Volume 29 Number 7 BioPharm · 2018-09-11 · Publisher Mike Tracey [email protected] National...

The Science & Business of Biopharmaceuticals

BioPharmINTERNATIONAL

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July 2016

Volume 29 Number 7

MANAGING BIOMANUFACTURING

CAPACITY EXPECTATIONS

QUALITY

MICROBIOLOGICAL

TESTING: TIME IS

OF THE ESSENCE

PEER-REVIEWED

BIOPROCESSING TECHNOLOGY

TRENDS OF RNA-BASED

THERAPEUTICS AND VACCINES

ANALYTICAL TESTING

FORCED DEGRADATION

STUDIES FOR

BIOPHARMACEUTICALS

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INTERNATIONAL

BioPharmThe Science & Business of Biopharmaceuticals

EDITORIAL

Editorial Director Rita Peters [email protected]

Senior Editor Agnes Shanley [email protected]

Managing Editor Susan Haigney [email protected]

Science Editor Randi Hernandez [email protected]

Science Editor Adeline Siew, PhD [email protected]

Community Manager Caroline Hroncich [email protected]

Art Director Dan Ward [email protected]

Contributing Editors Jill Wechsler, Jim Miller, Eric Langer, Anurag Rathore, Jerold Martin, Simon Chalk, and Cynthia A. Challener, PhD

Correspondent Sean Milmo (Europe, [email protected])

ADVERTISING

Publisher Mike Tracey [email protected]

National Sales Manager Steve Hermer [email protected]

East Coast Sales Manager Scott Vail [email protected]

European Sales Manager Linda Hewitt [email protected]

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

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

PRODUCTION

Production Manager Jesse Singer [email protected]

AUDIENCE DEVELOPMENT

Audience Development Rochelle Ballou [email protected]

© 2016 UBM. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical including by photocopy, recording, or information storage and retrieval without permission in writing from the publisher. Authorization to photocopy items for internal/educational or personal use, or the internal /educational or personal use of specific clients is granted by UBM for libraries and other users registered with the Copyright Clearance Center, 222 Rosewood Dr. Danvers, MA 01923, 978-750-8400 fax 978-646-8700 or visit http://www.copyright.com online. For uses beyond those listed above, please direct your written request to Permission Dept. fax 440-756-5255 or email: [email protected].

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EDITORIAL ADVISORY BOARDBioPharm International’s Editorial Advisory Board comprises distinguished

specialists involved in the biologic manufacture of therapeutic drugs,

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

editors and advise them on biotechnology trends, identify potential

authors, and review manuscripts submitted for publication.

K. A. Ajit-Simh President, Shiba Associates

Rory Budihandojo Director, Quality and EHS Audit

Boehringer-Ingelheim

Edward G. Calamai Managing Partner

Pharmaceutical Manufacturing

and Compliance Associates, LLC

Suggy S. Chrai President and CEO

The Chrai Associates

Leonard J. Goren Global Leader, Human Identity

Division, GE Healthcare

Uwe Gottschalk Vice-President,

Chief Technology Officer,

Pharma/Biotech

Lonza AG

Fiona M. Greer Global Director,

BioPharma Services Development

SGS Life Science Services

Rajesh K. Gupta Vaccinnologist and Microbiologist

Jean F. Huxsoll Senior Director, Quality

Product Supply Biotech

Bayer Healthcare Pharmaceuticals

Denny Kraichely Associate Director

Johnson & Johnson

Stephan O. Krause Director of QA Technology

AstraZeneca Biologics

Steven S. Kuwahara Principal Consultant

GXP BioTechnology LLC

Eric S. Langer President and Managing Partner

BioPlan Associates, Inc.

Howard L. Levine President

BioProcess Technology Consultants

Herb Lutz Principal Consulting Engineer

Merck Millipore

Jerold Martin Independent Consultant

Hans-Peter Meyer Lecturer, University of Applied Sciences

and Arts Western Switzerland,

Institute of Life Technologies.

K. John Morrow President, Newport Biotech

David Radspinner Global Head of Sales—Bioproduction

Thermo Fisher Scientific

Tom Ransohoff Vice-President and Senior Consultant

BioProcess Technology Consultants

Anurag Rathore Biotech CMC Consultant

Faculty Member, Indian Institute of

Technology

Susan J. Schniepp Fellow

Regulatory Compliance Associates, Inc.

Tim Schofield Senior Fellow

MedImmune LLC

Paula Shadle Principal Consultant,

Shadle Consulting

Alexander F. Sito President,

BioValidation

Michiel E. Ultee Principal

Ulteemit BioConsulting

Thomas J. Vanden Boom VP, Biosimilars Pharmaceutical Sciences

Pfizer

Krish Venkat Managing Partner

Anven Research

Steven Walfish Principal Scientific Liaison

USP

Gary Walsh Professor

Department of Chemical and

Environmental Sciences and Materials

and Surface Science Institute

University of Limerick, Ireland

4 BioPharm International www.biopharminternational.com July 2016

Contents


BioPharm International integrates the science and business of

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

peer-reviewed technical solutions to enable biopharmaceutical professionals

to perform their jobs more effectively.

COLUMNS AND DEPARTMENTS

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

BioPharm InternationalJTTFMFDUJWFMZBCTUSBDUFEPSJOEFYFEJOrBiological Sciences Database (Cambridge Scientifi c Abstracts)rBiotechnology and Bioengineering Database (Cambridge Scientifi c Abstracts)rBiotechnology Citation Index (ISI/Thomson Scientifi c)rChemical Abstracts (CAS) rŞScience Citation Index Expanded (ISI/Thomson Scientifi c)rWeb of Science (ISI/Thomson Scientifi c)

The Science & Business of Biopharmaceuticals


July 2016

Volume 29 Number 7

MANAGING BIOMANUFACTURING

CAPACITY EXPECTATIONS

QUALITY

MICROBIOLOGICAL

TESTING: TIME IS

OF THE ESSENCE

PEER-REVIEWED

BIOPROCESSING TECHNOLOGY

TRENDS OF RNA-BASED

THERAPEUTICS AND VACCINES

ANALYTICAL TESTING

FORCED DEGRADATION

STUDIES FOR

BIOPHARMACEUTICALS

www.biopharminternational.com

Cover: JurgaR/Getty Images

6 From the Editor

CPhI Pharma Awards seek nominations for excellence in biopharma.Rita Peters

8 US Regulatory Beat

Agency guidance and industry standards aim to reduce lapses and improve quality operations. Jill Wechsler

12 Perspectives on Outsourcing

CDMOs need to be aware that unfavorable public markets put emerging bio/pharma R&D spending at risk in 2017. Jim Miller

44 Troubleshooting

The author provides a review of the concepts of design and qualification that apply to single-use systems.Jerold M. Martin

49 New Technology Showcase

49 Product Spotlight

49 Ad Index

50 Biologics News Pipeline

CAPACITY

Managing Biomanufacturing

Capacity Expectations

Randi HernandezCapacity for complex

therapeutics is becoming

increasingly difficult to predict. 14

QUALITY

Microbiological Testing:

