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BioPharmThe Science & Business of Biopharmaceuticals

INTERNATIONAL

www.biopharminternational.com

INTERNATIONAL

OPTIMIZING RESIN USE TO

ACHIEVE COST-EFFECTIVE

BIOPROCESSING

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March 2016

Volume 29 Number 3

UPSTREAM PROCESSING

BIOPHARMA TAKES

ON RAW MATERIAL

VARIABILITY

PEER-REVIEWED

A RISK-BASED GENETIC

CHARACTERIZATION

STRATEGY FOR RECOMBINANT

CHO CELL LINES

REGULATIONS

GENERIC-DRUG PRODUCTION

AND OVERSIGHT CHALLENGE

FDA AND MANUFACTURERS

COMPLEX SUPPLY, WITHOUT ALL THE CHAOS

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where the challenges are in the supply chain and safeguarding against the disruption

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EDITORIAL

Editorial Director Rita Peters [email protected]

Senior Editor Agnes Shanley [email protected]

Managing Editor Susan Haigney [email protected]

Science Editor Randi Hernandez [email protected]

Science Editor Adeline Siew, PhD [email protected]

Community Manager Caroline Hroncich [email protected]

Art Director Dan Ward [email protected]

Contributing 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

Publisher Mike Tracey [email protected]

West/Mid-West Sales Manager Steve Hermer [email protected]

East Coast Sales Manager Scott Vail [email protected]

European Sales Manager Chris Lawson [email protected]

European Sales Manager Wayne Blow [email protected]

C.A.S.T Data and List Information Ronda Hughes [email protected]

Reprints 877-652-5295 ext. 121/ [email protected] Outside US, UK, direct dial: 281-419-5725. Ext. 121

PRODUCTION

Production Manager Jesse Singer [email protected]

AUDIENCE DEVELOPMENT

Audience Development Rochelle Ballou [email protected]

© 2016 UBM. 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 UBM for libraries and other users registered with the Copyright Clearance Center, 222 Rosewood Dr. Danvers, MA 01923, 978-750-8400 fax 978-646-8700 or visit http://www.copyright.com online. For uses beyond those listed above, please direct your written request to Permission Dept. fax 440-756-5255 or email: [email protected].

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EDITORIAL ADVISORY BOARDBioPharm International’s Editorial Advisory Board comprises distinguished

specialists involved in the biologic manufacture of therapeutic drugs,

diagnostics, and vaccines. Members serve as a sounding board for the

editors and advise them on biotechnology trends, identify potential

authors, and review manuscripts submitted for publication.

K. A. Ajit-Simh President, Shiba Associates

Rory Budihandojo Director, Quality and EHS Audit

Boehringer-Ingelheim

Edward G. Calamai Managing Partner

Pharmaceutical Manufacturing

and Compliance Associates, LLC

Suggy S. Chrai President and CEO

The Chrai Associates

Leonard J. Goren Global Leader, Human Identity

Division, GE Healthcare

Uwe Gottschalk Vice-President,

Chief Technology Officer,

Pharma/Biotech

Lonza AG

Fiona M. Greer Global Director,

BioPharma Services Development

SGS Life Science Services

Rajesh K. Gupta Vaccinnologist and Microbiologist

Jean F. Huxsoll Senior Director, Quality

Product Supply Biotech

Bayer Healthcare Pharmaceuticals

Denny Kraichely Associate Director

Johnson & Johnson

Stephan O. Krause Director of QA Technology

AstraZeneca Biologics

Steven S. Kuwahara Principal Consultant

GXP BioTechnology LLC

Eric S. Langer President and Managing Partner

BioPlan Associates, Inc.

Howard L. Levine President

BioProcess Technology Consultants

Herb Lutz Principal Consulting Engineer

Merck Millipore

Jerold Martin Independent Consultant

Hans-Peter Meyer Lecturer, University of Applied Sciences

and Arts Western Switzerland,

Institute of Life Technologies.

K. John Morrow President, Newport Biotech

David Radspinner Global Head of Sales—Bioproduction

Thermo Fisher Scientific

Tom Ransohoff Vice-President and Senior Consultant

BioProcess Technology Consultants

Anurag Rathore Biotech CMC Consultant

Faculty Member, Indian Institute of

Technology

Susan J. Schniepp Fellow

Regulatory Compliance Associates, Inc.

Tim Schofield Senior Fellow

MedImmune LLC

Paula Shadle Principal Consultant,

Shadle Consulting

Alexander F. Sito President,

BioValidation

Michiel E. Ultee Principal

Ulteemit BioConsulting

Thomas J. Vanden Boom VP, Biosimilars Pharmaceutical Sciences

Pfizer

Krish Venkat Managing Partner

Anven Research

Steven Walfish Principal Scientific Liaison

USP

Gary Walsh Professor

Department of Chemical and

Environmental Sciences and Materials

and Surface Science Institute

University of Limerick, Ireland

4 BioPharm International www.biopharminternational.com March 2016

Contents

BioPharmINTERNATIONAL

BioPharm International integrates the science and business of

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

peer-reviewed technical solutions to enable biopharmaceutical professionals

to perform their jobs more effectively.

COLUMNS AND DEPARTMENTS

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.

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INTERNATIONAL

www.biopharminternational.com

INTERNATIONAL

OPTIMIZING RESIN USE TO

ACHIEVE COST-EFFECTIVE

BIOPROCESSING

March 2016

Volume 29 Number 3

UPSTREAM PROCESSING

BIOPHARMA TAKES

ON RAW MATERIAL

VARIABILITY

PEER-REVIEWED

A RISK-BASED GENETIC

CHARACTERIZATION

STRATEGY FOR RECOMBINANT

CHO CELL LINES

REGULATIONS

GENERIC-DRUG PRODUCTION

AND OVERSIGHT CHALLENGE

FDA AND MANUFACTURERS

Cover: BullStorm/Getty Images; Dan Ward

6 From the Editor

Thought leaders tackle drug shortages and biomanufacturing challenges. Rita Peters

8 US Regulatory Beat

Policy makers debate strategies for promoting access to less costly medicines. Jill Wechsler

10 Perspectives on Outsourcing

The outsourcing market starts 2016 with company expansions, acquisitions, and new offerings Susan Haigney

49 Ad Index

50 Ask the Expert

The auhors discuss how to create a robust CAPA system and how to identify root cause.Susan Schniepp

and Andrew Harrison

BIOPROCESSING

Achieving Cost-Effective

Bioprocesses

Randi HernandezExperts in the field share some best

practices for optimizing process

economics in bio-manufacturing. 14

UPSTREAM PROCESSING

Biopharma Takes On

Raw Material Variability

Cynthia A. ChallenerCollaborative efforts are

underway between suppliers

and drug manufacturers. 20

DOWNSTREAM PROCESSING

Adherent Cell Culture

in Biopharmaceutical

Applications: The Cell-

Detachment Challenge

Marcos Simon and Juan J. Giner-CasaresThe necessity to detach cells from a

culture substrate during cell harvesting

remains one of the most challenging

steps in a cell-culture process. 26

PEER-REVIEWED

A Risk-Based Genetic

Characterization Strategy

for Recombinant CHO Cell

Lines Used for Clinical and

Commercial Applications

Luhong He and Christopher FryeThe authors provide a comprehensive,

risk-based transgene characterization

strategy. 32

DATA INTEGRITY

How Important is Data

Integrity to Regulatory Bodies?

Bob McDowall and Joanne RatcliffData integrity is a widespread, global

problem that must be addressed. 42

COLD CHAIN

Cold Chain:

Going the Extra Mile

Agnes ShanleyReal-time GPS technology, better

IT connections, and more conservative,

controlled shipping temperatures

are improving the shipment of

sensitive pharmaceuticals. 45

Volume 29 Number 3 March 2016

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From the Editor

Thought leaders

tackle drug

shortages and

biomanufacturing

challenges.

Keynote Series Addresses Crucial Industry Issues

At INTERPHEX 2016 at the Javits Center in New York, BioPharm

International and Pharmaceutical Technology will host a Keynote Series on

leading bio/pharma industry issues. Sessions will be presented on the

Innovation Stage in the exhibit hall. Admission is free to any attendee with an

exhibit hall pass. The following is a preview of the topics.

Overcoming Bottlenecks in Biopharmaceutical DevelopmentIn a panel discussion, industry experts will discuss how technology advances

are addressing challenges in biopharmaceutical development including qual-

ity control of raw materials, implementation of single-use technologies, process

monitoring, and downstream purification. The panelists are Marian L. McKee,

PhD, director, US development services, MilliporeSigma; Mike Goodwin, director

of R&D, SUT, Thermo Fisher Scientific; Alex Perieteanu, PhD, director, biophar-

maceutical services-Life Sciences, SGS Mississauga; Elizabeth Goodrich, director

of applications engineering, MilliporeSigma. (Tuesday, April 26, 10:15—11:45 AM)

Contract Services Market: 2016 UpdateHow will consolidation in the bio/pharmaceutical and contract services mar-

ket, a changing financial market, and an active political and regulatory year

shape the fortunes of the contract services market? In his annual presentation,

Jim Miller, founder and president, PharmSource Information Services will

offer his perspectives on the contract services landscape for the next few years.

(Wednesday, April 27, 10:30–11:30 AM)

Strategies and Innovations to Reduce Drug Shortages and Improve

Availability of Medicines Aging facilities and equipment, inadequate operator training, a lack of quality con-

trol, tighter regulatory enforcement, and business decisions to eliminate unprofit-

able product lines contribute to ongoing shortages of vital drug products. In this

session, industry thought leaders will identify triggers for drug shortages, methods

to avoid production line shutdowns and update facilities, and innovative industry

efforts to fulfill demand for needed therapies. (Wednesday, April 27, 1:30–3:15 PM)

An Interdisciplinary Approach to Address Drug Shortages. The effects

of drug shortages on patients, caregivers, hospitals, and medical professionals are

often not observed or understood by the bio/pharmaceutical manufacturing seg-

ment. This presentation will explore alternative, innovative, and cost-effective

ways to provide needed therapies to patients.

BARDA Innovation Initiatives in Medical Countermeasure

Manufacturing. This presentation will describe the Biomedical Advanced

Research and Development Authority’s (BARDA) program initiatives in manufac-

turing technologies for medical countermeasure advanced development, includ-

ing opportunities with the Centers for Innovation in Advanced Development &

Manufacturing and in continuous manufacturing.

Panel Discussion: Addressing Sterile Manufacturing Challenges Sterile injectables have been in extremely short supply, and industry efforts

have been focusing on root causes involving infrastructure, quality, and

efficiency. Experts involved in this work discuss recent initiatives, and offer

insights into what must be done to prevent injectables shortages in the future.

(Wednesday, April 27, 3:30–4:30 PM)

For more information about the Keynote Series, including sessions for

small-molecule manufacturing, visit: www.biopharminternational.com/bp/

Interphex2016. X

Rita Peters is the

editorial director of

BioPharm International.

Let us keep your process development running smoothly.

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Regulatory Beat

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It’s well known that generic drugs account

for 88% of prescription drug sales in the

United States and have saved billions for

patients and healthcare systems since Congress

enacted the Hatch-Waxman Act more than 30

years ago. That growth, though, has created

difficulties for FDA in processing the hundreds

of resulting abbreviated new drug applications

(ANDAs) and in inspecting an expanding num-

ber of generic-drug manufacturers and ingredi-

ent producers all over the world.

Concerns about ensuring the quality and

safety of medical products, moreover, have led

to the closure of outdated and noncompliant

facilities, contributing to shortages and price

spikes for certain widely used generics, particu-

larly sterile injectables. These developments have

raised questions about whether government reg-

ulatory policies limit competition in certain

drug classes and support monopoly pricing.

FEES ADD RESOURCESFDA’s ability to expeditiously approve new

generics was compromised by a budget squeeze

over many years. The Prescription Drug User

Fee program (PDUFA) of 1992 bolstered fund-

ing for new drug review by the Center for

Drug Evaluation and Research (CDER), but also

shifted resources away from generics.

ANDA approvals slowed to a crawl,

resulting in an enormous backlog of

pending applications.

Generic-drug makers finally agreed

to pay user fees in 2012 to strengthen

FDA regulation of generic-drug devel-

opment, review, and inspection. In its

first three years, the Generic Drug User

Fee program (GDUFA I) has generated

nearly $1 billion to support CDER’s

Office of Generic Drugs (OGD) and

certain operations of the new Office of

Pharmaceutical Quality (OPQ). FDA’s

field force also has increased inspections of over-

seas producers to help level the playing field

between US and foreign manufacturers.

As FDA and industry negotiate GDUFA

renewal in 2017, Congressional committees

and the broader healthcare community are

examining FDA policies and programs govern-

ing generics and the prescription drug market,

as seen at a January 2016 hearing before the

Senate Health, Education, Labor and Pensions

(HELP) Committee. Chairman Lamar Alexander

(R.Tenn.) cited concerns about “unnecessary

regulatory burdens” that can slow drug develop-

ment and the importance of a pharmaceutical

marketplace that “remains competitive” (1).

The panel also is developing a Senate version

of the “21st Century Cures” legislation, which

the House approved in July 2015. Instead of

combining multiple proposals into a compre-

hensive bill, Alexander and ranking Democrat

Patty Murray (D-Wash) are considering numer-

ous individual measures on FDA policies, disease

research, and expanded use of electronic data

technology to support broader research goals.

With deliberations running through April 2016,

though, there’s not much chance that Congress

will adopt any final “Cures” legislation this year,

but will wait until 2017 when action is required

to reauthorize FDA user fee programs.

NO MORE BACKLOGA main issue explored at the HELP hearing is

whether too-slow FDA approval of new generics

limits drug access and competition. Some legisla-

tors suggested that a pharma company would be

less likely to buy up a small drug firm with the

intent of boosting product prices if it knew that

FDA could quickly approve a new competing drug.

Despite complaints from generics makers about

still-delayed ANDA approvals, CDER director

Janet Woodcock made a strong case for agency

progress in addressing the backlog problem,

Generic-Drug Production and Oversight Challenge FDA and ManufacturersPolicy makers debate strategies for promoting access to less costly medicines.

Jill Wechsler is BioPharm

International’s Washington editor,

Chevy Chase, MD, 301.656.4634,

[email protected].

March 2016 www.biopharminternational.com BioPharm International 9

Regulatory Beat

speeding important new gener-

ics through the approval process

and expanding timely inspections

of manufacturing facilities. She

explained that generic-drug makers

submitted nearly 2500 applications

in 2013 and 2014, making it difficult

for OGD to process those documents

and to tackle long-pending submis-

sions, while also restructuring and

expanding its program (2).

Even so, in the past three years

CDER was able to “take action” on

approximately 85% of 4600 over-

due ANDAs and post-approval

supplements, Woodcock stated.

She promised that all the backlog

would be gone by 2017 and that

OGD would meet its goal for tak-

ing a “first action” within 10

months on ANDAs submitted this

year. No applications in the backlog

are first generics, she emphasized,

and OGD’s “express lane” policy

moves these products to the front

of the queue. She also highlighted

CDER efforts to promote advanced

manufacturing in the generic-drug

industry, as continuous, computer-

controlled production systems

would enable fast ramp-up of new

production.

Key to achieving these goals is an

FDA-industry effort to achieve more

first-cycle approvals. A “right-the-

first-time” policy permits rejection

of notably incomplete applications

when they first come in. CDER

also is issuing more guidance on

what data it wants from sponsors

and encouraging manufacturers

to conduct all necessary tests and

processes before sending in appli-

cations. A new pre-ANDA process

that addresses approval challenges

for particular drugs prior to applica-

tion submission may be included in

GDUFA II.

SUPPORTING COMPETITIONIn highlighting FDA efforts to

quickly approve first generics,

Woodcock acknowledged that mul-

tiple drugs per innovator may drive

down costs and facilitate patient

access to more affordable therapies.

Yet FDA does not approve a new drug

or generic in response to rising prices,

she noted, and does not have the

expertise to calculate what qualifies

as a “price hike:” would that involve

doubling a price from 10 cents to

20 cents, or possibly raising a list

price by more than 1000%?, she

queried, adding that a new report

from the US Department of Health

and Human Services (HHS) better

addresses generic-drug pricing (3).

The agency does keep a close eye

on sole-source products and those

with only one or two competitors,

as part of efforts to anticipate drug

shortages and supply disruptions.

Woodcock presented data indicating

that 99 innovator drugs have only

one generic competitor; 66 drugs

have two generics; and 623 drugs

have 3 or more generics. Of particu-

lar interest is the segment of 125

innovator drugs with no approved

generics (and no patent or exclusiv-

ity protections) (4).

These drugs may have limited

competition, Woodcock explained,

because they are orphans or spe-

cialized therapies that serve small

patient populations. Many topical

products, inhalants, and complex

substances also lack well-understood

methods for testing and document-

ing bioequivalence. To support the

development of generic versions of

such therapies, GDUFA provided

FDA with approximately $35 mil-

lion for research on new bioequiva-

lence test methods and guidances to

“open up previously blocked path-

ways” for new generics.

Woodcock acknowledged that, in

some cases, innovator firms take

steps to block and delay generic drug

entry. Generic-drug makers have

complained loudly about problems

in obtaining supplies for bioequiv-

alence testing of brand products

that are subject to Risk Evaluation

and Mitigation Strategies (REMS).

Woodcock said that FDA has advised

brand firms that REMS don’t war-

rant withholding drugs for research

purposes, and indicated that

Congressional action would help

address this problem more directly.

The Gener ic Pharmaceut ica l

Association also wants the legisla-

tors to repeal a recent budget provi-

sion that boosts Medicaid rebates

on generic drugs (5).

One strategy Woodcock strongly

opposed is to turn to drug com-

pounders to provide less costly

alternative medicines when gener-

ics fail to meet demand. She

emphasized that there are “very

great risks” in such proposals, cit-

ing two examples of compounded

drugs that sickened dozens of peo-

ple. Mass production of these drugs

without adherence to GMPs, she

warned, “could have put thousands

of people in the hospital.”

REFERENCES 1. US Senate Committee on Health,

Education, Labor & Pensions, “Alexander: Despite Extra $1 Billion to Speed Generic Drug Approvals, FDA Process Still Too Slow,” Press Release, Jan. 28, 2016, www.help.senate.gov/chair/newsroom/press/alexander-despite-extra-1-billion-to-speed-generic-drug-approvals-fda-process-still-too-slow., accessed Feb. 2, 2016.

