BioPharm International - PharmTechfiles.pharmtech.com/alfresco_images/pharma/2018/09/11/15b7908e … · BioPharm INTERNATIONAL The Science & Business of Biopharmaceuticals EDITORIAL

The Science & Business of Biopharmaceuticals

INTERNATIONAL

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ASEPTIC PROCESSING:

KEEPING IT SAFE

QUALITY/ANALYTICALVIRAL DETECTION

TECHNOLOGIES MUST CONTINUE TO EVOLVE

PEER-REVIEWEDENDOTOXIN

AS A QUALITY INDICATOR

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The Science & Business of Biopharmaceuticals

INTERNATIONAL

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INTERNATIONAL

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July 2015

Volume 28 Number 7

ASEPTIC PROCESSING:

KEEPING IT SAFE

QUALITY/ANALYTICAL

VIRAL DETECTION

TECHNOLOGIES MUST

CONTINUE TO EVOLVE

PEER-REVIEWED

ENDOTOXIN

AS A QUALITY

INDICATOR

REGULATIONS

BREAKTHROUGH DRUGS

RAISE DEVELOPMENT AND

PRODUCTION CHALLENGES

ES639436_BP0715_cv1.pgs 07.07.2015 04:15 ADV blackyellowmagentacyan

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INTERNATIONAL

BioPharmThe Science & Business of Biopharmaceuticals

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

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

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

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

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

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

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

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

K. A. Ajit-Simh President, Shiba Associates

Rory Budihandojo Director, Quality and EHS Audit

Boehringer-Ingelheim

Edward G. Calamai Managing Partner

Pharmaceutical Manufacturing

and Compliance Associates, LLC

Suggy S. Chrai President and CEO

The Chrai Associates

Leonard J. Goren Global Leader, Human Identity

Division, GE Healthcare

Uwe Gottschalk Vice-President,

Purification Technologies

Sartorius Stedim Biotech GmbH

Fiona M. Greer Global Director,

BioPharma Services Development

SGS Life Science Services

Rajesh K. Gupta Vaccinnologist and Microbiologist

Jean F. Huxsoll Senior Director, Quality

Product Supply Biotech

Bayer Healthcare Pharmaceuticals

Denny Kraichely Associate Director

Johnson & Johnson

Stephan O. Krause Principal Scientist, Analytical

Biochemistry, MedImmune, Inc.

Steven S. Kuwahara Principal Consultant

GXP BioTechnology LLC

Eric S. Langer President and Managing Partner

BioPlan Associates, Inc.

Howard L. Levine President

BioProcess Technology Consultants

Herb Lutz Principal Consulting Engineer

EMD Millipore Corporation

Jerold Martin Sr. VP, Global Scientific Affairs,

Biopharmaceuticals

Pall Life Sciences

Hans-Peter Meyer 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 Managing Director

Arlenda, USA

Paula Shadle Principal Consultant,

Shadle Consulting

Alexander F. Sito President,

BioValidation

Michiel E. Ultee Principal

Ulteemit BioConsulting

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

Hospira, Inc.

Krish Venkat CSO

AnVen Research

Steven Walfish Principal Statistician

BD

Gary Walsh Professor

Department of Chemical and

Environmental Sciences and Materials

and Surface Science Institute

University of Limerick, Ireland

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4 BioPharm International www.biopharminternational.com July 2015

Contents

BioPharmINTERNATIONAL

BioPharm International integrates the science and business of

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

peer-reviewed technical solutions to enable biopharmaceutical professionals

to perform their jobs more effectively.

COLUMNS AND DEPARTMENTS

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

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

Cover: Andreas Dalmann/EyeEm/Getty Images; Maria Toutoudaki/Getty Images; Dan Ward

6 From the Editor As biopharma enjoys success, it cannot ignore pressing patient access questions Rita Peters

8 US Regulatory Beat Manufacturers and FDA look for innovative strategies to meet accelerated timeframes. Jill Wechsler

12 European Beat The European Union has a challenging task ahead as it strives to harmonize regulations on advanced therapy medicinal products. Sean Milmo

16 Perspectives on Outsourcing While all market signs are pointing up, memories of past setbacks may discourage CDMOs from expanding. Jim Miller

40 Analytical Best Practices Approaches to the generation of process models, optimization techniques, and application of a design space are explored. Thomas A. Little

45 Product Spotlight

45 New Technology Showcase

46 Gene Therapies Update

46 Ad Index

Aseptic processing

Aseptic Processing: Keeping it SafeRandi Hernandez

Humans represent the greatest

risk for microbial contamination

in an aseptic process. 18

UpstreAm processing

Optimizing the Delivery of Cell-Culture AdditivesAshley Roberts

When using media supplements

in biologics, it is important to have

a key understanding of both the

supplement and the base medium

to ensure high titer and stability. 22

DownstreAm processing

Using Single-Use Technologies in Downstream ProcessingSusan Haigney

The use of single-use systems in

downstream processing offers benefits in

filtration and sampling and may reduce

the risk of contamination. 24

peer-revieweD

Endotoxin Test Concerns of Biologics: The Role of Endotoxin as a Quality Indicator in Biologic Manufacturing ProcessesKevin L. Williams

Low endotoxin recovery represents

an opportunity to add value to the

characterization of biologic

drug products. 28

risk Assessment AnD mitigAtion

Risk Assessment and Mitigation in Biopharmaceutical ManufacturingSusan Haigney

The challenges and strategies of assessing

and mitigating risk in biopharmaceutical

manufacturing are discussed. 34

QUAlity/AnAlyticAl

Viral Detection Technologies Must Continue to EvolveCynthia A. Challener

Advances in adventitious agent detection

methodology are bringing benefits, but

more work needs to be done. 37

Volume 28 Number 7 July 2015

fEATURES


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From the Editor

As biopharma

enjoys success,

it cannot ignore

pressing patient

access questions.

For Big Ideas, Big Action Is Needed

At the 2015 Biotechnology Industry Organization (BIO) convention, held

in Philadelphia in mid-June, all indicators showed that the industry was

riding a record wave of success.

According to a BIO post-event press release (1), more than 15,800 industry

players from 69 countries, 47 states, the District of Columbia, and Puerto Rico

attended. A record number of partnering meetings—27,279 meetings between

3100 companies—illustrated the convention’s business focus.

Business was good for the biotech industry in 2014; in fact, it was a record-

setting year, reports Ernst & Young (E&Y) in its 29th annual report, Beyond

Borders, Biotechnology Industry Report 2015 (2). The report cites record revenues,

profitability, financing, and drug approvals as signs of a healthy market.

In the established biotech areas of the United States, Europe, Australia, and

Canada, revenues increased 24% in 2014; R&D spending increased by 20%. A

robust stock market and large number of initial public offerings pushed the

biotech industry capitalization in the US to more than $1 trillion for the first

time, E&Y reports.

The report authors, however, warned that the biotechnology industry can-

not afford to become complacent, but must work with patients, payers, provid-

ers, and governments to develop new products for unmet medical needs, as

well as ways to improve care delivery and health outcomes. The industry must

also play a role in developing payment schemes to give patients better access

to breakthrough drugs.

In a keynote discussion, Tom Brokaw, the award-winning journalist, reiter-

ated the need for a discussion about patient access to crucial drugs. A long-time

observer of US business, politics, culture, and society, Brokaw has added first-

person experience as a patient undergoing treatment for multiple myeloma.

Brokaw noted that patients, drug companies, and doctors do not speak the

same language. The nation, he says, needs an all-inclusive health plan that

makes the price of drugs affordable to all.

To achieve this, the US needs a “big idea” debate on healthcare, Brokaw

said. However, in the current American political system, there are no big

ideas. Partisan politics, cable news, social media, and professional and amateur

political commentators contribute to a lack of direction and desire to solve the

nation’s pressing issues.

To illustrate how the desire to achieve a “big idea” requires cooperation

and sacrifice, Brokaw recounted the efforts of US paratroopers in the D-Day

invasion of Europe in 1944. When the soldiers dropped behind enemy lines

in France, they were separated from their units and were scattered across the

countryside. They assembled with others near them to form makeshift units

and accepted their new orders. The soldiers did not stop to argue about what

platoons or units the fighters were from; they simply went about the business

of liberating Europe, a “big idea” of those times. In his 1998 best-selling book,

The Greatest Generation, Brokaw related that the soldiers who fought in World

War II did so because it was the right thing to do.

The current generation of politicians, payers, patients, and drug companies

are challenged with determining “the right thing to do” to achieve one of

today’s “big ideas”—affordable, effective drug therapies for all patients. This

time, the enemy may be us.

References

1. Biotechnology Industry Organization, “The 2015 BIO International Convention Closes

in Philadelphia,” Press Release (Philadelphia, PA, June 18, 2015).

2. Ernst & Young, Beyond Borders, Biotechnology Industry Report 2015 (2015). ◆

Rita Peters is the editorial director of

BioPharm International.


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Regulatory Beat

Vis

ion

so

fAm

eri

ca

/Jo

e S

oh

m/G

ett

y Im

ag

es

The FDA program to expedite the devel-

opment and approval of innovative

drugs for serious and life-threatening

conditions is a great success, but the abbrevi-

ated development timeframe involved raises

numerous difficulties for manufacturers seek-

ing to ensure product quality and timely sup-

ply. Expert review teams in the Center for

Drug Evaluation and Research (CDER) and the

Center for Biologics Evaluation and Research

(CBER) are meeting deadlines and goals for

assessing breakthrough designation requests

and for expediting reviews of these drugs, but

the process is resource intensive and has raised

questions about how FDA can keep up with a

growing number of candidates.

When the breakthrough program was estab-

lished as part of the FDA Safety and Innovation

Act of 2012, stakeholders envisioned about two to

three designations a year. By the end of May 2015,

FDA had received 308 requests for breakthrough

status and had granted the designation for 90,

approximately 30%. Nearly 15 important new

therapies have come to market more quickly as a

result, contributing to the recent rise in new drug

approvals. FDA acting commissioner

Stephen Ostroff pointed out at the

annual meeting of the Food & Drug

Law Institute (FDLI) in April 2015 that

two-thirds of 2014’s near-record 51

new molecular entities (NMEs) took

advantage of at least one expedited

review program, and many were first-

in-class therapies.

Achieving fast approval of a break-

through therapy creates challenges

for manufacturers looking to develop

CMC data in roughly half the

time, noted Brian Kelley, vice-presi-

dent for bioprocess development at

Genentech. The process, he explained

at the April 2015 CMC workshop sponsored

by the Drug Information Association (DIA), is

resource intensive, and accelerated timelines

necessitate new approaches to product and pro-

cess development to ensure a reliable supply of

a quality product at launch. The breakthrough

designation “does not mean that sponsors can

do less,” he said; they just “need to start sooner.”

This may involve front-loading of crucial prod-

uct and process characterization activities, and

reaching agreement with FDA on which actions

for optimizing process and methods can wait

until after launch.

HigH priority for fDAExpedited quality assessments raise difficulties

for FDA, as well. New drug applications (NDAs)

for breakthrough therapies often contain less

manufacturing information than usual, requir-

ing innovative risk-mitigation strategies to ensure

product safety. Agency reviewers are agreeing to

less stability data at submission, accepting amend-

ments during the review cycle, and increasing

postmarketing commitments to cover residual

risk, explained Dorota Matecka, acting branch

chief in the Office of New Drug Products in

CDER’s Office of Pharmaceutical Quality (OPQ),

at the DIA workshop and again at the ISPE/FDA/

PQRI Quality Manufacturing Conference in June

2015. Matecka noted that CDER will schedule

CMC-specific meetings during development to

Breakthrough Drugs Raise Development and Production ChallengesManufacturers and FDA look for innovative strategies to meet accelerated timeframes.

Jill Wechsler is BioPharm

International’s Washington editor,

Chevy Chase, MD, 301.656.4634,

[email protected].

Achieving fast approval

of a breakthrough

therapy creates challenges

for manufacturers.


July 2015 www.biopharminternational.com BioPharm International 9

regulatory Beat

advise on these issues, often includ-

ing CDER upper management and

subject matter experts.

Robert Wittof t, pharmacist

in OPQ’s Office of Process and

Facilities (OPF), similarly urged early

discussion of residual product qual-

ity risks. Manufacturers need to

decide dosage form and methods

validation strategies much sooner,

he said at the CMC workshop, and

should “plan for the unexpected,”

such as facility qualification fail-

ures and changes in manufacturing

schedules. Effective communication

with contract manufacturers is cru-

cial, as is a transparent presentation

in the application of design evolu-

tion and a rationale for commercial

manufacturing process and controls.

John Groskoph, senior director

at Pfizer, observed that for most

breakthrough therapies, market

applications are being filed with

FDA after Phase II studies, approxi-

mately two years ahead of a tradi-

tional NDA that is based on Phase

III data. The time reduction pres-

ents “significant challenges to the

development team,” he commented,

and may be further complicated if

the firm seeks to file simultaneous

applications in Europe, Japan, and

emerging markets, as well as in the

United States.

Japan, for example, has estab-

lished the SAKIGAKE designation

program for innovative medicines

and medical devices that are devel-

oped first in Japan and offer “radical

improvement” over existing ther-

apies to treat critical diseases,

explained Yoshihiro Matsuda of

Japan’s Pharmaceut ica ls and

Medical Devices Agency (PMDA), at

the CMC workshop. He described

a greatly accelerated development

and approval process for such

therapies, combined with stronger

postmarketing oversight. The initia-

tive, he noted, requires risk-based

assessment strategies and a prod-

uct quality lifecycle management

plan, combined with clear analysis

of what can be evaluated during

review, and what can be analyzed

later after approval.

Groskoph noted that success-

ful launch of a breakthrough drug

involves addressing numerous

issues: data availability, meaning-

ful and practical specifications,

robust manufacturing processes,

clinical or commercial site produc-

tion, site readiness for pre-approval

inspection, deferral of Phase III

studies to post approval, and the

need for comparability protocols

to facilitate postapproval changes.

Communicat ion with FDA is

important throughout the break-

through development process, he

added, to facilitate agreement on

strategies for dealing with unex-

pected production problems.

For biologics, breakthrough des-

ignation may prompt greater focus

on the reliability of the Phase I cell

line, process and formulation, as

shorter pivotal trials may truncate

optimization of the Phase III pro-

cess, added Kelley of Genentech. A

key decision for manufacturers is

whether to devote more resources to

the project early to front-load pro-

cess characterization and validation

activities, even before gaining the

breakthrough designation. Such an

approach may involve testing lots

before assay validation is completed;

filing with broader specifications

with the aim of tightening them

post-launch; launching from the

clinical site and transferring to com-

mercial post-launch; and including

a postapproval lifecycle manage-

ment plan in the application to

support deferral of certain activi-

ties. But, Kelley commented, “you

can’t place bets” on potential break-

throughs too frequently without

overly straining company resources.

