STERILITY ASSURANCE - PharmTechfiles.pharmtech.com/alfresco_images/pharma/2018/09/11/4e868232 … · The global supply chain for bovine and porcine heparin and regulatory considerations

The Science & Business of Biopharmaceuticals

INTERNATIONALINTERNATIONAL

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November 2015

Volume 28 Number 11

STERILITY

ASSURANCE

UPSTREAM PROCESSING

IMPLICATIONS OF CELL

CULTURE CONDITIONS ON

PROTEIN GLYCOSYLATION

PEER-REVIEWED

ESTABLISHING PROCESS DESIGN

SPACE FOR A CHROMATOGRAPHY

PURIFICATION STEP

SUPPLY CHAIN

DIVERSIFYING THE

GLOBAL HEPARIN

SUPPLY CHAIN

www.biopharminternational.com

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PHARMACEUTICAL n HEALTH SCIENCES n FOOD n ENVIRONMENTAL n CHEMICAL MATERIALS

©2015 Waters Corporation. Waters, ACQUITY QDa and The Science of What’s Possible are registered trademarks of Waters Corporation.

Gain confidence in glycan, peptide, and

oligonucleotide analysis with mass detection.

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INTERNATIONAL

BioPharmThe Science & Business of Biopharmaceuticals

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

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© 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].

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

K. A. Ajit-Simh President, Shiba Associates

Rory Budihandojo Director, Quality and EHS Audit

Boehringer-Ingelheim

Edward G. Calamai Managing Partner

Pharmaceutical Manufacturing

and Compliance Associates, LLC

Suggy S. Chrai President and CEO

The Chrai Associates

Leonard J. Goren Global Leader, Human Identity

Division, GE Healthcare

Uwe Gottschalk Vice-President,

Chief Technology Officer,

Pharma/Biotech

Lonza AG

Fiona M. Greer Global Director,

BioPharma Services Development

SGS Life Science Services

Rajesh K. Gupta Vaccinnologist and Microbiologist

Jean F. Huxsoll Senior Director, Quality

Product Supply Biotech

Bayer Healthcare Pharmaceuticals

Denny Kraichely Associate Director

Johnson & Johnson

Stephan O. Krause Director of QA Technology

AstraZeneca Biologics

Steven S. Kuwahara Principal Consultant

GXP BioTechnology LLC

Eric S. Langer President and Managing Partner

BioPlan Associates, Inc.

Howard L. Levine President

BioProcess Technology Consultants

Herb Lutz Principal Consulting Engineer

Merck Millipore

Jerold Martin Independent Consultant

Hans-Peter Meyer Lecturer, University of Applied Sciences

and Arts Western Switzerland,

Institute of Life Technologies.

K. John Morrow President, Newport Biotech

David Radspinner Global Head of Sales—Bioproduction

Thermo Fisher Scientific

Tom Ransohoff Vice-President and Senior Consultant


Anurag Rathore Biotech CMC Consultant

Faculty Member, Indian Institute of

Technology

Susan J. Schniepp Fellow

Regulatory Compliance Associates, Inc.

Tim Schofield Senior Fellow

MedImmune LLC

Paula Shadle Principal Consultant,

Shadle Consulting

Alexander F. Sito President,

BioValidation

Michiel E. Ultee Principal

Ulteemit BioConsulting

Thomas J. Vanden Boom VP, Biosimilars Pharmaceutical Sciences

Pfizer

Krish Venkat Managing Partner

Anven Research

Steven Walfish Principal Scientific Liaison

USP

Gary Walsh Professor

Department of Chemical and

Environmental Sciences and Materials

and Surface Science Institute

University of Limerick, Ireland

ES701449_BP1115_003.pgs 11.06.2015 01:03 ADV blackyellowmagentacyan

4 BioPharm International www.biopharminternational.com November 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 Scientif c Abstracts) • Biotechnology and Bioengineering Database (Cambridge Scientif c Abstracts) • Biotechnology Citation Index (ISI/Thomson Scientif c) • Chemical Abstracts (CAS) • Science Citation Index Expanded (ISI/Thomson Scientif c) • Web of Science (ISI/Thomson Scientif c)

Cover: PLAINVIEW/Maria Toutoudaki/Getty Images; Dan Ward

6 From the Editor Biopharma and contract providers must tread carefully amid changing market dynamics. Rita Peters

8 Regulatory Beat New program emphasizes quality, risk, and global collaboration. Jill Wechsler

10 Perspectives on Outsourcing Better process develop-ment is creating industry benchmarks for bioprocessing. Eric Langer

48 Compliance Notes How to ensure archive records can be retrieved. Siegfried Schmitt

49 Troubleshooting There are many factors to consider when choosing viral clearance methods.Cynthia A. Challener

53 New Technology Showcase

53 Ad Index

54 Biologics News Pipeline

FILL/FINISHBest Practices for Sterility Assurance in Fill/Finish OperationsRandi HernandezExperts discuss best practices to achieve acceptable sterility assurance levels for aseptically filled products. 14

UPSTREAM PROCESSINGImplications of Cell Culture Conditions on Protein GlycosylationRichard Easton and Michiel E. UlteeThe authors present a review of the techniques commonly used for glycosylation analysis. 20

DOWNSTREAM PROCESSINGThe Development ofProcess Chromatographyin BioprocessingSusan HaigneyIndustry experts discuss the development of process chromatography in bioprocessing. 26

PEER-REVIEWEDEstablishing Process Design Space for a Chromatography Purification Step: Application of Quality-by-Design PrinciplesHui XiangThis case study reviews how quality-by-design principles can be implementedin an intermediate chromatography purification step that usescation-exchange chromatography. 28

GLOBAL SUPPLY CHAINDiversifying the Global Heparin Supply Chain: Reintroduction of Bovine Heparin in the United States?David Keire, Barbara Mulloy, Christina Chase, Ali Al-Hakim, Damian Cairatti, Elaine Gray, John Hogwood, Tina Morris, Paulo A.S. Mourão, Monica da Luz Carvalho Soares, and Anita SzajekThe global supply chain for bovine and porcine heparin and regulatory considerations are examined. 36

SUPPLY CHAIN

Piloting Track-and-Trace ImplementationRobert CelesteVirtual pilot programs examine scenarios that may occur while implementing serialization requirements for theUS Drug Supply Chain Security Act. 43

QUALITYInvestigating BiologicsSusan Schniepp and Andrew HarrisonThe authors discuss performing investigations of biological products. 46

Volume 28 Number 11 November 2015

fEATURES

ON THE WEBwww.biopharminternational.com

Future of Bioprocessing eBook

BioPharm’s The Future of Bioprocessing eBook features articles on advanced biologics, single-uses systems, market demand, patent reviews, automation, and more!

To read the eBook, visit:

BioPharmInternational.com/FutureofBioprocessingeBook

BioPharmINTERNATIONAL

The Science & Business of Biopharmaceuticals

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October 2015

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From the Editor

Biopharma and

contract providers

must tread carefully

amid changing

market dynamics.

Next Steps in Outsourcing Relationships

In an industry where change is the norm, biopharma companies must learn

to successfully navigate the financial, business, regulatory, and scientific

ups and downs of the market. In the fourth part of the 2015 CPhI Annual

Industry Report, Looking beyond the Global Pharma Horizon (1), industry repre-

sentatives commented on dynamics in biologics development and the contract

services market, and how challenges and strategic approaches in the two sectors

may direct the industry moving forward.

Increased funding in the emerging bio/pharma sector, changing customer

attitudes and business practices, regulations, a global supply chain, niche

technology offerings, and untapped markets will shape the contract services

market, writes Gil Roth, president of the Pharma and Biopharma Outsourcing

Association. Most critical, however, is the ways in which contract manufactur-

ing organizations (CMOs) and contract development and manufacturing orga-

nizations (CDMOs) learn from the industryÕs past.

In ÒCMO/CDMO Challenges and Opportunities,Ó Roth notes that recent

acquisitions in the contract services market were motivated by the desire to

integrate service offerings, or acquire niche technologies to attract earlier phase

clients with the goal of retaining that business through commercial manufac-

turing. At the same time, the improving economy has enabled more capital

investment in facilities at biomanufacturing firms, with a resulting shift of

some operations in house. In addition, a focus on orphan drugs with smaller

batch sizes may shift technology requirements and outsourcing relationships.

Hedley Rees, managing consultant at PharmaFlow, highlights differences in

the manufacture and supply of large-molecule biologic products versus small-

molecule drugsÑpotential pitfalls in the drug development processÑin ÒWhat

Does the Future Hold for Biopharmaceutical Outsourcing?Ó

Rees cites the effects of even minor changes in the production process, chal-

lenges in sourcing raw materials, analytical methods to detect changes during

manufacture, product sensitivity to environmental factors, and the current

model of pharmaceutical distribution as potential opportunities for failure. In

addition, advanced therapy medicinal productsÑgene therapies, somatic cell

therapies, and tissue-engineered productsÑwill demand closer ties between the

manufacturer and healthcare system versus the one-size-fits-all batch process-

ing traditionally used with current blockbuster therapies.

In the present fee-for-service outsourcing model, projects are directed by a

contract; changes must be negotiated, with both cost and time implications.

The risk for the contract service provider is low, versus a risk- and-reward-

sharing model.

ÒThe banana skin waiting for the unsuspecting pharmaco is that this new era

of biologics needs a different approach to outsourcing,Ó Rees warns.

Contractors can offer technical expertise that biologics companies need;

however, some biopharma companies are considering more in-house operations.

A move away from outsourced operations may drive the contract service market

to think more about a risk-sharing model.

Rees identifies factors that will drive discussions between drug owners and

contractors including the use of a quality-by-design approach; supply chain

reporting and control; patenting of process knowledge; the cost in of commer-

cialization; and the availability of qualified personnel.

For both parties, a careful eye on market changes and development needs, as

well as some strategic hand-holding, may avert some nasty slips or falls.

Reference 1. CPhI, Annual Report 2015, Part IV, Looking beyond the Global Pharma Horizon, online

www.cphi.com/europe/networking/cphi-pharma-insights, accessed Nov. 2, 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

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ett

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After two years of planning and analy-

sis, FDA officials are moving forward

with implementation of the Program

Alignment plan to better coordinate agency

field inspections with product reviews from

FDA research centers. The aim is to reduce

redundant processes and to provide more exper-

tise in evaluating today’s more complex and

varied production systems for drugs and bio-

logics. The growing number of pharmaceutical

ingredients and finished products imported

from abroad, moreover, heightens the need for

risk-based oversight and increased collaboration

with foreign regulatory counterparts to avoid

duplicate inspections.

Transforming oraThe reorganization of FDA’s 5000-person field

force represents the most important change

since the Office of Regulatory Affairs (ORA)

was formed, says Melinda Plaisier, ORA chief

and associate commissioner for regulatory

affairs. This Program Alignment initiative,

announced in September 2013 and further clar-

ified in February 2015 (1), is dissolving ORA’s

five regional offices and establishing

commodity-based and vertically inte-

grated inspection programs for drugs,

biologics, medical devices, tobacco

products, food, and bioresearch mon-

itoring that will operate out of ORA’s

20 district offices.

For drugs, Pla isier expla ined

at the PDA/FDA Joint Regulatory

Conference in Washington, DC in

September 2015, Alonza Cruse will be

director for Pharmaceutical Quality

Operations, which will have a cadre

of pharmaceutical inspectors divided

into four management teams. Anne

Reid is acting director for Biological

Operations, with two management teams, and

Jan Welch heads up three teams for medical

devices. Some product team directors also will

head district offices.

These teams of specialized investigators will

gain greater technical expertise through train-

ing, which should help them keep pace with

manufacturing changes and new technology,

especially those inspectors with sub-specialties

in, for example, sterile drugs, compounding,

APIs, or combination products. Pharmaceutical

inspectorate members also will be part of

the Center for Drug Evaluation and Research

(CDER) product review teams so that they will

fully understand development and manufactur-

ing issues involved in a new therapy and can

produce pre-approval inspection reports that

reflect a common understanding of pertinent

production and quality concerns.

Plaisier emphasized that the ORA overhaul

is a “work in progress,” and that many final

decisions and individual assignments are still

to come. Questions remain about the num-

ber of field management teams for each pro-

gram, where these will be located, and how to

align some 2000 investigators into the different

review programs, she explained. These transi-

tion activities will continue through the com-

ing year, with the goal of starting up the new

FDA Overhauls Inspection OperationsNew program emphasizes quality, risk, and global collaboration.

Jill Wechsler is BioPharm

International’s Washington editor,

Chevy Chase, mD, 301.656.4634,

[email protected].

fDa also seeks to halt violative

imports more quickly by

de-linking import alerts

from warning letters.


November 2015 www.biopharminternational.com BioPharm International 9

regulatory Beat

model in fiscal year 2017. ORA is

looking to develop metrics to mea-

sure the impact of these changes

internally, along with enhanced

training programs, new work plan-

ning systems, and more central-

ized laboratory operations.

CoorDinaTing ComplianCeClear, coherent enforcement

strategies with reduced layers of

review involve closer collabora-

tion between Center staff and field

inspectors to eliminate duplicate

case workups and to speed inspec-

tion findings to manufacturers,

explained Tom Cosgrove, direc-

tor of the Office of Manufacturing

Quality (OMQ) in the CDER Office

of Compliance (OC). These changes

should accelerate re-reviews of

plants looking to regain compli-

ance status and “not leave firms

in OAI (official action indicated)

status for a long time,” Cosgrove

commented at the PDA/FDA con-

ference. He emphasized the impor-

tance of complete documentation

of operations to demonstrate com-

pliance with GMPs. He also noted

that documentation by itself “is not

enough” to demonstrate full com-

pliance and that FDA inspectors are

being trained to do a “deeper dive”

into actual production practices.

FDA also seeks to halt violative

imports more quickly by de-linking

import alerts from warning letters.

Expeditious action against non-

compliant imports is important,

Cosgrove pointed out, because many

of these products raise data integ-

rity issues, including data that have

been deleted, back-dated, copied,

and fabricated. FDA is highlighting

data-integrity failures because such

problems also are linked to GMP vio-

lations and other problems that rep-

resent “real risk to patients.”

OMQ also is looking hard at

contract manufacturers and how

well their pharma clients moni-

tor contract operations for quality

and compliance. Clients need “to

get out there,” perhaps put a person

in the plant, to uncover GMP and

compliance problems “before we do,”

Cosgrove advised. He noted that the

manufacturer holding the approved

license for a medical product is

responsible for ensuring quality at all

its production facilities—including

those overseas or operated by part-

ners and suppliers.

Amidst all these organizational

changes, FDA is developing a new

model for assessing plant operations

based on standardized measures of a

facility’s state of quality and compli-

ance. The New Inspection Protocol

Project (NIPP) will apply to pre-

approval, GMP surveillance, and for-

cause inspections. CDER’s Office of

Pharmaceutical Quality is develop-

ing the new protocols and planning

pilot NIPP inspections with ORA.

The aim is to obtain quantitative

scores that can help compare sites,

while also reducing variability in

observations by different inspectors

and providing manufacturers with a

clearer idea of what they need to do

to maintain quality. While continu-

ing to document observed deficien-

cies, inspections also will identify

practices that exceed basic compli-

ance requirements to reward positive

behaviors.

gloBal CollaBoraTionEfforts at home to develop met-

rics for evaluating manufacturing

operations and to streamline and

target inspections also are being

applied to foreign manufacturers

producing medical products for the

United States. Because FDA lacks the

resources to monitor the growing

global pharmaceutical market, US

and European Union officials are

looking for greater “mutual reliance”

on each other’s inspection reports.

US officials have explored such

options for more than a decade, only

to be stymied by legal requirements

and confusing goals. Now authorities

are renewing efforts to reduce the

number of inspections conducted

by FDA investigators in the EU, and

by European inspectorates in the US,

to better target resources to areas of

greater risk, explained Dara Corrigan,

FDA associate commissioner for

global regulatory policy, at the PDA/

FDA conference.

FDA conducts thousands of for-

eign inspections each year, many

in Europe, Corrigan pointed out,

and reliable information indicat-

ing that a facility meets GMPs and

is a low-risk operation could help

avoid unnecessary site visits. To

move forward with a mutual reli-

ance initiative, FDA investigators

are observing audits of EU inspec-

torates, which are conducted by

other EU member states as part

of their own internal mutual reli-

ance inspection program. At the

same time, EU officials are audit-

ing ORA district operations to sup-

port increased EU reliance on FDA

inspection practices and reports.

Corrigan noted that FDA offi-

cials have been impressed with the

high level of discussion taking place

during these audits, but a num-

ber of important issues have to be

addressed for the initiative to move

forward. One is that US law requires

FDA inspection reports to redact

trade secret information before being

shared with other regulatory authori-

ties, a policy that rankles EU officials.

And while the vast majority of reg-

istered European drug facilities and

imported products come from six

EU member states (Germany, France,

Italy, United Kingdom, Spain, and

Ireland), it’s not clear if a mutual

reliance program could be lim-

ited to those countries. The path

forward, Corrigan said, involves

assessing the variability of EU

inspectorates and their expertise.

This is a high priority for both FDA

and the EU, and, Corrigan stressed,

“we want to succeed.”

referenCe 1.J. Wechsler, Pharma. Techn. 39 (5)

(2015). ◆



Perspectives on Outsourcing

Do

n F

arr

all/G

ett

y Im

ag

es

Biomanufacturing efficiency is on every-

one’s minds, being the single most

important area of focus for global bio-

processing. And contract manufacturing orga-

nizations (CMOs) are on the leading edge as

they implement performance improvements.

CMOs must remain efficient if they are to be

competitive—so this is no surprise. Results

from BioPlan Associates’ 12th Annual Report

and Survey of Biopharmaceutical Manufacturing

Capacity and Production (1) offer some clues as

to what CMOs are doing to remain competitive.

CMOs’ love affair with single-use devices

has been well documented. Indeed, single-use

implementation and integration is a much larger

focus for CMOs than it is for biotherapeutic

developers. And as the results in Figure 1 indi-

cate, it’s easy to see why: nine out of 10 CMOs

agree that biomanufacturing improvements over

the past year are coming from the use of dispos-

able and single-use devices.

Given that CMOs have long been at the fore-

front of single-use adoption, it’s perhaps more

interesting to look at factors that are rising in

importance for CMOs. One such factor is better

process development, cited by 81.8% of CMO

respondents as contributing to improved bioman-

ufacturing performance, up from

two-thirds of respondents in 2014.

This is a notable result, as pro-

cess development outsourcing has

been on the rise in recent years.

