The Science & Business of Biopharmaceuticals

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February 2016

Volume 29 Number 2

FINE TUNING THE

FOCUS ON BIOPHARMA

ANALYTICAL STUDIES

CELL THERAPIES

ADVANCES IN ASSAY

TECHNOLOGIES FOR

CAR T-CELL THERAPIES

PEER-REVIEWED

THE EMERGING VIEW

OF ENDOTOXIN

AS AN IIRMI

REGULATIONS

INNOVATIVE THERAPIES

REQUIRE MODERN

MANUFACTURING SYSTEMS

www.biopharminternational.com

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BioPharmThe Science & Business of Biopharmaceuticals

EDITORIALEditorial Director Rita Peters rpeters@advanstar.comSenior Editor Agnes Shanley ashanley@advanstar.comManaging Editor Susan Haigney shaigney@advanstar.comScience Editor Randi Hernandez rhernandez@advanstar.com Science Editor Adeline Siew, PhD asiew@advanstar.comCommunity Manager Caroline Hroncich chroncich@advanstar.comArt Director Dan Ward dward@media.advanstar.comContributing Editors Jill Wechsler, Jim Miller, Eric Langer, Anurag Rathore, Jerold Martin, Simon Chalk, and Cynthia A. Challener, PhD Correspondent Sean Milmo (Europe, smilmo@btconnect.com) ADVERTISING

Publisher Mike Tracey mtracey@advanstar.comWest/Mid-West Sales Manager Steve Hermer shermer@advanstar.comEast Coast Sales Manager Scott Vail svail@advanstar.comEuropean Sales Manager Chris Lawson clawson@advanstar.comEuropean Sales Manager Wayne Blow wblow@advanstar.comC.A.S.T Data and List Information Ronda Hughes rhughes@advanstar.comReprints 877-652-5295 ext. 121/ bkolb@wrightsmedia.com Outside US, UK, direct dial: 281-419-5725. Ext. 121 PRODUCTION Production Manager Jesse Singer jsinger@media.advanstar.com AUDIENCE DEVELOPMENT Audience Development Rochelle Ballou rballou@advanstar.com

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© 2016 Advanstar Communications Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical including by photocopy, recording, or information storage and retrieval without permission in writing from the publisher. Authorization to photocopy items for internal/educational or personal use, or the internal/educational or personal use of specific clients is granted by Advanstar Communications Inc. for libraries and other users registered with the Copyright Clearance Center, 222 Rosewood Dr. Danvers, MA 01923, 978-750-8400 fax 978-646-8700 or visit http://www.copyright.com online. For uses beyond those listed above, please direct your written request to Permission Dept. fax 440-756-5255 or email: mcannon@advanstar.com.

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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

Madhavan Buddha Senior Manager

Biocon Research Limited

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 February 2016

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

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BioPharm InternationalJTTFMFDUJWFMZBCTUSBDUFEPSJOEFYFEJOrBiological Sciences Database (Cambridge Scientific Abstracts)rBiotechnology and Bioengineering Database (Cambridge Scientific Abstracts)rBiotechnology Citation Index (ISI/Thomson Scientific)rChemical Abstracts (CAS) rŞScience Citation Index Expanded (ISI/Thomson Scientific)rWeb of Science (ISI/Thomson Scientific)

The Science & Business of Biopharmaceuticals

INTERNATIONALINTERNATIONAL

February 2016

Volume 29 Number 2

FINE TUNING THE

FOCUS ON BIOPHARMA

ANALYTICAL STUDIES

CELL THERAPIES

ADVANCES IN ASSAY

TECHNOLOGIES FOR

CAR T-CELL THERAPIES

PEER-REVIEWED

THE EMERGING VIEW

OF ENDOTOXIN

AS AN IIRMI

REGULATIONS

INNOVATIVE THERAPIES

REQUIRE MODERN

MANUFACTURING SYSTEMS

www.biopharminternational.com

Cover: moodboard/Getty Images; Dan Ward

6 From the Editor Biologics contribute to rebirth of biopharma innovation. Rita Peters

8 US Regulatory Beat FDA and industry see progress and challenges in bringing cutting-edge medicines to patients.Jill Wechsler

10 Perspectives on Outsourcing Heightened global uncertainty could slow bio/pharma development activity. Jim Miller

50 Biologics News Pipeline

50 Ad Index

ANALYTICAL ADVANCES

Fine Tuning the

Focus on Biopharma

Analytical Studies

Cynthia A. ChallenerTime and sensitivity are essential for

analytical technologies in all phases of

biopharma development. 12

PROTEIN ENGINEERING

Antibody Production

in Microbial Hosts

Anurag S. Rathore and Jyoti BatraThe authors review the status

of expression of antibodies in

microbial hosts. 18

PEER-REVIEWED

The Emerging View of

Endotoxin as an IIRMI

Kevin WilliamsThis article gives a perspective

for understanding potential risks

from low endotoxin recovery. 24

CELL THERAPIES

Advances in Assay

Technologies for

CAR T-Cell Therapies

Alison ArmstrongRapid methods to test CAR-T

therapies for potential contamination

are on the horizon. 32

GENE THERAPY

Use of an E. coli pgi Knockout Strain as a

Plasmid Producer

Cláudia P. A. Alves, Sofia O. D. Duarte, Gabriel A. Monteiro, and Duarte Miguel F. PrazeresThe authors describe the impact of the

knocking of the pgi gene of the wild type

MG1655 strain on the growth kinetics of

plasmid-free and plasmid-bearing cells. 38

FILTER INTEGRITY TESTING

Failure Mode

Effects Analysis for

Filter Integrity Testing

Magnus SteringUnderstanding of the risks associated

with FMEA is crucial in lot release testing.

43

PROTEIN AGGREGATION

Complementary Techniques

for the Detection and

Elucidation of Protein

Aggregation

Lisa Newey-KeaneThe author reviews some of the

techniques that can yield valuable

information on protein stability

during characterization studies. 46

Volume 29 Number 2 February 2016

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6 BioPharm International www.biopharminternational.com February 2016

From the Editor

Report:

Biologics

contribute

to rebirth of

biopharma

innovation.

Biopharma Innovation Born Again?

Despite a jump in new drug approvals in the past two years, the pace at

which new drugs are brought to market is extremely slow compared

with the introductions of technologies in other markets. While consum-

ers camp out overnight to be the first to buy the latest smartphone, patients

often wait years for effective therapies.

Still, biopharma companies are making a mark in the global innovation

arena. In a compilation of the top innovator companies worldwide, pharma-

ceutical companies claimed seven spots, an increase from four companies

in 2014. Now in its fifth year, the Thomson Reuters 2015 Top 100 Global

Innovators list (1) identifies corporations that successfully invest in R&D,

develop protected, commercialized products, and outperform companies with

lesser innovation efforts in revenue and R&D spend.

To select the leading 100 innovative companies, Thomson Reuters analyzed

patent and citation data across four criteria: volume, success, globalization, and

influence. To qualify, a company must have 100 unique inventions protected by

a granted patent over the most recent five-year period. The success criteria mea-

sures the ratio of inventions described in published applications to inventions

protected with granted patents. The globalization factor evaluates inventions pro-

tected in multiple regions: China, Japan, Europe, and the United States. Influence

measures the impact of an invention: how often it is cited by other organizations.

When ranked by the number of companies on the list, the pharmaceutical

industry trailed only the chemical, semiconductor and electronic compo-

nents, and automotive segments. The award report noted advances in genom-

ics, emergence of targeted therapies, and use of biologics as contributing to a

“pseudo rebirth, despite the decline of the Blockbuster drug era.”

The pharmaceutical companies on the Top 100 Global Innovators list are:

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In addition to the global innovators list, Thomson Reuters compiled a list of

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as a hub for the semiconductor and electronics development, biotechnology

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Innovation is also apparent in the types of instruments and methods avail-

able for today’s bio/pharma research and quality control. In this issue, industry

experts review advances in analytical technology, and present a wish list of

new capabilities they would like to have.

With two major trade shows and conferences for analytical research on

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(FSNBOZGSPN.BZoUIFCJPQIBSNBJOEVTUSZDBOFYQFDUOFXUFDIOPM

ogy introductions from instrument vendors over the next few months. Perhaps

these new tools can further accelerate bio/pharma innovation, and cut the

wait times for patients seeking cures and treatments.

Reference

1. Thomson Reuters, Top 100 Global Innovators, online http://top100innovators.

stateofinnovation.thomsonreuters.com/, accessed Jan. 28, 2016. X

Rita Peters is the

editorial director of

BioPharm International.

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8 BioPharm International www.biopharminternational.com February 2016

Regulatory Beat

Vis

ion

so

fAm

eri

ca

/Jo

e S

oh

m/G

ett

y I

ma

ge

s

FDA set a 19-year record in 2015 in approv-

ing more new drugs and biologics, and

agency officials expect this pace to continue.

Manufacturers are testing a full pipeline of impor-

tant, new therapies to treat both rare diseases and

widespread serious conditions such as high cho-

lesterol, diabetes, multiple myeloma, and a range

of cancers. More biosimilars are poised to come to

market in 2016 following FDA approval of the first

such therapy in 2015. At the same time, though,

the development of such innovative and targeted

therapies heightens the importance of establishing

production systems and processes capable of fast

scale-up of high-quality complex products.

The surge in new drug development will be

encouraged by increased public support for bio-

medical research and innovation, led by President

Obama’s campaign to cure cancer. Presidential

hopefuls are championing medical cures, as seen

in Hillary Clinton’s proposal to boost Alzheimer’s

research. The 2016 budget bill approved by

Congress at the end of 2015 increased funding

significantly for the National Institutes of Health

(NIH) and other government research programs,

while providing a small, but encouraging increase

in FDA’s appropriation.

The spending measure directs NIH to allot por-

tions of its $2 billion budget boost to

Alzheimer’s disease research, preci-

sion medicine, the Brain Research

through Advancing Innovat ive

Neurotechnologies (BRAIN) initiative,

and development of antimicrobial med-

icines. The development of new anti-

biotics gained from several spending

provisions: in addition to $100 million

to NIH for that purpose, the Centers

for Disease Control and Prevention

(CDC) received $160 million to prevent

and monitor superbug outbreaks, and

the Biomedical Advanced Research

and Development Authority (BARDA)

received nearly $100 million to help test

new therapies and diagnostics to protect against

infectious disease threats.

FDA also was instructed to improve its over-

sight of antibiotics as part of its $2.7 billion appro-

priation for 2016. Most of its $132 million budget

increase goes to expanding food safety programs,

but small amounts are allotted to ensuring the

accuracy of genetic tests important to precision

medicine, expanding foreign inspections of high-

risk operations, and supporting orphan drug devel-

opment. The funding bill further instructs FDA to

finalize guidance on biosimilar development and

to do more to prevent drug shortages (1). And it

puts a hold on FDA consideration of new therapies

that use genome-editing tools capable of modify-

ing the DNA of human embryos.

Legislative action further made permanent

the federal R&D tax credit, a long-sought change

expected to boost private sector investment in

biotech companies. Medical device makers gained

a two-year delay in paying a new tax authorized

by the Affordable Care Act (ACA), which manu-

facturers say will promote device innovation. And

the legislators extended the rare pediatric disease

priority review voucher program until September

2016 to keep it going until Congress can review

and reauthorize it.

MANUFACTURING CONCERNSThese developments generate optimism that scien-

tists finally will begin to transform the treatment

of disease based on decades of important genomic

research. FDA’s Center for Drug Evaluation and

Research (CDER) approved 45 new molecular enti-

ties (NMEs) and biotech therapies in 2015, beat-

ing the near-record set in 2014. FDA’s Center for

Biologics Evaluation and Research (CBER) also

approved more than a dozen new biologics, vac-

cines, blood products, and diagnostics.

In the process, FDA met nearly all the review

timeframes and goals set by the Prescription Drug

User Fee Act (PDUFA V), reported John Jenkins,

director of CDER’s Office of New Drugs (OND), at

Innovative Therapies Require Modern Manufacturing SystemsFDA and industry see progress and challenges in bringing cutting-edge medicines to patients.

Jill Wechsler

is BioPharm International’s

Washington editor,

Chevy Chase, MD, 301.656.4634,

jwechsler@advanstar.com.

February 2016 www.biopharminternational.com BioPharm International 9

Regulatory Beat

the FDA/CMS Summit in December

2015. Jenkins noted that most

NMEs were approved in one review

cycle; more new drugs reached the

market first in the United States;

and CDER continues to receive

hundreds of requests from man-

ufacturers for breakthrough drug

designations, a program that has

led to the approval of more than 20

innovative products (2).

Jenkins voiced concern, however,

about the continued emergence of

new therapies, noting the need for

FDA to assist pharmaceutical man-

ufacturers seeking to accelerate the

production timeframes for more tar-

geted and complex medical products.

In developing and bringing to mar-

ket important new therapies, Jenkins

noted, it’s “often manufacturing and

inspections that are the rate-limiting

steps” for expedited approvals, not

clinical development. CDER often

faces difficulties in scheduling timely

preapproval inspections within the

six-month review timeframe for

breakthrough drugs, he explained,

and small biotech companies have

run into difficulties with contract

manufacturers that have quality and

data problems uncovered during

plant inspections.

FDA seeks to avoid such issues

by clarifying policies and offering

new solutions to drug development

and manufacturing challenges. A

proposed rule, for example, aims to

clarify requirements for producing

fixed-combination and co-packaged

drugs and over-the-counter medi-

cines (3). The agency also is support-

ing the development of innovative

in-vitro diagnostics and combination

products through improved coor-

dination of oversight for combina-

tion therapies. CDER further aims

to encourage manufacturer adop-

tion of cutting-edge pharmaceutical

production technology by provid-

ing assistance in meeting regulatory

requirements for innovative systems.

A draft guidance document advises

manufacturers on how to request

meetings with experts on CDER’s

Emerging Technology Team to dis-

cuss regulatory issues related to sub-

mitting chemical, manufacturing,

and controls (CMC) data for new

manufacturing systems involving

both innovator and generic prod-

ucts. The aim is to avoid delays that

could discourage the adoption of new

production methods able to improve

drug safety and quality (4).

GLOBAL HARMONIZATIONFurther global harmonization

of drug-development policies

also is on the horizon following

major changes at the International

Council for Harmonisation (ICH).

The organization now seeks more

involvement of regulatory authori-

ties and manufacturers in Asia,

Africa, and the Americas in the ICH

standards-setting process. FDA, the

European Medicines Agency, and

other established authorities, more-

over, are collaborating more to

avoid duplication in drug facility

inspections and in the registration

of innovative medicines for rare

conditions and for children.

FDA also is under pressure from

Congress to expand its capacity for

conducting foreign drug inspections.

The House Energy & Commerce

(E&C) Committee recently asked the

Government Accountability Office

(GAO) to examine the effectiveness

of FDA foreign drug inspections and

overseas offices in the face of ever-

expanding imports of drugs and APIs.

Both Republicans and Democrats

want the GAO to update its 2010

report on FDA’s overseas regulatory

capacity, with a focus on agency

progress in implementing a risk-

based inspection system and in staff-

ing up its foreign offices (5). FDA’s

ability to process drug applications

efficiently and to ensure drug safety,

efficacy, and quality will continue to

draw scrutiny on Capitol Hill, along

with drug pricing issues. A broad

group of legislators has requested

multiple documents from the agency

on generic-drug approval times,

action dates, user-fee payments, and

other items to help assess market

competition issues related to generic-

drug price increases.

Biosimilar development will

remain in the spotlight, along with

efforts to ensure the safety and qual-

ity of “complex” generic drugs that

are not regulated as biologics, but

are more diverse than conventional

drugs. The legislators have asked

GAO to ensure that FDA requires

appropriate scientific analytic meth-

odologies to fully identify the struc-

ture and properties of such products

and to ensure their bioequivalence,

safety, and efficacy (6).

REFERENCES 1. Division A-Agriculture, Rural Development,

Food And Drug Administration, And

Related Agencies Appropriations Act,

2016 Congressional Directives, http://

docs.house.gov/meetings/RU/

RU00/20151216/104298/HMTG-114-

RU00-20151216-SD002.pdf, accessed

Jan. 5, 2016.

2. J. Jenkins, MD, CDER New Drug Review:

2015 Update, FDA/CMS Summit, Dec. 14,

2015, http://www.fda.gov/downloads/

AboutFDA/CentersOffices/Officeof

MedicalProductsandTobacco/CDER/

UCM477020.pdf, accessed Jan. 5, 2016.

3. FDA, 21 CFR Parts 300, 330, and 610,

Fixed-Combination and Co-Packaged

Drugs: Applications for Approval and

Combinations of Active Ingredients Under

Consideration for Inclusion in an Over-the-

Counter Monograph, Federal Register, 80

(246), Dec. 23, 2015, www.gpo.gov/

fdsys/pkg/FR-2015-12-23/pdf/2015-

32246.pdf, accessed Jan. 5, 2016.

4. FDA, Advancement of Emerging Technology

Applications to Modernize the

Pharmaceutical Manufacturing Base

Guidance for Industry, Draft Guidance

(CDER, December 2015).

5. US House of Representatives Energy &

Commerce Committee, Bipartisan

Committee Leaders Enlist Government

Watchdog on FDA’s Foreign Inspection

Program, Dec. 18, 2015, http://

energycommerce.house.gov/press-

release/bipartisan-committee-leaders-

enlist-government-watchdog-fdas-foreign-

inspection, accessed Jan. 5, 2016.

6. US House of Representatives Energy &

Commerce Committee, Letter to GAO

Requesting a Study on FDA’s Pathway to

Review Generic Non Biologic Complex

Drugs, Dec. 11, 2015, http://

energycommerce.house.gov/letter/letter-

gao-requesting-study-fda%E2%80%99s-

pathway-review-generic-non-biologic-

complex-drugs, accessed Jan. 5, 2016.

10 BioPharm International www.biopharminternational.com February 2016

Perspectives on Outsourcing

Do

n F

arr

all/G

ett

y I

ma

ge

s

Bio/pharmaceutical companies, and the

companies that serve them, tend to

think they are immune from broader

macroeconomic and political developments.

As populations age, emerging middle classes

expand, and scientific knowledge progresses,

research on new drugs and demand for

new therapies seem to follow an inexorably

upward trend.

However, the global financial crisis of 2008

demonstrated that the industry is not isolated

from macro events. Early-stage companies,

which drive the early development engine,

had great difficulty raising money as the

availability of venture capital declined and

the window for initial public offerings (IPOs)

closed altogether. It took nearly five years for

drug-development funding to become readily

available again, and for the pharmaceutical

services industry to once again thrive.

It’s worth reminding industry participants

of the vulnerability of the bio/pharmaceutical

industry to macro events because memories

tend to be short, and the global

environment is becoming uncer-

tain again. The coincidence of

two events, in particular, threaten

the global economic equilibrium:

the collapse of commodity prices

and the resumption of interest

rate hikes by the central bank

of the United States, the Federal

Reserve.

Most people are keenly aware

of the steep decline in oil prices,

but prices have also collapsed

for a broad range of commodi-

ties including industrial metals

such as copper and agricultural

products such as soybeans. The

falling prices are a result mainly

of increased supply (thanks to fracking for oil

and robust harvests) coming at a time when

demand, especially from China, has slowed

considerably. In affected economies, govern-

ment tax and royalty revenues have decreased

sharply, as have employment, foreign cur-

rency reserves, and investment.

