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BioPharmwww.biopharminternational.com

The Science & Business of Biopharmaceuticals

INTERNATIONAL

April 2011

FINAL WORD: Supply-Chain Analytics and Profitability 50

QUALITY

New Binary Gas Integrity Test Improves Membrane Quality AssuranceThe authors developed a test for defects in filter membranes 24

DRUG SUBSTANCE

Practical Considerations for Demonstrating Drug Substance Uniformity The authors describe considerations and best practices for meeting drug substance uniformity 30

GENOMICS TECHNOLOGY

Accelerating Bioprocess Optimization A series of advancements has changed the way bioprocesses are developed and optimized 38

REGULATORY BEATHealth-reform controversies pose new challenges 14

BURRILL ON BIOTECHIndustry starts the year with a positive spin 20

PERSPECTIVES ON OUTSOURCINGA look at third-party external supply networks 22

Volume 24 Number 4

Bio

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4 BioPharm International www.biopharminternational.com April 2011

Contents

BioPharm International integrates the science and business of

biopharmaceutical research, development, and manufacturing. We provide practical,

peer-reviewed technical solutions to enable biopharmaceutical professionals

to perform their jobs more effectively.

ON THE WEBwww.biopharminternational.com

PEER-REVIEWED FEATURES FEATURE

COLUMNS AND DEPARTMENTS

8 From the Editor Research and development headed for divorce.Michelle Hoffman

10 Global News

14 Regulatory Beat Health-reform controversies pose new challenges.Jill Wechsler

20 Burrill on Biotech Industry starts the year with a positive spin.G. Steven Burrill

22 Perspectives on Outsourcing A look at third-party external supply networks. Gregg Brandyberry

46 New Technology Showcase

47 Spotlight on: Fermentation & Cell Culture

48 Ad Index/Calendar

50 Final WordSupply-Chain analytics can lead

to increased profitability.Eugene Jones

QUALITY

New Binary Gas Integrity Test Improves Membrane Quality Assurance Sal Giglia and Mani Krishnan

The authors developed a test for defects in

filter membranes. 24

DRUG SUBSTANCE

Practical Considerations for Demonstrating Drug Substance Uniformity Sushil Abraham, Eric Rydholm, and Phil Wagner

The authors describe considerations

and best practices for meeting drug

substance uniformity. 30

GENOMICS TECHNOLOGY

Accelerating Bioprocess OptimizationLen van Zyl and Michael Zapata

A series of advancements has changed

the way bioprocesses are developed and

optimized. 38

BioPharm International ISSN 1542-166X (print); ISSN 1939-1862 (digital) is published monthly by Advanstar Communications, Inc., 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: Pitney Bowes, P. O. Box 25542, London, ON N6C 6B2, CANADA. Canadian GST number: R-124213133RT001. Printed in U.S.A.

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Volume 24 Number 4 April 2011

Social MediaFollow the BioPharm editors on Twitter:

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and manufacturing of biotech drugs

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Special Issue: Downstream Be sure to check out our Advances

in Separation & Purification special

supplement, inside this issue, for

articles on downstream bioprocessing

challenges, anion exchange, and

vaccine manufacturing.

Cover: Courtesy of Lonza

BioPharmINTERNATIONAL

Advances in

Separation & Purification

April 2011

BioPharmINTERNATIONAL

www.biopharminternational.com


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© 2011 Millipore Corporation. All rights reserved.

EDITORIAL

Editorial Director Michelle Hoffman [email protected]

Senior Managing Editor Angie Drakulich [email protected] Managing Editor Susan Haigney [email protected]

Editor (Europe) Rich Whitworth [email protected]

Scientific Editor Amy Ritter [email protected]

Assistant Editors Erik Greb & Stephanie Sutton [email protected], [email protected]

Art Director Dan Ward [email protected]

Washington Editor Jill WechslerContributing Editor Jim MillerCorrespondents Hellen Berger (Latin & South America, [email protected]), Jane Wan (Asia, [email protected]), Sean Milmo (Europe, [email protected])

ADVERTISINGPublisher Allen Basis [email protected]

Associate Publisher Pat Venezia, Jr. [email protected]

European Sales Manager James Gray [email protected]

Market Development, Classifieds, and Recruitment Tod McCloskey & Melissa Brown [email protected], [email protected]

Direct List Rentals Tamara Phillips [email protected]

Reprints The YGS Group [email protected], 800.290.5460 ext 100 or +1.717.505.9701 ext 100

Sales Assistant Daisy Roman-Torres [email protected]

PRODUCTION, MARKETING, CIRCULATIONProduction Manager Dave Erickson [email protected]

Marketing Promotions Specialist Cecilia Asuncion [email protected]

Audience Development Manager Wendy Bong [email protected]

President, Chief Executive Officer Joe Loggia; Vice-President, Finance & Chief Financial Officer Ted Alpert; Executive Vice-President, Corporate Development Eric I. Lisman; Executive Vice-President, Pharma/Science

Group Ron Wall; Chief Administrative Officer Tom Ehardt; Vice-President, Information Technology J. Vaughn; Vice-President, Electronic Media Group Mike Alic; Vice-President, Media Operations Francis Heid; Vice-President, Human Resources Nancy Nugent; Vice-President, General

Counsel Ward D. Hewins; Vice-President and General Manager, Pharma/Science Group Dave Esola; Director of Content Peter Houston

Right to privacy: Advanstar Communications, Inc., provides certain customer contact data (such as customers’ names, addresses, phone numbers, and email 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 Advanstar Communications 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 am and 5 pm CT, and a customer service representative will assist you in removing your name from Advanstar’s lists. Outside of the United States, please phone 218.740.6395. BioPharm International welcomes unsolicited articles, manuscripts, photographs, illustrations, and other materials but cannot be held responsible for their safekeeping or return.

© 2011 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 Permissions: Maureen

Cannon, 440.891.2742 or toll-free 800.225.4569, [email protected].

INTERNATIONAL

BioPharm

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-SimhPresident, Shiba Associates

Fredric G. BaderVice President, Process SciencesCentocor, Inc.

Rory BudihandojoManager, Computer ValidationBoehringer-Ingelheim

Edward G. CalamaiManaging PartnerPharmaceutical Manufacturing and Compliance Associates, LLC

John CarpenterProfessor, School of PharmacyUniversity of Colorado Health Sciences Center

Suggy S. ChraiPresident and CEOThe Chrai Associates

Janet Rose ReaVice President, Regulatory Affairs and QualityPoniard Pharmaceuticals

John CurlingPresident, John Curling Consulting AB

Rebecca DevineBiotechnology Consultant

Mark D. DibnerPresident, BioAbility

Leonard J. GorenGlobal Director, Genetic IdentityPromega Corporation

Uwe GottschalkVice President, Purification TechnologiesSartorius Stedim Biotech GmbH

Rajesh K. GuptaLaboratory Chief, Division of Product Quality Office of Vaccines Research and ReviewCBER, FDA

Chris HollowayGroup Director of Regulatory AffairsERA Consulting Group

Ajaz S. HussainVP, Biological Systems, R&D Philip Morris International

Jean F. HuxsollSenior Director, QA ComplianceBayer Healthcare Pharmaceuticals

Barbara K. Immel President, Immel Resources, LLC

Denny KraichelyPrincipal Research ScientistCentocor R&D, Inc.

Stephan O. KrausePrincipal Scientist, Analytical Biochemistry, MedImmune, Inc.

Steven S. KuwaharaPrincipal ConsultantGXP BioTechnology LLC

Eric S. LangerPresident and Managing PartnerBioPlan Associates, Inc.

Howard L. LevinePresidentBioProcess Technology Consultants

Herb LutzSenior Consulting EngineerMillipore Corporation

Hans-Peter MeyerVP, Innovation for Future TechnologiesLonza, Ltd.

K. John MorrowPresident, Newport Biotech

Barbara PottsDirector of QC Biology, Genentech

Tom RansohoffSenior ConsultantBioProcess Technology Consultants

Anurag RathoreBiotech CMC ConsultantFaculty Member, Indian Institute of Technology

Tim SchofieldDirector, North American Regulatory Affairs, GlaxoSmithKline

Paula ShadlePrincipal Consultant, Shadle Consulting

Alexander F. SitoPresident, BioValidation

Gail SoferConsultant, Sofeware Associates

S. Joseph TarnowskiSenior Vice President, Biologics Manufacturing & Process DevelopmentBristol-Myers Squibb

William R. TolbertPresident, WR Tolbert & Associates

Michiel E. UlteeVice President of Process SciencesLaureate Pharma

Krish VenkatPrincipal AnVen Research

Steven WalfishPresident, Statistical Outsourcing Services

Gary WalshAssociate Professor Department of Chemical and Environmental Sciences and Materials and Surface Science InstituteUniversity of Limerick, Ireland

Lloyd WolfinbargerPresident and Managing PartnerBioScience Consultants, LLC

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From the Editor

Michelle Hoffman is the editorial director of

BioPharm International.


Pharma will be

shopping among

later-stage biotech

companies ready for

development.

Celebrity Couples:

Research and Development Headed for Divorce

Psst. I’ve got a bit of gossip for you, a piece of news you won’t hear on TMZ or

Entertainment Tonight. Early reports seem to indicate that a huge celebrity

couple, seen everywhere at all the big industry firms is headed for splitsville.

That’s right. That perennial couple, the so-called jewel in every pharma companies’

crown—R&D—looks like they’re on the outs. R seems to be leaving D. Or is it getting

kicked out? An article in the Feb. 10, 2011 issue of Nature reported cuts to the research

staffs at both Pfizer and GSK. Pfizer, the article said, is shutting down its Sandwich,

UK, facility, thus cutting 2400 mostly research scientists. The company, according to

the article, is also eliminating 1100 jobs from its Groton, CT, research facility, and is

slashing research budgets from a previously stated target of $8.0 to $8.5 billion down

to between $6.5 and $7.0 billion. For its part, GSK was quoted in the same article

saying that it would be “externalizing parts of early-stage discovery; dismantling

development in areas with low financial and scientific return.” Judy Slinn, a busi-

ness historian at Oxford Brooks University in UK was also quoted as saying “Pharma

companies will still do development work. They won’t do discovery.”

OK. But then who will do discovery? Pharma is looking to small biotechs to fuel its

discovery engines. By most accounts, pharma will be shopping among later-stage bio-

tech companies ready for development. Yet there still seems to be a significant fund-

ing gap to develop promising technologies out of the universities and into at least

proof-of-concept stage; that is, there seems to be little money to develop technologies

to the point that they become attractive to large pharma.

This funding gap—the so-called Valley of Death—is nothing new. It’s just that

in an environment where everyone is cutting back on research, the problem of

developing promising technologies seems more acute. The Biotechnology Industry

Organization (BIO) is addressing the problem in what it is calling their “Big Thinking

Project.” BIO recently commissioned the firm headed by former NIH Director Elias

Zerhouni to collect suggestions for optimal government and industry initiatives from

key industry leaders. They asked for policy proposals that would support early innova-

tion to a point where risk-averse venture investors and big pharma firms would pick

up the tab for continued development. Their responses include a mix of reforms that

would reduce regulatory and financial risks.

The Obama administration is also keen on promoting innovation. In his 2011

State of the Union address, the president wasted little time pinpointing the source of

America’s future financial security. “The first step in winning the future is encourag-

ing American innovation,” he said. And the administration’s 2012 budget backs that

up with funding increases for biomedical research and FDA activities. But then it curi-

ously sounds a counter-innovative note. The very same administration “now proposes

to jettison the biotech exclusivity deal,” that had previously been approved as part of

the healthcare reform package, notes Jill Wechsler in Regulatory Beat this month. In

shrinking the exclusivity period from 12 to 7 years, the move threatens to disincent

investor support for the very innovation it otherwise hopes to stimulate.

Interestingly, in the same State of the Union Address, the president warned of com-

petition from overseas. “Meanwhile,” he cautioned, “nations like China and India

realized that with some changes of their own, they could compete in this new world.

And so they started educating their children earlier and longer, with greater emphasis

on math and science. They’re investing in research and new technologies.” Indeed

they are. The Chinese government is spending a reported $2.4 billion to support drug

development, while it introduces policies to promote its biotech sector, strengthen its

intellectual property protection, and strengthen tax and lending policies. In other

words, research, jilted by US pharma, may find solace in China’s warm embrace.

Sources: D. Cressey, Nature 470 (Feb. 10, 2011) p. 154; H. Jia, Nature Biotech. 28 (Oct. 2010), p. 990;

Podcast, A Conversation with Jim Greenwood, www.BiopharmInternational.com. ◆

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Stem cells are pluripotent cells derived from adult or embryonic tissue that show promise as cell-based therapies for a range of conditions, including cardiovascular disease, bone and cartilage repair, and neuronal regeneration. ELA cells are a unique class of progenitor cell, developed as a therapeutic by Parcell Laboratories of Massachusetts. ELA cells are a pure population of cells, isolated from healthy adult donors, and are found distributed in many tissues throughout the body.

ELA cells are phenotypically distinct from mesenchymal stem cells, and express a largely nonoverlapping set of unique genetic markers. They can be induced to differentiate into cells of ectodermal (epithelial-like), mesodermal (bone or cartilage-like), or endodermal (neuronal-like) phenotype, indicating that they potentially can be used to treat injuries in a wide range of tissues. The therapeutic benefit from ELA cells is derived from their role as regenerative cells. They promote wound healing by modulating the immune response, suppressing the release of cytokines at the site of injury. They also promote the release of trophic factors, including vascular endothelial growth factors that facilitate tissue repair, and can be used to rebuild structural tissues at the site of injury.

This therapy differs from other stem-cell products in that the cells are not culture expanded: rather, they can be harvested from donors in sufficient abundance that they can be purified and packaged at therapeutic concentrations without expansion. Parcell Laboratories has partnered with Alphatec Spine to deliver the ELA cells in vials for use during fusion procedures to enhance bone regeneration of the spine. They also maintain an active preclinical research program, exploring the potential benefit of these cells in models of spinal cord injury, wound healing, and soft tissue repair. —Amy Ritter

China’s contract research industryChina’s contract research industry has come a long way. In fact, it reached a milestone

when Shanghai-based WuXi PharmaTech listed its initial public offering (IPO) on the New

York Stock Exchange in September 2007. In the same year, Hutchinson Medipharma

established an agreement with Eli Lilly to work on novel compounds in oncology and

inflammation. Looking back, China’s contract research industry consisted mainly of

chemistry-based companies, largely as a result of a limited talent pool and accessibility

of quality deliverables in the early 1990s. As the regulatory and business environment

improved in the mid-1990s, however, foreign CROs found their way to Beijing and

Shanghai. MDS Pharma Services was the country’s first foreign-based contract research

organization (CRO) followed by Quintiles Transnational, Covance, and Kendle.

