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The Science & Business of Biopharmaceuticals

INTERNATIONAL

REALIZING

THE POTENTIAL

OF CAR-T CELL

THERAPIES

Bio

Ph

arm

Intern

atio

nal

MA

Y 2

016

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inish

I Sin

gle

-Use

Syste

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May 2016

Volume 29 Number 5

PEER-REVIEWED

IDENTITY TESTING OF

MULTI-COMPONENT

CELL-CULTURE MEDIA

QUALITY

ENSURING THE

VIRAL SAFETY OF

BIOTHERAPEUTICS

OUTSOURCING

OUTSOURCING OF

BIOMANUFACTURING

IN 2016

www.biopharminternational.com

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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]

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East Coast Sales Manager Scott Vail [email protected]

European Sales Manager Linda Hewitt [email protected]

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

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AUDIENCE DEVELOPmENT

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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

ES780670_BP0516_003.pgs 05.04.2016 01:52 ADV blackyellowmagentacyan

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

BioPharm International�JT�TFMFDUJWFMZ�BCTUSBDUFE�PS�JOEFYFE�JO��r�Biological Sciences Database (Cambridge Scientifi c Abstracts)�r�Biotechnology and Bioengineering Database (Cambridge Scientifi c Abstracts)�r�Biotechnology Citation Index (ISI/Thomson Scientifi c)�r�Chemical Abstracts (CAS) rŞScience Citation Index Expanded (ISI/Thomson Scientifi c)�r�Web of Science (ISI/Thomson Scientifi c)

6 Guest Editorial Policies for patient access to life-saving therapies must keep pace with biomedical innovation. James C. Greenwood

8 US Regulatory Beat FDA outlines its recommen-dations on some of the industry’s hottest topics.

10 Perspectives on Outsourcing Growth may be slowing, but outsourcing activity remains healthy.Eric Langer

46 Troubleshooting Material compatibility, material sourcing, facility layout, and training are crucial aspects of a disposable fill-finish system.Jennifer Markarian

48 Product Spotlight

48 New Technology Showcase

49 BIO Exhibitor Guide

50 Ask the Expert How to report quality metrics to FDA.

Siegfried Schmitt

50 Ad Index Cover: mevans/National Institutes of Health/Stocktrek Images/

Getty Images; Dan Ward

CELL THERAPIES

AND BIOPROCESSING

Realizing the Potential

of CAR-T Cell Therapies

Cynthia A. ChallenerEarly successes drive the need to

overcome safety issues, increase efficacy,

and address manufacturing challenges. 13

UPSTREAM PROCESSING

Designing a

Biomanufacturing Facility

Incorporating Single-Use

Technologies

Peter Genest and John JosephAsking the right questions is crucial. 20

SHIPPING SERVICES

Qualification and

Validation of Single-Use

Shipping Systems

Nicolas Voute, Elisabeth Vachette, Delphine Audubey, Stephane Baud, and Frederic BazinThe authors provide their

perspectives on shipping validation. 24

PEER-REVIEWED

Platform Approach for

the Identity Testing of Multi-

Component Cell-Culture Media

Satish Mallya, Benjamin Lay, Lihong McAleer, Alexandria Emory, and Nataliya AfoninaSeven cell-culture media with essentially

similar composition were examined. 30

RAW MATERIALS TESTING

An Integrated Approach

to Ensure the Viral Safety

of Biotherapeutics

Mark PlavsicThis article proposes integrated

solutions for systemic and proactive

viral risk mitigation. 40

Volume 29 Number 5 May 2016

FEATURES

The Science & Business of Biopharmaceuticals

INTERNATIONAL

REALIZING

THE POTENTIAL

OF CAR-T CELL

THERAPIES

May 2016

Volume 29 Number 5

PEER-REVIEWED

IDENTITY TESTING OF

MULTI-COMPONENT

CELL-CULTURE MEDIA

QUALITY

ENSURING THE

VIRAL SAFETY OF

BIOTHERAPEUTICS

OUTSOURCING

OUTSOURCING OF

BIOMANUFACTURING

IN 2016

www.biopharminternational.com

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Guest Editorial

Policies for

patient access to

life-saving therapies

must keep pace

with biomedical

innovation.

The Value of Saving Lives

Our nation is in the midst of an important debate regarding medical inno-

vation and how we should be paying for these advances. The irony is that

much of this discussion is being sparked by breakthrough cures and thera-

pies that have been developed for diseases that affect large patient populations—

exactly the kind of medicine that society needs.

While these are exciting and important developments, particularly for the

patients whose lives will be transformed by these therapies, the reality is that

access to our healthcare system is failing to keep pace with the work occurring in

labs across the country. In recent years, we have seen incredible advances in medi-

cal innovation and science, such as:

� t� Hepatitis C. In the past few years, new treatments for Hepatitis C came

onto the market with clinically demonstrated cure rates above 90%, up

from a mere 6% in the 1990s (1,2). These incredible innovations have the

potential to help millions of people lead healthier and more productive

lives, and to save potentially billions of dollars in healthcare costs related

to liver transplants and other costly and difficult medical procedures.

� t� Cancer. Since the early 1990s, the death rate for cancer has fallen by over

20%, and cancer patients have enjoyed 50 million additional years of

life collectively, thanks in large part to new medicines (3–5).

� t� HIV/AIDS. A near-certain death sentence not long ago, HIV/AIDS is now a

manageable chronic condition for many patients thanks to the develop-

ment of highly active antiretroviral therapies.

In 2015, FDA approved 48 new drugs, including many new cancer treatments

as well as treatments for cystic fibrosis, heart failure, high cholesterol, and many

other conditions. For the second year in a row, FDA approved more drugs to treat

rare diseases than ever before.

New drugs are the result of decades of hard work in labs and billions of dollars in

investments. These vast sums must be raised from investors—ranging from retire-

ment funds to individual purchasers of stocks—who are willing to take significant

risks. They will only do so if there is an expectation of reasonable returns. Only one

in 10,000 potential medicines researched goes on to become an approved treatment.

Yet the ecosystem that makes the United States the leader in medical innova-

tion is under attack. We are seeing more calls for innovation-crushing price con-

trols and increased government intervention. Some critics have even suggested

that insurance companies and federal and state programs restrict which medicines

patients may access, with cancer treatments frequently coming under attack. This

approach is extremely short-sighted and would be devastating for patients, who

rightly expect their physician to recommend, and their insurance provider to

cover, the medicine that is best for their particular situation.

We need to look at the outdated, cumbersome, and inefficient framework for

accessing life-saving and enhancing treatments and cures. Insurance companies

and policymakers must keep pace with biomedical innovation by ensuring access

to the latest medical breakthroughs for patients today and encouraging sustained

medical innovation for those who will need it tomorrow. Patients deserve no less.

References 1. E. Lawitz, et al., The Lancet, 383 (9916) 515-523 (Nov. 5, 2013).

2. R.T. Marinho and D.P. Barreira, World J Gastroenterol 19 (40) 6703-6709 (Oct. 28, 2013).

3. E.C. Sun, et al., “An Economic Evaluation of the War on Cancer,” National Bureau of

Economic Research (November 2009).

4. F.R. Lichtenberg, et al., “Has Medical Innovation Reduced Cancer Mortality?” National

Bureau of Economic Research (Revised October 2013).

5. T. Philipson, et al., Health Affairs 31 (4) 1–9 (April 2012). X

James C. Greenwood is president

and CEO of the Biotechnology

Innovation Organization (BIO),

www.convention.bio.org.

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New Brunswick™ is a trademark of Eppendorf AG, Germany.

U.S. Design Patents are listed on www.eppendorf.com/ip. All rights reserved, including graphics and images. Copyright © 2016 by Eppendorf AG.

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Regulatory Beat

Vis

ion

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fAm

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/Jo

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FDA issued a number of new guidance

documents in March and April of 2016

addressing some of the industry’s most

debated topics. The following provides a brief

overview of a few of these documents.

BIOSIMILAR LABELINGIn March 2016, FDA released draft guidance

on the labeling of biosimilar products (1). The

guidance states that information concerning

a clinical study of a biosimilar should only be

included in the product’s labeling if it is “neces-

sary to inform safe and effective use by a health

care practitioner.” This will only be required

in certain situations, FDA wrote, because stud-

ies done on biosimilars are generally meant to

show there are no clinically meaningful differ-

ences between the biosimilar and the reference

biologic, and may not be relevant to prescribers.

FDA recommends that information on a

biosimilar product label should incorporate the

relevant data from reference product’s labeling

with “product-specific modifications.” These

modifications may differ depending on the

indications for which the biosimilar is approved.

Information related to administration, prepara-

tion, storage, or safety should also be included

in the biosimilar label if different from the ref-

erence product, wrote FDA.

THERAPEUTIC PROTEINSIn April 2016, FDA released guidance (2)

addressing the development and validation of

immune assays for assessment of the immuno-

genicity of therapeutic protein products during

clinical trials.

The guidance provides recommendations for

the development and validation of screening

assays, confirmatory assays, titering

assays, and neutralization assays for

detection of anti-drug antibodies

and, on a case-by-case basis, some combination

products. The guidance does not apply to in

vitro diagnostic products and does not address

product and patient risk factors that may con-

tribute to immunogenicity.

DATA INTEGRITYAlso in April 2016, FDA published Data

Integrity and Compliance with CGMP (3), which

addresses the role of data integrity in CGMP

for drug manufacturing, finished pharmaceuti-

cals, and positron emission tomography drugs.

The guidance has been issued in response to

an increasing amount of data integrity vio-

lations found by the agency during CGMP

inspections. According to FDA, data integrity

CGMP violations have led to FDA warning let-

ters, import alerts, and consent decrees. The

new guidance answers data integrity questions

in the hope of clarifying what FDA expects

from manufacturers.

The guidance states that data should be

reliable and accurate, and companies should

implement effective strategies to manage data

integrity risks. Along with clarifying the defi-

nition of data integrity terms, the guidance

addresses the exclusion of CGMP data, work-

flow validation, data access, audits, electronic

records, and personnel training. FDA also clari-

fies sampling and testing requirements and how

to address data integrity problems.

REFERENCES 1. FDA, Labeling for Biosimilar Products, Guidance for

Industry, Draft Guidance (CDER, CBER, Silver Spring,

MD, March 2016).

2. FDA, Assay Development and Validation for

Immunogenicity Testing of Therapeutic Protein Products,

Guidance for Industry, Draft Guidance (CDER, CBER,

CDRH, Silver Spring, MD, April 2016).

3. FDA, Data Integrity and Compliance With CGMP

Guidance for Industry, Draft Guidance (CDER, CBER,

CVM, Silver Spring, MD, April 2016). ◆

FDA Releases Guidance on Biosimilars, Therapeutic Proteins, and Data IntegrityThe agency outlines its recommendations on some of the industry’s hottest topics.

The Editors of

BioPharm International

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Perspectives on Outsourcing

Do

n F

arr

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s

Outsourcing budgets have rebounded in

recent years, swinging from contrac-

tions to expansion after the 2008 reces-

sion. The budget growth of recent years appears

to be slowing, though, according to BioPlan

Associates’ 13th Annual Report and Survey of

Biopharmaceutical Manufacturing Capacity and

Production (1). Even so, the breadth of outsourc-

ing activity continues to increase, as a greater

proportion of companies undertake outsourcing

across a range of activities.

BUDGETS STILL GROWING, BUT MORE SLOWLYResults from BioPlan’s annual report indicate

that outsourcing budgets continue to grow, but

at a slower pace than observed in the past cou-

ple of years. Indeed, only slightly more than

one-third (35.7%) of respondents this year

report an increase in funding for outsourced

biopharmaceutical manufacturing, which is the

smallest proportion of all the areas examined.

For example, about 7 in 10 respondents are

hiking budgets for new capital equipment this

year, and close to two-thirds expect to increase

their funding for new technologies to improve

efficiencies for both upstream and downstream

production (see Figure 1).

On average, it is est imated

that outsourcing budgets will

rise by 2.3% this year, with the

next-smallest increases seen for

new facility construction (3.6%)

and operations for current prod-

ucts (3.7%). Moreover, the 2.3%

planned increase for 2016 is a step

back from the 3.8–3.9% range in

2014 and 2015.

The trend is a little different,

however, when factoring R&D into

the equation. Indeed, this year, a

majority (56.5%) of respondents

intend to increase spending on outsourcing of

R&D or manufacturing in the next 12 months,

up from 52.6% in 2015. More than one-quarter

expect to increase their budgets by more than

10%, with the overall estimated increase in

spending on outsourced R&D being somewhere

around 14%, which would represent a step up

from not only last year, but the prior couple of

years, also. This suggests that budgets for out-

sourced R&D are growing more quickly than for

outsourced biomanufacturing.

WHERE WILL THE BUDGETS BE ALLOCATED?Growing budgets spread across several activi-

ties within bioprocessing. This year, data indi-

cate that the activities being outsourced to at

least some extent by the largest proportion of

respondents include:

t� Analytical testing, bioassays (89.7%)

t� Toxicity testing (76.3%)

t� Fill/finish operations (74.2%)

t� Validation services (70.1%).

In each case, save for validation services, the

percentage of companies engaged in outsourc-

ing of these activities grew from 2015.

The greatest increases this year appear to

be for host-cell protein analysis testing (55.7%

outsourcing to some extent, up from 42.1% last

year); upstream process development (45.4%,

up from 38.9%); and cell-line stability testing

(62.9%, up from 55.8%).

However, breadth of outsourcing does not

necessarily equate to depth. In other words,

while many companies may be outsourcing

these activities, they may be only doing so to

small degrees. To determine just how much

activities are indeed being outsourced, respon-

dents were asked to estimate the extent to

which they were engaged in outsourcing them,

and the results were averaged out to reach an

estimate.

Outsourcing of Biomanufacturing in 2016Growth may be slowing, but outsourcing activity remains healthy.

Eric Langer

is president of

BioPlan Associates,

tel. 301.921.5979,

[email protected].

May 2016 www.biopharminternational.com BioPharm International 11


Fill-finish operations are the

most heavily outsourced, with an

estimated average of 35.6% (up

from 34.5% in 2015) of these oper-

ations being outsourced. Fill-finish

is the only area in which at least

one-third of all activities are per-

formed by contract manufacturers,

indicating that while more compa-

nies are engaging in outsourcing,

few are doing so heavily.

Beyond fill-finish, other activi-

ties that are relatively heavily out-

sourced include analytical testing

of other bioassays, toxicity testing,

and plant maintenance services.

Compared to last year, however,

the greatest change in outsourcing

depth are seen in:

t� GMP training (13.2% of this

activity overall being outsourced

up from 8.3% last year)

t� Regulatory services (10.5% share

of activities, up from 6.7%)

t� Cell line stability testing (18.1%,

up from 13.4%)

t� Upstream production operations

(10%, up from 7.5%).

Interestingly, the increase in

the depth of outsourcing of GMP

training and regulatory services

is not the result of more com-

panies outsourcing these activi-

ties. Instead, the proportion of

companies outsourcing them has

remained relat ively f lat, with

just a 3.9% relative increase for

GMP training and no increase

for regulatory services. This sug-

gests that those companies that

outsourced these activities last

year were sat isf ied with their

results and are doubling down

this year.

Looking ahead, the industry

can expect more outsourcing of

analytical testing of bioassays, fill-

finish operations, and API biolog-

ics manufacturing, per industry

respondents, at least 20% of whom

expect to outsource these activities

at significantly higher levels in the

next 24 months.

OFFSHORING PROJECTIONS REMAIN FLAT AT BESTOne of the key outsourcing trends in

recent years has been the globaliza-

tion of the outsourcing market, as

biomanufacturing clusters increas-

ingly emerge around the world.

Indeed, approximately 40% of global

biopharmaceutical manufacturing

capacity exists outside of the tradi-

tional hubs of North America and

Europe, with China and India alone

accounting for almost one-sixth of

global capacity (2).

However, many of these devel-

oping markets and hubs lack

regulatory approval for CGMP

manufacture in developed coun-

tries. Perhaps as a result, we have

yet to see an uptick in future off-

shoring projections, as evidenced

by this year’s study results.

Survey respondents were asked

to estimate the percentage of

operations currently done at their

facility that would be outsourced

internationally in five years to

India, China, or another lower-cost

country. This year, the industry

expects to off shore:

t� An average of 10.2% of clinical

trials/operations within the next

five years, a figure up slightly

from five-year projections made

from 2013–2015 (ranging from

9.3%–10.1%)

t� An average of 9.4% of biomanu-

facturing operations, down from

10.3% last year and 11.3% the

year prior

t� Just 3.9% of process develop-

ment for biomanufacturing,

outside of the 4.4–8.8% range

observed in the previous five

years.

It’s perhaps not too surprising

to see only a small percentage

of process development marked

for future off-shoring, if only as

these tend to be high-value activi-

ties, and that outsourcing them

to lower-cost countries may bring

up quality concerns or managerial

problems. By comparison, clini-

cal trials activities, where in-coun-

try trials may be mandatory and

where there is a long track record

for international contract research

organization activities, fewer con-

cerns are expected. It’s also worth

remembering that only 7.6% of

upstream process development

activities are currently outsourced

anywhere (not just offshoring),

and an even smaller percentage

(6.2%) of downstream process

development activities are cur-

rently outsourced.

Figure 1: Budget trends for outsourced biopharmaceutical manufacturing.

Average outsourced manufacturing budget change, 2009-2016

Data source: BioPlan Associates’ 13th Annual Report and Survey of BioPharmaceutical Manufacturing Capacity and Production, April 2016

2016

2015

2014

2013

2012

2011

2010

2009

2.3%

3.8%

3.9%

1.7%

0.8%

-0.4%

-1.2%

-1.3%

2009 t

o 2

016 (

Years

)Percentage of increase


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CONCLUSIONThe biopharmaceutical manufac-

turing community is continuing to

increase its budgets on outsourced

manufacturing, but those budget

increases are slowing in favor of

other areas such as new capital

equipment and innovative tech-

nologies. At the same time, there

continues to be double-dig it

growth in outsourcing of R&D and

manufacturing, suggesting that

outsourcing budgets are growing

more quickly for R&D than for

manufacturing.

Data do not show a huge drop

off in off-shoring projections—and

in fact there is a slight increase in

potential off shoring of clinical tri-

als and operations—but the trend

for off shoring of biomanufactur-

ing operations and process devel-

opment is flat at best.

Nevertheless, there appears to

be wider comfort with outsourc-

ing of various activities, with

a majority of those tracked this

year being outsourced by a greater

share of respondents than last year.

Traditionally outsourced areas such

as fill-finish operations and analyt-

ical testing of bioassays continue

to be the most heavily outsourced,

but there are indications that out-

sourcing of newer areas, such as

regulatory-related activities, are on

the rise.

SURVEY METHODOLOGYBioPlan Associates’ 2016 Thirteenth

Annual Repor t and Sur vey of

Biopharmaceutical Manufacturing

Capacity and Production yields a

composite view and trend analysis

from more than 200 responsible

individuals at biopharmaceutical

manufacturers and contract man-

ufacturing organizations in 30

countries. The methodology also

included more than 150 direct sup-

pliers of materials, services, and

equipment to this industry.

REFERENCES 1. BioPlan Associates, 13th Annual Re-

port and Survey of Biopharmaceutical

Manufacturing Capacity and Produc-

tion (Rockville, MD, April 2016), www.

bioplanassociates.com/12th

2. Top 1000 Global Biopharmaceutical Fa-

cilities Index, BioPlan Associates, online

database at www.top1000bio.com. ◆


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ard

Numerous, remarkable results

in early clinical trials have

driven significant invest-

ment in cell therapies, both

by large biopharmaceutical companies

and startup biotech firms backed by

venture capital. Jain PharmaBiotech

identified more than 500 companies

involved in cell-therapy technology (1).

Adoptive cell transfer (ACT), which

uses a patient’s T cells (T lymphocytes)

that are harvested and genetically engi-

neered to produce chimeric antigen

receptors (CARs) and recognize specific

proteins (antigens) on tumor cells, is

receiving a good portion of that atten-

tion. The CAR-T cells are expanded and

then reinfused back into the patient,

where they multiply and attack the tar-

geted cancer cells.

There are challenges to ACT using

T cells. T cell expansion and the per-

sistence of infused cells can vary sig-

nificantly from patient to patient, and

both directly influence the treatment

outcome. Conditioning of the patient

in advance of infusion can have an

impact, as can the tumor microenvi-

ronment. Selection of the most effec-

tive, longest-lasting T cells and the

right antigen targets is a key focus of

research efforts today. On-target, off-

tumor toxicity and cytokine release

syndrome are important safety issues

that must be addressed. Technologies

for the manufacture of therapies based

on living cells on a commercial scale

must also be developed.

