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BioPharmThe Science & Business of Biopharmaceuticals

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INTERNATIONAL

BioPharmThe Science & Business of Biopharmaceuticals

EDITORIALEditorial Director Rita Peters [email protected] Editor Susan Haigney [email protected] Editor Randi Hernandez [email protected] Science Editor Adeline Siew, PhD [email protected] Editor Ashley Roberts [email protected] Director Dan Ward [email protected] Editors Jill Wechsler, Jim Miller, Eric Langer, Anurag Rathore, Jerold Martin, Simon Chalk, and Cynthia A. Challener, PhD Correspondent Sean Milmo (Europe, [email protected]) ADVERTISING

Publisher Mike Tracey [email protected]/Mid-West Sales Manager Steve Hermer [email protected] Coast Sales Manager Scott Vail [email protected] Sales Manager Chris Lawson [email protected] Sales Manager Wayne Blow [email protected] List Rentals Tamara Phillips [email protected] 877-652-5295 ext. 121/ [email protected] Outside US, UK, direct dial: 281-419-5725. Ext. 121 PRODUCTION Production Manager Jesse Singer [email protected] AUDIENCE DEVELOPmENT Audience Development Rochelle Ballou [email protected]

UBm LIfE SCIENCES

Joe Loggia, Chief Executive Officer Tom Ehardt, Executive Vice-President, Life Sciences Georgiann DeCenzo, Executive Vice-President Chris DeMoulin, Executive Vice-President Rebecca Evangelou, Executive Vice-President, Business Systems Julie Molleston, Executive Vice-President, Human Resources Mike Alic, Executive Vice-President, Strategy & Business Development Tracy Harris, Sr Vice-President Dave Esola, Vice-President, General Manager Pharm/Science Group Michael Bernstein, Vice-President, Legal Francis Heid, Vice-President, Media Operations Adele Hartwick, Vice-President, Treasurer & Controller

UBm AmERICAS

Sally Shankland, Chief Executive Officer Brian Field, Chief Operating Officer Margaret Kohler, Chief Financial Officer

UBm PLC

Tim Cobbold, Chief Executive Officer Andrew Crow, Group Operations Director Robert Gray, Chief Financial Officer Dame Helen, Alexander Chairman

© 2015 Advanstar Communications Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical including by photocopy, recording, or information storage and retrieval without permission in writing from the publisher. Authorization to photocopy items for internal/educational or personal use, or the internal/educational or personal use of specific clients is granted by Advanstar Communications Inc. for libraries and other users registered with the Copyright Clearance Center, 222 Rosewood Dr. Danvers, MA 01923, 978-750-8400 fax 978-646-8700 or visit http://www.copyright.com online. For uses beyond those listed above, please direct your written request to Permission Dept. fax 440-756-5255 or email: [email protected].

UBM Life Sciences provides certain customer contact data (such as customers’ names, addresses, phone numbers, and e-mail addresses) to third parties who wish to promote relevant products, services, and other opportunities that may be of interest to you. If you do not want UBM Life Sciences to make your contact information available to third parties for marketing purposes, simply call toll-free 866-529-2922 between the hours of 7:30 a.m. and 5 p.m. CST and a customer service representative will assist you in removing your name from UBM Life Sciences’ lists. Outside the U.S., please phone 218-740-6477.

BioPharm International does not verify any claims or other information appearing in any of the advertisements contained in the publication, and cannot take responsibility for any losses or other damages incurred by readers in reliance of such content.

BioPharm International welcomes unsolicited articles, manuscripts, photographs, illustrations, and other materials but cannot be held responsible for their safekeeping or return.

To subscribe, call toll-free 888-527-7008. Outside the U.S. call 218-740-6477.

EDITORIAL ADVISORY BOARDBioPharm International’s Editorial Advisory Board comprises distinguished specialists involved in the biologic manufacture of therapeutic drugs, diagnostics, and vaccines. Members serve as a sounding board for the editors and advise them on biotechnology trends, identify potential authors, and review manuscripts submitted for publication.

K. A. Ajit-Simh President, Shiba Associates

Rory Budihandojo Director, Quality and EHS Audit

Boehringer-Ingelheim

Edward G. Calamai Managing Partner

Pharmaceutical Manufacturing

and Compliance Associates, LLC

Suggy S. Chrai President and CEO

The Chrai Associates

Leonard J. Goren Global Leader, Human Identity

Division, GE Healthcare

Uwe Gottschalk Vice-President,

Purification Technologies

Sartorius Stedim Biotech GmbH

Fiona M. Greer Global Director,

BioPharma Services Development

SGS Life Science Services

Rajesh K. Gupta Vaccinnologist and Microbiologist

Jean F. Huxsoll Senior Director, Quality

Product Supply Biotech

Bayer Healthcare Pharmaceuticals

Denny Kraichely Associate Director

Johnson & Johnson

Stephan O. Krause Principal Scientist, Analytical

Biochemistry, MedImmune, Inc.

Steven S. Kuwahara Principal Consultant

GXP BioTechnology LLC

Eric S. Langer President and Managing Partner

BioPlan Associates, Inc.

Howard L. Levine President

BioProcess Technology Consultants

Herb Lutz Principal Consulting Engineer

EMD Millipore Corporation

Jerold Martin Sr. VP, Global Scientific Affairs,

Biopharmaceuticals

Pall Life Sciences

Hans-Peter Meyer VP, Special Projects Biotechnology

Lonza, Ltd.

K. John Morrow President, Newport Biotech

David Radspinner Global Head of Sales—Bioproduction

Thermo Fisher Scientific

Tom Ransohoff Vice-President and Senior Consultant

BioProcess Technology Consultants

Anurag Rathore Biotech CMC Consultant

Faculty Member, Indian Institute of

Technology

Susan J. Schniepp Fellow

Regulatory Compliance Associates, Inc.

Tim Schofield Managing Director

Arlenda, USA

Paula Shadle Principal Consultant,

Shadle Consulting

Alexander F. Sito President,

BioValidation

Michiel E. Ultee Principal

Ulteemit BioConsulting

Thomas J. Vanden Boom Vice-President, Global Biologics R&D

Hospira, Inc.

Krish Venkat CSO

AnVen Research

Steven Walfish Principal Statistician

BD

Gary Walsh Professor

Department of Chemical and

Environmental Sciences and Materials

and Surface Science Institute

University of Limerick, Ireland

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4 BioPharm International www.biopharminternational.com May 2015

Contents

BioPharmINTERNATIONAL

BioPharm International integrates the science and business of

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

peer-reviewed technical solutions to enable biopharmaceutical professionals

to perform their jobs more effectively.

COLUMNS AND DEPARTMENTS

BioPharm International ISSN 1542-166X (print); ISSN 1939-1862 (digital) is published monthly by UBM Life Sciences 131 W. First Street, Duluth, MN 55802-2065. Subscription rates: $76 for one year in the United States and Possessions; $103 for one year in Canada and Mexico; all other countries $146 for one year. Single copies (prepaid only): $8 in the United States; $10 all other countries. Back issues, if available: $21 in the United States, $26 all other countries. Add $6.75 per order for shipping and handling. Periodicals postage paid at Duluth, MN 55806, and additional mailing offices. Postmaster Please send address changes to BioPharm International, PO Box 6128, Duluth, MN 55806-6128, USA. PUBLICATIONS MAIL AGREEMENT NO. 40612608, Return Undeliverable Canadian Addresses to: IMEX Global Solutions, P. O. Box 25542, London, ON N6C 6B2, CANADA. Canadian GST number: R-124213133RT001. Printed in U.S.A.

BioPharm International is selectively abstracted or indexed in: • Biological Sciences Database (Cambridge Scientifc Abstracts) • Biotechnology and Bioengineering Database (Cambridge Scientifc Abstracts) • Biotechnology Citation Index (ISI/Thomson Scientifc) • Chemical Abstracts (CAS) • Science Citation Index Expanded (ISI/Thomson Scientifc) • Web of Science (ISI/Thomson Scientifc)

Cover: Xiaoke ma/Getty Images; Dan Ward

6 From the Editor The biopharma industry needs to work on operational issues. Rita Peters

8 US Regulatory Beat Drug manufacturers face added pressure and incentives. Jill Wechsler

12 Perspectives on Outsourcing CMOs and CROs based in emerging markets continue to capture market share. Eric Langer

16 Inside Standards The European Pharmacopoeia defines the format and content of monographs for biologicals. Stephen Wicks

42 Troubleshooting Choosing the optimal protein expression vector depends on a number of factors. Siavash Bashiri, David Vikström, and Nurzian Ismail

46 Compliance Notes How to keep up with changing regulations.Siegfried Schmitt

47 Clinical Trials Update

48 Product Spotlight

48 New Technology Showcase

49 Ad Index

50 Biologics News Pipeline

Modular SySteMS

Modular Manufacturing Platforms for BiologicsRandi Hernandez

The costs and benefits of integrating

modular concepts for on-demand

bioprocessing are explored. 18

downStreaM ProceSSing

Filtration Technologies Advance to Meet Bioprocessing NeedsCynthia A. Challener

Higher cell densities, greater demand

for high-performance viral clearance,

and desire for large-scale single-use

technologies are driving development

of filtration technologies. 26

Peer-reviewed

Differentiation and Characterization of Protein Aggregates and Oil Droplets in Therapeutic ProductsCiaran Murphy

This article demonstrates how resonant

mass measurement can be used not only

to detect and quantify the formation of

protein subvisible particles in a critically

important size range but also to detect

and quantify any silicone oil droplets

in the formulation. 30

SuPPly chain

Innovations and Adaptations in the Cold ChainNils Markmann

Manufacturer supply chain needs are

changing in response to widening product

temperature ranges. 35

analytical teSting

Streamlining Raw Materials TestingRandi Hernandez

Rapid testing of biologic raw materials

can lead to greater efficiency. 39

Quality MetricS

An Update on the Quality Metrics InitiativeSusan J. Schniepp

Industry and regulatory agencies

continue to make progress in

establishing quality metrics for

the pharmaceutical industry. 40

Volume 28 Number 5 May 2015

fEATURES


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From the Editor

The biopharma

industry has had

some successes

but still needs

to work on

operational issues.

Quality Counts, Too

Arecent report from the Pharmaceutical Research and Manufacturers

of America (PhRMA) lists impressive contributions of biopharma com-

panies to patients, healthcare, and the economy. The report, 2015

Biopharmaceutical Research Industry Profile (1), indicates that in 2014, PhRMA

member companies spent an estimated $51.2 billion in research and develop-

ment, down slightly over the 2013 level of $51.6 billion (1).

Other key statistics from the report show that biopharmaceutical companies

on average invest as much as six times more in R&D, relative to sales, than

the average US manufacturing firm; the industry supports 3.4 million jobs in

the United States, 810,000 positions through direct employment. The report

emphasized the value that the biopharmaceutical industry provides to patients

and society; however, it failed to look at manufacturing-related shortcomings

that impact patient health.

Prescription drugs, which represent only nine cents per 2013 healthcare dol-

lar, Òplay a central role in improving the health outcomes of patientsÓ when

used appropriately, PhRMA reports; limited access to medicines and complex-

ity of treatment regimens, however, are barriers to the optimal use of medi-

cines. Two other threats that limit patient access to drugs and pose health risks

were not mentioned in the report: drug shortages and counterfeit drugs.

While the number of new drug shortages has dropped off since 2011, ongo-

ing shortages persist. With a limited number of manufacturing lines available to

produce generic drugs, especially injectable drugs, there is little margin for error.

Quality problems (40%) were the most-frequently cited cause for all drug

shortages from January 2011 through June 2013, according to a US General

Accountability Office report (2). Manufacturing delays and capacity issues

involving shutdowns or slowdowns of facilities to perform maintenance or

remediation were cited in another 30% of the drug-shortage cases. API-related

problems contributed to 9% of the drug shortages. In addition, 12% of drug

shortages were due to product discontinuation, a business decision.

Counterfeit medicines present another quality issue, both within and

beyond US borders. According to the National Institutes of Health (NIH) ÒPoor

quality medicines are a real and urgent threat that could undermine decades

of successful efforts to combat HIV/AIDS, malaria, and tuberculosisÓ (3).

Implementation dates for serializationÑthe first step of lengthy track-and-

trace efforts to prevent counterfeit and falsified medicinesÑare not far off, but

many companies have been slow to take action, reported experts at a BioPharm

International seminar at INTERPHEX 2015.

FDA has actively addressed the drug shortage issue, promoting risk assess-

ment, quality metrics, and facility and equipment improvements. Industry asso-

ciations, including the Parenteral Drug Association and International Society of

Pharmaceutical Engineers, have championed the cause of quality improvements.

The wheels of change turn slowly, however, and can be slowed by regulatory,

financial, and operational hurdles, delaying manufacturing and quality improve-

ments. Statistics can be presented to paint any performance picture. Patient

safety, through drug quality and serialization efforts, needs to be figured into

the bottom line.

References

1. PhRMA, 2015 Biopharmaceutical Research Industry Profile (Washington, DC, 2015).

2. General Accountability Office. “Drug Shortages, Public Health Threat Continues,

Despite Efforts to Help Ensure Product Availability” (Washington, DC, February 2014).

3. National Institutes of Health, “Global pandemic of fake medicines poses urgent risk,

scientists say,” Press release (Bethesda, MD, April 20, 2015). ◆

Rita Peters is the editorial director of

BioPharm International.


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Regulatory Beat

Vis

ion

so

fAm

eri

ca

/Jo

e S

oh

m/G

ett

y Im

ag

es

The globalization of drug production

has prompted FDA to overhaul its

strategies for ensuring the safety and

quality of drugs and biologics coming into

the United States, as well as those produced

at home. Recent legislation enhancing FDA

inspection authority supports stiffer enforce-

ment of manufacturing standards, along with

added incentives for establishing high quality

operations. Increased FDA reliance on infor-

mation submitted by manufacturers about

their operations and products also means

more focus on data integrity and voluntary

compliance with the rules.

Former FDA commissioner Marga ret

Hamburg emphasized how the rise in inter-

national biopharmaceutical production has

affected the agency in a series of speeches

marking her recent exit from FDA. She high-

lighted the importance of collaboration with

the European Medicines Agency (EMA), par-

ticularly in sharing reports from each other’s

inspection programs, at a meeting in London

to mark the 20th anniversary of EMA. And in

a farewell speech at the National Press Club in

Washington, DC, Hamburg reiterated how glo-

balization has required FDA to change how it

does business, most visibly by estab-

lishing outposts around the world to

support overseas field operations.

At t he s a me t i me, Howa rd

Sklamberg, deputy commissioner for

global regulatory operations and pol-

icy, traveled to India with Cynthia

Schnedar, director of the Center

for Drug Evaluation and Research’s

(CDER) Office of Compliance, to dis-

cuss the importance of tackling drug

safety and regulatory challenges with

local authorities and drug manufac-

turers. Their visit reflected efforts by

FDA to ensure the quality of the vast array of

drugs imported to the United States from that

country; CDER has sponsored workshops to

help Indian drug manufacturers understand

and meet US standards and avoid curbs on

imports.

New iNspectioN approachesDuring the trip, Sklamberg and Schnedar also

explained that Indian manufacturers may qual-

ify for less frequent inspections and reduced

oversight by achieving high quality ratings

from plant inspections and quality metrics

under development (1). FDA sought to off-

set complaints that it’s singling out India for

stricter enforcement by emphasizing increased

cooperation, as seen in joint inspections with

local officials.

FDA’s broader aim, Sklamberg and Schnedar

noted, is to standardize inspection methods in

all regions, including the US, so that “quality

will be understood and aspired to by manufac-

turers—no matter where they are in the world.”

A new inspection questionnaire is being devel-

oped to harvest uniform data in all regions to

support accurate measures of quality.

These efforts fit the FDA Program Alignment

(PA) initiative, which is restructuring FDA’s

Office of Regulatory Affairs (ORA) to shift from

a geography-based operation to one with spe-

cialized cadres of inspectors for pharmaceuti-

cals and for other regulated products, Sklamberg

explained at the IBC Pharmaceutical Compliance

Congress in March 2015. ORA’s pharmaceutical

inspectorate will work closely with CDER’s Office

of Pharmaceutical Quality as part of a team

approach for reviewing applications and main-

taining post-market surveillance of manufactur-

ers, incorporating a risk-informed process for

setting inspection priorities based on improved

data on firm operations and compliance histories.

FDA Revises Field Inspections to Reflect Global Market and Quality InitiativesDrug manufacturers face added pressure and incentives for meeting new FDA compliance policies and priorities.

Jill Wechsler is BioPharm

InternationalÕs washington editor,

chevy chase, MD, 301.656.4634,

[email protected].


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regulatory Beatregulatory Beat

An FDA pilot is testing the PA

program for domestic and for-

eign inspections of human and

animal drugs (overseen by FDA’s

Center for Veterinary Medicine)

to see if it reduces duplicative

work and minimizes disagree-

ment between FDA centers and

the field over inspection findings.

The team approach will involve

pre-inspection briefings, real-time

communication of inspection

findings to ORA and CDER, joint

development of recommendations,

and enhanced communication of

inspection findings.

The aim is to transition by late

2015 to multi-year, r isk-based

inspection planning based on staff

resources, performance-based met-

rics, and compliance outcomes.

FDA centers will establish enforce-

ment strategies and compliance

policies, and they will collaborate

with ORA on modernizing enforce-

ment standards and determining

the underlying reasons for quality

failures found during inspections.

In addition to citing problems,

inspections will document where

a firm’s quality management sys-

tem exceeds requirements and

thus open the door to reduced

oversight. The payoff for quality

achievement could be less fre-

quent inspection of that facility

and regulatory flexibility related

to post-approval manufacturing

changes where risk of manufactur-

ing problems in a facility is mini-

mal. FDA officials also propose to

reduce oversight of high quality

operations on occasion by request-

ing documents and posing ques-

tions online, instead of actually

visiting the site.