Today, the CRO industry in China has developed tremendously. According to Jan-

Willem Eleveld, vice-president for management consulting (Asia–Pacific and Japan) at

IMS Health, the CRO industry has evolved from preclinical offerings to clinical studies.

Established players such as Wuxi PharmaTech are also opening new business in the

clinical-trial arena, and there has been a rise in the number of firms that have established

current good laboratory practice (cGLP) facilities to carry out studies in China.

Adds Eleveld, “CROs are facing a new reality that Big Pharma [is] moving away from a

pure out-sourcing model and establishing fully integrated R&D centers in China. Unlike

past R&D outfits designed for local clinical trials for local approval, new R&D investments

are [aiming to reach the] global pipeline. As a result, they [are building] more in-house

capabilities in all aspects of R&D and [using] CROs (preclinical) to manage capacity

overshoot. On the other hand, clinical CROs have a great future because more and more

new drugs will be developed in China as part of a global R&D hub.”

Recently, there have been a number of active new establishments and acquisition

activities, resulting in the doubling of foreign CROs in the country last year alone. Several

foreign companies have expanded their services into central laboratories and drug-

supply management. To remain competitive in the changing business environment,

many local companies have embraced the mergers and acquisitions (M&A) approach

by opening their services to international drug developers. Some are also gaining

accreditation from organizations, such as the American Association for the Accreditation

of Laboratory Animal Care, to gain a better market position in the eyes of international

clients.

The Chinese government plays a paramount role in developing the contract

research industry in the country. Its Contract Research Organization Union China

(CROU) developed the first industry standard, Clinical Trial Services of Contract Research

Organizations, for the sector. Johnny Huang, senior consultant on healthcare practice

for Frost & Sullivan China adds, “The Chinese government is adopting a market policy

to improve its regulatory environment and CRO industry development. Its initiatives for

drug discovery in past years, included strengthening of intellectual propoerty rights and

focusing on shrinking evaluation time for drugs. The government has also focused on its

infrastructures by creating technology parks ... establishing a large talent pool in the field

of pharmaceuticals, and formulating numerous government bodies that help develop

drug discovery in China.”

Perhaps the challenge for the government going forward will be to harmonize

local CRO culture and international drug-research standards, he says. Some leading

pharmaceutical companies have taken initiatives to help local CROs improve their

practices in this area. Overall, to achieve success in the Chinese market, CROs need to

adopt a cost-effective approach. Huang says, “Flexibility and scalability of operations

allow companies to be more nimble and respond to change faster. In the long term,

CROs in China have to create their advantages in local talent pool, unique service, and

strong partnership with local clinical experts. These are the key ingredients to success

especially in multicentered clinical trials in China.”

—Jane Wan, a freelance writer based in Singapore

Discovery Pipeline


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European Union and India Battle over Free Trade Agreement Ongoing Free Trade Agreement (FTA) negotiations

between the EU and India have hit a hurdle as

some stakeholders urge the Indian government

to fight against certain provisions in the FTA amid

fears that access to generic drugs may be affected.

According to a statement from Doctors Without

Borders, the EU is “pushing for harmful intellectual

property provisions” to be included in the FTA that

will “hinder access to quality, affordable generic

medicines produced in India.”

India produces an enormous number of generic

products, many of which are particularly popular

in developing countries as cheaper alternatives to

branded medicines. The country has been able to

produce so many affordable versions of medicines

patented elsewhere because it did not grant patents

on medicines until 2005.

“The EU is pushing for intellectual property

provisions in the FTA that exceed what international

trade rules require. The most damaging measure

is so-called ‘data exclusivity,’ which would act

like a patent and block more affordable generic

medicines from the market, even for drugs that are

already off patent, or do not merit a patent to begin

with,” explained Doctors Without Borders.

India and the EU have been discussing an FTA

since 2007. India is one of the EU’s most important

trade partners; however, India still maintains

“substantial tariff and nontariff barriers that hinder

trade with the EU,” according to the European

Commission. A FTA would help to increase trade in

both goods and services, but the EU has outlined

a number of provisions that India must submit to,

including, among others, strengthening its patent

laws, which some believe will affect the supply of

generic medicines.

A release from the EU Delegation to Tanzania

acknowledged these fears, but believes they

are based on a misunderstanding of the EU’s

objectives and negotiation position. According

to the statement, the EU has proposed a clause

in the negotiations to ensure that nothing in the

FTA prevents India from producing and exporting

medicines to developing countries. “The EU is

fully aware that India is an important provider of

generic medicines to other developing countries;

the Free Trade Agreement currently under

negotiations between India and EU is certainly

not intended to restrict the ability of India to

continue doing the same for domestic and

international consumption.” The EU–India FTA is

expected to be finalized in spring 2011.

—Stephanie Sutton

Japan Halts Use of Pfizer and sanofi VaccinesJapan suspended the use of two pediatric vaccines, one made by Pfizer

and one by sanofi, following the deaths of four children in three days. The

deaths occurred shortly after vaccination with Pfizer’s Prevenar vaccine

(known as Prevnar in the United States) and sanofi’s ActHIB vaccine.

Although a panel of experts at Japan’s Health Ministry have found no link

between the deaths and vaccination, the suspension will remain in place

while further studies are conducted.

Other national regulatory agencies are aware of the situation. In Hong

Kong, the Department of Health said in a statement that it has “no record

of untoward event report[s] associated with Prevenar, but we will inform all

medical practitioners of the Japanese incident and request them to bring

up any anomaly detected.” sanofi’s vaccine is not used in Hong Kong, but

both are widely used in the US. According to Reuters, FDA is monitoring the

safety of both vaccines, but has not observed any safety concerns in the US.

This is not the first time that Prevenar has faced safety suspicions. In 2009,

a batch of Prevenar was pulled from the market in the Netherlands following

the deaths of three infants after vaccination. In February 2010, however, it

was announced that there was no link between the deaths and vaccination.

—Stephanie Sutton

Hamburg Describes Efforts to Develop Medical CountermeasuresAt a conference on preserving national security at the University of

Pittsburgh Medical Center in early March, FDA Commissioner Margaret

Hamburg stressed the importance of medical countermeasures for

responding to natural and deliberate threats to public health. The Obama

administration has mandated the development of these countermeasures,

and FDA will contribute to the initiative in three ways, Hamburg said.

First, the agency will foster enhanced review and novel manufacturing

approaches for medical countermeasures of the highest priority. From the

development process onward, FDA will collaborate with developers and

government partners to define viable regulatory pathways. The collaboration

will aim to anticipate and resolve bottlenecks and to identify and resolve

scientific problems, thus speeding drugs’ progress toward product approval.

Second, FDA will work with government agencies, including the

US Department of Health and Human Services, to examine the legal

framework, including regulatory and policy approaches, for developing

medical countermeasures and making them available. The agencies will

assess the adequacy of this framework and identify improvements that

could support preparedness and response to public-health threats.

Third, the agency will try to advance regulatory science and improve

the development and evaluation of countermeasures by strengthening

FDA’s own scientific capacity and establishing partnerships with

government, industry, and academia. Scientific opportunities are

“outstripping our ability to translate new discoveries and opportunities

into new products,” said Hamburg. Advancing regulatory science will

help develop new ways to evaluate product efficacy, flexible approaches

to product development and manufacturing, new methods to improve

product stability, and new statistical approaches to assessing efficacy

with limited data, she added.

FDA also will seek partners with whom to pool products, resources,

and knowledge to develop medical countermeasures.

—Erik Greb


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Regulatory Beat

Dig

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Alittle more than a year ago, leaders of

the pharmaceutical industry negoti-

ated a deal to provide billions of dol-

lars in discounts and fees designed to make

drugs more affordable to Americans. In return,

manufacturers anticipated a larger market for

prescription medicines in a reformed national

healthcare system, plus favorable policies gov-

erning research and development (R&D) and

marketing— without explicit price controls.

Now there’s considerable uncertainty about

how the Obama healthcare-reform program

will be implemented, and how well the system

will support biomedical innovation and new

drug development. Federal courts are weigh-

ing the constitutionality of the Affordable Care

Act (ACA), while reform critics in Congress

are challenging specific policies and curbing

funds needed to implement reform initiatives.

Some states face serious budget problems and

are looking to limit Medicaid programs, includ-

ing drug benefits. The Obama administration’s

budget plan for 2012 offers extra funding for

biomedical research and for FDA operations, but

it’s uncertain whether these proposals will sur-

vive the budget-cutting battle on Capitol Hill.

Killing the dealThe search for additional funds to

pay for healthcare-reform initiatives

and government health programs,

moreover, is driving the Obama

administration to ask Big Pharma

to ante up even more. During the

healthcare-reform debate of 2009,

the Pharmaceutical Research and

Manufacturers of America (PhRMA)

agreed to pay higher Medicaid

rebates and additional taxes, and to

subsidize the cost of drugs prescribed

to seniors caught in the “doughnut

hole” of the Medicare drug benefit— all adding

up to some $80 billion over 10 years. A pri-

mary gain for biomedical companies was the

promise of substantial protection for innovator

biotech therapies in the face of more aggressive

generic competition.

The administration now proposes to jettison

the biotech exclusivity deal and boost con-

sumer access to generic drugs to help gain some

of the $54 billion needed to support Medicare

payments to physicians. Shrinking the exclu-

sivity for innovator biologics from 12 to seven

years and thus speeding less costly biosimilars

to patients, according to Obama’s 2012 budget

plan, would save about $2.3 billion over 10

years. The Biotechnology Industry Organization

(BIO) warned that such “questionable short-

term budgetary savings” could jeopardize devel-

opment of new breakthrough cures.

John Castellani, president of PhRMA, said

in a press release that the proposal “flies in

the face” of the administration’s talk about

supporting “innovation, biomedical research,

jobs and US competitiveness.” But US Health

and Human Services (HHS) Secretary Kathleen

Sebelius told the House Energy and Commerce

(E&C) Committee last month that the admin-

istration now feels that a seven-year exclusivity

Jill Wechsler is BioPharm

International’s Washington editor,

Chevy Chase, Md, 301.656.4634,

[email protected].

the Obama administration

may jettison the biotech

exclusivity deal

to boost consumer access

to generic drugs.

Health-Reform Controversies Pose New ChallengesCourts and Congress seek to reshape policies and programs affecting drug costs and access.

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Regulatory BeatRegulatory Beat


period will permit innovator firms

to realize an appropriate return on

investment, while ensuring that

new breakthrough medicines are

widely available and affordable.

Another administration pro-

posal would end pay-for-delay

deals between brand-name and

generic drug makers that post-

pone when a new generic prod-

uct comes to market. The Generic

Pharmaceutical Association (GPhA)

applauded the shorter biotech

exclusivity period, but criticized

the curb on settlements as “mis-

guided.” Castellani agreed with

the generic-drug makers, noting

that these “pro-consumer settle-

ments” do not delay generic entry

and often bring low-cost drugs

to market sooner. Federal Trade

Commission officials, however,

have been pushing hard to curb

such arrangements, which they

insist are anti-competitive and

costly to consumers. The numbers-

crunchers predict that banning

pay-for-delay deals will save the

government $540 million next

year and nearly $8.8 bi l l ion

through 2021.

The generic drug gains together

provide only a small portion of the

resources needed to finance the

“doc fix.” Most of the money would

come from proposed reductions in

federal payments to state Medicaid

programs, stiffer scrutiny of cer-

tain Medicare reimbursement to

insurers, and proposals to reduce

Medicare fraud and abuse. The plan

also proposes to increase tracking

of high prescribers in Medicaid

programs to reduce excessive drug

utilization by states. And manufac-

turers would be hit with additional

penalties if they fail to pay appro-

priate Medicaid drug rebates and

to comply with rebate rules and

FDA policies for listing drugs on

databases. But these policies gener-

ate virtually no tangible savings,

and it’s questionable whether the

squeeze on biotech exclusivity is

worth the rather small budgetary

gain to the government.

MORe OveRsightAlthough pharmaceutical com-

panies t rad it iona l ly look to

Republican allies in Congress to

champion patent protection and

product exclusivity, GOP leaders

still are smarting over PhRMA’s

support for Obama’s healtchare

plan. As part of its investiga-

t ion into HHS implementa-

tion of healthcare reform, House

E&C Committee Chairman Fred

Upton (R–MI) is looking hard at

the “secret negotiations” between

the White House and healthcare

interest groups, including phar-

maceutical companies, leading to

enactment of the reform legisla-

tion. E&C Committee Republicans

complained in a Feb. 18, 2011 let-

ter to White House Aide Nancy-

Ann DeParle (formerly head of

the White House Office of Health

Reform) that instead of the open

and transparent debate on health-

care legislation that Obama had

promised, deals were made behind

closed doors with providers, drug

companies, and others.

Upton and his colleagues are

scrutinizing the HHS process for

determining whether states and

healthcare providers and payers

should receive waivers from com-

plying with specific ACA rules,

a process they believe indicates

widespread problems associated

with implementing the reform

law. The E&C Committee also

wants to know more about HHS

support for establishing state-

based insurance exchanges and for

developing new rules governing

insurers, including standards for

“essential benefits” that will shape

medical and drug coverage.

FDA operations and policies

are also under scrutiny by GOP

leaders. The E&C Health subcom-

mittee held a hearing in February

to examine whether FDA’s slow

process for approving more com-

plex medical devices for market is

harming US device makers. And

Republicans once more are exam-

ining the three-year-old heparin

crisis, complaining in a Feb. 23,

2011 letter to FDA Commissioner

Margaret Hamburg that st i l l

no one knows the source of the

adulteration nor the Chinese cul-

prits—all while imports in phar-

maceutical ingredients from that

China are booming.

UnRaveling the pieCesIn addition to hauling adminis-

tration officials up to Capitol Hill

to explain their actions, Congress

is moving forward with efforts to

revise portions of the ACA, after

failing in January to repeal the

legislation. In the low-hanging

fruit department, Democrats and

Republicans agree on the need to

repeal the 1099 reporting policy,

a burdensome rule that requires

businesses to report to the IRS any

expenditure over $600—a require-

ment that has little to do with

healthcare. President Obama has

signaled support for killing the pro-

gram, but the challenge is to find

the $22 billion or so needed over

the next 10 years to offset potential

revenue gains from the policy.

