BioPharmfiles.pharmtech.com/alfresco_images/pharma/2018/09/11/bfcf5030-… · BioPharm INTERNATIONAL The Science & Business of Biopharmaceuticals EDITORIAL Editorial Director Rita

BioPharmThe Science & Business of Biopharmaceuticals

INTERNATIONAL

www.biopharminternational.com

INTERNATIONAL

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December 2015

Volume 28 Number 12

MOVING UP THE

BIOPHARMA

CAREER LADDER

LYOPHILIZATION

QUANTITATIVE POST-PROCESSING

CHARACTERIZATION TECHNIQUES

FOR FREEZE-DRIED PRODUCTS

PEER-REVIEWED

VARIABLE PATHLENGTH FIBER-

OPTIC SPECTROPHOTOMETRY

FOR PROTEIN DETERMINATION

IN IMMUNOGLOBULIN

CONCENTRATES

REGULATIONS

GMP CHALLENGES FOR

ADVANCED THERAPY

MEDICINAL PRODUCTS

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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] Manager Caroline Hroncich [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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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

4 BioPharm International www.biopharminternational.com December 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�JT�TFMFDUJWFMZ�BCTUSBDUFE�PS�JOEFYFE�JO��r�Biological Sciences Database (Cambridge Scientific Abstracts)�r�Biotechnology and Bioengineering Database (Cambridge Scientific Abstracts)�r�Biotechnology Citation Index (ISI/Thomson Scientific)�r�Chemical Abstracts (CAS) rŞScience Citation Index Expanded (ISI/Thomson Scientific)�r�Web of Science (ISI/Thomson Scientific)

6 From the Editor

Biopharma employees in different market segments note subtle differences in job satisfaction. Rita Peters

8 US Regulatory Beat

FDA confirmed quality focus while Congress moved to bolster biomedical innovation. Jill Wechsler

12 European Regulatory Beat

Finalizing GMP requirements and quality standards for ATMPs in the EU is proving to be a complex task. Sean Milmo

16 Perspectives on Outsourcing

CMO industry consolidation may be frustrated by a dearth of attractive assets.Jim Miller

65 New Technology Showcase

65 Product Spotlight

65 Ad Index

66 Biologic News Pipeline

2015 EMPLOYMENT SURVEY

Moving Up the

Biopharma Career Ladder

Rita PetersLimited career and salary growth

complicate a somewhat positive

employment picture. 20

UPSTREAM PROCESSING

Combining Microbioreactors

and Advanced Statistical

Techniques to Optimize a

Platform Process for a New

Host-Cell Line

Colin Jaques and Daniela LegaUse of a subspace model is a viable

method to characterize process

space variables and optimize process

performance. 28

DOWNSTREAM PROCESSING

Bespoke Bioprocessing Resins

Randi HernandezAvitide’s Kevin Isett discusses the

company’s approach to purification resins. 38

PEER-REVIEWED

Variable Pathlength Fiber-

Optic Spectrophotometry

for Protein Determination

in Immunoglobulin

Concentrates

Alfred Weber and Heinz AnderleSoloVPE fiber-optics spectrophotometry

may be able to substitute or replace the

current Kjeldahl protein determination

procedure for 10% IgG in the future. 42

LYOPHILIZATION

Quantitative Post-Processing

Characterization Techniques

for Freeze-Dried Products

Katriona ScoffinMechanical tests complement visual

techniques for the post-processing

characterization of freeze-dried products. 51

ANALYTICAL TESTING

Rapid Mycoplasma Testing:

Meeting the Burden of Proof

Cynthia A. ChallenerExpectations are high for rapid

testing methods, but demonstration

of comparability proves challenging. 56

QUALITY TESTING

N-Glycan Composition

Profiling for Quality Testing

of Biotherapeutics

Rebecca Duke and Christopher H. TaronAdvances in glycan analysis are

enhancing biologics development

and quality control processes. 59

Volume 28 Number 12 December 2015

FEATURES

Cover: Don Bishop/Cocoon/Getty Images; Dan Ward

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From the Editor

Biopharma

employees in

different market

segments note

subtle differences

in job satisfaction.

Greener Pastures in Biologics?

The large molecule segment of the biopharmaceutical market has been

receiving more media and investment attention recently, creating a per-

ception that there may be more opportunities for skilled workers in the

biologics segment.

A session at the 2015 annual meeting of the American Association of

Pharmaceutical Scientists in October—How to Survive the Paradigm Shift of

the Pharmaceutical Industry: From Small Molecules to Biologics—featured

presentations and an audience discussion about the potential and pitfalls for

scientists to migrate from positions in the small-molecule segment of pharma-

ceutical development to positions in the biologics field. The discussion cen-

tered around the challenges of making such a switch and the positions most

suitable for a transition.

How do these perceptions translate to the job market? In the 2015 BioPharm

International employment survey (1), the editors sampled opinions about the

current job market and job satisfaction for workers in the biopharmaceutical

industry. Pharmaceutical Technology, a sister publication to BioPharm International,

conducted a similar survey (2) measuring opinions of employees developing

and manufacturing both small-molecule and large-molecule therapies. A com-

parison of professionals working in the small-molecule drug segment from the

Pharmaceutical Technology survey with those responding to the biologics-focused

BioPharm International survey, showed job and salary satisfaction between the

two audiences is similar.

Respondents from the small-molecule segment survey reported longer work

experience in the bio/pharma industry; 72% reported more than 10 years of

experience compared to 63.1% for large-molecule segment workers. The major-

ity of workers in both segments are contracted to work 40 hours per week.

More biologics-segment workers (14.8%) said they worked fewer hours in 2015

than they did two years ago, compared to the small-molecule segment (11.4%);

however, a higher percentage of workers in the biologics segment (20.3%)

feel more secure in their position than the small-molecule audience of the

Pharmaceutical Technology survey (15.8%).

On compensation, the small-molecule segment respondents were slightly

more satisfied than their counterparts in the biologics segment. Nearly 40%

of the small-molecule workers said they were paid fairly, compared to 38% on

the large-molecule segment. More than 20% of the biologics segment respon-

dents—compared to 16.5% of the small-molecule segment—said they were

paid below market value considering their level of expertise and responsibility.

In general, workers in both segments agreed that their work is valued by

their employers, they feel secure in their positions, they do not face discrimi-

nation at work, and have opportunities for advancement. The biologics seg-

ment workers, however, were slightly less positive in responses about using

their skills and training to the fullest extent and saw fewer opportunities to

engage in professional development.

Workers in the two market segments had similar opinions about changing

jobs; 20.4% of the biologics-segment workers said they would like to leave their

job, if given the opportunity, compared to 19.9% of the small-molecule work-

ers. Almost one-third of the biologics workers said they do not expect to leave

their job in the coming year compared to 37.7% of small-molecule workers.

References

1. BioPharm International 2015 Employment Survey.

2. Pharmaceutical Technology/Pharmaceutical Technology Europe 2015 Employment

Survey.

Rita Peters is the

editorial director of

BioPharm International.

ON-DEMAND WEBCAST

Originally aired December 8, 2015

Register for free at

www.biopharminternational.com/bp/iCE

EVENT OVERVIEW:

Determination of isoelectric point and charge isoforms are

critical steps in characterization for biopharmaceutical and

vaccine development. Charge variants or heterogeneity

ÏŅĵĵŅĺĬƼ� ŅÏÏƚųŸ� ±Ÿ� ±� ųåŸƚĬƋ� Ņü� ŞŅŸƋěƋų±ĺŸĬ±ƋĜŅĺ±Ĭ� ĵŅÚĜĀ-

cation including glycosylation, deamidation, sialylation and

oxidation. Changes may also occur during the manufacturing

ŞųŅÏåŸŸØ�±ýåÏƋĜĺč�ÆĜŅĬŅčĜÏ±Ĭ�±ÏƋĜƴĜƋƼ�±ĺÚ�Úųƚč�ŸƋ±ÆĜĬĜƋƼţ

In this webinar, experts present examples of method devel-

opment and transfer for charge heterogeneity analysis of

biopharmaceutical products, such as the Antibody-Drug

Conjugates (ADCs). Key parameters and considerations

will be highlighted for development of cIEF methods for

Maytansinoid ADCs using the iCE platform.

Key Learning Objectives

Q� Key considerations in cIEF method development

Q� Method optimization for ADCs

PRESENTERS:

KARAN K. SHAH, MS

Analytical and Pharmaceutical

Sciences

ImmunoGen, Inc.

Waltham, MA

MODERATOR:

RITA PETERSEditorial DirectorBioPharm International

Who Should Attend

Q� Group leads

Q� Team leads

Q� QC analysts

Q� Method Development Scientists

Q� Analytical Scientists

Q� �Reviewers

Sponsored by Presented by

cIEF Method Development

and Optimization for

Antibody-Drug Conjugates

For questions contact Sara Barschdorf at [email protected]


Regulatory Beat

Vis

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ca

/Jo

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m/G

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In the past year, the biopharmaceuti-

cal industry faced numerous challenges

on the regulatory, political, and legislative

fronts. Congress approved legislation seeking

to encourage the development of “21st Century

Cures” by incentivizing and streamlining manu-

facturer regulation. FDA revised its operations

for overseeing drug production and quality,

with an eye to reducing shortages and facilitat-

ing the development and approval of more new

therapies. Patient groups gained more visibility

as advisors on drug development and advocates

for more effective R&D programs. In recent

months, however, the uproar over escalating

drug prices raised questions about the value of

new pharmaceuticals, threatening support for

innovation policies.

MORE NEW DRUGS, BIOLOGICSThere was good news for important, life-saving

therapies coming to market, including addi-

tional cures for hepatitis C, new treatments for

seriously high cholesterol, and new therapies for

cancer and rare diseases. In 2014, FDA approved

a near record 41 new drugs and 10 biologics,

many designated as “breakthrough”

to treat serious and rare conditions.

This year appears headed for similarly

noteworthy achievements: the agency

had approved 35 new therapies as

of early November, with a number

of applications scheduled for final

assessment by year-end.

FDA’s breakthrough drugs program

continued to prove valuable, as the

agency approved 10 new therapeutics

in that category. That rate is likely

to continue, as FDA received nearly

100 sponsor requests for the break-

through designation, as of October

2015, and had granted 25. While

there’s strong support for the program, its con-

tinued growth may require more resources to

support the additional meetings and advisories

FDA provides manufacturers of promising treat-

ments. An important milestone in 2015 was

FDA’s approval of the first biosimilar therapy,

Zarxio from Sandoz. This unofficially launched

biosimilars in the United States, which have

been anxiously awaited by patients and payers

eager for access to less costly biotech therapies

since authorized by Congress in 2010. FDA has

invested considerable resources in developing

guidance for manufacturers on how best to

analyze and document comparability of new

biosimilars to reference products, a process

that relies on advanced assessment of product

structure and production methods to ensure

comparable quality, safety, efficacy, purity, and

potency. FDA has received six biosimilar appli-

cations to reference products, and 57 biosimilar

products for 16 reference drugs were enrolled

in FDA’s Biosimilar Product Development pro-

gram as of mid-September; sponsors were seek-

ing preliminary advice on another 27 projects.

The biopharmaceutical industry further dem-

onstrated its R&D capabilities by responding

quickly to the need for new vaccines and treat-

ments to combat the Ebola virus. While the

National Institutes of Health and other public

health agencies played critical roles in paving

the way for animal and clinical testing of prom-

New Drugs and New Initiatives Shaped 2015FDA confirmed quality focus while Congress moved to bolster biomedical innovation.

Jill Wechsler is BioPharm

International’s Washington editor,

Chevy Chase, MD, 301.656.4634,

[email protected].

FDA’s breakthrough

drugs program

continued to

prove valuable.

December 2015 www.biopharminternational.com BioPharm International 9

Regulatory Beat

ising new therapies, accelerated

product formulation and manufac-

turing proved valuable to this and

other international efforts to pro-

duce and distribute treatments for

lethal diseases around the world.

EMPHASIS ON QUALITYFDA’s campaign to reduce drug

shortages and to improve oversight

of growing foreign production and

import of drugs and active ingre-

dients was bolstered by the official

establishment of the new Office of

Pharmaceutical Quality (OPQ) in

the Center for Drug Evaluation and

Research (CDER). The new orga-

nization aims to provide a more

coordinated and effective process

for ensuring the consistent pro-

duction of high-quality medicines,

both brands and generics. This

effort involves establishing a more

predictable and timely application

approval process, with possibly

fewer inspections of those facili-

ties able to document low risk and

adherence to GMPs. FDA also col-

laborated with industry on devising

a set of quality metrics designed to

indicate a facility’s quality status,

but that program ran into objec-

tions from manufacturers, and fur-

ther guidance is not expected for

several months. The ultimate goal

is for OPQ programs and policies

to encourage industry adoption of

modern manufacturing systems

able to detect quality problems

before they occur, and to limit rou-

tine oversight in the process.

OPQ was officially established

in January 2015 after two years of

planning for the extensive organi-

zational changes in staff and func-

tions involved. The program makes

notable changes in the review of

drug chemistry, manufacturing,

and controls (CMC) submissions

from pre-clinical and clinical test-

ing, through application review, to

postapproval changes and generic-

drug development. While the

Office of Biotechnology Products

continues to oversee innovator and

biosimilar therapies, an Office of

New Drug Products now evaluates

the quality aspects of new drugs

during development and approval.

After a year on the market, those

products shift to the Office of

Lifecycle Drug Products, which

handles postmarketing changes

and generic-drug development.

An Office of Process and Facilities

brings together oversight of manu-

facturing operations, microbiology

reviews, and preapproval inspec-

tions, in coordination with CDER’s

Office of Compliance and the FDA

field force.

These changes facilitate team

reviews by staffers from a range of

CDER functions and field offices

to coordinate evaluations, inspec-

tions, and compliance actions.

There is an emphasis on adopting

risk-based models to target over-

sight to facilities, processes, and

products where quality failures

are most likely to harm patients. A

central project management staff

oversees whether OPQ operations

meet timelines and objectives, and

a new policy office manages the

development and publication of

quality-related guidance docu-

ments and rules. Important for

all these programs is a new unit

establishing a comprehensive data

system that lists the location and

operations of all facilities around

the world producing drugs and

their ingredients for the US market.

In September 2015, CDER director

Janet Woodcock signaled that the

new super office was firmly estab-

lished by stepping down as OPQ

acting director and handing over

the reins to former Novartis Vice-

President, Mike Kopcha.

Delays in bringing new generic

drugs to market, which had created

an immense backlog in abbrevi-

ated new drug applications (ANDAs)

awaiting approval, also prompted

reorganization of CDER’s Office of

Generic Drugs (OGD).

Woodcock had formed a new

OGD super office to manage the

ANDA review process, while also

shifting to OPQ the review of

generic-drug CMCs and manu-

facturing processes. Although

generic-drug makers have com-

pla i ned about CDE R’s s low

progress in whittling down the

backlog and speeding up ANDA

approvals, the agency appeared to

be making progress on both fronts

by year’s end.

CHANGES AT THE TOPIndustry comments on FDA reg-

ulations and lobbying on legis-

lat ion to bolster biopharma

innovat ion are coming f rom

different voices following the

appointment this year of new

execut ives to lead both the

Pharmaceutical Research and

Manufacturers of America and

the Gener ic Pha r maceut ica l

Association. Even more notable

is change at the top of FDA since

the departure in March 2015 of

commissioner Margaret Hamburg.

Duke University card iolog ist

Rob Califf came to FDA early in

the year as Hamburg’s unoffi-

cial replacement, but his formal

nomination as commissioner did

not come until this fall and only

began to move forward in the

Senate last month.

FDA chief sc ient ist Steven

Ostroff has been a capable place-

holder for Califf much of the

year and has kept agency pro-

grams on track. But a confirmed

commissioner is important for

initiating any important innova-

tions in agency policies, espe-

cially at a time when Congress

is weighing major revisions in

agency programs, and negotia-

t ions are moving forward for

renewal of user fees in 2017. All

these developments wil l con-

tinue to shape pharmaceutical

and biotech operations in the

coming year. ◆

10 BioPharm International December 2015 ADVERTORIAL

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European Regulatory Beat

Vis

ion

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A consultation period on GMP guide-

l ines (1) in the European Union,

specif ical ly for advanced therapy

medicinal products (ATMPs), comprising

gene- and cell-therapy products and tissue-

eng ineered t reatments, was due to be

completed in November 2015. The guidelines,

which will be drawn up by the European

Commission, however, are taking a long time

to complete. They are a requirement of an EU

regulation on ATMPs (2) which was approved

in 2007.

The objective behind the guidelines is to

bring together in a single document GMP

standards from a variety of sources, mainly

other pieces of EU legislat ion. But even

when the first dedicated GMP guidelines for

ATMPs are finalized, probably in 2016, they

are likely to have to be constantly revised.

Compliance with GMP standards has been

a major challenge for ATMP developers in

Europe. In fact, these difficulties could be

a prime reason why, up until 2014, only

four medicines under the ATMP regulation

had been given marketing authorization

in the EU’s centralized medicines licensing

procedure. Just how GMP problems for

advanced medicines are sorted out in Europe

could be a key influence over the

future of personalized medicines

and other new pharmaceut ica l

technologies in the region.

THE CURRENT R&D STRUCTUREAt the core of the difficulties with

GMP is the R&D structure in the

advanced medicines sector. Much

of the development work on ATMPs

is carried out by academic scientists

and clinicians attached to hospitals

of universities and other research

institutes. They lack the regulatory expertise

and resources to establish and operate GMP-

compliant manufactur ing processes. In

particular, they often do not have adequate

systems in place for evaluating the quality

of starting and raw materials, which is a

vital necessity in the production of advanced,

usually biological, medicines.

Academics seeking to arrange preclinical

and cl inical tr ials for their innovations

are so concerned about the complexities of

compliance with GMP that they try to avoid

having them classif ied as ATMPs by the

regulatory authorities, according to AGORA,

an EU-funded research project on solutions

to GMP problems in the sector (3).

LACK OF HARMONIZATION IN APPLYING GMP STANDARDSThe difficulties with GMP are aggravated by

the lack of harmonization in the way GMP

standards are applied by the EU’s member

states. “Research [has] found substantial

heterogeneity in the regulatory practice

across member states, which is leading to

confusion and uncertainty and is creating

a severe barr ier to the development and

delivery of [ATMP] medicines,” said AGORA

in a prog ress repor t to the European

Commission on its project (3). “[This is]

GMP Challenges for Advanced Therapy Medicinal ProductsFinalizing GMP requirements and quality standards for the development, manufacture, and clinical testing of ATMPs in the EU is proving to be a complex task.

Compliance with GMP

standards has been a

major challenge for ATMP

developers in Europe.

Sean Milmo is a freelance

writer based in Essex, UK,

[email protected].


European Regulatory Beat

weakening the position of the

EU academics and industry to

collaborate and compete globally

in this expanding field.”

Advanced medicines may need

the adoption of a different strat-

egy to manufacturing standards

within the sector. The consulta-

tion document (1) itself does not

call for any radical changes in

GMP, but stakeholders in their

comments on the document may

take a different view.

DEFINING MANUFACTURING QUALITYSenior members of the European

Medicines Agency (EMA), which

i s r e spons ible for t he EU’s

centralized approval procedure,

have h i nte d t hat d i f f e r e nt

concepts of manufacturing qual-

ity may need to be applied to

advanced medicines. “ATMPs are

complex pharmaceuticals, for

which tradit ional approaches

may not be possible,” Pau la

Salmikangas, chair of the EMA’s

committee for advanced thera-

pies (CAT), told an European

Directorate for the Quality of

Medicines & Healthcare (EDQM)

workshop on Paving the Way

for the Future—Biologicals at

Strasbourg, France, in 2014 (4).

Because AT MPs a re in the

front line of scientific discovery,

new standards become outdated.

