing and supply (5). The gov-
ernment changed its requirements
so this information could not be
shared with distributors.
Even so, the challenges of data
integration at that level can be for-
midable, and standards can help.
This was shown in Roche’s recent
pilot project, which focused on
special products and medicines
for cancer, and involved collabo-
ration with the systems integra-
tor SPI and TraceLink. The project,
which won the 2015 GS-1 Brazil
Automation Award, used EPCIS to
Figure 2. Most life-sciences companies see investments in serialization as
enhancing product security, according to UPS’ 2015 Pain in the Chain survey (1).
Product Security
IT investment: bar coding,
serialization, etc.
Cooperation with law
enforcement
Visible authentication: visible
holographs, security inks, etc.
67%
41%
38%
Q.You indicated that you've been successful at addressing product security. What strategies
have made you successful?
Contin. on page 46
ES722126_BP0116_040.pgs 01.14.2016 17:40 ADV blackyellowmagentacyan
January 2016 www.biopharminternational.com BioPharm International 41
Mo
nty
Rakuse
n/C
ultu
ra/G
ett
y Im
ag
es
An industry workgroup com-
prised of toxicologists and
manufacturing equipment
c lea n ing subjec t mat te r
experts was formed to examine the
European Medicines Agency (EMA)
shared facilities guideline, specifically
the provision for alternative approaches
to establishing limits and how to prac-
tically implement any proposed alter-
natives (1). This paper describes the
outcomes of these evaluations, includ-
ing alternatives for estimating health-
based exposure limits using available
assessments and a strategy to priori-
tize existing commercial products for
health-based exposure limit evaluation.
It provides a screening tool to identify
products that require calculation of a
health-based cleaning limit, and those
where cleaning to existing cleaning limits
(e.g., based on 1/1000th the minimum
therapeutic dose) may continue. The pre-
sumption is that all newly introduced
products will have formally documented
health-based exposure limits. EMA was
consulted during development of this
paper, which is intended to support imple-
mentation of the European Union GMP
Chapter 3 and 5 revisions for prevention
of cross-contamination (2).
EMA published a guideline on setting
health-based exposure limits for use in
risk identification in the manufacture of
different medicinal products in shared
facilities in November 2014 (1). The
European Commission (EC) also revised
related General GMP Chapters 3 and 5
in January 2015 by updating sections
on prevention of cross-contamination;
EMA Guideline on Setting Health-Based Exposure Limits
Andrew Teasdale, Bruce D. Naumann, Gretchen
Allison, Wendy Luo, Courtney M. Callis, Bryan
K. Shipp, Laura Rutter, and Christopher Seaman
The results of an industry
workgroup’s examination of
EMA’s guide on shared
facilities are presented.
Andrew Teasdale is a principal scientist
at AstraZeneca, andrew.teasdale@
astrazeneca.com; Bruce D. Naumann
is executive director, Toxicology, Merck &
Co.; Gretchen Allison is senior director,
Global Quality Operations, Pfizer Inc.;
Wendy Luo is associate director, Drug
Safety Evaluation, Bristol-Myers Squibb;
Courtney M. Callis is a research scientist,
Health/Safety/Environmental, Lilly
Research Laboratories; Bryan K. Shipp
is director, Risk Management, Pfizer
Inc.; Laura Rutter is SM manager,
Analytical Services, GlaxoSmithKline; and
Christopher Seaman is senior manager,
Occupational Toxicology, GlaxoSmithKline.
Health-Based Exposure Limits
ES722130_BP0116_041.pgs 01.14.2016 17:40 ADV blackyellowmagentacyan
42 BioPharm International www.biopharminternational.com January 2016
these revisions became effective
on March 1, 2015 (2). The EMA
guide effective date was June
1, 2015 for new products and
Dec. 1, 2015 for existing prod-
ucts. These timelines represent
a significant challenge for the
industry.
The EMA guide implementation
timelines are aggressive consider-
ing the current number of exist-
ing products. The implications of
the EMA guide are that current
cleaning limits would need re-
evaluation in relation to newly
derived health-based cleaning
limits. If revisions are necessary,
then cleaning validation activi-
ties may need to be re-started for
impacted manufacturing equip-
ment. Cleaning sampling and ana-
lytical test method detection levels
may need to be re-evaluated, and
potentially lower detection limits
established/validated. Because re-
evaluation may consume signifi-
cant resources, efforts need to be
prioritized according to impact on
patient safety.
The EMA guide uses permitted
daily exposure (PDE) values as a
basis for establishing appropriate
limits but states that PDE values
are considered synonymous with
the acceptable daily exposure
(ADE) values. Both represent a dose
that is not expected to cause an
adverse effect in an individual,
even with a lifetime of exposure.
The EMA guide also states that
“the use of other approaches to
determine health-based exposure
limits could be considered accept-
able if adequately and scientifically
justified.”
This paper describes the poten-
tial to adopt scientifically jus-
t i f ied and hea lth-protec t ive
options for estimating ADE val-
ues from available assessments. It
also provides examples on how to
prioritize existing products and
evaluate currently used clean-
ing limits to identify those prod-
ucts that require calculation of
an ADE- or PDE-based cleaning
limit, and those where cleaning
to existing cleaning limits (e.g.,
1/1000th the minimum thera-
peutic dose) already provides suf-
ficient patient protection. The
presumption is that all newly
introduced products will have
documented health-based expo-
sure limits.
ALTERnATIvE APPROACHES fOR SETTInG HEALTH-BASED ExPOSuRE LIMITS: ESTIMATIOn Of ADE vALuES fOR SMALL MOLECuLESThe goal of applying alternative
approaches should be to dem-
onstrate whether or not current
cleaning practices provide a suf-
ficient margin of safety for patient
health. The approach proposed in
this document provides a ratio-
nale for estimation of ADE values
using existing potency and toxi-
cological evaluations performed
for worker safety purposes, fol-
lowed by an evaluation of facil-
ity attributes and product mix.
For example, many pharmaceuti-
cal organizations establish occu-
pational exposure limits (OELs)
for their compounds or use a
control banding approach (occu-
pational exposure bands [OEBs])
to categor i ze compounds of
increasing severity based on their
inherent pharmacological and tox-
icological properties and/or limited
dataset. ADE values can be esti-
mated using OELs or OEBs, and
if an adequate margin of safety is
identified between cleaning limits
based on the estimated ADE value
and maximum possible carryover
based on existing cleaning limits,
a formal comprehensive ADE value
monograph may not be required.
Adequate documentation could
initially include a brief summary
or spreadsheet reflecting the sci-
entific rationale, and formal ADE
values would only be needed for a
subset of compounds where facil-
ity attributes and product mix do
not afford an adequate margin of
safety. The evaluation approach
should be documented for each site
and included in plans available for
review by regulatory inspectors.
Identifying the Most Hazardous Drugs
Prior to issuance of the EMA
guideline on setting health-based
limits, categories of drugs were
identified that have the greatest
concern (e.g., “certain hormones,
certain cytotoxics,” etc.); however,
specific criteria were never pro-
vided. The International Society
for Pharmaceutical Engineering
(ISPE) Risk-MaPP baseline guide
outlines example characteristics of
compounds of traditional concern
that could benefit from further
review (3):
• Genotoxic (specificallymuta-
genic) compounds that are
known to be, or highly likely to
be, carcinogenic to humans.
• Compounds that can produce
reproductive and/or develop-
mental effects at low dosages.*
• Compounds that can produce
serious target organ toxicity,
anaphylaxis, or other signifi-
cant adverse effects at low dos-
ages.*
Health-Based Exposure Limits
If effective protein
degradation and
inactivation could not
be demonstrated in
the cleaning studies,
cleaning limit should
be based on PDE.