P r e s i d e n t O b a m a a n d

Congressional leaders also are eye-

ing medical-liability reform as a

way to reduce spending on unnec-

essary healthcare services incurred

as a defense against malpractice

charges. A bill recently approved

by the House Judiciary Committee

would cap noneconomic and puni-

tive damages, limit the time for

filing suits and curb attorneys’

contingency fees. Such proposals

face considerable opposition from

lawyers and some patient advo-

cates, yet Obama expressed interest

in revising malpractice policy in

his State of the Union speech and

included $250 million in his 2012

budget plan to support grants to

Biological APPLICATION NOTE

High Sensitivity Physicochemical Characterization of a Thera-peutic Protein using the Agilent 1260 Infinity Bio-inert Qua-ternary LC with Agilent SEC and IEX Columns

Katja Kornetzk, Agilent Technologies

In-depth physicochemical characterization of therapeutic pro-

teins is required during all phases of drug development to en-

sure drug safety and efficacy. In this application note the new

Agilent Infinity Bio-inert LC was used for peptide mapping, SEC

and IEX of a therapeutic protein.

In this article we show results for size exclusion chromatog-

raphy of P128, a therapeutic protein under development at Gan-

gagen, India. SEC can demonstrate protein integrity or prove the

absence of dimers or multimer formation in its native conforma-

tion. A disadvantage of size exclusion is its lack of resolution.

Small particle SEC columns produce superior resolution with

minimal secondary interaction, providing a powerful tool for de-

tecting impurities or multimer formation. Results were compared

for two 5-μm particle columns and a 3-μm particle column. The

second compound present in the mixture could only be detected

with the Agilent Bio-SEC 3-μm.

The Agilent 1260 Infinity Bio-inert Quaternary LC system in

combination with the Agilent Bio HPLC column portfolio pro-

vides a powerful and versatile tool for characterizing the physico-

chemical properties of the therapeutic protein, drug P128.

The Agilent solution for analysis of therapeutic proteins

achieves bio-inertness, superior resolution, corrosion resistance,

high sensitivity and fast separation speed.

Figure 1: Size exclusion of a P128 therapeutic protein sample performed on the Agilent 1260 Infinity Bio-Inert Quaternary LC using different SEC columns.


Regulatory BeatRegulatory Beat

help states rewrite their malpractice

laws and establish health courts.

Reform could move forward if

there’s clear evidence that damage

caps would save money by reducing

defensive medicine and other costs.

But there’s little bipartisan sup-

port for improving ACA implemen-

tation. President Obama recently

voiced support for legislation

that permits states to opt out of

exchanges and the individual man-

date, provided the state can devise

alternative ways to cover more

uninsured individuals. Although

this action was considered a major

concession by ACA supporters,

Republicans labeled it a “fig leaf”

that didn’t increase local choices.

The bat t le cont inues over

Republican efforts to limit federal

spending overall. Policymakers

avoided a government shutdown in

early March by agreeing to a short-

term fix on funding the federal

government for the current (2011)

fiscal year. But Republicans still

demanded some $100 billion in

cuts for this year, as well as curbs

on many ACA provisions, includ-

ing individual coverage require-

ments, medical-loss ratio rules,

health insurance exchanges, and

Medicaid expansion plans.

Critics also are looking to elimi-

nate a number of entities established

by ACA, but seen by Republicans

as examples of a federal govern-

ment over-reach into state and

private sector activities. Although

health prevention has broad appeal,

Republicans want to dissolve the

Prevention and Public Health Fund,

which is supposed to dispense some

$15 billion over 10 years to sup-

port state and local prevention and

health initiatives. This prevention

“slush fund,” say Republicans, is

excessive—and its resources could be

tapped to offset the cost of repealing

the 1099 IRS reporting requirement.

Republ icans a lso don’t see

a need to spend $10 billion over

10 years to fund the Center for

Medicare and Medicaid Innovation

(CMMI), which was established by

ACA to support research and test-

ing of reimbursement and coverage

approaches for Medicare and other

health programs. There’s also skep-

ticism among Republicans about

federal investment in comparative

effectiveness research (CER), when

private plans and local organi-

zations have funded technology

assessment on their own. Some

Republicans would like to shutter

the Patient-Centered Outcomes

Research Institute (PCORI) and use

its $500 million a year in appro-

priated funds for other purposes.

Pharma companies generally sup-

port development of standards

and policies for CER research, but

probably won’t expend much effort

fighting to preserve PCORI.

There’s not much good news

for the pharmaceutical indus-

try regarding the battle to repeal

and revise healthcare reform.

Manufacturers already are pay-

ing stiffer Medicaid rebates and

absorbing 50% discounts on drugs

for Medicare Part D beneficiaries

caught in the coverage gap. The

IRS is establishing rules for collect-

ing some $2 to $3 billion in new

industry taxes beginning this year,

based on company sales of branded

drugs. At the same time, more

onerous legislation is on the table.

Once again, there is bipartisan

support for a Senate bill to permit

reimportation of high-cost medi-

cines from Canada. And Democrats

want to eliminate tax deductions

for direct-to-consumer advertising.

FUnding FdaStiffer FDA user fees are on the

hor izon as well. The Obama

administration has requested a $4.3

billion budget for the agency in

2012, a reasonable increase given

the tight funding environment in

Washington. But most of the gain

would come from higher user fees

on medical product manufacturers,

and most of any added appropria-

tions would fund a huge expansion

in food safety oversight authorized

by Congress, but without support

from food industry fees.

Any extra money for FDA’s

Center for Drug Evaluation and

Research is designated to support

development of medical coun-

termeasures and biosimilars, to

expand monitoring of imported

medical products, and to improve

the safety of certain high-risk

products such as vaccines and

human tissue. New generic-drug

user fees, which at long last appear

to be moving toward reality, would

improve the review of generic

drugs, while other proposed fees

would support more field inspec-

tions. There’s a small amount of

money earmarked for improving

regulatory science at FDA, which

would primarily complete work on

a new laboratory complex for drugs

and biologics at the agency’s White

Oak, Maryland campus. Without

an extra $24 million to get the new

laboratory operational, FDA would

have a new facility with no equip-

ment, and still have to pay rent on

an old, obsolete laboratory.

FDA is negotiating with manu-

facturers on its next five-year plan

for collecting fees from industry

under by the Prescription Drug

User Fee Act (PDUFA). Meanwhile,

the agency wants drug and bio-

tech firms to ante up some $850

million in PDUFA fees in 2012,

up more than $275 million over

2010. About $600 million will

fund drug oversight, and $125

million will support regulatory

activities involving vaccines and

other biologics. Combined with

all the added costs imposed by

healthcare reform, and apparent

threats to expanding healthcare

coverage to some 30 million unin-

sured Americans, manufacturers

have to worry about the erosion

of resources to support new drug

development. ◆

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Burrill on Biotech

Dig

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The biotech industry has followed in close

step with the surging capital markets so far

this year, and by close of trading business

at the end of February, the Burrill Biotech Select

Index was up 3.6 %, year-to-date. For the same

period, the Dow Jones Industrial average was up

5.6 % and the Nasdaq Composite index was up

almost 5 %.

The industry got off on the right foot with a

generally upbeat mood pervading the annual

JPMorgan Healthcare Conference in San

Francisco in early January, reflecting the fact

that many biotech and pharma CEOs in atten-

dance felt that 2011 would be a good year for

their companies.

There was also a general consensus at the

meeting that biotech mergers and acquisitions

activity would increase significantly as a result

of larger biotechnology and Big Pharma com-

panies looking to bolster their product pipe-

lines and catalyze growth. We certainly didn’t

have to wait too long to see this scenario begin

to unfold. In late January, Amgen announced

that it would pay $1 billion to acquire BioVex,

a privately held biotechnology company based

in Woburn, Massachusetts, with additional

operations in Abingdon, United Kingdom. The

transaction provides Amgen with BioVex’s lead

product candidate, OncoVEXGM-CSF, an investi-

gational oncolytic vaccine currently

in phase III clinical development

as a treatment for metastatic mel-

anoma. A phase III multinational

study in head and neck cancer is

also ongoing.

Biotech’s second largest company

by market cap, Gilead Sciences, also

announced an acquisition, saying

that it will pay $375 million cash

to acquire privately-held Calistoga

Pharmaceuticals, a company focused

on cancer and inflammatory dis-

eases. Factoring in potential milestones, the

total deal could be worth around $600 million.

Commenting on the rationale for the deal, Gilead

said that it serves to further broaden their pipe-

line and expertise in the areas of oncology and

inflammation.

In February, sanofi-aventis and Genzyme

finally reached an agreement approximately

seven months after sanofi made its initial offer

of $69 per share to acquire Genzyme. In the end,

sanofi agreed to pay $74 per share in cash, and

give each Genzyme shareholder one contingent

value right for each share they own, which will

entitle them to additional cash payments tied

to specified milestones related to Genzyme’s

Lemtrada, Cerezyme, and Fabrazyme drugs,

according to the released terms of the deal. The

final closing of this deal caused a surge of investor

interest in the sector and fueled speculation about

other “blue-chip” biotech companies that could

be in the crosshairs of Big Pharma companies.

Another transaction of note was Novartis’

acquisition of San Diego-based diagnostics com-

pany Genoptix for $470 million. Genoptix is

focused on delivering personalized and compre-

hensive diagnostic services to community-based

hematologists and oncologists.

Biotech got off on the

right foot with a generally

upbeat mood pervading the

annual JPMorgan Healthcare

Conference in San Francisco

in early January.

G. Steven Burrill is chief executive

officer at Burrill & Company, San

Francisco, CA, 415.591.5400,

[email protected].

Biotech Starts the Year PositivelyMergers and acquisitions expected to increase, as big companies bolster piplines by acquiring smaller biotech companies

April 2011 BioPharm International www.biopharminternational.com 21

Burrill on Biotech

Company TickerIPO

PriceAmount Raised

($M)Price

2/28/11%

Change2/28/11 Market

Cap($M)

AcelRx ACRX $5.00 40 $3.62 -27.60% 70

Fluidigm FLDM $13.50 75 $14.15 4.81% 268

Gevo** GEVO $15.00 107 $19.71 31.40% 486

BG Medicine BGMD $7.00 35 $8.55 22.14% 157

Endocyte** ECYT $6.00 75 $7.33 22.17% 203

Pacira Pharmaceuticals PCRX $7.00 42 $6.90 -1.43% 119

Table I: Performance of Biotech IPOs completed in 2010.

Table II: February Burrill indices.

**Burrill & Company is an investor in the company. IPO is initial public offering.

Index 12/31/2010 1/31/2011 2/28/2011 % Change (Month)% Change

(year)

Burrill Select 365.12 366.06 378.21 3.32% 3.59%

Burrill Large Cap 526.55 526.81 527.28 0.09% 0.14%

Burrill Mid-Cap 218.1 208.49 212.22 1.79% -2.70%

Burrill Small Cap 94.97 91.88 93.91 2.21% -1.12%

Burrill Diagnostics 158.05 161.07 169.7 5.36% 7.37%

Personalized Medicine 106.26 109.28 110.19 0.83% 3.70%

NASDAQ 2652.87 2700.08 2782.27 3.04% 4.88%

DJIA 11577.51 11891.93 12226.34 2.81% 5.60%

We have seen plenty of deals

like this in the personalized medi-

cine space during the previous 12

months. As described in the just

released 25th anniversary issue of

the annual report on the industry,

Biotech 2011*, this sector is attract-

ing a great deal of attention. This

is because we are rapidly moving

to a world that embraces not only

the possibility of using a person’s

genetic information to identify their

risks for disease, but also being able

to construct individualized strategies

for prevention or treatment through

the use of biomarkers, molecular

diagnostics and targeted therapeu-

tics. Not surprisingly, companies in

the healthcare sector are positioning

themselves for this evolving trend.

A November 2010 study by the

Tufts Center for the Study of Drug

Development reported that per-

sonalized medicine is occupying a

growing role in the clinical pipelines

of drug developers and causing com-

panies to change their research and

development paradigms as a result.

Not only are we seeing the

impact that the field of personal-

ized medicine is having on drug

development, but also that it is part

of a wider discussion on how these

evolving technologies will have a

direct impact on helping to bring

healthcare costs under control.

To drive healthcare costs down,

we need to focus on encouraging

wellness rather than simply treat-

ing sickness. Interestingly enough,

personalized medicine is moving

to a “participatory” phase as peo-

ple become much more involved in

decisions about their own health-

care. Driving this trend is the fact

that healthcare is being impacted

by the convergence of informa-

tion technology, wireless technol-

ogy, and the proliferation of mobile

devices. WiFi-enabled devices

are allowing patients to connect

with their physicians remotely.

Smartphones are becoming per-

sonal healthcare assistants capable

of collecting and transmitting vital

body signs for analysis and subse-

quent results. This “digital health”

world is just evolving and will bring

about a radical change in how medi-

cine is practiced in the next decade.

BioteCH initiAl PuBliC oFFeringS (iP0S)Several biotech companies made

their US market debut in February.

With the exception of renewable

fuels and chemical developer Gevo,

all other biotech companies had

to significantly lower their pricing

expectations to get IPO deals done.

The average aftermarket perfor-

mance of the biotech IPOs at the

end of February was 8.6 %. ◆

* Biotech 2011–Life Sciences:

Looking Back to See Ahead is

G. Steven Burrill’s 25th annual

publication on the state of the

biotechnology industry. This special

anniversary edition examines how the

industry has developed into the global

enterprise it is today. It describes what

companies will need to do in order to

remain competitive in a world being

reshaped by technology, globalization

and emerging markets. In addition, it

provides comprehensive analysis on

the global industry’s performance in

2010. Details: www.burrillandco.com/

resources.


Perspectives on Outsourcing

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Ihad dinner recently with an old and very

insightful Big Pharma colleague of mine and

after speaking together, he revealed an impor-

tant concern. “Gregg, I’m worried,” he said. “I

think the complexities of our current global sup-

ply chain outweigh our capability to control the

network,” he continued. He was talking about his

company’s third-party external supply network.

It’s a scary thought, the idea of being too big and

complex to adequately control an organization’s

supply chain. But as my colleague and I discussed

this in more detail, I definitely understood why he

felt that way. For the past 20 years and especially

the past decade, the industry has watched the evo-

lution into “gigantic pharmaceutical drug-makers,”

such as Johnson & Johnson, Pfizer, Roche, Merck &

Co, Novartis GlaxoSmithKline, and sanofi-aventis,

all $40-billion-plus annual revenue behemoths.