2. Implementation of the Generic Drug User Fee Amendments of 2012 (GDUFA), Testimony of Janet Woodcock, MD, Before the Committee on Health, Education, Labor and Pensions, Jan. 28, 2016, www.help.senate.gov/imo/media/doc/Woodcock5.pdf, accessed Feb. 2, 2016.

3. Office of the Assistant Secretary for Planning and Evaluation, Understanding

Recent Trends in Generic Drugs, Jan. 27, 2016, https://aspe.hhs.gov/pdf-report/understanding-recent-trends-generic-drug-prices, accessed Feb. 2, 2016.

4. FDA, Slides from FDA GDUFA presentation before the Senate Health, Education, Labor and Pensions Committee, January 2016, www.biopharminternational.com/generic-drug-production-and-oversight-challenge-fda-and-manufacturers-0.

5. GPhA, “GPhA to Congress: Embrace Five Opportunities for More Generic Drug Savings,” Statement by Chip Davis, President and CEO, GPhA, Feb. 1, 2016, www.gphaonline.org/gpha-media/press/gpha-to-congress-embrace-five-opportunities-for-more-generic-drug-savings, accessed Feb. 2, 2016. ◆


Perspectives on Outsourcing

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A survey of drug manufacturers reveals

that cost considerations, as well as

mergers and acquisitions, will con-

tinue to drive the use of outsourced services

(1). Distribution activities was the top function

biopharmaceutical companies will outsource,

followed by packaging and labeling, and drug

product manufacturing.

For the broader pharmaceutical market, the

top five activities that drug companies out-

source are drug product manufacturing,

packaging and labeling, distribution, small-mol-

ecule manufacturing, and holding and storage,

according to Kate Hammeke of ISR Reports (1).

Service providers are expanding their ser-

vices and capabilities to keep up with the

high demand. The following are some exam-

ples of the growth the outsourcing industry is

experiencing.

COMPANY DEVELOPMENTS, EXPANSIONS, AND ACQUISITIONSThe first few months of 2016 has seen an array

of investments, expansions, and acquisitions

in the pharmaceutical outsourcing market.

Outsourcing companies appear to be looking

to the early-phase development and clinical

trial markets to increase their portfolios.

Austrianova completed a new facility, add-

ing GMP cell-banking and fill-and-finish

services for cell therapy products to its encap-

sulation services and technology, the company

announced in a Jan. 25, 2016 press release (2).

Austrianova offers the production of master

cell and working cell banks (MCB and WCB)

at the scale required for Phase I and II stage

clinical trials using its isolator-based produc-

tion facility. The company can also fill bulk

cell product into syringes or vials in its GMP

facility. This new cell banking and

filling service is called GMP4Cells.

MCBs and WCBs are required for

all cell therapy products like stem-

cell therapies and biologics produced from

cells such as vaccines, antibodies, and recom-

binant proteins.

LabConnect, a Seattle-based provider of lab-

oratory services to biopharmaceutical, medical

device, and contract research firms, has built a

new 5000-sq-ft biorepository in Johnson City,

TN, that includes space for ambient, refriger-

ated, cold (-20 °C), and ultra-low temperature

(-70 to -80 °C) storage as well as liquid nitrogen

vapor phase storage (-190 °C) (3).

The facility includes storage capacity for

more than eight million samples, validated

and mapped backup freezers and generators,

redundant HVAC systems, building and biore-

pository security systems, and a temperature

monitoring system for freezers and refrigera-

tors with a 21 Code of Federal Regulations Part 11

compliant audit trail. LabConnect also tracks

sample locations and consolidates data within

a centralized database.

Catalent Pharma Solutions announced on

Feb. 2, 2016 (4) an investment of $4.6 million

to expand its Singapore clinical supply facility

by building GMP space for secondary packag-

ing. The investment doubles the ambient stor-

age space and quadruples cold-storage capacity,

the company reports.

The site provides clinical supply services

including project and supply-chain manage-

ment, comparator sourcing, clinical label print-

ing, secondary packaging, clinical storage,

import/export management, importer of record

service, and returns and destruction manage-

ment services. It has served as a regional hub

for studies in Australia, Singapore, Korea, Hong

Kong, and other countries in Southeast Asia.

SGS, a bio/pharmaceutical analytical and

bioanalytical contract solutions provider,

announced on Jan. 19, 2016 that after the acqui-

sition of Quality Compliance Laboratories in

December 2015, its global network now offers

chemistry and microbiology testing services

Biopharma Outsourcing Market Expands The biopharmaceutical outsourcing market starts 2016 with company expansions, acquisitions, and new offerings.

Susan Haigney is managing editor of


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Perspectives on Outsourcing

for cosmeceuticals, natural health

products, and medical marijuana

from facilities in Canada (5).

New capabilities within the

two facilities include an induc-

tively coupled plasma-mass spec-

trometer and inductively coupled

plasma-optical emission spec-

trometer for elemental impurities

analysis, automated tablet disso-

lution apparatuses, flow-through

USP dissolution apparatus, and

Vitek for bacterial identification.

Vet te r a nnounced on Ja n.

28, 2016 that the company’s

Schuetzenstrasse multi-functional

building in Ravensburg, Germany,

has been completed on schedule

and departments crucial to its

operation have started to move in

(6). The $32 million (€29 million)

investment is part of a $331 mil-

lion (€300 million) total invest-

ment strategy announced by the

company in September 2015.

The continued demand by large

and small customers for enhanced

drug development services, as well

as the need for ever-more future-

oriented sophisticated IT systems

to protect their data, created the

need for the new facility, accord-

ing to the company.

The 91,500-sq-ft, six-story build-

ing contains non-cGMP labora-

tories for development support,

laboratory space for microbiologi-

cal analysis, office workplaces for

Vetter Development Service and

IT, and a data processing center

with enhanced security systems,

including a safety cell that protects

technology and data from external

physical hazards in the event of an

emergency.

Novasep is bui lding a new

synthesis laboratory and adding

kilogram-scale production at its

existing US facility in Boothwyn,

PA (7). This extension will allow

Novasep to offer both chemistry

and purification services and to

produce the initial kilogram-scale

batches of synthetic molecules

that are needed for biological test-

ing and preclinical trials.

The new laboratory, equipped

with reactors up to 50 L in size,

will start operation in May 2016.

It will offer cryogenic capacities

and standard chemistry, as well as

preparative purification chroma-

tography processes.

PBOA EXPANDS MEMBERSHIPT he Pha r ma & B iopha r ma

Outsourcing Association (PBOA),

founded in 2014, has been

advocating for the pharma out-

sourcing industry as the global mar-

ket changes and expands. “We’re

focused on working on the reautho-

rization of the Generic Drug User

Fee Amendment (GDUFA), while

keeping an eye on FDA’s Quality

Metrics initiative, and helping make

sure that CMO/CDMOs [contract

manufacturing organizations/con-

tract development and manufactur-

ing organizations] are prepared for

track-and-track/serialization regula-

tions as they roll out in the United

States and the European Union

in the next few years,” says PBOA

President Gil Roth.

I n Febr u a r y 2 016 , PB OA

expanded its membership (8). IDT

Biologika and Ei SolutionWorks

joined the PBOA as general mem-

bers; 3M Drug Delivery Systems

(DDS) joined as a sustaining mem-

ber. Diego Romeu, manufacturing

and supply chain director at 3M

DDS, was also voted to a three-year

term on the board of trustees, along

with Rajan Puri, director of business

development at Therapure, and Lee

Karras, CEO of Halo Pharmaceutical.

“As we continue our mission to

represent the CMO/CDMO indus-

try before FDA, Congress, and other

stakeholders, it’s critical that we

increase our membership and pro-

vide a true voice for our industry,”

said Roth. “We’ve been successful in

bringing the CMO/CDMO perspec-

tive to issues such as GDUFA, quality

metrics, and serialization, and we’re

delighted to bring in new member

companies and add fresh points of

view to our Board of Trustees.”

REFERENCES 1. A. Shanley, “Surveys Examine

Outsourcing Trend,” Pharmaceutical

Technology, Supplement: Partnerships

in Outsourcing, 40 (13), pg 32-33,

www.pharmtech.com/surveys-

examine-outsourcing-trend

2. BioPharm Editors, “Austrianova Offers

GMP Cell Banking and Fill/Finish

Services,” BioPharmInternational.com,

www.biopharminternational.com/

austrianova-offers-gmp-cell-banking-

and-fillfinish-services-0

3. LabConnect, “LabConnect Builds New

Biorepository, Expands Services,

Offers Absolute Sample Protection,”

Press Release, Feb. 3, 2016, www.

labconnectllc.com/Documents/

New%20Biorepository%20PR%20

22016%20-%20New%20

Biorepository%20Expands%20

Services%20Offers%20Absolute%20

Sample%20Protection.pdf, accessed

Feb. 16, 2016.

4. Catalent, “Catalent Invests $4.6M to

Further Expand Asia-Pacific Clinical

Trials Hub in Singapore,” Press

Release, Feb. 2, 2016, www.catalent.

com/index.php/news-events/news/

Catalent-Invests-4.6M-To-Further-

Expand-Asia-Pacific-Clinical-Trials-Hub-

In-Singapore, accessed Feb. 16, 2016.

5. SGS, “SGS Announces Expansion and

Integration of Chemistry &

Microbiology Testing Offer Following

Canadian Acquisition,”Press Release,

Jan. 19, 2016, www.sgs.com/en/

news/2016/01/sgs-announces-

expansion-and-integration-of-

chemistry-and-microbiology-testing,

accessed Feb. 16, 2016.

6. Vetter, “Vetter Announces Completion

of Multi-Functional Building for

Development Service and State-of-the-

Art IT,” Press Release, Jan. 28, 2016,

www.vetter-pharma.com/en/

newsroom/vetter-news/news-l-vetter-

announces-completion-of-multi-

functional-building-for-development-

service-and-state-of-the-art-it,

accessed Feb. 16, 2016. 7. Novasep, “Novasep Adds Synthesis

and Kilo Lab Extensions at US Facility,” Press Release, www.novasep.com/home/about-novasep/media-events/press-release/novasep-adds-synthesis-and-kilo-lab-extensions-to-us-facility.html, accessed Feb. 16, 2016.

8. PBOA, “PBOA Welcomes New Members and Trustees,” Press Release, Feb. 10, 2016, www.pharma-bio.org/news/pboa-welcomes-new-members-and-trustees/, accessed Feb. 16, 2013. ◆

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

infoAsiaPacifi [email protected] Q For EU and other international inquiries, please contact [email protected]

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Although many of the newest

methods to optimize process

economics focus squarely on

the implementation of single-

use systems and the realization of contin-

uous processing, there are other methods

of cost cutting that could help a biopro-

cess become more sustainable. Operators

should look to methods to optimize the

use of solvents, get the most from their

resins, explore hybrid approaches and

miniature bioreactors for improved pro-

cess understanding, and investigate alter-

natives to mammalian vectors to improve

cost calculations.

OPTIMIZATION OF MEDIA VOLUMESResin prices can range from low single-

digit thousands of dollars per liter for ion

exchangers to tens of thousands of dol-

lars per liter for affinity resins, estimates

Kevin Isett, CEO and founder of Avitide,

which makes high- affinity resins. The

costs associated with chromatography

resins can significantly contribute to

overall manufacturing costs, especially

if two or three chromatographic steps

are required in a bioprocess, according

to Alex Xenopoulos, principal research

scientist at Mil l iporeSigma. Isett

explains that resin manufacturers his-

torically demanded a “premium” price

for resins that could withstand rigor-

ous cleaning and sanitation conditions

and facilitated a better unit economy

by allowing manufacturers to use less

expensive sanitization buffers. According

to Cobra Biologics’ Technical Director

Tony Hitchcock, cost has to be delicately

balanced with overall purity achieved,

Achieving Cost-Effective Bioprocesses

Randi Hernandez

Experts in the field share

some best practices for optimizing

process economics

in bio-manufacturing.

Bioprocessing

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binding capacities, and the num-

ber of cycles that can be performed

with the resin.

One strategy to lower the over-

all cost of bioprocessing is to mini-

mize resin-related costs. “For a

large commercial process produc-

ing one ton of antibody per year,

chromatographic resins contribute

about 10% of the downstream cost,

while filters contribute up to 30%,

because of the single-use nature

of depth, virus, and sterile filters,”

notes Xenopoulos. “For a smaller,

single-use process producing 50 kg

of antibody per year, the percentage

of cost contributed by chromato-

graphic resins goes down to a few

percentage points, because capital

and labor now constitute a larger

fraction of the total cost.”

Rathore et al. estimate that in a

typical monoclonal antibody (mAb)

platform process, 60% of down-

stream costs come from chroma-

tography (1); the cost of a typical

Protein A resin is 50% higher than

the cost of traditional chromato-

graphic media. Although Gjoka et

al. agree that economic optimiza-

tion of chromatography is critical,

these authors estimate that Protein

A resin is closer to “an order of mag-

nitude more expensive than other

non- affinity sorbents (including

most ion exchangers)” (2).

The switch to single-use systems

has been highly cited as a way to

reduce costs in biomanufacturing.

Although Xenopoulos mentions

that the use of disposables generally

increases the consumable compo-

nent of the cost of bioprocessing,

single-use chromatography devices,

such as prepacked columns and

membrane adsorbers, could offer

overall “cost advantages because of

the elimination of column pack-

ing and media washing and stor-

age steps.” Hitchcock points out

that at smaller processing volumes,

single-use products can reduce the

amount of liquids that are typically

used in cleaning operations.

Resin reuse

Cycling of resins can occur, and

resin reuse typically lowers the

cost burden of a process. A resin

can be used for up to 200–300

cycles, and smaller columns are

typically used to facilitate resin

reuse. Validating even more reuses

of resins can reduce costs further,

says Xenopoulos, although he says

that the benefit above 50 reuses is

quite small and the cost of valida-

tion should be taken into account.

Not all resins have the same num-

ber of reuses, adds Isett: “Ligand sta-

bility, resin matrix, and chemistries

employed to immobilize the ligands

are key determinants to the reusabil-

ity of any bioprocess resin.”

Reusing resin, although poten-

tially cost-effective, increases

total purification time, decreasing

throughput (1). Long-term use of a

resin has also been associated with

a resin’s decreased efficiency in

terms of product recovery as a result

of resin fouling, ligand degradation,

or reduction in pore surface area.

Using a simple depth filter before

loading, notes Xenopoulos, can

reduce the incidence of resin foul-

ing and maximize the number of

potential resin reuses.

Improving resin selectivity

Improving resin selectivity could

potentially help operators eliminate

a downstream chromatographic

step entirely, says Xenopoulos, or at

the least, reduce the load on down-

stream steps. It’s a double-edged

sword, however: although selecting

a resin with a high binding capac-

ity can limit the amount of resin

used, “such high-performance res-

ins usually have a higher per-liter

cost,” asserts Xenopoulos. Newer

separation technologies may play

a major role in improving process

economics, notes Isett. “Identifying

highly selective, product-specific

resins can enable reduced purifica-

tion unit operations, afford efficient

step-elution schemes, and permit

flow-through polish applications,

which will ultimately have the larg-

est beneficial impact on resin usage

and media/buffer volume costs in

batch and continuous downstream

processing.”

Finally, other small changes in

a bioprocess (e.g., proper selec-

tion of wash and elution buffers to

maximize product recovery, opti-

mizing solution conditions such

as dilution or pH changes, using

f low-through conditions, and

potentially, overloading a column

beyond nominal binding capacity)

can reduce resin volume and could

possibly improve the performance

of some of the chromatographic

steps, says Xenopoulos.

Additionally, some manufactur-

ers use in-line mixing and dilution

of buffer components, which could

help reduce the amount of media

used in downstream processing,

notes Benoit Mothes, scientific and

innovation downstream processing

head at Sanofi in France. Mothes says

that regarding buffers, adoption of

“in-line dilution and in-line concen-

tration will be the next improvement

to reduce the amount of media in

downstream processing.”

Continuous operations

Harnessing continuous downstream

operations has been cited as a good

way to both improve the utiliza-

tion of chromatography resins and

decrease the demand on filters (3,

4). Multi-column systems, in par-

ticular, allow resins to enjoy the lon-

gest lifespan (3). Adds Xenopoulos,

“Continuous, multi-column chroma-

tography has certainly been shown

to reduce resin volume used by sig-

nificant amounts, up to 80%. The

benefits depend on factors such

as product titer, batch time, and

cycling schedule and are higher

when the resin is reused multiple

times during cycling.”

Klutz et al. found that in upstream

operations for the manufacture of

mAbs, however, continuous pro-

Bioprocessing


Although not all products normally produced in mammalian

cells can be produced in microbial vectors—as the

glycosylation step necessary for antibody-dependent

cell-mediated cytotoxicity does not occur in microbial

cells—fragments of monoclonal antibodies (mAbs) can

successfully be produced in microbial vectors, such as

Saccharomyces cerevisiae, Pichia pastoris, and Escherichia

coli (1). Although certain post-translational modifications

such as glycosylation don’t currently occur in microbial

models, a few glycoengineering experiments with the genes

in P. pastoris vectors have focused on the humanization of

the N-glycosylation pathway (2, 3). If successful, this gene-

editing venture would allow for the production of full-length,

glycosylated mAbs in P. pastoris (2).

The use of microbial vectors offers manufacturers a way

to avoid costly perfusion operations and hard-to-model

mammalian cell metabolism conditions (2). Mammalian cells

are also highly susceptible to shear stress and are typically

associated with low product yields, risk of viral contamination,

and a requirement for animal-based serum (2).

Microbial models, on the other hand, have historically

been associated with low cultivation costs (2), low cell-

culture media costs, and high yields. Rathore points out that

although some microbial vectors (e.g., E. coli) contain some

endogenous proteases that can cause protein degradation,

the use of protease-free strains and secretion of proteins

in the periplasm—where there are fewer proteases—have

been methods used to account for potential degradation

issues. In contrast with more traditional microbial models

that resulted in inclusion bodies—which produced significant

host-cell impurities—newer microbial fermentations

produce “purer product compared with mammalian cell

culture,” says Alex Xenopoulos, principal research scientist at

MilliporeSigma. Still, he says, secreted systems “suffer from

low productivity compared with inclusion body systems.” If

the biological product is not secreted, says Kevin Isett, CEO

and founder of Avitide, then “the feed streams can contain

considerable higher host-cell protein levels when compared

to mammalian or insect feed streams,” which can put a

“significant strain on downstream purification operations,

particularly the capture step.”