ES722136_BP0116_042.pgs 01.14.2016 17:41 ADV blackyellowmagentacyan
January 2016 www.biopharminternational.com BioPharm International 43
* e.g., clinical doses <1–10 mg/
day or dosages in animal studies
<0.1–1 mg/kg/day
The threshold of toxicological
concern (TTC) concept may also
be useful to estimate ADE values
and applies to products contain-
ing mutagenic active ingredients
(ADE=1 µg/day), those that are
potent or toxic (ADE=10 µg/
day), or those that have no
prior evidence of these prop-
er t ies (ADE =100 µg/day) (4).
In pr inc iple, each of these
approaches can be used to identify
hazardous drugs for risk assess-
ments and, if scientifically justi-
fied, can be used for the derivation
of health-based cleaning limits.
Identification of a compound as a
hazardous drug should not auto-
matically lead to strict control
solutions, because it addresses only
hazard. A risk assessment should
be completed before conclusions
can be made about risk and the
need to modify controls.
Alternative Approach 1: If I Have an OEL
ADE or PDE value derivation and
OEL derivation share the same sci-
entific basis (e.g., the same method
to define the point-of-departure
[PoD]), with some differences in
the respective adjustment factors
related to the different exposure
scenarios or regulatory context.
By comparing the respective stan-
dard adjustment factors, it is pos-
sible to define the systematic
difference between the two calcu-
lations. If this systematic differ-
ence is adequately evaluated and
the OEL derivation is scientifically
justified, estimation of the ADE
from the available OEL could meet
expectations to derive health-
based cleaning limits, at least on
an interim basis to support priori-
tization and assessment of existing
cleaning limits.
The EMA guide acknowledges
that “good quality human clini-
cal trial data is highly relevant”
to the assessment for ensur-
ing patient safety. Following the
publicat ion by Fourman and
Mullen in 1993, many firms have
used a minimum therapeutic
dose approach (e.g., 1/1000th of
the minimum therapeutic dose)
to calculate cleaning limits (5).
The existing OEL monograph, if
available, can be used as a screen-
ing tool to determine if this
approach is health-protective. OEL
monographs typically include a
potency and toxicological evalu-
ation of relevant nonclinical and
clinical data. In the workplace,
compound-related effects (includ-
ing intended pharmacology and
unwanted toxicity) are consid-
ered adverse. Many products have
a favourable therapeutic index,
which means that the pharmaco-
logical activity is the most sensi-
tive effect. However, EMA doesn’t
address derivation of an ADE from
clinical data. Because the OEL is
frequently derived from the mini-
mum therapeutic dose, it is rea-
sonable to deduce that the ADE
value based on 1/1000th the
minimum therapeutic dose is suf-
ficiently conservative for most
compounds. ISPE Risk-MaPP out-
lines science-based adjustment
factors that support this assump-
tion. For example, a lower com-
posite adjustment factor (i.e., 10
to account for intra-species differ-
ences and 3 to extrapolate to a no-
effect level) can be scientifically
justified if using the minimum
therapeutic dose as the PoD for a
product with a reasonably robust
dataset (e.g., late- to commercial-
stage compound). Therefore, when
the human dose information is
used as the PoD for OEL deriva-
tion, the minimum therapeutic
dose cleaning limit approach may
be considered health-protective,
and no additional value would be
derived by proceeding to a formal
ADE derivation.
The exception would be com-
pounds (e.g., classical oncology
compounds) with an unfavour-
able therapeutic index that may
cause unwanted or even serious
adverse health effects at expo-
sures near or below the indicated
minimum therapeutic dose. A
mutagenic carcinogen adminis-
tered at a dose of 750 mg/day, for
example, would result in a limit
of 750 µg/day using the 1/1000th
approach, which is almost three
orders of magnitude higher than
the TTC level of 1 µg/day using
Dolan et al. (or 1.5 µg/day under
ICH M7) (4, 6). In these cases, a
review by a toxicologist is rec-
ommended. When the OEL is
calculated using a nonclinical
PoD (e.g., repeat-dose toxicity or
reproductive toxicity in animals),
or involves route-to-route extrap-
olation, then further review by a
toxicologist is also recommended
to determine an appropriate ADE
value.
Example of an Estimated ADE Value
Calculation Using Data from the OEL
Monograph for Compound A.
The following example illustrates
the calculation of an OEL for
Compound A using the minimum
therapeutic dose of 2.5 mg/day as
the PoD. All relevant nonclinical,
clinical, and post-marketing data
were evaluated. The details are
provided to show the similarity to
ADE derivation while highlighting
Health-Based Exposure Limits
The threshold of
toxicological concern
(TTC) concept may
also be useful to
estimate ADE values.
ES722133_BP0116_043.pgs 01.14.2016 17:40 ADV blackyellowmagentacyan
44 BioPharm International www.biopharminternational.com January 2016
Health-Based Exposure Limits
that assessment factors used might
differ.
The OEL incorporates a compos-
ite adjustment factor (AF) of 30,
derived as follows (see Equation 1—
UF is uncertainty factor; LOAEL
is lowest observed adverse effect
level; NOAEL is no observed
adverse effect level).
For the calculation of the OEL,
a bioavailability correction fac-
tor of 0.3 is included in the
numerator to account for oral
bioavailability in humans (30%).
Therefore, an ADE est imated
from the OEL is conservative (i.e.,
approximately three-fold lower
than needed) if the oral route
of exposure is applicable for the
cross-contamination scenario (see
Equation 2—TWA is time weighted
average).
By comparison, 1/1000th the
minimum therapeutic dose is
2.5 µg/day (2.5 mg/day/1000),
which is 10 times lower than the
estimated ADE value. Therefore,
the existing cleaning limit is
acceptable, and development of a
formal ADE value is not a high pri-
ority at this time.
Alternative approach 2: What if I don’t
have an OEL but I do have an OEB?
Many companies assign new com-
pounds to an OEB, or equivalent
designation by airborne concentra-
tion ranges, to provide guidance on
safe handling before sufficient data
are available to establish a numerical
OEL. The OEB is assigned based on
the compounds’ inherent pharma-
cological and toxicological charac-
teristics, including the intended use,
mechanism-of-action, dose-response,
and safety pharmacology studies.
One option is to use the OEBs to esti-
mate ADEs for the most hazardous
subset of compounds (i.e., those with
the most restrictive OEBs).
Example OEB assignment crite-
ria are provided in Table I corre-
sponding to ADE values of 10 and
1 µg/day corresponding to respec-
tive OEBs of ≥ 1 <10 µg/m3 and
<1 µg/m3 because it is assumed
that 10 m3 are breathed during the
workday. These are the same values
that would be derived using the
TTC concept outlined in Dolan et
al. (4). Application of these default
values presumes that sufficient
compound-specific data are not
available to estimate an ADE (5).
The examples focus on “potent” or
“high containment” compounds,
but in practice, the principle
would still hold across less restric-
tive bands. As with application
of the OEL, a bioavailability cor-
rection could be considered if the
compound is anticipated to have
extremely low bioavailablity via a
relevant route.
• IntraspeciesDifferences(UFH) 10 DefaultAdjustment
• InterspeciesDifferences(UFA) 1 Human
• LOAELtoNOAEL(UFL) 3 Minimaltherapeutictono
therapeuticeffect
• Sub-chronictoChronic(UFs) 1 Experiencewithlong-termuse
• DatabaseCompleteness(UFD) 1 Completedatabase
UFC=(UFH)(UFA)(UFs)(UFL)(UFD)=(10)(1)(1)(3)(1)=30
Assumptions:
LOAEL=2.5mg/day
Bio-availabilityCorrectionFactor=0.3(30%oralbioavailabilityinhumans)
CompositeUncertaintyFactor(UFC)=30
ModifyingFactor(MF)=1(nomodifyingfactor)
V=Volumeofairbreathedin8hours(10m3) [Eq.1]
Table I. Example occupational exposure band (OEB) assignment criteria and corresponding estimated acceptable daily
exposures (ADEs).