Time is of the Essence

Cynthia A. ChallenerPressures to accelerate

current and next-gen therapies

are challenging traditional

microbiological testing methods. 20

ANALYTICAL TESTING

Forced Degradation Studies

for Biopharmaceuticals

Anette Skammelsen SchmidtThe author addresses critical issues to consider

prior to performing forced degradation studies

and provides best practice recommendations

for these types of studies. 24

PEER-REVIEWED

Bioprocessing Technology

Trends of RNA-Based

Therapeutics and Vaccines

Claire Scanlan, Priyabrata Pattnaik, Ruta Waghmare, Elina Gousseinov, Mikhail Kozlov, Aaron Hammons, Ling Bei, Youssef Benchek, and Karim PiraniThis article reviews the current dynamics

in the RNA therapeutics/vaccines market. 30

CLEANROOM STANDARDS

Revised ISO Cleanroom

Standards Improve Air

Cleanliness Classification

Jennifer MarkarianRevised versions of ISO 14644 Parts

1 and 2 introduce changes to sampling

procedures and monitoring plans for

cleanrooms and clean zones. 38

PACKAGING TRENDS

Raw Materials

Packaging Innovations

for Biopharmaceutical

Manufacturing

Nandu DeorkarRecent trends in raw materials packaging

may impact manufacturing, quality,

and cost of biopharmaceuticals. 40

Volume 29 Number 7 July 2016

FEATURES

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From the Editor

CPhI Pharma

Awards seek

nominations

for excellence

in biopharma

development and

manufacturing.

Recognizing Biopharma Industry Excellence

Each day, the editors of BioPharm International receive press releases promot-

ing potential: announcements of new products, services, methods, and

processes destined to solve a problem, cure a disease, save money, and—in

some cases of extreme hyperbole—change the world.

As natural cynics, the editors question the validity of these projections of

great success. We also appreciate the true successes, as recognized by indepen-

dent evaluations by industry experts. The CPhI Pharma Awards are one such

program to recognize excellence in biopharma development and manufacturing.

The awards program recognizes companies and individuals helping to accel-

erate the development of biopharmaceuticals through the introduction of

innovations, new technologies, and strategies that support drug development,

manufacturing, and distribution. The program is organized by CPhI Worldwide,

a global tradeshow for the pharma industry, scheduled for Oct. 4–6, 2016 in

Barcelona, Spain. CPhI and BioPharm International are UBM plc brands.

The 2016 awards are organized into 12 categories including:

t Excellence in Pharma: Bioprocessing. Technologies, products, processes, and

services for the manufacture of biologic drugs.

t Excellence in Pharma: Contract Services & Outsourcing. Contracted services

and processes for the bio/pharmaceutical industry including research,

development, formulation, manufacturing, analysis, and consulting.

t Excellence in Pharma: Regulatory Procedures and Compliance. Technologies,

products, processes, and services designed to ensure that bio/pharma com-

panies comply with standards, rules, and guidances established by regula-

tory and compendial authorities.

t Excellence in Pharma: Supply Chain, Logistics, & Distribution. Technologies,

products, processes, and services for ensuring the safe handling and track-

ing of drug substances, raw materials, and drug products.

t Excellence in Pharma: Analysis, Testing, and Quality Control. Technologies, prod-

ucts, processes, and services for the analysis and testing of drug substances, raw

materials, and drug products in a laboratory or production-line setting.

t Excellence in Pharma: Packaging. Technologies, products, processes, and ser-

vices related to primary and secondary packaging.

t Excellence in Pharma: Drug Delivery Devices. Technologies, products, pro-

cesses, and services related to the delivery of drug products to patients.

t Excellence in Pharma: Corporate Social Responsibility. Innovation in improv-

ing transparency and public outreach.

t Excellence in Pharma: CEO of the Year. The chief executive officer of an innova-

tor or generic-drug company is eligible for nomination. Attributes to be con-

sidered include financial performance, product performance, leadership skills,

management capability, marketing, acquisitions, and corporate strategy.

t Excellence in Pharma: API Development. Technologies, products, processes,

and services for the development and manufacture of APIs.

t Excellence in Pharma: Formulation and Excipients. Technologies, products,

processes, and services related to the formulation of drug products.

t Excellence in Pharma: Manufac turing Technology and Equipment.

Technologies, products, processes, and services for the manufacture of

solid, semi-solid, parenteral, inhalation, or other dosage drugs.

Applications for the 2016 CPhI Pharma Awards must be submitted by Aug. 14,

2016 via the awards website at awards.cphi.com. Finalists will be announced

in September 2016. The awards will be presented on Oct. 4, 2016 during the

Pharma Awards Gala at CPhI Worldwide. X

Rita Peters is the

editorial director of

BioPharm International.

Accelerate your bioprocess journey6SHHGDQGHFLHQF\DUHFUXFLDODVSHFWVRIELRPDQXIDFWXULQJ7KHULJKWVXSSOLHUFDQFRQWULEXWHWR\RXUVXFFHVV'LVFRYHU KRZRXUSLRQHHULQJWHFKQRORJLHVDJLOHVHUYLFHVDQGDELOLW\WRGHVLJQDQGFRQVWUXFWFRPSOHWHIDFLOLWLHVLPSURYHVVSHHG WRPDUNHW

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gelifesciences.com/bioprocess

*(DQG*(PRQRJUDPDUHWUDGHPDUNVRI*HQHUDO(OHFWULF&RPSDQ\*HQHUDO(OHFWULF&RPSDQ\)LUVWSXEOLVKHG$SU*(+HDOWKFDUH%LR6FLHQFHV$%%M|UNJDWDQ8SSVDOD6ZHGHQ

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Regulatory Beat

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FDA has long emphasized the importance

of reliable and accurate data in ensuring

drug safety, quality, and purity, but cur-

rent good manufacturing practice (CGMP) vio-

lations involving data integrity failings seem to

be on the rise, especially at overseas bio/phar-

maceutical operations. Current FDA policies for

ensuring that manufacturers maintain accurate

records and submit complete information stem

from its Application Integrity Policy, which

was established in the wake of the generic-drug

scandal of the 1980s. And while most data

integrity citations tend to involve sloppy prac-

tices and inadvertent violations, as opposed to

outright fraud, FDA officials are taking stronger

action to emphasize the importance of main-

taining secure systems for collecting and retain-

ing records to document the production of

quality drugs and biologics.

To highlight its concerns about the rise in

serious data and recordkeeping lapses in the

United States and abroad, FDA issued in April

2016 a long-awaited draft guidance (1). The

guidance uses a Q&A format to outline key

strategies for ensuring that manufacturing data

are reliable and accurate, and that companies

establish risk-based approaches for prevent-

ing and detecting problems in docu-

menting processes and tests and in

retaining records. FDA emphasizes

the need to ensure that all records are

complete and that effective systems

are in place for retaining and tracking

information and for preventing the

deterioration and loss of stored data.