By the time the standards are

implemented, the products for

which they have been drawn

up have been superceded by a

new technology. “Manipulation

of cells and use of recombinant

nucleic acids may bear unknown

risks, which may not be solvable

through standardization or qual-

ity control,” Salmikangas added.

A major problem area with

advanced medicines is the qual-

ity assessment of starting and

raw mate r ia l s , wh ich of ten

comprise high-risk raw materi-

als. The availability of these in

high-quality grades was limited

because they were mainly sold

“for research use only” and were

accompanied by little product

documentation from the suppli-

ers, according to Salmikangas.

Raw-material suppliers claim that

their products are “GMP compli-

ant,” but many ATMP developers

are sceptical about the value of

such claims.

Delegates at a symposium in

April 2013 on gene and cell ther-

apy raw materials at Strasbourg,

France, joint ly organized by

EMA and EDQM, complained

about the lack of a definition

of GMP compliance. The meet-

ing also acknowledged that the

term “GMP grade” meant differ-

ent things to different groups,

according to an EDQM report on

the conference (5).

ADAPTING GMP IMPLEMENTATION BASED ON RISKSome research organizations are

call ing for a relaxing of GMP

rules at the early stages of ATMP

development, for example, in

first-in-man (FiM) studies. “The

level of appl icat ion [of GMP

requirements] should be appro-

priate to the ultimate risk asso-

ciated with their use (e.g., the

number of patients in a Phase I/

FiM study can vary and so does

the risk),” says the cell therapy

unit of the government-funded

C atapu lt U K , wh ich r u ns a

ne t work o f l a t e - s t age R& D

ce nte r s i n t he cou nt r y, i n

comments (6) on a European

Commission review last year of

the ATMP regulation (7).

This view is consistent with

a growing support in the R&D

sector in Europe for a phased

approach to the application of

GMP standards, as detailed by

the Parenteral Drug Association

( PDA) i n a 2 012 te c h n ic a l

report (8). This proposed that

standards should become more

str ingent as new drugs move

from the discovery stage through

to Phase III clinical trials and

market launch. It is also in line

with the emphasis placed by the

European Commission’s consul-

tative document on the need for

a risk-based approach with the

application of GMP for ATMPs.

The document (1) is mainly

focused on conditions of GMP

c ompl i a nc e t h at wou ld b e

applied in the marketing autho-

rization of advanced medicines,

as well as those required in the

manufacture of ATMPs for clini-

cal trials. It, however, acknowl-

edges that with investigational or

experimental ATMPs, which are

often developed in an academic

or hospita l set t ing operat ing

under different quality systems

to those for conventional drugs,

“addit ional f lex ibi l it y is war-

ranted, in particular for early

phases of clinical trials” (1). The

document also says that under a

risk-based approach, the ATMP

manufacturer has a responsibil-

ity “to put in place additional

measures—beyond those sug-

gested in the [EU’s] GMP guide-

Some research

organizations are

calling for a

relaxing of GMP rules

at the early stages of

ATMP development,

for example, in first-

in-man studies.


Regulatory BeatEuropean Regulatory Beat

l ines—if that is necessary to

address the specific risk of the

product” (1).

THE AGORA PROJECTThe two-year AGOR A project

has been test ing schemes for

g iv ing personnel in academic

R&D facil it ies more expert ise

in basic GMP standards. “[Our

ow n data] ha s cons i s tent ly

shown that GMP manufacturing

knowledge remains a barrier for

academic t r ia l l ists a iming to

per form the f u l l cyc le f rom

prec l in ica l invest igat ions to

early and subsequent late-stage

clinical tr ials,” it says (3). As

a result, it has been designing

training course packages aimed

at specific target groups, such as

cell biology scientists who know

about ATMPs but need to learn

GMP skills.

The project, whose par t ic i-

pants a re ma in ly academics

f rom t he Un ite d K i ngdom,

German, and Dutch universi-

ties, has attempted to extend the

scope of its exploratory train-

ing courses by working together

with other researchers in bio-

log ica ls and w ith indust r ia l

partners with GMP experience.

It has had a close co-operation

with Pharmacell BV, Maastricht,

Netherland, a contract manufac-

turer of cell therapy and regen-

erative medicine products, which

has two GMP plants, one of

which was first certified in 2006.

The AGORA team has spoken

out against proposals to reduce

the produc t qua l it y requ i re -

ments for ATMPs in academic

development on the grounds this

would lead to the “creation of

a two-tier ATMP development

process, which would substan-

tially undermine the delivery

of sa fe and ef fec t ive AT MPs

across the EU.” A major issue to

be sorted out in the discussions

fol lowing the issu ing of the

consultative document is how

the f lexibility proposed by the

document in the application of

GMP standards in ATMP manu-

facturing can be made consistent

with the maintenance of existing

GMP rules.

National regulatory agencies,

which are responsible for the

enforcement of GMP standards

in clinical trials of ATMPs, tend

to apply them in ways they think

f it for indiv idua l medic ines.

“Concer ns about GMP [w ith

ATMPs] are handled on a case-

by-case basis,” says a spokes-

person for the Medicines and

Healthcare products Regulatory

Agenc y ( M H R A) of t he U K ,

which is a leading European

country in advanced medicines

development. In the many years

in which the agency had been

in discussions with ATMP devel-

opers, it s message had been

that GMP is about applying “an

appropriate quality standard” for

consistent manufacture in the

“wide range of situations under

which ATMPs are developed,”

the spokesman told BioPharm

International.

Clearly, a great deal of adapt-

abi l ity is required with GMP

in the early-stage development

of ATMPs. Just how this can

be balanced against the need

for greater consistency in the

implementation of GMP with

advanced medicines in the later

c l inica l t r ia l phases and the

post-marketing period could be

a matter of debate for some time.

REFERENCES 1. European Commission,

Consultative Document: Good

Manufacturing Practice for

Advanced Therapy Products

(Brussels, July 2015).

2. European Union, Regulation (EC) No

1394/2007 on Advanced Therapy

Medicinal Products (Brussels,

November 2007).

3. AGORA, Progress Report: Advanced

Therapy Medicinal Product Good

Manufacturing Practice, Open

Access Research Alliance (London,

2015).

4. P. Salmikangas, “Advanced

Therapies and the RCG Working

Party—Preliminary Aspects,”

presentation at workshop on 50

Years of EDQM Leadership in the

Quality of Medicines (Strasbourg,

October 2014).

5. EDQM, Symposium Report: Raw

Materials for the Production of Cell-

based and Gene Therapy Products

(Strasbourg, 2013).

6. Cell Therapy Catapult UK, EC

Review of the ATMP Regulation—

Cell Therapy Catapult Responses

(London, 2014).

7. European Commission, Report to

European Parliament and the

Council: Regulation (EC) No.

1394/2007 on Advanced Therapy

Medicinal Products, COM 2014/188

(Brussels, March 2014).

8. Parenteral Drug Association (PDA),

Technical Report No. 56:

Application of Phase-Appropriate

Quality Systems and CGMP for the

Development of Therapeutic Protein

Drug Substance (Bethesda, US,

2012). ◆

The AGORA project

has been testing

schemes for giving

personnel in academic

R&D facilities more

expertise in basic

GMP standards.

PHARMACEUTICAL Q� HEALTH SCIENCES Q� FOOD Q� ENVIRONMENTAL Q� 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.


Perspectives on Outsourcing

Do

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Consolidation is an obsession for con-

tact manufacturing organization (CMO)

executives, who worry about the com-

petitive challenges posed by larger players; and

for sourcing executives, who worry about the

implications for supply-chain security and rela-

tive pricing power. But will the CMO industry

consolidate, and at what pace?

Consolidation is the process by which a

shrinking number of companies control an

increasing share of the available revenues in

a given industry. It is a product of industry

maturity and typically indicates a period where

organic growth is slowing and where factors

like economies of scale or brand recognition

play an increasing role in competitive success.

Industry consolidation, in part, occurs organ-

ically as a result of better-run firms growing

faster than the industry and poorly run firms

going out of business. But the main mecha-

nism for industry consolidation is the acquisi-

tion of one industry participant by another.

Practically overnight, acquiring companies gain

market share, capacity, economies of scale, tech-

nical capabilities, and client relationships that

would otherwise take years to build

organically. In the current macro-

economic environment, where the

cost of capital is historically low,

mergers and acquisitions are espe-

cially attractive.

The drug-product CMO indus-

try has undergone a significant

amount of consolidation in recent

years; only 32 of the industry’s

more than 20 0 pa r t ic ipants

account for 67% of industry rev-

enues, according to PharmSource

analysis (1). Acquisitions have

been a major driver of that con-

solidation, especially the deals

that brought together Patheon and DSM

Pharmaceutical Products, and Aenova with

Haupt.

PACE OF CONSOLIDATIONThe consolidation process, however, has slowed.

Most of the deals taking place these days are

smaller transactions by which the buyer fills

gaps in its technology rather than gaining

broad capabilities or expanding its geographic

footprint. Notable examples include Catalent’s

acquisition of Micron Technologies (micron-

izing) in November 2014 and Patheon’s acqui-

sition of Agere (solubility enhancement) in

March 2015.

The pace of consolidation has slowed not

because buyers’ appetites have been sated but

rather because there are not a lot of attractive

assets to acquire. While small acquisitions can

fill in gaps in an acquirer’s technology portfo-

lio or geographic footprint, it is the large tar-

gets that have strategic impact, and there are

a diminishing number of attractive large busi-

nesses in play. In Europe, for instance, there are

a number of drug-product CMOs with revenues

in excess of $100 million, but most of them

The drug-product

CMO industry

has undergone a

significant amount

of consolidation in

recent years.

CMO Investors Have More Money Than Places to Spend ItCMO industry consolidation may be frustrated by a dearth of attractive assets.

Jim Miller is president of PharmSource

Information Services, Inc., and publisher

of Bio/Pharmaceutical Outsourcing

Report, tel. 703.383.4903,

[email protected],

www.pharmsource.com.


Perspectives on Outsourcing

are dependent on European mar-

kets where drug prices are declin-

ing and where there is too much

manufacturing capacity, especially

for standard solid dose and liquid

products; many of those European

CMOs lack even one FDA approval.

As a result, they struggle to grow

and suffer from low profit margins.

There are several significant CMOs

in Japan but they serve a local mar-

ket that is undergoing some signifi-

cant growth challenges.

A compounding problem is that

the most attractive independent

drug-product CMOs are either

owned by families with no appar-

ent interest in selling or by not-

for-profit foundations established

by their founders to keep the busi-

ness from changing hands. That

includes companies such as Vetter,

Almac, Rottendorf, and Fareva.

CONSOLIDATION OPPORTUNITIESOne development that could add

some more attractive opportunities

to the mix is divestitures of dose-

and API manufacturing assets by

larger entities. A prime example

is BASF’s sale of its API business

to Siegfried for $300 million in

October 2015. That deal included

three manufacturing sites and a

portfolio of generic APIs, and dou-

bled Siegfried’s revenues. There are

a number of situations around the

fine chemical and dose manufac-

turing industries that could lead

to similar opportunities, which

are attractive because they involve

whole businesses and not just an

individual facility.

The dearth of acquisition tar-

gets is a challenge for investors in

drug CMOs, because the opportu-

nity to consolidate a fragmented

industry is an important invest-

ment thesis for them. Private

equity investors in particular like

to do substantial deals and are

frustrated by the lack of opportu-

nities. The small number of attrac-

tive and saleable businesses has

created a seller’s market in which

valuations have been driven up

beyond what private equity buyers

are willing to pay.

Of course that doesn’t mean that

deals won’t get done; at least one

European CMO is going through

a formal sales process and another

may be shedding some assets in

the next year. But truly game-

changing deals where one drug-

product CMO acquires another

will be rare. Where will acquisi-

tion-minded executives look to

employ their capital?

One avenue has been the con-

solidation of all bio/pharmaceuti-

cal manufacturing including drug

product, drug substance, and pack-

aging. Acquisitions of drug-product

CMOs by drug-substance CMOs

and of API manufacturers by dose

manufacturers have dominated the

industry in the past year. These

deals have combined under sin-

gle ownership businesses that are

closely related but differ consider-

ably when it comes to technology

and operations. In the near term,

these API/dose combinations will

seek to leverage synergies in areas

like business development, regula-

tory, and finance, but in the longer

term, they will seek to change the

way bio/pharmaceutical companies

manage their manufacturing and

supply-chain requirements.

Consolidation in bio/pharma-

ceutical contract manufacturing

is imperative if the industry is to

generate the returns necessary to

attract capital, fund innovation,

and get an equal seat at the table

with the major bio/pharmaceuti-

cal companies. But consolidation

requires willing sellers of qual-

ity assets as well as willing buy-

ers, and the industry may have a

shortage of the former.

REFERENCE 1. PharmSource, Dose CMOs by the

Numbers: Composition, Size, Market

Share, Profitability and Outlook

(PharmSource, August 2015). ◆

Consolidation in bio/

pharmaceutical

contract manufacturing

is imperative.

CATALENT EXPANDS BIOLOGICS CAPABILITIES

Catalent Pharma Solutions has announced the multi-site expansion of its

analytical and process development capabilities for biologics including an

expansion of bioassay and protein characterization capabilities at its Kansas City,

MO, facility and additional integrated analytical capabilities at its Madison, WI,

bio-manufacturing facility.

The investments were made in response to increased industry demand for

large-molecule analytical services, regulatory expectations around new biologics

entities, and additional characterization requirements for biosimilars, the company

reported in a Nov. 20, 2015 statement. The investment in Kansas City enables

the facility to offer dual and complementary options for kinetic and quantitative

binding assays for characterization and GMP testing.

The expansion at the Madison site will be operational by January 2016. The

company has expanded process development capability at the Madison site,

including integration of the Ambr 15 microbioreactor system into its cell line and

upstream development.



With a proven track record of providing qual-ity testing services for the largest pharma-ceutical and biopharmaceutical companies

in the world, Eurof ins Lancaster Laboratories is a global leader in bio/pharmaceutica l labora-tory services providing comprehensive, innova-tive, and timely solutions to streamline all of your CMC test ing require-ments.

A s a m e m b e r o f Eu rof in s Sc ient i f ic ’s B i oPh a r m a P r o d u c t

Testing Group—the largest network of har-monized bio/pharmaceutical GMP product testing laboratories worldwide—Eurofins Lancaster Laboratories provides comprehen-sive laboratory services to support all func-tional areas of bio/pharmaceutical production.

Facilities

With a global capacity of more than 500,000 square feet, our network of GMP labora-tories operates under the same strict qual-ity procedures, LIMS, and centralized billing system across 14 locations worldwide to make working with any of our global operations seamless. In addition to these laboratory locations, we also have teams of scientists placed at more than 40 client facili-ties throughout the US and Europe through our Professional Scientific Services (PSS) insourcing program. We also provide secure 24-hour data access from all of our laborato-ries via LabAccess.comSM.

Markets Serviced

We provide complete CMC Testing Services to support more than 800 virtual to large

Eurofins Lancaster

Laboratories, Incbio/pharmaceutical companies and CMOs, including testing of all starting material, process intermediates, drug substance, fin-ished product, and manufacturing support through our broad technical expertise in Biochemistry, Molecular & Cell Biology, Virology, Chemistry, and Microbiology.

Comprehensive Services

We offer the f lexibility to manage your testing programs more efficiently through your choice of three unique service models, including our award-winning Professional Scient i f ic Ser v ices (PSS), Fu l l Time Equivalent (FTE) or traditional fee-for-ser-vice. You can choose the best, most cost-effective service solution for your project goals. Our breadth of services include:t� Method establishment, including method

development, feasibility, optimization, cGMP qualification and validation, as well as verification of compendial methods

t� Comprehensive stability and release pro-grams for clinical and marketed products

t� Complete biochemical and chemical char-acterization and microbial identification

t� Raw material and excipient testing (USP/

NF, EP, JP)t� Production and non-production cell bank-

ing including full characterizationt� Lot release/unprocessed bulk testingt� Process/facilities validation, including

viral clearance, residual impurities testing, extractables & leachables, water testing, environmental monitoring, disinfectant efficacy, and on-site sample collection

t� Consulting/protocol writing.

EUROFINS LANCASTER

LABORATORIES, INC.

2425 New Holland PikeLancaster, PA 17601

TELEPHONE

717.656.2300

EMAIL

[email protected]

WEBSITE

www.eurofinsus.com

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Leading experts in:

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Global Services:

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Characterization

Cell Banking

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Bioassays

Professional Scientific Services


Do

n B

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ag

es; D

an W

ard

In an industry with evolving scientific

development, business ownership

transformations, and a fluctuating

investment environment, it is no

surprise that participants in the 2015

BioPharm International annual employ-

ment survey (1) shared conflicting opin-

ions about the employment market.

Participants reported greater job security,

but also a greater desire to change jobs.

They said their work was valued by their

employer but are more dissatisfied with

their salary. While respondents reported

similar opinions about the employment

market in the 2014 survey (2), some new

findings and opinions were revealed.

More than 440 biopharmaceutical

professionals from around the globe

responded to the 2015 survey, which

was fielded in September and October

2015. Nearly one-third (32.2%) of the

respondents were from innovator bio-

pharmaceutical companies; 16.8%

were from generic-drug manufacturing

companies. Representatives of contract

research and manufacturing organiza-

tions, government/regulatory organiza-

tions, academia, equipment and raw

materials suppliers, and consulting

firms, also responded.

Nearly 20 job functions were rep-

resented; quality control/assurance and

validation (11%) and research/develop-

ment/formulation (23.8%) were the top

selections, followed by process develop-

ment (8%) and lab management (6.4%).

Geographically, 55.6% of the respondents

were from the United States; 19.6% were

from Europe, 14.1% from Asia, and 5.1%

from Central and South America.

Moving Up the Biopharma Career Ladder

Rita Peters

Limited career and salary

growth complicate

a somewhat positive

employment picture.

2015 Employment Survey



continued on page 24

More secure now Less secure now No changeIncrease Decrease No change

How secure do you feel in your job compared with last year?

62.8%

50%

58.0%

5.7%

36.3%

5.9%

31.4%

■ 2015

■ 2014

60%

■ 2015

■ 2014

22.0%

Does your current salary reflect a change over

last year’s salary?

50%

$ $ $ $ $

$$ $ $

$ $ $ $ $

$ $ $ $ $

$ $ $ $ $

$ $ $ $ $

$ $ $ $ $

$$ $ $ $

Please rate your satisfaction with your current salary. ■ 2015 ■ 2014

28.8%

31.1%

50.9%

46.9%

20.3%

I am paid below market value, considering my level of expertise and responsibility.

20.5%

21.8%

I am paid within market value for my job function, but at the low end of the range, considering my level

of expertise and responsibility.

38.9%

33.5%

I am paid fairly for my level of expertise and responsibility.

38.0%

44.7%

I am paid excessively for my level of expertise and responsibility.

2.6%

0.0%



What drives you to pursue novel medicines?With high costs, declining productivity, ever-more stringent regulations and increasing scientific and engineering complexity. Why do you do it?

We know why.Lives are depending on you. At EMD Millipore, we share your drive to make a difference. For nearly 350 years, our people

have been trusted partners to all the risk-takers at work in the Life Sciences community; from research institutions and

laboratories, to manufacturing facilities around the world.

Scepter, SteriTest, EMD Millipore, Merck Millipore, and the M mark are trademarks of Merck KGaA, Darmstadt, Germany. Milli-Q and Mobius are registered trademarks of Merck KGaA, Darmstadt, Germany.

© 2012 EMD Millipore Corporation, Billerica, MA USA. All rights reserved.