Control band associated with airborne concentration range
≥1<10 µg/m3 <1 µg/m3
Estimated ADE (lower end of band x 10 m3)
10 µg/day <1 µg/day
Compound characteristics
High pharmacological potency (0.1–1 mg/day). Effects in humans may be serious and/or slowly reversible, but are not life-threatening and easily managed medically.
Very high pharmacological potency (< 0.1 mg/day). Acute exposures at very low doses may be incapacitating, life-threatening, and require medical intervention. Immediate and heroic medical intervention may be needed. Chronic effects may be irreversible, disabling, or life-shortening.
OEL=( 2 . 5 m g / d a y ) ( 0 . 3 )
(30)(1)x10m3/day
OEL=2.5µg/m3(8-hrTWA)
EstimatedADEvalue(dailysystemic
dose)=OELxV=2.5µg/m3x10m3/
day=25µg/day [Eq.2]
ES722612_BP0116_044.pgs 01.15.2016 02:05 ADV blackyellowmagentacyan
January 2016 www.biopharminternational.com BioPharm International 45
How can consistent screening to
evaluate the effectiveness of existing
cleaning limits using the OEL/OEB be
implemented?
Figure 1 provides an example deci-
sion process for systematic deter-
mination of whether traditional
cleaning limit calculations are
adequate using the OEL and OEB.
The decision tree shows that use of
OEL and application of 1/1000th
the minimum therapeutic dose
approach could be acceptable
under the following conditions:
• For existing commercial prod-
ucts where ex ist ing c lean-
ing limits are based on the
1/1000th approach
• IfanOELmonographincluding
toxicological evaluation of rel-
evant nonclinical and clinical
data is available
• Ifthepharmacologicalactivityis
the most sensitive (critical) effect.
LARGE MOLECuLE COnSIDERATIOnSThe EMA guide states that “deter-
mination of health-based expo-
sure limits using PDE limits of the
active and intact product may not
Health-Based Exposure Limits
Figure 1. Example decision tree for evaluating effectiveness of existing cleaning limits using the occupational exposure
limits (OEL) or occupational exposure bands (OEB). PDE is permitted daily exposure. ADE is acceptable daily exposure. EHS
is environmental health and safety.
Start Cleaning ValidationScreen Tool
ADE or PDE ValueAvailable?
Yes
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Value Used inCurrent Cleaning
Limit?
Screen Complete. MaintainCurrent Cleaning Practice.Document Assessment and
Conclusions.
Calculate New Health -based Cleaning LimitUsing the ADE or PDE
Value
CurrentCleaning Limit
Based on 1/1000th
Minimum TherapeuticDose?
New Health - basedCleaning Limit > CurrentCleaning Limit or Within
One Order -of-Magnitude?
New Health - basedCleaning Limit > CurrentCleaning Limit or Within
One Order -of-Magnitude?
Screen Complete. Maintain CurrentCleaning Practice. Document
Alternative-based Approach Assessmentand Conclutions. No ADE Value
Required at this Time.
Screen Complete. Maintain CurrentCleaning Practice. Document
Alternative-based Approach Assessmentand Conclutions.
Screen Complete. Make AnyNecessary Change to the Current
Cleaning Limits. DocumentAssessment and Conclusions
Toxicologist to Prioritizeand Develop ADE Value
For Review and Approval
Notify ToxiCologist toPrioritize Development of
ADE Value forReanalysis.
Communicate Potentialfor Change to Current
Cleaning LimitRejected if Not in Alignment With the MinimumTherapeutic Dose Used for the OEL / OEB
EHS OEL/OEBBased on Clinical
Data?
Minimum Therapeutic Dose
“Cytotoxics”“Certain Antibiotics”“Highly Sensitizing Products”“Certain Hormones”
Product Classof Concern?
Stop Screen and ConsultToxicologist
Calculate New Health -based Cleaning LimitUsing the “Estimated
ADE Value”
CanToxicologist
ProvideAppropriate
“Estimate ADEValue”?
Review RiskAssessment /
Segregation AssessmentNo
Fig
ure
s 1 is
co
urt
esy
of th
e a
uth
or.
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46 BioPharm International www.biopharminternational.com January 2016
Health-Based Exposure Limits
be required” for macromolecules
(proteins and peptides) (1). When
macromolecules are exposed to pH
extremes and/or heat, they rapidly
degrade and denature and are con-
sidered pharmacologically inactive.
Only breakdown products such as
smaller peptide fragments or amino-
acid derivatives formed due to pro-
tein hydrolysis would be present after
cleaning under conditions that are
shown to effectively degrade and
inactivate protein-based products.
In these scenarios, health-based
exposure limits of the active products
are no longer relevant. An experi-
mental approach and analytical
methods for assessing degradation
and inactivation of the API during
cleaning, and an approach for set-
ting safety-based acceptance limits
for inactivated product and process
residuals are described in separate
industry publications (7, 8). However,
if effective protein degradation and
inactivation could not be demon-
strated in the cleaning studies, clean-
ing limit should be based on PDE.
COnCLuSIOnThe proposed screening tool and
methods for estimating ADE values
are appropriate for implementation
of the EMA guide with the goal of
ensuring health-protective clean-
ing limits. It is valuable to note that
compound-specific hazard is not
the only concern as the hazard of
all compounds in the matrix must
be considered in order to calculate
the worst-case cleaning limit for all
Product A-to-Product B changeover
permutations. Hence, implemen-
tation of worst-case permutations
affords an additional level of safety
for many limits. Further assurance
is provided on completion of the
cleaning; when the equipment is
dry, a visual inspection is typically
performed for those surfaces that
can be visually inspected.
REfEREnCES 1. EMA, Guideline on Setting Health-
Based Exposure Limits for Use in Risk
Identification in the Manufacture of
Different Medicinal Products in
Shared Facilities (EMA, 20 Nov.
2014), www.ema.europa.eu/docs/
en_GB/document_library/Scientific_
guideline/2014/11/WC500177735.
pdf.
2. EU, EudraLex—Volume 4 Good
Manufacturing Practice (GMP)
Guidelines, Chapters 3 (Premises and
Equipment) and 5 (Production),
Revisions (January 2015), http://ec.
europa.eu/health/files/eudralex/
vol-4/chapter_5.pdf and http://ec.
europa.eu/health/files/eudralex/
vol-4/chapter_3.pdf.
3. ISPE, Volume 7: ISPE Baseline Guide:
Risk-Based Manufacture of
Pharmaceutical Products (Risk-MaPP)
(ISPE, September 2010).
4. D.G. Dolan et al. Regul. Toxicol.
Pharmacol. 43, 1–9 (2005).
5. G.L. Fourman and M.V. Mullen,
Pharm. Technol., April, 54–59 (1993).
6. ICH, M7 Assessment and Control of
DNA Reactive (Mutagenic) Impurities
in Pharmaceutical to Limit Potential
Carcinogenic Risk (ICH M7), Step 4
version (ICH, 23 June 2014, Adopted
by EMA September 2014), www.ich.
org/fileadmin/Public_Web_Site/
ICH_Products/Guidelines/
Multidisciplinary/M7/M7_Step_4.
pdf.
7. A. Mott et al., Journal of Validation
Technology 19 (4) (2013).
8. R. Sharnez et al., Journal of Validation
Technology 17 (4) (2012). ◆
Serialization—Contin. from page 40
share information among Roche,
its distributors, and clinics. The
work was accomplished within
three months. Integrating supply-
chain links was the biggest chal-
lenge, says Rafael Schirmer, SPI’s
director, who managed systems
integration for the project.
The pi lot used TraceLink’s
cloud-based system, so no instal-
lation or local software config-
uration was required, he says.
Connect ing with pharmacies
and distributors proved difficult
initially, Schirmer says, because
many of them did not want to
invest in the systems required.
Roche set up portals for them
to me e t r ep or t i ng r e qu i r e -
ments, and SPI configured web
boxes to enable them to access
TraceLink’s tool.
The project will soon move into
its next phase, but Brazil’s man-
date is clear, Schirmer says: All sup-
ply-chain members will be jointly
responsible for the traceability of
medicines.