Separately, 43% of industry respon-

dents reported outsourcing at least

some upstream process develop-

ment activities to some degree,

up from just 17.1% back in 2010.

Additionally, 41% reported at least

some outsourcing downstream pro-

cess development activities to some

degree. Improvements in process

development, therefore, are an encouraging

sign for CMOs as this becomes a growing busi-

ness opportunity.

A similar pattern plays out in validation ser-

vices. This is also a growing area of opportunity

for CMOs, with validation services a more pop-

ular outsourcing activity than process develop-

ment. In the 2015 survey, for example, almost

three-quarters (73%) of industry respondents

reported outsourcing at least some validation

services, up from less than two-thirds in 2010.

Another area to which more CMOs attri-

bute internal performance improvements is

upstream production operations. In the 2015

survey, 64% of respondents said that these

improvements contributed to better overall

performance, up from 56% in 2014. In fact,

CMOs were almost as likely to credit upstream

improvements as downstream improvements

with better biomanufacturing performance.

That may partly be due to the current bottle-

necks being experienced in purification and

separation operations. And CMOs’ experience

with multiple products and campaigns provide

them expertise that in-house manufacturers

may not have.

Upstream biomanufacturing operations out-

sourcing has been growing more rapidly than

downstream operations, according to BioPlan’s

data. In the space of five years, the percentage

of industry respondents outsourcing upstream

operations has doubled, from 21% in 2010 to

42% in 2015. While outsourcing of downstream

operations has been on the rise, it hasn’t had

quite the same growth trajectory, up from 28%

in 2010 to 39% of respondents in 2015.

Upstream operational improvements are less

of an industry focus for both CMOs and in-

house manufacturers. Indeed, when BioPlan

surveyed the industry on the single most

important area or operational focus in 2015,

CMOs Continue to Improve Overall Biomanufacturing Performance Better process development is creating industry benchmarks for bioprocessing.

Eric Langer is president of

BioPlan Associates,

tel. 301.921.5979, elanger@

bioplanassociates.com.


For US inquiries, please contact [email protected] • For Asia Pacifi c inquiries, please contact infoAsiaPacifi [email protected]

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Answers that work

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Fig

ure

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Perspectives on Outsourcing

not a single CMO pointed to

upstream processing advances as

the top area; because downstream

production (DSP) operations issues

remain strong. A worrisome 64%

of CMOs said that downstream

processing is impacting capacity

and overall production by caus-

ing at least some bottleneck prob-

lems (noted by 64%). In fact, only

a quarter of respondents are cur-

rently enjoying no bottlenecks in

their downstream processing.

CMOs sPending tO Offset POtentiAl CAPACity CrunChNot surprisingly, CMOs are seeing

more problems than biotherapeutic

companies due to downstream pro-

cessing, and are experiencing more

significant production capacity con-

straints, too. The BioPlan study indi-

cated they will likely continue to

invest in better DSP technologies

as a way out of these problems, for

example.

Facility constraints are the most

common factor CMOs cite as creat-

ing capacity crunches at their facil-

ities over the next five years (cited

by more than two-thirds). Probably

by no coincidence, 7 in 10 CMOs

plan to increase their spending on

new facility construction this year,

by an average amount of 11.3%.

The next biggest culprit in

projected capacity constraints is

downstream purification capacity.

Spending plans for CMOs are posi-

tive: almost three-quarters would

be increasing their capital equip-

ment budgets, with an average

increase of 11.7%.

Expected budget hikes—for cap-

ital equipment (11.7%) and new

facility construction (11.3%)—were

the largest of all areas tracked. To

grow their businesses, CMOs are

dedicating funds to offset potential

capacity constraints in the future.

Not surprisingly, better down-

stream purification technologies are

also on the agenda. CMOs note that

downstream innovation is the lead-

ing way to avoid future capacity con-

straints. Spending projections aren’t

quite as buoyant for downstream

innovation, though they are solid.

In 2015, 6 in 10 will increase spend-

ing on new technologies to improve

efficiencies and costs for downstream

production, for an average budget

increase of 6.1%. This is likely due

to new technologies providing more

incremental increases in efficiencies

as opposed to new equipment that

can quickly provide access to more

capacity and avoid crunches.

COnClusiOnSingle-use equipment is help-

ing CMOs achieve performance

improvements, both for down-

stream purification and for man-

ufacturing productivity overall.

But CMOs are taking numerous

other factors into account as they

improve efficiencies and lower costs.

These range from better analytical

testing and product release services

to better operations staff training,

optimized media and improved

existing quality management sys-

tems. Better process development

is also a growing area of interest for

CMOs as they take on more process

development work—both upstream

and downstream—for clients.

Nevertheless, one of the main

routes to overall productivity

improvements for CMOs will be

better downstream operations.

Besides the use of disposable equip-

ment, a majority of CMOs are

developing downstream processes

with fewer process steps. Many are

also using or evaluating a number

of technologies, including:

• Membrane-based filtration tech-

nologies

• Ion-exchange membrane tech-

nologies

• Ion-exchange technologies with

higher capacity.

Biotherapeutic developers might

keep a close eye on these activi-

ties. CMOs, with their broad expe-

rience, multiple product lines, and

need for rapid changeovers, are

often at the forefront of innova-

tion. Though their requirements

clearly differ from those of bio-

therapeutic developers, the process

improvements sparked by innova-

tions and adopted by CMOs can

provide a recipe for the industry as

a whole. As such, it will be interest-

ing to monitor the activities and

technologies that CMOs adopt to

improve downstream production

operations and overall biomanu-

facturing productivity.

referenCe 1. BioPlan Associates, 12th Annual Re-

port and Survey of Biopharmaceutical Manufacturing Capacity and Produc-tion (Rockville, MD, April 2015), www.bioplanassociates.com/12th, accessed Oct. 12, 2015. ◆

Figure 1: Improving biomanufacturing performance for CMOs, 2015 v. 2014

(select responses).

Use of disposable/single-use devices

Better process development

Overall better control of process

Improved downstream production operations

Better analytical testing & product release services

Improved upstream production operations

86.4%

83.3%

81.8%

66.7%

68.2%

77.8%

68.2%

72.2%

63.6%

66.7%

63.6%

55.6%

2015 2014

Source: 12th Annual Report and Survey of Biopharmaceutical Manufacturing, April 2015, www.bioplanassociates.com/12th


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PL

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Dan W

ard

Whe t he r o ut s o u r c i n g

aseptic techniques to a

third party, or perform-

ing these tasks in an

academic setting or in an in-house

laboratory, certain tools, technolo-

gies, and standard operating proce-

dures are necessary to ensure sterility

across settings. Because many biolog-

ics cannot be terminally sterilized,

isolators and restricted access barrier

systems (RABS) are typically the go-to

tools manufacturers use to ensure

product sterility.

To gain some insight into how to

best prepare sterile, parenteral prod-

ucts, BioPharm International spoke to

experts in both the theory and the

practice of sterile drug preparation.

Specifically, the publication spoke to

Bivash Mandal, PhD, a senior research

specialist at the Plough Center for Sterile

Drug Delivery Systems in the University

of Tennessee Health Science Center,

and Bernd Stauss, senior vice-president

of production/engineering at Vetter

Pharma-Fertigung GmbH & Co.

The Plough Center for Sterile Drug

Delivery Systems announced in August

2015 that it is installing three PODs

from G-CON Manufacturing in a new

facility on campus to manufacture

drugs for sponsors and train profession-

als on cGMPs for the large-scale produc-

tion of pharmaceuticals (1). Although

the location currently has the capacity

to manufacture small-volume parenteral

preparations for clinical investigation,

the facility expansion, which began

in September 2015, will al low the

Best practices for sterility assurance in Fill/Finish Operations

Randi Hernandez

Two experts discuss best

practices to achieve

acceptable sterility

assurance levels for

aseptically filled products.

Fill/Finish



university to manufacture drugs

for preclinical and clinical trials.

The PODs are slated to be up and

running by 2016.

Vetter is an outsourcing com-

pany that has helped guide dozens

of product approvals for biophar-

maceutical compounds and spe-

cializes in the commercial filling

and packaging of parenteral drugs.

In the past few years, Vetter has

focused on innovation in the

field, combining the advantages

of isolators and RABS to create a

new approach in sterility assur-

ance, which the company calls

its “Improved RABS Concept.”

The technique features an accel-

erated process cycle and an auto-

mated decontamination function

for increased operational excel-

lence in aseptic processes (2).

EquiPmEnt trEndSBioPharm: What are the trends in

the use of RABS and isolators? Is

use of this type of equipment the

best way to ensure the sterility of

one’s fill/finish processes?

Mandal: Aseptic processing is a

complex manufacturing tech-

nology that can be achieved by

using aseptic cleanrooms (manned

human-scale cleanrooms), isola-

tors/restricted access barrier sys-

tems (RABS), or both. As far

as the industrial trends are con-

cerned, some firms have taken a

mix-and-match approach. RABS

and isolators can be used in the

manufacture of biologics, includ-

ing vaccines, gene therapies, and

protein-based drugs. Often, bio-

logic products are preservative-free,

contain growth media, and are

easily susceptible to contamina-

tion. Another area that demands

the use of RABS and isolators is

the manufacture of sterile drug

products with toxic, cytotoxic, and

highly potent molecules, which

require stringent barriers to pro-

tect personnel who are handling

these materials. In general, RABS

and isolators are being used for

smaller-volume and high-value

pharmaceuticals. The benefit/cost

balance has to be considered when

discussing the use of barriers: RABS

and isolators come with a high

price tag and are associated with

additional expenses related to the

operation of a cleanroom, such as

energy costs, operating costs, test-

ing costs, and gown costs.

Because it has been established

that the personnel working in

cleanrooms can be a major source

of contamination, RABS and isola-

tors are preferred as a means of a

physical barrier to separate people

from filling processes. According

to FDA guidance on aseptic pro-

cessing, isolators and closed RABS

are superior in their ability to con-

trol contamination and reduce

validation workload. Operators

must use these advanced tech-

nologies with caution because the

use of RABS and isolators alone

does not guarantee the sterility

of products. In both isolators and

RABS, for instance, operators use

glove ports, and glove ports need

to be inspected on a daily basis.

Moreover, gloves are considered

a primary route of contamina-

tion, and they are a common cause

of failure in isolator technology.

Complete automation and use of

robotic technology in conjunction

with isolators and RABS should be

Fill/Finish

BEST PRACTICES IN FREEZE/THAW OPERATIONS

BioPharm International asked Bivash Mandal, PhD, senior research specialist

at The Plough Center for Sterile Drug Delivery Systems in The University of

Tennessee Health Science Center, for a few tips to help ensure optimal freeze/

thaw operations.

BioPharm: What are the dangers associated with multiple freeze/

thaw operations?

Mandal: Multiple freezing and thawing of a biopharmaceutical product

could affect the chemical and physical properties of the product. In the case of

protein drugs, the procedure can stress and may irreversibly denature complex

macromolecular structures, altering their stability. The rate at which freeze/

thaw processes occur plays a significant role in product quality. Fast freezing

rates could lead to smaller ice-crystal formation. This process can result in their

partial unfolding, increased aggregation, and decreased biological activity. There

is also an increased risk of the entrapment of air during fast freezing, which can

denature proteins as air-liquid interfaces form. On the other hand, slow thawing

rates often result in ice recrystallization, and the shear stress generated by slow

freezing can damage biologics.

BioPharm: How does the geometry of vials or cryobags affect the fill/finish

process of allogeneic cells?

Mandal: For the fill/finish of allogeneic cells, one of the crucial steps is the

final freezing step for cryopreservation of the cells with an acceptable shelf life.

An optimal cooling rate is one of the critical parameters affecting the survival of

cells during cryopreservation. For cryovials, freezing patterns will be influenced

by the variation in container-base geometry. If a vial’s base is not flat and does

not have a uniform thickness, there may be uneven thermal contact between

a sample and the lyophilization shelf. The mechanism of heat exchange will be

affected based on the dimensions and geometry of the sample container and

whether the container rests directly on a shelf or is supported in a tray.

—Randi Hernandez



developed to eliminate the human

interventions that are performed

using glove/sleeve assemblies.

Stauss: There are two distinct

technologies dominating the fill/

finish process: isolators and RABS.

Each technology has its advantages.

With isolator technology, the pro-

cessing takes place in systems that

are entirely shut off from the outside

environment. As it pertains to steril-

ity assurance levels (SAL), isolators

are often considered the best solu-

tion due to the automatic decontam-

ination processes involved. However,

isolators need extensive decontam-

ination and preparation processes

following a batch to enable a safe

change in product.

RABS technology also achieves the

SAL currently required by regulatory

authorities. With this technology,

the physical barriers of a production

plant are limited; a RABS requires

installation in a higher-class envi-

ronment (at least ISO 7, with the

RABS located in an ISO 5 area).

Conversely, this system provides

flexibility and high-capacity utiliza-

tion for multi-product filling lines;

this is a reason why RABS are often

found at CDMOs [contract develop-

ment and manufacturing organi-

zations]. When choosing between

isolator and RABS technology, each

company has to make the decision

that best fits their production situa-

tion and needs.

BioPharm: What equipment is

common for those performing fill/

finish operations?

Mandal: For fill/finish operations,

liquid-filling equipment (manual/

semiautomatic/automatic), peristal-

tic pumps, filtration apparatuses,

a lyophilizer (if required), a vial/

ampoule sealer/crimper (semiauto-

matic/automatic), and a biosafety

cabinet (hood) are required. During

fill/finish operations, it is also

required to monitor the environ-

mental air quality by passive sam-

pling using settling plates and active

sampling using a centrifugal sam-

pler and an impactor-type sampler.

A laser particle counter can moni-

tor the total particulate count of the

environmental air.

Successful product

integrity testing

using deterministic

or probabilistic

methods is the basis

for enabling sterility

in manufactured drug

products.—Bernd

Stauss, Vetter

quAlity mEASurEmEntSBioPharm: What have been some

common performance gaps when it

comes to environmental monitoring?

Mandal: Some of the common

performance gaps in environmen-

tal monitoring include not follow-

ing standard operating procedures,

not monitoring in all aseptic pro-

cessing areas, inadequate corrective

actions, not responding in a timely

fashion to out-of-limit results,

inadequate personnel training,

failure to validate the cleaning and

sanitization procedures, failure to

trend environmental monitoring

data, failure to identify common

microorganisms, and inadequate

documentation of deviations.

BioPharm: How are aseptically

manufactured drug products best

evaluated for their sterility?

Stauss: Proving the sterility of

manufactured drug products is

crucial to a drug manufacturer.

In the first step, the design of the

applied primary packaging materi-

als needs to meet integrity require-

ments. Successful product integrity

testing using deterministic or prob-

abilistic methods is the basis for

enabling sterility in manufactured

drug products. After the integrity

of the package design is estab-

lished, incoming packaging mate-

rials are routinely tested to ensure

they meet specifications.

Equipment surfaces that come

into contact with sterilized drug

product or sterilized primary pack-

aging materials, as well as any cru-

cial equipment in the cleanroom,

needs to be sterilized by using vali-

dated sterilization methods. Moist-

heat and dry-heat sterilization are

the most commonly used steril-

ization methods. Furthermore, the

aseptic processing operations need

to be tested for their ability to pro-

duce sterile products via process sim-

ulations (media fill). During media

fill, microbiological growth medium

is exposed to product contact sur-

faces to simulate the exposure that

the product may undergo during

manufacturing. The sealed contain-

ers filled with the medium are then

incubated at defined temperatures to

detect microbial contamination.

During manufacturing, varying

controls like bioburden and endo-

burden testing of product and fil-

ter integrity testing are performed.

Another important aspect is the

environmental monitoring of the

surroundings. Before release of a

batch, a sterility test in an isola-

tor is performed to further demon-

strate sterility of the filled batch.

Mandal: Aseptically manufac-

tured drugs must be sterile, pyro-

gen-free, particulate-free, stable,

and isotonic. Sterility testing must

be conducted on every batch of

a product that is manufactured.

FDA consistently emphasizes that

sterility testing is to remain a cur-

rent good manufacturing practice.

Chapter <71> of the United States

Fill/Finish


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Fill/Finish

Pharmacopeia (USP) states that ste-

rility tests on parenteral dosage

forms are not intended to be used

as a single criterion for the accept-

ability of a product (3). Sterility

assurance is achieved primarily by

the validation of the sterilization

processes and the aseptic process-

ing procedures.

Aseptically

manufactured

drugs must be

sterile, pyrogen-

free, particulate-

free, stable, and

isotonic.—Bivash

Mandal, University

of Tennessee Health

Science Center

Ideal ly, every v ia l/syr inge/

ampoule manufactured must be

tested for its sterility. Because

sterility testing is a destructive

process, however, testing each

individual unit is not possible. USP

<71> provides guidance for the

minimum number of articles that

need to be tested from each manu-

factured batch.

The sterility test can be per-

formed by two different methods:

by the direct inoculation method

or by the membrane filtration

method. In the direct inoculation

method, a predetermined amount

of product is added directly to the

medium under aseptic conditions

and incubated. In the membrane

filtration method, the contents of

the product to be tested are filtered

through an appropriate-sized filter,

such that if any microorganisms

were to be present, they would be

retained on the filter. This filter is

then washed with specified solutions

to remove any retained product, and

finally, the filter is incubated with

medium at appropriate conditions

for at least 14 days.

Two different media must be

used for testing, irrespective of the

testing method used. Fluid thiogly-

collate medium (FTM) is used to

culture primarily anaerobic micro-

organisms, although it can support

the growth of aerobic microorgan-

isms as well. Trypticase soy broth

(TSB), also called the soybean

casein digest medium, is used to

test for the presence of fungi and

aerobic microorganisms. If a par-

ticular drug product inhibits the

growth of bacteria, such as is the

case with beta-lactam antibiotics,

the formulation of the medium

can be modified to include cer-

tain agents that can deactivate the

antibiotics, such as beta-lactamase.

Alternatively, the membrane filtra-

tion method can be used.

A failure of the sterility test is

indicated by a growth in one or

more of the incubated samples.

There is no such thing as a false

positive in the sterility testing of

an aseptically manufactured prod-

uct. A comprehensive written inves-

tigation follows, which includes

identification of the bacteria,

specific conclusions, and correc-

tive actions. A sterility test that is

positive may be indicative of pro-

duction, personnel, or laboratory

problems. The most commonly

found microorganisms in steril-

ity test failures include, but are not

limited to: Staphylococcus aureus,

Pseudomonas aeruginosa, Escherichia

coli, Enterobacter aerogenes, Neisseria

gonorrhoeae, Aspergillus niger, and

Candida albicans.