It is not surprising, however, if

drug manufacturers are skeptical

about real reductions in FDA over-

sight. Agency officials have been

talking for years about relief in

submitting post-approval changes

for low-risk activities, but so far

have failed to make significant

modifications in documentation

and preapproval requirements for

most changes in production sys-

tems, ingredients, and sites.

More eNforceMeNtWhile offering regulatory “carrots”

to compliant manufacturers, FDA

officials also predict strong action

against those that violate the rules.

Federal enforcement agents are tar-

geting drug companies that submit

inaccurate data to the agency—an

issue that has led to serious repercus-

sions for a number of Indian generic-

drug makers. Federal prosecutors are

looking hard at cases involving man-

ufacturers “doctoring their data” and

anticipate more charges related to

fraudulent reports, particularly from

small companies.

These efforts are supported by

the FDA Safety and Innovation Act

(FDASIA) of 2012, which provides

added resources through generic-

drug user fees for the agency to

expand inspections of overseas

producers. The legislation also pro-

poses stiffer penalties on firms that

refuse access to their manufactur-

ing facilities or fail to provide full

and complete documentation on

their products.

FDA issued a final guidance in

2014 that spells out how it will

interpret industry actions it regards

as “delaying, denying, limiting, or

refusing” a drug inspection. Such

behavior could be grounds for

declaring products from that facil-

ity as “adulterated” (2).

In response, a legal group is

quest ioning FDA’s interpreta-

tion and implementation of the

inspection policy, stating that it

goes beyond the agency’s statutory

authority. A legal backgrounder

by attorneys with Hyman, Phelps

& McNamara for the Washington

Legal Foundation (WLF) claims

that FDA’s approach could have

“draconian effects” on companies

that, for any reason, are not aware

of specific FDA inspection require-

ments and fail, even inadvertently,

to meet its demands (3).

A key point of dispute is whether

FDA inspectors have the right to

take photographs as part of an

inspection, a practice that indus-

try lawyers have long challenged.

Because FDA is asserting authority

not specifically provided by stat-

ute, the attorneys advise manufac-

turers to challenge demands from

agency inspectors to review records

kept in another facility, to photo-

graph any and all operations, or to

question any company employee,

especially those not trained to

deal with regulatory authorities.

A manufacturer could have legiti-

mate reasons for requesting a dif-

ferent date for a plant inspection

or to direct questions to certain

specialists, without fear that its

operations will be shut down and

products taken off the market, the

lawyers advise.

Much of the debate hinges

on how FDA interprets what

is “reasonable” and “timely” for

inspectors to request, or for a man-

ufacturer to challenge. The need to

clarify FDA’s expanded inspection

authority will be debated further

as Congress continues to weigh a

broad range of proposals for refin-

ing FDA programs and policies in

upcoming legislation.

refereNces 1. H. Sklamberg and C. Schnedar, “From

New Jersey to New Delhi, a Global

Focus on Quality,” FDAVoice, FDA.gov,

March 24, 2015, http://blogs.fda.gov/

fdavoice/index.php/2015/03/from-

new-jersey-to-new-delhi-a-global-focus-

on-quality/

2. FDA, Guidance for Industry,

Circumstances that Constitute Delaying,

Denying, Limiting, or Refusing a Drug

Inspection (CDER, October 2014), www.

fda.gov/downloads/

RegulatoryInformation/Guidances/

UCM360484.pdf

3. FDA Law Blog,“HP&M-Authored WLF

Legal Backgrounder Provides ‘Warning

Letter’ to Industry Regarding FDA’s

Inspection Practices,” FDALawBlog.net,

March 16, 2015, www.fdalawblog.net/

fda_law_blog_hyman_phelps/warning_

letters/. ◆


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Perspectives on Outsourcing

Do

n F

arr

all/G

ett

y Im

ag

es

Biopharmaceutical manufacturing clus-

ters continue to emerge outside of the

traditional hubs of North America and

Western Europe. Contract manufacturing

organizations (CMOs) in developing markets,

however, are unlikely to threaten the power-

house CMOs in the United States and Europe,

according to preliminary data from BioPlan

Associates’ 12th Annual Report and Survey of

Biopharmaceutical Manufacturing Capacity and

Production (1).

As part of the survey, biotherapeutic devel-

opers are asked to estimate the percentage

of operations at their facilities likely to be

outsourced internationally in the next five

years. A solid majority of biologics developers

indicated in the 2015 study that they will not

offshore any of their process development for

biomanufacturing over that period, a result

that is consistent with years past. Essentially,

the technical dominance of North American

and European CMOs appears to remain unmo-

lested, at least for the time being.

Larger biologics developers often prefer to

keep process development capabilities in-house,

even as they avoid investing in manufacturing

facilities. This strategy enables them to save on

up-front facility costs while still evaluating pro-

cesses to the point where they can

tech-transfer a process to a CMO.

Recent years have seen more

enthusiasm for offshoring bioman-

ufacturing operations and clini-

cal trials/operations. Data from the

2015 survey shows expected levels

of biomanufacturing operations

offshoring that are steady with

2014, with a greater proportion of

respondents (close to two-thirds)

envisioning offshoring of clinical

trials and operations in their future.

Separate data confirm that the geographic

location of a CMO has little influence on the

selection process. For example, in the 2014

study, a CMO being “local to me” was the

least important of 19 CMO attributes that

were measured.

Top ouTsourcing desTinaTionsOffshoring for biologics has been generally

equated with emerging hubs in Asia, espe-

cially Singapore, China, India, or Korea. This

trend is changing, as domestic CMOs focus

on being able to meet full US and EU cGMP

standards and be allowed to manufacture

commercial products for those markets.

Michael Yu, president and CEO of Innovent

Biologics (Suzhou, China), a biopharmaceuti-

cal company that produces affordable bio-

logics by manufacturing products locally,

reportedly, to international quality standards,

says, “We want to be the leading biophar-

maceutical company in China … Our pri-

mary concern is to ensure the quality of the

products we develop meet international stan-

dards, not only in China, but also for the

highly regulated markets outside of China. To

date, no Chinese biologic product has been

approved for marketing in highly-regulated

markets such as the US.”

Despite these emerging facilities, biothera-

peutic developers are most comfortable off-

shoring to established markets—the US and

Europe. In terms of the outsourcing destina-

tions that are most-cited as at least a possibil-

ity for outsourcing during the next five years,

the leading markets are:

The future looks brighter for

cMos in emerging markets.

Biomanufacturing Outsourcing Globalization Continues While the United States and Europe still dominate, CMOs and CROs based in emerging markets continue to capture market share.

Eric Langer is president of Bioplan

associates, tel. 301.921.5979,

[email protected].


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perspectives on outsourcing

• USA (78% are at least considering)

• Germany (76%)

• Singapore (74%)

• United Kingdom (73%).

The US and Germany remain

the leaders, even when the data

are narrowed to more positive

intent, with each considered by

roughly 3 in 10 respondents to

have a “strong likelihood” or

“likelihood” of being an interna-

tional outsourcing destination in

the next five years.

It ’s worth not ing that the

BioPlan study is international

in scope, and in years past there

have been significant geographic

differences in potential desti-

nations. In the 2014 study, for

example, European companies

considered China and the US as

likely destinations, whereas US

companies preferred Singapore

and Germany.

Preliminary data show that

China is again the most attractive

of the emerging destinations. In

fact, many see it as a likely out-

sourcing destination during the

next five years, most likely due

to perceived cost advantages, and

expectations for developing qual-

ity initiatives. Among the BRIC

countries (Brazil, Russia, India,

and China), China continues to

lead the pack, followed by India.

In the short term, however,

quality problems and percep-

tions continue to stunt India’s

appeal. In 2014, Mumbai-based

Glenmark announced a new

manufactur ing fac i l ity based

in the US. Sujay Shetty, leader-

pharma, PwC India, commented,

“Due to qual ity issues, more

domestic companies are trying

to de-risk strategy by … setting

up facilities close to US soil, in

Canada and Mexico. With tech-

nolog ica l advances and gov-

ernment incentives, the cost of

manufacturing in the US, par-

t icularly injectables, has also

reduced, which is a big draw” (2).

Quality concerns tend to gain

broad exposure, and the percep-

tion of quality problems can play

a factor in outsourcing consider-

ations. In the 2014 study, “being

local” was the least important

consideration, yet “complying

with a company’s quality stan-

dards” was the second-most criti-

cal attribute that a CMO could

demonstrate. In prior years, it has

been the most important attribute.

Nevertheless, the future looks

br ighter for CMOs in emerg-

ing markets, as they continue to

develop regional manufactur-

ing clusters. China (8.6%) and

India (8.1%) together account for

approximately one-sixth of global

biomanufacturing capacity (3).

Biocon’s Bangalore Plant is one

of the highest-ranked facilities

on BioPlan’s list of the top 1000

biopharma facilities. As quality

systems improve, these emerging

regions will take on an increas-

ingly important role on the global

biopharma stage.

Recent headlines are testament

to growth in China. For example,

GE Healthcare Life Sciences built

two facilities in China for CMO

JHL Biotech, including a KUBio

modular biopharma factory in

Wuhan that will house 2000-L

single-use bioreactors (4). Pall

Corp. opened its Life Sciences

Centre of Excellence in Shanghai,

which offers biopharma process

solutions (CMO work), technical,

and validation support as well as

training (5).

Beyond China and India, Latin

America (6.6% share of global man-

ufacturing) and Russia and Eastern

Europe (2.9%) are also emerging:

all of these regions combined now

comprise more than one-quarter of

global biomanufacturing.

conclusionCMOs and contract research

organizations based in emerging

markets will continue to capture

market share, albeit slowly. These

CMOs continue to face domestic

regulatory and legal hurdles, and

are far from obtaining approvals

for US and EU markets. Perceived

cost-effectiveness offered by CMOs

in these markets may be eroding

as other supply chain costs are fig-

ured in, and whatever advantages

could be gained from cost-compet-

itiveness also appear to be declin-

ing, judging by BioPlan’s recent

survey results that suggest cost-

effectiveness is waning as a selec-

tion attribute.

Never t he le s s , t he inte r na -

tionalization of biomanufactur-

ing outsourc ing markets can

be expected to grow. BRIC and

other developing countries have

yet to pose a significant threat

to US and European dominance.

However, simply based on the

weight of their emerging popula-

tions, their growing economic

power, and demand for better,

cheaper domestic biologics, it is

likely that their growing biopro-

cessing competence will result in

cGMP production and export of

biologics to US and EU markets

in the future.

references 1. BioPlan Associates, 12th Annual Report

and Survey of Biopharmaceutical

Manufacturing Capacity and

Production (Rockville, MD, April 2014),

www.bioplanassociates.com/12th.

2. R. Mukherjee, “Glenmark Joins Pharma

Companies Setting Up US Plants,”

Times of India, July 15, 2014, http://

timesofindia.indiatimes.com/business/

india-business/Glenmark-joins-pharma-

companies-setting-up-US-plants/

articleshow/38398901.cms

3. BioPlan Associates, Top 1000 Global

Biopharmaceutical Facilities Index,

www.top1000bio.com, accessed Feb.

25, 2015.

4. JHL Biotech, “JHL Biotech Partners with

GE Healthcare Life Sciences to Build

Monoclonal Antibody Manufacturing

Plant,” Press Release, Mar. 13, 2013.

5. Pall Corporation, “Pall Opens New Life

Sciences Centre of Excellence in

Shanghai(China),” Press Release, Oct.

15, 2013. ◆


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Inside Standards

12

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Ensuring the Quality of BiologicalsThe European Pharmacopoeia defines the format and content of monographs for biologicals to keep pace with recent approaches and meet the needs of its users.

The European Pharmacopoeia (Ph. Eur.), which

celebrated its 50th anniversary in 2014,

provides common standards throughout

Europe for the quality of medicines and sub-

stances used to manufacture those medicines.

The Ph. Eur. is recognized worldwide for its essen-

tial role in ensuring the quality of chemical APIs

and excipients, yet it also plays a vital role in pro-

viding quality standards for medicinal products

and substances of biological origin. Biologicals

are those products derived from biological

sources such as tissues and cell extracts, fermen-

tation using micro-organisms, plasma, plants, or

those produced by recombinant DNA technology.

The use of materials of biological origin in medi-

cine has a long history, and the quality control of

biologicals has been in place for many years.

The Ph. Eur. began elaborating monographs on

biologicals as an early priority with the first bio-

logical monographs on vaccines for human use,

immunosera, and immunoglobulins

appearing in Volume 2 of the first edi-

tion of the Ph. Eur. in 1971. The second

edition of the Ph. Eur. (first published

in 1980) saw the elaboration of mono-

graphs that concerned substances

extracted from animal tissues (such

as heparins) and human blood and

plasma-derived medicinal products. In

1992, a general monograph for prod-

ucts of recombinant technology was

included in the Ph. Eur. This was fol-

lowed by monographs for an increas-

ing number of biotechnology products

in the 1990s and 2000s. As the Ph.

Eur. moved into the 21st Century, it

increased its activity in the biological

field to address increasing numbers of

biologicals and challenges driven by

development of biosimilar products.

PharmacoPeial standards for biologicalsBiologicals are large and often highly complex

molecules. The Ph. Eur. has established general

monographs that cover common quality attri-

butes and that are applicable to a specific class of

biologicals such as monoclonal antibodies (mAbs),

rDNA products, or vaccines for human use and

veterinary use. The Ph. Eur. also contains indi-

vidual monographs that cover diverse biological

substances and products (e.g., insulin and insulin

analogues, peptide hormones, human growth fac-

tor, human granulocyte colony stimulating factor,

interferons, erythropoietin, recombinant vaccines,

and blood coagulation factors). These individual

monographs provide analytical procedures and

acceptance criteria to allow demonstration that a

substance or product meets the required quality

standards. The monographs are based on licensed

specifications (for the drug substance or finished

product) backed up by batch data.

Additionally, the Ph. Eur. monographs are sup-

ported by a large number of general chapters that

describe standard methods. These general chapters

give requirements for equipment and equipment

verification and instructions on how to perform

a method, such as peptide mapping. Ph. Eur.

monographs and general chapters for biologicals

make reference to the use of chemical reference

standards and biological reference preparations

(BRPs) for identification, purity testing, and assay

(content/potency) of biologicals. BRPs are estab-

lished in view of achieving standardization of

test methods for the quality control of biologicals.

Collaborative studies are undertaken to establish

these reference preparations leading to the refine-

ment of Ph. Eur. monographs. As regards to refer-

ence standards, a one-standard-fits-all approach

is not possible for all biologicals due to their het-

erogeneity (e.g., for glycoproteins). Therefore, for

glycoproteins, this issue has been overcome by

Stephen Wicks, PhD, is the

scientific regulatory policy

and intelligence officer of the

european directorate for the

Quality of medicines & healthcare

(edQm) of the council of europe,

[email protected]. edQm

is an organization that protects

public health and, among other

things, is responsible for the

European Pharmacopoeia.


May 2015 www.biopharminternational.com BioPharm International 17

inside standards

using the Ph. Eur. reference standard

to demonstrate system suitability,

and users are requested to establish

their own in-house reference prepa-

ration for glycan analysis.

biosimilarsA similar biological or biosimilar

is a biological medicinal product

that contains a version of the active

substance of an already authorized

original biological medicinal prod-

uct (reference medicinal product)

in the European Economic Area

(EEA). The first biosimilar was

approved in Europe in 2006, and

there are now 20 biosimilars autho-

rized in Europe. Importantly, the

Ph. Eur. provides a framework for

the quality requirements for biosim-

ilars of increasing complexity. As

stated in the European Medicines

Agency (EMA) Guideline on Similar

Biological Medicinal Products (1),

“Comparability studies are needed

to generate evidence substanti-

ating the similar nature, in terms

of quality, safety and efficacy, of

the similar biological medicinal

product and the chosen reference

medicinal product authorized in the

EEA.” With regard to quality, Ph.

Eur. standards have a role during the

development of biosimilars as they

are used for method qualification

and validation. Where biosimilars

have been approved for use in the

European Union, there are corre-

sponding Ph. Eur. monographs that

cover the product classes of human

growth hormone, granulocyte col-

ony-stimulating factor, erythropoi-

etin, and insulin. Although the Ph.

Eur. recognizes that monographs

provide public standards for the

quality of medicinal products and

their constituents, the monographs

are not sufficient to assess identity

and similarity of medicinal prod-

ucts that are required to establish

the comparability of a biosimilar to

an original biological product in the

context of a marketing authoriza-

tion application.

Even though users of the Ph. Eur.

are supportive of the existing gen-

eral chapters and monographs, the

Ph. Eur. acknowledges that there

are number of challenges to address

in the future development of indi-

vidual monographs for biologicals.

First, there is a need for flexibil-

ity within biological monographs

because of the requirement to cover

heterogeneous products manu-

factured by different production

methods. Nevertheless, there is a

need for monographs to provide

enough detail for a user to be able

to perform the analytical methods.

Second, there is a need to balance

the inclusion of new technologies

in monographs with the robustness

of existing analytical methods.

The Ph. Eur. is often challenged

on why individual monographs are

elaborated for biologicals due to

their unique complexity and inher-

ent heterogeneity. The elaboration

of biological monographs is a tiered

process starting with the originator

product, but the monograph may

be subsequently adapted to account

for biosimilar products. For com-

plex biologicals, the Ph. Eur.’s cur-

rent focus is to introduce classes of

quality attributes and have specific

acceptance criteria; this is exempli-

fied by the recent monograph for

recombinant human coagulation

factor IX where the glycan analysis

test is described in the production

section of the monograph and calls

for the use of an in-house reference

preparation shown to be represen-

tative of clinically tested batches

and batches used to demonstrate

consistency of production. Also,

no acceptance criteria are given for

the test; this decision on the cri-

teria is left to the discretion of the

competent authority. The rationale

is that there is a direct relation-

ship between glycosylation and the

manufacturing process. This new

approach is aligned with the devel-

opment approach for biosimilars

and involves communication chan-

nels between the main regulatory

players in Europe: EMA, national

authorities, and the European

Directorate for the Quality of

Medicines & HealthCare.