Creating a supply chain that is
traceable, end to end, may seem
like a holy grail, but Open-SCS’
Gifford believes that standards will
make it easier. As he notes, they’ve
worked in other industries such as
semiconductors, where Sematech,
a consortium of the top 10 manu-
facturers, set a foundation in the
1990s. “Today, at AMD, Intel or
Samsung, at any plant anywhere
in the world, they’re all using the
same interfaces,” he says.
At this point, pharma is making
the connections required for trans-
parency and traceability. However,
as experts caution, only a strategic,
long-term approach will yield the
best results.
REfEREnCES 1. UPS, Eighth UPS Pain in the Chain
Survey Snapshot, www.ups.com/
media/en/UPS-PITC-Executive-
Summary-North-America.pdf
2. V. McCain, “Establishing CMO
Serialization Support Strategy,”
presentation at the First Serialization
Roundtable for CMOs (Philadelphia,
PA, Oct. 8, 2015).
3. J-P. Allard, “Serialization Solutions
Through Innovations,” presentation at
the First Serialization Roundtable for
CMOs (Philadelphia, PA, Oct. 8, 2015).
4. B. Tiftikci, “Serialization: Internal and
External IT Connectivity,” presentation
at the First Serialization Roundtable for
CMOs (Philadelphia, PA, Oct. 8, 2015).
5. P. Taylor, “Brazil’s Drug Traceability Pilot
Phase ‘Suspended,’” Oct. 7, 2015, www.
securingindustry.com/pharmaceuticals/
brazil-s-drug-traceability-pilot-phase-
suspended-/s40/a2548/#.Vo_
MCZMrI_U, accessed Jan. 8, 2015. ◆
ES722135_BP0116_046.pgs 01.14.2016 17:40 ADV blackyellowmagentacyan
January 2016 www.biopharminternational.com BioPharm International 47
Sve
ta D
em
ido
ff/G
ett
y Im
ag
es
Troubleshooting
There is a growing need to accelerate bio-
process development for mammalian cell
culture. Major pharmaceutical and bio-
tech firms are facing challenges to reduce pro-
cess development costs and cultivation times
(1). The conventional method for mammalian
cell-line development usually involves a series
of shake flasks for screening the cell-line prior
to large-scale cultivation. The shortcomings of
this method include long development times,
laborious operation, and limited experimental
throughput, which result in slow bioprocess
development of mammalian cell cultures.
Various scale-down miniature bioreactors
have been designed to speed up the biopro-
cess development of mammalian cell cultures.
Generally, it is accepted practice to perform the
small-scale experiment in a high throughput
and highly parallel manner. Current technol-
ogy endeavours to enable high-throughput pro-
cess development include the use of microtiter
plates, miniature stirred-tank bioreactors, and
microbioreactors (2, 3, 4).
Miniature stirred-tank bioreactors (MBRs)—
based on the conventional stirred-tank reactor
(STR)—enable a rapid and scalable experimental
process development. Experiments are usually
carried out in 4–16 parallel reactors running
simultaneously at scales of 10 mL to 500 mL. The
main advantages of these reactors are reduced
cultivation times and costs, and the ability to
conduct continuous monitoring and real-time
visualization of key process parameters in each
single bioreactor (3, 4). Moreover,
the capacity of miniature stirred
bioreactors for inline monitoring
and control of pH, dissolved oxygen
(DO), and temperature could make
these reactors an excellent alter-
native to shake-flask systems for
early-stage mammalian cell-culture
bioprocess development.
Scale translation of miniature bioreactors
to benchtop reactors remains a crucial issue
for mammalian cell-culture processes. Mixing
directly affects the heat transfer, gas dispersion,
and blending of different media components
in the reactor. Furthermore, poor mixing in
bioreactors can result in pH, nutrient, and tem-
perature gradients, as well as poor control of
operating parameters (5).
Mixing in mammalian cell-culture reactors is
achieved using either marine or pitched-blade
impellers to minimize shear damage. These
impeller designs will create low shear stress and
gently mix the culture (6). Detailed engineer-
ing characterization, such as mixing time, is
imperative to understand the performance of a
bioreactor. By characterizing the system during
the typical operating range, suitable parameters
for scale translation can be identified. In this
study, mixing time has been determined in a
prototype version of a commercial MBR system
and used as a criterion for translation to bench-
scale stirred bioreactors.
Materials and Methods Cell lines and media
All experiments in this work were carried out
using a Chinese hamster ovary (CHO) cell line
expressing an IgG antibody. The medium used
was of an animal-free origin and was chemi-
cally defined.
Miniature bioreactors
All MBR work was carried out with a 0.5-L min-
iature bioreactor system (HEL Ltd, UK) run in
parallel with a working volume of 0.35 L. The
operating parameters of pH, DO, and temper-
ature were controlled at 7.1, 30%, and 37°C,
respectively. Agitation was provided either by a
single three-blade marine impeller (direct driven)
or a four-bladed marine impeller (magnetic-bot-
tom-driven) with an agitation rate set to match
Mixing Time as a Criterion for Scale Translation of Cell-Culture Processes The authors conclude that miniature bioreactors can adequately predict the cell culture kinetics in scaled-up reactors using equal mixing times.
Mohd Helmi Sani is research
engineer in the department
of Biotechnology and Medical
engineering at the Universiti
teknologi Malaysia; and
Frank Baganz is senior lecturer
in the department of Biochemical
engineering at the University
College london.
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48 BioPharm International www.biopharminternational.com January 2016
the mixing time in the 5L-STR.
Aeration in the bioreactor vessel
was achieved either by a horseshoe
sparger or singular hole sparger.
Bench-scale bioreactors
The experiments were carried out in
5-L stirred-tank bioreactors (Biostat
B-DCU, Sartorius) run in paral-
lel with a working volume of 3.5 L
using the same operating parame-
ters as given for the MBRs. Agitation
was provided by a single three-blade
segment marine impeller. Aeration
in the bioreactor vessel was achieved
via a horseshoe-type sparger.
In all experiments at both scales,
the mode of operation was batch
phase from day zero until day six,
followed by fed batch from day
seven onwards. The cultures were
bolus-fed once a day to maintain
the glucose concentration at 2 gL-1.
Mixing time
Mixing time was measured in
the HEL-BioXplore miniature
bioreactor using the pH tracer
method (6). The experiment was
conducted using two types of
impellers; direct driven and mag-
netic driven with the horseshoe
and singular hole sparger. The
mixing time in the 5L-STR as a
function of agitation rate was
previously determined by inves-
tigator Silk (6).
Cell number and viability
The cell cultures were sampled
daily and cell concentration and
viability were analyzed using an
automated cell-counting device,
VI-Cell XR (Beckman Coulter),
which automates the trypan blue
dye exclusion method.
IgG HPLC assay
The quantification of IgG pro-
duced was determined through
the use of a high-performance liq-
uid chromatography (HPLC) sys-
tem (Agilent Technologies) with a
Protein-G affinity column.
resUlts and disCUssionMixing time is a useful parameter
to measure the mixing efficiency
in a reactor and homogeneity of a
fluid when agitated by an impel-
ler. These mixing time values vary
depending on the impeller and/or
sparger designs and geometry of
the reactor. Because the MBR exhib-
its a similar geometry as a conven-
tional STR, an empirical correlation
proposed by Nienow (7) was applied
to compare experimental and pre-
dicted values for the different MBR
configurations (Equation 1).
Tm= 5.9 (ε
Tg)−0.33 D
T0.67
Di
DT
−0.33
Tm = mixing time (s)
εTg = Total energy dissipation rate
in gassed bioreactor (Wm−3)
Di = impeller diameter (m)
DT = tank diameter (m)
[Eq. 1]
In agreement with other stud-
ies (Xing et al., [6]), Figure 1 shows
that mixing time is inversely pro-
portional to agitation rate. The
mixing time achieved ranges from
5–14 seconds and 4–11 seconds for
experimental and predicted values,
respectively. Although both experi-
mental data sets show the same
trend as the calculated values, the
measured values are either slightly
higher (magnetic driven) or lower
(direct driven) compared with the-
oretical predictions. These devia-
tions may be due to differences in
average energy dissipation rate that
were not measured. Nevertheless, the
results suggest that the Nienow (7)
correlation can be used to predict the
mixing times in the MBR with good
accuracy considering the experimen-
tal variations.