Fill/FiniSh BESt PrActicESBioPharm: Can you describe some

best practices for decontamination?

Stauss: The goal of a service pro-

vider to the biopharmaceutical

industry is to provide its custom-

ers with reliable and efficient asep-

tic production processes, which

are supported by safe and effec-

tive cleaning and decontamination

processes.

Automated decontamination of

RABS reduces downtime, increases

capacity utilization, and improves

overall equipment effectiveness.

Prior to the start of the decontami-

nation process, format parts are

cleaned offline, in full, and auto-

matically to remove particles, sili-

con, or residues, for example. This

automated cleaning process rep-

resents an important advantage

as compared to isolators, where a

manual cleaning process is nor-

mally applied.

Mandal: As an alternative to

formaldehyde-based sterilization,

vaporized hydrogen peroxide (VHP)

was introduced in the mid-1980s

to clean and decontaminate equip-

ment and machinery in the health-

care industry. Since then, the use of

VHP has been steadily increasing

due to the following advantages:

•Efficacyinrapid

decontamination of machines at

ambient temperatures and low

concentrations

•Stronghistoryofuseand

positive efficacy data on a broad

range of bacteria, fungus, spores,

and viruses

•Provenefficacytestingwith

biological indicators and

chemical indictors

•Abilitytokillresistantspores

•Usewithinacontrolledprocess

with real-time concentration

monitoring

•Notoxicbyproductswithits

use (VHP is a green solution)

•Associatedwithlessexposurerisk

to personnel and products outside

of a decontamination zone



•Afavorablesafetyprofile

(Typical concentrations used

are 150–700 ppm as compared

with formaldehyde [8000–10000

ppm] and chlorine dioxide

350–1500 ppm)

•Nolengthyaerationperiod

•Noresidue

•Astrongmaterialand

component compatibility profile

•Registeredbythe

Environmental Protection

Agency (EPA)

•ApprovedbyFDA.

casE studiEsBioPharm: Can you describe some

of your most challenging fill/finish

projects and what you did to over-

come obstacles that were presented?

Mandal: The Plough facility at

the University of Tennessee has

been manufacturing small-scale

batches for preclinical and Phase

I clinical trials for sponsors. We

have been using an aseptic clean-

room with manual intervention

and semiautomatic filling lines.

Most of the challenges we have

faced were mechanical or instru-

ment-oriented.

One of the projects (manufacture

of a sterile solution of polysaccha-

ride) had issues with the filling line

clogging when the filling opera-

tion was halted to switch person-

nel. The formulated product was

good, however, and was still within

acceptable limits of viscosity. Upon

investigation, we found that resid-

ual solution—which is in contact

with the filling needle tips—evapo-

rated in the laminar flow. We were

unable to remove the clot with high

pressure. The problem was solved

by running the entire fill continu-

ously, without interruptions.

Another challenge was with a

project focused on a parenteral that

was made up of an oily solution.

The process required us to overlay

nitrogen to protect the product

from oxidation. After stoppering

the product, the vial stopper even-

tually became pushed out in time.

The solution to the problem was

to crimp the vial in a reasonable

amount of time after stoppering.

Recently, we had a project on the

preparation and aseptic fill/finish

of a liposomal product contain-

ing a cytotoxic chemotherapeutic.

Liposomal products are notoriously

challenging fill/finish projects

because of issues with filtration,

drug loading, filter compatibil-

ity, and particle-size distribution.

Compatibility of the filter was an

important issue due to the drug

being adsorbed in the filter. The

proper control of the filtration

pressure was crucial, because there

is an increased occurrence of drug

loss from liposomes during filtra-

tion at higher pressures.

Additionally, the containment

of the cytotoxic chemotherapeu-

tic proved challenging. Special

procedures should be adopted to

deactivate the drug contaminated

materials after fill/finish. Cleaning

validation of the equipment should

be conducted in order to obviate

cross-contamination.

Stauss: Based on our day-to-day

experiences in customer projects, we

see the overall market is increasingly

becoming more challenging, par-

ticularly in areas such as:

• An increase in high-value

products in smaller batch sizes

• The cont inuous increase

in regulatory requirements,

including anticounterfeiting

activities

• Ever-more complex supply

chains on the customer side,

which have resulted in more

compl ic ate d r e que s t s fo r

CDMOs.

High-value products are often

based on complex compounds.

They demand high accuracy on the

filling line and have an increased

sensitivity to manufacturing pro-

cesses and environmental condi-

tions. A good example of a difficult

fill/finish project is the handling of

a highly sensitive API that requires

very small fill volume in a syringe.

Small filling volumes in such cir-

cumstances create signif icant

demands on all production areas,

including process design, technical

equipment, and packaging mate-

rial. This, in turn, creates high

demands on the operating staff.

In such cases, packaging material

and processes need to be adapted to

meet the requirements of a product.

Using the correct application tech-

nique of the silicone coating on a

syringe is a good example of a com-

mon packaging challenge.

Comprehensive project manage-

ment is necessary to handle such

a project successfully, taking into

consideration the needs of both the

product and the customer. To pro-

actively enable a successful product

launch, every potential impediment

to the best outcome in fulfilling

product requirements—including

manufacturing processes, use of tech-

nical equipment, and proper staffing,

to name a few—must be taken into

account during the project phase.

rEFErEncEs 1. The University of Tennessee Health

Science Center, “New Plough

Center for Sterile Drug Delivery

Systems to Expand UTHSC’s

National and Global Position as

a Pharmaceutical Manufacturer,”

Press Release, http://news.uthsc.

edu/new-plough-center-sterile-

drug-delivery-systems-expand-

uthscs-national-global-position-

pharmaceutical-manufacturer/,

accessed Oct. 13, 2015.

2. Vetter, “Vetter Embarks on a 300

Million Euro Investment Strategy

for Further Development to its

Manufacturing Sites and to Make

Available Additional Manufacturing

Capacities,” Press Release, www.

vetter-pharma.com/en/newsroom/

press/publications/vetter-embarks-

on-a-300-million-euro-investment-

strategy-for-further-development-to-

its-manufacturing-sites-and-to-make-

available-additional-manufacturing-

capacities/vetter-embarks-on-a-300-

million-euro-investment-strategy/,

accessed Oct. 13, 2015.

3. USP, USP General Chapter <71>,

“Sterility Tests,” USP 29–NF 24

(US Pharmacopeial Convention,

Rockville, MD, 2006). ♦

Fill/Finish



Med

icalR

F.co

m/G

ett

y Im

ag

es

This article reviews the impli-

cations of cell-culture con-

ditions on biologic product

quality, focusing on glycosyl-

ation and analytical techniques for its

accurate assessment. Glycosylation can

potentially affect a protein’s half-life,

immunogenicity, binding activity, and

stability. It is a complex process that

consists of the attachment of carbohy-

drate moieties, with possible attach-

ment sites via asparagine (N-linkage)

or serine/threonine (O-linkage) amino

acids in protein structures. In mam-

malian cell culture processes, the use

of different species can potentially

produce significant differences in the

types of glycosylation that can occur.

These differences in glycosylation can

have significant effects on the quality

of the therapeutic protein produced, as

can the choice of cell clone, the basal

and feed media used, and the cell-

culture conditions.

The choice of host cell and the bioreac-

tor conditions used in bioproduction of

proteins significantly affects protein prod-

uct quality. This is due both to the struc-

tural complexity of proteins themselves

and also to species-specific post-transla-

tional modifications that may occur dur-

ing the cell-culture process, glycosylation

being of particular importance.

Protein theraPeutics and cell-culture effects on Protein qualityBiopharmaceutical drugs are proteins

with polymeric structures, built up

in a series of structural levels starting

implications of cell culture conditions on Protein Glycosylation

Richard Easton and Michiel E. Ultee

The authors present a

review of the techniques commonly

used for glycosylation

analysis.

Michiel E. Ultee, PhD, is principal

at ulteemit Bioconsulting, llc, and

Richard Easton, PhD, is team

leader, carbohydrate analysis,

sGs life science services.

upstream Processing


EMD Millipore Corp. is a subsidiary of Merck KGaA, Darmstadt, Germany

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Take advantage of this process accelerating combination of high-quality products and targeted insight. We help you find the fast track through the maze.

Find out how at:www.emdmillipore.com/emprove

EMD Millipore, the M mark and Emprove are registered trademarks of Merck KGaA, Darmstadt, Germany.

© 2015 EMD Millipore Corporation, Billerica, MA, SA. All rights reserved.

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Merck Millipore is a business of

Your fast track through regulatory challenges.The new Emprove® program. Does the constantly changing regulatory landscape sometimes feel like a maze? The new Emprove® program provides the answers you need, with a portfolio of 400 pharma raw and starting materials backed by information to support your qualification, risk assessment, and process optimization activities.

• Portfolio of products to address different risk levels• Elemental Impurity Information (ICH Q3D) • Online access to all dossiers in the new Emprove® Suite

Take advantage of this process accelerating combination of high-quality products and targeted insight. We help you find the fast track through the maze.

Find out how at:www.merckmillipore.com/emprove

Merck Millipore, the M mark and Emprove are registered trademarks of Merck KGaA, Darmstadt, Germany.

© 2015 Merck KGaA, Darmstadt, Germany. All rights reserved.

ES700042_BP1115_B21_FP.pgs 11.04.2015 02:42 ADV blackyellowmagentacyan


AL

L F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

RS

from the amino-acid sequence,

referred to as primary structure,

through folding of the amino

acid chains into local (second-

ary) and longer-range (tertiary)

three-dimensional conforma-

tions. Multi-chain proteins, such

as IgG antibodies, additionally

have a quaternar y st r uc ture

resulting from structural associa-

tions between the subunits.

the choice of host cell

and the bioreactor

conditions used in

bioproduction of

proteins significantly

affects protein

product quality.

The choice of host-cell line for

recombinant protein production

depends first on the protein’s

molecular properties. Certain bac-

teria can be used for production of

the simplest proteins, those that

are composed only of amino-acid

polymers, with no post-transla-

tional modifications (PTMs) such

as glycosylation, because most

bacterial strains are incapable of

glycosylation. Production is fast

using simple media; however, puri-

fication can be challenging. Rapid

production of proteins with primi-

tive glycosylation can be achieved

using yeast. Insect cells, generally

used with a baculovirus vector in

transient fashion, are used mostly

for R&D and niche products.

Mammalian cells are used for the

production of complex proteins

such as antibodies and enzymes,

requiring full PTMs, including the

production of complex carbohydrates.

Proteins are delicate molecules

compared with small-molecule

drugs and present multiple stabil-

ity challenges. A typical glycopro-

tein such as an IgG antibody has

many sites of variability within its

structure, which comprises four

chains with a total molecular

weight of 150,000 Da. Additionally,

there are several post-translational

modifications of the protein chain

that can occur, such as oxidation

and deamidation of specific amino

acids. Each heavy chain also

includes a site where glycosylation

takes place (Figure 1).

Why is Glycosylation imPortant?Many complex proteins such as

antibodies and enzymes are glyco-

proteins, containing from 2–30%

carbohydrate. Glycosylation is a

complex process, with the carbo-

hydrate attached to the protein

either via the amino acids aspara-

gine (N-linked) or serine/threonine

(O-linked). Multiple sugar types

are possible, each with multiple

attachment sites, and variation of

the mammalian cells used in pro-

duction can lead to subtle glycosyl-

ation differences (1).

The choice of cell clone affects

product quality. Each clone has

slightly different abilities for gly-

cosylation and other PTMs, and

viabilities vary, resulting in differ-

ences in released intracellular deg-

radative enzymes into the culture.

Therefore, it may be necessary to

select a cell clone that is not the

highest producer to achieve the

desired protein quality.

The extent of glycosylation can

also vary depending on the basal

and feed media, even with a single

clone producing a single monoclo-

nal antibody, or single basal media

with varied feeds.

effects of cell-culture conditionsCel l- cu lture parameters that

affect the type and extent of gly-

cosylation include pH and CO2

levels; the amount of dissolved

oxygen (dO2); the temperature;

the levels and types of nutrients;

the presence and types of gly-

can precursors; cell viability, as

dying cells release degradative

enzymes; and the level of process

control.

upstream Processing

Figure 1: Graphic showing IgG antibody structure, sites for glycosylation, and

potential variability.


Bioreactors offer much greater

control of pH and dissolved gasses

than shake flasks do, and hence,

better process control, but shake

flasks are more economical and

readily allow larger numbers or

arrays. Shake-flasks are, therefore,

commonly employed for early

scouting studies on media and

feed conditions. Optimum cell-

culture development is achieved

by working with bioreactors to

enhance growth and productiv-

ity via selection of basal media

and culture feeds and the timing

for these feeds; optimizing bio-

reactor oxygenation conditions,

such as CO2 levels, pH, agitation,

temperature, seeding densities,

and split ratios; extending the

production phase of cell culture

through the use of temperature

shift; and minimizing accumula-

tion of growth-inhibiting metabo-

lites such as ammonia.

More recently, the availabil-

ity of miniaturized bioreactors

has provided an effective and

e f f ic ient way of conduc t ing

multi-parameter studies. Thus,

‘ big- data’ approaches a l low-

ing design-of-experiment (DoE)

studies, in which multiple inter-

reacting bioreactor conditions

are evaluated simultaneously, are

made possible. Such studies require

the employment of automated,

large-number bioreactor arrays to

make the process feasible (2).

sPecial cell-culture considerations for BiosimilarsBiosimilars are “generic” ver-

sions of protein pharmaceuticals

that must be highly similar to

the innovator drug in order to

be classified as such. Regulatory

guidelines require extensive ana-

lytical testing side by side with

the innovator drug, including

full glycosylation profiles (3).

Similarity to the innovator drug

is paramount; this must begin

from clone selection and pro-

ceed throughout process devel-

opment. Therefore, rather then

only selecting for clones with

the highest titer, as is usually the

case for innovator drugs, selec-

t ion is f irst for biosimilar ity,

which may mean that some of

the highest producing clones are

not selected.

Figure 2: Stacked high pH anion-exchange chromatography procedure with

pulsed amperometric detection (HPAEC–PAD) chromatograms of glycans

released from three preparations of bovine fetuin.

125

100

75

50

25

-10

0

NanoCoulombs(nC)

1

2

3

13 25 38 50 63 75 88 100 120

min

BioPharm International magazine

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of biopharmaceutical research,

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upstream Processing

techniques for Glycosylation analysisN-glycosylation occurs when the

carbohydrate is attached to aspar-

agine in the consensus sequence

asparagine-X-serine/threonine,

where X is any amino acid except

proline. As a nascent protein is

being synthesized in the endo-

plasmic ret iculum within the

cell, an en bloc transfer of a pre-

formed, lipid-anchored conserved

glycan occurs. As the synthesized

protein makes its way through

the Golgi apparatus, the con-

served N-linked glyan is ‘pro-

cessed’ by enzymes (glycosidases

and glycosyltransferases). It is

the presence of these processing

enzymes, their relative levels, and

the accessibility of the glycosyl-

ation site on the protein to these

enzymes that determine the final

glycosylation of a protein.

O-glycosylation occurs on the

amino acids serine and threo-

nine, but not in accordance with

a l inear consensus sequence.

There are some rules governing

the process—for example, there are

often nearby proline amino acids

within the regions of glycosyl-

ation, and also quite often, tandem

repeats of serine and theonine.

analysis of n-GlycansAnalysis of N-glycans is the most

active area of research. The tech-

niques that apply to analysis

of N-glycosylation also apply to

O-glycosylation.

The most abundant mam-

malian N-glycan structure is the

complex type where a number of

N-acetylglucosamine structures

are appended to the molecule and

extended with galactose, fucose, and

sialic acid residues to give between

two and four antennal structures.

The exact structure reflects the

nature of the enzymes present in the

cell type used in the expression pro-

cess as well as the precise environ-

ment in which the cells are located.

International Conference on

Harmonisat ion ( ICH) Qualit y

Guidelines Q6B describes the type

of analysis that should be per-

formed in relation to these struc-

tures to understand the quantities

of the different types of monosac-

charides present, the nature of the

glycans in terms of their antennary

profile, and linkage of monosac-

charides in each structure but also

the structure of the glycans at the

different glycosylation sites on the

protein backbone (4, 5).

analytical Procedure: the detailsThe analytical procedure of gly-

cosylation involves a number of

physical and enzymatic steps to

release the glycans and then sepa-

rate them from the peptides and

any O-glycopeptides present in

the mixture. O-glycans can be

released chemically from these

O-glycopeptides and purified sep-

arately from the remaining pep-

tide chains.

A permethylation derivatization

procedure is then performed on the

separated N-glycans and O-glycans,

enabling them to be analyzed

using matrix-assisted laser desorp-

tion ionization–mass spectrometry

(MALDI–MS) analysis.

It is also possible to analyze the

samples chromatographically with-

out having to employ the permeth-

ylation procedure. For example, a

high pH anion-exchange chroma-

tography procedure with pulsed

amperometric detection (HPAEC–

PAD) can be used for the analy-

sis of N-glycans. An example of

this can be seen in the N-glycans

released from bovine fetuin, as

shown in Figure 2. This method

gave consistent data across three

preparations, a series of clustered

peaks representing di-, tri-, and

tetra-sialylated glycans (i.e., two,

three, and four sialic acid groups

on the N-glycans within each clus-

ter). The analysis gives some struc-

tural information in terms of an

idea of what has been produced

but little information in terms of

the precise nature of what the gly-

cans are. Techniques like this are,

therefore, useful for comparative

work but do not give much struc-

tural information for characteriza-

tion of the molecules.

one final step in

characterization is

to determine the

stereospecificity

of any linkages

within structures

where data indicate

that potentially

immunogenic

epitopes might be

present.

In an alternative approach, a

fluorescence tag—2-aminobenza-

mide—can be used to specifically

label the released N-glycans and

these labeled N-glycans can then

be chromatographically separated.

The chromatographic eluent is

then analyzed by mass spectrom-

etry (MS) to identify the masses

of individual structures. This is a

useful technique because the chro-

matographic profile not only gives

a batch-to-batch comparison but

also provides information on the

masses of the glycans in each peak.