The decision by the Federal Reserve to

begin raising interest rates is exacerbating the

problem for emerging markets. Many busi-

nesses in those countries had borrowed US

dollars heavily to fund investment because

the Federal Reserve had kept interest rates

so low. Those companies, and some govern-

ments as well, are now facing a financial crisis

brought on by rising interest rates, declining

revenues, and rising repayment costs caused

by the depreciation of their local currencies

against the US dollar.

BIO/PHARMA IMPLICATIONSSo what do these macroeconomic develop-

ments have to do with the prospects of bio/

pharma companies, CROs, and CDMOs?

There are at least three negative implications

of the deteriorating global financial outlook

for the bio/pharma services industry.

Bio/pharma industry growth has been

hurt by the problems in emerging markets.

Expansion in those countries, with their

rapidly growing middle classes, was a major

Bio/pharma industry

growth has been hurt

by the problems in

emerging markets.

Macro Matters Heightened global uncertainty could slow bio/pharma development activity.

Jim Miller is president of PharmSource

Information Services, Inc., and

publisher of Bio/Pharmaceutical

Outsourcing Report,

tel. 703.383.4903,

Twitter@JimPharmSource,

info@pharmsource.com,

www.pharmsource.com

February 2016 www.biopharminternational.com BioPharm International 11

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Scan here withyour cell phoneto learn more

For complete details contact:

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Perspectives on Outsourcing

element of the post-patent cliff

st rategy of many global bio/

pharma companies. Now, declin-

ing government revenues, sky-

rocketing debt service, declining

foreign currency reserves, and

deprec iat ing cur renc ies w i l l

severely limit the ability of those

countries to import and distribute

all but the lowest-cost generics.

CROs and CDMOs have not

been major participants in the

emerging market expansion plans

of their global bio/pharma cli-

ents, so their exposure to these

developments will be limited.

They could see lower product vol-

umes, however, as emerging mar-

ket countries limit imports and

encourage more local production.

A bigger concern is what hap-

pens to investor confidence. The

banking and investment com-

munities will take a big hit as

the financial condition of com-

panies and countr ies around

the world deteriorates. Further,

there is increased uncertainty

surrounding even the strongest

economies, as evidenced by the

turmoil in world stock markets

at the beginning of 2016. While

few financial observers expect a

crisis as severe as the 2008 finan-

cial meltdown, investor nervous-

ness could reduce willingness to

invest, negatively impacting com-

pany valuations, and the ability

of companies to float IPOs and

otherwise raise capital.

That could be a big problem for

the CRO and CDMO industries.

Freely f lowing investment capi-

tal for early-stage companies has

been a huge driver of demand for

CRO and CDMO services in the

past three years. As seen in 2007–

2008, just the fear that finding

new capital could be difficult can

make early-stage companies slow

their spending (e.g., by reduc-

ing the number of candidates in

active development).

US-based CROs and CDMOs

have an additional concern as

the US dollar has appreciated

considerably against most cur-

rencies. The euro has lost 17%

of its value relative to the US

dollar in the past year and that

can considerably alter the com-

parative advantage of CDMOs

in the US and Europe. It means

that a €1 million contract with a

European CDMO, which would

have cost a US client $1.3 million

in 2014, will now cost that US cli-

ent just $1.1 million. On the other

hand, a European client consider-

ing a $1 million manufacturing

or development contract with a

US-based CDMO will be facing a

€909,000 expense today versus

€750,000 just a year ago. Moreover,

the situation could get worse for

US-based CDMOs in the next year

or two as the euro is expected

to depreciate to parity (€1= $1).

US-based CDMOs that have ben-

efitted from a weak US dollar for

nearly 10 years are now facing

strong foreign exchange head-

winds.

The point here is not to wal-

low in doom and gloom about

the macro environment. CRO and

CDMO executives should not take

for granted the robust market con-

ditions they have enjoyed during

the past three years. They need to

be fully cognizant of the macro

environment in which the indus-

try operates and the risks it pres-

ents. As learned in the 2008–2012

period, the bio/pharma industry is

not immune from global economic

and political developments.

12 BioPharm International www.biopharminternational.com February 2016

mo

od

bo

ard

/Cultura

/Gett

y Im

ag

es

Although just a few decades

old, the biopharmaceutical

industry has evolved signifi-

cantly since its inception.

Many candidate biologics today—anti-

bodies and antibody fragments, highly

potent ant ibody-drug conjugates

(ADCs), virus-like particles, cell- and

gene-based therapies, etc.—are differ-

ent from the first simple, recombinant

proteins. Manufacturers have been con-

tinuously challenged to develop ana-

lytical methods for timely and accurate

determination of the chemical, physi-

cal, and therapeutic properties of these

different actives, as well as potential

contaminants throughout the produc-

tion process, from raw material selec-

tion to process analysis, formulation

development, and release testing.

The introduction of biosimilars and

the move toward continuous process-

ing are creating the need for more rapid

and sensitive analytical techniques. The

advent of quality by design (QbD) has

further increased the importance of

analytical methods/technologies within

a manufacturing environment, accord-

ing to Fiona Greer, global director of

biopharma services development at SGS

Life Science Services.

Newer versions of traditional meth-

odologies, such as capillary isoelectric

focusing (cIEF) versus IEF gels, peptide

mapping with liquid chromatography–

tandem mass spectrometry (LC/MS/

MS), and high-performance LC (HPLC)

are available today. Notably, mass spec-

trometry-based methods and next-gen-

eration sequencing technologies are

Fine Tuning the Focus on Biopharma Analytical Studies

Cynthia A. Challener

Time andsensitivity areessential for

analytical technologies in all phases

of biopharma development.

Cynthia A. Challener, PhD

is a contributing editor to

BioPharm International.

Analytical Advances

February 2016 www.biopharminternational.com BioPharm International 13

addressing the need for greater sensitivity in less time.

Automation and high-throughput technologies are

also having an impact. As the industry introduces

more complex and increasingly potent molecular for-

mats with novel, highly potent product-related impu-

rities, however, ongoing advances will be required.

MANY SENSITIVE MASS SPEC METHODSFor product characterization, the most appropriate

techniques will depend on the class of molecule:

protein, glycoprotein, pegylated, ADC, vaccine, etc.

“Improvements in biopharmaceutical mass spectrom-

etry in the past 10 years—in sensitivity, dynamic

range, resolution, mass accuracy, and user-friendli-

ness—have dramatically improved our ability to get

detailed protein molecular information,” says Byron

Kneller, director of analytical/formulation develop-

ment with CMC Biologics.

The continued development and deployment of

LC/MS-based applications are having a significant

impact on both the characterization and quality

testing of biopharmaceuticals, particularly for recom-

binant proteins and monoclonal antibodies, agrees

Mike Garrett, senior director of global marketing

for BioReliance. “While in the past these methods

were reserved for early research into the structure of

these molecules, today, methods are being developed

that bring this technology closer to the quality con-

trol lab,” he observes. Access to more sensitive and

detailed characterization data is allowing manufactur-

ers to better understand and more carefully control

the molecular structures of their products during the

manufacturing process. Garrett also notes that LC/MS

has enabled finer control of bioprocess optimization,

allowing for correlation of process changes to both

molecular structure and yield.

Some of the most recent advances in product char-

acterization techniques have, according to Greer, been

developed in response to challenges encountered

with biotherapeutic products and their post-transla-

tional modifications (PTMs). Glycosylation analysis

in particular has been advanced significantly with

the advent of high-resolution mass spectrometry and

the use of hydrophilic interaction liquid chromatog-

raphy (HILIC) columns for glycans. “Introduction

of the quadrupole orthogonal acceleration time-of-

flight (Q-ToF) geometry and the increased resolving

power of MS now allow the direct determination of

the monoisotopic mass of antibody heavy chains

including modifications such as deamidation,” Greer

explains. Kneller adds that current-generation Q-ToF

and Orbitrap instruments allow for high-resolution

intact mass and peptide mapping measurements for

both characterization and process-development sup-

Analytical Advances

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14 BioPharm International www.biopharminternational.com February 2016

port, and current software contin-

ues to make data processing easier

and faster.

State-of-the-art MS instruments

with markedly increased sensitiv-

ity are also providing profound

insights into the impurity profiles

of biotherapeutics and allowing

the identification of previously

unknown host-cell contaminants,

according to Harald Wegele, head

of analytical development and

quality control in Europe for

Roche. “Sensitive assessment of

specific host-cell proteins (HCPs)

and other contaminants provides

crucial guidance for the develop-

ment of impurity depleting process

steps, which ultimately helps to

warrant a maximum of product

safety,” he states. Additional devel-

opments such as sequential win-

dow acquisition of all theoretical

mass spectra (SWATH) and parallel

reaction monitoring are improv-

ing the quantitative assessment of

process-related impurities.

Greer also expects wider adop-

tion of numerous other MS-based

analytical techniques, includ-

ing ion mobility-MS, capillary

electrophoresis-MS (CE–MS), and

hydrogen-deuterium exchange-MS

(HDX–MS). Wegele adds that size-

exclusion chromatography (SEC)

coupled to native MS already pro-

vides—particularly for novel anti-

body formats like bispecifics—a

fast and easy means for gaining

unachieved levels of information

on, for example, the size-variant

distribution of biotherapeutic, as

early as at the onset of clinical

development. He also points to 2D–

HPLC as providing a convenient

and accurate method for charac-

terizing single product peaks, side

products, and excipients.

MORE RAPID ANALYSESThere is tremendous pressure

on biopharmaceutical compa-

nies to get products to the mar-

ket more quickly and at lower cost

without compromising safety.

Manufacturers are consequently

looking for alternatives to con-

ventional cell-based analytical

methods. Newer personalized

treatments such as cell-based ther-

apies, in fact, require more rapid

release testing because they do

not have long-term stability and

must be administered to patients

soon after they are produced.

Manufacturers are also moving

to continuous processing, which

requires process analytical technol-

ogy (PAT) that provides real-time

process monitoring data.

Several newer testing methods

have been developed and are in

the process of being implemented

by the biopharmaceutical indus-

try, largely in cooperation with

regulatory agencies such as FDA.

Improvements in real-time, quan-

titative polymerase chain reac-

tion (qPCR)-based methods have

allowed for broader detection of

known potential contaminants

with improved speed and accu-

racy, according to Garrett. Newer

nucleic acid detection technolo-

gies, such as next-generation

sequencing, are also being applied

to the quality control testing lab.

“Importantly, these technologies

will allow manufacturers to test

their biopharmaceutical products

for both known and unknown

advent it ious contaminants,”

Garrett says.

Advances in bioassays have also

made potency testing easier, faster,

and more reproducible, according

to Kneller. “The broader availabil-

ity of reporter-gene assays (e.g., for

antibody-dependent cell-mediated

cytotoxicity [ADCC]) testing has

decreased the difficulty of imple-

menting some potency assays,

while access to soluble enzyme-

l inked immunosorbent assay

(ELISA) formats and ready-to-use

analytical cell banks has decreased

both the time needed for potency

assays and assay variability,” he

explains. Wegele adds that novel

LC-, cell-, and surface plasmon res-

onance (SPR)-based assay formats

are facilitating the assessment of

the impact of PTMs on antibody/

bispecific antibody Fc (crystalliz-

able fragment) effector functional-

ity, including pharamacokinetic

(PK) properties (e.g., via FcRn [neo-

natal Fc receptor] affinity chro-

matography). The Fc region of a

therapeutic antibody interacts with

receptors on various types of cells

and is involved in immune-medi-

ated effector functions, such as

ADCC and complement-dependent

cytotoxicity (CDC). It is therefore

potentially important in determin-

ing drug safety and efficacy and

must be fully characterized.

Advances in chromatogra-

phy methods are also enabling

more rapid analyses, according

to Kneller. “The increased use of

ultra high-pressure liquid chroma-

tography (UHPLC) systems and

sub-2 μm columns has enabled

more rapid, higher-resolution chro-

matographic assays, which has

decreased testing time for many

release methods,” he comments.

GAINING THROUGHPUTImplementation of high-through-

put (HTP) methods and expanding

use of automation are additional

avenues the biopharmaceutical

industry is pursuing to achieve

more rapid testing. The challenge

has been to reduce testing times

without loss of accuracy, precision,

specificity, sensitivity, and robust-

ness. Several successes have been

achieved to date, however.

Microfluidic capillary electro-

phoresis (MCE) has, according to

Wegele, become a central pillar for

product quality analytics during

clone selection and bioprocess devel-

opment due to its ease of sample

preparation, robustness, and unri-

valed high-throughput capability.

“This HTP method is indispensable

for meeting the steadily growing

Analytical Advances

February 2016 www.biopharminternational.com BioPharm International 15

Analytical Advances

demand for the shortest possible

sample turnover time and enhanced

time efficiency in present-day bio-

logics development,” he says.

Automated high-throughput

quantification of process-related

impurities (e.g., HCPs and Protein

A), titer, and fermentation broth

supplements such as insulin,

LongR3, etc., via electrochemilu-

minescence immunoassay (ECLIA)

is also now used at Roche to sup-

port process development, process

characterization/process valida-

tion studies, manufacturing, and

in-process control/release test-

ing, according to Wegele. “This

technology is high-throughput-

compatible and greatly reduces

hands-on time. As a result, it

enables novel insights for biopro-

cess development in near real-time

and facilitates the assessment of

process-related impurities deple-

tion,” he says.

Higher-throughput screens for

formulation development coupled

with the use of design-of-experi-

ment (DoE) tools have also enabled

faster, more comprehensive screen-

ing of many formulation condi-

tions and excipients and decreased

the time required for formulation

optimization, according to Kneller.

Often, combinations of light-scat-

tering, intrinsic and extrinsic fluo-

rescence, and calorimetry are used

to rapidly deliver information on

protein stability in many excipient

combinations.

METHODS FOR EMERGING BIOLOGICSRecent years have seen growing

interest in newer types of biologic

actives. Significant numbers of

antibody-based treatments have

been commercialized, and many

more, including those based on

antibody fragments and ADCs,

are in advanced stages of devel-

opment. Successful initial studies

with cell- and gene-based thera-

pies are attracting interest in these

therapies, many of which are now

in clinical trials. While many of

the analyses required to charac-

terize these different classes of

biologic drug substances are the

same, their characterization does

in many cases require different

analytical techniques.

For newer antibody formats,

both Garrett and Wegele note that

LC/MS is a relatively fast method

for gaining high levels of infor-

mation on the size-variant distri-

bution of biotherapeutics at early

development stages. CE is also

providing deeper insights into

the structure of these molecules,

according to Garrett. “Use of these

techniques has led to numerous

improvements in the manufactur-

ing of antibodies and antibody

fragments, particularly when con-

sidering the variables that can now

be investigated and controlled as

part of the manufacturing develop-

ment process,” he asserts.

For cell-based therapies, Garrett

notes that the development and

adoption of rapid, molecular-

based testing methods for both

process and product safety will

enable cell therapy products to

be manufactured in the time-

frames necessary to both manipu-

late patient-derived cells and then

deliver them safely. The develop-

ment of methods for assessing the

safety of the viral backbones used

to produce gene therapies has also

kept pace with their advancement

into the clinic. “Virology-based

tests have been refined such that

they now provide information on

the specific properties and qual-

ity of vector backbones, which

is crucial for ensuring the safety

of these advanced therapeutics,”

Garrett states. He also notes that

molecular methods such as next-

generation sequencing are being

employed to investigate the iden-

tity, purity, and stability of virus-

based gene therapies.

BIOSIMILAR SOLUTIONSFull analytical characterization

of branded biotherapeutics and

potential biosimilar products is

fundamental to the development

of biosimilars, and the pathway

for analytical method develop-

ment for biosimilars is somewhat

different from that of novel bio-

therapeutics, according to Jun Lu,

director of analytical development

for Catalent Pharma Solutions.

“Both release and characterization

methods are required at the very

early stage of biosimilar develop-

ment, because the reference prod-

uct from multiple lots must be

extensively characterized to estab-

lish the target product profile,” he

says. More specifically, analytics

are essential to defining the criti-

cal quality attributes (CQAs) that

form the quality target product

profile (QTPP).

Demonstration of similarities

between the biosimilar and ref-

erence product through side-by-

side comparison (i.e., physical,

biological, and chemical charac-

terization) is required before pro-

gressing into the clinic, according

to Greer. Matching of the amino

acid sequence and PTMs of the

reference product determined by

using LC/MS/MS and other pro-

tein characterization methods

must be performed as a clone selec-

Advances in

bioassays have also

made potency

testing easier, faster,

and more

reproducible.

16 BioPharm International www.biopharminternational.com February 2016

tion criterion, because upstream

and downstream development has

minimal impacts on changing

these CQAs, adds Lu.

Use of orthogonal methods

for biosimilar assessment is also

emphasized by regulators, because

subt le dif ferences between a

biosimilar and the reference prod-

uct may be difficult to detect

using only one analytical method.

FDA in particular has introduced

the concept of “fingerprint-like”

analyses, according to Greer.

“This approach entails the use of

a carefully selected portfolio of

characterization techniques for

primary and higher-order struc-

ture, together with biological and

potency assays producing data

that, when combined, add up to

more than the sum of the parts,”

she says.

For instance, Lu notes that for

analysis of high-molecular-weight

(HMW) species, which present

a high-risk safety concern, sup-

plementing SEC with analyti-

cal ultracentrifugation (AUC) is

strongly recommended. For the

determination of higher-order

structure, a combination of at

least two techniques from a list

including c i rcular dichroism

(CD), Fourier-transform infrared

(FTIR), differential scanning calo-

rimetry (DSC), and HDX–MS can

be used to potentially elucidate

any detailed structure differences.

Garrett adds that advanced cell-

based potency assays are impor-

tant for determining whether

the in-vitro effects of biosimilars

are similar to the originator mol-

ecule. Greer observes, however,

that the link between higher order

structure and biological activity

remains to be explored. She does

note, though, that several tech-

niques are emerging from research

backgrounds to address these

questions, such as HDX–MS and

2D-nuclear magnetic resonance

(NMR) imaging.

Statistical analysis of analyti-

cal data for the determination of

biosimilarity is also required by

FDA to ensure confidence in the

data. One consequence, according

to Lu, has been the replacement of

imaging methods such as sodium

dodecyl sulfate polyacrylamide gel

electrophoresis (SDS–PAGE) and

IEF gels with CE–SDS and cIEF,

respectively, which allows greater

analysis of the data output.

MORE WORK TO DODespite the numerous advances in

MS, CE, next-generation sequenc-

ing, and other rapid assays, fur-

ther deve lopments a re s t i l l

needed. Adoption of many new

analytical technologies takes time

given the need for extensive con-

firmation and validation of per-

formance. Many of these newer

methods are gaining acceptance

further down the development

pathway and closer to quality con-

trol, but are not yet widely used.

Regulators are, however, starting

to explore the potential advan-

tages these technologies can pro-

vide, according to Garrett.

One specific issue for Kneller

is HCP quantitation, which for

early clinical work is typically per-

formed using commercially-avail-

able ELISA kits, but then requires

transition to costly custom assays

later in development. “This transi-

tion can be difficult if kits do not

provide adequate coverage of all

HCPs potentially present in the

product. Orthogonal approaches

to HCP quantitation (e.g., mass

spectrometry) are not yet feasible

or widely-adopted, however,” he

notes. Wegele points to the need

for tools that enable the assess-

ment of the criticality (e.g., safety,

immunogenicity, PK, potency) of

various product-related impurities/

CQAs (e.g., HMW species, dimers,

fragments, PTMs, charge variants,

etc.) to identify control strategies

that make sense and do not lead to

excess testing burdens.