Matched mixing time between
miniature bioreactors and bench-
stirred tank reactors
In this case study, a typical fed-
batch CHO cell culture process was
chosen and mixing time was used
as a scale-translation criterion. The
troubleshooting
Figure 1: Comparison of experimental mixing times with Nienow correlation. A
dashed line represents the direct-driven impeller, the dotted line represents the
magnetic-driven impeller, and the solid line represents the Nienow correlation.
Mix
ing
tim
e (
s)
Agitation (rpm)
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January 2016 www.biopharminternational.com BioPharm International 49
troubleshooting
operating conditions for each reac-
tor system with regard to agitation
speed, aeration rate, and feed addi-
tion were adjusted accordingly.
The CHO cell-growth profile and
percentage viability for the two
types of reactors, miniature biore-
actors, and bench 5-L stirred-tank
bioreactors are depicted in Figure 2.
Both cultures had a prolonged expo-
nential phase but reached the peak
viable cell concentration (VCC)
at different days (Figure 2A). The
parallel MBR cultures exhibited a
slower exponential growth between
70–168 hours of cultivation com-
pared with the benchtop cultures.
The peak viable cell concentrations
for the two reactors, however, are
almost identical (Table I).
The benchtop culture showed
a considerably longer stationary
phase compared with the MBR and
achieved higher cell viability on day
14. Final percentage viability of the
MBR was at 60%, while the viability
in the 5-L stirred-tank reactor was
at 80% after >300 hours (Figure 2B).
The higher viability observed in
the stirred-tank reactor on day 14
might be due to the better monitor-
ing and control of gas sparging in
the larger reactor. Besides that, the
feed addition, which was performed
daily from day seven, enabled pro-
longed cell viability and antibody
production until the harvest day.
Figure 3 shows the final anti-
body production for both bioreac-
tor cultures. Based on the HPLC
analyses, the MBR culture reached
a final IgG titer of 0.69 gL-1, which
is 17% lower than that in the 5 L
STR with 0.83 gL-1. The derived
growth parameters for both sys-
tems, however, show excellent
agreement in terms of specific
production rate (qP) and generally
good comparability with regard
to the cumulative integral viable
cell concentration (CiVC) (Table I).
Figure 2: Chinese hamster ovary (CHO) growth kinetics in fed-batch cultures for
two reactors: a 5-L benchtop (solid line) and a miniature bioreactor (MBR) (dashed
line). Graph (A) represents viable cell concentration; graph (B) shows cell viability
for each type of bioreactor. The arrows (↓) indicate the points in the process in
which feed is added.
0
5
10
0 200 400
VC
C
(x1
06 m
L-1)
Time (hours)
50.0
100.0
0 200 400
Cell v
iab
ilit
y (%
)
Time (hours)
(B)
(A)
Contin. on page 55
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50 BioPharm International www.biopharminternational.com January 2016
Analytical Best Practices
Imag
e: P
AS
IEK
A/S
cie
nce P
hoto
Lib
rary
/Gett
y Im
ag
es
Out-of-Trend Identification and Removal in Stability Modelling and Regression AnalysisThis article defines the concept, justification, and method of removal of out-of-trend points in stability modelling and shelf-life prediction.
Outliers in regression modelling may
cause incorrect and invalid results
to occur when predicting stability. A
clearly defined out-of-trend (OOT) protocol is
needed to correctly and consistently identify
and remove outliers from expiry and stability
modelling and prediction where technically
warranted. OOT is a point (measurement) in a
regression analysis that has statistically greater
error at a defined risk factor from a regression
line or multiple-factor regression model than
other determinations; it is a time-dependent
result that falls outside a predicted statistical
interval (see Figure 1). Simply put, an OOT event
is an outlier in a regression analysis. This article
provides an overview of OOT from literature,
guidance, and best analytical practice and the
procedure to be used during stability modelling
and analysis.
OOT points are considered to be non-repre-
sentative of the test sample and are due to ana-
lytical, transcription, or other sources of error.
Failure to remove OOT point(s) if they exist will
produce calculated rates of change that will not
be representative of the drug product nor drug
substance. The following are indications that
OOTs are present in the stability analysis:
• Pointsdonotlineupontheregressionline
• Confidenceintervalsofthefitareexcessively
wide
• Root-mean-squarederror(RMSE)oftheresid-
uals has excessively expanded well beyond
the characterized analytical error
• Expiryfromonetimepointtoanotherhasa
large amount of difference
• R2has a large amount of changewith and
without the OOT time point.
“OOT stabi l ity data can be
describedasaresultorsequenceof
results that are within specification
limits but are unexpected, given
the typical analytical and sampling variation
and a measured characteristic’s normal change
over time (e.g., an increase in degradation prod-
uct on stability)” (1).
Regression analysis is normally used
to determine change over time and associ-
ated 95% confidence limits relative to rates of
change and expiry. InternationalCouncil on
Harmonization (ICH)Q1A(R2) Stability Testing
of New Drug Substances and Product (2) states,
“the nature of any degradation relationship will
determine whether the data should be trans-
formed for linear regression analysis. Usually
the relationship can be represented by a linear,
quadratic, or cubic function on an arithmetic
or logarithmic scale. Statistical methods should
be employed to test the goodness of fit of the
data on all batches and combined batches
(where appropriate) to the assumed degradation
line or curve.”
OOT evaluation and elimination should be
used for the following applications and predic-
tion:
• Shelf-lifeestimation
• Storageevaluation
• Impurityformationandtrending
Failure to remove OOT
point(s) if they exist will
produce calculated rates
of change that will not be
representative of the drug
product nor drug substance.
Thomas A. Little, PhD, is president
of Thomas A. Little Consulting, 12401
North Wildflower Lane, UT 84003
USA, drlittle@dr-tom.com.
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January 2016 www.biopharminternational.com BioPharm International 51
Analytical Best PracticesA
ll F
igure
s a
re c
ourt
esy o
f auth
ors
.
• In-processmonitoring and pre-
diction
• Trackingandtrendingoflotper-
formance.
LIKELY ROOT CAUSES FOR OOTThe following are typical possible
sources and mechanisms for OOT
events that may occur during sta-
bility evaluation:
• Sample selection and sample
handling errors
• Dilutionandsample-preparation
errors
• Samplematerialsandplateerrors
• Temperature, reaction time, and
pH effects errors
• Vendorandlotvariationoncrit-
ical reagents
• Flow rates and process time
errors
• Analysterrors
• Instrument variation and cali-
bration error
• Nonstandard test procedures
and not following the method
standard operating procedure
(SOP)
• Drift in standards or reference
materials
• Stability of test samples or criti-
cal reagents
• Calibration or compensation
errors
• Interactionandcompositeerrors
• Expiryofbulkmaterials.
HISTORICAL APPROACHES TO OOTThe following are typical histori-
cal approaches to OOT identifi-
cation and removal, though they
are not recommended approaches.
They are not considered to be sta-
tistically sound procedures for
OOT identification and removal.
Using procedures that are not sta-
tistically sound may remove time
points from a stability analysis
that cannot be defended nor jus-
tified upon review. There is no
statistical basis for the following
definitions of OOT and these do
not take into account process nor
method variation:
Figure 1: Out of trend (OOT) illustration and infuence of OOT.
Figure 2: Closed loop out of trend (OOT) identifcation and resolution.
