Volume 31 Number 12 BioPharm - files.alfresco.mjh.groupfiles.alfresco.mjh.group/alfresco_images/pharma/... · 12/10/2018 · lab and manufacturing space, our network of facilities

The Science & Business of Biopharmaceuticals

BioPharmINTERNATIONAL

December 2018

Volume 31 Number 12

KEEPING QUALITY AT THE FOREFRONT

SUPPLY CHAIN

GOING BEYOND THE

SURFACE TO ENSURE

SUPPLIER QUALITY

PEER-REVIEWED

SEPARATION AND

PURIFICATION OF A TISSUE-

TYPE PLASMINOGEN ACTIVATOR

FROM TRICHODERMA REESEI

LOT RELEASE TESTING

UNPROCESSED

BULK TESTING FOR

BIOPHARMACEUTICALS

www.biopharminternational.com

For personal, non-commercial use

http://www.biopharminternational.com

2 BioPharm International December 2018 ADVERTORIAL

CORPORATE CAPABILITIES www.biopharminternational.com

Company DescriptionWu X i Biolog ic s i s a le ad ing open-access technology platform contract ser-

v ices company. A s a premier contract discov-ery, development, and manufacturing organi-zation (CDMO), WuXi Biologics provides our worldwide clients with the necessary expertise, quality, and capacities to develop biologic drugs

from concept to commercialization. Along with our WuXi AppTec affiliates, we provide the world with the one true, single-source approach. The eff iciencies gained from this approach saves our clients critical time and money. Our company track record and achievements demonstrate our commitment to providing a truly one-stop service offering and value proposition to our global clients.

Markets Served We support biologics drug developers of all sizes in all major markets, from top 20 pharmaceutical companies to virtual biotech companies by providing an integrated CMC package from concept to IND filing through to BLA and beyond. We also provide stand-alone services to overcome the challenges at a particular stage of development. Our expertise covers all facets of development for various types of biologics, including mono-clonal and bispecific antibodies, recombinant and fusion proteins, and antibody drug con-jugates.

Services and CapabilitiesOccupying more than 1,000,000 sq. ft. of lab and manufacturing space, our network of facilities operates under global regulatory standards and provides our clients unparal-leled capacities across the discovery, develop-ment, and manufacturing continuum. We

WUXI BIOLOGICS

108 Meiliang Road. WWuxi MaShan Binhu District214092Chinatel. +86 (510)-8183-1200

EMAIL

[email protected]

WEBSITE

www.wuxibiologics.com

WuXi Biologicsoffer GMP clinical and commercial manu-facture of Drug Substance and Drug Product in multiple sites including the world’s largest mammalian cell culture GMP facility using disposable bioreactors. All the facilities are located within 1–2 hours’ drive from each other and provide a simpli-fi ed supply chain for our global clients.

WuXi Biologics offers open-access tech-nology platforms and a unique single-source discovery, development, manufacturing, and testing service for protein-based thera-peutics.

Our comprehensive one-stop offerings include:• Antibody discovery• Custom small-scale, research-grade protein

generation• Cell line development• GMP cell bank manufacture/characteriza-

tion• Assay development, qualification, and vali-

dation• Bioconjugation development• Cell culture process development• Purification process development• Formulation development• Protein characterization• Drug substance manufacture• Drug product formulation and fill• Biosafety testing (e.g., viral clearance)• GMP lot release testing• Stability studies• Worldwide regulatory submission support.



mailto:510)-8183-1200EMAILinfo@wuxibiologics.comWEBSITEwww.wuxibiologics.comofDrugSubstanceandDrugProductinmultiplesitesincludingtheworld%E2%80%99slargestmammaliancellcultureGMPfacilityusingdisposablebioreactors.Allthefacilitiesarelocatedwithin1%E2%80%932hours%E2%80%99drivefromeachotherandprovideasimpli-fiedsupplychainforourglobalclients.WuXiBiologicsoffersopen-accesstech-nologyplatformsandauniquesingle-sourcediscovery

https://www.wuxibiologics.com/

mailto:[email protected]

https://www.wuxibiologics.com/

© 2018 UBM All r ights reserved. No par t 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 wr i t ing f rom the pub l isher. Au thor i z a t ion to photocopy i t ems for in terna l /educat iona l or persona l use, or the in terna l /educat iona l or persona l use of specific clients is granted by UBM for libraries and other users registered with the Copyright Clearance Center, 222 Rosewood Dr. Danvers, MA 01923, 978-750-8400fa x 978-646-8700 or v is i t h t tp: //w w w.copyr ight .com onl ine. For uses beyond those l is ted above, please direc t your wr i t ten request to Permission Dept. fax 732-647-1104 or email: [email protected]

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

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

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

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

EDITORIAL

Editorial Director Rita Peters [email protected]

Senior Editor Agnes M. Shanley [email protected]

Managing Editor Susan Haigney [email protected]

European Editor Felicity Thomas [email protected]

Science Editor Feliza Mirasol [email protected]

Manufacturing Editor Jennifer Markarian [email protected]

Associate Editor Amber Lowry [email protected]

Art Director Dan Ward [email protected]

Contributing Editors Jill Wechsler, Eric Langer, Anurag Rathore, and Cynthia A. Challener, PhD

Correspondent Sean Milmo (Europe, [email protected])

ADVERTISING

Publisher Mike Tracey [email protected]

National Sales Manager Scott Vail [email protected]

European Sales Manager Linda Hewitt [email protected]

European Senior Sales Executive Stephen Cleland [email protected]

C.A.S.T. Data and List Information Michael Kushner [email protected]

Licensing and Reuse of Content: Contact our official partner, Wright’s Media, about available usages, license fees, and award seal artwork at [email protected] for more information. Please note that Wright’s Media is the only authorized company that we’ve partnered with for Advanstar UBM materials.

PRODUCTION

Production Manager Jesse Singer [email protected]

AUDIENCE DEVELOPMENT

Audience Development Christine Shappell [email protected]

Thomas W. Ehardt

Executive Vice-President, Senior Managing Director,UBM Life Sciences Group

Dave Esola

VP/Managing Director, Pharm/Science Group

UBM Life Sciences

K. A. Ajit-SimhPresident, Shiba Associates

Madhavan BuddhaFreelance Consultant

Rory BudihandojoDirector, Quality and EHS Audit

Boehringer-Ingelheim

Edward G. CalamaiManaging Partner

Pharmaceutical Manufacturing

and Compliance Associates, LLC

Suggy S. ChraiPresident and CEO

The Chrai Associates

Leonard J. GorenGlobal Leader, Human Identity

Division, GE Healthcare

Uwe GottschalkVice-President,

Chief Technology Officer,

Pharma/Biotech

Lonza AG

Fiona M. GreerGlobal Director,

BioPharma Services Development

SGS Life Science Services

Rajesh K. GuptaVaccinnologist and Microbiologist

Denny KraichelyAssociate Director

Johnson & Johnson

Stephan O. KrauseDirector of QA Technology

AstraZeneca Biologics

Steven S. KuwaharaPrincipal Consultant

GXP BioTechnology LLC

Eric S. LangerPresident and Managing Partner

BioPlan Associates, Inc.

Howard L. LevinePresident

BioProcess Technology Consultants

Hank LiuHead of Quality Control

Sanofi Pasteur

Herb LutzPrincipal Consulting Engineer

Merck Millipore

Hanns-Christian MahlerHead Drug Product Services

Lonza AG

Jerold MartinIndependent Consultant

Hans-Peter MeyerLecturer, University of Applied Sciences

and Arts Western Switzerland,

Institute of Life Technologies

K. John MorrowPresident, Newport Biotech

David RadspinnerGlobal Head of Sales—Bioproduction

Thermo Fisher Scientific

Tom RansohoffVice-President and Senior Consultant

BioProcess Technology Consultants

Anurag RathoreBiotech CMC Consultant

Faculty Member, Indian Institute of

Technology

Susan J. SchnieppExecutive Vice President of

Post-Approval Pharma

and Distinguished Fellow

Regulatory Compliance Associates, Inc.

Tim SchofieldSenior Fellow

MedImmune LLC

Paula ShadlePrincipal Consultant,

Shadle Consulting

Alexander F. SitoPresident,

BioValidation

Michiel E. UlteePrincipal

Ulteemit BioConsulting

Thomas J. Vanden BoomVP, Biosimilars Pharmaceutical Sciences

Pfizer

Krish VenkatManaging Partner

Anven Research

Steven WalfishPrincipal Scientific Liaison

USP

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.

INTERNATIONAL

BioPharmThe Science & Business of Biopharmaceuticals



















http://www.copyr


Table of Contents

4 BioPharm International December 2018 www.biopharminternational.com

BioPharm International integrates the science and business of biopharmaceutical research, development, and manufacturing. We provide practical, peer-reviewed technical solutions to enable biopharmaceutical professionals to perform their jobs more effectively.

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

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

COVER STORY

12 Focus on QualityThis issue features articles on a variety of quality control topics including validation of suppliers, lot release testing, and lab operations. See below for more information.

Cover Design by Dan WardImages: Michail Petrov/Stock.Adobe.com

SPECIAL QUALITY SECTION

SUPPLY CHAIN MANAGEMENTGoing Beyond the Surface to Ensure Supplier QualityAgnes Shanley

Success depends on supplier

communication and transparency, but it’s

up to buyers to demand the right

information and to look at the vendor’s

overall business. . . . . . . . . . . . . . . . . . .12

LOT RELEASE TESTINGUnprocessed Bulk Testing for BiopharmaceuticalsKatherine Marotte

Unprocessed bulk material harvested

directly from the bioreactor should

be tested for contamination prior to

downstream processing. . . . . . . . . . . .18

LAB OPERATIONSNIST Fluorescence-BasedMeasurement ServicesPaul C. Derose and Lili Wang

Fluorescence has long been used to detect

biological targets. As these measurements

are becoming more and more quantitative,

standards are needed to ensure accuracy

and reproducibility. . . . . . . . . . . . . . . . .24

FEATURES

PEER-REVIEWEDSeparation and Purification of a Tissue-Type Plasminogen Activator from Trichoderma reeseiJinhua Shao, Yufei Zhang, Zhiyong

Zhu, Fulin He, and Xiaoming Chen

This research proposes a method

for separating and purifying t-PA

from this fungal cell source. . . . . . . . . .30

UPSTREAM PROCESSINGAchieving Process Balance with Perfusion BioreactorsCynthia A. Challener

Advances in single-use technologies,

sensors, and cell retention systems

facilitate processes designed for the

long run. . . . . . . . . . . . . . . . . . . . . . . . .39

MANUFACTURINGOvercoming Challenges in ADCBioconjugation at Commercial ScaleCynthia A. Challener

Bioconjugation requires aseptic

manufacturing and containment for

cytotoxic payloads. . . . . . . . . . . . . . . . .42

OUTSOURCINGContract Organizations Expanded in AutumnSusan Haigney

CMOs and CDMOs made investments

in new and expanded facilities and

services in the last quarter of 2018. . . .44

COLUMNS AND DEPARTMENTS

FROM THE EDITOR

Innovation may capture headlines,

but quality programs are the

foundation to biopharma success.

Rita Peters. . . . . . . . . . . . . . . . . . . . . . . . .5

REGULATORY BEAT

Despite ongoing efforts to address

drug shortages, FDA reports a

rise in active shortages and in the

duration of supply problems.

Jill Wechsler . . . . . . . . . . . . . . . . . . . . . . .8

AD INDEX . . . . . . . . . . . . . . . . . . . . . . . .49

ASK THE EXPERT

A required time frame should

not be the driving force behind

root-cause investigations.

Susan Schniepp . . . . . . . . . . . . . . . . . . .50



December 2018 www.biopharminternational.com BioPharm International 5

From the Editor

Rita Peters is the

editorial director of

BioPharm International.

Innovation may

capture headlines,

but quality

programs are the

foundation to

biopharma success.

Bringing Quality into the Forefront

The end of the year is the traditional time to assess achievements and

shortfalls of the past 12 months. A preliminary review of the bio/pharma

industry accomplishments shows some impressive results. By the end of

November 2018, FDA had approved 53 novel drugs, setting a pace for the most

approvals on record.

The approvals included notable advances in science and targeted medicine. On

November 26, FDA approved Vitrakvi (iarotrectinib), the second cancer treatment

approved based on a biomarker for different types of tumors rather than tumors

originating in a specific organ or part of the body. The therapy is the first to

receive a tumor-agnostic indication at the time of initial FDA approval (1).

In announcing the approval, FDA Commissioner Scott Gottlieb noted that the

therapy marked “a new paradigm in the development of cancer drugs that are ‘tis-

sue agnostic.’” Gottlieb also credited increased knowledge of cancer mutations,

breakthrough therapy designation and accelerated approval processes, and a mod-

ern framework of clinical trial designs as contributing to more targeted and effec-

tive cancer treatments.

Uncovering quality issuesWhile the approvals marked progress in innovation, the global recall of the

API valsartan due to the discovery of impurities N-nitrosodimethylamine

(NDMA) and N-nitrosodiethylamine (NDEA) in drug products raised questions

about the quality of drug substances in the supply chain.

Quality problems were a mixed bag this year. The number of warning letters

issued by FDA were on a slightly slower pace compared with 2017; however, the

number of drug recalls were up.

While innovative achievements such as new drug approvals get the headlines

and are recognized and rewarded, quality programs are essential but rarely get

attention until something goes wrong. In this issue, and in supplemental online

coverage, the editors present multiple views of quality issues, throughout the drug

development and manufacturing continuum.

In this issueValidating supplier quality: consultants and experts in risk assessment share

best practices that will help ensure the quality of APIs, ingredients, and pro-

cess aids and materials.

Lot release testing: Recommended testing—bioburden testing, mycoplasma

testing, in-vitro viral screening, and virus-specific qPCR—for unprocessed bulk

material collected from a bioreactor is described.

Fluorescence standards: As fluorescence measurements of cell structures,

proteins, and DNA are becoming more and more quantitative, standards are

needed to ensure accuracy and reproducibility.

Online quality resourcesThe editors have added new features on mock inspections, risk assessment

and mitigation, standard operating procedures, technology transfer, regula-

tory authority actions, and other quality-related practices to the BioPharm

International website. Bookmark www.biopharminternational.com/quality to get

easy access to this information.

Reference1. FDA, “FDA Approves an Oncology Drug That Targets a Key Genetic Driver of Cancer,

Rather Than a Specific Type of Tumor,” Press Release, Nov. 26, 2018.X



http://www.biopharminternational.com/quality



Sartorius Stedim Biotech is a leading part-ner of the biopharma industry. Our solutions are supporting our customers to produce drugs safely, timely, and economically. The

key product catego-ries of our company are cel l cultivation, fermentation, f i ltra-tion, purification, and f lu id management. S a r t o r iu s S t e d i m Biotech has a world-wide presence, with manufacturing, sales, and R&D sites in more than 20 coun-tries in Europe, North

America, and Asia. More than 5100 employ-ees across the globe focus on single-use tech-nologies and added-value services to meet the rapidly changing technology requirements of the industry it serves.

At Sartorius Stedim Biotech, we empower scientists and engineers to simplify and accel-erate progress in life science and bioprocess-ing. As pioneers, we serve as a magnet and a dynamic platform attracting the leading experts in our sectors. We bring creative minds together to work towards shared goals: technological breakthroughs that lead to bet-ter health for more people.

Sartorius Stedim Biotech offers instru-ments, consumables, and services for biopharmaceutical, chemical, food, and aca-demic labs.

Confidently address increasing demands for enhanced quality, efficiency, accuracy, compliance, and production with instrumen-tation, intuitive software, hands-on training, and comprehensive services for the biophar-maceutical, chemical, food and beverage industries, and academic sector.

Experience quick start-up and streamline workf lows with our product and platform solutions ideally suited to meet your labora-

SARTORIUS STEDIM BIOTECH

5 Orville Drive Suite 200Bohemia, New York 11716USA

TELEPHONE

+1.800.368.7178

FAX

+1.631.254.4253

EMAIL

[email protected]

WEBSITE

www.sartorius.us

Sartorius Stedim Biotech

tory and bioprocess application needs. From consumables and laboratory water purifica-tion systems to industry-leading weighing technology, our sophisticated solutions also include liquid handling, fermentation, filtra-tion, and fluid management. Find out how to accelerate clinical pipelines, increase drug manufacturing efficiency, and reduce costs with our bioprocess solutions.

The biopharmaceutical industry can also reduce the time and cost required to bring products to the clinic and the market by taking advantage of single-use production platforms. These platforms integrate proven services and technologies for the develop-ment and manufacture of mAbs, vaccines, and bioconjugates.

Companies can streamline early stage process development activities by leverag-ing the expertise and experience Sartorius has acquired over many years while estab-lishing biomanufacturing processes for cli-ents. Furthermore, Sartorius can expedite the engineering of single-use and hybrid pro-cesses for scale-up during late-stage activities using process templates that reduce design costs and increase standardization.






http://www.sartorius.us

https://www.sartorius.com/en/company/investor-relations/sartorius-stedim-biotech-sa-investor-relations

The Foundations for

Single-Use Manufacturing.

Redefined from A–Z.

In the past, biopharma companies were struggling with various risk

factors which kept them from implementing single-use solutions.

With our solid single-use foundation for biomanufacturing processes

we are solving all of these challenges simultaneously. Our fully

integrated single-use platform connects an exclusive approach in

biocompatibility, state-of-the-art integrity control and testing as

well as a unique automation platform and supply network.

This strategy provides flexibility and acceleration which leads to

a cost-effective process that ensures the quality of your biologics

and enhances patient safety.

www.sartorius.com/single-use-redefined


http://www.sartorius.com/single-use-redefined

https://www.sartorius.com/en/company/investor-relations/sartorius-stedim-biotech-sa-investor-relations

8 BioPharm International www.biopharminternational.com December 2018

Regulatory Beat

Vis

ion

so

fAm

eri

ca

/Jo

e S

oh

m/G

ett

y I

ma

ge

s

Patients, providers, and policy mak-

ers are up in arms over persistent and

more prevalent shortages of important

medicines, primarily generic sterile injectables

needed to treat critical diseases and infections,

provide emergency care, and enable surgery

and multiple aspects of medical care. Despite

ongoing efforts to address the problem, FDA

sees a rise in active shortages and in the dura-

tion of supply problems, according to data pre-

sented at a public meeting on drug shortages

in late November 2018, sponsored by FDA and

the Duke Margolis Center for Health Policy (1).

Some shortages have lasted more than eight

years, and solutions remain elusive.

