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BioPharmwww.biopharminternational.com

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

[email protected]

Editor in Chief Laura Bush

[email protected]

Managing Editor Chitra Sethi

[email protected]

Associate Editor Haydia Haniff

[email protected]

Art Director Dan Ward

[email protected]

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.

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Single Use. Visit us at

www.millipore.com/smartmobius

© 2010 Millipore Corporation. All rights reserved.


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


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.



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


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.



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.

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DISPOSABLES BIOREACTORS: MABS


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.

[email protected].



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


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.



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.



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.



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.



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.



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


DISPOSABLES BIOREACTORS: VACCINES


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


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



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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This benchtop bioreactor combines all

the convenience of single-use technology

with the trusted performance, advanced

process management and true scalability

of a stirred-tank design.

Offered with 5 liter and 14 liter single-use

vessels, pre-sterilized and ready for use, out

of the box.

Move over bags. The next big wave in

single-use cell culture technology is here!

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a culture of innovationCERTIFIE

D



• 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



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



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



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



• 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.



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,

[email protected].

DISPOSABLES ECONOMICS


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


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.



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.



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



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



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


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DISPOSABLES FILTRATION


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


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.



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



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.



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.



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)



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



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


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


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Convenient. Scalable. Disposable.FiltrationInnovative

The Zeta Plus™ Encapsulated System

The System of Choice For Disposable Depth Filtration

• The pivoting holder design facilitates ergonomic loading and unloading

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• Translucent plastic capsule shell enables liquid level detection

• Accommodate single and two-stage depth filtration

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