The Ph. Eur. intends to under-

take further refinement of the for-

mat and content of monographs

for biologicals to keep pace with

recent approaches and meet the

needs of its users (e.g., the new

approach to include “Glycan anal-

ysis” in the Production section of

the monograph).

advanced theraPy medicinal ProductsThe Ph. Eur. has also worked

to address the emergent field of

advanced therapy medicinal prod-

ucts (ATMPs), or cell based and

gene therapy medicinal products,

and has developed a chapter that

addresses quality requirements

of the biological raw materials

that are used for the production

of these types of products. This

informational chapter, which is

intended to be published by the

end of 2015, has undergone public

enquiry and gives examples of the

critical quality attributes specific

to each class of raw material with

the aim of harmonizing the vari-

able practices that are currently

adopted for biological raw materi-

als. The development of this chap-

ter has been performed in close

collaboration with members of

the EMA Committee for Advanced

Therapies and the Biolog ics

Working Party.

Thus, the exist ing Ph. Eur.

monographs and chapters and the

continued development of mono-

graphs and chapters for biologi-

cals supports the Ph. Eur.’s mission

to facilitate patient access to high

quality medicines.

reference 1. EMA, Guideline on Similar Biological

Medicinal Products (Committee for

Medicinal Products for Human Use,

April 2013), CHMP/437/04 Rev 1. ◆



xiao

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a/G

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es

It should come as no surprise that

the good design principles govern-

ing the execution of pharmaceu-

tical manufacturing in modules

were birthed in Sweden, home of IKEA,

and neighbor to Denmark, home of the

Danish modern furniture movement.

While the idea of modular construc-

tion has been used in other industries

for decades, it has only started gaining

traction for pharmaceutical applications

during the past 20–30 years.

Modular systems can refer to a vari-

ety of different physical configurations,

which can be a source of confusion.

Whether something is called a pod or

module seems to largely depend on

where the unit is actually constructed

(on site or off site) and where all of the

cleanroom components live (inside the

actual unit that is shipped or inside

the existing facility infrastructure).

There are modular room enclosure sys-

tems (such as from Daldrop, AES, or

Plascore); modular room enclosure sys-

tems with utility systems (such as from

G-CON Manufacturing or SmartFit

Modular); building modules with util-

ity systems (such as from Pharmadule/

Morimatsu); or building modules with

process systems (such as GE Healthcare

Life Systems’ KUBio FlexFactory plat-

form). According to Pär Almhem, presi-

dent of ModWave, modular design

and construction in pharmaceutical

process facilities can include either

breaking a facility and its manufactur-

ing processes into functional build-

ing blocks/modules or into physical

building blocks/modules. Functional

blocks “create opportunities to sim-

plify, standardize, verify, and reuse

Modular Manufacturing Platforms for Biologics

Randi Hernandez

The costs and benefits of integrating

modular concepts for on-demand

bioprocessing are explored.

Modular Systems


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designs and modules in different

implementations,” while physical

modules allow for the construction

of modules off site, reducing “con-

gestion and disturbances on site,”

notes Almhem.

Blocks—also called modular

suites, mobile or modular clean-

rooms, pods, or flexible or expend-

able cleanrooms—can include

stand-alone modular buildings or

those that can be installed into

an existing building shell. They

can also include configurable

skids; process modules/skids based

on single-use equipment; or unit

operations within modules, such as

suites made exclusively for aseptic

filling of prefilled syringes or con-

tinuous manufacturing lines (1).

Prefabricated fill line/cleanroom unit

combinations via pods—or autono-

mous containment systems—can be

introduced into an existing process

easily, says Maik Jornitz, president of

G-CON Manufacturing.

A common choice for modu-

larization has been in the form

of ut i l it y suites, says J. Lee

Emel, executive director of CRB

Consulting Engineers. “Over the

past 10–15 years, the industry has

been experimenting with vari-

ous ways of incorporating mod-

ular design into project delivery

of capital programs,” says Emel.

“Modular design of process sys-

tems (superskids), utility systems,

spaces (penthouses and electrical

rooms), and wall-panel systems

have been utilized with much suc-

cess,” he adds. According to Emel,

speed to market is no longer the

only business driver behind using

modular systems; asset flexibil-

ity, cost of goods, and investment

deferment are now key significant

drivers of modular construction.

A full module typically is fash-

ioned with its own mechanical,

electrical, HVAC ductwork, high-

efficiency particulate air filters,

and plumbing systems—and pro-

cess equipment is usually already

part of the module (2). There are

numerous benef its related to

quality control with building

and qualifying units off site. If a

full module is unrealistic because

of budget or existing architec-

tural constraints, a piecemeal

approach to modularity could

incorporate turnkey process and

ut i l it y equipment, including

clean-in-place skids, wall-mount-

ing systems for piping, and air

handling/temperature control

units (2). For best results, modular

solutions need to be planned dur-

ing the early stages of a project

(3).

Advantages of modular systems

include shorter time to market,

higher cost predictability for each

module, the ability to standardize

operations, reduction of manu-

facturing disruption to current

operations, less waste, and the

flexibility to set up and test com-

plete product runs before they

are commercially deployed (1, 2).

In addition, closed modular sys-

tems are said to reduce required

ma nu fac t u r i ng a r ea , H VAC

requirements, chilled water and

steam demands associated with

cleaning, construction and start-

up times, and potentially, cost

of goods (4). A modular facility

is said to be associated with an

approximately 15% reduction in

energy consumption and, after

accounting for risk, is estimated

to be 5% more cost effective than

a traditional facility (5, 6). Not

only are modular production

facilities more cost effective to

operate, they also take a shorter

time to construct: While a tra-

ditional facility can take three

to seven years to complete,

its possible for a modular facil-

ity for oral solid dose, biologics

bulk, or aseptic filling to be built

in only 12 months from project

start to operational qualification,

estimates Almhem. Pods can be

built in an even shorter amount

time, says Jornitz. “If one utilizes

a shell building/cleanroom pod

scenario, the shell building can

be erected in six to eight weeks;

the cleanroom pods are built par-

allel to the shell building and

require 15–18 weeks.” The 15–18-

week timeline likely aligns with

process equipment and utility

bundles, he adds.

Standardized But cuStoMizedA key element of early project

planning deals with the cus-

tomization of modules to meet

a client’s needs. While modular-

ity supports standardization and

repeatability, it can also sup-

port customization if done cor-

rectly, says Almhem. “Taking a

fixed process and installing it

in a building module does not

promote c ustomizat ion,” he

says, but “div iding a process

into well-defined process steps—

with def ined inputs, outputs,

and transformations—is the best

way to allow for customization

while maintaining quality and

efficiency.” As Emel notes, dif-

ferent modular approaches will

work for different manufacturers,

especially if one has a large-scale

high-throughput facility using

large steel tanks while another

has a smaller facility focused

on lean manufacturing and sin-

gle-use systems. Robert Dream,

consultant at HDR Company

says that while true modularity

helps to reduce capital expendi-

ture and operating expenses, and

helps ensure better manufactur-

ing practices, standardization is

not necessarily a requirement of

modularity. Though not required,

standardization is likely desired,

counters Jornitz: “Even when a

project pod is designed, if such a

pod is replicated, we would always

recommend to clone the system

to reduce the qualification and

validation burden.”

Modular Systems



current Modular facilitieSWhile there are numerous exam-

ples of facilities that have been

built with a modular concept in

mind—such as Genentech in San

Francisco, CA, and Singapore;

Lilly in China, Egypt, Ireland,

the United Kingdom, the United

States, and Puer to R ico; and

Merck in Singapore, Ireland, the

United States, and Puerto Rico—

Almhem points out that these

units have become permanent

facilit ies once they were con-

structed. There are fewer exam-

ples of modular facilities that

have been moved and reconfig-

ured, he asserts. Mike Booker,

manager of business develop-

ment at PortaFab, a manufacturer

of cleanroom wall systems and

other modular cleanroom prod-

ucts, says the company has a

Mid-West pharmaceutical client

that uses modular construction

to meet its “ever-changing facil-

ity requirements.” Modular con-

struction helps PortaFab’s clients

reduce backend costs due to the

reusability and flexibility of exist-

ing materials.

Contrac t development and

manufactur ing organizat ions

(CDMOs) and contract manufac-

turing organizations (CMOs) have

tradit ionally v iewed modular

approaches as too expensive and,

as a result, have been reluctant to

adopt them, notes Almhem, but

this may change as it becomes

more clear that modular concepts

are competitive models. Because

the CMO business model may put

more cost pressure on delivery,

adds Emel, CMOs may “be willing

to try newer methods or technolo-

gies if they have the possibility to

reduce their initial capital invest-

ment.” While single-use equip-

ment lacks some flexibility in a

traditional facility, Jornitz says

that podular systems combined

with single-use systems, in par-

ticular, could be ideal for CMOs,

as CMOs typically run different

products, volumes, and processes

depending on individual clients.

The addition of single-use systems

reduces turnover and allows for

more f lexibility in production

planning. With single-use sys-

tems, “The changeover to another

product can be swift, and poten-

tial cross-contamination can be

avoided, since the product con-

tact layers are disposable,” says

Jornitz. Plus, if “capacity demands

flex, [the CMO] could potentially

add or deduct some of the clean-

room areas.”

PodS: PortaBle facilitieSLocal diseases often require local

solutions, and getting produc-

tion online quickly is a major

concern in emergency situations.

Therapeutic treatments produced

via a modular vaccine suite—a

pandemic-ready modular facil-

ity—could be a good option in

the future when dealing with the

proliferation of tropical diseases

and regional outbreaks. Smaller

manufacturing subsystems are best

suited for emerging markets where

local resources are limited and

speed of production is a crucial ele-

ment (1). A surge capacity is neces-

sary to rapidly scale up production

in case of emergencies, and a tradi-

tional manufacturing facility may

not be able to meet quickly escalat-

ing demands (4).

Modular bioprocessing facili-

ties may be best suited to countries

that struggle with GMPs. Pods, in

particular, may be especially use-

ful for emerging markets, points

out Emel, “where local construc-

tion expertise or materials are not

readily available.” In well-devel-

oped markets, where the process

can be fully closed, “other deliv-

ery methods may prove to be more

cost effective and flexible,” Emel

determines.

A pod (also known within the

industry as a POD, although the

word is not an acronym in this

context) is a prefabricated clean-

room box designed and built off

site, explicates Jornitz. The pod

hosts the air handlers, fire sup-

pression system, and controls—

which are all in the mechanical

space—and the mechanical space

is accessible from the gray space

of the host facility. “The clean-

room space is accessible via air

locks within the pod, which are

connected to a classified corri-

dor,” he says. This infrastructure

is unlike stick-built cleanrooms,

which are interconnected via

ductwork that is often as large

as the building structure itself.

In essence, instead of building

a shell around the process, the

process is being integrated into

the shell.

Jornitz says that his company

could produce antiv irals and

containment systems via pods

in approximately 12–18 weeks.

Caliber Therapeutics, originally

a subsidiary of G-CON, LLC

until the formation of G-CON

Manufactur ing Inc. in 2014,

was established with Defense

Department funding from the

Defense Advanced Resea rch

Projects Agency (DARPA) to be able

to respond to biological and pan-

demic threats. Caliber has received

inquiries from government offi-

cials about whether it could help

manufacture ZMapp, a treatment

to fight the Ebola virus. Caliber

uses G-CON’s pods for its entire

downstream processing, says

Jornitz. The work with G-CON

through Caliber’s Nicotiana ben-

thamiana plant expression system

“shows that podified downstream

processing st ructures can be

deployed and redeployed rapidly

without compromising required

quality attributes.”

Cleanroom pods can be used

for entire process streams, as in

the Caliber example, or can be

used as a single pod for filling

Modular Systems



Modular Systems

purposes, such as with PaxVax,

which uses a 24-foot-wide pod

for this purpose. PaxVax installed

the pod at its GMP vaccine pro-

duction facility located in San

Diego, California in 2013 for the

clinical and commercial manu-

facturing of its single-dose oral

cholera vaccine. The pod, custom-

ized specifically for PaxVax, was the

first controlled non-classified (CNC)

cleanroom environment to be deliv-

ered into the state of California,

which the company admitted pre-

sented a few “regulatory and engi-

neering challenges” (7). Because

pods are prefabricated and must

be introduced into an existing

shell building, once they arrive at

a facility, wall panels, doorways,

and existing pillar structures can

limit their movement within the

facility, notes Jornitz. For this rea-

son, a number of different sized

pods can be clustered or put into

place as a single unit, he adds.

While pods may be good solu-

tions as far as individual clean-

rooms or laboratory spaces go,

Almhem says they are typically

not good “for an integrated pro-

cess suite or facility that extends

over more than three to four mod-

ules.” Scalability of pods is also

a concern, and pods are gener-

ally more expensive than other

modular implementations. Emel

concurs that compared with open,

f lexible manufacturing spaces

using closed processing, podu-

lar systems are less flexible and

adaptable. Sid Backstrom, direc-

tor of business management at

G-CON, disagrees with the senti-

ment that pods cannot be inte-

grated in concert successfully,

and says that a recent delivery

by G-CON “belies that opinion.”

He says, “We delivered six pods

clustered together that integrated

a complete OSD [oral solid dos-

age] line from GEA Pharma that

includes continuous mixing and

direct compression, wet granula-

tion, drying, and a tablet press.

The unit will be used for R&D

work at f irst, and then com-

mercial manufacturing, either

domestically or disassembled and

redeployed overseas. Another five-

pod configuration is in fabrica-

tion now,” he says. “Our very first

customer purchased six pods, and

these are connected via a clean

corridor. Caliber Biotherapeutics

employs six pods for every step of

its downstream purification pro-

cess,” he adds. Backstrom says

that the flexibility of the OSD line

allows site managers to seamlessly

add pods for other process opera-

tions, such as encapsulation, with-

out affecting cleanroom operations.

Jornitz notes that there has

been recent interest in the use

of pods for analytical, micro-

bial, polymerase chain reaction,

and assay development, as well

as interest in the use of pods for

broader healthcare purposes,

including transmissible disease

containment. He says G-CON

has been approached to design a

mobile containment system to be

able to segregate patients. “The

transmissible disease contain-

ment systems range from patient

transfer to installed patient care

units,” he says.

Modularization of BiologicS and BioSiMilarSEmerging markets and the increas-

ing demand for biosimilars may

prompt pharmaceutical manu-

facturers to adopt a “local sourc-

ing” attitude that has historically

been reserved for other consumer

goods. As a result, biosimilar

approvals may inf luence facil-

ity design needs, and these facil-

ities may crop up in new areas.

“Biosimilar production site [man-

agers] look for a high degree of

flexibility and often try to run

multiple products through the

same process, especially in an in-

country/for-country situation,”

notes Jornitz. Biosimilar end users

are keen to incorporate single-use

process designs and flexible clean-

room spaces, he says. “Robust con-

tainment is a key element when

one wants to use multiple products

within the same process.”

Depending on certain key fac-

tors, prefabricated buildings or

manufacturing pods may also be

viable solutions for fast-track proj-

ects. Emel points out that schedule

flexibility associated with modu-

lar manufacturing could be a sig-

nificant competitive advantage,

especially in crowded therapeutic

markets where similar products are

in a race for regulatory approval.

Emel writes on CRB’s website,

“Being able to compress project

schedules also allows manufactur-

ers the opportunity to delay invest-

ment, allowing them to further

scrutinize market data, or con-

versely, seize a market opportunity

before their competition” (8).

A lmhem says that increas-

ing pressures to shorten time to

market, decrease operating cost,

and improve efficiency will push

biosimilar developers toward

facilities that are smaller, more

efficient, and more flexible. The

answer may not only lie with

modular models, but with a com-

bination of cost-saving efforts.

While the industry wil l con-

tinue to look for ways to reduce

capital investment when bring-

ing a drug to market, Emel says

that “the holy grail may very

well be continuous processing,

which could make manufactur-

ing facilities significantly smaller,

faster to build, and much less

costly.” Indeed, Richard Steiner,

business development manager

at ConsiGma at GEA Pharma

Systems and a proponent of

PCM&M or PCMM (portable, con-

tinuous, miniature, and modu-

lar) systems, told Pharmaceutical

Technology, “continuous process-

ing is enabling miniaturization;



Modular Systems

Best Practices in Facility Design

Multiple business and market forces are driving change in facility design and operation including the requirement to

establish Òin-countryÓ manufacture for local supply as authorities in smaller, yet faster, growing markets push a localization

agenda. Another driver of change is the need to achieve faster delivery of new drugs to clinical trials followed by faster speed

to market if approved. This change means more agile approaches to supporting the supply of clinical and development lots

during pre-commercial phases are needed. Downward payer pressure on prices is yet another motivation to transform current

manufacturing practice resulting in the necessity to pursue lower up-front build costs followed by lean low-cost operation.

The challenge of meeting these business and market needs is being met via lower cost, flexible, and quicker-to-build

facilities while achieving world-class quality standards. Innovative approaches and disruptive technologiesÑsuch as modular

facilities, continuous bioprocessing, closed systems, single-use systems, and traditional processesÑare being combined and

incorporated into these plants.

Modular facilities

Modular biofacility design is an approach that subdivides the architectural and bioprocessing system into smaller parts called

modules or skids that can be independently created and then combined and used for different purposes. Modular approaches

can be explained as separating out discrete, scalable, reusable elements, rigorous use of well-defined interfaces, and making

use of industry standards for those interfaces. Modularity offers benefits such as reduction in design and manufacturing cost

from less customization, shorter lead times, and flexibility in design, as well as the ability to increase mobility and deployment in

environments less accustomed to biopharmaceutical manufacturing.