Reed Harris, senior staff scientist

in Pharma Technical Development

at Genentech, would like to see

more effective methods for iden-

tifying the causes of excipient

degradation, which may be due

to trace-level impurities that are

below current detection capabil-

ities. He also points to the need

for better resolution of higher-

molecular-weight species using

SEC. SEC aggregate resolution is

needed because there is growing

evidence that antibody aggregates

are not as immunogenic as origi-

nally believed, and further work

is necessary to establish the true

patient risks for different aggre-

gate types. “Current SEC columns

resolve monomers from dimers,

but do not resolve different dimer

types or multimers such as trimers,

tetramers, etc., very effectively,” he

says. Furthermore, he notes that

while CE–SDS is an advance over

SDS–PAGE, further improvements

are needed. The presence of SDS

makes it difficult to analyze CE–

SDS peaks with mass spectrometry,

and therefore, most peak assign-

ments are performed by spiking

forms prepared using other meth-

ods into samples, which is time

consuming.

Particle analysis is another issue

for Wegele. He notes that currently

available methods are mostly

Analytical Advances

Despite the

numerous advances

in analytical

technologies, further

developments

are still needed.

February 2016 www.biopharminternational.com BioPharm International 17

Analytical Advances

insufficient for precise and robust

assessment of subvisible particles,

particularly translucent protein-

aceous particles and particles with

diameters less than approximately

2 μm. Roche has developed a mod-

ified light-obscuration sensor that

monitors the signal width rather

than length, leading to improved

detection of very small subvis-

ible particles, reduction of arti-

facts during the analysis of low

concentrations of translucent pro-

tein particles, and higher counting

accuracy compared to flow imag-

ing microscopy and standard light

obscuration measurements.

Other ongoing needs, accord-

ing to Wegele, include replace-

ment of ce l l - ba sed potenc y

a s says w it h nove l , c e l l - f r ee

assay formats in the qua l it y

control environment; methods

for the evaluation of the impact

of combined admin ist rat ion

of biotherapeut ics; and more

automated testing solutions to

cope with the steadily increas-

ing sample load of ever more

complex biolog ics and next-

generation biologics, which are

often highly potent therapeu-

tics with novel, highly potent

product-related impurities. “It

is important to address novel

and critical product-related side

produc ts (e .g., immune- ce l l -

activating side products acting

at the crossroads of immunol-

og y and oncolog y) to ensure

maximum pat ient sa fety and

guarantee efficacy,” he asserts.

Finally, Harris notes that the

industry is struggling to balance

the needs for comprehensive test-

ing, including testing to account

for unexpected events, and more

rapid product development. “Risk-

based (i.e., QbD) test strategies

will lead to a reduced set of tests,

but it is also necessary to include

tests that detect variation out-

side of process models. The two

approaches present a fundamental

conflict,” he states.

Indeed, biopharmaceutical man-

ufacturers remain challenged to

increase the speed and accuracy

of product development while still

ensuring safety in the face of more

rigorous regulatory scrutiny, novel

biologic molecules, and evolving

manufacturing strategies. “All of

these factors are adding complex-

ity to analytical testing programs,”

Garrett concludes. X

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18 BioPharm International www.biopharminternational.com February 2016

luis

mm

olin

a/E

+/G

ett

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es

The biopharmaceutical indus-

try is growing exponentially,

driven by an ever-increasing

demand for monoclonal anti-

bodies (mAbs) and related products that

are capable of treating complex, life-

threatening diseases such as cancer, as

well as other infectious and autoim-

mune diseases. With more than 200

recombinant proteins on the market

today, the biopharmaceutical industry

is expected to reach approximately $180

billion by 2016 (1). Therapeutic mAbs

and their derivatives—such as antibody

fragments (Fab), single-chain Fv (scFv),

and diabodies—represent the fastest-

growing class of approved therapeutic

proteins because of their high specific-

ity, increased serum half-life, and low

toxicity (2). These products have been

used in biosensors, protein purification,

and bioimaging (3).

Since the advent of an era of antibody-

based therapeutics, the major focus of

the industry has been on the genera-

tion of antibodies with glycosylation pat-

terns similar to the naturally occurring

immunoglobulin Gs (IgGs). Mammalian

cells have been primarily applied as

the hosts of choice for mAb expression.

However, clinical success of mAbs has

tremendously increased their demand,

and this in turn has fueled interest in

other cost-effective alternative produc-

tion systems such as microbial hosts.

Further, mAb production in microbial

hosts such as bacterial cells completely

abrogates the nuisance of glycosylation

control and speeds up production time-

lines, further simplifying bioprocessing.

Antibody Production in Microbial Hosts

Anurag S. Rathore and

Jyoti Batra

The authors review the

status of expression of antibodies in

microbial hosts and present

the recent advances in the

production of aglycosylated

antibodies in bacteria.

Anurag S. Rathore* (pictured) is a

professor, Department of Chemical

Engineering, Indian Institute of

Technology, New Delhi, India. Jyoti

Batra is a post-doctoral fellow at

the Indian Institute of Technology

Delhi, New Delhi, India.

Protein Engineering

February 2016 www.biopharminternational.com BioPharm International 19

AL

L F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

RS

The groundbreaking discovery of

hybridoma cell generation technol-

ogy leading to mAb production is

one of the key developments in the

past decade (4). Generation of anti-

bodies in microbial hosts was ini-

tially met with skepticism due to

potential immunogenicity issues.

Recent developments in aglycosyl-

ated antibodies possessing similar

antigen-binding affinity and engi-

neered-specific FcγR binding has

led to an enormous interest in these

products. In this article, the authors

review the status of expression of

antibodies in microbial hosts and

present the recent advances in the

production of aglycosylated anti-

bodies in bacteria.

STRUCTURE OF A MONOCLONAL ANTIBODY AND ITS DERIVATIVESAntibodies are glycoproteins con-

sisting of two heavy chains and

two light chains that are linked

w it h d i su l f ide bonds . T h i s

Y-shaped molecule possesses an

antigen binding fragment (Fab)

and a crystallizable fragment (Fc).

Presently, different formats of anti-

body fragments, such as single-

chain variable fragments (scFv),

the fragment (Fab’)2, diabody frag-

ments (dAb), and single-chain anti-

body fragments (scFab) have been

generated, which possess similar

antigen binding affinities, but dif-

ferent effector functions, due to

the lack of an Fc region. These dif-

ferent antibody formats have been

illustrated in Figure 1.

MOTIVATION FOR ANTIBODY PRODUCTION IN MICROBIAL HOSTSMammalian cells have been pre-

dominantly employed for the

expression of antibodies and related

products because of their ability to

introduce post-translational modi-

fications similar to that of human

cells. However, use of mammalian

expression system also has few dis-

advantages, which has propelled

manufacturers to search for alterna-

tive host systems. One of the most

important challenges is predicting

the manufacturing behavior of cell

lines at early stages and selection

of clones with appropriate growth

characteristics. Process improve-

ments have generally been expen-

sive and time consuming. Medium

complexity, serum requirements,

and shear sensitivity are some of

the other drawbacks that character-

ize mammalian systems (5). Cell-

culture process parameters affecting

the glycosylation level of mAbs also

need to be controlled to ensure con-

sistent product quality (6). Selection

of a stable antibody-producing

clone is also a tedious and cum-

bersome exercise. In addition, viral

contamination of a therapeutic pro-

tein preparation can be altogether

avoided with cultivation in micro-

bial system. While mammalian

systems have been predominantly

used for the expression of therapeu-

tic proteins, advances in cellular

engineering of microbial hosts have

resulted in an increasing acceptance

in the use of these hosts as an alter-

native for the production of mAb

therapeutics.

CHALLENGES ASSOCIATED WITH THE PRODUCTION OF ANTIBODIES IN MICROBIAL HOSTSMicrobial cells such as yeast and

bacteria possess many advantages,

such as fast growth, well-known

genetics, and low cultivation costs.

Glycoengineering platforms, dis-

play technologies and library

creation, and robust manufac-

turing process development sup-

port the use of microbial cells as

suitable candidates for pharma-

ceutical development. Although

bacterial cells do not have the gly-

cosylation machinery necessary

for mAb production, recent suc-

cesses in the expression of anti-

body-related products demonstrate

the application of a bacterial host

as an alternative choice. A num-

ber of bacteria, such as Escherichia

coli, Corynebacterium glutamicum,

Pseudomonas putida, and Bacillus

Protein Engineering

Figure 1: Structure of an antibody and its fragments: A) Antibody (IgG class);

B) antibody fragments (VH, scFv, Fab, scFab, dAb, scFv-CH3). Abbreviations

are: variable heavy chain (VH), variable light chain (V

L), constant light chain (C

L),

constant heavy chain domain 1 (CH1), constant heavy chain domain 2 (C

H2),

constant heavy chain domain 3 (CH3), antigen-binding fragment (Fab), and

crystallizable fragment (Fc).

20 BioPharm International www.biopharminternational.com February 2016

megaterium have been used for the

production of recombinant pro-

teins (7). Of these, E. coli is the

most popular host and two of the

commercially available antibody-

f ragment products—Lucent is

(ranibizumab injec t ion) and

Cimzia (cerolizumab pegol)—are

expressed in E. coli (8).

Antibody molecules contain

disulfide bonds in their structure,

and proper formation of these

bonds is highly crucial for fold-

ing, solubility, and epitope bind-

ing. Protein folding and disulfide

bond formation require an oxidiz-

ing environment, which is pro-

vided by the periplasm, the main

compartment for the production

of functional therapeutic proteins.

Co-expression of foldases and

molecular chaperones (Skp, FkpA,

DsbA, and DsbC) helps in disulfide

bond formation, prevention of mis-

folding, and aggregation of heterol-

ogous proteins (9). E. coli has several

cytoplasmic endogenous proteases

that can cause proteolytic degrada-

tion; this represents a critical dis-

advantage of E. coli as a production

vector. However, this problem has

been successfully obviated by either

use of protease-deficient strains or

secretion of protein into periplasm,

where there are fewer proteases. In

E. coli, the Sec-dependent pathway

has been used for protein secretion

because of its abundance, although

accumulation of protein aggregates

may occur due to the generation

of premature proteins before secre-

tion. The formation of the inclusion

bodies can be avoided by co-expres-

sion of cytoplasmic chaperones and

utilization of a signal recognition

particle (SRP)-dependent pathway.

In addition, periplasmic inclu-

sion bodies may also form due to

incorrect folding of translocated

proteins in the periplasm. Based

on the twin-arginine transloca-

tion system (TAT), a new transport

system enabling translocation of

fully folded proteins in bacterial

cells has been developed (10). Many

advances such as translation region

initiation (TIR) optimization, engi-

neering of secretion pathways, and

co-expression of molecular chaper-

ones have been made for enhancing

antibody productivity in bacteria

and these have been reviewed else-

where (7, 9, 11). Major focus in anti-

body production in E. coli has been

on the generation of scFv and Fab

fragments. Full-length antibody

(IgG) production continues to be a

challenge due to low productivity,

and various strategies have been

employed to counteract this issue.

Eukaryotic cells such as yeasts

offer additional advantages of post-

translational modifications—such

as disulfide bond formation, which

facilitates proper folding. While

Pichia pastoris is the major strain

used for expression of recombinant

antibodies, other strains such as

Protein Engineering

Table I: Examples of antibody fragment production in microbial hosts such as Escherichia coli (E. coli) and

Pichia pastoris (P. pastoris).

Organism Antigen

Antibody

Format

Key points Reference

E. coli

Digoxin scFv Co-expression of molecular chaperones 14

Tlh scFv Co-expression of Skp chaperone, TrxA fusion 15

TNF α scFvSubcloning scFv gene into pBV220 under control of tandem PRPL promoter system

16

PA63 scFvEngineered SRP pathway, use of DsbA signal peptide & coexpression of YidC

17

β-galactosidase scFv Cytoplasmic expression 18

Human prion scFv Comparable binding affinity to Fab and full antibody 19

Tubulin, core histones, Syk & Aurora-A protein kinase, Papillomavirus E6 protein

scFv Phage display library for isolation of humanized and functional scFvs

20

HIV capsid protein FabUse of bacterial cell line containing tRNA rare codons, mutagenized Fab fragment

21

CD18/CD11b Fab Use of protease-deficient host strain 22

P. pastoris

Leukemia Inhibitory factor (hLIF) scFv First production of scFv in P. pastoris 23

Cell-surface glycoprotein A33 scFv Co-expression of chaperones BiP & PDI 24

P185 HER-2 scFv Use of alternative expression vector 25

F4 fimbriae scFv Prolonged glycerol feeding 26

HBsAg Fab Co-integration of light and heavy chain in yeast genome 27

HIV1 Fab GAP promoter and overexpression of HAC1 and PDI 28

* scFv=single-chain variable fragment, Fab=antigen-binding fragment.

February 2016 www.biopharminternational.com BioPharm International 21

Protein Engineering

Saccharomyces cerevisiae, Yarrowia

lipolytica, and Schizosaccharomyces

pombe have also been used to a

lesser extent. Yeast cells tend to

hyperglycosylate the recombi-

nant proteins (even at non-native

positions), and this may result in

altered pharmacokinetics, activa-

tion of complement system, and

generation of anti-glycan antibod-

ies. P. pastoris cells—cells that have

been glycoengineered and express

antibodies with superior volumet-

ric productivity—have been devel-

oped to mitigate this problem (12).

Yields of up to 1.4 g/L of human-

ized IgG have been reported with

glycoengineered P. pastoris (13),

which is far superior to mamma-

lian systems. Still, the use of this

strain in antibody bioprocessing

suffers from disadvantages, such

as the occasional addition of het-

erogeneous O-linked glycans and

slight differences in the N-glycan

structure. A few examples of anti-

body and antibody derivative

expression in microbial hosts that

appear in current literature are pre-

sented in Tables I and II.

AGLYCOSYLATED ANTIBODIES AS ALTERNATE THERAPEUTIC IMMUNOGLOBULINSAntibodies constitute an impor-

tant class of serum glycoproteins,

and the IgG isotype is the most

common isotype used for pharma-

ceutical applications. Natural IgG

antibodies are glycosylated at the

Asn297 amino acid position of the

Fc fragment. The complex bian-

tennary glycan structure depends

on multiple glycosyltransferases

and glycosidases located in the

endoplasmic reticulum and Golgi

bodies. These glycan molecules

impart effector functions—such

as antibody-dependent cell cyto-

toxicity (ADCC) and complement-

dependent cytotoxicity (CDC)—to

the antibody molecules. Absence

of post-translational modifica-

tions in bacterial host cells leads

to a complete abolition of glyco-

sylation of antibodies, resulting in

structural changes such as open

conformation, enhanced flexibil-

ity, and complete loss of effector

functions. In early experimental

studies on X-ray structures of wild-

type glycosylated IgG molecules

and truncated Fc domains, glycan

Table II: Production of full-length immunoglobulins in Escherichia coli (E. coli) and Pichia pastoris (P. pastoris).

Organism Secretion Yield Strategy employed Remarks Reference

E. coli

Periplasm 150 mg/LUse of two cistron systems with optimized light- and heavy-chain translational levels

First successful production of IgG

29

Periplasm > 1 g/LDsbA/DsbC co-expression system and optimized translation initiation region (TIR)

Highest yield of full length IgG production

30

Periplasm12.6 μg with increase in wet cell mass

A low copy number of plasmids and a low concentration of inducer

Optimization of culture conditions

31

Periplasm 62 mg/L

A) Co-expression of periplasmic foldase; B) combination of SRP/Sec-dependent pathway, c) co-expression of Ffh cofactor for enhancing secretion of heavy and light chains in bacterial periplasm

Engineered E. coli host-vector system

32

Periplasm 1–4 mg/L

Effect of different promoters and co-expression of molecular chaperones: use of synonymous codon in 5’-region of heavy chain; high-throughput screening of clones by flow cytometry

Comprehensive engineering of bacterial strain for enhanced expression with dicistronic expression system

33

Periplasm 40–50 mg/LNovel bacterial display and flow cytometry screening method

Screening of aglycosylated IgGs exhibiting selective binding to FcγRI

34

Periplasm 362 mg/LCo-expression of periplasmic foldase and modification of 5’ untranslated region sequence

Highest volumetric productivity 35

Cytoplasm followed by in-

vitro refolding50 mg/L

Refolding of inclusion bodies of light- and heavy-chain culture to get fully assembled IgG

Advantage of combinatorial shuffling to obtain desired specificity and affinity

36

Cytoplasm 1–25 mg/LDomain swapping and remodeling of Fc framework; expression of DsbC in cytoplasm

First successful production of full-length IgG in bacterial cytoplasm

37

P. pastoris

Extracellular > 1 g/L Optimization of fermentation parametersSimilar antigen binding affinity and size to that of marketed antibody

38

Extracellular 1.6 g/LDesign of experiments based optimization of process parameters

Production of IgG at Industrial scale (1200 L)

39

22 BioPharm International www.biopharminternational.com February 2016

molecules were considered to pro-

vide the necessary flexibility to the

CH2 domain of an IgG molecule,

and removal of a glycan moiety

was thought to be associated with

the “closed IgG” conformation (40).

However, recent small-angle X-ray-

scattering experimental observa-

tions by researchers have suggested

that aglycosylated Fc has a more

flexible CH2-CH3 interface than

previously imagined. This is due

to their larger radius of gyration

than their glycosylated counter-

parts, and the crystal packing in

aglycosylated structures may result

in a “closed upper CH2 region” in

their X-ray crystal structures (41).

Figure 2 illustrates the conforma-

tional difference between glycosyl-

ated IgG and bacterially expressed

aglycosylated IgG. The wild-type

aglycosylated IgGs show almost

no affinity towards FcγRs and

don’t display any effector func-

tions, but their engineering have

enabled them to selectively bind

to receptors. The need of aglycosyl-

ated antibodies with engineered

Fc regions is highly crucial for

anti-tumor therapies, where tumor

cells exhibit many features simi-

lar to normal cells and selective

engagement of FcγR receptors is

essential to prevent deleterious

effects (such as multi-organ fail-

ure or septicemia) resulting from

nonspecific activation of dendritic

cells. This effect has been predomi-

nantly observed in the case of the

aglycosylated form of trastuzumab

(Herceptin), which exhibited a 160-

fold superior binding affinity to

FcγRIIa than clinical grade drug

and displayed enhanced antibody-

dependent cellular phagocytoxic

activity (42). This work deserves

a special mention as FcγRIIa and

FcγRIIb have high sequence identi-

ties (96%) but completely opposite

roles. Aglycosylated antibody frag-

ments showing selective binding

to FcγRIIa were isolated by yeast

Protein Engineering

Figure 2: Conformation of (A) glycosylated IgG (CHO cells) and (B) aglycosylated

IgG expressed in bacteria. Absence of carbohydrate moieties results in enhanced

flexibility of the CH2 domain of the aglycosylated IgG.

Table III: List of advances in the engineering of effector functions in aglycosylated antibodies.

Escherichia coli=E. coli; Pichia pastoris=P. pastoris.