The draft guidance defines key

terms, such as “backup” and “audit

trail,” and discusses methods for

restr ict ing unauthorized access

to computer IT systems. There’s

an explanation for why “testing

into compliance” is an inappropriate way to

achieve desired test results, a practice that is

cited increasingly in inspection reports. At the

same time, FDA seeks to avoid adding onerous

requirements or to complicate efficient drug

production. The regulators are all too aware

that a hard slap on a large producer could lead

to production delays that create drug short-

ages or reduce competition that helps maintain

lower drug prices.

INSPECTIONS FIND PROBLEMS FDA notes in the guidance that it is observing

an increased number of violations involving

data integrity in CGMP inspections, including

instances of poor records, inadequate written

procedures, and deficient systems for ensuring

effective production processes and controls at

manufacturing facilities all over the world. FDA

inspections cite a range of serious deficiencies

in how employees handle important records

and documents. There are reports of records

found in trash bins, data that do not match test

results, data manipulation, sample retesting to

achieve desired results, and deletion of undesir-

able results. These violations have led to warn-

ing letters, import alerts, and consent decrees,

particularly at facilities in India and China.

A scathing letter was sent in April to

Mumba i -based A PI producer Pol id r ug

Laboratories, following an in-depth inspection

in March 2015 (2). FDA cited the firm for failing

to record or investigate quality-related customer

complaints and for production deviations and

inadequate controls on computerized systems

to prevent unauthorized access or changes to

manufacturing data. FDA banned all imports

from this site in September 2015, as has Health

Canada and other regulatory authorities (3, 4).

Another warning letter to contract manufac-

turer Sri Krishna Pharmaceuticals highlights

FDA and Manufacturers Intensify Concerns about Data IntegrityAgency guidance and industry standards aim to reduce lapses and improve quality operations.

Jill Wechsler is BioPharm

International’s Washington editor,

Chevy Chase, MD, 301.656.4634,

[email protected].

July 2016 www.biopharminternational.com BioPharm International 9

Regulatory Beat

data integrity violations involv-

ing incomplete laboratory records,

inappropriate controls on com-

puter systems, a lack of written

procedures, plus a failure to fol-

low those procedures that are in

place (5). The letter cites multiple

situations where the firm deleted

non-conforming test results and

repeated tests to gain desired

results. FDA wants Sri Krishna to

conduct a comprehensive inves-

tigation into the extent of record

inaccuracies and to develop a

global corrective action and pre-

vention plan.

Of 28 warning letters issued by

FDA’s Center for Drug Evaluation

and Research (CDER) from January

2015 to May 2016, 21 cite data

integrity issues, reported Thomas

Cosgrove, acting director of CDER’s

Office of Compliance, at the ISPE/

FDA/PQRI Quality Manufacturing

conference in Bethesda, MD, in

June 2016. He noted that FDA is

seeing fewer problems at “top

tier” pharma companies, but more

violations in China and other

foreign countries. FDA and indus-

try experts further discussed key

components of FDA data integrity

requirements and industry best

practices at a special data integrity

workshop held in conjunction with

the quality conference.

Dealing with such problems

can carry high legal costs, noted

attorney Neil DiSpirito of Ballard

Spahr LLP at the May 2016 annual

meeting of the Food and Drug Law

Institute (FDLI) in Washington,

DC. Consequently, the importance

of fixing data problems is draw-

ing more attention in executive

offices and prompting more corpo-

rate initiatives to prevent and fix

data problems. The International

S oc ie t y for Pha r maceut ic a l

Engineering (ISPE) is developing

a white paper to make a strong

business case for investing in sys-

tems able to ensure that all data

are appropriately recorded and

reviewed and that appropriate con-

trols can detect any problems. The

impact of data breaches and com-

pliance problems are now “hitting

the bottom line” at pharma com-

panies, pointed out Frances Lipp,

president of Lachman Consultant

Services, at the FDLI conference.

Situations involving falsified data,

she noted, can lead to delays in

product launches, recalls, and

major overhauls of information

systems.

PDA OFFERS GUIDELINESThe Parenteral Drug Association

(PDA) has formed a task force to

address the “spectrum of issues”

related to the complexities man-

ufacturers face in ensuring the

integrity of processes generating

key production and regulatory

information, reported PDA presi-

dent and CEO Richard Johnson

at the FDLI conference. A lack of

accountability in production sys-

tems has led to improper data

manipulation, adjustment of time

clocks, record backdating, exclu-

sion of adverse informat ion,

and trashing of original records,

Johnson observed.

To remedy these problems, the

PDA group has developed guide-

l ines to help manufacturers

develop internal codes of conduct

for ensuring data integrity. Such

policies can emphasize to employ-

ees, suppliers, and contractors the

importance of meeting require-

ments for ensuring the accuracy

of information and records. They

apply to organizations that con-

duct clinical trials and laboratory

tests and that contract to provide

services to bio/pharma companies,

as well as to manufacturers.

The PDA task force also is pre-

paring a points-to-consider docu-

ment on the fundamental concepts

for data integrity, as well as tech-

nical reports on ensuring data

accuracy in laboratory systems.

The aim is to assist manufactur-

ers in establishing mechanisms for

detecting and remediating non-

compliance situations, to encour-

age harmonized standards for

ensuring data integrity in different

regions, and ultimately to restore

confidence of regulators and the

public in quality production sys-

tems, Johnson said. PDA will dis-

cuss its guidelines for company

codes and other related initiatives

at a workshop on data integrity

in September in Washington, DC

(in conjunction with its annual

FDA/PDA regulatory conference)

and at similar workshops in Berlin,

Germany, and San Diego.

In the old days of paper records,

it was relatively easy to destroy or

replace production files. Today’s

computerized systems require

audit trails for every operation,

which make discrepancies easier

to detect—by both manufacturers

and by FDA investigators. Thus it is

important for biopharma compa-

nies to ensure that all contractors

and suppliers—for IT, manufactur-

ing, and clinical trials—understand

and follow the rules, and report

quickly when problems emerge.

REFERENCES 1. FDA, Data Integrity and Compliance With

CGMP Guidance for Industry, Draft

Guidance (CDER, CBER, CVM, April

2016), www.fda.gov/downloads/

Drugs/GuidanceCompliance

RegulatoryInformation/Guidances/

UCM495891.pdf.

2. FDA, Warning Letter to Polydrug

Laboratories Pvt. Ltd. Corporate Office,

April 14, 2016, www.fda.gov/ICECI/

EnforcementActions/

WarningLetters/2016/ucm496623.

htm.

3. FDA, Import Alert 66–40, FDA.gov,

www.accessdata.fda.gov/cms_ia/

importalert_189.html

4. “FDA Bans Drugs From India’s Polydrug

Labs, Citing GMP Issues, FDANews.

com, www.fdanews.com/

articles/173104-fda-bans-drugs-from-

indias-polydrug-labs-citing-gmp-

issues?v=preview

5. FDA, Warning Letter to Sri Krishna

Pharmaceuticals Ltd.–Unit II, April 1,

2016, www.fda.gov/ICECI/Enforcement

Actions/WarningLetters/2016/

ucm495535.htm.