T cells are ideal vehicles for immu-

notherapy because they are central to

Realizing the Potential of CAR-T Cell Therapies

Cynthia A. Challener

Early successes drive the need

to overcome safety issues,

increase efficacy, and

address manufacturing

challenges.

Cynthia A. Challener, PhD,

is a contributing editor to

BioPharm International.

Cell Therapies and Bioprocessing



cell-mediated immunity and are

involved in long-term, antigen-

specific responses. They have, in

fact, been used in the past to treat

various viral infections. Once T

cells bind to cells expressing the

target antigen, they acquire the

specif ic functional properties

necessary for eliminating the tar-

get cells and generate long-last-

ing memory T cells that provide

a similar response if any target

cells reappear. CAR-T cell therapy

leverages these natural behaviors

of T cells. T cells can also be engi-

neered to express modified T-cell

receptors (TCRs) as an alternative

type of cell therapy.

AUTOLOGOUS VS. ALLOGENEICCAR-T cell therapies in clini-

cal development today are largely

autologous therapies; the genetically

modified cells originate from tissue

taken from an individual patient

and are returned to that patient

once expanded. There is some con-

cern in the industry that the need

Advancing cell therapy safety

Two main safety issues have been identified in the early-

phase clinical trials conducted to date for chimeric antigen

receptor (CAR)-T cell therapies. Cytokine release syndrome

(CRS) occurs in some patients, particularly those with

high tumor loads, when the CAR-T cells expand rapidly

and cause the release of large quantities of cytokines

(interferon, interleukins, etc.) that can lead to low blood

pressure and other problems that can be fatal if not treated.

On-target, off-cancer toxicity can also occur when the

CAR-T cells attack healthy cells due to the presence of

similar antigens.

“The ability to regulate the activity of CARs and find

new ways to proactively manage CRS is an area of

research interest across academia and industry,” observes

Eric Althoff, head of global media relations with Novartis.

Present approaches to minimizing CRS include the use of

lower initial doses for patients with greater tumor density

to reduce the cancer load and combined treatment with

cytokine blockers. “As we gain confidence with the efficacy

of CAR-T cell therapies, they will be used to treat patients

at a much earlier stage of disease, and thus there will be

reduced likelihood of CRS due to the reduced presence of

tumor cells,” notes Hyam “Hy” Levitsky, executive vice-

president and chief scientific officer of Juno Therapeutics.

Because on-target, of f-cancer toxici t y in l ive

patients is difficult to predict in the laboratory, initial

tests in human beings are performed using low doses

and careful monitoring. CAR-T cells are also being

engineered to include safety switches via transduction

of T cells with marker or suicide genes that allow for

their selective destruction in the event of severe toxicity.

“For example, CAR-T cells can be marked for destruction

via expression of a ligand on the cell surface, such as a

truncated epidermal growth factor receptor-like (tEGFR)

protein or the synthetic RQR8 peptide. If severe toxicity is

observed following administration of CAR-T cells, biologic

drugs specific for tEGFR (e.g., cetuximab) or RQR8 (e.g.,

rituximab) can be administered, resulting in destruction of

the marked CAR-T cells,” explains Bruce McCreedy, senior

vice-president of cell therapy at Precision BioSciences.

Another type of safety switch involves dimerization of a

fusion protein consisting of a small-molecule drug binding

domain with an apoptotic protein intermediate such as the

iCasp9/AP1903 (Bellicum Pharmaceuticals). Administration

of an inert small-molecule dimerizer induces activation of

the apoptotic pathway, leading to selective death of the

CAR-T cells.

Bispecific CAR-T cell constructs that express two CARs

are also being evaluated to address on-target, off-cancer

toxicity. The activity of these CAR-T cells can be amplified

or inhibited upon binding of each CAR expressed on the

surface of the T cell. For instance, according to McCreedy,

a bispecific CAR-T cell that expresses an activating CAR

specific for a tumor cell antigen and an inhibitory CAR

that recognizes an antigen expressed on normal tissues

can limit potential for on-target, off-tumor damage. “Both

an activating and an inhibitory signal would be delivered

upon binding to normal tissues if the tumor antigen-

specific CAR is also engaged (e.g., due to cross-reactivity),

whereas only an activating signal would be delivered to

a tumor cell that lacks expression of the normal tissue

antigen,” McCreedy says.

CAR-T cells are also being designed that include inducible

expression and/or signaling capabilities such that upon

administration, the CAR-T cell activity can be induced

through the activity of an inducible promoter sequence

incorporated into the CAR construct or by administration of

a small-molecule drug that allows the CAR to be expressed

at the cell surface or enhances activation of CAR-T cells

upon binding of target tumor cells. “Inducible expression of

the CAR or potentiation/attenuation of CAR-T cell activity

using small molecule modulators may allow for better

control of CAR-T activity in vivo with the goal of enhancing

both the safety and efficacy of CAR-T therapies,” notes

McCreedy. Many of these advanced CAR-T cell therapies are

being evaluated in Phase I/II clinical trials.

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to perform the same level of exten-

sive quality control and testing on

such small-scale product lots as for

large-scale production will prevent

these treatments from achieving

commercial viability. Starting mate-

rial variability for autologous CAR-T

products also presents a challenge;

the T cells from each patient differ

depending upon the extent of their

disease, previous therapies, genet-

ics, and the status of their immune

system at the time of cell collection.

“These patient-specific issues will

always be a challenge to the ability

to manufacture a CAR-T product for

every patient and to the consistency

of performance observed among

CAR-T products,” says Derek Jantz,

chief scientific officer for gene edit-

ing company Precision Biosciences.

Allogeneic cell therapies, in

which the T cells are derived from

healthy donors that have been

screened for desirable characteris-

tics rather than individual patients,

have the potential to allow for

larger-scale manufacturing and

minimize the heterogeneity associ-

ated with using raw material from

individual patients. “In addition

to transduction to introduce the

CAR, gene editing using CRISPR/

cas9 (EDITAS, Intellia), TALEN

(Cellectis), ZFN (Sangamo), or hom-

ing endonucleases such as ARCUS

(Precision Biosciences) is also nec-

essary to knock out expression of

the endogenous T-cell receptor,”

says Jantz. “Such gene-edited allo-

geneic CAR-T cells would have sig-

nificantly less potential to cause

graft-versus-host disease upon

adoptive transfer to the patient and

can be reproducibly manufactured

without the variability inherent in

the manufacturing of an autolo-

gous CAR-T product,” he adds.

These CAR-T products could also

be manufactured at large scale and

stored frozen, ready to be delivered

to the patient when needed.

One downside of allogeneic ther-

apies is the need for the additional

gene editing step, which reduces

somewhat the economic advantage

that may be achieved due to larger-

scale manufacturing. In addition,

while off-the-shelf allogeneic treat-

ment products would be available

for treating patients immediately,

given that autologous therapies are

now produced in 2–3 weeks, time-

liness of delivery is not an issue

in most cases, according to Hyam

“Hy” Levitsky, executive vice-pres-

ident and chief scientific officer of

Juno Therapeutics. He also notes

that the expansion of T cells is lim-

ited. “In order to make a quantity

of an allogeneic product sufficient

to treat large numbers of patients,

there is a concern that the exten-

sive expansion needed will rapidly

lead to senescence of the alloge-

neic T cells such that when infused

into patients, their ability to further

expand would be limited,” he says.

Finally, even though editing the

T-cell receptors on allogeneic CAR-T

cells will reduce the risk of graft-ver-

sus-host disease, they are still for-

eign cells and prone to be rapidly

rejected by the host immune system,

which is much less of an issue with

autologous therapies. Furthermore,

once rejected, there is no possibil-

ity of delivering a second dose. The

question of how long these cells per-

sist following administration to the

patient must be addressed in clinical

trials of allogeneic CAR-T products.

“Important issues to be determined

in clinical trials are whether destruc-

tion of allogeneic CAR-T cells occurs

in a time frame and to an extent that

limits anti-tumor activity and the

number of observed complete and

durable responses,” Bruce McCreedy,

senior vice-president of cell therapy

at Precision BioSciences states.

BETTER CELL SELECTION“Over the past ten years there has

been a tremendous increase in our

understanding of how T cells func-

tion and how the immune system

regulates itself. This knowledge has

now enabled us to develop highly

effective T-cell-based immunother-

apies,” Levitsky observes. He adds

that one of the key challenges is to

identify and select the most effec-

tive T-cell subsets to develop into

efficacious therapies. “From an effi-

cacy perspective, the main chal-

lenges continue to be expansion and

persistence of CAR-T cells following

administration to the patient and

activity of the CAR-T cells within

the tumor microenvironment where

numerous immunosuppressive fac-

tors are at work,” agrees Jantz.

Some companies are moving

toward manufacturing schemes in

which a defined mix of CD4+ and

CD8+ CAR-T cells with naïve and

memory phenotypes (i.e., not termi-

nally differentiated and exhausted

cells that do not expand well and

persist following administration) are

represented in the final product.

Strep-tag technology (2) devel-

oped by researchers at the Fred

Hutchinson Cancer Research Center,

Technical University of Munich,

and San Raffaele Scientific Institute

in Milan looks like a promising

approach to the problem. The small

protein tag can be used to separate

out T cells carrying a CAR protein

to yield highly pure samples that

can then be expanded to provide

more potent therapies with high

regenerative potential in less time

than is needed for mixed cell sam-

ples. In addition, the researchers

have shown that by using a special

antibody that binds the Strep-tag,

engineered cells can be rapidly and

repeatedly expanded. The Strep-tag

when used in combination with a

different antibody may also serve

as a “kill switch” if cytokine release

syndrome (CRS) or other toxic events

occur. Once infused into patients, T

cells with the Strep-tag can also be

tracked using a fluorescent antibody

specific for the tag.

Juno Therapeutics, which funded

the work at the Hutchinson Center,

has an exclusive license to the




tag technology for uses related to

oncology (as well as a non-exclu-

sive license for other purposes). “We

have a significant program focused

on the development of technology

for the physical selection of specific

cells in order to generate defined

cell products. These investments are

allowing us to select and steer cells

at the early manufacturing stage,”

Levitsky states.

Another approach involves the

engineering of “armored” CAR-T

cells that are genetically modified

to express a pro-inflammatory cyto-

kine (e.g., interleukin 12, IL-12) in

addition to the CAR. The localized

secretion of IL-12 recruits help from

other immune cells and supports the

activity of CAR-T cells within the

immunosuppressive tumor microen-

vironment, according to McCreedy.

In addition, replacement of the

murine scFv (tumor targeting por-

tion of the CAR that is exposed on

the outer surface of the cell) with

human sequences that do not nega-

tively impact the binding affinity

of the scFv is expected to reduce

the frequency of patient immune

responses directed against CAR-T

cells and hopefully improve their

persistence. Gene-editing technolo-

gies are also being employed to

genetically modify CAR-T cells in

ways that render them more capa-

ble of trafficking to tumor sites and

make them less susceptible to immu-

nosuppression within the tumor

microenvironment.

MANUFACTURING CHALLENGESManufacturing of CAR-T cell thera-

pies involves multiple steps, includ-

ing collection of the raw material,

separation of the T cells, transduc-

tion with a viral vector (typically

gammaretrovirus or lentivirus) to

introduce the CAR receptor and

other genetic modifications, expan-

sion of the engineered cells, cryo-

preservation, and eventual infusion

into the patient. While effective

small-scale bioprocessing methods

have been developed to meet the

product needs for early-phase clinical

trials, because these treatments are

based on living cells (and thus the

cells must be isolated as the product,

not a recombinant protein), larger-

scale manufacturing presents unique

challenges. “An incredibly high level

of organization and standardiza-

tion of processes are both essential,”

Levitsky notes.

In a poster presented at the

American Society of Hematology

Annual Meeting in December 2015,

Novartis reported on how it has suc-

cessfully transferred cell process-

ing technology from the University

of Pennsylvania to the company’s

cell manufacturing center in Morris

Plains, NJ (3). Novartis was the first

healthcare company to initiate

Phase II CAR-T therapy trials in the

United States, Europe, Canada, and

Australia, and the manufacturing

facility now supports their global

clinical trial program, according to a

company spokesperson.

Commercial-scale cel l-ther-

apy production processes must be

designed as cost-effective, closed

manufacturing systems that are flex-

ible, yet meet cGMP manufactur-

ing requirements, and allow the use

of simple techniques for cell recov-

ery on a large scale. Cell expansion

is particularly challenging at larger

Role of contract manufacturing in cell therapy development and manufacturing

Developers of CAR-T cell therapies with products showing

successful early-stage clinical results are currently seeking

manufacturing capacity that will enable the production of

the larger quantities of material needed for Phase III trials

and eventual commercialization. Contract development and

manufacturing organizations (CDMOs) will play a key role

in helping the cell-therapy industry to realize its potential,

according to Mark Bamforth, president and CEO of Brammer

Bio, which was formed in late March 2016 through the merger

of Brammer Biopharmaceuticals and Florida Biologix as a

CDMO focused on offering cell- and gene-therapy development

and manufacturing services.

Not just any CDMO will do, however. “To tackle the

challenges posed by these novel technologies and help

accelerate their transition from the clinic to patients in need,

CDMOs must have the necessary laboratory and production

capabilities and a highly skilled team of scientists with

specialized development, manufacturing, and analytical

expertise, and a robust quality system to ensure compliance

and product safety,” Bamforth says.

While there are a number of CMOs in the United States and

Europe with experience in manufacturing retroviral, lentiviral,

and adeno-associated virus vectors under GMPs, there are

few GMP manufacturers with experience in the manufacture

and release of cellular therapies, notes Bruce McCreedy, senior

vice-president of cell therapy at Precision BioSciences. He adds

that most of the larger companies involved in the development

of CAR-T products have elected to build or acquire their own

facilities and manufacture their products in-house. Brammer

Bio and the few other CDMOs in this space (WuXi PharmaTech,

which is building its third cGMP cell-therapy production facility

in Philadelphia; PCT; and Lonza) are targeting smaller biotechs

and large manufacturers that prefer to work with strategic

contract partners.



Cell therapy growth and pains: Investment, collaboration, and controversy

Cell therapy companies are attracting interest from

investors, and drug companies are seeking partnerships and

acquistions to accelerate development. Juno Therapeutics,

Kite Pharma, and Novartis are considered the leading

developers of engineered T-cell therapies, although most

large biopharma firms have initiated research efforts and

are partnering with small biotech companies specializing

in chimeric antigen receptors (CAR)-T cell and gene-editing

technologies.

Juno’s initial public offering (IPO) of $264.6 million was

the largest biotech IPO in 2014, and within one month

the company’s valuation more than doubled from $2

billion to $4.7 billion (1). In May 2015, Juno expanded

its pipeline with the acquisition of German biotechnology

company Stage Cell Therapeutics for approximately $59

million. Juno then entered into a 10-year partnership with

Celgene to develop and commercialize immunotherapies

for the treatment of cancer and autoimmune diseases in

June 2015, for which Celgene paid a total of $1 billion.

In January 2016, Juno acquired acquired AbVitro, a

privately held biotechnology company based in Boston.

In early April 2016, Juno announced that it formed a

new cell therapy company in China—JW Biotechnology

(Shanghai) Co.—with WuXi AppTec (2). Juno is also in

collaborations with the Fred Hutchinson Cancer Research

Center, the Seattle Children’s Research Institute, and the

Memorial Sloan Kettering Cancer Center in New York and

has additional agreements or partnerships with Editas

Medicine, Sanofi, Five Prime Therapeutics, and Sutro

Biopharma, among others.

Novartis created the Novartis Cell and Gene Therapy

Unit (CGTU) and has an exclusive global collaboration with

the University of Pennsylvania (Penn) to research, develop,

and commercialize targeted CAR immunotherapies for

the treatment of cancers. The collaboration was initiated

based on CAR research conducted by Carl June and Penn

with CTL019, which targets a protein called CD19 and is

under investigation in a number of B-cell malignancies. For

the manufacture of CTL019, Novartis purchased the first

FDA-approved GMP-quality site for cell-therapy production

in late 2012 from Dendron Corporation. According to Eric

Althoff, head of global media relations with Novartis, the

company has a goal to file a biologics license application

(BLA) for CTL019 in pediatric r/r ALL and r/r DLBCL with

FDA in 2017. Penn recently unveiled its new $27-million-

dollar Novar tis-Penn Center for Advanced Cellular

Therapeutics, which will focus on CAR-T cell therapies.

Novartis invested $20 million in the center (3).

The rapid development of the cell therapy market

segment is not without controversy. June, who was widely

recognized for his role in developing T cell therapies, in

March 2016 made corrections to three articles in the New

England Journal of Medicine to acknowledge that the actual

DNA was developed by researchers at St. Jude Children’s

Research Hospital (4).

In April 2015, Juno Therapeutics reached a settlement

with Novartis over the T-cell manipulation technology

used in the creation of CAR-T immunotherapies. The

litigation began as a contract dispute in 2012 between St.

Jude’s Research Hospital and Penn and was expanded

to include a patent. Juno became a party to the litigation

through a 2013 license agreement with St. Jude to use

the patent. Novartis entered into a partnership with Penn

to develop CAR-T therapies in 2012. Novartis will pay

Juno $12.25 million upfront and milestone payments and

royalties from net sales of Novartis’ CTL019. Juno will

share the payments with St. Jude based on the terms of

their contract (5).

In February 2016, Precision Biosciences announced that

it is also involved in a global collaboration with Baxalta to

develop a broad series of allogeneic CAR-T cell therapies.

The partnership combines Precision BioSciences’ ARCUS

gene-editing technology with Baxalta’s global infrastructure,

expertise, and growing immuno-oncology portfolio with the

goal of developing disruptive treatments for underserved

cancers. Precision BioSciences will receive an upfront

payment of $105 million from Baxalta, with additional

option fees, developmental, clinical, regulatory, and sales

milestones, potentially totaling up to $1.6 billion, plus

royalties on worldwide sales (6).

References

1. T. Soper, “Juno Stock Skyrockets After Celgene Invests $1 Billion

to Change the Way Cancer Is Treated,” GeekWire (June 29, 2015).

2. Juno Therapeutics, Press Releases, www.junotherapeutics.com.

3. University of Pennsylvania, “Novartis-Penn Center for Advanced

Cellular Therapeutics Unveiled at Penn Medicine,” Press Release

(Philadelphia, PA, Feb. 16, 2016).

4. A. Regalado, “T-Cell Pioneer Carl June Acknowledges Key

Ingredient Was’t His,” MIT Technology Review, March 14, 2016.

5. R. Hernandez, “Novartis and Juno Settle Over CAR-T Therapy

Technology,” BioPharmInternational.com, April 7, 2015.

6. Precision Biosciences, “Baxalta and Precision BioSciences form

Global Genome Editing Collaboration in Immuno-Oncology,“ Press

Release (Durham, NC, Feb. 25, 2016).



scales because cell culture must be

achieved while maintaining the phe-

notype and function of the cells. At

small scale, 2D culture processes are

widely used and understood. They

are not suitable, however, for the pro-

duction of trillions of cells, which

may be the typical lot size for alloge-

neic therapies. For autologous treat-

ments, however, planar technologies

using adherent 2D culture flasks,

multilayer vessels, or multiplate bio-

reactors may be sufficient. Advances

in the automation of these systems

can be advantageous as well.

Suspension on microcarriers using

3D culture in typical bioreactors

is the most likely way forward for

the large-scale expansion of alloge-

neic CAR-T cells. The challenge is

to choose a microcarrier with the

appropriate surface characteristics

and to establish the optimum micro-

carrier concentration, cell seeding

density, media, and shear conditions

for each cell system. The use of

microcarriers is attractive at large

scale because they provide greater

surface area to volume for higher cell

densities, and because the expansion

can be performed in traditional bio-

reactors, control of various process

parameters is possible.

Harvesting of the cells from the

microcarriers is typically achieved

via treatment with an enzyme,

although some microcarriers are

being developed that allow non-

enzymatic removal. Once harvested,

a volume reduction step is per-

formed, followed by product filling.

Development of effective methods

for the reduction of larger volumes (<

5–10 liters) is a focus area for many

companies, with tangential flow

filtration (TFF) and single-use flu-

idized-bed centrifugation two tech-

nologies of interest.