No OOT
0 5 10 15 20 25Time (Months)
Linear Fit
Linear Fit
Summary of Fit
RSquare
RSquare Adj
Root Mean Square Error
Mean of Response
Observations (or Sum Wgts)
RSquare
RSquare Adj
Root Mean Square Error
Mean of Response
Observations (or Sum Wgts)
0.974613
0.970987
0.245665
19.05007
9
0.043636
-0.09299
3.63802
20.35897
9
Summary of Fit
Linear Fit
Linear Fit
0 5 10 15 20 25Time (Months)
Pro
tein
Co
nce
ntr
ati
on
ug
/mL
Protein Concentration ug/mL =
21.129718 - 0.1733043*Time (Months)
Protein Concentration ug/mL =
19.297252 + 0.0884766*Time
(Months)
Pro
tein
Co
nce
ntr
ati
on
ug
/mL23
22
21
20
19
18
17
16
30
25
20
15
10
OOT
OOTDetermination
OOTIdentifcation
OOTVerifcation
New TimePoint Data
StabilityPrediction
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52 BioPharm International www.biopharminternational.com January 2016
Analytical Best Practices
• The difference between consec-
utive results is outside of half
the difference between the prior
result and the specification
• The result isoutside±5%of ini-
tial result
• Theresultisoutside±3%ofpre-
vious result
• The result is outside±5%of the
mean of all previous results.
CLOSED-LOOP APPROACH TO OOT IDENTIFICATION AND REMOVAL
A best-practice approach to OOT
determination and removal is to
see it as a part of a closed-loop con-
trol system during stability moni-
toring and expiry prediction (see
Figure 2). The five steps to a closed
loop system for OOT are:
• Additionofnewtimepointsand
data
• OOTidentification
• OOT determination and point
removal where warranted
• OOTverificationandevaluation
of OOT influence
• Stability and performance pre-
diction.
Addition of New Time Points and
Data, Closed Loop Step 1
As each new time point is added
to the stability analysis, the time
point should be checked for OOT
potential. If they are within the
criteria for OOT identification,
then rates of change, expiry, etc.
are determined. OOT identifica-
tion, determination, and verifica-
tion are used if new time points
appear to be suspect.
OOT Identification,
Closed Loop Step 2
Some CMC teams recommend
the percent change from point-
to-point as well as from the ini-
tial time point to indicate an OOT
(3). For example,more than a 5%
change from baseline may be con-
sidered a possible OOT event (4).
Analytically, there are four
methods to identify a point as
OOT: visually, outlier boxplot
of the residuals, multivariate
Jackknife distances, and control
chart of the residuals (see Figures
3–6). Jackknife distances are the
most sensitive in identification of
OOT points in a regression anal-
ysis as they include and remove
each time point in the analysis
to evaluate their influence in the
model. Once an OOT has been
identified, the next step is to test
it to determine if the OOT will be
removed. Root cause as to why
the point is OOT is a secondary
investigation once the point has
been determined to be OOT. Once
the point is determined to be a
possible OOT, the point is tested
Figure 3: Visual analysis.
Figure 4: Outlier box plot of residuals.
Protein Concentration ug/mL By Time (Months)
Time (Months)
Pro
tein
Co
nce
ntr
ati
on
ug
/mL
25
24
23
22
21
20
19
18
17
160 5 10 15 20 25
2
1
0
Time (Months)
Co
un
t
Residuals Protein Concentration ug/mL
-3 -2 -1 0 1 2 3 4 5
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January 2016 www.biopharminternational.com BioPharm International 53
Analytical Best Practices
statistically to determine if it is or
is not OOT.
OOT Determination,
Closed Loop Step 3
The following is the recommended
procedure for OOT outlier determi-
nation (see Figures 7 and 8):
• Exclude andhide the suspected
OOT point in the data analysis.
• Fit a linear regression linewith
the potential OOT time point
excluded.
• Savethepredictedresponse(con-
centration) from the linear fit.
• Calculate the difference (Delta)
at each time point.
• Calculateazscoreforeachtime
point (see Equation 1).
z score= (Measurement-predicted)/
s tde v (r e s idu a l s w i t h p o i nt
removed)
[Eq.1]
Once the z score for the OOT
has been calculated, it can be com-
pared with a risk threshold. A
k-sigma of 2.576 (or 99% risk) is
used to set the limit for OOT detec-
tion. Abs(z)> 2.576 (99%) isOOT,
so in this example, z is -27.238;
therefore, it is OOT. A z score with
a limit is the best method of OOT
detection. The key difference with
this procedure and other z score
procedures written in literature is
the z score is evaluated with the
point removed. This correctly
scales the residual error so that the
influence of the OOT point is not
included in the residual error and
the OOT time point can be cor-
rectly evaluated based on the other
measurements error.
OOT Verification
To verify the influence of the OOT,
the following measures are recom-
mended:
• ChangeinR2
• ChangeinRMSE
• Changeinexpirycalculation.
Comparingwithorwithout the
OOT time point will verify the
influence of the time point and
confirm the need for removal. If
the change in the three identi-
fied measures is trivial, then the
OOT has not been verified and
its removal is not warranted.
Differences inR2,RMSE,or expiry
of 3% or less are generally not
practically important to drug-sub-
stance or drug-product expiry or
stability evaluation. Verification
is performed by including, then
removing, the OOT point in the
stability evaluation and then mea-
suring the change in the key per-
formance metrics of the fit and the
prediction.Also,RMSEerrorcanbe
compared to the repeatability of the
analytical method to determine if
the residual error is primarily due
to analytical measurement error.
A control chart of the residuals
with the OOT time point excluded
will be a secondary confirmation
of OOT identification and removal
as an outlier (see Figure 9).Residual
Figure 7: Out of trend (OOT) determination.
1
Time(Months)
PredictedConcentration
Concentration z Score OOTDelta
2
3
4
5
6
0
3
6
9
12
18
0.02
0.0197
0.0195
0.0193
0.0177
0.0188
0.0199264151
0.0197320755
0.0195377358
0.0193433962
0.0191490566
0.0187603774
0.000074
-0.000032
-0.000038
-0.000043
0.000040
1.3832 OK
OK
OK
OK
OOT
OK
-0.6029
-0.7093
-0.8157
-27.2382
0.7448
Figure 5: Multivariate Jackknife distances.
Figure 6: Control chart of residuals.
Row Number
Jackknife Distances
Dis
tan
ce
50
0 1 2 3 4 5 6 7 8 9
30
10
-10
UCL = 3.67
6
A
A
B
B
C
C
1
UCL = 3.51
Avg = 0.00
LCL = -3.51
Sample
Resi
du
als
Pro
tein
Co
nce
ntr
ati
on
ug
/mL 4
2
1 2 3 4 5 6 7 8 9 10 11 12
0
-2
-4
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54 BioPharm International www.biopharminternational.com January 2016
Analytical Best Practices
points from the regression fit are
plotted onto a control chart, points
that are within the control limit
may be due to random or analyti-
cal method variation, points out-
side of the limits confirm points
are likely to beOOT. Remember
to exclude the OOT point when
building the chart so it does not
influence the limits.
Stability Prediction
Once the OOT point has been
removed and verified, stability
prediction can then be performed
per ICH Q1E (5). OOT points
should always be included in the
plot to indicate the measurement
but tagged as OOT to indicate
the point was included in graph
but not included in the analysis.
Including it in the plots will pro-
vide full disclosure as to all obser-
vations at all time points. Once a
point has been determined to be
OOT, it does not render the analy-
sis as additional data are added to
the stability prediction.
Single Factor, Single Batch,
and Multiple-Factor/Multiple-
Batch OOT
Thetechniqueforidentifyingand
removing an OOT point described
in this paper works equallywell
for single -batch/single -fac tor
versus multiple-batch/multiple-
factor stability modelling and
expiry prediction. The example
provided was for a single-batch,
single-factor analysis. For the mul-
tiple-factor and/ormultiple-batch
condition, the same approach
would be used. The model would
be fit with the OOT removed and
then the model would be saved, z
scores would be calculated for all
time points, and then OOT would
be determined based on a 2.576
(99%) threshold.