SuStAinABle progrAM?The growth in breakthrough des-

ignation requests is prompting

FDA and stakeholders to examine

options for refining breakthrough

criteria so that FDA will be able to

manage the program. The agency

is examining past designation

decisions and why it turned down

certain requests to see if the bar is

too low; a goal is to better educate

manufacturers on which promising

experimental products really qualify

for breakthrough status.

FDA “can’t sustain a program

where everything is a break-

through,” commented John Jenkins,

director of CDER’s Office of New

Drugs, at an April 2015 workshop

on breakthrough therapy desig-

nation criteria organized by the

Brookings Institution. FDA offi-

cials explained that extensive

resources are involved in determin-

ing designations and in support-

ing development and accelerated

review of breakthrough candidates.

Manufacturers acknowledged that

designation denials could decrease

if sponsors sought breakthrough

status only for therapies that offer

truly substantial improvements in

patient care. And they indicated

that additional resources from

industry are warranted to sup-

port the unexpectedly large break-

through program.

While FDA can quickly approve

products with clear outstanding

value, Jenkins noted that such

efforts may be stymied by man-

ufacturing problems and inspec-

tion delays. There are situations

where the clinical data are good,

but where sponsors “have to get

manufacturing and facilities in

line,” he said. Sites for inspections

need to be identified early, Jenkins

advised, especially for overseas

facilities that may raise travel diffi-

culties. Kay Holcombe, senior vice-

president of the Biotechnology

Industry Organization, urged close

examination of ways to prevent

approval delays due to difficul-

ties in making a drug according to

specifications. “If this is a hurdle

at the end,” she said, “we need to

deal with it more effectively.” ◆


10 BioPharm International July 2015

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registered trademark of Eppendorf, Inc., USA. US Design Patents are listed on www.eppendorf.

com/ip. All rights reserved, including graphics and images. Copyright © 2015 by Eppendorf AG.

Eppendorf AG

Eppendorf AG22331 Hamburg, Germany 22331

Email: [email protected]

www.eppendorf.com


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

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All rights reserved, including graphics and images. Copyright ©2015 by Eppendorf AG.

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European Beat

The European Medicines Agency is

approving a growing number of advanced

therapy medicinal products (ATMP)

despite claims that their commercialization is

being hampered by increasingly complex regula-

tory and standards requirements. The creation of

ATMPs by a 2007 European Union regulation (1),

backed by a specialist committee for advanced

therapies (CAT) within EMA, aimed to boost

development of medicines derived from progress

in cellular and molecular biology.

Initially, the regulation seemed to have little

impact on the number of advanced medicines on

the market after the start of its implementation

in early 2009. By mid-2013, there were only four

marketing authorizations from 10 applications

in the three ATMP categories of gene therapy,

somatic cell therapy, and tissue engineering (2).

Over the past few years, however, there have

been signs of a surge in ATMP development. The

number of medicine applications recommended

by CAT to be classified as advanced therapies rose

by 26% in 2014 (3). In late 2014, EMA recom-

mended for EU approval the first advanced ther-

apy medicine containing stem cells. It is also the

first drug for the treatment of moderate to severe

limbal stem cell deficiency (LSCD), a rare eye con-

dition due to physical or chemical burns to the

eyes that can result in blindness.

complEX REgUlationsAt the same time, the quality, safety,

and efficacy rules under existing and

proposed EMA guidelines on ATMPs

have been becoming more complex.

One reason is that expanding knowl-

edge about the new therapies has raised

new concerns, particularly relating to

issues regarding the quality of starting

materials and drug substances. The reg-

ulators have gradually become more

aware of the biological variability and intricacy of

ATMPs. This tightening of standards seems to be

deterring big pharmaceutical companies rather

than small- and medium-sized enterprises (SMEs)

from developing advanced therapy products.

In a 2014 report (2) on the application of the

2007 regulation, the European Commission, the

Brussels-based EU executive, found that the

majority of ATMP research was being done by

small companies and entities. Approximately 70%

of sponsors of ATMP clinical trials were SMEs or

not-for-profit organizations, while large pharma-

ceutical companies accounted for less than 2%.

The report concluded that because there are

“still many unknowns” with advanced therapies,

“it is important to put in place adequate controls

to prevent detrimental consequences for public

health” (2). Nonetheless, it is also acknowledged

that “too burdensome requirements” could have

adverse consequences for public health because

they could prevent the marketing of valid treat-

ments for unmet medical needs.

One onerous requirement is the amount of

data needed on starting materials, such as the

source and history of cells, and their detailed

characterization. In addition, a complete descrip-

tion, including source, characteristics, and testing

details, of all materials used during the manu-

facture of products is needed. Some developers

of ATMP products complain about the regulators

making demands for data that existing analytical

technologies cannot yet provide. There have also

been complaints about EMA wanting unnecessary

high levels of purity in cell-therapy treatments,

especially those comprising mixtures of undiffer-

entiated cells.

Another matter of contention has been EMA’s

insistence that marketing authorization appli-

cants for tissue-engineered products must demon-

strate through pharmacokinetics the longevity or

persistence of their medicines. “From the point of

Unravelling the Complexity of EU’s ATMP Regulatory FrameworkThe European Union has a challenging task ahead as it strives to harmonize regulations on advanced therapy medicinal products.

Sean Milmo is a freelance

writer based in Essex, Uk,

[email protected].


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Regulatory BeatEuropean Beat

view of our members, pharmacoki-

netics does not include longevity,

but resorption, distribution, and

excretion of a drug,” Matthias

Wilken, head of European drug reg-

ulatory affairs at the German

Pharmaceutical Industry Association

(BPI), told BioPharm International.

“The requirement to demonstrate

longevity might lead to extensive

clinical studies that would be an

undue burden to pharmaceutical

entrepreneurs,” he explained.

Also in some cases with ATMPs,

the regulators are seen as taking too

much of a “generic” approach to

advanced technologies and not

making a strong enough distinction

with conventional pharmaceuticals.

“The assessors and members of EMA

scientific committees often come

from the field of conventional

medicinal products,” said Wilken.

“Initially, there was a lack of under-

standing of the peculiarities of

ATMPs. But this [understanding] is

getting better, as is shown, for exam-

ple, by the fact that EMA, along

with the Commission, is currently

working on tailoring GMP require-

ments for ATMPs.”

Risk managEmEntThe big regulatory differences

between ATMPs and chemical-

based pharmaceuticals is the greater

emphasis needed with biological

products on quality issues, mainly

because with many of them, there

are gaps in knowledge about ways of

managing their risks. However, EMA

has acknowledged the limitations of

applying uniform rules to ATMPs by

adopting a risk-based approach that

allows the products to be assessed

on a case-by-case basis.

The distinct approach needed

for ATMPs has been highlighted

by the latest EMA guidance (4) on

advanced therapies, which covers

the quality, preclinical, and clinical

aspects of gene therapy. The draft

guideline (4) on gene therapy was

issued in May 2015 for a period

of public consultation ending in

August. It replaces a guidance note

(5) published in the early phase of

gene-therapy development in 2001.

Since the 2007 ATMP regulation

was implemented, EMA has had

to deal with three applications

for gene-therapy authorizations,

only one of which has so far been

successful. “[From a quality perspec-

tive], there were no major changes

or inconsistencies in the 2001

guideline that required an immedi-

ate revision,” an EMA spokesman

informed BioPharm International.

“However, some updates were

necessary, for example, to reflect

novel methodologies for testing and

characterization, and also to ensure

cross references to new legislation

and guidelines that were developed

separately.”

Also, the format of the sections

on quality and manufacturing

aspects in the revised guideline has

been changed to follow that in the

harmonized Common Technical

Document (CTD) for marketing

authorization application dossiers,

according to EMA. “This is expected

to be helpful for the small develop-

ers of gene-therapy products when

compiling their dossiers,” said the

EMA spokesman. As a result, 40% of

the 42-page draft guideline covers

quality matters, 30% non-clinical

issues, many of which relate to

assessing risks linked to quality

management, and only 10–15% to

clinical development.

A lot of the obligations in the

guideline requirements relate to the

quality of the components in the

vectors or delivery systems of the

products. Details of the quality of all

starting materials and their sources

have to be provided, including virus

seed as well as mammalian and

bacterial cell banks. All raw materi-

als used during manufacture have to

be tested and characterized.

Hospital-BasEd REsEaRcHPartly due to the detailed EU qual-

ity and safety requirements for

advanced therapies, companies

developing ATMPs are critical of

an exemption to EU rules granted

to hospitals involved in R&D and

the manufacture of the products.

Hospital-based research and pro-

duction in the sector are increasing

rather than contracting in Europe.

This trend is mainly because some

EU states are using these hospitals

as ATMS development centers at

the core of national regenerative

medicine programs.

The United Kingdom, which is

seeking global leadership in the sec-

tor has, for example, a network of

cell therapy centers of excellence

based in leading hospitals. “The

establishment [of these centers] is

essential if we are to build a con-

centrated critical mass of knowledge,

skills, and therapeutic know-how,”

according to a UK government-

commissioned report on regenera-

tive medicine (6).

Under the 2007 EU regulation on

ATMPs, member states are allowed

to give hospitals exemption from

the legislation as long as the hospi-

tal’s advanced therapies are being

provided on a “non-routine basis” to

its own individual patients. Some

organizations are calling for the

“non-routine” provision, which is

open to different interpretations, to

be extended to cover products only

when a fully validated, EU-approved

advanced therapy alternative cannot

be used.

“While the hospital exemption

rule allows the early development

and delivery of ATMPs that meet

an otherwise unmet clinical need

in a patient, the exemption should

only be used to deliver a product if

there is no licensed alternative, with

proven efficacy and safety, available,”

says Michael Werner, executive

director of the US-based Alliance

for Regenerative Medicines, a

Contin. on page 23


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Perspectives on Outsourcing

Do

n F

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These are high times for contract devel-

opment and manufacturing organiza-

tions (CDMOs) and contract research

organizat ions (CROs). A record f lood of

external financing is flowing into the bio/

pharmaceutical industry. Global bio/pharma-

ceutical companies are outsourcing more of

their development activity, and FDA is being

especially accommodating. R&D spending is

growing, and the clinical development pipe-

line is really coming to life.

The explosion of development activity is

pushing the contract services industry capac-

ity to its limits, particularly for early devel-

opment. Providers of preclinical research

services, such as Charles River Laboratories

and Covance, are rushing to reactivate capac-

ity that was mothballed following the finan-

cial crisis. CDMOs that were fighting for

survival two years ago are now telling clients

there is a three- to six-month wait for a pro-

duction slot.

To expand or noT To expandDespite the strong market envi-

ronment, the decision to expand

capacity is not an easy one for

CDMO execut ives, who were

burned twice in the past decade.

After a period of robust activity

in the late 1990s, funding and

development activity declined

sharply in the early 2000s as a

result of the dotcom bust and

some major c l in ica l fa i lures .

Then just as things were recov-

ering in mid-decade, the global

f inancial crisis once again cut

the product development pipe-

line to a trickle.

New manufacturing and analytical capac-

ity can take a year or more to construct,

equip, and validate, and in that time an

upset in industry or macroeconomic condi-

tions can leave CDMOs with a lot of unused

capacity that still has to be paid for. So it

is not surprising that CDMO executives are

careful in committing to new capacity.

Executives’ concerns are warranted because

the surge in funding that is propell ing

demand is driven by the skyrocketing valu-

ations of biopharma companies. Valuations

of publicly-traded bio/pharma companies (as

measured by the Nasdaq Biotech Index) have

climbed 300% since 2010, three times faster

than the broader stock market (as measured

by the S&P 500). Thanks to that surge in

equity prices, nearly 60% of the increased

external funding f lowing into early stage

bio/pharma companies has come from initial

public offerings (IPOs) and secondary offer-

ings by companies that are already public (see

Figure 1). But the rapid run-up in bio/pharma

stock prices has given rise to increased con-

cern about whether the “biotech bubble” is

about to pop.

The explosion of

development activity

is pushing the contract

services industry

capacity to its limits.

CDMOs Cautiously Address Expansion While all market signs are pointing up, memories of past setbacks may discourage CDMOs from expanding capacity.

Jim Miller is president of

pharmSource Information

Services, Inc., and publisher of Bio/

pharmaceutical outsourcing report,

Twitter@JimpharmSource,

[email protected],

www.pharmsource.com.


tel. 703.383.4903,


perspectives on outsourcing

FundIng STaBIlITyPharmSource has been

looking closely at the

funding issue, and

while the possibility of

the biopharma bubble

bursting is a concern,

a disruption in indus-

try funding activity

is not likely to be as

damaging today as it

was in 2008. This opti-

mism is based on sev-

eral key observations:

Global bio/pharma

companies inc reas -

i n g l y d e p e n d o n

ea r ly- s t age compa-

n ie s to feed t he i r

ow n pipe l i nes , so

they have a st rong

interest in suppor t ing them.

Pa r tnered or acqu i red prod-

ucts account for 50% or more

of approvals received by global

bio/pharma companies in recent

years, while upfront payments

f rom pa r t ner ing dea l s w it h

globa l biopharma companies

provided 20% of the total fund-

ing received by early-stage com-

panies. Investment in partner

relationships, including licens-

ing, may exceed 30% of total

R&D spending at the global bio/

pharmaceutical companies.

Venture capital is not nearly

as volatile as public financing.

Venture capital funding for bio/

pharma companies stayed fairly

consistent through the financial

crisis and has risen only grad-

ually in the past several years.

Globa l bio/pharma l icensing

activity will continue to provide

an exit for venture capital inves-

tors even if public equity mar-

kets shrink.

T h e e a r l y - s t a g e c o m p a -

n i e s h av e p l e nt y o f c a s h .

PharmSource analysis indicates

that 70% of the publ ic bio -

pharma companies have more

than two year’s cash on hand,

a s su m i ng c u r r e nt l eve l s o f

spending.

C D M O s , t h e r e f o r e , c a n

expand with the confidence that

demand for their services should

remain robust for the foreseeable

future. Capital for expansion

should be readily available given

market conditions and a growing

willingness on the part of bank-

ers to lend, but the biggest chal-

lenge for CDMOs will be getting

enough technical and project

management staff to meet the

growing demand. CDMOs and

contract labs are a lready hir-

ing aggressively, and poaching

of staff, fed by r ising salaries,

has become a big problem. This

poaching is especially true for

people with the higher-order

technical skills needed for prime

growth segments like advanced

formulations and analytical ser-

vices for biopharmaceuticals.