In fact, disposable systems are

highly preferred for CAR-T and other

cell-therapy production processes

due to the need for low-cost, flex-

ible, closed systems that minimize

contamination. McCreedy notes that

several systems are in development

that can separate desired cells (e.g.,

via elutriation or magnetic beads

coated with antibodies), electropor-

ate and/or transduce cells, wash,

resuspend, and culture large num-

bers of cells, including removal of

spent media and addition of fresh

media that is designed to stimulate

the proliferation and expansion of

CAR-T cells with specific desired

phenotypes. “Such instrumenta-

tion to automate the process and

minimize the space required in a

manufacturing facility should have

a positive impact by increasing the

consistency and reducing the cost of

GMP manufacture of cell therapies,”

he states.

The logistics involved in autolo-

gous CAR-T cell therapies are often

raised as an important issue, but

Levitsky believes they are an engi-

neering problem that is not without

technical challenges, but certainly

not the biggest challenge facing

developers of these next-generation

treatments. Eventually, he believes it

may even be possible to have CAR-T

cell therapies produced at the hospi-

tal using automated instrumentation

that can perform all of the neces-

sary steps. “Such a solution is not out

of the realm of possibility; there is

nothing to indicate it can’t be done,”

Levitsky asserts.

It is also important to note that

CAR-T products require the sepa-

rate manufacture of viral vectors

for delivery of the CAR transgene in

addition to cell expansion and har-

vesting. “Challenges associated with

process development and validation

include establishment of transduc-

tion conditions that reproducibly

result in an acceptable percentage

of T cells that express the CAR at

defined levels on the T cell surface,”

says McCreedy.

PRODUCT CHARACTERIZATION AND RELEASE TESTING ISSUESProduct character izat ion and

release testing present additional

challenges to GMP manufacturing

of CAR-T products. The creation of

master cell banks from customized

cell lines that express specific ligands

and/or reporter molecules for use in

expanding CAR-T cells in culture

and for use in characterizing the

potency and specificity for release of

GMP-manufactured CAR-T products

are beginning to make their way into

the manufacturing process, which

should help to provide additional

consistency in the process, according

to McCreedy.

Because CAR-T products are con-

sidered to be both cellular and gene

therapy by FDA and the European

Medicines Agency, genetic stabil-

ity studies are required in addition

to traditional stability upon storage

documentation. The required test for

replication-competent lentivirus is

particularly onerous due to the cost

and time required. Alternatives for

delivery of genetic data that avoid

the use of viral vectors are in devel-

opment, such as the introduction

of the CAR as a transiently express-

ing messenger RNA (mRNA), plasmid

DNA transfection, and the use of

transposable elements (transposons)

to replace existing genes with new

ones, according to Levitsky. He also

believes it is possible that as the field

matures and experience with CAR-T

cell therapies increases, there will

eventually no longer be a need for

the test.

REFERENCES 1. Jain PharmaBiotech, Cell Therapy—

Technologies, Markets, and Companies

(March 2016), http://pharmabiotech.

ch/reports/celltherapy/, accessed April

12, 2016.

2. R. Tompa, Crafting a Better T Cell for

Immunotherapy (Feb. 22, 2016), www.

fredhutch.org/en/news/center-

news/2016/02/crafting-a-better-t-cell-

for-immunotherapy.html, accessed April

18, 2016.

3. J.A. Boyd, et al., Successful Translation

of Chimeric Antigen Receptor (CAR)

Targeting CD19 (CTL019) Cell

Processing Technology from Academia

to Industry, Poster, American Society of

Hematology 57th Annual Meeting

(Orlando, FL, December 2015). X


Scie

nce P

hoto

Lib

rary

/Gett

y Im

ag

es

The benefits of adopting single-

use technologies in the pro-

duction of biopharmaceuticals,

such as lower capital invest-

ment and increased flexibility, are now

well documented and widely recognized

in the industry. But when building a

new facility based on single-use technol-

ogies, or incorporating single-use into

an existing facility, how do companies

ensure they fully realize the benefits?

Facility design is a complex, multi-

faceted, multi-step process, and early

decisions can cause unforeseen limita-

tions as the project progresses or, later,

when further development of the facil-

ity is required. Asking the right ques-

tions at the outset and having the depth

of experience and knowledge to under-

stand the consequences of the answers

are vital to establishing the right specifi-

cations during the design phase.

Identifying a partner or partners to

support the design and build of a facil-

ity and the process that sits within it is

the first key decision. Traditionally, an

architectural and engineering firm and

one, or possibly multiple, single-use

process-equipment supply partners are

selected. Working with a single exter-

nal point of contact can help drive effi-

ciencies in project-management and

delivery. To be successful, however,

the lead partner will need an under-

standing of biomanufacturing facility

design, engineering, qualification, and

validation, as well as the operational

aspects of combining process hardware,

single-use consumables, and automa-

tion platforms.

Designing a Biomanufacturing Facility Incorporating Single-Use Technologies

Peter Genest and

John Joseph

Asking the right questions

is crucial.

Peter Genest is global operations

manager, FlexFactory, tel: 1.860.670.3014,

[email protected], and John Joseph

is engineering project leader, both at

GE Healthcare’s Life Sciences business.

Upstream Processing


AL

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RE

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RE

CO

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TE

SY

OF

TH

E A

UT

HO

RS

Overall, there are four sets

of requirements to consider:

product(s) to be made, process

technologies, facility design, and

supporting services. In each case,

a series of questions will help iden-

tify objectives, design specifica-

tions, and potential constraints.

CONSIDERING THE PRODUCTProduct class

The first element that defines any

biopharmaceutical manufactur-

ing facility is the product itself.

Will the facility be manufactur-

ing monoc lona l ant ibod ies ,

recombinant proteins, vaccines,

antibody-drug conjugates, or frag-

ment antibodies? Also, will the

products be mammalian cell-

derived or microbial cell-derived?

While these questions are most

pertinent for the selection of

the bioprocessing technologies

required, they are also important

for the design of the facility itself.

The promise of flexibility and

simplification are often major

deciding factors for choosing sin-

gle-use technology. Removing the

need for cleaning and sanitization,

for example, means that switching

between one product and another

becomes quicker and easier. One

way to take advantage of this flex-

ibility is by making the facility

multi-purpose (i.e., the manufac-

ture of two or more products) to

drive greater facility utilization.

Deciding between a single- or

multi-product facility impacts

facility design considerations.

Factors such as avoiding cross-

contamination between products

and ensuring that process-specific

equipment can be moved around

efficiently or housed nearby for

rapid changeover need to be built

into the design.

Regulations

With the plethora of regulatory

guidelines and associated compli-

ance requirements to adhere to

when building a facility, it must

be clear whether the product is for

research and development pur-

poses (pre-clinical), clinical trials,

or commercial scale, as this will

define the relevant GMP require-

ments. Also, if producing at com-

mercial scale, which regulatory

standard is needed? Is the product

approved by FDA, the European

Medicines Agency (EMA), the

China FDA, Brazi l’s National

Hea lth Su r ve i l lance Agenc y

(ANVISA), or other agencies? In

some cases, local requirements go

beyond global ones. For example,

Chinese fire regulations demand a

greater level of fire resistance than

is typical globally, and in countries

such as Korea and Japan earth-

quake-proofing measures may have

to be implemented.

Capacity

To define the necessary capacity of

the facility, the primary question

is how many batches per product

per year are needed? However,

this number has not always been

defined when the facility design

stage is reached. Alternatively,

it should be possible to consider

what quantity (in kilograms) of

the bulk API needs to be produced

for each product within the facil-

ity per year to meet clinical trial or

Upstream Processing

Figure 1. An example of a production process from cell culture to bulk drug substance.

Project management

Centralized monitoring and control

Vial

Cell culture seed train

Purification Bulk formulation

Production bioreactor Harvest and viral inactivation

HyClone mediaand supplements

WAVEBioreactor

Xcellerex XDRbioreactor

UniFlux crossflow filtration

Normal flowfiltration

Viralinactivation

ÄKTA chromatographysystem and

AxiChrom columns

ÄKTA chromatographysystem and

AxiChrom columns

Nano-filtration

Ultrafiltration/diafiltrationconditioning

Bulk drugsubstance

Sterilefiltration

Single-use tubing sets/assemblies


commercial market requirements,

and then work back to the number

of batches.

For example, one can con-

sider 2 x 2000-L bioreactors run-

ning a typical 14-day incubation

period staggered a week apart,

which equates to one batch pro-

duced each week. A typical batch

at 2 g/L with a 70% overall yield

in downstream processing and a

95% production success rate will

therefore yield 138 kg/yr in total.

The final yield here is determined

by the product titer of the produc-

tion bioreactor, combined with the

efficiency of the downstream puri-

fication steps, both of which will

be driven by the details of the bio-

process itself.

SELECTING PROCESS TECHNOLOGIESThe next step is to drill down into

the discrete unit operations of the

biomanufacturing workflow. If

the production process is already

defined, it should be listed out, but

if not, then the contracted partner

may be able to provide an equip-

ment list with f lexible process

capability. Figure 1 shows an exam-

ple of a production process from

cell culture to bulk drug substance.

Starting with upstream, the sta-

tus of the cell line and whether the

process should be batch, fed-batch,

or perfusion needs to be decided.

Details about the nature of the

process also need to be captured,

including the bio-safety level and

lengths of culture time for the seed

and production bioreactors.

Moving to downstream, the

overall yield of the purification

process from post-cell culture har-

vest through to purified bulk API

should be provided, along with an

estimate of the step yield of each

unit operation. If chromatography

columns are used in the process

flow, also specify the column vol-

ume and diameter required along

with the desired number of cycles

for each step.

Ma ny s i ng le -u se consu m-

able supply partners now offer

large customized system designs

that can be tailored exactly to a

specific workflow. Having an all-

encompassing single-use system for

a unit operation may seem to be

the most efficient option. However,

manufacturing a large single-use

system comes with challenges.

Packaging size and transportation

integrity, sterility validation, com-

ponent supply, handling and stag-

ing, installation, and operational

use can all become more difficult

and lead to greater risk levels. In

some cases, defining and select-

ing smaller and simpler single-use

systems to function in a modu-

lar workflow can be beneficial for

minimizing risks.

Another important consider-

ation in selecting single-use con-

sumables is ensuring the supply

chain is robust. Switching out

any element of a validated pro-

cess requires significant addi-

tional work. Therefore, make sure

the supply partner has a proven

track record, a materials policy in

place, transparency on how they

work with raw material suppliers,

and a proactive communication

program, and that they can pro-

vide examples of how they have

dealt with previous situations of

raw material changes. Also check

the robustness of the qualification

and validation package supplied,

and make sure it meets all relevant

regulatory requirements.

BREAKING NEW GROUND OR RENOVATING?The crucial point in designing a

new facility is whether it will be a

brownfield/renovation or a green-

field site. If it is brownfield, then

designers and engineers will need

to know if the footprint is fixed

and whether there are any restric-

tions on the space, such as floor

strength, ceiling height, or door

and elevator sizes. When thinking

about the layout, are there exist-

ing personnel, product, or mate-

rial flows already in place? Also, is

there existing support infrastruc-

ture, such as utilities, warehous-

ing, or laboratory space, that can

be accessed? If possible, plans for

future plant expansion at the site,

or at other sites, should be taken

into account, particularly if they

will have an impact on the product

requirements of the facility being

built now.

If it is a greenfield site, then

there is increased flexibility in

what can be built. However, sourc-

ing an engineering firm with the

relevant experience for a stick-built

biopharmaceutical facility design

can be challenging in some parts

of the world. In response to this,

another option that has emerged

is the modular facility, made from

standardized prebuilt units deliv-

ered to the greenfield site. This

approach can have benefits in

ensuring consistent standards of

quality and reduction of time to

first batch. This modular approach

to building allows site excavation

to run in parallel with module

construction and validation of unit

operations to begin offsite.

For those on a brownfield site

or those building a new facility

adjacent to an existing one, any

current centralized automation

platform for data archiving and

process monitoring may need to

be linked to the new facility. In

other cases, a standalone automa-

tion platform will be appropriate.

Finally, the need for any addi-

tional support functions or build-

ings should be decided (e.g., fill

and finish building, a black utility

generation building, a warehouse,

quality control [QC] laboratories,

or a waste treatment plant).

The needs here can sometimes

run counter to expectations. For

example, when embarking on a

first foray into single-use, many

presume that the removal of the

Upstream Processing


Upstream Processing

hard piping and utilities needed

for clean in place of stainless steel

will result in a reduced footprint

requirement. What is not always

anticipated is the warehousing

requirements for the stock of sin-

gle-use consumables, which also

need to be unpacked and prepared

in a staging area. While having

adjacent warehousing on a site

may fulfill this need, more effi-

cient tracking, set-up, and speed

of changeover will be achieved if

some consumables staging and

storage sits within the facility

itself, in close proximity to, or as

part of the cleanroom environ-

ment. In total, the footprint is

likely to be reduced in switching

from stainless steel to single-use,

but the change is not always as sig-

nificant as expected.

When adding a single-use train

to complement existing stainless-

steel production facilities, the

flexibility of single-use can help

reduce the need for additional util-

ities. In one case, when design-

ers and engineers looked at which

existing underutilized utilities

could be shared with a new single-

use set-up, it turned out to be a

significant amount. For example,

the flexibility of single-use meant

that single-use unit operations

requiring a water supply could be

scheduled for the downtime or

periods of low water consumption

of the stainless-steel process. The

reduced consumption of utilities

required to operate the single-use

process allowed for easier integra-

tion of additional capacity into the

existing infrastructure of a pro-

duction site.

SAFETY AND TIME CONSIDERATIONSThe ability of operators to safely

work with biologic and potentially

hazardous materials at any stage

during the process is a key facil-

ity design consideration. Knowing

where to place biosafety cabinets,

if aseptic connections are required,

and knowing any special design

modifications to the single-use sys-

tem (e.g., extra clamps, material

selection, handling of highly toxic

excipients) is vital.

Next, if known, specify the buf-

fer and media requirements of

each unit operation step in the

production process, including

whether any solutions require spe-

cial handling (e.g., 70% ethanol),

if steps are time-constrained (e.g.,

a highly-labile product that must

be processed in a specified period),

or if temperatures other than room

temperature are required (e.g.,

temperature-sensitive media for

upstream or cold purification pro-

cessing).

The buffer preparation sched-

ule for downstream purification

can have a significant impact on

facility design. Whether it is just-

in-time preparation, one day in

advance of use, or before any puri-

fication is started, will influence

how much space is required for

buffer storage or whether a system

of built-in piping is required.

PLANNING FOR THE FUTUREFacility design is a multifaceted,

interlocking web of needs, wants,

and risks, and it must be prop-

erly managed from the outset to

accommodate and account for all

requests. Management includes

being able to step back and take

a holistic view. The prime driver

and desired outcome, whether it

is shortest time to market, lowest

overall cost, or capital preserva-

tion, will significantly direct the

decisions made at all stages of the

design and building process.

For example, for a small biotech

that was particularly concerned

about reducing capital expendi-

ture, the ultimate recommendation

was to buy-in ready-made buffer

and media in single-use liquid

delivery bags. The overall scale and

output of the facility was relatively

low, and therefore the additional

infrastructure required for in-

house preparation was not going

to drive significant savings in the

longer term. This change in pro-

cessing methodology minimized

both footprint and utility needs.

Another element to consider at

this point is how much “future

flexibility” to account for during

the design and build phase. Do

you want to allow for the possi-

bility of adding more production

bioreactors to expand manufac-

turing capacity? Do you want to

add 10% more communication

drops for the integration of future

equipment? The balance to be

struck is between too much and

not enough.

One reason such flexibility is

important is that future manufac-

turing needs are always uncertain.

Factors such as increased pro-

ductivity and titer, coupled with

increased market competition due

to products coming off patent, has

led to some stainless-steel facilities

becoming underutilized and end-

ing up shut down or sold.

The facility itself is only the

beginning. Operational training

will be required, as a minimum,

but many supply partners can

offer a much wider range of ser-

vices. Validation requires signif-

icant experience and know-how

and has the potential to consume

significant internal resources.

Outsourcing this element to an

experienced partner can be a cost-

effective option.

Ultimately, of course, budget is

a crucial factor, along with when

production needs to commence.

But these should be considered

alongside a close appraisal of the

experience and depth of knowl-

edge of the team that will be

delivering the project. By map-

ping skills against requirements,

it is possible to identify key attri-

butes external partners need to

have to make a project a success,

first time. ◆


As the biotech industry evolves,

there are mounting concerns

about transportation, security,

and robustness of cell-culture

media, intermediate, or bulk drug sub-

stance (BDS). Safe, stable, and closed sys-

tems are needed when sterile products

are shipped in single-use bags (1). In this

article, the authors look at the limitations

of validation for a single-use shipping

system, and provide perspective on what

shipping validation means.

INCREASING NEED FOR SHIPPING PRODUCT IN THE SINGLE-USE MARKETThe complexity of biopharmaceutical man-

ufacturing processes requires continuous

improvement. The expansion of manufac-

turing capacity worldwide has resulted in

the multiplication of links between produc-

tion facilities as well as the increasing need

for storage or transportation of media, inter-

mediate, BDS, and drug products.

Outsourcing to contract manufactur-

ing organizations (CMOs) offers a solution

to the capacity constraint. CMOs bring to

the biopharma industry valuable technical

expertise and flexible capacity and reduce

the total risks associated with building

internal capacity; however, a robust and

validated manufacturing process (2), includ-

ing product transportation between facili-

ties, is required.

Single-use technology (SUT) contin-

ues to expand because of its potential

for reducing both capital and operating

expenses (3). The growing adoption of

single-use, especially in critical process

steps, has increased the need for product

quality, robustness, and integrity. The

biotechnology industry is now expand-

ing its implementation of single-use bags

into all bioprocess steps for applications

including cell-culture preparation (4),

filtration (5), purification (6), storage (7),

mixing (8), freeze-thaw operations (9),

and fill-finish (10).

Depending on the manufacturing pro-

cess organization and the level of outsourc-

ing, the challenge of safe and robust BDS

transportation becomes a crucial step from

a risk analysis point of view (11, 12).

ACHIEVING SAFE SHIPMENT Supplier and user requirements

To comply with modern manufacturing

requirements, SUT must offer similar lev-

els of security and robustness as multi-use

technology (MUT). A MUT shipping con-

tainer is designed to withstand the different

static and dynamic forces to which it is sub-

jected during transportation, handling, and

storage operations. The shipped product

must also be protected from climatic condi-

tions, such as temperature and humidity

(13). Reusable products must:

t� #F� SPCVTU� JO� UFSNT�PG�QSPWJEJOH�QSP-

tection to the shipped product against

rigorous environmental and handling

conditions

t� .BJOUBJO�QSPEVDU�TUFSJMJUZ

t� .BJOUBJO�QSPEVDU�TUBCJMJUZ

t� &OTVSF�PQFSBUPS�TBGFUZ

t� "MJHO�XJUI�DVSSFOU�MPHJTUJD�PQFSBUJPO

t� #F�DPNQBUJCMF�XJUI�WBSJBCMF�WPMVNFT�

Stainless-steel tanks for bulk freezing

and distribution between drug substance

sites and drug product sites were the pro-

cessing units of choice until recently,

when the technology was challenged by

SUT (9, 14, 15). It is important to note that

there is a simpler supply chain with SUT

shippers because there is no need to man-

age the return of empty tanks or to clean

and verify them.

In addition to the aforementioned stan-

dard requirements, shipping with SUT

requires the following additional needs:

t� .BJOUFOBODF�PG�UIF�TUSVDUVSBM� JOUFHSJUZ�

of the single-use bag (i.e., no leaks)

Qualification and Validation of Single-Use Shipping Systems

Nicolas Voute, Elisabeth

Vachette, Delphine

Audubey, Stephane Baud,

and Frederic Bazin

The authors provide their perspectives on shipping

validation.

Nicolas Voute is marketing consultant,

[email protected], Tel.

+33.4.42.84.60.69, Fax: +33.4.42.84.69.68;

Elisabeth Vachette is product

manager; Delphine Audubey is senior

inside application specialist; Stephane

Baud is R&D program leader container;

and Frederic Bazin is R&D program

manager bags, all at Sartorius Stedim

Biotech, Fluid Management Technologies,

Sartorius Stedim FMT S.A.S., Z.I. Les

Paluds – Avenue de Jouques, CS91051,

13781 Aubagne Cedex, France.

Shipping Services


t� 2VBMJGJDBUJPO�PG�QSPEVDU�JO�DPOUBDU�

with the single-use material (i.e.,

extractable and leachable testing).