10 BioPharm International July 2016

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Perspectives on Outsourcing

Do

n F

arr

all/G

ett

y I

ma

ge

s

All

fig

ure

s a

re c

ou

rte

sy o

f th

e a

uth

or.

Contract research organizations (CROs)

and contract development and manufac-

turing organizations (CDMOs) may not

be feeling it yet, but the downturn in external

financing for early-stage bio/pharma companies

is real. The impact on CDMOs and CROs will be

delayed, but there is no doubt that service pro-

viders will be feeling it in coming months.

Two recent articles in the financial press

underscore what has been happening. An item

on Bloomberg.com chronicled the challenges

early-stage public companies are facing as they

go out for further funding beyond their initial

public offerings (IPOs) (1). The article high-

lights the plight of two bio/pharma companies,

Aldeyra Therapeutics and Ovascience, whose

stock prices took big hits when they floated

secondary public offerings of their stock on

May 26th. The shares of Ovascience went down

30% on the day of the offering while those of

Aldeyra fell 10%.

The Bloomberg article noted that Aldeyra and

Ovascience’s experiences are reflective of what

has been happening to many bio/pharma com-

panies trying to tap public markets.

According to the article, the num-

ber of secondary offerings from

bio/pharma companies is down

40% in 2016, with 64 offerings

vs. 106 in 2015; but the amount

raised is down 70% from $9 bil-

lion to $2.6 billion. So it’s not just

the decline in the number of offer-

ings that is hurting early-stage bio/

pharma; they are also raising fewer

dollars per offering.

The market has also not been

friendly to young bio/pharma com-

panies trying to tap public markets

for the first time. There have been

half the number of IPOs, 8 in 2016

vs. 17 in 2015, but the amount raised, just $483

million by Bloomberg’s count, is down 75%.

Data compiled from the PharmSource Lead

Sheet (Figure 1) confirms the Bloomberg analysis

and also shows that venture capital (VC) invest-

ment has held up well despite the public market

travails. Nevertheless, an article in the New York

Times described how the balance of power has

shifted from entrepreneurs to investors in the

venture capital world (2). According to the arti-

cle, VC firms have been able to demand much

tougher terms from companies they are invest-

ing in, including lower valuations and the hir-

ing of more experienced executives. The article

focuses on Internet companies, but the pinch is

being felt across the start-up spectrum.

Bio/Pharma Funding Challenges Could Hurt CDMOs in 2017CDMOs need to be aware that unfavorable public markets put emerging bio/pharma R&D spending at risk in 2017.

Jim Miller is president of PharmSource

Information Services, Inc., and

publisher of Bio/Pharmaceutical

Outsourcing Report,

tel. 703.383.4903,

Twitter@JimPharmSource,

[email protected],

www.pharmsource.com.

Figure 1: Venture capital investment 2014–2016.

VC

2014

$12.0

$10.0

$8.0

$6.0

$4.0

$2.0

$-

2015 2016

IPO Secondary Offering

US $

Billio

n

The market has not been

friendly to young bio/pharma

companies trying to tap public

markets for the first time.


Perspectives on Outsourcing

IMPACT ON EARLY-STAGE COMPANIESIt’s not surprising that CROs and

CDMOs may not be feeling the

impact of the funding down-

turn quite yet. After the 2008

global f inancial crisis, it took

two years for investigational new

drug (IND) filings and Phase I

clinical trial starts to reflect the

funding decline (Figure 2). That’s

because early-stage companies

focused their remaining cash on

getting their lead candidates into

the clinic as quickly as possible

in hopes of demonstrating proof

of concept (POC). POC is typi-

cally the prerequisite for licens-

ing deals and other partnering

arrangements from larger bio/

pharma as well as funding from

public sources. Funding from

partnering arrangements is prob-

ably the most secure funding

source because large bio/pharma

companies now depend on in-

licensed and acquired candidates

for at least half of the products

they ultimately take to commer-

cial markets.

Ea rly- stage companies t r y-

ing to get into the clinic are an

important source of business

for CDMOs because most are

dependent on service providers

to manufacture APIs and formu-

lated dose forms. Those compa-

nies represent the majority of

customers at most CDMOs, but

they typically have just a small

number of new drug candidates,

so CDMOs need to constantly

replenish their customer port-

folios to thrive. That replenish-

ment is highly dependent on a

rebound in public bio/pharma

equity markets: venture capital

might get candidates through

discovery and into preclinical,

but emerging bio/pharma compa-

nies need the larger tranches of

public funding to sustain a clini-

cal development program.

IMPACT ON CROS AND CDMOSThe most recent downturn in

external financing is barely a year

old, so CROs and CDMOs aren’t yet

feeling the pinch. Emerging bio/

pharma companies are using the

funds they have to get their can-

didates into the clinic, as in past

funding cycles, which is good for

CDMO business in the near term.

But without public markets, the

industry could see a sharp drop in

IND filings, and in the demand for

CDMO services, as it did in 2010.

One way for CDMOs and CROs

to prepare themselves for the

worst case is to focus on resur-

recting their business develop-

ment skills. After several years of

just answering unsolicited exter-

nal inquiries, they will soon have

to be prospecting for new busi-

ness and selling the customer on

why they should use them rather

than a competitor. As they were in

the last downturn, new business

development skills will be a key to

sustained success.

REFERENCES 1. M. Nilsen, “Biotech’s Vicious Second-

ary Cycle,” Bloomberg.com, May 26,

2016, www.bloomberg.com/gadf ly/ar-

ticles/2016-05-26/biotech-secondary-

offerings-get-punished

2. K. Benner, “Start-Ups Once Showered

With Cash Now Have to Work for It,”

New York Times, May 20, 2016, www.

nytimes.com/2016/05/21/technology/

start-ups-once-showered-with-cash-

now-have-to-work-for-it.html?rref=col

lection%2Ftimestopic%2FVenture%20

Capital&action=click&contentCollec

tion=timestopics&region=stream&m

odule=stream_unit&version=latest&

contentPlacement=5&pgtype=collect

ion&_r=0

Figure 2: Investigational new drug filings 2009–2015.

900

800

700

600

500

400

300

200

100

0

2009 2010 2011 2012 2013 2014 2015

Nu

mb

er o

f I

ND

Fil

ing

s

Early-stage companies

trying to get into

the clinic are an

important source of

business for CDMOs.


Jurg

aR

/Gett

y Im

ag

es

Demand for any given new prod-

uct is typically only known

after significant investments

have already been made.

Because executives commonly plan capac-

ity requirements based on launch fore-

casts, there are many factors that can lead

to miscalculations of capacity, making

it challenging to know what capacity to

build into a facility. According to a new

survey by BioPlan Associates, more than

half of respondents (60%) expect facility

constraints to create biopharmaceutical pro-

duction capacity constraints by 2021 (1).

BioPlan found that the development of

more efficient single-use products, better

downstream purification technologies, the

introduction of continuous downstream

operations, and increased modularization

of production systems were identified as

the top things the industry must do to

avoid further capacity restrictions at bio-

manufacturing plants (Figure 1). Analytical

testing concerns and adequate hiring of

qualified personnel to manage the facili-

ties are among the other top issues that are

expected to create capacity limitations in

the future (Figure 2).