Continuous bioprocessing

Continuous bioprocessing is a production method used to process biological drug substance without interruption. It contrasts

markedly with the majority of biological drug manufacturing that is batch based. The advantage of continuous processing is the

potential to remove hold steps, reduce factory footprint, and the prospect of effective scale out, ultimately lowering capital and

operating cost. Continuous processing can be coupled with process intensification, which seeks to maximize biomass produced

per cubic meter of processing capacity per unit time, through better expression systems and higher yielding processes.

Closed systems

Ensuring systems are closed through the manufacturing process provides opportunity to rethink environmental control to the

extent that space around the process equipment need not be housed in traditional cleanroom conditions. Closed systems further

reduce the complexity and cost of facility design and simplify operational running. Physical segregation of unit operations becomes

unnecessary except for pre- and post-viral clearance steps. In time, even this separation will be tested for necessity.

Single-use technologies

Single-use technologies are routinely selected as solutions for bioprocessing requirements at lower-end commercial scale.

Advances are centering on making their consumption repetitively effective from a supply-chain perspective, creating economic,

consistent, and sustainable use to scale-out production volumes. Speed of startup, flexibility, and elimination of sterilization/

cleaning requirements are benefits accruing from this approach.

Traditional vs. new

Integration of some or all of these facility designs plus targeted advances in online process monitoring, robotics, automation, and

real-time release are transforming the biologics manufacturing landscape. Step-change implementation is not straightforward in

a highly regulated and conservative industry, but innovative approaches to scientific risk evaluation, mitigation, and elimination

hold the key to success. The past and current state for manufacturing biological drugs is one featuring large-scale, high capital

cost, fixed assets, and location in western markets (e.g., United States, Ireland, and Singapore). The bioprocessing equipment is

stainless steel, and significant time and cost is required to validate and scale up these facilities. When fully utilized and producing

limited variety of lower titer products, they are relatively unit-cost efficient. These types of facilities are not at the end of their

usefulness; in fact 95% of manufacturing capacity is still in this format. Investment continues to be channeled into large-scale

stainless steel capacity including in the Asian region.

Revolutionary facility design and operation is a complementary addition to the industryÕs array of capability that meets differing

and emerging needs and offers to help expand access to biologics in new markets.

ÑSimon Chalk, director of the BioPhorum Operations Group



miniaturization is allowing pro-

cessing systems to become por-

table and modular” (9).

PCM M systems, lau nched

in partnership with Pfizer, GEA

Pharma, and G-CON, are virtu-

ally factories within a pod. The

first example of this type of unit,

constructed to produce oral solid-

dosage drugs, was delivered on

March 17, 2015. Final site accep-

tance and commissioning are

in process. The continuous pro-

cessing skid was prefabricated in

Belgium, integrated into a por-

table cGMP pod that had been

prefabricated in College Station,

TX, and then re-assembled into

Pfizer’s gray warehouse space

in Groton, CT. According to

Michael O’Brien, vice-president

of Pfizer Pharmaceutical Sciences

Technology & Innovation Group,

who spoke about the units at the

2015 Parenteral Drug Association

A n nu a l ( PDA) Me e t i ng on

March 16–18, 2015 in Las Vegas,

NV, PCMM systems help facili-

tate production and distr ibu-

tion on demand, rather than via

forecast models. Using the same

equipment for development and

manufacturing could help mini-

mize technical transfer costs and

resources, he wrote in his PDA

presentation notes, and could

reduce inventory overheard while

still allowing patients to gain

access to crucial medications (10).

In fact, DARPA’s Battlefield

Medicine program seems to take a

similar approach to PCMM in that

it focuses on the flexibility of a

modular reaction design. According

to the Battlefield Medicine website,

“In developing a flexible, miniatur-

ized synthesis and manufacturing

platform, Battlefield Medicine will

leverage continuous flow approaches

that will, if successful, pave the path

forward for enabling distributed, on-

demand medicine manufacturing

capabilities in battlefield and other

austere environments” (11).

Another example of modu-

lar system that is available is via

a Lödige continuous line. In this

example, the line itself is modu-

lar, says Almhem, and can be

installed into existing cleanroom

or mechanical areas. Alternatively,

the line can be implemented as a

complete processing suite in part

of an integrated facility, rather

than just in a stand-alone pod.

“Continuous manufacturing has

great potential to significantly

reduce manufacturing cost for OSD

products,” he says.

By using modularization and

the other aforementioned tech-

nologies such as continuous man-

ufacturing, facilities to produce

monoclonal antibodies (mAbs)

can also be operational 12 months

after project initiation, Almhem

says, and there are several modu-

lar mAb facilities that are already

in operation. In fact, modular

systems are best for mAbs and

are likely better than any other

system design for these prod-

ucts, states Jornitz, as most new

mAb processes are at the scale of

2000 L, which fits within the pod

design. While large-scale bioreac-

tor systems may require a tradi-

tional facility design, he says that

once harvest volumes are concen-

trated via ultrafiltration, podular

processes can resume. “Because it

is compact and highly integrated,

a continuous line lends itself very

well to a modular implementa-

tion,” Almhem adds.

challengeS aSSociated with ModuleS and PodS“Building one module is easy;

building an integrated modular

facility takes a lot more skill and

expertise,” observes Almhem.

Some of the oft-mentioned chal-

lenges associated with modular

models deal with technical con-

struction and the workforce plan-

ning and training required to

get a facility fully operational.

On-site construction and compli-

ance teams may need to engage in

testing and qualification activities

throughout the duration of each

project (2).

Pods are required to have a

shell building around them, as

they are critical cleanroom spaces,

notes Jornitz. The shell is required

to ensure the humidity and tem-

perature within the pod remain

stable, he adds. In the pod exam-

ple (sometimes known as “plug-

and-play”), this infrastructure

can be minimal and cheap, as all

of the critical cleanroom compo-

nents are contained within the

pod. In contrast, in a module-

based approach, an entire facility

is often built, shipped, and recon-

structed on site.

Governing regulatory agencies

for modules: Building codes

Regulatory bodies will require

some amount of infrastructure

around every cleanroom space,

notes Backst rom. Obta in ing

proper cleanroom protocol, regu-

latory objectives, and the desired

International Organization for

Standardization (ISO) classifica-

tions can introduce significant

obstacles and have the potential

to negatively affect the quality

of a module, comments Booker.

Closed processing is used in mod-

ules and is generally governed by

a grade D environmental clas-

sification (6). Open processing

areas—as required for seed prepa-

ration, final purification, and bulk

aseptic filling—are designed to

be grade C and are performed in

biosafety cabinets with laminar

air flow (6). Using CNC modules,

however, can allow manufactur-

ers to circumvent most cleanroom

classification requirements, says

Emel. As noted by Nelson, CNC

areas (noncritical areas in GMP-

manufacturing facilities) provide

some operation conditions, but

do not have a cleanliness class

Modular Systems



Modular Systems

assigned to the space and are not

monitored like grade-classified

rooms or ISO areas (4). Although

the goal of a cleanliness clas-

s i f icat ion through a Federa l

Standard 209 is to prevent con-

tamination, as PortaFab points

out on its website, “the presence

of viable particles (living organ-

isms) within the particle count

achieved by applying methods

described in Federal Standard

209 may affect operations of a

cleanroom” for biopharmaceu-

tical processes. Thus, the com-

pany writes, a measure of both

viable and nonviable particles

is required to provide enough

information about whether or

not a cleanroom is suitable for a

biologic (12).

The level of segregation between

processing areas should be deter-

mined using a risk-based approach

and should consider air handling,

viral processing, live organism

zones, and cell cultivation and cell

purification activities (6). The mod-

ule should also have areas for prod-

uct formulation; fill/finish; material

dispensing, washing, sterilization of

parts; and lyophilization.

A manufacturer has to con-

sider locat ion-spec i f ic cha l -

lenges related to the local code

requirements for construction

in that geographic region and be

well-versed in the rules govern-

ing expansions or upgrades. As

an alternative option, modules or

pods can be constructed off-site,

which is said to simplify site logis-

tics, enhance quality control, and

reduce waste (6).

Cost of building a

biomanufacturing module

Although some sources say that

modular facilities require a mod-

est upfront capital investment (3),

others, such as consultant Emel,

attest that a full modular suite

can be prohibitively expensive.

Although costs of building mate-

rials for pods may exceed that of

stick-built facilities, Almhem says

the cost differences are small and

can be “easily compensated by

the efficiencies of building in a

factory rather than in the field,

allowing for the same or poten-

tially lower cost for a modular exe-

cution.” Plus, modular design will

save clients costs by eliminating

architectural general contractor

fees, says Booker.

C os t c a lc u l at ion s a s s o c i -

ated with the incorporation of

modular processes often do not

take into account the total cost

of ownership, says Jornitz, and

are typically just accounting for

cost per square foot. Traditional

structures are designed for one

product lifecycle, whereas pods

have a lifespan of more than 20

years and can be repurposed or

relocated, he attests. Other ben-

efits of pods include compact

ductwork (which lowers the oper-

ating costs); they can be sanitized

robustly and therefore can run in

multi-product modes; and they

are easily scalable, so installation

is minimally disruptive to other

existing processes.

Although the most environ-

mentally friendly approach to

incorporating modules in a facil-

ity may be to salvage and convert

existing assets, and this method

may be “an important consider-

ation from a cost, schedule, and

investment depreciation perspec-

tive,” according to Emel, most

industry experts envision gut-

ting a building to a shell and

reusing only the utilities, park-

ing, access systems, and admin-

istration of legacy systems. The

prevailing attitude appears to be

that current facility architecture

and design is quickly becoming

obsolete. “The challenge today is

that many of the legacy facilities

were designed for single products

or platforms, as well as lower pro-

duction efficiencies, so they aren’t

always at the right scale or uti-

lize the appropriate technology to

address the current business needs

of many biotech manufacturers,”

says Emel, who points out that

most biotech companies are focus-

ing on specialty market products

requiring smaller batch produc-

tion requirements. “If existing

assets are able to be converted,

biotechs are creating flexible open

spaces leveraging closed process-

ing to meet their needs.”

referenceS 1. P. Almhem, Pharm. Process. 24

(4), pp. 30–32 (May 2014).

2. J. Gilroy and G. Martini, Pharmaceut.

Proc. 27, pp. 22–23 (2012).

3. A. Shanley and P. Thomas, Pharm.

Manufact. 11 (1), pp. 2–9 (2012).

4. K.L. Nelson, “Approaches for Flexible

Manufacturing Facilities in Vaccine

Production” supplement to BioPharm

Internat. 24, pp. s22–28 (2011).

5. H.L. Levine et al., BioProcess Int.

11 pp. 40s–45 (April 2013).

6. P. Almhem et al., “Modularisation in

Biologics Manufacturing,” Pharma

Focus Asia, www.pharmafocusasia.

com/manufacturing/modularisation-

biologics-manufacturing,

accessed March 25, 2015.

7. G-CON Manufacturing, “G-CON

Manufacturing Announces Delivery

and Operation of a Custom Made

POD for PaxVax, Developer of Oral

Vaccines for Infectious Diseases,”

Press Release, Jan. 18, 2013.

8. J.L. Emel, “Building A Thoughtful

Manufacturing Supply Chain,” CRB,

www.crbusa.com/blog/536-building-

a-thoughtful-manufacturing-supply-

chain, accessed March 25, 2015.

9. J. Markarian, Pharm. Tech. 38

(11), pp. 52–54 (2014).

10. M.K. O’Brien, “Portable, Continuous,

Miniature, & Modular (PCM&M)

Development and Manufacturing: The

Foundation for a Transformational

Development, Manufacturing, and

Distribution Model,” presentation

at the 2015 PDA Annual Meeting

(Las Vegas, NV, 2015).

11. DARPA, “Battlefield Medicine,”

www.darpa.mil/Our_Work/BTO/

Programs/Battlefield_Medicine.

aspx accessed April 1, 2015.

12. Portafab, “Bio-Pharmaceutical

Cleanroom Design Guidelines,”

Portafab, www.portafab.com/bio-

pharmaceutical-cleanroom-design.

html, accessed March 25, 2015. ◆



The harvesting of biologic actives

from cell media and other down-

stream separation processes, such

as viral clearance, rely on filtra-

tion technologies, in large part due to

the sensitivity of biomolecules to heat

and many chemical treatments. This sen-

sitivity precludes the use of alternative

methods. Filter manufacturers have been

challenged with increasing titers and cell

densities, greater expectations for viral

clearance, and the desire of biopharma-

ceutical manufacturers to employ single-

use technologies at an increasingly larger

scale. They have responded with higher-

capacity, higher-performance filters based

on new materials, membrane structures,

and filter designs.

Market sizeThe global pharmaceutical membrane fil-

tration market was estimated by Markets

and Markets to be valued at $3.69 billion

in 2013, and the market research firm

expects it will grow to $7.96 billion by

2018 (1). This estimate includes micro-

filtration, ultrafiltration, nanofiltration,

reverse osmosis, and ion-exchange filtra-

tion technologies used for cell separa-

tion, protein purification, sterilization,

virus removal, and water management.

In 2013, protein purification accounted

for the largest share of the filtration mar-

ket, but demand for membrane filters

for sterilization is growing the fastest.

Overall drivers of the market for mem-

brane filters include the growth of the

global pharmaceutical/biopharmaceu-

tical industry, particularly in the Asia-

Pacific region and in other emerging

markets. Increasing demand for single-

use technologies is a second important

driver of growth, according to Markets

and Markets.

the Move to single useThe adoption of single-use technologies

for downstream biomanufacturing pro-

cesses can present filtration challenges.

The use of disposable technology has

grown, however, to cover more process

operations and larger scales of produc-

tion. “Filtration technology has evolved

to adapt to these challenges as suppliers

leverage new materials and innovations

that enable higher capacities,” asserts

Richard Pearce, director of downstream

processing for EMD Millipore. He adds

that the integration of filtration technolo-

gies into larger scale single-use manufac-

turing processes must also be addressed;

as the trend for larger scale, single-use

manufacturing systems grows, filtration

technologies will need to be able to oper-

ate in this environment. Pearce believes

that products offering more efficient fil-

tration will lead to a reduction in the size

of filters, thus simplifying their integra-

tion with single-use technologies.

handling higher cell densitiesOne of the key trends in biopharmaceuti-

cal manufacturing over the past decade

has been the dramatic increase in titers

and subsequently, cell densities enabling

drug manufacturers to shift from large

10,000-L stainless steel reactors to smaller

disposable bioreactors, according to Rene

Faber, vice-president of filtration tech-

nologies at Sartorius Stedim Biotech. He

also notes that biopharmaceutical prod-

ucts are becoming more difficult to filter.

These higher cell densities and titers have

introduced challenges for initial clarifica-

tion in monoclonal antibody production.

“Existing filtration technologies don’t

have the capacity to handle these large

cell masses. In addition, manufacturers

are adding additives into the bioreactor in

Filtration technologies advance to Meet Bioprocessing needs

Cynthia A. Challener

Higher cell densities,

greater demand for high-

performance viral clearance,

and desire for large-scale

single-use technologies

are driving development

of filtration technologies.

Cynthia Challener, PhD,

is a contributing editor to

BioPharm International

downstream Processing



order to help with clarification steps,

and filtration technologies must

have the capacity to handle them,”

says Pearce.

At the same time, the demand

for higher standards of filter perfor-

mance, economy, safety, and impact

on final product quality continue to

challenge filter vendors, according

to Oliver Triebsch, senior director

of marketing with Pall Life Sciences.

He notes that this need for better

economy (filter capacity per cost) is

also driven by the higher-concen-

tration, more challenging-to-filter

fluids coming from bioreactors, or

alternatively, a need to process clean

fluids more rapidly.

Fi lte r manufac turer s have

responded to these challenges by

developing high-capacity filters with

the ability to process higher masses

of protein or cell mass with better

retention performance. For example,

EMD Millipore has introduced new

products for clarification that retain

cell mass, precipitants, and floccu-

lants, providing an excellent level of

protection to the downstream steps,

according to Pearce.

Innovative pleat geometries and

advanced membrane designs have

also been developed to address flow

rate or throughput demands, accord-

ing to Triebsch. “Such enhance-

ments enable quicker processing

at stable flow rates in combination

with high volumetric throughputs

until blockage, and provide consis-

tent processing with reduced filter

footprints and more manageable fil-

ter costs,” he says.

High-performance depth filter

media in disposable filter capsule

devices have also been introduced

that increase process efficiencies

and address the needs for simplicity,

safety, speed, and intuitive operation,

according to Triebsch. “These filters

remove whole cells and debris in

monoclonal antibody or recombinant

protein processes and improve exist-

ing filtrations while eliminating the

need for centrifugation,” he observes.

Tailor-made depth filter grades

for the vaccine industry also deliver

high virus yields with significantly

reduced turbidity, enabling efficient,

robust, and economic cell and cell

debris removal from the bulk reac-

tor output containing the virus or

virus-like particles directly after cell

culture.

Sartorius, for example, focused

on the need for large-scale single-

use filters so that customers could

have an integrated, modular, sin-

gle-use cell-harvesting system for

modern processes with high cell

densities, according to Faber. To do

so, the company adapted dynamic

body-feed (DBF) filtration technol-

ogy from the plasma and food and

beverage industries, which enables

the filtration of feedstreams with

high-particulate loads. “DBF is simi-

lar to depth filtration, but a high-

purity diatomaceous earth (DE) is

used with cell material to form a

cake that acts as a very efficient filter

layer, providing high productivity

and reproducibility for a variety of

feedstocks, even with high biomass,”

Faber says.