Host cells

Antibody

variant

Fc mutation Significance Reference

E. coli

Aglycosylatedtrastuzumabvariant

E382V, E382V/M428I in CH3 domain

Mutations resulted in enhanced stability of the CH2 domain in a aglycosylated Fc variant crucial for FcγRI binding; this variant is absent in wild-type aglycosylated IgG

A) Structure confirmed by small-angle X-ray scattering (SAXS) analysis

41

IgG1 B) Structure confirmed by smFRET analysis 46

E. coli IgG1F243L/T393A/H433P (variants generated through error-prone polymerase chain reaction)

Two-fold improved FcγRIIIa efficiency, hence, enhanced antibody-dependent cell-mediated cytotoxic activity

47

Yeast Aglycosylated IgG S298G/T299A Comparable binding to activating FcγRIIa and inhibitory FcγRIIb as that of wild-type glycosylated IgG Fc

48

E. coliAglycosylated trastuzumab-Fc5

E382V/M428I Selective high binding affinity toward FcγRI without any significant binding to other FcγRs

34

E. coliAglycosylated trastuzumab

Q295R/L328W/A330V/P331A/ I332Y/E382V/ M428I

120-fold high selective binding to FcγRI while retaining pH-dependent FcRn binding

49

E. coliAglycosylated trastuzumab

S298G/T299A/N390D/E382V/M428L

160-fold higher FcγRIIa binding affinity and 25-fold improved selectivity to FcγRIIa over inhibitory FcγRIIb.

42

February 2016 www.biopharminternational.com BioPharm International 23

Protein Engineering

surface display and flow-cytom-

etry screening. In another study,

researchers employed a knob-into-

hole technique to assemble E. coli-

derived aglycosylated half IgG and

CHO-cell derived glycosylated half

IgG to generate hemi-glycosylated

IgG. It was observed that hemi-gly-

cosylated IgG with a defucosylated

glycan was able to display ADCC

activity that was two-fold more

potent than hemi-glycosylated

IgG with a fucosylated glycan (43).

Advances in engineering of effector

functions in aglycosylated antibod-

ies are summarized in Table III, and

these efforts have paved the way

for possible emergence of aglycosyl-

ated antibodies as next-generation

therapeutics with high selectivity

and novel effector functions.

Aglycosylated antibodies are simi-

lar to their wild-type glycosylated

counterparts in terms of bioavail-

ability, pharmacokinetics, and epi-

tope binding. Several aglycosylated

antibodies are under clinical trials

and to date, no immunogenicity

issues have been reported. Tolerx

(MA, USA) has developed a series of

humanized aglycosylated antibodies

(TRX1, TRX4, TRX518) by knocking

out asparagine at the 297th position.

TRX1 is a monoclonal IgG1 anti-

CD4 mAb (N297A mutation) that is

being exploited for the suppression

of autoantibodies in autoimmune

disorders and neutralizing antibod-

ies induced by enzyme replenish-

ment therapy (44). Onartuzumab

(MET-mAb, Genentech) is the first

full-length monoclonal humanized

and affinity-matured aglycosylated

antibody expressed in E. coli for the

treatment of lung cancer. It inhib-

its hepatocyte growth factor (HGF)-

mediated activation and receptor

signaling and possesses similar phar-

macokinetic properties as that of its

glycosylated counterparts. Similarly,

yeast-expressed aglycosylated mono-

clonal antibody ALD518 (Alder

Biopharmaceuticals) exhibited a

half-life of 25 days (comparable to

the wild-type glycosylated form) and

significant improvements in patients

with rheumatoid arthritis (45).

Recent success in aglycosylated IgG

production and clinical efficacy data

indicates that they may have even

more applications in the future.

CONCLUSIONThe development of microbial

expression systems that are capable

of delivering projects that are not

immunogenic and retain antigen

specificity could pave the way for

the cost-effective production of mAb

products. Though substantial prog-

ress has been made in cellular engi-

neering and in the secretion pathway

of microbial cells, further progress

in metabolomic and proteomic tech-

niques is required to improve the

understanding of microbial systems

and the generation of host strains

with enhanced potential.

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Given the safety concerns

associated with the pres-

ence of microbial impuri-

ties in therapeutic proteins,

the preclusion of impurities at more

sensitive levels has been suggested

(1). The detection of endotoxin as

an innate immune response modu-

lating impurity (IIRMI) would occur

at levels that may be well below the

currently prescribed limits for endo-

toxin as a pyrogen—as per United

States Pharmacopeia (USP) <85> and

<151> (2). The term “IIRMI” is con-

tained in the FDA Center for Drug

Evaluation and Research/Center for

Biologics Evaluation and Research

(CDER/CBER) guidance document

on Assessment of Immunogenicity in

Therapeutic Proteins (3). Manufacturers

of therapeutic proteins seek to pre-

c lude mic rob i a l contaminant s

because they can increase immu-

nogenicity risks (4); however, this

preclusion is made from a pyrogen

perspective rather than an immuno-

genicity perspective. While fever is a

type of immune response that typi-

cally occurs as a result of infection,

there are many possible additional

immunogenic responses that occur

in the absence of fever or prior to

the development of fever. This paper

elaborates several ways in which the

two perspectives differ and how they

may merge over time. Adjuvant-type

activity (immune stimulation from

added impurities) is desirable for vac-

cines (5, 6) but is undesirable for ther-

apeutic proteins (see Figure 1) because

it can make the associated therapeutic

protein a target of antidrug antibodies

(ADA) and can result in either immu-

nogenicity or the neutralization of

antibody efficacy (7, 8, 9).

Section 5 of the 2014 FDA guidance

document, Immunogenicity Assessment

for Therapeutic Protein Products (3),

states the following:

“Impurities with adjuvant activity

Adjuvant activity can arise through mul-

tiple mechanisms, including the presence of

microbial or host-cell-related impurities in

therapeutic protein products (Verthelyi and

Wang 2010; Rhee et al. 2011; Eon-Duval et

ABSTRACTThe recognition that microbial artifacts are capable of modulating the mammalian immune system is an emerging view of biologic drug contamination control testing. The term IIRMI, or “innate immune response modulating impurity,” has been coined. It is important to recognize that pyrogenicity is only one potential risk of endotoxin

contamination and that immune activation is an inherent property of endotoxin, even in the absence of pyrogenicity. Immune stimulation of biologics is undesirable,

as it can stimulate anti-drug antibodies against administered recombinant proteins. Historically, many methods have been used to “detoxify” endotoxin

to remove the pyrogenicity of endotoxin while retaining its immune stimulation properties for adjuvant use in vaccines. This article gives a broad perspective for

understanding potential risks from low endotoxin recovery (LER) and other potential detoxification methods and presents a new paradigm to help drive future testing.

The Emerging View of Endotoxin as an IIRMI

Kevin Williams

Kevin Williams is senior R&D scientist at Lonza.

PEER-REVIEWED

Article submitted: Aug. 20, 2015.

Article accepted: Nov. 24, 2015.

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February 2016 www.biopharminternational.com BioPharm International 25

al. 2012; Kwissa et al. 2012). These innate immune

response modulating impurities (IIRMIs), includ-

ing lipopolysaccharide (LPS), β-glucan and flagel-

lin, high-mobility group protein B1 (HMGB1), and

nucleic acids, exert immune-enhancing activity by

binding to and signaling through toll-like receptors

(TLR) or other pattern-recognition receptors pres-

ent on B-cells, dendritic cells, and other antigen-

presenting cell populations (Iwasaki and Medzhitov

2010; Verthelyi and Wang 2010). This signaling

prompts maturation of antigen-presenting cells and/or

serves to directly stimulate B-cell antibody production.

Recommendations

It is very important for manufacturers to minimize

the types and amounts of such microbial or host-cell-

related impurities in therapeutic protein products.”

In terms of immunogenicity, therapeu-

tic proteins have become increasingly safe

over time with the realization that natural

animal-derived proteins may be recognized

as non-self, and recombinant human pro-

teins can become aggregated to bring about

immunogenic reactions. The “humaniza-

tion” of previously animal-based and chime-

ric monoclonal antibodies has also lowered

immunogenicity rates. But a low level (and

some not so low levels) of persistent pro-

clivity toward immunogenicity remains and

can be seen in clinical studies and marketed

package inserts (13).

ESTABLISHING ENDOTOXIN AS AN IIRMIThe FDA guidance document (3) references

a study by Verthelyi and Wang (1), which

shows that even low levels of microbial arti-

facts such as LPS, peptidoglycan, and deoxy-

ribonucleic acid (DNA) fragments can induce

the immunogenicity of therapeutic proteins.

Researchers have shown both in vitro and in

vivo that synergistically, IIRMIs are active at

lower levels than when present alone:

“This synergistic effect was then confirmed

in vivo, as studies showed that the combina-

tion of 10 ng of LPS and 500 ng of cytosine-

phosphate guanine oligodeoxynucleotides

(CpG ODN), which do not induce an immune

response when present individually, were suf-

ficient to promote the immunogenicity of

proteins and contribute to a clinically relevant

break in tolerance to self” (1).

Verthelyi and Wang noted that while low

levels of multiple impurities present in a

product can synergize to act as adjuvants

in mice, the levels are not expected to pre-

dict the levels that might be relevant in

humans, whom they state “are likely to be

much more sensitive to TLR agonists than

rodents” (1, 14). They discuss the relevant

levels of endotoxin viewed as an IIRMI to

those standardized for testing of pyrogens,

by either Limulus-based methods (limulus

amebocyte lysate [LAL] and recombinant fac-

tor C [rFC]) or rabbit pyrogen tests (RPT). The

authors write, “Of note, the current guide-

lines for setting limits on these impurities

are not based upon their potential impact on

product immunogenicity” (1). The response

to IIRMIs (here using LPS and bacterial DNA) is

thus amplified by the engagement of multiple

receptors, reminiscent of an engine firing on

multiple cylinders rather than a single cylinder.

The effects of low pyrogenic potency,

“detoxif ied” endotoxins, administered

with therapeutic proteins, can be seen in

the realm of vaccines—specifically, the use

of monophosphoryl lipid A (MPLA) as an

FDA-approved adjuvant that stimulates the

immune sensing of co-administered or sub-

sequently administered proteins: “MPLA is a

heterogeneous mixture of lipid A derivatives

created by successive acid and base hydroly-

sis of lipid A from Salmonella minnesota 595.

The predominant species created from that

process is 3-O-deacyl-4-monophosphoryl

lipid A. MPLA possesses attractive biological

characteristics as an immunoadjuvant such

as augmentation of T helper 1 (Th1) activity AL

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Figure 1: The basic mechanism of the innate immune response

modulating impurity (IIRMI) adjuvant effect. MPLA is monophosphoryl

lipid A. The immunogenicity phenomenon has been seen

historically in mild to severe adverse reactions (10,11,12).

vaccineprotein

therapeuticprotein

Detoxifiedendotoxin

= Adjuvant

Impurity

BAD...

GOOD...

26 BioPharm International www.biopharminternational.com February 2016

and antigen-induced T cell clonal expansion.

Yet, MPLA possesses approximately 1/1000th

of the systemic proinflammatory activity

of native E. coli [Escherichia coli] lipid A in

humans” (15).

Detoxified endotoxin is used (or being

developed for use) in several vaccines includ-

ing malaria (16, 17), hepatitis B (18), human

papilloma virus (19), and various cancer

vaccines (20). MPLA has low activity with

LAL and rabbit pyrogen while retaining its

adjuvant activity. While the LAL reduction

associated with O-deacylation and dephos-

phorylation has long been known, what is

less recognized is that after “detoxification,”

both LAL and RPT activity are greatly muted

(“when MPLA and lipid X were similarly

tested, they showed very low pyrogenicity”

[5]), and the adjuvant activity remains (21).

The mechanism of adjuvant type response

versus the historically recognized proinflam-

matory response to LPS is believed to be

due to activation of the TRIF versus MyD88-

dependent pathway (22, 23).

MPLA is not the only vaccine adjuvant

using LPS being developed (24, 25, 26).

There is a widespread interest in developing

nontoxic LPS types for use as adjuvants of

peptide and protein components of disease-

causing organisms to complement proteins

that typically elicit low levels of immune

stimulation (unlike live attenuated vaccines),

yet “adding MPLA to vaccine preparations

boosts serum antibody titers by 10–20 fold

compared to vaccine alone” (15). The grow-

ing importance of nonpyrogenic LPS struc-

tures can be seen in the development of

nontoxic lipid A derivative drugs (27). An

example is the anti-sepsis drug candidate

Eritoran, which has been shown to block

the TLR4 receptor by displacing active lipid

A with the inactive lipid A form. Eritoran is

a synthetic molecule derived from natural

Rhodobacter sphaeroides lipid A (28). Despite

providing valuable information on the inter-

action of the antagonist with the endotoxin

receptor TLR4 and co-receptor MD-2 (29), the

drug candidate failed its Phase III trial, as it

did not provide a clear survival benefit (30).

CONTROL OF CONTAMINANTS FROM AN IIRMI VANTAGEThe IIRMI view is one of endotoxin and

other artifacts of microorganisms being able

to elicit an immune response in mammalian

systems at very low levels. The relevance to

the administration of therapeutic proteins is

seen as an adverse event producing capabil-

ity that mirrors the effect of an adjuvant as

paired with a clean recombinant protein.

Thus, the occurrence of immunogenicity

can be viewed as a problem of the past that

is not entirely in the past. Biologics are life-

saving drugs, but some (depending upon

the dose and indication), still have infusion

reaction incidences approaching 25%, with

half of those being said to be Grade 3 (severe)

or 4 (life-threatening) (12). A caveat is that

endotoxin control is only one aspect of the

overwhelmingly complex issue of immuno-

genicity. The importance of general micro-

biological control in the manufacture of

biologics can be seen from many refer-

ences (31). Such control largely revolves

around traditional efforts to control bio-

burden during processing, process valida-

tion that includes more extensive testing,

cleaning validation, and the assurance of

the quality of high-purity water systems.

The emerging view of endotoxin as an

IIRMI—while straightforward in concept—

has ramifications extending across a broad

spectrum of current activities associated

with the manufacture and administration of

a therapeutic protein drug compound. Some

items that seem common place today may

require review in light of this emerging para-

digm, including:

t Determination of the relevant level of

endotoxin reactions in humans from the

IIRMI perspective

t Consideration of low potent LPS types that

may present an adjuvant question mark

wherein historically they have been irrel-

evant from a pyrogen perspective

t Treatment types including depyrogena-

tion that do not incinerate or completely

remove endotoxin

t The pairing of biologics with large-volume

parenterals (LVPs) or small-volume paren-

terals (SVPs) possessing historical quality

requirements

t LER can be viewed as a “detoxified” form

of endotoxin from the IIRMI vantage.

RELEVANT LEVELSThe levels of endotoxin Verthelyi and Wang

identified as significant for adjuvant activity

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February 2016 www.biopharminternational.com BioPharm International 27

of LPS was stated to be as low as 1–10 ng/mL

in the presence of sub-stimulatory levels of

bacterial DNA (CpG), which does not easily

translate from mouse models. An endotoxin

unit (EU) is 1/5th the activity needed by E.

coli reference standard to bring about the

threshold pyrogenic response (K=5 EU/kg=1

ng/kg) (32). The 1–10 ng/mL range is likely

much lower due to the known differences

between mice and human response. Relative

to the human response, mice are highly resil-

ient to inflammatory challenge. For example,

the lethal dose of endotoxin is 5–25 mg/kg

for most strains of mice, whereas a dose that

is 1,000,000-fold less (30 ng/kg) has been

reported to cause shock in humans (33). The

purpose here is not to suggest specific levels,

but rather to point to a characteristic of the

IIRMI view, which is that relevant IIRMI lev-

els are expected to be lower than the levels

precluded by traditional pyrogen and bacte-

rial endotoxin test (BET) testing. Further

experimentation will be needed to authori-

tatively inform manufacturers and regulators

of relevant levels for humans.

Historical pyrogen and BET testing always

considers the dose to be a critical param-

eter of drug administration as it pertains to

endotoxin test preclusion. A large dose should

contain less endotoxin than a small dose.

This relationship is described in the tolerance

limit (TL) calculation expressed as TL=K/M

where K is the threshold pyrogenic response

constant (K=5 EU/kg/hr for parenterals) and

M is the relevant dosage of a specific drug (34).

Today’s biologic drugs are expected to be

cleaner than that required by historical pyro-

gen standards. This requirement can be seen

in FDA biologics license application (BLA)

requests to lower BET limits as well as the

FDA Q&A Guideline expectation that drugs

be tested at a “…dilution just above the level

that neutralized the interference” (35).

The practice of pre-dosing before thera-

peutic protein administration with anti-fever

and a steroid drug prior to some monoclonal

therapy shows the expectation of adverse

responses that includes fever (36). The large

amounts of various solutions being admin-

istered to patients can be seen in the use

of one and sometimes more than one LVP

infusion. The expectation of lower-than-cal-

culated TL specifications in BET is built into

the administration of such large volumes of

solutions and may point to an already occur-

ring encroachment of the IIRMI-based view

onto traditional BET preclusion activities.

This mixing or blurring of lines of expected

endotoxin exclusion levels for contaminants

(the pyrogen versus immunogenic potential

of contaminants) can be expected to con-

tinue as the IIRMI view advances.

ENDOTOXIN TYPESThe use of MPLA here as an example of LPS

adjuvant activity is analogous because it is

not naturally occurring; however, there are

many natural, low pyrogenic LPS types that

are associated with waterborne-type Gram-

negative bacteria of the non-hexaacyl type

(that differ from the prototypically pyro-

genic E. coli LPS) (37). According to Darveau

and Chilton, “Naturally occurring low bio-

logically reactive lipopolysaccharide (LBR

LPS) forms are known to function through

TLR4, which directly activates B cells and

indirectly activates naive T cells through

APCs [antigen-presenting cells]. Therefore,

LBR LPS forms are attractive candidate mol-

ecules for future adjuvant study. Although

various structure/function studies have

established key components of the lipid A

structure required for potent immunostim-

ulatory activity without toxicity, it is still

not possible to reliably predict how a spe-

cific alteration in the LPS structure might

affect the ability to function as an effective

immune adjuvant” (38).

The basic assumption that the effect of a

detoxified endotoxin adjuvant may equate

to “low potency natural endotoxin” (LPNE)

activity should be explored experimen-

tally. If LPNE possesses adjuvant activity,

then testing for such varieties of LPS (e.g.,

from genuses that include Pseudomonas and

Burkholdaria) could be done by testing at

levels well below current standards if these

types are shown to be prevalent in a par-

ticular process. Such efforts would repre-

sent a significant change that would not

be enacted lightly. IIRMI testing, however,

could be advantageous for select processes

and products based on risk assessment, for

example, processes containing LPNE from

such bioburden types.

Given that the types of bacteria likely

to proliferate in water systems include

Gram-negative bacteria with LPNE, such

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28 BioPharm International www.biopharminternational.com February 2016

as Pseudomonas (which is 50–70 times less

pyrogenic than E. coli [37]) and Burkholderia

(some spec ies were prev iously c lassi-

fied as Pseudomonas), it is worth explor-

ing the preclusion of these less potent

types. For example, overgrowth of a spe-

cific LPNE in bioburden or water purifi-

cation systems may not be detected by

conventional testing but could present a

significant amount of LPS by mass. This

level of “cleanliness” is not an issue in non-

biologics if they are not co-administered

with a therapeutic protein. Munford lists

LPNE types (39) occurring in soil, water,

or plant habitats as including those from

Burkholderia, Acinetobacter, Enterobacter,

Chromobacter ium, Erwinia, Rhodobacter,

Rhizobium, Xanthomonas, and Pseudomonas.