FLEXIBLE SOLUTIONSEven if demand is accurately predicted,

changes to a development plan can also

occur that require facility changes, says

Christian Wyss, attorney at Vischer AG,

who specializes in drafting and negotiat-

ing contracts for clients in the life sciences.

These developments can arise because an

opportunity presents itself to improve a

drug or add more indications—or, scien-

tific issues may have to be addressed that

Managing Biomanufacturing Capacity Expectations

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AL

L F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

BIO

PL

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S.

were not planned. Wyss notes that

there could also be problems with a

technology transfer. “Either the man-

ufacturing process was not as robust

as the sponsor thought it was, or the

tech transfer failed to successfully

convey all subtleties to the contract

manufacturing organization [CMO].”

To alter technical capacity, a facil-

ity has to have “solution-oriented

professionals that are willing and

able to find room for flexibility in

a highly regulated environment,”

says Wyss. The change can become

more complicated if there is a change

in product type, which may even

require a completely different facil-

ity, says Tom Ransohoff, vice-pres-

ident and principal consultant at

BioProcess Technology Consultants.

It is more difficult to respond to shift-

ing demands if process equipment

and clean utility systems are hard

piped into the infrastructure, says

Parrish Galliher, CTO for upstream

at GE Healthcare’s Life Sciences busi-

ness. Multiple closed-off cleanroom

sections in facilities, numerous heat-

ing/ventilation/air conditioning

zones, and low ceilings (which limit

types and scales of new equipment)

can also serve as barriers to rapid

capacity expansion, Galliher states.

Forecasting long-term demand

during the transition from clinical

to commercial is challenging, says

Ransohoff. He adds that to meet

uncertain or changing demands,

one strategy is to “number up,” or

use multiple single-use bioreac-

tors to achieve a range of upstream

scales. Galliher concurs that adding

extra operating shifts to an existing

facility helps rapidly expand capac-

ity, as well as overlapping or “stag-

gering” of batches to meet need.

According to a report compiled by

Patheon, ORC International, and

PharmSource, demand and capac-

ity forecast inaccuracies have

prompted biopharmaceutical com-

panies to embrace the use of out-

sourcing with more fervor than

ever before (2).

Capacity

Figure 1: The top 10 areas to address to avoid capacity constraints, according to

a survey of biomanufacturers.

Figure 2: The top 10 factors creating future capacity constraints, as identified by

a survey of biomanufacturers in 2016.

Avoiding capacity constraints“If this industry is to avoid significant capacity constraints, the

most important areas to beaddressed are:”

46.0%

38.8%

37.4%

36.0%

33.8%

30.9%

29.5%

28.1%

27.3%

26.6%

Develop more cost-effective disposable, single-use products

Source: Figure adapted from the Thirteenth Annual Report and Survey of BiopharmaceuticalManufacturing Capacity and Production, BioPlan Associates Inc., 2016. Used with permission.

Develop better-performing disposable, single-useproducts

Develop better continuous bioprocessing - downstream technologies

Develop better downstream purificationtechnologies

Develop more ‘modularized’ production systems

Standardize international regulatory processes

Streamline FDA regulatory process

Optimize cell culture systems to increaseupstream performance

Fund more research to maximize productionefficiencies

Optimize systems to improve downstreampurification performance

Which factors are likely to create biopharmaceuticalproduction capacity constraints at your facility

in 5 years (by 2021)? (n=140)

Facility constraints

Analytical testing and drug product release

Inability to hire new, experienced technical and productionstaff

Inability to retain experienced technical and production staff

Physical capacity of downstream purification equipment

Inability to hire new, experienced scientific staff

Inability to retain experienced scientific staff

Costs associated with downstream purification

Physical capacity of fermentation/bioreactor equipment

Inability for me to optimize my overall system, given mycurrent technology and resources

60.0%

37.1%

30.7%

29.3%

27.1%

25.0%

23.6%

20.7%

20.0%

17.9%



FORECASTING CAPACITY NEEDSA multitude of unforeseen circum-

stances can skew capacity fore-

casts. Some of these could include

reports of a serious adverse event,

slow enrollment in clinical trials, sale

of a parent company that is devel-

oping the drug, an unusually suc-

cessful marketing strategy, provider

motives and incentives, final cost to

the patient, willingness for a payer or

pharmacy benefit manager to reim-

burse a drug, a change in raw mate-

rial availability, availability of new

therapeutic alternatives, or new regu-

latory legislation.

In a Nature Reviews Drug Discovery

study from 2013, investigators

concluded that more than 60% of

companies miss their demand fore-

casts by at least 40% (3). A signifi-

cant number of companies were

also overly optimistic by more than

160% of the actual peak revenues

that a product could pull in. Even

up to six years post-launch, fore-

casts were still found to be off the

mark by percentages as high as 45%.

The researchers found that demand

for oncology drugs was most com-

monly underestimated, most likely

because of the additional indica-

tions for which these drugs earned

approval by FDA after initial launch.

This demand underestimation is an

important finding considering the

large number of biologic, immune-

oncology therapeutics (with various

proposed indications) that are cur-

rently in the pipeline. The authors

found that analyst forecasts for

generic therapies were also mark-

edly off-target (3). These findings

could have implications for future

demand calculations for biologics, as

well as biosimilars with numerous

market competitors—especially if the

Centers for Medicare and Medicaid

Service’s proposal to use reference

pricing for all groups of therapeu-

tically equivalent drugs under

Medicare Part B (even for biosimilars

that are not interchangeable) comes

into effect.

A 2007 article in Pharmaceutical

Executive estimated that a launch

delay costs an average of $15 mil-

lion per drug per day (4). This num-

ber changes, however, depending on

the market demand of the drug in

question. “The general rule is that

a biologic will generate, on average,

$300 million per year. So, each day

delayed is a loss of $1 million,” esti-

mates Galliher. “I have seen much

larger numbers in print for blockbust-

ers,” he adds.

Including post-approval R&D

costs, as well as costs associated with

unsuccessful projects, the estimate

for the average out-of-pocket cost to

develop a new compound was found

to be $2870 million (in 2013 dollars),

according to an analysis by DiMasi

et al. that appeared in the May

2016 issue of the Journal of Health

Economics (5). Even though there

have been slight methodological

differences in DiMasi et al.’s studies

since 2003—when the authors began

looking at the cost of bringing a drug

to market—this cost of development

has still increased substantially since

2003. Additionally, said the authors

of the study, “clinical success rates

are substantially lower for the stud-

ies focused on more recent periods”

(5). Thus, because failure rates have

increased and the cost of developing

a drug has also increased so mark-

edly, it is increasingly difficult to

accurately predict the demand for a

drug—as well as that drug’s associ-

ated capacity requirements.

Indeed, many industry experts

agree that predicting capacity will

become even more problematic for

pharmaceutical manufacturers in the

future because of market access issues.