While SUT shipping can offer sub-

stantial advantages compared to MUT

shipping, there are challenges with

SUT shipping as summarized in Table

I. Several considerations are related

to the material of construction of

SUT. Moreover, the end users’ require-

ments for shipping depend largely on

the application as shown in Table II.

REGULATORY ASPECTAs indicated in the Parenteral Drug

Association’s (PDA) Technical Report

(TR) N°66 (16), the supply of process

solutions in large-volume bags, from

point of manufacture to point of

use is a well-established practice that

involves the following elements:

t� "�CBH�EFTJHOFE� UP� GJU�B� SJHJE�XBMM�

outer container

t� "�SJHJE�XBMM�PVUFS�DPOUBJOFS�TVDI�BT�

a plastic drum or tote or a stainless-

steel bin

t� 4FDPOEBSZ� QBDLBHJOH� NBUFSJBMT�

(e.g., dunnage) and lids or other

mechanical devices to suppress the

fluid wave action in the bioprocess

bag.

Transportation of process solution

in small-volume bags (nominal vol-

ume less than 20 L) is also a common

process that requires less complex

packaging solution (16). The excep-

tion is the transportation of frozen

materials that necessitates tempera-

ture-resistant materials and cold-

chain logistics (17).

Shipping systems must be quali-

fied for their intended use through

proper design and testing in con-

sultation with a packaging engi-

neer. The International Safe Transit

Organization (ISTA) (18) and the

American Society for Testing and

Material (ASTM) D4169 (19) are good

references for testing standard. These

standards are complex with many dif-

ferent protocols, and the selection of

a relevant protocol linked to an appli-

cation is not trivial. It must be ana-

lyzed with a packaging and transport

expert. The following are some key

considerations for end-users:

t� 8IBU� JT� UIF� TIJQQJOH�VOJU� TJ[F �

weight, and construction)?

t� 8IBU� BSF� UIF� TIJQQJOH� SPVUFT��

intercontinental (truck/air/boat),

national (long-distance truck), or

continental (short-distance truck)?

t� 8IBU� JT� UIF�BTTVSBODF� MFWFM� UIBU�

should be established? Level I >

level II > level III

t� %FGJOF� UIF� MJGFDZDMF�QIBTFT�PG� UIF�

shipped unit (storage, transport,

handling, transport, use)

t� 8IBU�BSF� UIF�BTTPDJBUFE�FOWJSPO-

mental conditions for each phase of

the lifecycle (temperature, humid-

ity, compression, vibration, shock,

free fall, bump, and pressure)?

t� 8IBU�BSF�UIF�IBOEMJOH�DPOEJUJPOT�

(forklift, tarmac, roads)?

t� 8IBU�BSF�UIF�UZQF�PG�TIPDL �TIBLF �

and vibrations associated with the

shipping routes?

t� $BO�JU�CF�TUBDLFE

t� 8IBU�JT�UIF�BQQSPQSJBUF�OVNCFS�PG�

samples for validation?

t� 8IBU�JT�UIF�BEFRVBUF�TFWFSJUZ�PG�UIF�

simulated shipping test?

t� 8IBU� TBGFUZ�NBSHJOT�BSF�BDDFQU-

able?

t� 8IBU�BSF� UIF�BDDFQUBODF�DSJUFSJB��

product is damage-free, package is

intact, or both?

Based on the projected distribu-

tion, the end user should define a

test plan using the distribution cycle

(DC) defined in Table I of the ASTM

D 4169 (19). The DC should correlate

with the projected lifecycle phases of

the shipped unit (20).

In addition, many pharmaceuti-

cal or biotechnological products

are temperature sensitive and

require specific precaution during

storage and transportation (21).

Transport and storage conditions

have to be determined considering

Shipping Services

Table I: Advantages and challenges of single-use technology (SUT) shipping.

Table II: End-user requirements for shipping according to the application.

SUT advantages SUT challenges

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(WFI)t��3FEVDFE�SJTL�PG�DPOUBNJOBUJPO�EVF�UP�DMPTFE�TZTUFNt��/P�DSPTT�DPOUBNJOBUJPO�EVF�UP�TJOHMF�VTFt��$"1&9�SFEVDUJPOt��3FEVDUJPO�PG�NBJOUFOBODF�DPTUt��3FBEZ�UP�VTF�OP�DMFBOJOH �OP�TUFSJMJ[BUJPO �OP�WFSJGJDBUJPO�QSJPS�UP�VTFt��-FTT�SFTPVSDFT�BOE�UJNF�SFRVJSFE�GPS�NBJOUFOBODF �DMFBOJOH �BOE�WBMJEBUJPO��MFTT�TUBGG�JOUFOTJWFt��1PUFOUJBM�GPS�POF�XBZ�MPHJTUJDTt��-FTT�JOGSBTUSVDUVSF�BOE�QFSJQIFSBM�FRVJQNFOU�SFRVJSFE

t��"QQSPWBM�BOE�RVBMJGJDBUJPO�PG�NBUFSJBM�PG�DPOTUSVDUJPO�SFRVJSFT�FYUFOTJWF�BOE�TQFDJGJD�FYUSBDUBCMF�MFBDIBCMF�TUVEJFTt��1PUFOUJBM�DIBMMFOHFT�PG�HVBSBOUFFE�TVQQMZt��$IBOHF�NBOBHFNFOU�t��3JTL�PG�CSFBLBHF�PG�CBHT�EVSJOH�USBOTQPSU�BOE�MPTT�PG�TUFSJMJUZt��7FSJGJDBUJPO�PG�UIF�DPOUBJOFS�JOUFHSJUZ�BU�MPX�

QSFTTVSFt��8BTUF�NBOBHFNFOU

Application End-user requirement

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.POPDMPOBM�BOUJCPEZ�BOE�SFDPNCJOBOU�QSPUFJO t��7PMVNF��-t��4UFSJMFt��.VMUJ�VTF�TIJQQJOH�TPMVUJPO�MFTT�UIBO��SFVTFt��7BSJBCMF�TIJQQJOH�WPMVNF

1MBTNB�BOE�NFEJB t��7PMVNF��-t��4UFSJMFt��.VMUJ�VTF�TIJQQJOH�TPMVUJPO�MFTT�UIBO��SFVTFt��7BSJBCMF�TIJQQJOH�WPMVNF


the risks of product degradation

(22, 23).

PDA TR N°66 has highlighted spe-

cific factors of importance for trans-

portation that must be considered by

end-user (16). These factors are:

t� %JNFOTJPOBM� GBDUPS� J�F� �WPMVNF�

to be shipped and dimensions of

the shipper)

t� .PEF�PG�USBOTQPSUBUJPO �XIFUIFS�

it’s ground, air, rail, boat, or a

combination of more than one

mode. Metrics must include hold

time on tarmac.

t� 5IF� BTTPDJBUFE� FOWJSPONFOUBM�

conditions (temperature, humid-

ity, pressure, and variation)

t� 'VODUJPOBMJUZ� J�F� �GPSLMJGU�BDDFTT �

stack ability of outer container,

access to fill and drain port, sec-

ondary container to collect leak)

t� 3PPN� DMBTTJGJDBUJPO� TVDI� BT� GJMM�

and drain procedure to maintain

sterility

t� -PHJTUJDT� F�H� � FYUFSOBM� TIJQQFS �

cold-chain logistics).

Shipping Services

Protecting Precious Cargo: Cold Chain Shipping Services for Biopharmaceuticals

As the biopharmaceutical industry has grown, so has the

number of cold-chain logistics and shipping companies, and

technology developers, that serve it. Technology providers are

expanding the number and variety of solutions they offer. Here is

a short sampling of companies that offer specialized cold-chain

services, and some of their offerings.

Brink’s Global Services focuses on managing risk

Legendary for its armored cars, and in the transportation

business since 1859, this secure high-end-goods transport

company moved into pharmaceuticals in 2014 and offers

shipment solutions that include full risk assessment.

Brink’s Online is a secure web portal that provides shipping,

inventory management, and invoicing materials on demand.

www.brinkssecuredata.com/Sectors/high_tech_electronics_

pharmaceutical.aspx

Cryoport: liquid nitrogen, not dry ice

Cryoport uses liquid nitrogen dry vapor shippers to eliminate

the risk of temperature changes that can occur with dry ice.

Cryoportal, a logistics-management platform, allows users

to manage documentation from a single user interface.

It also features full data-monitoring and data-tracking

capabilities, including chain of custody and chain of condition.

www.cryoport.com

Patient-centered solutions

Marken offers a range of services for biopharmaceuticals,

clinical trials, and diagnostics, utilizing a global network and

technology such as its Sentry GPS-enabled sensor platform,

which allows data to be collected in real time, so that teams can

work most efficiently with ground transportation companies and

airlines. www.marken.com

Simulate “What Ifs”

Modality Solutions offers an integrated platform focusing on

cold-chain packaging, transportation validation, and logistics

solutions. Its Advantage Transportation Simulator allows users

to assess the impact of changes and stresses on shipments.

www.modality-solutions.com

Cold-chain solutions throughout the product lifecycle

A diversified contract services company, PCI Services offers

logistics and distribution, as well as storage and returns

capabilities for biopharmaceuticals. www.pciservices.com

Custom packaging, shipping, and testing services

Sonoco ThermoSafe provides cold-chain packaging and

shipping solutions for pharmaceutical manufacturers, biologics

developers and suppliers, clinical trials, and other healthcare

markets. The company’s ISC Labs offer custom packaging

solutions, as well as design, testing and validation services.

www.thermosafe.com

“It’s a patient, not a package”

UPS offers a wide range of shipping and logistics as well as

temperature-control options for biopharmaceuticals and clinical

trials. Its Temperature True service offers different service levels

depending on the shipping and storage temperature and speed

required for delivery. The company offers advanced monitoring

to allow it to intervene based on milestones, GPS readings,

and product conditions, to prevent delays and product recalls.

www.ups.com

Transparency and compliance support

World Courier offers logistics processes, storage and

distribution, temperature control solution, and shipping

processes for commercial pharmaceuticals and clinical trial

logistics. The company’s CTM-Star inventory management and

stock control tool allows customers remote access and visibility,

and access to source documents. www.worldcourier.com

Help ensure safety with thermal exposure indicators

Cryoguard Corp. offers indicators that can detect thermal

exposure from -40 to -150 ˚C, providing a red light warning

when materials have been exposed to potentially damaging

thermal exposures. They can be used to monitor materials in

liquid nitrogen dewers, dry shippers and tanks, foam coolers,

insulated boxes, freezer canes, and containers cooled by dry ice.

www.cryoguard.com

—Agnes Shanley


Shipping Services

In addition to PDA TR66, the analy-

sis of the regulatory requirements and

relevant references can be summa-

rized as follows:

t� 5IF� USBOTQPSUBUJPO� SPVUFT� NVTU�

be defined for international ship-

ment. A risk assessment for vibra-

tion, handling, delays, and seasonal

variation should be established (11).

t� 5IF� TVJUBCJMJUZ�PG� UIF�DPOUBJOFST�

(compatibility, safety, robustness)

and of the container-closure sys-

tem (material of construction,

integrity, interaction) must be

defined as well as a rational for

the choices of the material, the

barrier properties, the compatibil-

ity, and the safety (13).

t� 3FDPNNFOEBUJPOT� BSF� HJWFO� UP�

define storage and shipping con-

ditions, to assess risk of the envi-

ronmental parameter variation,

to define labeling, and to assess

short-term excursion outside

storage conditions according to

the International Council on

Harmonization (ICH) accelerated

testing (24).

t� 2VBMJGJDBUJPO�BOE�WBMJEBUJPO�PG�UIF�

shipped product can be performed

in real shipment with monitoring

(25–27) or in simulated shipment

according to ASTM D4169 (19) or

ISTA (18).

t� *O�BEEJUJPO �QSFDBVUJPOT� GPS� USBOT-

port of hazardous materials and

dangerous goods are also described

in additional regulations. The

&DPOPNJD�$PNNJTTJPO� GPS�&VSPQF�

has defined regulations applicable

to road transportation of chemical

dangerous goods (28). Part 3 of the

documents lists the chemical enti-

ties considered as dangerous goods

for which special precautions are

required.

t� 5IF�6OJUFE�/BUJPOT�IBT�FTUBCMJTIFE�

regulations for any type of trans-

ports (air, road) of biological and

infectious substances (29).

t� 5IF� *OUFSOBUJPOBM� "JS�5SBOTQPSU�

Association has established regula-

tions applicable to safe air transpor-

tation of dangerous goods (30).

Shipping is indeed complex and

users should verify the vendor’s

claims about some regulations. It

is important for the end-user to

understand what is behind the

claim and the relevance to its appli-

cation. As discussed in this article,

shipping validation protocol for

mechanical constraints needs to be

carefully defined with parameters

setting linked to the application in

close collaboration between end-

users and suppliers.

*U� JT� B� SFRVJSFNFOU�PG� '%"� ��

�� UIF�&VSPQFBO�.FEJDJOFT�"HFODZ�

�� UIF�&VSPQFBO�6OJPO� �� BOE�

other drug regulatory agencies that

the process produces consistently

similar and reproducible results that

meet the quality standard of the

QSPEVDU��"DDPSEJOH� UP� '%" �WBMJEB-

UJPO� JT� i&TUBCMJTIJOH�EPDVNFOUFE�

evidence that provides a high

degree of assurance that a specific

process—including shipping—will

consistently produce a product

meeting its pre-determined specifi-

cations and quality attributes” (33).

A properly designed system will pro-

vide a high degree of assurance that

every process step, including ship-

ping, has been properly evaluated

before its implementation.

In the biopharmaceutical indus-

try, qualification and validation are

intended to demonstrate that the

manufacturing process provides the

desired level of product quality and

specifically its activity, sterility, and

QPUFODZ��2VBMJGJDBUJPO�PG�B�TIJQQJOH�

system and shipping equipment is

part of the validation.

MECHANICAL ROBUSTNESS An SUT shipping system composed

of a bag and a stainless-steel bin

should ensure safe shipment (i.e.,

no loss of integrity and no loss of

product sterility). It can be granted

by the mechanical robustness of the

shipper. The objective is to verify

that no leaks occur during transpor-

tation. According to Tull, “Product

quality can be defined in terms of

the ability of a product to perform its

desired function despite the stresses

to which it has been exposed before

and during its intended use” (23).

Bag leakage can be analyzed fol-

lowing methods described in the

PDA TR N°27 (34). This document,

however, describes high-sensitivity

Table III: Main features of the American Society for Testing and Material (ASTM)

and International Safe Transit Organization (ISTA) standards.

ASTM D4169ISTA Procedure 3H (recommended by ISTA)

5ISFF�MFWFMT�EFTDSJCFE�JO�UIF�"45.�%��t��"TTVSBODF�MFWFM�*t��"TTVSBODF�MFWFM�**t��"TTVSBODF�MFWFM�***

"DDFQUBODF�DSJUFSJBt��$SJUFSJPO��o�1SPEVDU�JT�EBNBHF�GSFFt��$SJUFSJPO��o�1BDLBHF�JT�JOUBDUt��$SJUFSJPO��o�1SPEVDU�JT�EBNBHF�GSFF�BOE�JOUBDU

��EJTUSJCVUJPO�DZDMFT�%$��%$�TIPVME�CF�DIPTFO �XIJDI�DPSSFMBUFT�UP�UIF�QSPKFDUFE�EJTUSJCVUJPOt��1SFDPOEJUJPOJOH�BOE�DPOEJUJPOJOHt��)BOEMJOHt��4IPDL�IPSJ[POUBM�JNQBDU �SPUBUJPOBM�GMBU�ESPQ �BOE�FEHF�ESPQt��4JNVMBUJPO�PG�UIF�WJCSBUJPO�EVSJOH�USVDL�USBOTQPSUt��-PX�QSFTTVSF�t��"JS�WJCSBUJPOt��$PNQSFTTJPO�PQUJPOBM

5FTU�DPOTJTUT�PG��JOEJWJEVBM�UFTUT�UIBU�BSF�DBSSJFE�PVU�TFRVFOUJBMMZ�PO�UIF�TBNF�QBDLBHF�5IF�UFTUT�TJNVMBUF�UIF�IBOEMJOH�BOE�USBOTJU�SFRVJSFE�JO�B�MPOH�IBVM�SPBE�EJTUSJCVUJPO�OFUXPSL�BOE�DPWFS�USVDL�USBOTQPSU�POMZ��*U�JT�DPNQPTFE�PG�TFRVFODFT�JODMVEJOH�

t��1SFDPOEJUJPOJOH�BOE�DPOEJUJPOJOHt��4IPDL�IPSJ[POUBM�JNQBDU �SPUBUJPOBM�GMBU�ESPQ �BOE�FEHF�ESPQt��4JNVMBUJPO�PG�UIF�WJCSBUJPO�EVSJOH�USVDL�USBOTQPSUt��$PNQSFTTJPO�PQUJPOBM


methods adapted for final packag-

ing and not necessarily for inter-

mediate or BDS. More global test

methods such as diffusion of a dye,

detection of a liquid leak, or damage

of the bag (films and seals) may be

more relevant (35).

&OTVSJOH�B� TBGF� TIJQNFOU�NFBOT�

preventing leakage and loss of

integrity when the SUT shipping

system undergoes the mechanical

stresses generated during shipment.

It is difficult to define these stresses

and to determine the adequate

safety margin.

ASTM AND ISTA STANDARDSAs already mentioned, a well-known

and common practice is to apply the

ASTM or ISTA standard protocol on

shipping system and check its per-

formance according to these agency

guidelines. Table III briefly describes

the main features of the ASTM and

ISTA standards. It is, therefore, diffi-

cult to select the right parameters to

grant a safe validation.

SELECTING A PROTOCOLIt is important to choose a protocol

that correlates to the projected lifecy-

cle phase of the shipped unit. A typi-

cal distribution sequence between

two plants is depicted in Figure 1.

ASTM (19) proposes 18 DC for

modeling any type of transport by

carrying out accelerated and strin-

HFOU� UFTUJOH��'PS�FYBNQMF �%$��PG�

ASTM is representative of the typical

shipment shown in Figure 1. DC 12

includes five test programs adapted

to simulate each segment of the pro-

jected distribution (see Table IV) with

impact (horizontal impact, rotational

flat drop, and edge drop), low pressure

(representative of shipment by plane

or high altitude), and vibration tests.

A second step is to define the

severity of testing (level and dura-

tion). Some differences between the

three assurance levels are shown

in Table V, which is not exhaustive.

Duration is always a decision to be

taken by the end-user even though

standards may make recommenda-

tion, unless conditions dictate oth-

erwise. ASTM also recommends level

2 in that case. The level of severity

must be defined according to real

shipment condition in addition of

desired safety margin.

There is no official ASTM claim;

suppliers can only claim that they

pass ASTM selected tests described by

the standard. Therefore, knowing the

distribution cycle, schedule, duration,

severity level, and acceptance crite-

ria are mandatory to understand the

validation performed on the system.

Moreover, the suitability with the

intended use can only be proven by

end users; these conditions might dif-

fer from one site to another or from

one product to another. Transport

simulation test results performed

according to DC12 of ASTM D4169

as well as test results obtained in real

shipping conditions will be described

in a forthcoming paper. The paper

will present mechanical robustness

and vibration data test results in a

simulated and real-life scenario to

define and validate the conditions for

safe transportation, the safety mar-

gin, and the limits of the each system.

CONCLUSIONShipping is indeed complex and the

user should not be assuaged sim-

ply by vendors’ claims about regu-

lations (i.e., claims of being “ISTA

certified” or “ASTM compliant”).

It is important to also understand

what is behind each claim and verify

that it is applicable to the product’s

intended use. The end-user should

understand the trial conditions used

in the vendor tests and compare

them to its application. The accep-

tance criteria (bag and shipper), the

protocol, and trial conditions shall

be discussed. Shipping validation

needs to be carefully defined in

close collaboration between end-user

and vendor, with parameter setting

linked to actual use. Collecting vibra-

tion data on the real use will help the

end user and the vendor to under-

stand the physical constraints of the

Shipping Services

Figure 1: Typical transportation from plant A to plant B.

Plant A:

Handling

(forklift)

Plant B:

handling

(forklift)

Truck to

airport

Truck from

airport

Handling

(tarmac)

Handling

(tarmac)

Air shipment

FIG

UR

E 1

IS

CO

UR

TE

SY

OF

TH

E A

UT

HO

RS


Shipping Services

shipping mode and select the best

protocol to replicate them in labora-

tory testing. The limits of the system

should be defined with knowledge

of the safety margin and be tested

under real packaging and real trans-

port conditions.