Yeast vectors are beneficial in that they have high yields

and are capable of post-translational modifications. There

are many ways to optimize microbial vectors through

glycoengineering so that they produce proteins that function

properly, according to experts in the field (1, 2).

Despite the fact that microbial models offer some

exciting production alternatives that could slash costs,

Xenopoulos believes that the ability for mammalian cells to

produce proteins with complex secondary structures, their

continuously increasing titers, and the fact that they are

already well understood outweighs all of the benefits of

microbial models. Ultimately, concludes Xenopoulos, “the

selection of a production system really depends on the

structure of the desired protein, with cost being secondary

considerations.” Summarizes Cobra Biologics’ Technical

Director Tony Hitchcock, “ The perceived advantage of

microbial systems are around costs of fermentation media

and processing times, and this has to be balanced out

against more complex and capitally intensive recovery

operations.”

References1. A. Rathore and J. Batra, BioPharm Int. 29 (2),

pp. 18–23(February 2016).2. O. Spadiut et al., Trends Biotechnol. 32 (1),

pp. 54–60(January 2014).3. J.L. Corchero et al., Biotechnol. Adv. 31, pp. 140–153 (2013).

– Randi Hernandez

Bioprocessing

Microbial Vectors for the Production of mAbs

cesses use more fermentation

media for perfusion than did fed-

batch operations (84 pounds per

grams mAb vs. 59 pounds per

grams mAb, respectively), making it

more expensive to use continuous

processes upstream. Downstream

continuous operations, neverthe-

less, were more efficient from a cost

perspective—which the authors

attributed to better utilization of

chromatography resins in down-

stream operations. Plus, using fed-

batch operations upstream instead

of continuous perfusion did not

affect overall yield, according to

Klutz and his colleagues: “In all

cases, fed-batch fermentation is

more cost efficient than the perfu-

sion fermentation at the same level

of cell-specific productivity.” Klutz

et al. concluded that a hybrid

approach—consisting of fed-batch

operations upstream and continuous

chromatography downstream—was

the most cost-effective model, corre-

sponding to a 15% reduction in cost

of goods (CoGs) (3).

As a whole, it appears a hybrid

approach uses the least amount of

media. Although hybrid methods

“lack the elegance of completely con-

tinuous templates,” according to

Xenopoulos, they do address cost

pressures related to high cell-culture

media usage.


Bioprocessing

Although the aforementioned

study by Klutz el al. did not find

an increase in productivity asso-

ciated with continuous perfusion,

other experts in the field say that

the productivity increase out-

weighs the amount of media con-

sumption in continuous upstream

operations. “Continuous process-

ing provides clear advantages in

terms of space-time-yields,” says

Christel Fenge, vice-president of

marketing and product manage-

ment, fermentat ion technol-

ogy, Sartorius Stedim Biotech.

In other words, “the amount of

material produced per unit time

and volume is higher compared

with conventional fed-batch pro-

cesses.” Xenopoulos concurs that

while continuous perfusion uses

more media—which he estimates

can total 1–10 bioreactor volumes

per day—he says the increased

cost is “somewhat mitigated” by

the increased productivity, as mea-

sured by the cost per gram of prod-

uct. Scott Waniger, vice-president

of bioservices at the Cell Culture

Company, also asserts that gener-

ating greater production “makes

up for the additional raw material

consumption of relatively inexpen-

sive liquid tissue culture media.”

To control overall costs, “there

is also an effort to reduce the cost

of the media itself, with develop-

ment efforts directly targeted to

perfusion cell culture media,” notes

Xenopoulos. “Biomanufacturers

usually work with media vendors,

but sometimes invest in internal

development of media, showing

the importance of this aspect.” In

an attempt to reduce cell-culture

media volume used, some compa-

nies opt to use richer media, which

Xenopoulos says can unfortunately

also increase media cost per liter.

An additional strategy to optimize

media consumption, says Waniger,

is to perform an offline analysis

of spent media to characterize

the consumption and production

of media elements. “The resulting

information allows for identifica-

tion of the components that are

limited, and have been consumed

by the cell line,” Waniger articu-

lates. “With this knowledge, the

operator can add concentrated

amounts of nutrients as a supple-

ment to replace any specific ones

that have been exhausted.”

Buffer recycling

Jungbauer and Walch write that

buffer recycling positively impacts

process economics (5). The reuse

of buffers could decrease waste

streams and save money that

goes into wastewater treatment

efforts. The authors suggest the

use of multi-column separations,

integrated continuous counter-

current chromatography, and

countercurrent tangential f low

chromatography as effective ways

in which buffers can be recycled.

They note that continuous pro-

cesses and the introduction of filtra-

tion into chromatography systems

would allow for a substantial reduc-

tion in solvent consumption.

Unlike resins and other solvents,

expert consensus is that buffers

don’t significantly contribute to

overall biopharmaceutical manu-

facturing costs. “Cost savings of

recycling would most probably

be countered by the cost of the

recycling equipment and by the

need to validate and test the recy-

cled buffers,” notes Xenopoulos.

“Buffer recycling could address

environmental concerns of dis-

posal, especially if a particularly

exotic buffer is used.” Hitchcock

adds that the operation and vali-

dation challenges associated with

buffer recycling may not make it

“ worthwhile for a majority of pro-

duction processes.”

Rather than focus on salvag-

ing buffers, Sanofi’s Accelerated

Seamless Antibody Purification

(ASAP) platform focuses on avoid-

ing the use of nonvaluable buffers

as a technique to reduce down-

stream purification costs. “Most

of purification processes include

a minimum of three chromato-

graphic steps made in a sequence

of distinct unit operations,” says

Mothes, who runs the ASAP pro-

gram in France. He says that unit

operations cannot normally be

operated in a continuous mode, “as

adjustment of pH, molarity, and

protein concentration are neces-

sary between each chromato-

graphic or filtration step.” The

ASAP platform, however, elimi-

nates the need to perform what

he calls these “non-added-value

unit operations” and shortens pro-

cess cycle time to less than three

hours. Use of the system facilitates

a reduction in the buffer volumes

that are necessary for processing,

notes Mothes. While a typical

mAb purification relies on a total

of nine buffers, the ASAP model

requires only four. Mothes adds

that Sanofi’s process will “provide

an entirely purified mAb in a few

hours while reducing the volume

of resin used” and will enable

the reduction of buffer volumes

because of the platform’s small col-

umns and singular process skid.

OTHER FACTORS THAT INFLUENCE COSTAdditional costs related to biophar-

maceutical manufacturing can be

tied to events that occur after an

actual product is manufactured.

These factors can relate to drug

safety, speed to clinic, and time to

market, notes Fenge. Hitchcock esti-

mates that 80% or more of costs

are locked up in the manufactur-

ing design of a product; therefore,

“understanding the implications of

choices within the development

phase” and manufacturing pro-

cesses is key. He adds, “Once these

choices have been made, there is

often only a limited amount of

cost reductions that can really be

achieved.”


Bioprocessing

Miniature Bioreactors for Improved Process Understanding

Better process understanding has the potential to reduce

cost, and this is increasingly being achieved through the

use of miniature and microbioreactor systems. Christel

Fenge, vice-president of marketing and product management,

fermentation technology, Sartorius Stedim Biotech says

the miniature systems and their corresponding single-use

vessels “more precisely mimic larger-scale bioreactors” in

terms of stirring, gas, and pH control. Citing work by Lewis et

al. (1), Fenge comments that “these systems more accurately

predict cell-line productivity at larger scale, ensuring that

high-productivity cell lines (that also work at scale) are

selected at an early stage.” Multiparallel systems that

incorporate automated feeding and sampling have also driven

process efficiencies and allowed for speedier clone selection

and initial parameter selection, Fenge adds. “In combination,

these micro- and mini-bioreactor systems provide efficiency

gains as a result of automated high-throughput cell line,

media, and process development that can enable a faster

throughput of pipeline projects and allow larger experiment

designs, leading to a wider process understanding.”

Process efficiencies equal cost savings, says Cobra

Biologics’ Technical Director Tony Hitchcock, as reduced

development times, improved productivity, and enhanced

characterization time allow products to be brought to

clinical stages and the market more quickly. Scott Waniger,

vice-president of bioservices at the Cell Culture Company

stresses that cost-savings also come in the form of a

decrease in validation efforts. “As long as the miniature

bioreactors are scalable, the savings obtained from reduced

validation as the process scales up are drastically high,”

Waniger notes. “Using a system in which the cell-occupied

space does not change in form, fit, and function—and can

be scaled up with the insertion of more cell spaces in a

parallel manner—helps generate data that can be directly

extrapolated from small to large scale and prevents costly

and time-consuming revalidation at each step.”

Reference1. G. Lewis et al., Bioprocess J. 9 (1), pp. 22–25 (2010).

– Randi Hernandez

Fenge mentions that incorpo-

rating a fully integrated upstream

platform early—combined with a

similar downstream strategy—can

help reduce development-related

costs. “By selecting tools that are

efficient in their own right but that

have also been designed to work

together can significantly reduce

the resources and time needed to

develop processes and can ensure

low cost of goods in manufactur-

ing with reliable high productiv-

ity and consistent product quality.

For example, by deciding to use an

expression platform with a track

record of high-productivity cell

lines that has a proven performance

from small- to large-scale bioreac-

tors, the risk of delays and high

costs associated with determining

optimum process parameters and

control strategies are considerably

reduced, and a rapid path into [the]

clinic is provided.”

Costs re lated to outsourc-

ing must also be managed, and

Waniger suggests taking extra mea-

sures to ensure that the technol-

ogy transfer, method performance,

and manufacturing process are all

addressed in the original request

for proposal (RFP). “CDMOs [con-

tract and development manu-

factur ing organizat ions] may

unintentionally add costs to proj-

ects where the RFP does not fully

describe the needs of the manu-

facturing process,” Waniger says.

“Ultimately, these costs are passed

on to the patient.” Waniger also

suggests conduct ing compre-

hensive stability studies on the

final product, which he says will

maximize product shelf life and

reduce the frequency of batch pro-

duction.

“The pace of discovering novel

drugs and engineering highly

p ro duc t ive b iopha r mace ut i -

cal production systems, in large

par t, has outpaced the eng i-

neer ing of high-per formance

separation ligands and resins,”

concludes Isett, who bel ieves

that the industry should put an

increased focus on optimizing

downstream operations to make

biopharmaceutical manufactur-

ing more cost-effective overall.

“This is particularly true for vac-

cine and biosimiliar companies,

where sensitivities to manufac-

turing costs/pressures are more

pronounced.”

REFERENCES 1. A. Rathore et al., BioPharm Int. 28 (3),

pp. 28–33 (March 2015).

2. X. Gjoka et al., J. of Chrom. A 1416, pp.

38–46 (Oct. 16, 2015).

3. S. Klutz et al., Chem. Eng. Sci. 141, pp.

63–74 (2016).

4. A. Xenopoulos, J. Biotechnol. 213, pp.

42–53 (2015).

5. A. Jungbauer and N. Walch, Curr. Opin.

Chem. Eng. 10, pp. 1–7 (2015). ◆


Ted H

oro

witz/F

use/G

ett

y Im

ag

es

Raw material variability can

have impacts across the board,

causing inconsistent processes

and process yields, produc-

tivity/efficiency issues, and problems

with quality compliance. Any one of

these problems can ultimately impact

drug efficacy and patient safety. While

increased outsourcing of raw material

manufacturing to emerging regions can

contribute to greater issues regarding

raw material consistency and trace-

ability, overall raw material quality

and reliability has increased in recent

years. At the same time, the sensitiv-

ity of analytical instruments and the

awareness of the impact of raw material

variability on biologic drugs have also

increased, leading to a need for further

definition. Raw material suppliers and

biopharmaceutical manufacturers both

have roles to play in addressing this

issue. The key to success will be open

and transparent communication.

MANIFOLD AND SIGNIFICANT IMPACTSVariability in raw materials from media

to packaging can have an impact on the

characteristics and quality of drug prod-

ucts that potentially impact safety and

efficacy. More specifically, lot-to-lot vari-

ability of raw materials may impact drug

product critical quality attributes (CQAs)

such as identity, purity, quality, and sta-

bility and cause lot-to-lot variability of

the drug product during its lifespan,

according to Nataliya Afonina, presi-

dent of AN Biologics Consulting. “Such

variability may ultimately lead to out-

Biopharma Takes On Raw Material Variability

Cynthia A. Challener

Collaborative efforts are underway between suppliers

and drug manufacturers.

Cynthia A. Challener, PhD,

is a contributing editor to


Upstream Processing

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of-specification (OOS) results for

drug products and, in addition to

impacting patient safety, affect the

drug product clinical or commer-

cial supply chain and regulatory

submissions, which are all signifi-

cant consequences,” she asserts.

“The impact of raw material

variability can indeed be mani-

fold and very significant,” agrees

Raphael Gübeli, technical product

manager for liquid formulation at

Merck KGaA. Some effects, such

as reduced overall yield of the bio-

pharmaceutical manufacturing

process, mainly have a negative

economic impact. Unexpected

raw material impurities can, in

some cases, chemically alter the

drug substance or remain in the

final formulated drug product at

elevated levels, potentially affect-

ing efficacy and safety. “If not

detected during final release test-

ing by the manufacturer, there

could be harmful consequences for

the patient,” Gübeli says.

In some cases, minute quantities

of an impurity can have a measur-

able effect. “For example, increas-

ingly more information is being

gained on the effect of variable

elemental metal impurities on cell

health and the yield of cell-culture

processes. As more knowledge is

gained, it has become clear that

even at extremely low levels, a pro-

cess can still be impacted by the

presence of metal or other impuri-

ties,” observes Gary Perkins, head

of process solutions customer rela-

tions in the quality services group

of MilliporeSigma, the life-science

business of Merck KGaA.

Consequently, all raw materials

used to manufacture the API, plus

any excipients and other materials

used during formulation, must be

fully characterized and monitored

for changes in quality/properties.

Packaging materials must also be

monitored, as unexpected leach-

ables or other impurities can also

have an effect on product quality.

BETTER QUALITY, BUT GREATER SENSITIVITYConcerns raised about raw mate-

rial variability are not necessarily

due to a decline in raw material

quality, say the experts inter-

viewed for this article. There have

been some issues with increasing

raw material variability due to out-

sourcing of raw material manu-

facture to China, India, and other

developing countries and the use

of repackagers, which can reduce

transparency in the supply chain,

according to Afonina. She notes

that audits of even reputable ven-

dors must encompass the complete

chain of suppliers and repackagers.

In general, raw material vari-

ability hasn’t changed in recent

years, but analytical capabili-

ties have improved significantly,

increasing the ability of both sup-

pliers and drug manufacturers to

analyze, characterize, and under-

stand that variability, according

to Perkins. These improved capa-

bilities are a double-edged sword.

“We encounter more variability as

our ability to measure and analyze

it is enhanced. In fact, variabil-

ity that was previously invisible

can now be identified, pushing

manufacturers to further stream-

line processes to mitigate potential

impacts of even the slightest varia-

tions,” Perkins explains. He adds

that as detection limits continue

to reach ever lower levels, the

challenge becomes standardiza-

tion, as it often does in the phar-

maceutical industry.

It is also important to note,

according to Gübeli, that expec-

tations for raw material consis-

tency and control of processes

and final product quality have

increased dramatically, and thus

the variability has been reduced

in many cases. Parag Kolhe, group

leader and senior principal sci-

entist with Pfizer Biotherapeutic

Pharmaceutical Sciences, agrees

that raw-material suppliers are pro-

viding consistently better quality

materials. He provides one example

in the primary packaging space;

prefilled-syringe manufacturers

have acknowledged the sensitivity

of biotech products toward silicone

oil and tungsten and improved

their processes so that their pri-

mary packaging component specs

are more tightly controlled.

Raw materials such as formula-

tion excipients, however, which are

present in drug products in high

amounts, have received much less

attention than active biopharma-

ceutical ingredients, according to

Gübeli. “Many parameters defining

the quality aspects of excipients

are not yet routinely monitored or

included as part of supplier certifi-

cates of analysis, and thus are not

strictly under control,” he states.

MANY SOURCES OF VARIABILITYExcipients are just one of many

potential sources of raw material

variability that can impact bio-

pharmaceutical manufacturing. In

general, variability can arise from

inefficient/ineffective raw material

manufacturing controls and resid-

ual impurity controls in starting

materials. They can come from the

basic starting materials (e.g., nat-

ural sugars and other plant-based

compounds) used to manufacture

biopharmaceutical raw materials or

Upstream Processing

In general,

raw material variability

hasn’t changed in

recent years, but

analytical capabilities

have improved

significantly.


Upstream Processing

be an artifact of the manufacturing process itself (e.g.,

impurities in recycled solvents, chemicals released into

the raw material process fluid from the equipment, etc.).

The most common types of raw material variability

can be placed into three general categories, accord-

ing to Gübeli. The first group includes trace impuri-

ties that alter the biopharmaceutical API, either by

directly modifying it or by catalyzing its modification.

Examples include peroxides, aldehydes, reducing sug-

ars, and catalytically active metal ions. The second

category comprises trace impurities that are them-

selves toxic to humans, such as lead and aluminum.

The third class consists of microorganism contami-

nants (and their associated endotoxins) that lead to

variabilities in the bioburden of raw materials and can

cause severe immunological responses in patients.

As mentioned previously, raw material variability

often occurs due to inadequate control of raw mate-

rial manufacturing processes and/or analytical release

testing by suppliers, and is most common at small

outsourcing companies, according to Afonina. Specific

problems to watch for include contamination with

antibiotics or other foreign raw materials due to a lack

of appropriate segregation of processing/handling facil-

ities, equipment, or control of processes used for the

manufacture of raw materials; poor GMP and analyti-

cal practices resulting in the approval of out-of-specifi-

cation material; switching of suppliers for a given raw

material; and deficiencies in the auditing of raw mate-

rials suppliers by biopharmaceutical manufacturers.

SUPPLIER RESPONSIBILITIESVendors are responsible for controlling/minimizing

raw material variability, according to Kolhe. They must

control their own manufacturing processes and audit

manufacturers of any raw materials they purchase. If

manufacture of these materials is outsourced, they need

to audit all facilities in the supply chain. If changes are

made to manufacturing processes, comparability stud-

ies must be performed and the customer notified. “All

these actions are important for all raw materials, but

specifically for animal- and plant-derived materials for

which properties are difficult to control,” says Afonina.

“Overall,” she adds, “strong risk-based management

and quality systems should be in place.”