These global giants are the results of mergers,

acquisitions, and myriad comarketing, in-licens-

ing, and other growth strategies to make up for

the disappointing failures of traditional R&D. The

resulting manufacturing “mash-ups” have created

third-party external supply networks that are dif-

ficult to manage because of the number of CMOs

involved, long-term contracts (some with substan-

dard organizations), contracts with no provisions

for ongoing cost reductions, and other nuances

that result in the buying organization

having a higher risk profile than desired.

Now, there is no doubt that making

changes to the network can be difficult

and expensive due to product registra-

tions and other regulatory hurdles on a

country-by-country basis. But if a com-

pany is willing to play a longer game,

with a forward vision, it is possible for

it to optimize its third-party external

network to reduce all types of supply risk

and ensure the ability to manufacture

products cost effectively.

There is a new methodology, called

collaborative optimization, which I’ve become

familiar with through my work as senior advisor for

A.T. Kearney Procurement and Analytic Solutions.

The methodology has its roots in optimizing trans-

portation logistics. It’s been successfully used in

many direct and indirect categories of spend as

diverse as packaging, chemicals, and temporary

labor and also to simultaneously optimize multiple

categories of spend used in sequential manufac-

turing processes. It was designed for complex cat-

egories of spend when the complexity comes from

many specifications, many potential suppliers with

a multitude of capabilities, and regional or global

manufacturing and distribution needs.

Collaborative optimization has three key compo-

nents. First, it establishes deep analytics in the cost

makeup of a product/service through the creation

of a detailed cost breakdown. Second, it gives sup-

pliers the ability to quote in multiple ways, includ-

ing creatively (i.e., to quote on parts of the business

that play to an individual supplier’s strengths or

to provide new ways to meet requirements more

cost efficiently). This function is called expressive

bidding and makes ups the “guts” of a new type

of request for quotation that has an abundance of

cost-rich information and cost-reduction ideas.

The real differentiator with collaborative optimi-

zation is the ability to use combinatorial optimiza-

tion through an embedded combinatorial analyzer

that allows for scenario comparisons to be rapidly

generated. These comparisons determine the most

desirable outcome based on what a corporation

must have. This requirement might be the lowest

total price or the best total cost when certain con-

straints are added. This method overcomes many of

the limitations in today’s procurement approaches

by fostering creativity into the quotation process

and handling large amounts of data in a fast and

efficient way, thereby enabling the buying entity

to determine the best solution for its specific needs.

What I like the best about collaborative optimiza-

tion is that it does not pit supplier against sup-

Gregg Brandyberry is CEO of Wildfire

Commerce, and senior advisor for

A.T. Kearney Procurement and

Analytic Solutions, tel. 215-327-5739,

[email protected].

A Hard Look at Third-Party External Supply Networks The complexity of third-party external supply networks requires new ways to manage them

April 2011 www.biopharminternational.com BioPharm International 23

Perspectives on Outsourcing

plier the way traditional procurement

practice often does. Instead, it identi-

fies the best way forward that is ben-

eficial to buyers and suppliers.

So, how could this methodology

be applied to a third-party external

network? To start, a corporation

would need to decide it wants to

fix the “mess” (a word used by the

late, great systems thinker, Russell L.

Ackoff, PhD, to describe most cur-

rent state situations). The company

also would need to recognize that the

fix might take 5 to 10 years to fully

implement. For a corporation that

was willing to play the long game

using collaborative optimization, the

benefits would be substantial. One

benefit is that it allows a company

to identify where new contract-

manufacturing opportunities should

be sourced. It also allows a com-

pany to understand where existing

contract manufacturing should be

sourced and if there is a financial or

risk-based business case to support

working through regulatory and con-

tractual barriers. This method also

ensures that the best CMO network

had been identified to reduce risk and

provide real cost competitiveness.

When a company thinks about its

the third-party external supply net-

work, a crucial question to consider

is whether the company is using a

CMO’s total capabilities across its

total product requirements on a

global basis. From my experience,

which includes more than 30 years

in procurement, the supply chain,

and related operations, including 10

years as vice-president of procure-

ment of global systems and opera-

tions at GlaxoSmithKline, I’m fairly

confident that the answer would be

“no.” And that reply is really no fault

of an individual or an organization. It

goes back to what my colleague and

I were discussing: the complexities of

what an organization is dealing with

are greater than its ability to con-

trol unless there is a commitment to

develop a long-term vision and a will-

ingness to use different approaches in

managing a third-party external sup-

ply network. Collaborative optimiza-

tion offers the potential for annual

incremental savings and potentially a

lower overall risk profile.

The key factor for effective supplier

management is that once a company

has established what its end-state

third-party external network should

look like and what suppliers should

constitute that network, it is imper-

ative that there is an ongoing col-

laborative environment in place that

ensures that future supply partners

are always incented to improve and

innovate. This evaluation may make

an organization take a hard look at

the skills (or lack thereof) that it has

in place to support third-party exter-

nal manufacturing. My guess is that

there are some gaps in relationship

management. Identifying and filling

those gaps is a first crucial step in

improving supplier management. ♦


Membrane Integrity Test

Over the years, there have been

a number of nondestructive

integrity tests for filtration

membranes conducted as part

of manufacturing quality assurance. Tests

are typically implemented by the filter

manufacturer as an additional quality

assurance test prior to shipping the prod-

uct to users.

Millipore has developed a test designed

to be more sensitive to detection of

defects. The new high sensitivity binary

gas integrity test is based on the principle

of differing gas permeabilities through the

liquid layer of a wetted membrane that

results in a concentration enhancement

of the faster permeating gas.

The test makes use of the fact that

the permeate composition in an intact

(integral) membrane, with no defects,

can be predicted based on the trans-

port properties of the gases permeat-

ing through the liquid layer and the

known operating conditions. So, if

the test spots a deviation from the

expected concentration, it is a clear

indication of a defect or the presence

of open pores.

The binary gas test has low sensitivity

to membrane porosity, liquid layer thick-

ness, and membrane area. This means

that integral devices will exhibit a rela-

tively narrow range of test values, making

it easier to spot defects.

To “test the test”, researchers applied

both the binary gas integrity test and the

standard gas–liquid diffusion method to

a newly developed virus clearance filter.

Results demonstrated that the binary gas

test provided a significantly higher level

of virus retention assurance compared to

the air–water diffusion test.

TesTIng for defecTs ThaT coMproMIse perforManceIntegrity testing of microporous or ultra-

porous filters is routinely used to detect

the presence of oversized pores or defects

that can compromise the filter’s reten-

tion capability. Test choices include the

particle challenge test, the liquid–liquid

porometry test, the bubble point test, the

The authors have developed a test for defects in filter membranes based on

the principle of differing gas permeabilities through the liquid layer of a wetted

membrane. Both the binary gas integrity test and the standard gas–liquid

diffusion method were applied to a newly developed virus clearance filter. Results

demonstrated that the binary gas test provided a significantly higher level of virus

retention assurance compared to the air–water diffusion test. The authors conclude

that the binary gas test provides superior defect detection sensitivity in virus filters.

new Binary gas Integrity Test Improves

Membrane Quality assurance

Sal Giglia, Mani Krishnan

Sal Giglia*is principal applications engineer,

and Mani Krishnan is director of single use

technologies, both at

eMd Millipore, [email protected]

peer reviewed

article submitted: aug. 24, 2010.

article accepted: sept. 30, 2010. AL

L F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

RS



Figure 2: Binary gas diffusion through a wetted membrane:

(a) integral and (b) non-integral membrane.

Slow gas

Fast gas

(a)

(b)

Membrane

Liquid filled pores

Figure 1: Gas diffusion through a wetted membrane:

(a) integreal and (b) non-integral membrane.

Gas Molecules

Liquid filled pores

Membrane

Low pressure side

(a)

(b)

gas–liquid diffusion test, and diffusion tests measuring

tracer components. Although each test has benefits, there

are also compromises that must be made.

The particle challenge test is destructive and therefore

not applicable as a pre–use test. The liquid–liquid porom-

etry and bubble point tests are useful for ensuring that the

user has selected a membrane with the proper nominal

pore, but are not sensitive enough to identify small num-

bers of small defects, particularly for filters larger than 47

mm. With the gas–liquid bubble point test, a single or few

small defects may add only a small amount of gas flow

that cannot be distinguished from the background diffu-

sive flow rate through the integral part of the membrane in

a filter device.

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Figure 3: Normalized permeability of

common gases in water at 25 oC.

1

10

100

10002CO

2O

2N

6SF

2C

No

rma

lize

d p

erm

ea

bil

ity.

6F

The most commonly

applied nondestructive

integrity test for mem-

brane filters, especially

virus filters, is the gas–liq-

uid diffusion test. A wetted

membrane provides a liq-

uid layer across which dif-

fusive air flow occurs (see

Figure 1a).

As pressure is increased,

diffusive flow increases lin-

early until either the liquid

layer begins to thin or until

the bubble point is reached,

whereupon robust bulk air

flow commences. As shown

in Figure 1, a measured gas

flow rate more than that

predicted for an integral

membrane signals the pres-

ence of a defect.

The sensitivity of this test is limited by the

minimum detectable excess flow. There can be

significant device–to-device variability in gas

diffusion flow rates of integral membrane filter

devices due to differences in membrane area,

membrane thickness, membrane porosity, and

pore tortuosity (twists and turns). Other factors

such as thinning of the liquid due to evapo-

ration, liquid retention of membrane support

layers (porous non-wovens, for example), and

membrane movement or compression can also

affect measured gas diffusion rate. This variabil-

ity in gas flow rate acts as “background noise”

that can diminish the sensitivity of the gas–liq-

uid diffusion test.

New INTegrITy TesT developedThe binary gas test was developed to increase

the level of defect detection sensitivity, and has

been found to be particularly useful for evaluat-

ing virus filtration membranes. It is based on

the principle of differing gas permeabilites of

a gas mixture’s two components through the

liquid layer of a wetted membrane.

Unlike the single gas in the gas–liquid dif-

fusion test, the binary gas test relies primar-

ily on the measurement of downstream gas

composition rather than downstream flow

rate. In an integral membrane, the permeate

gas is depleted of the slower permeating gas.

However, if a defect is present, the leak through

the membrane will contaminate the permeate

stream, resulting in an elevated concentration

of the slower permeating gas.

A key advantage of the binary gas test is that

the permeate concentration expected through

an integral membrane is well defined and its

sensitivity is not compromised by most of the

background noise factors cited for the gas-liq-

uid diffusion test. Figures 2a and 2b illustrate

how the test works.

UsINg gases To MeasUre defecTsDiffusion can be specified for each compo-

nent in a gas mixture permeating across a

membrane. Assuming that the gas is com-

pletely mixed on both sides of the mem-

brane, the composition of the permeate gas

can be calculated from the ratio of diffusive

flow rates of the two components, and the

inlet side composition.

The composition of the permeate gas is inde-

pendent of membrane thickness, tortuosity,

porosity, and area. It is also independent of the

pressure difference across the membrane but

instead is dependant on the pressure ratio. The

permeate composition does, of course, depend

on the feed side composition. To maintain a

constant feed side composition, a constant

sweep flow must be applied.

A measured binary gas composition can be

used to estimate a defect size in a device. The

calculated defect size can, in turn, be used to

estimate the liquid flow rate through the defect

and the total volume of liquid passing through

the defect for a given time period. Therefore,

the permeate gas concentration can be used

to predict the loss in virus log reduction value

(LRV) due to the defect.

The sensitivity of the binary gas test is

related to the selectivity (ratio of permeabil-

ites) of the gases through the liquid layer. To

maximize the sensitivity of the binary gas test,

the gas pair should have a high selectivity.

Figure 3 shows normalized permeabilites of

some common gases in water. For this study,

researchers selected a carbon dioxide/ hexaflu-

oroethane (CO2/C2F6) pair, with a selectivity

of about 1000.

The selection of the concentration of the

gases in the mixture was influenced by a num-

ber of factors, including the ease of composi-

tion measurement, gas flow rate through the

membrane, and economic considerations. The

concentration selected enables convenient flow

and composition measurement for even rela-



Figure 4: Laser hole drilled through a

Viresolve Pro membrane disc.

tively small membrane areas (as low as 3 cm2

membrane area).

coMparIng how The TesTs MeasUre conTrolled defecTs and vIrUs reTenTIonThe research included conducting integrity

tests on virus filtration membranes using both

the air–water diffusion test and the binary gas

test. The virus filtration membrane used was

Millipore Viresolve Pro (asymmetric PES) mem-

brane in flat sheet or disc formats.

Researchers introduced defects by laser drill-

ing 2–10 μm diameter holes in the center of the

membrane discs (see Figure 4).

For the air diffusion test, pressurized air was

applied to the upstream side of the membrane

and downstream air flow rate was measured

using a mass flow meter.

For the binary gas test, the CO2/C2F6 test

gas was introduced to the membrane at 345

kPa, and a constant sweep gas rate at a 4:1 ratio

relative to the permeate flow rate was main-

tained through the vent port of the filter holder.

Gas composition was measured using fourier

transform infrared spectroscopy (FTIR) (inDuct

FTIR, MKS Instruments).

M e a s u r e m e n t s w e r e

recorded continuously until

an essentially steady state

permeate composition was

achieved, typically within

15–20 minutes, which was

the time required to fully

flush out the residual air

from the volume down-

stream of the membrane,

the sample lines leading

to the FTIR, and the FTIR

sample chamber. A small

volume custom cell was

procured for the FTIR in

order to minimize the total

internal volume downstream of the membrane

and thereby reduce the overall test time.

After the initial air diffusion and binary gas

testing, researchers challenged the membrane

devices with a solution consisting of a bacterio-

phage mixed with polyclonal human immu-

noglobulin in a buffer solution. The solution

was filtered through the membrane until flux

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Figure 5: Defect detection by the air diffusion test.

Laser hole size (microns)

Feed gas = Air

Feed pressure = 345 KPaG

Permeate pressure = 0 KPaG

Typical integral diffusive flow range

00

1

2

3

4

5

2 4 6 8 10 12

Data

Model

Pe

rme

ate

flo

w r

ate

(cc

/min

)

Figure 6: Defect detection by the binary gas test.