REFERENCES 1. N. Riesen, R. Eibl, “Single Use Bag

Systems for Storage, Transportation,

Freezing, and Thawing” in Single-Use

Technology in Biopharmaceutical

Manufacture, Dieter Eibl and Regine Eibl

Eds. (John Willey & Son, Hoboken, NJ,

2011).

2. S.D. Jones and H. Levine 2005,

BioExecutive International 3, 2–5 (2005).

3. L.Howard et al., BioProcess International 10

(11s) 20–30 (2012).

4. J. Wood et al., Biotechnol. Prog. 29 (6)

1535–1549 (2013).

5. T. Vicente et al., Eng. Life Sci. 14, 318–326

(2014).

6. M. Kuczewski et al., Biotechnol J. 6 (1)

56–65 (2011).

7. A. Shukla and U. Gottschalk, Trends in

Biotechnology 31 (3) 147–154 (2013).

8. A. Shukla et al., BioProcess International 10

(6) 34–47 (2012).

9. A. Goldstein et al., “Disposable Freeze

Systems in the Pharmaceutical Industry: A

journey from current stainless steel to

future disposable freeze systems in clinical

and large scale manufacturing,” American

Pharmaceutical Review, www.

americanpharmaceuticalreview.com/

Featured-Articles/126890-Disposable-

Freeze-Systems-in-the-Pharmaceutical-

Industry/, accessed 11 Feb. 2016.

10. E. Langer and R. Radler, Eng. Life Sci. 14

(3) 238–243 (2014).

11. EC, EU Guidelines for Good Manufacturing

Practice (Brussels, February 2014).

12. FDA, Guidance for Industry Q8(R2)

Pharmaceutical Development (Rockville,

MD, November 2009).

13. S. Kumar, Int. J. Res. Pharmaceut. Biomed.

Sci., 4 (4) 1400 (2013).

14. T. Matthews et al., BioPharm International

Supplement (November 2009), www.

biopharm international.com/freeze-bulk-

bags-case-study-disposables-

implementation

15. S. Singh et al., BioPharm International 23

(6) (June 2010).

16. PDA, Technical Report N°66: Application of

Single-Use Systems to Pharmaceutical

Manufacturing, 2014.

17. R. Srinivas Madhukar et al., Journal for

Clinical Studies 5 (3) 50-56 (2013).

18. ISTA, ISTA 3H: 2011, Products or

Packaged-Products In Mechanically

Handled Bulk Transport Containers

(January 2011).

19. ASTM, Standard Practice for Performance

Testing of Shipping Containers and Systems,

ASTM D4169-14 (November 2014).

20. M. Magendans, Transport Simulation Test,

www.sebert.nl/powerpoint/ASTM-

Simulated-Transport-Test.pdf, accessed 21

Feb. 2016.

21. C. Ammann, AAPS PharmSciTech. 12 (4)

1264–1275 (2011).

22. WHO, WHO Technical Report Series,

No.961, Guidelines time- and temperature-

sensitive pharmaceutical products (2011).

23. J. Tull, B.K. Nunnally, “Design and

Execution of a Shipping Qualification for a

Vaccine Drug Substance,” ivtnetwork.com.

2009, www.ivtnetwork.com/sites/default/

files/Process_Qualification_Special_

Edition.pdf#page=50.

24. USP, USP <1079> Good Storage and

Shipping Practices, 2012.

25. P.H. Singh et al., Packaging Technogy and

Science 20, 387–392 (2007).

26. B. Wallin, Developing a Random Vibration

Profile Standard, www.halthass.co.nz/

wp-content/uploads/technical-library/pdf/

Developing-a-random-vibration-profile-

standard.pdf, 2010.

27. E. Joneson, Int. J. Adv. Packaging Technol. 2

(1) 70–74 (2014).

28. UNECE, Part 3 dangerous goods list and

limited quantities exceptions, 2011, www.

unece.org/fileadmin/DAM/trans/danger/

publi/unrec/.../part3.pdf.

29. Regulations for UN3373, www.un3373.

com/info/regulations, 2011.

30. IATA, Dangerous Goods Regulations (DGR),

www.iata.org/publications/dg, 2010.

31. FDA, Guidance to the Industry: Information

on Container Closure System for Shipping

BDS as Biologics (Rockville, MD, May

2002).

32. EMA, Guideline on Process Validation for the

Manufacture of Biotechnology-derived Active

Substances and Data to be Provided in the

Regulatory Submission (London, April

2014).

33. FDA, Guidance for Industry Process

Validation: General Principles and Practices

(January 2011).

34. Pharmaceutical Drug Association, Technical

Report N°27: Pharmaceutical Package

Integrity, 1998.

35. M.J. Akers et al., Package Integrity Testing.

Parenteral Quality Control, 3rd Edition.

(Informa Healthcare, New York, 2007), p.

325. ◆

Table IV: DC12 of American Society for Testing and Material (ASTM) D4169.

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4JEF�IPSJ[POUBM�JNQBDU�UFTU��JNQBDU�BMM�GPVS�TJEFT�PG�UIF�TIJQQJOH�VOJU�

'PSLMJGU�USVDL�IBOEMJOH��POF�SPUBUJPOBM�GMBU�ESPQ�GSPN�FBDI�CBTF�FEHF

4DIFEVMF�*�MPX�QSFTTVSF�BJS�TIJQNFOU 1SFTTVSF�FRVJWBMFOU�UP��N�GPS�B�QFSJPE�PG��NJO

4DIFEVMF�&�WFIJDMF�WJCSBUJPO�USVDL�BOE�BJS 1FSGPSN�UIF�UFTU�VTJOH�SBOEPN�WJCSBUJPO

4DIFEVMF�"�IBOEMJOH�GPSLMJGU�IBOEMJOH�BOE�UBSNBD

4JEF�IPSJ[POUBM�JNQBDU�UFTU��JNQBDU�BMM�GPVS�TJEFT�PG�UIF�TIJQQJOH�VOJU�

'PSLMJGU�USVDL�IBOEMJOH��POF�SPUBUJPOBM�GMBU�ESPQ�GSPN�FBDI�CBTF�FEHF

4DIFEVMF�#�IBOEMJOH�JG�BQQMJDBCMF 8BSFIPVTF�TUBDLJOH��JOUFOEFE�UP�EFUFSNJOF�UIF�BCJMJUZ�PG�UIF�TIJQQJOH�VOJU�UP�XJUITUBOE�UIF�DPNQSFTTJWF�MPBE�UIBU�PDDVS�EVSJOH�XBSFIPVTF�TUPSBHF�PS�WFIJDMF

Table V: The three assurance levels for DC12 of the American Society for Testing

and Material (ASTM) D4169 for truck conditions.

Description Level 1 Level 2 Level 3

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GPS��-�BOE��-�

GPS��-

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In the past few years, there has

been an increase in the number

of therapeutic proteins in develop-

ment and those that have received

approval from regulatory agencies.

Proteins are produced by gene expres-

sion in bacterial or mammalian cell

culture. Cell-culture media are com-

posed of essential raw materials that

are required for cell growth and

expression. Similar cell-culture media

may be used simultaneously in one or

few biological processes at a drug prod-

uct manufacturing facility. As per Code

of Federal Regulations (CFR) 211. 84, each

media needs to be specifically identi-

fied from other media during release

testing prior to use in production (1, 2).

Cell-culture media contain mul-

tiple components such as hormones,

vitamins, folic acid, lipids, amino

acids, sugars, insulin, among others.

Therefore, development of a specific

identity test presents a challenge due

to matrix interference (3). Several spec-

troscopic non-destructive techniques,

including Fourier Transform Infrared

(FT–IR), near-infrared (NIR), and Raman

spectroscopy have been used for the

identification and characterization of

cell-culture media. FT–IR spectroscopy

(mid-IR region) differentiates materials

ABSTRACTCell-culture media are essential raw materials that are required for the

manufacture of biotherapeutic proteins. Cell-culture media are composed of multiple components, and therefore, it is difficult to develop specific identification

tests for media as required by Code of Federal Regulations (CFR) 211.84. The developed testing algorithm, which incorporates a combination of few relatively

simple analytical methods such as osmolality, quantitation of glucose, and folic acid, provides specific identity confirmation for seven cell-culture media

with essentially similar composition that were examined in this study. The methods are well suited for routine use in the quality-control environment and

the provided identification approach meets CFR, FDA, and other regulatory agencies requirements. As part of this approach, a platform glucose method

that utilizes a linear standard curve was validated using alternative cell-culture media, and all established acceptance criteria were met. The assay was shown to be specific for the detection of glucose in all studied multi-component media without matrix interference regardless of glucose concentration or the vendor.

A Platform Approach for the Identity Testing

of Multi-Component Cell-Culture Media

Satish Mallya, Benjamin Lay, Lihong McAleer,

Alexandria Emory, and Nataliya Afonina

Satish Mallya is senior research investigator;

Benjamin Lay is lab supervisor; Lihong McAleer

is scientist; Alexandria Emory is associate

manager; all at Bristol-Myers Squibb. Nataliya Afonina is president and principal consultant at

AN Biologics Consulting LLC.

PEER-REVIEWED

Article submitted: 12/2/2015.

Article accepted: 1/15/2016.

PA

SIE

KA

/Ge

tty I

ma

ge

sPeer-Reviewed


based on their chemical composition and thus,

provides specific chemical identity (4). This test

compares the spectrum of each media with

the spectrum of each corresponding control

and was successfully employed for identifica-

tion of different types of growth media with

diverse formulations used in vaccine produc-

tion (5). However, this approach cannot be

easily employed for media used in the manu-

facture of biologics, which might differ only

by one additional component or by a concen-

tration of similar components.

NIR spectroscopy has a light absorption

much weaker in intensity compared with

that of FT–IR and is based on the overtones

of major bands produced in the mid-IR

region. Therefore, NIR does not provide spe-

cific identity of the studied material and

requires a use of spectral libraries or mul-

tivariate data analysis (MVDA) for spectra

evaluation. NIR spectroscopy is a very popu-

lar method of analyzing solid and liquid raw

materials because it is sensitive to matrix

modifications such as surface area, sam-

ple morphology, and other sample proper-

ties (6, 7). NIR coupled with chemometric

analysis was used for the characterization of

soy raw material to evaluate the variability

and impact on product quality. The analysis

revealed that near-infrared spectra of differ-

ent soy lots contain enough physicochemical

information about soy hydrolysates to allow

the identification of lot-to-lot variability as

well as vendor-to-vendor differences (8). A

few publications related to NIR spectroscopy,

however, are associated with identification

of complex raw materials such as cell-culture

media. A combined approach of NIR and

MVDA was used for the identification and

qualification of basal medium powder com-

ponents (9) and was evaluated as an identity

tool for cell-culture media (10).

Raman vibrational spectroscopy provides

a unique chemical fingerprint of molecules

similar to that of FT–IR and is being used for

raw materials identification, characteriza-

tion, and quantitation (10, 11). However, as

a non-specific identification test, it requires

the use of chemometric analysis for unam-

biguous sample identification. Raman spec-

troscopy coupled with principle component

analysis was successfully applied as a single

integrated method for rapid identification

and characterization of five different chemi-

cally defined components of cell-culture

media used in a Chinese hamster ovary

(CHO) cell manufacturing process for recom-

binant proteins (11). Although, both NIR

and Raman spectroscopy are fast and non-

destructive, they require the use of chemo-

metric analysis for specific identification of

cell-culture media or media components,

which is not easily employed in the qual-

ity control (QC) environment. Therefore,

this approach (NIR and Raman spectroscopy

coupled with chemometrics analysis) is more

useful for the identification of media compo-

nents and screening consistency and char-

acterization of media that are not in the QC

environment.

The micellar electrokinetic capillary chro-

matography (MEKC) was used an alternative

approach to spectroscopic methods for media

identification. MEKC was successfully vali-

dated and employed for the identification of

cell-culture media from Invitrogen (12). It was

also used for simultaneous determination of

media components such as folic, mycophe-

nolic, nicotinic acids, hypoxanthine, and

other components in protein-containing

matrices from a monoclonal antibody manu-

facturing process (13). However, due to the

complexity of sample analysis, this approach

is more suitable for media characterization or

in-process monitoring rather than a use as an

identity test (ID) in a QC environment.

Another practical approach for media iden-

tification is the use of relatively simple tests

for the identification of process buffers (14).

In such case, few nonspecific identification

tests—such as compendial methods, pH,

osmolality, color/appearance used in combi-

nation with UV-visible spectrometry, high-

performance liquid chromatography (HPLC),

or other tests—may provide specific ID for

similar media. This approach necessitates

implementation of platform methods, which

are applicable for several media with dif-

ferent composition. This approach ensures

simplicity, cost effectiveness, and consistency

between operators in various global com-

panies. The approach also certifies quality

of media in addition to tests provided on a

vendor’s certificate of analysis (CoA) by con-

tinuously evaluating cell-culture media prop-

erties during release testing.

The goal of this work was to develop a

platform approach and an algorithm for spe-

Peer-Reviewed


cific identification of seven culture media

with essentially similar formulations. The

study also includes development and valida-

tion of the platform method for quantitation

of glucose as one of the key components of

studied media.

MATERIALS AND METHODSAll eight multi-component culture media

used in this study were custom manufac-

tured for Bristol-Myers Squibb Co. The media

samples were obtained as a powder and

were reconstituted to the liquid stage. Seven

media (M12, M51, M55, M10, M53, M57, and

M17) had essentially similar formulations

and were used in the development of identity

testing strategy. The eighth media—media

CDS, M12, and M57, with a target glucose

concentration after reconstitution of 6.3 mg/

mL, 2.0 mg/mL, and 3.0 mg/mL, respec-

tively—were used in the glucose method

validation.

The reagents used for glucose analysis

were purchased from Sigma-Aldrich (now

MilliporeSigma) in Saint Louis, MO: glu-

cose 100 mg/mL (catalog number G8644),

fructose (catalog number F0127), galactose

(catalog number G6404), mannose (catalog

number M2069), Sigma Protein-Free (SPF)

media (catalog number C5467), and glucose

hexokinase reagent (catalog number G3293).

HPLC-grade water (catalog number AH-365-

4) was obtained from Burdick and Jackson

(Muskegon, MI).

The reagents for folic acid analysis were

purchased from Fisher Scientific, Sigma-

Aldrich, and other vendors and are not dis-

cussed in this work in detail. The United

States Pharmacopeia (USP) reference standard

of folic acid was purchased from Sigma-

Aldrich (Saint Louis, MO) (catalog number

1286005).

OSMOLALITY METHODOsmolality was performed essentially follow-

ing USP <785> (15).

Glucose method

Glucose standard curve—A stock solution of

100 mg/mL glucose was diluted with HPLC-

grade water to produce a final concentra-

tion of glucose varying from 0.1 to 2.5 mg/

mL. The contents of the hexokinase kit were

reconstituted with 50 mL of HPLC-grade

water and 10 μL of each of the diluted glu-

cose sample was added to 1 mL of reconsti-

tuted hexokinase reagent. After incubation

for 15 min, the absorbance at 340 nm (A340)

was measured using an Agilent 8453 UV–VIS

spectrophotometer. A plot of the A340 vs. glu-

cose concentration (mg/mL) was analyzed by

linear regression.

Analysis of culture media—The SPF culture

media was analyzed as part of system suit-

ability (described in the following passages).

SPF culture media was diluted two-fold with

HPLC-grade water and 10 μL of diluted

media was analyzed in six replicates in a

manner similar to a glucose standard. The

average concentration and relative standard

deviation (%RSD) were reported.

Analysis of test articles (culture media)—The

target concentration of glucose in the test

article was used to calculate a dilution factor

and each culture medium was diluted with

HPLC-grade water to produce glucose con-

centration of ~2 mg/mL. Each test article was

analyzed in triplicate.

Sys t e m s u i t a b i l i t y r e q u i r e m e n t s —T he

method has the following system suitability

requirements:

t� 5IF� DPFGGJDJFOU�PG�EFUFSNJOBUJPO� 32) for

the standard curve must be ≥ 0.98.

t� 5IF� BWFSBHF� HMVDPTF� DPODFOUSBUJPO� JO�

SPF media must be ±15 % of the value

reported on the CoA supplied by the man-

ufacturer.

t� 5IF� �34%�PG� UIF� HMVDPTF� DPODFOUSBUJPO�

from the six replicates of SPF media must

be ≤ 15%.

The media sample analysis was initiated

only if all system suitability requirements

were met.

Folic acid method

The folic acid method utilized an ultra-

high performance liquid chromatogra-

phy (UHPLC) system from Waters Acquity

equipped with photodiode array detector

(PDA) and Acquity UHPLC column, BEH C18,

1.7 μm, 2.1 x 100 mm. A gradient separation

was performed using mobile phase A: 50 mM

phosphate, 4 mM 1-heptanesulfonic acid, pH

4.5 and mobile phase B: 30% acetonitrile

and 10% methanol in mobile phase A (other

method details are not included). The folic

acid in the media was determined from a

standard curve.

Peer-Reviewed


RESULTSGlucose method

development and validation

As mentioned previously, the authors’ goal

was to develop and validate a simple method

for quantitation of glucose that first, uses

common equipment available at various man-

ufacturing sites worldwide and second, may

serve as part of the specific identification

platform for multi-component cell-culture

media. For this purpose, a standard curve glu-

cose method was developed and validated by

UV-visible spectrometry, using similar prin-

ciples as the commercially available kit. In the

commercial kit, quantitative analysis of glucose

is based on two sequential enzymatic reactions

shown in the following passages (16, 17).

The first reaction is the phosphorylation of

glucose to glucose-6-phosphate (G-6-P). This

reaction is catalyzed by the enzyme hexo-

kinase and utilizes adenosine triphosphate

(ATP) as the source of phosphate. The G-6-P

formed in the first reaction is oxidized to

6-phosphogluconate (6-PG) by the enzyme

glucose-6-phosphate dehydrogenase (G-6-P

DH). During this oxidation, an equimolar

amount of nicotinamide adenine dinucleo-

tide (NAD) is reduced to form nicotinamide

adenine dinucleotide (NADH), which absorbs

light at 340 nm and can be quantitatively

assayed using UV-VIS spectrometry. The reac-

tion scheme is shown in Equations 1 and 2.

Glucose + ATP G-6-P + ADPHexokinase

[Eq. 1]

G-6-P + NAD 6-PG + NADHG-6-P Dehydrogenase

[Eq. 2]

Based on the stoichiometry of the reaction,

the molar concentration of glucose is equiva-

lent to the molar concentration of NADH.

The concentration of NADH is determined by

using Beer-Lambert’s Law.

A validation of the assay was performed

in accordance with USP–NF Genera l

Chapter <1225>, International Council

on Harmonization (ICH) Q2(R1) Tripartite

Guideline Validation of Analytical Procedures:

Text and Methodology, and FDA Guidance for

Industry, Bioanalytical Method Validation (18,

19, 20). The parameters of the validation

included specificity, linearity, accuracy, pre-

cision, range, limit of detection (LD), limit of

quantitation (LQ), and robustness (21).

Specificity was demonstrated by analysis

of three different multi-component media

that contained glucose ranging from 2.0 to

6.4 mg/mL (Table I). In addition, the specific-

ity of the method for glucose as compared

with other hexoses was demonstrated by

analysis of fructose, mannose, galactose ana-

lyzed at 2.5 mg/mL, and sucrose (a com-

monly used excipient) analyzed at 5 mg/mL.

The results presented in Table I indicate that

the experimentally determined glucose con-

centration in the three culture media (CDS,

M12, and M57) are within 97 –103% of the

expected values, which meets the method

acceptance criteria of 85–115% of the

expected concentration. Among the hexoses

and sucrose that were tested, only glucose

was detected by this method, thereby demon-

strating specificity for glucose (Table I). A sec-

ond aspect of specificity was to demonstrate

that media components do not interfere with

Peer-Reviewed

Table I: Validation of the glucose method with specificity as a parameter.