CONCLUSIONHaving a well-defined and sta-
tistically valid OOT protocol is a
powerful addition to any stabil-
ity program and needs a clearly
definedSOPtoconsistentlyapply
the logic to day-to-day stability
evaluation. Including an OOT
protocol to stability testing and
data analysis will produce more
stat ist ica l ly rel iable stabi l ity
determination and expiry predic-
tion. OOT determination based
on the protocol described in this
paper opens the door for the
automation of OOT determina-
tion and removal from any stabil-
Figure 8: Stability analysis with out of trend (OOT) removed.
RSquare
RSquare Adj
Root Mean Square Error
Mean of Response
Observations (or Sum Wgts)
0.986058
0.981411
6.143e–5
0.01946
5
Summary of Fit
Linear Fit
Concentration = 0.0199264 - 6.478e–5*Time
(Months)
Linear Fit
Time(Months)
Bivariate Fit of Concentration By Time (Months)
Co
nce
ntr
ati
on
OOT
0
0.021
0.02
0.019
0.018
0.017
5 10 15 20
Figure 9: Control chart of residuals.
Individual Measurement of Delta
8
Sample
UCL = 0.67
Avg = -0.00
LCL = -0.67
Delt
a
6
4
2
0
1 2 3 4 5 6 7 8 9 10 1211
1
-2
A
ABBC
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January 2016 www.biopharminternational.com BioPharm International 55
Analytical Best Practices
ity analysis and may be the best
approach to systematically eval-
uate all time points in stability
analysis.
REFERENCES 1. PhRMA CMC Statistics and Stability
Expert Teams, “A Review of the
Potential Regulatory Issue and
Various Approaches,”
Pharmaceutical Technology 27
(2003).
2. ICH, Q1A(R2) Stability Testing Of
New Drug Substances (ICH,
February 2003).
3. T-J, Torbovska, “Method for
Identification of Out-of-Trend
Stability Results,” Pharmaceutical
Technology 37 (2003).
4. PhRMA CMC Statistics and Stability
Expert Teams, “Identification of
Out-of-Trend Stability Results, Part
II,” Pharmaceutical Technology (Oct
2, 2005).
5. ICH, Q1E Evaluation for Stability
Data (ICH, February 2003). ◆
CONCLUSIONThis study has demonstrated the
potential of miniature bioreac-
tors for scale translation using
equal mixing t ime as a cr ite -
rion. The mixing times obtained
for experimental and predicted
values in the miniature biore-
actors were comparable, which
suggested that the correlation
can be applied to predict mix-
ing t imes at this scale. Scale
comparison cultivations for fed-
batch CHO cell cultures were
carried out using miniature bio-
reactors and compared with a
standard 5-L stirred-tank reac-
tor at matched mixing time. The
results show that theMBR gave
comparable CHO cel l growth
and product kinetics compared
to that at the 5-L scale. The
results of this investigation sug-
gest that MBRs can be used as
sca le -down models of bench-
scale bioreactors.
ACKNOWLEDGEMENTSMohd Helmi Sani thankful ly
acknowledges the funding by the
Ministry of Higher Education,
M a l a y s i a a n d Un i v e r s i t i
TeknologiMalaysia.
REFERENCES 1. H. Zhang et al., Cur. Pharm. Biotech.
11 (1), pp. 103–112 (2010).
2. M.A. Hanson and G.Rao,
“Miniaturization of bioreactors,” in
Encyclopedia of Industrial
Biotechnology: Bioprocess,
Bioseparation and Cell Technology,
M.C. Flickinger, Ed. (John Wiley and
Sons, 2010).
3. J. Betts and F. Baganz, Microbial
Cell Factories, 5 (1), pp. 21–35
(2006).
4. R. Bareither and D. Pollard, Biotech.
Prog., 27 (1), pp. 2–14 (2011).
5. Z. Xing et al., Biotech. Bioeng. 103
(4), pp. 733–746 (2009).
6. N.J. Silk, “High Throughput
Approaches to Mammalian Cell
Culture Process Development,”
(EngD thesis), University College
London (2014).
7. A. Nienow, App. Mech. Rev. 51 (1), pp.
3–32 (1998). ◆
Figure 3: Antibody production in Chinese hamster ovaries (CHO) growth kinetics
in fed-batch culture for two reactors: a 5-L stirred-tank reactor (solid line) and a
miniature bioreactor (dashed line).
0.00
0.50
1.00
0 200 400
An
tib
od
y ti
ter
(gL
-1)
Time (hours)
Type of reactor MBR STR
Peak viable cell concentration
(x 106 cell mL-1)
9.56 10.04
Cumulative integrated viable cell
concentration (x 108 cell d-1 mL-1)
4.05 4.83
Max. IgG antibody titer (gL-1) 0.69 0.83
Specifc IgG production rate
(pg cell-1 d-1)
10.2 9.7
Table I: Derived growth and product parameters of fed-batch Chinese hamster
ovary (CHO) cell culture in a 0.5-L miniature bioreactor (MBR) and a 5-L stirred-
tank bioreactor (STR) with equivalent mixing time.
Troubleshooting—Contin. from page 49
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56 BioPharm International www.biopharminternational.com January 2016
PRODUCT SPOTLIGHT
Ultra High-Performance Liquid Chromatograph System The Prominence-i LC-2030 LT is a new model of Shimadzu’s i-Series integrated high-performance liquid chromatograph and ultra high-performance liquid chromatograph systems. The new system comes without a PDA and UV detector, allowing users to select a detector of their choice. The system provides a 14-second injection cycle time and 1536 continuous, unattended analyses using 4 x 384 well micro plates. The new model contains a color touch panel that supports control of the analysis, monitoring instrument status, and usage of the system and consumables. The panel also assists with maintenance and displays a chromatogram. The system contains auto-startup, auto system suitability test, quick-batch function, auto-shutdown, and auto-validation for system qualification. Instrument power can be turned off upon shutdown, saving more than 95% of standby electricity. Solvent and sample consumption can also be reduced to one-fifth of the usual amount without sacrificing data quality.
Shimadzu www.shimadzu.com
Plastic Union Tri-Clamp The BioClamp Plastic Tri-Clamp, from BioPure Technology, part of the Watson-Marlow Fluid Technology Group, is a patented plastic union tri-clamp meant to reduce distortion on polymeric fittings when subjected to heat.
The clamp is molded from reinforced Nylon 66 USP Class VI, FDA, includes a tamper-evident feature, and is suitable for sterilization by autoclave and gamma irradiation. The instrument is manufactured and packed in an ISO Class 7 cleanroom and has lot numbers molded in for full traceability.
BioPure Technologywww.biopuretech.com
New Technology Showcase
WUXI BIOLOGICS EXPANDS CGMP
MANUFACTURING CAPACITY FOR
CLINICAL AND COMMERCIAL SUPPLY
WuXi Biologics is adding 460,000 sq. ft. of clinical
and commercial drug substance and drug product
cGMP manufacturing facilities to its already
extensive CDMO capabilities. The new campus
will house the world’s largest mammalian cell
culture production plant using disposable bioreactors (14 X 2000L fed-batch
and 2 X 1000L perfusion). The facilities will be operational by H2 2016.