Laser Hole Size (microns)

Pe

rme

ate

C2F

6 C

on

cen

tra

tio

n (

pp

mv

)

0

10

100

1000

10000

100000

2 4 6 8 10 12

Data

Model

Feed gas = 90/10 CO2/C2F6

Feed Pressure = 345 KPaG

Permeate Pressure = 0 KPaG

Integral layer range

FilterAir-Water Flux (cm3/min-m2)

Binary Gas Test Value

ØX-174 LRV

Device no. 1 12 72 5.9

Device no. 2 11 760 5.0

Device no. 3 12 284 5.6

Table I: Comparison of integrity test sensitivity between

the air-water and binary gas tests on prototype Viresolve

Pro devices.

had declined by 75% compared to the clean buffer. They

then collected feed and permeate samples, determined the

infectious titer, and calculated the virus LRV.

Figures 5 and 6 show the air diffusion and binary gas

test results as functions of defect size. The solid lines are

the model predictions for the air diffusion and binary

gas tests. The shaded regions in each graph show typical

test value ranges for integral membranes. These regions

represent background noise against which a signal for

a defect must be compared. Figure 5 shows that a 2 μm

defect was not “visible” to the air diffusion test because

the additional flow rate due to the defect was not large

enough to increase the total flow rate beyond the range

typically measured for integral membranes. In contrast,

as shown in Figure 6, the elevated C2F6 concentra-

tion in the permeate is a clear signal for the same 2 μm

defect. This result was an unambiguous demonstration

of the binary gas test’s superior defect detection sensitiv-

ity. For defect sizes larger than 2 μm, both tests provided

a strong signal for a defect.

The research also showed that the loss in LRV com-

pared to an integral membrane due to a single defect

can be predicted from the binary gas value. This means

that a maximum allowable binary gas value can be

established for a desired level of LRV assurance based on

a worst case assumption of a single defect.

In addition to using the test in conjunction with

controlled defects, researchers looked at defect detec-

tion sensitivity in manufactured devices. Both tests

were applied to a set of prototype Viresolve Pro devices.

A portion of the two–layered membrane used to manu-

facture the devices was tested for virus retention and

the LRV of the three discs was determined to be 5.9.

The LRV of the devices, however, ranged from 5.0 to

5.9. While the air diffusion test did not differentiate

among these devices, the binary gas test showed clearly

elevated values for the two devices with lower LRV val-

ues (see Table I).

coNclUsIoN Compared to the conventional air-water diffusion test,

the binary gas test provides superior defect detection

sensitivity in virus filters. While the air diffusion test

provided an LRV assurance of about 4.5–5.0 for the

virus filters studied, the binary gas test can provide an

LRV assurance of greater than 6.0.

The greater sensitivity of the binary gas test is due to

a much more favorable signal–to–noise ratio than the

air-water diffusion test. Unlike the gas–liquid diffusion

test, the binary gas test has low sensitivity to mem-

brane porosity, liquid layer thickness, and membrane

area. Other factors that can confound the sensitivity of

the air diffusion test such as thinning of the liquid due

to membrane asymmetry or evaporation, liquid reten-

tion of membrane support layers (porous non–wovens,

for example), and membrane movement or compres-

sion have a much lower impact on the sensitivity of

the binary gas test. ◆

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Drug-Substance Uniformity

The final step in drug-substance

(active pharmaceutical ingredi-

ent, API) manufacture is typi-

cally a 0.2 µm filtration step.

This filtration step serves as a final clari-

fication of the process pool and also as a

bioburden control measure prior to the

storage and further processing of drug

substance (DS) to drug product (DP).

There are many technical considerations

to ensure a consistent and robust bulk fil-

tration step, for example:

• Selection and sizing of the filter

• Materials of construction for the DS

containers and tubing for transfers

• Filtration equipment procedures

• Filter preparation to reduce extractable

and leachable components

• Container–closure integrity.

One aspect that must not be overlooked

is the DS uniformity (or homogeneity)

which is discussed in this article. When

designing validation studies, it is impor-

tant to consider the stringent regulatory

expectations for ensuring batch unifor-

mity and integrity of drug products (1).

The DS may be filtered into single

or multiple vessels. In the former case

(single agitated vessel), uniformity con-

siderations are mixing speed and mix-

ing time prior to taking a sample that is

representative of the entire contents of

the vessel. In the latter case, it is neces-

sary to demonstrate uniformity between

the DS containers. The initial concentra-

tion of product effluent from the filter

may be expected to be slightly lower

than the rest of the pool due to dilu-

tion with residual flush solutions in the

filter pores and housing assembly, as

well as due to non-specific protein or

excipient adsorption to the filter. Thus,

uniformity acceptance criteria should

take into consideration an asymptotic

increase in concentration during a rea-

sonable initial product volume. This

paper describes risk-based approaches

to establish rigorous acceptance criteria

abstract

Drug-substance uniformity is an important consideration for the final step in the

manufacture of drug substance/active pharmaceutical ingredient. Uniformity studies

are necessary to ensure that the entire contents of the batch are homogenous and

that the drug substance specification sample is representative of the batch. this

paper describes considerations for drug-substance uniformity, such as selection

of appropriate test parameters and sample points, and approaches to establishing

acceptance criteria. additionally, operational considerations and best practices to

ensure robust and consistent drug substance filtration and uniformity are described.

Practical Considerations for Demonstrating Drug-

Substance Uniformity for Biological Products

Sushil Abraham, Eric Rydholm, and Phil Wagner

Sushil Abraham* is director of process development, Eric Rydholm, is principal

engineer, and Phil Wagner is senior engineer, all at Amgen, Longmont CO, [email protected].

PEER REviEWEd

Article submitted: Oct. 20, 2010.

Article accepted: Feb. 11, 2011.



and define operational parameters

to ensure consistent DS uniformity.

RelevAnt PeRFORmAnCe PARAmeteRS FOR A DRUg-SUBStAnCe UniFORmity StUDyThe DS specification parameters

for biological products confirm the

identity, purity, potency, quality

and safety of the API. The ultimate

aim of a DS uniformity study is to

ensure that individual DS containers

are consistent with respect to all of

these critical quality attributes (CQAs)

or specification parameters. For the

purpose of demonstrating unifor-

mity, it is not necessary to test each

of the DS specification parameters,

since a subset of the parameters may

be used as a surrogate for the others.

Typically, the quantitative specifica-

tion parameters can be used to dem-

onstrate uniformity. Representative

assays may include protein concen-

tration by absorbance (e.g., UV280),

high performance liquid chromatog-

raphy (HPLC) or bioassay. Of these,

the UV280 is the simplest and fast-

est measurement with an acceptable

degree of accuracy and precision.

Protein concentration also allows

evaluation of possible mechanisms

for introduction of non-uniformity

to the DS batch (e.g., dilution due to

residual flush liquid, protein adsorp-

tion to the filter material, or inher-

ent uniformity challenges with the

upstream pool). Other parameters,

such as pH, osmolality, conductivity

and/or purity (e.g., aggregate concen-

tration) may also be used as measures

for DS uniformity. It is important to

consider the potential failure modes

for uniformity when selecting per-

formance parameters such that they

are sensitive enough to pick up a lack

of uniformity. For example, if dilu-

tion with residual water is a poten-

tial source for non-uniformity, pH

may not be the best parameter to use

because water for injection (WFI) may

not significantly impact the buffering

capacity of the drug substance formu-

lation buffer. Additionally, stabilizing

agents such as polysorbate may be

a critical component of the DS and

it may be necessary to demonstrate

that the concentration of such agents

is within required limits. Other fac-

tors, such as shear could impact DS

product quality (particle size, aggrega-

tion, potency), and a risk assessment

should be considered to identify and

mitigate these potential outcomes.

Validation provides evidence of suffi-

cient uniformity such that any sample

location across the fill is representa-

tive of the entire DS lot with respect

to the CQAs. Validation allows batch

release and stability testing to be

performed on a representative sam-

ple collected from a single location

within the DS fill operation.

Another key question is when to

take the samples to demonstrate uni-

formity and how to establish appro-

priate validation acceptance criteria.

Typically, samples may be taken at

the beginning, middle, and end of

bulk filtration, and if they meet pro-

spective acceptance criteria, the entire

batch can be considered to be homog-

enous. The sample collection strategy

is an important consideration: Does

one take a point sample (i.e. directly

from the filter bell), or a pool sample

from the actual container? The lat-

ter is more relevant but needs to be

balanced against the potential risk of

contamination during sampling, and

mixing considerations prior to taking

the sample. Typically, the beginning

sample is taken as a pool sample from

the first container, as this reflects how

the contents of the DS will be for-

ward processed. If the DS lot-release

and stability samples are taken as

point samples at the middle of bulk

filtration, the use of point samples

during a uniformity study may be

favorable for consistency and to mini-

mize potential for contamination.

There are several ways to establish

acceptance criteria to demonstrate DS

uniformity, which may be used in iso-

lation or in combination, as appro-

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Table I: Typical processing steps in drug substance �ltration operation.

Operational Step Purpose of Step

Assemble filter and housing —

Weight check Establish dry weight of filter assembly

Flush filter with WFI and/or bufferTo remove potential extractables/leachables and to wet filter

for pre-use integrity test

Pre-use integrity test To confirm filter is integral prior to product processing

Air purge Remove residual liquid from filter assembly

Weight check To confirm that residual liquid in filter assembly is minimal

and within process expectations

Autoclave/SIP filter assembly Bioburden control

Buffer flush If required to equilibrate filter

Optional air purge and weight check (may not always be necessary)

Remove residual liquid from filter assembly

Product processing – product flush to drain (may sometimes be necessary)

To minimize dilution impact and thereby ensure that product collected as part of the batch is uniform

Product filtration Bioburden control measure

Post-use integrity test To confirm that the filter was integral during processing

Bulk 0.2µm

filter

Single drug

substance

vessel

Uniformity parameters:

Agitation speed (N)

Mixing time (Tm )

Bulk 0.2µm

filter

Uniformity parameters:Multiple drug substance containers

Feed tank

Feed tank

Protein concentration or other

parameters, e.g., between

beginning, middle and end of

filtration

Figure 1: Flow diagram of drug substance �ltration.

priate. Prior to selecting a preferred

strategy, it is important to understand

how the DS will be used during the

subsequent DP formulation and test-

ing, and to ensure that the require-

ments for these steps are addressed in

the DS approach. Several methods for

DS testing, and their pros and cons,

are briefly discussed below.

Calculation based on DP specifica-

tion limits

This method is based on a back cal-

culation of the requirements for DP

processing and DP specification cri-

teria. The DS batch may be divided

and used over several DP batches. In

order to maintain this flexibility, it is

necessary to ensure that any DS batch

or part thereof used for DP processing

will meet the DP specification require-

ments. It is necessary to take into

account the smallest DP stock keeping

unit (SKU), including product concen-

tration and manufacturing volume,

which will be used. The advantage

of this method is that it is based on

processing needs. However, it may

be complicated by the existence of

multiple SKUs and the desired safety

margin between a passing uniformity

result for DS compared to the associ-

ated specification range for DP.

Requirements at or tighter than the

DS specification limits

The criteria for DS uniformity may

be established to be the same as the

DS specification limits. While this is

a relatively simple way of setting the

uniformity process validation accep-

tance criteria (PVAC), it does carry

the undesirable risk of failure for DP

specification and uniformity limits

if the sample measurements are at

the DS specification limits, especially

if there are stability concerns during

storage. One way to reduce this risk is

to set the validation acceptance crite-

ria within the DS specification limits,

thereby providing a safety margin.

One should also ensure that the docu-

mented analytical method precision is

able to achieve results of the reduced

range. The advantage of this approach

is its simplicity in applying a safety

margin relative to the specification

limits, which ensures robustness in

meeting the process needs. The down-

side is that the safety margin allow-

ance may be subjective and may not

align with the method precision.

Inclusion of analytical method vari-

ance (to account for potential mea-

surement error)

This strategy takes into consideration

all potential errors that could con- AL

L F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

RS


where:– value for each sample: Tstart = X1; Tmiddle = X2; Tend = X3

– calculated average for all samples (X1, X2, and X3);

n – number of samples

tribute variability to the uniformity

results, such as analytical method

variability and volume measure-

ment for the product and additives,

in order to ensure acceptable DS uni-

formity. The root mean squares of

the potential errors are calculated to

derive the acceptance criteria. The

acceptance criteria may be established

based on the analytical method vari-

ability or precision when the analyti-

cal method variance is significantly

greater (more than one order of mag-

nitude) than the process variance. It

should be emphasized that the pro-

spective method validation accep-

tance criteria should be used in the

calculation, rather than the results

from the analytical method valida-

tion exercise. One must also ensure

that sound technical justification

exists for how the method validation

acceptance criteria are developed. The

latter is typically based on a fairly

small data set and could result in

overly tight acceptance criteria, result-

ing in a failure of the uniformity

study. The acceptance criteria is set so

that the percentage relative standard

deviation (RSD) of the sample points

is less than or equal to the analytical

method precision. An assessment on

process impact in using this method

is recommended to ensure that vali-

dation acceptance criteria are not set

too wide.

Percentage of the feed stream con-

centration

Another simple way to establish uni-

formity study validation acceptance

criteria is to evaluate individual sam-

ples against a percentage of the feed

material concentration. Selection of

the actual percentage value could use

a similar approach to that described

the two previous methods. This

method is based on the assump-

tion that typically, there may be a

small amount of product adsorption

onto the bulk filter or dilution from

flush water/buffer retained in the fil-

ter apparatus. As such, this source of

variability is expected during the ini-

tial phase of the bulk filtration step.

This method is applicable where the

principal uniformity failure mode is

based on dilution. It is fairly simple to

implement, but can be problematic if

the method variability is high.

Tolerance interval

This approach may be used if ade-

quate historical filtration data

exists to calculate a tolerance inter-

val and capture the expected long-

term behavior of the DS fill process.

Typically, the tolerance interval con-

tains 99% of the population (cover-

age) with a 95% confidence limit.

However, the confidence and popula-

tion coverage depends on the size of

the historical data set. Care should be

taken in combining data from labo-

ratory or pilot scale filtrations with

full–scale or manufacturing data, as

the non-recoverable volume of the

systems may result in substantial dif-

ferences in terms of batch uniformity.

Additionally, the calculated unifor-

mity limits should not be wider than

the DS/DP specification limits. While

this method is based on actual histor-

ical experience, it does require addi-

tional sampling and testing during

clinical lots.