Innovative new technologies for

primary and secondary clarification

will still be needed as cell densities

coming out of bioreactors continue

to increase, according to Pearce.

Better retention with those high cell

masses will also be important. EMD

Millipore, Sartorius, Pall, and other

filter manufacturers are investigat-

ing new materials, new variations

on existing materials, and new filter

designs.

Focus on tangential-Flow FiltrationTangential-flow filtration (TFF) has

faced challenges as well, and in par-

ticular the need to recirculate the

concentrating sample through a TFF

cassette using a pumping system

has been problematic. Recirculation

results in a constantly varying sol-

ute concentration in the recir-

culating feed, extending process

times—which can impact product

stability—and causing potential

exposure and damage to shear-sen-

sitive biological products during the

recirculation process, according to

Triebsch.

To address this issue, Pall Life

Sciences has developed single-pass

TFF systems based on proprietary

cassette designs and flow paths that

enable the concentration of tar-

get bioproducts without the need

to recirculate the retentate. “With

these systems, the consistency of

the feed, retentate, and permeate

is maintained during the entire

filtration process, minimizing the

specific filtration process time and

eliminating the potential issues

associated with recirculation of

the retentate and feed,” Triebsch

explains. In addition, he notes that

the filters are suitable for monoclo-

nal antibodies, antibody fragments,

immunoglobulins, and enzymes;

are reliably scalable; can be effec-

tively coupled with chromatography

(column and membrane) steps and

other unit operations; and can oper-

ate continuously.

other harvesting challengesOther challenges related to filtra-

tion still need to be addressed.

Pearce notes that there is a need for

filtration technology for the primary

clarification step that will work with

a variety of feedstocks. “We cur-

rently have good technologies for

mammalian feedstock and mam-

malian feedstock with added floc-

culants and precipitants, but there

is a wide range of flocculants and

precipitants that must be addressed.”

Filtration of high-concentration

final protein formulations also poses

challenges. The high viscosities of

these solutions require pressures

that make it difficult to use exist-

ing ultrafiltration (UF) technolo-

gies, and as a result, the recovery of

those systems is more involved and

a greater burden is placed on the

filtration steps, according to Pearce.




To address this issue, EMD Millipore

has developed a range of filters that

can process concentrations over

200g/L.

The growing numbers of anti-

body-drug conjugates (ADCs) in

development also require advanced

filtration systems, according to

Faber. “These biopharmaceuticals

are particularly challenging because

they are highly potent and require

special handling to protect opera-

tors and reduce the amount of wash

water needed, because water used

for processing highly potent com-

pounds must be burned for disposal,

which is very costly,” he explains.

For example, ADC manufacturers

are looking for fully closed, single-

use crossflow cassettes and fully

automated systems that are compat-

ible with organic solvents needed for

ADC production, Faber notes.

Meeting deManding virus Filtration needsIn the area of virus filtration, cur-

rently available high-capacity virus

filters are now challenged with

higher log-reduction values for

the retention of viruses and small

organisms while maintaining per-

formance under variable process

conditions, such as depressurization

during process interruptions, accord-

ing to Pearce. To address this issue,

membrane morphologies have been

carefully adapted to combine high

viral clearance of small viruses like

parvovirus with high-throughput

capacity and been demonstrated to

provide constant, stable flow rates

in both dilute and more complex

and concentrated biological fluids,

according to Triebsch. For these

next-generation virus removal

and sterile filter membranes, EMD

Millipore uses a patented selec-

tive layer technique, while Pall has

implemented an innovative laid-

over pleat filter construction and

narrower cores. Sartorius uses an

advanced asymmetric hollow-fiber

membrane structure that ensures

high capacities with no impact

on virus retention through pres-

sure variations, high loads, or pro-

cess interruptions. The filter comes

ready-to-use, which eliminates

costly sanitization and is gamma

compatible for easy implementation

in single-use mAb processes, accord-

ing to Faber.

new BioProducts, continuous ManuFacturing, and MoreAs newer bioproducts are designed

and developed, there will always be

a need to generate relevant appli-

cation support information and

new products. “The preference is to

employ existing, well-established

and industry-accepted products, but

as newer molecules such as antibody

constructs and conjugates, viral vac-

cines, nanoplexes, and cell thera-

pies, etc., are developed, there may

be a need to supplement existing

filtration products with additional

membranes or housing components

with different properties, including

porosity or chemical and biological

compatibility,” states Triebsch.

Customers are also beginning

to look at the overall cost of filtra-

tion processes with the desire to

reduce them. “The filter is only a

small part of the filtration process

costs. Minimizing WFI and energy

consumption is becoming impor-

tant for many biopharmaceutical

manufacturers, as is the better uti-

lization of existing manufacturing

infrastructure. Most are also looking

to improve the yields of their filtra-

tion processes, because in general,

biologic substances are very valu-

able, and even a small increase in

recovery can significantly improve

the overall process economy,” com-

ments Faber. He notes that filter

manufacturers can help address

these issues by modifying the sur-

face chemistry of filters to reduce

the adsorption of biomolecules on

their membranes.

Legacy filtration processes come

increasingly under scrutiny of

regulatory agencies looking for

more information on the design

and development of f iltration

processes. “We are finding that

if appropriate quality-by-design

approaches have not been applied

properly, in some cases, filtra-

t ion processes lack suff icient

reproducibility and must be fur-

ther optimized to meet regulatory

requirements,” Faber observes.

The ability of filtration technol-

ogy to be employed for continuous

manufacturing is another issue that

filter producers are tackling today,

according to Pearce. “We expect

that as the adoption of integrated

continuous processes proceeds, the

application of continuous filtration

processes will become more estab-

lished, and in fact, enable continu-

ous operations,” asserts Triebsch.

At the same time, he believes that

continuous, integrated downstream

processing will facilitate new appli-

cations and modes of use for filtra-

tion processes.

For the longer term, filter manu-

facturers are developing filtration

technologies that can provide both

separation and purification through

combination of the size exclusion

and physical removal capabilities

of filtration with the charge and

affinity capabilities of purification,

according to Pearce. “The creation

and use of new materials will be

required, and the challenge will be

to develop these technologies in a

cost-effective manner and in forms

that can be used for single-use and

large-scale manufacturing,” he

states. “There are new materials that

enable very high-surface-area filtra-

tion with high permeability, and

it will be interesting to see if they

can be produced cost effectively for

downstream manufacturing,” Pearce

adds.

reFerence 1. Markets and Markets, “Pharmaceutical

Membrane Filtration Market worth $7,960.3 Million by 2018,” Press Release, March 2014. ◆



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The formation of protein aggre-

gates is of crucial concern in

biopharmaceuticals for paren-

teral administration. Protein

aggregates can elicit an immune

response in the recipient and there is

an expectation from the regulators that

companies will monitor and, where

necessary, reduce, levels of subvisible

particles of all kinds in biotherapeutic

formulations. A related issue is the pres-

ence of aggregates in formulations of

silicone oil, a widely used lubricant in

prefilled syringes. Silicone oil droplets

have the capacity to act as nucleation

points for aggregate growth.

The ability to distinguish between

and quantify aggregates and oil drop-

lets is challenging for most analytical

systems. This article demonstrates how

the technique of resonant mass mea-

surement (RMM) can be used not only

to detect and quantify the formation of

protein subvisible particles in a critically

important size range but also to detect

and quantify any silicone oil droplets in

the formulation.

bRidging the subvisible gaPEnsuring protein stability through-

out the lifecycle of a biopharmaceu-

tical product, from formulation right

through to patient administration, is a

complex challenge. Proteins are natu-

rally unstable and are prone to dena-

ture, self assemble, and agglomerate with

little external stimulus required. Quality

control (QC) and in-use process studies

that comprehensively characterize the

presence of subvisible aggregates within

ABSTRACT

As naturally unstable molecules, proteins are prone to denature, self assemble, and

agglomerate with little external stimulus required. Protein aggregation presents a

key challenge in the production of biopharmaceuticals for parenteral administration.

There is an expectation from the regulators that companies will monitor and, where

necessary, reduce, levels of subvisible particles in biotherapeutic formulations.

A related issue is the presence of aggregates in formulations of silicone oil, a

widely used lubricant in prefilled syringes. Silicone oil droplets have the capacity

to act as nucleation points for aggregate growth. This article demonstrates

how resonant mass measurement can be used not only to detect and quantify

the formation of protein subvisible particles in a critically important size range

but also to detect and quantify any silicone oil droplets in the formulation.

differentiation and Characterization of Protein

aggregates and oil droplets in therapeutic Products

Ciaran Murphy

Ciaran Murphy is head of product

management at malvern instruments;

email: [email protected];

tel: +44 (0) 1684 892456;

malvern instruments ltd,

enigma business Park,

grovewood Road, malvern,

worcestershire wR14 1XZ,

united kingdom

PEER-REVIEWED

article submitted: dec. 17, 2014.

article accepted: Feb. 12, 2015.

L

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UN

A D

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IGN

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SPeer-Reviewed: Resonance mass measurement



biopharmaceuticals are, therefore, essential to

ensuring product performance and safety.

Subvisible particles are usually defined as

particles that are not visible to the naked eye

and have a size of <100 µm. They can fur-

ther be defined into a micron (1–100 µm) and

submicron (1 µm) size range. United States

Pharmacopeia <788> requires the quantifica-

tion of subvisible particles that are ≥10 µm

and ≥25 µm in size (1). Particles at this size are

typically characterized using light obscuration

and/or imaging techniques. Meanwhile smaller

aggregates (<0.1 µm), typically caused by oligo-

merization, are characterized using size exclu-

sion chromatography (SEC). It is, however, also

recognized that particles in the 0.1 to 10 µm

size range have a strong potential to be immu-

nogenic. This is a measurement range that is

becoming increasingly important to FDA and

there are very few characterization techniques

that can provide quantitative sizing details in

this measurement space.

The application of RMM to detect and count

particles in the size range 50 nm–5 µm and

to measure particle mass and size provides a

system that is uniquely positioned to quanti-

tatively measure protein aggregates within the

range of 250 nm–5 µm. This technology is also

capable of providing information on sample

concentration, viscosity, polydispersity, density,

and volume. Moreover, it is able to distinguish

between negatively buoyant proteinaceous par-

ticles and positively buoyant contaminating sil-

icone oil droplets. The technology allows users

to determine how much silicone oil is injected

along with the protein, whether this amount

impacts aggregation, and whether the intended

administration method is fit for purpose.

Resonant mass measuRement RMM exploits the relationship between the A

LL F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

R.

Peer-Reviewed: Resonance mass measurement

Figure 1: A resonant mass measurement instrument set-up.

PneumaticsFluidics

Optics

Sensor

FrequencyMeasurement

and Feed

Pressuresource

sample

waste

Signal Processing

PC

Figure 2: Following shear induced by syringing, the number of

aggregates detected in a biopharmaceutical increases markedly.

#/mL is the number of particles per mL.

1.4e+05

1.3e+05

1.2e+05

1.1e+05

1e+05

90000

80000

70000

60000

50000

40000

30000

20000

10000

0

0 0.5 1 1.5 2 2.5 2.72

Co

nce

ntr

ati

on

[#

/mL]

Diameter [μm]



buoyancy of a particle within solution and

its size. The technique works by passing the

sample through a microfluidic channel embed-

ded within a resonating cantilever (see Figure

1). The frequency of cantilever resonance shifts

either up or down as particles with densities

that are different from that of the carrier solu-

tion flow through it. By precisely measuring

the magnitude in excursion from the cantile-

ver’s base line frequency, an accurate measure-

ment of particle size can be derived for the

particles within solution.

Applying resonant mass measurement to par-

ticles that have very small mass and whose size

falls within the submicron region requires the

use of low mass resonators. These can be engi-

neered using micro electric-mechanical system

(MEMS) sensors. Each sensor chip comprises a

microfluidic network and a minute cantilever

that resonates with a particular frequency. As

the instrument’s fluidics system pushes sample

through this channel, the resonant frequency

of the cantilever alters. This shift in resonance

frequency is measured by a laser focused on the

tip of the cantilever, which is then transmitted

to a split photodiode detector.

Each particle that passes through the sensor

causes a change in the resonant frequency, giv-

ing an accurate and precise measurement of the

individual particle’s buoyant mass. In this way,

the mass and size can be calculated for each

individual particle. As protein aggregates are

denser than the solution, they will produce a

negative change in resonance. Silicone oil drop-

lets, however, are buoyant within what are typi-

cally aqueous solutions, and result in a positive

shift in resonance. This difference in buoyancy

allows users to distinguish between silicone oil

particles and those of proteinaceous origin, and

characterize and quantify particles in relation

to these discrete populations.

The following case studies illustrate how this

innovative analytical set up enables subvisible

particle characterization in bioformulation QC

and process studies.

Case study 1: FoRmation oF

subvisible PRotein aggRegates

in ResPonse to sheaR stRessThe shear force exerted on a biopharmaceutical

formulation during syringing has been identi-

fied as having the potential to cause aggrega-

tion. Quantifying the presence of aggregate

species within formulation, pre-, and post-

syringing is, therefore, essential for monitoring

the safety of administration.

To illustrate the proficiency of RMM in this

area, subvisible particles within a biotherapeutic

formulation were quantified before and after

being subjected to syringe-induced shear stress.

Measurements were made using the Archimedes

system from Malvern Instruments. Figure 2

compares the number of subvisible particles

within a control sample (blue) with those in

a sample that has undergone syringe-induced

shear (green). Prior to syringe stress, the number

of particles detected is low, demonstrating that

the sample is reasonably pure, and contains

only a few large aggregates. Following the appli-

cation of shear stress, the number of particles

detected increases significantly, with particle

sizes ranging from 500 nm up to 1700 nm. This

is important data that indicate a formulation’s

response to the type of stress induced. It can be

used to compare different stress conditions to

provide an overall picture of the degradation

profile for the biopharmaceutical of interest.

The study also compared the number of sub-

visible particles produced following injection of

a formulation using syringes from two different


Figure 3: The number of subvisible aggregates present within a

formulation after shear stress from syringe 1 (red) is much greater

than with syringe 2 (blue). #/mL is the number of particles per mL.

1.2e+06

1.1e+06

1e+06

9e+05

8e+05

7e+05

6e+05

5e+05

4e+05

3e+05

2e+05

1e+05

0

0 0.5 1 2 2.5 2.941.5

Co

nce

ntr

ati

on

[#

/mL]

Diameter [μm]



manufacturers using RMM. Figure 3 shows that

the number of aggregates formed using syringes

from manufacturer 1 (red bars) is many orders

of magnitude greater than with syringes from

manufacturer 2 (blue bars). This finding sug-

gests that the protein is more compatible with

the latter administration technology.

The next step in this investigative study was

to identify the root cause of aggregation. Here,

the ability to distinguish silicone oil particles

from proteinaceous material is a significant

benefit of employing RMM.

Case study 2: deteCtion and quantiFiCation oF siliCone oil FRom two diFFeRent syRinge manuFaCtuReRsSilicone oil is non-toxic and its delivery

during parenteral administration of a drug

is not dangerous in itself. However, the

presence of silicone oil particles, together

with other inorganic contaminants, has been

shown to induce aggregation. RMM enables

the quantification of silicone oil content,

which can have a detrimental impact on the

protein itself, and/or promote an increase in

immunogenicity following administration.

Figure 4 shows variations from the frequency

baseline caused by protein and/or silicone oil

particles passing across the cantilever. The

positive peaks are associated with the detection

of the more buoyant silicone oil particles while

the negative excursion indicates the presence

of the more dense proteinaceous material.

Figure 5 shows the data from RMM mea-

surement following injection using the dif-

ferent syringe systems. The data indicate that

syringes from manufacturer 1 (red) introduce

significantly higher levels of the lubricating sil-


Figure 5: The number of silicone oil particles present in solution

following administration is much higher for syringes from

manufacturer 1 (red) than from manufacturer 2 (blue).

#/mL is the number of particles per mL.

1.8e+06

1.6e+06

1.4e+06

1.2e+06

1e+06

8e+05

6e+05

4e+05

2e+05

0

0 0.5 1 1.5 2.22

Diameter [μm]

Co

nce

ntr

ati

on

[#

/mL]

2

Figure 4: Resonance mass measurement can distinguish between negatively and positively buoyant particles

allowing discrete quantifcation of the silicone oil and protein populations within a sample. “Double” refers to the

sensor detecting two particulates going through it, indicating that two particulates have been seen but only one has

been counted/measured.

Acquisition

Fre

qu

en

cy [

Hz]

998.0

997.8

997.6

997.4

997.2

997.0

996.8

996.6Double?



icone oil during administration. The increased

levels of silicone oil, rather than the induced

shear, may be driving the protein aggregation

mechanism in formulations delivered with

syringes from manufacturer 1.

The ability to detect and quantify silicone

oil content using RMM provides additional

insight into a biotherapeutic product and more

knowledge about possible protein degradation

pathways. Developers can, therefore, quickly

determine whether or not injected silicone

oil is contributing to aggregation, or focus on

identifying an alternative cause.

the gRowing bioFoRmulation toolkitFDA Guidance for Industry: Immunogenicity

Assessment for Therapeutic Protein Products indi-

cates that “…the use of any single method for

assessment of aggregates is not sufficient to pro-

vide a robust measure of protein aggregation”

(2). As such, FDA recommends an orthogonal

approach to biopharmaceutical characteriza-

tion whereby a variety of different and comple-

mentary techniques are used. Comprehensive

characterization of the components of a bio-

pharmaceutical formulation that lie within the

subvisible size region is currently beyond the

capabilities of a single analytical technique.

RMM is one of the few techniques that can

bridge the sizing gap between SEC and light

obscuration and provide quantitative particle

information within this space, making it an

important part of a bioformulation toolkit.

Moreover, its unique ability to distinguish pro-

tein aggregates from silicone oil and to quan-

tify each is greatly beneficial in understanding

product stability and immunogenicity.