Even repackagers must have the ability to trace spe-

cific raw material lots back to any changes that were

made at their suppliers. “If a client asks a supplier for

more information in order to investigate batch-to-

batch variability in raw material quality, the supplier

should be able to provide supply-chain documenta-

tion, including origins and analysis. If the answers

are not there, more analysis should be done on the

material,” Perkins says. All these key points should

be covered in the quality agreement between the raw

material vendor and the drug product manufacturer,

according to Afonina.

To best aid manufacturers as they deal with raw

material challenges, suppliers can, in addition to

meeting specifications of pharmacopoeia mono-

graphs, provide in-depth raw material characteriza-

tion data including customer-specific parameters and

historical data that can be used for the prediction of

future batch-to-batch consistency and information on

appropriate handling and storage conditions, accord-

ing to Gübeli. Trust can also be built by assuring

independent supplier auditing and certification, such

as is offered through the EXCiPACT voluntary interna-

tional certification scheme for excipients.

Most manufacturers select raw materials that are

excipient-grade or of similar quality due to the fact that

the raw material or its impurities have the potential to

be transferred to the final formulated biopharmaceuti-

cal product. For raw materials classified as excipients

(e.g., according to US or European pharmacopoeia), the

most important types of variability are part of the phar-

macopoeia monographs and are controlled and speci-

fied in the certificate of analysis (CoA) by the supplier,

according to Gübeli. Unfortunately, he also notes that

the pharmacopoeia monographs generally lag behind


state-of-the-art knowledge about

raw material variability.

COMMUNICATION IS KEYThere is, however, a shared respon-

sibility between the vendor and

the client. “Clients need to be

aware of their needs with respect

to factors that are critical to the

performance of their processes and

make sure that suppliers under-

stand these factors,” Perkins states.

Biopharmaceutical companies also

need to understand if the typi-

cal variability in a raw material

is acceptable for a given process/

product or if more controls are

needed, according to Kolhe.

In general, it’s the responsibility

of the manufacturer of a biophar-

maceutical to select the appropri-

ate raw material that fulfills the

requirements for the production

of a safe and high-quality bio-

pharmaceutical under GMP. “Each

biopharmaceutical and associated

manufacturing process is unique

and influenced by many raw-mate-

rial associated factors to a different

extent. These phenomena are only

known by the manufacturers of

the biopharmaceuticals. Therefore,

we encourage manufacturers to

cooperate with us as suppliers and

exchange information on critical

raw material parameters. Only then

can we as a supplier assure consis-

tency of these particular param-

eters by introducing them into

custom CoAs,” Gübeli comments.

Biopharmaceutical manufactur-

ers need to implement raw-material

management strategies, including

processes/systems for the release

of raw materials for use based on

specifications, quality agreements,

routine audits, and raw-material

change communications, according

to Kolhe. Drug product manufac-

turers should also have segregated

areas for receiving/handling raw

materials to avoid the potential for

contamination from other areas in

of the manufacturing plant and

have a system in place to moni-

tor trends in the analytical data,

according to Afonina.

Many drug companies are tak-

ing risk-based and science-based

approaches to managing raw mate-

rial variability. Such approaches

require significant product under-

standing regarding the impact of

raw-material attribute variability.

“Formal risk assessment exercises

are conducted to evaluate the poten-

tial risks associated with raw mate-

rial availability with respect to final

product quality, safety, and efficacy.

The obtained results are then used to

understand the risks and determine

any actions needed in terms of raw-

material quality attributes,” Kolhe

explains. Integration of control strat-

egies for final drug substances and

drug products with raw-material

control measures also help ensure

consistent product quality.

“One of the issues here is that

there is no standard or specific guid-

ance on the management of raw

material variability,” says Perkins.

“In addition, the patchwork of reg-

ulatory guidance documents that

are applicable make references

to science-based and risk-based

approaches to the minimization

of raw material variability, but

most are open to interpretation.”

MilliporeSigma and other compa-

nies, as participants in initiatives

of associations like the BioPhorum

Operations Group, are opening up

lines of communication, even across

competitors. “A consistent indus-

try approach to variability analy-

sis will enable greater supply chain

transparency, standardized process

control, and key materials charac-

terization,” Perkins asserts. “The

general idea is that manufacturers

would be accountable for a set of

clear material definitions, so cus-

tomers know that they are choosing

the right raw materials,” he adds.

Transparency between a buyer

and supplier is key so that man-

ufacturers can understand and

address variability issues with the

potential to affect product quality

and patient safety. “If the buyer

provides information on the end

use of a material, the supplier

can then determine what infor-

mation they need to know about

where the material is coming from

(if repackaging) or about key raw

materials needed for its manu-

facture, as well as what analyses

may be necessary,” states Perkins.

Suppliers can in turn assist bio-

pharmaceutical customers with

risk assessment processes by pro-

viding streamlined documentation

involving detailed raw material

processing and characterization

information, according to Gübeli.

He adds that drug manufacturers

will have the greatest success in

obtaining raw materials that meet

their detailed demands if they

begin interacting with suppliers

early in the development process.

MilliporeSigma is also work-

ing with customers and indus-

try groups to develop an eData

exchange format. The system will

enable secure electronic sharing of

comprehensive raw material pro-

duction and test data. Not only

are the data priceless; multi-variant

analysis techniques provide a bet-

ter understanding of variability

and its impact on final drug prod-

ucts, according to Perkins. X

Upstream Processing

Transparency

between a buyer

and supplier is key so

that manufacturers

can understand and

address variability

issues.

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Cell culture is widely employed

in biomedical applications and

has numerous applications,

spanning from diagnosis, ther-

apy, and the production of biological

drugs. Cells used for biopharmaceuti-

cal applications are mainly of animal

origin and can be cultured in suspen-

sion or adherent cell cultures. A unique

characteristic of adherent cells is their

dependence on an anchorage surface

on which to attach to exert their nor-

mal metabolic activity and prolifer-

ate. While cell attachment may prove

advantageous for some purposes, such

as supernatant harvesting, cell detach-

ment may become an enormous chal-

lenge when cells are to be harvested.

Cell detachment is probably one of the

most relevant processes hindering a

faster development for cell-based bio-

technology. In this article, the authors

focus on different strategies available for

animal adherent cell detachment.

Animal adherent cell cultures are

derived either from tissue explants

or from cell suspensions. In a stan-

dard culture process, once cells have

attached to the culture support, they

undergo a lag phase and then start

Adherent Cell Culture in Biopharmaceutical Applications: The Cell-Detachment Challenge

Marcos Simon and

Juan J. Giner-Casares

The necessity to detach cells from a culture

substrate during cell harvesting

remains one of the most challenging

steps in a cell-culture

process.

Marcos Simon, PhD, is founder of The

Bolt-on Bioreactor project and Technology

Transfer Manager at CIC biomaGUNE.

Juan J. Giner-Casares, PhD, has

been a postdoctoral researcher in

the BioNanoPlasmonics Lab led by

Luis Liz-Marzán at CIC biomaGUNE.

Downstream Processing


growing exponentially at a high

metabolic activity, until conflu-

ency is reached. Then, growth

stops and the culture reaches a

stationary phase. Most often, cells

are harvested when population

density suppresses growth. The

on-demand release of cultured

cells, however, would be greatly

beneficial for certain biomedical

applications in which a certain

development step of the living

cells is pursued, rather than sim-

ply the pursuit of a large num-

ber of collected cells. This release

should be efficient and triggered

by a simple stimulus.

CHOICE OF CELL-DETACHMENT METHOD: FACTORS TO CONSIDERCell behavior and the degree of

adhesion of cells to the culture sup-

port are greatly affected by several

culture parameters. However, adhe-

sion degree is not the only factor

to be considered when choosing

the most convenient cell detaching

method within a given cell-culture

process. The authors consider dif-

ferent factors and requirements that

influence the choice of the right

cell-detachment method.

Degree of cell adhesion

Vigorous cell-culture processes,

such as culture on microcarriers,

require strong cell adhesion that

needs be matched by the harshness

of the detaching step. On the other

hand, cell culture on t-flasks is

more suitable for cells that establish

weak interactions with the support.

Further use of detached cells

When subsequent reattachment

is expected from detached cells

(e.g., in a subpassage step during

cell expansion or for therapeutic

cells production), cell viability

and membrane component integ-

rity are more important than in

the case of cell harvesting for fur-

ther preparation of cell extracts.

Similarly, when a cell sheet is to

be obtained, cell-to-cell interac-

tions must remain intact, but not

when cell suspension is the target

product. In addition to physical

integrity of the cells, an optimal

functionality/metabolic state of

such cells is also desirable.

Process compatibility

Some chemicals necessary for par-

ticular cell-detachment procedures

may interfere with subsequent

downstream processing steps that

are unaffected by other alternative

detachment methods. Therefore,

detachment based on physical

processes is certainly preferable

to chemical treatments, and help

mitigate concerns related to the

impact of process additives on a

final product.

Culture support

Cell-detaching methods where

direct access to the support is nec-

essary, such as cell scraping, are

not compatible with cell-culture

supports such as microcarriers or

hollow fibers.

Process scale

Manual cell-detachment tech-

niques such as cell scraping may

be adequate at laboratory scale but

unfeasible at industrial scale due to

the laboriousness of the method.

Reusability of

culture substrates

Some re spons ive subst rate s

designed to promote cell detach-

ment under the effect of a particu-

lar stimulus undergo an irreversible

structural modification that pre-

vents repeated cell culture on the

same substrate.

Regulatory constraints

Reg u lator y rest r ic t ions may

exclude some cell-detachment

methods from consideration in

particular applications or require

extensive validation before autho-

rization of use is granted.

Spatial resolution

Applications such as co-culture of

different cells in a given geometri-

cal pattern or single-cell harvesting

require the ability to detach cells

only from selected areas without

affecting cells growing in other

areas of the support.

Temporal resolution

Some stimuli used to promote cell

detachment are deleterious for

cells. In these cases, it is important

to control the temporal resolution

of the cell-detachment technique.

Compatibility with

sterilization methods

In case sterilization of the cell culture

device is necessary prior to use, care

should be taken to ensure that the

sterilization method does not affect

the characteristics of the cell detach-

ment technique (e.g., when using

supports with modified surfaces).

Shelf-life

Cell-culture devices are often pro-

vided as ready-to-use devices that

are stored for a long period of time

before use. Some components of

chemical and biological origin

contained in sophisticated cell-

detachment systems are unstable

and have a short shelf-life.

Production costs

Expensive cell-detachment systems

negatively affect the overall cell-

culture process cost and determine

the applicability of the technique.

ALTERNATIVE CELL-DETACHMENT METHODSFor many years, treatment with the

protease enzyme trypsin has been

the standard method for cell detach-

ment. This method, widely named

trypsinization, is based on the addi-

tion of an active concentration of

the enzyme to the cell culture and

subsequent digestion of cell-mem-

brane proteins that establish the

interaction with the support surface.



Trypsinization variants have been

developed along the years to try and

circumvent the drawbacks associ-

ated to the deleterious effect of this

enzyme on the cells and other limi-

tations derived from the chemical

nature of the stimulus employed to

disrupt the interaction of the cells

with the support.

The successful development of

biopharmaceuticals produced from

animal cell culture and other appli-

cations of adherent cells in the bio-

pharmaceutical industry has spurred

research on alternative cell-detach-

ment methods that address deficien-

cies of the trypsinization technique

and help realize the huge potential

of adherent cells to enable therapeu-

tic and diagnostic solutions.

Cell attachment requires the inter-

action of cell membranes with the

culture support, and in order to dis-

tort this interaction, different cell-

detachment methods use different

types of stimuli to target either the

cells, the membrane support, or both.

Besides, the effect of different stim-

uli on the target may have a differ-

ent degree of reversibility, having an

impact on the applicability of the dif-

ferent cell-detachment alternatives.

The following is a brief description

of these alternative cell detachment

methods, currently on different

degrees of development, with poten-

tial impact in biopharmaceutical

applications. This information is

summarized in Table I.

In cell scraping, a rubber or plastic

spatula is used to physically remove

the cells from the culture support.

This manual method based on a

mechanical stimulus is limited to

devices with a smooth culture sur-

face accessible to the laboratory

technician and has important auto-

mation limitations. The method is

quick and easy when performed in

a reduced number of devices but dis-

ruptive to the cells and may result in

significant cell death. The absence

of chemical reagents, surface mod-

ifications, or complex equipment,

however, makes this technique an

attractive alternative for laboratory

scale cell detachment from t-flask-

like culture devices.

An alternative to cell scraping for

use in microcarrier-based cell cul-

ture is the combination of vigorous

shaking with a mild trypsinization

treatment (1). This cell-detachment

technique combines two types of

stimuli: mechanical and chemical.

The method also has the potential

for scalability. Careful analysis of

the combined effect of both stim-

uli on the cells and on the subse-

quent processing of the product,

however, is necessary. Apart from

concerns raised by the use of proteo-

lytic enzymes, this method is easy to

implement in large-scale microcar-

rier cell culture. A variation of this

method exploits the strong shear

forces in microfluidic systems (2).

In such a method, cells are cultured

on the internal walls of channels in

microfluidic chips. When a strong

liquid flow is passed through the

microfluidic channels, the cells are

subjected to a high shear force that

provokes an efficient detachment.

This method is considered a harsh

detachment procedure, however,

often resulting in significant damage

and even death of the cells.

Enzymatic treatment with alterna-

tive proteolytic enzymes such as col-

lagenase or Pronase, a commercially

available mixture of several nonspe-

cific endo- and exoproteases pro-

duced by Streptomyces griseus, are

alternatives to the traditional tryp-

sinization method. These enzymes

digest proteins exposed in the cell

surface to distort the interaction of

the cells with the culture support.

Enzymatic activity on the cell sur-

face can be deleterious to the cells

and irreversible damage such as the

apoptotic effect induced by the tryp-

sin treatment (3) can be expected

from other proteases. This method

has poor temporal resolution, lead-

ing to extended activity of the

enzyme on the cells and subsequent

cell damage. Strong regulatory con-

siderations are also associated with

the use of enzymes in biopharma-

ceutical applications. On the other

hand, this method provides an effi-

cient way to disrupt cell-to-cell inter-

actions, a useful feature of interest

for harvesting cell suspension rather

than cell sheets.

Non-enzymatic chemical treat-

ment is also an option. Enzymatic

treatment is usually combined with

Ethylenediaminetetraacetic acid

(EDTA), a chelating agent for diva-

lent cations that helps trypsin do

its task and inhibits the interaction

of some of the proteins involved in

cell-to-cell interactions and cell-to-

support interaction. EDTA is also

applied alone when cells are loosely

attached to the culture support. The

citric saline method (4) has also been

reported as a very gentle treatment

for cell detachment. Non-enzymatic

chemical treatment provides a gen-

tle cell-detachment method. Besides

poor spatial and temporal resolu-

tion, these methods suffer from

poor performance in terms of the

ratio between required amount

of chemical and cell detachment.

Therefore, applicability of this tech-

nique is restricted to a limited num-

ber of cell lines.

Thermoresponsive substrates

have been used to promote the

detachment of adhered cells. In this

method, the cell-culture support

is coated with a thermoresponsive

polymer such as poly(N-isopro-

pylacrylamide) (pNIPAAm) fol-

low i n g d i f f e r e nt c he m ic a l

strategies. Cells are cultured on the

pNIPAAm-coated support, and cell

detachment is achieved by drop-

ping the temperature of the culture

below the lower critical solution

temperature (LCST) of pNIPAAm

(32 °C). Below LCST, pNIPAAm

undergoes a phase transition from

a shrunken state to a swollen state

that induces cell detachment due

to changes in the hydrophobicity of

the polymer. This strategy, initially




Cell-detachment technique

Stimulus Major advantages Major drawbacks Comments

Cell scraping Mechanical scratch of cells from surface

High temporal resolution, simplicity, low cost

Cell damage, poor scalability, highly invasive

Simple method with poor applicability beyond research

Vigorous shaking Shear forces on cells combined with proteolytic digestion of adhering proteins on cells

Scalable, low to medium cost

Cell damage, impact on downstream processing

Applicable to microcarrier cell culture to diminish deleterious effect of enzymatic treatment

Enzymatic treatment Proteolytic digestion of adhering proteins on cells

Well established, tunnable to adhesion strength, can be used on different types of supports

Cell damage, impact on downstream processing, regulatory concerns

Trypsinization and its variants are well established, straightforward methods. Cell-to-cell interactions are affected.

Non-enzymatic treatment

Molecular conformational changes on adhering molecules on the cells

Mild effect on cell integrity Applicable only to loosely attached cells

Alternative to enzymatic treatment with reduced impact on cell viability, but poor detachment efficency

Thermoresponsive substrates

Temperature change on the substrate

Noninvasive, automation potential can be used on different types of supports

Potential issues with leachables and extractables, cost of surface modifications

Technique with high potential for automation that can be adapted to most culture surfaces

pH-responsive substrates

pH change in substrate surface

Automation potential can be used on different types of supports

Very sensitive to process parameters, requires surface modification of the substrates

Future potential for this technique depends on availability of compatible pH-sensitive polymers

Electro-responsive substrates

Electric discharge on substrate

High temporal and spatial resolution, noninvasive, automation potential

Very complex surface modifications required, limited to small areas, not valid for microcarrier culture

Sophisticated technique with poor potential application beyond research

Photo-responsive substrates

Light-induced molecular modifications on substrate surface

Noninvasive, automation potential, scalable

Regulatory concerns due to required surface modifications, complex chemistry

Highly applicable technique once substrate modification issues are solved

Plasmonic substrates

Light-induced temperature change in localized nanoparticles on substrate surface

Noninvasive, automation potential, scalable, simple surface modification

Requires substrate modifications

Besides attractive features of photo-responsive substrates, easy surface modification allows for use on different types of substrates

Magnetic detachment

Magnetic field pull-on cells Allows for precise re-location of detached cells

Chemical modification of cell surface is required prior to application, regulatory concerns, poor scalability

Interesting technique for research applications

Shock-waves Shear forces on cells High spatial and temporal resolution

Cell damage, complex implementation, only for localized cell detachment

For use in localised cell detachment but complex compared to alternative techniques

Freeze-thaw Freezing cells Noninvasive, easily implemented

Cell damage This method is of application on processes where cell damage is of no concern. Scalable only when culture is performed on microcarriers following a supernatant separation step

Table I: Cell-detachment methods with potential impact on biopharmaceutical applications.


applied to the recovery of cell sheets

from t-flask type culture devices (5),

has been extended successfully to

microcarrier-based cell cultures (3).