Equivalency acceptance criteria

The objective of the equivalency

approach is to test the null hypothesis

of non-equivalence (non-uniformity)

within a DS batch. If the null hypoth-

esis is rejected, evidence of unifor-

mity within a batch is demonstrated.

Multiple samples are required for each

sample point within the batch, and

the means are calculated for each

sample location. Uniformity within




80%

85%

90%

95%

100%

0 100 200 300 400 500 600 700 800 900 1000

Perc

en

tag

e in

itia

l co

nce

ntr

ati

on

Volume (mL)

0.1 M NaCl run #1

0.1 M NaCl run #2

Product filtration run #1

Figure 2: Concentration curves for surrogate (0.1 M NaCl) and

product �ltrations.

a batch is demonstrated when proscribed con-

fidence intervals (typically at 90% or 95%) of

the difference between the means are within

the calculated acceptance criteria. The benefit

of this equivalence acceptance criteria (EAC)

approach is a statistically defined proof of

uniformity within the allowable variability.

However, this method does require a larger

number of samples to be collected from each

sample point in order to have sufficient statisti-

cal power to make the acceptance criteria mean-

ingful.

–EAC<μ1 – μ2<EAC



where:

μ1, μ2, μ3 are the mean of the sample test

parameter over the course of bulk filtration

(e.g. protein concentration at the beginning,

middle and end), and EAC is the equivalency

acceptance criteria. It is beyond the scope of

this paper to describe equivalence testing;

References 2 and 3 provide general sources on

statistical equivalence.

OPeRAtiOnAl PARAmeteRSPrior to entering uniformity validation, it is

necessary to have robust control of the prepara-

tion operations for the bulk filtration step. If the

bulk filter is autoclaved or sterilized in place,

this may result in retention of steam condensate

on the filter, filter housing, and/or system pip-

ing, which in turn could result in dilution of

the DS and increased variability in uniformity

results. Additionally, the filter may be integrity

tested prior to use (either prior to or after ster-

ilization) or preflushed with buffer to remove

potential extractables/leachables and equilibrate

the filter. The buffer flush is especially impor-

tant if the formulation buffer contains polysor-

bate or other agents which are known to bind

to the filter. The buffer flush could alternatively

be performed post autoclave/SIP of the filter,

however, this adds complexity to the operation

to ensure that the aseptic state of the equipment

is not compromised during the flush. The integ-

rity test procedure requires wetting of the filter

with water. Although the majority of water is

removed during the integrity test (diffusion or

bubble point), there may be sufficient residual

water retained in the filter. A good practice is

to implement an in-process control point to

ensure that the residual liquid from the integ-

rity test flush, buffer flush, or steam condensate

is reduced to an acceptable level prior to process-

ing. This approach could be carried out in the

form of an air-purge step, a filter-drying step

(in an oven),or a simple comparison of the filter

weight before and after preparation steps. The

allowable residual fluid in the filter can be cal-

culated based on the uniformity requirements.

Such control measures provide confidence that

the potential sources of dilution are removed or

minimized prior to validation.

These controls are especially important to

ensure that the DS meets specification limits in

the event of reprocessing across the bulk filtra-

tion step (e.g., if the post-use integrity test fails).

Table I highlights typical steps in preparation

for the drug substance filtration operation. It

should be noted that not all the steps shown

in Table I may be required; some steps may be

eliminated depending on the need for opera-

tional simplicity, for example, by minimizing

manipulations such as the air purge or buffer

flush to the filter assembly post autoclave. The

final decision on the number and order of steps

should ensure that the potential failure modes

are adequately addressed while maintaining a

robust and consistent process to meet the uni-

formity requirements.

In addition to the controls described above,

it may be necessary to send a pre-determined

product flush to drain in order to reduce dilu-

tion effects. While it is desirable to minimize

this loss of product, it is imperative to ensure

that yield optimization does not override the

quality consideration for demonstrating the

uniformity of the batch. Regarding uniformity

performance parameters and acceptance criteria

discussed previously, it should be noted that it is

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not necessary to demonstrate that the first drop

emerging from the DS filter is absolutely identi-

cal/uniform with the rest of the batch as long

as the lot release samples are not taken at this

point. The quality consideration should take

into account acceptance criteria that are scien-

tifically justifiable and based on how the drug

substance will be forward processed.

Another important consideration with

respect to the product flush is the size of the

filter and filter housing. While it is appropriate

to size the filter with a safety margin so that it

can process batches in a robust manner and in

a reasonable time frame, oversizing the filter

could result in challenges from a bulk unifor-

mity perspective. One way to address this issue

is to perform a prefiltration step with a larger

filter to remove particulates which may foul the

0.2 µm filter. The prefiltration can be performed

as part of the preceding unit operation so as to

collect a clarified feed stream for the bulk filtra-

tion step. A further variation is to perform a

batch-wise bulk filtration into a single collection

vessel, thereby allowing for a buffer chase to

maximize product recovery and also any dilu-

tion that may be necessary to achieve the target

product concentration. This option could also

be adapted into a continuous mode by the use

of a surge tank between the filter and collection

containers to avoid dilution effects. Following

adequate mixing, the product may be dispensed

aseptically into appropriate collection vessels.

This obviates the need for the product flush,

and thus maximizes product recovery during

processing.

Other considerations to achieve uniformity

at the bulk filtration step are the design and

operation of the upstream feed vessel. Mixing

studies should be used to establish set point and

ranges for agitation speed and time to ensure

thorough mixing of the contents to be filtered

(e.g., top, middle and bottom of vessel). The

design of the vessel is also important to ensure

that dead zones and holdup volumes (both

line and sample port) are minimized. This is

especially important if dip tubes are present,

which may be used for product introduction

into the vessel and/or subsequent withdrawal

for the bulk filtration step. Both examples may

impact uniformity if lines are not efficiently

flushed, or if hydrostatic pressure within the

tube results in concentration changes over the

course of the filtration. It is preferable to use

separate routes for product introduction and to

locate dip tubes such that they do not result in

a dead zone within the tank. The vessel loca-

tion and piping required to transfer the product

from the feed vessel through the filter should

also be evaluated to minimize holdup volume

and ensure proper drainage (to account for con-

densate drainage following product transfer line

steaming) in order to minimize the possibility

of dilution.

BeSt PRACtiCeSCharacterization studies to confirm that prep-

aration procedures are adequate and robust

should be performed prior to performing uni-

formity studies with product. These can be

performed using buffers or salt solutions with

pH and/or conductivity as convenient indica-

tors to assess uniformity through filtration.

Careful assessment of the buffer used for such

studies is necessary to ensure that the buffer

selected is a representative model to use, and

parameters such as density and viscosity which

could impact the kinetics of filtration should

be considered. Figure 2 shows the result from

one such study where a sodium chloride solu-

tion was used to determine whether a prod-

uct flush would be required. As expected, the

initial samples during the filtration step have

a slightly lower conductivity but this quickly

stabilizes to greater than 97% of the initial

concentration. The results from replicate stud-

ies are consistent, showing that the operation

is reproducible. An alternative strategy would

be to utilize a representative protein surrogate,

if possible.

It would be prudent to also perform a (non-

GMP) engineering run with product prior to the

process validation studies. This provides added

assurance that the uniformity validation study

acceptance criteria will be met. Figure 3 also

compares the product profile with the surrogate

salt filtration runs, confirming the results and

conclusions drawn from the wet testing. If there

is a potential for reprocessing at the bulk filtra-

tion step (e.g., as a result of failed filter integrity

test post use or operational errors which may

have compromised the aseptic nature of the

batch), it is recommended to test the uniformity

of the batch at the reprocessing step during the

engineering run.

The standard practice in industry is to per-

form uniformity validation during the con-

formance runs (also referred to as process

performance qualification, and historically



referred to as process validation). Future process

changes also require an assessment of the vali-

dated state of the step and whether revalidation

is necessary to confirm that uniformity is not

affected. However, since ongoing uniformity

testing is not typically performed, subtle shifts

or trends in the process would not be detected.

The use of the filter weight checks as inpro-

cess controls along with robust maintenance

of operational parameters provide assurance

that DS uniformity is maintained. It may be

desirable to perform uniformity testing on a

periodic basis to provide further confirmation.

The approach of using the “percentage of feed

stream concentration,” described above, pro-

vides a simple means to confirm uniformity on

an ongoing basis.

COnClUSiOnDrug-substance uniformity is an important

consideration for the final step in the manu-

facture of API. Uniformity validation studies

are necessary to ensure that the entire contents

of the batch are homogenous and that the

drug substance specification and stability sam-

ples are representative of the batch in terms

of critical quality attributes. Using risk-based

approaches and comprehensive process char-

acterization studies, appropriate test param-

eters (e.g., protein concentration) and sample

points (beginning, middle, and end) can be

selected. Scientifically sound strategies which

may be used in combination for developing

uniformity acceptance criteria include those

based on specification limits, measurement

error, tolerance intervals of historical data and

equivalency of sample means. Additionally,

operational considerations, such as filter

weight checks, pre-processing air drying, prod-

uct flush, and the use of wet testing and/or

engineering runs provide greater assurance of

robust and consistent drug substance filtration

and uniformity. ◆

ReFeRenCeS 1. 21 CFR 211 (Government Printing Office, Washington

DC), section 110.

2. G.B. Limenati, Analy. Chem. 6, 1A–6A, (2005).

3. S. Richter and A. Richter, Qual. Engin. 14 (3), 375–

380 (2002).

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Bioprocess Optimization

The introduction of new technol-

ogy often means a significant

step forward in the performance

of one subprocess, but only

an incremental improvement in the full

product development process. In the case

of Next Generation Genomics (NGG)

Technologies, a series of incremental tech-

nology advancements has brought about

the ability to radically change the way bio-

processes are developed and optimized.

Bioprocessing in pharmaceuticals and in

industrial biotech have significantly differ-

ent economic drivers, but both can real-

ize significant economic benefit from the

application of these new technologies.

Pharmaceutical applications are driven

foremost by the cost of development,

regulatory approval, and compliance, and

only secondarily by process productivity.

In contrast, the primary market driver of

industrial bioprocesses is productivity, par-

ticularly in commodity and biofuel appli-

cations. In this paper, we focus on the

practical application to biopharmaceuti-

cals, which need to increase emphasis on

productivity of manufacturing due to the

continual rise in health care costs, and the

expansion of access to pharmaceuticals in

developing countries. In addition, there is

the potential for significant impact of NGG

in the emerging FDA initiative, known as

quality by design (QbD).

For new biologics to be profitable, they

must be developed in a cost-effective man-

ner and optimized to produce the high-

est possible titers. For existing biologics

to remain profitable, especially with the

emergence of biosimilars, they must be

efficiently optimized in order to improve

productivity and scales, with the resultant

lowering of cost–of–goods. Remarkably,

most of the research currently conducted

uses outdated tools and is performed gener-

ally on model cell lines that have been sub-

jected to numerous population doubling

events that, over time, induce extensive

genetic polymorphisms, ultimately decreas-

ing product quality and process stability

(1). When it comes to production of these

newly developed biologics, total economic

pressure is a key driver of success. Aside

from the inherent complexity (structural,

glycosylation, folding, stability, etc.) of the

biopharmaceutical products themselves,

bioprocess engineers are also faced with the

intricacy of the production process itself.

For each product, a cell line with sufficient

production phenotypes has to be devel-

oped. Current strategies involve time con-

suming, labor–intensive steps, from the

introduction of the product genes to the

isolation and characterization of candidate

clones. Cell-line development spans sev-

eral months, or in some cases, years, and

involves the screening of several hundred

cell clones for high productivity before a

few dozen are selected as candidate pro-

duction lines. The process typically lasts

for up to six months for each candidate

Accelerating Bioprocess Optimization Through the Use of Next Generation

Genomics TechnologiesLen van Zyl, Michael Zapata III

An understanding of changes in gene expression can be used to fine–tune bioprocessing

Len van Zyl*, PhD, is the CEO and CSO of

ArrayXpress and a faculty member at NC

State University. Michael Zapata III is the

chairman of the board at ArrayXpress Inc.

[email protected]



before it can enter the evaluation

phase, where its efficacy and safety in

animal and human subjects are deter-

mined (2). Once the process has been

established and approved, follow–on

improvements become very costly,

each of which must address FDA’s

requirements for quality and safety.

In the past, insufficient knowledge of

the biology of the production organ-

ism and the impact of the conditions

it is grown under made it difficult to

maintain stabile product quality attri-

butes when variables had changed.

We believe that NGG provides a mod-

ern and comprehensive approach to

address this gap.

There is also a regulatory benefit to

implementing NGG techniques. In

an innovative and forward–thinking

move, FDA’s QbD initiative empha-

sizes the achievement of product

quality by thorough process under-

standing, monitoring, and control.

The approach allow manufacturers

to identify critical process parame-

ters (CPPs) and the direct effects they

have on product quality. Adopting

QbD principles and process analyti-

cal technology (PAT) guidelines, can

help ensure an overall understanding

of the bioprocess, ultimately assist-

ing manufactures to achieve process

robustness, stability and quality. PAT

has been defined by FDA as a mecha-

nism to design, analyze, and control

pharmaceutical manufacturing pro-

cesses through the measurement of

CPPs that affect various critical qual-

ity attributes (CQA). The belief is that

with more complete understanding

comes the ability to not only develop

products more quickly, but also to

knowledgably and safely optimize

products and processes downstream,

while continuing to maintain higher

levels of quality control than previ-

ously achievable.

UNdErSTANdING ThE BIOlOGy Of ThE PrOdUCTION OrGANISmIn contrast to small molecules, the

production of biopharmaceuticals

is a process–within–a–process, with

the cultivation process supporting

the metabolic production process

within the cell. In order to develop

a detailed process understanding,

one must understand the biology of

the organism and its environment.

In short, what is required is a sys-

tems biology approach, with a focus

on building a solid understanding

regarding the biology of the cells

themselves. Ultimately, the cells are

the primary production vehicles, and

by following PAT guidelines and QbD

principles, one can develop a clear

understanding of the biology of the

cells and control of the overall bio-

process. By identifying both genomic

and metabolic factors that play into

the production of therapeutic bio-

logics, process engineers can identify

CPPs and characterize the impact that

each of the variable CPPs (i.e., media

conditions, fermentation conditions,

pH, temperature, dissolved oxygen

content) has on titers and quality.

