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Supplement to:INTERNATIONAL
Guide to
Disposables Implementation
November 2010
Guide to
Disposables Implementation
CONTENTS
EXTRACTABLES & LEACHABLES
Regulatory Expectations and Consensus Industry
Recommendations for Extractables Testing of
Single-Use Process Equipment BPSA eases confusion over extractables and leachables
testing through guides.
Jerold Martin 6
BIOREACTORS: MABS
Evaluation of a Single-Use Bioreactor for the
Fed-Batch Production of a Monoclonal Antibody Despite different material, agitation, and aeration, the performance of
the disposable bioreactor is similar to that of stainless steel bioreactors
Emmanuelle Cameau, Georges de Abreu, Alain Desgeorges,
Elodie Charbaut, Henri Kornmann 12
BIOREACTORS: VACCINES
Disposable Bioreactors for Viral Vaccine
Production: Challenges and Opportunities Switching to single-use bioreactors can have fi nancial
and performance benefi ts.
Jean-François Chaubard, Sandrine Dessoy, Yves Ghislain,
Pascal Gerkens, Benoit Barbier, Raphael Battisti, Ludovic Peeters 22
ECONOMICS
An Economic Analysis of Single-Use Tangential
Flow Filtration for Biopharmaceutical Applications Single-use TFF offers the greatest savings in clinical and
contract manufacturing, where the scale is low and
changeovers are frequent.
Michael LaBreck, Mark Perreault 32
FILTRATION
Implementing Single-Use Technology in Tangential
Flow Filtration Systems in Clinical Manufacturing A case study evaluates the performance, control of operations,
productivity, and cost savings of a single-use system.
Keqiang Shen, Be Van Vu, Nikunj Dani, Bryan Fluke,
Lei Xue, David W. Clark 40
Cover credit: GLaxoSmithKline group of companies
November 2010 The BioPharm International Guide 3
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-SimhPresident, Shiba Associates
Fredric G. BaderVice President, Process Sciences Centocor, Inc.
Rory BudihandojoManager, Computer Validation Boehringer-Ingelheim
Edward G. CalamaiManaging PartnerPharmaceutical Manufacturing and Compliance Associates, LLC
John CarpenterProfessor, School of PharmacyUniversity of Colorado Health Sciences Center
Suggy S. ChraiPresident and CEO,The Chrai Associates
Janet Rose ReaVice President, Regulatory Affairs and QualityPoniard Pharmaceuticals
John CurlingPresident, John Curling Consulting AB
Rebecca DevineBiotechnology Consultant
Mark D. DibnerPresident, BioAbility
Leonard J. GorenGlobal Director, Genetic Identity, Promega Corporation
Uwe GottschalkVice President, Purification TechnologiesSartorius Stedim Biotech GmbH
Rajesh K. GuptaLaboratory Chief, Division of Product QualityOffice of Vaccines Research and Review, CBER, FDA
Chris HollowayGroup Director of Regulatory Affairs, ERA Consulting Group
Ajaz S. HussainVP, Biological Systems, R&DPhilip Morris International
Jean F. HuxsollSenior Director, QA ComplianceBayer Healthcare, Pharmaceuticals
Barbara K. Immel President, Immel Resources, LLC
Stephan O. KrausePrincipal Scientist, Analytical Biochemistry, MedImmune, Inc.
Steven S. KuwaharaPrincipal ConsultantGXP BioTechnology LLC
Eric S. LangerPresident and Managing PartnerBioPlan Associates, Inc.
Denny KraichelyAssociate Director, CMC Team Leader, Portfolio Management & Technical Integration, Johnson & Johnson Pharmaceutical R&D, Inc.
Howard L. LevinePresident, BioProcess Technology Consultants
Herb LutzSenior Consulting EngineerMillipore Corporation
Hans-Peter MeyerVP, Innovation for Future Technologies, Lonza, Ltd.
K. John MorrowPresident, Newport Biotech
Wassim NashabehGlobal Head, Technical Regulatory Policy & Strategy, Genentech, A Member of the Roche Group
Barbara PottsDirector of QC Biology, Genentech
Tom RansohoffSenior Consultant, BioProcess Technology Consultants
Anurag RathoreBiotech CMC ConsultantFaculty Member, Indian Institute of Technology
Tim SchofieldDirector, North American Regulatory Affairs, GlaxoSmithKline
Paula ShadlePrincipal Consultant, Shadle Consulting
Alexander F. SitoPresident, BioValidation
Gail SoferConsultant, Sofeware Associates
S. Joseph TarnowskiSenior Vice President, Biologics Manufacturing & Process Development,
Bristol-Myers Squibb
William R. TolbertPresident, WR Tolbert & Associates
Michiel E. UlteeChief Scientific OfficerLaureate Pharma
Krish VenkatPrincipal, AnVen Research
Steven WalfishPresident, Statistical Outsourcing Services
Gary WalshAssociate ProfessorUniversity of Limerick, Ireland
Lloyd WolfinbargerPresident and Managing PartnerBioScience Consultants, LLC
www.biopharminternational.com
BioPharmINTERNATIONAL
Group Publisher Michael Tessalone
Editor in Chief Laura Bush
Managing Editor Chitra Sethi
Associate Editor Haydia Haniff
Art Director Dan Ward
Associate Pat Venezia, Jr.
Publisher [email protected]
European James Gray
Sales Manager [email protected]
Production Kim Brown
Manager [email protected]
Marketing Cecilia Asuncion
Promotions Specialist [email protected]
President, Chief Executive Officer Joe Loggia; Vice
President, Finance & Chief Financial Officer Ted Alpert; Vice President, Information Technology J. Vaughn; Executive Vice President, Corporate
Development Eric I. Lisman; Vice President, Electronic
Media Group Mike Alic; Vice President, Media
Operations Francis Heid; Vice President, Human
Resources Nancy Nugent; Vice President, General
Counsel Ward D. Hewins; Executive Vice President,
Healthcare & Pharma/Science Group Steve Morris; Vice President and General Manager, Pharma/
Science Group Dave Esola
© 2010 Advanstar Communications Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical including by photocopy, recording, or information storage and retrieval without permission in writing from the publisher. Authorization to photocopy items for internal/educational or personal use, or the internal/educational or personal use of specific clients is granted by Advanstar Communications Inc. for libraries and other users registered with the Copyright Clearance Center, 222 Rosewood Dr. Danvers, MA 01923, 978-750-8400 fax 978-646-8700. For uses beyond those listed above, please direct your written request to Permission Dept. fax 440-891-2650 or email: [email protected].
4 The BioPharm International Guide November 2010
We believe that evolution is essential to delivering smarter single-use
technology. Our fully integrated solutions are backed by over 50 years of
expertise gained from close collaboration with our clients. Are you ready to
take your bioprocessing operations to the next level?
Mobius. Smart people, smart products, smart services.™
Evolution at work.
Learn more about Mobius
Single Use. Visit us at
www.millipore.com/smartmobius
© 2010 Millipore Corporation. All rights reserved.
6 The BioPharm International Guide November 2010
DISPOSABLES EXTRACTABLES & LEACHABLES
ternational Guide November 2010
Regulatory Expectations and Consensus Industry Recommendations for Extractables Testing of Single-Use
Process Equipment
Abstract
The demand for single-use bioprocess
systems in biotech and pharmaceutical
manufacturing has expanded significantly
over the past few years. Applications for
single-use systems in biomanufacturing
range from upstream media preparation
single-use bioreactors, buffer preparation,
and intermediate processing, to storage of
active pharmaceutical ingredients (APIs),
bulk formulations, and filling of final dos-
age containers. Despite their
increased acceptance and
implementation, a pri-
mary concern with single-
use disposable polymeric
equipment continues to
be that of extractables
and leachables. In con-
trast with final dosage
container and closure
systems, the absence of specific regulatory
guidance for process equipment extract-
ables and leachables has many drug
manufacturers unsure of what data must
be submitted.
In response to the interest in single-
use disposable manufacturing and to
address the challenges of associated
problems affecting the industry, the Bio-
Process Systems Alliance (BPSA), the
single-use biomanufacturing trade associ-
BPSA eases confusion over extractables and leachables testing through guides.
Jerold Martin
Pa
ll C
orp
ora
tio
n
Jerold Martin is the senior
vice president of global scientifi c
affairs at Pall Life Sciences, Port
Washington, NY, 516.801.9086,
[email protected]. He also
is the chairman of the Board and
Technology Committee at
Bio-Process Systems Alliance.
EXTRACTABLES & LEACHABLES DISPOSABLES
November 2010 The BioPharm International Guide 7
ation, has developed best practice educa-
tional guides on a range of topics includ-
ing component quality tests, disposal
issues, irradiation and sterilization, the
economics of single-use technology, and
extractables and leachables. These guides
tie together the expertise and leadership
of BPSA’s supplier and end-user member
companies to provide recommendations
for industry best practices.
In December 2007 and January 2008,
BPSA published its fi rst white papers on ex-
tractables and leachables testing of single-
use process equipment.1,2 This was the fi rst
independent consensus guide that provided
basic concepts of extractables and leach-
ables from single-use equipment, summa-
rized the existing regulatory requirements,
and recommended a risk-based approach
that could reduce the amount of testing re-
quired by users. As part of this initiative,
BPSA also conducted a training seminar
in February 2008, at the headquarters of
the FDA and CBER near Washington, DC,
where we differentiated single-use process
equipment from fi nal dosage container
and closures, discussed the BPSA recom-
mendations for extractables and leachables
testing of single-use equipment, and the
proposed risk-based approach. BPSA also
has encouraged its supplier member com-
panies to develop more generic and compa-
rable extractables data that would reduce
the burden on users for redundant testing.
Regulatory Expectations for Extractables and Leachables Data
In the ensuing period, BPSA has gotten
positive feedback from users and regulators
about the risk-based approach. However,
FDA 483 observations and warning letters
continued to cite insuffi ciencies in extract-
ables data submitted by biopharmaceutical
manufacturers in drug product applications.
For example, in an April 2008 prelicensing
inspection 483, the FDA stated, “Besides the
0.22 μm sterilizing filters, there was no leach-
able and extractable testing performed for the
equipment and (--redacted--) materials used
in (--redacted--) purification process includ-
ing purification of (--redacted--).”
The drug manufacturer submitted a
validation project plan defining require-
ments for the evaluation of extractables
and potential leachables from the prod-
uct contact components of the process
equipment, filters, and chromatography
media used to manufacture (the) drug
substances and products, and the risk
assessment. FDA’s subsequent 483 re-
sponse review memo stated that the draft
protocols and proposed corrective ac-
tions were considered to be adequate.1
In a related inspection, the FDA similarly
observed, “There were no leachable and ex-
tractable testing performed for (--redacted--)
materials used in buffer preparation.”
The drug manufacturer agreed to imple-
ment an extractables and leachables assess-
ment policy that included risk assessment,
safety assessment, and model solvent study
design, along with generic family-approach
studies for leachables and extractables for
the storage of (- -redacted--) used in buffer
preparation activities.2 This is consistent
with BPSA’s published recommendations.
BPSA encouraged its member
companies to develop generic
and comparable extractables
data to reduce the burden on
users for redundant testing.
DISPOSABLES EXTRACTABLES & LEACHABLES
8 The BioPharm International Guide November 2010
At IBC’s 7th International Single Use
Applications for Biopharmaceutical Manu-
facturing Conference, held in La Jolla, CA,
on June 14, 2010, Destry M. Sillivan of the
FDA provided an update and overview of
types of data to be reviewed and specifi c
areas of concern to the FDA.