A key issue in this approach is raised

by regulatory concerns associated

with the use of current thermo-

responsive polymers (6). Chemical

synthesis of such polymer brushes

onto the surfaces of cell-culture sup-

ports requires a number of steps and

reagents that might hinder their

applicability. Otherwise, these sys-

tems are interesting due to their

potential for automation, scalability,

and noninvasive cell detachment.

The use of pH-responsive poly-

mers grafted on cell-culture sup-

ports has allowed researchers to

promote cell detachment by low-

ering environmental pH from 7.4

to 6.5 (7). Cell detachment can

occur following structural changes

that take place in the pH respon-

sive polymers upon pH decrease.

Changes in the polymer charge

f rom posit ive to negative in

response to a pH increase have been

used to detach cells from chitosan-

coated supports (8). This method

could potentially be employed to

detach cells from microcarriers or

from t-flask-like cell culture devices,

but similar to thermoresponsive

substrates, treatment with chemi-

cals, and enzymatic treatment,

requires the homogeneous modifi-

cation of the overall culture envi-

ronment conditions, which could

result in poor temporal resolution.

Sophisticated gold-coated elec-

tro-responsive substrates have been

employed to hydrolyze—under a

transient electrical potential—the

ether bond that retained the cell-

adhesive ligands to which cells are

attached (9). In this example, the

complex structure of the system is

a drawback for large-scale and mass

production. Besides, the reaction is

irreversible, because the cell-adhe-

sive ligand molecules remain bound

to the cells after the ether bond is

hydrolyzed. Moreover, the applied

electric potential might induce paral-

lel electrochemical reactions. Despite

the aforementioned drawbacks, the

potential automation of the system

may result in future efforts to further

develop electro-responsive systems.

Photo-responsive substrates based

on different designs and with dif-

ferent features have been studied.

Some of these systems are based

on changes on the hydrophilicity

of the substrate by light illumina-

tion (10). Some are based on revers-

ible structural changes in molecules

grafted on the surface of the sup-

port (11), and others use the irrevers-

ible cleavage of a photolabile linker

to release a cell-adhesive molecule

bound to the substrate (12). In all

cases, the high spatial and tempo-

ral resolution of light sources is an

important advantage of these cell-

detachment systems. Some of these

systems induce an irreversible modi-

fication on the substrate or a surface

modification on the cell membrane.

Complex modifications of the sub-

strate are necessary to sensitize cul-

ture supports and prepare them to

respond to light. Otherwise, use of

light as stimulus for cell detach-

ment is one of the most promising

alternatives for automated, nonin-

vasive cell detachment for biophar-

maceutical applications, including

large-scale production of biophar-

maceuticals using different types of

culture supports.


Figure 1: Cell detachment from plasmonic substrates. Transmitted light images of

HeLa cells grown on plasmonic substrates before (A) and after (B) irradiation with

a near-infrared laser at 980 nm. Power density of 340 mW/cm2 during 20 min.

(A)

(B)

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Plasmonic substrates incorporate a

particular type of light-induced cell-

detachment properties. In this type

of substrate, a transient hyperther-

mic effect results from the interac-

tion of light of a given wavelength

with nanoparticles integrated in the

support. To date, available systems

were based on the use of near-ultra-

violet light, resulting in an aggressive

treatment for the growing cells and

in loss of spatial and temporal resolu-

tion due to the formation of reactive

oxygen species (ROS) that diffuse in

the culture medium (13). Efforts in

this direction have led to the devel-

opment of a plasmonic substrate that

promotes cell detachment upon a

short illumination with near-infra-

red (NIR) light (14). This technique

is based on plasmonic substrates pro-

duced by embedding gold nanoparti-

cles on the surface of the cell-culture

support. The controlled size and

geometry of the nanoparticles, as

well as their close contact, leads to

an intense plasmonic phenomena

upon interaction with NIR light—

better suited for cell treatment than

ultraviolet light—widely known to

induce mutagenic alterations in cel-

lular DNA. Besides, the system com-

bines the attractive properties of

light as a controlled stimulus with

a simple and robust support readily

transferable to different cell culture

devices. Most advantageously, NIR

light can be applied from outside the

cell-culture device, thus providing a

noninvasive cell-detachment system.

The effect of NIR light on cells grow-

ing on plasmonic substrates is shown

in Figure 1.

Cells labeled with magnetic

nanoparticles or liposomes have

been used to demonstrate the via-

bility of magnetic systems for cell

detachment. In these systems, cells

lightly adhered on the culture sup-

port are treated with positively

charged liposomes that readily bind

to the negatively charged cell mem-

branes (15). Then, a magnetic field

is applied to detach the cells from

the culture surface. The good spatial

and temporal resolution associated

to the localized application of the

magnetic field and the possibility to

deliver detached cells to designated

locations are of potential interest.

However, the complexity associated

to this method and other consider-

ations, such as the specific features

required for the cell membrane to

interact with the liposomes, preclude

its application beyond dedicated

research experiments.

Shock waves originated from a

piezoceramic source adapted from a

commercial lithotripter have been

used to detach adherent cells (16).

In this work, the ability of surviving

detached cells to reattach and propa-

gate was not assessed. However, cells

detached by laser-induced shock-

waves have been shown to adhere

again (17). This technique has good

spatial and temporal resolution.

There are few advantages to this com-

plex and shear-intensive technique,

however, when applied to the culture

of adherent cells for biopharmaceuti-

cal applications.

For particular applications that are

more related to research and produc-

tion of cell fragments, freeze/thaw is

a traditional method for cell detach-

ment, where cell viability is not of

major concern (18). This method has

been proposed for cell harvesting

from microcarriers (19), especially for

harvesting protease-sensitive biologi-

cal materials, and it is often used to

obtain cell fragments. Freeze/thaw

has a detrimental effect on cell via-

bility and integrity and a poor tem-

poral resolution. On the other hand,

regulatory concerns related to this

technique are scarce.

CONCLUSIONSome of the discussed methods for

cell detachment are already avail-

able commercially, while others are

in different stages of development.

In coming years, the authors expect

to see commercial variants of these

methods implemented in existing

devices for cell culture, as is the case

of the ongoing efforts of the authors

to incorporate plasmonic cell-

detachment features into the Bolt-on

bioreactor (20). Refinement of the

methods that have great potential

for enabling efficient cell detach-

ment is also necessary, as many of

the newer techniques are still too

complex and/or sophisticated to be

widely used by the cautious biophar-

maceutical industry.

REFERENCES 1. W.N. Nienow et al., Biochem. Eng. J. 85,

pp. 79–88 (2014).

2. K.W. Kwon et al., Lab Chip 7, pp. 1461–

1468 (2007).

3. H.S. Yang et al., Cell Transplant. 19, pp.

1123–1132 (2010).

4. R. Pape et al., Arterioscler. Thromb. Vasc.

Biol. 28, pp. 534–540 (2008). DOI:

10.1161/ATVBAHA.107.159483.

5. H.E. Canavan et al., J. Biomed. Mat. Res.

Part A 75A (1), pp. 1–13, (2005). doi:

10.1002/jbm.a.30297

6. M. Patenaude and T. Hoare, “(302c)

Injectable, Degradable Thermoresponsive

ploy(N-isopropylacrylamide) Hydrogels,”

presentation at the 12th Annual Meeting

of the AIChE (2012).

7. X.-Q. Dou et al., Soft Matter 8, pp. 9539

(2012).

8. Y. Chen et al., Biomaterials 33 (5), pp.

1336–1342 (February 2012).

doi:10.1016/j.biomaterials.2011.10.048

9. W.-S. Yeo et al., Chembiochem 2 (7–8),

pp. 590–593 (Aug. 3, 2001).

10. Y. Hong et al., Biomaterials 34 (1), pp.

8–11 (January 2013). doi:10.1016/j.

biomaterials.2012.09.043

11. A. Higuchi et al., Biomacromolecules 5, pp.

1770–1774 (2014).

12. M. Wirkner at al., Adv. Mater. 23 (64), pp.

3907–3910 (2011) doi: 10.1002/

adma.201100925.

13. T.A. Kolesnikova et al., ACS Nano 6 (11),

pp. 9585–9595 (2012).

14. J.J. Giner-Casares et al., Angew. Chem. Int.

Ed. 55, pp. 974–978 (2016).

15. A. Ito et al., Tissue Eng. 10 (5-6), pp. 873–

880 (May–Jun 2004).

16. C.-D. Ohl et al., Biochimica et Biophysica

Acta 1624, pp. 131– 138 (2003).

17. Y. Hosokawa et al., Appl. Phys. A 79, pp.

795–798 (2004).

18. N. Nishishita et al., Am. J. Stem Cells 4 (1),

pp. 38–49 (2015).

19. V.V. Ranade and J.B. Cannon, Drug

Delivery Systems, Third Edition, (CRC

Press, April 25, 2011).

20. The Bolt-on Bioreactor Project. www.

boltonbioreactor.com. ◆


ABSTRACT

The Chinese hamster ovary (CHO) cell lines used for the production of recombinant

therapeutic proteins are immortalized cells with a relatively high degree of genetic

plasticity. Given this inherent genetic flux, recombinant genes (or transgenes,

also referred to as the expression construct) expressed in CHO cells—used for

therapeutic bioproduct development—can be subject to genetic alteration that

may potentially impact the integrity and/or stability of those transgenes and, in

turn, impact drug substance production. Provided is a comprehensive, risk-based

transgene characterization strategy; its implementation is based on chemistry,

manufacturing, and control (CMC) development phases to ensure that the

integrity and stability of the transgene is maintained for clinical and commercial

CHO production cell lines. Early-phase assessment includes characterization

of the expression plasmid prior to cell-line generation (transfection); evaluation

of transcript integrity of those transgenes expressed transiently and stably in

CHO cells after transfection but prior to single-cell cloning of the candidate

production cell lines using single-cell sorting (or alternative methods); and

profiling of transgene copy number in cell-line populations across cell generations

spanning the manufacturing window. Mid-phase assessment includes further

characterization of the integrity and stability of the integrated transgenes using

the defined commercial cell-culture processes. Finally, the presented strategy

includes the late-phase characterization of the expression construct using cells

at the limit for in vitro cell age harvested from the commercial cell-culture

process to support the marketing authorization applications. Together, the

presented strategy is integrated with other existing drug substance analytical

control and product characterization strategies to ensure the integrity and

consistency of the drug substance used for clinical and commercial applications.

A Risk-Based Genetic Characterization Strategy for Recombinant CHO Cell Lines Used for Clinical and Commercial Applications

Luhong He and Christopher Frye

Luhong He, PhD, is senior research

scientist, and Christopher Frye is research

advisor, both in the department of Bioprocess

Research and Development at

Eli Lilly and Company.

Email: [email protected]

PEER-REVIEWED

Article submitted: Nov. 30, 2015.

Article accepted: Dec. 17, 2015.

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Over the past several decades,

numerous recombinant proteins

have been approved as therapeu-

tic drugs by regulatory authorities,

and many more are currently undergoing

clinical development (1). Chinese hamster

ovary (CHO)–derived cell lines have become

the predominant host for the manufactur-

ing of glycosylated therapeutic proteins.

During this period of explosion in the appli-

cation of CHO expression systems, consid-

erable efforts have been made to improve

recombinant protein production to meet

the demands of high quantity and consis-

tent quality of biopharmaceutical products.

Those efforts can be categorized into two

fundamental areas: improving therapeutic

protein developability by protein engineer-

ing based on structure-activity relationship

(SAR) studies, and improving therapeutic

protein production systems with a focus on

host-cell engineering, cell-culture medium,

and process development.

While the aforementioned efforts suc-

cessfully introduced clinical candidates

with more desirable therapeutic traits (e.g.,

enhanced activity, chemical and physical sta-

bility) and boosted the productivity of CHO

cell lines from < 100 mg/L to > 10g/L, unin-

tended consequences have resulted from the

extensively-engineered transgenes expressed

in CHO hosts, which inherently possess a

relatively high degree of genetic instability.

The authors’ internal development experi-

ence, which is consistent with published

literature, indicates that transgenes inte-

grated into the CHO genome can, in some

cases, result in unintended protein species

caused by a number of potential mecha-

nisms including transgene RNA (transcript)

aberrant splicing (2), genetic mutation (3, 4),

and amino acid misincorporation (5–7) dur-

ing cell-culture processes. If not removed by

the purification process, these unintended

byproducts are typically considered prod-

uct-related impurities (PRI), as defined by

International Council for Harmonisation of

Technical Requirements for Pharmaceuticals

for Human Use (ICH) Q6B (8), and could

have potential implications on the safety and

efficacy of the intended products. In addi-

tion, the integrated transgenes may be par-

tially or completely lost or silenced (through

epigenetic mechanisms) during the CHO

cell-culture process (9–13), which may poten-

tially lead to a lack of robustness of the drug

substance manufacturing process.

ICH Q5B (14) recommends that, “segments

of the expression construct should be ana-

lyzed using nucleic acid techniques in con-

junction with other tests performed on the

purified recombinant protein for assuring the

quality and consistency of the final product.”

This manuscript presents a comprehensive

genetic characterization strategy developed

and implemented based on bioproduct chem-

istry, manufacturing, and control (CMC)

development phases to ensure that the integ-

rity and stability of the transgenes is main-

tained during the cell-culture process and in

the cells at the limit for in vitro cell age. It is

crucial to note that the genetic characteriza-

tion strategy presented here is not an isolated

practice but rather an important part of a

holistic, integrated strategy assembled dur-

ing bioproduct development, including an

appropriate analytical control strategy cou-

pled with extensive product characterization.

In addition to meeting the regulatory

requirements that ensure the safety and

efficacy of biopharmaceuticals for clinical

and commercial applications, the strategy

presented here also provides an approach

that addresses business considerations. CMC

development is a complex, expensive, and

resource-intensive process. There are differ-

ent strategies to addressing business needs.

At one extreme would be initiation of early

clinical development using drug substance

from a manufacturing process that would

have to change significantly for commercial-

ization. This approach will require additional

effort to demonstrate comparability between

the early-phase and commercial product,

which potentially creates a risk of the need

for additional clinical trials. An alternative

approach is to develop a “commercializable”

process early and leverage that process for all

clinical development. This approach has the

potential advantage of lowering risk to prod-

uct comparability assessment, but requires

additional early process development invest-

ment. The strategy presented in this paper

is based on the latter approach, where the

process development objective is to develop

a commercial production cell line for first

human dose (FHD) or first-in-human (FIH)

application, and leveraging a “commercial-

Peer-Reviewed


izable” platform for cell culture and down-

stream process development. At the heart of

this strategy is the view that the production

cell line is the foundation of any bioprocess,

and thus, appropriate genetic characteriza-

tion of the production cell line is absolutely

crucial to the success of process development.

PHASE–APPROPRIATE STRATEGY FOR CHARACTERIZATION OF TRANSGENES EXPRESSED IN CHO CELLSThe CMC development phases discussed in

this manuscript refer to the three phases com-

monly associated with clinical and commer-

cial development. The early phase begins from

generation of the clonally derived production

cell line and ends with the release of the for-

mulated drug product for FHD/FIH. The mid-

phase involves activities including product

resupply for ongoing clinical trials (first effi-

cacy dose [FED]) and development of the com-

mercial manufacturing process. Late-phase

development starts from the manufacture of

product for the first registration dose (FRD)

using commercial manufacturing processes

and ends with process validation and sub-

mission of the product licensing applications.

Table I summarizes the CMC development

phase–appropriate activities for the character-

ization of the transgenes encoding the thera-

peutic proteins expressed in CHO cells.

For clarity, two terms, genetic suitability

and genetic stability, are used to describe the

characterization of the transgenes expressed

in CHO cells. The genetic suitability of a pro-

duction cell line refers to the acceptability

or appropriateness of the cell line for clini-

cal drug substance manufacturing. In con-

trast to genetic suitability, more traditional

genetic stability studies refer to the charac-

terization of the production cell lines used for

commercial drug substance manufacturing

followed the ICH Q5B guidance. In conjunc-

tion with protein characterization and quality

assessment, the genetic stability studies using

nucleic acid techniques examine the integ-

rity and stability of the expression construct,

which could potentially impact protein integ-

rity and process consistency. The genetic sta-

bility studies are performed in two phases: the

mid-phase study, referred to as the “genetic

stability risk assessment,” which evaluates the

integrity and stability of the expression con-

struct under defined commercial cell-culture

process, and the late-phase study, referred to

as the “expression construct characterization,”

which generates the genetic characterization

data package (based on the commercial cell-

culture process) that forms part of the market-

ing authorization application (MAA).

TRANSGENE CHARACTERIZATION ACTIVITIES DURING EARLY-PHASE DEVELOPMENT As shown in Table I, four genetic characteriza-

tion activities are implemented during early-

phase development to minimize the risk of

choosing inappropriate production cell lines.

Those activities are summarized in the fol-

lowing passages.

EVALUATION OF THE EXPRESSION PLASMID USED FOR TRANSFECTIONThe cloning of genes coding for therapeutic

proteins within an expression plasmid back-

bone is usually a straightforward procedure.

It is a common practice and a recommenda-

tion of ICH Q5B to confirm the nucleotide

sequence of the coding region of the gene(s)

of interest and associated flanking regions

that are inserted into the plasmid backbone.

After the sequence of the expression plasmid

is confirmed, the expression plasmid DNA

is usually isolated at larger scale to prepare

sufficient plasmid DNA to enable cell-line

generation. From time to time, researchers

encounter plasmid instability, such as loss

of plasmid or changes in plasmid structure

during large-scale bacterial cultivation (15).

Through the evaluation of expression plas-

mid batches prior to transfection, the authors

identified plasmid structural changes and

point mutations that resulted from a larger-

scale preparation, even though the expression

plasmid was previously confirmed from a

small-scale preparation (mini-prep). Because

the issues were discovered and corrected prior

to transfection, they did not cause significant

delays in the generation of the production cell

line.

EVALUATION OF INTEGRITY OF THE TRANSGENE MRNA EXPRESSED IN CHO CELLSRNA splicing is a natural process in

mammalian cells that removes introns and

joins exons in a primary transcript to create

mature messenger RNA (mRNA) for transla-

tion. For biopharmaceutical development, the

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recombinant genes coding for the mature

mRNA transcripts (i.e., cDNA versions) are com-

monly used for the production of drug sub-

stance to avoid complexity of RNA splicing in

the production host. However, with the imple-

mentation of SAR studies and advancements

in DNA manipulation techniques, more and

more recombinant genes undergo extensive

and complex genetic manipulation to improve

the encoded candidate’s therapeutic traits.