Restrained growth of capacity

may not be the worst thing for

CDMOs, however. Tight capac-

ity conditions are likely to help

CDMOs improve their prof it-

ability, just like they have for

the airlines. After years of being

beaten up on price by clients,

especially the global biopharma

companies, CDMOs and con-

tract labs finally find themselves

with some pricing power and the

ability to improve their bottom

lines. A healthy and profitable

CDMO sector is in the best inter-

est of the bio/pharmaceutical

industry as outsourcing becomes

increasingly core to its business

model. ◆

Figure 1: External fnancing for early-stage bio/pharmaceutical companies.

$-

$5.0

$10.0

$15.0

$20.0

$25.0

$30.0

$35.0

2008 2009-12 2013/14

Upfront license fees

Venture capital

Secondary offering

IPO

Private equity

Other

global bio/pharma

licensing activity

will continue to

provide an exit for

venture capital

investors.



And

reas

Dalm

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Maria T

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Dan W

ard

Aseptic processing has garnered

some increased scrutiny from

FDA in recent years, primarily

because it is considered a high-

risk activity by the agency, says Rainer

Newman, consultant at Aseptic Process

Technology, LLC. In addition, says Satish

Singh, research fellow and group leader at

Pfizer, “Microbiologically-related recalls

have always been a significant portion

of the enforcement actions by FDA.” In

fact, more than 75% of FDA recalls dur-

ing 2004–2011 involved sterile products,

and approximately 80% of these recalls

were linked to “lack of sterility assurance.”

Many of the remaining 20% of recalls were

attributed to microbial contamination or a

failed fill/finish product test (1). Although

packaging failures factored into a majority

of the “lack of sterility assurance” cases,

many of the remaining contamination

cases were associated with GMP issues or

manufacturing errors such as incomplete

sterilization or non-sterile components

being added to sterile products (1).

The large-scale meningitis outbreak in

2012 has been attributed to poor aseptic

processes, when contaminated vials in lots

of preservative-free methylprednisone ace-

tate sickened 751 people and killed at least

64 people (2). Although states currently

oversee the activities of compounding

pharmacies, FDA released new draft guid-

ance documents in February 2015 seeking

to exert more regulatory control over drugs

produced by state-licensed pharmacies,

federal facilities, and outsourcing facilities.

The documents released by the agency

state that medications compounded in

an outsourcing facility “that meet certain

Aseptic Processing: Keeping it Safe

Randi Hernandez

Humans represent the greatest risk

for microbial contamination in an aseptic

process.

Aseptic Processing



conditions may be entitled to exemp-

tions from certain provisions of the

Federal Food, Drug, and Cosmetic

Act (FD&C Act), including the new

drug approval requirements and the

requirement to label drug products

with adequate directions for use” (3).

Just because validation of a sterile

effluent typically occurs under the

“worst-case scenario” tenet, it does

not mean that facilities should oper-

ate with this frame of mind when

ironing out their aseptic processing

capabilities. Negative agency inspec-

tion, increasing operating costs, and

poor media fill results are among

the reasons many manufacturing

organizations are deciding to update

or replace aging aseptic processing

equipment. Many newer facilities,

such as SAFC’s new antibody-drug

conjugate-focused floor in its St.

Louis, MO campus, have spaces that

are designed to handle media fill for

various types of products and have

isolators for both highly potent com-

pounds and biologic products.

While investigational medicinal

products are not typically expected

to be validated at the same stan-

dards as are products for rou-

tine production, sterile products

must have validated processes “of

the same standard as for products

authorized for marketing,” accord-

ing to the European Commission’s

EU Guidelines to Good Manufacturing

Practice: Medicinal Products for

Human and Veterinary Use Annex 13,

Investigational Medicinal Products (4).

PhySicAl environmentEnvironmental monitoring, no mat-

ter how necessary it may be, is always

an intervention in the process, notes

Newman. There are various schemes

and equipment, however, that can

support minimized interventions

when manufacturing sterile biologics

in a controlled environment, he says.

Environmental monitoring, when

it involves personnel moving in

and out of the suite, can actually

increase contamination risks if per-

formed too frequently, asserts Singh.

“Suites are qualified to handle a

certain maximum number of peo-

ple, but it does not mean that the

maximum personnel load should be

used, especially for long periods of

time, whereby the air handling sys-

tems may be overwhelmed.” Singh

adds, “Routine monitoring activity

is an integral part of the normal pro-

cesses and should be qualified as

such.” Smoke studies are often per-

formed to ensure a facility is meet-

ing expectations for air balance and

airflow in aseptic areas.

Clean water requirements

The demand for high-quality water

for aseptic processing is of para-

mount importance, as water is not

only an ingredient in many aseptic

formulations, but is also as a cleaning

agent in cleanrooms (5). Microbial,

chemical, or endotoxin contami-

nants in feed water sources can occur

in excess of a system’s ability to clear

them, and this has periodically been

a problem with water sourcing, says

Newman. A concise environmental

monitoring program can help a man-

ufacturer assess product bioburden

that may be attributable to water-

derived organisms.

oPerAtor interventionSThe most common introduction

of contaminants by operators in

aseptic processing is during set up,

during interventions, and during

material additions, notes Newman.

Failed media fills are often the result

of an operator action, he adds. Singh

says that humans most frequently

introduce contaminants when inter-

ventions are required on the line,

during filling. “Interventions can be

repeated, for example, if sampling for

fill-weight check is not automated,”

Singh notes. “Or, they can be epi-

sodic, [such as] if a vial falls over or

gets stuck in transport, for example.

Compounding, if not carried out

properly, can also introduce micro-

bial loads. Normally, these would be

filtered out, but can [still] result in

high prefiltration bioburden levels.”

Future GMP initiatives should

focus on the minimization of human

intervention in the aseptic process,

suggests Bill Hartzel, director of stra-

tegic execution at Catalent Pharma

Solutions, who says, “Humans are

among the highest contributors

to microbial and particulate con-

tamination.” According to Hartzel,

expanded use of isolators and

increased automation will help miti-

gate the risk of product adulteration.

Protecting operators

Protecting drug product from micro-

bial contamination is a key concern

when making biologics, which is why

aseptic filling typically occurs in a

positive pressure isolator. In a closed

system, positive pressure is generally

used within an isolator (wherein air

flows out of the isolator) to protect

biologics from outside contaminants

that may be airborne in the environ-

ment. Conversely, negative pressure

inside an isolator is used when deal-

ing with a highly potent product to

prevent egress of toxic material out of

the closed system. Concerns related

to operator safety exist when deal-

ing with highly potent or cytotoxic

drugs, such as is the case with the

manufacture of antibody drug con-

jugates (ADCs) with cytotoxic pay-

loads. To protect the worker making

these specific types of ADCs, a more

complex, negative pressure isolator

is typically used, notes Newman.

Gary Partington, technical sales

and marketing manager of Walker

Barrier Systems, says that an isolator

blower or fan pulls air through a safe-

change high-efficiency particulate air

(HEPA) filter into the isolator and out

through a safe-change double HEPA

filter into the facility exhaust system.

“If there is a leak in this isolator, the

negative flow keeps the potent mate-

rial from escaping,” notes Partington.

For highly potent materials, says

Singh, “isolator technology is a basic

requirement.” Bioconjugates must be

Aseptic Processing



assessed for compatibility with pro-

cessing equipment, Singh adds, and

for these products, as well as with all

biologics, “light sensitivity, interfa-

cial stresses, and temperature impact

must be assessed.”

Blow-fill-SeAl technologyThe chances of contamination by

operator are greatly reduced by

incorporating automated blow-fill-

seal (BFS) technology for the aseptic

preparation of sterile pharmaceuti-

cals. The container closure is auto-

matically formed, filled, and sealed

within the confines of a class A

internal environmental in a matter

of seconds, which drastically reduces

the risk of contamination. While the

manufacturing of the final prod-

uct may benefit from this contained

process, Hartzel highlights that fea-

sibility studies of small quantities of

drug product comparing the bulk

substances to the filled product in a

BFS container may be beneficial, as

the inherent steps in the BFS pro-

cess are significantly different than

they are in traditional vial filling.

“First, the BFS process uses heat to

convert the virgin plastic pellets into

a vial and the filling takes place sec-

onds after the container is formed,”

says Hartzel, emphasizing that the

heat of the process could potentially

impact thermally sensitive biologic

drugs. “The second caveat is that

plastics are semipermeable and are

not impervious like glass,” Hartzel

adds. Manufacturers must under-

stand how storage conditions affect

permeation rates and design appli-

cable stability programs.

Singh agrees that products manu-

factured using BFS have to be com-

patible with exposure to momentary

high temperatures as well as long-

term exposure to the polymer,

including migration of oxygen in

and water out. BFS technology is

characterized by an efficient heat

transfer and rapid cooling process

within the body of the container,

hence in a matter of a few seconds,

the temperature of the molten plas-

tic (~385 ˚F to 450 ˚F) equilibrates

to the mold temperature (80–90 ˚F),

says Hartzel. “There are multiple fac-

tors influencing temperature of the

drug product during fill, including

wall thickness, fill speed, surface-

to-volume ratio, and temperature of

the incoming variables. By control-

ling these variables, you can keep the

temperature from spiking to less than

90 ˚F,” he says.

Singh adds that in spite of the con-

cerns about thermal stress on the

formulation of biologics, the short

heat stress in BFS may be acceptable,

as long as it has been evaluated and

addressed during the development

process—but the long-term compat-

ibility concerns remain. Systems can

be added to a standard BFS system

that can minimize the heat impact,

notes Tim Kram, general manager,

rommelag. “How much heat is added

to the system is a function of con-

tainer design, type of plastic resin

used, and the fill volume,” he says.

“The product temperature can be

controlled to the point of fill and

heat added to the system can be

removed after filling. For most prod-

ucts and fill volumes, it is possible

to keep the final filled product tem-

perature under 20 ˚C (68 ˚F).” Kram

says that regardless of the presence

of systems to minimize the impact of

heat on product formulation, some

biologics may still not be compatible

with a BFS system.

cleAning And diSinfection of ASePtic AreASDaily or weekly cleaning of aseptic

processing areas is usually appro-

priate, says Newman, depending

on the level of activity in the area.

He explains that cleaning regimens

are rather detailed, have to be val-

idated, and must periodically be

requalified. Current disinfection

protocols for an isolator commonly

make use of vaporized hydrogen

peroxide (VHP), although according

to Newman, it is “questionable if

VHP should be considered a steril-

ization process or a high-level dis-

infectant.”

Partington notes that isolators are

generally validated to a 106 steril-

ity assurance level (SAL), which he

says “is far better than a cleanroom.”

VHP reduces the availability of the

isolator, however, due to the time

needed to expose the isolator, evacu-

ate the VHP, and allow for aeration.

Hydrogen peroxide (H2O2) cleaning

of an isolator, therefore, reduces the

output of the isolator line, at least for

a small time. “H2O2 is absorbed by

plastic materials in the isolator and

[these materials] need time to outgas

so that the residual H2O2 level in

the isolator is 1 ppm or less before

processing can begin,” Partington

explains. Singh points out that

newer technologies, such as cata-

lytic converters, are being developed

that can speed up the cycle time

to achieve the target residual levels

of H2O2, improve turnaround time,

and consequently, improve the over-

all utilization of isolators.

Single-use containers for aseptic

processing—such as filters, tubing,

connectors, and bags to hold bulk

products—are already relatively

common in aseptic filling, and can

also help keep contamination events

low. Most of these items are pre-ster-

ilized. Other product contact parts,

such as pumps or needles, are often

dedicated to their specific processes,

says Singh, but “even in these opera-

tions, single-use systems are being

introduced.”

The introduction of new containers

While all containers and closure sys-

tems require a material compatibility,

extractability, and container-closure

integrity evaluation, according to

Newman, problems can arise when a

manufacturer is considering changes

in container-closure design. “New

containers that have unusual or

novel dimensions, shapes, or other

attributes may impact the design and

function of filling and other han-

Aseptic Processing



Aseptic Processing

dling equipment,” Newman asserts.

“Depending on the change, there

may be more fundamental challenges,

e.g., sterilization of materials and

components.” To determine if any

of the packaging components have a

negative impact on the product, the

product is typically tested in its final

packaging format under controlled

conditions, says Kram.

New container presentations and

closures can offer innovative drug-

delivery solutions, but they can also

introduce variables into the process

of sterile filling. “The market space

has changed, and the delivery of the

medication is rising in importance,”

Hartzel observes. “In addition, the

recent manufacturing challenges of

traditional glass vials have opened the

minds of [people] in the market to

explore new technologies to improve

drug manufacturing and container-

closure systems.”

Despite the willingness of industry

to try new materials, Singh says that

new containers/closures are fairly rare

in sterile products, and Type 1 glass

is likely to remain the industry work-

horse for a while. “For new containers/

closures with new contact materials,

development studies would have to

be performed to determine compat-

ibility (chemical and physical) with

the intended final product, provid-

ing adequate stability with low risk

[for] leachables.” The new container

or closures would also have to fit with

the processing facility with change

parts as required, as well as provide a

significant added benefit, Singh adds.

New product contact materials or

components are generally tested for

extractables under exaggerated-use

conditions of solution composition,

temperature, and time of contact,

says Singh. “Preferably, this is some-

thing that the vendor of the compo-

nent performs and has available as

a data packet for the user. The user

of the component can then assess

these compounds (i.e., extractables)

for safety and toxicity, and make a

risk-based decision to monitor specific

species under actual-use conditions

(i.e., leachables). In this case, method

development for detection and quali-

fication of the leachable in the prod-

uct solution would be required once

the compounds of concern have been

identified.”

Filter integrity testing

According to Newman, there are

two camps of thought on when fil-

ter integrity testing should be done.

Testing post-use is something that is

done every time. Pre-use testing of

filters, however—especially pre-use,

post-sterilization testing—is only

required depending on whom you

ask, as Europe and the United States

have differing views regarding the

risks and benefits associated with pre-

use testing of filters. Europe requires

pre- and post-testing for filters for

some products, and the US is expected

to follow suit. Singh says that for ster-

ilization filters, both pre-use and post-

use testing is common practice. For

bioburden reduction filters, however,

post-use is most common, and pre-use

“may be dependent on the procedures

adopted by the company.”

A filter’s bacterial retention capabili-

ties post-use must be tested through

qualification and validation proce-

dures, by the manufacturer and the

final user, respectively. Validation of

a sterile filter to see how it affects a

processing stream can include vari-

ous elements, including integrity

testing, “fit-for-use” requirements,

sterilization, stability, binding, com-

patibility, extractables and leachables,

and retention (6). Filters, in particu-

lar, should be analyzed for removal

of bacteria (such as challenge organ-

ism Brevundimonas diminuta) from the

stream per ASTM 838-05, and investi-

gators should be able to demonstrate

that the process stream does not nega-

tively impact the filter. The presence

of extractables and leachables from

compounds that may have moved

from the filter to the process stream

should also be assessed (6). Historical

successes with “similar formulations,

filtration dynamics, membrane types,

and process parameters,” could help

a sponsor satisfy FDA Phase I GMP

Guidance requirements, but may not

satisfy EU guidance requirements (6).