Glucose method validation, specificity

SampleGlucose (mg/mL)

experimental valueGlucose (mg/mL)

target value%

of target value

CDS, Lot 1 6.3 6.4 98

CDS, Lot 2 6.3 6.4 98

CDS, Lot 3 6.6 6.4 103

M12 2.0 2.0 100

M57 2.9 3.0 97

Sucrose (5 mg/mL) Not detected Not detected N/A

Fructose (2.5 mg/mL) Not detected Not detected N/A

Mannose (2.5 mg/mL) Not detected Not detected N/A

Galactose (2.5 mg/mL) Not detected Not detected N/A


the detection of glucose. This specificity can

be accomplished by showing that there is

no signal from media that contains all com-

ponents except glucose. Because glucose-

free media—which would work as a negative

control for each studied media—is not com-

mercially available; an alternate approach is

described in the following passages.

Linearity of the signal was demonstrated

from the standard curve (Table II and Figure 1A).

The signal was linear as a function of glucose

from 0.1 to 2.5 mg/mL in water. The coef-

ficient of determination (R2) was 0.99, hence

meeting the acceptance criteria of ≥ 0.98.

It was also necessary to confirm that the

signal was linear as a function of glucose

concentration in multi-component cell-cul-

ture media. The concentration of glucose in

the cell-culture medium CDS is 6.4 mg/mL.

Therefore, it was not feasible to spike in glu-

cose at the levels that were studied in water.

Therefore, an alternative approach was used

to demonstrate linearity. As discussed previ-

ously, the method measures the absorbance of

NADH, which is formed at an equimolar ratio

with glucose (Equations 1 and 2). The linearity

of signal produced at low levels of glucose in

the media can, therefore, be assessed by spik-

ing the media with NADH at concentrations

that correspond to those typically generated

by glucose at 0.1 to 2.5 mg/mL. The values of

absorbance at 340 nm for NADH spiked in the

CDS media are shown in Table II. A graph of

absorbance vs. concentration of NADH (mM)

is shown in Figure 1B. The graph is linear with

a coefficient of determination (R2) of 0.99.

This experiment demonstrates the linearity of

detection of NADH in the culture media.

An overlay of the absorbance of NADH

spiked in media and the linearity curve of glu-

cose in water (Figure 1B) indicates that linearity

in media is comparable to that in water, as the

corresponding slopes were linear (0.0575 vs.

0.0552 absorbance units/mM, respectively).

The media components did not interfere with

the detection of NADH, thereby demonstrat-

ing assay specificity. This approach confirmed

that the assay was specific for glucose, and

data generated for linearity with glucose in

water can also be used to determine accuracy,

range, and LQ of the method.

Accuracy was determined by calculating

the percent recovery of glucose spiked in CDS

culture medium at concentrations ranging

from ~50% to ~150% of the glucose concen-

tration in each medium. The spike recov-

ery was 104–107% and within 85–115% of

specified value. The precision (repeatabil-

ity) was demonstrated by showing that the

%RSD of six replicates of the measured con-

centration of glucose in CDS medium was

1%. Intermediate precision was measured by

the analysis of three independent lots of CDS

medium by two analysts over three differ-

ent days. The %RSD for analyst 1 for days 1,

2, and 3 was 1% for each lot. The %RSD for

analyst 2 for days 1, 2, and 3 was 3% for each

lot. The overall RSD for three lots over three

Peer-Reviewed

Figure 1: Linearity of the glucose method. 1A illustrates a glucose standard curve, and 1B shows an

overlay of a glucose standard curve with the curve for nicotinamide adenine dinucleotide and hydrogen

(NADH) spiked in CDS cell-culture media.

1.0

0.8

0.6

0.4

0.2

0.0

1.0

0.8

0.6

0.4

0.2

0.00 0 5 10 151 2 3

y = 0.3065x + 0.0075 R2 = 0.9988

y = 0.0552x + 0.0073 R2 = 0.9988

y = 0.0575x - 0.0079 R2 = 0.9988

Glucose

Glucose

NADH

Ab

sorb

ance

at

34

0 n

m

Ab

sorb

ance

at

34

0 n

m

Concentration (mg/mL) Concentration (mM)

1A 1B

AL

L F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

RS


days within two analysts was 2%, which met

the acceptance criteria of ≤ 15%. The LQ

was experimentally shown to be 0.15 mg/

mL. Based on the linearity, accuracy, and

LQ, the range of the assay is 0.15 mg/mL to

2.5 mg/mL. The calculated LD was 0.07 mg/

mL. Because the assay is based on the detec-

tion of NADH, which is generated in equimo-

lar amounts to glucose, it was important to

ensure that all the reactions go to completion.

Therefore, a critical parameter is the time

of incubation of the culture media with the

hexokinase assay reagent kit. Robustness was

demonstrated by showing that the results of

three different media, in which the incuba-

tion time was varied from 15 min to 3 hours,

were comparable (data not shown).

As ment ioned prev iously, the g lu-

cose method was applied to M12 and

M57 media (Table I) and the experimen-

tally determined concentrations of glucose

were within 15% of the expected values. An

applicability of this method was also dem-

onstrated for all other media used in the

study (data not shown). Overall, the vali-

dated method is specific for a detection of

glucose without matrix interference and may

be used as a platform approach for multi-

component cell culture media analysis.

Identification of culture media

For the development of an identification

strategy, a few steps were taken to evaluate

the methods and results for seven culture

media, M12, M51, M55, M10, M53, M57,

and M17, used in this study. All compen-

dial microbiological tests performed for each

media as part of release testing were excluded

from this evaluation. In the first step, simple

tests also provided in the vendor’s CoA, such

as color and appearance, were evaluated. The

results revealed that appearance of all media

in powder form prior to reconstitution was

essentially similar and varied from off-white

orange-beige to pale orange color (data not

shown). Therefore, this test cannot serve as a

differentiator of media.

In the next step, FT–IR with attenuated

total reflection (ATR) accessory was performed

on all media in powder form as per USP <197>

(4). The spectra did not show any significant

difference and, therefore, cannot be used

for specific identification of media (data not

shown). The evaluation of solubility and pH

values of media also did not show any sig-

nificant difference and cannot be used for the

discrimination of media (data not shown).

In the next step, the authors assessed osmo-

lality values of seven media and developed

respective specifications based on analysis of

several lots of material (Table III). The osmolal-

ity values are also provided on vendor’s CoA.

The specification range for the tests plays a

crucial role in media identification, because it

serves to differentiate the media containing the

same components at different concentrations.

A unique identification of each media is based

on the principle that the specification range of

each media is not overlapped with others. For

the purpose of this study, the individual testing

results are less important as long as their values

fall within the specification range. A similar

approach was employed to identify the buffers

used in vaccine production (14).

Based on the specification range presented

in Table III, three media—M12, M51, and M55—

were segregated from others, because their

osmolality values were in the range of 125–165,

Peer-Reviewed

Table II: Validation of the glucose method with linearity as a parameter.

Glucose method validation, linearity

Glucose (in water) analyzed per method NADH spiked in CDS cell-culture medium

mg/mL mM A340

mg/mL mM A340

0.1 0.56 0.02649 0.4 0.56 0.02111

0.3 1.39 0.08230 1.0 1.41 0.07404

0.5 2.78 0.16198 2.0 2.82 0.15678

1.0 5.56 0.32482 3.0 4.23 0.23823

1.5 8.33 0.48042 4.0 5.64 0.31568

2.0 11.11 0.62087 6.0 8.46 0.47327

2.5 13.89 0.76171 8.0 11.28 0.64298


Peer-Reviewed

800–1050 and 366–496 mOsm/kg, respectively.

They also were also distinguished from four

other media (M10, M17, M53, and M57), which

were identified as a group given that their over-

lapped osmolality specification values ranged

from 225–348 mOsm/kg (Table III).

In the next step, the authors assessed target

glucose values determined by the platform

glucose method and developed respective

specifications (Table III). The data and specifi-

cations were evaluated for all studied media

including media individually discriminated by

osmolality tests as well as media segregated as

a group. Based on glucose specification ranges

of 8.5–11.5, 2.5–3.5, and <0.05 mg/mL, three

media—M53, M57, and M51, respectively—

were individually segregated from others. Two

media, M12 and M55 (identified from each

other by osmolality test), had similar target

content of glucose (2 mg/mL) and overlapping

specification in the range of 1.7–2.3 mg/mL

and 1.6–2.4 mg/mL, respectively, and were

therefore segregated as a group. Two remain-

ing media, M12 and M17, with overlapping

glucose specification range of 3.8–6.4 mg/mL

and 4.1–6.1 mg/mL, respectively (segregated

as a group), were identified by the folic acid

method. The specification ranges for folic acid

for the media M10 and M17 were 3.2–4.8 mg/

mL and 5.1–7.7 mg/mL, respectively.

DISCUSSIONAspects of cell-culture

media identification

The authors’ approach for specific identifica-

tion of cell-culture media as well as other

complex non-compendial raw materials is

based on evaluation of available tests pro-

vided by the vendor CoA, in-house methods,

and established strategies for release test-

ing, which comply with FDA, CFR 211.84,

and other regulatory requirements (1, 2).

Several aspects need to be considered in the

development of the strategy. It is important

to emphasize that simplicity related to the

method performance in the QC environ-

ment, aspects of global technology trans-

fer, and equivalency of the equipment used

in testing need to be considered when this

strategy is being developed. Also, the meth-

ods, which provide data that can be used

without any further data processing, must

be considered first. Prior to establishing a

release testing scheme and specifications, all

culture media properties with close formula-

tions from one vendor or multiple vendors

need to be evaluated. In addition, they need

to be compared with the properties of other

media used at the same manufacturing facil-

ity to ensure that all media are specifically

identified. This approach would help support

quality compliance of the facility. It is also

important to pay attention to release tests

of media from a single supplier, because it

is easier to accidently substitute one media

for another if they have similar formulations

and labels and are from the same vendor.

Based on these aspects, the authors devel-

oped a general tool for cell-culture media

identification, which include a combina-

tion of the following methods: osmolality

(compendial), platform glucose by UV-visible

spectroscopy, and complementary folic acid

by UHPLC. The osmolality method estab-

lished an alignment of all acquired data with

that from the vendor’s CoA. Although, solu-

Table III: Characteristics of cell-culture media by osmolality, glucose, and folic acid methods.

Cell-culture media characteristics

MediaOsmolality (mOsm/

kg)Glucose (mg/mL)

Folic acid(mg/L)

SpecificationTarget

concentrationSpecification Specification

M12 125–165 2.0 1.7–2.3 N/A

M51 800–1050 <0.05 N/A N/A

M55 366–496 2.0 1.6–2.4 N/A

M10 258–348 5.0 3.8–6.4 3.2–4.8

M53 225–280 10.0 8.5–11.5 N/A

M57 225–265 3.0 2.5–3.5 N/A

M17 290–330 5.1 4.1–6.1 5.1–7.7


Peer-Reviewed

bility, pH, and color appearance tests did not

distinguish the media from one another by

established specifications, all of these tests

were used as part of release testing.

In light of the aforementioned approach, it

was important to develop a simple platform

test for quantitation of glucose, which is pres-

ent in each one of the studied culture media,

except one. Also, by employing a linear stan-

dard curve for a calculation of glucose concen-

tration in media, the method is independent of

the UV-visible spectrometer being used, which

helps simplify method transfer. This method

can also be adapted to a 96-well plate for high-

throughput media screening. Validation of the

assay for quantitation of glucose in the culture

media includes the demonstration of specific-

ity. This is normally performed by demonstrat-

ing the absence of a signal in the media that

contains all components except the analyte

of interest, which in this case is glucose. The

lack of availability of glucose-free media neces-

sitated an alternate approach to demonstrate

specificity. As described in the introduction,

the glucose levels are quantitated by measuring

the NADH that is generated in presence of glu-

cose (Equations 1 and 2). Thus, the absorbance of

commercially purchased NADH spiked in water

was shown to be comparable to the absorbance

of NADH spiked in culture media, indicating

that the media components do not interfere

with the detection of glucose. The folic acid

method was used as a supplementary tech-

nique for the two remaining cell-culture media

that could not be distinguished by a combina-

tion of osmolality and glucose methods.

Strategy and algorithm for specific

identification of culture media

Based on the acquired data and established

specifications discussed in the previous sec-

tion, a streamlined approach to identity testing

Figure 2: Strategy for identity testing of the studied cell-culture media using osmolality, glucose, and

folic acid methods. Non-specific identification of each media is defined by triangles.

Samplecell-culture

media

M51

M51 M53

M10 M17

Non-specific identification

M57M12M55

M10M17

M12 M55

Osmolality

Glucose

Folic acid

M10M17M53M57


Peer-Reviewed

was defined that will allow efficient, unam-

biguous identification of the seven cell-cul-

ture media used in this study. The strategy

for specific identification of studied media is

shown in Figure 2. The scheme demonstrates

how data acquired from osmolality and glu-

cose analysis, complemented with folic acid

test when needed, results in unambiguous

identification of all seven media. The osmo-

lality test differentiated three studied media

(M51, M12, and M55). The specifications of

four other media (M10, M17, M53, and M57)

overlapped and were between 225 mOsm/kg

and 348 mOsm/kg. Therefore, these media

were segregated together and identified as a

“group identity” because their specifications

overlapped (Table III). Glucose analysis of all

seven media further discriminated two media

(M53, M57) not segregated by osmolality

and identified two “group identity” media,

(M10, M17) and (M12, M55), which had over-

lapping specifications. The glucose speci-

fication and osmolality specification range

of M51 media distinguished it from others.

However, the M12 and M55 media discrimi-

nated from others by osmolality, belong to a

“group identity” based on the similar glucose

specification range (Table III). The remaining

media—M10 and M17—are effectively iden-

tified by folic acid as a complementary test;

only these two media were tested by folic acid.

It is important to emphasize that all tests used

in this strategy are non-specific and only a

combination of two or more tests would pro-

vide a specific identity of each cell-culture

media. Moreover, because the tests are non-

specific, the established specification range for

each test would serve as an actual discrimina-

tion tool in identification of media. Obviously,

all data acquired by all methods need to fall

within the respective specifications. Another

important point is that due to this “non-spe-

cific” identity of each media by a single test,

only a combination of two or more tests would

provide a specific identity as required per CFR

211.84. The intermediate step of “group iden-

tity” is essential, because it narrows down a

number of media for further identification.

The scheme presented in Figure 2 for studied

media can be extrapolated to suggest a gen-

eral algorithm for their identity testing. This

more general scheme is shown in Figure 3. A

similar approach was proposed for chroma-

tography resins in another, separate study, but

employed different methods (22). As men-

tioned previously, compendial microbiological

tests were not included in this algorithm, but

need to be performed as well as part of release

testing. Glucose analysis can be pursued in

parallel with the osmolality analysis to iden-

tify the media and confirm the quantity of

glucose within established specifications. The

quantitation of glucose is specifically impor-

tant for media with similar composition man-

ufactured and labeled by the same vendor.

Any sample not identified through the combi-

nation of the described two non-specific iden-

tity tests can be subjected to folic acid analysis

and quantity verification against established

specifications to provide a final discriminative

evaluation of sample identity.

Overall, the proposed testing scheme can

incorporate a few relatively simple analytical

Figure 3: General algorithm for identity testing of a collection of cell-

culture media using a combination of non-specific methods including

osmolality, glucose, and folic acid. In addition, label check, color

and appearance, and pH and solubility tests are performed for each

culture media.

Sample culture media

Label check

Color and appearance

Solubility, pH

Osmolality

Glucose

Unambiguous identity? Folic acidNo

Yes

Identity defined


Peer-Reviewed

methods into an efficient testing algorithm

to provide definitive identity confirmation

for cell-culture media from one or more ven-

dors. The methods are well suited for routine

use in a QC environment and the specific

identification approach meets regulatory

requirements. This is an example of a plat-

form tool for glucose, which may be used for

other strategies applied to similar media. It

is important to highlight that glucose, as a

more simple test compared with a folic acid

test, may be used alone for the identification

of five cell-culture media examined in the

study, while only two media needed to be

identified through the folic acid method.

CONCLUSIONThe developed testing algorithm, which

incorporates a combination of a few rela-

tively simple analytical methods such as

osmolality, quantitation of glucose, and folic

acid, provides specific identity confirma-

tion for seven studied cell-culture media

with essentially similar composition. The

methods are well-suited for routine use in a

QC environment and the provided identifi-

cation approach meets CFR, FDA, and other

regulatory agency requirements. This assay

used in this study was shown to be specific

for detecting glucose in all studied multi-

component media without matrix interfer-

ence, regardless of a glucose concentration or

the vendor.

ACKNOWLEDGEMENTSThe authors would like to thank Dr. Sam

Mathew, Michael Adamo, and Jaimin

Patel from Bristol-Myers Squibb, as well

as Dr. K.C, Cheng (currently at Actinium

Pharmaceuticals, Inc.) and Dr. Xiao-Ping Dai

(currently at Celgene Corporation) for pro-

viding valuable input.

REFERENCES 1. CFR Title 21, Part 211.84: Food and Drugs

(Government Printing Office, Washington, DC), pp.

148–149.

2. G. Beck et al., BioProcess Int. 8 (7), pp. 2–13

(2009).

3. N. Afonina, B. Lay, Z. Akhunzada, S. Mathew, S.

Mallya, and M. Grace, “Controlling Raw Materials in

Biological Manufacture: Regulatory and Analytical

Aspects,” presentation at Informa/IBC Life

Sciences (Cologne, Germany, June 27–28, 2012).

4. USP, USP General Chapter <197>,

“Spectrophotometric Identification Tests,” USP 38–

NF 33 pp. 220–221.

5. N. Afonina, J. Timmermans, and L. Bhattacharyya,

“A Novel Approach to the Development of ID Release

Tests for Resins and Growth Media,” presentation

at the 2nd Annual Meeting on Raw Materials and

Contract Services for Mammalian Cell Products (St.

Louis, MS, June, 11–13, 2000).

6. G. Reich, Adv. Drug Deliv. Rev. 57 (8), pp.1109–

1143 (June 15, 2005).

7. T. Strother, European Biopharm. Rev. EBR 12 (169)

article 3215, (2012).

8. H.W. Lee et al., Biotechnol. Prog. 28 (3), pp. 824–

832 (2012).

9. A.O. Kirdar, G. Chen, and A.S. Rathore, Biotechnol.

Prog. 26 (2), pp. 527–531 (2010)

10. C. Sharma et al., BMC Proceedings 5 Supplement

(8), P5 (2011), doi:10.1186/1753-6561-5-S8-P5

(2009).

11. B.P.W. Ryan et al., Biotechnol. Bioeng. 107 (2), pp.

290–301 (2010).

12. J.K. Simonelli et al., Chromatographia 66 (11–12),

pp. 977–981 (2007).

13. J. Zhang et al., Electrophoresis 30 (22), pp. 3971–

3977 (2009).

14. N. Afonina, K. Bhatt, L. Howson, and L.

Bhattacharyya, “ID Tests for Liquid Formulation Raw

Materials: Growth Media and Buffers,” presentation

at the 7th Annual Meeting on Viral Vectors and

Vaccines (Lake Tahoe, NV, Nov. 6–9, 2000).

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Osmolarity,” USP 38–NF 33 (US Pharmacopeial

Convention, Rockville, MD, Feb. 1, 2015) pp. 541–

543.

16. J.J. Carroll, N. Smith, and A.J. Babson, Biochem.

Med. 4, pp. 171–180 (1970).

17. M.W. Slein, Methods of Enzymatic Analysis, H.U.

Bergmeyer, Ed. (Academic Press, New York, NY

1963), pp. 117–123.

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of Compendial Procedures,” USP 38–NF 33 (US

Pharmacopeial Convention, Rockville, MD, Feb. 1,

2015) pp. 1445–1461.

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Analytical Procedures: Text and Methodology, Step 4

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Method Validation (Rockville, MD, May 2001).

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Jr., The Analyst (UK) 129, pp. 1091 –1098 (2004). ◆


Scie

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es

Use of continuous cell lines

in the manufacture of bio-

logical therapeutic prod-

uc t s , such a s vacc ines ,

recombinant proteins, and monoclo-

nal antibodies, is associated with the

concomitant risk of process/product

contamination with endogenous ret-

roviruses, latent viruses, or new and

emerging adventitious agents. Cell-

culture applications are impossible

without the use of nutrient media for

cell multiplication and subsequent

product generation. Although several

serum-free and chemically defined

nutrient media formulations are avail-

able for commercial use, many cell-

culture applications require use of

nutrient media supplementation with

serum or other animal-derived com-

ponents. Use of serum-supplemented

cell-culture media is considered a

point of entry for the introduction of

adventitious agents into a manufac-

turing process. Other raw materials

used in the manufacture of biothera-

peutics, especially those of animal

and human origin, could also present

a viral safety risk.