In Europe, because physicians seem

to be more accepting of biosimilars,

market penetration forecasts may be

a bit more clear—but in the United

States, physician acceptance and pre-

scribing practices (as well as the inter-

changeability status of a biosimilar)

may make launch and capacity pre-

dictions increasingly challenging.

HYBRID CAPACITY VS. OTHER MODELSWhile it seems like a number of phar-

maceutical companies still rely on a

largely in-house approach to manag-

ing capacity, most large firms have

been open to the concept of using

outside CMOs to meet short-term

requirements. Small firms often use

a completely outsourced model to

meet capacity. The percentage of pro-

duction that is outsourced at each

biomanufacturing firm depends

largely on what type of product is

being manufactured. According

to numbers from the 2016 BioPlan

report (1), approximately 59% of

respondents used at least some out-

sourced capacity for mammalian cell

culture, 55% used outsourcing for

microbial fermentation, 42% used

outsourcing for production in yeast,

33% outsourced for production in

plant cells, and 33% outsourced

capacity for production of therapies

in insect cells (see Figure 3).

Companies such as Amgen,

Bristol-Myers Squibb, and Roche use

hybrid approaches for the production

of their medications. Wyss estimates

that almost all biotech companies

that have several products on the

market use a mixed approach to

manufacturing, but most companies

keep the number of CMOs that they

work with to a minimum. An excep-

tion would be a small biopharma

company with few products, says

Wyss. “Drug development companies

with no product on the market or

one-product companies often rely on

CMOs only and do not use in-house

manufacturing. When the date for

market launch is set, these companies

will often look for additional CMOs

to back up their supply chain.”

EXCESS CAPACITYAs mentioned, it is common to over-

estimate or underestimate demand

for a drug. Overestimating can lead

to the manufacture of too much

product, which would then have

to be disposed at the manufactur-

Capacity


Capacity

er’s cost. Or, if an overestimation

becomes apparent prior to produc-

tion, gaps in revenue could appear,

and the company must somehow

fill capacity.

Idle capacity at CMOs can some-

times be handled without disruption

(provided that notice is given to the

CMO in advance), and the CMO can

use the capacity for other custom-

ers, says Wyss. If little to no notice is

given, Wyss says that costs for equip-

ment remaining idle “can be 80–90%

of the costs of manufacturing, at least

for a couple of weeks or months.”

While costs to maintain an idle facil-

ity may be high, says Ransohoff,

“they are much lower than the eco-

nomic losses associated with failing

to supply the market demand for a

highly profitable biopharmaceutical.”

Facilities can become idle for vari-

ous reasons, including the failure of

a late-stage product to get approval

by FDA or the failure of a new prod-

uct to gain market share. Ransohoff

points out that some large companies

also purposely keep some capacity

available to account for “unantici-

pated surges in demand.”

Ransohoff notes that he has

seen some companies using their

excess capacity for the production

of biosimilars, citing Biogen’s

manufacture of Biogen/Samsung

Bioepis’ etanercept biosimilar

Benepali as an example of this

trend. Sometimes CMOs use pro-

visions that are built into con-

tract agreements to resell unused

capacity, which Ransohoff says

helps CMOs mitigate the “costs

of typical ‘take or pay’ provi-

sions for clients,” or the costs to

reserve facility time regardless of

if capacity was used.

Wyss argues, however, that he

does not expect excess capacity

in-house to be used for the pro-

duction of biosimilars too often:

“To my knowledge, even pharma

companies having both original

products and biosimilars strictly

separate the supply chain man-

agement for original products

and biosimilars.”

CAPACITY REDUCTIONS: DECREASING VOLUMES WITHOUT COMPROMISING QUALITY OR REVENUEIn general, a decrease in capacity is

viewed as a negative event, and com-

panies are more reticent to announce

capacity reductions. “Decreases in

capacity/moth-balling facilities are

generally not positive developments

for companies since they represent

inefficient utilization of capital, often

resulting from a failure of a product

candidate (or candidates) in clinical

trials or of a manufacturing business

model to develop as planned,” states

Ransohoff. “By contrast, increases

in capacity signify optimism for the

future of the company’s products or

manufacturing business model.”

Indeed, as facilities age, they may

have to be updated or taken offline

entirely. “Retirement of very old

plants is being exceeded by new

capacity growth, [and] overall capac-

ity needs are growing,” notes Greg

Guyer, leader in biologics develop-

ment and operations at Bristol-Myers

Squibb (BMS). Announcements of

capacity reduction are indirectly seen

when sites are sold between compa-

nies, he says. In fact, the sale of these

types of facilities can have a posi-

tive spin, notes Galliher. When the

sale of an older facility occurs, “fis-

cal responsibility is also being dem-

onstrated by closing unnecessary

capacity and selling off underutilized

assets, in which case positive finan-

cial/investor outcomes can result.”

There is new evidence that a

capacity decrease may not neces-

sarily have negative connotations.

For example, concentrated fed-batch

(CFB) cell culture has been shown

in early experiments to yield prod-

ucts of similar quality compared

with those made through traditional

fed-batch culture (6). Not only could

these concentrated fed-batch runs

Figure 3: Current percent production outsourced; by system.

“What percent of biomanufacturing organization’s productioncurrently outsourced for each?” 2016

MammalianCell Culture

MicrobialFermentation

Yeast

Plant Cells

Insect Cells 11.1% 22.2%

16.7%

16.7%8.3%8.3%8.3%

12.9%3.2%

5.9%5.9%

2.0%7.8%23.5%13.7%

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

3.2%3.2% 12.9%19.4%

16.7%



be manufactured at lower volumet-

ric capacities (meaning smaller facili-

ties could accommodate volumes

typically seen at larger facilities), the

resulting products were also shown

to enhance cell-line charge hetero-

geneity, proving that concentrated

fed-batch could be associated with

“both process and product quality

benefits” (6).

Despite these benefits, concen-

trated fed-batch used more perfusion

and feed media, required numerous

filters, and also overloaded down-

stream processes, causing filter

fouling in some cases. The yields

obtained in the Yang study (6) were

not sufficient enough to suggest

totally replacing larger facilities, but

the technique has potential for some

slow-growing cell lines. While it may

not be economically feasible for a leg-

acy system to be converted to CFB,

Yang et al. wrote that new compa-

nies seeking flexibility in capacity

operations might want to consider

trying CFB to meet their production

needs. The authors concluded, “The

key to unlocking the cost and capac-

ity savings of concentrated fed-batch

is increasing the specific productivity

of the process through cell line and

process development.”

WORKING WITH CMOSThe use of CMOs can be helpful

when there are fluctuating capac-

ity and demand conditions, but

sometimes a biopharma company

may be wary of the CMO model. A

biopharma company may initially

choose an “in-house” approach

because it fears the leverage a CMO

can gain over its business. “The

leverage stems from the fact that

the CMO/contract development

and manufacturing organization

[CDMO] has all manufacturing

knowledge, and there is always a

substantial risk that technology

transfer will not be successful imme-

diately,” states Wyss. Thus, he says,

detailed technology transfer plans

are crucial.