Stakeholders participating in the FDA pub-

lic meeting looked to address the systemic

root causes of shortages, with a focus on the

economic incentives likely to drive product

quality, supply chain resiliency, and appropri-

ate reimbursement for drugs. In some cases,

said FDA Commissioner Scott Gottlieb, “the

prices reimbursed on these drugs have been

driven down to such low levels, that it makes

it hard to manufacture them profitably and

have enough margin left over to

invest in modern manufacturing

upgrades” (2). Adam Kroetsch, deputy

director of the Office of Program and

Strategic Analysis in the Center for

Drug Evaluation and Research (CDER),

linked shortages to older generic

drugs where the market fails to

reward product quality and reliability.

Consolidation throughout the sup-

ply chain was cited as a key factor

in aggravating shortages. Mergers in

the generic-drug industry can limit

production of a drug to one firm and

minimize sources of key ingredients. A

handful of hospital buying groups and

large distributors, moreover, limit competitive

bidding opportunities and overall reimburse-

ment for common, but important, injectables.

SEEKING NEW SOLUTIONSA full range of issues and proposals will be

weighed by an FDA Drug Shortages Task Force,

formed in July 2018 in response to rising con-

cerns from members of Congress (3). The panel

includes representatives from the Centers for

Medicare and Medicaid Services, the Federal

Trade Commission, and other federal agencies,

as well as senior leaders from FDA. The group

has been meeting with manufacturers and other

stakeholders leading up to the public meeting

with the goal of developing a report to Congress

outlining a policy framework to resolve the

problems underlying persistent drug shortages.

Manufacturers at the meeting cited unclear

or changing FDA product development stan-

dards and noted that efforts to scale up produc-

tion to address a shortage can be stymied by

FDA’s complex post-approval changes require-

ments. Several speakers urged greater transpar-

ency in agency warning letters and inspection

reports to provide information earlier on where

quality problems at one manufacturer may lead

to limited production and create an opening

for added competition. To avoid delays in gain-

ing FDA approval of new or upgraded facilities

to produce alternative products, the agency

Quality Manufacturing Key to Reducing Drug ShortagesDespite ongoing efforts to address drug shortages, FDA reports a rise in active shortages and in the duration of supply problems.

Jill Wechsler

is BioPharm International’s

Washington editor,

[email protected].

FDA continues to urge

industry to adopt more

reliable, high-quality

manufacturing systems.





Regulatory Beat

noted its adoption of a more

efficient inspection program for

sterile drug facilities, with more

transparent quality standards that

will make the oversight process

more predictable.

There was disagreement among

participants on some issues, with

certain manufacturers seeking

incentives to produce drug com-

ponents in the United States to

shorten supply chains, while oth-

ers maintained that high-quality

products can be obtained reliably

overseas. Generic-drug makers

urged flexibility in meeting prod-

uct standards that FDA revises

after drug development and test-

ing is underway. David Gaugh

of the Association for Accessible

Medicines (AAM) proposed federal

grants or other assistance to help

manufacturers upgrade or build

new manufacturing facilities to

provide excess capacity for when

shortages occur and to establish

a national contingency plan to

stockpile critical medicines (4).

FDA officials continue to urge

industry to adopt more reliable,

high-quality manufacturing sys-

tems to avoid production break-

downs, but ga ins have been

elusive. A CDER quality metrics

initiative has been mired in dis-

pute for months over what and

how to measure quality opera-

tions and a firm’s “quality culture.”

CDER’s emerging technology pro-

gram offers advice and support

to companies looking to adopt

continuous manufacturing and

other advanced production tech-

nologies, but uptake has been

limited. Efforts to streamline the

post-approval changes process also

have fallen short in modifying

agency oversight of manufactur-

ing revisions. At the same time,

newer just-in-time manufacturing

systems that reduce vendor inven-

tories may increase vulnerability

to unexpected shortages. All these

issues are before the FDA taskforce

and will be considered in its report

on the multiple challenges for all

supply chain parties in preventing

drug shortages to ensure public

health.

REFERENCES 1. Duke, Margolis Center for Health

Policy, Identifying the Root Causes of

Drug Shortages and Finding Enduring

Solutions, Public Meeting, Nov. 27,

2018, Washington, DC, https://

healthpolicy.duke.edu/sites/default/

files/atoms/files/duke-fda_drug_

shortages_agenda.pdf.

2. FDA, Remarks by Scott Gottlieb, MD,

at the Public Meeting on Identifying the

Root Causes of Drug Shortages and

Finding Enduring Solutions public

meeting, Nov. 27, 2018, www.fda.gov/

NewsEvents/Speeches/ucm626706.

htm.

3. FDA, “Statement by FDA Commissioner

Scott Gottlieb, MD, on Formation of a

New Drug Shortages Task Force and

FDA’s Efforts to Advance Long-Term

Solutions to Prevent Shortages,” Press

Release, July 12, 2018, www.fda.gov/

NewsEvents/Newsroom/Press

Announcements/ucm613346.htm.

4. David Gaugh, “Preliminary

Recommendations for Addressing

Drug Shortages,” AAM Blog post,

https://accessiblemeds.org/

resources/blog/preliminary-

recommendations-addressing-drug-

shortages. X

The biggest hurdle for advancing gene therapy, for both

treating rare diseases as well as addressing more common

conditions, is difficulties in achieving efficient scale-up of

production processes, says Peter Marks, director of FDA’s

Center for Biologics Evaluation and Research (CBER). Marks

noted that there are now more than 700 investigational

new drug applications (INDs) filed with the agency, and

more arriving daily. But to move forward with development,

he observed at Prevision Policy’s Biopharma Congress in

Washington in November 2018, requires a “quantum leap” in

manufacturing capabilities.

Marks added that FDA often is “very lenient” about

companies meeting manufacturing standards for testing

gene therapies in Phase I trials. But scaling up to larger

production for further studies is difficult and can delay

product development for years.

Clinical trials for gene therapies are not a problem,

Marks noted. A manufacturer can gain proof of concept

in a study of only 10 patients. To move forward, however,

requires manufacturing at scale, and few companies can

produce the necessary vectors, and they need to do so

more efficiently.

While there remain many uncertainties about developing

and testing gene therapies, particularly related to concerns

about immune reactions to vectors, Marks seeks to avoid

“regulatory paralysis” arising from safety concerns.

“Things will happen,” he conceded, and sponsors and

regulators need to understand the risks and work to

develop therapies that patients and providers can trust.

The vision, Marks observed, is that gene therapies

eventually will provide important treatments for a broader

range of diseases, but only if they can be proven to be

safe and can be produced efficiently, which also is key to

reducing product costs.

—Jill Wechsler

Manufacturing Challenges Limit Gene Therapy DevelopmentFor personal, non-commercial use


https://healthpolicy.duke.edu/sites/default/files/atoms/files/duke-fda_drug_shortages_agenda.pdf

https://www.fda.gov/NewsEvents/Speeches/ucm626706.htm

https://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm613346.htm

https://accessiblemeds.org/resources/blog/preliminaryrecommendations-addressing-drugshortages



The Parenteral Drug Association (PDA) is the leading global provider of science, tech-nology, regulatory information, and education

for the pharmaceutical and biopharmaceutica l com-munity. For more than 70 years, since its founding as a non-profit in 1946, PDA has been committed to developing scientif ically sound, practical technical information and resources to advance science and regu-lation through the expertise

of our more than 10,500 members worldwide.PDA is a truly global organization, with

membership nearly equally divided between those working in the US and in other regions across the globe. Represented in this diverse membership is a growing number of young professionals and students.

Recognized for our expertise and authority in the field of parenteral science and technol-ogy, PDA is leading the way in promoting the exchange of information on rapidly evolving technology and regulations to ensure high-quality pharmaceutical production.

PDA supports its mission to advance phar-maceutical and biopharmaceutical science and regulation so members can better serve patients by:• Providing global forums for the scientific

community, regulators, and industry pro-fessionals on emerging trends within the industry

• Delivering unique, hands-on education and training through PDA’s manufacturing training facility

• Fostering career-long learning and profes-sional development

• Encouraging scientific information shar-ing among industry peers

The Parenteral Drug Association

• Serving as a leading contributor of infor-mation and expertise to influence global industry and regulatory solutions.

PDA draws its strength from its members, which includes a corps of 2,500 active volun-teers. Our conferences, meetings, and courses bring together pharmaceutical manufacturers, suppliers, end users, academics, and regula-tory officials for an unprecedented level of exchange on timely issues of mutual interest and concern.

Through the development of Technical Reports on pressing industry topics and responses to regulatory initiatives, PDA and its members influence the future course of phar-maceutical products technology.

A natural progression was for PDA to expand this expertise into the world of creating standards that provide guidance to industry on best practices for drug manufacturing for patient use. In 2017, PDA was accepted as an ANSI-accredited standards developer, and work is currently underway on PDA’s first standards.

Our internationally recognized publications, PDA Journal of Pharmaceutical Science and Technology and PDA Letter, keep pharmaceutical manufacturing professionals up to date on the latest science and current industry and regula-tory news.

Together, these activities promote the advancement of pharmaceutical science in the interest of the ultimate end-user—the patient.

PDA—Connecting People, Science, and Regulation®

PARENTERAL DRUG

ASSOCIATION (PDA)

4350 East-West HighwaySuite 600Bethesda, MD 20814USA

TELEPHONE

+1.301.656.5900

FAX

+1.301.986.0296

EMAIL

[email protected]

WEBSITE

www.pda.org

NUMBER OF EMPLOYEES

45

YEAR FOUNDED

1946




http://www.pda.org

https://www.pda.org/

2019 PDAAnnualMeetingSolving Manufacturing and Supply Challenges

for Current and Future Medicinal Products

Join industry and regulatory experts in

San Diego at the 2019 PDA Annual Meeting.

This solutions-oriented Conference brings together industry and regulatory experts to shed

light on a wide range of topics that matter most to you and your company in the face of a

rapidly changing pharmaceutical manufacturing landscape.

You don’t want to miss these engaging sessions addressing today’s most pressing

challenges, including:

• Navigating the Global Regulatory Landscape

• Accelerating Pharmaceutical Innovation

• Bridging Current Technology with the Future of Medicine

• Disaster Recovery

$G�QPG�QH�VJG�ƒ�TUV�VQ�TGIKUVGT�HQT�VJKU�HNCIUJKR�2&#�OGGVKPIЯ�0QY�KU�VJG�VKOG�VQ�GZRNQTG�

unique challenges, analyze current capabilities, and envision future possibilities of

DKQ�RJCTOCEGWVKECN�OCPWHCEVWTKPIЯ

Learn more and register today at pda.org/2019Annual

MARCH 11-13, 2019 | SAN DIEGO, CA

EXHIBITION: MARCH 11-13

PRACTICAL APPLICATIONS OF STERILE MANUFACTURING WORKSHOP: MARCH 13-14

TRAINING COURSES: MARCH 15

#PDAAnnual

Save up to$600 when you

register byFebruary 1, 2019

pda.org/2019Annual




Quality: Supply Chain Management


AUREMAR/STOCK.ADOBE.COM

Going Beyond the Surface to Ensure Supplier Quality

Success depends on supplier communication and transparency, but it’s up to buyers to demand the right information

and to look at the vendor’s overall business.

AGNES SHANLEY

As niche, global markets grow and increase the com-

plexity of pharmaceutical manufacturing, vendor man-

agement has become more challenging; the API and

excipient supplier base has moved offshore, and more core

operations are being outsourced to contract partners. Today,

a typical pharmaceutical manufacturer works with 100–200

contract manufacturing organizations (CMOs) (1). A 2013

study found that supply and supplier issues account for 40%

of the pharmaceutical industry’s top supply-chain risks (2).

Adding to the difficulty have been corporate mergers and

acquisitions, both on the manufacturer and on the supplier

sides. Mergers shift the focus away from manufacturing, as

Steve Cottrell, president of Maetrics, wrote in April 2018 in

the PharmaPhorum blog (3). This, in turn, limits “the ease

with which supply chain gap analyses, supplier assessments,

and quality assurance checks (e.g., non-conformance or out-

of-stock issues) can be carried out,” he wrote.

The results have been clearly seen in an overall increase

in drug shortages, recalls, and regulatory citations for insuf-

ficient quality management and vendor oversight. Overall,

supply reliability issues cost biopharmaceutical companies

$2 billion in revenue each year, according to the Boston

Consulting Group (4). Pharmaceutical manufacturers still

have limited visibility into their supply chains, and fairly

loose, ad hoc connections with many of their vendors, in

sharp contrast to the close supplier-manufacturer partner-

ships and data exchange programs that exist in the automo-

tive, aerospace, and electronics industries.

INDUSTRY EFFORTSManufacturers have been working individually and in con-

cert to address these issues, through initiatives such as the

Pharmaceutical Supply Chain Initiative (PSCI), a group of 33

manufacturers that has developed best practices, self-assess-

ment guidelines, and an audit protocol based on the principles

of sustainable sourcing and traceability, transparency, business

resilience, and management capability and systems.

Contin. on page 16



ADVERTORIAL December 2018 BioPharm International 13


THERMO FISHER SCIENTIFIC

4815 Emperor Boulevard Suite 300Durham, NC 27703

USA

+1.919.226.3200

GLOBAL

+1.866.728.4366

WEBSITE

thermofisher.com/patheon

Human studies in as little as 12 months. You can meet important milestones, such as filing the IND, or secure additional funding with confidence.

• Our deep process development skills sig-nificantly increase the batch yield and reduce processing time for your molecule. Applying Design of Experiment methodol-ogy to both Upstream and Downstream Processing, we define the CPPs and CQPs that enable robust processes, maximize yields, and optimize throughput.

• We have a fully integrated global network of cGMP facilities across Europe, North America, and Australia, and are a leader in manufacturing monoclonal antibodies and recombinant proteins using single-use technologies.

• We offer a range of purification processes that ensure your drug substance is of the highest quality and yield.

• Our analytical capabilities include rapid identification and characterization of your recombinant protein or antibody, devel-opment and implementation of cGMP methodologies, and data generation for regulatory submissions.

• In late clinical phases, and as part of the establishment of your commercial supply, we provide a complete validation package according to regulatory and cGMP guide-lines.

• We are driven by your deadlines, flexible in our approach, and determined to get it Right-The-First-Time.

Company DescriptionThermo Fisher Scientific is the world leader in serving science, with revenues of more

than $20 bi l l ion and approx imately 70,000 e mp l oy e e s g l o b a l l y. We help our customers accelerate l ife-sciences research, solve complex ana ly t ica l cha l lenges, improve patient diagnos-tics, deliver medicines to market and increase labo-

ratory productivity.Thermo Fisher Scientific’s Patheon and

Fisher Clinical Services brands provide indus-try-leading solutions for drug development, clinical trial logistics, and commercial manu-facturing to biopharma clients of all sizes. With more than 40 locations globally, the company can provide integrated, end-to-end capabilities across all phases of development, including API, biologics, formulation, clinical trials solu-tions, and commercial manufacturing.

Markets ServedThermo Fisher Scientific has biologics man-ufacturing locations in the US, EU, and Australia. We bring deep scientific exper-tise to every challenge and our proven track record of scaling up biologics helps ensure you gain cost and time savings at every stage of the biologic development process.

We understand the uncertainty associated with forecasting demand. We think strategi-cally to offer biomanufacturing options that meet the unique needs of your molecule. We can work with your cell lines. Our contracts are individualized to meet your requirements. Our strong reputation is built on successfully transferring complex molecules.

Services and Capabilities• Quick to Clinic™ program can deliver your

large-molecule drug substance for First-in-

Thermo Fisher Scientifi c



https://www.thermofisher.com/in/en/home.html

https://www.patheon.com/en-us?cid=fl-bid-patheon

API BIOLOGICS EARLY DEVELOPMENT

ĝ��7KHUPR�)LVKHU�6FLHQWLð�F�,QF��$OO�ULJKWV�UHVHUYHG�


TRIALS & TRIUMPHS

MADE

FOR

Find out more at�[OLYTVÄ�ZOLY�JVT�WH[OLon

HOW A CONTRACT MANUFACTURER NEVER LOSES

:0./;�6-�>/(;�0;»:�9,(33@�4(205.�

-VY�TVZ[�JVTWHUPLZ�� [PTL�HUK�TVUL`�HYL�HS^H`Z�VU� [OL� SPUL��([�;OLYTV�-PZOLY�:JPLU[PÄ�J�� [OL�

Z[HRLZ�HYL�T\JO�OPNOLY��@V\�ZLL��^L�RUV^�[OH[�V\Y�IPVWOHYTH�JSPLU[Z»�IYPSSPHU[�KPZJV]LYPLZ�ZH]L�

lives. Millions of them. But only if we can help them meet clinical development milestones to get

[OLPY�KY\NZ�[V�WH[PLU[Z��;OH[»Z�^O`�^L�JVTIPUL�ZVWOPZ[PJH[LK�ZJPLUJL�HUK�LUNPULLYPUN�Z[YH[LNPLZ�

with industry-leading experience and, most importantly, our passion for making a difference. We

call it the power of human ingenuity. The results? More than 1,000 new molecules developed for

clinical trial, and 117 new drug approvals launched in the last decade – more than the next six

manufacturers combined. But with millions more patients awaiting new treatments every day, we

know our work is just getting started.

CLINICAL TRIAL SOLUTIONS COMMERCIAL MANUFACTURING


https://www.thermofisher.com/in/en/home.html

https://www.patheon.com/en-us/patheon-now-part-of-thermo-fisher-scientific



The organization, which started up

in 2005 with five members, has trained

190 auditors and 150 staffers at phar-

maceutical industry suppliers in best

practices and principles and is pro-

moting the concept of shared supplier

audits to reduce costs for manufactur-

ers and their suppliers. The number of

shared PSCI audits more than doubled

from 61 in 2016 to 152 in 2017, accord-

ing to Enric Bosch Radó, a manager in

Boehringer-Ingelheim’s environmen-

tal health and safety department, who

presented a progress report at Salon

International de la Logistique (SIL),

the international logistics meeting in

Barcelona on June 5, 2018 (5).

REAL-TIME DATA EXCHANGE BioPhorum, a collaborative industry

effort, is working on a blueprint for

21st century supply chain management

as part of its Technology Roadmap

program. Newer technologies, such

as single-use systems for cell culture,

Protein A, and chromatographic res-

ins, require a significant investment

from biopharmaceutical manufacturers,

while the cost of poor quality (evident

in raw material variability and lack of

understanding and control of the sup-

ply chain) is high and must be driven

out, according to the group’s latest

report (6). Close collaboration between

manufacturers and vendors will be

increasingly important for ensuring

supplies of single-use technologies,

and as more companies evaluate con-

tinuous bioprocessing, said Jonathan

Haigh, head of downstream processing

at FujiFilm Diosynth, a company that

is both a manufacturer and a contract

manufacturer, in a video (7) discussing

roadmapping efforts.