Sillivan stated that, “the responsibil-
ity for establishing that single-use ma-
terials selected for the manufacturing
process do not adversely impact the
product falls on the manufacturer of the
drug product under review,” and recom-
mended that drug sponsors, “submit
suffi cient information to provide evidence
that the product contacting material
does not introduce contaminants into the
product so as to alter the safety, identity,
strength, etc.”
Following closely the recommendations
made in BPSA’s 2008 Extractables Guide
and FDA CBER training seminar, Sillivan
stated that, “CBER recommends a risk-
based approach be taken in evaluating ex-
tractables and leachables where you take
multiple aspects into account (e.g., indica-
tion, safety issues, product characteristics,
dosage, formulation, and stability profi le).”
If there is no relevant risk associated with
the (material in question), “vendor data can
be cross referenced and a detailed justifi ca-
tion for the applicability of these data and a
justifi cation for no additional testing should
be submitted,” he added.
According to Sillivan, “Where there is
relevant risk, the drug sponsor may have
to determine toxicity based on maximum
dosage of potential leachables based on ex-
tractables data. If the maximum dosage of
potential leachables presents a safety risk,
leachable evaluation and testing may be nec-
essary. Additionally, if product quality could
be affected by potential leachables, studies
may need to be performed to assess the ef-
fect on product quality, including effi cacy.”
New products contacting single-use ma-
terials often are reported in the product an-
nual report with no information regarding
material composition, no extractables or
qualifi cation studies performed in support
of use of the new material, and no written
justifi cation of why the studies that were
submitted were appropriate to support suit-
ability for use of the new materials with the
drug product. The FDA and CBER consider
this level of information to be insuffi cient
to determine if the change was submitted
appropriately.
The FDA and CBER generally recom-
mend that either the drug sponsor or
the material manufacturer demonstrate
through suffi cient testing that the mate-
rial is suitable for the submitted processes
and product.
BPSA Introduces Updated Extractables Guide
Recognizing the need for further education
on extractables testing, including extrac-
tion and analytical methods, BPSA began
to develop a follow-up guide providing
more extensive information on methods
of extraction and extractables analysis in
2009. The resulting BPSA 2010 Extractables
Guide white paper, “Recommendations for
Testing and Evaluation of Extractables
from Single-Use Process Equipment”6 was
The FDA and CBER generally
recommend that the drug spon-
sor or the manufacturer demon-
strate that the material is suitable
for the submitted product.
EXTRACTABLES & LEACHABLES DISPOSABLES
November 2010 The BioPharm International Guide 9
developed by an expert team representing
suppliers, users, and independent testing
laboratories.
The 2010 BPSA Extractables Guide begins
with a review of the key concepts introduced
BPSA’s 2008 white paper, then expands
on the previously proposed risk-based ap-
proach, providing recommendations for ex-
traction conditions, explanations of analyti-
cal methods, and suggestions for users on
how best to evaluate and compare supplier-
generated data.
The fi rst section covers three revisions
made to the 2008 extractables white paper.
• Two levels of risk evaluation are now
differentiated—the fi rst, for materi-
als, includes information on polymer
chemical compatibility, generic extract-
ables data, and suitability for use. The
second risk evaluation considers drug
product-related toxicity and quality
risk, evaluating the extractables data
as presumptive leachables by calculat-
ing worst-case levels in downstream
process fl uids and fi nal drug product,
and applying recognized toxicological
assessment methods.
• A change was made to the decision
tree fl ow chart originally published in
the 2008 white paper, which included
an option to test only for leachables in
intermediates or fi nal products in the
absence of adequate supplier extract-
ables data. Subsequent discussions
with CBER reviewers highlighted the
need to more strongly address concerns
that leachables could be masked in pro-
tein solutions. BPSA recognized that to
analyze for potential leachables in bio-
pharmaceuticals, knowing the extract-
ables from process equipment was not
an option. In the 2010 BPSA Extractables
Guide, the option to skip extractables
testing if unavailable and go directly to
leachables testing is omitted.
• The third revision in the 2010 BPSA Ex-
tractables Guide is the introduction of the
term, migrants, to refer to potential leach-
ables from process equipment. Several
end users have noted that in FDA and
EMA documents, the term leachables
only appears in reference to fi nal product
containers and packaging, not to process
equipment, and looked to BPSA to recog-
nize this distinction. In response, BPSA
has introduced the term migrants when
referring to chemicals that leach from
process equipment, but may or may not
be detected as “leachables” in fi nal prod-
uct dosage containers.
A revised risk decision tree highlights ma-
terials risk evaluation, which incorporates a
review of available generic extractables data
and determining suffi ciency, followed by ap-
plication of that data to the drug product and
performing a risk evaluation for toxicity and
product quality. After the risk assessments
are done, a decision can be made whether
the available extractables data, when con-
sidered as potential migrants, are suffi cient,
or whether further testing for migrants
from the process equipment into the actual
process fl uid, where they may be detected
as leachables, is merited. In the latter case,
both data on extractables and migrant (i.e.,
process-derived leachables) should be sub-
mitted in regulatory fi lings.
After the risk assessments, a
decision can be made whether
the available extractables data
are suffi cient, or whether further
testing for migrants is merited.
DISPOSABLES EXTRACTABLES & LEACHABLES
10 The BioPharm International Guide November 2010
The next section of the 2010 BPSA Ex-
tractables Guide provides detailed consid-
erations on extraction conditions, includ-
ing selection and preconditioning of test
articles such as autoclaving, irradiation,
and fl ushing. BPSA recommends water
and ethanol as primary worst-case sol-
vents representing most bioprocess fl u-
ids, and also discusses options for other
extraction fl uids. Model extraction con-
ditions are differentiated by component
types and summarized in a detailed table
covering fi lter capsules, tubing, sterile
connectors and fi ttings, biocontainers,
mixing bags, and bioreactors.
This is followed by an extensive section
on analytical methods, including detailed
descriptions of applicable techniques—
what they do, what they detect, and what
their limits of detection are. This section
on “what you need to know about analyti-
cal methods” is intended to guide suppliers
in generating appropriate data, and enable
users of single-use systems to better under-
stand suitable extractables data and the im-
plications to their process and product.
Lastly, the guide provides recommenda-
tions for user actions when extractables
data from suppliers are incomplete or inad-
equate. Suggestions also are provided for
comparing data from different suppliers of
comparable components where extractions
may have been conducted under differing
pretreatment or extraction conditions or dif-
ferent analytical methods.
Availability of supplier-generated core
(generic) extractables data generated ac-
cording to consensus recommendations,
as proposed by BPSA, can minimize du-
plicate testing by users for individual and
multiple drug products. These conditions
and analytical methods are intended to
guide suppliers and users to develop more
comparable extractables data that facili-
tate user and regulatory evaluations. Cop-
ies of the 2010 BPSA Extractables Guide
can be purchased from BPSA at www.bp-
salliance.org. BP
References 1. BPSA Extractables and Leachables
Subcommittee. Recommendations for
extractables and leachables testing
of single-use–part 1: Introduction,
regulatory issues, and risk assessment.
BioProcess Int. 2007 Dec;5(11):36–49.
2. BPSA Extractables and Leachables
Subcommittee. Recommendations for
extractables and leachables testing of
single-use–Part 2: executing a program.
BioProcess Int. 2008;6(1):44–53.
BPSA. Available from: http://www.
bpsalliance.org.
3. FDA 483 response review memo.
Available from: http://www.fda.
gov/biologicsbloodvaccines/
bloodbloodproducts/approvedproducts/
licensedproductsblas/fractionatedplasma
products/ucm161014.htm.
4. FDA 483 response review memo.
Available from: http://www.fda.
gov/biologicsbloodvaccines/
bloodbloodproducts/approvedproducts/
licensedproductsblas/fractionated
plasmaproducts/ucm161016.htm.
5. Sillivan, DM. Review of single use
processes and materials: overview
of types of data to be reviewed and
specific areas of concern. IBC’s 7th
International Single Use Applications for
Biopharmaceutical Manufacturing, La
Jolla, CA: 2010 Jun 14.
6. BPSA Extractables Subcommittee.
Recommendations for testing and
evaluation of extractables from
single-use process equipment. 2010:
Washington, DC. Available from:
http://www.bpsalliance.org.
The 2010 BPSA Extractables
Guide provides recommendations
for user actions when extract-
ables data from suppliers are
incomplete or inadequate.
Engineering expertise you can trust: From URS to final qualification.
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www.sartorius-stedim.com/engineeringturning science into solutions
12 The BioPharm International Guide November 2010
DISPOSABLES BIOREACTORS: MABS
ternational Guide November 2010
Evaluation of a Single-Use Bioreactor for the Fed-Batch Production Process
of a Monoclonal Antibody
Abstract
In this study, we tested the combination of a
disposable bioreactor and a disposable dis-
solved oxygen sensor as a replacement for our
standard bioreactors. The possibility to run
a fed-batch cell culture process developed for
the production of a monoclonal antibody in a
50-L single-use bioreactor was investigated.
The single-use bioreactor was assessed
both as a seed train and as a production
bioreactor. Therefore, three confi gurations
corresponding to different combinations of the
50-L disposable bioreactor
and the reference 5-L glass
bioreactor (fully scalable up
to 300 L) were compared.
In the past decade, biopharmaceutical
manufacturing processes have un-
dergone multiple changes resulting
in signifi cant improvements in effi ciency.
In parallel with the development of high
producing cell lines and robust chemi-
cally defi ned media for cell culture, the
constant evolution of disposables has led
to simpler operations. The use of dispos-
ables eliminates cleaning and steriliza-
Despite different material, agitation, and aeration, the performance of the disposable bioreactor is
similar to that of stainless steel bioreactors.
Emmanuelle Cameau, Georges de Abreu, Alain Desgeorges,
Elodie Charbaut Taland, Henri Kornmann
Me
rck S
ero
no S
A
Emmanuelle Cameau is a biotech pro-
cess sciences upstream specialist,
Georges De Abreu is a biotech central
services manager, Alain Desgeorges,
PhD, is a biotech process sciences
upstream coordinator, Elodie Char-
baut Taland, PhD, is a biotech pro-
cess sciences manager, and Henri
Kornmann, PhD, is a biotechnology
production director, all at Merck
Serono SA, Aubonne, Switzerland,
+41(0)218217111, emmanuelle.
DISPOSABLES BIOREACTORS: MABS
14 The BioPharm International Guide November 2010
tion steps, as well as cleaning validation,
thus reducing costs and the time of opera-
tion per batch. Traditional disposable de-
vices such as fi lters, tubing, bags, bottles,
and syringes have commonly been used in
biopharmaceutical manufacturing since
the 1990s. The breakthrough in dispos-
able bioreactor technology development
in terms of larger capacities was the use
of bag systems for cell culture. The 20-L
rocker system was commercially available
in 1998 and the technology became a suc-
cess, especially in cell expansion opera-
tions. It allowed working with complete
sterility, thereby securing the industrial
cell culture processes. Further develop-
ment of this system led to higher volumes
and to the equipment available today on
the market.