Although the protein engineering process has

been successful in that regard, the full impact

of some nucleotide sequence modifications is

difficult to predict. It has been observed that

several engineered antibody genes expressed in

CHO cells produced unintended transcripts—

resulting from aberrant RNA splicing at cryptic

splicing sites—in addition to the expected full-

length transcript (2, 16). Aberrant mRNA splic-

ing can lead to unexpected low-level expression

of the recombinant transgenes (2) and/or give

rise to truncated product-related impurities

(16). In one case, the authors experienced an

antibody-producing cell line, which had cryp-

tic aberrant mRNA splicing that gave rise to a

truncated light chain (LC) product. Although

the truncated LC was effectively removed

through the downstream purification process,

the overall purification yield was significantly

reduced (to approximately 10%). A new pro-

duction cell line was subsequently generated

for Phase II/III and commercial applications.

The aberrant splicing was eliminated in the

new cell line by site-specific mutagenesis at the

cryptic splicing sites.

To mitigate the risk of unintended splicing,

the authors identified and eliminated cryptic

aberrant splicing proactively during clinical

candidate selection to avoid potential delays

to the clinical development or the need to

switch cell lines for commercial applications.

The authors’ experiences and the literature

indicated that several publicly available

splice-site-recognition programs were unable

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CMC development phase

Timing Characterization activity

Early (gene to FHD)

Prior to transfection

Evaluation of the integrity of the expression plasmid used for transfection to ensure the DNA used has the expected gene coding sequence. Assays include restriction mapping and DNA sequencing of the expression cassette.

Prior to single-cell cloning

Evaluation of integrity of the transgene mRNA expressed in CHO cells to ensure engineered genes do not give rise to unintended aberrant mRNA splicing or other modifications. Assays include RT-PCR and DNA sequencing.

Prior to production cell-line selection

Genetic suitability evaluation of transgenes in the clonally derived cell lines to eliminate cell lines that display significant changes in their transgene population profiles due to cell aging. Assays include single-cell qPCR.

Prior to drug product release for clinical applications

Confirmation of nucleotide sequence of transgenes in MCB to ensure the MCB carries expected transgene sequence. Assays include RT-PCR and DNA sequencing.

Mid (FHD to FRD)Prior to commercial cell-culture process determination

Genetic stability risk assessment of the chosen production cell line under defined cell-culture process at various cell ages from bench and pilot scales to ensure the cell line and cell-culture conditions are acceptable for commercial development. Assays include coding sequence confirmation, restriction endonuclease mapping for integration pattern analysis and transgene copy number determination.

Late (FRD to MAA)Prior to commercial process validation at manufacturing sites

Expression construct characterization in MCB and EOPC at the limit for in vitro cell age from fully representative commercial manufacturing process for MAA. The assays developed during genetic stability risk assessment will be applied.

Table I: Phase-appropriate characterization activities for therapeutic transgenes expressed in

Chinese hamster ovary (CHO) cells. FHD=first human dose, FRD=first registration dose,

PCR=polymerase chain reaction, qPCR=quantitative polymerase chain reaction, MCB=master cell

bank, RT–PCR=reverse transcription polymerase chain reaction, EOPC=end-of-production cells,

MAA=marketing authorization application.


to identify the aberrant splicing sites used

in the CHO cell environment (2). The most

effective methods to identify the presence

of aberrant splicing events are reverse

transcription polymercase chain reaction

(RT–PCR) and Northern blot. The authors’

data indicate that RT–PCR is a more sensitive

method to detect low level of aberrant splic-

ing compared to Northern blot (unpublished

data). The potential bias due to PCR primer-

binding capability can be overcome by

applying multiple pairs of primers.

RT–PCR-based methods have been devel-

oped and implemented to screen all new clini-

cal candidates for the aberrant splicing events

in both transiently and stably transfected

CHO cells before they are chosen to gener-

ate production cell lines supporting clinical

development. This early screening approach

has identified cryptic aberrant splice sites in

multiple therapeutic candidates including

monoclonal antibodies, bispecific antibod-

ies, and fusion proteins, therefore preventing

significant investment in the development

of certain candidate-encoding genes (unpub-

lished data).

EVALUATION OF TRANSGENE DISTRIBUTION PROFILES IN CHO CELL LINESThe inherent adaptive ability of CHO

cells and their capacity for the expression

and secretion of recombinant proteins

have been the most important factors

that have enabled the adoption of CHO

cells as the industry’s predominant host

for development and manufactur ing

of therapeutic proteins (17). However, as

immortalized cells, CHO cell lines exhibit

a high level of genetic and phenotypic

diversity and instability (17). In spite of

repeated rounds of single-cell cloning by

limiting dilution or florescence-activated

cel l sor t ing (FACS), c lonal ly-der ived

CHO cell lines have often been observed

to diverge, becoming a heterogeneous

populat ion over long per iods of sub-

culturing (17–22). Extensive efforts have

been devoted to the screening process

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Figure 1: Transgene copy number distribution profiles of cell lines expressing recombinant protein at

generations of 0, 30, 45, and 60. Generation 0 (G0) represents the generation of a master cell bank, G30

represents the generation of cells harvested from a 5000-L bioreactor, and G60 represents the limit for

in vitro cell age designed for a commercial manufacturing process. The distribution profiles shown in the

scatterplot and histograms (1A: cell line 3E4; 1B: cell line 7H2) were generated by Oneway platform of JMP

software (SAS Institute, Inc, Cary, NC) as outlined in (12). The cycle threshold (Ct) values of transgene were

generated by a single-cell quantitative polymerase chain reaction (qPCR) assay. The number of tested

single cells (number), mean and standard deviation (std dev) of the Ct values are shown below the plots.

1A: Cell line 3E4

40

35

30

25

20

3E4-G

0

3E4-G

30

3E4-G

45

3E4-G

60

3E4-6

0

3E4-G

30

3E4-G

45

3E4-G

60

Ct

Sample Name

Means and std DeviationsMean Std DevNumberLevel

3E4-G0

3E4-G30

3E4-G45

3E4-G60

46474848

30.430.330.730.6

0.60.60.70.6

1B: Cell line 7H2

40

35

30

25

20

Ct

7H

2_G

O

7H2_GO7

H2

_G

3O

7H2_G3O

7H

2_G

45

7H2_G45

7H

2_G

60

7H2_G60

7H

2_G

0

7H

2_G

30

7H

2_G

45

7H

2_G

60

Sample Name

Means and Std Deviations

Level Number Mean Std Dev

47484546

29.030.631.230.8

2.11.51.00.8

FIG

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.


to identify desired characteristics and

suitability of recombinant CHO cell lines.

Historically, suitability has been evalu-

ated utilizing phenotypic measures (pro-

ductivity) assessing cell-line productivity

across a generational span encompassing

the manufacturing window. Given the vari-

ability of the phenotypic assessment, the

current and preferred measure of suitability

is to assess the genetic profiles of the produc-

tion cell-line population, thus obtaining a

measure of the genetic consistency of the cell

line across generations. The profiles provide

an indication of significant loss of the trans-

gene (% negative cells) or significant levels

of genetic heterogeneity within the cell-line

population (e.g., broad transgene copy dis-

tribution or standard deviation) across gen-

erations (12). Cell lines displaying genetic

heterogeneity may have a higher risk of not

meeting commercial manufacturing require-

ments and, therefore, should be eliminated

during production cell-line screening. The

methodology used for testing transgene het-

erogeneity was initially developed and imple-

mented to evaluate the genetic suitability of

clonally-derived CHO cell lines expressing

IgG1 and IgG4 monoclonal antibodies. It

has now been adapted to permit the evalu-

ation of candidate cell lines expressing Fab,

Fc fusion proteins, proteins (with or without

glycosylation), as well as bispecific or bifunc-

tional molecules. Undesired cell lines—mea-

sured by three parameters including the

percentage of negative cells, standard devia-

tion of cycle threshold (Ct) value (a measure

of population heterogeneity), and mean Ct

changes during aging (a measure of popula-

tion drift) (12) —were identified regardless

of the type of transgenes being used. In the

authors’ experience, approximately 20% of

clonally-derived candidate production cell

lines analyzed showed significant transgene

population heterogeneity over generations.

As examples, Figure 1 shows the transgene

distribution profiles of two cell lines express-

ing identical recombinant therapeutic pro-

teins obtained from the same transfection.

The cell line in Figure 1A (designated 3E4)

represents a relatively homogenous trans-

gene population across the generational span

needed for the manufacturing process, while

the cell line in Figure 1B (designated 7H2)

displayed a significant population shift when

the cells were aged, as indicated by the Ct

mean change of >1 and overall large stan-

dard deviations across generations.

CONFIRMATION OF NUCLEOTIDE SEQUENCE OF TRANSGENES IN THE MASTER CELL BANKAlthough it has been a common practice to

verify the nucleotide sequence of transgenes

encoding the therapeutic proteins in mas-

ter cell banks (MCB) for the MAA as rec-

ommended by ICH Q5B guidance, it was

only recently recommended by the European

Medicines Agency (EMA) that the sequence

of the coding region should be confirmed

prior to the initiation of clinical trials (23).

For recombinant CHO cell lines, trans-

genes are integrated into CHO chromosome.

The most common technique to verify the

nucleic acid sequence encoding the product

is sequencing of coding regions amplified by

the polymerase chain reaction (PCR) from

pooled cDNA isolated from the production

cell line. The nucleic acid sequence of the

predominant transgene transcripts should be

identical—within the limits of detection of

the methodology—to the expected sequence

encoding for the protein.

In summary, the early-phase characteriza-

tion activities focus on evaluating and mini-

mizing the potential risks associated with a

transgene’s integrity and consistency when

expressed in CHO cells. By implementing

these activities, manufacturers can ensure that

potential issues are identified during cell-line

generation and are prevented from posing

risk to the development of the manufacturing

process. It should be noted that the transgene

expression system used for the generation

of a production cell line may also impact its

integrity and stability. Two of the most com-

mon expression systems leveraged for the pro-

duction of therapeutic proteins in CHO cells

are the dihydrofolate reductase (DHFR)-based

methotrexate (MTX) selection system and

the glutamine synthetase (GS)-based methi-

onine sulfoximine (MSX) selection system.

Observations have been reported of genomic

DNA mutations in the cell lines utilizing the

DHFR/MTX system (24), and the mutation

rates measured by 6-thioguanine (6-TG) assay

positively correlated with the MTX concen-

trations used to select the recombinant cell

lines (3). Because multiple rounds of ampli-

fication are often applied using the DHFR/

Peer-Reviewed


Peer-Reviewed

MTX system—which can result in as much as

a 1000-fold increase in transgene copy num-

ber (25)—more detailed DNA and amino acid

sequence analyses may be necessary to ensure

consistent product quality (3). In contrast, the

GS–MSX expression system typically does not

require multiple rounds of amplification and,

thus, usually results in relatively low trans-

gene copy numbers. The authors’ internal data

indicate that the average transgene copy num-

ber is approximately 5 in 62 clonally-derived

production cell lines generated leveraging the

Lonza GS expression system. The relatively

low transgene copy number may reduce the

risk of DNA alterations, although it does not

eliminate the possibility of modification (26).

TRANSGENE GENETIC STABILITY RISK ASSESSMENT DURING MID-PHASE DEVELOPMENTAlthough significant efforts are devoted to the

identification of production cell lines with

the fewest potential risks for commercializa-

tion, many of these studies are performed

using cells grown in shake flasks or small

bench-scale bioreactors under nonoptimized

cell culture conditions. Therefore, it is cru-

cial to further evaluate the integrity and sta-

bility of the transgenes using commercial

cell-culture conditions during the mid-phase

development. This approach can enable cor-

rective actions to be taken to avoid costly

changes later. It has been reported that unex-

pected genetic alterations were identified in

the MCB, the manufacturing working cell

bank (MWCB), the end-of-production cell

bank (EPCB), and in production cells (27).

Deta i ls of this eva luat ion, termed

the “genetic stability risk assessment” can

be found in Table I. Mid-phase assessment

focuses on evaluating the impact of cell age

and cell-culture process using DNA and RNA

isolated from the MCB or premaster research

cell bank (pmRCB), end-of-production cells

(EOPC) from a typical defined cell-culture

process, and EOPC inoculated from the pro-

posed limit for in vitro cell age. It includes

assessment of integrity of predominant cod-

ing transcripts, consistency of integration

patterns, and average transgene copy

numbers for the aforementioned samples.

The established assessment methods will be

applied to characterize the expression con-

struct needed for future MAA. The tested

limit for in vitro cell age will be used to pro-

pose the commercial manufacturing operat-

ing space for marketing applications.

CHARACTERIZATION OF THE EXPRESSION CONSTRUCT FOR MAAThe ultimate goal of genetic characterization

of the production cell line is to demonstrate

the integrity and stability of the expres-

sion construct carrying the transgenes. This

includes demonstrating that these transgene-

carrying constructs are stably maintained from

the starting cell banks (MCB and/or WCB) to

the EOPC at the limit for the in vitro cell age

inoculated from a WCB and harvested from

the commercial drug substance manufacturing

process. This characterization—which con-

firms that the commercial cell culture process

does not lead to unintended changes in the

transgenes—is performed not only to meet

regulatory expectations for the MAA, but also

to provide assurance of safety and manufactur-

ing consistency when coupled together with

product characterization and an analytical

control strategy. The characterization, focused

on the integrity and consistency of the expres-

sion construct, consists of three aspects:

t� 7FSJGJDBUJPO�PG�QSPUFJO�DPEJOH�TFRVFODF�JO�

production cells through the end of pro-

duction. This is commonly accomplished

by nucleotide sequence analysis of the

transgene-specific PCR products amplified

from pooled cDNA.

t� "TTFTTNFOU�PG� JOUFHSBUJPO�QBUUFSOT �XIJDI�

provides insight into potential insertions

and/or deletions of the expression con-

struct. The most common methodology

for this assessment is restriction endonu-

clease mapping analysis by Southern blot.

t� %FUFSNJOBUJPO�PG� BWFSBHF� USBOTHFOF� DPQZ�

number. The current common method is

quantitative PCR (qPCR). Transgene-specific

qPCR assays and a normalizer qPCR assay

targeting a host genome region are usually

applied. The characterization, focused on

the integrity and consistency of the expres-

sion construct, consists of three aspects,

which are methods established during the

mid-phase “genetic risk assessment”.

The in vitro cell age is defined by ICH

Q5B as “measure of time between thaw of

the MCB vial(s) to harvest of the produc-

tion vessel measured by elapsed chronologi-

cal time in culture, by population doubling


Peer-Reviewed

(PD) of the cells, or by passage level of the

cells when sub-cultivated by a defined pro-

cedure for dilution of the culture” (14). In

experience with the authors’ CHO cell lines,

a typical cell age from a 5000-L production

bioreactor is found to be approximately 30

PD including the cell age of a WCB, seed

train, and bioreactor expansion and growth

in the production vessel. Extra cell culture

passages in the cell expansion stages are

added to generate the cells at the limit for in

vitro cell age to increase flexibility of manu-

facturing operation. The typical limit for in

vitro cell age is in total approximately 45–60

PD, depending on the growth rate of each

individual cell line and the specific cell-cul-

ture expansion process. The mid-phase risk

assessment enables the design of limit for in

vitro cell age for the commercial process.

The impact of a change in cell-culture pro-

cess after the commercial process has been

defined should be evaluated to determine if

re-testing of the cells at the limit of in vitro

cell age is necessary. Changes in cell-culture

process scale and/or manufacturing site may

not require re-testing of cells at the limit for

in vitro cell age, provided the available data

meet the predetermined acceptance criteria

and were obtained using cells with sufficient

in vitro cell age to cover the additional cell gen-

erations resulting from the increased scale and

cell-culture performance. Changes in media

components and growth conditions often

result in changes in cell culture profiles, and

one should consider re-testing the integrity

and stability of the expression construct at the

limit for in vitro cell age in those situations.

Although a two-tier cell-banking system

(MCB and WCB) is commonly established

for the commercial manufacturing of drug

substance, it is considered that the character-

ization of the expression construct for each

WCB is unnecessary if the following criteria

are met:

t� 5IF�.$#�IBT�CFFO�GVMMZ�DIBSBDUFSJ[FE�BOE�

the expression construct is confirmed to

be stable. If the characterization cannot be

carried out on the MCB, it should be car-

ried out on each WCB.

t� 5IF�DFMM�BHF�PG�UIF�DVSSFOU�BOE�UIF�GVUVSF�

WCB, as well as the EOPC derived from

the future WCB, will be controlled within

the previously approved limit for in vitro

cell age.

By implementing a development-phase

appropriate genetic characterization strat-

egy, manufacturers can be assured that the

integrity and stability of transgenes in all

clonally derived CHO cell lines intended

for drug substance commercial manufactur-

ing meet regulatory requirements for MAA.

Harnessing this strategy has facilitated the

early identification of unsuitable/unstable cell

lines, thus enabling effective investments of

time and resources only on those cell lines

that are appropriate candidates. This strategy

also meets business needs consistent with

aggressive development timelines and the

industry-wide movement toward more effi-

cient practices, from therapeutic candidate

selection to product launch.

POTENTIAL APPLICATIONS OF NEW TECHNOLOGIES FOR THE CHARACTERIZATION OF TRANSGENESAs the biotechnology industry continues to

mature, so does the ability to understand the

processes used to manufacture biopharma-

ceutical products. Part of this understand-

ing involves the recognition and ability to

characterize production cell lines as popu-

lations of cells exhibiting various levels of

genetic and phenotypic heterogeneity. The

described strategy includes characterization

of production cell-line populations in addi-

tion to more traditional genetic characteriza-

tion methodologies. Although this strategy

has historically served well in accomplish-

ing its intended purpose, it is also recognized

that there is a need to continue to monitor

and assess the potential value of new nucleic

acid-based technologies, such as next-genera-

tion sequencing (NGS), to potentially further

enhance the capability for genetic character-

ization (28). NGS can be utilized as a com-

plementary/orthogonal analysis tool for the

investigation of bioproduct-related impuri-

ties. NGS can also provide a means of better

understanding impurities if they are related

to genomic mutations or nutrient limitations

in a cell-culture process. New approaches and

technologies are becoming available and hold

promise for permitting better characterization

of cell lines and cell-culture processes, which

could lead to improved insight into how to

develop manufacturing processes more holis-

tically. Implemented together, the described

phase-appropriate genetic characterization


Peer-Reviewed

strategy, orthogonal product characterization,

and appropriate product control strategies will

ensure the safety and efficacy of clinically vali-

dated therapeutic proteins.