BioPhorum has called for changing

the way manufacturers and vendors

interact, and to promote an atmo-

sphere of trust and harmonized meth-

ods using electronic data exchange.

The group is also working on improv-

ing tools, such as forecasting and plan-

ning software.

SHARING BEST PRACTICESOne method that suppliers and their

customers are using to attempt to bal-

ance rapid growth in demand and

the need for careful planning is the

sales and operational planning pro-

cess, which examines inventory replen-

ishment and distribution needs, and

assesses manufacturing, including man-

ufacturing and quality equipment and

warehousing and how well it can sup-

port customer requirements, said Aida

Tsouroukdissian, head of demand plan-

ning at MilliporeSigma in a 2018 video

(8). Other important methods include

supply chain business continuity plan-

ning and supply chain mapping as well

as change management.

There is a need to go beyond the

superficial level, noted Roger Estrella,

senior risk manager for supplier busi-

ness continuity at Genentech, in an

Rx-360 workshop addressing challenges

in the pharmaceutical raw material sup-

plier chain (9). Estrella’s business conti-

nuity group works closely with Roche’s

corporate quality risk organization,

which handles audits, recalls and cus-

tomer complaints, and process capabil-

ity to be able to respond to and find the

root cause of quality issues.

Roche analyzes suppliers based on

their potential impact on the business

and the patient. The first question is

what would happen to patients if there

were a problem with the supply of this

particular product? What impact would

a supply interruption have on revenues

and on patients? Materials are classified

based on level of risk (e.g., oncology

products would be placed in a higher

category of risk than treatments for

rheumatology), he said.

Manufacturers must identify hazard-

ous conditions, assess risks and develop

contingency plans, get them approved

and then implement them.

Challenges to asserting full control

over supply chains include cognitive

bias, supply chain complexity, and

business change management, Estrella

said. Too often, employees may tend

to discount risks, especially those that

may occur in the future, he said, adding

that strengthening supplier risk assess-

ment requires a top-down approach

and senior-level management sup-

port if it is to succeed. He said that,

wherever possible, Roche avoids being

dependent on overseas suppliers.

GOING BEYOND THE SURFACEIt is no longer enough for manufactur-

ers to have transparency from most

important suppliers; they now need

insights into how these suppliers man-

age their supply chains, Estrella said.

Roche asks that suppliers provide

some idea of their business continuity

management by conducting manu-

facturing risk assessments at each rel-

evant manufacturing site; developing

mitigation plans for each risk; deter-

mining worst-case scenarios for the

most likely site risks; and estimating

the time that it would take for them to

return to normal after a supply upset.

If suppliers aren’t already doing this,

the company helps them with the pro-

cess and uses results to develop its risk

mitigation inventory levels for the par-

ticular material, he said.

Manufacturers must also look at

each supplier ’s business portfolio

and ask how each particular prod-

uct fits into that vendor’s big picture.

“Consolidations have complicated the

supplier-manufacturer picture. Every

time that a merger and acquisition

takes place, a rising star product can

become a dog, and the new owners

may decide not to invest in quality

and delivery performance initiatives,”

he said. In some cases, the new busi-

ness owners may even stop making a

product that has always been impor-

tant to the manufacturer’s business,

and biopharmaceutical manufactur-

ers must be prepared and develop

Supply Chain Management — Contin. from page 12




www.biopharminternational.com December 2018 BioPharm International 17

alternatives.“The supplier with whom

you have the greatest level of spend is

not necessarily the vendor that is most

impactful to your business,” Estrella

said. He also noted that a supplier may

be critical to an individual biopharma-

ceutical company’s business, but biop-

harmaceutical manufacturing may not

be a major market focus for them, so

manufacturers should be prepared.

In addition, he said, manufacturers

must work to minimize the number

of intermediates in the supply chain.

“The more handoffs you have, the more

potential points of failure you have,”

he said. So if there are handoffs it is

essential for manufacturers to know

who is involved, and determine how

they will handle any situations that

may come up in the future.

Stressing the importance of supply

chain mapping and risk management,

he noted that it is straightforward to

identify risk, but challenging to mit-

igate it. Biopharmaceutical manufac-

turers must be prepared to go beyond

the superficial level to understand and

communicate more closely with sup-

pliers to prevent quality and supply

problems from affecting patients and

their bottom line.

REFERENCES1. A. Alvarado-Seig, et al., “Threats to

Pharmaceutical Supply Chains, The Public-Private Analytic Exchange Program Research Findings,“July 2018, www.dhs.gov/sites/default/files/publications/2018_AEP_Threats_to_Pharmaceutical_Supply_Chains.pdf

2. M. Jaberidoost et al., DARU Journal of Pharmaceutical Sciences 21, 69 (2013).

3. S. Cottrell, “Gaining a Clear View of Supply Chain Visibility,” pharmaphorum.com, April 20, 2018, www.pharmaphorum.com/views-analysis-market-access/gaining-clear-view-supply-chain-visibility/.

4. A. Merchant et al., “How to Break the Vicious Cycle in Biopharma Supply,” bcg.com, March 16, 2017, www.bcg.com/en-us/publications/2017/healthcare-operations-how-to-break-the-vicious-cycle-in-biopharma-supply.aspx.

5. E.B. Radó, “Creating a Better Supply Chain in the Pharmaceutical Industry: The Pharmaceutical Supply Chain Initiative,” a presentation made at Salon International de la Logistique (SIL) (Barcelona, Spain, 2018).

6. BioPhorum, “Biopharmaceutical Manufacturing Technology Roadmap: Supply Partnership Management,” biophorum.org, December 2017.

7. BioPhorum, “The Importance of Supply Partners in the Technology Roadmap,” biophorum.com, www.biophorum.com/importance-of-supply-partners-in-the-technology-roadmap/.

8. A. Tsouroukdissian, “Supply Chain Forecasts and Capacity,” emdmillipore.com, Nov. 30, 2017, www.emdmillipore.com/US/en/20171130_131854.

9. R. Estrella, “Challenges to Raw Material and Supplier Risk Management,” Rx-360 Workshop, “Addressing Challenges in the Pharmaceutical Raw Material Supply Chain Through Industry Collaboration,” rx360.org, March 13, 2014, www.youtube.com/watch?v=gEcSpkfViWU. ◆

www.tosohbioscience.com

Tosoh Bioscience, TOYOPEARL and ToyoScreen are registered trademarks of Tosoh Corporation.

RoboColumn is a registered trademark of Repligen GmbH LLC

TOSOH BIOSCIENCE LLC • Customer service: 866-527-3587 • Technical service: 800-366-4875, option #3

Tosoh Bioscience:The Market Leader of HIC Resins

Ether

Phenyl

Butyl

O

OH

O n

PPG

Most hydrophobic

Least

hydrophobic (OCH2CH2)nOH

OCH2CH2CH2CH3

Hexyl

OCH2CH2CH2CH2CH2CH3

0

5

10

15

20

25

15 25

2

2

4

4

6

6

35 45 55

De

tec

tor

resp

on

se (

mA

U)

B-c

on

ce

ntra

tion

(%)

Residence time (minutes)

UV @ 280 nm

UV @ 495 nm

B%

60

65

70

75

80

85

90

95

100

Best-in-Class HIC Resins for ADC Purifi cation

Resins are available in these formats:RoboColumns®, MiniChrom columns, 96-well plates, ToyoScreen® columns, and in bulk volumes

The wide range of TOYOPEARL® HIC ligands enables protein and mAb separations

DARs were separated by TOYOPEARL PPG-600Mresin using a 3-step gradient






http://www.tosohbioscience.com


https://www.tosoh.com/

http://www.dhs.gov/sites/default/files/publications/2018_AEP_Threats_to_Pharmaceutical_Supply_Chains.pdf

https://pharmaphorum.com/viewsanalysis-market-access/gainingclear-view-supply-chain-visibility/

https://www.bcg.com/en-us/Error/PageNotFound/?aspxerrorPath=/en-us/publications/2017/healthcareoperations-how-to-break-the-viciouscycle-in-biopharma-supply.aspx&redir=true

http://www.biophorum.com/importance-of-supply-partnersin-the-technology-roadmap/

http://www.merckmillipore.com/IN/en/20171130_131854?bd=1

https://www.youtube.com/watch?v=gEcSpkfViWU


Quality: Lot Release Testing

SC

IEN

CE

PH

OT

O/S

TO

CK

.AD

OB

E.C

OM

Unprocessed Bulk Testing for Biopharmaceuticals

Unprocessed bulk material harvested directly from the bioreactor should be tested for contamination prior to downstream processing.

KATHERINE MAROTTE

Biopharmaceutical products are manufactured in living

systems, such as animal or human cells or tissues, which

pose a contamination risk from bacteria, yeast, mold,

viruses, or mycoplasma. Contamination can come from a vari-

ety of sources, such as raw materials—including cell culture

media and additives—the cells themselves, lab personnel, the

manufacturing process, or ineffective cleaning of the manu-

facturing facility. Contamination can be detrimental for a

product or a facility, potentially leading to drug recall, harm to

patients, or the complete shutdown of a facility. Products must

therefore be tested for the presence of contaminants through-

out the production process to ensure product safety and purity.

For each product, the master, working, and end of production

cell banks are evaluated for contaminants. In contrast to this

testing, which is performed once on each cell bank, every lot

of unprocessed bulk (UPB) material—the product that is har-

vested directly from the bioreactor—must be tested.

There is a high probability that, if present, adventitious

agents would be detected in the UPB material harvested at

this point in the manufacturing process because the bioreactor

provides highly enriched growth conditions, and the cells

could have amplified any adventitious agents that were present

at the beginning of the manufacturing process. In addition,

the material has not yet gone through further processing/

purification, which could lower levels of contaminating agents,

rendering them harder to detect in analytical testing.

ASSAYS USED FOR TESTING UPB MATERIALThe assays used for testing UPB material are often referred

to as lot release testing, because the manufacturer doesn’t

proceed with downstream processing of the UPB until

passing results are obtained in the lot release testing. If a

contaminant is present, then the contamination is not spread

to the downstream processing equipment. This testing typi-

cally includes bioburden or sterility testing, mycoplasma

testing and in-vitro viral screening as well as a few optional

assays, such as virus-specific quantitative polymerase chain

KATHERINE MAROTTE is principal scientist, viral safety testing

services department, at Eurofins Lancaster Laboratories. She has 14

years of virology experience and specializes in assay development

and GMP viral testing.





reaction (qPCR) or transmission elec-

tron microscopy (TEM) testing. The

unprocessed bulk product then goes

through multiple purification steps

resulting in the final drug substance.

Lot release testing also encompasses

the testing of animal-derived products.

Bioburden testing is recommended

over sterility testing, because manufac-

turing is not always a sterile process.

Some clients choose, however, to test lot

release products for sterility. Bioburden

testing involves the enumeration of the

microbial content of a product. The

methodologies involve the plating of

the product using one or more solid

nutrient media types, incubating for

three to five days, and visually observ-

ing and counting the microbial growth

at the conclusion of the incubation

period. Depending on the material or

process, additional testing may be per-

formed to enumerate potential anaero-

bic bacteria or detect the presence of

specified microorganisms of concern.

Specifications for the acceptable levels

of bioburden are determined based on

the risk to the manufacturing process,

considering the abundance and type

of organisms that may be detected, the

potential impact of metabolic by-prod-

ucts or toxins, and the subsequent puri-

fication steps that may follow. Method

suitability is performed for individual

products or a representative of com-

mon in-process samples in order to

demonstrate that the material does not

interfere with the recovery of potential

microbial contaminants. The method

parameters are challenged by introduc-

ing low levels of microorganisms to the

product under the conditions of the test

and enumerating the recovery.

Mycoplasma testing is a general

screening assay performed to test for

the presence of mycoplasma and other

mollicutes, including acholeplasma and

spiroplasma. The assay is comprised

of two qualitative methods for the

presence or absence of mycoplasma.

Each sample is tested by both a direct

culture method and an indicator

cell-culture method that are each

required by the compendia. The direct

culture method uses specialized broth

and agar to promote the growth of

a wide range of metabolic growth

rates of mycoplasma, alcholeplasma,

and spiroplasma. The indicator cell

culture method uses an indicator cell

line to promote the growth of alpha

cultivari species that will only grow in

the presence of cells. Mycoplasmastasis

must be performed to determine

whether the sample matrix will have

any mycoplasmastatic effect.

In-vitro adventitious agent (IVAA)

testing is a general screening assay

performed to test for the presence of

adventitious viruses (Figure 1). This

assay cannot determine the type or

amount of virus present, but rather

detects the presence or absence of a

virus in a sample. The assay has the

ability to test for a wide range of

possible contaminants, which include

cytopathic, hemagglutinating, or

hemadsorbing viruses. Each sample

is tested in a minimum of three cell

lines that are chosen based upon

the cell-line source used to prepare

the biopharmaceutical product and

theoretical susceptibility to particular

viruses of concern.

Cell lines required include a human

diploid cell line, a monkey kidney cell

line, and monolayer cultures of the

same species and tissue type as that

used for production. Prior to testing

in the IVAA, clinical-phase samples

should be tested in an interference

assay to ensure that the sample matrix

will not cause toxicity to the indicator

cells or cause any interference with

detection of viruses that could be pres-

ent in the sample. The interference

assay includes spiking the sample

with one concentration of virus that

is comparable to the positive con-

trol. Commercial products require a

full matrix qualification study. These

studies differ from interference test-

ing because they require multiple virus

spike levels in order to understand the

sensitivity of the virus detection in

the sample matrix. Matrix qualifica-

tion testing must also be performed on

three lots of product.

Figure 1. An analyst (Eurofins Lancaster Laboratories) observes cells for cytopathology caused by the presence of adventitious agents.

Fig

ure

co

urt

esy

of

the a

uth

or.

Contin. on page 22





Ajinomoto Bio-Pharma Services is a fully integrated contract development and manu-facturing organization, with sites in Wetteren and Balen, Belgium and San Diego, California,

USA, providing comprehen-sive process development services, cGMP manufactur-ing and drug product fi ll/fi nish services of small mol-ecule and biologic APIs and intermediates. Ajinomoto Bio-Pharma Services off ers a broad range of innovative platforms and capabilities to rapidly scale from clinical

and pilot programs to commercial quantities, including: Corynex technologies, oglionucleo-tide synthesis, antibody drug conjugations (ADC), high potency APIs (HPAPI), continu-ous fl ow manufacturing, and more. Ajinomoto Bio-Pharma Services is dedicated to providing a high level of quality and service to meet our client’s needs.

Large Molecule ManufacturingOne of the industry’s top leaders in microbial biologic manufacturing, using patented tech-nologies and providing impeccable quality.

• cGMP Protein and Plasmid Production• Microbial Cell Banking & Characterization• Fermentation & Product Recovery• Purification & Formulation• Corynex® Protein Expression System

Highly Potent & ADC ServicesAjinomoto Bio-Pharma Services off ers dedi-cated production and laboratory suites for high containment ADC bioconjugates and highly potent compounds, as well as formulation and fi ll/fi nish services:

• Conjugation & Purification• Fill/Finish• Lyophilization• Dedicated labs and manufacturing

AJINOMOTO BIO-PHARMA

SERVICES

11040 Roselle StreetSan Diego, CA92121USA

EMAIL

[email protected]

WEBSITE

www.AjiBio-Pharma.comwww.facebook.com/AjiBioPharmawww.linkedin.com/company/ajibio-pharma-services/https://twitter.com/AjiBioPharma

TELEPHONE

+858.882.0123

FAX

858.882.0133

Ajinomoto Bio-Pharma Services

Oligo & Peptide SynthesisAn industry leader in the synthesis of high quality, scalable, custom oligonucleotides, and peptides

• AjiPhase® Synthesis Technology• Fast Solid Phase Synthesis

Drug Product ManufacturingA leader in aseptic fi lling, off ering a wide range of capabilities for vials and syringes

• Aseptic Formulation• Aseptic Fill/Finish• Packaging, Serialization, & Aggregation

Services

Development ServicesOur highly knowledgeable Process Development team will establish and characterize a robust manufacturing process to ensure consistent cGMP manufacturing performance:

• Process Development • Optimization & Scale Up

Product Quality & Analytical Services Our well-designed product quality and analyti-cal programs work to ensure the success of your small and large molecule products through their life cycle:

• On-Site Laboratories• Phase Appropriate Analytical Method

Development & Lot Release Testing• ICH–Compliant Stability Programs




http://www.AjiBio-Pharma.com

http://www.facebook.com/

http://www.linkedin.com/

https://twitter.com/

https://ajibio-pharma.com/

A HEALTHY WORLD

WHAT DO YOU

WANT TO MAKE?

CDMO SERVICES:

WITH AJINOMOTO BIO-PHARMA SERVICES,

YOU HAVE THE POWER TO MAKE.

You have the power to make a difference by delivering

therapeutics that improve quality of life and inspire a healthier

world. You need a manufacturing partner who has the power

to make your every challenge their own and who shares your

unwavering tenacity and dedication from clinical studies through

commercial success. Together, we have The Power To Make.

Small & Large Molecules

High Potency & ADC Services

Process Development

Drug Product Manufacturing

Oligos & Peptides

www.AjiBio-Pharma.com


http://www.AjiBio-Pharma.com

https://ajibio-pharma.com/



OPTIONAL ASSAYSThere are a few optional assays that

may be performed on bulk harvest

material, depending on the type of

product. These assays include virus-

specific qPCR testing, TEM, and in-

vivo testing. Not all viruses will be

detected in the IVAA test, so there is

a consideration for performing virus-

specific qPCR assays, particularly if

a risk assessment of the process has

identified particular agents of concern.

The downside to these assays is that

the specific gene sequences can be

detected, but the assay cannot deter-

mine whether or not a virus is infec-

tious. Quantitative PCR testing is

performed on each lot of UPB material.

In-vivo adventitious agent testing

can detect viruses that are not able to

grow in cell cultures or do not cause

noticeable effects in the IVAA indi-

cator cells that were used for testing.

The animal systems used for in-vivo

testing include suckling mice, adult

mice, embryonated chicken eggs, and

guinea pigs. In-vivo testing is only

performed on unprocessed bulk prod-

ucts if this testing was not performed

on the working or end-of-production

cell banks. This testing is performed

on only one lot of material. TEM

testing may also be performed on

UPB samples and includes a quan-

titation of the number of retrovirus-

like particles present. TEM testing

is usually performed on one or more

lots of early phase products and on

at least three lots of material for later

phase products.