Potent types listed are largely members of

Enterobacteriaceae (e.g., E. coli, Salmonella,

etc.) and are natural inhabitants of the

human gut. General methods used in pro-

cesses to remove LPS regardless of the bac-

terial type would be unaffected. However,

for processes relying solely upon LAL to

gauge the efficacy of endotoxin removal,

for example, this philosophy could change

depending upon tools developed to gauge a

wider spectrum of LPS types.

It is an unsettling prospect that LPNE

could add to adjuvant activity of therapeutic

proteins (38). Historically, there has been a

singular focus on precluding the bacteria

that produce proinflammatory, “endotoxic”

endotoxins (Enterobacteriaceae, i.e.,

E. coli) as per USP <151> and <85>

(2), rabbit pyrogen, and bacterial

endotoxin testing, respectively.

This fits an underlying, longstand-

ing theme that microbial artifacts

injected into the blood stream

may have significant effects that

do not necessarily correlate with

our ability to “see” them, analyt-

ically speaking, or correlate with

their ability to produce fever. The

mammalian physiological view of

endotoxin is ultra-sophisticated

when it comes to the detection of

microbe invaders and their artifacts.

The basic Lipid A PAMP should be

viewed as a set of dials (phosphate,

sugar, number and types of acyl

chains–either symmetrical or asym-

metrical, substitutions, etc.) rather

than an “on-off” button (pyrogenic or non-

pyrogenic) (39, 40, 41). The activity of LPS

at low levels is being borne out in studies of

the low-dose effect of endotoxin in various

disease states—such as sepsis (42), inflam-

mation (43), cancer (44), and cardiovascular

disease (45).

TREATMENTS—DETOXIFICATION/DEPYROGENATION Detoxification does not remove the adju-

vant effect of MPLA, but rather, signifi-

cantly diminishes the proinflammatory

effect. This is seen in other kinds of “detoxi-

fication” efforts, as Gamma irradiation of

Salmonella typhimurium is known to remove

its pyrogenicity, while allowing it to retain

its immunogenicity inducing capability

(46). Similarly, irradiated LPS retains the

adjuvant activity of LPS, and it serves as

a good adjuvant for inactivated virus vac-

cines. A wide variety of historically accumu-

lated means of detoxification are shown in

Figure 2. References include chemical (47, 48,

49) ionizing radiation (46, 50) use of sur-

factants (51) (reversible), enzymatic (52, 53)

mutation (54, 55, 56) (natural and induced),

antimicrobial peptides (57), natural low

pyrogenic forms, and LER. A review of prac-

tices that do not incinerate or completely

remove the functional LPS PAMP would be

in order from the “endotoxin as IIRMI” view

for therapeutic protein processing.

Peer-Reviewed

Figure 2: Low endotoxin recovery (LER) can be viewed as one

of a dozen general methods of “detoxifying” lipopolysaccharide,

historically performed for the purpose of adjuvant research.

Chemical

Ionizing radiation

Surfactants (reversible)

Enzymatic (i.e., deacylation)

Mutation (pathway alteration, natural and induced)

Antimicrobial peptides (host defense and synthetic)

Natural low pyrogenic forms

Surfactants with chelator (almost irreversible)=LER

DETO

XIF

ICA

TIO

N

Endotoxin

Immunogenic and pyrogenic

Immunogenic but reduced or no pyrogenicity

February 2016 www.biopharminternational.com BioPharm International 29

PAIRING BIOLOGICS WITH LVP/SVPS GOVERNED BY DIFFERING HISTORICAL REQUIREMENTS The most common symptoms associated

w ith m Ab in f usions a re endotox in-

l ike, dose-dependent, and include a

fever component (with chills, aches, and

neut ropenia). Package inser t s of ten

recommend including a pre-infusion

regimen of acetaminophen, antihistamine,

and steroid in preparation for the initial

mAb dose. Historically, too many drugs

being administered at once would be

considered potentially pyrogenic; however,

in a recent FDA Q&A document (35), testing

is recommended just over the level of

interference (below tolerance limit).

Many mAbs are administered in a LVP

infusion. LVPs have a rather permissive limit

of 0.5 EU/mL, although often tested at much

lower levels. SVPs also may have permissive

historical limits that may not be updated to

be in line with biologics drug expectations

that are often assigned lower limits as part of

the BLA review. A new quality designation of

“for use with therapeutic proteins” for LVPs

and/or SVPs to be used with biologics might

improve current safeguards. Such a designa-

tion would also allow specific preclusion of

some synergistic IIRMIs. Additionally, BET

limit calculations are based on a one-hour

criteria for K and M, where K is the thresh-

old pyrogenic response =5 EU/kg/hr and

M is the maximum human dose as dosed

in either mg or mL. Here, the 350 EU/dose

value is given using the routinely applied

patient weight of 70 kg (5 EU/kg X 70 kg/

dose=350 EU/dose limit. BET limit calacula-

tions may have little relevance to immuno-

genic concerns.

From a quality perspective, the practices of

some compound pharmacies seem out of line

with regulatory agency-approved biologics.

Given the new FDA draft guideline on Mixing,

Diluting, or Repackaging of Biologics Products

Outside the Scope of an Approved Biologics

License Application (58), there are many recent

warnings associated with compound phar-

macy testing in which no endotoxin test-

ing had been performed (59). Also often

cited are the poor aseptic conditions present.

Using solutions of low quality or from low

quality compounding environments in a

co-administered or concurrent manner with

painstakingly manufactured and tested ther-

apeutic proteins seems incongruent from an

IIRMI perspective.

LOW ENDOTOXIN RECOVERY (LER)Endotoxin subjected to LER solutions can be

considered a type of “detoxified” endotoxin

by the IIRMI view. The LER discussion is an

active one with industry participants split

on the characteristics of potential endo-

toxin contaminants that could come from

processes subjected to LER-causing condi-

tions. The concept of “detoxification” with

residual immune activation potential could

help inform the LER debate. The search for

compounds that utilize the immune stimu-

lation property of LPS without the induc-

tion of proinflammatory effects is ongoing,

as many subunit vaccines do not have the

ability to stimulate the immune system (38).

In the realm of endotoxin testing, if one is

singularly worried about the pyrogenicity

of a sample, then it may come to play out

that LER subjected drug formulations are

not particularly pyrogenic, although there

is conflicting rabbit pyrogen data (60, 61).

However, if one is worried that a given LER-

prone protein formulation could increase

the therapeutic protein immunogenicity if

such LPS monomers are present, then one

would want to detect and preclude the pres-

ence of LPS monomers or otherwise “detoxi-

fied” endotoxin solutions that retain the

potential to be recognized by mammalian

immune systems.

WHAT MAY THE IIRMI VIEW MEAN FOR THE USE OF LAL?One might assume that, given the IIRMI

view, cytokine-based tests such as a mono-

cyte activation test (MAT) or human toll-like

receptor test (h-TLR) would enhance current

LAL testing. A couple of facets of LAL, how-

ever, may be viewed as critical to its contin-

ued use. The first is the sensitivity of LAL. It

is more sensitive than any cytokine-based

test available commercially. The recognition

of endotoxin as an IIRMI is to acknowledge

that pyrogenic activity does not equate to

immunogenic potential. And the preclusion

of LPS, by far the most potent of IIRMIs,

could serve to preempt the possibility of

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30 BioPharm International www.biopharminternational.com February 2016

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synergism with another low-level IIRMI that

may not be able to be readily precluded.

Secondly, LAL has been found to be active

to under-acylated LPS in a way that mam-

malian-based cytokine assays are not (62).

This has been touted as an advantage, as

it is thought that these tests respond only

to what a human would respond to. But

this view only considers the proinflamma-

tory pathway of LPS and not the potential

for adjuvant-induced immune stimulation.

Under-acylated LPS is one type of LPS being

studied for its low pyrogenicity but high

immunogenicity potential. Because LAL is

better, although not perfect, at detecting

these types, the use of mammalian-based

assays that cannot detect them present a

flawed strategy.

CONCLUSIONThe emerging view of endotoxin as an

IIRMI highlights several new concerns to

consider, including: (a) the level and types

of endotoxin contaminant required to pro-

duce fever versus the level and types capable

of stimulating the immune system, (b) the

pairing of therapeutic proteins with large-

volume or small-volume drugs possessing

lower-quality standards as compared with

biologics requirements, and (c) the delivery

of biologics with or without additional exter-

nal handling, such as compound pharmacy

manipulation.

The last thing biologics manufacturers

intend is to introduce impurities with an

adjuvant effect to therapeutic proteins. As

illustrated is this article, endotoxin adjuvants

(including detoxified endotoxin) adminis-

tered with vaccine proteins are capable of

eliciting nonpyrogenic endotoxin responses.

The need for an updated view on immuno-

genicity is well stated by Haile, et al.: “It is

only the more recent understanding of the

innate immune system’s biology that dictates

the need of assessing a broader spectrum of

known and unknown IIRMIs in order to con-

trol or reduce the risk of unwanted immuno-

genicity by therapeutic proteins” (63). These

biologics manufacturing concerns contrast

with historical, purely pyrogen-centric activi-

ties that represent an important—but more

minimal—standard that is typically associ-

ated with nonbiologic medications.

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Supplements,” in Endotoxins: Pyrogens, LAL

Testing and Depyrogenation, K.L. Williams, Ed.

(Informa Healthcare USA, Inc., New York, NY, 3rd

ed., 2007), pp. 294.

35. FDA, Guidance for Industry, Pyrogen and

Endotoxins Testing: Questions and Answers

(CDER, CBER, CVM, CDRH, ORA) (Rockville, MD,

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(January 1969).

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U.S.A. 100 (14), pp. 8146–8150 (Jul. 8, 2003).

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13726–13735 (May 4, 2007).

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24, 2014).

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(Jul. 1, 2011).

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(2015).

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Biochemistry 47 (24), pp. 6468–6478 (2008).

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Scope of an Approved Biologics License

Application (CDER, CBER) (Rockville, MD, Feb.

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other Actions, www.fda.gov/Drugs/

GuidanceComplianceRegulatoryInformation/

PharmacyCompounding/ucm339771.htm,

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2015), http://biopharma-asia.com/magazine-

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(Apr. 22, 2015), doi:10.1371/journal.pone.

32 BioPharm International www.biopharminternational.com February 2016

LA

GU

NA

DE

SIG

N/G

ett

y Im

ag

es

Extensive testing is required

throughout the drug-devel-

opment process and during

manufacturing to ensure the

safety and efficacy of marketed medic-

inal products. Numerous assays for

the characterization of biopharma-

ceuticals and determination of any

biologic contaminants have been

developed and are highly effective for

most biotherapeutics. For many cell-

based therapies, such as chimeric anti-

gen receptor (CAR) modified T-cells

(also known as CAR-T), however, these

conventional methods often take too

long and require excessive sample

quantities. Consequently, developers

of these novel treatments have been

faced with a number of challenges.

The development of new rapid meth-

ods designed to provide comparable

results while meeting the need for

high-throughput performance show

signif icant promise for addressing

these issues.

VECTOR/CELL-TESTING REQUIREMENTSCAR T-cell therapies are produced by

harvesting blood cells from a patient,

selecting and growing the desired

T-cell population, and then transduc-

ing them with a viral vector (typically

lentivirus) carrying the CAR-T gene

cassette. Transfecting cell lines with

plasmids produces the viral vectors,

and special care is taken to ensure that

no replication-competent lentiviruses

(RCLs) are generated. After CAR T-cell

expansion, the cells are reintroduced

into the patient.

Advances in Assay Technologies for CAR T-Cell Therapies

Alison Armstrong

Rapid methods to test CAR-T

therapies for potential

contamination are on the

horizon.

Alison Armstrong, PhD, is

senior director, development

services at BioReliance, UK.

Cell Therapies

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and retraining temps by retaining our scientists at your

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winning Professional Scientific ServicesSM (PSS):

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t Delivers a 50-year history of regulatory compliant

technical expertise in your lab

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insourcing service provider for the past 10 years

Choose the PSS Insourcing solution that enables

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34 BioPharm International www.biopharminternational.com February 2016

AL

L F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

R.

Microbial and viral testing is

performed to determine whether

any microorganisms (e.g., bac-

teria, viruses) are present dur-

ing the pharmaceutical process,

including in intermediates, active

ingredients, the manufacturing

environment, and formulated

drug products. Test methods are

employed for detection, screening,

enumeration, and identification

purposes. Examples include steril-

ity testing for detection, screen-

ing for specified microorganisms,

determination of the total aerobic

microbial count for enumeration,

and analysis to identify specific

microbes. Other tests to deter-

mine the long-term stability (e.g.,

genomic and epigenetic stabil-

ity, X-chromosome inactivation)

and quality and function (e.g.,

potency, efficacy, lot-to-lot vari-

ability) are also required.

A l l raw mater ia ls must be

sourced from approved suppliers

and subjected to extensive test-

ing to ensure there is no presence

of microbial or viral contami-

nation, as are master and work-

ing cell banks (MCBs and WCBs,

respectively). Plasmids produced

in bacteria are tested for sterility

and endotoxin levels, and viral

vectors are subjected to identity,

purity, adventitious agent, and

potency testing before they can

be released for the transduction

of cells. While no screening of

patient blood cells for adventi-

tious agents is required, the trans-

duced cells are tested for sterility,

mycoplasmas, RCLs, and endo-

toxins. The specific tests required

for the control of critical raw

materials and throughout the pro-

duction process are determined

by regulatory guidel ines and

are designed to ensure that cell

therapy products are well char-

acterized and free of microbial

contamination. The required level

of testing depends on the phase

of the drug-development cycle

and the step of the production

process; for instance, cell banks

and plasmids differ in their test-

ing requirements. Figure 1 presents

a schematic of the manufactur-

ing steps and associated testing

regimes for a cell therapy produc-

tion process.

FDA, the European Medicines

A g e n c y ( E M A ) , t h e U S

Pharmacopeial Convention (USP),

and the European Pharmacopoeia

(Ph. Eur.) have all published guid-

ance materials related to the pro-

duction and testing of cell-based

therapies. Some examples include:

t &." HVJEBODFPO UIFEFWFMPQ-

ment and manufacture of lenti-

viral vectors (1)

t '%"HVJEBODF GPS UIFNBOVGBD-

ture and ex-vivo use of retroviral

gene therapy vectors (2, 3)

t USP (4) and Ph. Eur. (5) general

guidance on the manufacture

and quality control of therapy

viral vectors

t '%" TVQQMFNFOUBM HVJEBODF

on testing for replication-com-

petent retroviruses in retrovi-

ral-vector-based gene therapy

products (6)

t '%" HVJEBODFPO QPUFODZ UFTUT

for cellular- and gene-therapy

products (7)

t '%" HVJEBODF PO QSFDMJOJDBM

assessment of investigational

ce l lu la r- and gene-therapy

products (8).

STANDARD METHODSA broad range of standard, well-

recognized methods that are

accepted by regulatory agencies

around the world are employed

to characterize raw materials, bio-

logic actives, and formulated drug

products. For cell-based therapeu-

tics, in addition to the analysis

of typical raw materials such as

media, assays must also be per-

formed to characterize any cell

lines and viral vectors used.

Cell Therapies

Figure 1: Schematic of the manufacturing steps and associated testing

regimes for a cell therapy production process.

Vector/Cell

Master CellBank (MCB)

Working CellBank (WCB)

Process Development(Growth/Production/Modification)

Drug Substance

Identity

Identity

Identity

Safety

Safety

Safety

Stability Shipping

QA/QC LRT Container Closure

Purity

Purity

Expression

In-processtesting

In-processtesting

In-processtesting

Drug Product

Master-WorkingVirus Bank (MVB/WVB)

February 2016 www.biopharminternational.com BioPharm International 35

Standard identity tests for cell

lines include cytochrome oxidase

or short tandem repeat (STR) pro-

filing and DNA fingerprinting,

while those for vectors include

determination of genetic identity

by sequencing of transgenes and

restriction enzyme digestion.

The absence of microbial con-

taminants in cell lines and vec-

tor products (bulk harvest [BH]

purified) is confirmed through

sterility and mycoplasma test-

ing. Due to the large number of

different viruses that can poten-

tially contaminate biologic drugs,

numerous assays, both in vivo and

in vitro, must be conducted on

cell lines and vectors to demon-

strate the absence of adventitious

viral agents. These tests include

those that detect ranges of viruses

(broad specificity) and those that

target specific viruses that have

been known to be an issue (e.g.,

bovine, porcine). Virus-specific

testing of cell lines is typically

accomplished using polymerase

chain reaction (PCR)-based meth-

ods and are often required if there

is a known risk of contamination

associated with the components

used in a given process. Cell lines

may also be subjected to trans-

mission electron microscopy or

product-enhanced reverse tran-

scriptase assays.

For CAR T-cell therapies pro-

duced using lentivirus as the viral

vector, the absence of replication-

competent vectors, particularly

RCLs, must also be demonstrated.

These assays are performed on

vector products (BH, purified vec-

tor, and ex-vivo transduced cells).

Purified vectors are subject to

further tests, including determi-

nation of the viral titer, resid-

ual bovine serum and plasmid,

osmolality pH, endotoxin, host-

cell DNA, and protein assays. The

vector titer and endotoxin tests

are also conducted for the ex-vivo

transduced cells.

UNIQUE TESTING NEEDS OF CAR AND OTHER CELL-BASED THERAPIESAlthough cell-based therapies have

been in development for more than

two decades, they still face a num-

ber of challenges. Regulatory scru-

tiny is particularly high. Because

these drug products contain live

cells, terminal sterilization is not

possible; therefore, demonstration

of the absence of contaminants is

essential. Early failures and ques-

tions about the safety of initial

treatments have also led to intense

interest from regulators.

Testing can be difficult, how-

ever. Often, there are limited

supplies of the key raw materi-

als required for process, product,

and test method development.

In addition, drug-substance and

drug-product lot sizes are often

quite small; thus, sample volumes

are typically small, leading to the

need to use modified test proto-

cols, which must be validated for

the same specificity and sensitiv-

ity as the original test.

Further complicating the issue

is the limited shelf life of most

cel l-based therapies. In some

cases, the transduced cells can be

frozen, allowing for completion

of testing prior to product release.

It is not possible, however, to

freeze most CAR T-cell therapies.

The difficulty lies in the lengthy

nature of most conventional ste-

rility assays and tests for determi-

nation of the absence of bacteria,

adventitious viruses, and RCL.

Most assays and tests are cell-

cu lt u re -based methods w ith

extended incubation times to

allow turbidity formation in liq-

uid culture and colony forma-

tion on solid media, and require

up to two to four weeks to com-

plete. In addition, they involve

many manual procedures (e.g.,

sampling, dilution, dispending,

incubation, reading, recording,

subculture, and microorganism

identif ication), which all take

time. Overall, these tests can take

as many as 40 days from start to

finish, assuming up to one week

for sample delivery to the test

lab, two to four weeks for testing,

and up to an additional week for

delivery of the final report.

Rapid tests (PCR- and rapid-

ce l l -g row th-based mic robio -

logical and RCL assays) must be

performed on cell-based therapies

that cannot be frozen. Generally,

conventional assays must also

be run for confirmation of the

results of the rapid tests, even

though the results won’t be

received until well after the treat-

ment has been administered.