ReFeRenCes

1. USP, United States Pharmacopeia—National

Formulary, General Chapter <788>, “Particulate

Matter in Injections” (US Pharmacopeial

Convention, Rockville, MD, 2011).

2. FDA, Guidance for Industry: Immunogenicity

Assessment for Therapeutic Protein Products

(Rockville, MD, Aug. 2014). ◆


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Jaso

n B

utc

her/

Gett

y Im

ag

es

The rise of targeted therapies

and the globalization of spe-

cialty pharmaceutical com-

mercialization present a host

of opportunities for manufacturers.

The foundational premise is clear:

More commercial and clinical trial

drugs are being shipped to more

patients in more countries than ever

before. Healthcare is becoming more

innovative and more accessible at the

same time.

With these opportunities, however,

come great challenges. The increase

of global clinical trials for high-value

cold chain products means that the

stakes—and the costs—for each trial

have risen dramatically. The distri-

bution of specialty medications to

emerging markets means that the total

supply chain must be evaluated more

stringently. The industry now oper-

ates in an environment where there

is no “acceptable loss” of product or

samples. As a result, global manufac-

turers must constantly evaluate the

advances in technology, processes, and

resources that keep cold chain prod-

ucts safe. They must remain vigilant

over the growing and diverse risks in

the supply chain and understand the

need for increased expertise from their

partners. They must stay focused on

continuous improvement across all

parts of their supply chain to ensure

that drugs are delivered safely and

effectively, while risks are mitigated

appropriately.

Here, we explore three macro -

trends in the global pharmaceutical

Innovations and Adaptations in the Cold Chain

Nils Markmann

Manufacturer supply chain

needs are changing

in response to widening

product temperature

ranges.

Nils Markmann is global director

of operations for World Courier.

Supply Chain



supply chain—specif ically the

temperat u re - cont rol led sup -

ply chain—and key takeaways

that manufacturers should con-

sider as they work to drive new

growth globally.

TArgeTed TherApIeS And unIque TemperATure needSPharmaceutical manufacturers

have seen a significant shift in

their development goals and pro-

cesses. The pursuit of the next

blockbuster, small-molecule prod-

uct has given way to excitement

around new, targeted therapies,

biologics, and personalized medi-

cines—all of which are driving

demand for better temperature

control in the supply chain.

Increasingly, there is a need

for more than just cold shipping.

Instead, there is an expanded

need for a highly connected and

functional supply chain. In fact,

“cold chain” no longer accurately

describes the full suite of solu-

tions that manufacturers need.

The industry is moving toward

“temperature control” as the all-

encompassing term, in recogni-

tion of the wider range of new

temperature requirements for

biologic products. From samples

and biologics shipped at “body

temperature” (close to 37 °C) to

control led room temperature

(about 20 –25 °C), all the way

down to ultra-frozen products

stored at -80 °C, the new spec-

trum of temperature needs makes

it clear that the traditional cold

chain (a range of 2–8 °C) is no

longer sufficient to accommodate

all product needs.

New temperature requirements

necessitate new packaging solu-

tions. It’s difficult to maintain

product temperatures in a labora-

tory or storage setting, and the

difficulty increases exponentially

when products must travel at a

consistent temperature across

thousands of miles over the

course of hours or days. The right

packaging is essential to ensuring

product integrity.

Fortunately, the options for

p ac k ag i n g a nd mon ito r i n g

technolog ies have expanded

signif icantly in recent years.

Semi-active packaging, includ-

ing expanded polystyrene (EPS)

and expanded poly urethane

(EPU) systems, is still common

and is often is the more eco-

nomical choice for local, short-

distance transports. Semi-active

solutions are unable to regulate

their own internal temperatures

and require different packaging

configurations seasonally, even

within the same route. For this

reason, semi-active packaging is

best suited for controlled, short-

haul routes. Many semi-active

solutions are only intended for

use a single time and must be dis-

carded afterward, causing them

to be somewhat of an environ-

mental liability.

New options are focused on

offering longer durations of tem-

perature control with l ighter

mater ials that have a smaller

environmental impact. Passive

packaging solutions are more

technologically advanced and

provide proven temperature con-

trol over long distances. These

solutions rely on specific engi-

neering to create a highly stable

inter ior storage environment.

Independent test ing indicates

these solutions perform five to

seven times more efficiently than

semi-act ive solut ions. Phase-

change mater ial (PCM), com-

prised of paraffin or salt-based

solutions, allow for more precise

temperature control to main-

tain product stability over long

distances or through extreme

climates. Many packaging man-

ufacturers are now developing

thei r own vacuum-insu lated

panel (VIP) containers, combined

with PCM solutions, for easier

handling and storage.

On the monitoring side, GPS

technologies and tracking equip-

ment that include automat ic

start-up and shutdown mecha-

nisms (so they can be used safely

on flights) offer a real-time view

into a shipment’s status and data-

driven peace of mind throughout

a product’s entire journey.

A l t h o u g h m o n i t o r i n g

technology can be a benefit, it can

also be a disadvantage for some

products. Manufacturers should

partner with organizations that

can help them find the solutions

that align with their product-

specif ic needs. Se lec t ing too

much technology (e.g., using

a container with an electr ic-

powered ref r igerat ion system

when a PCM-based solut ion

w o u l d w o r k ) , c a n c r e a t e

unnecessary expense. Selecting

not enough protection can risk

the integrity of a product.

globAl ClInICAl TrIAlS And expAndIng mArkeT preSenCeIn tandem with the shift in the

types of drugs being created,

there’s a lso a shif t in where

drugs are being developed. It is

estimated that pharmaceutical

sales in Brazil, Russia, India, and

China will grow to nearly a quar-

ter of a trillion dollars by 2016 (1).

Supply Chain

“Cold chain”

no longer accurately

describes the

full suite of

solutions that

manufacturers need.


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EVENT OVERVIEW:

Due to observed collision induced dissociation (CID) fragmen-

tation inefciency, developing sensitive liquid chromatography

tandem mass spectrometry (LC-MS/MS) assays for CID-resistant

compounds is especially challenging. As an alternative to tra-

ditional LC-MS/MS, a methodology that preserves the intact

analyte ion for quantifcation by selectively fltering ions while

reducing chemical noise is presented. Utilizing a quadru-

pole-Orbitrap MS, the target ion is selectively isolated while

interfering matrix components undergo MS/MS fragmentation

by CID, allowing noise-free detection of the analyte’s surviving

molecular ion. In this manner, CID afords additional selectivity

during high-resolution accurate mass analysis by elimination of

isobaric interferences, a fundamentally diferent concept than

the traditional approach of monitoring a target analyte’s unique

fragment following CID.

Key Learning Objectives:

n The benefts of high resolution accurate mass (HRAM) in

LC-MS/MS

nMethodology for preserving the intact analyte ion for

quantifcation

nHow CID afords additional selectivity during HRAM analysis

Presenter:

EUGENE CICCIMARO

Research Investigator II

Bristol-Myers Squibb

Moderator:

RANDI HERNANDEZ

Senior Editor

BioPharm

Who Should Attend:

n Researchers interested in Biopharma

LC-MS technologies

n Biologists working on protein

characterization

n Bioanalytical researchers

Sponsored by Presented by

For questions, contact Kristen Moore at [email protected]



There are more than 186,000 reg-

istered clinical trials taking place

across the globe, and global phar-

ma’s volume has more than dou-

bled over the last decade (2).

New patient populations are

needed to test the latest in phar-

maceutical advances. Increasingly,

regulatory bodies mandate tests

need to be conducted within the

specific countries in which manu-

facturers seek approval to launch

and market a product. As a result,

the importance of efficient, global

clinical trials will increase dramati-

cally in the coming years.

As the global market for clini-

cal t r ia ls expands, there are

significant hurdles that manufac-

turers must clear to ensure prod-

ucts arrive at the right place, at

the right temperature, at the right

time. Manufacturers and logistics

partners often must navigate geo-

political roadblocks to maintain

a high-performing clinical and

commercial supply chain. Apart

from global concerns in hot-but-

ton countries, manufacturers must

consider all local, regulatory land-

scapes in which they do business.

They must evaluate the existing

capabilities in the market and dis-

ruptions in the supply chain to

determine how all of these factors

may impact shipping time.

Improper customs paperwork,

for example, can delay or com-

promise even the most optimally

packed shipments. Delayed flights,

weather issues, or airport per-

sonnel strikes can cause product

transport issues that could delay

or even derail entire clinical stud-

ies. Manufacturers can avert these

issues by deploying knowledgeable,

trained logistics personnel within

their global markets. Placed at

varying locations around the world

and operating within a proven

inf rast ructure, these exper ts

should be able to mobilize local

knowledge and expertise on a real-

time basis—substantially reduc-

ing risk of temperature excursions.

Global expansion may be the goal,

but global support has to come

along with expansion. When con-

ducting clinical trials in emerging

markets with time- and tempera-

ture-sensitive medications, manu-

facturers should consider working

with specialty logistics experts in

each specific country. Local, in-

market knowledge of regulations

and transport infrastructure can

mean the difference between keep-

ing the clinical supply chain mov-

ing and grinding a productive trial

to a halt.

FoCuS on rISk mITIgATIonThe financial risks of mishandled

shipments are fairly clear for com-

mercial drugs that must remain

temperature-controlled: lost sales

and revenue, lost productivity,

and lost opportunity to improve

patient lives. The risks are no less

severe on the clinical side, where

transportation failures involving

clinical product or patient sam-

ples could result in studies being

compromised. Delays and other

disruptions in trials impact the

expected time to market for a prod-

uct, potentially putting millions or

billions of future revenue dollars

at risk.

As a first step, it is crucial for

manufacturers to identify the

needs for all of their shipped

products, especially those of high

value. Meeting risk mitigation

and quality objectives within the

framework of an effective cost/

performance strategy can be a

simple matter of combining the

right packaging fit with the right

service fit for each individual

application.

Considerations for manufactur-

ers include:

• Size of package needed

• Package type/temperature-control

mechanisms

• Environmental concerns (reusable

packaging or one-time use)

• Temperature needs and variables

(weather, location)

• Best route (fastest, cheapest,

ground vs. air, best controls)

• Cost of shipping

• Value of the ingredients and

product.

Beyond that, though, global

manufacturers are a lso chal-

lenged with needing to under-

stand worldwide political issues

and infrastructure limitations that

may impact logistics. Take the fol-

lowing situations as examples:

• C a mb o d ia ’s Ph nom Pe n h

International Airport has no

freezer available and only one

refrigerator set to one range

that is neither qualified nor

control led. In conjunct ion

with technolog ica l rest r ic-

tions, clearing customs can take

many days. It may be possible

for logistics partners to refresh

refrigerants, but there are no

guarantees.

Supply Chain

Contin. on page 49

global

manufacturers

are challenged

with needing

to understand

worldwide

political issues

and infrastructure

limitations.



To ensure the quality of raw

materials used in biologic pro-

cesses, the materials must be

tested for identity and purity.

Testing is the best way to determine if

a vendor is meeting the needs of a drug

manufacturer. BioPharm International

spoke to Foster T. Jordan, corporate

senior vice-president of endotoxin and

microbial detection at Charles River,

to learn more about streamlining drug

development by bringing safety test-

ing directly to the manufacturing floor.

BioPharm: Please describe the shift in

safety testing location for biologic raw

materials from the laboratory to the

manufacturing floor. What is driving

this change?

Jordan: Controlling the quality of

raw materials used in cell culture-based

biotech manufacturing processes is a

complex process that needs to be mea-

sured consistently. The raw materials

used in biomanufacturing—from inor-

ganic salts and recombinant proteins to

animal-derived serum—run the risk of

becoming contaminated by adventitious

agents, which can cause significant dis-

ruptions in the manufacturing process

and availability of product. Yet biologics

also have much higher financial value

than small-molecule drugs. By shift-

ing testing to the manufacturing floor,

manufacturers can obtain results more

quickly that ultimately lead to more effi-

cient manufacturing practices.

BioPharm: What are some drawbacks

and benefits associated with testing raw

materials on the manufacturing floor?

Jordan: One major drawback is the

short-term investment that companies

will incur to make sure their manufac-

turing practices meet regulatory require-

ments for GMP testing outside the

central lab. But the quicker results will,

over the long term, help to reduce both

financial and regulatory risk.

BioPharm: Which endotoxin and

microbial detection products are most

commonly used to test biologic raw

materials?

Jordan: Kinetic LAL products such as

Endochrome-K, KTA1, and PTS (all from

Charles River) are examples of products

used in biologics testing for endotoxins.

The majority of biologics are still largely

subjected to traditional microbiological

testing methods, but interest in rapid

testing methods is emerging.

BioPharm: Historically, what kinds of

problems with biologic raw materials

have been revealed through quality test-

ing?

Jordan: Environmental microbiologi-

cal organisms as well as gram-negative

bacterial endotoxin are common con-

tamination risks in biological manufac-

turing. By nature, biologics are subject

to microbial contamination for many

reasons. Manufacturers can validate the

aseptic manufacturing process to pre-

vent microbial contamination.

BioPharm: What special considerations

must be taken into account when mea-

suring bioburden in raw materials for

biologics? What about when measuring

bioburden for finished products?

Jordan: Manufacturers must exercise

extreme caution when validating test

procedures. Biological products can-

not be terminally sterilized due to their

natural instability to heat; therefore,

much greater attention may be required

to eliminate and/or prevent viable

microbial organism contamination that

could compromise the finished product.

Because final product biological mate-

rials cannot be terminally sterilized,

any contaminated products are a huge

financial loss. In short, there must be a

comprehensive, risk-based monitoring

program—from raw materials, products

in-process, and finished products—and

these programs should all be supported

by a solid quality assurance program. ◆

Streamlining Raw Materials TestingRandi Hernandez

The rapid testing of

biologic raw materials can

lead to greater efficiency.

Analytical Testing



In October 2005, Janet Woodcock

stated FDA wanted “a maximally

efficient, agile, flexible, pharma-

ceutical manufacturing sector that

reliably produces high quality drug prod-

ucts without extensive regulatory over-

sight” (1). The groundwork to achieve

this realization started with the adop-

tion of Title VII, Sections 705 and 706

of the Food and Drug Administration

Safety and Innovation Act (FDASIA) in

July 2012. It has been almost 10 years

since Dr. Woodcock revealed her vision

and almost three years since the industry

began to hear rumblings that FDA was

going to establish quality metrics that

would be used to determine the suitabil-

ity of a manufacturer’s ability to provide

quality products to the patient. So how

far have FDA and the industry come in

establishing a set of quality metrics appli-

cable to the pharmaceutical industry?

The aforementioned sections of FDASIA

gave FDA the ability to establish crite-

ria to perform risk-based inspections of

bio/pharmaceutical manufacturers and

to request certain documents be pro-

vided in advance or, in some cases, in

lieu of inspections. On Feb. 12, 2013, FDA

published a notice in the Federal Register

asking for assistance from industry in

developing a strategic plan and quality

metrics to prevent drug shortages (2).

FDA’s objective seems to be to reduce

companies’ regulatory burden allowing

for innovation while maintaining respon-

sibility of protecting the public health.

Industry respondsIn response to the Federal Register notice

and during the subsequent three-year

review period, several organizations have

made recommendations to the agency

on what they perceived as fair, reason-

able, and equitable quality metrics.

Organizations that independently par-

ticipated in the development of qual-

ity metrics include the Parenteral Drug

Association (PDA), the International

Society for Pharmaceutical Engineering

(ISPE), the Generic Pharmaceutical

Association (GPhA), Pharmaceutical

Researchers and Manufacturers of America

(PhRMA), and Consumer Healthcare

Products Association (CHPA). These orga-

nizations held conferences, wrote white

papers, and polled their members to try

and define what specific quality metrics

would be appropriate for the intended pur-

pose. This effort culminated in a meeting

facilitated by the Brookings Institute in

May 2014. Representatives from the above

organizations, covering all aspects of the

pharmaceutical industry, met to try and

come to a consensus on exactly what qual-

ity metrics could be and how they could

be used as an indicator of a company’s

ability to continuously provide high qual-

ity medicine to patients. The outcome of

the Brookings meeting resulted in a set of

proposed consensus metrics that included

lot acceptance rate, product complaint

rate, confirmed out-of-specification rate,

and recall rate.

The proceedings from the meet-

ing clarified that the “consensus set of

metrics are somewhat rudimentary, and

provide limited information about the

culture of quality at a given organiza-

tion” (3). According to the published pro-

ceedings, “many [participants] remarked

that a strong quality culture is a critical

component in driving the system and

processes that underpin the quality con-

trol and assurance infrastructure at an

organization. However, quality culture is

also difficult to capture through metrics.”

It was at this point where industry took

off in different directions; PDA and ISPE

have been the most engaged organizations

An update on the Quality Metrics Initiative

Susan J. Schniepp

Industry and regulatory agencies

continue to make progress in establishing

quality metrics for the

pharmaceutical industry.

Susan J. Schniepp is a fellow at

regulatory Compliance Associates.

Quality Metrics



in subsequent efforts. ISPE designed

and asked members to participate

in a pilot program for quality met-

rics, while PDA held a conference on

quality metrics that focused on how

to define a mature quality culture.