TESTING ON DIFFERENT BIOTHERAPEUTIC TYPESAdditional time and work may need

to be allocated for different types of

products before testing can begin. For

instance, some gene therapy prod-

ucts that contain live virus cannot be

tested for in certain cell-based assays

because the product would infect and

subsequently kill the indicator cells. In

cases like this, the virus in the prod-

uct may need to first be neutralized

with an antibody before testing for the

presence of adventitious agents. The

antibody itself would also need to be

evaluated to make sure it is not toxic

to the indicator cells in the assays or

that it doesn’t interfere with the abil-

ity to detect virus. When testing viral

vectors for gene therapy products, vec-

tors may also exhibit some degree of

toxicity to the indicator cells. This is

typically resolved by dilution of the

product in the assay. Viral vectors

also have the potential to generate

a false positive result in a cell-based

assay. The vector may be able to infect

certain cell types, giving the appear-

ance of the presence of an adventitious

agent; however, the infection may not

be productive because the vector is

replication defective.

Viral vaccines also present chal-

lenges when testing for contaminants

using cell-based assays. The vaccine

virus may be able to infect the cell

lines that are used for testing, and neu-

tralization with antibody is often not

effective. Therefore, uninfected control

cells are typically cultured in parallel

with infected cells. The in-vitro testing

is then performed on the control cells.

Expediting results for UPB lot

release testing is important. A lot

of material cannot be released for

downstream purification until it tests

negative for adventitious agents. If a

contaminant is identified during puri-

fication, the entire manufacturing

plant must be decontaminated. This

comes with a great expense and delay

for the client. If testing is expedited,

the lot of material can go into down-

stream purification faster with less risk

of facility contamination. The manu-

facturing facility would also know as

soon as possible if there is contamina-

tion at their site. Additionally, time

waiting for test results equates to time

and money lost by the client. To keep

the client informed during testing, the

testing lab can provide interim assay

results and testing status updates

throughout the entire testing process.

Clients should be notified immedi-

ately of any concerning results or assay

issues so that a plan of action can be

implemented as soon as possible. Close

attention should also be paid to turn-

around-time monitoring so that time-

lines can be met.

There are a number of steps that

can be implemented to expedite and

streamline the testing of lot release

products. First, as much information

as possible must be obtained from the

client before the sample even arrives

at the testing facility. Information

such as the sample volume require-

ments, turnaround times, and expecta-

tions should be discussed early in the

process. The testing facility will also

need to know the stage of the prod-

uct, whether interference and stasis

testing can be performed concurrently,

or whether antibiotics are present in

the sample upfront to minimize test-

ing delays. Defined shipment sched-

ules, special sample labels, customized

sample submission forms, and specific

entry processes allow for expediting

availability of the sample to the testing

laboratory.

CONCLUSIONEach lot of UPB material produced at

a manufacturing facility must undergo

testing for contaminating agents.

Because each batch of product cannot

be released for purification before this

testing is completed, it is beneficial

for the client that the testing labora-

tory completes the lot release testing as

soon as possible.

To expedite the testing as much as

possible, the laboratory should have a

streamlined, well-defined process from

before the sample arrives until the data

are reported. If a contaminating agent

is detected, proactive communication is

vital to establishing a path forward. ◆

Lot Release Testing — Contin. from page 19



Sponsored by Presented by

EVENT OVERVIEW:

Decisions regarding clone selection and cell culture optimization

are often based on titer alone. N-glycan data can provide additional

valuable information. However, up until now high throughput

N-glycan sample preparation, separation, and data analysis were

simply too time consuming.

Learn how you can now obtain actionable glycan data from >300

samples per day in your own lab. Introducing the SCIEX C100HT with

Fast Glycan technology. 96-well plate sample prep, compatible

with automation. Simply put the plate and the cartridge in the

instrument; no other reagents to add. The software will identify the

glycans for you. From the makers of the SCIEX PA 800 Plus.

Key Learning Objectives

■ Get introduced to a more advanced way for clone selection and

cell culture optimization

■ Discover how fast you can prepare and separate N-glycan

samples

■ See how C100HT software can:

• Identify the glycans for you

• Separate samples by your N-glycan profile pass/fail criteria

Who Should Attend

■ Bioprocessing laboratory managers

and scientists at biopharmaceutical

companies and contract research labs

performing clone selection and cell culture

optimization

■ LC and CE users looking for increased

screening capabilities

For questions contact Kristen Moore at [email protected]

Screen Hundreds of N-Glycans per Day More Samples — Better Decisions — Faster

Presenter

Dr. Mark Lies

Senior Product Manager

Application Scientist

SCIEX

Moderator

Rita Peters

Editorial Director

BioPharm International

View this free webcast at www.biopharminternational.com/bp_p/faster

ON-DEMAND WEBCAST Aired December 7, 2018


http://www.biopharminternational.com/bp_p/faster


https://sciex.com/

http://www.chromatographyonline.com/

http://www.biopharminternational.com/


Quality: Lab Operations

The National Institute of Standards and Technology (NIST)

and the National Institutes of Health (NIH) are leading a

flow cytometry quantitation consortium to serve the flow

cytometry communities (1). The consortium members are a

diverse group, including manufacturers of reagents and instru-

ments, as well as users in industry, government, and academ-

ics. The present objective of this group is for NIST to provide

fluorescence intensity assignments to reference microspheres

submitted by the consortium members. This service assures

consistency and traceability of the value assignment and enables

the standardization of the fluorescence intensity scale and per-

formance characteristics of flow cytometers.

Antigens on a cell surface or within a cell can be tagged

through binding with fluorescently labeled monoclonal antibod-

ies and measured using flow cytometry. Antigen expression level,

indicative of the presence of a functional gene, is then measured

via the fluorescence intensity of these cell-bound antibodies. In

principle, the measured intensity should be directly proportional

to the number of antibodies bound per cell (ABC). Moreover, if

ABC is determined, the number of antigens on the cell surface

can then be calculated using an antibody-antigen binding model

that accurately relates the two quantities. Being able to measure

antigen expression accurately and correlate it with gene expres-

sion level would be a boon to disease diagnostics, drug develop-

ment, clinical trials, and therapy monitoring, just to name a few

high impact areas.

CALIBRATION OF FLUORESCENCE INTENSITY FOR FLOW CYTOMETERSUnfortunately, the issues that complicate this determination

are many, leading to large inaccuracies in practice. The most

PAUL DEROSE and LILI WANG are both senior scientists in

the Biosystems and Biomaterials Division at NIST, focusing on

developing standards underpinning measurement assurance in flow

cytometry.

Fluorescence has long been used to detect biological targets. As these measurements are becoming more and more quantitative,

standards are needed to ensure accuracy and reproducibility.

PAUL C. DEROSE AND LILI WANG

NIST Fluorescence-Based Measurement Services

IBR

EA

KS

TO

CK

/ST

OC

K.A

DO

BE

.CO

M

Contin. on page 28





About VironovaVironova is founded on combined expertise in areas of virology, image analysis, electron microscopy, and mathematics.

This expertise has enabled Vironova to offer a world-leading nanoparticle charac-

terization analysis service with specialized expertise in vaccines, gene ther-apy, and drug delivery particles such as viruses, virus-like particles, and l iposomes. Experience from providing an EM service for gene therapy vectors, and of the type of features that need to be cha rac ter i z ed f re -

quently and in high volume, has inspired Vironova to develop comprehensive hard-ware and software solutions. Vironova revo-lutionizes access to transmission electron microscopy–based image analysis in bio-pharmaceutical development. Our solution enables automated analyses for faster and better informed decisions to secure robust bioprocessing and final product quality.

EM ServicesOur electron microscopy service team tailor the analysis to address the specific questions of the customer. It will deliver reports that contain:

• Representative EM-images• Quantitative analysis• Concluding summary with comments

from the experts.

Vironova Analyzer Software (VAS)The part 11-compliant Vironova image analysis software, VAS, is designed for the analysis of transmission electron microscopy (TEM) images of nanoparticles. The soft-ware automates the extraction of morphologi-cal data and measurements from the TEM

VIRONOVA AB

Gävlegatan 22113 30 StockholmSweden

EMAIL

[email protected]

WEBSITE

www.vironova.com

Vironova ABimages and transfers them into graph-plot-ting and report-generating tools. Using VAS for automated nanoparticle detection can save up to six hours per sample compared with manual approaches.

The VAS software in the hands of highly expert microscopists allows Vironova EM services to be cost effective and enables fast delivery of both high quality images and reli-able quantitative data.

MiniTEMMiniTEM is a low-voltage, tabletop micro-scope that can be placed in any standard laboratory. The system software enables con-trol of the microscope and automatic image acquisition, particle detection, and classifica-tion. MiniTEM provides bioprocess work-ers with their own in-house solution that enables non-experts in electron microscopy to acquire meaningful nanoparticle charac-terization data quickly and easily.

Vironova Biosafety, part of the Vironova group of companiesVironova Biosafety is a contract research orga-nization (CRO) specialized in viral clearance studies for biological drugs. The office and laboratories are co-located with the mother company Vironova. Viral clearance studies are performed in BSLII- and BSLIII- certi-fied cell and virus laboratories, in compliance with good laboratory practice (GLP) prin-ciples. The services range from early R&D to market release. Literature-based viral risk assessment is also offered by experts with extensive scientific and regulatory experience.




http://www.vironova.com

https://www.vironova.com/

SPECIAL SPONSORED SECTION

Front Center&Timely, Accurate Analytical Purity Data Helps to Avoid Late Surprises When Scaling Up

The safety and efficacy of a drug

product can often be directly cor-

related with sample purity as well

as particle characteristics like the size,

shape, and overall morphology of the

particles that compose the active ingre-

dient and excipients.

To avoid product variability and con-

tamination, drug manufacturers should

periodically monitor their compounds’

primary particles and purity profiles

throughout the development and manu-

facturing processes. Transmission Elec-

tron Microscopy (TEM) is the best tech-

nology for visualizing and understanding

nanoparticle characteristics and sample

purity, said Josefina Nilsson, Head of

EM services at Vironova, in a recent Bio-

Pharm International webcast.

“TEM and image analysis are great

techniques to really get to know your

product, understand the design space,

and determine how changes in e.g. the

formulation, or upstream and down-

stream processes affect particle charac-

teristics and purity,” said Nilsson.

Nonetheless, conventional TEM is

not a routinely used analytical method

in biopharmaceutical process develop-

ment because the equipment requires

a highly skilled operator and is costly

to maintain. In addition, conventional

TEM must be housed in a laboratory

facility with robust concrete flooring to

minimize vibrations, raised ceiling, and

a low electromagnetic environment.

“During discussions with our part-

ners and clients, we realized that they

would benefit from being able to con-

duct TEM and image analysis in-house,”

said Nilsson. In response, Vironova de-

signed the MiniTEM™, a highly auto-

mated tabletop microscope for routine

bioprocessing testing in standard labo-

ratory settings.

Benefits of New TEM System

The MiniTEM produces high-resolution

images with a spatial resolution of about

1 nanometer (nm). Unlike the conven-

tional TEM, the MiniTEM does not need

special housing, a cooling system, or a

highly trained operator. Because image

acquisition, particle detection and clas-

sification and analysis are automated, the

microscope can be “operated on a gener-

alist level, which makes it accessible for

more personnel,” said Martin Ryner, Stra-

tegic Development Manager at Vironova,

who spoke during the same presentation.

Automation allows the MiniTEM user to

perform other tasks during image collec-

tion and analysis, he said.

Ryner added that Vironova calcu-

lated the amount of time (Figure 1)

required for sample preparation, mi-

croscope operation, data analysis, and

data reporting when MiniTEM and

conventional TEM with manual analy-

sis were used. All activities, particularly

data analysis, were less time-consuming

when MiniTEM was used. “The online

image analysis takes about an hour to

sample in the MiniTEM, providing

about 60,000 registered particles,” said

Ryner. “This is not at all a task that is

feasible to perform manually. Extrapo-

lated data show that manual analysis

would require about two weeks of labor.”

Nilsson pointed out that the analy-

sis performed by MiniTEM can alert

drug developers that a formulation has

not been optimized to maintain particle

integrity over time. Levels of intact par-

ticles, broken particles, debris, and ag-

gregates in the sample are indicators of

product integrity that are tracked via

MiniTEM. Intact particles can char-

acterize efficacious products. The de-

tection of host cell debris in a sample

��

��

��

��

��

� ��

� ��

� ��

��

��

Figure 1: Comparison of MiniTEM versus conventional TEM.


SPECIAL SPONSORED SECTION

BROUGHT

TO YOU BY

is evidence of a failure in downstream

purification process. Debris can foster

the development of aggregates that can

induce unwanted immunogenicity. Ag-

gregates of particles can be formed dur-

ing upstream operations or as a result of

unsuitable downstream conditions.

MiniTEM analysis can accommo-

date unstained as well as negatively

stained samples. Because the micro-

scope detects particles in areas of good

staining quality, consistent and reliable

results are produced.

Using the MiniTEM

The operation of MiniTEM involves the

following steps: selecting imaging way-

points, imaging, particle classification,

and quantitative data output. The user

identifies imaging waypoints as well as

the number of images that the automat-

ed system should collect at each selected

point. The microscope collects a set num-

ber of images at each waypoint.

“The user can instruct the software to

visit preconfigured waypoints with a cer-

tain pattern or, based on a low magni-

fication assessment screening, can iden-

tify the waypoints to investigate,” said

Ryner. “Using the inbuilt operational

automations, the system then automati-

cally screens these areas. Each waypoint

will be visited and each area will be im-

aged in a structured way. This would

take a few hours if done manually.”

In image analysis, particles and struc-

tures of interest are detected, measured,

and classified. Particle analysis can be

automated or conducted manually. Im-

ages and particle measurements are plot-

ted and presented graphically to support

decision making to optimize product de-

velopment and manufacturing.

Characterization of

Viral Gene Delivery Platforms

MiniTEM has been used to analyze adeno-

associated virus (AAV) samples to de-

termine the overall morphology of their

primary particles and identify debris and

other unwanted structures, Nilsson said.

The analysis is crucial because AAV par-

ticles must be intact for the gene therapy

to be biologically effective, she pointed

out. In gene therapy, viral vector stability

and integrity must be carefully investi-

gated before scaling up production.

Nilsson showed three micrographs

of AAV samples (Figure 2). The rather

smooth background of the micrograph

on the left indicates that the product is

quite clean, she said. However, the sam-

ple does contain some smaller debris as

well as proteasomes. In the center micro-

graph, the AAV sample contains a much

higher concentration of small debris,

some proteasomes and large aggregates.

Although the AAV sample in the right

micrograph has a very clean background,

broken particles referred to as doublet

and triplet formations are present.

An AAV sample containing numer-

ous proteasomes (Figure 3) also was

shown. Image analysis software detected

and classified the structures, which then

were presented in a simple size distribu-

tion histogram. Nilsson said that the size

distribution of all potential structures in

the sample can indicate its purity.

The histogram in Figure 3 also shows

a comparison of dynamic light scatter-

ing (DLS) and MiniTEM analysis of

AAV particle sizes. Unlike MiniTEM,

DLS could not distinguish AAV particles

and proteasomes, Nilsson said.

Nilsson added that the detailed char-

acterization of viral particles by electron

microscopy and image analysis during

early product development provides in-

formation that can improve drug formu-

lation and production. The results of the

MiniTEM analysis can enable the manu-

facturer to adjust the process parameters.

To watch the full presentation on demand,

please visit: www.biopharminternational.

com/bp/puritydata.

Debris (proteasomes) Aggregates and background debris Particle integrity

1 �m

AAV particle

proteasome

A comparative study of particle size analysis of a AAV samples using DLS and MiniTEM™

Figure 2: Overall sample morphology of AAV samples by nsTEM.

Figure 3: MiniTEM analysis of AAV samples reveals hidden contaminants.


http://www.biopharminternational

https://www.vironova.com/

http://www.biopharminternational.com/



basic one and possibly the most impor-

tant one to overcome is a fluorescence

scale on flow cytometers that changes

in magnitude between different cytom-

eter platforms and when different ref-

erence microspheres with fluorescence

intensities assigned by their respective

manufacturers are used. These reference

microspheres, or “beads”, over a range of

fluorescence intensities are used to cali-

brate the fluorescence signal of a flow

cytometer. The NIST assignment of

equivalent reference fluorophore (ERF)

units to the mean fluorescence signal,

typically referred to as the mean fluores-

cence intensity (MFI) in flow cytometry,

using calibration beads in suspension

helps to define the ERF scale. Figure

1 illustrates a calibration curve of ERF

units versus fluorescence signal.

DETERMINATION OF ERF UNITS WITH REFERENCE FLUOROPHORE SOLUTIONSBecause every fluorescence detec-

tion system produces a different signal

intensity for the same sample, reference

solutions have commonly been used in

flow cytometry and other fluorescence-

based techniques (2) to relate the mea-

sured fluorescence intensity to the

reference fluorophore concentration. A

fluorescence spectrometer is used to mea-

sure the fluorescence emission spectra of

a set of serial-diluted reference solutions

with known fluorophore concentrations,

producing a calibration curve of the inte-

grated fluorescence intensity versus the

concentration of the reference fluoro-

phore (see Figure 2).

Standard Reference Mater ia l

(SRM) 1934, a set of four fluores-

cent dye solutions covering the spec-

tral range from 440 nm to 800 nm,

was released by NIST in May 2016 to

enable users to produce such calibra-

tion curves with high accuracy using

high purity dyes (see Figure 3, top)

(3). The SRM solutions were certified

for values of concentration and corre-

sponding uncertainty. This was needed

because most commercially available

fluorescent dyes are supplied with an

approximate purity and no estimated

uncertainty, making a set of standard

dyes necessary.

The fluorescence emission spectrum

of each bead suspension, used for fluo-

rescence intensity calibration of flow

cytometers as described in the previous

section, is then measured on the same

fluorescence spectrometer as that used

for the reference solutions with the

same measurement conditions. In this

way, the fluorescence intensity of each

suspension can be expressed in ERF

units, as illustrated in Figure 2 by the

dye-labeled bead suspension data point

Figure 1. Calibration curve of equivalent reference fluorophore (ERF) units versus the fluorescence signal of five reference bead suspensions measured using a flow cytometer.

Figure 2. Calibration curve of fluorescence intensity of five serial diluted reference solutions measured on a fluorescence spectrometer versus dye concentration. The intensity of one fluorescent bead suspension is also measured, and the curve is used to express it as an equivalent dye concentration.

FIG

UR

ES

CO

UR

TE

SY

OF

TH

E A

UT

HO

RS

Lab Operations — Contin. from page 24





on the linear calibration curve. Because of

an increasing need for reference fluoro-

phores covering other fluorescence chan-

nels in the emission range from 400 nm

to 900 nm, we are currently working on

three candidate reference fluorophores,

Pacific Orange, Alexa Fluor 700, and

Alexa Fluor 750 to add to those of SRM

1934. Certain commercial equipment,

instruments, or materials are identified in

this paper to foster understanding. Such

identification does not imply recommen-

dation or endorsement by the National

Institute of Standards and Technology,

nor does it imply that the materials or

equipment identified are necessarily the

best available for the purpose.