The technique is so sensitive that it



is possible to resolve and identify

the two different forms of the so-

called “G1F” structure, the mono-

galactosylated biantennary glycan

found on antibodies. The two com-

ponents have a galactose structure

on either arm of the biantennary

structure and these can be sepa-

rated out. Further use of mass spec-

trometry, however, is necessary to

fully characterize what these gly-

cans are.

characterization usinG mass sPectrometryThe aforementioned permeth-

ylat ion procedure a l lows the

separation of glycans with sim-

ilar masses in the non-deriva-

t ized state. For example, two

glycans—with identical struc-

tures apart from one contain-

ing a single N-aetylneuraminic

acid (sialic acid) residue and the

other containing two fucose resi-

dues—differ in molecular weight

by only 1 Da in the native state

but differ by 13 Da when deriva-

t i z e d . Mat r i x- a s s i s te d l a s e r

de sor p t ion ion i z at ion – ma ss

spectrometry (MALDI–MS) anal-

ysis gives a population profile

of the glycans that are present.

Using the peaks (masses) of the

spectrometric analysis, in com-

bination with the knowledge of

the N-glycan, the core structure,

and the biosynthetic pathways

that are present, it is possible

to arrive at glycan monosaccha-

ride compositions. However, this

leaves the arrangement of the

glycans within the structure to

be determined.

Next, electrospray–mass spec-

trometry (ES–MS) can be used

to fragment the permethylated

glycans. This div ision act ion

is useful, as the fragmentation

pathways are well defined, well

understood, and produce spe-

cific masses depending on where

the fragmentation takes place.

This process depends on the

exact nature of the structure of

the N-glycans so these fragment

masses allow the determination

of the antennary structures pres-

ent on the glycans.

A third method, gas chroma-

tog raphy–mass spec t romet r y

(GC–MS), can be performed to

determine how the monosaccha-

rides are linked to one another

within the glycan st ructure.

These structural variations can

have a large effect on the drug’s

efficacy and properties.

By c he m ic a l ly mo d i f y i ng

the permethylated glycans and

attaching particular “reporter

groups” wherever each monosac-

charide is linked one to another,

der ivat ives are produced that

provide key structural informa-

t ion when analyzed by mass

spectrometry. Using an electron-

impact mass spectrometer, the

f ragmentat ion pat terns show

characteristic fingerprints for the

different linkages. Furthermore,

the GC apparatus on the front

end of the mass spectrometer

can identify each of the dif-

ferent l inked st ructures with

their different reporter groups,

because these have different elu-

tion times from the GC column.

The combination of GC reten-

tion time and mass spectral fin-

gerprint fragmentation pattern

identifies the different monosac-

charide linkages that are in the

glycan population.

One final step in characteriza-

tion is to determine the stereo-

specificity of any linkages within

structures where data indicate

that potentially immunogenic

epitopes such as Ga lαGal (a

known immunogenic epitope)

might be present. Treatment of

the glycan with a specific exo-

glycosidase enzyme and analy-

sis by MS pre- and post-exposure

can confirm the presence of an

alpha-linked galactose by reveal-

ing a mass shift upon incubation.

conclusionProteins are inherently complex

and much larger in size than small-

molecule drugs, and multiple types

of PTMs, including glycosylation,

are a feature of most protein thera-

peutics. Their occurrence depends

on the molecular nature of the

protein; the selection of cell type

and individual clone; and cell-

culture and bioreactor conditions.

Extensive analytical assays are

needed to characterize a protein

therapeutic. Biosimilars require

even more analytical testing than

do innovator drugs because of the

need to prove biosimilarity to the

given innovator molecule.

Mass spectrometric analysis pro-

vides structural information on the

composition, antennal structures,

and linkages present in N-glycan

molecules and the use of chroma-

tography provides a unique glycan

profile due to the precise structure

and associated interactions of the

glycans with the column matrix.

This profile can be used as a refer-

ence against which other batches

can be compared. In addition, the

use of LC/ES-MS of proteolytic

digests allows sites of glycosylation

to be isolated and identified. These

sites can then be analyzed using a

number of techniques to determine

the nature of the glycans at each

site and the specific populations of

the glycans on the biopharmaceuti-

cal molecule.

references 1. D. Ghaderi et al., Biotech Gen. Eng.

Rev. 28, pp. 147–176 (2012).

2. S.D. Jones et al., Am. Pharma.

Rev. 18 (1), pp. 44–48 (2015).

3. FDA, Guidance for Industry, Scientific

Considerations in Demonstrating

Biosimilarity to a Reference Product

(Rockville, MD, April 2015).

4. FDA, Guidance for Industry, Quality

Considerations in Demonstrating

Biosimilarity of a Therapeutic

Protein to a Reference Product

(Rockville, MD, April 2015).

5. ICH, Q6B, Specifications: Test

Procedures and Acceptance Criteria for

Biotechnological/Biological Products,

Step 5 version (Sept. 1999). ♦

upstream Processing



Ro

bert

A P

ears

/Gett

y Im

ag

es

BioPharm International spoke with

Mark A. Snyder, PhD, Manager,

Applications R&D Group, and

Kim Brisack, R&D Applications,

at Bio -Rad Laborator ies’ Process

Chromatography Division about the devel-

opment and challenges of process chroma-

tography in bioprocessing.

BioPharm: How has the use of chroma-

tography changed since it was first intro-

duced for bioprocessing?

Snyder: Almost everything is different!

Column design has changed significantly,

having evolved from pure manual pack-

ing to fully automated systems. Also, very

large (1–2 meter) columns are now being

routinely used. Resins have made sig-

nificant progress on several fronts with

expanded bead chemistries and particle

sizes, a wider catalog of ligands, higher

binding capacities, and faster flow rates.

Other forms of downstream matrices, such

as monoliths and membranes, have also

been developed. Another area of progress

is the reduced number of purification steps

for typical processes, from an historical

average of five, down to three, with some

manufacturers even trying out two-step

processes. Economic pressures to do more

with less has led to increased use of soft-

ware-aided process decisions. Modeling

software is increasingly used to minimize

the use of buffers, time, and staging. Even

the modes of chromatography modes have

grown from simple flow-through (vs. bind/

elute) to weak partitioning, displacement

chromatography, and so on.

Brisack: Even as the fear of using very

large columns has been eliminated, many

downstream processes are moving to

The Development of Process Chromatography in Bioprocessing

Susan Haigney

Industry experts discuss the

development of process

chromatography in bioprocessing.

Downstream Processing



smaller columns. The use of smaller

columns has been enabled both by

higher binding capacities and by

biotherapeutics with higher per-mg

activities than before. In some cases,

the impetus is on incorporating pre-

validated, prepacked columns with

minimal impact to the processing

area during changeover. Continuous

chromatography, which uses smaller

columns by design, by maximizing

the loading on each column, is also

an option to maximize productivity

in processing areas in lieu of massive

stainless steel columns.

BioPharm: What are the typical

challenges involved in process chro-

matography?

Snyder: The advent of faster protein

production rates during fermenta-

tion has created issues such as higher

target protein aggregation. Today,

aggregates have to be eliminated

more aggressively than in the past.

In the same vein, regulatory scrutiny

of quality outputs such as isoforms,

glyco-heterogeneity, and dimer con-

tent has increased. Consequently,

the need for an even more uniform

product has put additional pressure

on the downstream process. At the

same time, the globalization of bio-

tech, coupled with pressures to lower

healthcare costs, requires processes

that have a smaller footprint, take

fewer steps, use less consumables,

and can be easily transferred from

one manufacturing site to another.

All of these challenges are being

met the same way as always—through

careful consideration of chemistry

and matrix for each step, a thorough

understanding of how the step works,

and intelligent overall design for easy

transition from step to step. I should

add a note of thanks to those compa-

nies who sell viral clearance solutions.

The advent of viral filtration mem-

branes and other technologies has

allowed processes to maintain their

viral clearance safety margins while at

the same time shrinking the number

of overall steps needed.

Brisack: Historically, high recovery

was one of the most important vari-

ables to consider when selecting a

media. With the focused attention

on product homogeneity as outlined

above, clearance of product-related

and process-related impurities often

takes precedence over pure recovery.

Incremental differences in dimer

clearance, for example, can impact

the overall product profile owing to a

reduced immunogenicity of the final

product.

BioPharm: Which chromatographic

tools are most commonly used in

downstream processing?

Snyder: The tools most commonly

used in downstream processing are

software packages. As mentioned,

modeling software designed to tease

out inefficiencies, as well as plan

footprints and use of utilities, have

significantly helped in overall pro-

cess economics and facility plan-

ning. In addition, the introduction of

quality by design (QbD) has fostered

the use of design-of-experiments

(DoE) programs, which have aided

process developers in making reg-

ulatory submissions that allow for

easier process changes after licensure.

Typically, these process changes rep-

resent incremental process improve-

ments, [which would have made]

submissions to regulatory agencies

[...] too burdensome in the past.

However, DoE software could be mis-

used if the end user doesn’t under-

stand exactly how the results can

and cannot be interpreted. Finally,

statistical packages are increasingly

being used to perform multivariate

analysis on large amounts of data to

discern relationships between input

and output parameters from multiple

manufacturing cycles that might not

have been revealed during process

development.

BioPharm: Are there any new

novel/innovative techniques or tools

currently being used by the industry

or in development?

Snyder: New chromatographic

purification products are being intro-

duced all the time. Also, I think

that new methodologies for making

inline, online, or at-line measure-

ments of various quality outputs are

slowly making their way into the

industry. These will help fulfill FDA’s

[goal] of making more use of process

analytical technology to help ensure

quality products by increasing pro-

cess control.

Brisack: Automated column pack-

ing has become a reality but it is still

not a ‘push-button’ technology. New

advances in software and hardware

have, however, been able to reduce

variability in this historically chal-

lenging procedure. There has also

been an increased interest in contin-

uous processing to maximize resin

capacity and reduce core cycle

time. Whether this can realistically

be implemented at process scale

remains to be seen. ◆

Downstream Processing

The need for an even

more uniform product

has put additional

pressure on the

downstream process.

— Mark A. Snyder, PhD,

Bio-Rad Laboratories

Automated column

packing has become a

reality ... — Kim Brisack,

Bio-Rad Laboratories



Since FDA introduced the risk-

based approach as a means to

improve the regulation of phar-

maceut ica l manufac tur ing

and product quality (1), the concept

of quality by design (QbD) has been

gradually implemented into biologics

development processes. In contrast to

the traditional quality-by-testing (QbT)

mentality, the QbD approach requires

that quality “be built in by design”

based on the knowledge of the product

and process (2). It is a holistic approach

that involves a thorough understand-

ing of the relationship between process

inputs and process outputs so that any

potential risks affecting product quality

can be mitigated. The aim is to ensure

that the final drug product meets the

pre-established quality requirements

defined by a set of critical quality attri-

butes (CQAs), which will ultimately

determine its clinical performance.

This article presents a case study in

which a QbD approach is applied to

establish the design space for an inter-

mediate chromatography purification

step. The drug substance is a protein

molecule expressed in microbial

host cells, which is purified by a pro-

cess consisting of capture, intermedi-

ate purification, and polishing steps.

The intermediate purification step is

achieved by cation-exchange chro-

matography (CEC) (see Figure 1). This

step removes both process-related and

ABSTRACT

This case study reviews how quality-by-design principles can be implemented

in an intermediate chromatography purification step that uses cation-

exchange chromatography. The drug substance is a protein molecule

expressed in microbial host cells. The purification process involves a capture

step and an intermediate purification step, followed by a polishing step.

Establishing process design space for a chromatography purification step:

application of Quality-by-design principles

Hui Xiang

Hui Xiang is principal scientist

at allergan inc., 2525 dupont drive, irvine,

ca 92612, united states; tel: 714.246.5330;

[email protected].

PEER REVIEWED

article submitted: may 19, 2015.

article accepted: June 22, 2015.

Ra

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speer-reviewed: Quality by design



product-related impurities. The product-

related impurities are inactive variants that

are closely related to the drug substance; a

delicate design of conditions is, therefore,

required to separate the product-related impu-

rities from the drug substance. The objec-

tive of development is to achieve maximum

purity and recovery at this intermediate puri-

fication step.

ExpErimEntal approachScreening experiments were performed to

identify the resins and chromatography con-

ditions that separate the impurities from the

drug substance molecule. The process was

developed, scaled-up, and transferred to the

manufacturing group to perform production

batches. A scale-down process model was

developed and qualified, and baseline stud-

ies with small-scale runs were performed to

generate a history of step performance, which

helped to establish the performance accep-

tance criteria for step yield and purity.

Process characterization was performed

based on a series of design-of-experiment

(DOE) studies. A screening design was

employed to identify the ranges of parameters

with potential risks. The parameters identified

to have significant impact were further stud-

ied using a full factorial design. The results

refined the parameter ranges based on the

responses of purity and step recovery. Least

square fit models of the results were used to

perform Monte Carlo-based simulations to

identify the process capability and the poten-

tial number of failures using the JMP software

version 9.0 (3). Based on the regression model

and Monte Carlo simulation results, targets

and ranges for the parameters were proposed

for this intermediate purification step.

matErials and mEthodsResins used for screening were obtained

from suppliers such as GE Healthcare, Toso

Bioscience, Bio-Rad, and Applied Biosystems.

Chemicals used in buffers were purchased

from Mallinckrodt, J.T. Baker, or EMD.

Chromatography runs were performed on

the AKTA explorer (or purifier) and custom-

built chromatography skid. Protein concen-

tration was measured with a DU 720 UV/

vis spectrophotometer (Beckman Coulter).

Buffer pH and conductivity were measured

with a pH/conductivity meter (Mettler

Toledo). Product yield was calculated based

on enzyme-linked immunosorbent assay

(ELISA) results, and purity was measured

by reverse-phase high-performance liquid

chromatography (RP–HPLC). Both ELISA and

RP–HPLC were qualified for their intended

purposes.AL

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peer-reviewed: Quality by design

Figure 1: A schematic illustration of the production process

of a protein molecule expressed in microbial host cells. The

intermediate purifcation is achieved by a cation-exchange

chromatography step, which removes both process-related and

product-related impurities.

Figure 2: A cation-exchange chromatography (CEC)

chromatogram. Product-related impurities elute during the Wash

2 step as a single peak before the API elutes, whereas process-

related impurities elute during the cleaning step.

Upstream process

Downstream process

Fermentation culture

Harvesting and conditioning

Capture

Intermediate purifcation

Polishing

Tangential fow fltration

Terminal fltration

Drug substance

mAU

2000

1500

1000

500

00 100 200 300 400 500

CEC Eluate

Wash 2 Peak

Flowthrough/Wash1

UV A280

Post-elution Clean Peak

ml



rEsults and discussion Screening and early development

The screening experiments identified a POROS

resin to separate the product-related impurities

from the drug substance molecule. With low

pH and low salt concentration, the capture step

eluate was suitable for loading directly onto the

CEC column. The dynamic binding capacity at

10% breakthrough (DBC10) was determined to

be approximately 25 g/Lbed by frontal analysis,

which was well above the load under the pro-

cessing conditions.


Process parameter Target Low High

Flow rate (cm/hr) 240 180 300

Protein load (g/Lbed) 3.1 2.0 4.3

Load pH 4.8 4.6 5.0

Load conductivity (mS/cm) 4 3 5

Wash 2 pH 4.8 4.6 5.0

Wash 2 conductivity (mS/cm) 18 16 20

Elution pH 4.8 4.6 5.0

Elution conductivity (mS/cm) 27 24 30

Table I: Process parameters and test ranges in

a design-of-experiment screening study.

Figure 3: The effect of gradient slope and fow rate on separation resolution (Rs) between the product-related

impurities and the API. The chromatogram peak profles with different gradient slopes and different fow rates are

illustrated in Panels A and C, respectively. It can be observed that resolution increases with a shallower gradient slope

(B), but decreases when the fow rate is increased (D).

Figure 4: Fish-bone diagram of the cation exchange (CEX) chromatography step; HETP is height equivalent to a theoretical plate.

A B

DC

Peak 2

0 - 1 M NaCl

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

0

1

0.5

1.5

2.5

2

3

0 100 200 300 400 500 600

5 10 15 20 25

Reso

luti

on

(R

s)

Gradient CV

Reso

luti

on

(R

s)

Flow Rate (cm/hour)

y = 0.0604x + 0.9172R2 = 0.9573

y = -0.003x + 3.0439R2 = 0.8153

30 35

10 CVs

15 CVs

20 CVs

25 CVs

30 CVsPeak 1

mAU

150

100

50

0

mAU

140

120

100

80

60

40

20

0

40

55 60 65 70 75

100 cm/hour

200 cm/hour300 cm/hour

400 cm/hour500 cm/hour

0 - 1 M NaCl in 20 CVs

mL

50 60 70 80 90 100 mL

Column Preparation

Sanitization contact time

Post-sanitization hold time

Column HETP / asymmetry

Column volume

Flow rate

Feed pH

Feed conductivity

Feed volume

Feed ConcentrationFlow rate

Buffer conductivity

Buffer pH

Flow rateCollection A280 start/stop

Dilution buffer pH,conductivity and volume

Product pool hold timeand temperature

Buffer conductivity

Buffer pH

Flow rateFlow rate

Buffer conductivity

Buffer pH

Buffer volume

Buffer volume

Wash 1 Wash 2

CEX Eluate

ElutionEquilibrationLoad



Output

Relative

importance

(1–5)b

Relative size of effect (Pareto Plot) Total effecta

Flow

rate

Protein

load

Load

pH

Load

cond.

Wash 2

pH

Wash 2

cond.

Elution

pH

Elution

cond.

Flow

rate

Protein

load

Load

pH

Load

cond.

Wash 2

pH

Wash 2

cond.

Elution

pH

Elution

cond.

Product

purity5 0.12 1.33 0.48 0.50 2.87 4.08 1.24 2.95 0.60 6.65 2.40 2.50 14.35 20.40 6.20 14.75

Step

recovery3 0.58 0.85 0.53 0.22 2.19 2.47 1.27 1.42 1.74 2.55 1.59 0.66 6.57 7.41 3.81 4.26

a Total effect = size of effect x relative importance Total score 2.34 9.2 3.99 3.16 20.92 27.81 10.01 19.01

b Relative importance: 1 (least important) – 5 (most important)c Control factor: 1 (easy to control) – 3 (difficult to control)d Adjusted score = total score x control factor

Control factor (1–3)c 1 2 2 2 1 1 1 1

Adjusted scored 2.34 18.4 7.98 6.32 20.92 27.81 10.01 19.01

Rank 8 4 6 7 2 1 5 3

e Cond. = Conductivity Include in next design of experiment? No Yes No No Yes Yes No Yes

Table II: Impact of operating parameters on the cation-exchange chromatography (CEC) intermediate purifcation step.

A series of linear salt gradient elution

studies were performed to screen the elution

profiles of bound proteins from the column.