Traditionally, the management of

inadvertent virus contamination is

achieved through the incorporation of

various measures aimed to preclude,

detect, and inactivate adventitious viral

agents from the biological products

(i.e., selection, testing, and clearance).

A lthough a plethora of regula-

tory guidance documents have been

enacted governing product safety from

adventitious agents (1–16), complete

An Integrated Approach to Ensure the Viral Safety of Biotherapeutics

Mark Plavsic

Testing product and process intermediates

alone is helpful, but does not

provide a complete solution to viral safety. This

article proposes integrated

solutions for systemic and

proactive viral risk mitigation.

Mark Plavsic, PhD, DVM, was

head of Corporate Product Biosafety,

Genzyme, a Sanofi Company,

Framingham, MA, USA. Presently,

Mark is head of process development

and manufacturing with Torque

Therapeutics, [email protected].



AL

L F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

R

risk elimination has not yet been

achieved. Several examples of

bioproduction process contam-

ination have been documented

over the years, implicating min-

ute virus of mice (MVM), epizo-

otic hemorrhagic disease virus

(EHDV), reov i rus 2 (REO-2),

cache valley virus (CVV), and

calicivirus 2117 (17–23). Instances

of final product contamination

with adventitious agents have

also been published (24, 25).

A recent Pharma IQ study (26)

revealed that 37.5% of respon-

dents—who were from the bio-

t e c h no lo g y m a nu f a c t u r i n g

industry in the United States and

Europe—named viruses as their

biggest concern, despite the fact

that nearly two thirds (62.5%) of

surveyed participants already had

strategies in place to mitigate the

risk of viral contamination. More

than a third (37.5%) of partici-

pants said their organization was

satisfied with the solutions they

currently have in place. These

strategies so far appear to be work-

ing on the whole, as 87.5% of com-

panies also reported they have not

had to deal with a contamination.

The purpose of this article is to

discuss some holistic, interlock-

ing approaches across the manu-

facturing chain to reduce the risk

of adventitious viral agent con-

tamination and to ensure unin-

terrupted supply of safe biological

products to patients in need.

PRODUCT SAFETY AND QUALITY BY DESIGN (QBD)Tradit ional ly, product sa fety

has relied on the incorporation

of three key measures into the

manufacturing process: selection,

testing, and viral clearance. These

measures are collectively known

as the “safety triangle” (Figure 1).

The elements of the safety trian-

gle include the selection of source

materials and release based on

prior material and supplier qual-

ity approval and qualification test-

ing for adventitious contaminants;

testing for various adventitious

contaminants at appropriate stages

of the manufacturing process from

raw materials, starting materials

(e.g., cell banks, viral and bacte-

rial seeds), and manufacturing

intermediates; and viral clearance,

employed either in raw material

control or evaluation of the capac-

ity and capability of the down-

stream purification process to

clear (remove or inactivate) poten-

tial adventitious contaminants.

Although the safety triangle still

represents a central dogma in the

viral safety of biological products,

it has been generally accepted that

the safety triangle alone may not

be sufficient, and some enhance-

ments may be warranted.

Today’s industry and regula-

tory expectations require that

an effective viral risk mitigation

strategy be built into the whole

manufacturing chain, spanning

from the suppliers of crucial raw

materials to the fill/finish and

contract manufacturing organi-

zations (CMO), where applicable

(Figure 2).

In this context, viral risk mitiga-

tion should be an integral part of

the overall quality system and qual-

ity risk management strategy (12).

Viral safety needs to be designed

into the overall drug-development

process and QbD approach.

The concept of product “safety

by design” (SbD) represents an

integrated, holistic approach to

viral safety across the manufac-

turing chain. The goal of SbD is

to protect a manufacturing pro-

cess from interruptions caused by

viral contamination and ensure

product and patient safety by pre-

venting virus introduction, ensur-

ing early detection, and enabling

rapid response to ensure contain-

ment/elimination of viruses if

introduced into the manufactur-

ing process. It typically spans the

following five areas:

t� 3BX�NBUFSJBMT�3.

t� /FX� QSPDFTT�QSPEVDU� EFWFMPQ-

ment (PD)

t� .BOVGBDUVSJOH�QSPDFTT

t� 2VBMJUZ�TZTUFN�24

t� %FUFDUJPO�UFTUJOH�

In the context of this article,

animal-origin (AO) and chemi-

cally defined (CD) raw materials

are defined as follows.

Animal (including human) ori-

gin materials are derived from

va r ious spec ie s of a n ima l s ,


Figure 1: Illustration of the biotherapeutics safety triangle.

Selection

Testing Clearance

Productbiosafety


including humans, and they can

be either primary (direct) or sec-

ondary (indirect).

Primary (direct) animal origin

materials are derived from animals

or their tissues with or without

further processing. Unprocessed

primary animal origin materi-

als are derived with minimal or

no further processing. Examples

include: whole blood, serum, cells,

tissue extracts, and intestinal

mucosa. Processed primary animal

origin materials are derived after

a series of processing steps such as

extraction, precipitation, or purifi-

cation. Examples include: bovine

serum albumin (BSA), human

serum albumin (HSA), porcine

trypsin, purified enzymes, wool-

derived cholesterol, heparin, gela-

tin, casein, and collagen.

Secondary (indirect) animal

origin materials are recombinant

proteins derived from manufac-

turing (e.g., fermentation) pro-

ce s se s where a n i ma l - or ig i n

materials were added.

Chemically defined raw mate-

rials have all components iden-

tif ied; components are in the

chemical form, and the structure

and concentration of all com-

ponents is known and of high

purity with only minimal levels

of trace chemical impurities.

The following sections address

viral risk mitigation across the

five areas in more detail.

RAW MATERIALSRaw materials have been regarded

as one of the main portals of viral

entry into a GMP manufacturing

environment. The main goal of

viral risk mitigation at this level

is to prevent virus introduction

into a manufacturing process

via raw materials. The following

measures should be considered in

addressing this level of viral risk

remediation:

t� *NQMFNFOU� B� QSPDFTT� PG� JEFO-

tification and segregation of

all critical (e.g., animal and

human origin) raw materials.

t� *OUSPEVDF� B� S JTL� BTTFTTNFOU�

for animal- and human-origin

components.

t� %FWFMPQ� B�QPMJDZ�PG� iUISFF�3Tw�

(replacement/reduction/refine-

ment) for animal- and human-

origin components.

t� .BJOUBJO� TPMJE� LOPXMFEHF� PG�

raw materials origin, sourcing,

manufacturing, testing, stor-

age, and traceability.

t� 3FWJFX�PG� DSJUJDBM� SBX�NBUFSJBM�

specifications for adequacy and

viral safety acceptance criteria.

t� .BJOUBJO� B� EFUBJMFE� TVQQMJFS�

auditing and qualification pro-

gram that includes biosafety

considerations.

t� &TUBCMJTI� B� TVQQMJFS� EFWFMPQ-

ment and improvement pro-

gram addressing key areas of

qual ity, biosafety, and r isk

management.

t� 5SFBU � SBX� NBUF S JB M T � F �H � �

through ultraviolent [UV-C]

irradiation, gamma irradiation,

heat, pH, solvent detergent,

nanofiltration, etc.) to clear

viruses. Although treatment

options are helpful mit iga-

tion tools, they are not equally

effective against all viruses.

From that perspective, raw mate-

rial treatment provides a certain

level of risk mitigation, but not

complete risk elimination.

Some examples of raw mate-

rial (animal origin and chemi-

cally defined) treatment options

include:

t� 8IFO� TFSVN� JT� VTFE� JO� NBO-

ufacture, serum treatment by

gamma i r rad iat ion (30 – 50

KGy), UV-C (30–150 mJ/cm2),

or other modalit ies may be

considered to be practical risk

mitigation tools.

t� 8IFO� QPSDJOF� USZQTJO� JT�VTFE�

in manufacturing processes,

replacement with recombinant

trypsin can be investigated.

Alternatively, liquid porcine

trypsin solution could be nano-

filtered using 15–20-nm pore

size filters (circo- and parvo-

virus removal) or treated by

UV-C rays.

t� #VML� QPXEFS� NBUFSJBM� PG� BOJ-

mal/human origin (e.g., serum

albumin, transferrin) can be

gamma irradiated in its final

packaging. Alternatively (or

additionally), nanofiltration

(20 -nm pore size) of l iquid

solutions can be considered at

the point of use.

t� -JRVJE� DFMM�DVMUVSF� NFEJB� DBO�

be treated by nanofiltration

(20-nm pore size), UV-C, or

heat (e.g., high-temperature for


Figure 2: Effective viral risk mitigation across the whole manufacturing process

(example: r-proteins, a typical recombinant protein manufacturing process).

SUPP & RM Media prep USP DSP DS/DP Fill/Finish

CMO

SUPP: Suppliers of raw materialsRM: Raw materialsUSP: Upstream process (cell-culture operations)DSP: Downstream purification processDS/DP: Drug substance/drug productCMO: Contract manufacturing organization



short-time treatment, HTST) at

the point of use. Gamma irra-

diation of the media powder

before reconstitution may also

be investigated realizing that

certain media components may

be radiosensitive and thus not

compatible with gamma irra-

diation.

t� 0UIFS� SBX� NBUFSJBMT� JODMVE-

ing formulation buffers can be

nanofiltered (20-nm pore size)

as liquid solutions.

BUILDING VIRAL SAFETY INTO A QUALITY SYSTEMEffective virus risk mitigation

should be part of the overall

quality system. That way, the

viral safety is “system-driven”

instead of being people-depen-

dent. Prevention of virus intro-

duction, viral risk understanding/

mitigation, and effective response

to potential viral contamination

are the main objectives of this

TUFQ��8IFO�CVJMEJOH� WJSBM� TBGFUZ�

into the overall quality system,

a written, comprehensive virus

mit igat ion program is neces-

sary. The main purpose of this

program is to promulgate a sus-

tainable, long-term policy as a

foundation for viral safety based

on the SbD principles. It will

form the basis for incorporation

of viral safety into the overall

quality system for both commer-

cial processes and new products

in development.

Additionally, a viral risk assess-

ment must be performed. The

purpose of a viral risk assessment

is to promote ongoing and pro-

active viral risk identif ication

and management. It should be

conducted using a risk analysis

tool suitable for viral risk (e.g.,

failure mode and effect analysis

[FMEA], preliminary hazard anal-

ysis [PHA], and risk ranking and

filtering [RRF]) with periodic risk

re-evaluation, addressing the fol-

lowing areas at minimum:

t� 3JTL�PG�WJSVT�FOUSZ�XJUI�BQQSP-

priate controls:

o Starting materials (e.g., cell

banks, viral and bacterial

seeds, animals used in pro-

duction)

o Raw mater ia ls (e.g., cel l-

culture media, serum, plant

extracts)

o Personnel

o Equipment

o Manufacturing process (e.g.,

type of cells, type of process,

open vs. closed cell-culture

steps, duration, containment)

o Manufacturing plant internal

environment and utilities

o Outside plant environment

t� 4QFDJGJD� WJSVT� DPOUSPMT� JO� UIF�

product manufacturing process:

o Virus testing (bulk harvest,

drug substance, drug product)

o Viral clearance afforded by

downstream purification.

A written emergency (contami-

nation) response plan is an impor-

tant part of the SbD method. The

objective of a viral response plan

is to specify necessary steps in

the response process, delineate

clear roles and responsibilities,

and ensure rapid response to a

suspect or confirmed viral con-

tamination. It ought to be spe-

cific in terms of clearly addressing

the questions of what, why, who,

how, when, and where. A success-

ful response plan achieves effec-

tive area containment, allows

rapid virus elimination through

effective disinfection, and enables

speedy facility return to the rou-

tine manufacturing regimen.

Existing procedures may need

to be modified to incorporate

viral safety. Certain quality pro-

cedures may need to be refined to

integrate elements of viral safety,

as appropriate. Such procedures

may include aseptic training, pur-

chasing of suitable raw materials,

raw material supply-chain man-

agement, and cleaning and saniti-

zation, for example.

Biosafety should be incorpo-

rated into a company’s quality

audit program. Incorporat ion

of viral safety elements into the

internal and external (supplier)

audits helps identify weaknesses

and strengths of the firm’s qual-

ity systems. Importantly, it helps

drive improvements in the prac-

tices of the suppliers of critical

raw materials.

-BTUMZ � B� USBJOJOH� NPEVMF� PO�

viral safety and its impact on

product and patients would be

prudent. This training brings

awareness and drives employee

behavior in specif ic units of

operations that are more suscep-

tible to viral contamination. In

concert, these measures would

greatly enhance a quality-based

viral risk mitigation program and

a firm’s readiness to respond to a

contamination event.

PRODUCT DEVELOPMENT/FX�QSPEVDU�EFWFMPQNFOU�QSFTFOUT�

an ideal opportunity to incorpo-

rate all the relevant principles of

SbD into the new process. Here,

product safety is intentionally

designed into the new process with

the goal of preventing introduc-

tion of adventitious viruses into

the process and designing mean-

ingful product testing strategy,

while enabling rapid detection and

containment in any area where a

problem has occurred. The follow-

ing points should be considered

when building SbD into new prod-

uct/process development:

t� 4FMFDUJPO� BOE� FOHJOFFSJOH� PG�

cell lines

t� %FWFMPQNFOU�PG� BOJNBM�PSJHJO�

free/chemically defined cell

banks (e.g., from transfection

to master and working cell

CBOL�<.$# �8$#>�HFOFSBUJPO

t� %FWFMPQNFOU�PG� BOJNBM�PSJHJO�

free/chemically defined manu-

facturing processes, devoid of

animal and human origin com-

ponents


t� *ODPSQPSBUJPO�PG�BO�BQQSPQSJBUF�

and meaningful testing strategy

t� 6TF� PG� VQTUSFBN� WJSBM� CBSSJFS�

technologies (e.g., UV-C, HTST,

nanofiltration) for media/raw

material treatment

t� 6TF� PG� DMPTFE� QSPDFTT� TZTUFNT�

where appropriate

t� *ODPSQPSBUJPO�PG�FGGFDUJWF �WBMJ-

dated viral clearance steps in

downstream processing includ-

ing two orthogonal viral clear-

ance steps (e.g., an inactivation

step and a viral removal by

nanofiltration) for drug-sub-

stance generation

t� %FTJHO � PG� DMPTFE � QSPDFTTFT�

units of operat ion, making

them inaccessible to environ-

mental adventitious agents

t� 6TF� PG� EJTQPTBCMF � TJOHMF�VTF�

equipment whenever feasible

t� &YQMPJUBUJPO�PG�B�DMFBS�TBNQMJOH�

plan and well-defined testing

plan for adventitious agents

t� *ODMVTJPO� PG� QSPDFTT� BOBMZUJ-

cal technology (PAT) to enable

early detection of cell culture

contamination

t� *NQMFNFOUBUJPO�PG� BMM� UIF� SBX�

materials and quality system

principles discussed in the pre-

vious sections.

COMMERCIAL PRODUCT MANUFACTURINGControl measures at this level

serve primarily to prevent virus

int roduc t ion and to ensu re

v irus containment. The con-

trols employed at this level may

include:

t� $POUJOVPVT� QSPDFTT� JNQSPWF-

ment, considering some of the

elements discussed for new

product development

t� &GGFDUJWF� GBDJMJUZ� BOE� FRVJQ -

ment cleaning and sanitization

procedures using proven viru-

cidal and sporicidal chemicals

t� "O� POHPJOH� GBDJMJUZ� JNQSPWF-

ment plan and proper facility

design to prevent contamina-

tion, including area contain-

ment to prevent virus spread

from the affected area; seg-

regation of various activities

such as raw material handling,

media and buffer preparation,

cell-culture operations, down-

stream operations, and post-

viral clearance operations; air

pressure differential; HVAC;

treatment of production water;

control and appropriate f i l-

tration of production gases;

waste treatment and waste dis-

posal; and a pest control pro-

gram

t� 1SPQFS� FNQMPZFF� USBJOJOH� BOE�

gowning, inc lud ing pol icy

on managing employees with

apparent communicable (respi-

ratory, gastrointestinal, cutane-

ous) diseases in GMP areas.

VIRAL TESTING/DETECTION LEVELAs the name indicates, the main

purpose of this level is to detect

adventitious viruses. Early detec-

tion is essential in order to deploy

an adequate response to contain

and eliminate the virus. Various

testing methods are used at sev-

eral stages of process/product

development and manufacture.

The following testing principles

should be considered:

t� 5FTUJOH� EFTJHO� TIPVME� CF� TVJU-

able for the process and, at a

minimum, in compliance with

the current regulatory require-

ments.

t� 5FTUJOH� TIPVME� CF� DPOEVDUFE�

at the most meaningful pro-

cess steps using appropriate

samples, sample volumes, and

suitable testing methods.

t� -JNJUBU JPOT � PG � UIF � FYJTU JOH�

testing methods used for raw

materials, cell banks, seeds,

harvests, and product test-

ing should be understood and

addressed appropriately.


Figure 3: The author presents an augmented safety triangle that can help

ensure a holistic and integrated approach to product safety.

SUPP: Suppliers of raw materialsRM: Raw materialsUSP: Upstream process (cell-culture operations)DSP: Downstream purification processDS/DP: Drug substance/drug productCMO: Contract manufacturing organization

SUPP & RM

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t� 3JTL�CBTFE� UFTUJOH� BVHNFOUB-

tion: specific virus testing (e.g.,

by polymerase chain reaction

[PCR] or other technology)

should be implemented, if jus-

tified by risk assessment. This

testing should include new and

emerging agents.

t� /FX� EFUFD U JPO � UFDIOPMPHZ�

(13) with broad detection and

identification capability (e.g.,

deep sequencing, microarrays)

should be adopted to augment

the overall testing and charac-

terization program, to address

certain testing gaps, or to sup-

port risk assessment program.

SUMMARY OF VARIOUS VIRUS RISK-MITIGATION OPTIONSAlthough the sa fety t r iangle

plays a central role in ensuring

viral safety of the final product,

various other options (Figure 3)

are available to further augment

the safety tripod and reduce the

overall risk of viral entry into the

QSPEVDU� TUSFBN�� 8IFO� QSPQFSMZ�

integrated, these measures can

bring residual viral safety risk to

a very low level.

CONCLUSIONSViral safety is an important qual-

ity attribute of a drug product.

Endogenous or adventitious viral

agents are generally regarded as

product impurities and are not

acceptable in the final drug dos-

age. Ensur ing f reedom f rom

adventitious or endogenous viral

agents is therefore crucial for the

safety of biological drug recipi-

ents. Traditionally, the safety

triangle (selection–testing–viral

clearance) has been helpful, but

it may not be sufficient to meet

today’s expectations. An inte-

grated viral risk-mitigation pro-

gram (i.e., safety by design) across

the supply chain, therefore, is

important to provide a high level

of viral safety of biological prod-

ucts. Safety by design should not

only be incorporated into new

product development, it should

also be part of a larger process-

control strategy for both develop-

ment and commercial products.

Zero risk in the manufacture of

biological products is not achiev-

able; aiming for risk that is “as

low as reasonably achievable”

"-"3"� TIPVME� CF� BO� BDDFQU-

able goal—and it can be achieved

through use of a holistic, inte-

g rated, and produc t- spec i f ic

safety by design program.

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Serum in the Manufacture of Human

Biological Medicinal Products (London,

May 30, 2013).

2. EMA, Requirements and Controls

Applied to Bovine Serum Used in the

Production of Immunological Veterinary

Medicinal Products (London, Nov. 9,

2009 [Revised]).

3. EMA, Guideline on the Use of Porcine

Trypsin Used in the Manufacture of

Human Biological Medicinal Products

(London, Feb. 20, 2014).

4. EMA, Note for Guidance on Minimising

the Risk of Transmitting Animal

Spongiform Encephalopathy Agents via

Human and Veterinary Medicinal

Products (London, 5.3.2011).

5. OIE Terrestrial Animal Health Code,

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(Chapter 11.5, 2011), http://web.oie.

int/eng/normes/MCode/en_

chapitre_1.11.5.htm, accessed on

March 1, 2016.

6. CFR Title 9, parts 113.46, 113.47,

113.50, 113.51, 113.52, 113.52,

113.54, 113.55 (Government Printing

Office, Washington, DC, Jan. 1, 2006

edition), pp. 640–645.

7. USP, <1024> Bovine Serum Appendix

1, USP 38–NF 33 (US Pharmacopeial

Convention, Rockville, MD, Oct. 1,

2015), pp. 719.