DETERMINATION OF BEAD SUSPENSION CONCENTRATIONNote that the ERF value for the bead

suspension shown in Figure 2 is

obtained for all beads suspended in a

buffer solution. In flow cytometry, indi-

vidual beads or cells are detected one

at a time, so the ERF value per bead

needs to be determined (see Figure 3).

To calculate this quantity, the concen-

tration of the bead suspension must be

determined. NIST has demonstrated

the determination of the concentra-

tion of spherical bead suspensions

using light obscuration see Figure 3

(bottom) (4). This technique counts

particles by measuring a decrease in

the intensity of laser light when a par-

ticle passes through its beam. The val-

ues obtained are International System

of Units-traceable with an uncertainty

of approximately 2% for beads with

diameters from 2 micrometers to 5

micrometers.

Additionally, flow cytometry serves as

an orthogonal method for bead concen-

tration measurement for enhancing the

confidence of the measurement. NIST

is working to expand the size range to

hundred(s) of nanometers and the pres-

ent measurement service to include con-

centration measurement of particles and

bioparticles because of the application

needs.

ASSIGNMENT OF FLUORESCENCE INTENSITY PER BEAD Figure 3 explains the ERF assign-

ment process of a single bead. (Left)

Calibration beads from different man-

ufacturers intended to cover the same

fluorescence channels give fluorescence

intensities that are not comparable. (Top)

SRM 1934 is comprised of three dye

solutions (coumarin 30, fluorescein, Nile

red) and one suspension of allophyco-

cyanin (APC), covering the emission

spectral ranges 440 nm to 550 nm with

405 nm laser excitation, 500 nm to 580

nm and 570 nm to 700 nm with 488

nm laser excitation, and 640-800 nm

with 633 nm laser excitation, respectively.

These three lasers are most commonly

used in flow cytometers.

A calibrated fluorometer and reference

solutions produced from SRM 1934 are

used to express the fluorescence intensity

of a bead suspension as the number of

fluorophores in solution with the same

intensity under the same instrument

conditions. (Bottom) A light obscura-

tion (LO) instrument and a flow cytom-

eter (FC) are both used for counting the

number of particles in a suspension. For

LO, a decrease in the detector signal is

counted as a particle, and the volume

that passes through the detection region

is also measured. These two quantities

enable the particle concentration to be

determined. For FC, an increase in fluo-

rescence signal is counted as a particle

using flow and optical components that

are similar to the LO components shown

here. The FC density plot, also shown

here, compares the number of calibra-

tion beads (P1) to that of an internal

standard (P2), allowing an independent

determination of the bead concentration.

(Right) The combination of fluorescence

intensity and bead concentration mea-

surements of a suspension allow an ERF

assignment of an average single bead.

Calibration beads assigned in this way are

comparable to one another, independent

of the manufacturer.

Members of the Flow Cytometry

Quantitation Consortium can choose to

have NIST assign fluorescence intensi-

ties to beads using the components of

SRM 1934. NIST supplies these assign-

ments through a separate cooperative

research and development agreement

(CRADA) with members individually,

addressing their specific needs. NIST

has assigned ERF values to reference

beads from several companies and has

Figure 3. Schematic of the equivalent reference fluorophore (ERF) assignment process.

Contin. on page 46




Peer-ReviewedD

ES

IGN

CE

LLS /

ST

OC

K.A

DO

BE

.CO

M

Jinhua Shao, Fulin He*,

[email protected], and

Xiaoming Chen are at the

School of Chemical and

Biological Engineering, Hunan

University of Science and

Engineering; Yufei Zhang is at the Key Laboratory

of Anti-fibrous Biological

Therapy, MuDanjiang Medical

University; and Zhiyong Zhu

is at the College of Computer

Science, Hunan University of

Science and Engineering.

*To whom correspondence

should be addressed.

PEER-REVIEWED

Submitted: April 10, 2018Accepted: May 3, 2018

Separation and Purification of a Tissue-Type

Plasminogen Activator from Trichoderma reesei

JINHUA SHAO, YUFEI ZHANG, ZHIYONG ZHU, FULIN HE, AND XIAOMING CHEN

ABSTRACTThis work was designed to establish methods for purifying a fibrinolytic enzyme with high purity and fibrinolytic activity from Trichoderma

reesei-submerged culture liquid. Presently, the majority of tissue-type plasminogen activator (t-PA) is separated and purified from biological tissues or anima cells. Separation and purification of t-PA from genetically recombined T. reesei, a fungus, has not been reported. This research proposes a method for separating and purifying t-PA from this fungal cell source. The method, characterized by low cost, high activity recovery, and simple operation, can be applied in clinical settings.

Thrombotic diseases have been among the most serious diseases threatening human health (1, 2). Thrombolytic therapy in mod-

ern medicine is a safe, effective approach for treating thrombotic diseases (3); how-ever, despite showing favorable therapeu-tic effects, thrombolytic agents applied in clinical practice across the world present side effects of differing severity and are also expensive.

Tis sue-t y pe pla sminogen ac t iva-tor (t-PA), an enzyme found in different types of tissues, can dissolve a thrombus by selectively changing the plasminogen in the thrombus to a fibrinolytic enzyme. Moreover, systemic hemorrhage is unlikely to occur with the thrombolysis induced by t-PA. Considering these advantages, t-PA has become a new thrombolytic agent (4–7). Due to its rapid growth, short activ-ity cycle, and its easily controlled microor-ganism growth conditions, large amounts of target product can be obtained by arti-ficially controlling the fermentation condi-tions. This benefit gives the production of t-PA-based thrombolytic agents broad potential application. A type of glycopro-tein, t-PA and the mature t-PA molecule

is a single strand (sct-PA) that contains 527 amino acid residues with the relative molecular weight of 6.8–7.2 ×104; there are four potential glycosylation sites, among which three can be glycosylated (namely Asn117, Asn184, and Asn448), while the glycosylation of the fourth site cannot yet be discovered in mammal cells.

Trichoderma reesei, a f ilamentous fun-gus with high cellulose content, shows a strong ability to synthesize and secrete proteins (8–10). Experiments have dem-onstrated that it shows preferable per-formance in producing proteins and is non-toxic to humans. Even in enzyme-producing conditions, T. reesei does not produce mycotoxin and antibiotics (11–14). Therefore, the plasminogen activator sepa-rated from this fungus is safe and reliable in application.

At present, the majority of t-PA is separated and purif ied from biological t issues (15–22) or animal cel ls (23). Separating t-PA from the genetical ly recombined T. reesei has not been reported. This research proposes a simple method for separating and purifying t-PA from genetically engineered fungus T. reesei. The method, characterized by low cost,





Peer-Reviewed

high activity recovery, and simple operation, can be applied in clinical settings.

MATERIALS AND METHODSMaterials and equipmentGenetical ly engineered fungus, T.

reesei 306, was obtained from the Laboratory of Applied Microbiology, Tianjin University of Science and Technology, China. The strain was genetically engineered using T. reesei

RutC-30, as the host connected to a human melanoma cell line, where t-PA cDNA, an exoglucanase I gene , was posit ioned and inte-grated into the T. reesei chromosome. This guided the secretory expres-sion of t-PA with the leader peptide sequence CBHI under the control of the CBHI promotor, encoded by a single copy gene, which can direct protein production to an amount as high as 50% of the total amount of T. reesei extracellular secretory pro-tein (24, 25). The growth media consisted of agarose (Sino-American Biotechnology), f ibrinogen (Sigma), sodium dodecyl su l fate (Sigma), acrylamide (Sigma), bis-acrylamide (Sigma), and thrombin (Institute of Hematology, Chinese Academy of Medical Sciences). Protein molecu-lar weight markers (Marker, 14,400 to 97,400 Da), protein markers for testing isoelectric points (pI) (3.5–9.3) (Shanghai Sibas Biotechnology Deve lopment), an ion-exchange chromatography (Q-sepharose High Performance Column, Pharmacia Biotech), gel f iltration chromatog-raphy medium (Superdex 75 prep grade, Pharmacia Biotech), and a protein purif ication system (AKTA prime, GE Healthcare) were used in this experiment. Other chromato-graphic- or analytic-grade reagents used were imported or made in China.

Activity/protein concentrationThe act iv it y of the f ibr inoly t ic enzyme was measured according to a f ibrin plate method proposed by

Astrup (26). To test the concentra-tion of the protein, the Folin-phenol method (27) was used with bovine serum albumin as standard protein.

Fibrin agarose plateTaking the preparation of 10 plates as an example, the researchers added 50 mL of normal saline into 0.25 g of agarose, placed it into the 45 °C water bath for 30 min of thermal insulation after the agarose was dissolved, and then half branch of the 500U/branch thrombin was added and blended (26). Next the researchers dissolved 0.2 g of bovine fibrinogen into the barbital sodium buffer solution (pH = 7.4) and placed it into the 45 °C water bath for 30 min of thermal insulation. Next 5 mL of agarose thrombin solution and 5 mL of fibrinogen solution were each blended and immediately poured onto a 9-cm diameter plate. The plates were then reserved in the refrigerator (under 4 °C) for use after 30 min of stewing.

Enzyme activity unitThe plasminogen activator standard conf iguration (the series concentra-tions of which was 0.5 U/ml, 1 U/ml, 3 U/ml, 5 U/ml, 10, U/ml 20 U/ml, 30 U/ml, and 50 U/ml) was diluted with normal saline; 10-uL samples of each concentration were placed onto the prepared fibrin plates, with three plates for each concentration. Then they were placed into the incubator for 6 h of thermal insulation at 37 °C, and the dissolving circle diameters were observed and measured verti-cally three times to take the average. The regression equation about the enzyme activ ity (C) and the dis-solving circle diameter (A) was con-structed as logC=7.7428 logA-6.2443, where C was the t-PA enzyme activity (U/mL) and A was the dissolving cir-cle diameter (mm). The same method was used to measure the sample, and the resulting dissolving circle diam-eter was substituted into the regres-sion equation to calculate the enzyme activity of t-PA.

Preparation of crude enzymeThe fermentation process proposed by Tian Ming et al. was adopted to prepare crude enzyme (26). T. reesei

was inoculated in an Erlenmeyer f lask (inoculation amount: 10%) to be cultured at 28 °C and a rotation rate of 150 rpm for 48 h. Then the fermentation broth was centrifuged at 4 °C and 8000 rpm for 25 min to remove the cells and solid impuri-t ies, such as those residues pres-ent. In this way, the supernatant containing t-PA was obtained for later use.

Decolorization of crude enzymeA D296 strong anion-exchange resin column (Φ 1.5 × 30 cm) was used to decolorize the clear supernatant from which the cel ls and solid impuri-ties had been removed, with a linear velocity maintained to within 0.5 cm/min. When the resin was almost satu-rated with pigment, the addition of the enzyme was stopped, followed by the replacement of the enzyme using 0.02 mol/L phosphate buffer, so as to regenerate the resin.

Ammonium sulfate precipitationA f te r b e i ng ad ju s ted to 45% saturation using ammonium sulfate, the decolorized fermentation broth was placed in a refr igerator and stored at 4 °C for 8 h. Then, the fermentation broth was centrifuged at 4 °C and 8000 rpm for 25 min to separate the supernatant and the precipitate. Afterwards, the collected precipitate was dissolved using 0.02 mol/L phosphate buffer (pH 7.4) for later use.

Desalination through dialysisA dia lysis bag (w ith a molecu-lar weight cut-off of 10,000 Da) of an appropriate size was boiled in 1 mol/L ethylene diamine tetraacetic acid (EDTA) solution, 2% sodium bicarbonate (NaHCO

3) solution, and

distilled water for 30 min in total (10

Contin. on page 34





Company DescriptionSGS Life Sciences is a leading contract ser-vice organization providing clinical research services, analytical development, biologics

characterization, utilities qualif ication, biosafety, and quality control test-ing. SGS provides Phase I-IV clinical trial man-agement and ser v ices encompassing PK /PD simulation and modeling, data management, phar-macovigilance, and regu-latory consultancy. SGS also offers contract labo-ratory services (detailed

below) that include analytical chemistry, microbiology, stability studies, method devel-opment, and protein analysis. SGS is the world’s leading inspection, verification, test-ing, and certification company.

Technical Services• Quality control testing of raw materials,

APIs, and finished products• Monograph testing (USP, EP, BP, and JP)• Analytical method development and

validation• Microbiological testing• Container testing (extractables and

leachables)• Stability testing according to ICH guide-

lines or customer specifications• Utilities qualification (air, gas, water, &

surface)• Medical device testing• Protein/peptide analysis and quantification• Glycosylation analysis• Biologics safety testing (endotoxin, virus,

and mycoplasma)• Cell-line characterization• Host-cell impurity testing (residual DNA)• Virus testing (cell bank and virus seeds

characterization)

SGS Life Sciences

• Antibody product analysis• Bioanalysis (mass spectrometry, immuno-

and cell-based assays)

FacilitiesWith truly global coverage and a strong local footprint in North America, SGS has facilities in Lincolnshire (Illinois), Fairfield (New Jersey), West Chester (Pennsylvania), Mississauga (Ontario), and Markham (Ontario), as well as a clinical trial manage-ment office in Germantown (Maryland). SGS’ laboratories operate according to high quality standards (cGMP, GLP, ISO 17025) and have been inspected by the US-FDA or local regulatory authorities.

Markets ServedSGS services various life-science companies including pharmaceutical, biopharmaceutical, biotechnology, and medical device manufac-turers. SGS operates a global, wholly-owned network of 18 life-science laboratories, with facilities in the US, UK, Canada, Belgium, France, Germany, Italy, Switzerland, China, and India. The Top 20 pharmaceutical com-panies trust SGS as a partner for their quality control testing needs.

SGS LIFE SCIENCES

75 Passaic AvenueFairfield, NJ 07004 US

TELEPHONE:

+1.973.244.2435

FAX:

+1.973.244.1823

EMAIL:

[email protected]

WEBSITE:

www.sgs.com/lifescience

NUMBER OF EMPLOYEES:

Global: 95,000Life Science: 1850

YEAR FOUNDED:

1878




http://www.sgs.com/lifescience

https://www.sgs.com/

RELIABILITY

NETWORK

QUALITY

LIFE INSPIRED, QUALITY DRIVEN

LIFE SCIENCES

LABORATORYSERVICES

GET TO MARKET QUICKLY, SAFELY & EFFICIENTLY

SGS Life Sciences enables the medical and health

innovators of the world to deliver life-changing

solutions in the quickest, safest and most efficient

way, helping improve the lives of many.

SERVICES INCLUDE:

• Biologics Characterization

• Extractables & Leachables

• Stability

• Biosafety

• Microbiology

• Analytical Chemistry

CONTACT

[email protected]

www.sgs.com/lifescience



http://www.sgs.com/lifescience

https://www.sgs.com/


Peer-Reviewed

min in each solution). Then the dis-solved solution, precipitated using ammonium sulfate, was poured into the dialysis bag, which was tied and placed in a beaker containing dis-tilled water, for dialysis. Every five to 10 min, barium chloride was added, drop-wise, to the distilled water to check whether or not any precipitation was generated.

ChromatographyStrong anion-exchange chromatogra-phy (Q-Sepharose High Performance Column, Pharmacia Biotech) was applied to separate and purify the decol-orized crude enzyme to a greater extent. The process was conducted using a col-umn measuring Φ 2.6 × 7 cm with a sample volume of 23 mL. A 0.02 mol/L phosphate buffer saline (PBS) (pH 7.4) was then used as the eluent at a f low rate of 2 mL/min. Next, 4 mL of each product was collected in a separate tube. The gradient elution ratio was 300 mL to 300 mL. Afterwards, the activity and the optical density (OD) (at 280 nm) of the fibrinolytic enzyme were detected, followed by the collection and lyophiliza-tion of the active components.

Detecting purityA prote in pu r i f ic at ion s y s tem (AKTA prime, GE Healthcare) was used to detect purity. The f iller of the column (Φ 1.0 × 60 cm) was the gel f i ltration chromatography medium (Superdex 75 prep grade, Pharmacia Biotech). A 2-mL sam-ple was applied at a f low rate of 1.0 mL/min. A sodium chloride solution with 0.2 mol/L PBS (pH = 7.4 and containing 0.02 mol/L phosphate

buffer) was used as the eluent, in which the protein concentration was 0.0018 g/mL.

SDS-PAGE and PAGES od iu m dodec y l su l f a te –p oly-ac r y lamide ge l e lec t rophores i s (SDS–PAGE) (27) and PAGE were performed to measure the relative molecular weight and purif ication of the purif ied t-PA. For the elec-trophoresis, 12% separation gel and 5% stacking gel with a cross-linking degree of 2.6% were applied to pour the vertical slab gel. The molecu-lar weight of the protein marker was between 14,400 and 97,400 Da.

Fibrin-autographyFibrin-autography was detected using a SDS–PAGE f ibrin-autography method (28).

Preparation of gelsPolymerized gels with a crosslink degree of 2.6% were prepared by orderly pouring of the resolving gel containing 12% acrylamide and the stacking gel containing 5% acrylamide into the clamped vertical glass plates.

Electrophoresis conditionsAll electrophoreses started at a con-stant current of 10 mA. When the samples had completely entered the resolving gel, the running conditions were set as constant 30 mA for three to four hours.

Fixing, staining, and destainingWhen the blue dye front reached the bottom of gels, signaling that the electrophoresis was f inished, gels

were removed and f ixed for two to four hours. Fixed gels were stained at 60 °C for 10 minutes, followed by an overnight destaining. The results of SDS-PAGE were analyzed using an ultraviolet transilluminator gel imaging system, and the pro-tein molecular weights are showed in Table I.

Sample preparationThe protein concentrations were appropriately adjusted so that a suit-able amount of protein could be loaded onto the gels. Then 10-μL, appropriately diluted protein sample was well-mixed with an equal volume of sample buffer (2×) containing 4% SDS, 20% glycol (w/v), and 0.2% bro-mophenol blue and incubated at 30 °C for 2 h.

Protein reactivationTo remove the SDS bound to the pro-teins after electrophoresis and recover the proteins, the obtained gels were removed and irrigated by 2.5% deter-gent (Triton X-100) for an hour.

Gel visualizationAfter an adequate wash, gels were placed on the pre-prepared f ibrin plates and kept at 37 °C in the incu-bator for several hours. The dissolved bands were dynamically monitored.