EXPECTATIONS FOR RAPID METHODSThe potential benefits of effec-

tive rapid-test methods have led

to interest in their implemen-

tation for many more therapies

than those that are just cell-based

therapies that cannot be frozen.

Not only do these assays enable

reduced product-release cycle

t imes, they general ly require

small sample volumes and provide

higher-quality results. In addi-

tion, most can be automated and

combined with electronic data

capture, reducing opportunities

for human error and the introduc-

tion of contaminants, and further

decreasing the overall test time to

approximately nine days (one day

for sample delivery, seven days for

testing, and one day for delivery

of the final report).

New microbiological testing

methods achieve the same results

as corresponding classical meth-

ods, but within a shorter time

period, for example, less than

three days for sterility testing,

and 24 hours or less for microbial

counts and ID tests. The ultimate

rapid tests are completed in real

time (e.g., one to three hours).

The ultimate goal is to develop

Cell Therapies

36 BioPharm International www.biopharminternational.com February 2016

Cell Therapies

methods than can be completed in

hours rather than days.

Faster access to test results can

also improve the manufacturing

process, because potential problems

can be investigated/addressed much

sooner than is possible when con-

ventional methods are employed.

Very rapid methods may also enable

in-process and raw material testing.

There are, however, several key

requirements that must be met

by any rapid-test method that is

intended to replace an existing com-

pendial method. Most importantly,

a rapid method must meet or exceed

the performance of the existing

assay in terms of both specificity

and sensitivity. Extensive validation

is required by regulatory agencies to

support the use of a rapid method

by demonstrating comparability to

the standard method. Parameters

that are considered part of such an

evaluation can include accuracy,

precision, linearity, specificity, the

detection limits, operational range/

sample volume, robustness, repeat-

ability, and intermediate precision.

Particular laws, regulations and

guidance regarding rapid testing for

biopharmaceutical manufacturing

include:

t &%2.T i&1 4UFSJMJUZw

which discusses approaches to

sterility testing (9)

t &%2.Ti&1 .ZDPQMBTNBw

includes information on nucleic

acid detection for mycoplasma in

Europe (10)

t Code of Federal Regulations

(CFR) 610.12 update (11)

t 5IF 1BSFOUBM %SVH "TTPDJBUJPO

( P D A ) p u b l i s h e d P D A

Technical Report Number 33

(TR33): Evaluation, Validation

and Implementat ion of New

Microbiological Testing Methods (12)

t EurPhTi&1"MUFSOBUFNFUI-

ods for control of microbiological

RVBMJUZw

In 2011, FDA’s Center for

Biologics Evaluation and Research

(CBER) investigated matrix effects

through the evaluation of three

rapid microbial test systems:

Millif lex Detection (Millipore),

BacT/ALERT (bioMerieux), and

BACTEC (BD) (14).

It should be noted that when

rapid test methods are approved,

they are approved as part of the fil-

ing for a specific drug product. In

addition to equivalent performance

and significantly faster turnaround

times, rapid methods should also be

easy to use and be less costly than

the corresponding standard meth-

ods. They must also be designed to

address potential matrix effects.

The technologies on which rapid

test methods are based are divided

into four categories for convenience:

t (SPXUICBTFENFUIPET

t 7JBCJMJUZCBTFENFUIPET

t $FMMVMBS DPNQPVOEPS BSUJGBDU

based technologies

t /VDMFJDBDJEUFDIOPMPHJFT/"5T

Examples of rapid microbiologi-

cal assays and rapid methods for the

detection of adventitious agents are

presented as follows.

RAPID MICROBIAL ASSAYSA variety of technologies have been

developed for rapid microbiologi-

cal assays. Methods derived from

blood-culture methods employed

in clinical microbiology are attrac-

tive because they are based on tech-

niques that have been approved

by regulatory authorities, albeit for

different applications. Examples

include methods that rely on car-

bon dioxide sensors (pH-sensitive

f luorescence and colorimetric

response) and the use of a pressure-

sensitive transducer to measure

changes in headspace pressure.

Many methods have also been

developed that are based on technol-

ogies that have not previously been

used in a clinical setting. Examples

include determination of the elec-

trical impedence of the media sup-

porting growing microorganisms,

solid-phase fluorescence laser scan-

ning microscopy, flow cytometry of

fluorescently labeled organisms, and

ATP bioluminescence.

ATP bioluminescence is of inter-

est because the sample preparation

is similar to that of compendial

methods: It is compatible with a

wide range of product types, and

the results can be read and reported

automatically using compliant soft-

ware. Nonsterile product release

is possible within 23 to 48 hours;

however, this method does not pro-

vide enumeration of contamina-

tion levels. Automation of current

compendial cell-culture methods

is, however, making rapid microbial

enumeration possible. Detection

of positive cultures is faster with

automated interpretation of culture

results (via image processing), the

general workload is reduced, and

computerized data management

provides documentation control.

Microscopy, solid-phase fluores-

cence laser scanning, and ATP bio-

luminescence have also been

applied for total aerobic microbial

count enumeration.

Nucleic-acid-based methods have

also been developed for rapid steril-

ity and mycoplasma testing. One

concern with NATs is that nonvi-

able DNA can provide false positive

results. Careful design of test sys-

tems to ensure sterile environments

for samples is crucial. Such methods

can be run after immunomagnetic

separation to achieve targeted sepa-

ration using magnetic beads linked

to antibodies or lectins that bind

specific organisms. Real-time detec-

tion is possible with such systems

given that within 20 minutes, 36 to

48 nucleic acid amplification cycles

can be achieved for a typical bacte-

rium using PCR.

DNA contamination issues can be

minimized through the use of NAT

methods based on RNA. Several

such methods have been devel-

oped for microbial identification.

Genotypic identification of bacte-

rial organisms can be completed in

eight hours to three days depending

February 2016 www.biopharminternational.com BioPharm International 37

on the specific technology. However,

the equipment required for these

tests is expensive, and specialized

skills are necessary to perform them.

Biochemical methods and methods

involving gas chromatography anal-

ysis of the fatty-acid content in cell

membranes have also been devel-

oped for rapid microbial identifica-

tion.

RAPID METHODS FOR ADVENTITIOUS AGENT DETECTIONAs mentioned previously, due

to the large number of potential

adventitious viral agents that are

possible, many different assays are

required to ensure detection of all

likely viruses. In addition to being

lengthy, cell-based methods may

give false negatives, because in

some cases, replicate viruses may

not give any signs of cytopathic

effects, or an infectious virus may

not replicate in the cell lines cho-

sen for the assay.

PCR-based assays are much simpler

and more rapid, with turnaround

times of hours compared with weeks.

Each PCR test, however, detects the

DNA sequence from a specific virus.

While accurate, the results include

both viable and nonviable DNA, and

are not specific to live virus particles.

In addition, PCR alone is not appli-

cable for the detection of multiple

viruses in a single assay.

Combining PCR with degenerate

probes enables the detection of a

broad range of viruses. Information

on the virus family, subfamily, and

genus is obtained using these meth-

ods. However, these degenerate/

virus-family PCR assays are limited

by the fact that contaminant DNA

(nonviable) can influence the results.

PCR is also being combined with

mass spectrometry and micro-

arrays for lot-release testing. New

cell-based methods in development

using engineered cells are designed

to detect v iral contaminants

within 48 hours. Massive parallel

sequencing, or deep sequencing,

can be used to detect multiple DNA

sequences from different viruses.

Notably, multiplexing allows the

rapid detection of virus families.

These next-generation sequencing

technologies help to minimize the

risks associated with conventional

and simple PCR-based methods for

adventitious agent detection.

Automation is also playing an

important role in advancing rapid

methods for the detection of adven-

titious viruses. For nucleic acid

extraction, specially designed soft-

ware can aid in primer/probe selec-

tion, while bioinformatics tools help

facilitate multiple sequence align-

ments. Automated PCR assembly

leads to improved design princi-

ples and chemistries, and real-time

PCR amplification and analysis

dramatically reduce assay times,

as does the ability to immediately

compare results to existing viral

sequence records in databases.

Furthermore, these databases are

continually updated with infor-

mation on new viruses soon after

they are identified, enabling more

comprehensive analyses.

CONCLUSIONThe development of CAR-T and

other cell-based therapies has cre-

ated opportunities for patients

with diseases that previously had

no treatment options. However,

extensive safety testing of such

drug products is necessary, as they

are based on live cells that cannot

undergo a final sterilization step.

Not only is comprehensive char-

acterization of the cells necessary,

rigorous testing to demonstrate the

absence of any microbial or viral

contamination is paramount. In

addition, testing strategies must

be designed to meet the unique

requirements of each cell-based

therapy. Advances in rapid test-

ing methods for use throughout

the entire manufacturing process

for CAR-T therapies will not only

provide even greater assurance of

drug product safety, but also may

facilitate the further development

of novel, effective treatments for

patients with unmet medical needs.

REFERENCES 1. EMA, Guideline on Development and

Manufacture of Lentiviral Vectors

(London, May 2005).

2. FDA, Guidance for Industry: Guidance

for Human Somatic Cell Therapy and

Gene Therapy (Rockville, MD, Mar.

1998).

3. FDA, Guidance for FDA Reviewers and

Sponsors: Content and Review of

Chemistry, Manufacturing, and Control

(CMC) Information for Human Somatic

Cell Therapy Investigational New Drug

Applications (INDs) (Rockville, MD, Apr.

2008).

4. USP, USP General Chapter <1046>,

“Cell and Gene Therapy Products” (US

Pharmacopeial Convention, Rockville,

MD, 2011).

5. EDQM, EurPh, Gene Transfer Medicinal

Products for Human Use 5.14 (EDQM,

Strasbourg, France, 2010).

6. FDA, Guidance for Industry:

Supplemental Guidance on Testing for

Replication Competent Retrovirus in

Retroviral Vector Based Gene Therapy

Products and During Follow-up of

Patients in Clinical Trials Using

Retroviral Vectors (Rockville, MD, Nov.

2006).

7. FDA, Guidance for Industry: Potency

Tests for Cellular and Gene Therapy

Products (Rockville, MD, Jan. 2011).

8. FDA, Guidance for Industry: Preclinical

Assessment of Investigational Cellular

and Gene Therapy Products (Rockville,

MD, Nov. 2012).

9. EDQM, EurPh, Sterility 2.6.1 (EDQM,

Strasbourg, France, 20601, 04/2011)

10. EDQM, EurPh, Mycoplasma 2.6.7

(EDQM, Strasbourg, France, 20607,

01/2008),

11. CFR Title 21, Part 610.12 (Government

Printing Office, Washington, DC), pp.

70–75.

12. PDA, Technical Report No. 33,

Evaluation, Validation and

Implementation of Alternative and Rapid

Microbiological Methods (Revised

2013).

13. EDQM, EurPh, General Text 5.1.6.

(EDQM, Strasbourg, France, 2011).

14. FDA, Center for Biologics Evaluation

and Research, “Identifying Faster

Sterility Tests for Biological Products:

Regulatory Research Seeks to Reduce

the Time Needed to Ensure the Safety

of Critical Products,” (Rockville, MD,

2011), www.fda.gov/downloads/

BiologicsBloodVaccines/

ScienceResearch/UCM266975.pdf,

accessed Jan. 29, 2016.

Cell Therapies

38 BioPharm International www.biopharminternational.com February 2016

Ato

mic

Im

ag

ery

/Gett

y Im

ag

es

The possibility of using plasmids

as biopharmaceuticals for gene

therapy and DNA vaccination

has gradually emerged during

the past 20 years (1). The plasmid DNA

(pDNA) molecules in these biopharma-

ceuticals should transfer genes to target

individuals (humans and animals) to

prevent or exercise control over diseases

such as AIDS, tuberculosis, and cancer.

A significant challenge in this context is

the development of manufacturing pro-

cesses capable of producing the required

material to run pre-clinical and clinical

trials (1, 2).

The manufacturing of pDNA com-

prises a series of interlinked activities

(see Figure 1) designed to consistently

obtain a defined amount of a safe and

effective product (1). The pDNA is typi-

cally produced by replication in Gram-

negative Escherichia coli. However, in

most cases, the strains used (e.g., DH5α,

JM101, BL21) were originally developed

for cloning or for the production of

recombinant proteins (1). Due to their

miscellaneous mutagenized genetic

backgrounds, such strains may thus

not be the best choice for producing

the large amounts of pDNA required

for clinical trials and eventual com-

mercialization (3, 4). A more rational

approach is to start from a wild-type

strain and select/mutate genes that are

likely to have an impact on the kinet-

ics of cell growth and on the synthesis

of pDNA (5, 6). Such engineered strains

should grow up to high cell densities

and produce large quantities of pDNA

(i.e., maximize volumetric pDNA yield,

Use of an E. coli pgi Knockout Strain as a Plasmid Producer

Cláudia P. A. Alves, Sofia

O. D. Duarte, Gabriel A.

Monteiro, and Duarte

Miguel F. Prazeres

The authors describe the

impact of the knocking of

the pgi gene of the wild

type MG1655 strain on the

growth kinetics of plasmid-free

and plasmid-bearing cells.

Cláudia P. A. Alves is research

assistant, Sofia O. D. Duarte is a

PhD student, Gabriel A. Monteiro

is associate professor, and *Duarte

Miguel F. Prazeres is full professor, all

at the iBB–Institute for Bioengineering

and Biosciences, Department of

Bioengineering, Instituto Superior Técnico,

Universidade de Lisboa, Av. Rovisco Pais,

1049-001 Lisboa, Portugal. *To whom all

correspondense should be addressed.

Gene Therapy

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mg pDNA/L) as quickly as possible

and at the lowest cost. Although

the growth/production medium,

bioreactor operating variables, and

culture strategies are key aspects at

this stage, starting from a robust,

high producer of pDNA is highly

recommended.

This article describes the growth

kinetics, pH profile, and pDNA

volumetric yield obtained with

GALG20, an endA, recA, and pgi

knockout of the wild type MG1655

strain (6, 7). One of the targets, pgi,

codes for phosphoglucose isom-

erase, an enzyme that catalyzes

the conversion of glucose-6-phos-

phate into fructose-6-phosphate.

This knockout allows the redi-

rection of the carbon flux to the

pentose phosphate pathway, lead-

ing to an increase in nucleotide

synthesis and pDNA production,

whereas the deletion of endA and

recA minimizes pDNA nonspecific

digestion and recombination (6).

Additionally, the authors show that

supercoiled (sc) pDNA produced

by these cells can be isolated from

impurities and from open circular

(oc) pDNA by an optimized hydro-

phobic interaction chromatogra-

phy (HIC) step.

MATERIALS AND METHODSStrains and plasmids

The GALG20 strain was con-

structed by deleting the genes

endA, recA, and pgi in the wild type

strain MG1655 by P1 transduction

as described previously (6). The

strain was then transformed with

the 3697 bp plasmid pVAX1GFP (8).

Cell growth

Inocula were prepared from fro-

zen stocks of transformed GALG20

and wild type MG1655 strains

in 15 -mL conica l centr i fuge

tubes with 5 mL of LB medium

(NZYTech, Lisbon, Portugal) sup-

plemented, when required, with

30 μg/mL kanamycin (Amresco,

Solon, OH). Cells were incubated

overnight at 37 ºC and 250 rpm

and used to inoculate 250-mL

baffled shake flasks containing 50

mL of complex medium (20 g/L

glucose, 10 g/L bacto peptone, 10

g/L yeast extract, 3 g/L ammo-

nium sulfate ((NH4)2SO4), 3.5 g/L

potassium hydrogen phosphate

(K2HPO4), 3.5 g/L potassium dihy-

drogen phosphate (KH2PO4), 200

mg/L thiamine, 2 g/L magnesium

sulfate (MgSO4), and 1 mL/L of a

trace element solution [9]) with 30

μg/mL kanamycin, pH 7.0, at an

initial optical density at 600 nm

(OD600) of approximately 0.1.

Plasmid purification

Transformed GALG20 cells were har-

vested after 10 hours by centrifuga-

tion and subjected to alkaline lysis

as described previously (7). Then,

plasmid in the clarified lysates

was precipitated with 0.7 volumes

of pure isopropanol (2 hours, -20

°C) and recovered by centrifuga-

tion (30 min at 18,514 g and 4 °C).

After drying at 4 ºC, pellets were

resuspended in 10 mM Tris-HCl,

pH 8, and solid ammonium sulfate

was added up to a concentration

of 2.5 M to precipitate proteins

(15 min on ice) and condition the

solution in preparation for HIC.

Precipitated proteins were removed

by centrifugation (30 min, 17,949

g, 4 °C). The pDNA in this solu-

tion was purified by HIC using

a column packed with 10 mL of

Phenyl Sepharose 6 Fast Flow resin

in an ÄKTApurifier100 system (GE

Healthcare). A mobile phase con-

taining mixtures of 2.2 M ammo-

nium sulfate in 10 mM Tris-HCl,

1 mM ethylenediaminetetraacetic

acid (EDTA), pH 8 (buffer A) and

10 mM Tris-HCl, 1 mM EDTA, pH

8 (buffer B) was used to run the

separation. The absorbance of the

eluate was continuously measured

at 254 nm with a UV detector posi-

tioned at the column outlet, and

the system was operated at 2 mL/

min. Following column equilibra-

tion with three column volumes

(CV) of 17% buffer B (≈ 204 mS/

cm), 1 mL of the pDNA-contain-

ing feed was injected. Unbound

material was washed out with 4

CV of 17% B, and two elution steps

were performed with 2 CV of 35%

B (≈ 173 mS/cm) and with 2 CV

of 100% B (≈ 2 mS/cm). Fractions

(1.5 mL) corresponding to peaks

were collected during the run and

dialyzed against 10 mM Tris-HCl, 1

mM EDTA, pH 8 for desalting prior

to analysis in 1% (w/v) agarose gels.

Plasmid quantitation

Analytical chromatography was

performed in an ÄKTApurifier10

system (GE Healthcare), using a

commercial Tricorn high-perfor-

mance column with a 1.7 mL bed

volume (SOURCE 15PHE 4.6/100

PE, GE Healthcare) and following

a modification of the HIC–HPLC

(high-performance liquid chro-

matography) method described by

Diogo et al. (10). Briefly, after col-

umn equilibration (2.5 min) with

1.5 M ammonium sulfate in 10

mM Tris-HCl pH 8, 50 μL samples

were injected and elution was per-

formed for 1 min with the same

buffer. Species bound to the matrix

were then eluted with 10 mM Tris-

HCl pH 8 for 0.8 min and column

was re-equilibrated for 5.5 min

Gene Therapy

Figure 1: Overview of main activities in plasmid manufacturing.

pDNA pDNAcell banks purification fill/finishcell

culture

bulk

pDNAE.coli

40 BioPharm International www.biopharminternational.com February 2016

with the initial buffer. The absor-

bance of the eluate was continu-

ously measured at 260 nm, and the

system was operated at 1 mL/min.

The plasmid was quantified using a

calibration curve constructed with

plasmids standards (purified using

the HiSpeed plasmid Maxi Kit from

Qiagen) prepared in a concentra-

tion range from 0 to 100 μg/mL.