Process engineers can identify how

well these variables can be controlled,

and subsequently establish the criti-

cality of these variables within the

overall bioprocess. By understanding

these CPPs and their importance in

the overall bioprocess (e.g., effects on

product yield, quality, and process

stability), process engineers can rap-

idly and effectively improve produc-

tion titers and process robustness. We

took the approach of initially focus-

ing on the biology of the produc-

tion cells. By focusing on the genetic

changes associated with the overall

bioprocess, one develops a very clear

understanding of how changes in

CPPs influence production yields and

overall product quality. Once manu-

facturers understand the intricacies

of the overall bioprocess, one can

consider bioprocess flexibility.

For existing processes and products

already in revenue–generating pro-

duction, the cost of one change, even

if it results in significant increases in

yield and stability, can often be too

high to be recuperated within the

product lifecycle. Process changes,

even if they could be made tech-

nically, are rarely made because of

the costs associated with requalifi-

cation and validation of the overall

process. Bioprocess flexibility in the

recent past has been associated with

increased safety risks, primarily due

to our lack of understanding of the

actual biology of the production cells

and how it affects process quality

and stability. Flexible manufacturing,

implemented with a systems–biol-

ogy understanding, fits well within

FDA’s current QbD initiative and

allows for ongoing process improve-

ment. We believe NGG is a key tool

for the acquisition and rational use

of this understanding. In a flexible

manufacturing environment, once a

design space has been defined, using

NGG and other tools, manufactur-

ers will have the flexibility to make

process changes within that design

space with no prior approval from

FDA regulators. Manufacturers that

adopt this approach will be able to

regularly improve their performance

and efficiencies, while maintaining

higher stability and quality control,

and while also reducing the costs of

recertification.

ThE PrACTICAl ImPlEmENTATION Of NGG fOr ACCElErATING BIOPrOCESS QBd One such project that the authors

have been involved with is a col-

laborative partnership between a

major pharmaceutical company and

ArrayXpress, a contract genomics

services company. While the specific

organism and target compound are

confidential, the tools, techniques

and processes utilized provide a great

example for demonstrating the ben-

efits of the NGG systems biology

approach. The primary objective of

the project under discussion was to

increase production titers of an essen-

tial target compound used in the

manufacturing process of a current

large revenue generating commercial

product. The secondary objective was

to build knowledge that will allow AL

L F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

RS



Figure 1: Fishbone diagram showing all the confirmed and putative CPPs

associated with the overall target compound production process.

Export of

product?

Envir

on

men

tal F

act

ors

Reg

ula

tio

n (

Tran

scri

pti

on)

Reg

ula

tio

n (

Tran

slati

on)

– Temperature

– Osmolarity

– pH

– Shear Stress

– Dissolved Oxygen

– Free Radicals

– Wrong Amount?

– Need in Feed?

– Wrong Form?

– Wrong Amount?

– Need in Feed?

– Missing?

– Missing?

– Wrong Amount?

– Wrong Amount?

– Missing?

– Wrong Amount?Carbon Source

Nitrogen

Amino Acids

Phosphorus

Nucleosides

Lipids

Minerals

Vitamins

– Wrong Amount?

– Need in Feed?

– Need in Feed?

– Missing?

– Chelator needed?

– Wrong Amount?

– Need in Feed?

– Missing?

– Wrong Amount?

– Need in Feed?

– Need in Feed?

– Wrong Form?

Product

Produced

– Local Regulators

MacroNutrie

nts

MicroNutrie

nts

– Codon Usage

– tRNA expression

– Ribosome

capacity

– Plasmid Copy Number

– Sigm

a Fa

ctor

s

– Re

gulato

rs

– stress

for faster and more efficient man-

ufacturing of other products using

the same organism/expression plat-

form. As with all bioprocesses, the

organism itself is only a single vari-

able influencing productivity, with

many environmentally tunable vari-

ables making up the remainder. We

have CPPs that influence production,

but due to prior technology limita-

tions, the manufacturing engineers

did not know their full impact on the

metabolic processes and production

efficiencies of the cells. Therefore,

these parameters had previously

simply been lumped together as an

unknown called “process variabil-

ity” or “biological variability”, and as

such, their manufacturing was com-

pletely at the mercy of the process

itself, with limited process stability

and dramatic product titer variability.

By bringing together the cells

and the CPPs in a systems model,

we can now see the entire equation.

The cells are the primary production

machinery; therefore our approach

was to evaluate the physiological

condition and the state of the cells

during the various media and fer-

mentation development stages. We

first generated a working hypotheses

by developing a fishbone diagram

that showed all the confirmed and

putative CPPs associated with the

overall target compound production

process (see Figure 1). This allowed

for the identification of critical areas

to be characterized in more detail,

which was subsequently experimen-

tally tested.

Our approach was to design

highly focused and statistically

sound microarray experiments with

complementing standard analytical

chemistry tests. We wish to empha-

size the importance of having a very

well thought out experimental design

and analysis strategy prior to proj-

ect initiation. This approach made

it possible to identify key genes and

their associated molecular path-

ways that were differentially affected

due to changes of various CPPs in

the overall production process. The

use of DNA microarrays provides a

detailed qualitative snapshot of the

state of the transcriptome at the time

of sampling, somewhat like a molecu-

lar fingerprint, that can reveal subtle

process variations in great detail. This

approach is especially useful in time

course experiments like the ones we

faced, to determine whole transcrip-

tome changes associated with differ-

ent CPPs, monitored across different

growth phases (different time points)

of the cells during the media and fer-

mentation optimization stages.

Strong bioinformatics, both in sta-

tistical design and data analysis and

mining, are the next key to success.

A particularly important aspect of

statistical inference in high through-

put problems, such as microarray

experiments, is the assessment of

statistical significance exhibited by

the data in the presence of a tre-

mendous multiplicity of hypothe-

ses. A single experiment can involve

tens of thousands of hypothesis

tests. This assessment requires effi-

cient estimation of experimental

error and careful control of false dis-

covery rates. We applied two inter-

connected analysis–of–variance

models: A normalization model

that accounts for experiment–wide

systematic effects that could bias

inferences made from the data on

individual genes, and a gene model

that is fit to the normalized data

from each gene, allowing inferences

to be made using separate estimates

of variability. Expression differences

are then parameterized as factorial

effects in linear mixed effects mod-

els appropriate to the experimental

design. These effects can be esti-

mated efficiently using statistical

softwaresuch as JMP Genomics or

SAS PROC MIXED. Resulting least

square estimates are then mapped

onto their associated metabolic path-

ways using KEGG metabolic path-

way maps (www.genome.jp/kegg/

pathway.html) in combination

with proprietary software mapping

tools (3).

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Figure 2: Mapping differentially expressed genes onto their associated metabolic

pathways led to the identification of a truly essential amino acid directly involved

with increased protein titers.

ALANINE AND ASPARTATE METABOLISM

Glycolysis

Pyruvate

1.2.4.1

1.2.4.11.2.1.51

4.1.1.15

4.1.1.11

6.3.2.11

3.4.13.3

4.1.1.12

6.1.1.7

5.1.1.1

2.6.1.12

2.6.1.2

2.6.1.44

1.8.1.4

2.3.1.12

6.4.1.1

3.5.1.3

1.4.3.1

1.4.3.2

1.4.3.16

5.1.1.13

3.5.1.15

2.6.1.14

3.5.1.1 3.5.1.38 6.3.1.1 6.3.5.4

6.1.1.22

6.3.5.6

6.1.1.23

4.3.1.1

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4.3.2.2

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1.2.1.18

2.6.1.18 2.6.1.19

Dihydro-lipoamide-E

S-Acetyldihydro-lipoamide-E

Pyrimidinemetabolism

ß-Alanine Camosine

D-Alaninemetabolism

L-Alanyl-tRNA (Ala) Protein

D-Alanine

Reductivecarboxylate cycle

Histidine metabolism

Nicotinate andnicotinamide metabolsim

Gly, Ser & Thr metabolism

Lysine biosynthesis

ß-Alanine metabolsim

Urea cycle

Arg & Pro metabolsimAdenylo-succinate

L-Arginino-succinate

L-Aspartate

L-Alanine

Selenoamino acidmetabolism

Cyanoamino acidmetabolism

Pantothenate andCoA biosynthesis

Fatty acidmetabolism

O-Acetyl-camitine

Nicotinate andnicotinamide metabolism

Malate

Furnarate Citrate cycle

2-Oxosuccinamate

Citrate

N-Acetyl-L-aspartate

L-Asparagine

L-Asparaginyl-tRNA (Asn)

L-Aspartyl-tRNA (Asn)2-Oxoglutarate

Oxaloacetate

Succinate

Reductivecarboxylate cycle

Acetyl-CoAMalonate semialdehyde

N-Carbamoyl-L-asparate

D-Aspartate

L-Aspartyl-tRNA (Asp)

2-Hydroxy-ethyl-ThPP ThPP

Lipoamide-E

The ability to map differentially

expressed genes onto their associated

biochemical pathways provides the

opportunity to “zoom in” on each

of the metabolic pathways associ-

ated with protein production. Key

metabolites that are either depleted

or produced are relatively easy to

identify, but true process understand-

ing comes from identifying how the

compounds are used in the metabolic

machinery. Amino acids, for exam-

ple, could be depleted by translation,

interconversion to other amino acids,

or detoxification by the cell. Each

of these routes has dramatically dif-

ferent impacts on cell health and

productivity. With the application of

NGG techniques you do not have to

wait until the end of the project to

begin seeing results. Each individual

experiment contributes to the “sys-

tems” knowledge but in the short

run provides specific information

on variables that can be tuned for

performance. Over the past three

years we have completed numerous

microarray experiments as part of

our primary media and fermentation

optimization objectives. A few exam-

ples will be highlighted here that will

demonstrate the power of microarray

technology to improve bioprocess sta-

bility and production yields as part of

a larger NGG initiative.

In the manufacturing process of

the target compound of interest,

the original growth medium com-

ponents were not well defined. As

a result, different medium lots var-

ied dramatically in protein yield and

product titers. One of the primary

objectives was to develop a chemi-

cally defined medium that would

yield consistent titers. In our experi-

ments, we evaluated whether stress

response mechanisms of the pro-

duction cells caused a reduction in

titer during phase transition, and

how media and fermentation condi-

tions impacted these stress responses.

We carefully designed time course

experiments to cover transition

through growth phases with trial

versions of different defined media.

Complimentary to this, we com-

pleted analytical chemistry tests to

assign putative roles to transcription

regulators that might be involved in

stress response. By ultimately corre-

lating differentially expressed genes

of sigma factors with their associ-

ated biochemical pathways, we were

able to optimize and change cer-

tain media components that led to

improved protein production.

The real success of the microar-

ray studies came, however, when

we discovered a medium supple-

ment that dramatically enhanced

protein production for a chemically

defined medium. This particular

medium was characterized by high

titers when monitored over time.

However, at a particular stage dur-

ing the fermentation process, pro-

tein yields suddenly dropped off.

We carefully designed a time course

microarray experiment, and mapped

the resulting differentially expressed

genes to their associated metabolic

pathways. To our surprise, analy-

ses of interconverting pathways led

to the identification of a particular

amino acid (see Figure 2). The ability

to map differentially expressed genes

to their associated pathways clearly

made it possible to “zoom” in” and

identify a key component that led

to medium optimization and that

targeted a truly essential amino acid.

We also used microarrays to survey

the dynamics of gene expression in

media with varying productivities,

as well as to examine process condi-

tions that enhanced productivity.

The results identified genes within

the cells cultured in media yielding

high product concentrations/titers

that are related to growth and cell

division and were expressed at sig-

nificantly higher levels compared to

those cells grown in media yielding

lower titers. This enabled the cells to

remain viable longer at the end of

cultivation, when the cell concentra-

tion is highest, thus allowing more

product molecules to accumulate.

Despite the utility and versatility

of DNA microarray technology, it

only provides for a qualitative pic-

ture of the overall transcriptome and

only reveals the activity of genes

for which probes are present on the

array. Furthermore, based on many

recent research reports for both

prokaryotic and eukaryotic organ-



Figure 3: A detailed breakdown of a typical mRNA-seq work fiow. This work

fiow is for a bacterial species for which comprehensive genome information is

available. Partially adapted from Wilhelm and Landry (2009).

isms, we know that cell physiology,

as well as many functional cellular

and biological processes, including

cell cycle progression and induc-

tion and suppression of apoptosis,

are not entirely dependant on the

level of gene expression, but rather

controlled by upstream regulatory

regions and the increasingly impor-

tant “non-coding” regions of the

genome (e.g., microRNAs/small

RNAs) (4,5). This is where NGG

comes to the forefront.

One of the key technolo-

gies in NGG is Next Generation

Sequencing (NGS). Recent techno-

logical advances in DNA sequencing

have dramatically improved overall

throughput and quality and have

led to the development of meth-

ods to characterize whole transcrip-

tomes of entire cell populations in a

way that was never before possible

(6–8). RNA–sequencing (mRNA–

seq) involves the direct sequencing

of complementary DNAs (cDNAs)

using high throughput, massively

parallel NGS technologies (Illumina’s

Genome Analyzer IIx; Illumina’s

HiSeq2000, Roche’s 454 FLX sys-

tem, to name a few), followed by

mapping of the resulting sequenc-

ing reads to a reference genome

(see Figure 3 for a detailed RNA–seq

work-flow diagram).

In a single RNA-Seq experiment,

one can derive not only an accu-

rate, quantitative measure of tran-

scriptome-wide gene expression

levels (as with real–time quanti-

tative polymerase chain reaction

technologies), but also discover

novel transcribed regions (new

exons/genes) in an unbiased man-

ner (as with a whole genome tiling

microarray approach), map their

boundaries, and identify the 5’ and

3’ ends of the genes (9,10). In addi-

tion, this methodology enables a

global survey of the usage of the

alternative splice sites (similar to

a custom designed splicing micro-

array). It allows for the identifica-

tion of transcription start sites, the

identification of new splicing vari-

ants, and the monitoring of allele

expression (9,10). Based on the

power of the RNA–Seq approach, it

is clear, that at least for comprehen-

sive studies in higher eukaryotes

where surveys of differential splic-

ing activity, antisense transcrip-

tion, and discovery of novel regions

of transcription are desired, high

throughput sequencing of RNA has

augmented and is beginning to

supersede microarray-based meth-

ods (9,10). Not only do the eco-

nomics of faster development and

better optimization support it, but

it also allows for a host of new qual-

ity control and bioprocess monitor-

ing capabilities after the research

and optimization is completed. All

of this dovetails perfectly with the

intent of FDA’s QbD guidelines.