The next step was the development of
stirred-tank bioreactors with a configu-
ration similar to conventional stainless
steel bioreactors. Compared to tradi-
tional wave bioreactors, stirred-tank
bioreactors offer the benefits of sparg-
ing, stirring of the suspension, and a
higher utilization rate of the bag size as
cultivation space. Such bioreactors are
considered for use in the industry in cell
amplif ication processes, to reach higher
cell densities, or as production biore-
actors. The single-use disposable bio-
reactor (SUB) from HyClone (Thermo
Fisher Scientif ic) entered the market in
2006. Currently, disposable bioreactors
up to 2,000 L (SUB, Hyclone and XDR,
Xcellerex) culture volume are commer-
cially available and plans are to develop
3,000 L bioreactor systems over the next
few years.1
In addition to disposable bioreactors,
innovative single-use sensors are cur-
rently being developed, which will allow
a cell culture process to run long-term in
a fully disposable system. Single-use sen-
sors also will solve technical problems
sometimes raised by using plastic bags
in single-use bioreactors, which can in-
terfere with the functioning of stainless
steel sensors because of static electricity
problems, and therefore create drifts es-
pecially for pH measurement.2
In this study, we tested the combina-
tion of a disposable bioreactor and a
disposable dissolved oxygen (DO) sen-
sor as a replacement of a standard bio-
reactors to run a fed-batch cell culture
process developed for the production of
a monoclonal antibody (MAb). The dis-
posable equipment selected was the 50 -L
stirred-tank SUB by HyClone coupled to
the TruLogic RDPD controller based on
DeltaV technology and the disposable
TruFluor DO probe by Finesse Solu-
tions.
The SUB was assessed both as a seed
train and as a production bioreactor.
Therefore, three configurations corre-
sponding to dif ferent combinations of
the 50 -L disposable bioreactor and the
reference 5 -L glass bioreactor (fully
scalable up to 300 L) were compared
(Figure 1): seed train and production in
a 5 -L glass bioreactor (named 5L/5L);
seed train in the 50 -L SUB; production
in 5 -L glass bioreactor (named SUB/5L);
and seed train and production in the
50 -L SUB (named SUB/SUB).
Material and Methods
Cell Culture and Bioreactor Operation
The cell line used in this study was a
Chinese hamster ovary (CHO) cell line
developed for the production of a MAb.
BIOREACTORS: MABS DISPOSABLES
November 2010 The BioPharm International Guide 15
The cell culture process was performed
using chemically defined cell culture
media and feeds. A typical seed train was
used for the production runs (Figure 1).
After vial thawing, cells were grown in
T-f lasks and shake f lasks for six days
at 37 °C, 5% CO2, and then transferred
into a 2-L Cultibag RM Optical (Sarto-
rius Stedim Biotech GbmH), followed by
a 50 -L wave bag. The two last steps of
the seed train were performed in biore-
actors (N-2 and N-1 steps). To properly
assess the performance of the SUB as a
seed train bioreactor and a production
bioreactor, the cell suspension from the
50 -L wave bag was split in a 50 -L SUB
(Hyclone, Thermo Fisher Scientif ic
Inc.) and a 5 -L benchtop-scale glass bio-
reactor (BIOSTAT B-DCU Quad, Sar-
torius-Stedim Biotech GbmH) for the
N-2 step. The N-1 step consisted of the
passage of the N-2 bioreactor, removing
part of the suspension, and adding fresh
media. Then, the seed train in the 5 -L
glass bioreactor was used to inoculate
two 5 -L glass bioreactors for the produc-
tion phase. The seed train in the SUB
was used to inoculate, in parallel, one
SUB and two 5 -L glass bioreactors for
the production phase. The experimental
plan is described in Figure 1 and was
performed twice.
Disposable 50-L bioreactor
Disposable 50-L bioreactor
Feed/harvest
2 x 5-L bioreactors
2 x 5-L bioreactors
2 X 5-L bioreactors
50-L rocker
Cell culture amplification in
T-flasks and shake flasks
2-L rocker
Seed train (N-2, N-1) Production Amplification
Figure 1. Description of the process and experimental scheme. The cell amplification
was performed in T-flasks and shake-flasks, followed by a passage in a 2-L
bag rocker and a 50-L bag rocker. The inoculum was splitted to inoculate the
N-2 bioreactors (SUB and 5-L glass vessel bioreactor). The N-1 SUB seed train
bioreactor was used to inoculate two 5-L glass vessel bioreactors and itself as
production bioreactor with the remaining inoculum. The 5-L glass vessel seed train
bioreactor was used to inoculate one other 5-L bioreactor and itself as production
bioreactor.
DISPOSABLES BIOREACTORS: MABS
16 The BioPharm International Guide November 2010
The process developed in the 5 -L glass
bioreactors was adapted for the SUB as
follows: pH, temperature, DO setpoints,
and feeding strategy were unchanged
compared to the 5 -L bioreactor. Values
for gas f low rates and agitation setpoint
were scaled up by keeping the head-
space renewal rate and the power per
volume constant, respectively. The pH
regulation was performed using CO2 in
overlay and sodium hydroxide for the
5 -L glass bioreactors.
Partial pressure of carbon dioxide
(pCO2), pH, and oxygen (pO
2) were
controlled off line on ABL5 (Radiometer
Medical). Viable cell density (VCD) and
viability were measured using a Vi-Cell
automatic cell counter (Beckman Coul-
ter). Metabolites and electrolytes were
controlled off line using a Nova Bioprofile
100+ (Nova Biomedical). Protein pro-
duction was determined using a Protein
A–based assay on the Gyrolab platform
(Gyros).
Using a Disposable DO Probe
The 50 -L SUB was operated using a Tru-
Logic RDPD (R&D and process devel-
opment bioprocess) controller (Finesse
Solutions). The disposable TruFluor
DO probe (Finesse Solutions) was
compared to the InPro6800 polaro-
graphic probe (Mettler Toledo) at a set-
point of 40%. The disposable DO probe
was inserted in a sleeve manufactured
with the SUB 50 -L bag. The sleeve con-
tained the disposable sensor, and the
probe contained a non-invasive reader
30
40
50
60
70
80
90
100
0
1
2
3
4
20 21 22 23 24 25 26
Via
bility
(%)
Via
ble
cell d
en
sity
(x10
6/m
L)
Working day
"SUB VCD"
"5L Bio VCD"
"SUB Viab"
"5L Bio Viab"
Figure 2. Profiles of viable cell density (SUB VCD and 5-L Bio VCD) and viability
(SUB Viab and 5-L Bio Viab, in %) of the seed train bioreactors. The passage of the
wave bag into the N-2 bioreactors was performed at working day 20, and the passage
from N-2 to N-1 bioreactor was performed at working day 23. VCD and Viab were
determined using the Vi-cell cell counter (Beckman Coulter). The graphs show mean
values with errors bars of two different runs. The variability of the viability is so low
that the error bars are not visible.
DISPOSABLES BIOREACTORS: MABS
18 The BioPharm International Guide November 2010
connected to the transmitter. The stan-
dard DO probe was inserted with a
Kleenpack connector (Pall Corporation)
after sterilization. After insertion, the
standard DO probe was calibrated as
usual. The DO was controlled using the
InPro6800 probe, but signals from both
probes were recorded on the controller.
Results
Process Scale-Up in the SUB
The seed trains performed in the 50-L SUB
and in the 5-L glass bioreactors resulted in
comparable cell density and viability (Fig-
ure 2). For all confi gurations, viable cell den-
sities at the end of the growth phase ranged
from 2.7 to 3.2 x 106 cells/mL, and viability
ranged from 95.5 to 98.7%.
The production phase performed in the
50-L SUB and in the 5-L glass bioreactors
also gave similar results for the maximum
VCD obtained at production day 7 (work-
ing day 33) and integral viable cells (IVC)
(Figure 3 and 4). The IVC curves (Figure
4) show comparable cell growth through-
out the process between different scales
and confi gurations, whether the seed train
material was coming from the 50-L SUB or
the 5-L bioreactor. The metabolites profi les
such as glucose, lactate, glutamine, and
glutamate were all comparable between
different scales and confi gurations (data
not shown).
The MAb titers obtained at produc-
tion day 7 (corresponding to the highest
point of viable cell concentration) were
comparable for all configurations (Fig-
ure 5), again demonstrating the similar
performance of the SUB and the 5 -L
bioreactor in seed train and production.
The titer was plotted against the IVC
(slopes: 5L/5L 22.62, SUB/SUB 21.81,
0
1
2
3
4
5
Viable cell density at production day 7
Via
ble
cell d
en
sity
(m
illio
ns
cells/
mL)
SUB/SUB
5L/5L
SUB/5L
Figure 3. Viable cell density bar graph at production day 7 (working day 33) of the
production bioreactors. The viable cell density reached a maximum value at production
day 7 (working day 33) and started decreasing on the next day. The values shown here
are means of two or three values with error bars.
BIOREACTORS: MABS DISPOSABLES
November 2010 The BioPharm International Guide 19
SUB/5L 19.23) and the relationship was
found to be linear, as expected.3 The
slopes were very close for the three
configurations, demonstrating that the
same specif ic productivity is reached in
dif ferent configurations.
The pH in the bioreactor was regulated
using CO2 in overlay in the 5-L glass biore-
-10
-5
0
5
10
15
20
25
30
35
1 2 3 4 5 6 7 8 9
Inte
gra
l vi
ab
le c
ells
(millio
ns
of
via
ble
cells.
day/m
L)
Production day
SUB/5L SUB/SUB 5L/5L
Figure 4. Integral viable cell density of the three configurations (SUB/SUB, SUB/5L,
and 5L/5L). The graphs are means of two or three runs with error bars.
0
100
200
300
400
500
600
700
Product titer on production day 7
Pro
du
ct t
iter
(mg
/L)
SUB/SUB
SUB/5L
5L/5L
Figure 5. Product titer bar graph at production day 7 (working day 33). The viable
cell density reached a maximum value at production day 7 (working day 33) and
started decreasing on the next day. The values showed here are means with error
bars of two or three values.
DISPOSABLES BIOREACTORS: MABS
20 The BioPharm International Guide November 2010
actors and in the 50-L SUB. The pCO2 levels
were compared between the SUB and the
5-L runs to assess the CO2 stripping per-
formance of the SUB (Figure 6). As shown
in Figure 6, the 50-L SUB cultures or the
5-L bioreactor coming from the SUB seed
train have appropriate pCO2 levels below
60 mmHg. In addition, to assess the impact
of pH regulation using CO2 instead of acid,
pCO2 data were compared to historical data
from a 5-L acid-regulated run and showed
that the 50-L SUB production run with the
50-L SUB seed train have even lower pCO2
levels than the acid-regulated runs (40–54
mmHg).
DO Probe Comparison
A drawback of single-use bioreactors is
the possible impairment of sensors based
on electric potential differences because
of the static electricity phenomena.4 How-
ever, disposable sensors now are available
based on fi uorescence, which may not
cause the same problems.
One of these probes was
tested in the single-use
bioreactor to monitor the
density of oxygen, in com-
parison to a reference stain-
less steel polarographic
probe. The trends of the
two probes are identical,
even if a small constant dif-
ference (<2%) is noted be-
tween the two curves, prob-
ably because of calibration
(data not shown). Thus, in
this specifl c confl guration,
no impairment of the stain-
less steel DO probe was ob-
served in the environment
of the SUB. The disposable
fi uorescence DO probe showed a compa-
rable performance, and could probably
be used to replace the reference stainless
steel probe.
Discussion and Conclusion
The aim of this study was to asses a dis-
posable bioreactor in combination with
a disposable probe for a fed-batch MAb
production process. The equipment cho-
sen was the HyClone SUB coupled to the
TruLogic RDPD controller and the dis-
posable TruFluor DO probe by Finesse
Solutions. The single-use DO sensor
showed comparable results to conven-
tional ones. The equipment was readily
implemented because the set-up time
was only one day.
The SUB gave results comparable to
the 5 -L glass vessel bioreactor (small-
scale reference for the process) for the
seed train and the production steps.