The metrics used in the ISPE

Quality Metrics Pilot Program

include the consensus metrics from

the Brookings meetings as well as

others. Some of the unique metrics

used by ISPE in their pilot include

timeliness of annual product qual-

ity reviews, recurring deviations

rate, corrective action and preven-

tive action (CAPA) effectiveness

rate, process capability, and quality

culture (4). The results of the pilot

program are expected to be avail-

able in April 2015. The preliminary

findings from the program seem to

indicate the collection, formatting,

and submission of the metrics to

FDA may be of a significant resource

burden to collect the metrics.

trAdItIonAl, enhAnCed, And other QuAlIty systeMsA conference held in December 2014

by PDA (2) and FDA focused on qual-

ity culture metrics (5). The hypoth-

esis for the meeting was that mature

quality attributes have a strong rela-

tionship to positive quality culture

behaviors. The consensus from the

meeting was that measuring a qual-

ity culture is subjected and defined

by a set of behaviors, beliefs, values,

attitudes, and governance and that

mature quality attributes go beyond

traditional quality systems in creat-

ing a framework for a strong qual-

ity culture. PDA divided quality

systems into three types: Traditional,

Enhanced, and Other. PDA defined

traditional quality systems by tradi-

tional metrics including deviations,

complaints, CAPA, etc. Enhanced

quality systems were defined as those

that had advanced programs such as

risk management, knowledge man-

agement, quality by design, quality

manual, etc. The other quality sys-

tems were much more evolved and

have programs like shared quality

goals, rewards and recognition pro-

grams, and cost of quality awareness

programs, etc. Conference attendees

defined the top five mature quality

attributes as the following:

• Program to show how employee’s

specific goals contribute to over-

all quality goals

• Program to measure, share, and

discuss product quality perfor-

mance and improvement from

shop floor to executive manage-

ment

• Continuous improvement pro-

gram/plans with active support

of CEO and corporate manage-

ment of quality management sys-

tems (QMS)

• Program that establishes qual-

ity system maturity model and

action plan and tracking to mea-

sure progress

• Internal survey measuring a com-

pany/site quality culture.

Meanwhile, FDA presented the

following metrics for measuring a

quality system:

• Lot acceptance rate (the number of

lots rejected by the establishment

in a year divided by the number of

lots attempted by the same estab-

lishment in the same year)

• Right-first-time rate (the number

of lots with at least one deviation

by the establishment in a year

divided by the number of lots

attempted by the same establish-

ment in the same year)

• Product quality complaint rate

(the number of complaints

received by the manufacturer

of the product concerning any

actual or potential failure of a

unit of drug product to meet any

of its specifications, divided by

the total number of lots released

by the manufacturer of the prod-

uct in the same year)

• Invalidated out-of-specification

(OOS) rate (the number of OOS test

results invalidated by the estab-

lishment, or contracted establish-

ment in a year divided by the total

number of tests performed by the

establishment in the same year).

the developIng future of QuAlIty MetrICsSo what is the future of quality met-

rics? Perhaps it is still too early to

tell. Xavier University (6) held meet-

ings on the topic of quality in March

2014 and again in March 2015. ISPE

has yet to reveal the results of their

pilot program, and PDA has yet to

publish the results of their surveys

from the December 2014 confer-

ence. The most important part of

the puzzle, FDA’s guidance on qual-

ity metrics, which is due to be pub-

lished in 2015, is still missing. But

whatever happens, it is important

to keep in mind that there are no

perfect quality metrics. The concept

of unintended consequences needs

to be addressed so everyone is on a

level playing field. Data trending can

be more valuable in determining the

robustness of a quality system than

direct comparisons, and an open,

honest quality culture will drive the

integrity of the quality metrics.

referenCes 1. J. Wechsler, Pharm. Technol.

29 (12), pp. 36–42 (2005).

2. FDA, Food and Drug Administration

Drug Shortages Task Force

and Strategic Plan; Request for

Comments, Federal Register, Vol.

78, No. 29 pp. 9928-9929 (Feb. 12,

2013), www.gpo.gov/fdsys/pkg/

FR-2013-02-12/html/2013-03198.

htm, accessed April 15, 2015.

3. Brookings Institute, Measuring

Pharmaceutical Quality through

Manufacturing Metrics and Risk-

Based Assessment, Meeting Summary

(Engelberg Center for Health Care

Reform at Brookings, May 1-2, 2014).

4. A complete listing of the metrics

used in the ISPE Pilot can be found at

www.ispe.org/quality%20metrics%20

defined, accessed April 15, 2015.

5. PDA, Quality Metrics, www.pda.org.

6. Xavier University, FDA/Industry

Collaborative Approach to Quality:

With the Patient in Mind (Xavier

University, Cincinnati, OH, April

12, 2014, http://xavierhealth.org/

wp-content/uploads/Xavier-Proposal-

for-CDER-Metrics-program.15-

April-2014.pdf,

accessed April 15, 2015. ♦

Quality Metrics



Troubleshooting

Production of proteins for manufac-

turing therapeutics and pharmaceuti-

cals is a complicated process, and its

optimization can be time-consuming. Many

problems are associated with protein expres-

sion, including toxicity, misfolding and deg-

radation, aggregation in inclusion bodies, low

yields, and difficulties in purification. One

of the most commonly used protein expres-

sion systems uses Escherichia coli as a protein

factory. E. coli has many advantages—includ-

ing rapid growth, ease of scale up, and low

costs—but it poses challenges as well. Here,

the authors discuss some parameters that can

influence protein yields and quality during

protein expression in E. coli.

Bacterial strain selectionThe choice of a bacterial strain for protein

expression is closely tied to the properties

of the target protein to be expressed and the

choice of expression vector. T7-based expres-

sion systems are based on expression from the

strong T7 promoter. The BL21(DE3) strain is

one of the most commonly used strains in both

industry and academia. It carries the phage

T7 gene 1—encoding T7 RNA polymerase—

in its chromosome under the control of the

lacUV5 promoter. Protein expression can be

induced with the addition of isopropyl β-D-1-

thiogalactopyranoside (IPTG).

Expression in the T7-based system tends to

be “leaky,” which is problematic for the expres-

sion of toxic proteins—but this can be resolved

by presence of pLysS or pLysE, which expresses

T7 lysozyme, an inhibitor of T7

RNA polymerase (1). The BL21-AI

strain might be a better alterna-

tive for tighter regulation, because

the expression of T7 RNA poly-

merase from the chromosome is

now under the control of the arabinose pro-

moter, which has lower basal expression than

the lacUV5 promoter.

In some cases, better yields of toxic pro-

teins may be obtained in the C41(DE3) and

C43(DE3) strains. These strains were derived

from BL21(DE3) and were specially selected

because they could tolerate the expression of

toxic proteins (2). Interestingly, the mutations

that were key to this tolerance converted the

strong lacUV5 promoter to one that resembled

the weaker wild-type lac promoter (3).

Controlling the intensity of expression is a

major problem with T7-based systems. One way

to do this is to lower the amount of inducer

added to the culture. However, this is problem-

atic with the “all-or-nothing” induction char-

acteristics of the T7-based systems, because low

inducer concentration leads to a mixed popula-

tion. This population is typically comprised of a

few cells expressing large amounts of the target

of low quality, and the majority of the cells not

expressing anything (4).

Codon usage differs among prokaryotes

and eukaryotes. Thus, it may be difficult

to obtain reasonable yields of nonbacterial

proteins expressed in E. coli. The Rosetta

strains, which carry a plasmid supplement-

ing tRNAs for six to seven rare codons in

E. coli, may help to overcome that. Strains,

such as Shuffle (New England Biolabs) and

Origami (Novagen), are specially engineered

to provide an oxidizing environment in the

cytoplasm, allowing the formation of disul-

fide bonds in the cytoplasm itself, and cir-

cumventing the need to target these proteins

to the periplasm. The Lemo21(DE3) (Xbrane

Biosciences) and Tuner (Novagen) strains con-

fer the capacity to tune the expression of tar-

get proteins, and this may favor higher yields

of better-quality protein. Lemo21(DE3) car-

Optimization of Protein Expression in Escherichia ColiChoosing the optimal protein expression vector depends on strain, promoter, and a number of other factors.

Siavash Bashiri is ceo; David Vikström is cto; and Nurzian Ismail is senior

scientist and project leader; all at

Xbrane Bioscience.



ries a plasmid harboring the gene

for T7 lysozyme, an inhibitor of

T7 RNA polymerase, under the

control of the titratable rham-

nose promoter. By regulating the

expression of T7 lysozyme, the

level of expression of the target

protein from the T7 promoter

may be tuned (3).

choice of promoter/vector systemThe pET vector series contains

either the T7 promoter or the T7/

lacO promoter, which has the

lac operator sequence inserted

between the T7 promoter and

translation initiation site to reduce

basal expression. Selection for pET-

harbouring cells may be performed

with either ampicillin or kanamy-

cin, depending on the pET vector

chosen. The choice of a specific

promoter system depends on the

strength of promoter desired, the

“leakiness” of the promoter system

(which is undesirable for highly

toxic proteins), and compatibility

with the bacterial strain selected.

The T7 promoter system requires

the use of T7 RNAP-containing

strains, but promoters such as lac,

lacUV5, T5, tac, trc, rhaBAD, and

araBAD may be used with any E.

coli strain.

The lacUV5 promoter, a deriva-

tive of the lac promoter, contains

two mutations in the -10 region

and an additional mutation at -66

within the catabolite gene activator

protein (CAP) binding site. These

mutations result in an increase in

promoter strength and reduced

catabolite repression of the lacUV5

promoter (5).

The tac promoter combines the -10

region of the lacUV5 promoter and

the -35 region of the trp promoter

and is at least five-fold more efficient

than the lacUV5 promoter (6).

The trc promoter has similar pro-

moter strength to the tac promoter

and varies in sequence only by one

base pair (7).

The lac, lacUV5, tac, and trc pro-

moters all include the binding site

for lacI repressor, thus, in order to

achieve efficient repression, the

plasmid must also carry the lacI

or lacIq gene, especially for high

copy plasmids. The lacIq gene con-

tains a mutation in its promoter

that enhances lacI expression by

tenfold (8).

The pBAD series of plasmids

allow protein expression from the

araBAD promoter. Repression of the

araBAD promoter is more efficient

than lac-derived promoters, reduc-

ing any unwanted basal expres-

sion. Protein expression from the

araBAD promoter may be modu-

lated to a limited extent by varying

inducer concentrations, but these

promoters also suffer from the “all-

or-nothing” induction characteris-

tics as previously described for the

T7-based systems (9).

If greater control is desired,

vectors containing the rhaBAD

promoter—including the two

regulatory genes RhaS and RhaR—

allow protein expression to be

tuned more efficiently (4).

The Rhamex vectors (Xbrane

Biosciences), which include the

regulatory genes RhaR and RhaS,

allow expression from the rhaBAD

promoter, and are available in a

range of copy numbers, provid-

ing an additional level of control

to protein yields. Depending on

the protein target, the ability to

tune the level of expression of

the target mRNA may give cer-

tain advantages, such as increased

protein accumulation, increased

protein solubility, and increased

cell fitness.

affinity taggingThe addition of affinity tags may

aid the detection and purification

of target proteins. Common affin-

ity tags include poly-His, FLAG,

c-Myc, poly-Arg, and StrepII tags.

The tag could be located in the

N- or C-terminus of the target

protein, but the position of the

tag should not affect localization

or topology of the protein in the

case of membrane proteins. For

example, for proteins synthesized

with targeting signal sequences,

the tag should be located at the

C-terminus to avoid mis-target-

ing and to ensure mature protein

capture, especially if the signal

sequence is cleaved.

Another advantage to insert-

ing the tag in the C-terminus is

that it allows detection of the fully

synthesized protein. The protein

can only be detected using the

tag if the entire polypeptide has

been synthesized. Depending on

the purpose of protein expression,

these tags may be left in the final

product or cleaved off with the use

of specific proteases during the

purification process.

Proteases such as Tev prote-

ase, enterok inase, thrombin,

and factor Xa recognize distinct

amino acid sequences, which

may be included after the tag for

N-terminally-tagged proteins or

before the tag for C-terminally-

tagged proteins. The choice of

protease depends on the speci-

ficity of the protease recognition

site, the amino acids that are left

in the mature protein after cleav-

age, ease of protease removal dur-

ing purification, and the cost. It

should be noted that tagging of a

target protein may interfere with

its correct folding, assembly of

complexes, the activity of protein,

and even expression yields, so it is

best to leave a protein untagged,

if possible.

fusion partnersRecombinant proteins expressed

in E. coli often end up in inclusion

bodies. This is not necessarily a

bad thing, as inclusion bodies are

easily isolated, and it is sometimes

possible to refold a protein that has

been isolated in this way to suffi-

ciently high quality. The refolding

troubleshooting



troubleshooting

step adds an additional stage in the

purification process, however, and

requires additional time for opti-

mization. This increases the cost

of production. In addition, only a

fraction of the isolated protein will

refold to give active protein, result-

ing in loss of yield.

The addition of such fusion

partners as maltose binding pro-

tein (MBP), glutathione-S-trans-

ferase (GST), ubiquitin, SUMO, or

thioredoxin (Trx), which are pres-

ent in plasmids supplied by vari-

ous companies, may aid solubility.

It may be necessary, however, to

screen several fusion partners,

because these proteins may not

enhance solubility for some tar-

gets, or they may affect solubility

to different levels.

Some of these fusion partners

also aid purification. For example,

MBP will bind to amylose-agarose,

while GST will bind to glutathi-

one-agarose for purification by

affinity chromatography. Similar

to affinity tags, these fusion part-

ners must be cleaved off during

purification. Unfortunately, in

some cases, the target protein may

not remain soluble after cleavage

of the fusion partner.

eXpression conditionsE x p r e s s ion c ond i t ion s c a n

have two different effects: First,

they can increase protein yield

per cell, and second, they can

increase cell densities per volume

of culture. The ideal scenario

would be obtaining a condition

in which both protein yield per

cell and cell densities per vol-

ume are high, but this cannot

always be achieved. It may be

necessary to optimize expression

conditions to obtain high cell

densities to compensate for a low

protein yield per cell. There are

many parameters that affect cell

growth and recombinant pro-

tein expression, such as choice

of medium, carbon source, tem-

perature, pH, aeration, inducer

concentrations, and length of

induction.

For batch cultivation, LB (Luria-

Bertani) medium is commonly

used. Although it is a rich medium,

it does not support growth to very

high cell densities, particularly

because it contains a low amount

of carbon source and divalent cat-

ions. Media such as 2xYT, Terrific

Broth (TB), and Super Broth (SB)

are better than LB for obtaining

high cell densities. However, nutri-

ents become limiting in batch

cultivations. Much higher cell

densities may be obtained in fed-

batch cultivations.

Another type of medium, the

auto-induction medium, elimi-

nates the need for the addition

of an inducer, specif ically for

lactose-induced systems. It relies

on having a mixture of glycerol,

D-glucose, and α-lactose in the

medium. D -glucose is gener-

ally preferred, and represses any

expression from lac-based promot-

ers. Once D-glucose is depleted,

lactose is taken up by the cells,

which then promotes the expres-

sion of target protein from the

lac promoter. The amount of

D-glucose in the medium deter-

mines the timing of the induc-

tion. The use of an auto-induction

medium simplif ies cultivation

procedures, and, in some cases,

improves protein yields.

Cultures are typically grown

at 30 –37 °C, but the tempera-

ture may be optimized for the

target being expressed. In some

cases, a greater fraction of sol-

uble protein is achieved when

cultures are grown at lower tem-

peratures, although the trade-off

is that the cultures grow more

slowly. Subjecting cultures to

high temperature for a short

time initiates the expression of

heat-shock chaperones, which

may be beneficial in promoting

higher yields of properly-folded

prote ins . Inducer concent ra-

tions and the induction period

are additional parameters that

can be optimized according to

the target protein, especially if a

titratable promoter system is used.

The optimal length of induc-

tion period can vary from 4–24

hours. Another parameter, aera-

tion, is affected by the choice of

vessels/flasks and the volume of

culture used. Ideally, the volume

of culture should not exceed 10%

of the total volume of the flask.

Increasing the shaking speed and

the use of baffled flasks also pro-

motes better aeration.

conclusionChoosing the best expression sys-

tem depends largely on the target

protein and the scale of manufac-

turing. There are many factors that

can be optimized. This article

merely touches on a number

of them, such as the choice of E.

coli strain; the promoter system;

the need for tags such as signal

sequences or solubility-enhancers;

and importantly, the culture and

induction conditions. It is impor-

tant to consider the entire process—

from vector and insert design to

bioreactor conditions—because

each part of the process will have a

huge impact on the final result.

references 1. F.W. Studier, J. Mol. Biol. 219, pp. 37-44

(1991).

2. B. Miroux and J.E. Walker, J. Mol. Biol.

260, pp. 289–98 (1996).

3. S. Wagner et al., Proc. Natl. Acad. Sci.

105, pp. 14371–14376 (2008).

4. M.J. Giacalone et al., BioTechniques 40,

pp. 355–64 (2006).

5. B. J. Hirschel et al., J. Bacteriol. 143,

pp. 1534–1537 (1980).

6. E. Amann et al., Gene 25, pp. 167-78

(1983).

7. M.E. Mulligen et al., J. Biol. Chem. 260,

pp. 3529–3538 (1985).

8. M.P. Calos, Nature 274, pp. 762–765

(1978).

9. D.A. Siegele, D.A. and J.C. Hu, Proc.

Natl. Acad. Sci. 94, pp. 8168–8172

(1997). ◆


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integrates the science and business of

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Compliance Notes

Q We are a medium-sized company and

our products are sold in nearly 70 coun-

tries world-wide. Consequently, we are

inspected by several agencies. These inspections

resulted in some observations for not complying

with cGMP regulations. Because our quality unit

is relatively small, it is challenging to stay up to

date with the latest regulatory changes. What are

the best ways to stay abreast of shifting regula-

tions to remain compliant?

AAs you are aware, companies must com-

ply with currently applicable regulations.

To do so, you will need individuals, ide-

ally from the quality unit, dedicated to gather-

ing regulatory intelligence. This process includes

collating information on existing and anticipated

laws and regulations. Such information is avail-

able from a variety of sources including from the

regulatory agencies themselves, free newsletters,

and commercial providers.

Regulatory intelligence does not stop there.