The parameters scouted included a linear

gradient slope (or column volumes [CVs]

from 0–1 M sodium chloride [NaCl]) and a

linear flow rate (100–500 cm/hour). The elu-

tion profiles are illustrated in Figure 2. Peak

1 contained the product-related impurities

and Peak 2 was the drug substance peak.

The linear gradient runs helped to elucidate

the effect of gradient slope and flow rate on

resolution (Rs) between the product-related

impurities and drug substance peaks (see

Figure 3). The Rs is calculated from the ratio

of the difference between the peak reten-

tion volumes (VR) to the average of the peak

base width at 10% of peak height (wb). The

results suggested that resolution increases

with shallower gradient slope, but decreases

with the increase of flow rate. A resolution

of 1.5 indicates baseline separation, which

requires greater than 10 CVs in 0–1 M NaCl

gradient and allows a linear flow rate of up

to 500 cm/hour.

Based on the linear gradient studies, the

washing step and elution conditions were

developed. The developed chromatogra-

phy scheme consisted of equilibration,

loading, wash 1, wash 2, elution, post-

elution cleaning, and decontamination

steps, as illustrated in Figure 2. The prod-

uct-related impurities were desorbed from

the column as the wash 2 peak, and the

product molecule was eluted as the CEC

eluate. The strongly bound impurities,

mostly process-related impurities, were

eluted under high salt conditions, shown

as post-elution clean peaks in Figure 2. The

column was decontaminated with 0.1 N

sodium hydroxide (NaOH) at the end of

chromatography.


Figure 5: Process characterization workfow. DOE is design of

experiment.

ProcessDevelopment

ScreeningDOE

AssessmentFollow-on

DOEIntegration

DOE

ProcessCharacterization

ProcessValidation

Process parameters

Previously screened

range

Follow-on test range

Target Low High

Protein load (g/Lbed)

2.0 – 4.3 2.0 – 4.3 3.2 2.0 4.3

Wash 2 pH 4.6 – 5.0 4.7 – 4.9 4.8 4.7 4.9

Wash 2 conductivity

(mS/cm)16 – 20 17 – 19 18 17 19

Elution conductivity

(mS/cm)24 – 30 26 – 28 27 26 28

Table III: Process parameters and test ranges in the follow-on

design-of-experiment study.



Scaling down the process

model and baseline runs

To prepare for the process characterization

studies, a small-scale process model with a

scale-down factor of 1/25 was established

and qualified to match the production-scale

batch performance. A baseline study was per-

formed to assess the performance variation

and thereby, establish performance baselines

under small-scale purification conditions. A

total of 15 small-scale runs were performed

under target process conditions. The CEC

results of the 15 runs gave a mean step yield

by ELISA of 70%, with a 6σ range of 53–87%,

and a mean purity by RP–HPLC of 96%, with

a 6σ range of 94–97%.

Process characterization strategies

The primary objective of this intermediate puri-

fication step was to remove closely related prod-

uct-related impurities from the capture step

eluate. Risk assessment was performed using

the failure mode and effect analysis (FMEA)

on the parameters illustrated in the fish-bone

diagram (see Figure 4); the parameters with

potentially high risks on the recovery and/or

purity step were selected for a DOE-screening

study. Based on the baseline study results and

risk assessment, the acceptance criteria of step

yield and purity were set at ≥ 65% and ≥ 95%,

respectively, as the goal for process character-

ization studies.

Among the parameters studied, those that

had a relatively large effect on recovery and/

or purity and were relatively difficult to con-

trol were selected for a higher resolution fol-

low-on DOE study, which resolved the main

effects, interactions, and quadratics. Monte

Carlo simulations were performed under dif-

ferent scenarios, using proposed parameter

ranges. At the same time, full-scale purification

was performed at the manufacturing site to

accumulate historical data, which were com-


Figure 6: Least square ft models of step yield (A) and purity (B).

A

120

110

100

90

80

70

60

50

98

97.5

97

96.5

96

95.5

95

94.594.5 95 95.5 96 96.5 97 97.5 98

50 60 70 80 90 100 110 120

Step Yield Predicted

Ste

p Y

ield

Act

ual

Pu

rity

Act

ual

P<.0001 RSq=0.99RMSE=1.8768

Purity Predicted P=0.0074RSq=0.96 RMSE=0.2867

BActual by Predicted Plot Actual by Predicted Plot

Summary of ft

R2 Adjusted R2

RMSE Response mean

Observations

0.99 0.98 1.88 87.87 18

ANOVA

Model Sum of squares

df Mean square

F p

Regression 3195.01 12 266.25 75.59 <0.0001*

Residual 17.61 5 3.52

Total 3212.62 17

Summary of ft

R2 Adjusted R2

RMSE Response mean

Observations

0.96 0.88 0.29 96.32 18

ANOVA

Model Sum of squares

df Mean square

F p

Regression 11.12 12 0.93 11.28 <0.0074*

Residual 0.41 5 0.08

Total 11.53 17

Term

Unstandardizedcoefficient

Standardized coefficient t ratio p VIF

B SE β

Intercept 91.031 0.912 0.000 99.819 <.0001*

Protein load (2,4.3) -1.384 0.469 -0.098 -2.949 0.0319* 1.000

Wash 2 pH (4.7,4.9) -10.739 0.469 -0.758 -22.887 <.0001* 1.000

Wash 2 conductivity (17,19) -3.500 0.456 -0.280 -7.675 0.0006* 1.210

Elution conductivity(26,28) 0.594 0.469 0.042 1.265 0.2615 1.000

Protein load*Wash 2 pH 0.355 0.469 0.025 0.757 0.4834 1.000

Protein load*Wash 2 conductivity -1.900 0.469 -0.134 -4.049 0.0098* 1.000

Wash 2 pH*Wash 2 conductivity -7.378 0.469 -0.521 -15.724 <.0001* 1.000

Protein load*Elution conductivity 3.138 0.469 0.221 6.687 0.0011* 1.000

Wash 2 pH*Elution conductivity 0.423 0.469 0.030 0.900 0.4091 1.000

Wash 2 conductivity*Elution conductivity -0.148 0.469 -0.010 -0.314 0.7659 1.000

Wash 2 conductivity*Wash 2 conductivity -3.208 0.708 -0.165 -4.530 0.0062* 1.210

Wash 2 pH*Wash 2 conductivity*Protein load 0.671 0.469 0.047 1.431 0.2119 1.000

Dependent variable: step yield by Hc-ELISA (%); *statistically significant effect (p < 0.05); VIF = variable inflation factor

Table IV: Fit for step yield: Summary of regression coefficients and colinearity.




Figure 7: Prediction profler and Monte Carlo simulation based on the constructed regression models. Monte Carlo

simulation was performed based on different scenarios, this fgure illustrates an example of simulation using proposed

parameter target and treating the proposed parameter ranges as 6σ ranges. The specifcations of step yield and step

purity were set at 65% and 95%, respectively.

Prediction Profler

Simulator

Responses

Simulate to Table

Spec Limits

Defect

Response LSL USL

Rate Mean SD

Ste

p Y

ield

Pu

rity

Desi

rab

ilit

y

00

.25

2

2.5 3

3.5 4

4.7

4.7

5

4.8

4.8

5

4.9 17

17

.5 18

18

.5 19

26

26

.5 27

27

.5 28 0

0.2

5

0.5

0.7

5 1

0.7

51

91.03125

96.55756

0.682252

3.15

ProteinLoad Wash 2 pH

Multivariate

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Multivariate Multivariate Multivariate

Wash 2Conductivity

ElutionConductivity Desirability

Step Yield

Step Yield Std Dev: 1.0178

0.1555Std Dev:

Add Random Noise

Add Random Noise

0

0.0003

0.0003

90.4489

96.5314

3.15

0.72

4.8

0.04

18

0.4

27

0.4

5.0335

0.3295Purity

Purity

Step Yield 6595

.

.Purity

N Runs: 10000

All

4.818 27

110

90

70

50

97.5

96.5

95.5

94.5

[88.687,93.3755]

[96.1994,96.9157]

Term

Unstandardizedcoefficient

Standardized coefficient t ratio p VIF

B SE β

Intercept 96.558 0.139 0.000 693.099 0.000*

Protein load (2,4.3) -0.388 0.072 -0.457 -5.409 0.003* 1.000

Wash 2 pH (4.7,4.9) 0.266 0.072 0.313 3.707 0.014* 1.000

Wash 2 conductivity (17,19) 0.122 0.070 0.163 1.752 0.140 1.210

Elution conductivity (26,28) -0.077 0.072 -0.090 -1.068 0.334 1.000

Protein load*Wash 2 pH 0.069 0.072 0.081 0.964 0.380 1.000

Protein load*Wash 2 conductivity -0.014 0.072 -0.017 -0.200 0.850 1.000

Wash 2 pH*Wash 2 conductivity 0.320 0.072 0.376 4.458 0.007* 1.000

Protein load*Elution conductivity 0.069 0.072 0.081 0.964 0.380 1.000

Wash 2 pH*Elution conductivity -0.119 0.072 -0.140 -1.661 0.158 1.000

Wash 2 conductivity*Elution conductivity -0.184 0.072 -0.217 -2.573 0.050* 1.000

Wash 2 conductivity*Wash 2 conductivity -0.183 0.108 -0.157 -1.688 0.152 1.210

Wash 2 pH*Wash 2 conductivity*Protein load 0.506 0.072 0.596 7.062 0.001* 1.000

Dependent variable: step purity by reverse-phase high-performance liquid chromatography (%); *statistically significant effect (p < 0.05); VIF = variable inflation factor.

Table V: Fit for step purity: Summary of regression coefficients and collinearity.




pared to the Monte Carlo simulation results.

The process capability and failure rate, with

proposed parameter ranges and pre-defined

recovery and purity desirability, were calculated,

which helped to establish the design space of

processing conditions. The process character-

ization workflow is illustrated in Figure 5. This

article focuses on DOE screening and follow-on

DOE studies; the integration DOE study will be

reported in a future article.

DOE screening study

A two-level, eight-factor, fractional facto-

rial design was performed to screen the

main effects of protein load, loading pH and

conductivity, wash 2 pH and conductivity,

elution pH and conductivity, and flow rate.

The parameters and test ranges are summa-

rized in Table I. A total of 14 small-scale runs

were performed, which included 12 experi-

mental runs and two control (parameters set

at the center points) runs. The experiments

were considered valid, as the control runs

met the acceptance criteria on step recov-

ery and purity, based on previous baseline

studies using qualified scale-down process

model. The results were analyzed using JMP

software, which indicated that at the ranges

tested, wash 2 pH, wash 2 conductivity, and

elution conductivity had the largest effect

size and the effects were statistically sig-

nificant. Based on the results, the param-

eters were scored based on their effect size,

response importance, and control factor,

and those with high scores were selected for

a follow-on DOE study, as summarized in

Table II. The ranges of other parameters were

set at the test ranges.

Term Effect sizea Marginb (%)

Step recovery (%) Step purity (%) Step recovery (%) Step purity (%)

Protein load (2,4.3) 2.77 0.78 7.91 15.50

Wash 2 pH (4.7,4.9) 21.48 0.53 61.37 10.62

Wash 2 conductivity (17,19) 7.00 0.24 20.00 4.88

Elution conductivity (26,28) 1.19 0.15 3.39 3.06

Protein load*Wash 2 pH 0.71 0.14 2.03 2.76

Protein load*Wash 2 conductivity 3.80 0.03 10.86 0.58

Wash 2 pH*Wash 2 conductivity 14.76 0.64 42.16 12.78

Protein load*Elution conductivity 6.28 0.14 17.93 2.76

Wash 2 pH*Elution conductivity 0.85 0.24 2.41 4.76

Wash 2 conductivity*Elution conductivity 0.30 0.37 0.84 7.38

Wash 2 conductivity*Wash 2 conductivity 6.42 0.37 18.33 7.30

Wash 2 pH*Wash 2 conductivity*Protein load 1.34 1.01 3.84 20.24

a Effect size is defined as the difference made on the step recovery or purity as the parameters move across the test ranges.b Margin is defined as the % difference, over the acceptance range of the step recovery or purity, made as the parameters move across the test ranges.

Table VII: Effect size and margin of the parameters on step recovery and purity.

Process parameters

Proposed targets

Proposed parameter

ranges

Current specifcations

Results from 10,000 simulated runs

Predicted step yield and purity

Failure rate and process capability

Mean ± standard deviation

Failure rateProcess

capability index

Protein load (g/Lbed)

3.2 ≤ 4.3 Step yield:≥ 65%

Step yield: 90.50% ± 5.06%

0.24 ppm 1.68

Wash 2 pH 4.8 4.7–4.9

Wash 2 conductivity 18 17–19 Step purity:≥ 95%

Step purity: 96.52% ± 0.33%

1.57 ppm 1.55Elution conductivity 27 26–28

Table VI: Proposed process conditions and predicted results from 10,000 simulated runs.




Follow-on DOE study

A two-level, four-factor, full factorial DOE

study was performed to further characterize

the effect of protein load, wash 2 pH, wash 2

conductivity, and elution conductivity. The

test ranges for these parameters are summa-

rized in Table III. The DOE responses are step

yield (%) by ELISA and purity (%) by RP–HPLC.

The linear regression model was refined by

backward stepwise regression taking away the

model terms with statistically insignificant

effect (p-value greater than 0.05).

The refined regression model, illustrated

in Figure 6, explained 99% of the variation

of the step yield (R2 = 0.99, F (12,5) = 75.59,

p < 0.0001) and 96% of the variation of

the step purity (R2 = 0.96, F (12,5) = 11.28,

p = 0.0074). The R2 value depicted the good-

ness of fit of the model, and the adjusted

R2 was a modification of R2 that adjusted

for the number of explanatory terms in the

model. In the models for step yield and step

purity, both the R2 (0.99 and 0.96, respec-

tively) and the adjusted R2 (0.98 and 0.88,

respectively) are high, indicating good model

fitting with statistical significance (p-value of

<0.0001 and 0.0074, respectively, at α = 0.05)

(see Figure 6). The multi-collinearity was

assessed by the variable inflation factor (VIF).

In the constructed regression models, all the

terms have VIF values around 1 (see Tables

IV and V), indicating lack of over-fitting.

The characteristics of the regression model

are summarized in Tables IV and V, which

include unstandardized coefficient (B), stan-

dardized coefficient (β), t-ratio, p-value, and

VIF of the independent variables.

The regression model and available data

from actual manufacturing batches were

used in the Monte Carlo simulation. Due

to limited data from production batches,

Monte Carlo simulations were performed

under different scenarios to assess how well

the simulated model outputs meet the pre-

established acceptable response ranges. A

simulation example is illustrated in Figure 7.

The simulation was done under the follow-

ing scenario:

• The current parameter settingwas set as

target

• Theproposedparameter rangewas treated

as the 6σ range, thus, the parameter stan-

dard deviation (SD) was calculated by divid-

ing the parameter range by 6.

Based on 10,000 simulated runs, the pre-

dicted step yield was 90.50 ± 5.06%, and

the predicted step purity was 96.52 ± 0.33%,

with failure rates of 0.24 ppm and 1.57 ppm

and process capability index (Cpk) of 1.68

and 1.55, respectively (see Table VI). The

effect size and margin of the parameters, as

well as their interactions and quadratics, on

step recovery and purity are summarized

in Table VII, which shows that Wash 2 pH

and its interaction with Wash 2 conductiv-

ity have the biggest effect on step recov-

ery, while protein load and the three-level

interaction of protein load, Wash 2 pH, and

Wash 2 conductivity have the biggest effect

on step purity.

Figure 8 illustrates a contour plot to show

the effect of Wash 2 pH and conductivity on

Figure 8: Contour plot illustrating the effect of Wash 2 pH and conductivity on step yield and purity,

at the protein load of 2 g/Lbed (A), 3.2 g/Lbed (B), and 4.3 g/Lbed (C). Step yield below 65% is

shown in red, purity below 95% is shown in blue, and the proposed control range is shown in light

green.

A5

UCL 4.9

4.8

LCL 4.7

4.6

4.5

4.4

4.3

Wa

sh 2

pH

Wash 2 Conductivity

16 16.5 LCL 17 17.5 18 18.5 UCL 19

5

UCL 4.9

4.8

LCL 4.7

4.6

4.5

4.4

4.3

Wa

sh 2

pH

Wash 2 Conductivity

16 16.5 LCL 17 17.5 18 18.5 UCL 19

5

UCL 4.9

4.8

LCL 4.7

4.6

4.5

4.4

4.3

Wa

sh 2

pH

Wash 2 Conductivity

16 16.5 LCL 17 17.5 18 18.5 UCL 19

B C

Contin. on page 45



Ima

ge

: C

ou

rt

es

y o

f u

s P

ha

rm

aC

oP

eIa

l C

on

ve

nt

Ion

Heparin regulates hemo-

stasis at various points

of the coagulation cas-

cade mainly through

its interaction with antithrombin

and heparin cofactor II. Because

of these properties, heparin is a

life-saving anticoagulant drug

used in renal dialysis, cardiac sur-

gery, and treatment for deep vein

thrombosis. The drug also binds

to platelets, inhibiting platelet

function and contributing to the

hemorrhagic effects of heparin.

Bovine heparin, first approved

in 1939, was widely used in the

United States for more than 50

years (see Figure 1). Like all drugs,

heparin can cause adverse effects,

but overall, bovine heparin prod-

ucts were found to be safe and

effective during that period.

In the late 1980s, bov ine

spong i for m encepha lopathy

(BSE , or “mad cow disease”)

was reported first in the United

Kingdom and later in several

other countries, raising concerns

about the use of bovine-sourced

heparin products in humans.

Because of t hese concer ns ,

manufacturers of bovine hepa-

Diversifying the Global Heparin Supply Chain: Reintroduction of

Bovine Heparin in the United States?David Keire, Barbara

Mulloy, Christina Chase, Ali Al-Hakim, Damian

Cairatti, Elaine Gray, John Hogwood, Tina

Morris, Paulo A.S. Mourão, Monica da Luz

Carvalho Soares, and Anita Szajek

The global supply chain

for bovine and porcine heparin and

regulatory considerations are examined.

David Keire is acting laboratory chief, Branch I, Division of

Pharmaceutical Analysis, FDA; Barbara Mulloy is visiting

professor at Institute of Pharmaceutical Sciences, King’s

College London; Christina Chase is senior scientific writer

at US Pharmacopeial Convention (USP); Ali Al-Hakim

is acting director of Division of New Drug API at the FDA;

Damian Cairatti is head of Latin American Regulatory

Affairs at USP; Elaine Gray is principal scientist at

National Institute of Biological Standards and Control

(NIBSC) in the UK; John Hogwood is research scientist

at NIBSC; Tina Morris is senior VP of Science Global

Biologics at USP; Paulo Mourão is professor at Federal

University of Rio de Janeiro; Monica Da Luz Carvalho

Soares is a member of the Deliberative Council of the

Brazilian Pharmacopeia at ANVISA and a visiting fellow

at the University of Maryland Baltimore County (UMBC);

and Anita Szajek is principal scientific liaison at USP.