This shows that despite dif ferent ma-
0
50
100
150
200
250
300
1 2 3 4 5 6 7
pC
O2 (m
mH
g)
Production day
SUB/SUB
SUB/5L
5L/5L
Figure 6. The pCO2 profiles for the different
configurations (SUB/SUB, SUB/5 L, and 5 L/5 L),
measured with ABL-5 gas analyzer (Radiometer). The
values showed here are means with error bars of two
or three values.
BIOREACTORS: MABS DISPOSABLES
November 2010 The BioPharm International Guide 21
terial, agitation, and aeration, the dis-
posable bioreactor had a performance
similar to standard bioreactors, at least
for the fed-batch process tested here.
The scale-up to 50 -L also was straight-
forward. Given that all HyClone dispos-
able bioreactors have the same overall
reactor geometry ratio up to 2,000 L,
it can be expected that the scale-up to
larger volumes such as 300 L could be
performed using the same principles.
The scale-up to a higher volume such
as 1,000 L could be more complex be-
cause process scale-up is rarely linear
between such dif ferent scales.
The single-use bioreactor showed the
capacity to be used either as a seed train
bioreactor or a production bioreactor, or
both. If this double use is to be imple-
mented, the bag aeration configuration
should be carefully defined to be able
to cope with different oxygen demand
in cell expansion and production. In this
case, the same bag was used for both
phases, a limitation in oxygen f low rate
appeared toward the end of the culture.
The bioprocess container bag used
for these experiments was equipped
with a 20 -mm sparger membrane. The
bubbles released by this system were
small enough to have sufficient oxygen
transfer to the culture, but big enough
to strip CO2. The bag is now available
with a dual sparge system, consisting
in a 20 -mm porous frit for the oxygen
transfer and an open pipe for CO2 strip -
ping, enlarging the range for pCO2 strip -
ping. Many dif ferent disposable bioreac-
tors systems coexist on the market and
new versions are frequently released,
showing the high dynamism of single-
use technology. Each system presents
its own features and advantages. Some
other disposable bioreactors currently
are being assessed in our company.5
This study enabled us to demonstrate
the applicability of using a single-use
bioreactor for producing a MAb at
50 -L scale, and we can expect that fur-
ther scale-up to at least 300 -L can be
achieved. In the future, it can be ex-
pected that disposable bioreactors will
become far more common in biophar-
maceutical manufacturing. Their use is
of specific interest when producing ma-
terial for early clinical trials to avoid a
capital investment early, when the final
production bioreactor volume, as well as
the future of the molecule, are unknown.
Some people claim that the use of dispos-
able bioreactors also has big advantages
when building a new facility, because
the need for utilities might be reduced
in a fully disposable environment, there-
fore reducing start up time, installation
costs, and campaign turnaround.6 On
the other hand, some concerns exist
about the environmental impact of dis-
posables, although assessing the latter
is far from simple. The reduced use of
purified water, clean and pure steam,
and cleaning chemicals compared to
stainless equipment has to be balanced
with the increased plastic waste. One
way to reduce the impact of such waste
could be to convert back part of the
32.6 GJ/ton of energy stored in plastic
in waste-to-energy incineration facili-
ties, not necessarily solving the issue of
carbon footprint.7 The ultimate solution
might reside in recycling these dispos-
able products, requiring further devel-
opment on innovative transformation
Continued on... p. 31
22 The BioPharm International Guide November 2010
DISPOSABLES BIOREACTORS: VACCINES
ternational Guide November 2010
Abstract
Disposables historically have been used in
biotechnology processes for the past three
decades, initiated with the use of single-
use plastic support for cell culture (e.g.,
vials, shakers, T-fl asks, and roller bottles).
Another step was made more recently by the
implementation of plastic
bags into these processes,
either used in the process
itself or in supportive steps,
such as media and buffer
preparation and storage.
One of the key trends for biotech
manufacturing is the develop-
ment of disposable bioreactors.
This trend was initiated by the intro-
duction of the Wave system. The imple-
mentation of this technology was in line
Disposable Bioreactors for Viral Vaccine Production:
Challenges and Opportunities
Switching to single-use bioreactors can have financial and performance benefits.
Jean-François Chaubard, Sandrine Dessoy, Yves Ghislain,
Pascal Gerkens, Benoit Barbier, Raphael Battisti, Ludovic Peeters
2010 G
laxoS
mithK
line g
roup o
f com
panie
s. A
ll rig
hts
reserv
ed
Jean-François Chaubard is a
director, Sandrine Dessoy and Yves
Ghislain are expert scientists,
Benoit Barbier and Raphael
Battisti are technicians, and
Pascal Gerkens, PhD, and Ludovic
Peeters are associate scientists,
all at Viral Industrial Bulk, GSK
Biologicals, Rixensart, Belgium,
jean-francois.x.chaubard@
gskbio.com.
BIOREACTORS: VACCINES DISPOSABLES
November 2010 The BioPharm International Guide 23
Thermo Fisher Xcellerex
Cultibag STR Nucleo Hyclone XDR10 L glass
Reference
Disposable bioreactor technologies selected
Sartorius-Stedim Biotech ATMI – Pierre Guerin
with the use of disposable plastic bags.
The Wave bag technology quickly cap-
tured the interest of the biotech commu-
nity. These systems mainly are used for
cell expansion to replace shake f lasks or
intermediate small- and pilot-scale bio-
reactors, simplifying the process. De-
spite many advantages, this technology
presents some limitations for its use as
a final production-scale bioreactor, such
as scalability, specificity of the agitation,
and the bioreactor geometry.
Because of these limitations, an in-
dustrial need drove the development of
more conventional and scalable dispos-
able bioreactors. One of the f irst sys-
tems introduced on the market was the
Single-Use Bioreactor (SUB) from Hy-
clone. The SUB opened a new range of
applications because of its potential use
either as seed vessels or as production
vessels for cell-based processes.
Today, single-use bioreactors are used
extensively for the production of mono-
clonal antibodies (MAbs) and recom-
binant proteins. Based on this market
trend, vaccine manufacturers such as
GSK Biologicals decided to investigate
the potential use of this emerging tech-
nology for vaccine manufacturing. The
focus of this article will be on the use of
disposable bioreactors in the context of
viral vaccine production.
Viral Vaccines Specificity
Viral vaccine manufacturing process-
es present some specific constraints
as compared to other biotech products
linked to the cell substrate used and to the
viral production. These specificities are:
• Multiple cell lines are used for these
productions such as VERO, MDCK,
MRC5, BHK, and CHO cells, making
it more challenging to develop a plat-
form process.
• Cell substrates for viral production
often are cell-anchored cell lines,
such as VERO cells, requiring the
use of micro-carriers for bioreactor
process steps.
• Viral production must be handled in
the right biosafety containment, i.e.,
biosafety level 2 or 3 environments.
• Production scales generally are
Figure 1. Disposable bioreactors from four companies were evaluated
DISPOSABLES BIOREACTORS: VACCINES
24 The BioPharm International Guide November 2010
Despite many advantages,
single-use technology presents
limitations for its use as a fi nal
production-scale bioreactor.
smaller compared to MAb processes
(ranging from 500 to 2,000 L).
Main Drivers for Implementing
Disposable Bioreactors
in Vaccine Production
There are many benefits of using dispos-
able systems in biotech processes. Two
of these benefits justify the evaluation of
disposable bioreactors for viral produc-
tion processes.
• Maximizing facility output because
of the fast turnover of disposable sys-
tems (no clean-in-place and steam-
in-place operations in production
vessels).
• The reduction of capital investments
linked to the reduction and simpli-
f ication of the facility design and to
the reduction of equipment invest-
ment.
In addition to these points, other driv-
ers specific to vaccine manufacturing
were considered.
• Simplified biosafety level 2 and 3 pro-
duction areas (because of the smaller
footprint, lower ceiling height, and
removal of water-for-injection and
steam utilities).
• Minimizing the harvest size to re-
duce the size of purification equip-
ment and suites.
Methodology
As previously mentioned, generic pro-
cesses are dif f icult to define in the con-
text of viral vaccine production. There-
fore, a worst-case process that would
cover all other company viral vaccine
processes was defined. The disposable
bioreactor technology selection based
on this process will then be recommend-
ed as a standard single-use bioreactor
platform for viral vaccine applications.
The following set of parameters was
used to define the worst-case process:
• animal-free media to decrease shear
protection and nutritive support from
serum containing formulations
• cell cultures using micro-carriers (at
a high concentration) because cells
grown in this condition are more
sensitive to shear stress compared to
suspension cultures
• VERO-adherent cell lines
• medium renewal by sedimentation
• lytic virus.
The combination of these process
criteria made this process challenging
enough to cover a large range of current
and future in-house processes.
Single-Use Bioreactor Selection
Based on market availability, system ma-
turity, available scale, and mixing sys-
tems, four disposable bioreactors were
selected (Figure 1).
• Cultibag STR from Sartorius Stedim
Biotech
• Nucleo from ATMI—Pierre Guerin
• Hyclone SUB from ThermoFisher
• XDR from Xcellerex.
Criteria for Selection
To evaluate these four disposable biore-
actor technologies, several criteria were
selected. These criteria can be divided
in two sections:
ThinkOutsidethe Bag
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DISPOSABLES BIOREACTORS: VACCINES
26 The BioPharm International Guide November 2010
• Process criteria such as cell culture
and viral production performances,
mixing and aeration characteristics,
and scale-up predictability.
• General criteria such as film type,
biosafety, procurement, assurance of
supply, and price.
Characterization Results
Mixing and aeration performance evalua-
tion remains mandatory to ensure robust
scale-up of cell culture processes, espe-
cially for adherent cell line applications
(microcarrier use). In this study, these per-
formances were evaluated for the four bio-
reactor technologies that were identifi ed.
Mixing
Three tools for mixing characterization
were used: mixing time experiments,
correlation software, and the particle
image velocimetry (PIV). Typical re-
sults of the PIV technique are shown in
Figure 2.
Mixing characterization was per-
formed on the four selected technolo-
gies; their mixing configurations are
shown in Figure 3. Mixing performanc-
es were evaluated based on several cri-
teria such as mixing time, maximum
shear levels, etc. These performances
were then compared to the application’s
specif ic requirements: minimizing
Flow pattern in the impeller area Shear pattern in the impeller area
Stirred SUB technologies
10 L glass
Reference
Figure 2. Results generated by the particle image velocimetry technique
Figure 3. Mixing con� guration of the four single-use bioreactors (SUB) evaluated
BIOREACTORS: VACCINES DISPOSABLES
November 2010 The BioPharm International Guide 27
shear stress while assuring good homo-
geneity levels and maintaining all the
microcarriers in complete suspension.
Some results of this study are depicted
in Figure 4.
This fi gure shows the combination of
maximum shear stress (represented by the
tip speed) and mixing time in the minimum
operating conditions necessary to maintain
microcarriers in complete suspension. In
this graph, two technologies seem to show
better results in terms of acceptable mix-
ing time combined with a low maximum
shear stress (tip speed).
Aeration
Aeration performances also were com-
pared based on gas transfer capacity
measurements (kLa) in our end-of-cell-
growth conditions for each bioreactor.
Gas transfer capacities in our op-
erating conditions (minimum agita-
120
100
80
60
40
20
0
0 0.5 1 1.5
Tip speed at 200 L scale (m/s)
95
% m
ixin
g t
ime
(s)
Ref = Stainless steelbenchmark
Ref
Operating conditions:Minimum speed to maintainmicrocarriers in suspension
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0 20 40 60 80 100 120
Time (h)
Ce
ll d
en
sity
(1
06/m
L)
2
3
4
5
Ref.