Your success is our success. Every day the people of EMD Millipore focus our

passion and energy to helping customers advance

their work towards better human health.

www.emdmillipore.com

EMD Millipore: one company, two names

EMD Millipore is a division of Merck KGaA, the original pharmaceutical

and chemicals company founded in 1668 in Darmstadt Germany. For legal

reasons Merck KGaA operates in the U.S. and Canada under the name

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employees in 67 countries, is known as EMD Millipore in the U.S. and

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Our portfolio

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Why do leading pharma companies place their trust in our sterilizing filters?Because lives depend on their drug

Put the expertise gained from billions of doses and 50 years of technical

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EMD Millipore and the M logo are registered

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©2015 EMD Millipore Corporation, Billerica, MA

USA. All rights reserved.

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

Why do leading pharma companies place their trust in our sterilizing filters?Because lives depend on their drug

Put the expertise gained from billions of doses and 50 years of technical

leadership to work for you. Visit emdmillipore.com/sterile.

Merck Millipore and the M logo are registered

trademarks of Merck KGaA, Darmstadt, Germany.

©2015 EMD Millipore Corporation, Billerica, MA

USA. All rights reserved.

Merck Millipore is a business of



continued from page 21

Bio/pharma workers contemplate job and career changes.

My company provides adequate training for basic jobs skills.

Due to rounding, some percentages may not add up to 100%. Some questions allowed multiple answers.

Results based on 2015 BioPharm

International

employment survey.

● Strongly Agree

● Somewhat Agree

● Somewhat Disagree

● Strongly Disagree

● Strongly Agree

● Agree

● Disagree

My company provides advanced training for employee professional growth.

● Strongly Agree

● Agree

● Disagree

I would like to leave my job, given the opportunity.

I do not expect to leave my job in the coming year.

I would like to change careers and leave the bio/pharma industry.

If it were necessary for you to change jobs this year, how would you assess the job market?

2015 2014

It would be straightforward to find a job comparable to the one I have now.

20.5% 21.7%

It would take a while, but I would be able to find a job comparable to the one I have now.

51.6% 46.0%

It would be straightforward to find a job, but it prob-ably wouldn't be as good as the one I have now.

11.6% 15.5%

I would have to search hard and be pre-pared to take what I could get.

16.3% 16.8%

■ 2015 ■ 2014


Nearly two-thirds of the respon-

dents were over age 40, and 71%

were male. The respondents had

a range of experience in the bio-

pharma industry; 36.9% have less

than 10 years of experience, 31%

have 10–20 years, 26.1% have 20–35

years of experience, and 6.1% have

worked in the industry for more

than 35 years. More than half of the

respondents (42.5%) worked in other

industries besides bio/pharma for less

five years.

JOB SECURITY AND SATISFACTIONJob security continued an upward

trend. In 2013, 33.9% said they

felt less secure in their positions

than the previous year; in 2014,

31.1% reported feeling less secure;

in 2015, the percentage dropped

to 28.8%. A slightly smaller per-

centage of respondents (20.3%)

said they feel more secure in their

positions compared with last year,

down from 22% in 2014, but up

from 18.3% in 2013. Overall,

74.6% agreed or strongly agreed

that their job was secure.

Intellectual stimulation, challeng-

ing projects, a good work/life bal-

ance, and the company’s potential

for success were the most frequently

cited as “the main reason I come

to work.” Low pay was the greatest

factor identified for quitting a job.

Major sources of job dissatisfaction

included issues with management,

negative workplace attitudes, limited

budgets, and discrimination.

MORE HOURS ON THE JOBWhile reported workloads remain

stable or decreased slightly com-

pared with 2014, most respon-

dents are working more hours than

they are contractually obligated.

In 2015, 63.7% of the respondents

reported an increased workload,

down slightly from the reported

64.3% in 2014. Nearly 40% of

the respondents said they worked

more hours in 2015 than two years

ago, an increase from 2014, when

only one-third of the respondents

reported additional hours worked.

Business increases without staff

increases (72.5%), new technolo-

gies (32.4%), staffing cuts (31.2%),

and increased regulatory pressure

(28.7%) were the leading reasons

for increased workloads.

While more than 53.2% of the

respondents reported they are

contracted to work approximately

40 hours per week, only 24.7%

reported working 40 hours. More

than one-third (34.4%) are con-

tracted for more than 40 hours

per week; more than 69.7% of the

respondents said they work 40 or

more hours per week.

COMPENSATION SLIPS In 2015, compensation discontent

continued to increase with almost

60% reporting dissatisfaction

with their salaries; 38.9% of the

respondents said they were paid

at the low end of the salary range

for their job function, consider-

ing their expertise and responsibil-

ity. Another 20.5% said they were

paid below market value. In 2014,

55.3% said they were paid at the

low end or below market value.

Fewer pay increases may contrib-

ute to the dissatisfaction. In 2015,

58% reported a salary increase; a

drop from the 62.8% of respon-

dents reporting increases in 2014.

Nearly two-thirds reported receiv-

ing a cash bonus. Despite the

unhappiness with compensation, a

strong majority of respondents said

their work is fully valued by their

employer (35.1% strongly agree;

44.7% agree).

CAREER ADVANCEMENT?Respondents had mixed opin-

ions about the types of training

offered by employers. More than

three-quarters agreed or strongly

agreed that their company pro-

vided adequate training for basic

job skills. Nearly half, however, felt

their companies did not provide

advanced training for employee

professional growth.

Opportunities for growth were

simil iarly l imited; 41.9% did

not feel there is room for career

advancement in their present

companies; 32% did not feel there

are opportunities for professional

development. Still, 78% agreed

or strongly agreed that they were

using their skills and training to

the fullest extent.

The survey respondents were not

impressed with the expertise and

training of industry newcomers.

More than three-quarters said the

new hires were adequately trained,

but not exceptional; 16.1% said the

new hires were poorly trained.

Compared with 2014, more

respondents in 2015 (59.2%) agreed

somewhat or strongly that they

would like to leave their job, given

the opportunity, up from 51.1%

in the previous year. A majority of

respondents (65.1%) plan to stay

with their positions next year, com-

pared to 63.6% in 2014. More than

28.6% of the respondents, however,

agreed or strongly agreed that they

would like to change careers and

leave the biopharma industry.

Confidence levels of those seek-

ing new positions within the

industry in 2015 were slightly

more posit ive than the 2014

responses; 20.5% said it would be

straightforward to find a compa-

rable new job; 51.6% said it may

take a while, but they would be

able to find a comparable position.

Of the less optimistic responses,

11.6% said it would be straightfor-

ward to find a job, but it probably

would not be as good as the cur-

rent position; and almost 16.3%

anticipated a difficult search and

they would have take the position

that was available.

REFERENCES1. 2015 BioPharm International

Employment Survey.

2. 2014 BioPharm International

Employment Survey. X




Who We Are

Emergent is a fully integrated specialty phar-maceutical company seeking to protect and enhance life. It is also a recognized leader in the production of sterile biopharmaceuticals. Emergent serves as the client’s single source

of ser v ice . Emergent mainta ins compliance with regulatory, customer and corporate standards. From clinica l through commercial production, Emergent has the expe-rience, knowledgeable personnel and involved management team to ensure our partners’ prod-ucts proceed from project

initiation through manufacturing quickly with an emphasis on quality. We operate with integrity, committed to the safety of our products and the service of our customers!

Major Markets

Emergent’s Contract Development and Manufacturing capabilities support bulk drug substances and finish drug products for Phase I through Phase III clinical trials and provides commercial production for its clients. Incorporating research, full prod-uct/process development, and non-clinical/clinical skill sets, this experienced group is responsible for discovering, developing, and/or commercializing innovative therapeutic products and technologies. Emergent’s state-of-the-art single use manufacturing facility enables turnkey upstream and downstream development for microbial, mammalian, and viral cell lines (Bioreactors range from 50L to 2000L). Current Fill/Finish capacity includes vials and syringes, for both liquid and lyophi-lized products, including lyo cycle devel-opment. Emergent can accommodate vials from 3mL to 100mL and pre-fi lled syringe products (0.5–20mL). Emergent’s facilities

EMERGENT BIOSOLUTIONS

INC.

400 Professional Drive, Suite 400 Gaithersburg, MD 20879 USA

TELEPHONE

800.441.4225

FAX

301.795.1899

WEBSITE

www.emergent biosolutions.com

NUMBER OF EMPLOYEES

1400

DATE FOUNDED

1998

Emergent BioSolutions Inc.

meet the international regulations required to service clients from all over the world. Emergent has submitted a Drug Master File with the FDA as well as many other agencies and currently manufactures 20 commercial products approved for distribution in over 50 countries including the United States, Canada, Japan, Brazil, and most of Europe. Emergent’s manufacturing facilities (located in Baltimore, MD) have supported over 200 clinical drug product candidates.

Services Offered

Drug Substance Manufacturet� Clinical & Commercial Scalet� Single-Use Platform (up to 2000L)t� Process Developmentt� Upstream & Downstream

Drug Product Manufacturet� Clinical & Commercial Scalet� Vials & Syringest� Lyophilizationt� Lyo Cycle Developmentt� Terminal Sterilization

emergentcontractmanufacturing.com

Emerging Capability

Emergent BioSolutions is a fully integrated

Contract Development & Manufacturing

Organization, supporting both bulk drug

substances and sterile injectable drug

products at clinical and commercial scale.

BDSManufacture

Single-use Platform

Aseptic Fill/Finish

Vials & Syringes

Clinical & Commerical

Emergent Contract Manufacturing:

Enhancing Life in Every Single Dose.


Lere

n L

u/P

hoto

dis

c/G

ett

y Im

ag

es

Over the past decade, two

improved capabilities have

changed the face of biopro-

cess design. First, the avail-

ability of high-throughput miniature

bioreactors has made it possible to eval-

uate many more conditions. This has

enabled design-of-experiment (DoE)

approaches, such as response surface

optimization, to be used. Second, bet-

ter systems for collecting, storing, and

analyzing process data have facilitated

the application of multivariate data

analysis (MVDA) to historical biopro-

cess data. These developments have

been timely, as regulatory authorities’

requirement for better understanding

of product and process has encour-

aged a process-space approach to bio-

process development.

CHARACTERIZING HIGH-DIMENSIONAL PROCESS SPACESHistorically, bioprocess outputs were

generally characterized one factor at a

time for a moderate number of values

Combining Microbioreactors and Advanced Statistical Techniques to Optimize a Platform Process

for a New Host-Cell LineColin Jaques and Daniela Lega

Use of a subspace

model is a viable method to characterize process space

variables and optimize

process performance.

Upstream Processing

Colin Jaques is senior principal scientist,

research and technology, and Daniela

Lega is lead scientist, research and

technology; both at Lonza Biologics.


AL

L F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

RS

.

for each factor (see Figure 1). The

different levels of a factor that are

being investigated are best imag-

ined as points along a line (i.e.,

the x-axis). This line equates to a

one-dimensional process space. A

model can be fitted to the data,

and the optimal value for that fac-

tor can be predicted. That optimal

value obtained is only valid, how-

ever, when all other parameters

are equal to the values used in

the experiment.

In reality, many factors influ-

ence process performance and

these may interact with each other.

If many different variables are in

play, characterizing the process

space is necessary to evaluate per-

formance for each factor level com-

bination. For the two-dimensional

example shown in Figure 2, which

investigates pH and dissolved oxy-

gen tension (DOT), this requires 52

experimental points. As the num-

ber of factors to be investigated

increases, the number of experi-

mental points increases as a power

of the number of factors. A typical

bioprocess for a mammalian cell

line may have 170 process factors.

Using this approach, a 170-dimen-

sional space would require 3.3 x

10119 cultures for full characteriza-

tion, which is not feasible.

By using DoE, it is possible to

reduce the number of cultures nec-

essary to characterize the process

design space. By reducing the abil-

ity of the model to detect higher-

order interactions—so that main

effects, two-factor interactions, and

quadratic behavior are the only

items characterized—the number

of cultures can be reduced by many

orders of magnitude (see Table I).

Even with high-throughput min-

iature bioreactors and efficient

experimental design; however, it is

still not feasible to characterize the

effects of 170 factors.

One approach to explor ing

high-dimensional spaces is to con-

sider only a subspace of the over-

all space. An example of this in

practice is best illustrated by the

creation of maps that are used for

navigational purposes. We live in

a four-dimensional world of space

and time; however, carrying a

four-dimensional map around is

not convenient. Over small dis-

tances, the surface of the earth

approximates to a two-dimen-

sional plane in three-dimensional

space. The features of the earth

don’t change much over short

periods of time. By collapsing the

two dimensions with the least use-

ful information (i.e., height and

Upstream Processing

Figure 1: Characterization of a model one-dimensional process space spanning

a range of pH values.

5

4

pH

Tit

er

(g/L

)

3

2

1

07.1

Artificial data for illustrative purposes only

7.2 7.37.06.96.8

Figure 2: Characterization of titer at harvest for a model two-dimensional process

space spanning a range of dissolved oxygen tension (DOT) and pH values.

Artificial data for illustrative purposes only

5

Tit

re (

g/L

) 4

3

2

20

DOT (%)

pH

30

40

50

60 6.86.9

7.07.1

7.2

1

Contin. on page 32



Company Description

SGS Life Science Services is a leading con-tract service organization providing clini-cal research services, analytical development,

biologics characterization, utilities qualification, bio-safety, and quality con-trol testing. SGS provides Phase I-IV clinical trial management and services encompassing PK /PD simulation and modeling, data management, phar-macovigilance and regu-latory consultancy. SGS also offers contract labo-ratory services (detailed

below) that include analytical chemistry, microbiology, stability studies, method devel-opment, and protein analysis. SGS is the world’s leading inspection, verification, test-ing, and certification company.

Technical Services

t� Quality control testing of raw materials, APIs, and finished products

t� Monograph testing (USP, EP, BP, and JP)t� Analytical method development and

validationt� Microbiological testingt� Container testing (extractables and

leachables)t� Stability testing according to ICH guide-

lines or customer specificationst� Utilities qualification (air, gas, water &

surface)t� Preformulation and formulation

developmentt� Medical device testingt� Protein/peptide analysis and quantificationt� Glycosylation analysist� Biologics safety testing (endotoxin, virus

and mycoplasma)t� Cell-line characterizationt� Host-cell impurity testing (residual DNA)

SGS Life Science Services

t� Virus testing (cell bank and virus seeds characterization)

t� Antibody product analysist� Bioanalysis (mass spectrometry, immuno-

and cell-based assays)

Facilities

With truly global coverage and a strong local footprint in North America, SGS has facili-ties in Lincolnshire (Illinois), Fairfield (New Jersey), West Chester (Pennsylvania), Carson (California), and Mississauga (Canada), as well as a clinical trial management office in Germantown (Maryland). SGS’ laboratories operate according to high quality standards (cGMP, GLP, ISO 17025) and have been inspected by the US-FDA or local regulatory authorities.

Markets Served

SGS serves various lifescience companies including pharmaceutical, biopharmaceutical, biotechnology, and medical device manufac-turers. SGS operates a global, wholly-owned network of 18 Life Science Services labo-ratories with facilities in the US, UK, Canada, Belgium, France, Germany, Italy, Switzerland, China, India, and Singapore. The Top 20 pharmaceutical companies trust SGS as a partner for their quality control testing needs.

SGS LIFE SCIENCE SERVICES

75 Passaic AvenueFairfield, NJ 07004 US

TELEPHONE:

+1.973.244.2435

FAX:

+1.973.244.1823

EMAIL:

[email protected]

WEBSITE:

www.sgs.com/lifescience

NUMBER OF EMPLOYEES:

Life Science Services worldwide: 1600 SGS Group: 80,000

YEAR FOUNDED:

1878


time), a useful navigational map

can be made in a two-dimen-

sional subspace.

A subspace approach can be

useful for reducing the number of

cultures required to characterize a

process space, but how should an

investigator select which factors to

characterize? Historically, figuring

which factors to characterize has

relied heavily on the knowledge of

process experts. MVDA provides

tools for summarizing complex

process data, which can help in

the identification of the relevant

process factors.

EXAMPLE OF A PROCESS OPTIMIZATIONThe aforementioned approach to

process optimization can be illus-

trated with an example of the

optimization of an existing plat-

form process to better fit a new

host-cell line. When a new host-

cell line is introduced, the starting

point for the process will usually

be the existing platform process

that was originally designed for

the previous host-cell line. In this

example, the existing process gave

satisfactory performance with the

majority of cell lines derived from

the new host; however, a small

proportion of cell lines displayed

poor metabolism and poor viabil-

ity towards harvest. While it is

possible to weed the poorly per-

forming cell lines out during the

cell-line selection process, it is

desirable to have a platform pro-

cess that is as broadly applicable

as possible. To this end, a pro-

gram of process optimization

was undertaken.

Data generated in the old plat-

form process during the develop-

ment of the cell-line construction

(CLC) procedure for this host were

analyzed by the MVDA technique

ca l led pr inc ipa l components

analysis (PCA). PCA f inds an

alternative set of orthogonal axes

(principal components) through

the data. These principal compo-

nents are arranged such that the

first principal component (PC1)

captures the maximum possible

variance from the data set (equiv-

alent to the line of best fit though

the data set). The second princi-

pal component (PC2) captures the

maximum possible variance left

in the data set once the variance

associated with PC1 is removed.

In this way, most of the behav-

ior present in a high-dimensional

data set can be summarized in a

lower number of dimensions.

Multiple CLCs were performed,

with each round generating mul-

tiple cell lines. Data were available

for 15 measurements over 16 time

points for 20 cultures (19 Chinese

hamster ovary cell lines) making

the same recombinant mono-

clonal antibody (mAb) giving a

240-dimensional data set. The

first two principal components

(PC1 and PC2) accounted for

46% of the behavior in the data

set. The scores and loadings for

these PCs are plotted in Figure 3.

The PCA scores clustered accord-

ing to the different CLC rounds.

The CLC rounds each used slight

variations of the CLC method.

The ability of PCA to distinguish

between the rounds indicated that

the method was summarizing bio-

logically relevant behavior.

The loadings of the PCA model

were examined to gain insight

into the behavior of the cultures.

Loadings on PC1 were heavily

influenced by growth, osmolality,

and amino acid concentrations.

The feed system of the platform

process was linked to the viable

cell concentration. The correla-

tion observed with cell concentra-

tion osmolality and amino acid

concentrations suggested that

the feeding regime might not be

optimal for cell lines derived from

the new host. As a result, amino

acid analysis was performed on

supernatant samples from these

cultures and concentrations of

a number of amino acids were

selected for optimization.

Loadings on PC2 were heavily

influenced by culture metabolism

and viability. Loadings for lactate

concentration pCO2 and sodium

concentration were inversely cor-

related with loadings for viabil-

ity. Sodium concentration and

pCO2 are determined by lactate

metabolism, and as all three were

inversely correlated with viability,

Upstream Processing

Experimental strategy Cultures required Resolution of design

Full characterization 3.3 x 10119 All main effects, interactions, quadratic

curvature, and cubic curvature

Central composite

design

1.5 x 1051 All main effects, interactions, quadratic

curvature

I-optimal design 14716 All main effects, two-factor

interactions, and quadratic curvature

One factor at a time 850 All main effects, quadratic curvature,

and cubic curvature

Table I: Number of cultures needed to characterize a 170-dimensional process

space for different experimental strategies.

Contin. from page 29

A typical bioprocess

for a mammalian cell

line may have 170

process factors.


Figure 3: (A) Scores and (B) loadings plots from principal components analysis

(PCA) of historical cultures. Scores are colored by cell-line construction (CLC)

round.

20

(A)

(B)

0.15

Sco

res

on

PC

2 (

18%

)Sco

res

on

PC

2 (

18

%)

20

0.15

-20

-0.15

-0.15

Loadings on PC1 (32%)

-20

Scores in PC1 (32%)

CLC 1 CLC 2 CLC 3

Stro

ng

infl

ue

nce

of ce

llco

nce

ntra

tion

, osm

ola

lity

Str

on

g i

nfl

ue

nce

of

glu

tam

ine

, g

luta

ma

te

Strong influence of Viability and NH

4

Strong influence of lactate,

sodium, pCO2

0

0

0

0

Upstream Processing

lactate metabolism was selected

for further investigation. Lactate

metabolism is known to be influ-

enced by pH and by the concen-

trations of key trace elements. For

this reason, trace element analy-

sis was performed on supernatant

samples for the historical cultures,

and trace elements with subopti-

mal concentrations were selected

for optimization.