Global Supply Chain



AL

L F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

RS

rin products voluntarily with-

drew them from the US market

in the 1990s. Since then, hepa-

rin products approved for use in

the US and Europe have been

sourced solely from pigs, with

approximately 60% of the supply

of the drug coming from China.

Figure 2 illustrates steps involved

in manufacturing heparin from

porcine intestinal mucosa and

potent ia l impur it ies that are

inactivated and/or removed from

each manufacturing step.

Heparin is a lifesaving drug

that was safely used since the

1940s. However, in 2007, con-

taminated hepar in caused a

number of deaths in the US and

hundreds of adverse reactions

worldwide (1). The contaminated

heparin was found to contain

over-sulfated chondroitin sulfate

(OSCS). OSCS was an inexpen-

sive synthetic adulterant that

had some anticoagulant activ-

ity and was presumably added to

heparin to increase profit when

the drug was in short supply due

to a pig disease outbreak. This

“heparin cr isis” demonstrated

the vulnerability of drug sup-

plies produced from increasingly

global manufacturing chains and

highlighted the risks inherent in

reliance on one country and one

animal species as the primary

source for a crucial drug.

To mit igate these concerns

by diversifying the sources of

heparin drugs, FDA is consid-

ering reintroduction of bovine

heparin drug product to the US

market. In August 2015, the US

Pharmacopeial Convention (USP)

hosted the 6th Workshop on

the Characterization of Heparin

Products in São Paulo, Brazil,

Global Supply Chain

Figure 1: Historical development timeline of therapeutic heparin in United States.

Figure 2: Heparin manufacturing process.



with co-sponsors, the National

Institute for Biological Standards

a nd C ont r o l ( N I B S C , U K ),

Nat ional Health Survei l lance

Agency (ANVISA, Brazil), and

Sao Paulo State Pharmaceutical

M a nu f a c t u r e r s A s s o c i a t io n

(SINDUSFARMA, Braz i l). The

focus of the workshop was an

examination of the global hepa-

rin supply chain, specifically the

risks of heparin shortages, adul-

teration, and contamination.

The following is an overview

of scientific research and clini-

cal experience presented at the

workshop to generate improved

understanding of the differences

between porc ine and bov ine

heparins, the clinical implica-

t ions of reintroducing bovine

hepar in in the US, and the

broader ramifications of bovine

heparin in the US market and

worldwide.

PoRCINe VS. BoVINe HePARINSHeparin is a natural product,

extracted from animals. Just as

pork and beef are different from

each other, heparin products

made from pigs and cattle are

similar but not identical. Two ses-

sions of the USP workshop focused

on laboratory tests used to under-

stand the differences in structure

and biological activity between

bovine and porcine heparin.

The structure of heparin is

that of a linear polysaccharide

consist ing of repeating disac-

charide motifs in which uronic

acids alternate with glucosamine.

The polysaccharide chains can

vary in length and in substitu-

tion with sulfates and N-acetyl

groups. Structural analysis tech-

niques range f rom relat ively

simple, st ra ightforward spec-

troscopic and chromatographic

analyses to sophisticated appli-

cations of techniques in nuclear

magnetic resonance and mass

spectrometry.

T he biolog ica l ac t iv it y of

heparin, including its abilities

to inhibit the enzymes of blood

clot formation in v ivo and in

vitro, can be quantified by sev-

eral methods. Research suggests

that molecular weight and disac-

charide composition both play

an important role in biological

activity. High molecular weight

fractions of heparin, for exam-

ple, have a greater effect than do

lower molecular weight fractions

on anticoagulation potency.

Overall, the combination of

structural and functional infor-

mation available provides a clear

picture of heparin products from

different species. Numerous sam-

ples have been tested by hepa-

rin manufacturers and academic

and regulatory labs revealing

clear and consistent differences

in the structures (see Figure 3)

and biological activity profiles

of porcine mucosal and bovine

mucosal heparin. In addition,

the few samples of bovine lung

heparin tested were quite dis-

tinct from either mucosal sample

type.

Impor tant ly, the data pre -

sented show that bovine hepa-

rin was significantly less potent,

weight for weight, than por-

cine heparin. The relationship

between laboratory testing and

clinical experience is not straight-

Global Supply Chain

Figure 3: Part of the proton NMR spectra of (lower, red spectrum) porcine mucosal

and (upper, blue spectrum) bovine mucosal heparin. Though the two spectra are

similar, they are not exactly the same; some differences are indicated by arrows. The

NMR spectra refect the chemical structures of porcine and bovine heparin, showing

that heparin samples from the two sources are similar but not identical.

Bovine intestine

Porcine intestine

PPM 5.70 5.60 5.50 5.40 5.30 5.20 5.10 5.00 4.90 4.80 4.70

FDA is considering

reintroduction of

bovine heparin drug

product to the US

market.



Global Supply Chain

forward for such complex prod-

ucts, yet a difference in potency

could have important clinical

relevance (2). Therefore, further

investigation including clinical

research may be warranted.

BoVINe HePARIN AND SAFety There are two main safety con-

cerns associated with bovine

hepa r i n . T he f i r s t i s hepa -

r in- induced thromboc y tope -

nia (HIT), an inf requent but

potentially devastating adverse

event (3, 4). HIT is an immune

response in which the body

makes antibodies to large com-

ple xes for med be t ween t he

highly sulfated heparin chains

and platelet factor 4 (5). HIT

occurs in 0.2 –5% of pat ients

regardless of the type of hepa-

rin administered; porcine and

bovine heparin appear similar in

terms of HIT risk (6, 7). Another

common adverse event associ-

ated with heparin is bleeding,

which can be controlled through

the neutralization of heparin by

protamine sulfate (8).

The second safety concern,

specific for bovine heparin, is

the possible presence of BSE

infectious agents (9). During the

BSE epidemic in the UK, some

people consumed BSE-infected

beef. From 1999–2000, after a

long incubation period, some

of these individuals developed

variant Creutzfeldt-Jakob disease

(vCJD); 229 cases have occurred

worldwide as of May 28, 2015

(10). Since its peak in 2000,

vCJD has declined significantly

but has not been eradicated, as a

few cases are still detected every

year. No known cases of vCJD,

however, have been linked to use

of bovine heparin. In addition,

in India, Brazil, and Argentina—

where bovine heparin products

have been in continuous use—no

cows have tested positive for BSE

and no cases of vCJD have been

observed. In the US, only three

atypical BSE cases (i.e., differ-

ent from the distinct BSE strain

from the UK that causes vCJD) in

cattle have been identified (11).

Since the 1990s, much has been

learned about how BSE leads to

vCJD in humans. In addition,

methods for minimizing BSE

r isk in bovine mater ials have

advanced (12, 13). General ly,

r isks f rom t issue spongiform

encephalopathy (TSE) agents are

controlled in three steps: ani-

mal origin of species and supply

chain control, tissue harvest con-

trols, and chemical treatments

that remove infectious agents

(11). If bovine heparin is reintro-

duced, these steps will be instru-

menta l for ensur ing pat ient

safety. Because of the safety con-

cerns noted previously, bovine

heparin is likely to have its own

USP monograph and a separate

label (Physician Labelling Rule)

that differentiates bovine hepa-

rin from porcine heparin.

PoSSIBLe ReINtRoDUCtIoN oF BoVINe HePARIN INto tHe US MARKetThe only approved source of hep-

arin in most of the world is pig

intestine, but the global pig sup-

ply is limited geographically. In

addition, there is little growth

potential for porcine heparin

products to be manufactured in

other parts of the world. Thus,

FDA is concerned about potential

shortages due to pig disease or

possible geo-political instability.

After considering the available

options, FDA hosted a meeting

with its Science Board in June

2014 to discuss the possible rein-

troduction of bovine heparin in

the US. Reintroduction of bovine

heparin would no longer limit

the source to one animal spe-

cies and would extend the geo-

graphic distr ibution of source

animals. I f d isease occurs in

one animal source and/or there

is geo-political instability in a

major source country, the risk of

supply shortages could be more

readily mitigated.

T he or ig ina l US -approved

heparin drugs from the 1930s

were from a bovine source (cow

lung) and upon approval of por-

cine heparin products, both were

used interchangeably until the

1990s without major safety risks

or concerns. Notably, bovine

mucosa heparin drug product is

currently available and manu-

factured in South America (par-

ticularly Brazil and Argentina)

and India. In some countries,

bovine heparin is preferred for

rel ig ious reasons. Thus, there

have been more than 50 years of

safe and effective use of bovine

lung heparin in patients in the

US, and bovine mucosal heparin

has been used safely in South

America and India. However,

because the heparin character-

ization technology has advanced

in the inter im, the specif ica-

tions for the proposed bovine

heparin should be modernized

in a manner similar to the recent

update of the USP porcine hepa-

rin monograph.

Bovine heparin is

likely to have its own

USP monograph and

a separate label that

differentiates bovine

heparin from porcine

heparin.



BoVINe HePARIN USe woRLDwIDeHeparin derived from bovine

lungs was in general use in Europe

and the US until it gradually fell

out of use for two reasons: the

commercial advantages of the

more potent porcine product, and

concerns in the 1990s about trans-

mission of BSE to humans through

contaminated beef products.

Wo rk s ho p s p e a ke r s f r o m

Argent ina, Braz i l , and India

emphasized that bovine heparin

has been approved and used for

decades in their countries. The

Argentinian Pharmacopeia is dis-

cussing how to manage bovine

and porcine heparins, and the

Brazilian Pharmacopeia is cur-

rently devising specific mono-

graphs for bovine and porcine

mucosal heparin.

The ava i labi l it y of bov ine

and porcine heparins in Brazil

has varied considerably in recent

years. In 2008, 42% of heparin

was of bovine origin, but this was

followed by a decrease and then

total removal from the market in

2013. In Argentina, the bovine

source accounts for 70% of the

total heparin; this has remained

unchanged in recent years.

No ser ious adverse e f fec t s

have been associated with the

use of bovine heparin in these

two countries or in India. Some

excess adverse events associ-

ated with heparin in cardiovas-

cular surgery, however, were

repor ted to ANVISA in 2008

when, on short notice, bovine

heparin replaced porcine hepa-

rin for cardiovascular surgery in

Brazil. The Brazilian Society of

Cardiovascular Surgery (14) and

ANVISA (15) published warning

notes suggesting careful mon-

itoring of anticoagulant levels

when using bovine heparin.

There are no reports of clinical

trials comparing heparins from

bovine versus porcine intestine.

Therefore, although bovine hep-

arin has been used successfully

in several countries for many

years, caution may be required

when porcine heparin is replaced

with bovine hepar in without

warning.

BoVINe HePARIN USe IN tHe MANUFACtURe oF LMwHsLow molecular weight heparins

(LMWHs) are manufactured from

heparin sodium (also known as

unfractionated heparin sodium,

or UFH) using chemical or enzy-

matic depolymerization meth-

ods (16). Currently, in the US, all

forms of LMWH are made from

porcine UFH. Therefore, their

composition and properties are

known based on their biological

starting material.

The structure and composition

of bovine UFHs differ from that

of porcine; therefore, a LMWH

product made from bovine UFH

could have different properties

f rom the same product made

from porcine UFH. For example,

if enoxaparin (a LMWH) is pro-

duced from bovine heparin in the

future, this product could not be

called enoxaparin because the

structure and properties would be

different and the activity would

probably be different as well.

IMPLICAtIoNS FoR PUBLIC StANDARDSThe current USP Heparin Sodium

monograph descr ibes system

suitability and acceptance cri-

ter ia for identity, purity, and

s t r e ng t h t hat a r e de s ig ne d

strictly for porcine heparin (17,

see Table I).

The workshop presentations

showed clear differences in the

structures and biological activ-

ity profiles of porcine mucosal,

bovine lung, and bovine muco-

sa l hepar in samples, thereby

suggesting the need to have a

separate monograph for bovine

heparin sodium. The structural

d i f ferences would necessitate

separate ident i f icat ion refer-

ence standards (RS) for 1H NMR,

chromatographic identity, and

molecular weight determina-

tion tests for bovine heparin (see

Table I). Depending on the tis-

sue source(s) of bovine heparin,

there may be a need to estab-

lish separate identification RS for

bovine lung and bovine mucosal

heparins.

Based on the testing of numer-

ous samples, the anticoagulant

act iv ity of bovine hepar in is

significantly lower than that of

porcine in the laboratory. Most

of the current bovine products

gave potencies of about 100 IU/

mg and some batches were esti-

mated to be as low as 70 IU/mg.

Consider ing the monograph

specification for porcine hepa-

rin is 180 IU/mg, it is likely that

the clinicians will need to give

more bovine heparin than por-

cine heparin by weight. Many

years of safe bovine heparin use

suggest that the higher amounts

needed, as compared with por-

cine heparin, do not impact clin-

ical efficacy. Clinical issues with

bovine heparin, however, will

need to be monitored carefully

because wider clinical use could

identify unforeseen differences

Global Supply Chain

the anticoagulant

activity of bovine

heparin is significantly

lower than that

of porcine in the

laboratory.



Global Supply Chain

Table I: Pharmacopeial requirements for porcine and bovine heparin. RS is reference standard.

Monograph section Test Porcine heparin Bovine heparin

Identification 1H NMR

No unidentified signals greater than 4% of the mean of signal height of 1 and 2 are present in the following ranges: 0.10–2.00, 2.10–3.20, and 5.70–8.00 ppm. No signals greater than 200% signal height of the mean of the signal height of 1 and 2 are present in the 3.75–4.55 ppm for porcine heparin.

• New acceptance criteria need to be generated based on batch data.

Strong anion exchange high performance liquid chromatography (SAX-HPLC) as chromatographic identity

The retention time of the major peak from the Sample solution corresponds to that of the Standard solution.

• Chrom ID method needs to be qualified/validated for bovine heparin. Depending on resolution, a new method may be needed.

• New RS may be needed

Anti-factor Xa to Anti-factor IIa ratio

Acceptance criteria: 0.9–1.1

• New acceptance criteria may be needed.• New potency standard may be needed.• Potency method needs to be qualified/

validated for bovine heparin.

Molecular weight (MW) determinations

Acceptance criteria: M24000 is NMT 20%, Mw is between 15,000 Da and 19,000 Da, and the ratio of M8000–16000 to M16000–24000 is NLT 1.0.

• MW method needs to be qualified/validated for bovine heparin.

• New acceptance criteria may be needed

SodiumIt meets the requirements of the flame test for sodium.

It meets the requirements of the flame test for sodium.

Species and tissue identification

Disaccharide analysis Add for species identification Add for species and tissue identification

Assay Anti-factor IIa Potency Not less than (NLT) 180 USP Heparin Units/mg• Needs to be determined using batch data• New RS may be needed• New acceptance criteria are needed.

Other componentsNitrogen Determination, Method I <461>

1.3%–2.5% 1.3%–2.5%

Impurities Residue on Ignition <281> 28.0%–41.0% 28.0%–41.0%

Galactosamine in Total Hexosamine

Not more than (NMT) 1% NMT 1%

Nucleotidic Impurities and Protein Impurities

NMT 0.1% NMT 0.1%

Absence of OSCS References Identification Test A and B References Identification Test A and B

BSE/TSE N/A

Specific testsBacterial Endotoxins Test <85>

NMT 0.03 USP Endotoxin Unit/USP Heparin Unit

NMT 0.03 USP Endotoxin Unit/USP Heparin Unit

Loss on Drying <731> Loss of NMT 5.0% of weight Loss of NMT 5.0% of weight

pH <791> 5.0–7.5 in a solution (1:100) 5.0–7.5 in a solution (1:100)

Sterility Tests <71>Where it is labeled as sterile, it meets the requirements

Where it is labeled as sterile, it meets the requirements

Additional requirements Packaging and StoragePreserve in tight containers, and store below 40 ˚C, preferably at room temperature.

Preserve in tight containers, and store below 40 ˚C, preferably at room temperature.

LabelingLabel to indicate the tissue and the animal species from which it is derived

Label to indicate the tissue and the animal species from which it is derived

USP Reference Standards <11>

USP Heparin Sodium Identification RSUSP Oversulfated Chondroitin Sulfate RSUSP Dermatan Sulfate RSUSP Galactosamine Hydrochloride RSUSP Glucosamine Hydrochloride RSUSP Heparin Sodium for AssaysUSP Heparin Sodium Molecular Weight Calibrant RSUSP Adenosine RS

New RS needed:USP Bovine Heparin Sodium Identification RS



between porc ine and bov ine

heparin. Furthermore, bovine

heparin requires higher doses of

protamine for neutralization.

Future bovine heparin produc-

tion sites (both drug substance

and drug product) should be

under cGMP compliance. Supply

chains including farms, slaugh-

terhouses, and facilities that iso-

late, treat, store, and ship the

bovine tissue need to follow the

same steps and tests described for

porcine heparin (18). These steps

include, but are not limited to:

• Determine the species origin

to verify that the ingredient

comes only from the correct

species.

• Confirm the absence of OSCS

and ruminant material contam-

inants from another species.

• Ensurethatallheparinsuppli-

ers are audited and inspected

regularly regarding their docu-

mentation practices and com-

pliance with cGMP.

• Rejec t any lots conta ining

mucosa from another species.

CoNCLUSIoNHeparin is an essential, life-sav-

ing drug that is needed world-

wide. To avoid drug shortages,

FDA is considering reintroduc-

tion of bovine heparin, which

would diversify the supply chain

by adding a bovine source to the

currently used porcine heparin.

Sourcing heparin from two spe-

cies could greatly reduce vulner-

ability to shortages when disease

strikes one species, and could

also reduce reliance on one coun-

try as the primary source. The

risks involved in reliance on one

species from one country were

clearly illustrated by the hepa-

rin crisis of 2007–2008, when

adulterated porcine heparin from

China caused numerous deaths

and hundreds more adver se

effects (1). Currently, China is the

source for roughly 60% of crude

porcine heparin used in the US

and Europe.

Bovine heparin is currently

being used in some countries

and was used safely for more

than 50 years in the US before

manufacturers voluntarily with-

drew it from the market during

the BSE crisis in the UK. Despite

concerns about bovine products,

there are no known cases of BSE

contamination of bovine heparin.