Figure 4. Mixing performances comparison of the four disposable bioreactors
Figure 5. Cell growth profiles in one disposable bioreactor
DISPOSABLES BIOREACTORS: VACCINES
28 The BioPharm International Guide November 2010
tion speed required to maintain mi-
crocarriers in complete suspension)
were evaluated for the four bioreactor
technologies. A high gas transfer
capacity is useful to decrease the
amount of oxygen needed to maintain
the dissolved-oxygen concentration to
its set point. With low sparging f low-
rates, shear induced by bubble break-up
at the liquid surface will be decreased.
These low f low rates also have a positive
effect on foaming.
Cell Growth and
Viral Production Results
Figure 5 shows a typical example of
four cell growths obtained in one of the
selected disposable bioreactors. These
data demonstrate that the cell growths
obtained in this system are consis-
tent and also equivalent to our control
bioreactor (the control bioreactor
is a small-scale 10 L bioreactor that
was validated as a representative
scale-down model of the larger stainless
steel vessels).
A critical feature of microcarrier-
based cell culture is the homogeneity
of cell adhesion to the beads. This point
was monitored in all experiments per-
formed in the different disposable sys-
tems selected and compared to the con-
trol bioreactor. Microcarrier pictures
by microscopy at different time points
(days 0, 2, and 5) were analyzed for each
culture. These pictures show that a ho-
mogenous cell adhesion to the bead can
be achieved using the right disposable
bioreactor system, and the level of ho-
mogeneity is similar to that obtained in
a stainless steel bioreactor.
The most important process criterion
for evaluating the performance of these
systems was their ability to support the
same level of viral production as in a
conventional bioreactor. To evaluate this
point, several serotypes were produced
in the different disposable bioreactors
selected. Figure 6 shows an example of
the results obtained for one viral sero-
type with the three disposable bioreac-
tor systems. Viral production obtained
1 2 3 Reference
120
100
80
60
40
20
0
Disposable bioreactor (number)
Figure 6. Viral production in the three disposable bioreactors evaluated
BIOREACTORS: VACCINES DISPOSABLES
November 2010 The BioPharm International Guide 29
with two systems are equivalent to the
one obtained with the control bioreactor.
By the end of this evaluation, we dem-
onstrated that with the right disposable
systems, it is possible to achieve process
performances equivalent to stainless
steel bioreactors.
Risk Assessments
Disposable bioreactors are a new tech-
nology in the biotechnology field. To
evaluate potential risks associated with
implementing this new technology in
future manufacturing processes, risk
assessments based on the FMEA meth-
odology were performed. The main risk
assessments performed were related to
the two critical risks: assurance of sup-
ply, and biosafety, a risk specific to viral
vaccine applications. The biosafety risk
assessment will be used as an example.
Biosafety is one of the main concerns of
viral vaccine production, especially when
biosafety level 2 and 3 viruses must be
produced at large scale. One concept of
biosafety is that the equipment itself is
considered the fi rst barrier to isolate the
pathogenic micro-organism from the envi-
ronment. The second barrier is the room
where the equipment is located. The move
from stainless steel to disposable equip-
ment has weakened the fi rst barrier. The
main problem in terms of biosafety is the
loss of integrity of the disposable bag lead-
ing to a leak of the viral contaminant, and
potentially operator contamination. To
identify all potential root causes for the
loss of integrity of the disposable biore-
actor, a risk assessment was conducted.
Risks were scored according to four cri-
teria, each scaled from 1 to 3: impact, oc-
currence, detection, and action response
time. A risk priority number (RPN) was
calculated as the multiplication of these
four criteria. Based on the associated
RPN, risks were classifi ed as follows:
Operator infection
Operator contamination
Liquid projectionsLiquid spell in BL3 area
Leak of contamination liquid/gas
Rupture of bag integrity
Contact with cutting objects Contact with high temperatures Friction Overpressure
Medical follow-up
Retention vessel
Additional external protections
Cut duringpreparation and
operations
Contactwith steam
Doublejacket
Filteroverh eat
Contact withmovingpieces
Liquidoversupply
GasOver supply orfilter blocked
31 2 1 6 3 1 1 1 3 31 1 2 6 3 1 1 1 3 3 1 1 1 3 31 1 2 6 3 1 1 1 3
Figure 7. Summary of biosafety risk assessment and associated risk priority numbers
DISPOSABLES BIOREACTORS: VACCINES
30 The BioPharm International Guide November 2010
• 1 to 3 RPN: low risk
• 4 to 6 RPN from: medium risk
• >7 RPN: high risk.
Figure 7 gives a summary of the risks
identified associated with their respec-
tive RPN. Based on this risk assessment,
a set of corrective actions were defined.
The following gives examples of im-
provements made to the systems to miti-
gate potential biosafety issues:
• automation, aeration, and pump stops
when an overpressure is detected
• external protection was developed
to avoid liquid projections in case of
leak and to avoid contact with cutting
objects
• a retention vessel will be part of the
system skid to keep the liquid con-
tained in case of spill
• integrity testing of the disposable
bag is under development using pres-
sure to detect bag defaults.
Implementing all of the corrective ac-
tions identified in the risk assessment
will help secure the disposable system
for manufacturing operations.
Cost of Goods
Most of the studies performed today are
in favor of disposable implementation
from a cost perspective. Despite this,
two scenarios should be considered in
which the effect of disposable use on
cost can be significantly different.
• Introducing disposables to an exist-
ing process in an existing facility.
• Introducing disposables to a new facility.
In the first scenario (existing facility),
the effect of disposables may be margin-
al and can sometimes even result in in-
creased cost of goods. The reason is that
savings linked to disposable implemen-
tation are minimized because operating
costs are fixed. Costs linked to full time
employees will not be affected because
production teams are in place. Building
depreciation and maintenance also will
be equivalent.
In the second scenario (new facility),
the effect of implementing disposables
can be more significant if the new facil-
ity is designed for using disposables. In
this case, the facility footprint can be
significantly reduced, utility sizing and
distribution can be minimized, and pro-
duction headcount can be adjusted.
At GSK Biologicals, we decided to
compare two greenfield manufacturing
plants for viral bulk production, one us-
ing the old process as a reference, the
other one using a similar process but im-
plementing disposable bioreactors along
with other disposable systems for media
and buffer preparations and purification
intermediates. It is important to mention
that the manufacturing scheduling was
changed along with disposable bioreac-
tor implementation. This point has a ma-
jor effect on costs.
The model used to make this cost cal-
culation was developed in-house and
was validated on existing marketed vac-
cines. The f irst component of the cost
analysis was establishing a bill of ma-
terial analysis. Regarding the new pro-
cess, 50% of the raw material cost was
linked to the medium, ~25% was linked
Cost of goods analyses show
savings when implementing
disposable technologies in new
facilities and redesigning the
manufacturing schedule.
BIOREACTORS: VACCINES DISPOSABLES
November 2010 The BioPharm International Guide 31
to micro-carriers, and the disposable
bioreactor represents 6% of our raw ma-
terial cost.
The output of our model shows that 35%
can be saved on facility investment, and
the manufacturing headcount (production
and maintenance) can be reduced by 30%.
If we consider the effect on total direct
cost, 25% can be saved on the cost per dose
driven by saving on building depreciation
and labor. As mentioned previously, a sig-
nifi cant amount (~50%) of the savings are
linked to the optimization of manufactur-
ing scheduling.
Conclusion
We demonstrated the feasibility to
achieve equivalent process performance
using the right disposable bioreactor
systems compared to stainless steel bio-
reactors, even in the case of challenging
processes, such as the one described in
this article.
Additionally, cost of goods analyses
show a significant savings when dispos-
able technologies are implemented in
new facilities, along with redesigning
the manufacturing schedule.
Disposable bioreactors are an attrac-
tive technology for viral vaccine produc-
tion if biosafety risk can be mitigated.
One major point is that supply assurance
is still a major problem because back-up
supply is difficult to establish because of
the specificity of these disposable biore-
actors. BP
Continued from p. 21
methods. Some other interesting future
directions with respect to single-use
bioreactors could be the development of
systems for perfusion process applica-
tions, as well as more insights on leach-
ables and extractables. BP
References 1. Brecht R. Disposable Bioreactors:
Maturation into pharmaceutical glycoprotein manufacturing. In: Eibl R, Eibl D, editors. Disposable Bioreactors. Springer: Advances in Biochemical Engineering/Biotechnology; 2009. p. 1–31.
2. Selker M, Paldus B. Single-use sensors for Upstream applications. Next Gen Pharm. 2009; 16. Available from: http://www.ngpharma.com/article/Single-use-Sensors-for-Upstream-Applications/.
3. Smolke C, editor. The metabolic pathway engineering handbook. Boca Raton, FL: CRC Press; 2009.
4. Parmeggiani L. Encyclopaedia of occupational health and safety: A-K. Switzerland: International Labour Office; 1983.
5. Poles A, et al. Comparison of fed batch cell culture performances between stainless steel and disposable bioreactors, submitted to BioPharm Int.
6. Ravisé A, Cameau E, De Abreu G, Pralong A. Hybrid and disposable facilities for manufacturing of biopharmaceuticals: Pros and cons. In: Eibl R, Eibl D, editors. Disposable bioreactors. Springer: Advances in Biochemical Engineering/Biotechnology; 2009. p. 185-219.
7. Porter R, Roberts T, editors. Energy savings by wastes recycling, Commissioned by European Economic Communities. Elsevier, London; 1985.
Evaluation of a Single-Use Bioreactor
32 The BioPharm International Guide November 2010ternational Guide November 2010
Abstract
Tangential f low filtration (TFF) is a
common processing step in concentra-
tion and diafiltration (buf fer exchange)
operations in the downstream processing
of biopharmaceutical products. Using a
presanitized, disposable TFF membrane
makes it possible to reduce the number
of process steps and thus reduce labor by
50% or more and reduce buf fer and water
usage by 75% or more. In addition to the
cost savings realized from the reduced
labor and buf fer usage, single-use TFF
can increase productivity,
by >45% in many cases.
This article outlines
an economic model for
comparing the costs of
reusable and single-use
TFF in biopharmaceuti-
cal applications.
Over the last decade, the biopro-
cessing industry has recog-
nized that single -use products
can provide signif icant savings in time,
labor, and capital. As a scalable and
f lexible technology, single -use systems
have increased production capacity,
eliminated clean-in-place (CIP) steps,
cleaning validation, steam-in-place
sterilization, and reduced the use of
caustic chemicals and water for injec-
An Economic Analysis of Single-Use Tangential Flow Filtration for Biopharmaceutical Applications
Single-use TFF offers the greatest savings in clinical and contract manufacturing, where the scale is low and changeovers are frequent.
Michael LaBreck, Mark Perreault
Novasep
Michael LaBreck is the global product
manager for TangenX technology,
and Mark Perreault is the director
of membrane application develop-
ment for TangenX technology,
both at Novasep, 781.545.5756,
DISPOSABLES ECONOMICS
November 2010 The BioPharm International Guide 33
Clarified feedstream
Afinity chromatography
UF retentate vessel
UF retentate vessel
Ultrafiltration
Ultrafiltration
Anion/cationchromatography
Virus removal
0.2 μm asepticfiltration
Final formulation
tion. In addition, single -use products
can reduce the risk of cross contami-
nation between batches or campaigns.
These benefits apply to single -use tan-
gential f low ultraf iltration as well.
Tangential f low filtration (TFF) is a
common processing step in concentra-
tion and diafiltration (buffer exchange)
operations in the downstream process-
ing of biopharmaceutical products. As
shown in Figure 1, typically there are
several ultrafiltration TFF steps in down-
stream processing, such as following the
affinity chromatography, anion or cation
exchange chromatography steps.