These findings from PCA were

used to guide selection of a process

subspace to characterize and opti-

mize. Eleven factors were selected:

two pH parameters, four amino

acid concentrations in the feed,

and five trace element concentra-

tions in the feed. An I-optimal

response surface with three levels

for each factor was designed. An

I-optimal design is an experimen-

tal design derived using an algo-

rithm that minimizes the average

predict ion var iance over the

design space for a given number of

experimental points. The experi-

ment was executed in 96 cultures

over two ambr miniature biore-

actor systems (TAP Biosystems).

More than 90 variants of the main

nutrient feed were prepared. More

than 50 process outputs were mea-

sured and response surface models

were built for each.

As examples of the response sur-

faces generated, the response of

viability at harvest to initial and

final pH are presented in Figures

4A and 4B. Based on an analysis of

the results, pH had the single larg-

est impact on viability at harvest

(Figure 4A) of all the factors inves-

tigated. However, by optimizing

the levels of the other nine factors

in the experiment, it was possible

to increase the viability at harvest

further and to make the viability at

harvest less sensitive to process pH

opening up a larger process design

space for further process optimiza-

tion and simplification (Figure 4B).

Contin. on page 36



HAMILTON COMPANY

4970 Energy WayReno, NV 89521

TELEPHONE

775.858.3000800.648.5950

FAX

775.856.7259

EMAIL

[email protected]

WEBSITE

www.hamiltoncompany.com

NUMBER OF EMPLOYEES:

Over 1000 employees

YEAR FOUNDED:

1953

Company Description

Hamilton Company is an industry leader in the design and manufacture of liquid handling, process

analytic sensors and systems, robotics, and automated storage solutions. For more than 60 years, Hamilton Company has been satisfy-ing customer needs by com-bining quality materials with skilled workmanship to ensure the highest level of performance. Hamilton’s life-long commitment to preci-sion and quality has earned

us global ISO 9001 Certification.Founded on the technology of analytical

Microliter™ and Gaslight® syringes, Hamilton has a broad offering of laboratory products including precision fluid measuring instruments, chromatography products, process sensors, labo-ratory electrodes, pipettes, and more.

Measurement Techniques Supported

Online Measurement of:Viable Cell Density (Capacitance)Total Cell Density (Optical)Dissolved Oxygen, Optical and PolarographicpH ORPConductivity

Process Applications

CIP, SIPCell CultureFermentationBuffer ProductionChemical SynthesisDownstream ProcessingWater TreatmentFacility Boilers, Cooling, and Wastewater Treatment

Major Product Innovations

Hamilton Company was founded on the tech-

Hamilton Company

nology of analytical syringes and has evolved with advances in scientific techniques to provide a broad offering of products.

Analog and Digital Sensors

Our electrochemical pH, ORP, Conductivity and DO sensors are reliable tools for application-oriented use and are characterized by high qual-ity and long life. Along with standard analog sensors, our unique Arc technology incorporates a digital/analog transmitter into the sensor head and also enables wireless Bluetooth communica-tion directly to cell phones, tablets, and similar devices.

Leadership in Dissolved Oxygen Measurement

Our Optical DO sensors have brought large gains in accuracy and longevity while eliminat-ing routine maintenance and polarization wait-ing times. Both trace and standard measurement ranges are offered. Polaragraphic sensors are offered in both trace and standard ranges too. All DO sensors are available with Arc technol-ogy and are offered in all sizes and mounting configurations.

Cell Density

Hamilton’s inline Cell Density sensors are avail-able for both Viable and Total Cell Density. Incyte is the viable cell density sensor based on capacitance. It measures only living cells, ignoring dead cells, debris, changes in media, and microcarriers. Online measurement allows detection of events in real time without sampling. Dencytee is the total cell density sensor that utilizes optical density measurements in the NIR range. Dencytee increases light source intensity to compensate for increasing turbidity. Thus it can measure across a much larger range than conventional OD sensors.

Smart Process ControlDissolved Oxygen, pH, and Conductivity measurements connect directly to your PLC

Robust 4-20 mA and Digital signals generated directly from the sensor

Eliminate weak signal problems and transmitter costs with direct connection to PLC

In lab pre-calibration and digital sensor management

Several monitoring options including wireless monitoring of multiple sensors

Optical and amperometric Dissolved Oxygen sensors for all applications

pH sensors with self-pressurized reference for best performance and longevity

For more information, visit www.ham-info.com/1059 or contact us toll free below.

Web: www.hamiltoncompany.com

USA: 888-525-2123To fi nd a representative in your area, please visit hamiltoncompany.com/contacts.


Due to a number of cultures

having zero values, it was not

possible to develop a robust

response surface model for lac-

tate concent rat ion. Instead,

a PCA model was developed to

study the correlation patterns

in the data. The loadings of the

PCA model indicated that lactate

concentration at harvest was not

influenced by culture pH but was

strongly influenced by trace ele-

ment concentrations and amino

acid concentrations.

The derived models were used

to perform simultaneous in-silico

optimization of multiple process

outputs. Optimization focused

on increasing viability at harvest,

reducing lactate accumulation at

harvest, and maintaining antibody

concentration at harvest. The opti-

mized process was evaluated with

two cell lines in 10-L airlift reac-

tors. Cell line 1 was known to dis-

play acceptable lactate metabolism

and culture viability at harvest.

Cell line 2 was known to display

unacceptable lactate metabolism

and culture viability at harvest.

Profiles for lactate concentration

and culture viabil ity in both

processes for both cell lines are

presented in Figures 5A and 5B.

Lactate concentration at harvest

was decreased for both cell lines,

and the onset of late-lactate accu-

mulation was delayed in the new

process. Viability at harvest was

substantially increased for both

cell lines with positive implica-

tions for the downstream process.

The availability of automated

miniature bioreactor systems has

made the execution of large exper-

iments like this straightforward.

However, it exposes new bottle-

necks in the development process.

The design and coding of such

large experiments is demand-

ing and the preparation of large

numbers of feeds and/or medium

var iants requi res innovat ive

approaches. The large number of

samples produced challenges the

throughput of existing analyti-

cal technologies. Assimilating all

of the knowledge generated can

be so overwhelming that the full

benefit of the techniques is not

extracted.

CONCLUSIONAutomated miniature bioreac-

tors and advanced stat ist ica l

techniques such as multivariate

data analysis and response sur-

face optimization now available

to the bioprocess developer are

changing the face of bioprocess

development. By applying these

tools in a single round of opti-

mization, it is possible to make

substantial improvements to a

mature plat form process and

optimize process conditions for

cell l ines derived from a new

host. However, advances in high-

throughput analytics, data pro-

cessing, and medium and feed

preparation are also warranted. x

Upstream Processing

Figure 4: Response surface model predictions of viability at harvest as a function

of initial and final pH for (A) the original process, and (B) the optimized process.

7.1

(A) (B)

7.0

6.9

6.8

7.1

7.1

7.0

Fin

al

pH

Fin

al

pH

7.0

6.9

6.96.8

Initial pH Initial pH

6.87.17.06.96.8

90 95

9080

8060

60

40

20

Figure 5: Comparison of (A) culture viability profiles and (B) lactate

concentration profiles for the original and optimized process in 10-liter

cultures.

100

(A) (B)12

10

8

6

4

2

0

Cu

ltu

re v

iab

ilit

y (

%)

La

cta

te c

on

cen

tra

tio

n (

g/L

)

90

80

70

60

50

40

30

20

10

24 48 72 96 120 144Elapsed time (h)

168 192 216 240 264 288 312 336 3600

0 24 48 72 96 120 144Elapsed time (h)

168 192 216 240 264 288 312 336 3600

Cell line 1 optimizedCell line 2 optimized

Cell line 1 optimizedCell line 2 optimized Cell line 1 optimized

Cell line 2 optimizedCell line 1 optimizedCell line 2 optimized

Cell line 1 standardCell line 2 standard

Cell line 1 standardCell line 2 standard

Contin. from page 33Lactate metabolism

is known to be

influenced by pH and

by the concentrations

of key trace elements.

ADVERTORIAL December 2015 BioPharm International 37


Company Description

The Abzena g roup, wh ich inc lude s PolyTherics and Antitope, has undergone a significant transformation following the acquisition of US-based Pacif icGMP in

September 2015. The addition of PacificGMP’s manufactur-ing and process development capabilities allows Abzena to support customers’ projects seamlessly from lead optimi-sation and selection through to GMP manufacturing for clini-cal trials.

T he compa n ie s in t he Abzena group have been devel-oping and offering their tech-nologies and services for over 10 years and their scientists have provided expertise to a wide

range of companies, including most of the top 20 biopharmaceutical companies, many large and small public and private biotech, and academic groups, across the world.

Services and Capabilities

Immunogenicity assessment

EpiScreen™ is an accurate and sensitive way to assess the potential immunogenicity of proteins and antibodies ex vivo by measuring CD4+ T cell responses, the primary driv-ers of memory-based immunogenicity. This method has been used across the industry to assess risk at the preclinical stage. Antitope also provides rapid high-throughput in silico analysis of MHC class II binding and T cell epitope location with iTOPE™ and TCED™.

Antibody humanization

and protein deimmunization

Antitope’s Composite Human Antibody™ and Composite Protein™ services are used to create biopharmaceuticals devoid of the T cell epitopes while minimizing the loss of antibody affinity or protein activity that can occur with standard protein engineering techniques.

Abzena Cell line development

High-expressing mammalian cell lines are developed for the production of specif ic partner antibodies and proteins, including biosimilars, using Composite CHO™, NS0, Sp2/0 or other cell lines. Antitope’s cell lines are suitable for commercial manufacture.

Antibody drug conjugates

PolyTherics has developed its range of novel ThioBridge™ linkers to eff iciently conju-gate drugs to antibodies to create less het-erogeneous ADCs with better stability. PolyTherics’ proprietary technology uses site-specific conjugation to naturally occurring inter-chain disulfides avoiding the need for antibody re-engineering.

Optimization of pharmacokinetics

PolyTherics’ proprietary PEGylation technol-ogies including TheraPEG™, HiPEG™ and CyPEG™ enable polyethylene glycol (PEG) or other polymers to be conjugated to specific sites to extend the in vivo half-life of thera-peutic proteins. PolyTherics’ has conjugated both linear and branched PEG and its pro-prietary low viscosity polymer, PolyPEG™ to a variety of therapeutic proteins.

Manufacturing process development

PacificGMP are experts in upstream and downstream process development. The focus is on improving yield and consistency in single use systems during the manufacturing process.

GMP manufacturing

PacificGMP provides an array of cGMP pro-duction services for recombinant proteins, antibodies, gene therapy, vaccines, and ex

vivo products for use in preclinical and clini-cal studies. PacificGMP has extensive experi-ence in the use of single single-use f lexible technology.

ABZENA

Babraham Research Campus Cambridge, CB 223AT

TELEPHONE

+1.223.903.498

EMAIL

[email protected]

WEBSITE

www.abzena.com


Ole

ksiy

Maksym

enko/G

ett

y Im

ag

es

The lessons that Kevin Isett

learned during his time at

Adimab, a therapeutic anti-

body discovery technology

company, were top of mind when

Isett embarked on the development

of a novel purification technology.

Rather than making the product first

and figuring out which markets to

target, working at Adimab gave him

the insight to understand the value

of building a technology platform to

address precise industry need. Despite

the fact that the bioprocessing indus-

try is conservative and highly regu-

lated, according to Isett, Avitide was

able to draw from community feed-

back to drive the development of its

service offerings and capabilities.

Isett says that to f ind an appro-

priate recovery process for biothera-

peutics—one that would produce a

biopharmaceutical that could meet

purity requirements for clinical and

commerc ia l produc t ion but a l so

would not compromise process yield

or development time—process engi-

neers “were forced to screen hundreds

of different resins and modalities.”

Although Protein-A resins to purify

Fc- conta ining molecules such as

monoclonal antibodies (mAbs) are

able to mainta in high y ield and

purity levels, Isett notes that there is

a hole in the market for a “Protein-A-

like” solution for other types of large-

molecule products. Thus, it became

Avitide’s goal to develop affinity res-

ins—suitable for any molecule—that

could employ high-yield, high-purity

capture steps within a reasonable pro-

cess development t imeframe. Isett

adds that the company also sees sig-

nificant value in being able to make

aff inity resins that can select for

product quality attributes.

Bespoke Bioprocessing ResinsRandi Hernandez

BioPharm

International

sat down with Kevin Isett, PhD, co-founder and CEO of Avitide, to find out why

he thinks the company’s

tailored approach to

purification resins will

change the face of bio-

pharmaceutical separation.



Av it ide adve r t i s e s t hat i t

can take a partner’s molecule

a nd c h a r a c t e r i z e t he mol -

ecule’s aff inity l igands, scale-

up ligand production, develop a

resin for the molecule, validate

the resin performance, ensure

technology transfer, and ship

the resin to the company part-

ner—all within a period of three

months. BioPharm International

spoke with Isett to learn more

about why he thinks process

tech nolog ie s a re pos it ioned

to improve therapeut ic t ime-

lines, costs, and access and how

Avitide’s technology will do for

the separation of al l types of

large molecules what Protein A

did for the downstream purifica-

tion of antibodies.

BioPharm: For which types

of biologics are there few (or no

good) options for purification?

Isett: For monoclonal anti-

bodies, Protein A is a true plat-

form. We were not interested

in developing another Protein

A-l ike resin, as that need is

technically well served by the

l i fe-science industry and well

adopted by the biopharmaceuti-

cal manufacturing community.

Outside of monoclonal anti-

bodies, there exist more than

350 vacc i ne s , t he rap eut ic s ,

and gene therapies that are in

development that do not have a

high-performing affinity puri-

fication approach similar to the

efficiencies realized through the

purification of monoclonal anti-

bodies by Protein A. Without a

true affinity-based purification

platform, the process eff icien-

c ies rea l i zed for the current

mAbs will never materialize for

other biological therapeutics and

vaccines. This will then restrict

global access for therapeutics

and vaccines, and continue to

exacerbate the high manufac-

turing costs, of which 50–80%

reside in purification operations.

BioPharm: Will your resins

be specifically useful for one or

more type of biologic (i.e., anti-

body-drug conjugates, bispecific

antibodies, etc.)?

Isett: Our goal was to develop

resins for any protein-based ther-

apeutic or vaccine. Oftentimes,

our partners ask that we solve

particularly difficult isoform sep-

aration problems. This type of

separation is quite literally not

solvable at scale with existing

lower selectivity ion exchange,

mixed mode, or hydrophobic

inte rac t ion ch romatog raphy

(HIC) resins. We have also been

asked to develop aff inity res-

ins that recognize a conserved

motif across multiple molecules,

thus enabling a platform affin-

ity purification solution for our

partners. Again, the scope of the

affinity resin and separation per-

formance is entirely driven by

our partners.

BioPharm: How can purifi-

cation be done in one or fewer

steps? If you had to estimate,

how many purification steps are

typical for an antibody product?

What about for other molecules?

Isett: Even though a resin can

produce the required purity in a

single chromatography step, regu-

lators will always insist that more

than one step be used in order to

meet viral clearance (and other)

requirements. For antibody prod-

ucts, it is typical for there to be

two bind/elute chromatography

steps along with a flow-through

or membrane-chromatography

step to meet final purity and viral

clearance specif ications. For a

non-mAb process, four steps are

the minimum seen today, with

the norm being five or six steps.

Our affinity-based platform can

create a two-step, mAb-like pro-

cess for challenging molecules.

BioPharm: How do Avitide’s

resins work, and why can your

l igands in part icular bind to

such a wide range of targets?

Isett: We have invested heav-

ily in specific technologies and

key areas of development that

allow us to deliver high-perform-

ing, molecule-specif ic aff inity

resins in three months. We adapt

our platform to discover novel

solutions to a diverse array of

molecules such as gene therapies,

therapeutic enzymes, vaccines/

virus-like particles, and bispe-

cific antibodies. Our proprietary

peptide-based affinity ligands are

tailored to find biopharmaceu-

tical molecular surface features

that are relevant to bioseparation,

are cost effective to manufacture,

and are robust enough to with-

stand the rigors of bioprocessing.

BioPharm: What is the most

challenging type of molecule for

which to f ind a good binding

ligand?

Isett: Frankly, we have yet

to fail to find highly selective


Many therapeutics

in development

do not have a

high-performing

affinity purification

approach similar

to the efficiencies

realized through

the purification of

monoclonal antibodies

by Protein A. –Isett


ligands and to convert those into

efficient affinity resins regardless

of molecule type. To date, Avitide

has discovered affinity ligands

and developed high performing

affinity resins to a diverse array

of biopharmaceutical molecules

for capture, and for product iso-

form separations. That said, prod-

uct isoform separations are the

most challenging simply due to

the fact that there is much less

spatial area to find ligands ade-

quate for separating each isoform.

BioPharm: How could Avitide’s

platform help speed up biosimilar

development in particular?

Isett: Outside of antibodies,

our affinity resins can signifi-

cantly reduce the cost of man-

ufacturing, allow the choice of

more cost-effective production

hosts, shape the product profile

by virtue of the affinity ligand’s

se lec t iv it y, and avoid down-

st ream manufac tur ing inte l -

lectual property (IP) that may

exist from legacy products. In

addition, the IP created with the

Avitide’s resin solution extends

past that of the drug product

composition of matter patents.

BioPharm: A re P rote in-A

separation conditions somehow

not gentle? Can you explain how

this purification method could

lead to product loss?

Isett: Protein A, which has

benefitted from more than 20

years of incremental develop-

ment, typically elutes antibod-

ies in conditions below pH 3.5.

Not all antibodies have the same

inherent stabilities, and elution

condit ions invar iably induce

unwanted aggregation and prod-

uct loss. In fact, in most antibody

recovery processes, the two major

contaminants post-P rote in-A

chromatography are aggregate

antibody and leached Protein A.

In many cases for other mole-

cules, low pH elution conditions

may compromise the molecule’s

structure and activity, and thus,

need to be avoided completely. We

specialize in producing affinity res-

ins that benefit from soft elution

conditions. This in turn enables

affinity purification of molecules

with narrow stability windows.

BioPharm: How many proj-

ects have you delivered to your

partners?

Isett: Avitide has developed

more than 100 novel affinity res-

ins and delivered 16 resins for

five partnered projects. Partner

projects vary and may require

different resins to be developed.

While one campaign may have

scope for one type of aff inity

resin, other campaigns may have

expanded scope for additional

selectivities or a larger survey

of resin development space. We

don’t expect our partners to find

which affinity ligands and resin

conditions will work best for their

process. Instead, our partners

clearly stipulate the performance

criteria for the affinity resin to

be developed, and then Avitide

works to meet those performance

specifications. We validate every

affinity resin performance prior

to shipment using our partners’

relevant bioprocess feed streams.

BioPharm: For 2016, what

are the company’s predictions

for some of the bioprocessing

trends that will gain traction?