In case of disruption

In the event of a contamination, the

question of when processes should

resume relies on the disruption,

according to Newman. It’s important

to account for all elements of the dis-

turbance—such as how long a room

may have lost positive pressure—to

determine the degree of the disrup-

tion and if a short disinfection process

would suffice, or if a full room qualifi-

cation will be necessary before manu-

facturing resumes. Partington points

out that in the event of a power loss

or if the blower on an isolator ceases,

the isolator interior is protected by

HEPA filters, so the disruption is not

immediately disastrous. Nonetheless,

he also says that both positive and

negative pressure isolators need to

be periodically tested for leaks, and

gloveboxes should be examined and

tested frequently.

referenceS 1. S. Sutton and L. Jimenez,

American Pharmaceutical Review

15 (1), pp. 42–57 (2012).

2. The United States Department of

Justice, “14 Indicted in Connection with

New England Compounding Center and

Nationwide Fungal Meningitis Outbreak,”

www.justice.gov/opa/pr/14-indicted-

connection-new-england-compounding-

center-and-nationwide-fungal-

meningitis, accessed May 30, 2014.

3. FDA, “FDA issues new draft documents

related to compounding of human drugs,”

www.fda.gov/NewsEvents/Newsroom/

PressAnnouncements/ucm434270.

htm, accessed June 1, 2015.

4. EC, EudraLex, Volume 4 EU, Guidelines to

Good Manufacturing Practice Medicinal

Products for Human and Veterinary Use,

“Annex 13, Investigational Medicinal

Products” (Brussels, Feb. 3, 2010).

5. J. Chua, “Pharma’s thirst for pure,

clean water,” www.eco-business.com/

news/pharmas-thirst-for-pure-clean-

water/, accessed May 22, 2015.

6. R.W. Acucena, “Defining a Strategy

for the Validation and Qualification

of Sterile Filtration Processes of

Investigational Medicinal Compounds,”

presentation at the PDA Metro

Chapter Dinner, March 4, 2014. ◆



Elk

or/

Gett

y Im

ag

es

Media supplements can be

used to support or stabi-

lize cell growth, increase

concentration, and main-

tain cell productivity when used in cell

cultures. BioPharm International spoke

with Becky Moore, PhD, and Mark J.

Stramaglia, RPh, MBA, Gibco, Thermo

Fisher Scientific, about the importance

of understanding the cell line of the bio-

logic when adding supplements, how

the physical state of the supplement can

affect the base medium, and how to get

the most out of the feeding process.

BioPharm: When are supplements

incorporated into the biologic formula-

tion process?

Moore and Stramaglia: Simple supple-

mentation is often incorporated earlier

in the formulation process to prepare

for more complex process development

efforts. More complex supplementation

is usually first engaged during process

development between stable produc-

tion cell-line development and Phase I

clinical research. Complete feeding

solutions extend fed-batch culture life

and volumetric protein production.

Media supplements encompass a range

of additives from simple additions like

L-glutamine to more complex additives,

like FunctionMAX TiterEnhancer, used to

help maintain cell productivity.

BioPharm: What is the most important

aspect to consider when determining if

a supplement will be compatible with a

medium?

Moore and Stramaglia: The cell line

being used is most important. Some

parental lines are more sensitive to highly

Optimizing the Delivery of Cell-Culture Additives

Ashley Roberts

When using media supplements

in biologics, it is important

to have a key understanding

of both the supplement and

the base medium to ensure high

titer and stability.

Upstream Processing



concentrated feeding versus others

that may be more metabolically

demanding. The key is having the

flexibility to deliver complete feed-

ing supplements at various concen-

trations based on the needs of the

cells. Options such as EfficientFeed

A+, B+, and C+ provide maximum

flexibility to super-concentrate

nutrient delivery up to 200 g/L

concentrations. Straightforward,

water-only reconstitution that

does not require pH adjustment

is an important aspect for work-

flow considerations. Additionally,

eliminating the need for pH adjust-

ment and reconstitution of mul-

tiple feeds can reduce risk of errors

associated with overshooting salt

addition, reconstitution errors,

or extreme pH handling that can

impact bioreactor control.

BioPharm: Is there a standard sup-

plement that you find works best

across an array of biologics?

Moore and Stramaglia: An ideal

feeding supplement would provide

the flexibility to deliver the opti-

mal level of nutrient supplemen-

tation while complementing the

original base medium and avoiding

unneeded salt content. A part of

delivering the optimal concentra-

tion is allowing super concentration

to either work within the bioreac-

tor constraints or to avoid product

dilution. There are commercially

available supplement options with

matched catalog basal formulations

that offer the flexibility needed to

super-concentrate up to 3X or 200

g/L without pH adjustments that

add salt content.

BioPharm: How does the supple-

ment’s physical state affect the

process of inclusion into the for-

mulation?

Moore and Stramaglia: Supplements

are most economically shipped

from vendors to end users in a dry-

format state. Methods for reconsti-

tution have an impact on the salt

or osmolarity contribution to the

culture. This can impact the total

amount of nutrients delivered

before having negative impacts on

the health of cells. To deliver the

most nutrient-rich components,

users should choose supplements

that only require water addition—

no acids or base—over those requir-

ing pH adjustments.

BioPharm: How does the type of

supplement, or amount added to

a base medium, affect a biologic’s

stability?

Moore and Stramaglia: More and

more we find customers analyz-

ing protein quality characteristics

when modifying feeding strategies

or supplements. To provide better

total-end biologic delivery, it is

important to ensure that the sup-

plement used to gain a higher titer

also hits the target product quality

profile. Therefore, use of supple-

ments shown not to impact prod-

uct quality from an original state

is essential. Further, the capability

to modify product quality param-

eters in a predictable and repro-

ducible way is becoming more

important.

BioPharm: Are there any advan-

tages or disadvantages associated

with using media kits?

Moore and Stramaglia: In sets

of media options for finding a

base medium, if a kit provides a

designed group of options to help

find the best composition for a

new production cell line, one must

test enough conditions to gain the

full benefit of the kit or panel.

High-throughput process develop-

ment is required to understand the

impact of multiple formulations,

as well as process parameters.

Design of experiments investigate

multiple conditions in the quest

to select the appropriate base and

feed solutions. ◆

global advocacy group representing

stakeholders in the ATMP sector.

Its European arm has been among

the leading critics of criteria applied

for the exemption, particularly those

relating to manufacturing stan-

dards. Sceptics about the potential

of exempted hospital-based devel-

opment systems contend that they

encourage the avoidance of the

strict EU data requirements because

the hospitals have to adhere only

to national quality and safety stan-

dards, although these standards

should be consistent with those at

the EU level.

Even the European Commission

in its report (3) on the impact of

the ATMP regulations concedes that

the exemption can enable hospital-

based centers to have lower develop-

ment costs than commercial ATMP

organizations because of the advan-

tages of being subject to less rigor-

ous standards. A major objective

behind the EU’s ATMP regulation

was the introduction of harmonized

standards across Europe. The way

the hospital exemption is operating

shows that there is still some dis-

tance to go before full harmoniza-

tion is achieved.

RefeRenCes 1. EC 1394/2007 Regulation on advanced

therapy medicinal products (Brussels,

November 2007).

2. European Commission, Repor t in

accordance with Ar ticle 25 of EC

1394/2007 on advanced therapy

medicinal products (Brussels, March

2014).

3. EMA, Annual Report 2014 (London, April

2015).

4. EMA, Guideline on the quality, non-clinical

and clinical aspects of gene therapy

medicinal products–draft (London, March

2015).

5. EMEA, Note for guidance on the quality,

preclinical and clinical aspects of gene

therapy medicinal products (London, April

2001).

6. UK Department of Health, “Building

on our own potential: a UK pathway

for regenerative medicine,” Report of

Regenerative Medicine Expert Group,

March 24, 2015 (London). ◆

Upstream Processing

European Beat—Contin. from page 14



The use of single-use systems

(SUS) in biopharmaceut i-

cal manufacturing has been

steadily increasing. While

the industry has used SUS in upstream

processing for some time now, the use

of single-use technologies is becom-

ing popular in downstream processes

as well. And it appears that the sin-

gle-use trend will continue to grow.

According to Nilesh Mehta, senior

process engineer, Merck GTO, Sterile

and Validation COE, “as new tech-

nologies become available or exist-

ing technologies are converted into

a single-use format, the number of

single-use systems used for biophar-

maceutical applications will continue

to grow.” The benefits of SUS may also

influence the switch to SUS. Mehta

states that the use of SUS in down-

stream processing may “reduce risk

of cross contamination and carryover

due to inadequate cleaning steps.”

BioPharm International spoke with

Nilesh Mehta about the benefits and

challenges of using SUS in down-

stream processing.

The BenefiTs of single Use in DownsTream ProcessingBioPharm: Are there specific aspects of

downstream processing that benefit

from the use of single-use systems?

Mehta: Yes, single-use systems pro-

vide significant advantage over tra-

ditional stainless-steel systems for

downstream processing. Some exam-

ples are f i lt rat ion systems, buffer

preparation systems, and sampling/

dispensing systems. Key advantages of

using single-use systems are speed of

implementation, which is crucial for

high-impact new product launches,

and increased operational efficiency

related to a reduction in the resources

required for cleaning and setup.

Normal flow filtration (NNF) sys-

tems are now available in capsule

format; these systems are entirely

single-use without any need for a

stainless-steel holder. Systems can

be designed, tested (with good scale-

down models), and implemented

fairly quickly without the need for

procur ing sta inless-steel par ts or

developing related cleaning cycles.

In addition to offering the same

advantages as NFF systems (speed,

reduced cleaning, etc.), tangential

f low f i ltration (TFF) systems with

completely single-use fluid paths also

allow for greater flexibility when try-

ing to achieve extremely low operat-

ing volumes for product concentration

steps that may not be possible with

traditional stainless-steel systems.

challenges of sUs in DownsTream ProcessingBioPharm: What specific challenges do

manufacturers face when using sin-

gle-use systems in downstream pro-

cessing?

Using single-Use Technologies in Downstream Processing

Susan Haigney

The use of single-use systems in

downstream processing

offers benefits in filtration and sampling and

may reduce the risk of

contamination.

Downstream Processing

Tangential flow

filtration systems with

completely single-use

fluid paths allow for

greater flexibility.




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Mehta: Extractables and leach-

ables a re usua l ly a cause of

concern with downstream pro-

cessing, especially since product

streams are fairly clean at this

point and there may not be fur-

ther processing that would clear

anything that is introduced into

the process stream from single-

use components. Most often, the

risk to a patient is fairly low and

can be justified through under-

standing the type and amount of

compounds that can potentially

leach into product streams.

Material compatibility could

also be an issue for downstream

steps where organic solvents or

harsh chemicals are used as part

of a purification process. If there

is not sufficient level of review

to confirm compatibility with

process streams or if all mate-

rials of construction in a given

single-use system are not known

upfront, this issue could easily

be missed during initial design

process and can result in re-work

and delays during implementa-

tion.

Another challenge with sin-

gle -use is re lated to amount

of waste that is generated due

to use of single-use systems.

Diligent design efforts should

focus on reducing amount of

pla s t ic a nd ot her mate r ia l s

used as part of manufacture of

each single-use system to what

is absolutely required to make

the design work. Special efforts

need to focus on minimizing

amount of packaging material

used to transport single-use sys-

tems from supplier to end-user.

Smart packaging designs and use

of recyclable/re-usable packaging

material should help in signifi-

cantly reducing waste generated

and minimize impact on the

environment.

TrenDs in single-Use sysTemsBioPharm: What trends are you

seeing in the use of SUS in down-

stream processing for the future?

Mehta: S t a nda rd i z at ion of

designs should allow for greater

adoption and easier implemen-

tation of single-use systems in

the future. This [standardiza-

tion] will also reduce the risk of

unanticipated fai lures related

to untested designs or products.

Having standards will also allow

suppliers to focus on improv-

ing in areas of higher risk with

respect to single-use systems

while maintaining key features

of standard design that have

been proven to be robust through

years of testing and use. ◆

Downstream Processing




Company DescriptionWith a proven track record of providing quality

testing services for the largest pharmaceutical

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Eurofins Lancaster Laboratories is a global leader

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FacilitiesWith a global capacity of more than 500,000 square

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locations, we also have teams of scientists placed at

more than 40 client facilities throughout the US and

Europe through our Professional Scientific Services

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“

Lipid A is … an information-

rich molecule, with many

possibilities for specific rec-

ognition by prokaryotic and

eukaryotic proteins,” says Christian

Raetz (1). Recent Parenteral Drug

Association (PDA) conferences in

Bethesda (October 2014) and Berlin

(February 2015) highlight the ongoing

disagreement among drug manufactur-

ers, Limulus amebocyte lysate (LAL) test

manufacturers, and regulators as to

the significance of the low endotoxin

recovery (LER) phenomenon. The con-

troversy on endotoxin detection can

be summarized by two different views

or philosophies of microbiological con-

taminant control (Figure 1). The first

philosophy would maintain that only

“biologically active” components of

bacteria are relevant to microbiological

control. The second would seek to con-

trol microbiological process ingress by

monitoring important markers or qual-

ity indicators such as endotoxin. Here,

endotoxin and lipopolysaccharide

(LPS) are used interchangeably. The lat-

ter philosophy does not need to answer

the question, “Is the microbiological

artifact biologically active?” to use it as

a gauge as to the purity or contamina-

tion status of a given aseptic biologics

process. In fact, the demonstration of

“biological activity” is not as clear-cut

as some would currently maintain.

Consider that the GMP context for

product contamination is the concept

of “adulteration,” which does not distin-

guish biologically active contaminants

from those that are not biologically

active. The mere proximity of “filth” is

the traditional criteria for adulteration.

The “old time” verbiage of the Code

of Federal Regulations (CFR) is, “(1) if

it consists in whole or in part of any

filthy, putrid, or decomposed substance;

or (2)(A) if it has been prepared, packed,

or held under insanitary conditions

whereby it may have been contaminated

with filth,” and the GMP concept of

“adulteration” via contamination portray

product/process contamination in the

context of probability, proximity, and

severity of its occurrence (2). A valid end-

product United States Pharmacopeia (USP)

Bacterial Endotoxin Test (BET) <85> test

of a finished drug or active pharmaceuti-

cal ingredient is a legal requirement.

ABSTRACT

Low endotoxin recovery represents an opportunity to add value

to the characterization of biologic drug products.

Endotoxin Test Concerns of Biologics: The Role of Endotoxin as a Quality Indicator in Biologic

Manufacturing ProcessesKevin L. Williams

Kevin L. Williams is senior

scientist of R&D at Lonza.

peer-revieWed

Article submitted: Apr. 22, 2015.