When TFF systems are operated as clean-
and-reuse systems, they typically involve
10 major process steps (setup, CIP, fl ush,
normalized water permeability [NWP],
equilibration, processing, CIP, fl ush, NWP,
and storage). Using presanitized, single-
use TFF membranes, however, reduces the
number of process steps from 10 to four
(set up, equilibrate, process, CIP).
As a result, single -use TFF can re-
duce labor and processing time by 50%
or more. In addition, by eliminating
many f lush and CIP steps, single -use
TFF can reduce water, CIP solution, and
buffer consumption by 75% or more. By
developing an economic model, compa-
nies can identify the costs savings as-
sociated with these reductions in labor,
buffer, and water. This article will
outline such an economic model
for comparing the costs of reusable
and single -use TFF in biopharmaceuti-
cal applications.
An Economic Model for Single-Use TFF
A typical TFF process contains basic
operations that include pre-use, pro-
cess, and post-use activities. Figures 2a
and 2b show the percentage of time re-
quired to perform each step. Typically,
in a process based on reusable TFF, only
50% of the total process time is devoted
Figure 1. In a typical downstream process, there are several ultrafiltration tangential flow filtration (TFF) steps following the affinity chromatography, anion or cation exchange chromatography operations
ECONOMICS DISPOSABLES
34 The BioPharm International Guide November 2010
Setup3%
Sanitize4% Flush
6%NWP4%
Equilibration2%
Process50%
Clean30%
Store1%
A) Reusable TFF
Setup8% Equilibration
2%
Process80%
Clean10%
B) Single-use TFF
to actual processing of the product. The
remaining 50% is spent in preparing and
cleaning the TFF system. In contrast,
in a single-use TFF system, 80% of the
total process time is devoted to process-
ing the product, thus increasing process
efficiency.
Two key factors that affect the econom-
ics of single-use TFF are membrane area
and the number of annual process cycles.
Figure 3 shows the relationship between
the number of annual process cycles and
the economic benefit (percent savings)
associated with single-use TFF at sev-
eral different process scales. As seen in
the figure, single-use TFF technology is
most beneficial at smaller scales, i.e., <5
m2. Larger-scale processes can benefit,
however, when the number of annual pro-
cess cycles is less than 20.
Process Example: Single-Use TFF Cassettes
The following economic analysis of an
ultrafiltration process demonstrates the
savings that can be achieved when using
single-use f iltration cassettes instead of
reusable ones. The analysis takes into
consideration the cost of consumables,
such as water, CIP solutions, and buffer
solutions, as well as labor and overhead
costs associated with running a TFF
process in a cGMP process. These costs
can vary from site to site, so individual
site costs can be input to create a cus-
tomized model.
The membrane surface area of the fil-
tration cassettes chosen for this model
was 2.5 m2, an area sufficient to process
batch sizes up to approximately 1,000
L. This amount of membrane surface
Figures 2a & 2b. The percentage of time required to perform each step of a reusable tangential flow filtration (TFF) process. Typically, only 50% of the total process time is devoted to actual processing of the product. The remaining 50% is spent in preparing and cleaning the TFF system. In contrast, in a single-use TFF system, 80% of the total process time is devoted to processing the product, thus increasing process efficiency.
DISPOSABLES ECONOMICS
November 2010 The BioPharm International Guide 35
90
80
70
60
50
40
30
20
10
00 5 10 15 20 25 30 35 40
Number of uses
Area: 0.1 m2
Area: 0.5 m2
Area: 5.0 m2
Area: 10 m2
Area: 20 m2
Pe
rce
nt
savin
gs
area typically would be used for one
ultrafiltration process step in clinical-
scale production. In clinical-scale and
contract manufacturing, often only four
to six batches of product are processed
per campaign and then the reusable cas-
settes are either placed in storage or dis-
carded. This scenario is represented in
this particular economic analysis.
Consumables, including the filtration
cassettes, buffers, water, and CIP solu-
tions used during the process, also are
accounted for in this model.
Cassette costs vary from different
manufactures, so an average cost of the
reusable cassettes was used. Typically
reusable cassettes cost approximately
$3,600 per m2. By way of comparison,
single-use cassettes are approximately
20% of the cost of reusable cassettes or
approximately $800 per m2.
In addition to the cassettes, various
buffer solutions are used for the f iltra-
tion process. Some of the buffers are
used for the membrane equilibration
and diafiltration operations. More im-
portantly, with reusable TFF, a signif i-
cant amount of these buffers are used
for the CIP portion of the process. In
addition to these CIP solutions, purif ied
water is used for f lushing the reusable
cassettes. These CIP solutions and the
need for purif ied water for f lushing the
cassettes are eliminated when single-
use cassettes are used.
Lastly, labor and overhead costs are
considered. These labor and overhead
costs (also referred to as factory over-
head, factory burden, and manufactur-
ing support costs) refer to both direct
and indirect factory-related costs that
are incurred when the product is manu-
Figure 3. The relationship between the number of annual process cycles and the economic benefit (percent savings) associated with single-use tangential flow filtration (TFF) at several different process scales. Single-use TFF technology is most beneficial at smaller scales, i.e., <5 m2. Larger-scale processes can benefit, however, when the number of annual process cycles is less than 20.
ECONOMICS DISPOSABLES
36 The BioPharm International Guide November 2010
0
20,000
40,000
60,000
80,000
100,000
120,000
140,000
160,000
Reusable Single-use
To
tal co
st (
USD
)
factured. Along with costs such as direct
material, the cost of labor and overhead
must be assigned to each batch produced
so that the cost of goods are valued and
reported accurately. The overhead in-
cludes such things as the electricity
used to operate the factory equipment,
depreciation on the factory equipment
and building, factory supplies, and fac-
tory personnel not included as direct
labor. Once these costs have been tabu-
lated, they have a significant impact on
the cost of the ultrafiltration process.
Typical labor and overhead costs for a
cGMP approved manufacturing facility
range from $2,000 to $3,500 per hour in
the United States. In our model, we as-
sumed this cost to be $3,500 per hour.
As mentioned above, implementing
single-use cassettes can eliminate many
of the non-value–added steps of pre-
use and post-use CIP and as a result
the overall time of the ultrafiltration
process is reduced. As a result, there is
increased productivity and the overall
cost of manufacturing is reduced. This
increase in productivity can be illus-
trated by tabulating the labor and over-
head costs associated with each process
step and calculating the total cost sav-
ings per batch. Table 1 lists each of the
ten primary activities associated with a
typical reusable ultrafiltration process.
Table 1 also shows the reduced number
of process steps needed for a single-use
ultrafiltration process. That 2.4 -h reduc-
tion in processing times translates into
total savings for the process of approxi-
mately $8,400 per batch.
After identifying the costs for mem-
branes, water, CIP solutions, and buffer, as
shown in Tables 2 and 3, we compare these
costs in Table 4. Although the cost of cas-
settes is higher in the single-use model,
these higher membrane costs are more than
offset by the reduction of costs associated
with water and buffer usage and overhead.
Over the course of a � ve-run campaign, the
accumulated costs saving is signi� cant in
favor of single-use TFF. Figure 4 provides a
Figure 4. The overall cost comparison between reusable and single-use tangential flow filtration, for a five-batch campaign with a membrane surface area of 2.5 m2
DISPOSABLES ECONOMICS
November 2010 The BioPharm International Guide 37
graphical depiction of this overall cost com-
parison of reusable and single-use TFF.
Conclusions
Single-use tangential-� ow fl ltration (TFF)
cassettes offer signifl cant improvements in
process economics when compared to re-us-
able TFF cassettes. Presanitized cassettes
are installed, equilibrated with buffer, and
used in processing. There is no need to � ush
them with deionized water or water for injec-
tion, or to measure water permeability rates
between runs, saving time and resources.
Thus, by eliminating several f lushing
and clean-in-place steps, single-use TFF
reduces processing time and labor and
overhead costs. Likewise, buffer and wa-
ter consumption reduced by up to 75%.
In addition to the economic advantag-
es described here, development work
and scale -up can be conducted much
more quickly. Moreover, cassette per-
formance is more consistent from run
to run because each process uses a new
membrane, and the risk of cross-con-
tamination is minimized with single -
use cassettes.
The economic benefits of single-use
TFF are greatest in clinical manufactur-
ing and contract manufacturing where
the scale of operation is low (i.e., with
a membrane surface area <2.5 m2) and
frequency of process changeover is high
(with ≤6 batches per campaign).
Removing non-value–added steps in-
creases the efficiency and productivity of
tangential f low filtration operations. This
improvement in economics and produc-
tivity will benefit the entire downstream
process.
Table 1. Labor and facility costs, per batch and per campaign (assuming five batches per campaign) for reusable and single-use tangential flow filitration steps, assuming an overhead cost of $3,500 per hour
Process Step
Reusable Single-use
Percent of
process
Time (h)
Cost (USD)
Percent of
process
Time (h)
Cost (USD)
Set-up 3 0.24 840 8 0.64 2,240
Sanitize 4 0.32 1,120
Flush 3 0.24 840
NWP 2 0.16 560
Equilibration 2 0.16 560 2 0.16 560
Process 50 4.00 14,000 80 4.00 14,000
Clean 30 2.40 8,400 10 0.80 2,800
Flush 3 0.24 840
NWP 2 0.16 560
Store 1 0.08 280
Total 100 8.00 28,000 100 5.5 19,600
Savings per batch 30 2.4 8,400
Savings per campaign 42,000
ECONOMICS DISPOSABLES
38 The BioPharm International Guide November 2010
In addition to the signif icant cost
savings realized from the reduced
labor, reduced buffer usage, and po-
tentially reduced validation (although
the latter is not accounted for in
this model), the major benefit of
single -use systems such as single -
use TFF is an increase in productiv-
ity, which may amount to >45% in
many cases. BP
Table 2. Membrane costs
Table 3. Cost of water, buffer, and clean-in-place solutions (for simplicity, the costs per liter of these solutions has been averaged)
Reusable cassettes
Single-use cassettes
Cost per liter (USD) 0.90 0.90
Consumption per batch (L) 103.6 27
Cost per batch (L) 518 135
Consumption per campaign (L) 575.55555 150
Total cost per campaign (USD) 2,588 675
Savings per campaign (USD) 1,913
Table 4. Comparison of the overall costs in US dollars of reusable and single-use tangential-flow filtration (TFF), for a process requiring a membrane surface area of 2.5 m2 and assuming five batches per campaign
Reusable TFF Single-use TFF
Cost per batch
Campaign cost
Cost per batch
Campaign cost
Labor and overhead 28,000 140,000 19,600 98,000
Buffers (including water and CIP solutions)
518 2,588 135 675
TFF cassettes 1,800 9,000 2,000 10,000
Total 30,318 151,588 21,735 108,675
Savings 8,583 42,913
Reusable cassettes
Single-use cassettes
Membrane surface area (m2) of the cassette 2.5 2.5
Cassette cost per m2 (USD) 3,600 800
Total cassette cost 9,000 2,000
Number of batches per campaign 5 5
Number of cassettes per campaign 1 5
Total membrane cost per campaign (USD) 9,000 10,000
Savings in membrane cost per campaign (USD) 1,000
DISPOSABLES ECONOMICS
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40 The BioPharm International Guide November 2010
DISPOSABLES FILTRATION
ternational Guide November 2010
Abstract
In a multiproduct cGMP clinical manu-
facturing facility, f lexibility and short
processing times are important operating
attributes. A critical aspect of a multi-
product facility is the procedures used to
minimize product cross-contamination.