Isett: For 2016, we will con-

tinue to see an increased need for

downstream manufacturing tech-

nologies for novel biologics drug

modalities, investment in exclu-

sive manufacturing technologies

in competitive disease and mol-

ecule areas, transition to plat-

form ‘mAb-like’ manufacturing

operations architecture for diverse

non-antibody therapeutics and

vaccines, and the practical imple-

mentation of continuous manufac-

turing processes for non-antibody

therapeutics. These bioprocess

trends relate to a larger biophar-

maceutical industry movement to

improve therapeutic and vaccine

timelines, costs, and global access,

which is largely defined by the

process technologies employed. x


Without a true

affinity-based

purification

platform, the process

efficiencies realized

for the current mAbs

will never materialize

for other biological

therapeutics and

vaccines. –Isett

Product isoform

separations are the

most challenging

simply due to the fact

that there is much

less spatial area to

find ligands adequate

for separating each

isoform. –Isett

ADVERTORIAL December 2015 BioPharm International 41


General Company Description

Tosoh Bioscience LLC is a major supplier of chromatography products worldwide,

particularly to the phar-m a c eut i c a l , b io t e c h -nology, and chemica l industries. The company is a division of Tosoh Corporation, a globa l chemical company with headquarters and manu-facturing faci l it ies in Japan. We provide bulk chromatographic resins for the purif ication of biopharmaceutical drugs

in commercial manufacturing processes and we manufacture analytical HPLC columns. Tosoh’s portfolio of over 500 TSKgel® and TOYOPEARL® products encompasses all common modes of liquid chromatography. CaPure-HA™, a hydroxyapatite resin for biomolecule purification, is a unique resin from Tosoh in that it is both the ligand and base bead.

Facilities

t� Tosoh Corporation (Bioscience Division) serves Japan

t� Tosoh Bioscience LLC serves North and South America

t� Tosoh Bioscience GmbH serves Europe, Middle East, and Africa

t� Tosoh Bioscience Shanghai Co., Ltd. serves China

t� Tosoh Asia PTE (Singapore) serves Asia-Pacific and India

Services

t� Onsite packing assistance for process-scale columns

t� Methods development assistancet� Regulatory expertiset� Redundant validated manufactur-

ing lines for process media productst� Custom analytical, semi-prep, and

prep-scale HPLC columns

TOSOH BIOSCIENCE LLC

3604 Horizon Drive, Suite 100King of Prussia, PA 19406

TELEPHONE

484.805.1219

FAX

610.272.3028

EMAIL

[email protected]

WEBSITE

www.tosohbioscience.com

Tosoh Bioscience LLCt� Professional technical services

for all of our product lines

Major Product Innovations

t� CaPure-HA, hydroxyapatite media for the purification of biomolecules

t� TOYOPEARL NH2-750F anion exchange resin for the purification of proteins in elevated salt conditions

t� TOYOPEARL AF-rProtein A HC-650F high capacity resin for the capture and purification of monoclonal antibodies

t� TOYOPEARL GigaCap® high capacity/high resolution low elution volume ion exchange resins for protein purifications:

ºTOYOPEARL GigaCap S-650S ºTOYOPEARL GigaCap Q-650S ºTOYOPEARL GigaCap CM-650S t� TSKgel-5PW type high resolution resins: º TSKgel SuperQ-5PW (20) for oli-

gonucleotide purification º TSKgel SP-5PW (20) for smaller

protein and peptide purification º TSKgel SP-3PW (30) for

insulin purificationt� �50:01&"3-�#VUZM�� 1IFOZM��

and PPG-600 resins for mAb purificationt� TOYOPEARL Hexyl-650C resin for

flow through polishing applications

Markets Served

t� &��$PMJ and mammalian cell expressed biologics such as monoclonal antibod-ies, cytokines, growth factors, insulin, blood factors, plasma, and other large and small proteins and peptides

t� Other markets served include oli-gonucleotides, DNA, RNA, and pegylated proteins.


For decades, spectrophotometry

has been used to accurately and

precisely measure purified pro-

tein concentrations, with the

extinction coefficient (usually at 280

nm) determined either experimentally

or calculated from amino-acid com-

position. To reduce the overall instru-

mental uncertainty in absorbance

measurement, samples require dilution

to an absorbance of 0.6–0.7/cm. The

uncertainty in the sample preparation

step can be minimized in gravimetric

dilution, but measurement of a larger

series of samples generally involves

considerable work to ensure flasks and

cuvettes are clean.

To overcome the limitations of

conventional cuvette-based spectro-

photometry, a spectrophotometric

technique based on measurement

of the incremental absorbance upon

path length variation has recently

been introduced (1). This variation is

achieved by moving an optical light-

guide fiber immersed into the sample

solutions in a stepwise fashion, with

the absorbance readout at every step

and the calculation of the slope from

the absorbance vs. distance plot. This

technique is called “slope spectros-

copy” by the manufacturer and is

intended for use in measuring con-

centrated sample (protein) solutions

without dilution. The SoloVPE system

(C Technologies, Inc.) has been com-

mercially available for a few years and

has been evaluated mainly for concen-

tration measurement of recombinant

proteins such as monoclonal antibod-

ies (2).

In plasma fractionation, an interest-

ing candidate for assessment of the

slope spectroscopy technique would

be immunoglobulin G for intravenous

administration (IVIG), highly purified

by chromatography and formulated as

a liquid concentrate. Gammagard liq-

uid is a polyvalent IgG product, con-

taining 10% protein of more than 98%

purity in a 250 mM glycine solution,

pH=4.8 (3). Currently, the protein is

ABSTRACT

As an innovative and time- and resource-saving single-step technique that avoids sample

pre-dilution, the use of hazardous reagents, and high-power consumption, SoloVPE

fiber-optics spectrophotometry may be able to substitute or replace the current

Kjeldahl protein determination procedure for 10% IgG in the future.

Variable Pathlength Fiber-Optic Spectrophotometry

for Protein Determination in Immunoglobulin Concentrates

Alfred Weber and Heinz Anderle

Heinz Anderle is senior scientist and

Alfred Weber is senior manager; both

at Baxalta Innovations GmbH.

PEER-REVIEWED

Article submitted: July 31, 2015.

Article accepted: Aug. 17, 2015.

Peer-Reviewed: Protein Characterization


IVIG

Lot no.

SoloVPE(mg/mL)

Kjeldahl(mg/mL)

Slope(1/cm)

E0.1% (1/cm)(Recalculated)

1 100.41 100.943 140.47 1.392

2 100.49 100.629 140.59 1.397

3 99.41 99.651 139.07 1.396

4 101.82 100.91 142.45 1.412

5 100.05 99.283 139.97 1.410

6 101.63 100.434 142.18 1.416

7 100.61 99.070 140.75 1.421

8 99.10 100.087 138.64 1.385

9 100.09 100.340 140.03 1.396

10 99.91 100.432 139.77 1.392

11 98.87 99.790 138.32 1.386

12 101.58 100.559 142.11 1.413

13 100.32 100.119 140.35 1.402

14 99.31 101.276 138.93 1.372

15 100.33 100.479 140.36 1.397

16 100.44 99.273 140.52 1.415

17 98.92 99.204 138.39 1.395

18 99.81 99.584 139.63 1.402

19 100.57 99.085 140.70 1.420

20 100.17 100.431 140.14 1.395

21 97.11 96.930 135.86 1.402

22 99.64 99.113 139.40 1.406

23 98.42 99.204 137.69 1.388

24 99.33 99.446 138.96 1.397

25 100.61 99.140 140.75 1.420

26 100.64 98.955 140.80 1.423

27 99.39 97.804 139.05 1.422

28 100.14 99.750 140.10 1.404

29 99.72 99.074 139.51 1.408

30 98.96 99.544 138.45 1.391

31 99.99 97.855 139.89 1.430

32 100.48 99.892 140.57 1.407

33 99.74 97.580 139.54 1.430

34 99.55 99.161 139.27 1.404

35 98.94 99.968 138.42 1.385

36 99.68 99.240 139.45 1.405

37 98.76 97.818 138.17 1.412

38 98.67 99.257 138.04 1.391

Table I: Comparison of SoloVPE and Kjeldahl protein

concentration results for 10% IgG batches with recalculation

of the extinction coefficient E as slope/Kjeldahl protein.

measured by differential Kjeldahl determina-

tion of the total and the non-protein nitro-

gen content (4). Despite the automation in

reagent dosing and titration, which min-

imizes handling of hazardous substances

such as sulfuric acid, sodium hydroxide solu-

tion, and hydrochloric acid, the Kjeldahl

technique remains a time- and energy-inten-

sive method and requires effective removal

of acid fumes.

W hi le not spec i f ica l ly in use for

Gammagard liquid, the other available nitro-

gen-based technique, the Dumas method,

does not require hazardous liquids, but

instead an oxygen-helium stream to sup-

port sample combustion in steel crucibles

or single-use tin capsules, which are heated

electrically in a furnace to approximately

1000 °C (5). Although the Dumas method,

with its higher throughput, has been almost

fully automated, the power requirement and

reagent consumption remain significant.

The batch-to-batch consistency of the pro-

tein composition allows total protein mea-

surement by ultraviolet (UV) absorbance as

an alternative to nitrogen-based techniques

in a sample dilution containing about 0.5

mg IgG/mL with physiological saline as the

diluent and blank. 10 percent IgG must be

diluted approximately 200-fold in a volu-

metric flask. The turbidity is corrected by

measurement at 320 nm, and the extinction

coefficient E0.1% is calibrated to 1.399/cm on

the Kjeldahl data.

For Kjeldahl digestion, Dumas combustion

of the protein solution, and UV absorbance

measurement with gravimetric dilution, the

sample density needs to be first measured

in an oscillating tube densimeter. If the

slope-spectroscopy technique (by way of the

of SoloVPE device) yields equivalent results,

time-consuming sample preparation and

measurement steps can be reduced to a sin-

gle-step procedure, cleaning of the equip-

ment may be eliminated, consumption of

reagents can be decreased, and the electrical

power obviated.

Equivalence of this assay technique with

the Kjeldahl method must be demonstrated

first by adherence to the AOAC acceptance

criteria for precision and accuracy: At an

analyte concentration of 10%, recovery must

remain within 98–102% and precision no

higher than 2.8% (6, 7); accuracy is con-



firmed by agreement with an independent,

validated reference method (such as the

Kjeldahl digestion). Inter- and intra-assay

precision must be determined by six-fold

repeated measurements as outlined in the

respective regulatory guidelines; linearity

should be at least at 80–120% of the working

level. Furthermore, due to the commercial

importance of IVIG, the bias (difference in

results) between both methods should be

as small as possible, as shown by the Bland-

Altman statistical analysis of the datasets (8).

A 20% subcutaneous IgG concentrate, for-

mulated in the same slightly acidic environ-

ment as the corresponding licensed 10%

IVIG concentrate, is now on the market,

thus, 5–25% IgG would cover the entire

range during production. For rheological

investigation, even higher IgG concentra-

tions (up to 38%) have been obtained exper-

imentally (9).

MATERIALS AND METHODST h i r t y- e ig ht batc hes o f 10 % I V IG

(Gammagard liquid), with known protein

concentration as determined by Kjeldahl

method with solution density measurement,

were obtained in the original bottles, from

which aliquots were drawn antiseptically

with a syringe. An experimental ultra-high

IgG concentrate (target: more than 30%)

was prepared by centrifugal ultrafiltration

(Centriprep 10 kDa) of an 0.03% azide-pre-

served aliquot, and the permeate retained to

mix dilutions (5%, 10%, 15%, 20%, and 25%

IgG) with the stock concentrate (32%). Of

these preparations, protein concentrations

were determined by UV absorbance mea-

surement at 280 nm (with turbidity correc-

tion at 320 nm) and by Dumas combustion

method in a VarioMax analyzer (Elementar),

with the permeate (glycine/azide) as the base

value. Solid acetanilide served as the stan-

dard for the nitrogen content, which was

assumed to be 16% for Kjeldahl and Dumas

determination (10).

For conventional UV-absorbance measure-

ment, a Hitachi U-3000 spectrophotometer

with standard 1-cm quartz cuvettes and a

Hitachi U-3310 high-sensitivity spectro-

photometer with 0.1-mm thin-layer quartz

cuvettes in a spring-clamp holder were used.

The SoloVPE fiber-optic spectrophotom-

eter, comprising the variable pathlength

fiber/detector unit and an Agilent Cary 60 as

the light source/spectrometer unit, was pro-

vided by C Technologies, Inc., together with

the single-use plastic well cuvettes and the

polyimide-coated 0.6-mm diameter quartz

“fibrettes” as the consumables.

VARIABLE PATHLENGTH VS. CONVENTIONAL SPECTROPHOTOMETRY In the SoloVPE device, monochromatic light

fed from the fiber-optic light guide passes

through the quartz fibrette and is absorbed

by the sample solution in the adjustable path

between the fibrette end window and the

plastic well cuvette (Figure 1). A silicon pho-

todiode at the bottom of the cuvette cavity

measures the transmitted light intensity. In

the solution, the light pathlength d can be

increased in increments as small as 0.05 μm,

and the absorbance values A are read out at

all 10 measurement points. To remain within

a linear regression (R2≥0.999), at least five

results are required for slope calculation. The

slope ΔA/Δd obtained from the absorbance

vs. pathlength regression calculation is then

divided by the mass specific or molar extinc-

tion coefficient (E or ε) to calculate the mass

or molar concentration, respectively.


Figure 1: Photograph of a light path from the

SoloVPE fibrette dipping into fluorescein solution.

AL

L F

IGU

RE

S A

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SY

OF

TH

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UT

HO

RS

.


Spectrophotometric measurement is inher-

ently limited by both dark signal and noise of

the detector as well as by the bit-depth of the

analog/digital signal converter. A deviation

from linearity caused by monochromator stray

light may be readily observed above 3 absor-

bance units (i.e., 0.001 residual transmission).

High-sensitivity spectrophotometers such as

the Hitachi U-3310 with its additional auxil-

iary stray light-eliminating monochromator

may extend the upper absorbance range limit

from about three to four absorbance units (i.e.,

from 0.001 to 0.0001 residual transmission),

above which the thermal detector noise of the

common red-sensitive photomultiplier (PMT)

becomes dominant. For routine ultraviolet–vis-

ible spectrophotometry (UV–VIS) instruments,

equipping the detector with a blue-sensitive

PMT and cooling it from ambient temperature

to –20 °C for example, is not a viable option.

A straightforward and presumably user-

friendly approach to overcome these instru-

mental limitations was proposed through

pathlength reduction by the use of thin-layer

cuvettes. Several manufacturers offer cuvettes

with a pathlength of 0.5, 0.2, and even 0.1

mm, and a detachable cover window. The

suitability of such a technique for measur-

ing a monoclonal antibody up to a concen-

tration of 90 mg/mL has been described by

Watson and Veeraragavan (11). Although an

absorbance value of 1.4 for 10% IgG in a 0.1-

mm cuvette would remain unaffected by spec-

trophotometer interference from stray light,

dark signal, or noise, the authors’ experience

discourages from routine use of such cuvettes

because of the delicate handling and cum-

bersome cleaning involved, and the practical

impossibility of applying sample concentra-

tions above 20% IgG due to their high viscosity.

RESULTS AND DISCUSSION 10% IgG solutions and

inter-assay precision

For all 38 batches of 10% IgG, protein con-

centration data obtained using the SoloVPE


TestConcentrations in mg/mL (batch no.)

3 4 26 30 31 36 38

1 99.82 100.96 99.95 99.46 99.48 99.66 98.79

2 99.57 101.01 98.71 99.45 99.35 100.03 98.56

3 99.46 101.22 98.72 99.51 99.41 99.49 98.40

4 99.45 101.18 98.71 99.48 99.65 99.43 98.29

5 99.46 101.18 98.80 99.47 99.74 99.38 98.13

6 99.56 101.19 98.69 99.52 99.72 99.38 98.10

Mean 99.55 101.12 98.93 99.48 99.56 99.56 98.38

RSD (%) 0.13 0.10 0.46 0.03 0.15 0.23 0.25

Target % IgG

Dumas (mg/mL)

TP UV (mg/mL)

Concentrations in mg/mL (batch no.) RSD(%)

E0.1%(calc.)Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Mean

5 49.6 50.47 49.88 49.81 49.77 49.13 49.80 49.78 49.70 0.51 1.402

10 100.9 100.47 99.98 99.89 100.51 100.21 102.18 99.56 100.39 0.85 1.392

15 152.9 149.16 148.24 146.84 145.30 147.63 146.26 151.83 147.68 1.41 1.351

20 205.7 204.18 197.68 196.08 197.11 196.60 196.27 195.39 196.52 0.37 1.337

25 256.7 256.84 248.81 245.00 243.13 246.45 245.93 245.00 245.72 0.70 1.339

32 331.2 321.88 313.80 314.39 308.63 310.76 311.84 303.14 310.43 1.22 1.311

Table II: Intra-assay precision results for six 10% IgG batches (lot numbers the same as in Table I). RSD=relative standard

deviation.

Table III: Protein concentrations as measured by Dumas, total protein ultraviolet (TP UV), and SoloVPE method, and

SoloVPE inter-assay precision results for six experimental 5–32% IgG solutions. RSD=relative standard deviation.

Contin. on page 48



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and the Kjeldahl method were in good

agreement (see Table I). The recalculated

average extinction coefficient of E0.1% as

1.404 ± 0.013/cm corresponds well to the

Kjeldahl-calibrated value of E0.1%=1.399/

cm for the total protein (TP) UV mea-

surement in 200-fold diluted solution,

especially when considering the inherent

measurement uncertainty.

A statistical Bland-Altman comparison

between SoloVPE and Kjeldahl protein con-

centration data was performed. The results

indicate that both methods agree well (at a

95% confidence interval). The mean bias was

-0.33% with 95% limits of agreement rang-

ing from 1.56% to 2.21%, both of which do

not exceed the acceptable recovery limits of

98% and 102% for an analyte concentration

of 10%.

The intra-assay precision test for 10% IgG

solutions, run in a separate series, consisted

of six consecutive readouts of the same sam-

ple well cuvette with the same fibrette. As

the sample solution can be used directly

without any preparation procedure, the

coefficient of variation did not exceed 0.5%

(see Table II).

Experimental 5–25% IgG

solutions and intra-assay precision

The dilutions were prepared by weighing

from the 32% stock concentrate and the

permeate, based on the initial protein con-

centrations and density values. For com-

parison, the dilutions were analyzed by the

Dumas method in a VarioMax combustion

automat with density-based sample dosage.

The stock TP UV concentration was deter-

mined using the single TP UV measurement

as 325.46 mg/mL, and averaged from all six

TP UV measurements (five dilutions and the

stock), based on the respective overall dilu-

tion factor, as 321.88 ± 3.59 mg/mL. For the

SoloVPE measurements without dilution,

this particularly viscous stock was difficult

to fill into the sample well cuvettes without

producing bubbles.

While the 5% and 10% solutions gave an

excellent slope regression for the SoloVPE

measurement with all 10 data points on the

regression curve, the higher concentrations

required—with increasing concentration—

moderate to considerable “slope tweaking”

by omission of visually deviant data points,

usually at absorbance values >1.6 and at

times also of the lowest value at the “foot”

(see Figure 2).

Only with this recalculation of the slope

can the difference between the SoloVPE and

the other methods be reduced for the high-

est concentrations, to approximately –7%.

Intra-assay precision, determined for these

samples by measurement on six days, was

reasonable, with relative standard deviations

(RSDs) below 1.5% (see Table III). Table III also

shows the extinction coefficient E0.1%, recal-

culated using these data.

Interestingly, the variation in the recal-

culated extinction coefficient E0.1% (based

on the TP UV concentrations) suggests a

concentration-dependence with a possible

optical effect of macromolecular crowding,

in particular a reduced increase in scatter

(and turbidity) at extremely high concentra-

tions (12, 13). A corresponding shift in light

attenuation (i.e., the sum of absorbance and

turbidity) has to our knowledge not been

described so far.


DayWavelength in nm A (slope) in 1/cm

278.11 287.40 Run 1 Run 2 Run 3 Run 4 Run 5

1 277.6 286.7 104.30 105.99 n.d. n.d. n.d.

2 277.4 286.5 104.76 104.41 104.28 104.74 105.49

3 277.3 286.4 103.90 104.46 n.d. n.d. n.d.

4 277.3 286.3 103.39 103.58 n.d. n.d. n.d.

5 277.4 286.6 104.78 104.46 n.d. n.d. n.d.

6 277.4 286.5 104.70 104.50 n.d. n.d. n.d.