CONCLUSIONPotential risks related to the integrity

and stability of recombinant transgenes

expressed in CHO cells are inevitable during

biopharmaceutical development. These risks

include unintended aberrant mRNA splicing

in the coding regions due to the bioengi-

neering process and potential instability of

the integrated recombinant genes as a result

of the global genetic instability inherent

in CHO cells. The genetic characterization

strategy described here can play a crucial

role in assessing and managing these risks.

The authors’ implementation of this strategy

has prevented problematic expression con-

struct/cell lines from being developed inap-

propriately, thereby enabling more efficient

and effective process development programs.

The data presented here demonstrate that

the CHO production cell lines developed by

the authors have relatively low integrated

transgene copy numbers, and those genes

are stably maintained from their respective

master cell banks to the end of production

cells at the limit for in vitro cell age used for

drug substance manufacturing. To ensure

the most effective genetic characterization

strategy, new technologies will continue to

be evaluated for potential future applications.

ACKNOWLEDGMENTThe authors would like to thank Christal

Winterrowd and Monica Myers for provid-

ing copy number analysis and Dennis Gately

and Matthew Hilton for providing technical

input to the characterization strategy. We’d

also like to thank Matthew Hilton, Michael

De Felippis, Tongtong Wang, and Anli

Ouyang for critical review of the manuscript

and helpful discussions. In addition, the

authors wish to recognize and thank Lonza

Biologics (Slough, UK) for the licensing of

and permission to modify the GS-CHO

expression system.

REFERENCES 1. J.M. Reichert, MAbs 7 (1), pp. 1–8 (2015).

2. S.D. Wijesuriya, R.L. Cotter, and A.H. Horwitz,

Protein Expr. Purif. 92 (1), pp. 14–20 (2013).

3. D. Guo et al., Biotechnol. Bioeng. 107 (1),

pp. 163–171 (2010).

4. P. Zhang et al., MAbs 4 (6), pp. 694–700 (2012).

5. L. Feeney et al., Biotechnol. Bioeng. 110 (4),

pp. 1087–1097 (2013).

6. Y. Yang et al., MAbs 2 (3), pp. 285–298 (2010).

7. D. Ren et al., J. Chromatogr. B 879 (27),

pp. 2877–2884 (2011).

8. ICH Q6B, Specifications: Test Procedures and

Acceptance Criteria for Biotechnological/Biological

Products, Step 4 version (1999).

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11. V. Paredes et al., Biotechnol. Lett. 35 (7),

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12. L. He et al., Biotechnol. Bioeng. 109 (7),

pp. 1713–1722 (2012).

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pp. s16–27 (June 2009).

14. ICH, Q5B, Quality of Biotechnological Products:

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Step 4 version (1995).

15. F.A. Al-Allaf et al., 3 Biotech. 3 (1), pp. 61–70

(2013).

16. J.M. Reichert, N.M. Jacob, and A. Amanullah,

Biotechnol. Prog. 25 (4), pp. 1198–1215 (2009).

17. F. Wurm, Processes 1 (3), pp. 296–311 (2013).

18. M. Derouazi et al., Biochem. Biophy. Res.

Communications 340 (4), pp. 1069–1077 (2006).

19. L.M. Barnes, C.M. Bentley, and A.J. Dickson,

Cytotechnology, 32 (2), pp. 109–123 (2000).

20. N.S. Kim, S.J. Kim, and G.M. Lee, Biotechnol.

Bioeng. 60 (6), pp. 679–688 (1998).

21. P.M. O’Callaghan and D.C. James, Brief Funct.

Genomic Proteomic 7 (2), pp. 95–110 (2008).

22. M. Kim et al., Biotechnol. Bioeng. 108 (10),

pp. 2434–2446 (2011).

23. EMA/CHMP/BWP/534898/2008 Committee

for Medicinal Products for Human Use

(CHMP), Guideline on the Requirements for

Quality Documentation Concerning Biological

Investigational Medicinal Products in Clinical Trials

(March 15, 2012).

24. R.J. Harris et al., Biotechnol. (NY) 11 (11),

pp. 1293–1297 (1993).

25. R.J. Kaufman and P.A. Sharp, J. Mol Biol. 159 (4),

pp. 601–621(1982).

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pp. 1077–1085 (2015). ◆

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Data integrity is a major regula-

tory topic in GMP-regulated

laboratories. The problem is

widespread, as cases of non-

compliance have also been observed

in laboratories regulated by good labo-

ratory practice (GLP) and good clini-

cal practice (GCP), not just GMP.

Non-compliances from the European

Medicines Agency (EMA) or warning

letters from FDA show examples from

companies in the United States, Canada,

the United Kingdom, and Italy, as well

as China and India. So, it is not just a

problem for Asia—it is a global issue.

The non-compliances are not con-

fined to falsification and fraud. In fact,

in the majority of labs, the main data

integrity issues concern poor data man-

agement, which account for 95% of

non-compliance cases; only 5% are a

result of falsification or fraud. Problems

arise from basic errors such as rely-

ing on paper as raw data and failing to

protect electronic records, rather than

deliberate manipulation of data. It is

the latter, however, that gets the major

headlines. In July 2014, FDA issued a

stern warning that data integrity was a

key focus of its enforcement efforts (1).

REGULATORY GUIDANCE ON DATA INTEGRITYIn January 2015, the UK Medicines

and Healthcare products Regulatory

Agency (MHRA) went further and issued

Guidance for Industry on Data Integrity

(revised in March 2015) (2). This guid-

ance consists of descriptions, defini-

tions, and expectations based around

those definitions, which include raw

data, original records, file structures,

and audit trail. The regulatory expecta-

tions presented in this guidance are use-

ful. However, the definitions are simply

presented as a shopping list; a diagram

explaining and linking key definitions

would be more beneficial.

The guidance presents data integrity

as the extent to which all data are “com-

plete, consistent, and accurate” through-

out the data lifecycle, which covers the

period from data acquisition through to

interpretation, reporting, and archiving

and then destruction after the record

retention period. There are similarities

in the US regulations; a 20-year-old FDA

definition of data integrity describes the

degree to which a collection of data is

complete, consistent, and accurate (3).

In European GMP regulations, doc-

umentation constitutes a key part of

quality assurance and, therefore, com-

pliance with GMP (4). An organization

must have good documentation, follow

standard operating procedures (SOPs),

and demonstrate compliance with the

applicable regulations. Several require-

ments focus on data integrity for com-

puterized systems (5). If critical data

are entered into a data system, a second

check is required, which can either be

a second person or can be automated

using the computer system itself. In

the US, the regulations for laboratory

records focus on the concept of com-

plete data (6).

A review of data integrity warning

letters reveals several citations for fail-

ure to have complete data including a

failure to have a complete procedure (7),

a failure to fully document the work

that is carried out (8), or being selective

in reporting data (e.g., using test sam-

How Important is Data Integrity to

Regulatory Bodies? Bob McDowall and

Joanne Ratcliff

Data integrity is a widespread,

global problem that must be addressed.

Dr. Bob McDowall is director of

R D McDowall Ltd., rdmcdowall@

btconnect.com. Dr. Joanne

Ratcliff is communications

project manager at Mettler Toledo

GmbH, [email protected].

Data Integrity


ples to “check” if an instrument is

working correctly) (9). Complete

data includes the actual observa-

tion, which can be visual or by

computerized system. Contextual

metadata, which puts the data or

result in context, is also required.

Examples of metadata include

operator ID, units of measurement,

sample information, identity,

batch number, instrument suit-

ability, and readiness. Audit trail

events are also part of the meta-

data. If a hybrid system or a fully

electronic system is used, FDA

and European regulatory agencies

require companies to review and

evaluate audit trail events to see if

data have been manipulated with-

out authorization.

FDA guidance comprises a list

of commonly asked questions

and answers that are crucial for

maintaining data integrity (10).

Detailed explanations are given

for:

t� 8IZ� QBQFS � SFDPSET � G SPN � B�

hybrid system should not be

defined as raw data—instead it

should be the underlying elec-

tronic records; Although the

reasoning focuses on chroma-

tography, it is applicable to any

computerized system.

t� 8IZ� TIBSJOH�PG�VTFS� JEFOUJUJFT�

in a computerized system is not

allowed—as it makes it impossi-

ble to attribute the work carried

out to a single individual.

t� 8IZ� BDUVBM� TBNQMFT� TIPVME�

not be used as system suitabil-

ity tests to see whether a batch

passes or not. FDA warning let-

ters reveal many citations for

this transgression.

DATA INTEGRITY CRITERIAThe criteria for data integrity are

defined by the acronyms ALCOA

and ALCOA+. ALCOA, which stands

for attributable, legible, contempo-

raneous, original, and accurate, was

developed by an FDA GMP inspec-

tor in the late 1980s. ALCOA has

been used in many areas that are

regulated both by FDA and other

regulatory agencies worldwide. In

2010, EMA published a paper on

electronic source data for clini-

cal records, in which another four

requirements relating to elec-

tronic data were added: complete,

consistent, enduring, and avail-

able (11). The Good Automated

Manufacturing Practice (GAMP)

Data Integrity Special Interest Group

(SIG) now refers to ALCOA+, which

includes the nine attributes or crite-

ria for data integrity (see Table I).

INSPECTION TRENDS In the past, an inspector would

review piles of paper; to view

a computer system, he would be

shown print-outs of the screen.

Now, the focus is on the comput-

erized systems and the electronic

records within; the paper output

is secondary. Inspectors will focus

on the electronic records, looking

at how they were generated and

manipulated within the applica-

tion. In the light of these changes,

consideration needs to be given to

the person who will manage the

system during an inspection. How

is the system configured to pro-

tect the electronic records? Are

electronic signatures being used in

the application, ensuring that the

configuration of the application is

documented and reflects the set-

tings within the software?

Annex 11 now requires audit

trail entries to be reviewed, and

FDA considers these entries as part

of the complete data. Many find-

ings of non-compliance during

inspection have been discovered

by looking at audit trail entries,

therefore, an approach to review-

ing audit trail events is needed.

Citations have noted when audit

trails were been turned off, the

audit trail had not been reviewed,

or user identities were shared,

which is not allowed under the

regulations. Additional citations

can be found in the September

2014 issue of LCGC (12).

THE TEN COMPLIANCE COMMANDMENTSThe 10 compliance command-

ments for computerized laboratory

systems described in Table II should

be considered (12).

Data Integrity

Table I: Good Automated Manufacturing Practice (GAMP) criteria for data integrity—ALCOA+.

ALCOA Term

Criteria Definition

A Attributable Who performed the action and when? If a record is changed, who did it and why? Link to the source data.

L Legible Data must be recorded permanently in a durable medium and be readable.

C Contemporaneous The data should be recorded at the time the work is performed and date/time stamps should follow in order.

O Original The information must be the original record or a certified true copy.

A Accurate No errors or editing performed without documented amendments.

+ Complete All data including any test, repeat, or reanalysis performed on the sample.

+ Consistent Consistent generation of records and application of date time stamps in the expected sequence.

+ Enduring Data should be recorded on controlled worksheets, in laboratory notebooks or in validated electronic systems.

+ Available Data needs to be available and accessible for review, audit, or inspection over the lifetime of the record.


WHERE SHOULD THE ELECTRONIC DATA TRANSFER BEGIN?Let’s consider preparation of stan-

dards by way of example. In an

analytical lab, standards are usu-

ally prepared using an analyti-

cal balance. The actual weight

is typically printed on a strip

printer and pasted into a lab jour-

nal. Afterwards, the standards are

widely used for analytical meth-

ods, such as high-performance liq-

uid chromatography (HPLC), gas

chromatography (GC), titration, etc.

Data management and documenta-

tion for analytical instruments are

usually managed by dedicated soft-

ware or a laboratory information

management system (LIMS). This

procedure is currently allowed by

regulatory bodies, which state that

print-outs representing original

data for simple devices (such as bal-

ances) are acceptable (2) but is not

the case for more complex devices.

Nevertheless, it is important that

reference standards are accurate

and traceable because they repre-

sent the starting point of many

BOBMZTFT��8BSOJOH�MFUUFST�IBWF�DJUFE�

“no details available on the prep-

aration of standards or solutions,

especially of analytical reference

standards” (13, 14). Independent of

process, it is important to ensure

that all the data are available.

This then begs the question:

8IFSF�TIPVME�UIF�FMFDUSPOJD�USBOT-

fer begin? In the process described

above, there is a gap in the data

transfer between the “simple

instrument” (the balance) and the

“complex instrument” (e.g., the

HPLC). Clearly, this is not recom-

mended because it introduces an

additional level of risk; it is obvi-

ous why no gaps should occur.

Capturing the data at the point

of origin and transferring the data

electronically throughout the

whole workflow is a much lower

risk approach and reduces the risk

of errors during the early stages of

a process, giving additional confi-

dence in the compliance of a work-

flow or laboratory.

WHAT DATA SHOULD BE TRANSFERRED?T h e n e x t q u e s t i o n t h e n

CFDPNFT�� ù8IBU�EBUB�OFFE� UP�CF�

transferred? It is essential to associ-

ate results with metadata to build

context around the values col-

lected. Although integrating even

a simple instrument can be tricky,

the advantages of an integrated

TPMVUJPO� BSF�PCWJPVT��8IFO� FMFD-

tronic data transfer starts from

the beginning of the process, each

piece of information needs to be

input only once for it to be avail-

able throughout the whole system.

This allows seamless movement of

data and other information from

the start of a process to the end,

without the need for manual effort,

such as manual transcription, creat-

ing an efficient work environment.

Data Integrity

Table II: The 10 compliance commandments.

Commandment Comment

1 Management is responsible. Management must take the lead in making certain that the integrity of data in the lab is managed and maintained.

2 Use a networked system, ideally with a database.

Stand-alone systems should not be used in a regulated environment.

3 Document the system configuration and manage all changes to it.

You must document and ensure the configuration protects the records.

4 Work electronically and use electronic signatures.

Try to work electronically wherever possible. The advantage is that the data are maintained within the system. Don’t use a hybrid because you have two incompatible formats (worst possible situation).

5 Allocate each user a unique identity and use adequate password strength.

Ensure that you have unique user identities, so that you can attribute the work to a single individual.

6 Separate roles to avoid conflict of interest. Separate the roles within any computerized system. Typically “Admin” should only be accessible by IT or a small group of people, not standard laboratory workers. Exception if there are only 1–2 users (in which case it is necessary to share the roles).

7 Define methods that can and cannot be adjusted.

Consider which methods within a system can actually be changed and which cannot.

8 Have a standard operating procedure for data manipulation.

For chromatography data systems, a standard operating procedure (SOP) for both automatic and manual integration is necessary.

9 Ensure staff are trained and competent. The need for an SOP is clear, but do people understand it and are they competent to use it? Ensure the people are trained, both in data integrity and the instrumental techniques they are using.

10 Carry out effective self-inspections or internal audits.

Make certain that internal audits don’t just focus on paper. Instead, they should go deeper and look at things within the computerized system.

Contin. on page 48


Mura

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es

Shipping temperature-sensitive

pharmaceuticals, such as biologi-

cal products, has never been easy.

Today, changing global require-

ments and a more complex supply chain

are driving more conservative approaches

to temperature control. For products such

as cell therapies and tissue culture, cryo-

genic shipping is now preferred. Other

biopharmaceuticals are being shipped at

lower tempertures, and even small-mol-

ecule-based pharmaceuticals that might

have been shipped at ambient tempera-

tures a few years ago, must now be kept at

controlled temperatures.

Transport has become much more

than simply moving product from one

place to another. It’s now being seen

as a “mobile form of storage,” says

Volker Kirschner, director of tempera-

ture control solutions for World Courier

Management Co, part of Amerisource

Bergen. This is especially true in Europe,

he says, where the European Union (EU)

began strengthening its good distribution

practice (GDP) guidelines three years ago.

Nothing can be taken for granted.

“From political unrest to pandemic

concerns, the possibility of changes in

shipping patterns and logistics strategies

is quite real,” he says, and developing

a global transport strategy has become

more complex.

Specialists in cold-chain shipment

are responding to regulatory challenges

and uncertainty by investing in new

technology and IT, and strengthening

partnerships with specialty logistics

providers. For their pharmaceutical and

biopharm customers, they caution, care-

ful risk-assessment is the only way to

avoid costly product shipment problems.

Cold Chain: Going the Extra Mile

Agnes Shanley

Real-time GPS technology,

better IT connections,

and more conservative,

controlled shipping

temperatures are improving the shipment

of sensitive pharmaceuticals.

Cold Chain


Many are adding services and prod-

ucts to help make this task easier.

THE RISE OF GOOD DISTRIBUTION PRACTICES The most pronounced recent

change in the cold-chain market

has been more complex global

regulations, from the EU’s GDP

guidelines for pharmaceuticals to

the Drug Supply Chain Security

Act in the United States. “It can be

difficult for companies, especially

smaller ones, to know where to

begin, and how to revamp compli-

ance processes to stay ahead,” says

Wanis Kabbaj, director of global

healthcare strategy for UPS.

Additional requirements (e.g.,

for controlled room temperature)

have increased compliance chal-

lenges. Many drugs that were once

shipped at ambient emperatures

must now be shipped at 15 °F to

25 °C, increasing costs at a time

when most pharmaceutical com-

panies are trying to reduce logis-

tic and supply chain operations

budgets, says Ariette Van Strien,

chief commercial officer at Marken

Global Life Science Supply Chain

Solutions, which specializes in cold

chain and logistics.

Compliance has become espe-

cially challenging for clinical trial

supply shipments, she says, noting

that suppliers must scrutinize their

global network, enhance tracking

capabilities, and improve quality

standards.

Of utmost importance, says

Kirschner, are quality management

systems (QMS) and documentation;

personnel and training; risk man-

agement, especially regarding stan-

dard operating procedures (SOPs);

facilities and storage requirements;

and transport. “Quality depart-

ments within manufacturers’ sup-

ply-chain operations are gaining

more and more influence,” he says,

noting that regulatory agencies

expect pharmaceutical manufac-

turers to take a risk-based approach

and enforce audits and quality

agreements.

MANAGING RISKThe most c r it ica l aspec t to

maintaining stability and integ-

rity is choosing the most appropri-

ate temperature conditions for the

product and, consequently, the best

packaging for maintaining those

conditions, says Mark Sawicki,

chief commercial officer at the cold

chain specialist, Cryoport.