Gel electrophoresis

Agarose gels were prepared with

1% (w/v) agarose (ThermoFisher

Scientific) in tris-acetate-EDTA

(TAE) buffer (40 mM Trisbase, 20

mM acetic acid and 1 mM EDTA,

pH 8) and loaded with samples

mixed with a 6X loading buffer

(40% w/v sucrose, 0.25% w/v bro-

mophenol blue), using NZYDNA

ladder I I I (NZYTech, Lisbon,

Portugal) as molecular weight

marker. Electrophoresis was run at

120 V for 90 min, using 1% TAE

as the running buffer. Gels were

stained in an ethidium bromide

solution (0.4 μg/mL), and images

were obtained with an Eagle Eye

I I gel documentat ion system

(Stratagene, La Jolla, CA).

RESULTSGrowth kinetics

The pgi gene of the wild type E.

coli strain MG1655 was knocked

out with the goal of redirecting

the carbon flux into the pentose

phosphate pathway to increase

nucleotide synthesis, NADPH

generation, and hopefully pDNA

production (5). The growth charac-

teristics of the new strain GALG20

(either non-transformed or trans-

formed) were studied and com-

pared with MG1655 (see Figure 2).

Experiments were performed in

baffled shake flasks using 20 g/L

glucose in a rich medium at an

initial pH of 7.0. The pH of the

medium was also measured dur-

ing the course of cell growth (see

Figure 3). The data show that dur-

ing the first four hours, there are

no differences between growth

profiles of the native MG1655 and

GALG20 strains. A significant drop

of the pH of the medium to 5.7,

however, is detected at four hours

for the case of MG1655 that is in

stark contrast with the lack of pH

variation observed for GALG20

(pH ~ 6.9). From the fourth hour

on, GALG20 continued to grow at

a rate that was significantly higher

Gene Therapy

Figure 2: Growth curves of Escherichia coli strains GALG20 and MG1655.

Complex medium with 20 g/L glucose at pH 7 was used to grow cells in baffled shake

flasks (37 ˚C, 250 rpm). Results are an average of three independent experiments.

Figure 3: Variation of medium pH during growth of Escherichia coli strains

GALG20 and MG1655. Complex medium with 20 g/L glucose at pH 7 was used

to grow cells in baffled shake flasks (37 ˚C, 250 rpm). Results are an average of

three independent experiments.

40

30

20

10

0

0 2 4 6 8 10

GALG20 GALG20+pDNA MG1655

OD

60

0

time (h)

7.5

6.5

5.5

4.5

pH

time (h)0 2 4 6 8 10

GALG20 GALG20+pDNA MG1655

February 2016 www.biopharminternational.com BioPharm International 41

Gene Therapy

when compared with MG1655. As

a result, optical densities (ODs)

of 30 were obtained for non-

transformed GALG20 after 10 h,

whereas MG1655 did not surpass

ODs of 10 at the same time instant.

After 10 h of growth, pH values

decreased to 4.8 for MG1655, 5.6

for non-transformed GALG20, and

6.3 for transformed GALG20. The

plasmid DNA produced by trans-

formed GALG20 cells was quanti-

fied by HIC–HPLC from clarified

alkaline lysates of cells harvested

after 10 h of growth as described

by Gonçalves et al. (7). Results

from four independent shake flask

cultures indicate a pDNA produc-

tion of 63.0 ± 11.7 mg/L.

Supercoiled pDNA isolation

Experiments were performed to

check if the knockout of the pgi gene

had an impact on the purification

and final quality of pDNA. Firstly,

transformed GALG20 cells grown as

described above were disrupted by

alkaline lysis to release pDNA. Then,

sequential precipitation with isopro-

panol and with ammonium sulfate

was used to concentrate nucleic acids

and remove protein and RNA impu-

rities, respectively (11). The resulting

high-salt solution (~2.5 M) was then

subjected to HIC (see Figure 4) using

a phenyl Sepharose column to iso-

late the sc isoform from the mix-

ture containing sc and oc pDNA

and also RNA (see lane F in Figure

5). Elution steps with decreasing

ammonium sulfate concentra-

tions were used to separate plasmid

topoisomers and RNA. The chro-

matogram (Figure 4) is character-

ized by two flowthrough peaks

emerging sequentially at 17% B

(~1.83 M ammonium sulfate), a

first elution peak at 35% B (~1.43 M

ammonium sulfate), and a second

peak at 100% B (0 M ammonium

sulfate). An agarose gel electropho-

resis analysis of the correspond-

ing fractions shows clearly that the

flowthrough contains oc pDNA

Figure 4: Purification of supercoiled plasmid DNA by hydrophobic interaction

chromatography in a phenyl Sepharose column. Stepwise elution with decreasing

ammonium sulfate concentration was used to separate plasmid topoisomers and

RNA. The numbers over peaks correspond to the collected fractions. Percentage of

buffer B (dashed line) and conductivity (dotted line) are also shown.

Figure 5: Agarose gel electrophoresis analysis of fractions isolated by

hydrophobic interaction chromatography. The numbers above each lane

correspond to fractions collected during the chromatographic run presented in

Figure 4. Lane F corresponds to the column feed.

160

120

80

40

0

250

200

150

100

50

0Ab

sorb

an

ce 2

54

nm

(m

AU

)

Co

nd

uct

ivit

y (

mS

/cm

); %

B

Elution volume (mL)

0 20

3-4

7-9

34-39 49-54

40 60 80

MW (bp)

10000

3000

2000

1000

200

F 3 8 37 53

OC

SC

RNA

42 BioPharm International www.biopharminternational.com February 2016

(lanes 3 and 8, Figure 5), whereas

sc DNA is obtained in the elution

peak at 35% B (lane 37, Figure 5). As

for RNA, it is removed only when

the column is eluted at a low salt

concentration (lane 53, Figure 5).

DISCUSSIONThe pgi knockout strain GALG20

is able to grow up to optical densi-

ties that are significantly higher

when compared with the MG1655

control strain (~30 vs. ~10 after

10 h). Furthermore, the acidifi-

cation of the culture medium

during growth of GALG20 is less

pronounced when compared

with MG1655. The sharp decrease

of pH in the case of MG1655 is

consistent with acetate produc-

tion, a phenomenon that occurs

in aerobic conditions when high

concentrations of glucose inhibit

respiration (Crabtree effect). In

the case of GALG20, however, the

results indicate that the knock-

ing out of pgi impaired the abil-

ity of the new strain to produce

acetic acid (and hence acidify

the medium). This result is con-

sistent with the down-regulation

of glycolysis and of the tricar-

boxylic acid cycle, and with the

redirection of the carbon flux to

the pentose phosphate pathway.

Although the main purpose of

the aforementioned knockout was

the increase in nucleotide synthe-

sis and, consequently, in pDNA

production, the low acetate pro-

duction by the GALG20 strain

makes it possible for cells to reach

a higher density (≈ 3-fold higher

than the wild type MG1655), espe-

cially when medium pH is not

controlled during cell growth. The

results obtained for pDNA volu-

metric yield (63.0 ± 11.7 mg/L)

also prove the effect of the pgi

knockout on pDNA production. At

shake-flask scale, the volumetric

yield of GALG20 is 7- to 10-fold

higher (7) than the one presented

by its parental strain MG1655

also deleted for the endA and recA

genes (MG1655ΔendAΔrecA), high-

lighting the favorable outcome of

the metabolic pathway redirection

imposed by the pgi knockout.

Supercoiled plasmid DNA pro-

duced by GALG20 was isolated

and purified by a process that

combines alkaline lysis with tan-

dem precipitation with isopro-

panol, ammonium sulfate and

purification by HIC. Agarose gel

analysis of collected fractions

show that the method applied

is able to separate sc pDNA from

the oc isoform and from RNA.

Densitometry analysis of the

bands in the agarose gel presented

in Figure 5 confirmed the suc-

cessful isolation of sc pDNA. The

column feed contained approxi-

mately 51.3% of sc pDNA and

48.7% of oc pDNA (lane F). The

oc pDNA was removed essentially

in the first (lane 3) and second

(lane 8) f lowthrough peaks. A

small amount of sc pDNA was lost

in the second flowthrough peak

(2.4% of the total pDNA present,

see lane 8). The elution of the sc

pDNA isoform occurred mainly

during the step at 35% B (~1.43

M ammonium sulfate). An analy-

sis of the corresponding fraction

(lane 37) shows that 99.2% of the

pDNA recovered is sc, a level of

homogeneity that is superior to

the FDA requirements for clinical-

grade pDNA vectors (12).

CONCLUSIONThis work highlights the advan-

tages of engineering E.coli strains

for improved pDNA production

as a mean to develop efficient

manufacturing processes able

to meet pre-clinical and clinical

trial requirements. Specifically,

the authors present a pgi knock-

out E.coli strain (GALG20) that is

able to reach higher cell densities

and pDNA yields than its parental

strain MG1655, as a consequence

of a metabolic pathway redirec-

tion. Additionally, the decrease in

the pH of GALG20 cell cultures

was less pronounced when com-

pared with the variation observed

for the wild type MG1655. These

results are especially interesting

when carrying cultures with no

external pH control, since impair-

ment of cell growth is reduced.

In addition, the authors present

a purification method relying on

HIC that is able to isolate the ther-

apeutically valuable sc pDNA iso-

form, which is virtually free from

RNA and oc pDNA.

ACKNOWLEDGEMENTFunding received by iBB-Institute

for Bioengineering and Biosciences

from FCT-Portuguese Foundation

for Sc ience and Technolog y

(UID/BIO/04565/2013 and doc-

toral grant SFRH/BD/84267/2012

awarded to Sofia Duarte), from

Programa Operacional Regional de

Lisboa 2020 (Project N. 007317),

and from the European Project

INTENSO (FP7-KBBE-2012-6) is

acknowledged.

REFERENCES 1. D.M.F. Prazeres, G.A. Monteiro,

Microbiol. Spectrum 2 (6) PLAS-0022-

2014 (2014).

2. K.J. Prather et al., Enzyme Microb.

Technol. 33 (7) 865 – 883 (2003).

3. D.M. Bower, K.L.J. Prather, Appl

Microbiol Biotechnol. 82 (5) 805-813

(2009).

4. A.R. Lara, O.T. Ramirez, Methods Mol.

Biol. 824, 271-303 (2012).

5. D.S. Cunningham et al., J. Bacteriol.

191 (9) 3041-3049 (2009).

6. G.A.L. Gonçalves, et al., App.

Microbiol. Biotechnol. 97 (2) 611-620

(2013).

7. G.A.L. Gonçalves et al., J. Biotechnol.

186, 119-127 (2014).

8. A. R. Azzoni, et al., J Gene Med. 9 (5),

392-402 (2007).

9. K. Listner, L.K. Bentley, M. Chartrain,

Methods Mol. Med. 127, 295-309

(2006).

10. M.M. Diogo, J.A. Queiroz, D.M.F

Prazeres, J. Chromatogr. A 998 (1-2)

109-117 (2003).

11. M.M. Diogo, et al., Biotechnol. Bioeng.

68 (5) 576–583 (2000).

12. FDA, Guidance for industry:

Considerations for Plasmid DNA Vaccines

for Infectious Disease Indications

(Rockville, MD, Nov. 2007).

Gene Therapy

February 2016 www.biopharminternational.com BioPharm International 43

The quality assurance of lot

release in the biopharmaceuti-

cal industry is based to a great

extent on integrity testing of

used sterilizing-grade filters. If the integ-

rity test fails, the product is put in quar-

antine and an investigation is conducted.

The consistency of this quality assurance

approach is based on the reliably exe-

cuted integrity testing of the filters. One

of the greatest risks are false-passed test

results, which put the patient in danger.

From a more global point of view,

the goal of failure mode effects analysis

(FMEA) for filter integrity testing (FIT)

is to align the risks as closely as possible

with its source. This analysis can iden-

tify the root cause of the risk and help

the quality assurance staff and operators

detect the occurrence of a particular

deviation. Additionally, the analysis also

helps define the adapted level of train-

ing to reduce operator mistakes.

This article identifies risks for achiev-

ing a higher level of FMEA for FIT and

for improved quality assurance.

SCOPE OF THE FMEA FOR FIT The purpose of FMEA for FIT is to estab-

lish documentary evidence to assure that

the manufacturing process is capable

of producing the pre-determined qual-

ity specifications when using a specific

tester for integrity testing of filters, while

guaranteeing the safety of the operator.

RISK IDENTIFICATIONIdentifying risks and unwanted events,

the potential consequences (sever-

ity/impact), the likelihood that the

unwanted event will occur (probabil-

ity), and the likelihood of detecting the

unwanted event (detectability) require

knowledge in FIT. The suppliers of the

filters and testing device should provide

supporting documentation that identi-

fies the following risks.

Selecting the incorrect program or

setting incorrect testing parameters

Selecting the wrong program or set-

ting the wrong testing parameters could

mean that the test pressure is wrong

and/or the test limit is not adapted.

Incorrect test pressure

Different suppliers use different test

pressures for the diffusion test, and dif-

ferent filter pore sizes require differ-

ent test pressures. Mixing test pressures

from different suppliers and test pres-

sures for different pore sizes when set-

ting the parameters for testing can lead

to potential quality deviations.

Fick’s law gives the relation between

applied differential pressure and diffu-

sion for filters, under condition that all

pores are filled with water. Fick’s law

demonstrates that the diffusion value is

not dependent on the pore size (μm) but

the porosity (percentage of void) as long

as the pores are filled with water. As long

as the pores are filled with water, it is

commonly considered to be a linear rela-

tion between the applied test pressure

and the resulting diffusion value (1–2).

The porosity can be quite similar

between a 0.1 μm and a 0.2 μm mem-

brane. To detect different pore sizes, the

diffusion test pressure for a filter with a

given pore size (e.g., 0.1 μm) is selected

so that a filter with a bigger pore size

(e.g., 0.2 μm) would give a failing test

result, even if the filter with the bigger

pore size (0.2 μm) is integer. In other

words, the diffusion test pressure of a

0.1 μm cartridge (4 bar) is close to or

above the expected bubble point (BP) of

a 0.2 μm membrane (BPmin = 3.2 bar;

expected BP = 3.7 to 3.9 bar).

Failure Mode Effects Analysis for Filter Integrity Testing

Magnus Stering

Understanding of the risks associated

with FMEA is crucial in lot

release testing.

Magnus Stering is product manager,

Integrity Testing Solutions, Filtration

Technology, Sartorius Stedim Biotech.

Filter Integrity Testing

44 BioPharm International www.biopharminternational.com February 2016

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If a 0.1 μm membrane cartridge

is tested at a pressure that is signifi-

cantly lower than the expected bub-

ble point for a 0.2 μm membrane

cartridge, one cannot say for sure

that the 0.1 μm membrane was not

an 0.2 μm membrane (see Figure 1).

The diffusion value of a diffu-

sion integrity test that has been

conducted at too high a pressure,

but still gives a conform test result,

could be extrapolated down to the

value one would have had if the

test had been conducted at the cor-

rect pressure without risking a false

conformity test result. The only

risk is an over estimation of the

diffusion value, thus risking a false

failed-test result; however, there

is no risk for any false passed-test

result. On the other hand, the dif-

fusion value of an integrity test

that has been conducted at too

low a pressure could not necessar-

ily be extrapolated up to the value

one would have had if the test had

been conducted at the correct test

pressure (see Figure 1). The risk of

conducting a diffusion test at too

low a pressure is a false passed test.

Wrong test limit

Filter suppliers also use different

test limits for a given size of filters.

The test methods may have the

same name such as water intrusion

test (WIT) but may measure differ-

ent things. The WIT from supplier

A expresses the measured value as

a gas flow; the WIT from supplier

B expresses the measured value as

a water flow. Supplier C uses car-

tridge-specific factors that are only

available from that supplier.

No test approach is better than

the other; however, the person

doing the programming must be

aware about the differences. The

WIT value from suppliers A and B

can easily be converted one to the

other by Equation 1:

WITA = WIT

B ∙ p

abs

Where pabs

= absolute test pressure in

bar (e.g., 3.5 bar absolute

for a test pressure

expressed as 2500 mbar)

[Eq. 1]

This means that if the person

doing the programming sets the

parameter for a WITB using the max

value for a WITA the risk is to get false

passed-test results. If the max value

for a WITB is used when program-

ming a WITA most likely the test

results will be repeatedly false-failed.

Using a barcode scanner when

entering data during programming

allows the operator to select the cor-

rect program, thus minimizing the

risk of using the incorrect test pro-

gram. The filter supplier can confirm

the correct test parameters to avoid

using the wrong test parameters.

INFLUENCE FROM THE TYPE OF TEST GASWhen performing a diffusion test

(forward flow test) or a pressure

drop test on a filter, the type of test

gas has to be considered. The max-

imum allowable diffusion value for

a cartridge is typically given for

air as the test gas if no other gas

is stated. As nitrogen (N2) has a

lower solubility in water than air,

the diffusion rate for a given filter

will be reduced as it follows well-

established laws of diffusion (see

Equation 2):

N = D ∙ H ∙ P ∙ φ / L

Where

N = diffusive flux of the test gas

D = diffusivity of the test gas through

the wetting liquid

H = solubility coefficient of the test

gas into the wetting liquid

P = applied differential pressure

φ = overall porosity of the membrane

structure (in %)

L = thickness of the wet layer

[Eq. 2]

If nitrogen is being used instead

of compressed air, the maximum

allowable diffusion rate must be

modified accordingly to avoid false

passed test results (see Equation 3):

Diffusion

= DiffusionmaxAir

x 0.82maxN

2

[Eq. 3]

In fact, the use of nitrogen

instead of air simply lowers the dif-

fusion but keeps the same overpro-

portional increase when the bubble

point is reached (see Figure 2).

The laws of bubble point do not

take solubility into account but

rather the surface tension of the

wetting liquid (see Equation 4).

BP = 4 ∙ k ∙ γ ∙ cosθ / d

Where

k = tortuosity factor (non-cylindrical pores)

γ = surface tension of the wetting liquid

θ = wetting angle of the wetting liquid on

the membrane material

d = biggest pore diameter

[Eq. 4]

Filter Integrity Testing

Figure 1: 0.1 μm vs. 0.2 μm pore size membrane filters. BP is bubble point.

DiffMax

0.1μm

DiffMax

0.2μm

PDiff

0.2μm

PDiff

0.1μm

Integer

0.2μm

Integer

0.1μm

Only a few 0.1μm cartridge types

can be tested for BP because the

BPmin is typically above the pressure

resistance of the cartridge

Failing

0.1μm

Dif

fu

sio

n m

L/m

in

BPmin

0.2μm

BPactual

0.2μm

Pressure

February 2016 www.biopharminternational.com BioPharm International 45

The water intrusion test is also

not influenced notably by the use

of nitrogen instead of air as test

gas because the contact surface

between the gas and the water is

limited. No significant amount of

gas is dissolved into the water dur-

ing the test.

If compressed air and nitrogen are

available at the point of use, color

coding must be used. Different con-

nection types may also be used to

avoid connecting the integrity testing

device to the wrong pressure source.

IMPACT FROM THE TEMPERATURE OF THE TEST GASAll filter integrity testing methods

using pressurized gas are bound

to the ideal gas law. There is no

significant difference if the test is

called diffusion or forward flow or

if the testing device is measuring

pressure decay or flow. All pressure-

based integrity testing is influenced

by temperature variations.

One of the prerequisite condi-

tions for integrity testing is stable

temperature. If the device is pres-

surized with cold compressed gas,

the heat exchange between the

test gas and the filter housing will

cool the housing and heat the gas.

As the housing is at a lower tem-

perature than ambient due to the

cooling effect of the test gas, the

environment will heat the housing

and slowly bring both the housing

and the test gas to ambient tem-

perature (see Figure 3).