Table IV: Wavelength results determined using the Cary 60 spectrophotometer and photometric check results from the

SoloVPE; n.d.=no data.

Peer-Reviewed—Contin. from page 45


2.4

2.0

1.6

1.2Ab

sorb

an

ce a

t 2

80

nm

0.80 5 10 15 20 25

Path length (μm)

326.11/1.399 = 233.10 mg/mL

Points for manual re-calculation (n = 4)

340.14/1.399 = 243.13 mg/mL

R² = 0.9995R² = 0.9997 y = 32.611x + 0.7648

y = 34.014x + 0.7401

Points valid for SoloVPE software (n = 7)

Points omitted (n = 3)

SoloVPE slope re-calculation for 25% IgG

30 35 40 45 50

Figure 2: Manual “slope tweaking” by omission of absorbance values

deviant from linearity (A>~1.7 AU) may improve accuracy for high-

protein concentrations.

While mathematical modeling and exper-

imental evidence of such effects for IgG

are beyond the scope of the present study,

the data obtained indicate that the extinc-

tion coefficient for the TP UV method of

1.399/cm, as calibrated for 10% IgG in 250

mM glycine, cannot be applied to a corre-

sponding 20% IgG concentrate and its even

higher-concentrated ultrafiltration stock

using the SoloVPE technique.

Instrument performance

and system suitability tests

Wavelength accuracy was determined daily

using the sealed cuvette containing the

Ho(ClO4)3 in perchloric acid in the stan-

dard cuvette holder, for which the deflection

mirror had to be shifted and the connector

cable changed at the start of every measure-

ment day. Alternatively, a separate SoloVCA

adapter (C Technologies, Inc.) can be con-

nected via the fiber-optic light guide to the

spectrophotometer. From days four to five

(during the weekend), the SoloVPE spectro-

photometer was turned off (as the Cary 60

does not include a high-voltage supplied

detector with a photomultiplier tube, it may

be left on overnight during the week). The

wavelength position was read out manually

from the software’s window with the tracer


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cursor. At the instrument’s fixed slit width

of 1.5 nm, the wavelength error was – 0.5 to

–0.8 nm for the 278.11 nm and –0.7 to –1.1

nm for the 287.40 nm absorption maximum

(see Table IV) (14).

To establish a photometric system suitabil-

ity test, the absorbance A of a water-soluble

phenolic compound solution was measured

at 280 nm in 0.1 mm cuvettes as 117.25/cm

with the Hitachi U-3310 spectrophotom-

eter and as 114.00/cm with the Hitachi U

3000 spectrophotometer, and in the 0.2 mm

cuvette as 113.00/cm in the U-3310. In the

SoloVPE, the range was consistently between

103 and 106/cm (see Table IV); the reason for

this difference is unknown.

CONCLUSIONA reasonable agreement between the

S oloV PE a nd Kje lda h l met hod was

obtained when measuring IgG concentra-

tions with the current release methods at

adequate precision. Although the extinc-

tion appears to be influenced by macro-

molecular crowding effects depending on

concentration, the extinction coefficient

E0.1% of 1.399/cm for 1 mg IgG/mL, as cali-

brated for the TP UV method, remains

valid using the SoloVPE technique at a

concentration of 10% to fulfill the AOAC

criteria for recovery and precision. As reca-

libration of the extinction coefficient is

not required, variable pathlength spectro-

photometry appears to be a viable instant

total protein method to determine the

current 10% IgG concentrate. For higher

concentrations such as a 20% IgG solu-

tion, however, specific recalibration of the

extinction coefficient is essential.

As evident from the raw measurement

data for high IgG concentrations, the vari-

able pathlength technique might benefit

from further instrumentation improve-

ments, such as a higher incident light

beam intensity or lower detector dark sig-

nal, to overcome the limited absorbance

range. Before implementation of such

modifications into a new instrument gen-

eration, however, the user-customizable

slope calculation algorithm should be set

so as to omit absorbance values >1.7/cm as

observed with the current version.

As an innovative and both time- and

resource-saving single-step technique that

obviates sample pre-dilution, the use of

hazardous reagents, and high power con-

sumption, SoloVPE fiber-optic spectropho-

tometry may be able to replace the current

Kjeldahl and Dumas protein determination

procedures for other protein species, with

a constant composition and extinction

coefficient.

ACKNOWLEDGEMENTSThe authors thank Joe Ferraiolo and Larry

Russo (C Technologies, Inc.) for SoloVPE

instrument setup, user training, and tech-

nical support; Laurence Plapied, Grégoire

Bouchez , a nd Chr i s tophe Ca r newa l

(Baxalta Lessines Technical Operations)

for providing the Gammagard liquid sam-

ples and the Kjeldahl protein concentra-

tion data; and Brigitte Neubauer-Watzal,

Daniel Bergmann, and Robert Ras (Baxalta

Vienna Quality Control Department) for

the Dumas analysis. Karima Benamara is

acknowledged for editing the manuscript.

REFERENCES 1. M.C. Salerno, T. Shih, and C. Harriston/C

Technologies, Inc., “Interactive Variable

Pathlength Device”, US patent application

US2009/0027678, January 2009.

2. S. Huffman, K. Soni K, and J. Ferraiolo,

Bioproc. Int. 12 (8), pp. 66–73 (2014).

3. W. Teschner et al., Vox Sang. 92, pp. 42–55

(2006).

4. J. Kjeldahl, Fresenius’ Z. Anal. Chem. 22, pp.

366–382 (1882).

5. P. Blondel and L. Vian, Ann. Pharm. Fr. 51 (6),

pp. 292–298 (1993).

6. L. Huber, LC–GC Int. 1998 (2), pp. 96–105

(1998).

7. I. Taverniers, M. de Loose, and E. van

Bockstaele, Trends Anal. Chem. 23, pp. 536–

551 (2004).

8. J.M. Bland and D.G. Altman, Lancet 8476 (1),

pp. 307–310 (1986 Feb. 8).

9. V. Burckbuchler et al., Eur. J. Pharm. Biopharm.

76, pp. 351–356 (2010).

10. E. Brand, B. Kassell, and L.J. Saidel, J. Clin.

Invest. 23, pp. 437–444 (1944).

11. L. Watson and K. Veeraragavan, Biopharm Int.

27 (2), pp. 26–37 passim (2014).

12. C. Fernandez and A.P. Minton, Anal. Biochem.

381 (2), pp. 254–257 (2008).

13. C. Fernandez and A.P. Minton, Biophys. J. 96,

pp. 1992–1998 (2009).

14. J.C. Travis et al., J. Phys. Chem. Ref. Data 34,

pp. 41–56 (2005). x


Sand

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ag

es

Vi sua l appearance, a pr i-

mary evaluation method

for freeze-dried products, is

subjective and likely to vary

between laboratories and over time.

This article discusses how visual meth-

ods can be quantified and presents

an overview of mechanical methods

of characterizing and quantifying the

properties of freeze-dried materials.

A well-dried product is one that

will reconstitute into a viable prod-

uct. It must have been frozen at a rate

that will produce a desirable struc-

ture, and then kept at a low enough

temperature during drying to main-

tain that structure without collapse.

The structure of the dried prod-

uct reflects how successful the drying

process has been; if there has been

a collapse, then parts of the prod-

uct will have been less thoroughly

dried. Defects in the structure and,

therefore, in the drying of a product

will have an impact on how well it

will rehydrate, whether any bioactiv-

ity will be preserved, and how long

and under what conditions it can be

stored. Even if the product has been

dried well, the structure of the dried

Quantitative Post-Processing Characterization Techniques

for Freeze-Dried ProductsKatriona Scoffin

Mechanical tests

complement

visual techniques

for the post-

processing

characterization

of freeze-dried

products.

Katriona Scoffin is a freelance writer

with extensive experience in the life

sciences. She works from Cambridge, UK.

Contact her at [email protected].

Lyophilization


Lyophilization

AL

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OF

BIO

PH

AR

MA

TE

CH

NO

LO

GY

LT

D.

product still has a bearing on

how easily it will rehydrate.

ASSESSING THE VISUAL APPEARANCEThe primary method of assess-

ing the quality of a freeze-dried

product is by visual inspection.

Catastrophic failure is visually

evident by a collapse of the prod-

uct (Figure 1A). Other product

defects are not readily apparent

but can still be determined by

an assessment of appearance. For

example, a partial collapse may

have occurred during drying, or

a suboptimal crystal structure

may have been achieved during

freezing so that the product will

not reconstitute evenly. Some

of these events may result in a

change of appearance (Figure 1B),

but it is not easy to relate changes

in appearance to specific defects.

Aspects of visual appearance

that can be assessed include

shrinkage, collapse, skin forma-

tion, color, contaminants, and

consistency. In order to analyze

changes, it can be helpful to give

a score to each of these attributes.

The scores can then be analyzed

and referred to during cycle devel-

opment, scale up, or equipment

changes. However, the process

remains subjective and the con-

sistency of scoring is likely to vary

by day, analyst, or laboratory.

MICROSCOPIC FEATURES REVEAL PRODUCT STRUCTUREA microscopic visual assessment

using scanning electron micros-

copy (SEM) or tunneling elec-

tron microscopy (TEM) reveals

more information about prod-

uct structure. SEM and TEM are

valuable methods for looking at

the microstructure to reveal pos-

sible micro-collapse and to deter-

mine the porosity of the product,

which has a direct bearing on

rehydrat ion. The SEM images

shown in Figure 2 reveal that fast

cooling creates a structure with

smaller crystals and therefore

lower porosity than slow cooling.

This information can be used

during cycle development for

determining the impact of dif-

ferent methods on product struc-

ture. For example, SEM clearly

shows the difference in structure

caused by adding an annealing

phase to the freezing of manni-

Figure 1: (A) Catastrophic failure is visually evident by the collapse of the freeze-dried product. (B) There can still be

differences between products that are not easy to relate to specific defects; the product is uniform and adhered to the vial

(left); uniform but not adhered to the vial (middle); has a peaked cake (right).

(A) (B)

Figure 2: Scanning electron microspcope (SEM) images of two samples of

freeze-dried mannitol reveal the differences in structure resulting from fast

and slow cooling.

Fast cool Slow cool

x1000

x9000

TM-1000_2707 2010/09/17 10:14 D1.9 x1.0k 100 um

TM-1000_2705 2010/09/17 10:11 D1.9 x9.0k 10 um

TM-1000_2711 2010/09/17 10:30 D1.9 x1.0k 100 um

TM-1000_2709 2010/09/17 10:26 D1.9 x9.0k 10 um


Lyophilization

tol (Figure 3). It is also an excellent

way of determining the quality of

an individual sample. However,

SEM still requires a visual assess-

ment of the images, which results

in variation due to subjectivity.

The rate at which gas

is adsorbed depends

on the surface energy

of the material.

It is possible to develop a dis-

crete classif ication with terms

that could be applied to differ-

ent regions in the same cake, but

assessing the microscopic fea-

tures remains a primarily quali-

tative method.

QUANTITATIVE CHARACTERIZATION METHODSThe challenge is to find a quan-

titative method that relates reli-

ably to the product structure.

The ideal would be a mechanical

measurement that relates consis-

tently to pore size, structure, and

any presence of micro-collapse.

The following mechanical tests

can be applied alongside a visual

and microscopic analysis:

t� �(BT� BETPSQUJPO� UP� EFUFSNJOF�

the specific surface area and

the mean pore diameter

t� �9�SBZ�EJGGSBDUJPO� UP� DIFDL� GPS�

different polymorphic forms

t� �9�SBZ�NJDSPDPNQVUFE� UPNPH-

raphy scanning to measure the

porosity and heterogeneity

t� �1SFTTVSF� UFTUJOH� UP� BTTFTT� UIF�

mechanical properties.

GAS ADSORPTION METHODSThe specific surface area and the

mean pore diameter of a mate-

rial can be determined by mea-

sur ing the rate of adsorption

and desorpt ion (evaporat ion)

of nitrogen gas to and from the

material’s surface at low tempera-

tures and under varying degrees

of pressure.

T he rate at wh ich gas i s

adsorbed depends on the surface

energy of the material, which

itself depends on the surface

structure. A rough surface has a

larger surface area than a smooth

surface. There also will be more

surface atoms that are incom-

pletely bound, which will increase

the adsorption rate of a gas. The

Brunauer-Emmett-Teller (BET)

equation allows the determination

of specific surface area from the

gas adsorption rate.

The overall porosity of the mate-

rial can be related to the difference

between the adsorption and the

desorption rate of the nitrogen.

The Barrett-Joyner-Halenda (BJH)/

Kelvin equations can be applied to

the data to determine the pore-size

distribution.

The following example shows

how this method can be applied

to samples of freeze-dried man-

nitol. Nitrogen adsorption and

desorption isotherms were mea-

TVSFE�BU��¡$�VTJOH�BO�"4"1�

Tr i s t a r 30 0 0 ( M ic romer it ic s

Inst r u ment Cor p.) volu met-

r ic adsor pt ion system for a

fast-cooled mannitol solution,

a slow-cooled mannitol solu-

tion, and liquid nitrogen (LN2)-

quenched mannitol.

Figure 3: Scanning electron microscope (SEM) images of two freeze-dried

mannitol–sucrose mixtures. The mixture on the left has been annealed and

reveals a rougher structure. The mixture on the right was not annealed and shows

a smoother, amorphous structure.

Figure 4: The adsorption and desorption of nitrogen (LN2) under pressure by

different samples of mannitol.

Isotherm linear plot

FAST-COOLED MANNITOL

SLOW-COOLED MANNITOL

LN2-QUENCHED MANNITOL

40

35

30

25

20

15

10

5

00.75 0.8 0.85 0.9 0.95 1

Qu

an

tity

ad

sorb

ed

(cm

3/g

STP)

Relative pressure (P/Po)


Lyophilization

Isotherms were simi lar for

each method (Figure 4), but the

hysteresis between the adsorp-

tion and the desorption curves

was wider for the LN2 quench-

cooled mannitol. This result

indicates that the energy needed

for evaporation from the pores

was distinctly different from the

energy associated with conden-

sation within it, which implies

that desorption was inhibited

due to constriction, thereby sug-

gesting that this sample had a

smaller pore size (Figure 5).

X-RAY DIFFRACTION TO DETERMINE POLYMORPHIC FORMS9�SBZ�EJGGSBDUJPO� JT� B� XFMM�FTUBC-

l ished method of determining

crystal structure. When used on

lyophilized materials, the dif-

f ract ion spectrum can reveal

the different polymorphic forms

resulting from different cooling

methods.

Figure 6 shows the different

spectra obtained from samples

of dried mannitol that were fro-

zen using different cooling rates.

The spike at a diffraction angle

of 9.5°2ĭ for the slow-cooled

solution is characteristic of the į

polymorphic form.

MICRO-CT SCANNING FOR POROSITY AND HETEROGENEITYMicro - computed tomography

(CT) scanning is a noninva-

sive method of visualizing the

internal micro -st ructure of a

three-dimensional (3D) object.

$SPTT�TFDUJPOBM�9�SBZT� BSF� UBLFO�

at smal l interva ls across the

product. CT software takes these

cross sections and builds them

into a 3D-visualization of the

internal structure.

Computational methods have

been developed for quantifying

the 3D-images, allowing mea-

surement of porosity, pore-size

distribution, pore connectivity,

Figure 5: Determining the characteristics of mannitol lyophilized with different

cooling methods; (A) The Brunauer-Emmett-Teller (BET) adsorption analysis to

determine the surface area of the cake; (B) The Barrett-Joyner-Halenda (BJH)

adsorption analysis to determine the mean pore diameter.

600

500

400

300

200

100

0

Slow cool Fast cool LN2 cool

ads pore diam

des pore diam

Me

an

po

re d

iam

ete

r, A

ng

stro

ms

8

7

6

5

4

3

2

1

0

Slow cool Fast cool LN2 cool

SA ads

SA des

SA BET

Sp

eci

fic

surf

an

ce a

rea, m

2/g

ram

(A)

(B)

Figure 6: X-ray diffraction (XRD) spectra of mannitol lyophilized with different

freezing rates. The spectra reveal the different polymorphic form in the sample

cooled at 0.5°C per minute (bottom).

5000

4000

3000

2000

1000

0

Inte

nsi

tyIn

ten

sity

Inte

nsi

ty

2500

1000

1500

1000

5000

0

2000

1500

1000

500

0

0 15 20 25 30

Slow cool (0.50C/min)

Fast cool (Precooled shelf-450C)

LN2 cooled

35 40 35 50

0 5 10 15 20 25 30 35 40 35 50

0 5 10 15 20 252Φ

2Φ

2Φ

Characteristic of δ

β, α30 35 40 35 50

5 10


Lyophilization

and particle size throughout the

sample matrix. The nondestruc-

tive and penetrating nature of

the analysis allows observation

of the structure throughout the

entire sample, without risk of

deforming the material by cut-

ting or other methods to access

the lower parts of the product

that could invalidate the results.

PRESSURE TESTING TO DETERMINE CAKE STRENGTHWork i s u nde r way be t ween

Imperial College London and

Biopharma Technology Ltd to

develop a miniature load cell to

measure stress and strain in a

lyophilized cake while it is still

in the vial. Measuring the stress

(ı) and strain (İ) gives the elas-

ticity, Young’s modulus (E) (see

Equation 1):

Young’s modulus (E) = strain ( ε )

stress ( σ )

[Eq. 1]

The following example shows

how this method works in prac-

tice. The aim was to compare

the strength of different cakes

through compression test ing.

The samples were tested in the

vial using a Stable Microsystems

Texture analyzer and Lloyds EZ50

column test stand (Figure 7). Three

different samples of mannitol

were tested: fast-cooled, slow-

cooled, and LN2-quench cooled.

The pressure was applied to the

cakes at a speed of 1 mm/sec,

tested in increments of 0.01 mm/

sec to a depth of 3 mm, starting at

1 g of force.

The results showed that the

fast-cooled lyophilized mannitol

was the least flexible (i.e., had the

greatest resistance to deforma-

tion when force was applied) and

that the slow-cooled lyophilized

mannitol was the least resistant

to deformation (Figure 8). This

result is likely to be due to mor-

phology; the slow-cooled mannitol

has larger pores, and therefore a

weaker structure. An SEM analysis

of the samples revealed small holes

that are visible in the LN2 and

slow-cooled samples; holes are the

weakest points in the cakes where

they can crack or break easily.

The clear differential shown

bet ween the Young ’s modu-

lus for these mannitol samples

mer it s f u r ther work on how

this method can be developed

to provide a quantitative assess-

ment of the mechanical prop-

erties of freeze-dried materials,

either to compare different for-

mulat ions or batches of the

same product.

CONCLUSION1SPEVDU�BQQFBSBODF� BTTFTTNFOU�

can be subjective. Microscopic

analysis is a valuable tool for

determining product structure,

but it still requires a subjective

visual analysis that is likely to

vary across operators, sites, and

time.

(BT� BETPSQUJPO �9�SBZ� EJGGSBD-

t ion, micro -CT, and pressure

testing are quantifiable methods

of measuring aspects of product

structure. These techniques can

be used to improve the consis-

tency of freeze-drying processes

across different sites and over

time. They can also improve the

understanding of the process or

formulat ion changes that are

required to achieve high quality

freeze-dried products. ◆

Figure 7: A miniature load cell is used

to apply pressure to the freeze-dried

cake while it is still in the vial.

50000

45000

40000

35000

30000

25000

20000

15000

10000

5000

0

Fast

-co

ole

d m

an

nit

ol

LN

2-q

uen

ched

man

nit

ol

E modulus (N/m2)

E modulus (N/m2)

Slo

w-c

oo

led

man

nit

ol

Figure 8: The results of the pressure test can be used to determine Young’s

modulus for different samples. The difference in results can be related to the

different structure of the samples.