Single-use (SU) technology enables f lex-
ibility, short process times, and limited
chances for cross-contami-
nation. An SU tangential
f low filtration (TFF)
system was implemented
in a cGMP clinical manu-
facturing facility. In this
article, we evaluate the performance, con-
trol of operation, productivity, and overall
cost savings of the system.
Improving the time it takes to bring
new drugs to the market continues
to be an important goal for pharma-
ceutical companies. There are several ap-
proaches that are taken with the overall
Implementing Single-Use Technology in Tangential Flow Filtration Systems
in Clinical Manufacturing
A case study evaluates the performance, control of operations, productivity, and cost
savings of a single-use system.
Keqiang Shen, Be Van Vu, Nikunj Dani, Bryan Fluke, Lei Xue, David W. Clark
Jo
hn
so
n &
Jo
hn
so
n
Keqiang Shen is a senior sci-
entist, Be Van Vu is an associate
engineer, Nikunj Dani is a senior
systems engineer, Bryan Fluke
is a senior associate engineer,
Lei Xue is a senior manager, and
David W. Clark is the global
head of supply execution, all in
pharmaceutical development and
manufacturing sciences at Johnson
& Johnson, Inc, Spring House, PA,
215.628.5953, [email protected].
FILTRATION DISPOSABLES
November 2010 The BioPharm International Guide 41
goal of bringing more drugs into com-
mercialization phase.1 Single-use (SU)
technology is one of the strategies being
adopted to reduce overall drug develop-
ment time. SU technology brings sig-
nifi cant advantages of reduced capital
costs, faster construction and installa-
tion, reduced processing cycle times,
and elimination of the need for post-use
equipment cleaning and verifi cation.2,5
Starting with the use of plasticware such
as pipettes, petri dishes, and t-fl asks,
disposable components are being in-
creasingly incorporated in the laboratory
environment. With the development of SU
bioreactors at the 2,000 L scale (Xcellerex
XDR), chromatography systems (GE Rea-
dytoProcess), and systems for micro- and
ultrafi ltration, disposable equipment
continues to replace fi xed stainless steel
equipment in manufacturing plants.
Demonstrating that process equipment
is adequately cleaned is a critical aspect
of any pharmaceutical plant operation.
Following some type of post-use clean-
ing, the presence of residual protein and
cleaning agent is typically assessed by
subjecting equipment rinse solutions and
swab samples to test methods including
pH, conductivity, and f luorescamine.
These procedures are time consuming
and reduce equipment utilization. In a
multiproduct facility, when equipment is
shared between products with high and
low potency, meeting the low acceptance
criteria for residual protein can be very
challenging.
To meet the demand of reducing produc-
tion cycle time and improving productiv-
ity, a general downstream production
process review was performed to identify
the bottlenecks that affect the overall
process efficiency. This process review
identified that an existing stainless
steel (SS) ultrafiltration–dialfiltration
(UF–DF) system used for an intermedi-
ate UF–DF process in the facility was a
bottleneck and presented an opportunity
to evaluate an SU UF–DF system. The SS
UF–DF system was designed for process
development and scale-up rather than
GMP production and had a limited reten-
tate tank capacity (25 L). Additionaly, the
clean-in-place (CIP) and post-CIP swab-
bing of the SS UF–DF system are very
complex and time-consuming.
Criteria for Evaluating SU UF–DF Systems
Currently there are several SU UF/DF
systems available. An evaluation was
made to choose an appropriate system
based on GMP production requirements,
operational needs, budget, and timeline.
These criteria included:
• Operation: The system should have the
capacity to handle 5 m2 membrane size,
10–50 L retentate tank working volume,
>80 L/h/m2 (LHM) feeding fl ux, and
differential pressure (∆P) and trans-
membrane pressure (TMP) controls at
0–20 psi. The retentate tank should have
a mixing device to avoid localized con-
centration during the operation.
• Process monitoring, control, and data
management: The process should be
able to be controlled by constant TMP
and ∆P. The pressures, fl ow rates, process
Because the whole fl ow path
is disposable, the risk of cross-
contamination during product
changeover was minimized, and
the time for CIP was reduced.
DISPOSABLES FILTRATION
42 The BioPharm International Guide November 2010
SS UF-DF System
Flowpath/membraneinstallation
Pre-use tank CIP/rinse
Pre-use rinse samples
Integrity test(Use auxiliary pump)
UF-DF(Manual)
Post-use tank CIP/rinse
Post-use membrane CIP/rinse
Rinse/protein swabsamples
Tank storage
Membrane storage
Pre-use membrane CIP/rinse
Single use UF-DF System
Membrane storage
Post-use membrane CIP/rinse
UF-DF(Automated)
Integrity test(Use feed pump)
Membrane CIP/rinse(No rinse samples)
Flowpath/membraneinstallation
phases, and other process parameters
should be able to be monitored and re-
corded in real time. Data management
should meet 21 CFR Part 11 compliance.
• Disposable parts: There should be no
or a limited number of bio-compatibil-
ity issues for any disposable parts that
contact product and process buffers.
Figure 1. Operation comparison of a stainless steel ultrafiltration–diafiltration (SS UF–DF) system and a single-use (SU) system
FILTRATION DISPOSABLES
November 2010 The BioPharm International Guide 43
The levels of leachables and extract-
ables should be in the acceptable safe-
ty range for clinical drug substances.
• Equipment availability and vendor
support: The product should be readily
available. An integrated and off-the-shelf
system is preferred because it saves time
on equipment validation and meets short
project timelines. The manufacturer
should have a good record for on-time
equipment delivery and reliable techni-
cal support. The manufacturer should
have the capability to consistently supply
high quality accessories and consum-
able items.
• Cost: The price of the equipment
and disposable items should be rea-
sonable to reduce the overall cost of
production when implementing a new
UF–DF system in a GMP production
facility.
The Millipore SU Mobius FlexReady
Solution for TFF model TF2 system was
selected after comparing three different
SU UF–DF systems that are currently on
the market. An ultrasonic permeate f low
meter and a retentate pressure control
valve (PCV) were included to enhance
process control. The length of the rods
on the cassette holder was extended to
increase the holder capacity from 2.5 to
5 m2 of membrane.
Operational Performance of the Single-Use System
A typical UF–DF operation includes fi ow
path and membrane installation, pre-use
CIP, integrity test, UF, DF, product recov-
ery, and post-use CIP. Figure 1 compares
a SS UF–DF system and a single-use UF–
DF system. The implementation of the
Millipore SU Mobius FlexReady Solution
for TFF offers some key advantages over
the existing SS UF–DF system in terms of
preparation, membrane integrity testing,
UF–DF general operation, critical param-
eters (feed fi ow rate, effl ciency of mixing in
retentate tank), and product recovery.
Equipment preparation and set up: The
disposable fi ow path is gamma irradiated
and ready for use when it arrives. With the
SS UF–DF system, cleaning occurs in two
steps: the UF–DF system and membrane.
The gamma irradiated retentate bag and
fi ow path have eliminated the system pre-
use and post-use CIP, rinse samples and
protein swabs, and storage steps, however
cassette cleaning must still be performed.
The disposable fi ow path is relatively easy
to install, and typically can be done in 60
minutes or less. The fi ow path is discarded
after each use, minimizing the potential for
product cross-contamination.
Membrane integrity testing: The feed
pump on the SS UF–DF system is a rotary
lobe pump. An auxiliary peristaltic pump
is required to perform membrane integrity
testing for the SS UF/DF system. The SU
UF–DF system eliminates the need for an
auxiliary peristaltic pump for membrane in-
tegrity testing.
UF–DF general operation: Four fully
automated operations (initial fl ll, fed-
batch, DF, and batch concentration)
make the system user friendly. Process
parameters are easy to input, and the re-
sulting data are captured in our data ac-
To increase the percent
recovery, a buffer � ush step
was performed to recover
product retained in the system
and membrane.
DISPOSABLES FILTRATION
44 The BioPharm International Guide November 2010
00.10.20.30.40.50.60.70.80.91
0 1 2 3 4 5 6
Re
ten
tate
re
sid
ue
(%
)
Diavolume
Diafiltration from 1.1 M NaCl to 0.15 M NaCl
Experimental Theoretical
quisition system, eliminating the need to
transcribe data into a paper-based GMP
batch record. This facilitates trending or
comparisons from batch to batch. Elec-
tronic data also simplify the technology
transfer from pilot plant to commercial
manufacturing.
Feed flow rate: One parameter with a
significant impact on the UF–DF opera-
tion is the feed f low rate. To maximize
the permeate f lux, UF–DF operations
typically require f low rates in the range
of 240–360 LHM to maintain specific
crossf low characteristics. With the SU
Mobius FlexReady Solution, the peristal-
tic feed pump can achieve 20 L/min with
30 psi back pressure. The feed pump is
either controlled by fiP or fixed pump
speed, which is correlated to the f low
rate. The f low rate meets our current
process feed f low rate specification, and
the automated TMP control using the re-
tentate PCV is relatively stable.
Mixing Efficiency: Mixing is critical
during the diafl ltration step to ensure a
homogeneous solution for effl cient buffer
exchange. With the previous SS UF–DF
system, retentate return ∆ ow distribution
was the only method for mixing in the re-
tentate tank. The SU Mobius TFF system
design incorporates ∆ ow distribution by
a retentate diverter plate and a magneti-
cally coupled agitator for enhanced mix-
ing in the retentate tank. Eliminating
dead legs from the ∆ ow path is critical to
achieving effl cient buffer exchange. To
this end, the design of the low dead-vol-
ume t-connectors for the pressure indica-
tors in the disposable ∆ ow path assembly
is important. In Figure 2, diafl ltration
from 1.1 M NaCl to 0.15 M NaCl was per-
formed at a constant-volume diafl ltration
(50 L) and 9% agitation speed with a 5
m2 30 kD Millipore Biomax membrane.
The maximum diafl ltration volume with
low agitation speed was considered the
worst-case scenario. The experimental
curve, is comparable to the theoretical
curve indicating good mixing.
Product recovery: To increase the per-
cent recovery, a buffer ∆ ush step was
performed to recover product retained in
the system and membrane. With the SU
Mobius FlexReady TFF system, buffer
Figure 2. Constant-volume diafiltration in the Millipore single-use Mobius FlexReady solution for TFF. The solid line is experimental and the dotted line is theoretical: Retentate residue (%) = 100 * e (R–1)*N, where R is the Donnan effect and N is diavolume.
FILTRATION DISPOSABLES
November 2010 The BioPharm International Guide 45
0
2
4
6
8
10
12
14
1 2 3 4 5 6
Dia
filt
rati
on
vo
lum
e (
DV
)
Run #
can be transferred to the retentate tank
and weighed directly. This saves time and
eliminates the step of weighing the fi ush
buffer, which was required with the previ-
ous SS UF–DF system.
System Process Control
To meet the requirement of process con-
trol, a retentate PCV was applied for con-
stant TMP control. The retentate PCV
position can be selected between 0 and
100% open for feed f low rate control. The
100% open position was chosen for PCV at
the start of diafiltration. Table 1 summa-
rizes the performance of constant TMP
control at diafiltration phase for six GMP
runs with a feed f low rate of 180 LHM.
The diafiltration started with the feeding
f low rate controlled by the pump speed.
Once the feed f low rate was in the recom-
mended operating range (ROP), constant
TMP control was applied. The TMP set-
ting was randomly selected in the TMP
ROP range (<20 psi). It was found that
TMP could be controlled at a narrow f luc-
tuation range with a coefficient of varia-
tion (CV) less than 10% in these runs.