Determination of pIIsoelectrofocusing (IEF) (27) (with a gel concentration, T, of 7.5% and crosslinking degree, C, of 3%) was applied to measure the pI of the t-PA. The pI of the protein maker was between 3.5 and 9.3.

Table I. The purification steps of tissue-type plasminogen activator (t-PA) from Trichoderma reesei.

Total activity (U) Total protein (mg)Specific activity

(U/mg)Purification factor Recovery rate (%)

Stoste 565958.46 3159.75 61.70 1.00 100.00

Desalting 494772.50 746.00 529.19 8.58 87.422

Dialysis 394959.05 259.00 2185.17 35.42 69.786

Q-Sepharose 247303.20 46.00 3202.24 52.44 43.696

Peer-Reviewed — Contin. from page 31




Peer-ReviewedF

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

RS

.

RESULTSDecolorization of crude enzymeThe decolorization results of the crude enzyme using the D296 strong anion-exchange resin are shown in Figure 1, where it can be seen that the protein peak and the activity peaks of the enzyme were basically consistent. There was only one protein elution peak without tailing. The level of purification and the activity recovery were 1.34-fold and 92.79%, respectively.

Ammonium sulfate precipitationThe decolorized crude enzyme was precipitated using ammonium sulfate of different degrees of saturation. The results are illustrated in Figure 2: t-PA precipitation was generated when the saturation of the ammonium sulfate was 25%. When the saturation was between 25% and 45%, the activity of the enzyme increased with rising saturation. Moreover, peak activity occurred when the saturation of the ammonium sulfate was 45%. While exceeding this degree of saturation, the activity of the enzyme decreased and only formed a peak when the sat-uration reached 75%. This may have been the result of precipitation of the enzyme because some small molecules were inhibiting enzymatic activity when the ammonium sulfate reached a certain degree of saturation (i.e., satu-ration that was sufficient to precipi-tate the enzyme). This explained why the activity of the enzyme decreased with increasing saturation after the latter exceeded 45%. As the degree of saturation of the ammonium sul-fate was increased to 65%, the small molecules inhibiting the activity of the enzyme were precipitated. Under such circumstances, the activity of the enzyme was enhanced once again. With completely saturated ammonium sulfate, the enzyme lost all activity. In this way, the optimal saturation of the ammonium sulfate in the precipita-tion was deemed to have been 45%. The precipitated t-PA purified from T. reesei showed an activity recovery of

20.38%, indicating that a great deal of activity was lost during the long precipitation period. Therefore, the precipitation time, during which the least activity was lost, was determined as 8 h based on the determined degree of saturation.

Desalination through dialysisThe precipitated crude enzyme was desalinated with a molecular weight cut-off of 10,000 Da. As shown in Table I, which summarizes the dialysis results, the dialysis removed not only the salts, but a lso some

impurities inhibiting the activity of the f ibrinolytic enzyme. By doing so, the tota l protein content was reduced while the specif ic activity grew signif icantly, contributing to the level of purif ication reaching 35.42-fold and high activity recovery. All of these indicated that a favor-able effect was obtained by dialysis.

ChromatographyThe separat ion resu lts obta ined by using a strong anion-exchanger (Q-Sepharose High Performance Column, Pharmacia Biotech) for

Figure 1. Decolorization curve of the crude enzyme by D296 anion exchange resin. OD is optical density.

Figure 2. The curve of ammonium sulfate salting-out. t-PA is tissue-type plasminogen activator.



Peer-Reviewed

the t-PA purif ied from T. reesei are shown in Figure 3. According to the chromatographic curves shown, the t-PA was completely adsorbed by the anion-exchanger. The activity peak of the enzyme in elution was found at 0.2 to 0.8 mol/L of sodium chlo-ride (NaCl). The activity peak of the target enzyme and the absorption peak of the protein at 280 nm were completely superimposed, and there

was only one protein elution peak. The result indicated that the enzyme undergoing strong anion-exchange chromatography reached high purity. The active components were collected to measure the purity through gel fil-tration chromatography.

Purification of t-PABy treating the t-PA fermentation broth separated from T. reesei through

the aforementioned processes, the t-PA was purified. The activity recov-ery and level of purif ication of the fermentation broth are summarized in Table I.

Measuring purityThe active components obtained from anion-exchange chromatography were separated by gel f i lt rat ion chromatography medium (Superdex 75 prep grade, Pharmacia Biotech), and the purity was assayed by a protein purif ication system (AKTA prime, GE Healthcare). The results are shown in Figure 4, where it was seen that the enzyme had only a single symmetrical peak, indicating that the target protease had reached chromatographic purity.

Purity and molecular weightSDS-PAGE and PAGE electropho-resis were used to measure the lyophi-lized and purified sample’s molecular weight and purity. Figures 5 and 6

reveal that the t-PA showed one band in both denatured gel electrophore-sis and native-PAGE, implying that the t-PA had reached electrophoretic purity.

Detecting molecular weightThe results of the SDS-PAGE fibrin-autography, shown in Figure 7, sug-gested that there were solution spots on the bands, which indicated that the protein in the band was able to dissolve fibrin. The molecular weight of the solution spots was 65,000 Da, implying that the substance that could dissolve the fibrin was present in the band with a molecular weight of 65,000 Da. The SDS-PAGE f ibrin-autography proved that the fermentation broth of T. reesei con-tained a substance that can dissolve fibrin, and that its molecular weight was 65,000 Da. The test also veri-f ied the correctness of SDS-PAGE and PAGE and proved that the molecular fragment with a molecular weight of 65,000 Da was the target enzyme.

Figure 3. Separation of using tissue-type plasminogen activator ion exchange chromatography with a strong anion-exchanger (Q-Sepharose High Performance Column, Pharmacia Biotech). NaCl is sodium chloride; OD is optical density.

Figure 4. The gel filtration chromatography (Superdex 75 prep grad, Pharmacia Biotech).




Peer-Reviewed

Detection of t-PA pIThe pI of the t-PA separated from T.

reesei was detected through isoelectric focusing (IEF), and Figure 8 shows

the results. The pI of the t-PA was 4.24, while that of natural t-PA is between 7.8 and 8.6, with the pI of the maximum component being 8.2. Compared with its natura l coun-terpart, the measured pI (4.24) of the t-PA obtained in this research was decreased by 4.8. The isoelec-tric point of protein was related to the number of the acidic amino acid and the alkaline amino acid. The ratios of the a lkaline amino resi-due and the acidic amino acid of the serum albumin and the hemoglobin were 1.2 and 1.7, with the isoelec-tric points of 4.7 and 6.7, respec-tively. As shown in Figure 8, the t-PA was mainly the substance with a molecular weight of approximately 60,000 Da. The aforementioned substance belonged to the protease domain with its activity having been acquired by the cracking of the pep-tide bond at 275 Arg–276 Ile in natu-ral t-PA. Based on the observed t-PA sequence, the ratio of the number of basic amino acid residues to that of acidic amino acid residues was 1.16. Therefore, the pI was reduced to 4.24.

DISCUSSIONIn order to expla in the nature , structure, and function of a cer-

tain enzyme, that enzyme should be acquired, and the method of separating and purifying it should be studied. Though microorgan-isms can generate various valuable enzymes, there are always several pigment substances within a micro-organism’s fermentation liquid that increase the diff iculty of enzyme separation and purif ication. This experiment employed D296 mac-roporous resin to remove pigment from the enzyme solution. Though there is a certain amount of chlo-rine ion (Cl-1)which enters into the enzyme solution during the decolor-ization of the enzyme and increases the alkalinity of the enzyme, desalt-ing v ia cont inuous pur i f icat ion may remove the Cl-1 in the solu-tion. Thus, the entry of Cl-1 does not affect the enzyme’s ability to purify during chromatography. An ion exchange resin can be used in the decolorization of a microorgan-ism’s crude fermentation enzyme liquid during separation and puri-f ication. An exchange resin that absorbs most pigment but not the target enzyme is used as the decol-orization medium. The pH value that maintains the stability of the target enzyme and has no electric charge, or that has the same electric

Figure 5. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of the t-PA produced by Trichodera reesei. A. Purified sample. B. Standard protein.

Figure 7. The picture of 16h sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) fibrin autography technique.

Figure 8. The picture of isoelectrofocusing of the recombinant t-PA. t-PA is tissue-type plasminogen activator.

Figure 6. The polyacrylamide gel electrophoresis (PAGE) of the t-PA produced by Trichodera reesei. t-PA is tissue-type plasminogen activator.For personal, non-commercial use



Peer-Reviewed

charge as that of the target enzyme, is also used as the starting pH value. In addition, the salt-type of the ion exchange functional group counter-ion is also used during the decolor-ization process.

The desalted and deposited pro-tein can maintain its native confor-mation and activity and can thus be re-dissolved. The experiment suggests that the neutral salt vitriol used in desalting is optimal because it has the highest solubility in water, and the temperature coefficient of solubility is relatively lower. Because the ion of the sample greatly affects the ion exchange chromatography, desalting should be conducted before under-going ion exchange chromatography. Desalting is conducted with gel filtra-tion and a dialysis bag. Compared to gel filtration, an enzyme’s specificity and ability to purify increases after the desalting process.

The nat ura l t-PA isoelec t r ic point is within 7.8–8.6, and the maximum component isoelectr ic point is 8.2. The pH has been shown to be relatively stable at 5.8–8.0 and optimal at 7.4 (29). This shows that natural t-Pa is an alkaline protein. Separation is conducted using positive ion exchange. However, the T. reesei-recombinant t-PA isoelectric point is different. Upon testing, it was shown that recombinant t-PA is acidic, and as a result, a strong negative ion exchange medium is use for separation.

This experiment yielded an active component with a molecular weight of about 65,000 Da through the recombination of tectotype plasmino-gen activator t-PA and by conduct-ing D296 decolorization, ammonium sulfate desalt, dialysis desalt, strong negative ion exchange resin chroma-tography, and gel f iltration chroma-tography purification. The component has a similar relative molecular mass as that of t-PA.

t-PA, a second-generation throm-bolytic, is limited in its ability to be used as a thrombolytic therapy.

It had a tendency to combine with plasminogen act ivator inhibitor (PAI) and form a compound after entering the plasma, which causes it to quickly lose therapeutic activity. In addition, a t-PA specif ic recep-tor in the liver cell membrane can quickly bind t-PA and shorten its half-life, resulting in increased risk of bleeding.

The mutant of t-PA, however, is a third-generat ion thromboly t ic and has a greatly improved half-life, increased affinity to fibrous protein, and stronger catalytic activity, mak-ing it a more desirable candidate for thrombolytic therapy than natural t-PA. The experiment shows that T. reesei is not only suitable for low-toxicity protein production, but also produces no fungal toxin or antibiotic under enzyme-producting conditions. This suggests that recombinant T.

reesei may be a useful, lower-cost, and less harmful alternative to producing thrombolytic therapies on an indus-trial scale.

ACKNOWLEDGMENTSWe greatly acknowledge the financial support from the Key Laboratory of Comprehensive Uti l ization of Advantage Plants Resources in Hunan South, Hunan University of Science and Engineering (XNZW14K02); Key project of Hunan Provincial Education Department (16A083).

AUTHOR CONTRIBUTIONSJ.H. Shao and F.L. He conceived and designed the experiments; Shao and Y.F. Zhang performed the experi-ments; X.X. Liu and H.Z. Li ana-lyzed the data; He contr ibuted reagents/materials/analysis tools; and Shao wrote the paper. Authorship is credited to those who have con-tributed substantially to the work reported.

CONFLICTS OF INTERESTThe authors declare there are no con-flicts of interest regarding the publica-tion of this paper.

REFERENCES 1. M. Hammes, et al., Clin Nephrol 84,

21–28 (2015). 2. A. Polimeni, et al., JACC Cardiovasc

Interv 10, 1819–1827 (2017). 3. Y. Hong, The Food Industry 36, 96–99

(2015). 4. N. Semeraro, et al., Thromb Res 129,

290–295 (2012). 5. M.J. Ahn, et al., Journal of

Microbiology and Biotechnology 25, 2090–2099 (2015).

6. A. Kovacs, et al., Thromb Res 133, 80–87 (2014).

7. S.T. Laing, et al., Thromb Res 130, 629¬635 (2012).

8. M.K. Narasimhan, M. Chandrasekaran, and M. Rajesh J. General and Applied Microbiology 61, 157–164 (2015).

9. L.M. Xia and P.L. Cen, Process Biochem. 34, 909–912 (1999).

10. S.L. Yuan, et al., Microbiology China 41, 1621–1628 (2014).

11. V. Ponnuswamy, et al., Electronic Journal of Biotechnology 20, 1–8 (2016).

12. H. Fang, C. Zhao, and X.Y. Song, Bioresour Technol 101, 4111–4119 (2010).

13. H. Fang, et al., Biotechnol Bioprocess 18, 390–398 (2013).

14. Z. Shuo, et al., Biological Chemistry 398, 765–773 (2017).

15. A.S. Todd, J Pathol Bacterio l78, 281–283 (1959).

16. M. Dahiya, G. Rajamohan, and K.L. Dikshit, FEBS Lett. 579, 1565–1572 (2005).

17. P. Wallen, N. Bergsdorf, and M. Ranby, Biochim Biophys Acta 719, 318–328 (1982).

18. R.B. Aisina, Biochemistry (Mosc) 74, 1104–1113 (2009).

19. P. Vijayaraghavan, et al., Bio Med Research International 17, 859–871 (2017).

20. N.C. EI-Ayache, et al., Bioorg Med Chem Lett. 20, 966–970 (2010).

21. R. Bos, L. Berger, and W. Nieuwenhuizen, Biochim Biophys Acta. 1117, 188–192 (1992).

22. T. Plug and J.C. Meijers, Thromb Res 146, 7–14 (2016).

23. J. Gamble, S. Kenny, and G.J. Dockray Regul Pept 185, 9–13 (2013).

24. G. Jie, Tianjin University of Science and Technology 12, 89–91 (2004).

25. D. Pennica, et al., Nature 301, 214–221 (1983).

26. M. Tian, L.F. Chen, and J. Gang, Amino Acids and Biotic Resources 29, 18–20 (2007).

27. R.J. Guo, Protein Electrophoresis Technology, pp. 92–118 (Science Press, Beijing, China, 2005).

28. J.D. Bai, Guidelines for Protein Purification (Science Press, Beijing, China, 2nd ed., 2011).

29. Y. Li, S.S. Chen, and W.C. Wang, Genetically Engineered Pharmaceutics, pp. 217–219 (Chemical Industry Press,

Beijing, China, 2002). ◆




Upstream Processing

Achieving Process Balance with Perfusion Bioreactors

Advances in single-use technologies, sensors, and cell retention systems facilitate processes designed for the long run.

CYNTHIA A. CHALLENER

Perfusion cell-culture processes have been used for more

than two decades for the production of unstable/labile

biologic drug substances such as recombinant blood factors.

With rising pressure to reduce costs and streamline bioprocess

development, biopharmaceutical manufacturers are exploring

the application of perfusion to a wider selection of biomolecules.

“Perfusion is not a new concept in this industry, but was

perceived as too complex to run for many years. With recent

availability of perfusion bioreactors and novel cell retention

technologies, there is a renewed interest in perfusion cell culture,”

notes Peter Levison, executive director of business development

at Pall Biotech.

The differences in processing conditions for batch and perfu-

sion cell culture lead to different requirements for bioreactors

used for these processes. Equipment suppliers are responding to

needs for more durable reactors and sensors, a greater variety of

monitoring technologies, and bioreactors that can integrate with

cell retention devices.

Perfusion processes are generally considered to be those in

which the product is continuously harvested from the bioreactor

and new media is fed. These processes can be run for weeks or

even months at a time, while batch processes may last for hours

or a few days at most.

Liquid management is different for batch and perfusion

processes, according to Yasser Kehail, Xcellerex global product

manager at GE Healthcare Life Sciences. “For a perfusion

process, you may need to add a vessel volume per day of cell-

culture media. For a perfusion process run in a 2000-L bioreactor,

2000 L of media may be needed per day vs. 2000 L per run for

a batch process. While the implications of media consumption

from an economic perspective can be determined at small-scale

rather effectively, it is in the scale-up phase where the system

design is imperative for realizing desired process economies at

larger scales,” he explains.

It is also important, according to Levison, to remember that

a perfusion system requires more than a bioreactor alone. “The

most specific need is that the cell retention device and sys-

tem to control the perfusion recirculation flow, cell bleed, and

product-containing flow must be integrated with the bioreac-

CYNTHIA A. CHALLENER, PHD is a contributing editor to

BioPharm International.



Upstream Processing

tor effectively to maintain the culture at

steady state,” he observes. The choice of

the cell retention system, its filter pore

size, and its integration or sterile connec-

tion to the bioreactor are of fundamen-

tal importance for all types of perfusion

processes, agrees Thorsten Adams, head

of bioreactor development at Sartorius

Stedim Biotech. The system must also be

operational under aseptic conditions for

the duration of the perfusion culture.

PERFUSION DESIGN NEEDSFor perfusion processes, the bioreactor

must be designed to withstand long pro-

cess times and support high cell den-

sities. “When looking at a bioreactor

design, there are hardware, consumables,

and automation differences,” Kehail

says. “Because perfusion is an intensified

process, there are more cells and thus a

higher oxygen uptake rate, which results

in a higher carbon dioxide (CO2) evolu-

tion rate,” he adds. Perfusion bioreactors

therefore need larger mass flow control-

lers sized accordingly. In addition, due to

the higher air flow rates, the bioreactor

consumables need a larger exhaust filter.

Sensor design and performance

are also crucial, according to Kehail.

Bioreactor probe reliability is crucial due

to fouling and longer process durations.

“Perfusion bioreactors require system inte-

gration, particularly for fine-tuning of the

media feed. The control strategy usually

involves connecting the media feed to

the vessel weight and feeding this infor-

mation back to the media feed pump.

In addition, to maintain a steady state,

the control strategy for cell bleeding via

the pump is also often fine-tuned using

feedback from a viable cell density probe,”

he comments. Appropriate flow sensors

(either clamp-on or inline) to measure

media feed or permeate and viable cell-

density probes are therefore important.

An extra-large retention port is also

needed in a perfusion bioreactor to con-

nect the cell retention device. This port

must be carefully designed in terms of

the connector type, size, and diameter,

according to Kehail. Seamless integration

of a cell retention device with the perfu-

sion bioreactor is crucial, agrees Levison.

Technologies for cell retention include

gravity settling, pumping through internal

filters, external loop flow-through filters,

and centrifugation. Alternating tangen-

tial flow (ATF) filtration technology

(developed by Refine Technology, now

Repligen) is commonly used.