Jup

iterim

ag

es/G

ett

y Im

ag

es

Mycoplasma contamination

of biopharmaceutical pro-

cesses and drug substances,

typically through the use

of contaminated raw materials or human

contact, is generally not readily apparent

but can affect cell growth and activity

(cell metabolism and gene expression),

and some species are pathogenic. Routine

analysis of raw materials, monitoring of

bioprocesses, and testing of final product

lots for mycoplasma is thus required by

regulatory agencies.

There are more than 200 known spe-

cies (class Mollicutes) of these tiny (< 1

μm in diameter) bacteria that lack cell

walls. The gold standard assay involves

direct cell culture in complex media

enriched with growth factors, often in

combination with an indirect culture/

staining method to detect fastidious

mycoplasma species that do not grow in

broth and agar. The method is highly

effective, but lengthy (28–35 days) and

labor-intensive, resulting in longer pro-

duction times and increased risk. It is also

inappropriate for unstable cell therapy

treatments.

Several rapid mycoplasma test kits

based on nucleic acid amplification and

other techniques are commercially avail-

able for use in research labs, and some are

also claimed to be effective for commer-

cial biopharmaceutical manufacturing

(raw material analysis, cell line qualifica-

tion, in-process monitoring, and product

release). Use of these methods is limited

in practice, however, due to the chal-

lenges associated with demonstrating

comparability to compendial methods.

RECOGNIZED NEEDThe length of standard mycoplasma

culture methods can be problematic

for biopharmaceutical manufacturers

on a number of levels. The long times

Rapid Mycoplasma Testing: Meeting the Burden of Proof

Cynthia A. Challener

Expectations are high for

rapid testing methods, but

demonstration of comparability

proves challenging.

Cynthia A. Challener, PhD,

is a contributing editor to

BioPharm International.

Analytical Testing


for raw material testing and cell

line qualification delay the start

of production processes. Lengthy

assay times for lot release extend

the time it takes to get products

available to patients.

For products that have short shelf

lives measured in hours or days,

such as patient-specific autolo-

gous cell therapy products, lengthy

methods are inappropriate, and

there is a strong argument that

the use of rapid methods will lead

to increased product and patient

safety, according to John Duguid,

director of process development for

Vericel Corporation. In addition,

cell-culture methods cannot be

used for some viral vaccines, and

thus alternative methods are nec-

essary. For products where expiry

or reactivity are not a concern,

Duguid notes that the decision to

implement a rapid method is gener-

ally driven by an assessment of the

return on investment needed for

purchase, development, validation,

and implementation.

With respect to process monitor-

ing, risk is increased as the result

of lengthy assay times. If contami-

nation is detected, all material

processed during that assay time

is lost, and the delivery of the for-

mulated product to the patient may

be delayed. “More timely access to

analytical results can significantly

improve the decision-making pro-

cess and minimize any necessary

downtime for investigation of the

contamination source and need for

cleaning and revalidation of equip-

ment,” says Garry Takle, executive

director of vaccines and biologics

commercialization with Merck.

HURDLES TO CLEARDespite the clear benefits of rapid

mycoplasma tests and the encour-

agement of their use by regulatory

agencies, several significant hurdles

remain to be cleared before they

are widely adopted for biophar-

maceutical manufacturing. First,

Duguid stresses, regulatory agen-

cies require extensive validation

packages to support use of alterna-

tive methods. Alternative methods

must provide comparable or better

sensitivities and breadth of detec-

tion of mycoplasma species when

compared to the standard cell-cul-

ture method. Parameters including

accuracy, precision, specificity, the

detection limits, operational range/

sample volume, robustness, repeat-

ability, and intermediate precision

must be evaluated.

“The validation expectations of

regulatory agencies for alternative

rapid mycoplasma testing are very

high but quite clear,” asserts Takle.

He recommends that any companies

developing rapid testing methods

for US approvals communicate with

FDA early on. “Representatives at the

agency are very knowledgeable about

mycoplasma testing and accessible,

through involvement in consortia

or other industry organizations such

as the Parenteral Drug Association

(PDA), and should be considered a

valuable resource,” he adds.

Rapid methods should also

be easy to use and allow for sig-

nificantly faster turnaround times,

all at a lower cost than the con-

ventional method. That can be a

challenge, according to Duguid,

because often rapid methods with

detect ion platforms dif ferent

from conventional methodology

require both analysts and review-

ers to have specialized expertise.

Most current tests are nucleic acid

tests (NATs)—mainly based on the

polymerase chain reaction (PCR)—

and require expertise in molecular

biology techniques. DNA-binding

with f luorescence is another

example. Biochemical (enzyme

assays, enzyme-linked immuno-

sorbent assays [ELISA], and cell-

surface receptor detection), mass

spectrometry, and immunological

techniques (detection of specific

antibody-antigen interactions) have

also been developed.

Changes in process condi-

tions can also impact many rapid

test methods. The performance

of a PCR-based method may, for

instance, be affected by changes

in the composition of the cell cul-

ture media or increases in sample

cell mass. Validation requirements

also differ for different testing

applications. For instance, when a

test is to be used as a complemen-

tary test for in-process monitoring,

demonstration of comparability

may not be necessary, while a com-

parability study is required if the

rapid method will be replacing an

official method for lot release. In

addition, NATs can be destructive

testing methods, and thus the use

of orthogonal methods may be pre-

cluded, according to Duguid.

Finally, demonstration of compa-

rability generally involves conduct-

ing parallel rapid and conventional

cell-culture assays using the same

sample. Such tests are not possible

in many facilities that manufac-

ture cell-based therapies because

the use of viable mycoplasma cul-

tures is prohibited. In these cases,

Duguid notes that performance of

the assays required to demonstrate

equivalence to the conventional

methodology must be outsourced

to a qualified testing laboratory,

which may further complicate the

method development, validation,

and technology transfer processes.

THE IMPORTANCE OF SAMPLE PREPARATION AND TEST PROTOCOLSRapid PCR-based mycoplasma

testing methods were first intro-

duced in the 1980s and are the

most common assays being inves-

tigated today. “PCR is a very well-

understood technology that is

used in other approved assays, and

it meets all of the important crite-

ria for rapid mycoplasma testing,”

Takle observes.

Several different PCR techniques

have been employed for the devel-

Analytical Testing


opment of rapid mycoplasma

methods, including real-t ime

PCR, touchdown PCR, transcrip-

t ion mediated ampl i f icat ion,

microarrays, and hybrids with

a growth step prior to PCR. In

addition to providing adequate

specificity and sensitivity, some

rapid tests also offer quantita-

tion and identification. The latter

take longer—up to 7 days—and

are, therefore, not appropriate for

applications where a very rapid

turnaround is required, but are

suitable in applications where

other longer-term testing is nec-

essary, such as 14-day sterility

testing for bacteria and fungi.

Regardless of the specific PCR

technique, successful testing can-

not be accomplished without

careful sample preparation and

proper protocols, according to

Takle. Unlike culture-based meth-

ods, which only detect viable

organisms, NATs detect all nucleic

acids from viable, non-viable, and

lysed organisms. The presence of

nucleic acid from sources other

than viable organisms can lead to

false-positive results.

One potential way to avoid

this issue is to develop methods

based on RNA, rather than DNA,

detection because only organ-

isms undergoing protein synthe-

sis will be identified. Even with

this approach, proper sample

preparation and rigorous testing

protocols that include appropri-

ate measures for the prevention

of DNA contaminat ion are a

necessity. An initial culture step

can also help in some cases to

identify viable organisms. “False

positives pose equal risk for the

manufacturer, because it is very

di f f icult to prov ide ev idence

that an initial positive result was

actually false,” says Takle.

False negatives, on the other

hand, present a risk to patients

and are of greatest concern to

regulatory agencies. This concern

is alleviated by ensuring that

the rapid mycoplasma method

has the necessary level of sensi-

tivity for a wide range of myco-

plasma species. The preparation

and characterization of quality

mycoplasma stock samples for

determination of comparability is

crucial for successfully achieving

this goal. Details are outlined in

the World Health Organization

I n t e r n a t i o n a l S t a n d a r d t o

Harmonize Assays for Detection

of Mycoplasma DNA (1).

SIMILAR ADOPTION RATE EXPECTED GOING FORWARDThe adoption of rapid myco-

pla sma test ing for b iopha r-

maceut ica l manufac tur ing is

expected to continue at the slow

rate observed in recent years. To

date, only a handful or so (6–12)

methods have been approved,

and mainly for new products,

according to Takle. He expects,

though, that as more new bio-

logic drug products are approved

with rapid mycoplasma testing as

part of their filings, the number

of methods used in the industry

will increase, even for existing

products.

“Because the compendia method

takes so long, most biopharmaceu-

tical companies are investigating

the potential of rapid mycoplasma

testing in some form for various

applications. It will take longer for

implementation of these newer

methods for existing products,

though, because the expectations

for demonstrating comparability

are very high,” Takle explains.

Advances in PCR technology

are helping as well. The panels

of mycoplasma organisms that

can be detected with PCR con-

tinue to expand, and improve-

ments in PCR reagents have led

to improvements in the sensitiv-

ity, accuracy, and robustness of

many rapid mycoplasma meth-

ods, according to Takle. He also

notes that many newer methods

have been designed specifically

to address ease-of-use issues. The

more ef fect ive automation of

sample preparation steps has also

been important. “These advances

h ave s i g n i f i c a nt ly r e duc e d

the amount of hand-on t ime

required to perform some rapid

tests,” comments Duguid.

BIOPHARMA WEIGHS IN-HOUSE VS. OUTSOURCED MYCOPLASMA TESTINGA number of testing labs now

offer rapid mycoplasma PCR test-

ing services, but in general, larger

biopharmaceutical manufacturers

tend to prefer to keep this test-

ing in-house at present. “Since

both false-positive and false-neg-

ative results can have far-reach-

ing impacts, companies with the

resources to do their own rapid

mycoplasma testing often do so

to retain control over the pro-

cess,” Takle comments. As rapid

mycoplasma testing becomes more

commonplace and both manufac-

turers and testing labs gain more

experience with these methods,

he expects that outsourcing will

likely increase.

Merck is currently investigat-

ing the use of rapid mycoplasma

testing for new products and is

also looking at its application for

some existing products. Vericel

uses nucleic acid-based rapid

mycoplasma testing for raw mate-

rial testing, in-process testing,

and final product release test-

ing of autologous cell therapy

products, and has validated and

implemented a rapid mycoplasma

test for an autologous cell ther-

apy approved for marketing in

Europe. This test will likely also

be included with future US regu-

latory submissions.

REFERENCE 1. M. Nübling et al., Appl. Environ.

Microbiol. 81(17), pp. 5694-702. ◆

Analytical Testing


The pharmaceutical industry is

being transformed by the suc-

cess of protein-based therapeu-

tics (biologics). In 2012, biologics

sales in the United States topped $63 bil-

lion, up 18.2% from 2011 (1). Additionally,

it is estimated that half of new drug

approvals in 2015 will be biologics, with

this trend forecasted to increase over the

next decade (2).

Many classes of biologics are recom-

binant secretory proteins (e.g., antibod-

ies, fusion proteins, growth factors,

cytokines, etc.) that possess post-trans-

lational carbohydrate modifications of

certain asparagine residues (N-glycans)

or serine/threonine residues (O-glycans).

The structure and composition of gly-

cans can dramatically affect the stability,

bioactivity, and pharmacokinetics of a

biologic drug. Most biologics are recom-

binant N-glycosylated proteins that are

derived from non-human protein expres-

sion systems such as Chinese hamster

ovary (CHO) cells (3). Glycan structure

and composition are particularly sensitive

to changes in a biologic’s manufacturing

environment, such as process improve-

ments, scale-up, and method transfers.

As such, glycan structure is designated a

critical quality attribute (CQA) that must

be monitored during manufacturing, and

the glycan profile of a finished product is

used to assess the consistency of a manu-

facturing process from batch to batch.

STRUCTURAL ANALYSIS OF N-GLYCANSWorkflows that enable N-glycan struc-

tural analysis are complicated and have

historically involved sample-process-

ing methods that are slow, labor inten-

sive, multi-stepped, and dimensional.

Conventional preparation of N-glycans

derived from monoclonal antibod-

ies (mAbs) and immunoglobulin Gs

(IgGs) generally proceeds as outlined in

Figure 1. Typically, fluorescently labeled

N-glycans are produced and analyzed

using liquid chromatography (LC) cou-

pled to fluorescent detection (FLR), LC

with detection using mass spectrom-

etry (MS), or capillary electrophoresis

(CE) with laser-induced fluorescence

(LIF). There is currently no single stan-

dardized protocol within the pharma-

ceutical industry for N-glycan analysis

and most organizations have developed

customized workflows to suit their

individual needs (4). This landscape

is rapidly changing, however, as

industry demands have increased for

more standardized workf lows that

permit faster, more accurate, higher

throughput, reproducible, and quan-

titative structural characterization of

N-glycans. Recent advances in sample

preparation and analysis of N-glycans

that are enabling better quality testing

of N-glycans of biologics are reviewed

here.

ADVANCES IN N-GLYCAN SAMPLE PREPARATIONThe field has seen remarkable recent

advances in the range, supply, and

quality of reagents, consumables, and

analytical instrumentation aimed at

improving glycan analysis. An impor-

tant area of improvement has been in

N-Glycan Composition Profiling for Quality Testing

of BiotherapeuticsRebecca Duke and

Christopher H. Taron

Advances in glycan analysis are enhancing

biologics development

and quality control

processes.

Rebecca Duke is a postdoctoral

researcher, and Christopher H. Taron

is scientific director, Protein Expression

and Modification Division, both at

New England Biolabs, Ipswich, MA.

Quality Testing


Quality Testing

AL

L F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

RS

sample preparation methods that

have been considerably stream-

lined from day to hour times-

cales. New methods are now also

higher throughput, and analyses

in 96-well formats are feasible

using robotic liquid handling plat-

forms. A 384-well sample prepa-

ration method was reported for

analysis of N-glycans derived

from IgG using LC–FLR (5). This

study marked a significant step

toward bringing the throughput

of glycomics closer to the realm of

genomics. Additionally, the field

has seen the recent emergence of

turnkey commercial sample prepa-

ration kits that permit glycans to

be rapidly liberated from glycopro-

teins and labeled for analysis.

Enzymatic release of N-glycans

A key early step in N-glycan com-

position profiling involves com-

plete and unbiased removal of

N-glycans from a target glycopro-

tein. N-Glycan release typically

involves denaturing a protein

with a reducing reagent (e.g.,

dithiothreitol [DTT]), sodium

dodecyl sulfate [SDS], and an

alkylating reagent (e.g., iodo-

acetamide [IAA]), respectively,

prior to enzymatic release of gly-

cans with peptide N-glycosidase

F (PNGase F). This process has

severa l technica l drawbacks:

SDS inhibits the funct ion of

PNGase F, long PNGase F diges-

tions are typically required (two

hours to overnight) and often

not all N-glycans are completely

released, and the presence of

detergent or SDS in a sample is

detrimental to downstream anal-

ysis using MS.

The field has moved to eliminate

these technical complications. A

first improvement was the devel-

opment of RapiGest SF (Waters

Corporation), an acid (H+) cleav-

able MS and PNGase F ‘friendly’

labile surfactant. RapiGest SF dena-

tures a mAb in approximately

three minutes in the absence of

DTT, eliminating the need for

time consuming clean-up steps

prior to MS analysis. RapiGest SF

has also been reported to reduce

the amount of time needed for

PNGase F digestion from overnight

to approximately two hours (6);

although in this study, N-glycan

release was not entirely complete.

A second improvement has been

the development of Rapid PNGase

F (New England Biolabs), a novel

MS-compatible composition of the

enzyme that permits complete and

unbiased release of all N-glycans

from mAbs and fusion proteins in

10 minutes (7).

Figure 1: Schematic illustrating the steps of a typical sample processing method for N-glycan analyses of monoclonal

antibodies (mAbs) and IgGs. Most N-glycan sample preparation workflows consist of these general steps, but additional

technical options could also be employed.

Fab

Fc

2. De-glycosylation

1. Denaturation

8� Monoclonal antibody (e.g., mouse, chimeric, humanized)

8� Digestion with peptidases (e.g., trypsin)

8� Reduction of disulfide bonds using carbomethylating agents (e.g., dithiothreitol

[DTT] or mercaptoethanol [ME])

8� Alkylation of cysteine (Cys) residues using reagents such as iodoacetamide (IAA)

8� Ionic and non-ionic detergents (e.g., sodium dodecyl sulfate [SDS], NP-40 and Triton)

8� Pre-labeling clean up using Solid phase extraction (SPE) with reverse phase (RP)

resins such as C8 and C18

8� Desalting prior to labeling using porous graphitized carbon (PGC)

8� Precipitation of proteins using organic solvents

8� Fluorescent labeling using Schiff base or carbamate chemistry

8� Solid phase extraction (SPE) to remove excess free label using hydrophilic resins

functionalized with amide, diol or microcrystalline cellulose

3. Purification

4. Labeling

5. SPEDiol

HO H2N

HO

O

R RSi

O

O

R

R

RSi

HILIC

8� Treatment with endoglycosidases or Peptide N-Glycosidase F (PNGase F)

8� Chemical release of N-glycans using hydrazinolysis or -eliminationβ


Quality Testing

In addition to PNGase F, several

other endoglycosidases are now

available for application in gly-

can profiling workflows (8). For

example, peptide-N-glycosidase A

(PNGase A) can be used for pro-

filing N-glycans from proteins

derived from plant or insect cells

and that contain a core Į(1,3)-

linked fucose, a modification

that is recalcitrant to PNGase F

digestion. Additionally, endogly-

cosidases (e.g., Endo D, H, F1, S,

and S2) cleave within the chitobi-

ose core of N-glycans (Figure 2A).

Endoglycosidases S and S2 work

more eff iciently than PNGase

F under native conditions and

can selectively remove classes of

N-glycans from the fragment crys-

tallizable (Fc) region of IgG (i.e.,

hybr id versus high mannose

N-glycans). This method of degly-

cosylation is often useful for inves-

tigation of the role glycosylation

plays in the biological activity of a

target glycoprotein. Additionally,

antibody subunit fragmentation

proteases (e.g., single site hinge or

upper hinge region cleavage) are

commercially available and can be

used in combination with endogly-

cosidases to permit separate analy-

sis of N-glycans derived from the Fc

or fragment antigen-binding (Fab)

regions of antibodies (Figure 2B).

These tools are expanding the

breadth of applications for which

N-glycan profiling can be used.

Fluorescent labeling of glycans

Recent advances in N-glycan

labeling chemistries have yielded

tremendous improvements in

both labeling speed and ana-

lytical sensitivity. There are two

basic labeling strategies that are

typically employed in analytical

workflows: Schiff base (SB) con-

densation and reactive carbamate

chemistry (Figures 3A and 3B).

Both methods are used after gly-

cans are released from a protein by

PNGase F. The PNGase F reaction

produces a glycan product with

a transient glycosylamine moi-

ety on the N-acetylglucosamine

(GlcNAc) that was formerly linked

to asparagine. This GlcNAc can be

converted from a glycosylamine

form to a reducing sugar form

thereby enabling use of SB con-

densation with amine functional-

ized fluorophores (Figure 3A). The

most frequently used fluorophore

is 2 -aminobenzamide (2 -AB);

Figure 2: (A) Schematic summarizing the general specificity of selected endoglycosidases. Endo S removes Fc N-glycans

from IgG only. (B) Antibody fragmentation enzymes currently available (New England Biolabs, Promega, and Genovis). IdeZ/

IdeE (Steptoccocus equi) cleaves the upper hinge region of human IgG’s but has higher activity towards mouse IgG’s than

IdeS (Streptoccocus pyogenes), particularly mouse IgG2a. GingisKHAN (KGP) cleaves human IgG1 at a single site above

the hinge region. SpeB (Streptoccocus pyogenes) digests IgG’s from many species and subclasses. (C) Hinge sequence

showing cleavage specificity. The cysteine residues of disulfide bonds are indicated in red.