Article accepted: May 12, 2015.

Peer-Reviewed: Endotoxin Testing



EnDoToxIn In ITs MAny foRMsSome have sought to solve the LER conun-

drum by substituting naturally occurring

endotoxins (NOEs) that are not purified

standards and that often show better recov-

ery from spiked biologics subject to LER.

However, when viewed by the second philos-

ophy, that of choosing a quality indicator, it

can be seen that only switching out the posi-

tive control (reference standard endotoxin

[RSE] or control standard endotoxin [CSE] for

NOE) does not provide any additional char-

acterization of the drug process itself. A NOE

spike may recover better than a standard

CSE or RSE spike, but the recovered positive

control does not represent the endotoxin

content of the non-spiked sample. Rather, it

assumes that the non-spiked sample contains

monomers that are not biologically active

with the LAL reagent and thus, concludes

that the sample is not contaminated. The

basic theory of LER is that LPS disaggrega-

tion to monomers occurs via the chelation of

ions needed by LPS to maintain its aggregate

form in solution by buffer (citrate or phos-

phate). The disaggregation is followed by the

subsequent coating of LPS monomers with

abundant polysorbate molecules, thereby

forming a non-LAL-reactive or masked endo-

toxin-drug solution that does not allow LPS

to react with LAL.

The assumption that resides in the first

philosophy is that “monomers don’t mat-

ter,” because they are not active with LAL

(although the monomers are sometimes

active and sometimes inactive with the

rabbit pyrogen test)(3) and thus, are of no

concern to patients. However, the effect

of monomers in the mammalian body is

still unknown. What is known is that the

monomer is the active endotoxic principle

for all endotoxins (RSE, CSE, NOE; aggre-

gated and disaggregated). This fact, that the

sub-monomer Lipid A is the active princi-

ple of endotoxin response, has been estab-

lished beyond doubt by various studies of

the toll-like receptor 4 (TLR4) and associated

co-receptor myeloid differentiation protein

(MD-2), which show how the monomer fits

into the hydrophobic pocket of MD-2 and

how MD-2 with LPS fits into the TLR4 dimer

to bring about the transmembrane signaling

event that instructs the cell nucleus to pro-

duce cytokines (4).

MD-2, as the co-receptor of TLR4, holds

the prototypically configured endotoxic

Lipid A (hexa-acylated) in a hydrophobic

pocket. Five of the six acyl group fingers

rest inside the MD-2 hydrophobic pocket

(glove) and the sixth finger (in the proto-

typical agonistic Escherichia coli LPS) must

stick out to attach to a second TLR4 to

help form the dimer. There are 10 func-

tional TLRs in humans (TLR11, 12, and 13

have been lost from the human genome)

(5), which are mixed and matched in like

or different dimers (homodimers and het-

erodimers, respectively) to detect dozens AL

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Figure 1: Alternative philosophies of microbial contaminant detection via the endotoxin marker.

Philosophy 1 relies on the tenet that “monomers don’t matter,” whereas Philosophy 2 is based on

the fact that “monomers may matter.”

Philosophy 1: Extrapolation to perceived patient health

• Seems biologically inactive• No harm to the patient• “Unnatural” to "x it

• Low Endotoxin Recovery (LER)=nuisance (bother)• “Monomers don’t matter”

Philosophy 2: Drug manufacturing characterization

• Testing is masked in production• Can’t gauge product quality or process change effects• Need valid release tests

• Lipopolysaccharide (LPS) remains an important quality indicator• “Monomers may matter”



of microbial artifacts. The TLR structure is

based upon the leucine-rich repeat (LRR)-

type sequence of alternating loops of

hydrophobic and basic amino acids. Lipid

A antagonists can prevent the activation of

TLR4 by displacing the active monomer in

the dimer structure and thus, preventing

fulfillment of the conditions for activation.

It is the fine structural detail of the Lipid

A molecule that determines the endotoxin

response, with a wide degree of variants

demonstrating a wide spectrum of mam-

malian host responses (from agonistic to

antagonistic).

The first TLR was discovered by the knock-

out of a receptor in Drosophila that subse-

quently allowed the fly to be invaded and

overgrown by fungal hyphae (6), thereby

beginning the quest for various additional

TLRs and the various associated microbial

artifacts that activate them. It should be

noted that monomer structure of Lipid IVa

(a precursor to Lipid A in bacterial Lipid A

metabolism) and Eritoran are antagonists

and do not fit into MD-2 as well as the E. coli

LPS, and thus, cause blockage of the TLR4

receptor and its activity. The drug candidate

Eritoran was hoped to be used to treat sep-

sis, but the drug failed in Phase III clinical

trials. This shows that while the knowledge

of what happens to endotoxin within the

body is advanced, it is not yet complete. Also,

the blocking of the TLR4 pathway does not

negate the potential for the activation of the

complement–coagulation system.

ThE BLA AnD ThE unIQuE RoLE of LPs As A QuALITy InDICAToRThe biological license application (BLA) sub-

mission has become the focal point for the

demonstration of time-dependent LPS spike

recovery as a quality indicator in biologic

products and constituents subject to LER.

According to FDA, “Sponsors of BLA sub-

missions have reported unacceptable time-

dependent recovery of endotoxin spiked

into undiluted drug product using the

LAL USP <85> methods for endotoxin”(7).

Manufacturers that submit BLAs are now

being required to perform follow-up studies

to demonstrate recovery of LER-prone sam-

ples and to perform rabbit pyrogen testing

in lieu of LAL testing for such samples on an

interim basis, until such a time as an alterna-

tive detection protocol may be developed.

In its simplest form, a quality indicator

such as LPS provides information on a manu-

facturing process. It answers critical ques-

tions, such as:

• Was a microbial contaminant present?

• Where did it come from?

• Which type of microbial contaminant

was it?

• How much of it was present?

• How might its presence (and the quantity

present) have affected product quality?

Therefore, as a quality indicator of a manu-

facturing process, it is not always the bio-

logical activity of the indicator itself that

tells the story, but rather, the mere presence

of the artifact. For example, the presence

of coliforms indicates poor sanitary condi-

tions (i.e., from waste water) in certain food

processing environments or in non-sterile

drug manufacturing. Coliforms are defined

as Gram-negative rods that ferment lactose

and produce acid and gas at 35 °C, while

fecal coliforms are further distinguished

by their ability to ferment lactose at higher

temperatures (44.5–45.5 °C). Whether a spe-

cific coliform is a disease- or illness-causing

organism does not negate its use as a qual-

ity indicator. Likewise, endotoxin found in

a manufacturing process (in various forms)

should still tell a story specific to its own

historical occurrence in the process. It will

be difficult to detect contaminants of a pro-

cess when the analyte (LPS) of such testing

is masked. Losing the visibility of what has

occurred in a specific drug manufacturing

process means losing the ability to assure

characterization of the product itself. The

ability to gauge LPS content via LAL testing,

therefore, is diminished. Again, an indica-

tor is significant not only for what it is (i.e.,

its own biological activity), but for what it

means (i.e., microbial ingress has occurred

and thus biological activity at the time of

occurrence could have adversely affected the

product). This nuanced distinction doesn’t

seem widely appreciated. If monomers can be

re-aggregated and detected, then the meaning

of such a measurement can serve its historical

purpose, namely, that of indicating the occur-

rence of Gram-negative bacterial ingress.

Therefore, LPS, even as a monomer, is

significant both in its mere presence and,

more importantly, as a microbial artifact,




given its historical complexity—eons of

history in prokaryotic and metazoan inter-

actions, including shaping the genome of

man. Nobel Prize-winner Bruce Beutler,

who discovered TLR4, said, “Particularly

because they strike down so many people

before or during reproductive age, microbes

constitute the strongest selective pressure

with which our species must contend, and

we may assume that microbes have shaped

the human genome more than any other

selective pressure in recent times” (8). The

LPS monomer is not a depyrogenated or

incinerated fragment of LPS, but rather, the

entire bacterial pathogen-associated molec-

ular pattern (PAMP, now largely called a

MAMP or microbe-associated molecular

pattern) that has served for an estimated

one billion years (beginning in plants) as

a target of metazoan immune systems in

terms of recognition and response to pro-

karyotic invasion. If LPS were to be lost as

a quality indicator during drug process-

ing, it is hard to envision what might take

its place. A risk-assessment philosophy (in

terms of process testing points using alter-

nate methods) of some sort would have

to be used to justify the improbability of

microbial ingress during processing of

LPS-masked solutions; this represents a

less-than-desirable situation. What is unde-

sirable here is that contamination is a time-

linked occurrence, and by its very nature, is

unpredictable. At present, there is no good

substitute for endotoxin detection.

BIoLogIC MoLECuLEs AnD MAnufACTuRIng PRoCEssEsSome have argued that the presence of

monomers causes no harm to the patient.

While this has not been scientifically proven,

the harm envisioned from LER is not neces-

sarily directly derived from drug solutions

containing monomers of LPS, but rather,

from poorly or under-characterized biolog-

ics as end-products. Biologics are typically

large and complex molecules. The molec-

ular weight of a monoclonal antibody is

approximately 150,000 Daltons; compare

that with an early therapeutic protein, such

as r-human insulin, at approximately 5800

Daltons. Therefore, the characterization of

the process that produces these biologics

is paramount. A review of life-threatening

adverse events that have occurred from first-

in-human (FIH) doses of modern biologics

(mAbs and therapeutic proteins) shows some

tragic instances that arose from less-than-

perfectly characterized drug products (Table

I). In other words, some slight change in the

manufacturing process produced a product

that was slightly different at the molecular

level. These molecules appeared to be charac-

terized at the time, but had somehow, during


Table I: Summary of points from references on the adverse events associated with frst-in-human

(FIH) dosage of a monoclonal antibody (mAb) development compound.

“Giving Monoclonal Antibodies to Healthy Volunteers in Phase I Trials: Is it Safe?” from the British Journal of Clinical Pharmacology (10).

• “We identified 70 completed trials of mAbs in healthy volunteers, but the published data were too sparse to allow confident assessment of the risks of mAbs in healthy volunteers. Our best estimate of risk of a life-threatening adverse event was between 1:425 and 1:1700 volunteer-trials, but all such events occurred in a single trial (of TGN1412).”

• “There is substantial under-reporting of phase I trials of mAbs in healthy volunteers, so their safety record is difficult to assess.”

• “The risk of life-threatening adverse reactions in FIH trials of mAb exposed by the TGN1412 incident…”

• Six healthy volunteers experienced life-threatening cytokine storm from which it took six months to recover.

• The authors compared events with small-molecule trials to risks in everyday life of ~1:1,000,000.

• Researchers also noted a “Lack of animal models to predict immunotoxicity.”

“Cytokine Storm in a Phase I Trial of the Anti-CD28 Monoclonal Antibody TGN1412”

from the New England Journal of Medicine (11).

• “Cytokine release had not been observed in the preclinical studies of TGN1412…”

• “Regulatory authorities, who tested TGN1412 from the same batch as the infused drug, found no errors in its manufacture, formulation, or administration and found no contamination with endotoxin, pyrogen, or microbiologic or other agents.”

• Today, clinicians would not dose such patients simultaneously.



the manufacturing process, accrued slight

changes in fine structure (e.g., 3D structure,

glycosylation, charge, hydrophobicity, fold-

ing, heterogeneity, bioactivity, truncation,

oxidation, deamination, or aggregation) (9).

ThE CLInICAL MAnIfEsTATIon of EnDoToxIn MonoMERs vs. AggREgATEs CAnnoT BE REsoLvEDThe two questions presented as a duality

of competing hypotheses from Figure 1:

“Monomers don’t matter” versus “Monomers

matter”, in terms of biological activity when

presented in drug products, may seem criti-

cal to the ongoing LER debate, but this issue

cannot be easily solved. For every determina-

tion that “only” aggregates are biologically

active, there are two other studies indicating

that under specific physiological conditions,

monomers are indeed the active physical

unit of endotoxin (12, 13, 14). Levin showed

one such example where, in the presence of

hemoglobin, LPS disaggregation produced

increasing LAL gelation times (15, 16). Aside

from hemoglobin, “there are more than

200 acute phase proteins (APP) in mammals

responding to endotoxin” (17). Short of pro-

ducing and giving endotoxin monomers to

compromised patients, such as those with

sepsis, disseminated intravascular coagu-

lation (DIC), or via the intrathecal route—

something which would never be done—the

harm or lack thereof of monomers cannot

be established with any certainty. It is worth

highlighting that there are challenges in

determining “biological activity” given that

the endotoxin response is partitioned in the

body amongst different tissue types—for

example, the gut tolerates a huge load of

endotoxin-bearing Gram-negative bacteria,

whereas the spinal column is very sensitive

to extremely minute levels of endotoxin.

Moreover, the body’s response may vary with

different disease states such as inflammation,

infection or DIC, for example.

Additionally, some small molecules that

mimic Lipid A but are of different (sometimes

widely unrelated) structures have been found

to ameliorate the host response to LPS, hence

making it difficult to rationalize that only the

aggregate is active during the initial recogni-

tion of LPS. If only the aggregate were the

active configuration of LPS, one wonders how

small molecules could find their way to TLR4/

MD-2 to mimic the LPS behavior in a disrup-

tive manner. The corresponding aggregate

behavior of small molecules seems unlikely

to mimic that of LPS. Neal et al. used a small

molecule (MW of ~390 versus ~1700 for Lipid

A) to inhibit the host reaction to LPS both in

vitro and in vivo (18). Slivka et al. used a 17-res-

idue peptide (MD2-I), synthesized to repro-

duce the TLR4-binding region of the MD2

protein that contains all the critical interact-

ing residues, and showed evidence that this

sequence targets TLR4 directly as an antago-

nist (19). At any rate, the biological activity

question cannot yet be answered definitively

and does not affect the use of LPS as a biolog-

ics manufacturing process quality indicator.

LPS is viewed as a fever-causing substance

first and foremost; however, as seen by F.

Bang in 1955, it was the gelation of the

Limulus blood—the coagulation dysfunction

caused by endotoxin—that prompted the

development of LAL as a detection method

(gel clot). Therefore, careful consideration

should be given to blood dysfunction pos-

sibilities that may come through the com-

plement-activation pathway via monomeric

endotoxin, as well as singularly focusing on

the fever-causing activation of TLR4 (20). It is

the TLR4 receptor-activated route that brings

about the production of fever-causing cyto-

kines and is the route that many presume

requires an aggregated endotoxin presenta-

tion to activate. While rabbit pyrogen test

can detect fever, it cannot detect blood-clot-

ting dysfunction, nor can it be relied upon to

consistently predict the occurrence of fever

from masked or disassociated endotoxin,

which may process differently and along a

different timeline compared with traditional

drug injections into rabbits.