If the measurement phase starts

before the temperature is stable,

there could be a significant impact

on the measured value. A tempera-

ture change of only 1 °C inside the

filter housing during the measure-

ment phase of 5 minutes may induce

an error of approximately 20–40%

depending on the test value.

Calculation:

t 5IF UFTU QSFTTVSF JT NCBS

gauge or approximately 3500

mbar absolute.

t 5IF UZQJDBMEJGGVTJPOWBMVF GPS

a 10” filter is 12 mL/min for a

max diffusion value of 18 mL/

min.

t 5IFUZQJDBMUFTUUJNFJTNJO

t 5IF UZQJDBMOFUWPMVNF JT

mL.

For diffusion, Equation 5 is used:

Diffusion = Δp ∙ V / (pref

∙ t)

Where

Δp = pressure drop (during pressure

decay measurement)

or cumulated pressure drop

(during flow cell measurement)

V = net volume

pref

= 1000 mbar

t = test time in minutes

[Eq. 5]

The pressure drop under iso-

therm conditions is:

Δp = 12 ∙ 1000 ∙ 5/1400 = 43 mbar

The pressure change under iso-

volumetric conditions due to tem-

perature variation is expressed by

Equation 6:

p1 / T

1 = p

2 / T

2 Δp

T = p

2 – p

1

= p1 ∙ T

2 / T

1 – p

1

Where:

p1 = start pressure in mbar absolute

p2 = end pressure in mbar absolute

T1 = temperature in Kelvin at start

(before change e.g. 293 K)

T2 = temperature in Kelvin at end

(after change 294 K)

ΔpT = pressure change due to

temperature variation

[Eq. 6]

Filter Integrity Testing

Figure 3: Changes in housing and test gas temperature.

Ambient temperature

Housing temperature

Test gas temperature

Tº

Co

ldW

arm

t

Figure 2: Comparison of air and nitrogen as the test gas. BP is bubble point.

BPmin

0.2μm

BPactual

0.2μm

Pressure

Tested

with air

Tested with

nitrogen

Dif

fu

sio

n m

L/m

in

Contin. on page 50

46 BioPharm International www.biopharminternational.com February 2016

Cost-efficient biopharmaceuti-

cal development must adhere

to the crucial principle “fail

early and fail cheap” when

eliminating unpromising candidate

molecules from the pipeline. To do this

in an informed way, it is vital to have

sound information to make decisions

about which therapeutics offer the most

potential. Protein instability, typically

caused by aggregation or degradation,

is a pre-eminent concern for biologics

because of the associated risks of immu-

nogenicity and reduced efficacy. In the

earliest stages of candidate validation

and formulation development, informa-

tion that helps detect a tendency toward

instability can deliver considerable

cost and time savings. As development

advances, the requirement shifts to a

need to elucidate and control protein

stability within an evolving, increas-

ingly complex formulation, and the

associated analytical requirements alter

accordingly.

In this article, the author reviews some

of the techniques that can yield valuable

information on protein stability, focus-

ing specifically on protein aggregation.

Emphasis is placed on the enhanced

information made available when tech-

nologies are used orthogonally, and the

alignment of different approaches with

specific stages of the biopharmaceutical

development workflow.

CANDIDATE SCREENING: RAPID AND ROBUST ELIMINATION OF LOW-POTENTIAL MOLECULES The progressive refinement of potential

therapeutics to develop an optimal bio-

pharmaceutical product often begins

with the assessment of a substantial

number of candidates. Early screening

Complementary Techniques for the Detection and Elucidation

of Protein AggregationLisa Newey-Keane

The author reviews some of the techniques that can yield valuable information on protein stability during characterization studies.

A key emphasis is the data delivered by alternative techniques, the enhanced information produced when technologies are used orthogonally, and the alignment

of different approaches with specific stages of the biopharmaceutical development workflow.

Lisa Newey-Keane, PhD, is life

science sector marketing manager,

Malvern Instruments, Enigma Business

Park, Grovewood Road, Malvern,

Worcestershire, WR14 1XZ, United

Kingdom, Tel: +44 (0) 1684 892456,

Fax: +44 (0) 1684 892789,

www.malvern.com

Protein Aggregation

February 2016 www.biopharminternational.com BioPharm International 47

AL

L F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

R.

criteria relate to the assessment of

factors including bioefficacy, devel-

opability, and manufacturability,

and are designed to identify candi-

dates that are not only efficacious

but will also lend themselves to

simple formulation and profitable

manufacture. Propensity to aggre-

gate is one of the screening tests

associated with developability,

because of the long-term impact of

aggregation on product stability.

The most relevant analyt i-

cal techniques at this early stage

combine automation and high

throughput with minimal sam-

ple volume requirements. At this

point, candidates have limited

availability, so maximizing the

information gained from every

microgram of sample is crucial.

Lack of sample also results in a

need for measurements at low con-

centration, raising the question of

whether results are representative

of how the molecule will behave

in its final formulation concentra-

tion. The requirement for robust

and reliable selection means that

simple and clear indicators of

promising performance are prefer-

able. The measurement of hydro-

dynamic size (RH), for example, is

one of the most effective ways of

detecting potential problems with

molecular conformation and struc-

ture, and stability in solution.

Dynamic light scattering (DLS)

and size-exclusion chromatogra-

phy (SEC) are established tech-

niques for measuring RH and

also parameters such as molecu-

lar weight, which are extremely

helpful in screening candidates for

potential protein aggregation. In

addition, Taylor dispersion analysis

(TDA), a much newer technique,

is also now proving valuable and

adding beneficial orthogonality at

this point in the pipeline.

TDA is a microcapillary flow-

based technique that enables rapid

and accurate determination of

the diffusion coefficients of tar-

get molecules, and consequently

their RH. Instruments that imple-

ment TDA alongside UV detection,

such as the Viscosizer TD (Malvern

Instruments), measure the size of

the target molecule directly in its

formulation without interference

from other species present, includ-

ing peptides, formulation excipi-

ents, and surfactants. TDA is,

therefore, a powerful complemen-

tary method to DLS and SEC for

identifying outliers on the basis of

their size in formulation, possibly

due to self-association or conforma-

tional changes, which may prove

problematic further down the pipe-

line. For example, Figure 1 shows

how TDA can sensitively detect

the reduction in hexameric insulin

associated with decreasing insulin

concentration. In addition, instru-

ments such as the Viscosizer TD

provide relative viscosity screening

within the same samples, facili-

tating the removal of candidates

with abnormal/unhelpful viscos-

ity profiles—a distinct issue when

developing injectable therapeu-

tics—from the pipeline at the earli-

est opportunity.

EARLY FORMULATION: DETECTION, QUANTIFICATION, AND CHARACTERIZATION OF AGGREGATES TO IMPROVE STABILITYThe successful progression of

promising candidates through

early-stage formulation relies on

a more rigorous assessment of

protein aggregation and stability.

Because candidate numbers remain

high, long-term stability studies

for every formulation are infeasi-

Protein Aggregation

Figure 1: Taylor dispersion analysis (TDA) is highly complementary to dynamic

light scattering (DLS) for size measurement. The data show how TDA is able to

sensitively detect the reduction in hexameric insulin associated with decreasing

insulin concentration. This reduction is less clear from DLS data, which is strongly

influenced by the presence of large hexamers.

3

2.5

2

1.5

1

0.5

0

Hyd

rod

yn

am

ic r

ad

ius

(nm

)

Insulin concentration (mg/mL)

0.5mg/mL 1mg/mL 2mg/mL 3mg/mL

TDA

DLS

Hexamer Rh

~ 2.7nm

Uses and benefits of Taylor dispersion analysis

t 4J[FBOEWJTDPTJUZNFBTVSFNFOUJODMVEJOHBHHSFHBUJPOEFUFDUJPO

t 6MUSBMPXTBNQMFWPMVNFT

t "VUPNBUFE XBMLBXBZBOBMZTJT

t -BCFMGSFFBOBMZTJTXJUINJOJNBMTBNQMFQSFQBSBUJPOSFRVJSFNFOUT

48 BioPharm International www.biopharminternational.com February 2016

ble at this point; fast, automated

measurements will, therefore,

be a priority. Differential scan-

ning calorimetry (DSC) is widely

used for the assessment of ther-

mal stability (e.g., for measuring

changes in the melting transition

midpoint temperature that can be

linked directly with stability) and

for detecting changes in confor-

mation, via measurement of the

associated enthalpy. Highly speci-

fied DLS systems, on the other

hand, such as the Zetasizer Nano

(Malvern Instruments), deliver

automated measurement of stabil-

ity prediction parameters such as:

t #22

, the second virial coefficient:

an indicator of the strength of

electrostatic and hydrophobic

bonding

t ,D, the DLS interaction param-

eter: a function of both ther-

modynamic and hydrodynamic

interactions

t ;FUBQPUFOUJBMBNFBTVSFPGUIF

overall strength of intermolecu-

lar electrostatic interactions.

Higher (either positive or nega-

tive) zeta potentials are associated

with increased repulsion between

molecules and a lower risk of native

aggregate formation, which, though

often reversible, increases the risk

of aggregation of the denatured pro-

tein. More positive values of B22 and

,D are also associated with stability.

Complementary to the applica-

tion of DLS at this stage is TDA,

because of its ability to detect

aggregates in the presence of excip-

ients or other larger particles, and

also SEC, especially when imple-

mented with multiple detectors.

Unlike DLS and TDA, SEC is not an

“in-solution” technique, so it offers

an orthogonal approach to build-

ing an understanding of aggrega-

tion mechanisms as a prelude to

exerting effective control.

SEC involves the separation of a

sample into fractions on the basis

of hydrodynamic size, followed by

analysis of each eluting fraction. It

is typically deployed, often with a

single detector, as a quality control

release assay to measure the oligo-

meric state of a protein. However,

with a multiple detector array, SEC

becomes a far more powerful tool

for investigating the composition

and aggregation state of a sample

(see Figure 2). The resulting data

aid the development of a detailed

understanding of the degradation

pathways and products associated

with aggregation.

A multiple angle light scattering

(MALS) detector determines the

absolute molecular weight of each

species in the sample, thereby char-

acterizing any aggregates present

in terms of the number of mono-

mer units involved. Light scattering

detection also enables the measure-

ment of hydrodynamic radius and

provides an assessment of polydis-

persity. A UV detector, on the other

hand, measures the concentration

of chromophore-containing species

to reveal the percentage of aggrega-

tion. Adding a viscometer to the

detector array brings intrinsic vis-

cosity data, which, in combination

with molecular weight measure-

ments, can be used to quantify the

structural characteristics of any

protein species present to further

elucidate aggregation mechanisms.

LATE FORMULATION: UNDERSTANDING STRUCTURAL STABILITY TO ACHIEVE EFFECTIVE CONTROLBy late-stage formulation, although

the number of drug candidates

would have dropped, drug devel-

opers are required to provide

more information on the for-

mulation. Here, there is a need

for analysis that provides deeper

insight into aggregation mecha-

nisms and the factors that influ-

ence them, including the impact

of possible excipients. This require-

Protein Aggregation

Figure 2: Applying a light-scattering detector in size exclusion chromatography

(SEC) analysis (Malvern SEC-MALS 20—orange trace) reveals high molecular

weight pepsin aggregates (molecular weight data overlaid in black) that are not

detected with a single RI detector (red trace).

400

300

200

100

0

109

106

1000

1

6

4

2

0

-2

6 8 10 12 14 16 18 20 22 24 26 28 30

Retention volume (mL)

2013-05-03_06;16;09_Pepsin_35_kDa_01.vdt: All Peaks

Re

fra

ctiv

e i

nd

ex (

mV

)

MA

LS s

ign

al

90

º(m

V)

Mo

lecu

lar w

eig

ht (

Da

)

Use and benefits of multi-detector size exclusion chromatrography

t )JHIJOGPSNBUJPOBMPVUQVUGSPNMPXTBNQMFTJ[FPGBQQSPYJNBUFMZOH

t "CTPMVUFNPMFDVMBSXFJHIU JOUSJOTJDWJTDPTJUZ DPODFOUSBUJPO BOEIZESPEZOBNJD

TJ[FNFBTVSFNFOU

t 2VBOUJGJDBUJPOPGQSPUFJOTUSVDUVSBMQBSBNFUFST

February 2016 www.biopharminternational.com BioPharm International 49

Protein Aggregation

ment is essential for the adoption

of the rigorous quality-by-design

approach, integral to biopharma-

ceutical development.

Previously mentioned techniques

continue to have applications here.

For example, the ability of TDA to

measure the size of the target mol-

ecule in an increasingly complex

formulation is extremely helpful

in ensuring the structure-function

relationship of the target protein is

not altered. The detailed informa-

tion and structural insights offered

by SEC are also valuable. But along-

side these various approaches,

other complementary techniques

are added to the mix for more

detailed investigation.

One such technology is the res-

onant mass measurement (RMM),

which, with a measurement range

of 50 nm to 5 μm and the ability to

differentiate particles on the basis

of their buoyancy in suspension,

is deployed to distinguish protein-

aceous particles including aggregates

from other species present, such

as silicone oil, glass, or air bubbles.

Other novel techniques of proven

value at this point include exten-

sions to DLS; for example, DLS com-

bined with Raman spectroscopy in

instruments such as the Zetasizer

Helix (Malvern Instruments), which

boosts DLS with its chemical identi-

fication capabilities.

A primary strength of DLS

is its high sensitivity monitor-

ing of protein size and its ability

to detect low levels of aggregated

material. Light scattering intensity

scales with molecular diameter to

the power of six, so the hydrody-

namic size data reported by DLS

is strongly influenced by the size

of the largest aggregates present.

This means that DLS systems can

rapidly detect the onset of aggrega-

tion. Adding Raman spectroscopy

enables the detailed investigation

of detected aggregates to uncover

unique insights into protein fold-

ing, unfold ing, aggregat ion,

agglomeration, and oligomeriza-

tion. Figure 3, for example, shows

how Raman spectroscopy can

be used to investigate structural

changes associated with protein

instability.

In combinat ion, DL S and

Raman spectroscopy also enable

an orthogonal approach to DSC

for the determination of melt-

ing temperature (TM) and van’t

Hoff enthalpies, which are cru-

cial parameters relating to ther-

modynamic behavior and thermal

stability. All experiments can be

carried out with minimal dilution

to study the protein in its native

state within the formulation.

CONCLUSIONInformational requirements along

with practical constraints, such

as sample availability, mean that

certain analytical techniques are

optimally suited to specific stages

of the drug-development pipeline.

Verifying that the beneficial struc-

ture–function relationship of a bio-

logic is maintained as formulations

become more complex is essential,

but it presents an increasing ana-

lytical challenge because of the

associated need to differentiate the

molecule of interest, gather data

for it, and understand the impact

of added excipients. As instrumen-

tation suppliers continue to inno-

vate, new techniques are being

commercialized to answer to these

evolving needs and deliver the

information needed to progress.

Understanding what can be mea-

sured, and how, is key to applying

complementary methods in a cost-

effective way to advance biophar-

maceutical development.

Figure 3: Raman spectroscopy is a powerful tool for investigating the structural

changes associated with protein instability. Here, clear differences are observed

in bovine serum albumin samples measured at 20 °C (blue) and 90 °C (red)

respectively (50 mg/mL, at pH 7.4, in phosphate buffer saline), indicating thermal

instability.

1.2

1

0.8

0.6

0.4

0.2

0

No

rm

ali

ze

d i

nte

nsit

y

Raman shift cm-1

800 1000 1200

BSA 20 C BSA 90 C

Skeletal

Amide III

Amide I

1400 1600 1800

Use and benefits of DLS combined with Raman spectroscopy

t .FBTVSFNFOUPGWBOU)PGGFOUIBMQJFTBOEQSPUFJONFMUJOHUFNQFSBUVSF5M

t 2VBOUJGJDBUJPOPGIJHIFSPSEFSQSPUFJOTUSVDUVSFJODMVEJOHTFDPOEBSZBOEUFSUJBSZ

TUSVDUVSFNBSLFST

t %FUBJMFEJOTJHIUJOUPDPMMPJEBMBOETUSVDUVSBMTUBCJMJUZ

50 BioPharm International www.biopharminternational.com February 2016

IN THE PIPELINE

Catalent Biologics and Roche

Announce Research Collaboration

Catalent Biologics and Roche announced a collabora-

tion to develop molecules coupling different therapeu-

tic modalities using SMARTag technology, Catalent’s

programmable protein-modification platform. Roche

will gain non-exclusive access to the SMARTag plat-

form and will have an option to take commercial

licenses to develop molecules. The SMARTag platform

will be combined with the hydrazino-Pictet-Spengler

conjugation platform and will permit evaluation of

alternative sites of drug conjugation.

Roche will pay Catalent an up-front fee of $1 mil-

lion and provide additional research funding during

the initial phase of the collaboration. Catalent has the

potential to receive up to $618 million in development

and commercial milestones, plus royalties on net sales

of products, if Roche pursues commercial licenses and

all options are exercised.

Cell Therapy Catapult and Synpromics to

Collaborate on Viral Vector Manufacture

Synpromics and Cell Therapy Catapult announced

the launch of a collaboration to reduce the cost and

increase the scale and efficiency of viral vector manu-

facturing, thus removing a barrier to the development

of the cell and gene therapy industry.

The collaboration will use Synpromics’ synthetic

promoter design technology and the Cell Therapy

Catapult’s flexible manufacturing platform to create sta-

ble producer cell lines for the high titer and large-scale

manufacture of viral vectors. The work will be funded

in part by a €2 million grant from Innovate UK.

Ad Index Company Page

BINSWANGER 11

CHI 17

CREATIVE MARKETING ASSOCIATES 13

EPPENDORF NORTH AMERICA 5

EUROFINS LANCASTER LABORATORIES 33

INTERPHEX 7

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BIOLOGICS NEWS PIPELINE

Filter Integrity Testing—Contin. from page 45

A temperature change of 1 K will

give the following:

ΔpT = 3500 ∙ 294/293 – 3500 = 12

mbar

The percentual influence Δ% is

calculated as follows:

Δ% = ΔpT / Δp ∙ 100% = 12 / 43 ∙

100% = 28%

Based on the above, it is of

utmost importance to have iso-

therm conditions for all used

components and fluids. But even

if the temperature is stable, the

temperature must be within a

certain range compared to the

validated test conditions. The

temperature influences the solu-

bility of the gas and the surface

tension of the wetting liquid and

will have an impact on the test

value as shown in Equation 2.

Test ing cond it ions can be

greatly improved with longer sta-

bilization time for temperature

equilibration rather than using a

long measurement time.

CONCLUSIONThe establishment of a compre-

hensive FMEA for FIT is often

beyond the reach of the end user

alone due to the complexity of

evaluating the impact from exter-

nal elements on the test result.

The supplier of the integrity test

device may have a pre-established

FMEA that could help. An audit

by the supplier upon installation

of the devices would allow fur-

ther identification of specific risks

and could contribute to setting up

comprehensive standard operat-

ing procedures.

Solid training for the end user

is needed to provide a full under-

standing of factors such as envi-

ronmental influences. Training is

also mandatory from a regulatory

point of view.

REFERENCES 1. Parenteral Drug Association, Sterilizing

Filtration of Liquids, Technical

Report 26 (Bethesda, MD, 2008).

2. Parenteral Drug Association,

Sterilization Filtration of Gases, Technical

Report 40 (Bethesda, MD, 2005). X

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