4β 4β

4β 4β3

6α

α

4β 4β3

6α

α

4β 4β3

6α

6α

α

3

6α

α

4β

2β

2β

2β

4β

3

6α

α

3

6α

α

Chitobiose core – ‘endo’ cleavage site

(A) (B) Antibody Subunit Fragmentation

KGP

SpeB,

Fab and Fc

Fab

Fc

IdeS

IdeZ/IdeE

Fc + F(ab’)2

Endo S

upper lower

Hinge sequence: S-C-D-K-T-H-T-C-P-P-C-P-A-P-E-L-L-G-G-P-S-V(C)

KGP SpeB IdeS /IdeZ /IdeE

Pauci-mannose

High-mannose

Hybrid

Endo D

Endo H, F1

Endo S

(IgG Fc)

Endo S2

(IgG Fc)


Quality Testing

however, several other f luores-

cent compounds are commercially

available that are tailored for vari-

ous downstream applications (e.g.,

triple charged 8-aminopyrenetri-

sulfonic acid [APTS] for CE, and

protonatable procainamide (Proc)

for enhanced ionization in positive

mode MS) (Figure 3C).

An important advance has

been the development of a new

generation of ‘instant’ labels.

These compounds are based on

the reaction of activated fluores-

cent carbamates (e.g., InstantAB

from Prozyme, 6-aminoquinoyl

N-hydroxysuccinimidyl carba-

mate [AQC], and RapiFluor from

Wate r s) w ith g lycosylamine

(Figure 3D). Compared to SB con-

densation that has a reaction

time of 2–4 hours at approxi-

mately 65 °C, this chemistry

takes place at room temperature

in just minutes. The RapiFluor

label was rationally designed to

combine the strong fluorescence

emission of AQC and the ioniza-

tion benefits of a tertiary amine

(Figure 3D). This label allows a sin-

gle sample preparation to enable

two-dimensional (2-D) glycan

analysis using off line LC–FLR

and MS or online LC–FLR–MS.

It is conceivable that many cur-

rently used f luorescent glycan

labeling reagents could be syn-

thesized in an ‘instant’ format by

reaction with N-N-disuccinimidyl

carbonate. Procainamide is now

commercially available as an acti-

vated carbamate, referred to as

InstantPC (from Prozyme).

Solid-phase extraction (SPE)

Another important aspect of

N-glycan sample preparation is

solid-phase extraction (SPE). SPE

is commonly used to separate gly-

cans that have been fluorescently

derivatized from excess free fluo-

rophore that can obscure glycan

peaks during chromatography.

Often the mAbs analyzed in bio-

pharmaceutical quality control

environments are pure finished

products. In this case, develop-

ment of an effective SPE method

can be relatively straightforward,

especially when the expected rep-

ertoire of glycan structures (e.g.,

glycan size and charge state) is

known. When the sample matrix

is more complicated and biological

analytes and impurities are pres-

ent, an effective SPE strategy that

does not result in a large decrease

in glycan yield must be deter-

mined empirically. In fact, such

samples may require more rigorous

clean up, and pre-labeling SPE may

also be required.

Recently, an alternative to the

traditional cartridge-based SPE

was developed. It involves the

extraction of free N-glycans from

solution using hydrazide function-

alized beads (9). This approach,

termed glycoblotting, was used

to profile N-glycans from human

serum proteins and IgG, cells,

organs, and plant tissues in high

throughput (HTP) formats. Its suc-

cess led to the commercialization

of high-density hydrazide beads,

BlotGlyco (S-BIO). With the emer-

gence of HTP N-glycan profil-

ing, SPE in a 96-well format has

become more commonplace with

options including HyperSep Diol

(Thermo Scientific) and hydro-

philic interaction liquid chroma-

tography (HILIC) (Waters and Nest

Group).

ADVANCES IN GLYCAN SEPARATIONS AND DATA ANALYSISAdvances in separation technolo-

gies have played a critical role in

the progress of glycoanalytics.

Several instrument configurations

permit separation and analysis

of fluorescently labeled glycans.

Common approaches involve first

separating labeled glycans by LC or

CE ahead of their structural analy-

sis. Both technologies have signifi-

cantly improved in resolution and

throughput over the past decade.

Figure 3: (A) 2-Aminobenzamide (2-AB) and (B) InstantAB N-glycan labeling

using Schiff Base and reactive carbamate chemistry, respectively. (C) Selection

of amine functionalized fluorophores frequently used for Schiff Base labeling. (D)

Features of instant labels and the examples AQC and RapiFluor.

H2NH2N

H2N

H2N

H2N

N

N

H

H2N

H2N

NH2

NH2

NH2

H2N

HO

HOOH

(A) (C)

(B)

(D)

Rapid Labels

2-aminobenzamide (2-AB)

Schiff base condensation

Activated carbamate chemistry

Activated carbamate

6-aminoquinolyl-N-hydroxysuccinimidylcarbamate(AQC)

fluorophore

RapiFluor

Charge Tag

InstantAB

glycosylamine

reducing sugar

Procainamide (Proc)

8-aminopyrene-1,3,6 -

trisulfonic acid (APTS)

2-aminobenzoic acid

(2-AA)

2-aminoacridone (AMAC)

2-aminopyridine (AP)

OH

OH

OH

NH

NH

HN

RO

RO

O

O

O

O

O

O

O

O O

O O

S

S S

Na+

Na+ Na+

-O

-O-O

HO

HO

HO

OH

OH

OH

NH

NH

HN

HN

N

HCLHN

RO

RO

O O

HN

N

O

O

O

O

HN

N

O

RR = or

O

O

O

HN

N

N

O

O

O

O HN

N

N

OHN

N

O

O

O

O

O

O

O

OO

O

O


Quality Testing

Ultra-High Performance

Liquid Chromatography

A major advance has been the

development and commercializa-

tion of ultra-high performance

liquid chromatography (UHPLC),

liquid chromatography systems

capable of functioning at high pres-

sures (e.g., 10,000 psi). These sys-

tems offer shorter sample run times

and greater resolution than older

HPLC systems. Additionally, in

the past, characterization of com-

plex N-glycan structures typically

required multiple offline chromato-

graphic techniques (referred to as

2- and 3-D analyses). Considerable

time is now being saved by online

coupling of UHPLC systems fit-

ted with HILIC columns to both

fluorescence detection (FLR) and

tandem mass spectrometry (MS/

MS) systems. Such powerful ana-

lytical systems are now becoming

mainstream and are amenable for

use in biopharma for simultaneous

relative quantification of N-glycans

(FLR) and structure confirmation

using accurate mass and fragmen-

tation. Adding to the strength

of HILIC–UPLC platforms is the

relational database GlycoBase3+

(NIBRT), which contains normal-

ized retention times for glycans

expressed as glucose unit (GU)

values (10). This resource contains

more than 600 GU values and is

a powerful tool that can assist in

structural assignments.

Capillary electrophoresis

Capillary electrophoresis (CE) is

an established analytical tech-

nique routinely used in the bio-

pharmaceut ica l indust r y for

the separation of f luorescently

labeled N-glycans. CE utilizes an

applied voltage to separate ions

based on their electrophoretic

mobility. Typical instrumenta-

tion consists of a high-voltage

power supply; a sub-millimeter,

micro, or nanofluidic capillary;

and a LIF or MS detector. For

N-glycan profiling, CE sample

run times are fast, and the tech-

nique is ideally suited to mul-

t iplex ing and r unning h igh

numbers of samples with low

concent rat ion, wh i le ma i n-

taining high resolution. Multi-

capillary CE, as designed for DNA

sequencing, is now routinely

combined with on-column LIF

detection. Direct structural infor-

mation on the glycan peaks in

the electrophoretic profile, how-

ever, is not obtained. This is

changing with the development

of glycan mobility databases and

related bioinformatics tools to

help assist structure assignments.

Currently, character izat ion is

performed using co-migration

w ith commerc ia l ly ava i lable

standards and exoglycosidase

treatment followed by analysis of

migration shifts. In 2014, Agilent

and Picometrics described the

first online CE–LIF–MS instru-

mentation for biopharmaceuti-

cal applications (11). Also, recent

efforts have focused on develop-

ing methodology to deliver CE–

MS profiles that can be compared

to those generated using standard

CE–LIF of ATPS labeled glycans,

so that unidentified peaks can be

more easily assigned (12).

Exoglycosidase sequencing

The structures in each chromato-

graphic peak of a glycan profile

must be sequenced to ensure the

quality of a biopharmaceutical.

This is often done with fragmen-

tation MS. Treatment of samples

w ith exoglycosidases hav ing

Figure 4: (A) Exoglycosiadase enzymes typically used to digest fluorescently

labeled N-glycans derived from a mAb produced in Chinese hamster ovary

(CHO) cells. Enzymes are Neuraminidase A from Arthrobacter ureafaciens

(ABS) for removal of α(2-3,6,8,9) linked sialic acid, Bovine Kidney Fucosidase

(BKF) for removal of core and outer arm fucose in α(1-2,3,4,6) linkages, β-N-

Acetylglucosaminidase S from Streptoccocus pneumoniae (GUH) for removal

of β(1-2,3,4,6) linked N-acetylglucosamine and Bovine Testes Galactosidase

(BTG) for removal of β(1-3,4) linked galactose. (B) hydrophilic interaction liquid

chromatography-fluorescent (HILIC–FLR) profiles depicting enzyme array

digestion of 2-AB labeled human serum IgG N-glycans.

(A) (B)

UND

ABS

ABS+BKF

ABS+BKF+BTG

ABS+BKF+BTG+GUHM3

HILIC Retention Time (RT)

N-Acetylneuraminic acid (NANA)

N-Acetylglucosamine (GlcNAc)

Mannose (Man)

Fucose (Fuc)

Galactose (Gal)

Neuraminidase A

(ABS)

-N-Acetylglucosaminidase S

(GUH)

-Galactosidase

(BTG)

Sequential

Exoglycosidase

Digestion

‘Core’ N-glycan (M3)

Fucosidase

(BKF)6 4

4 4

4

2

3

26

6

6

β

β

β

β

β

β

β β

α

α

α

α

4 43

6β β

α

α

α


Quality Testing

highly defined specificities, how-

ever, can enhance st ructural

interpretations. Exoglycosidases

sequentially remove monosac-

charides from the non-reducing

end of a glycan, and give infor-

mation about the type of sugar

removed and how it was linked

(anomer and linkage) to the gly-

can (Figure 4) (2). The use of exo-

glycosidase digestion panels to

decipher N-glycan structure is an

experimentally straightforward

approach that was first described

in the 1980s (13). For many years,

however, the supply and purity of

these reagents was inconsistent.

An import advance has been the

development of recombinant and

highly pure versions of the field’s

preferred exoglycosidases for gly-

can sequencing applications that

are manufactured under ISO9001

and ISO13485 quality standards

(New England Biolabs).

FUTURE TRENDS IN GLYCAN ANALYSISTechnical advances in glycan

analysis will enable better defini-

tion of the guidelines for assess-

ing the glycan composition of

biologics. Currently, there is

still a lack of clarification on the

level of glycan characterization

required for any given biologic,

or what deviations in glycosyl-

ation are acceptable, generating

potential commercial and legal

risks for companies (14). Federal

agencies such as FDA, European

Medicines Agency (EMA), and

National Institute for Standards

and Technology (NIST), as well as

academic organizations like the

Minimum Information Required

for a Glycomics Exper iment

(MIRAGE), are improving guide-

lines and analytical standards.

These improved guidelines have

aided many companies in devis-

ing custom in-house programs to

show that they understand, mea-

sure, and control the glycosylation

of their products. However, more

rigorous industry standardization

is needed.

Advances in N-glycan analysis

will continue to enable impor-

tant possibilities in biotech and

d r ug manufac t u r ing beyond

quality control of biotherapeu-

tics. The ability to more read-

ily assess glycan structure opens

a world of opportunit ies that

a f fec t many aspec ts of drug

development. In the next decade,

it is likely that faster and simpler

glycan analysis will be routinely

used on-line to address glycan

composition during a manufac-

turing process. This will allow

faster troubleshooting of altered

glycosylation and the effect of

varying bioprocessing parame-

ters on glycan structure to be

bet ter understood. Improved

analytics will allow the tailor-

ing (glycoengineering) of glycan

composition to create more effi-

cacious biologics or bio-betters

to become more commonplace

in the drug-discovery process.

Glycoengineering may also pro-

vide new options for developing

intellectual property positions

a round novel drugs. Fina l ly,

inc reases in the throughput

of glycan analysis promises to

improve cell line development

processes, whereby manufac-

turing lines that have the best

yields and most desired glycan

profiles can be identified.

With regards to glycan analysis

workflows and instrumentation,

the field will continue improving

on speed, automation, through-

put, and sensit iv it y. Sample

preparation methods will likely

continue toward higher through-

put platforms (e.g., 384 well) and

those capable of profiling highly

hete rogeneous g lycosylat ion

from complex sample matrices.

Additionally, use of exoglycosidase

arrays as part of sample prepara-

tion will likely become more com-

monplace and will improve the

quality of data interpretation.

Finally, instrumentation may

move closer toward the dream of

fully automated sample prepara-

tion, analysis, and interpretation

of data in a single machine. For

example, HPLC chip technology

systems have implemented re-

usable microfluidic chips for the

online de-glycosylation of mAbs,

followed by enrichment, separa-

tion, and ionization of glycans. It

is anticipated that future methods

may further explore miniaturiza-

tion and automation of sample

preparation and analysis. Such

concepts may one day help move

glycan analysis from the realm

of a specialty skill to that of a

routine analysis that can be per-

formed by any technician.

REFERENCES1. S. Aggarwal, Nat. Biotechnol.

32 (1), 32-39 (2014).

2. S. A. Berkowitz, J. R. Engen, J. R.

Mazzeo, and G. B. Jones, Nat. Rev.

Drug Discov. 11(7), 527-540 (2012).

3. O. Hossler, S.F Khattak, and

Z.J. Li, Glycobiology. 19

(9), 936–949 (2009).

4. K. Mariño, J. Bones, J. J. Kattla,

and P. M Rudd, Nature Chem.

Biol. (6), 713-723 (2010).

5. H. Stöckmann, R. M. Duke, S. M.

Martín, and P. M. Rudd, Anal. Chem.

87 (16), 8316–8322 (2015).

6. Y. Q Yu, M. Gilar, J. Kaska, and J.

C. Gebler, Rapid Commun. Mass

Spectrom; 19, 2331–2336 (2005).

7. A. Bielik et al., “Novel Formats

of PNGase F for N-Glycan

Removal Workflows and

Applications”, DOI: 10.13140/

RG.2.1.3263.3442, abstracts of

Well Characterized Biotechnology

Pharmaceutical (WCBP) Conference

(Washington, DC, Jan, 2015).

8. M. Collin and, A. Olsen, EMBO J.

20 (15), 3046-3055 (2001).

9. M. Abe; Polym. J. 44, 269−277 (2012).

10. M. P. Campbell, L. Royle, C. M.

Radcliffe, R. A. Dwek and, P. M. Rudd,

Bioinformatics. 24, 1214−1216 (2008).

11. L. A. Gennaro and O. Salas-

Solano, Anal. Chem. 80 (10),

3838-3845 (2008).

12. S. C. Bunz, E. Rapp, and C. Neususs,

Anal. Chem. 85, 10218-10224 (2013).

13. P. M. Rudd, et al, Nature.

388, 205-207 (1997).

14. J. Fournier, BioPharm International

28 (10), 32-37 (2015). ◆


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BIOLOGICS NEWS PIPELINE

IN THE PIPELINE

Amgen’s Biosimilar BLA for ABP 501 Submitted to FDA

Amgen announced the submission of a biologics

license application (BLA) with the FDA for ABP 501,

a biosimilar candidate to Humira (adalimumab).

This represents Amgen’s first BLA submission using

the 351(k) biosimilar pathway.

ABP 501 is a biosimilar candidate to adalimumab, an

anti-TNF-α-monoclonal antibody, which is approved

in many countries for the treatment of various inflam-

matory diseases. Amgen’s BLA submission includes

analytical, clinical, and pharmacokinetic data. Phase

III comparative efficacy and safety studies were con-

ducted in both moderate-to-severe plaque psoriasis

and moderate-to-severe rheumatoid arthritis.

The Phase III studies met their primary endpoints,

showing clinical equivalence to adalimumab. Safety

and immunogenicity of ABP 501 were also compa-

rable to adalimumab. Data to support the transition of

adalimumab patients to ABP 501 are included in the

submission.

It was initially unclear whether or not Amgen was in

support of the development of biosimilars. The com-

pany previously said minor changes in formulation

may have significant affects on patient safety. During

an Oct. 28, 2015 third-quarter earnings call, Amgen

CEO, Bob Bradway, said the company was preparing

to file clinical trial data with FDA for ABP 501, and

expected legal pushback from AbbVie, the original

developer of Humira.

AbbVie CEO Richard Gonzalez has said the com-

pany plans to aggressively defend its patents for

Humira from biosimilar competitors, many of which

will protect the product until 2022. AbbVie currently

has 70 patents protecting Humira, and the company

expects nearly $18 billion in global sales will come

from Humira alone. Amgen currently has nine biosim-

ilar molecules in development and is expected to

launch ABP 501 in 2017.

Rice University Scientists Develop

Method to Control Infectivity of Viruses

Scientists at Rice University have developed a

method that uses light to control the infectivity of

viruses and gene delivery to the nuclei of target cells.

The method uses two shades of red light to control

the level and spatial distribution of gene expression

in cells via an engineered virus.

The scientists at Rice built custom adeno-associated

virus (AAV) vectors by incorporating proteins that

naturally come together when exposed to red light,

and break apart when exposed to far red. These natu-

rally light-responsive proteins help the viral capsids—

the hard shells that contain genetic payloads—enter

the host cell nuclei.

The protein pair comprises phytochrome B and its

binding partner phytochrome interacting factor 6

(PIF6), found in thale cress. The researchers generated

host cells that express phytochrome B tagged with a

nuclear localization sequence, a small peptide known

to help shuttle proteins into the nucleus more effec-

tively. The smaller PIF6 was then attached to the out-

side surface of the virus capsid. This is the first time

optogenetic proteins have been used to control the

infectivity of viruses.

According to Suh, the platform may be used in

the future to control what cells and tissues express,

and at what level. The strategy could also find use in

tissue-engineering applications like bioscaffolds for

implantation.

Adaptimmune and Universal Cells Collaborate

to Develop Allogenic T-Cell Therapies

Adaptimmune Therapeutics, a clinical-stage biophar-

maceutical company focused on cancer immuno-

therapy products, and Seattle-based Universal Cells,

a genome-editing company developing universal

donor stem cells, have entered into a collaboration

and license agreement for the development of alloge-

neic T-cell therapies.

The enhanced T-cell technology involves the

selective engineering of cell surface proteins (T-Cell

Antigen Receptors) and class I and class II human leu-

kocyte antigen proteins, without the use of nucleases,

to develop universal T-cell products. The companies

are planning to develop off-the-shelf allogeneic affin-

ity-enhanced T-cell therapeutics to treat large patient

populations.

Under the terms of the agreement, Universal Cells

will grant Adaptimmune an exclusive, sub-licens-

able, worldwide license to use, sell, supply, manu-

facture, import, and develop products and services

utilizing Universal Cells’ technology within the

T-cell immunotherapy field. Universal Cells will

receive an upfront license and start-up fee of $5.5

million, and will be eligible for up to $41 million

in milestone payments for certain development

and product milestones. Universal Cells would also

receive a profit-share payment for the first product

and royalties on sales of other products utilizing its

technology.

From R&D to production—

all from one source.

www.eppendorf.comEppendorf® and the Eppendorf logo are registered trademarks of Eppendorf AG, Germany. New Brunswick™ is a trademark of Eppendorf AG, Germany.

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