Teams must analyze whether regulations will

impact your company’s quality system, oper-

ations, or regulatory dossier/application.

Sometimes new or changed regulations come

with a defined implementation (“grace”) period,

while in other instances they are immediately

enforceable upon publication. The European

Union EudraLex Volume 4, Chapter 3 on Premises

and Equipment, for example, was revised in

August 2014 but was not enforced

until March 1, 2015 (1). In some cases

there is only an expectation, which

may be embedded in guidance docu-

ments or in concept papers published

by a regulatory agency. For example,

the Medicines and Healthcare Products

Regulatory Agency first issued a guid-

ance document in January 2015 that

sets a 2017 deadline for compliance

to certain data integrity aspects (2).

Similarly, 21 Code of Federal Regulations

Part 11 Electronic Records; Electronic Signatures

in the United States has an effective date of Aug.

20, 1997 (3).

Once the regulatory intelligence team has

reviewed and analyzed changes to the legis-

lation for applicability and impact, it is then

tasked with communicating and disseminating

it to all the relevant parties within the company

including the quality unit, production depart-

ments, logistics teams (e.g., for good distribution

practices), and regulatory affairs. Process owners

in these various departments must implement

the changes to comply with the new require-

ments. The quality unit needs to verify timely

compliance through internal audits throughout

the process.

At the very minimum, companies must estab-

lish a regulatory intelligence function to gather

information and proactively help shape new

legislation. Most new laws and regulations go

through an ‘open comment’ period where inter-

ested parties—either individuals or members of

industry associations—can provide feedback and

ask clarifying questions to help eliminate errors,

point out omissions, or suggest changes.

Establish a dedicated regulatory intelligence

function, put the right communication channels

in place, and empower your quality unit to verify

compliance in a timely manner. Doing so will

help you stay current with applicable regulations

and compliant with new and amended laws.

RefeRences 1. European Commission, EudraLex, The Rules Governing

Medicinal Products in the European Union, Vol. 4, EU

Guidelines for Good Manufacturing Practice for Medicinal

Products for Human and Veterinary Use Part 1, Chapter 3:

Premises and Equipment.

2. MHRA, Good Manufacturing Practice: Data Integrity

Definitions And Guidance, Jan. 23, 2015, Re-issued

March 13, 2015.

3. FDA, Title 21 CFR Part 11, www.accessdata.fda.gov/

scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=11

and http://www.fda.gov/downloads/

RegulatoryInformation/Guidances/ucm125125.pdf,

accessed April 13, 2015. ◆

Siegfried Schmitt is principal

consultant at PAReXeL.

How to Stay Abreast of Shifting Regulations and Remain CompliantSiegfried Schmitt, principal consultant, PAREXEL, discusses how to keep up with changing regulations.


Oncobiologics Readying

Avastin Biosimilar Candidate

for Phase III TrialsOncobiologics announced on April 15, 2015 that it

expects to initiate Phase III trials for its biosimilar

to Genentech’s Avastin (bevacizumab) following the

successful conclusion of a pharmacokinetic study

comparing the biosimilar to versions of Avastin that

are approved in the United States and European

Union. The study will also compare the reference

products to each other.

Avastin, which is a vascular endothelial growth

factor, is used to treat various cancers. It is also

used off-label for the treatment of age-related

macular degeneration and has been shown to be just

as effective as Lucentis for this indication. When

diluted to the concentrations called for in ophthalmic

indications, Avastin is one-twentieth the price of

Lucentis, which sells for approximately $2000 per

injection.

According to calculations from BioWorld, Avastin

captured $7 billion in revenue in 2013—so it is no

surprise that there are more than 20 biosimilars

for the medication in various stages of discovery

and development. Actavis and Amgen’s candidate,

ABP 215, will complete Phase III clinical trials in

early 2016. Boehringer Ingelheim GmbH’s BI 695502

will begin Phase III trials versus bevacizumab plus

chemotherapy in patients with lung cancer. Pfizer’s

PF-06439535 completed Phase I trials in August 2014,

and Phase I trials of Mabxience S.A.’s BEVZ92 are

expected to be completed by September 2016.

Oncobiologics is developing the Avastin biosimilar

in partnership with inVentiv Health. Oncobiologics

has 11 other biosimilars in development, including

products for adalimumab, cetuximab, rituximab, and

trastuzumab.

NIH Announces Positive Results

for Experimental Ebola VaccineOn April 1, 2015 the National Institutes of Health (NIH)

and the Walter Reed Army Institute of Research (WRAIR)

announced positive results from VSV-ZEBOV when tested

in 40 healthy adults. The vaccine, meant to protect against

the Ebola virus, was found to be safe and triggered a

strong antibody response in all 40 volunteers who were

given the vaccine.

The investigational vaccine, developed by scientists

at the Public Health Agency of Canada and licensed to

NewLink Genetics in collaboration with Merck, is based on

a genetically modified and attenuated vesicular stomatitis

virus (VSV). According to a press release, a gene for a VSV

protein is replaced with a gene segment from a key protein

in the Zaire strain of the Ebola virus.

The study involved 52 volunteers, 26 of which were at the

NIH Clinical Center, while the other 26 were at a WRAIR

clinic. Of the volunteers, six at each site were given a

placebo and the remaining 40 received one of two different

dosages of the investigational vaccine. The volunteers’

blood was tested after the vaccination to confirm antibody

development against the Zaire strain of Ebola.

“The prompt, dose-dependent production of high levels

of antibodies following a single injection and the overall

favorable safety profile of this vaccine make VSV-ZEBOV

a promising candidate that might be particularly useful

in outbreak interventions,” said Richard T. Davey, Jr.,

MD, investigator at the National Institute of Allergy and

Infectious Disease, in a press release.

Positive Phase III Results

End Pfizer’s Ibrance Trial Early Pfizer announced on April 15, 2015 that its Phase III

trial, PALOMA-3, for Ibrance (palbociclib) was ended

early because it met its primary endpoint in improving

progression-free survival (PFS) for the combination of

Ibrance with fulvestrant. The trial compared fulvestrant

with placebo in women with hormone receptor-positive

(HR+), human epidermal growth factor receptor 2-negative

(HER2-) metastatic breast cancer following disease

progression during or after endocrine therapy, according

to a press release. The trial was ended early due to efficacy

based on an assessment by an independent Data Monitoring

Committee on the anti-cancer medicine with novel cyclin-

dependent kinase 4/6 (CDK 4/6) inhibition.

“The results of this trial are especially important because

they help us understand the potential of Ibrance to improve

outcomes in patients with this difficult to treat cancer. We’re

gratified to be able to stop the trial early and are engaging

in discussions with health authorities regarding a regulatory

path forward,” said Mace Rothenberg, MD, senior vice-

president of clinical development and medical affairs and

chief medical officer for Pfizer Oncology, in a press release.

The drug received FDA Priority Review in October 2014

when in combination with letrozole, which reduces the

amount of estrogen produced by the body. In February

2015, FDA granted accelerated approval of Ibrance in

combination with letrozole for postmenopausal women with

metastatic estrogen receptor positive, HER2- advanced

breast cancer who are treatment naïve.


Clinical Trial Materials Development Update

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PRODUCT SPOTLIGHT

Diaphragm Valve Streamlines InstallationITT’s Pure-Flo EnviZion valve is designed with tool-less installation capabilities and lessened maintenance time. With a breakthrough mount and turn design, and no tools or torqueing required, the valve maintenance time has been reduced from 23 minutes to three. Effects of thermal cycling are eliminated by a constant sealing force provided by the integrated thermal compensation system.

The model comes in sizes from 0.5 to 1 inch with a pressure rating of 150 psi and manual and actuated designs. Various surface finishes are available and interior and exterior electropolish is available, with a 25 roughness average as the standard polish.

ITT

www.itt.com

Single-Use Biocontainer Increases CustomizabilityMeissner’s single-use biocontainer, FlexGro, is designed for use with rocker-style bioreactors. The presterilized container is available with design options to accomodate up to 50 L and features Meissner’s TepoFlex polyethylene multi-layer film. TepoFlex is a clean film platform that does not have any slip agents ensuring consistency in the cell cultivation process.

A variety of connection, sampling options, and assembly designs allow the user to customize the biocontainer to fit specific end-user requirements. Different process needs can be met with a variety of available standard assembly designs.

Meissner

www.meissner.com

New Technology Showcase

TECHNOLOGY AND SERVICES

A strategic biosimilar development

partner, Catalent offers unique

technologies and integrated services

to help you get more, better products

to clinic, faster. Catalent expertly and flexibly combines triplet fix codon

optimization, GPEx® cell line engineering technologies, integrated

analytical, fill-finish and clinical supply services. Catalent engineered cell

lines supported the launch of five commercial biosimilar projects, with many

more in clinical trials. Catalent, tel. +1.888.765.8846, www.catalent.com

CELEBRATE PERFORMANCE WITH THE

NEW EX-CELL® ADVANCED™ CHO

FED-BATCH PLATFORM BY SAFC®

Introducing the next generation in chemically-defined CHO fed-batch media. This contemporary media and feed platform was

developed across a wide range of CHO cell lines commonly used in industrial bio-manufacturing with an emphasis on simple adaptation (regardless of cell bank medium), demonstrated performance with sustained high biomass and maximum titers, and formulations allowing for flexibility to adjust protein quality attributes as needed. For more information or to try a sample, please visit us at www.Sigma-Aldrich.com/CHOperformance, SAFC®

INTEGRATED LC/MS PLATFORM

The Waters Biopharmaceutical Platform

Solution with UNIFI brings together UPLC/

MS characterization technology with

the UNIFI Scientific Information System

for intact protein mass analysis, peptide

mapping, glycan analysis workflows,

and bioseparations. The platform now supports a mix of Q-Tof MS and

optical detectors within a networked workgroup. For peptide and protein

bioanalysis, configurations are available with tandem quad MS.

Waters, tel. 508.478.2000, www.waters.com/biopharm

SINGLE-USE BIOREACTORS

EMD Millipore’s Mobius CellReady 200-L

bioreactor integrates several features

that are intended to provide ease of use,

reliability, and operational flexibility.

The unit contains a working volume of

40–200 L, which allows it to function as

both a seed and production vessel, and its standard design is optimized for

the cultivation of mammalian cells in suspension.

EMD Millipore, tel. 800.548.7853, www.millipore.com



Ad IndexCompany Page Company Page

AnTITope lImITed 7

bIo InTernATIonAl ConVenTIon 29

CATAlenT phArmA SoluTIonS 52

emd mIllIpore 15

eppendorF norTh AmerICA 9

ge heAlThCAre lIFe SCIenCeS 5

pArker hAnnIFIn domnICk hunTer 19

SAFC bIoSCIenCeS SIgmA AldrICh 51

SgS lIFe SCIenCe SerVICeS 11

Thermo FISher SCIenTIFIC 37

VeTTer phArmA-FerTIgung gmbh 13

WATerS Corp 2

Supply Chain

• In Laos, the rainy season lasts

f rom August to September.

With limited airport facilities,

products can be left on the run-

way. Manufacturers can avoid

unnecessary damage by send-

ing products before or after the

rainy season.

• With the growing population

and spend on healthcare in Latin

America, it’s an ideal location for

a growth plan, but each country

within the region has different

regulations and resources.

Although conducting trials and

commercializing in new markets

represents opportunities to expand

needed therapies to new patients, it

also represents millions of dollars

worth of risk when working with

cold-chain products. Proactive

planning— combined w ith a

knowledgeable logist ics part-

ner that can help navigate global

nuances—will help protect prod-

ucts, ensure that temperature con-

trols are met, and increase effective

use for patients.

A SoluTIon For eVery TherApyManufacturers worldwide are

investing more in their supply

chains. A $15-billion increase in

spending for global biopharmaceu-

tical logistics is expected between

2012 and 2018 (3). As global pre-

dictive growth explodes, shippers

and trial sponsors can expect an

increase in regulatory and overall

logistics complexities that foster in-

market resources across the globe.

The spend on cold chain logistics

was more than $8 billion worldwide

in 2014 and is expected to exceed

$10 million by 2018 (3).

The rise of targeted therapies

and solutions for rare diseases has

created even more challenges for

manufacturers, as the demand for

personalized, high-value drugs

with more active pharmaceutical

ingredients (APIs), shorter shelf-

l ives, and st r ic t temperature

r e qu i r e me nt s i nc rea se s . A s

products become more targeted,

so will the logistics solutions. This

trend indicates pharmaceutical

companies are (or will become)

more invested in supply chain

operations.

For t u nate ly, soph i s t icated

logistics providers are investing

in new technologies and improv-

ing their capabilit ies in order

to ensure they can successfully

handle the most innovative new

products on a global basis. As

the supply chain continues to

expand in complexity, the rela-

tionships between manufacturers

and their logistics partners must

also grow.

reFerenCeS 1. C. Ross, ÒBuilding BRICs: pharmaÕs

key emerging markets are becoming

giants,Ó PMLive, http://www.

pmlive.com/pharma_intelligence/

building_brics_pharmas_

key_emerging_markets_are_

becoming_giants_483972,

accessed Apr. 1, 2015.

2. ClinicalTrials.gov, https://

clinicaltrials.gov/ct2/resources/

trends#RegisteredStudiesOverT

ime, accessed Apr. 1, 2015.

3. M. Lipowicz and N. Basta, Ò2014

Biopharma cold chain forecast,Ó

Pharmaceutical Commerce (2014),

http://www.pharmaceuticalcommerce.

com/index.php?pg=supply_chain_log

istics&articleid=27206&keyword=

biopharma-cold%20chain-logistics-

forecast, accessed Oct. 30, 2014. ◆

Contin. from page 38



BIOLOGICS NEWS PIPELINE

IN THE PIPELINE

Innate Pharma Announces $1.3 Billion

Collaboration with AstraZeneca

Innate Pharma announced on April 24, 2015 that

it signed a co-development and commercialization

agreement with AstraZeneca and MedImmune,

AstraZeneca’s biologics research and development

branch.

The agreement will help accelerate and expand

the development of Innate’s anti-NKG2A antibody,

IPH2201, including its development in combination

with MedImmune’s anti-PD-L1 immune checkpoint

inhibitor, MEDI4736. IPH2201 is a Phase II, first-in-

class humanized IgG4 antibody that inhibits the anti-

cancer functions of natural killer and cytotoxic T cells

via NKG2A.

As part of the collaboration, plans have been

made to begin Phase II combination tr ials of

IPH2201 and MEDI4736 in solid tumors. Innate is

also planning Phase II to study IPH2201 as a mono-

therapy, in combination with other approved treat-

ments for various cancers, and in the discovery of

associated biomarkers.

Under the terms of the agreement, AstraZeneca

will pay Innate up to approximately $1.3 billion

in cash and double-digit royalties on sales. An ini-

tial payment of $250 million was paid to Innate

for the consideration of exclusive global rights

to AstraZeneca to co-develop and commercialize

IPH2201 in combination with MEDI4736, as well as

access to IPH2201 in monotherapy and other com-

binations.

AstraZeneca will pay an additional $100 million

to Innate prior to Phase III development, with a

potential of additional regulatory and sales-related

milestones up to $925 million.

Sartorius Stedim Biotech Acquires BioOutsource

Sartorius Stedim Biotech (SSB), a supplier for the

biophar maceut ica l indust r ies , has acqu i red

BioOutsource, a CRO based in Glasgow, United

Kingdom. This move expands SSB’s portfolio with

the addition of contract testing services to support

biopharmaceutical clients and help fast-track biolog-

ics through the development pipeline as well as facil-

itate lot release testing in large-scale manufacturing.

BioOutsource recorded EUR9 million in revenue

over the past 12 months. The privately owned

Scottish company, which employs approximately

85 staff, has been operating in the biotech space

since 2007. It provides contract testing services to

the biopharmaceutical industry, including assays to

monitor the safety and quality of biologic products.

BioOutsource’s services are used in early stage drug

development for characterization, comparability,

and lot release testing of biotherapeutics. Details of

the transaction were not disclosed.

Combination of Yervoy and Opdivo Shrinks

Melanoma Tumors Drastically in NEJM Case Report

A case report published on April 20, 2015 in the

New England Journal of Medicine (NEJM) inves-

tigating the use of monoclonal antibodies Yervoy

(ipilumumab) and Opdivo (nivolumab) found that

the combination of drugs reduced the tumors by

80% in more than half of melanoma patients. Even

though there were only 13 patients in the Phase I

trial, the researchers observed a “remarkable” tumor

response in one patient whose large chest-wall mela-

noma almost completely disappeared in three weeks

after only one dose of the drug combination.

While the results of the immunotherapy treat-

ment are impressive, the study investigators warn

that they have concerns about the “overly vigor-

ous antimelanoma” response they observed and

say that “such an antitumor effect occurring in a

transmural metastasis in the small bowel or myo-

cardium, common sites for metastatic melanoma,

could have grave consequences.” Despite these

warnings and a treatment delay due to a rash, the

patient in question who had remarkable results has

resumed treatment.

The results of the case study were published

alongside a larger double-blind study involving

142 patients with metastatic melanoma with BRAF

V600 wild-type tumors. In that study, 61% (44 of

72) patients receiving the drug combination of

Yervoy and Opdivo responded to the drug, and 22%

(16 patients) reported a complete response. Eleven

percent (four of 37 patients) in the group that

received Yervoy and placebo showed a response

of some kind, indicating that the combination

of drugs is more efficacious than Yervoy alone.

Progression-free survival was also significantly

higher in the Yervoy/Opdivo group than in the

Yervoy group alone.

The success of the drug combination of Yervoy

and Opdivo comes at a price, however, as serious

drug-related adverse events were reported in more

than half of patients in this arm (54%), which is

significantly more than the 24% of those on Yervoy

plus placebo who experienced side effects.


Introducing a next generation, chemically-defined CHO fed-batch media platform from SAFC. Developed

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