STERILITY ASSURANCEimages2.advanstar.com/PixelMags/biopharm/pdf/2015-11.pdf · 756-5255 or email: [email protected]. UBM Life Sciences provides certain customer contact data (such

The Science & Business of Biopharmaceuticals

INTERNATIONALINTERNATIONAL

Bio

Ph

arm

Intern

atio

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BER 2

015

Pro

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ultu

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

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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

BioProcess Technology Consultants

Anurag Rathore Biotech CMC Consultant

Faculty Member, Indian Institute of

Technology

Susan J. Schniepp Fellow

Regulatory Compliance Associates, Inc.

Tim Schofield Senior Fellow

MedImmune LLC

Paula Shadle Principal Consultant,

Shadle Consulting

Alexander F. Sito President,

BioValidation

Michiel E. Ultee Principal

Ulteemit BioConsulting

Thomas J. Vanden Boom VP, Biosimilars Pharmaceutical Sciences

Pfizer

Krish Venkat Managing Partner

Anven Research

Steven Walfish Principal Scientific Liaison

USP

Gary Walsh Professor

Department of Chemical and

Environmental Sciences and Materials

and Surface Science Institute

University of Limerick, Ireland

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

oh

m/G

ett

y Im

ag

es

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 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

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 ,

“Sterility Tests,” USP 29–NF 24

(US Pharmacopeial Convention,

Rockville, MD, 2006). ♦

Fill/Finish



Med

icalR

F.co

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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


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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.

ES700057_BP1115_A21_FP.pgs 11.04.2015 02:42 ADV blackyellowmagentacyan

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 CO2levels; 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

wh

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