For perfusion processes used to manu-

facture chimeric antigen receptor (CAR)

T-cell therapies, the size and ease of use of

perfusion systems—in addition to robust-

ness and sterility—significantly impact

the scale-out of these processes, in which

bioreactor farms consisting of many doz-

ens to hundreds of bioreactors are oper-

ated in one facility, according to Adams.

STAINLESS STEEL VS. SINGLE USELong process durations, high oxygen

transfer rates, and short mixing times

are not a challenge for quality stainless-

steel bioreactors equipped with double

mechanical seals or magnetic couplings,

redundant sensors, and exhaust systems,

according to Adams.

Advances made in process analytical

technology (PAT) tools and bioprocess

software also enable implementation of

perfusion processes in stainless-steel bio-

reactors. Adams points to glucose and

lactate sensors, which allow fully auto-

matic glucose feeds, while biomass mea-

surements are suitable for automatic

control of perfusion rates and cell bleed.

Off-gas analysis (O2/CO

2) is now also

used in cell culture to calculate metabolic

flux. “Multivariate data analysis (MVDA)

using software such as our SIMCA

online system allows efficient pro-

cess control and early fault detection by

looking for differences from the ‘golden

batch’,” Adams says. Sartorius Stedim

Biotech also introduced a sampling sys-

tem for stainless-steel bioreactors that

ensures a closed sampling process.

The benefits of single-use technology

align well with continuous processing.

“The inherent advantages of single-use

systems compared to stainless-steel are

speed and flexibility, which are also the

main goals of perfusion applications,”

asserts Kehail. For new processes, for

instance, an extra perfusion port or feed

line for additional media supplements

can be easily added.

In addition, the ability to assure ste-

rility and no introduction of contami-

nants is essential in the longer running

perfusion bioprocesses; sterility also is

facilitated by employing disposable sys-

tems, according to Levison. In addition,

change-over processes are also simplified

using single-use systems.

As with batch and fed-batch pro-

cesses, single-use bioreactors offer numer-

ous other advantages over stainless steel.

“Users can eliminate most of the clean-

ing and validation steps that are associ-

ated with stainless-steel systems, saving

both time and cost. They can also realize

economic benefits with the reduction of

de-risking in the startup phase of a cell

culture process, which is a higher risk and

more time-intensive part of the opera-

tion,” Levison states.

Additionally, Pall Biotech has seen

growing interest in N-1 perfusion biore-

actors to increase the seed density of the

N bioreactor (which is often fed-batch).

The goal, according to Levison, is to

intensify the process and reduce overall

upstream process time, which delivers

even more cost and time savings.

ENSURING PROCESS STABILITYThe longer process durations can be more

challenging for single-use bioreactors.

“The industry has called for continued

innovation in the applications of single-

use technologies,” notes Levison. “Since

perfusion processes can run upwards of

60+ days (some even 120+ days), all of

the equipment and consumables must

be fully reliable, because the biocontainer

cannot be changed partway through the

cell-culture process,” he observes.

Equipment suppliers are address-

ing this issue in a number of ways. Pall

Biotech has optimized its single-use

technologies and focused critical devel-

opment resources on improving controls




Upstream Processing

and sensing to provide real integrity and

guaranteed stability for the bioreactor

run, according to Levison. He adds that

the company has also invested significant

resources on assuring the integrity of its

disposable biocontainers.

Sartorius Stedim Biotech has intro-

duced a bioreactor design with a magneti-

cally driven stirrer shaft that is fixed at the

top and bottom position in the bag holder

to reduce unwanted movements and

ensure long process stability, according to

Adams. It has also developed a biocon-

tainer film that is both robust and flexible

and offers a low and defined leachables

profile. Adams notes that the company’s

single-use bioreactors for perfusion also

incorporate the same sensor and software

platforms provided with its stainless-steel

bioreactors, allowing for the same process

control strategies.

Fluid paths that decrease shear are

key as well, according to Levison. “It is

not just the design of the biocontain-

ers, but also the impellers and aera-

tor that can have a great impact on how

the cells are handled in the process,” he

explains. There have been bioreactor bag

improvements to handle high air flows

by optimizing the seam and its angle,

according to Kehail. He also points to

advances in sparge design for optimiz-

ing oxygen transfer and CO2 stripping.

GE Healthcare Life Sciences’ disposable

bioreactor platform has a sparger plate

for aeration and a separate (Tee-sparger)

for CO2 stripping with its own mass flow

controller, which allows better process

control. Sartorius Stedim Biotech has

developed the combisparger, which com-

prises a microsparger part for efficient O2

transfer combined with a ring-sparger

part to provide an automatically regulated

air stream for CO2 stripping.

Advances have also been made with

respect to the reliability of the one-inch

sterile connectors on bioreactor bags

required for some retention devices,

according to Kehail. New flow sensors

with improved accuracy for measure

media addition and the permeate have

also been introduced.

“Suppliers and many others in the

industry are looking at how further design

and development can optimize the pro-

duction environment. As the industry

pushes towards process intensification and

even continuous processing solutions, this

is the next logical step,” Levison asserts.

The fact remains, however, that run-

ning a perfusion process with a produc-

tion bioreactor and an external perfusion

system is still complex and requires expert

skills that are not available ubiquitously in

the industry, according to Adams.

Foam formation has also yet to be

addressed, according to Kehail. Foam can

be generated due to the high air flow rates

in perfusion processes, and new, reliable

foam sensors are needed to prevent pro-

cess failures.

SIMPLIFYING CELL RETENTIONTo maintain steady-state conditions, the

perfusion rate, cell bleed rate, and prod-

uct harvest rate must all be synchronized,

monitored, and controlled. One way to

simplify perfusion processes is to have

the cell retention device closely connected

to the perfusion bioreactor, yet have the

flexibility to operate with multiple com-

mercially available perfusion bioreactor

solutions, according to Levison.

Pall Biotech is working on adopting

acoustic wave separation (AWS) tech-

nology, originally developed for batch

applications, for perfusion processes.

The company is gathering data at the

process development and intermediate

scales with the goal of commercial-scale

application. “Our team has focused on

running AWS perfusion technology con-

nected with, but agnostic to, the bioreac-

tor design so there would be no impact

on the product quality or continuous cell

culture,” Levison comments.

AWS retains recirculating cells from

a perfusion bioreactor without the need

for a hollow fiber filter device, which is

a widely used approach for cell retention.

No membrane means no fouling or loss

of product, according to Levison. “The

results we have seen so far show success-

ful, simplified integration of the AWS

cell retention technology with perfusion

bioreactors at cell densities of up to 100

million cells/mL with 100% product

transmission under typical process condi-

tions used in the continuous production of

biologics,” he notes.

Sartorius Stedim Biotech is work-

ing with Repligen to develop integration

of the Repligen ATF system into the

Sartorius bioreactor with respect to both

process control and consumables, accord-

ing to Adams. “The goal is to create a

unique plug-and-play technology that

allows the running of high-end perfu-

sion and concentrated fed-batch processes

with predefined templates, thus reducing

the complexity and unlocking the power

of perfusion for the ‘novice’ user,” he says.

PROCESS DESIGNIn addition to the design of bioreactors,

the design of perfusion processes impact

their successful implementation. “When

it comes to systems integration and con-

trol, cells are most happy in a steady state;

control response, sensor accuracy, and so

on can have an impact, and this is critical

to keep in mind for scaling considerations,”

Kehail asserts. He points to the feed rate

and its effect on pH and osmolarity as

important issues to consider. “It is impor-

tant to design a process with a balanced

feed rate that ensures the target is reached

without compromising the critical quality

attributes of the product,” he states.

Running longer processes requires

additional mitigation steps, according to

Levison. When perfusion processes are

run for 90 days or longer, mitigation of

the cost of the perfusion media over time

is crucial, as is the need to prevent the

fouling of cell retention devices. In the

former case, he notes that the industry is

working to address the issue with the goal

of optimizing the cell culture conditions

needed to run perfusion processes reli-

ably and successfully. To address the latter

problem, Levison recommends having

a remediation plan in place along with a

redundant system as a backup to ensure

the continuity of a continuous upstream

process. X




Manufacturing

SV

ETA

/S

TO

CK

.AD

OB

E.C

OM

Overcoming Challenges in ADC Bioconjugation at Commercial Scale

Bioconjugation requires aseptic manufacturing and containment for cytotoxic payloads.

CYNTHIA A. CHALLENER

Antibody-drug conjugates (ADCs) comprise one of

the fastest growing segments of the biopharmaceuti-

cal market. Consisting of antibodies linked to highly

potent (cytotoxic) small-molecule “payloads,” ADCs are

designed for the targeted delivery of therapeutic actives. The

antibodies are engineered to bind to specific cells—most

often tumor cells—and then release the active agent, avoid-

ing the damage to healthy cells observed with traditional

systemic treatments. This specificity has attracted significant

interest.

Today there are nearly 200 ADCs in development, with

60% in the discovery and preclinical stages and 20 products

approved or in late stages of clinical development (Phase

II and above) (1). The global ADC market is expected to

expand at a compound annual growth rate of 19.4% between

2017 and 2030. The newer ADCs are being developed with

novel conjugation approaches, more potent warheads, and

modified linker technologies. They are also being tested in

combination with other novel therapies, such as immune

checkpoint inhibitors and epigenetic modulators (1).

Initial conjugation strategies suffered from a lack of speci-

ficity. These issues have been addressed using a number of

different approaches. Examples of third-generation ADC

conjugation platforms include avoiding/limiting retro-

Michael drug de-conjugation, cysteine re-bridging, enzyme-

assisted ligation, glycan re-modeling, and ligation at the

nucleotide-binding site of the antigen-binding fragment (1).

As more products are approved and other candidates

move into later-stage clinical trials, the capability for large-

scale production of ADCs is increasing. Because these

complex therapies include both large- and small-molecule

components and are highly potent, they present many manu-

facturing challenges.

SCALING UPDesigning the correct process equipment for larger-

scale clinical and commercial good manufacturing prac-

CYNTHIA A. CHALLENER is a contributing editor to BioPharm

International.




Manufacturing

t ice (GMP) bioconjugat ion of

payloads to antibodies for ADC

production must be based on the

outcome of appropriate process

development activities, according

to Jyothi Swamy, associate director

of ADC/Bioconjugation Contract

Manufacturing at MilliporeSigma.

Understanding the critical process

parameters drives the design of the

process equipment. “Volume-range

requirements, the order of addition,

mixing needs, and in-process mon-

itoring instrumentation are key fac-

tors in equipment design,” she notes.

Multi-use fixed equipment must also

be designed to ensure validated decon-

tamination and cleaning.

“Development campaigns should

begin with the final scale and equip-

ment in mind, and all development

trials should be designed accordingly,”

Swamy adds. “If the development

strategy has a process for controlling

scale changes from the earliest batches,

challenges should be minimal for com-

mercial processes,” she says.

Assuming reaction stoichiometry,

pH, buffer composition, temperature

control, reaction time and fluid sheer

rates, and mixing time are all under

control, process hold times, protec-

tion of employees from cytotoxic raw

materials, and product change-over for

multiproduct facilities become the next

series of challenges associated with

scale-up, according to Thomas Rohrer,

associate director of commercial devel-

opment for bioconjugates at Lonza

Pharma & Biotech.

“Process hold times during the mod-

ification and conjugation reactions

must be tested to determine whether

holding the antibody-linker complex

for a period of time following the anti-

body modification reaction causes any

change in the reaction stoichiometry,”

he explains.

A comprehensive occupational

hygiene monitoring program that can

detect cytotoxins in the 10-9 g range

in room air and on process surfaces is

also required to protect manufacturing

personnel from residual cytotoxins fol-

lowing a manufacturing campaign. In

addition, the cytotoxins used for ADC

manufacturing may remain unchanged

or may undergo degradation or deac-

tivation during the product changeover

cleaning, so a unique detection method

and cleaning procedure may need to be

developed, according to Rohrer.

ENSURING CONTAINMENTADCs for human clinical applica-

tions must be manufactured in an

aseptic biological environment operat-

ing under current GMP. This aseptic

environment must also isolate man-

ufacturing personnel from exposure

to cytotoxic chemicals. “The equip-

ment used in ADC manufacturing

must protect employees from exposure

to cytotoxins and prevent microbial

contamination of the process inter-

mediates and drug product during the

conjugation process,” Rohrer says.

Designing a facility

(both clinical and

commercial) that can

meet both GMP and

safety requirements

is the key.

Designing a facility (both clinical

and commercial) that can meet both

GMP and safety requirements is the

key, agrees Swamy. “For ADC pro-

cessing, a cleanroom environment is

required while maintaining contain-

ment for potent compounds using

engineering controls,” she says.

Both primary and secondary con-

tainment of the toxin are required.

The primary containment strategy

includes handling the unconjugated

cytotoxin in a closed front isolator

cabinet under negative pressure in

a room with negative pressure in

relation to surrounding cleanrooms,

according to Rohrer. Hermetically

sealed process vessels and equipment

are then used to execute the process

while protecting the valuable raw

materials from contamination.

If the primary containment should

fail , the secondary containment

reduces the exposure of manufacturing

personnel to the cytotoxin by exchang-

ing the room air in the process suite

more than 30 times each hour. The

high-efficiency particulate (HEPA)

filters that serve the manufacturing

area are designed as safe-change filters

to protect personnel during preventive

maintenance. Personnel working in

the manufacturing area wear one-way

over-gowning that is removed before

exiting the protective air locks.

Specific modifications for pro-

duction equipment used in biocon-

jugation processes include double

mechanical seals and overflow trays,

which strengthen the primary con-

tainment envelope, according to

Rohrer. In addition, all waste gener-

ated during the manufacturing pro-

cess that may contain cytotoxin must

be inactivated.

If inactivation conditions are

not compatible with stainless steel,

Rohrer points out that incineration

of all liquid and solid process waste

is recommended to prevent introduc-

tion of cytotoxic substances into the

environment.

Other important considerations

for larger commercial-scale biocon-

jugations noted by Swamy include

the use of larger amounts of toxic

payloads and typically large volumes

due to the dilute nature of most con-

jugation chemistry.

TRAINING OPERATORSThe need to understand contain-

ment practices is just one type of

Contin. on page 48




Outsourcing

Iun

limit

3d

/Ad

ob

e.c

om

Contract Organizations Expanded in Autumn

CMOs and CDMOs made investments in new and expanded facilities and services in the last quarter of 2018.

SUSAN HAIGNEY

Contract manufacturing organizations (CMOs) and con-

tract research and development organizations (CDMOs)

invested in expanded facilities and services in late 2018.

The following are some recent investments for biologic drug

development and manufacturing.

NEW AND EXPANDED FACILITIESAGC Biologics, a clinical and commercial manufacturer of

therapeutic proteins, announced on Sept. 20, 2018 that it

will establish a new process development and manufacturing

facility at its CDMO facility in Chiba, Japan, as part of its

ongoing program to expand its production capacities globally

(1). The new facility will contain single-use bioreactors at the

500- and 2000-L scale and will be suited for the production

of monoclonal antibodies (mAbs), fusion proteins, and other

types of therapeutic proteins. The facility is expected to be

operational in the second half of 2019.

COLLABORATIONSFujifilm Diosynth Biotechnologies (FDB) announced on

Nov. 1, 2018 that its collaboration with UK-based Centre

for Process Innovation (CPI), for the project AMECRYS:

Revolutionizing Downstream Processing of Monoclonal

Antibodies by Continuous Template-Assisted Membrane

Crystallisation, has hit an important milestone with the

successful technology transfer of the expression and purifi-

cation of model mAbs from FDB to CPI (2). AMECRYS

is a research project funded by the European Commission

under the Horizon 2020 program in the framework of

Future and Emerging Technologies actions (FET-OPEN),

which supports early stages of science and technology

research and innovation.

Charles River Laboratories International announced on

Oct. 25, 2018 that it has entered into an exclusive partner-

ship with Distributed Bio, a company specializing in com-

putational design and optimization of antibody discovery

platforms, that grants Charles River access to Distributed

Bio’s antibody libraries and integrated antibody optimization

technologies (3). Distributed Bio’s libraries are computation-

ally optimized for sequence diversity and engineering fitness

through the analysis of thousands of human antibody rep-

ertoires and all known monoclonal therapeutics in clinical



You drive development.

We’ll offer directions.If laboratory roadblocks have you seeing double, our insourcing solutions at your site will surpass your wildest expectations on your way to market approval.

Eurofins Lancaster Laboratories’ award-winning PSS Insourcing Solutions® offers the most advanced, sophisticated biopharmaceutical managed laboratory testing services from early phase development to finished product testing, as well as comprehensive laboratory management, including:

• GMP LEAN Laboratory Design and Validation • Regulatory and Technical Training • LEAN Project Support/Management • Upstream and Downstream Services

Partner with PSS and enjoy the ride.

www.EurofinsLancasterLabs.com


http://www.EurofinsLancasterLabs.com

https://www.eurofins.in/


Outsourcing

trials. The companies expect to create

an end-to-end platform for therapeutic

antibody discovery and development.

NEW INVESTMENTSADC Biotechnology (ADC Bio) has

secured additional funding of $3.18

million (£2.5 million) from existing

investors and company management

to ensure the achievement of specific

business goals within the company’s

overarching corporate strategy.

“We are delighted to have obtained

this additional injection of funds

that will be used to support the com-

pany’s strategic aspirations, including

conceptual design of a downstream

formulation and filling operation to

complement our existing bioconjuga-

tion operations at the Deeside facility.

We are also looking to fully exploit our

core Lock-Release technology to create

a transformative manufacturing para-

digm that will significantly streamline

ADC manufacturing supply chains,”

said Charlie Johnson, CEO of ADC

Bio, in a company press statement (4).

REFERENCES1. AGC Biologics, “AGC Biologics Adds

Mammalian Production Capacity in Japan,” Press Release, Sept. 20, 2018, www.agcbio.com/resource-center/

news/agc-biologics-adds-mammalian-production-capacity-in-japan

2. Fujifilm, “Fujifilm Announces that

AMECRYS Collaboration Reaches Project

Milestone to Develop New Technologies

to Improve Downstream Processing of

Monoclonal Antibodies,” Press Release,

Nov. 1, 2018, www.fujifilmusa.com/press/

news/display_news?newsID=881545

3. Charles River, “Charles River and

Distributed Bio Enter Exclusive

Partnership to Create an Integrated

Antibody Discover and Development

Platform,” Press Release, Oct.