If bovine heparin is reintroduced

in the US, methods for inactivat-

ing BSE could be applied to fur-

ther reduce any risk. The other

main safety issue with heparin

is a severe adverse effect called

HIT, but bovine heparin does not

appear to have higher rates of HIT

than does porcine heparin.

Porcine and bovine heparins

are distinctly different in terms

of their structures and biological

activities. These complex products

may behave differently in clinical

use than in laboratory testing, but

if potency does in fact differ sig-

nificantly in clinical use, this will

need careful evaluation and per-

haps dosage adjustment to avoid

giving patients too much or too

little heparin. If bovine-sourced

heparin is reintroduced, supply-

chain control will be critical, with

frequent inspections of slaughter-

houses and processing facilities for

cGMP compliance. From the data

presented at the meeting, bovine

heparin and porcine heparin are

not equivalent drugs, and there-

fore, they will require two differ-

ent compendial monographs. The

next steps are for manufacturers

to bring bovine products to the

regulatory agencies for evaluation

and possible reintroduction to the

market after a 15-year absence.

ACKNowLeDGeMeNtSThe participants in the USP 6th

Workshop on the Characterization

of Heparin Products are acknowl-

edged with gratitude.

ReFeReNCeS 1. A.W. McMahon et al.,

Pharmacoepidemiol Drug Saf., 19,

921-933 (2010).

2. A. Tovar et al., BMC Reasearch Notes

6, 230 (2013)

3. T.E. Warkentin et al., Blood 106,

3791-3796 (2005).

4. T.E. Warkentin and A. Greinacher, Ann

Thorac Surg 76, 2121-2131 (2003).

5. A. Greinacher et al., Arterioscler

Thromb Vasc Biol 26, 2386-2393

(2006).

6. J. E. Ansell et al., Chest 88, 878-882

(1985).

7. J.L. Francis et al., Ann . Thor. Surgery

75, 17-22 (2003).

8. G. Costantino et al., PLoS One 7,

e44553 (2012).

9. J.L. Harman and C.J. Silva, Journal of

the American Veterinary Medical

Association 234, 59-72 (2009).

10. EuroCJD Network, Creutzfeldt-Jakob

Disease International Surveillance

Network (2015), available at www.

eurocjd.ed.ac.uk/surveillance%20

data%201.html#vcjd-cases.

Accessed Aug. 27, 2015.

11. FDA, Bovine Spongiform

Encephalopathy (2015), www.fda.gov/

animalveterinary/

guidancecomplianceenforcement/

complianceenforcement/

bovinespongiformencephalopathy/

default.htm, accessed Aug. 27, 2015.

12. I. DeVeau et al., “Analytical

Microbiology Expert Committee. The

USP Perspective to Minimize the

Potential Risk of TSE Infectivity in

Bovine-Derived Articles Used in the

Manufacture of Medical Products.,”

Pharmacopeial Forum 30, 1911-1921

(2004).

13. D. Taylor, Comptes rendus biologies

325, 75-76 (2002).

14. W. J. Gomes and D.M. Braile. Rev

Bras Cir Cardiovasc. 24(2):3-4

(2009).

15. ANVISA, information on contaminated

Heparin (2008), http://s.anvisa.gov.

br/wps/s/r/ZVo, accessed Aug. 27,

2015.

16. H. Liu et al., Nat Prod Rep 26, 313-

321 (2009).

17. USP, USP38–NF33, First Supplement,

Heparin Sodium monograph, p.3748

(USP, Rockville, MD).

18. FDA, Heparin for Drug and Medical

Device Use: Monitoring Crude

Heparin for Quality ( 2013 ), www.

fda.gov/downloads/drugs/

guidancecompliance regulatory

information/guidances/ucm291390.

pdf Accessed Aug. 27, 2015. ◆

DISCLAIMeRThis article reflects the views of

the authors and should not be

construed to represent US FDA’s

views or policies.

Global Supply Chain



Henrik

Jo

nss

on/E

+/G

ett

y Im

ag

es

The pharmaceut ica l indus-

try faces complex issues in

its effort to meet the require-

ments of the US Drug Supply

Chain Security Act (DSCSA) (1). Pilot

programs are needed to determine the

feasibility of solutions, but the number

of pilots required will be costly, in terms

of money, time, and human resources.

Virtual pilots, which use computer

software to project or simulate physi-

cal pilots, can reduce this burden by

providing some measure of learning or

proof that a particular set of solutions

would be scalable and workable.

Traditionally, databases, spreadsheets,

dashboards, and test systems are used

to understand systems, processes, and

information and to predict the expected

outcome of a physical pilot. Simulation

software, however, is a more efficient

technique that can be used to create and

execute a virtual pilot for any process,

including implementing the DSCSA in

the pharmaceutical supply chain.

Simulation software has traditionally

been used in manufacturing environ-

ments to replicate complex processes

and provide a way to compare variations

on specific scenarios to gain insights

beyond algorithmic calculations found

in spreadsheets. Due to the increase

in computing power, simulation soft-

ware has become easier to use and is

increasingly seen in many industries.

Simulation software is used to study

processes within and between systems,

departments, companies, and indus-

tries, including flow of products, peo-

ple, cash, and information. Simulations

Piloting Track-and-Trace Implementation

Robert Celeste

Virtual pilot programs examine

scenarios that may

occur while implementing serialization

requirements for the US Drug

Supply Chain Security Act.

Robert Celeste is a founding partner of

RC Partners healthcare consultancy,

[email protected], tel. 1.215.584.7374.

Supply Chain



can also address human behavior,

time considerations, and the physical

environment.

The software has matured to the

point that it can be used during the

information collection phase of a

process study; it can provide mean-

ingful results, both at a summary

level and in the details and nuances

of a process. Simulations can be used

to create multiple scenarios to exam-

ine alternatives before large amounts

of resources are expended to plan,

develop, and execute a physical pilot.

In fact, the virtual pilot can become

the blueprint for the physical pilot.

ConSIdeRIng SPaCe and TIme ConSTRaInTSCertain trading partners have physi-

cal space or time constraints that

must be considered while imple-

menting DSCSA requirements.

Production lines, for example, vary

in terms of space available to include

serialization equipment. Simulation

can provide insight into how best to

use existing space, such as whether

to serialize on the production line or

serialize label-stock prior to the pro-

duction run. Because case and pallet

packing may be accomplished else-

where in the facility, a simulation of

the environment could help address

product and staff flow.

Likewise, wholesalers may have

time constraints in their window of

operations for picking and packing

orders to be delivered the next day.

Timing and positioning of product

and staff can be simulated to give

projections of what to expect upon

scale-up.

In addition to product and staff

flow, information flow is crucial

to meeting DSCSA requirements.

Creating, supplying, and archiving

DSCSA data can be simulated in

order to gain experience with the

latency, scalability, and feasibil-

ity of making data available from

other systems within the orga-

nization. All of these flows can be

studied in one simulation, to gain

better insight on how they inter-

act with each other and depend

on each other. For example, the

DSCSA law sets requirements for

the timing of transaction informa-

tion. A virtual pilot may be cre-

ated to examine the process flow

of information, product, and

staff, should if the product arrives

prior to the required Transaction

Information, Transaction History,

and Transaction Statement.

Another potential problem is that

during the transition to 100% seri-

alized products, companies in the

supply chain may receive some

serialized product and some non-

serialized product. To better under-

stand the transition, simulation

techniques could examine varying

numbers of products received, the

mix or percentage of serialized vs

non-serialized products on hand,

and daily variability of serialized

product and associated traceability

data. Simulations can also examine

the latency of delivery and number

of suppliers.

aSSeSSIng human faCToRS For complex systems or processes,

it’s often difficult to assess the

impact of individual tasks or

even understand these tasks and

their variation throughout the

workweek with enough clarity to

confidently declare that a pilot

and ensuing implementation will

be successful. Operations under

the various stages of DSCSA will

require supplier, customer, and

internal process changes as new

steps are added to existing pro-

cesses, such as receiving, pick-

ing, packing, and shipping. A

virtual pilot could demonstrate

how these new processes will

affect daily tasks as well as iden-

tify what changes to support sys-

tems must be made. Simulations

could also provide insight as to

the activity balance needed to

keep up with throughput of prod-

uct and information.

Simulations can

be used to create

multiple scenarios to

examine alternatives

before large amounts

of resources are

expended to plan,

develop, and execute

a physical pilot.

A study of process and infor-

mation will include interview-

ing or speaking to the people

that perform the process. It is

imperative that they are actively

engaged in the information col-

lect ion and analysis process.

There are often nuances of why

a par t icu lar step occurs and

opportunities to explore with

the process expert how newly

accessible information, equip-

ment, or adjacent processes

might improve their process.

Some of this information may be

difficult to analyze. Using a vir-

tual pilot, however, allows inclu-

sion of behaviors, preferences,

and other information points

that can d i rec t ly a f fec t the

outcome of the study or pilot.

Also, the process expert often

is drawn into the simulat ion

process because it is interactive

and easily understood. The end

result is a more robust picture of

the process under study that can

be viewed at many levels and

replicated with different scenar-

ios (e.g., five people performing

a task instead of four). Lastly,

Supply Chain



Peer-Reviewed: Quality by design—Contin. from page 35

simulations can take into con-

sideration the physical environ-

ment itself, such as the layout

of equipment and materials, size

of rooms, and distance between

process points.

InCoRPoRaTIng buSIneSS ChangeSBusinesses a re incorporat ing

DSCSA-tr iggered changes into

established operations that are

constant ly chang ing due to

changes in business pract ices

and economic conditions (e.g.,

new customers, the loss of exist-

ing customers, acquisitions and

divestitures). Virtual pilots based

on simulations are an economic

way of re-running DSCSA sce-

narios, given an ever-changing

business environment. Adding a

newly acquired warehouse or sys-

tem to the simulation, for exam-

ple, offers an opportunity to take

advantage of existing work and

test it against the new reality.

ConCluSIonUnlike a static diagram, a simulated

environment actually runs; pro-

cesses require certain input, and if

that input is not there, the process

simulation stops until that problem

is fixed. Each glitch that is fixed

in the simulated environment is a

glitch that won’t have to be fixed

in the final physical pilot, during

implementation, or in production.

Not every pilot requires an enor-

mous investment in time, staff, or

funds. A pilot may merely demon-

strate proof-of-concept or experi-

ment with connecting systems to

those of trading partners in a test

environment. A company may be

interested in piloting what to do

with all the new traceability data

made possible due to the DSCSA

law. A simulation or virtual pilot

is capable of generating a lot of

data and regenerating it based on

changes in input parameters.

Implement ing the DSC SA,

with its serialized traceability

system that is interoperable with

thousands of trading partners, is

a complex challenge. There are

many issues yet to be understood

and many details that need to be

incorporated into a company’s

plans and investment. There are

also many benefits and oppor-

tunities to be gained by early

adopters who realize the value

that a more connected and vis-

ible supply chain brings. Virtual

pilots, with simulation at their

core, a l low for better contin-

gency planning, “what if” analy-

sis, and even training of staff in

developing an awareness of new

departmental dependencies and

contributions toward the new

reality that serialization brings.

RefeRenCe 1.R. Celeste, “Global Serialization:

Could Virtual Pilots Be in Our

Future?,” www.pharmtech.

com/global-serialization-could-

virtual-pilots-be-our-future,

accessed Oct. 12, 2015. ◆

Supply Chain

the step yield and purity under

different protein loads. The pro-

posed parameter target and ranges

were labeled, and the design space

was represented as a rectangle

area in green, located within a

white area surrounded by pink

and blue. The white area repre-

sented the desired outputs of the

CEC step that meet both the step

yield and step purity require-

ments. It is clear that the Wash 2

pH and conductivity need to be

tightly controlled to achieve the

desired step performance under

different protein load conditions.

ConCluSIonThe process condit ions were

developed for a CEC intermedi-

ate purification step to separate

product-related impurities from

the drug substance. The process

was characterized by applying a

QbD approach using risk assess-

ment, DOE screening, and follow-

on DOE studies. The proposed

process condit ions would be

able to ach ieve t he des i red

performance requirements for

th is pur i f icat ion step, based

on the Monte Carlo simulation

results. It is necessary to demon-

strate the proposed process con-

ditions at production scale and

across the entire pur if icat ion

pro ce s s . T h i s work w i l l b e

reported in a future article.

RefeRenCeS 1. FDA, Pharmaceutical cGMPs for

the 21st Century—A Risk-Based

Approach: Final Report (Rockville,

MD, September 2004).

2. A.S. Rathore and R. Mhatre, Quality

by Design for Biopharmaceuticals:

Principles and Case Studies,

(John Wiley & Sons, Hoboken,

New Jersey, 2009).

3. SAS Institute Inc., “JMP Statistics

and Graphics Guide,” www.

jmp.com/support/downloads/

pdf/jmp_stat_graph_guide.

pdf, accessed Oct 5, 2015.

aCknowledgemenTThe author would like to thank

the Biopharmaceutical Process

Sciences Group for performing

t h e e x p e r i m e n t s a n d t h e

Biopharmaceut ica l Analy t ica l

Sciences Group for testing the

samples.



Contract manufacturing orga-

n i zat ions that manufac-

ture traditional and biotech

pharmaceutical products are

responsible for performing investi-

gations and reporting the results to

their clients. But are there difference

between performing investigations

for a biological product vs. a tradi-

tional pharmaceutical product? The

short answer is there is no process

difference when performing devia-

t ion invest igations for tradit ional

pharmaceutical products vs. biotech

products. The differences lie in the

complexity of the manufactur ing

processes and thus the variables that

need to be considered regarding what

could have impact on the deviation.

Chemical processes, although some-

times quite complex, often have fewer

variables even though many of the

categories are the same. For instance,

when invest igat ing an unknown

impurity in a biological process from

a simple oligopeptide fermentation

process , the considerat ions may

include fermentation conditions (e.g.,

t ime, temperature, oxygen uptake,

byproduct production), potential con-

tamination of reactants including

master cell banks and fermentation

reagents, equipment integrity, and

performance. Further considerations

for the downstream purification pro-

cess variables and the effect of a final

configuration (e.g., folding) also need

to be considered.

Performing root cause investigationsThe purpose of performing an inves-

tigation into a deviation is to deter-

mine why the deviation happened

and what its impact was on the prod-

uct quality. To determine the impact

of the deviation on the product qual-

ity, it is important to determine the

root cause of the deviation. The pro-

cess used in the industry to determine

root cause is, of course, the inves-

tigation procedure. This procedure,

regardless of whether the product you

are investigating is biotech or tradi-

tional, should require the investigator

to review various systems and deter-

mine whether they were the cause of

the deviation being investigated.

It is important to remember when

pe r for m i ng a n i nves t igat ion to

keep in mind a few general rules.

Remember, one size does not fit all.

Simple errors require simple correc-

tions while serious deviations require

broader investigations. The complex-

ity of the investigation is related not

only to the seriousness of the inves-

tigation but also to the complexity of

the factors that could influence the

outcome.

The best tool to have during any

invest igat ion i s inqu is it iveness .

Continuing to ask questions and avoid

assumptions will lead to a better out-

come. Using other tools, such and fish-

bone diagrams and determination of

most probable number (MPN), are to

be encouraged, but they do not take

the place of asking questions. In per-

forming an investigation, it is impor-

tant for the investigator to widen

their perspective and look for ways to

relate similar issues. The best way to

ensure events are not related is to try

and relate them, not the other way

around. Keep in mind that human

error is rarely a true root cause. There

is usually something in the process

that causes that human error.

And finally, always verify the facts

of the investigation. It is also impor-

tant to include a historical review.

investigating Biologics

Susan Schniepp and Andrew Harrison

The authors discuss

performing investigations of biological

products.

Susan Schniepp is distinguished fellow,

and Andrew Harrison is chief regulatory

affairs officer and general counsel, both

of regulatory compliance associates.

Quality



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This review should determine

if the deviation occurred with

this or other products, with the

specific manufacturing line or

other manufacturing lines, and/

or with the operators. The his-

torical review can help to pri-

oritize the resources and detailed

system review. In addition, some

companies make use of tools

(fishbone diagram, MPN) to help

prioritize resources. These tools,

if used correctly, can be helpful

in determining root cause, but

remember, they are just tools and

do not take the place of thinking.

The deta i led invest igat ion

should include a review of vari-

ous systems. The systems most

often reviewed are equipment

and machinery, the manufactur-

ing process, the raw materials

used in manufacturing, the speci-

fications, the environment, and

finally, the operators. This is not

to imply that these systems are

the only areas you should look

at during the investigation but

that these are the most probable

areas where you will uncover the

root cause of the deviation. Each

investigation must address the

following elements: root cause,

impact to the material or product,

the immediate correction taken,

the corrective action to prevent

re-occurrence for specific prod-

uct/operation, and the preventive

action taken to prevent re-occur-

rence for all products/operations.

Once these e lements have

been investigated, results from

the investigation must be docu-

mented. The written narrative

should clearly explain what hap-

pened, when it happened, and

who was involved or observed

what happened. The narrative

documents the solut ion and

rationale for the root cause that

was determined through the

investigation process.

successful investigationsThe key to any successful inves-

tigation is not stopping too soon

and assuming you have the solu-

tion prior to completing the inves-

tigation. Ask questions until you

can think of no more questions to

ask and be sure to document the

answers to your questions. If you

follow your investigation proce-

dure and thoroughly document

your results, you should have an

acceptable investigation regardless

of whether you are manufacturing

a traditional product or a biotech

product. ◆

Quality



Compliance Notes

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Q : We will be replacing some instruments in

our laboratory, and the data on the equip-

ment will be archived. We are looking for

a low-cost solution, such as cloud-based archives,

to accomplish this while remaining compliant.

Can you offer any recommendations?

A: Your approach to archive records that are

not required to run the daily business is

good business sense. It is also compliant

with European regulations, which state: “Data

may be archived. This data should be checked for

accessibility, readability, and integrity. If relevant

changes are to be made to the system (e.g., com-

puter equipment or programs), then the ability to

retrieve the data should be ensured and tested” (1).

The corresponding US regulations can be found

in 21 Code of Federal Regulations (CFR) Part 211.180

(c) and Part 11, which detail essential records

and retention periods. You should note that the

US requirements of 21 CFR Part 11 also apply to

archived data and records (2). In general, the US

and EU regulations for records are very similar.

What do these requirements mean for you?

Once you have transferred the data to your

archive (e.g., a cloud-based archive), you will

confirm if the data can be accessed. This exer-

cise must be repeated, as it is necessary to verify

that you can actually access the data

throughout the data lifecycle, which

is typically several years (often in the

range of 5–15 years). For this reason, it

is essential to maintain the access infor-

mation somewhere safe for several years.