8. EDQM, European Pharmacopoeia,

Monograph: Bovine Serum,

01/2008:2262, pp. 1506–1507.

9. ICH, Q5A (R1) Quality of

Biotechnological Products: Viral Safety

Evaluation of Biotechnology Products

Derived from Cell Lines of Human or

Animal Origin, Step 5 version (1997).

10. FDA, Memorandum: Points to Consider

in Characterization of Cell Lines Used

to Produce Biologics (Rockville, MD,

Jul. 12, 1993).

11. FDA, Memorandum: Points to Consider in

the Manufacture and Testing of

Monoclonal Antibody Products for Human

Use (Rockville, MD, Feb. 28, 1997).

12. ICH, Q9 Quality Risk Management

(Rockville, MD, January 2011).

13. PDA, Technical Report #71: Emerging

Methods for Virus Detection, PDA 2015.

14. ICH, Q5D Quality of Biotechnological

Products: Derivation and

Characterisation of Cell Substrates

Used for Production of

Biotechnological/Biological Products

(March 1998).

15. WHO, Recommendations for the

Evaluation of Animal Cell Cultures as

Substrates for the Manufacture of

Biological Medicinal Products and for

the Characterization of Cell Banks

(2010).

16. EMA, Guideline on Virus Safety

Evaluation of Biotechnological

Investigational Medicinal Products

(London, July 24, 2008).

17. R.L. Garnick, Dev. Biol. Stand. 88, pp.

199–203 (1996).

18. H. Rabenau et al., Biologicals 21, pp.

207–214 (1993).

19. R. Nims et al., BioPharm Int. 21 (10),

pp. 89–94 (2008).

20. A. Oehmig, J. Gen. Vir. 84 (12), pp.

2837–2845 (2003).

21. A. Kerr and R. Nims, PDA J. Pharm.

Sci. Tech. 64, pp. 481–485 (2010).

22. M. Plavsic et al., Bioprocess. J. 9 (2),

pp. 6–12, (2011).

23. Y. Qiu et al., Biotechnol. Bioeng. 110

(5), pp. 1342–1351 (2013).

24. P.P. Pastoret, Biologicals 38, pp.

332–334 (2010).

25. J.G. Victoria et al., J. Vir. 84 (12), pp.

6033–40 (2010).

26. Pharma IQ, “62.5% of Biologics

Professionals Have Strategy in Place

to Mitigate Risks of Contamination,”

www.pharma-iq.com/manufacturing/

articles/625-biologics-professionals-

have-strategy-place-mitigate-risks-

contamination, accessed June 2,

2015. ◆

Traditionally, the

safety triangle has

been very helpful,

but it may not be

sufficient enough

to meet today's

expectations.


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Troubleshooting

Single-use systems (SUS), also called dis-

posable technology, can improve manu-

facturing efficiency by reducing the time

needed for cleaning and cleaning validations.

In addition, using SUS reduces the cost associ-

ated with cleaning. For a multiproduct facil-

ity, SUS mitigate cross-contamination risks.

Contract manufacturing organization Grand

River Aseptic Manufacturing (GRAM) recently

expanded its disposable technology capabilities

at its FDA-approved manufacturing facility in

Grand Rapids, Michigan and has identified addi-

tional benefits. “Compared to stainless-steel sys-

tems, process development times with SUS are

reduced and there is greater flexibility in process

design, which contributes to an overall lower

capital cost,” says Steve Nole, GRAM’s direc-

tor of manufacturing. It is important, however,

to understand the challenges associated with

using SUS and to have plans in place to mitigate

risk. Nole and Amanda Hawkins, GRAM’s man-

ager of sterile operations, spoke with BioPharm

International about some of the best practices the

company has implemented in using SUS.

KEYS TO SUCCESSBioPharm: What have you found are the keys to

successfully using disposable technology?

GRAM: GRAM is an advocate of disposable

technology, but some customers may be hesi-

tant to use it, especially if they have an existing

process. Open-mindedness from the client is,

therefore, the first step in using disposable tech-

nology for a project. We have found that clients

with processes at the earlier stages are more

open to using disposable technology because of

its benefits.

The next step is checking for

compatibility between the cli-

ent’s material and the disposable

technology materials. GRAM can

conduct early-phase testing internally before a

project moves forward. Once a project reaches

the commercial scale, a formal leachable and

extractable study must be performed; these

studies are typically outsourced to an indepen-

dent, third-party testing laboratory.

Another key is careful planning for material

sourcing. Standard bags, for example, are read-

ily available, but custom-built configurations

can have long lead times. We must forecast and

have these in stock, because a client may not be

able to wait four months to start a project. On

the other hand, materials have an expiration

date. There is a fine balance between having

the right amount of inventory in stock and hav-

ing an excess that’s at risk of expiring and going

unused. As a best practice, we are developing

a ‘library’ of what sizes of bags and what types

of tubing and connectors will work best with

certain products, so that we can have these on

hand and know what to use.

HANDLING COMPONENTSBioPharm: What best practices have you identi-

fied for handling the tubing and fluid path?

GRAM: We have standardized the fluid path,

although there are some slight variations

depending on the specific process. All areas

where the tubing will be are designated and

consistent, so that the operators are familiar

“All the operators have

had ‘touch time’ with the

connectors and have practiced

how to use them ... ”—GRAM

Using Single-Use Systems in Aseptic Fill-FinishMaterial compatibility, material sourcing, facility layout, and training are crucial aspects of a successful disposable fill-finish system.

Jennifer Markarian is

manufacturing editor at

BioPharm International.


Call for Papers * Call for Papers * Call for Papers

BioPharm International integrates the science and business of biopharmaceutical development and manufacturing. We provide

practical, peer-reviewed technical solutions to enable biopharmaceutical professionals to perform their jobs more effectively.

We are currently seeking novel research articles for our peer-reviewed journal as well as manuscripts for our special issues.

Submitted manuscripts should be sufficiently novel to be of interest to an experienced audience. Articles should be data driven

and provide sufficient technical detail to support the main thesis or should offer a novel synthesis of existing data. Topics should

be timely and useful and should focus on the development of peptides, monoclonal antibodies, fusion proteins, other thera-

peutic proteins, nucleic acids, vaccines, cells for cell therapy, and any other class of biotechnologically generated molecular class.

For peer-reviewed papers, members of BioPharm International’s Editorial Advisory Board and other industry experts review

manuscripts on technical and regulatory topics. The review process is double-blind. Manuscripts are reviewed on a rolling basis.

Our single-themed issues, which include literature reviews and tutorials, cover a range of topics. Upcoming issues address out-

sourcing and bioprocessing.

BioPharm International readers are involved in product and process development, manufacturing, quality control/quality assur-

ance, analytical technologies, regulatory affairs, plant and project engineering and design, and corporate management for the

entire scope of biopharmaceutical products, including therapeutic peptides, proteins, nucleic acids, and cells for cell therapies

and regenerative medicine, as well as both therapeutic and prophylactic vaccines.

Please visit our website, www.BioPharmInternational.com, to view our full Author Guidelines. Manuscripts may be sent to

Editorial Director Rita Peters at [email protected].

WWW.BIOPHARMINTERNATIONAL.COM

with and comfortable working

around the tubing. In the aseptic

processing area, the tubing is ele-

vated to avoid trip hazards.

It is important to plan an effi-

cient layout. Long tubing lines

can be minimized by placing the

formulation vessel in the formula-

tion room as close as possible to

the receiving vessel in the aseptic

processing area. Another practice

is to filtrate in bulk rather than

continuously, so that a one-time

transfer from formulation vessel to

receiving vessel can be performed

and the tubing removed after the

transfer.

Connectors are a key compo-

nent and there are many differ-

ent options. Some are genderless,

and others have male/female ends

and are more complicated to put

together. Some are more difficult

than others to make sure they are

fully connected. All the operators

have had ‘touch time’ with the

connectors and have practiced how

to use them outside of the aseptic

processing areas, as well as during

media fill validation. There is a

technique to pulling straight down

and not too fast, for example.

BioPharm: What are the con-

cerns for storing, handling, and

inspecting the supply of dispos-

able components?

GRAM: Since some disposable

technology can be used for dif-

ferent purposes, it is essential to

organize and label all material

correctly in storage so that opera-

tors can identify and choose the

correct equipment.

All personnel that will be han-

dling the disposable technology

should be trained on the impact

of damaged material. It is crucial

to inspect the disposable materi-

als for tears, rips, and particles.

The inner and outer bags should

be inspected at receiving to make

sure they are integral. Extra

attention must be paid to follow-

ing correct processes for moving

equipment into the cleanroom

environment. Bags containing

the disposable equipment should

not be opened until they are in

the GMP-controlled environment.

Once transferred to the sterile area,

bags should be inspected again

and checked to make sure there

are no particles before the dispos-

able equipment comes into contact

with the product.

Some components and even

preassembled systems come prest-

erilized using gamma irradiation.

Or, for a more flexible approach,

the user can assemble the system

and then autoclave it. Disposable

components must have data

records and certificates of com-

pliance from the vendor. We also

audit the disposable technology

material manufacturers and their

validated steril ization gamma-

irradiation process.◆

Troubleshooting


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PRODUCT SPOTLIGHT

Filling and Closing System Reduces Decontamination Cycle

The FlexPro 50, from Groninger,

is a modular filling and closing

system designed to process

vials, cartridges, and syringes,

as well as vials in bulk and trays.

FlexPro 50 lines can be executed

with manual or fully automated

process steps. Nested vials,

cartridges, and syringes can be processed on one line

configuration by changing format parts. Additional flexibility

is given by exchanging the machine trolleys to process

vials in trays or in bulk in the same line configuration.

The FlexPro 50 can produce an output of up to 4700

objects per hour. The isolator works independently

of a facility’s HVAC system. Gloves on the front

and back of the main and add-on isolators allow

machine access from both sides. The new Direct-

Injection-System reduces the decontamination cycle

by approximately 50%. The line can be changed to

process different products in around two hours.

Groninger www.groninger.de

Ultracentrifuge Allows for Multi-Wavelength Analysis The Optima AUC from Beckman

Coulter Life Sciences is an

ultracentrifuge that determines

molecular weight, size shape, and

polydispersity. The Optima AUC has

a 38.1-cm (15-inch diagonal) touch-

screen with the ability to export data

locally or remotely. The optics are

contained outside the rotor chamber, reducing the impact

of the g-force on the optics. The Optima AUC also allows

molecules to float free and unbound so that researchers

are able to characterize them in their native state. The

ultracentrifuge can perform multi-wavelength analysis and

allows for the development of new detection systems.

Beckman Coulter Life Scienceswww.beckmancoulter.com

LABORATORY SERVICESAs a member of Eurofins’ BioPharma

Product Testing Group—the

largest network of harmonized

bio/pharmaceutical GMP product

testing laboratories worldwide—

Eurofins Lancaster Laboratories supports all functional areas of bio/

pharmaceutical manufacturing, including method development,

microbiology, process validation, and quality control throughout

all stages of the drug development process. Eurofins Lancaster

Labs, tel. 717.656.2300, www.EurofinsLancasterLabs.com

BIONE—THE SIMPLEST SINGLE-USE BIOREACTOR SYSTEM ON THE MARKET TODAY!You don’t need to make a large capital

investment to convert your existing benchtop

glass bioreactor to a single-use bioreactor.

Simply remove your existing headplate and

place the preassembled and sterile Distek BIOne

System into the glass vessel. The bioreactor liner

molds to your existing glass system allowing

you to continue using your cabinet, probes, motor, heating blanket,

water jacket, and recipes. Distek, www.distekinc.com

NEW INTEGRATED BIOLOGICS SOLUTION CENTERWuXi has started construction of a state-of-

the-art, integrated R&D and manufacturing

center at the company’s headquarters (see

press release). This 250,000 sq. ft. facility

will be operational in 2017 and can accommodate 800 scientists. The center will

provide comprehensive concept to clinic biologics discovery, development, and GMP

manufacturing services on one consolidated campus. This facility utilizes WuXi’s

single-source technology platforms. WuXi Biologics (A wholly owned subsidiary

of WuXi AppTec), [email protected], www.wuxibiologics.com/news-events.

ONLINE VIABLE CELL DENSITY MONITORING BY HAMILTONHamilton’s Incyte, viable cell density sensor,

enables measurement of viable cells without

influence from changes in the media,

microcarriers, dead cells, or debris. It is designed

for mammalian cell culture, yeast, and high density bacterial fermentation. Its 12

mm diameter, PG13.5 mounting thread, and 120, 225, 325, and 425 mm lengths

fit all reactor sizes. Either 2 or 4 sensors connect to the Arc View Controller, which

displays, records, and exports measurement data in 4-20 mA, OPC, or Modbus

formats. Hamilton Company, tel. 888.525.2123, www.hamiltoncompany.com

New Technology Showcase

INDUSTRY PIPELINE


VISIT US AT BIO INTERNATIONAL CONVENTION 2016

NEW PRODUCTS AND SERVICES

BIO 2016 EXHIBITOR GUIDE

AND TECHNOLOGY SHOWCASE

BIO Convention

2016 EXHIBITOR

GUIDE

STAY CURRENT ON BIOTECHNOLOGY

Visit BioPharm International sponsors that are exhibiting at the 2016 BIO

International Convention. See descriptions and booth information below.

KT

SD

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/SC

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PH

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ES

As a premier Contract Discovery, Development

and Manufacturing Organization (CDMO),

WuXi Biologics offers our global clients the

necessary expertise, quality, and capacity

to develop biologic drugs from concept to

commercialization. Along with our WuXi AppTec

affiliates, we provide the world’s ONE true single-

source approach that saves our clients critical

time and money. WuXi Biologics, A wholly owned

subsidiary of WuXi AppTec t 288 Fute Zhong

Road, Waigaoqiao Free Trade Zone, Shanghai

200131, China t www.wuxibiologics.com t�tel.

+86.400.820.0985 t [email protected]

BIO Booth # 5876

Hamilton CompanyHamilton’s Incyte,

viable cell density

sensor, enables

measurement of viable

cells without influence

from changes in the media, microcarriers, dead

cells, or debris. It is designed for mammalian

cell culture, yeast, and high density bacterial

fermentation. Its 12 mm diameter, PG13.5

mounting thread, and 120, 225, 325, and 425

mm lengths fit all reactor sizes. Either 2 or 4

sensors connect to the Arc View Controller, which

displays, records, and exports measurement

data in 4-20 mA, OPC, or Modbus formats.

Hamilton Company t�tel. 888.525.2123

t��www.hamiltoncompany.com

SAFC®Introducing the next

generation in chem-

ically-defined CHO

fed-batch media. This

contemporary media

and feed platform was

developed across a wide range of CHO cell lines

commonly used in industrial bio-manufacturing

with an emphasis on simple adaptation (regardless

of cell bank medium), demonstrated performance

with sustained high biomass and maximum titers,

and formulations allowing for flexibility to adjust

protein quality attributes as needed. For more

information or to try a sample, please visit us at

www.Sigma-Aldrich.com/CHOperformance, SAFC®.

BIO Booth # 5633

Eppendorf is a leading life-science company

that develops and supplies lab instruments

and bioprocess systems for microbial and

cell-culture applications. The Eppendorf

Bioprocess portfolio includes scalable

platforms in stand-alone, parallel, and single-

use systems with working volumes from 60mL

to 2400L. Lab products include liquid handling,

centrifuges, shakers, incubators, sample prep,

and detection all from Eppendorf.

Eppendorf, 102 Motor Parkway, Hauppauge, NY

11788 t�www.eppendorf.com t�Info@eppendorf.

com t�tel. 800.645.3050

Emergent BioSolu-

tions provides con-

tract manufacturing

services for both bulk

drug substances and

sterile injectable drug products. Emergent’s state-

of-the-art, single-use BDS facility enables turnkey

upstream and downstream support for microbial,

mammalian, and viral cell lines. Emergent’s Fill/

Finish service offering comprises of vials and

syringes, for both liquid and lyophilized products.

Emergent’s manufacturing facilities currently

produce 20 commercial products, and a host of

clinical stage programs. Emergent BioSolutions t�

400 Professional Dr, Suite 400 t�Gaithersburg, MD

20879 t�tel. 240.631.3200 t�emergentbiosolutions.

com t�[email protected]

BIO Booth # 5556

Tosoh Bioscience CaPure-HA™ from

Tosoh Bioscience LLC

is for the purification

of multiple classes

of biomolecules

including monoclonal

and polyclonal

antibodies, antibody

isoforms, isozymes, antibody fragments, and

the isolation of single-stranded from double-

stranded DNA. The highly selective and robust

nature of CaPure-HA offers the flexibility to

use this resin at any stage in a process from

capture to final polishing. Tosoh Bioscience,

LLC, 3604 Horizon Drive, Suite 100, King of

Prussia, PA 19406 t�www.tosohbioscience.

com t�tel. 484.805.1219 t [email protected]


Ask the Expert

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Siegfried Schmitt is principal consultant at

PAREXEL International.

Siegfried Schmitt, Principal Consultant, PAREXEL International, discusses how to report quality metrics to FDA.

Q: We are a contract manufacturing orga-

nization (CMO), specializing in chemi-

cal synthesis of APIs. Since the publication of

FDA’s draft guidance, Request for Quality Metrics,

Guidance for Industry (1), in July 2015, we have

been debating how to best capture and report

quality metrics in our organization. What do

you recommend?

A: As with all regulations or guidance

documents, there can be var ious

approaches to achieving compliance. Quality

metrics need to be specific to each business

and circumstance. For example, metrics will

be different for a site that manufactures a large

variety of products in multipurpose equipment

in small numbers of batches, compared with

a site that produces large volumes of just a

couple of products in dedicated process trains.

In addition, it is crucial to ensure that all

parties involved understand what will be mea-

sured and how to report these metrics. Ideally,

metrics should be checked for completeness

and correctness before they are presented to

be signed. Quality metrics should also be col-

lated, measured, and reported in real-time, not

months later in an annual report. Real-time

reporting not only saves time and is more

efficient, but it reduces error in reporting and

allows real-time reaction of the site to metrics,

as needed.

The following are a few additional best prac-

tices to keep in mind when implementing

quality metrics:

t�Avoid using quality metrics to drive finan-

cial rewards, which can easily lead to

behavior that is aimed at benefit maximi-

zation rather than improving quality.

t�Understand that quality metrics alone do

not drive improvements—it requires senior

management to provide the required

resources for these improvements.

t�Achieving 100% perfection is rarely achiev-

able, nor feasible. For example, reducing

the number of deviations may be a laudable

goal, but trying to have zero deviations is

not plausible.

Measuring quality metrics is not only impor-

tant because it is becoming a regulatory expecta-

tion, but because it is good practice to have in

place for the benefit of your company. Therefore,

it is important to implement quality metrics, but

you must ensure that these are applicable to your

unique circumstances and are measurable.

REFERENCE 1. FDA, Draft Guidance, Guidance for Industry: Request for

Quality Metrics (Rockville, MD, July 2015). ◆

Reporting Quality Metrics to FDA

Ad Index

Company Page

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Quality metrics need

to be specific to each

business and circumstance.

Accelerate your bioprocess journey6SHHG�DQG�H�FLHQF\�DUH�FUXFLDO�DVSHFWV�RI�ELRPDQXIDFWXULQJ��7KH�ULJKW�VXSSOLHU�FDQ�FRQWULEXWH�WR�\RXU�VXFFHVV��'LVFRYHU� KRZ�RXU�SLRQHHULQJ�WHFKQRORJLHV��DJLOH�VHUYLFHV��DQG�DELOLW\�WR�GHVLJQ�DQG�FRQVWUXFW�FRPSOHWH�IDFLOLWLHV�LPSURYHV�VSHHG� WR�PDUNHW�

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gelifesciences.com/bioprocess

*(�DQG�*(�PRQRJUDP�DUH�WUDGHPDUNV�RI�*HQHUDO�(OHFWULF�&RPSDQ\��*HQHUDO�(OHFWULF�&RPSDQ\��)LUVW�SXEOLVKHG�$SU��*(�+HDOWKFDUH�%LR�6FLHQFHV�$%��%M|UNJDWDQ��8SSVDOD��6ZHGHQ

��$$��

The world’s ONE true single-source platform

from concept to commercialization.

4785

40 IND-enabling global CMC

projects completed 99percent of the CMC and

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stable cell lines developed

for GMP Manufacturing

cell-based assays developed

including 15 qualified for GMP

Since 2014

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