If a CMO/CDMO has its own pro-

prietary manufacturing platform, a

biopharma company cannot easily

transfer the process back in-house

or to another CMO, Wyss points

out. “If the contract manufacturing

agreement does not give the sponsor

a license to this technology solely

for the continued production of this

specific biologic drug, the manu-

facturing process will have to be

partially re-designed, which is prac-

tically not feasible from a time and

cost perspective.”

Intellectual property

barriers to capacity outsourcing

Manufacturers typically choose an

in-house model to keep better con-

trol of their supply chain, handle

development risks, manage speed

of development and launch, and for

tax purposes, says Ransohoff—but

another important reason to keep

production in house is to ensure pro-

tection of a company’s intellectual

property (IP).

There seems to be mixed responses

about whether or not the protection

of IP is a significant problem when

working with CMOs. Guyer says

when BMS outsources, it establishes

clear contract provisions to protect its

IP, and if it cannot reach agreeable IP

terms with CMO partners, it simply

does not work with that CMO. He

says BMS rarely finds IP to be a bar-

rier to successful relationships with

outsourcing partners. Conversely,

GE’s Galliher sees IP as a “major

issue” for CMOs when joint owner-

ship of technology platforms and/

or inventions exist. “The customer

usually wants to retain its rights to

the drug and cell line and how it is

made, especially if proprietary tech-

niques are used. The CMO wants the

business freedom to use the process

techniques for other customers and

its own cell line if it is providing it.”

Although there many be signif-

icant advantages in terms of labor

costs when using foreign outsourc-

ing operations, specifically, concerns

about keeping IP secure often prevail,

says Wyss, and as a result, pharma

companies rarely outsource to coun-

tries with perceived weak patent pro-

tection or in areas where national

laws provide for mandatory licenses

to local generic drug manufacturers.

“It seems that originator companies

have been able to solve all quality

related issues when manufacturing

in those countries, but are still reluc-

tant to expose themselves to these

legal risks before patent expiry.” Wyss

tells this publication that intellec-

tual property issues come into play

most when a manufacturer is decid-

ing between different CMOs in vari-

ous parts of the country. He adds,

“many countries have regulations

requiring that at least a part of the

manufacturing of the drugs sold is

accomplished within that country,

either directly by relevant legislation

(such as in Russia), or indirectly by

making this a requirement to obtain

research funding or collaborate with

public academic institutions (e.g.,

the standard Cooperative Research

and Development Agreement in the

United States requires manufactur-

ing in the US).” On the other hand,

Galliher mentions that it is also rela-

tively common for some companies

to choose a foreign CMO to handle

capacity specifically to avoid local

IP legislation.

REFERENCES 1. BioPlan Associates, Inc., Thirteenth

Annual Report and Survey of

Biopharmaceutical Manufacturing

Capacity and Production, April 2016.

2. ORC International, Patheon, and

PharmSource, “Impact of Incorrect

Forecasts on New Product Launches,”

Industry Report, 2016.

3. M. Cha, B. Rifai, and P. Sarraf, Nat. Rev.

Drug Disc. 12, pp. 737–738 (2013).

4. T. Noffke, “Successful Product

Manager’s Handbook”, a supplement

to Pharmaceutical Executive (March

2007), www.pharmexec.com/no-time-

delay, accessed April 30, 2016.

5. J.A. DiMasi, H.G. Grabowski,

and R.W. Hansen, J. Health Econ.

47, pp. 20–33 (2016).

6. W.C. Yang et al., J. Biotechnol.

217, pp. 1–11 (2016).

Capacity


Sto

ckb

yte

/Gett

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Effective microbiological test-

ing during biopharmaceutical

drug development and manu-

facturing is crucial for ensur-

ing sterility, determining antimicrobial

effectiveness, detecting microbial con-

tamination or bioburden levels, ana-

lyzing endotoxins, and implementing

environmental monitoring programs.

Growing pressures to increase produc-

tivity, flexibility, and cost-effectiveness

and the unique properties of many

next-generation therapies are challeng-

ing today’s microbiologists.

CONVENTIONAL PROTOCOLS NO LONGER SUITABLE FOR TRADITIONAL BIOLOGICSMicrobiology testing of pharmaceutical

products is performed with a view to

detection, enumeration, and identifica-

tion of microbial contaminants, accord-

ing to Marian McKee, senior director of

BioReliance operational development

services at MilliporeSigma. Traditional

microbiological methods for sterility

testing take 14 days from inoculation to

detection and conclusive results, while

culture-based methods for mycoplasma

take 28 days. “The time to obtain results

for these traditional microbiological test

methods is lengthy,” McKee notes.

For release of traditional biologics that

are produced in large lots with longer

stability profiles, the turnaround time on

conventional microbiological tests, which

are sensitive and robust, is not a concern

with respect to the ability to obtain reli-

able results. The faster release of bulk drug

substances and batches during in-pro-

cess testing, however, is desirable. “More

rapid alternatives to traditional methods

are needed to speed the manufacturing

Microbiological Testing: Time is of the Essence

Cynthia A. Challener

Pressures to accelerate

current and next-gen

therapies are challenging

traditional microbiological testing methods.

Cynthia A. Challener, PhD

is a contributing editor to

BioPharm International.

Quality: Microbiological Testing

July 2016 BioPharm International 21

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process while ensuring process and

product safety,” says McKee.

Protein-based biologic drugs can

also present real technical challenges

with respect to reading assay results.

“Occasionally the therapeutic mol-

ecule can have toxic effects on the

detector cells that are used to detect

viruses in in-vitro assays that comply

with ICH Q5A (1) and related regu-

latory guidelines,” observes Archie

Lovatt, scientific director of biosafety

for SGS. He does add, however, that

this issue can usually be overcome

by dilution of the sample. Results of

the tests performed on diluted sam-

ples can also be supported by viral

clearance data generated during pro-

cess development.

For biopharmaceutical manufactur-

ers including contract manufacturers

that produce many different prod-

ucts, the time and resources involved

in performing required microbial

testing is a challenge, says Aaron

Ortiz, QC manager of microbiology

at GSK Biologics’ GMS Rockville site.

“Qualifying the bioburden method

for each step of each process is

extremely time consuming. In addi-

tion, the many different matrices of

the different in-process samples lead

to challenges with recovery of differ-

ent organisms depending on the pro-

cess step,” he explains.

NEXT-GEN THERAPIES HAVE UNIQUE NEEDSThe lengthy time to obtain results

for traditional microbiological test

methods, while not a technical issue

for more stable proteins, antibodies,

and other older types of biologics,

is typically unacceptable for newer

cell-based therapeutics. “Some next-

generation products have short shelf

lives and require novel technologies

to detect microbes that are more

rapid than traditional methods,”

says Lovatt. Cellular- and gene-ther-

apy products, which not only have

short shelf lives, but also nontradi-

tional lot sizes, are driving the need

for rapid microbiological methods

(RMMs) to reduce testing times and

sample volumes, agrees McKee.