A perfect example, Sawicki says, is

the distribution of critical biomarker

samples. Under currently accepted

practice, these samples are shipped

on dry ice. However, he notes, this

shipping method has been con-

nected with a 15–30% product com-

promise or failure rate. As a result,

more companies are opting to use

cryogenic temperatures to ship bio-

markers, to help eliminate potential

risks associated with transit times

and temperature excursions.

PCI Pharma Services, a diversi-

fied contract services firm that offers

cold-chain expertise, has seen a dra-

matic increase in demand for storing

product at ultra-low temperatures,

from -40°C down to -196°C, and has

adapted its products and services to

focus on supporting these temper-

atures for storage, packaging, and

shipments, says Samantha James,

associate director of clinical services.

Pharmaceutical manufactur-

ers need to conduct more critical,

comprehensive shipping studies

on all materials transported in

support of a clinical program,

says Sawicki, not only looking at

sample failure but the impact of

the selected transport medium on

assay or product performance.

“We consult with every client

to select the best system and tem-

perature-control solution, whether

dry ice, liquid nitrogen, or phase-

change materials (PCMs) to ship

pharmaceutical products, diagnos-

tic specimens, biotherapeutics, and

tissue samples,” says Van Strien.

Generally, the most crucial fac-

tors to consider include therapeutic

indication, route, mode of trans-

portation, and product sensitivity,

she says. “Because a pharmaceuti-

cal’s stability is not always known

in early trial phases, additional

caution may be necessary, and pro-

tection for any changes in environ-

mental conditions.”

Van Strien advocates developing

a thorough risk assessment to help

ensure product stability from ori-

gin to destination, including stud-

ies of the following:

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strategy.

For clinical products that are

only available in small samples,

incorrectly handled shipping and

transportation can put millions or

even billions of revenue dollars at

risk by compromising clinical stud-

ies, potentially delaying product

commercialization, notes Kirschner.

When minimizing risk, it is impor-

tant for manufacturers to consider

the package type and choice of tem-

perature-control mechanisms, envi-

ronmental concerns, value of the

ingredients in the product, temper-

ature requirements and variables,

and overall shipping costs.

Suppliers need to master non-

technical issues as well, including

local politics and security issues,

especially when conducting clini-

cal trials in new geographic mar-

kets. Proactive planning is required,

Kirschner says, to help protect the

integrity of products and, ulti-

mately, the safety of patients who

use those pharmaceuticals.

Generally, he notes, up-to-date

methods should be used to assess

risk. For example, he says, failure

mode and effects analysis (FMEA)

can be conducted, and risk prior-

ity numbers (RPNs) assigned, based

Cold Chain


on the probabilities and severities

of certain adverse events occur-

ring during transport. These RPNs

would help determine whether mit-

igation is required, and what type

would work best for the specific

product, market, and application.

Temperature is not the only

variable to consider. Many phar-

maceuticals and biopharma man-

ufacturers require a great deal of

stability protection from temper-

ature, shock, vibration, humid-

ity, and light, explains Kabbaj.

“Healthcare logistics has little

room for error, so specialized pack-

aging, advanced temperature and

location monitoring leveraging

global control towers, and in-tran-

sit intervention are all critical.”

In add it ion, Kabbaj says ,

extended time in transit when

product is being transported inland

may expose the packaging to tem-

perature fluctuations that increase

risks. Ocean transportation risk fac-

tors include container placement

on the vessel, sunlight exposure,

container insulation, and dwell

time on the dock—all introducing

additional packaging stressors.

He suggests that pharmaceuti-

cal manufacturing professionals

take the following steps to ensure

that they are covering all the bases

required for risk mitigation:

t� Test packaging, including seals,

for endurance, and put prototypes

t hrough compression, impac t ,

vibration, temperature, humidity,

and shock tests. Package optimi-

zation should include validation

through design qualification,

operational qualification, and

per for mance qua l i f icat ion

protocols.

t� Optimize packaging and transpor-

tation together. Do not develop

a cold-chain solution sequen-

tially with assumptions based

on transportation, and then

the transportation solution that

comes with pack-out expiry.

Pairing a lighter and cheaper

cold-chain packaging with expe-

dited air transportation, for

example, may prove to be more

effective than opting for a slower

mode of transportation (e.g.,

ground shipping) with complex,

heavy, and bulky packaging.

t� Innovate continually to create

bet ter ef f iciencies. Opt imize

the shipping carton, mini-

miz ing unused space, and

select more precise packaging

configurations.

“Pharmaceutical manufactur-

ers that do not continually review,

evaluate, and update packaging

best practices leave themselves

open to product risk and cost inef-

ficiencies,” Kabbaj says. “Leave

room for new ideas, material inno-

vations, and new packaging manu-

facturers by engaging cold chain

partners in a collaborative mode.”

TECHNOLOGIES ENABLE FASTER RESPONSECold-chain service providers have

been developing new technologies

to help pharmaceutical companies

better manage risk and respond to

problems. “New requirements are

driving developments in packag-

ing and monitoring technologies,”

says Kirschner. As an example, he

points to newer semi-active pack-

aging technologies that allow for

longer durations of temperature

control with lighter materials that

have less environmental impact.

In addition, he says, passive pack-

aging solutions have been devel-

oped that can provide proven

temperature control over long dis-

tances. Independent testing has

shown these solutions to perform

five to seven times more efficiently

than semi-active solutions, he says.

Kirschner also points to new phase-

change materials (PCMs), comprised

of paraffin or salt-based solutions,

that allow for more precise tem-

perature control to maintain prod-

uct stability over long distances or

through extreme climates.

Many packaging manufactur-

ers are now developing their own

vacuum-insulated panel (VIP)

containers, combined with PCM

solutions, for easier handling and

storage. Additionally, he says, on

the monitoring side, developments

in global positioning software

(GPS) and tracking equipment

now include automatic start-up

and shutdown mechanisms that

can provide a real-time view into

a shipment’s status. Many tech-

nology providers have invested in

real-time, GPS-enabled data log-

gers. Combined with improved

data access, through customer

portals and better IT, the technol-

ogy offers immediate insights into

bottlenecks and delays.

LOGGING MORE THAN JUST TEMPERATURE“These data loggers provide the

ability to track both condition and

chain-of-custody, in real time,”

says Sawicki. They can track, not

just temperature, but also monitor

the impact of external influences

on container integrity including

orientation and package damage,

he adds, and can also tell whether

the container has been opened.

Examples of new cold-chain

data loggers include Sensitech’s

TempTale and Nexleaf’s ColdTrace

systems. Contract service suppli-

ers are also offering enhanced IT

connections. PCI Pharma Services,

for instance, has developed a por-

tal to allow clients to access real-

time data, 24/7. “They can monitor

stock levels, and view and trace

all live shipments associated with

their project, with direct links to

courier tracking,” says James.

Last year, Marken introduced

Sentry, a GPS-enabled sensor plat-

form, designed specifically for

pharma with real-time track and

trace capability through custom-

designed software that monitors,

records, and reports on location,

temperature, motion, shock, expo-

Cold Chain


Cold Chain

sure to light (i.e., when the box

opens), atmospheric pressure, and

remaining battery life. The device

transmits in real time and commu-

nicates through customized cloud-

based software that connects with

Marken’s Maestro IT system, says

Van Strien.

Today, major cold-chain trends

include increased use of temper-

ature-managed shipping, and an

interest in evaluating transport

methods and equipment critically,

to maximize sample integrity, says

Sawicki. There is also an increased

emphasis on protecting the prod-

ucts of cell, gene, and immuno-

therapy, says Van Strien, and the

complex supply chain logistics that

these products require.

CRYOGENIC SHIPMENT FOR CELL THERAPIESA growing number of biopharm

companies must now ship cell- and

gene-based therapies, says Kabbaj.

Such products require cryogenic

storage at temperatures -238 °F

and below. In the past, shippers

relied on dry ice (which performs

best at approximately -108.4 °C),

he explains, but dry ice emits car-

bon gas, which can damage pro-

teins in biologic shipments, and

might be harmful to handlers and

to the environment. “Not only is

dry ice shipping subject to a wide

range of international regulations,

it simply does not provide a cold

enough temperature for many new

specialty therapies” he says.

Yet another important trend,

says Van Str ien, is increased

demand of remote shipping, for

example, of clinical trial dosages

to patients’ homes. “We will con-

tinue to develop further services

to allow patients to participate

in clinical trials from home, or

remote sites of their choosing,”

she says.

Marken has also introduced an

enhanced online booking app

for investigator sites that is pre-

programmed by study. It has also

integrated enhanced scanning

technologies throughout its global

network. “More mobile technol-

ogy will help treat patients at

locations of their choosing,” says

Van Strien. ◆

This transfer of data is achiev-

able using a laboratory execu-

tion system (LES), such as LabX,

which enables a variety of instru-

ments to be directly connected

( ba lances , t it rator s , densit y

meters, refractometers, thermal

analysis instruments, and pH

meters). Useful features such as

SOP-user guidance on the instru-

ment terminal, automatic results

capture in a database, and real-

time data access support trace-

ability of data and compliance

with the GAMP data integrity cri-

teria in ALCOA+.

As regulators continue to tighten

their inspection approaches, it is

crucial for managers and scientists

in regulated GXP laboratories to

understand these criteria for data

integrity and to assess and improve

laboratory data management

processes to ensure compliance

with current regulations. Only

after all these points have been

addressed can data integrity truly

be achieved.

REFERENCES 1. C. Rosa, “Current Regulatory/Inspection

Issues Related to Supply Chain,” Food and Drug Law Institute (FDLI), Conference Understanding cGMPS–What Attorneys Need to Know, Washington DC, July 15, 2014, www.fdli.org/docs/cgmps/carmelo-rosa.pdf?sfvrsn=0

2. MHRA, Guidance for Industry on Data

Integrity (MHRA, March 2015), www.gov.uk/government/uploads/system/uploads/attachment_data/file/412735/Data_integrity_definitions_and_guidance_v2.pdf

3. FDA, “Glossary of Computer System Software Development Terminology,” 1995, www.fda.gov/iceci/inspections/inspectionguides/ucm074875.htm

4. European Commission, EudraLex, The

Rules Governing Medicinal Products in

the European Union, Volume 4, Good

Manufacturing Practice, Chapter 4 Documentation (June 2011), http://ec.europa.eu/health/files/eudralex/vol-4/chapter4_01-2011_en.pdf

5. European Commission, EudraLex, The

Rules Governing Medicinal Products in the

European Union, Volume 4, Good

Manufacturing Practice, Annex 11 Computerised Systems (January 2011), http://ec.europa.eu/health/files/eudralex/vol-4/annex11_01-2011_en.pdf

6. US Electronic Code of Federal

Regulations, 21 CFR 211.194(a). 7. FDA, FDA Warning Letter to Trifarma

S.p.A, July 2014, www.fda.gov/ICECI/enforcementactions/warningletters/ 2014/ucm404316.htm

8. FDA, FDA Warning Letter to Ipca Laboratories Ltd., January 2016, www.fda.gov/ICECI/enforcementactions/warningletters/ucm484910.htm

9. FDA, FDA Warning Letter to Micro Laboratories Ltd., January 2015, www.fda.gov/iceci/enforcementactions/warningletters/2015/ucm431456.htm

10. FDA, Questions and Answers on Current

Good Manufacturing Practices, Good

Guidance Practices, Level 2 Guidance–Records and Reports, www.fda.gov/Drugs/ Guidance ComplianceRegulatoryInformation/Guidances/ ucm124787.htm

11. EMA, GCP Inspectors Working Group publication, Reflection paper on expectations for electronic source data and data transcribed to electronic data collection tools in clinical trials (London, June 2010), www.ema.europa.eu/docs/en_GB/document_library/Regulatory_and_procedural_guideline/2010/08/WC500095754.pdf

12. R.D. McDowall, LCGC 27 (9) (September 2014), www.chromatographyonline.com/role-chromatography-data-systems-fraud-and-falsification

13. FDA, FDA Warning Letter to RPG Life Sciences Ltd., May 2013, www.fda.gov/ICECI/EnforcementActions/WarningLetters/2013/ucm355294.htm

14. FDA, FDA Warning Letter to Wockhardt Ltd., July 2013, www.fda.gov/ICECI/EnforcementActions/WarningLetters/ 2013/ucm361928.htm ◆

Data Integrity—Contin. from page 44


Ask the Expert

retrieved and sent for analysis. The

green gel was identified as contain-

ing copper. The company was still

convinced that the contamination

was due to poor quality control on

the part of the vial manufacturer

and continued operations and work-

ing with the vial manufacturer to

determine the source of the copper.

To help facilitate the investigation,

the firm hired a consultant to go to

the glass manufacturer and reaudit

their facility. The auditor could not

identify the source of the copper at

the vial manufacturer and recom-

mended that the company reevalu-

ate their operations for the presence

of copper. The company continued

to manufacture product but agreed

to reevaluate the facility for potential

sources of copper.

This time the facilities and main-

tenance personnel opened up the

line as they would during a routine

maintenance shut down. When they

opened the depyrogenation tunnel,

they discovered the presence of cop-

per on the top of the HEPA filters as

well as a coating of green on the side

doors to the tunnel. Manufacturing

was finally halted on the line until

the in-depth evaluation could be

performed.

The root cause of the problem was

determined to be a faulty cooling

valve in the depyrogenation tunnel,

which was identified as a potential

root cause but ultimately not pur-

sued because it was at the bottom of

the probability list and rated as pos-

sible but highly unlikely. The tunnel

in question was 20 years old and was

bulit with copper piping above the

line and the HEPA filters.

Although the line was routinely

maintained and checked, there was

no alarm associated with the cool-

ing valves to indicate a failure. The

failing valve caused liquid to con-

dense on the copper lines and drip

onto the HEPA filters. As more and

more liquid collected on the fil-

ters, the stress caused the filters to

breach, relieving the pressure. This

breaching happened on a predict-

able seven-day cycle. Ultimately, the

incident involved the investigation

of lots manufactured on the line

over a sixth-month period, which

was the last time the cooling valve

was inspected and determined to be

functioning to standards. Once the

problem was properly identified, the

effective corrective action could be

taken; however, by that time, 28

lots of product manufactured for

several different clients during the

two-month period were rejected

because of the presence of green

vials intermittantly detected dur-

ing the inspection process. If the

site had conducted a thorough

investigation and cleared their

equipment, personnel, etc., by pur-

suing the identified root causes

before jumping to the conclusion

the vial manufacturer was at fault,

they could have stopped manufac-

turing and corrected the problem

before the loss of 28 lots of product.

CONCLUSIONThe bottom line is there are many

perspectives on what constitutes

a good CAPA system, but the real-

ity is the quality and thoroughness

of the investigations ultimately

drive the effectiveness of the CAPA.

When conducting the investiga-

tion, it is important not to jump

to conclusions on what caused the

non-conformance. The investiga-

tion should use root-cause analy-

sis tools and should address why

potential areas are either elim-

inated as the root cause or are a

potential cause of the non-con-

formance. If you can conduct a

complete investigation, you will

ultimately have a robust CAPA

program. ◆

Your opinion matters.

Have a common regulatory or compliance question? Send it to [email protected] and it may appear in a future column.

The key to a robust

CAPA system lies in

the thoroughness

and quality of the

investigation.

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Ask The Expert—Contin. from page 50


Fa

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Susan Schniepp and Andrew Harrison, both of Regulatory

Compliance Associates, discuss how to create a

robust CAPA system and how to identify root cause.

Q: I work for a contract manufacturer,

and I think we have a fairly robust cor-

rective actions and preventive actions (CAPA)

system. Sometimes we close our CAPAs before

we have completed our effectiveness check due

to the timeline needed to implement and mea-

sure the effectiveness of the preventive action.

Occasionally, we fail our effectiveness checks,

requiring us to reopen our CAPA. This creates a

lot of concern during audits. Is there something

I can do to prevent this from happening?

A: The key to a robust CAPA system lies

in the thoroughness and quality of the

investigation. Based on the limited informa-

tion, it sounds like you are having trouble rec-

ognizing the root cause(s) identified during the

investigation. The investigation process should

make use of root-cause analysis tools designed

to examine the impact of the equipment, pro-

cess, people, materials, environment, and man-

agement on the identified non-conformance.

The investigation process should review each

possible root cause; the investigators should

either eliminate it or it should become part of

the corrective action. As the elimination pro-

cess progresses, the investigation will naturally

and logically hone in on the root cause(s) of

the non-conformance. Any potential root cause

that can’t be eliminated needs to be remedi-

ated. Most companies make the mistake of

stopping too soon and not pursuing all the

possible root causes that can’t be eliminated in

the investigation stage, which leaves them in

the situation you have described.

ROOT CAUSE CASE STUDYIt might help if we look at a situation where

a probable root cause was identified but not

pursued as part of the preventive action. The

incident involved the detection of a discolored

vial during inspection of lyophilized vials. The

vials inspectors discovered several green-hued

vials before the packaging phase of the opera-

tion. What seemed to be a simple issue isolated

to one batch grew and ultimately affected more

than 28 batches produced over a two-month

period. Several green-hued vials were discov-

ered during inspection of a lyophilized batch

of product that was produced on a 20-year-old

automated line.

The inspectors that discoved the vials imme-

diately informed the quality department, and

an investigation was opened. The lot was put

on quality assurance (QA) hold, and the vials

were sent out for analysis. Manufacturing on

the line was continued while the investigation

was being performed. The results of the analysis

indicated the green color in the vial was due to

the presence of copper. The firm was unable to

determine the source of copper in their opera-

tion and concluded the copper was most prob-

ably due to contamination of the vial at the vial

manufacturer and the investigation was closed.

Seven days after the detection of the first

green vials during the manufacturing of a

different lyophilized product, the same issue

occurred. The original investigation was re-

opened and a for-cause audit was performed at

the vial manufacturer. The results of the for-

cause audit were inconclusive with no defini-

tive source of the copper identified. During

the investigation into the vial manufacturer,

the company continued to manufacturer other

products and implemented a 100% inspection

of incoming vials before use.

Seven days later, a line operator noticed a vial

exiting the depyrogenation tunnel that con-

tained a green, gel-like blob in it. The manu-

facturing run was stopped, and the vial was

Creating Robust CAPA Systems

Susan Schniepp is distinguished fellow, and Andrew

Harrison is chief regulatory affairs officer and general

counsel, both of Regulatory Compliance Associates.

Ask the Expert

Contin. on page 49

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