Drug regulators, along with each thera-

peutic protein manufacturer, will have to

determine the safeguards that will consti-

tute cGMP activities for LER-prone solu-

tions, be it increased process controls

(increased microbial monitoring, hazard

analysis, or validation of process hold-

times) and/or a form of pretreatment for

BET samples to re-aggregate disassociated

endotoxin. It should at the very least be

agreed that philosophically viewpoint 2

of Figure 1 would be the more appropriate

paradigm for drug manufacturing control

of contaminants.




ConCLusIonRegulators, drug manufacturers, and LAL pro-

viders are still adjusting to LAL testing subse-

quent to the knowledge of the occurrence of

LER. The BLA is the choke point for efforts to

commercialize a biologic drug relative to LER,

as BLA approval requires the demonstration of

valid hold-time endotoxin spike studies that

may demonstrate masked recovery due to LER.

LER is most relevant when viewed from a

drug product characterization perspective.

Important questions from a risk-analysis per-

spective regarding LER include:

• What is the time-course profile of the

process constituents in regard to microbial

ingress? Does it lend itself to transient

contamination events from a microbial

control strategy vantage (21)?

• Could theproduct be contaminatedwith

endotoxin and could this contamination

be subsequently masked by an LER or LER-

like effect?

• How could a contamination event of live

Gram-negative bacteria, as evidenced by

the detection of LPS, affect a drug’s prop-

erties as they relate to patient safety?

Each specific drug manufacturing pro-

cess flow puts LER in context relative to risk

management given the formulation constitu-

ents, hold times, temperatures, and container

types. The “no patients are being harmed”

argument associated with philosophy 1

seems premature. Even if this assumption

turns out to be valid, there does not appear

to be a valid substitute or replacement for a

longstanding quality indicator such as LPS.

The risk to patients from poorly character-

ized biologics is not negligible. It is well known

that the presence of both Gram-negative bacte-

ria and endotoxin can induce changes to drug

molecules during expression and purification

processes. The new LER paradigm is reminis-

cent of the early mycoplasma debate, wherein

the effect of mycoplasma on cell cultures used

to produce drug products was up for debate

(22, 23). Subsequently, as better methods of

detection were developed, product charac-

terization improved, and today mycoplasma

testing is viewed as a critical routine quality

indicator. Analogously, new sample treatments

are needed to reveal the presence of endotoxin

regardless of the aggregation state. LER repre-

sents an opportunity to add value to the char-

acterization of biologic drug products.

REfEREnCEs 1. C. Raetz, J. Bacteriol. 175 (18), pp. 5745–5753

(Sept. 1993). 2. CFR Title 21, Part 351, (Government Printing

Office, Washington, DC), pp. 114. 3. P. Hughes, “FDA Regulations and Endotoxin

Detection,” presentation at the PDA Conference (Bethesda, MD, Oct. 21, 2014).

4. T. Scior et al, Comput. Struct. Biotechnol. J. 7 (9), pp. 1–11 (May 2013).

5. T. Kawai and S. Akira, Nature Immunol. 11 (5), pp. 373–384 (2010).

6. B. Lemaitre et al., Cell 86 (6), pp. 973–983 (September 20, 1996).

7. P. F. Hughes et al., “Low Endotoxin Recovery: An FDA Perspective,” BioPharm Asia, http://biopharma-asia.com/magazine-articles/low-endotoxin-recovery-an-fda-perspective/, accessed June 12, 2015.

8. B. Beutler, “How Mammals Sense Infection: From Endotoxin to the Toll-like Receptors,” Nobel lecture presentation at the Center for Genetics of Host Defense, (The University of Texas Southwestern Medical Center, Dallas, TX, Dec. 7, 2011).

9. E. Narke, “Journey in the Development of Biologics Through End of Phase III,” slide presentation for raps.org, www.slideshare.net/enarke/cmc-biologics-pathwaydraft8, accessed June 12, 2015.

10. E. Tranter et al., Br. J. Clin. Pharmacol. 76 (2), pp. 164–172.

11. G. Suntharalingam, et al., N. Engl. J. Med. 355, pp. 1018–1028 (September 7, 2006).

12. M. Mueller et al., J. of Bio. Chem. 297, pp. 26307–26313 (2004).

13. H. Sasaki and S.H. White, Biophys. J. 95 (2), pp. 986–993 (2008).

14. K. Takayama et al., J. of Bio. Chem. 269, pp. 2241–2244 (1994).

15. R.J. Roth and J. Levin, “Effects of Human Hemoglobin on Bacterial Endotoxin In Vitro and In Vivo,” in Endotoxin in Health and Disease, H. Brade et al., Eds., (Marcel Dekker, Inc., New York, NY, 1999), pp. 389–402.

16. J. Levin, “The Original Description of the Limulus Amebocyte Lysate (LAL) Test: From Past to Present,” presentation at the PDA 9th Annual Global Conference on Pharmaceutical Microbiology (Bethesda, MD, 2014).

17. S. Chemonges, J.P. Tung, and J.F. Fraser, Proteome Sci. 12 (1), pp. 12 (2014).

18. M.D. Neal et al., PLoS ONE, 8 (6), pp. e65779 (June 2013).

19. P.F. Slivka, et al., Chembiochem. 10 (4), pp. 645–649 (Mar. 2, 2009).

20. P. Libby and D.I. Simon, Circulation 103 (13), pp. 1718–1720 (Apr. 3, 2001).

21. K. Suvarna et al., American Pharm. Rev. 14 (1) (January/February 2011), www.americanpharmaceuticalreview.com/Featured-Articles/36755-Case-Studies-of-Microbial-Contamination-in-Biologic-Product-Manufacturing/, accessed June 12, 2015.

22. D.K.F Chandler, D.V. Volokhov, and V.E. Chizhikov, American Pharm. Rev., 14 (4) (May/June 2011), www.americanpharmaceuticalreview.com/Featured-Articles/37370-Historical-Overview-of-Mycoplasma-Testing-for-Production-of-Biologics/, accessed June 12, 2015.

23. R.J. Geraghty et al., Br. J. Cancer 111, pp. 1021–1046 (2014). ◆




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BioPharm International spoke with

Stephan Krause, director of qual-

ity assurance, Laura Jeannel, asso-

ciate director of quality assurance,

and Paul Davis, senior manager of business

resilience, at AstraZeneca Biologics to get

the latest on performing risk management

in biopharmaceutical manufacturing.

Risk Assessment ChAllenges in BiophARmABioPharm: What top challenges do compa-

nies face when performing risk assessment

in biopharmaceutical manufacturing com-

pared with solid-dosage manufacturing?

AstraZeneca: In biopharmaceutical

manufacturing, microbial contamina-

tion is usually the greatest overall risk.

The primary factors that affect the risk of

microbial proliferation during hold times

are the growth-promoting properties of

the in-process materials, initial bioburden

level, and storage conditions. In general,

when risk ranking is performed for a par-

ticular manufacturing site, microbial fail-

ure events are only infrequently observed,

and similar, future events are difficult

to predict. It is challenging when poten-

tial risks are scored and then ranked.

Available risk scoring tools such as failure

mode and effects analysis (FMEA) should

be properly modified to allow suitable risk

ranking of high-impact risks with respect

to other less-impact risks due to the exist-

ing uncertainty factor(s) of some of the

high-impact risks.

Management reviews need to capture

and aggregate risks across manufacturing

campaigns to mitigate potential risks dur-

ing changeover. In general, a biopharma-

Risk Assessment and mitigation in Biopharmaceutical manufacturing

Susan Haigney

The challenges and strategies of

assessing and mitigating risk in

biopharmaceutical manufacturing are discussed.

Risk Assessment and mitigation



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ceutical manufacturer faces similar

business continuity risks as those of

traditional pharmaceutical manu-

facturers.

BioPharm: Does the use of single-

use systems (SUS) and/or equipment

present any specific challenges or

considerations when performing a

risk assessment?

AstraZeneca: The use of SUS

removes some of the risks that

would otherwise come with the

required cleaning process (e.g.,

cross-contamination and cleaning

residues). Risks that may arise with

the use of SUS may be the depen-

dency on the SUS supply/vendor

from a business continuity perspec-

tive. From a quality risk manage-

ment (QRM) perspective, the risks

are usually around product/material

compatibility (e.g., leachables, etc.).

While one risk source may effec-

tively be transferred outside, the

internal focus then turns toward

ensuring comprehensive controls are

in place for ensuring vendor quality

and continued supply.

Risk mitigAtion stRAtegiesBioPharm: What strategy does your

company use to mitigate risk in the

manufacture of biopharmaceuticals?

AstraZeneca: To obtain the most

valuable risk mitigation plans out of

the formal risk-scoring process, we

review and manage all risks cross-

functionally. Whenever possible, we

use the same risk identification, col-

lection, and analysis tools, regardless

of whether they may impact product

quality and/or business continuity.

Once collected, we primarily sepa-

rate risks based on either potential

quality/patient impact or production

impact (only). Depending on the

risk priority numbers, higher-scor-

ing risks are mitigated and results

reviewed and monitored by senior

management. Lower-scoring risks,

although individually of lesser con-

cerns, can be trended within a given

category and/or among different cat-

egories as needed.

For business continuity, all risks

are assessed and have action deci-

sions made by management, regard-

less of scoring level. The documented

actions are tracked through to com-

pletion. Multiple risk identifying

methods are maintained on an ongo-

ing basis. To account for changes in

the business environment, previ-

ously accepted risks are periodically

reviewed to determine if mitigation

is now required. Risk management

should be a continuous process to be

effective.

Using the same risk management

tool and a standardized process for

QRM, and all other business conti-

nuity risks, allows employees to

communicate risks as they are iden-

tified within all functional areas,

in a consistent manner. It is part

of our quality culture to encourage

all employees to submit their per-

ceived risks. It is important that all

CAPAs [corrective actions and pre-

ventive actions] identified fully

address the corresponding risks and

failure modes. Within our company,

we have a tiered approach, which

includes risk registers, for both qual-

ity and business continuity, at the site

and corporate level.

RegulAtoRy RequiRementsBioPharm: What are the specific

regulatory requirements for risk

assessment and mitigation in bio-

pharmaceutical manufacturing?

AstraZeneca: We are required to

follow International Conference on

Harmonization (ICH) Q9 for QRM

and ICH Q10 (1, 2) for the risk over-

sight by senior quality manage-

ment. Regulatory expectations exist

that risk assessments are performed

as part of individual quality sys-

tems including proactive systems

(e.g., training programs, self-inspec-

tions, change control, validation,

etc.) and reactive systems (e.g., devi-

ations, out of specification, supplier

complaints, etc.).

It should be in the interest of the

manufacturer to invest time and

resources into the QRM process as

the QRM results may ultimately

allow for less testing, auditing, con-

trol elements, etc. Although QRM

should not be used entirely for the

purpose of avoiding work, it can

reduce time and work required com-

pared with when QRM is not used

and all risks are equally addressed.

Risk Assessment toolsBioPharm: What current tools are used

in risk assessment and mitigation in

biopharma manufacturing? Are there

tools in development?

AstraZeneca: We often use a modi-

fied version of the FMEA tool, similar

to what is published in Parenteral

Drug Association (PDA) Technical

Report (TR) 44 and PDA TR 54 (for

biopharmaceuticals) (3, 4). An FMEA

is appropriate for managing risks for

processes in late-stage clinical and/

or commercial manufacturing. A

modification of the standard FMEA

table(s) may be required, or a differ-

ent/simpler QRM tool can be used

when assessing risks with an associ-

ated high uncertainty component.

For example, when assessing initial

and post-mitigation risks as part of

the manufacturing plant’s control

strategy for virus contamination pre-

vention, there is likely no previous

occurrence within a specific plant

to score likelihood based on history

(microbial contamination) and/or

detectability. Although the impact

score would likely be at its maxi-

mum, the occurrence/likelihood can

be difficult to scale in a meaning-


it is important that

all CApAs identified

fully address the

corresponding risks

and failure modes.



ful way. In addition, the detectabil-

ity score also contains a relatively

high uncertainty score as we may

not have data for all critical sampling

points and/or not all viruses may be

detected by the available method(s).

Within development, simpler and

less formal QRM tools are preferred.

For example, it can be challenging

to score and rank meaningfully the

critical quality attributes (CQA) when

developing a control strategy. Most

often, there is not much patient data

from the clinical studies or adverse

events cannot be directly linked to

specific CQA levels. The uncertainty

factor often outweighs any avail-

able data or knowledge and typically

drives the need for a low occurrence

rating in manufacturing prior to set-

ting the (final) control strategy.

Regardless of the tools being uti-

lized, the most important element

for success is fostering a risk-manage-

ment culture throughout the orga-

nization. Risk management should

be seen as the responsibility of every

employee. All employees should be

empowered and accountable for iden-

tifying and mitigating risk.

the pRoBlem of DRug shoRtAgesBioPharm: What role does risk mitiga-

tion play in drug shortages? Does this

role affect biopharmaceuticals more or

less than solid-dosage manufacturing?

AstraZeneca: For drug shortages,

risk mitigation is important for

many drugs, and biopharmaceuti-

cals are no exception. Some of the

reasons leading to supply short-

ages could be increased market

demand, poor product quality and/

or GMP practice, increasing regu-

latory expectations, or limited or

competing manufacturing capac-

ity. PDA TR 68, Risk-Based Approach

for Prevention and Management of

Drug Shortages, provides an excel-

lent overview of the QRM process

to prevent/manage supply shortages

for biopharmaceuticals (and other

drugs) (5).

When mitigating the risk of drug

shortages, it is important to take a

global view of the overall supply

chain, in addition to establishing

comprehensive risk-management

programs at each manufacturing

location. Each location and/or critical

supply function then becomes a link

in the overall supply chain. It is then

a matter of developing recovery plans

in the event of a disruption.

RefeRenCes 1. ICH, Q9 Quality Risk Management

(ICH, June 2006).

2. ICH, Q10 Pharmaceutical Quality

System (ICH, 2009).

3. PDA, Technical Report 44, Quality

Risk Management for Aseptic

Processes (PDA, 2008).

4. PDA, Technical Report 54, Implementation

of Quality Risk Management for

Pharmaceutical and Biotechnology

Manufacturing Operations (PDA, 2012).

5. PDA Technical Report 68, Risk-Based

Approach for Prevention and Management

of Drug Shortages (PDA, 2014). ◆


When mitigating the

risk of drug shortages,

it is important to take

a global view of the

overall supply chain.

Call for Papers * Call for Papers * Call for Papers

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We are currently seeking novel research articles for our peer-reviewed journal as well as manuscripts for our special issues. Submitted manuscripts should be sufficiently novel to be of interest to an experienced audience. Articles should be data driven and provide sufficient technical detail to support the main thesis or should offer a novel synthesis of existing data. Topics should be timely and useful and should focus on the development of peptides, monoclonal antibodies, fusion proteins, other thera-peutic proteins, nucleic acids, vaccines, cells for cell therapy, and any other class of biotechnologically generated molecular class.