We have developed methods to

use various current and next gen-

eration genomics tools to increase

the performance and cost effective-

ness of bioprocesses during manu-

facturing. NGS technologies, in

particular, have several potential

applications in this scenario. These

include the generation of valuable

genomics resources, development of

molecular fingerprints for improved

product yield and quality and bio-

process monitoring, quantitative

expression analysis (RNA–seq), and

identification of metabolic bottle-

necks, all leading to evidence–based

bioprocess optimization (see Figure

4). For example:

(1) Sequencing genomic DNA,

mRNA, micro/smallRNAs, and

immunoprecipitated DNA frag-

ments from production strains or

cell lines provides genomic resources

that have a direct impact on under-

standing the overall biology of an

organism. Specifically, it enables the

understanding of gene regulation

(e.g., the role of noncoding regula-

tory RNA elements and transcrip-

tion factors/sigma factors in gene

regulation) and genome structure

and dynamics (chromosomal rear-

rangements, alternative splicing

events, etc.). These resources have a

direct impact on understanding the

genome architecture of the produc-

tion species, laying the foundation

for intelligent, biology-guided pro-

cess development.



Figure 4: Integration of NGG technologies to develop a species specific

genomics platform that can be used to optimize bioprocesses, while building a

solid understanding of the biology of the particular production species.

(2) To develop functional gene-based

markers, NGS of mRNA of contrast-

ing phenotypes for the biomolecule

of interest (for example, yield and

quality) can be used to identify can-

didate genes involved in or associ-

ated with the production phenotype.

These genetic markers can then be

used as molecular fingerprints to

assist with the selection of produc-

tion lines, and to monitor process

development and optimization with

the goal of guiding early stage biopro-

cesses. At later stages, these molecular

fingerprints can also be used to mon-

itor process scaleup and manufactur-

ing. Active monitoring throughout

the entire process l i fe cycle

maximizes product yield and quality

while minimizing associated costs.

(3) Coupled to metabolic path-

way analysis, RNA–seq of produc-

tion strains/cell lines in different

growth and environmental condi-

tions sheds light on key metabolic

pathways controlling biomolecule

production and identifies potential

metabolic bottlenecks. The ability to

zero in on the control points govern-

ing metabolic flow towards increased

production allows for process manip-

ulation based on empirical knowl-

edge instead of large DOE fishing

expeditions and brute force methods.

CONClUSIONIntelligently designed NGG experi-

ments have become the hallmark of

research and manufacturing design

and optimization. With the advent

of NGG technologies, the old and

the new can be combined for very

powerful results. Short term gains

in production (in our case, some-

times more than 300%) were recog-

nized from DNA microarray and/or

mRNA-seq experiments that have

provided foundational information

for our NGG initiatives. We have

dramatically improved our ability to

rapidly optimize both growth media

and fermentation conditions associ-

ated with the production of a key

protein used in the manufacturing

of a major commercial product. The

technology has enabled us to better

understand the overall bioprocess,

as well as the physiology of the pro-

duction cells themselves. We have

incorporated various current and

next generation genomics tools to

form the basis of a bioprocessing

genomics platform that will enable

us to ultimately support FDA’s QbD

initiative. Not only will this improve

our understanding of bioprocesses

and the effect of all CPPs on protein

yield, quality and process stability,

but it will also make flexible biopro-

cessing possible and safe. ◆

rEfErENCES 1. A. Kantardjieff et. al., Biotechnol. Adv. 27,

1028–1035 (2009).

2. N.M. Jacob et. al., Chemical Engineering

Progress 105 (11), 35–42 (2009).

3. M. Kanehisa, Trends Genet. 13 (9), 375–

376 (1997).

4. P. Brodersen and O. Vionnet, Nat. Rev.

Mol. Cell. Biol. 10, 141–148 (2009).

5. L.S. Water and G. Storz, Cell 136, 615–

628 (2009).

6. N.M. Jacob et. al., Biotechnol. Bioeng.

105, 1002–1009 (2009).

7. D.J. Turner et. al., Mamm. Genome. 20,

327–338 (2009).

8. P.K. Wall et. al., BMC Genomics. 10,

347–366 (2009).

9. Z. Wang, M. Gerstein, and M. Snyder, Nat.

Rev. Genet. 10, 57–63 (2009).

10. B.T. Wilhelm and J.R. Landry, Methods.

48, 249–257 (2009).


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Ad IndexIndustry Calendar

For event details and a complete calendar list, visit

www.BioPharmInternational.com/events

Continued from P. 50

Final Word

decades of experience in col-

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Send your Final Word story ideas to Editorial Director Michelle Hoffman at [email protected].

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BioPharm International www.biopharminternational.com April 2011 49

Tod McCloskey

(800) 225-4569, ext. 2739

[email protected]

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Smart editorial. Smart marketing.

Optimizing the cul-t u r e me d iu m i s an integral part of upst ream process development, and is

essential for efficient biopharma-

ceutical manufacturing. The aim

is to design a robust, economi-

cal, and reproducible system that

enhances the overall performance

of the specific cell line. Typically,

cell culture performance is assessed

using a number of parameters,

including cell density and viability.

However, the defining parameter of

any successful production system is

increased protein expression. Traditionally, optimal mamma-

lian cell growth was achieved by

adding animal sera, such as fetal

bovine serum (FBS) at a concen-

tration of 5–20% to defined basal

media. Although sera may provide

important growth and regulatory

factors, their composition is com-

plex and undefined, which can

lead to batch-to-batch variability

and downstream processing chal-

lenges. Furthermore, the potential

for contamination by adventitious

agents, such as viruses, prions,

and bacteria, poses serious bio-

safety risks. This has led regula-

tory authorities such as the US

Food and Drug Administration

and European Medicines Agency

(EMEA) to issue guidelines that

urge biomanufacturers to avoid

ingredients of animal or ig in.

Regulatory pressures related to

safety concerns are therefore driv-

ing the biopharmaceutical industry

away from the dominance of serum

as a media supplement, and toward

the use of serum-free, animal-com-

ponent free, or even chemically

defined media (CDM) for both new

and older manufacturing processes. Serum-Free Media

Plant-derived hydrolysates have

been routinely used to reduce or

eliminate serum from traditional

basal media formulations, often

in combination with a variety of

addit ional supplements. These

hydrolysates are composed of a

mixture of peptides, amino acids,

carbohydrates, and lipids, and as a

multitude of unidentified compo-

nents with indeterminate biologi-

cal activity. They are produced by

the enzymatic or acidic digestion

of a given raw material from var-

ious plant sources including, but

not limited to soy, wheat, and cot-

ton. Some process scientists have

been reluctant to use plant-derived

protein hydrolysates as medium

supplements because of their lack

of definition, which impairs their

ability to assess the root causes

of variability in their production

processes. Recent improvements,

including novel enzyme digestion

techniques, refined processing tech-

niques, automation, and formal

cleaning validations have resulted

in more consistent hydrolysates sold

under the trade name of HyPep and

UltraPep.1 These improved plant

protein hydrolysates are widely

accepted as performance-enhanc-

ing substitutes for animal-derived

media components for a variety of

cell lines (e.g., hybridoma, BHK,

CHO, Vero, MDCK).2–4 Several

biopharmaceuticals produced using

plant-derived protein hydrolysates

have reached the market and many

more are in various stages of devel-

opment.As an alternative solution to

traditional basal media supple-

mented w ith an ima l- der ived

serum, high-performing, richly

for mu lated CDM have been

developed for biopharmaceuti-

cal production as stand-alone

substrates. The optimized mix-

tures of biochemical constitu-

ents in CDM have been carefully

designed to stimulate cell growth,

maintain good cell viability, and

promote high protein y ie lds.

Although CDM have been used

Figure 1. Cell viability of Chinese hamster ovary cells cultured in chemically

defined medium with and without supplementation with HyPep. The plant-derived

hydrolysate extended cell viability. 100

90

80

70

60

50

40

30

20

10

0

Via

ble

ce

lls (

%)

0 2

4 6

8 10

Day

100% CDM-C80% CDM-C

100% CDM-C + 8 g/L HyPep80% CDM-C + 8 g/L HyPep

CDM-C: single chemically defined media

Hydrolysate supplements may provide constituents that are beneficial for performance.

Partial Replacement of Chemically

Defined Media with Plant-Derived

Protein Hydrolysates

Plant-derived hydrolysates can be used as valuable and

practical tools to improve cell culture performance.

JAMES BABCOCK, CHRISTOPHER WILCOX, HANS HUTTINGA

ABSTRACT

Protein hydrolysates are routinely used as cell culture sup-

plements to enhance the overall performance of many

biopharmaceutical production systems. This enhance-

ment is subject to the additive effect of the native hydro-

lysate components and the supplemented growth or

production medium. Therefore, it is necessary to experi-

mentally determine the proper hydrolysate dosage for a

given hydrolysate medium combination that provides the

desired optimization effect such as better growth pro-

motion, enhanced cell viability, increased target protein

production, or a combination of all three. In mammalian

systems, hydrolysates have been used in combination

with a variety of other supplements to help reduce or

eliminate serum requirements in systems using traditional

basal media. Today, many high-performing, richly formu-

lated chemically defined media have become available as

stand-alone substrates for biopharmaceutical production.

This article shows that these chemically defined media

can benefit from the addition of hydrolysates and other

supplements. It also demonstrates that in other cases, plant-

derived hydrolysates can partially replace a significant por-

tion of the active ingredients in these rich media.

Sh

effi

eld

Bio

-Scie

nce C

en

ter

for

Cell

Cu

ltu

re T

ech

no

log

y

JAMES BABCOCK, PHD, is the global applications manager of cell culture at

the Sheffield Bio-Science Center for Cell Culture Technology. CHRISTOPHER

WILCOX, PHD, is the global market segment manager of cell culture and

HANS HUTTINGA is the global business development director of cell

nutrition, both at Sheffield Bio-Science, a Kerry Group Business, Beloit, WI,

800.833.8308, [email protected].

June 2010

Volume 23 Number 6



Final Word

Supply-Chain Analytics: Solving the Future Stronger analytics can lead to greater profitability

As the pharmaceutical industry weath-

ers some of its biggest challenges in

decades, supply-chain analytics may

offer the solution to some of the industry’s

biggest problems. Accenture’s Global Pharma

Industry Supply Chain & Tech Ops study—

completed in May 2010—offers some key

insights into how analytical capabilities could

help transform supply chain operations.

While most study participants—25 pharma

and biotech companies from around the

globe and across industry segments—would

agree that “supply chain analytics are a cru-

cial part of our strategic priorities,” their

efforts are largely focused on developing

greater visibility into supply chain perfor-

mance. There is certainly room to improve in

this area, but the true value of analytics goes

far beyond simple performance management.

Indeed, companies that indicated stronger

analytical capabilities (e.g., closer integration

with customers on demand forecasting) also

consistently demonstrated higher margins.

Data availability is the most fundamental

requirement for strong analytic capabilities.

This is an area in which the pharma indus-

try continues to struggle. Supply-chain data

is typically scattered throughout a f rag-

mented landscape of manufacturing, enter-

prise resource planning (ERP), and laboratory

information management systems (LIMS),

which do not exchange information. Pharma

companies’ ability to pull information from

outside the organization is not much better.

Few have been able to develop tight links

with customers, and even where these links

are in place, the customers’ data are often of

less-than-sterling quality.

But having the data, while necessary, is

far from sufficient to develop strong ana-

lytics. In fact, organizational factors which

break the link between data and decisions

are often the biggest obstacles to overcome.

Too often, supply-chain organizations in the

pharma industry operate in disconnected

functional silos which encourage decision

making based on tradition, rather than data.

Traditional thinking, for example, might

dictate a decision to build inventory to

ensure that all orders are filled. Analytical

thinking might instead suggest tracking cus-

tomer inventory levels and patient demand

to optimize order performance.

As organizational capabilities mature and

data quality improves, focus will shift from

using analytics to enhance the effectiveness

of traditional processes to building new ways

of operating. In the consumer goods indus-

try, for instance, manufacturers are increas-

ingly turning to point-of-sale data from their

retail customers to design algorithms which

allow product manufacturing and replenish-

ment strategies to be tailored to the stages of

the product life cycle in real time.

The utility of such an approach for pharma

companies facing tougher generic competi-

tion and lengthening research and develop-

ment timeframes is obvious. What’s more, the

industry’s current focus on improving prod-

uct traceability and supply-chain security will

tend to build exactly the kind of links with

customers and distribution partners that can

provide the data to drive more analytically

oriented forecasting and replenishment.

For guidance on how analytics can be best

deployed in the pharma industry, it’s help-

ful to look at how analytics have driven

improved performance in other industries:

• A leading big-box retailer in the United

States has been able to leverage two

Eugene Jones, is a senior executive at Accenture, a

global management consulting, technology services, and

outsourcing company. He leads the supply-chain practice

for Accenture’s Life Sciences industry group.

s

M.

Fre

em

an

/Ph

oto

Lin

k/G

ett

y I

ma

ge

s

Continued on P. 48

Parenteral Drug AssociationTraining and Research Institute (PDA TRI)

Upcoming Laboratory and Classroom Training for

Pharmaceutical and Biopharmaceutical Professionals

June 2011

Sterile Pharmaceutical Dosage Forms: Basic Principles June 1-2, 2011 | Bethesda, Maryland | www.pdatraining.org/sterilepharma

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• Fundamentals of Lyophilization (June 20-21)

• Economical Design of Lyophilization Experiments Workshop – New Course (June 22)

• Validation of Lyophilization – New Course (June 23-24)

Fermentation/Cell Culture Technologies Training Workshop June 28-30, 2011 | Bethesda, Maryland | www.pdatraining.org/fermentation

July 2011

Biotechnology: Overview of Principles, Tools, Processes and Products July 11-12, 2011 | Bethesda, Maryland | www.pdatraining.org/biotechnologyoverview

A Risk Based Approach to Technology Transfer July 25, 2011 | Bethesda, Maryland | www.pdatraining.org/riskbasedapproach

Practical Applications of Risk Management – New Course July 26, 2011 | Bethesda, Maryland | www.pdatraining.org/practicalapplications

Laboratory Courses

The PDA Training and Research Institute is accredited by the Accreditation Councilfor Pharmacy Education (ACPE) as a provider of continuing pharmacy education.

For more information on these and other upcomingPDA TRI courses please visit www.pdatraining.org

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