The system uses an ultrasonic perme-
ate fi ow meter to calculate cumulative di-
afl ltration volume (DV), which is used to
target the diafl ltration process endpoint.
During the factory acceptance testing,
the accuracy of the permeate fi ow meter
was tested using four solutions with dif-
ferent densities (2 M guanidine HCl, 2 M
urea, 100 mM NaCl, and pure water). The
results suggest that the solution densities
have little impact on the fi ow meter read-
ing (data not shown).
In GMP production, the accuracy of the
fi ow meter was evaluated by comparing DV
values using a fi ow meter versus those ob-
tained form a weight scale. The comparison
of results is summarized in Figure 3, where
the dark columns represent DV results ob-
tained from the weigh scale and the light
columns represent DV values obtained
from the permeate fi ow meter. The average
difference between these two measure-
ments is 3.3%.
Product Safety
System integration, product quality, and
product safety impact factors (leachables,
Figure 3. Comparison of measuring diafiltration volume by permeate flow meter (light columns) versus by weigh scale (dark columns)
DISPOSABLES FILTRATION
46 The BioPharm International Guide November 2010
extractables, and biocompatibility) are
major concerns. It is benefi cial for a com-
pany to use a family of SU products from
the same manufacturer to save the time
and reduce the cost of safety evaluations
and validation.
Before introducing an SU UF–DF sys-
tem to our facility, several other SU sys-
tems made by Millipore, including buffer
and media mixing tanks and containers,
were already evaluated and used in GMP
production. The f lowpath and associated
retentate tank liner for the Millipore SU
Mobius FlexReady Solution for TFF are
made of the same film and material as
those used for the associated containers
for the Millipore mixing system, which
are widely used in our facility. The risk
of leachables and extractables affecting
product quality and the compatibility of
buffers and protein were previously as-
sessed,3, 4 saving both time and cost on
those safety evaluations. Unlike SU UF–
DF systems, the Mobius FlexReady Solu-
tion for TFF is a fully integrated system
for operation, process monitoring and
control, and data management. In the
preliminary evaluation, two systems had
the same assessment score based on op-
eration specifications, process monitor-
ing, and control. System integration and
product quality and safety impact were
the differentiating factors. Ultimately,
the Millipore Mobius FlexReady Solu-
tion for TFF was chosen because of its
better integrated system and the exis-
tence of previous safety evaluation as-
sessment.3, 4
Summary and Conclusions
The operation of this single-use system
is easier than our existing SS UF–DF
systems and the performance is compa-
rable. The disposable f low path pieces
can be quickly installed, resulting in
short equipment turnaround times.
The retentate diverter plate and mag-
netically coupled mixing device helped
avoid the concentration gradients in the
retentate tank and improved the UF–DF
performance. Product yield and purity
of the intermediate UF/DF step using
the Mobius FlexReady Solution for
TFF are comparable to the results from
previous runs (data not shown). Also,
the low system hold-up volume (0.6 L)
and working volume (2.0 L) allowed for
a wider operating range. The strong
feed pump maintained a 20 L/m2 f low
rate even at 30 psi back pressure. Be-
cause the whole f low path is disposable,
the risk of cross-contamination during
Table 1. Summary of constant transmembrane pressure control (TMP) control
Run # TMPavg
(psi) TMPmax
(psi) TMPmin
(psi) SD CV (%)
1 12.5 13.5 10.5 0.5 3.9
2 12.0 12.5 11.0 0.3 2.4
3 13.2 13.8 12.7 0.3 2.4
4 13.8 14.3 12.9 0.4 3.1
5 14.3 16.2 12.0 0.6 4.1
6 10.6 13.0 9.4 0.7 6.9
SD: Standard deviation; CV: coefficient of variation
FILTRATION DISPOSABLES
November 2010 The BioPharm International Guide 47
product changeover was minimized, and
the time and effort for CIP were reduced
in our multiproduct facility.
The features of process monitoring,
control, and data management enhance
the process automation capability and
reliability of data recording. The Allen
Bradley Controllogix system and the
associated human–machine interface
(HMI) software were user-friendly. The
PID-like display provided real-time infor-
mation of recipe parameters and process
data. The control system was interfaced
with our production information manage-
ment system for data collection and ar-
chiving. This feature enables the future
implementation of electronic batch re-
cords in a GMP production environment.
It also enables easy, batch-to-batch com-
parison for technology transfer.
The PCV and permeate fi ow meter in-
creased process monitoring and control
capabilities. We observed fi uctuations of
TMP while using the PCV for constant
TMP control. It was found that the fi uctu-
ations could be reduced to an acceptable
level (Table 1) by initiating the operation
with feed fi ow control by pump speed,
then switching to constant TMP control
by PCV once the feed fi ow rate is within
the ROP range. During the diafl ltration,
total diafl ltration volume target can be
preset and monitored with the included
permeate fi ow meter. Although there was
a measurement difference of approxi-
mately 3% in the total diafl ltration volume
versus the weight scale measurement,
the result is acceptable because the ROP
for total diafl ltration volume was quite
wide and diafl ltration completion was pri-
marily based on the conductivity and pH
of permeate fl ltrate.
Implementing this single-use ultrafil-
tration-diafiltration system doubled the
retentate capacity of the intermediate
UF–DF system, and therefore shortened
the unit process time and improved pro-
ductivity. By eliminating the six pre- and
post-use CIP steps, the usage of water for
injection and caustic solutions for CIP
also was reduced. Furthermore, the 16
corresponding rinse samples and swab
sampling testing for CIP steps were
eliminated. The overall cost saving will
depend on actual utilization. We found
the payback period to be 3.8 years when
performing ten campaigns per year.
Acknowledgement
The authors would like to thank the Mil-
lipore team for technical and logistic sup-
port for introducing this SU UF–DF to
our facility. The authors also would like
to acknowledge the Centocor pilot plant
purifl cation team for their support in
implementing the SU UF–DF system for
GMP production. BP
References1. Paul MS, Mytelka SD, Dunwiddie TC,
Persinger CC, Bernard H, Stacy MR, et
al. How to improve R&D productivity:
the pharmaceutical industry’s grand
challenge. Nature Rev Drug Discov.
2010;9:203–14.
2. Charles I, Lee J, Dasarathy Y. Single-
use technologies—a contract
biomanufacturer’s perspective. BioPharm
Int. Suppl. Guide to Disposables. 2007
Nov; 31–6.
3. Millipore, Inc. PureFlex: Extractables,
bioreactivity safety evaluation
approach. Technical brief.
4. Millipore, Inc. Extractables bioreactivity
safety evaluation of PureFlex film for
Centocor. 2007.
5. Maigetter RZ, et al. Single-use (SU)
systems. Encyclopedia of industrial
biotechnology: bioprocess, bioseparation,
and cell technology. 2010: 1–39.
NAME OF GUIDE CHAPTER NAME
For Client Review Only. All Rights Reserved. Advanstar Communications Inc. 2008
48 The BioPharm International Guide November 2010
BioPharm: Acceptance of disposables
in the industry is rapidly increasing as
companies gain experience with the de-
vices. What is the state of disposables
implementation at Pfi zer?
Barnoon: We have played around
with disposables for many years now.
We have been early adopters and
have tested lots of different technolo-
gies in various parts of our business
on the healthcare, biotechnology, and
vaccines side. We have even imple-
mented some as part of our platform
processes so that they are a standard
mode of operation for us.
BioPharm: In an article that you pub-
lished with BioPharm International,
you said that although single-use sys-
tems help reduce capital costs, in many
applications, capital savings are offset
by increased operating costs. In which
applications do you think disposables
make the most economic sense?
Barnoon: Let me start with a disclaim-
er. You really have to perform your
own analysis to see if it makes sense
to implement disposables in your
own facility and your own process. It
varies so much depending on which
disposable technology you use. It’s
not one size fi ts all. It also depends
on how you envision the operation
of that facility; scale and run rate.
These factors have a profound effect
on the way the economics turn out.
That disclaimer aside, the article that
you referenced looked at a particular
facility that was envisioned to operate at
a very high run rate and we found that
for instances where the operating cost
associated with disposables were more
than the traditional stainless solution
because of the high run rate, we were
actually offsetting the cost savings as-
sociated with capital (initial implemen-
tation) pretty quickly. So where it ended
up making sense were areas where we
were using fairly simple disposables that
don’t cost a lost on a per unit basis such
as standard bio bags, rocker bioreac-
tors, and that sort of technology.
BioPharm: What approach can bio-
pharmaceutical manufacturers follow to
evaluate the lifecycle costs of single-use
systems?
Barnoon: The approach that worked really
well for us was to use a net present value
calculation to quantify the difference be-
tween a disposable option and a baseline
stainless steel option. We looked at both
side by side and treated the disposable
Do Single-Use Technologies Make Economic Sense?
Live Interview from the Intephex 2010 Mainstage with Barak Barnoon, director of process engineering, Pfizer Global Manufacturing, Pfizer Inc.
”“
You have to perform
your own analysis
to see if it makes
sense to implement
disposables.
NAME OF GUIDE CHAPTER NAME
For Client Review Only. All Rights Reserved. Advanstar Communications Inc. 2008
option as though it were a new invest-
ment, comparing it to stainless steel. We
carved up the facility, looked at different
options such as buffer prep and media
prep, and evaluated both what the initial
costs were and also what the operating
cost would be, and basically evaluated
those cash fi ows for the whole lifecycle
of the facility. That allowed us to get the
full lifecycle cost for the disposables.
We were able to evaluate different op-
tions—not only disposables compared
to stainless but also different versions of
disposables—and see the full cost of the
solution. Additionally, what it allowed us
to do was to get answers fairly quickly in
a short time.
BioPharm: Is the industry still hesitant
about using disposables in large-scale
chromatography steps due to cost
constraints?
Barnoon: I think so. I think there is a
hesitancy to throw out a very expensive
resin after a single use when you could
regenerate it and use it multiple times. I
think there might be a game changer in
a sequential serial multicolumn simu-
lating moving bed operation, where you
design it such that you reach the full
resin cycle lifetime within one batch.
That allows you to size the columns
where you can use them once and throw
them away. That potentially could
make it a no-brainer for disposables.
BioPharm: To what extent is cost
a factor in making decisions about
whether or not to implement single-use
technologies?
Barnoon: Cost is obviously is one
consideration and the most important
one on the commercial manufacturing
side. There are other considerations
that absolutely have to be taken such
as the technical risks, where you are
looking to implement them, the scale,
the regulatory framework, and so on.
The weighting of these considerations
will be different whether you are talk-
ing about clinical manufacturing or a
commercial facility, whether you are
talking multiproduct operations or
single products. But on the commer-
cial side, cost is a prominent concern,
especially when you consider that we
have an obligation as manufacturers
to make our products accessible and
affordable to people around the world.
BioPharm: How close is the biopro-
cessing industry to implementing a
fully disposable process stream?
Barnoon: I think the time is now.
There is no real substantial techni-
cal hurdle to implementing it now. A
better question would be, Where does
it make sense to implement a fully dis-
posable process? It makes more sense
in certain applications than others.
In pandemic fi u vaccine manufactur-
ing, it seems to be a good fl t for rapid
rollout. And certainly as the industry
evolves and new disposable technolo-
gies are introduced, as cost profl les
change, as the biopharmaceutical seg-
ment changes and we move to smaller
volumes in personalized medicine,
as those economics change, where it
makes more sense to use a fully dis-
posable train will change.
This conversation has been edited for length and clarity.
Watch more vidcasts from Interphex and BIO 2010 on our web site, at
www.biopharminternational.com/vidcasts
50 The BioPharm International Guide November 2010
Say hello to
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