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BioPharm International readers are involved in product and process development, manufacturing, quality control/quality assur-ance, analytical technologies, regulatory affairs, plant and project engineering and design, and corporate management for the entire scope of biopharmaceutical products, including therapeutic peptides, proteins, nucleic acids, and cells for cell therapies and regenerative medicine, as well as both therapeutic and prophylactic vaccines.

Please visit our website, www.BioPharmInternational.com, to view our full Author Guidelines. Manuscripts may be sent to Editorial Director Rita Peters at [email protected].

WWW.BiophARminteRnAtionAl.Com



Jaso

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Gett

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ag

es

The raw materials used in the

manufacture of biologic drugs

come from different sources:

animals (although use of such

materials is decreasing), plants, and—

more frequently—chemically derived

ingredients. As a result, it is not pos-

sible to know all of the possible viral

contaminants that may be present.

The biopharmaceutical industry has

consequently taken extensive mea-

sures to prevent contamination and

continues to develop advanced ana-

lytical methods to detect both known

and unknown viruses. Traditional

cell-based assays are broad-based and

generally effective, but do suffer limi-

tations, such as lengthy test times.

Rapid nucleic acid-based techniques

have been developed as alternatives,

but they often only target specific

viral agents. The methods under inves-

tigation today enable the identifica-

tion of multiple viruses, but issues

must be addressed before they can be

fully adopted by industry.

Technology advances drive inTeresT in new meThodsAdventit ious agents are microbial

contaminants that are introduced

inadvertently into a biopharmaceu-

tical manufacturing process. Even

if they are not harmful to patients,

they are impurities and are undesired.

Although current industry processes

and analytical techniques have been

effective in keeping biologic drugs safe

from viral contamination, there has

been significant scientific progress

viral detection Technologies must continue to evolve

Cynthia A. Challener

Advances in adventitious

agent detection

methodology are bringing benefits, but more work

needs to be done.

Cynthia A. Challener, PhD,

is a contributing editor at

BioPharm International.

Quality/analytical



with respect to viral detection,

and these advances are attracting

the interest of the biopharmaceu-

tical industry.

Several important steps have

been taken by the industry to

prevent contamination, includ-

ing the movement away from

a n ima l -based to chemica l ly

der ived mater ia ls, the use of

well-characterized cell lines, and

the implementation of risk-miti-

gation and control strategies for

sourcing activities. The imple-

mentation of process steps that

minimize the risk of viral infec-

tion, such as heat treatment and

viral f i ltration, has also been

highly effective.

In fact, it is important that

these approaches be combined

with extensive analytical testing,

according to Ivar Kljavin, direc-

tor of adventitious agent man-

agement with Genentech. “It is

not possible to prove a negative

result with analytical testing, and

therefore, testing, while a critical

part of an effective solution for

viral contamination prevention,

is not sufficient alone,” he asserts.

T he convent iona l in v i t ro

adventitious virus assay, which

uses various indicator cell lines

for detection of viruses, is sensi-

tive and can potentially detect

one infectious virus particle, but

the virus must replicate in at least

one of the indicator cell types

and produce some type of detect-

able effect (visual for cytopathic

effects and/or hemadsorption

or hemagglutination for specific

red blood cells). False negatives

are possible if a virus replicates

but does not cause a detectable

effect or is present but does not

replicate in the specific indica-

tor cells. There is also some vari-

ability with cell-based assays, and

these tests typically take 14–28

days, during which time con-

taminated materials may be sent

downstream for further process-

ing. The need to decontaminate

a production facility can lead to

significant disruption of the drug

supply, a situation that is unac-

ceptable, according to Kljavin.

Despite t hese l imitat ions ,

when used in conjunction with

other risk-mitigating steps like

viral clearance validation, cell-

based assays are effective. New,

broader, more sensit ive, and

more rapid assays have been

developed, however, that are

attracting the interest of the bio-

pharmaceutical industry.

sTaTe of The arTThe Parenteral Drug Association

(PDA) has been work ing on

a technical report represent-

ing some of the best thinking

of technology developers, users,

and regulators on viral detec-

t ion methods . Accord ing to

Paul Duncan, senior principal

scientist for vaccine analytical

development in vaccine biopro-

cess R&D with Merck Research

Laboratories, based on this work,

it appears that the profile for the

polymerase chain reaction (PCR)

mass spectrometry approach has

decreased somewhat in recent

years, perhaps due to a shift in

focus to clinical diagnostic appli-

cations. He also notes that vari-

ous microarray opt ions ex ist

and have interesting advantages,

including rapid turnaround, but

their accessibility is limited. A

compelling ‘product presenta-

tion’ that would fit into factories

as in-process rapid decision-mak-

ing tools (not necessar i ly for

product release, though) is still

lacking. “On the other hand,” he

says, “massively parallel sequenc-

ing is now readily available, and

some commercial test ing labs

are quite good at processing the

datasets and reaching reasonable

conclusions. Of course, several

large pharma companies are also

developing their own expertise

and capabilities.” He does note,

though, that the turnaround

time for the sequencing approach

is longer, and bioinformat ic

analysis is still an area of active

development.

gmP validaTion a key issueUse of new viral detection meth-

ods in commercial biopharma-

ceut ica l manufac tur ing in a

GMP testing environment may

require system validation, lock-

down, and archival, business and

regulatory acceptance, which

are challenging issues to address

at this time. “There is a need to

continue evolving these systems

to keep up with expanding data-

bases, improvements in comput-

ing infrastructure, analysis tools,

and algorithms, yet at the same

time maintain GMP change con-

trol,” Duncan explains. In addi-

tion, he points out that replacing

existing tests with any of these

newer techniques involves the

added complexity of defending

the suitability (breadth and sen-

sitivity of detection) of the new

methods to regulators in not just

one, but every intended market.

Manufacturers would also have

to come to terms with new types

of specifications and manage the

risk of false positives.

Having said that, at the present

time newer viral detection analy-

ses do have a use in biopharma-

ceutical development and perhaps

even manufacturing as character-

ization and investigation tools,

according to Duncan. “These new

analytical tools certainly have

a place where scientific validity

through suitable controls is the

main concern,” he says.

PreParaTion is of Primary imPorTanceSample selection and process-

ing determine what is possible to

detect and how results must be

interpreted, according to Duncan.

Quality/analytical



In particular, sample selection

requires understanding the differ-

ent ‘compartments’ and the types

and packaging of nucleic acids to

be expected in each. Cell pellets

and whole culture lysates present

different challenges and oppor-

tunities than cell-free superna-

tants or raw material solutions,

for instance. “There are tradeoffs

in the selection of any given

compartment, and also tradeoffs

in sampling all compartments at

once,” he observes.

In add it ion, the se lec t ion

of just one sample type with-

out adequate controls can make

interpretation of results unnec-

essarily complex. On the other

hand, the preparation of sepa-

rate RNA and DNA extractions

instead of combined total nucleic

acid extractions may help differ-

entiate the relevance of some sig-

nals. Concentrating samples from

some matrices to achieve higher

sensitivity can also be surpris-

ingly challenging; there may be,

for example, differential effects

on recovery of different viruses,

according to Duncan.

For e ac h c a s e , t he r e a r e

likely many viable alternative

approaches, but defending any

selected approach requires a sig-

nificant effort, because the goal is

to detect all possible viruses—or

adding even more complexity—

the detection of all possible bac-

teria and fungi as well. Sample

processing also creates an oppor-

tunity for cross-contamination,

and therefore, Duncan notes that

significant care is needed to pre-

vent false positives and unneces-

sary investigation.

huge role for bioinformaTicsBioinformatic analysis must parse

meaningful information from

a noisy background. The chal-

lenge is detection of not only

glaringly obvious viral signatures

(high levels of complete cover-

age of known viral genomes),

but also hints of signatures of

agents that are unknown and/or

unexpected, that may be pres-

ent at low levels in the midst of

tens to hundreds of millions of

other sequence reads. The com-

putational infrastructure must be

sufficiently scaled, or else even

terrific software will have lim-

ited success, given the analysis

challenges. That isn’t the only

issue. “Not all software can be

readily translated across different

computational platforms, so per-

haps bioinformatics analysis solu-

tions may need to be developed

to some extent in the context of

the specif ic high-performance

computational infrastructure,”

Duncan adds. He also notes that

once detected, some interpreta-

tion of these signatures is then

required to determine if they sug-

gest actual infectious contamina-

tion or just normal inactivated

process residual nucleic acid from

input materials.

enhancing accePTanceBeyond further improvement in

the newer, advanced viral detec-

t ion technologies themselves,

Duncan believes that there are

additional steps the biopharma-

ceutical manufacturing industry

can take to improve their accep-

tance. Better control of upstream

bioprocessing could make it less

necessary to rely solely on con-

ventional non-real-time testing

for assurance of culture integ-

rity, and perhaps allow for more

in-line or near-real-time process

monitoring approaches. “Such

an approach may be a tougher

sell in the viral vaccines space,

but could be a realistic oppor-

tunity for some biologics where

there are extremely robust down-

stream viral inactivation/clear-

ance systems in place,” he says.

“Examples of such advances

cou ld a rg uably inc lude, for

instance, single-use processing

and otherwise better use of closed

systems, chemica l ly- de f ined

media, and a better understand-

ing of other inputs including cell

substrates,” Duncan continues.

He adds that longer-term perfu-

sion cultures and continuous

downstream processing will also

require better in-line and near-

real-time means for defending

culture integrity.

PracTical fuTureFor practical use in the future,

Duncan would also like to see

factory-friendly presentations of

systems that do not rely on de

novo sequencing and would like

to have the opportunity to evalu-

ate in some detail how well they

work at detecting real contamina-

tions, because they might be the

most rapid options in the near

term. “It is possible that such sys-

tems already approach the sensi-

tivity needed to be meaningful

and practical,” he notes.

In the longer term, Duncan

be l ieve s t hat shor te r t u r n-

a round t imes w ith sequenc-

ing might be achievable, but at

the cost of generating an astro-

nomica l amount of data for

which archiving could become

a real issue. “For applications of

sequencing that may not require

such rapid turnaround, such as

cell-substrate characterization in

early development, there is also

still a need to raise the under-

stand ing of many technica l

issues among technology devel-

opers, service laboratories, bio-

pharmaceutical companies, and

regulators in order to assure that

potential adventitious or endog-

enous agent-related issues in bio-

logical production systems can

be detected and addressed,” he

asserts. An interest group spon-

sored by PDA is actively pursu-

ing this goal and welcomes active

contributors. ◆

Quality/analytical



Analytical Best Practices

Imag

e: P

AS

IEK

A/S

cie

nce P

hoto

Lib

rary

/Gett

y.

Robust Optimization, Simulation, and Effective Design Space Approaches to the generation of process models, optimization techniques, and application of a design space are explored.

Developing product knowledge and

process understanding is at the

heart of modern drug development.

Establishing a clear line of sight between crit-

ical quality attributes (CQAs), process param-

eters, and material attributes is a primary

goal of drug development. Even though there

are ICH guidance documents such as Q8 and

Q11 that define what a design space is, there

is still a poor understanding of the meaning

and application of a design space.

ICH Q8, Pharmaceutical Development (1)

defines a design space as:

“The multidimensional combination and

interaction of input variables (e.g., material

attributes) and process parameters that have

been demonstrated to provide assurance of

quality. Working within the design space is

not considered as a change. Movement out of

the design space is considered to be a change

and would normally initiate a regulatory post

approval change process. Design space is pro-

posed by the applicant and is subject to regula-

tory assessment and approval.“

This paper explores technically rigorous

approaches to the generation of process mod-

els, optimization techniques for selection of

set points, and application of a design space to

defined CQAs and safe operational ranges.

Design of experiments (DOE) and other mul-

tivariate analysis techniques assist the developer

in mapping out the design space and building

process models. Once the DOE is complete, the

developer can use the DOE to build a process

model, define the design space, and run simula-

tions for various optimums and to determine

effective factor ranges where the out-of-specifi-

cation (OOS) rates will be acceptable.

In reference to modern drug development

ICH Q11, Development and Manufacture of Drug

Substances states (2):

“R isk assessment can be used dur ing

development to identify those parts of the

manufacturing process l ikely to have an

impact on potent ia l CQAs. Fur ther r isk

assessments can be used to focus devel-

opment work on areas for which bet ter

understanding of the link between process

and quality is needed. Using an enhanced

approach, the determination of appropriate

material specifications and process param-

eter ranges could follow a sequence such as

the one shown below:

• Identify potential sources of process vari-

ability.

• Identify the material attributes and pro-

cess parameters likely to have the greatest

impact on drug substance quality. This

can be based on prior knowledge and risk

assessment tools.

• Design and conduct studies (e.g., mecha-

nistic and/or kinetic evaluations, multi-

variate design of experiments, simulations,

modelling) to identify and confirm the

links and relationships of material attri-

butes and process parameters to drug sub-

stance CQAs.

• Analyze and assess the data to establish

appropriate ranges, including establish-

ment of a design space if desired.”

The following are generally accepted key

steps for building a process model and using

the model for development of product knowl-

Optimization works to find

the best solution that meets

all cQA requirements.




edge, process understanding, and

regulatory submission:

1. State all CQAs of interest and

their limits (upper specifica-

tion limit [USL] and lower

specification limit [LSL]).

2. Define the scale (small and/

or at scale).

3. Def ine a l l processes and

materials that will be used.

4. Complete a risk assessment

(high level for all unit opera-

tions and low level for indi-

vidual unit operations and

materials).

5. Develop all single-factor and

multiple-factor study designs

and DOEs, include interac-

tions and quadratics where

indicated by the risk assess-

ment.

6. Bui ld the process model

from the analysis of experi-

mental data and determine

all crit ical process param-

eters and critical material

attributes.

7. Optimize the process and

def ine the recipe and set

points at their best value

(robust optimization).

8. Evaluate the set points using

the design space to evaluate

margin.

9. Evaluate the design space

using simulation and evalu-

ate parts per million (PPM)

OOS.

10. S e t n o r m a l o p e r a t i n g

ranges and proven accept-

able ranges with margin.

11. Verify the small-scale and

at-scale results. Rescale the

small-scale model to match

the at scale process.

12. Define the effective design

space used for process con-

trol and define the purpose

of the design space.

Steps 7–12 will be discussed in

detail in this paper.

SteP Seven: OPtimize the PrOceSS And define the Set POintSW hen de te r m i n i ng t he r e c-

ipe for a formulat ion or pro -

cess (set points) there are two

methods that can be used. The

f irst is optimization, and the

second is robust optimization.