25, 2018, http://ir.criver.com/

phoenix.zhtml?c=121668&p=irol-

newsArticle&ID=2373437

4. ADC Bio, “ADC Bio Secures Additional

Equity Round Investment,” Press Release,

Nov. 14, 2018, http://adcbio.com/aer/ ◆

determined total expanded uncertain-

ties in these values to be between 5%

and 13%, depending on the spectral

region and type of bead (5). By using

these beads and their assigned values,

users calibrate their flow cytometers to

a traceable fluorescence intensity scale

that can then be used to measure ERF

values for any fluorescence signal on

that instrument.

ANTIBODY QUALITY AND CHARACTERIZATIONIt has been estimated that more than

$800 million a year are wasted due

to poor quality antibody reagents (6).

To address this problem, an NIH-

led working group has proposed that

reagent and document standards

should be developed to determine the

quality of reagents and ensure repro-

ducibility in outcomes involving their

use (6). NIST is responding to this

need by measuring quantitative fluo-

rescence characteristics of antibodies,

such as antibody-cell binding kinet-

ics, lifetimes, quantum yields, and

corrected spectra, and using these mea-

surement quantities to determine anti-

body binding characteristics through

mathematical modeling. The objective

of these measurements is to identify

a set of antibody quality parameters

that are quantitatively correlated with

the different fluorescence intensities

measured by flow cytometry for these

fluorescently labeled antibodies bound

to the same cell-associated antigens.

When this correlation is well under-

stood, these antibody parameters may

be used as quantitative criteria for

determining antibody quality.

NIST will continue to work with

stakeholders in the flow cytometry

community and other key applica-

tion areas (e.g., quantitative poly-

merase chain reaction, enzyme-linked

immunosorbent assay, and fluores-

cence microscopy) where fluores-

cence detection is important to ensure

more reproducible and accurate mea-

surement science for improving the

quality of commercial products and

services. An ultimate goal of quantita-

tive flow cytometry is to measure the

number of antigens or ligand-binding

sites associated with a cell through

the measurement of ABC (7,8). To

have sufficient measurement assurance

in flow cytometry, many sources of

uncertainty in the measurement pro-

cess have to be taken into consider-

ation, including cytometer calibration

and standardization, antibody qual-

ity, cell count and viability, process

control materials, and cell population

gating, just to name a few. Offering

a set of fluorescence-based measure-

ment services is one of the ways NIST

is using its expertise to work with

stakeholders to improve the measure-

ment confidence in fluorescence-based

application fields including, but not

limited to, flow cytometry.

REFERENCES1. NIST, “Flow Cytometry Quantitation

Consortium,” Fed. Regist. 2016; 81(136), 46054-46055 (July 15, 2016), www.nist.gov/programs-projects/quantitative-flow-cytometry-measurements

2. ASTM E 578 -01, “Linearity of Fluorescence Measuring System,” Annual Book of ASTM standards, Vol 03.06 (2001, original version 1983).

3. NIST, Certificate of Analysis, Standard Reference Material 1934,

“Fluorescent Dyes for Quantitative Flow Cytometry (Visible Spectral Range)” (2016), wwws.nist.gov/srmors/view_cert.cfm?srm=1934

4. D. Ripple and P.C. DeRose, J.Res. NIST, 123 -002 (2018).

5. L. Wang, P.C. DeRose, and A.K. Gaigalas, J.Res. NIST, 121, 264-281 (2016).

6. Nature Methods, September 2016, Special Issue on Antibody Reagent Quality.

7. L. Wang, et al., Curr. Protoc. Cytom. 75:1.29.1-1.29.14 (2016).

8. L. Wang, A.K. Gaigalas, and J. Wood, Flow Cytometry Protocols, (Humana Press/Springer, New York, NY, 4th ed., 2018) pp. 93-110. ◆

Quality: Lab Operations — Contin. from page 29


http://adcbio.com/aer/

http://www.nist


http://www.agcbio.com/

http://www.agcbio.com/

https://www.fujifilmusa.com/press/news/display_news?newsID=881545

http://ir.criver.com/phoenix.zhtml?c=121668&p=irol-irhome

http://www.nist



Eurofins Lancaster Laboratories Professional Scientific Services® (PSS) is a global, award-win-ning insourcing solution that places our people at your site dedicated to running and manag-

ing your laboratory services while eliminating headcount, co-employment and pro-ject-management worries.

We infuse our 55-year track record of scientific and

laboratory operations expertise, as well as HR and great place to work best practices, to recruit, hire, train, and manage highly qualified scien-tists to perform laboratory services at your site, using your quality systems and equipment. Our teams will even help you set up your laboratory and validate equipment according to your SOPs and Lean laboratory practices as needed.

Eurof ins Lancaster Laboratories PSS employs and manages full-time employees and provides a comprehensive benefits package, as well as training, development, and career advancement opportunities. Offering these additional benefits allows us to attract, retain, and motivate high-caliber employees to serve you. Our on-site dedicated leaders manage our full-time employees and provide you with scientific insourced services free from co-em-ployment. PSS is not staff augmentation, so it solves the challenges associated with the EU Temporary Agency’s Workers Directive 2008/104.

Major Products/ServicesOur PSS Insourcing Solution® provides ser-vices for clients who require lab services at their facility. We have proven success provid-ing dedicated teams for a variety of technical disciplines that span the drug development pipeline, including:• Analytical chemistry• Microbiology

EUROFINS LANCASTER

LABORATORIES

PROFESSIONAL SCIENTIFIC

SERVICES®

2425 New Holland Pike, Lancaster, PA, 17601, United States

TELEPHONE

717.656.2300

E-MAIL

[email protected]

WEB SITE

www.EurofinsLancasterLabs.com/PSS

NUMBER OF EMPLOYEES

<1600

YEAR FOUNDED

2002

Eurofi ns Lancaster Laboratories

Professional Scientifi c Services®

• Environmental monitoring• Proteomics• Biochemistry• Quality assurance• Sampling• Stability• Biopharmaceuticals• Process development• Biomedical engineering• Method development/method validation• Microscopy• Bioinformatics• Genomics• Cell and molecular biology• Immunochemistry• Sample management• Medical device testing• Engineering• Project management• Bioassay characterization.

FacilitiesWith more than 1600 employees worldwide, Eurofins Lancaster Laboratories PSS pro-vides services at more than 70 sites in over 15 countries throughout North America and Europe and is part of Eurofins BioPharma Product Testing, which operates 34 laboratory locations, totaling more than 1,600,000 ft2 across 17 countries worldwide.

Markets ServedWith more than 70 client sites worldwide, our PSS Insourcing Solution® provides the optimal external service provider in house for pharmaceutical and biopharmaceutical com-panies around the world.




http://www.EurofinsLancasterLabs

http://www.EurofinsLancasterLabs

https://www.eurofinsus.com/


Manufacturing

expertise required by operators per-

forming bioconjugations to produce

ADCs. Knowledge of both small-

molecule and biologic manufacturing

is needed, because aspects of both are

involved, according to Swamy. Lonza

has found that site manufacturing

personnel with aseptic or high-

potency operations experience are

ideal candidates for ADC bioconju-

gations operators. “Employees with

this base level of experience can rap-

idly transition into ADC operations

with some additional ADC-specific

training,” Rohrer says.

AGGREGATE POTENTIALThe linkage of small-molecule cyto-

toxic payloads to larger biomolecules

creates some additional challenges

with respect to the stability of the

products. All protein therapeutics

made for c linical or commercial

applications may harbor high-molec-

ular-weight variants (i.e., aggregates)

that have the potential to reduce the

potency of the drug when scale-up is

performed, according to Rohrer.

“This issue is magnified in ADCs

because many of the cytotoxic drugs

conjugated to the antibodies are hydro-

phobic, thus increasing the chances of

aggregate formation during the manu-

facturing of the drug substance and

subsequent storage of the drug prod-

uct,” he explains. “Analytical methods

used to detect high-molecular-weight

variants and residual amounts of free

drug must have the precision, sensi-

tivity, and selectivity to detect these

process-related impurities,” he adds.

CUSTOMIZED ANALYTICAL SOLUTIONSAll analytical methods used dur-

ing the bioconjugation process must

be robust and suitable for valida-

tion, according to Swamy. The most

appropriate analytical methods for a

given ADC depend on the proper-

ties of the drug, the linker, and the

choice of conjugation sites, notes

Rohrer.

“The clinical efficacy of an ADC

reflects the target site specificity and

binding properties of the antibody,

the in-vitro and in-vivo stability of

the linker, and the potency of the

drug payload. These unique proper-

ties require an in-depth understanding

of the physicochemical properties of

the individual and combined elements

of the ADC, so appropriate analyti-

cal and bioanalytical methods must

be selected to monitor the functional

attributes of the resulting drug sub-

stance,” he observes.

The most

appropriate

analytical methods

for a given ADC

depend on the

properties of the

drug, the linker,

and the choice of

conjugation sites.

On-line and at-line PAT testing

can be added at later stages of the

development process to enable better

monitoring—and hence understand-

ing—the progress of a bioconjuga-

tion process, according to Swamy.

These techniques must, however, be

verified against existing methods. “As

the bioconjugation process matures,

the addition of data-trending and

automated processing can also reduce

the off-line testing required,” she

comments.

PROPER CLEANINGBecause bioconjugations processes

for ADC production are complex

and require specialized expertise in

the handling and processing of cyto-

toxic materials—combined with the

need for aseptic manufacturing and

expertise in biologic and chemical

APIs—many pharmaceutical compa-

nies outsource these activities to con-

tract development and manufacturing

organizations (CDMOs). CDMOs

operate multiproduct facilities, which

present additional challenges for

ADC bioconjugations.

“One of the biggest challenges with

ADC manufacturing is cleaning and

decontamination of the processing

equipment. This challenge is mitigated

by the use of complete single-use tech-

nology, including the processing equip-

ment,” Swamy asserts. She adds that

the use of complete single-use technol-

ogy also provides operator safety.

Rohrer agrees: “When reaction

conditions permit, introduction of

single-use equipment into the ADC

manufacturing paradigm can help

with product change-over.”

Notably, problems associated with

the introduction of new cytotoxic

drugs that are hydrophobic and

therefore difficult to remove from

product contact surfaces using tra-

ditional cleaning strategies can be

solved by using disposable equipment.

“Single-use equipment is well-

established at the laboratory scale, and

the lower operating/validation cost at

manufacturing scales combined with

the development of polymer films

engineered to meet the solvent require-

ments of ADC manufacturing has

encouraged wider use,” notes Rohrer.

REFERENCE1. Roots Analysis, Antibody Drug

Conjugates Market (4th Edition), 2017-2030, Oct. 27, 2017, www.rootsanalysis.com/reports/view_document/antibody-drug-conjugates-market-4th-edition-2017-2030/180.html ◆

Manufacturing — Contin. from page 43


http://www.rootsanalysis



Ask the Expert — Contin. from page 50

Ask the Expert

10 times over the course of four

months. You finally decide to ques-

tion the ability of the operator to

do the job correctly and bring your

concerns to management.

Your boss asks if anyone has

interviewed the operator directly

to find out why he is having this

issue with the batch record. You

say no, that you have relied on the

opinion of the supervisor. The boss

recommends you interview the

operator before demoting him.

When you talk to the operator, he

informs you that in order to sign

the batch record when it needs to be

signed, he needs to exit the aseptic

core, degown, sign the batch record,

and regown, leaving the product

unattended during that time. The

operator tells you he chose to stay

with the product and sign the batch

record later but sometimes forgot

after the manufacturing run. In this

simple exchange with the operator

you realize that the root cause of

the repeat deviation is not a result

of human error but a result of poor

process flow.

The question you need to address

now is how were other operators

handling the situation? By not tak-

ing the time to perform the initial

investigation thoroughly, you have

created a data integrity nightmare

because you now need to review all

the batch records completed by the

other operators to determine if the

product is still acceptable.

Admittedly, this is a simplistic

example, but it certainly exemplifies

the importance of opting to perform

a complete and thorough investi-

gation over meeting an artificially

imposed time frame. Explaining to

an inspector during an audit that

you didn’t perform a thorough

investigation because you needed

to meet an arbitrary time frame is

not a position you want your com-

pany to be in. You also don’t want

to explain why you closed an inves-

tigation to meet the time frame and

then felt compelled to reopen it after

the batch was released because you

had concerns about its conclusions.

QUALITY OVER BREVITYThe other element that needs to

be addressed is that of the preva-

lent culture existing in the orga-

nization. It is good to set a time

goal for performing investiga-

tion, thus ensuring their timely

completion. It is not acceptable to

have the time frame be the driv-

ing force behind the investigation.

Management needs to emphasize

their commitment to having thor-

ough investigations as opposed to

incomplete investigations that meet

the self-imposed time frame. It is

ideal when an investigation is com-

pleted and a true root cause identi-

fied in the specified time frame but,

if that is not achievable, manage-

ment needs to be clear that they

prefer the identification of the true

root cause over the rushed investi-

gation that merely checks the box

for completion in a timely manner.

Without this management com-

mitment, the premature closing of

investigations will likely continue.

Investigations need to focus on

determining root cause in a timely

manner. The length of time it

takes to complete an investigation

depends on the complexity of the

investigation. The primary driver

for avoiding compliance and data

integrity risks concerning investi-

gations is arriving at a root cause

in a timely manner. This allows

you to be confident in presenting

your investigations during inspec-

tion and avoiding unnecessary

scrutiny when the investigation is

rushed and a conclusion is reached

prematurely.

REFERENCES 1. FDA, 21 CFR 211.22(a), Current Good

Manufacturing Practice for Finished

Pharmaceuticals, Responsibilities of

Quality Control Unit, Sept. 29, 1978.

2. FDA, 21 CFR 211.192, Current Good

Manufacturing Practice for Finished

Pharmaceuticals, Production Record

Review, Sept. 29, 1978.

3. European Commission, Eudralex,

Volume 4, Good Manufacturing Practice

(GMP) guidelines, Chapter 1–

Pharmaceutical Quality System (EC,

January 2013). ◆

Ad Index

Company Page

AB SCIEX 23

AJINOMOTO ALTHEA 20,21

EAGLE STAINLESS CONTAINER 51

EUROFINS LANCASTER LABORATORIES 45,47

PDA 10,11

SARTORIUS STEDIM N AMERICA INC 6,7

SGS 32,33

THERMO FISHER SCIENTIFIC 13,14-15

TOSOH BIOSCIENCE 17

VIRONOVA AB 25,26-27

WUXI BIOLOGICS US 2,52



50 BioPharm International www.biopharminternational.com December 2018

Ask the Expert

Investigation Timeliness vs. Thoroughness: Finding the Right BalanceA required time frame should not be the driving force behind root cause investigations, says Susan Schniepp, executive vice-president of Post-Approval Pharma and Distinguished Fellow, Regulatory Compliance Associates.

Contin. on page 49

Fa

na

tic S

tud

io/G

ett

y I

ma

ge

s

Susan Schniepp is executive vice-president,

Post-approval Pharmaceuticals and

distinguished fellow at Regulatory Compliance

Associates.

Q. I have just been promoted to be in

charge of investigations for my com-

pany. Our standard operating procedure (SOP)

requires us to complete investigations in 30

days. Depending on the nature of the investiga-

tion and to meet the SOP requirement, I have

started to close investigations at the 30-day

time point even though I think the investiga-

tion might not be complete. Sometimes I have

had to re-open investigations because the prob-

lem recurs, confirming that the investigation

was not completed. Do I have a compliance risk

if I continue with this practice?

A. The short answer is yes, you have a com-

pliance risk. You probably also have a

data integrity issue and a quality culture issue

to accompany your compliance risk.

There is no time element associated with

conducting investigations. Thirty days is an

arbitrary number pharmaceutical companies

impose on themselves. The US Code of Federal

Regulations states “… if errors have occurred,

that they have been fully investigated” (1), and

“Any unexplained discrepancy … shall be thor-

oughly investigated, whether or not the batch

has already been distributed” (2). Europe’s

EudraLex also addresses investigations by stat-

ing, “An appropriate level of root cause analysis

should be applied during the investigation of

deviations …” (3). None of these citations indi-

cate a time for completion of an investigation.

What they do imply is that investigations need

to be thorough and determine root cause. In

some cases, the investigation and root cause

can be easily determined in the defined SOP

time frame of 30 days. In other cases, the inves-

tigation may be more complicated and could

exceed the time frame requirement of 30 days.

To address this potential discrepancy, your SOP

should allow for investigation extensions. The

length of the extension request should be made

based on the complexity of the investigation.

DATA INTEGRITY PROBLEMSWhen an investigation is rushed, the organiza-

tion leaves itself vulnerable. Suppose, for example,

you have a second shift manufacturing opera-

tor who continually forgets to sign a step in the

batch record for a specific product. This opera-

tor is the only one who seems to have this issue.

Your initial investigation into the first occurrence

of the issue determines a root cause of human

error. Because the operator works on the second

shift, it is inconvenient to interview him directly,

so you rely on the word of his supervisor that

this was just a case of human error. You decide to

retrain the operator on the proper use of filling

out the form and skip the operator interview in

order to complete the investigation and perform

the retraining in the allotted 30-day time frame.

A few weeks later, the same operator makes

the same mistake. You review the previous

investigation, arrive at the same conclusion, and

perform the retraining of the operator empha-

sizing the importance of correctly filling out

the batch record. This scenario repeats itself

SSSusan SSSchhhniiiepp iiis

When an investiga-

tion is rushed, the

organization leaves

itself vulnerable.



Highly-Polished SanitaryStainless Steel Vessels FOR THE PHARMACEUTICAL INDUSTRY

CTL SERIES

(10L TO 300L)

BTB SERIES

(0.5L TO 30L)

STB SERIES

(1mL TO 750mL)

CTH SERIES

(4L TO 500L)

ISO 9001 CertifiedEagleStainless.com | 215.957.9333

I TT SERIES SPOTLIGHT> Slanted Bottom with

Tri-Clamp Drain Port

> Optional Matching Dolly

> Optional CTH/CTL Lid with PSIL Gasket

> Certifications Available- 316L MOC Lot Traceability- 10 Ra Surface Finish

KTT SERIES - INSIDE DETAIL

(20L TO 200L)


https://www.eaglestainless.com/

https://www.eaglestainless.com/

latform PA new era in bispecifics

™

9-18 months faster development and no CMC challenges

Our highly-flexible, proprietary system:

o Reduces the drug development timeline by 6 to 18 months

o Lowers the cost of manufacturing by as much as 90%

[email protected]

www.wuxibiologics.com

HKEX: 2269.HK



http://www.wuxibiologics.com

http://www.wuxibiologics.com

https://www.wuxibiologics.com/services-solutions/discovery/wuxibody-bispecific-antibody-platform/

Documents

Volume 31 Number 12 BioPharm - files.alfresco.mjh.groupfiles.alfresco.mjh.group/alfresco_images/pharma/... · 12/10/2018 · lab and manufacturing space, our network of facilities