WuXi Biologics, info@wuxibiologics.com, www.wuxibiologics.com
LABORATORY SERVICES
As a member of Eurofins’ BioPharma
Product Testing Group—the
largest network of harmonized bio/
pharmaceutical GMP product testing
laboratories worldwide—Eurofins
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
PRECLINICAL CYTOKINE
STORM ASSAY
Therapeutic proteins and antibodies
can be associated with cytokine
release-mediated toxicity. Use of in vitro and in vivo animal studies
have failed to be predictive of such severe reactions. Abzena has
developed and validated an enhanced in vitro assay to evaluate
the risk of a drug to stimulate a cytokine storm prior to clinical
trials. Multiple pro-inflammatory cytokines IL-6, IL-8, IL-10, IFN-γ,
and TNF-α are accurately measured. Abzena, www.abzena.com
VETTER-JECT® SYRINGE
CLOSURE SYSTEM
Vetter-Ject® is a patented, ready-
to-use closure system for prefilled
syringes specially designed
for complex, silicone-sensitive
compounds. The new tamper-evident
closure system combines an integrated staked needle with a baked-
in siliconization process for standard glass barrels. The closure
Vetter-Ject® is steam sterilized separately and mounted to the
syringe prior to filling. Vetter Pharma, www.vetter-ject.com
ES722122_BP0116_056.pgs 01.14.2016 17:39 ADV blackyellowmagentacyan
January 2016 www.biopharminternational.com BioPharm International 57
BIOLOGICS NEWS PIPELINE
IN THE PIPELINE
UK Pharmaceutical Companies Collaborate
on CAR-T Cell Immuno-oncology Therapies
Cell Therapy Catapult, University of Birmingham, and
Cancer Research Technology announced the launch
of a collaboration to develop a new immuno-oncology
cellular therapy created modifying the genes of T-cells
to target solid tumors.
The project is aimed at translating an academic
discovery program into a commercially viable cell
therapy. It is funded by Cancer Research United
Kingdom (UK) and was developed by Steven Lee,
PhD, and Professor Roy Bicknell, both of whom are
from the University of Birmingham. The collaborating
partners have launched a new company—Chimeric
Therapeutics Limited—to manage all future intellec-
tual property rights of any of the resulting discoveries.
The project is based on a new generation chimeric
antigen receptor T-cell (CAR-T) immuno-oncology ther-
apy for solid tumors. This involves directing the CAR-T
cell toward a new, highly specific marker of tumor angio-
genesis, CLEC14a. This therapy will act as a vasculature
disruptive agent, compromising oxygen supply to the
tumors and inhibiting growth. The technology is cur-
rently undergoing the final stages of preclinical develop-
ment will enter into clinical trials in the near future.
According to an announcement from the Cell Therapy
Catapult, the company will assist in accelerating the
translation of the academic discoveries around CAR-T
immunotherapies for solid tumors and the CLEC14a tar-
get toward a commercially available cell therapy.
Ionis Pharmaceuticals Receives
Orphan Drug Designation for HTTRxFDA has granted Ionis Pharmaceutical’s orphan drug
designation for IONIS-HTTRx, for the treatment of
patients with Huntington’s disease (HD). IONIS-
HTTRx is the first therapy to enter clinical develop-
ment that is designed to directly target the cause of
the disease by reducing the production of the protein
responsible for HD.
IONIS-HTTRx is a Gen. 2.0+ antisense drug in devel-
opment for the treatment of Huntington’s disease. The
drug is designed to reduce the production of all forms
of the HTT protein, which is the protein responsible
for HD. HD is referred to as a triplet repeat disorder
and is one of a large family of genetic diseases in which
certain gene sequences are mistakenly repeated. In HD,
the gene that encodes for the HTT protein contains
a trinucleotide sequence that is repeated in the gene
more than 36 times. The resulting HTT protein is toxic
and gradually damages neurons in the brain.
The drug has also been granted orphan drug des-
ignation by the European Medicines Agency for the
treatment of patients with HD.
Merck KGaA, Pfizer, and Syndax
Collaborate on Ovarian Cancer Treatment
Merck KGaA Darmstadt, Germany; Pfizer; and Syndax
announced a collaboration to evaluate the combina-
tion of avelumab and entinostat for heavily pretreated,
recurring ovarian cancer patients.
Avelumab is an investigational fully human anti-
PD-L1 IgG1 monoclonal antibody developed by Merck
KGaA and Pfizer. In November 2014, the companies
announced a collaboration to develop the antibody,
which is thought to potentially enable the activation
of T-cells and the adaptive immune system. Avelumab
is currently under clinical investigation across a broad
range of tumor types.
Syndax’s entinostat is an investigational oral
small molecule that targets immune regulatory cells
(myeloid-derived suppressor cells and regulatory
T-cells). According to Syndax, the delivery of entino-
stat in combination with hormone therapy can result
in improvements in overall survival in advanced HR+
breast cancer patients.
Merck KGaA, Pfizer, and Syndax have entered into
an exclusive agreement to study the combination of
these two investigational agents in ovarian cancer.
Syndax will be responsible for conducting Phase Ib/II
clinical trials in ovarian cancer.
AD INDEX Company Page
EPPENDORF 5
EUROFINS LANCASTER LABORATORIES 9, 29
FREESLATE INC 2
NOVO NORDISK PHARMATECH A/S 23
PARENTERAL DRUG ASSOCIATION 19
UNCHAINED LABS 39
WATERS CORP 59
WUXI APP TEC 60
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58 BioPharm International www.biopharminternational.com January 2016
Ask the Expert
Q: Our company has been growing and
expanding into new markets in recent
years. Therefore, we need to revise the contents
of our quality system documentation, such as
standard operating procedures (SOPs), to reflect
the changed circumstances. However, we find it
difficult to change SOPs that require approval by
more than one department. Do you have any sug-
gestions for improving this process?
A: Market expansion provides a good oppor-
tunity to review your documentation man-
agement process, ensuring timely and efficient
revisions of documents, such as SOPs. The best
approach for changing SOPs that require approval
by more than one department is to restrict the
contents of SOPs. Process documentation should
be limited to only those steps included within the
remit and under the control of just one group or
department.
The modern approach to a pharmaceutical qual-
ity system (PQS) recommends ‘identification of the
pharmaceutical quality system processes, as well as
their sequences, linkages, and interdependencies.
Process maps and flow charts can be useful tools
to facilitate depicting pharmaceutical quality sys-
tem processes in a visual manner’ (1). This guide-
line has been adopted by the European Union,
Japan, and the United States, as well as many other
countries. These processes—such as change man-
agement, material management, or sample man-
agement—often involve several departments or
groups in an organization (see Figure 1).
Covering steps 3 and 4 in Figure 1 in one SOP
would require the collaboration of departments
A and C, plus their respective approvals. While
this approach is compliant with the applicable
regulations, it is discouraged for various reasons,
including:
• Proposed changes to the process (e.g., timing)
can be different among departments and thus
can lead to conflicting situations.
• There is a potential risk that one department
will impose upon another how to perform a
task. This can potentially lead to a suboptimal
or defective process.
• The approval process may differ among the
involved parties, leading to frustrating delays
and/or complications in achieving timely revi-
sions of the procedures or instructions.
These concerns point to the importance of
restricting the contents of SOPs. In the example
above, one would therefore separate the cur-
rent SOP for steps three and four into separate
SOPs—and the process-flow approach needn’t or
shouldn’t stop here. Preparing the detailed flow
for the activities within the SOP before writing any
text will drive a logical and sequential descriptive
procedure. All too often, this recommendation is
not followed, and the sequence is reversed (i.e., the
process flow follows the creation of the SOP’s text).
This rarely results in a well-written and easy-to-fol-
low SOP. An additional benefit of the process flow-
based approach is that it is also aligned with other
quality system approaches, such as International
Organization for Standardization (ISO) 9001:2015
Quality Management Systems–Requirements (2).
REFERENCES 1. ICH, Q10 Pharmaceutical Quality System (ICH, April 2009).
2. ISO, 9001:2015 Quality Management Systems–
Requirements (ISO, Sept. 15, 2015), www.iso.org.◆ Fa
na
tic S
tud
io/G
ett
y Im
ag
es
Fig
ure
1 is c
ou
rte
sy o
f th
e a
uth
or
Siegfried Schmitt,principal consultant,
PAREXEL
Managing Market Expansion’s Effect on ProceduresSiegfried Schmitt discusses how to streamline thedocument management process during market expansion.
Figure 1: Quality system processes.
Sample Management Process version 12
In Process Control Samples
Step 1
Dep
art
men
t A
Dep
art
men
t B
Dep
art
men
t C
Step 4
Step 2
Step 3
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