Opt imizat ion work s to f ind

the best solution that meets all

CQA requirements (see Figure

1), robust optimizat ion works

to assure the minimum trans-

mitted variation occurs for all

CQA goals. The dif ference in

the two approaches is optimiza-

tion works to achieve all goals

and limits for all CQAs; robust

opt imizat ion does the same

but in addition it works to find

the point in the design space

where the first derivative (SAS/

JMP) (see Figure 2) equals zero,

also known as the sweet spot.

Mathematically, the sweet spot

is found where the f irst deriv-

at ive of each response w ith

respect to each noise factor are

zero. Software programs such

as SAS/JMP have these features

built in. Robust optimization

reduces var iat ion at the oper-

ational target and is generally

preferred over other optimiza-

tion strategies.

Figure 1: Robust optimization for a target concentration.

Figure 2: Partial derivative for robust optimization.

Optimization

Optimization

Optimization

Optimization

11

10

9

8

7

6

5

4

22 23 24 25 26 27 28 29 30Temp

NaCl

Robust

Optimization

AL

L F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

R.




To achieve superior results and to

find the robust optimum, two-fac-

tor interactions and quadratic terms

must be included in the model.

Main effects, quadratics, and inter-

actions must be considered during

the risk assessment and part of the

DOE design. Main effects only and/

or screening type experiments will

not result in a robust solution.

SteP eight: evAluAte the Set POintS uSing the deSign SPAceOnce the set points have been

selected, the visualization of the

design space can be generated.

Every DOE can create a design

space. Care needs to be exercised

in understanding and interpret-

ing a design space. The visualiza-

tion of the design space is of the

mean (average) in the response

surface (see Figure 3) relative to

the limits of the CQAs. Many

think that being anywhere in the

white space will achieve a good

result and being in the colored

or shaded area is bad. That is an

incorrect interpretation of the

graph. Just being in the white

area is no assurance that each

batch, vial, or syringe will be

in specification, only that the

average from the process model

will be within the limits. Also

any visualization of the design

space is static; the actual design

space is dynamic depending on

the settings of the other factors.

Only simulation (3) can explore

and evaluate settings within the

design space, examine potential

failure rates, and evaluate the

dynamic nature of the process.

SteP nine: evAluAte the deSign SPAce uSing SimulAtiOnTo simulate batch-to-batch, unit-

to-unit, or vial-to-vial variation

at the set point, Monte Carlo

simulation is used. OOS capabil-

Figure 3: Visualization of the design space.

3

4

5

6

7

8

9

10

11

Titer

Concentration

%HMW

100 125 150 175 200

Load (OD)

NaC

l

NOR Ranges 4.5 Sigma PAR Ranges 6 Sigma PAR Ranges

FactorsSet

Point

% of

Mean

1 Std at

Set PointParameters -3 Nominal 3 -4.5 Nominal 4.5 -6 Nominal 6

Load OD 150.2 5% 5 Load OD 135.20 150.20 165.20 127.70 150.20 172.70 120.20 150.20 180.20

NaCl 10 1% 0.15 NaCl 9.55 10.00 10.45 9.33 10.00 10.68 9.10 10.00 10.90

Temperature 28.6 1% 0.1 Temperature 28.30 28.60 28.90 28.15 28.60 29.05 28.00 28.60 29.20

Flow Rate 21.07 5% 1 Flow Rate 18.07 21.07 24.07 16.57 21.07 25.57 15.07 21.07 27.07

Nominal PPM* 0 2 10

Success Rate 100.0000% 99.9998% 99.9990%

Based on 100,000 simulated purfication runs

*parts per million

Table I: Normal operating range and proven acceptable range evaluation.




ity in PPM should be targeted to

less than 100 for each CQA or

lower. The simulation includes

three key sources of variation:

the mathematical expression or

model from the characterized

product or process; variation of

each factor at the targeted set

point; and the residual variation

not accounted for by the model

(4). The residual variation is the

root mean squared error (RMSE)

f rom the model and includes

the variation from the analyti-

cal method as well as any other

uncontrolled factor when build-

ing the model.

A good understanding of the

process or equipment capability

will aid the developer in build-

ing the simulation (see Figure

4). Normal, truncated, and non-

normal distr ibutions are used

to inject the simulated noise

plus the RMSE and ref lect it

onto the model to predict the

CQA response. The more accu-

rately the variation at set point is

understood, the more accurately

it will reflect OOS release rates of

drug substance or drug product.

An important addition to the

design space is the edge of failure

graph (see Figure 5). The edge of

failure graph is recommended to

visualize the design margin and

failure rates that will occur from

the process in volume. Red dots are

OOS, and green are in specification.

SteP ten: Set nOrmAl OPerAting rAngeS And PrOven AccePtABle rAngeS To evaluate normal operating ranges

(NOR) and proven acceptable ranges

(PAR), the simulation injects varia-

tion at set point, 3 sigma, 4.5 sigma,

and 6 sigma ranges (see Table I) are

typically evaluated for their asso-

ciated PPM. Normal, non-normal,

actual re-sampling from measure-

Figure 4: Simulation using the process model.

Prediction Profler

1000

1300

16001271.53

[1247.31,

1295.75]

2.352.452.552.652.539901

[2.50962,

2.57018]

0

10154.545436

[3.33438,

5.75649]

0.69388

Desi

rab

ilit

y

5

150.20817

Load (OD)

Random

Normal

Mean

SD

150.208

5

28.636767

Temp

Random

Normal

Mean

SD

28.6368

0.15

10

NaCl

Random

Normal

Mean

SD

10

0.1

21.077257

Flow Rate

Random

Normal

Mean

SD

21.0773

1

Desirability

Defect

Titer

Concentration

%HMW

All

Rate

0

0.00001

0

0.00001

PPM

0

10

0

10

M ean

1270.64

2.53998

4.6453

SD

29.8877

0.00643

0.32733

Simulator

Responses

Titer Add Random Noise Std Dev: 1.6329932

Concentration Add Random Noise Std Dev: 0.0020412

%HMW Add Random Noise Std Dev: 0.0816497

N Runs: 100000

Simulate to Table

Spec Limits

Response

Titer

Concentration

%HMW

LSL

1000

2.51

.

USL

.

2.57

8




ments, and uniform distributions

can be used to evaluate PPM rates.

Typically, the limits are set to assure

the CQA PPM failure rates are below

100. Uniform distributions should be

used if processing to range; normal

distributions are typically used when

processing to target; however, other

distributions are possible based on

the product and the problem.

SteP eleven: verify the SmAll-ScAle And At-ScAle reSultSVerification and validation (5)

runs at the robust optimum are

performed to verify the model

prediction and the actual mea-

surements a re in ag reement.

Typical acceptance criteria con-

firm the small- or at-scale mea-

surements are within the 99%

quantile interval from the simu-

lated results. If there is a detected

shift between the small-scale and

at-scale data, the model can be

rescaled/calibrated to match the

at-scale results. Some mechanistic

understanding of the scale differ-

ence is generally recommended

when scale effects are detected.

SteP twelve: define the effective deSign SPAce thAt will Be uSed fOr PrOceSS cOntrOlFinal ly, there is a d i f ference

between the v isual izat ion of

the design space and the effec-

tive design space an applicant

may want to file with the health

authorities. The effective design

space is the region where no

OOS events occur and/or the

applicant will adjust to correct

for processing conditions, raw

material potency, and/or dose

or formulation requirements. In

most cases, the effective design

space is much smaller in range

than the visualized design space.

SummAryKnowing how to complete a risk

assessment and design an appro-

priate experiment are only two

key steps in a series of develop-

ment activities. Knowing how to

complete the development, select

the robust optimum, simulate

potential OOS rates for all CQAs,

determine and evaluate design

margin, find the NOR and PAR

limits, and define and defend the

effective design space are essen-

tial skills that all those that work

in drug development. These skills

should be gained by instruc-

tion and by practical experience

working on drug substance and

drug product and with the health

authorities on filing the design

space and associated control plans.

referenceS 1. ICH, Q8, Pharmaceutical

Development (ICH, November 2005).

2. ICH, Q11, Development and

Manufacture of Drug Substances

(ICH, November 2012)

3. SAS/JMP Software, Profilers and

Simulation, Version 12, May 2015

4. T. Little, Robust Optimization, Design

Space and Tolerance Design (Course

Notes, 2015)

5. FDA, Process Validation, General

Principles and Practices (FDA,

January 2011). ◆

Figure 5: Edge of failure and simulated design margin.

Edge of Failure

1000

110011601220128013401400

2.52.512.522.532.542.552.56257

2.5

3.5

4.5

5.5

6.5

8

144 162 180

Load (OD)

28.3 28.9

Temp

4.65 4.95 5.25

NaCl

22 25 28

Flow Rate

there is a difference

between the

visualization of the

design space and

the effective design

space an applicant

may want.



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NEW BENCH TOP TANGENTIAL

FLOW PERFUSION SYSTEM

The KML™-100 [KrosFlo® MagLev] Benchtop

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1.5 to 100 L working volume bioreactors

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flow-paths. The system uses a single-use, non-

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BIOPHARMACEUTICAL CONTRACT

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Cytovance® Biologics is a

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CELEBRATE PERFORMANCE WITH THE

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CaPure-HA™ from Tosoh Bioscience LLC is for the purification of multiple classes of biomolecules including monoclonal and polyclonal antibodies, antibody isoforms, isozymes, antibody fragments, and the isolation of single-stranded from double-stranded DNA. The highly selective and robust nature of CaPure-HA offers the flexibility to use

this resin at any stage in a process from capture to final polishing. Tosoh Bioscience, LLC, 3604 Horizon Drive, Suite 100, King of Prussia, PA 19406 • www.tosohbioscience.com • tel. 484.805.1219 • [email protected]

New Technology Showcase

PRODUCT SPOTLIGHT

Bioreactor Harnesses

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clarification system

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mammalian cell

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cell densities

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Ultrapure

diatomaceous earth (DE) is available in prefilled

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kg. The system allows DE to be mixed into the cell

culture fluid via a quick-connect adapter for dust-free

powder transfer, while filter blockage is prevented

by a porous filter aid. The system’s fully single-use

process enables harvesting of cultures up to 2000 L.

Sartorius Stedim Biotech

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High-Pressure Homogenizer

Reduces MaintenanceGEA’s Panther NS3006L high-pressure homogenizer is a standalone unit designed to reduce particle size to nanometer ranges for injectable drugs. Continuous operation allows up to 1500 bar for limited productions in pilot plants. The ergonomic unit is made from Super Duplex SS alloy, fitted with wear-resistant parts, and has an analog pressure gauge in a sanitary design with built-in pulsation damper. The unit also has a rupture type, sharp-edge homogenizing valve (Stellite) in special abrasion and wear-resistant material.

GEA Niro Soavi

www.nirosoavi.com


GSK Invests $95 Million in Gene Control ResearchOn June 16, 2015, GlaxoSmithKline (GSK) announced it

would invest $95 million to launch an independent, non-

profit in Seattle, WA. The new company, Altius Institute

for Biomedical Sciences, will research and develop new

technologies and approaches “for decoding how genes are

controlled and how a cell’s ‘operating system’ functions in

health and disease,” according to a press release.

Led by John A. Stamatoyannopoulos, MD, leader in gene

regulation and professor of genome sciences and medicine

at the University of Washington School of Medicine, the

group will be independent from GSK, operating with its own

management, board of directors, and external advisors. Per

the agreement, GSK and Altius have entered a 10-year, $95

million collaboration in which GSK will provide cash and

resources to Altius during the first five years to further the

institute’s research. There is the potential for additional

funding from GSK to apply discoveries from Altius to a range

of projects identified by GSK. The agreement also allows GSK

to “retain first rights to option the Institute’s inventions, and

to invest in commercialization of the discoveries via spinout

companies,” according to a press release. The collaboration

is intended to enable rapid translation of genetics research

technology to the drug-discovery process. The ability to

understand and control a cell’s genes may increase the

probability that a drug will succeed in targeting the right

disease once in late-stage development.

“Dramatic breakthroughs in understanding how the human

genome functions are still in their infancy in terms of how

they can be applied to drug discovery, but we can see their

potential to transform the process. This is not an incremental

change. We are aiming for transformative outcomes that could

improve our ability to bring innovative and more effective new

medicines to patients,” said Lon Cardon, senior vice-president

of Alternative Discovery and Development at GSK, in the

press release.

Penn Announces Gene Therapy Collaboration with WuXiThe University of Pennsylvania (Penn) announced on June 15, 2015 that it had entered into a manufacturing collaboration with WuXi AppTec. The collaboration was developed to combine Penn’s viral vector production with the knowledge of manufacturing and infrastructure from WuXi. Penn will work with WuXi on manufacturing processes within cGMP guidelines for the production of viral vectors in WuXi’s 145,000 ft2 facility at the Navy Yard in Philadelphia, PA.

The Gene Therapy Program at Penn researches gene therapies to develop gene vectors derived from recombinant viruses, with a focus on developing new adeno-associated virus (AAV) vectors, as well as adenovirus and lentivirus research. According to a press release, the “current clinical focus is in the area of orphan and infectious diseases,” and collaboration with academic and biopharmaceutical partners helps with clinical translation of the research.

“Advances in the technology of gene transfer developed at the University of Pennsylvania have ushered in an era of exciting translational research in gene therapy. A critical step toward successful commercialization of these products is the development of processes and analytics for scalable production of vectors,” said James M. Wilson MD, PhD, professor and director of the Gene Therapy Program and the Orphan Disease Center at the Perelman School of Medicine, in a press release.

REGENXBIO and WuXi AppTec Collaborate on Gene Therapy REGENXBIO entered into an exclusive strategic manufacturing collaboration with WuXi AppTec, the companies announced June 11, 2015. REGENXBIO plans to establish efficient, scalable cGMP manufacturing processes for adeno-associated virus (AAV) gene therapy treatments incorporating REGENXBIO’s NAV Technology.

Under the terms of the agreement, REGENXBIO and WuXi will establish a proprietary production process for NAV Technology treatments designed to enable the rapid advancement of REGENXBIO’s and licensees’ therapeutic programs from clinical trials through commercialization in WuXi’s 145,000 ft2 cGMP facility in Philadelphia, PA.


Gene Therapies Development Update

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Ad Index

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COVANCE Cover Tip, 48

CYTOVANCE BIOLOGICS 2, 25

EMD MILLIPORE 7

EPPENDORF NORTH AMERICA 10-11

EUROFINS LANCASTER LABORATORIES 27

GE HEALTHCARE LIFE SCIENCES 5

PATHEON PHARMACEUTICAL SVC INC 15

SAFC BIOSCIENCES SIGMA ALDRICH 47


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Documents

BioPharm International - PharmTechfiles.pharmtech.com/alfresco_images/pharma/2018/09/11/15b7908e … · BioPharm INTERNATIONAL The Science & Business of Biopharmaceuticals EDITORIAL