Laboratory data may be in some

proprietary format depending on the

instrument on which they were gener-

ated. If this is the case, consider how the

data can be read in years to come. It is

neither practical nor feasible to maintain

software programs that would enable these data to

be read. It is highly unlikely that the software will

still run on operating systems many years from

now, nor will there be many operators who would

know how to use the software. A potential solution

may be to standardize the data, transferring it into

a generic data format.

A key issue with archived data is maintaining a

description or inventory of where the data belongs.

Just because a file is named ‘injection 01 29 Oct

2011,’ this does not tell someone years from now

that these data belong to the impurity profile for a

stability sample for product xyz in month three for

an accelerated study. How will you know what the

data are, whether they are complete, and who cre-

ated them and when? This demonstrates that there

is no benefit in ‘dumping’ data in an archive—this

must be done with the same diligence needed for

archiving a paper document.

It’s also important to consider how long you will

need to store the data and what your defined reten-

tion periods are. It is well known that paper can be

stored for hundreds of years and will still be legible.

Experience with electronic archives extends to

years, at best decades. This is where you will have

to keep in mind the longevity of the electronic

archive. Low-cost solutions may be tempting, but

is there sufficient assurance that these will still

exist at the end of your data lifecycle?

A key issue with archived data is maintaining a

description or inventory of where the data belongs.

Siegfried Schmitt is principal

consultant at PAREXEL.

Good Documentation Practice: Saving Data for the Long TermSiegfried Schmitt, principal consultant, PAREXEL, answers a reader’s question on how to ensure archive records can be retrieved.

Contin. on page 53



Troubleshooting

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The introduction of adventitious viral

agents is a recognized, inherent risk of

biologic drug production that is well

addressed by international regulatory require-

ments for analysis of upstream inputs and viral

clearance during downstream processing. The

success of viral clearance depends on the selec-

tion of appropriate methods that ensure the

removal or destruction of any viruses without

affecting the target protein. Several factors must

be considered when making this selection: the

clearance requirements, the properties of the

target protein, the mechanism(s) of the clearance

method(s), and the impact of process parameters.

Many Methods to choose froMThere are numerous methods that can be

employed to achieve viral clearance during down-

stream biopharmaceutical processing, some that

are better understood than others. “Viral clear-

ance steps can be broadly classified into three

basic categories: well-understood steps that are

known to consistently provide robust virus reduc-

tion; lesser understood steps that have been some-

what characterized but provide lower or variable

virus reductions; and steps that are not character-

ized, are not particularly effective, or are unpre-

dictable,” says Daniel Strauss, a senior scientist

with Asahi Kasei Bioprocess America.

Ideally, methods from the first category,

which include inactivation mechanisms such

as low pH, detergent, or temperature hold

steps and robust removal steps, such as virus

filtration that clear a broad range of poten-

tial contaminants, are used. “These dedicated

viral clearance steps are well established in

the industry, and as long as they are operated

within established ranges and the

product quality is not impacted,

virus reduction can usually be

assured,” Strauss notes.

Processes from the other categories are typi-

cally steps optimized for product purification and

not dedicated for viral clearance. These steps can,

according to Strauss, provide acceptable clearance

but they often require more effort to optimize and

validate while providing lower log reduction values

(LRVs). Literature data can help in evaluating and

planning effective optimization of these steps.

a Matter of balanceImplementation of viral clearance steps must

be accomplished without affecting the integ-

rity of the protein product, which can be a

challenge because physiochemical methods

can induce the aggregation, fragmentation, or

other undesired effects on some molecules. It is

essential to have an understanding of the pro-

tein and its characteristics, according to Kathy

Remington, a principal scientist in BioReliance’s

Development Services group. “Knowing the

protein’s tolerance for low pH, detergent, heat,

etc., will help to direct the selection of a viral

inactivation step. Understanding the size of the

protein and its filterability is also necessary for

selecting an appropriate virus reduction filter,”

she explains.

Selecting the Right Viral Clearance Technology Whether taking an upstream, downstream, or holistic approach, there are many factors to consider when choosing viral clearance methods.

Cynthia A. Challener, PhD is a

contributing editor to

BioPharm International.

“Understanding the size

of the protein and its

filterability is necessary for

selecting an appropriate

virus reduction filter.”–Kathy

Remington, BioReliance



troubleshooting

Nanofiltration is added to most

processes (except for viral gene

therapies) and provides excellent

clearance of small (parvovirus) to

large viruses (e.g., murine leukemia

virus [MuLV]), according to Frank

Riske, a senior consultant with


and previously a senior director with

Genzyme. “Nanofiltration has sim-

plified the method selection process

because it is almost always included

unless the product molecule is too

large to pass through a 20-nm pore.

It is a gentle, effective method that

works universally on small, large,

enveloped, non-enveloped DNA and

RNA viruses,” he observes.

A second inactivation/removal

step is also frequently included, and

the specific type is determined by

the tolerance of the target protein

for low pH (~3–4), solvent (tri-(n-

butyl) phosphate; TNBP)/detergent,

etc. In some cases, Riske notes

that this inactivation step is incor-

porated into a chromatography

method, such as in a solvent wash.

“Column chromatography will fre-

quently be effective for virus reduc-

tion, but the degree of reduction is

dependent on the resin type (mode

of action), the conditions used for

the separation, and the character-

istics of the target. Typically, anion

exchangers in either a flow-through

or bind/elute mode are effective for

separating viruses from biologic

products,” he remarks.

the iMportance of early data developMentIdeally, according to Remington, the

development of a viral clearance

strategy should be done in con-

junction with development of the

protein purification strategy using

actual viral reduction data. Because

viral clearance studies are typically

outsourced, particularly by smaller

companies, these data are often not

generated until they are needed to

support a regulatory submission.

“One common pitfall is to make

decisions about viral clearance

methods at early stages of devel-

opment without fully considering

the implications if all goes well and

the molecule advances to late-stage

development and commercial pro-

duction,” agrees Strauss.

For well-understood steps, such

as detergent or low pH inactivation

steps, Remington notes that this

approach is usually successful, but

for others, and particularly chro-

matography steps, the lack of devel-

opment clearance data may prove

problematic. Riske adds that these

problems generally arise because

the column and purification condi-

tions are chosen based on the abil-

ity to produce a purified protein

that meets specifications and are

developed to maximize purity with

reasonable recovery. Only then is a

chromatography step tested for viral

clearance. “One solution is to add

a step specifically to reduce viruses

flow-through anion exchange may

work—or the separation conditions

can be modified,” Riske says.

“It must be understood that

viruses are complex proteins that are

impacted by pH, conductivity, and

other operating parameters, just as is

the protein product. Determination

of the mechanism of virus removal/

inactivation and understanding the

impact of process parameters on

viral removal/inactivation is the best

way to ensure good clearance, but

the development of viral clearance

studies are often required to obtain

this information,” Remington says.

She adds that mapping the design

space of a process step for viral clear-

ance is the best way to optimize the

step for virus reduction and is the

approach generally used to under-

stand the clearance potential of a

step that will be included in a man-

ufacturing platform.

Lot-to-lot-variation in both prod-

uct feedstocks and consumables is

another factor that is often not con-

sidered during development because

it does not generally have an impact

when preparing a small number

of batches to supply clinical trials.

“When those processes advance to

commercial production with regular

manufacturing and carefully orches-

trated facility constraints, however,

any variation in performance from

batch to batch can disrupt produc-

tion timelines,” asserts Strauss. The

selection of robust, highly scalable

methods that are not affected by

variations in feedstocks and consum-

ables is essential for achieving reliable

and consistent processes in terms of

both their viral clearance capability

and performance in the plant is cru-

cial for avoiding these issues.

a need for defined clearance targetsHaving a broad perspective on vari-

ous regulatory requirements and

defined clearance targets is also

important when selecting viral clear-

ance methods. Regulatory require-

ments vary by country, individual

”typically, anion

exchangers in either

a flow-through or

bind/elute mode

are effective for

separating viruses

from biologic

products.”–Frank

Riske, BioProcess

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agency, the phase of development,

and the contamination risk profile

for a particular product, according

to Strauss. Target clearance values

also depend on whether certain

viruses are known to be present in

the host organism or if the process

is intended to clear a broad range of

virus types. “An inadequate under-

standing of the clearance require-

ments can result in failure to hit

the needed clearance targets and

the need for additional validation

studies or viral clearance steps.

Alternatively, resources may be

wasted achieving excessive removal

values,” he says.

iMproveMents in filtrationAdvances in filtration technology

are having the biggest impact on

viral clearance, according to indus-

try experts. The newer generation

virus filters have higher fluxes, larger

product capacities, and increased

virus removal capabilities, with

some also offering steam-in-place

(SIP) capabilities, which enable their

use in closed processes. Others do

not require prefiltration to avoid fil-

ter clogging. “These advances allow

for implementation of high capacity,

cost-effective virus filtration steps

that easily achieve required virus

reduction results,” Strauss states.

New chromatography mem-

branes also simplify the viral

clearance assessment, according

to Remington. “These disposable

membranes remove the need for

evaluation of sanitization steps and

the need to evaluate aged resins.

In addition, the risk of a potential

viral contaminant being carried

over from run to run is eliminated

with disposable technologies,” she

says. The increased availability

and understanding of membrane

adsorbers as viral clearance tools,

such as anion exchange (AEX)

membranes, allow these technolo-

gies to serve as good backup solu-

tions for virus removal that can be

dropped into many processes with

minimal development work and

without many of the concerns of

adding an additional chromatogra-

phy step, according to Strauss.

Configurations of older methods,

such ultraviolet–C (UV–C) high-

temperature short-time (HTST)

inactivation are also facilitat-

ing the implementation of these

older methods into current down-

stream biopharmaceutical processes,

according to Remington.

holistic approachUnfortunately, there is no one-

size-fits-all method that provides

complete clearance of all viruses

in all processes. The industry is,

however, developing a much bet-

ter understanding of the mecha-

nisms of virus reduction by many

of the commonly-used meth-

ods. Remington believes that this

increased understanding will facil-

itate the design of processes that

optimize viral safety.

Taking a holistic approach is the

most effective means of achiev-

ing viral control, according to

Riske. “While traditionally viral

clearance has been achieved dur-

ing the downstream processing of

biopharmaceuticals, increasingly

there is a focus on ensuring that

raw materials are free of adventi-

tious agents. This holistic approach

tackles potential contamination

by adventitious agents throughout

the entire process.” Filter steriliza-

tion of production media using

nanofilters is one possible measure

that companies can take, but Riske

notes that nanofilter prices are cur-

rently too high and their through-

put too low for practical use in

upstream processes at large scale.

Filter manufacturers are working

to address these issues, and Riske

looks forward to seeing solutions

on the market in the future.

Another important development

for the biopharmaceutical industry

has been the shift in perspective

of viral clearance. “For many years,

the industry approached viral clear-

ance as an exercise in validation

and not something that needed to

be developed, optimized, and fully

understood at a mechanistic level.

Recently, however, there have been

big improvements in available data

with many more articles in peer-

reviewed journals and an increase

in published conference proceed-

ings. These data have helped sig-

nificantly to improve method

selection, development, and valida-

tion, and they have also influenced

the stances of regulatory agencies in

ways that have eased the regulatory

process across the industry,” asserts

Strauss. He hopes that companies

will continue to be forthcoming

with their viral clearance knowl-

edge and experiences as a means to

demonstrate to the agencies and to

the public the industry’s commitment

to patient safety. ♦

“for many years, the

industry approached

viral clearance as an

exercise in validation

and not something

that needed to be

developed, optimized,

and fully understood

at a mechanistic

level.”–Daniel

Strauss, Asahi Kasei

Bioprocess America

troubleshooting



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Lancaster Laboratories supports all functional areas of bio/pharmaceutical

manufacturing, including method development, microbiology, process

validation, and quality control throughout all stages of the drug

development process. Eurofins Lancaster Labs, tel. 717.656.2300,

www.EurofinsLancasterLabs.com

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

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DBC of of TOYOPEARL AF-rProtein A HC-650F

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Ad Index Company Page

ANTITOPE LIMITED 13

BIO RAD LABORATORIES COVER TIP, 56

CHI 47

EMD MILLIPORE 7, 21

EPPENDORF NORTH AMERICA 5

EUROFINS LANCASTER LABORATORIES 51

GE HEALTHCARE LIFE SCIENCES 17

TOSOH BIOSCIENCE 55

VETTER PHARMA-FERTIGUNG GMBH 11

WATERS CORP 2

Compliance Notes—Contin. from page 48

Your data are valuable, so you will want to be able to

access and retrieve them as you please. Therefore, you

will need to know where your data are located. Cloud-

based archives may be perfectly appropriate for your

purpose, but only if you have appropriate controls over

their location and access to your data. If the data were

in a cloud with lax controls, who might have access to

your data? What happens if your archive (e.g., cloud)

changes ownership?

By all means, explore available archiving options,

including the cloud, but it is crucial to ensure your

data doesn’t become another example of what is often

widely labeled as the ‘digital landfill.’

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, Annex 11:

Computerised Systems, http://ec.europa.eu/health/files/

eudralex/vol-4/annex11_01-2011_en.pdf, accessed July 1,

2015.

2. FDA, Guidance for Industry Part 11, Electronic Records;

Electronic Signatures—Scope and Application (FDA, August

2003), www.fda.gov/downloads/RegulatoryInformation/

Guidances/ucm125125.pdf, accessed August 27, 2015. ◆



BIOLOGICS NEWS PIPELINE

IN THE PIPELINE

DTU Announces the Creation of a

Massive Protein Characterization Database

In an effort to minimize the risk associated with pro-

tein aggregation of aqueous biologics, the Technical

University of Denmark (DTU) announced it will

launch a public database containing information

about the properties and behaviors of proteins in

large-molecule pharmaceutical formulations. The proj-

ect, which will be run by PIPPI (Protein-excipient

Interactions and Protein-Protein Interactions in for-

mulation) and DTU, will be funded by a $30-mil-

lion stipend from Horizon 2020, the European

Commission’s EU Framework Programme for Research

and Innovation that is meant to improve Europe’s

competitiveness in pharmaceutical science. The proj-

ect will be rolled out over the next four years, and

DTU plans to hire 15 new PhD-level positions begin-

ning in 2016 to help support the project.

Information on the formulation of biologics, excipi-

ents, protein stability, and the activity of a product in

solution will be included in the database. The compre-

hensive protein library will include characterization

information on protein size, charge, hydrophobicity,

and a protein’s ability to interact with surrounding

substances.

The project’s current partners include the University

of Manchester, the Ludwig-Maximilian University

of Munich, Lund University, Novozymes A/S, Wyatt

Technology Europe GmbH, Medlmmune Ltd, the

University of Copenhagen, MAXIV Laboratory, and

Nano Temper Technologies GmbH.

University of Maryland Grants License

for Novel Antibody Platform to Glycocept

A new approach to antibody engineering believed to

increase the efficacy of therapeutic antibodies—origi-

nating from researchers at the University of Maryland,

Baltimore (UMB)—will be licensed to a small, up-and-

coming pharmaceutical company called Glycocept,

according to a release from the university. Glycocept’s

president and CEO, Ronald P. Dudek, recently served

as vice-president of commercial strategy of Juno

Therapeutics, a company which has drummed up

an impressive amount of industry attention for its

advances in chimeric antigen receptor T-cell (CAR-T or

CART) technologies.

Rather than working to increase the binding affin-

ity of an antibody to a cellular receptor that stimu-

lates cancer-cell apoptosis—a mechanism by which

many antibodies work—the novel antibody technol-

ogy licensed to Glycocept alters an antibody’s glycosyl-

ation sites so that the antibody is less likely to bind to

cellular receptors that inhibit immune response (spe-

cifically, the binding ability of an Fc receptor called

FcgR2b).

Glycocept will use UMB’s novel platform, dubbed

HyGly, to help outside biopharmaceutical companies

produce novel biotherapeutics and/or develop more

effective versions of existing antibody-based drugs

(i.e., biobetters) that are nearing patent expiration. The

company will also harness the technology for the pro-

duction of its own novel biologics in house.

USP Submits Comments to FDA

on Biologics Naming Guidance

The United States Pharmacopeial Convention (USP)

has submitted its comments to FDA regarding

Draft Guidance, Nonproprietary Naming of Biological

Products: Guidance for Industry, which details FDA’s

biologic naming proposal. FDA has proposed that

al l biological products have a nonproprietary

name that includes a manufacturer-specific FDA-

designated suffix.

“We understand the naming approach for biolog-

ics in the Draft Guidance reflects FDA’s interest in

preventing inadvertent substitution of and facili-

tating pharmacovigilance for biological products,”

said Jaap Venema, PhD, executive vice-president and

chief science officer of USP, in a press release. “At

the same time, USP believes it is critically impor-

tant to maintain a uniform and scientifically-based

approach that does not create unintended risks for

patients and practitioners, and encourages FDA to

consider alternative solutions to reach its goals.”

In their comments, USP states, “the existing sci-

entifically-based nonproprietary naming system for

biologics and other drugs has served patients and

practitioners well for over a century. The naming

approach proposed in the Draft Guidance represents

a departure from well-established scientific nam-

ing principles and could have unintended negative

consequences; and while USP shares FDA’s goal of

improving safe medication use, USP encourages

FDA to consider alternative solutions to achieve this

goal.”

USP plans on commenting separately to FDA’s

Proposed Rule, “Designation of Official Names and

Proper Names for Certain Biological Products,”

which provides proposed names for six products

based on the naming convention outlined in the

draft guidance.


www.tosohbioscience.com

Tosoh Bioscience and TOYOPEARL are registered trademarks of Tosoh Corporation.

Every mAb is unique.Every mAb is unique.

Your Protein A should be as well.

TOSOH BIOSCIENCE LLC • Customer service: 866-527-3587 • Technical service: 800-366-4875, option #3

TOYOPEARL® AF-rProtein A HC-650FHigh Capacity Protein A Resin for Monoclonal Antibody Purifi cation

0

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2 3.5 5

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DB

C f

or

IgG

(g

/L)

1 g/L

5 g/L

10 g/LResin: TOYOPEARL AF-rProtein A HC-650F

Column size: 5 mm ID × 5 cm

Mobile phase: 0.02 mol/L sodium phosphate, 0.15 mol/L NaCl, pH 7.4

Residence time: 2, 3.5, 5 min

Detection: UV @ 280 nm (10% breakthrough)

Sample: human IgG @ 1, 5, 10 g/L in mobile phase


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