In addition, for cellular thera-

pies that involve the modification

of patient cells followed by their

injection back into the patient,

the process is typically completed

within just a few days. As a result,

Lovatt points out that the use of

contract research organizations/

third-party testing laboratories may

not be practical. “Microbiological

testing for these therapies may

need to be performed at the pro-

duction site in order to avoid addi-

tional delays due to the need for

the shipment of samples,” he says.

Ortiz also notes that developing

and implementing appropriate tech-

nologies for the detection of organ-

isms in many of the new types of

biologic drug substances and drug

products classified as next-genera-

tion therapies is also a challenge.

VACCINES DRIVE TEST DEVELOPMENTThe need for a rapid response to a

possible influenza pandemic in the

first decade of the 2000s spurred

the development of RMMs, accord-

ing to McKee. Alternative strategies

for in-process release of vaccine

batches were needed to accelerate

production of flu vaccines, with

the 14-day sterility test seen as a

prime target for decreasing the

turnaround time.

The high cell content in many

vaccine batches can also interfere

with the ability to visually read

traditional test results. “Alternative

test methods based on detection of

metabolites or luminescence have

been developed that can overcome

this obstacle.” McKee notes.

Viral vaccines in particular can

cause challenges due to the need

to generate neutralizing antisera to

neutralize the cytopathic effect of

the vaccines on the cells that are

used to detect viruses/mycoplasma,

such as MRC-5 and Vero, etc. “The

generation of the antisera can sig-

nificantly extend the time required

for the testing process and, con-

sequently, manufacturing of viral

vaccines,” says Lovatt.


US and EU Regulators Seek to Reduce Inspections

FDA is assessing an information exchange with the European Medicines Agency

(EMA) that would identify facilities with strong records of compliance with good

manufacturing practices based on inspections by competent local inspectorates,

potentially reducing the number of facility inspections.

Although FDA has been receiving inspection reports from EMA for years, current

FDA policy prevents sharing of trade secret information that appears in field inspection

reports, explained Dara Corrigan, associate commissioner for global regulatory policy

at FDA at the ISPE/FDA/PQRI conference in Bethesda, MD, in June 2016. Other US

government agencies share sensitive, classified information with European Union (EU)

authorities, offering a precedent for FDA to act similarly. Legislation enacted in 2012

permits FDA to share confidential information in situations where the agency can

certify that the other country can keep this information secret.

FDA has been negotiating for three years to devise a system for mutual reliance

on inspection reports by local regulators found to meet acceptable standards. The

situation is complicated due to each of the 28 EU member states conducting its

own pharma facility inspections following different practices and standards.

Currently, regulatory officials from FDA, EMA, and EU member states are auditing

inspection programs by other authorities, with the goal of building confidence in the

capabilities of the other inspectorates and their inspection findings.


Influenza vaccines, for example,

must be manufactured in a very

limited time due to their seasonal

nature, and it is not possible to gen-

erate antisera quickly enough. As a

result, microbiological testing using

rapid nucleic acid methods is allowed

in the pharmacopeias, while poly-

merase chain reaction (PCR) methods

for mycobacteria detection, which

typically take one week, can be used

as alternatives to traditional culture-

based methods, which take up to 56

days, according to Lovatt. Nucleic

acid testing is also an effective alter-

native for microbial testing of viral

vaccines for which the viral drug sub-

stance is difficult to neutralize.

REGULATORY FLEXIBILITY CAN BE BENEFICIALSignificant changes to regulatory

requirements for microbial testing

have, in fact, been crucial for acceler-

ating the adoption of more advanced

methods. As mentioned previously,

the pharmacopeias have added chap-

ters to address the needs of next-gen-

eration therapies for RMMs that can

assure product safety. McKee points

to the European Pharmacopoeia (Ph.

Eur.) chapters 2.6.7 Mycoplasmas and

2.6.27 Microbiological Control of

Cellular Products as two important

examples. Ph.Eur. 2.6.27 outlines an

alternative to traditional compendial

sterility testing. “The 2.6.27 method

is suitable for qualification and vali-

dation of rapid methods for sterility

testing and incorporates conditions

and control organisms that yield a

more sensitive and broader range of

detection for cellular products,” she

explains. Ph.Eur. 2.6.7 Mycoplasmas,

meanwhile, includes specific guid-

ance for validation of nucleic acid

amplification techniques (NAT) that

may be used as alternatives to the

28-day culture and indicator cell

methods for detection of adventi-

tious mycoplasmas.

In addition, FDA has made

changes to its microbial testing

requirements that have had a posi-

tive impact on the development

of more rapid, advanced microbio-

logical methods (2). Amendments

to the Sterility Test for Biological

Products rule (21 Code of Federal

Regulations 610.12), which estab-

lishes FDA’s microbiological testing

requirements for biological products,

have been particularly important,

according to McKee. “The changes

to the rule provide manufacturers

of biologic products greater flexibil-

ity. Biopharmaceutical manufactur-

ers are in fact encouraged to use the

most appropriate and state-of-the-art

test methods to assure the safety of

biologic products,” she explains.

In addition, McKee notes that the

changes to the rule promote innova-

tion in the development of sterility

testing and are thus paving the way

for the use of novel methods such

as adenosine triphosphate (ATP) bio-

luminescence, chemiluminescence,

and even non-growth based RMMs.

“Not only was the use of a mandated

method for sterility testing removed,

the change in the rule allows greater

flexibility in sampling, putting the

onus of sample size on the manu-

facturer; the sample must be appro-

priate to the material tested both in

volume and representation of the lot

size,” she says.

There are some changes to regula-

tions that have created challenges,

however. Ortiz points to the rela-

tively recent low endotoxin recovery

(LER) phenomenon. For biologics,

FDA is requesting studies to deter-

mine if drug substances or drug

products demonstrate LER when

performing endotoxin analyses (3).

Due to the recent discovery of this

phenomenon and the various formu-

lations of drug substances and drug

products, there have been difficulties

in developing proper studies for LER.

RAPID SOLUTIONS: AUTOMATED VS. ALTERNATIVEThe development of new test meth-

ods has been pursued using two

fairly different strategies. Some

methods are still culture-based, but

with automation to help accelerate

the process. Others involve com-

pletely new testing technologies.

“Some automated RMMs use

a culture phase coupled with an

automated end-point to reduce the

overall testing time, but are still

in keeping with the traditional

tests described in the compendia.

Alternative methods, on the other

hand, are different from traditional

methods and often do not incorpo-

rate a culture phase,” McKee says.

The results for alternative methods

are also often reported in units other

than colony forming units (CFUs),

which is the typical format for cul-

ture-based assays. The results of these

tests, therefore, are not directly com-

parable with those obtained using

traditional or automated growth-

based methods, according to McKee.

“The prevailing concern is that

these alternative methods require

extensive validation studies to dem-

onstrate equivalency to traditional

microbiological methods. The need

for such extensive method vali-

dation in addition to the capital

expenditures necessary to develop

and implement these new technolo-

gies poses a hurdle for most bio-

pharmaceutical manufacturers that

wish to employ rapid microbial

methods,” McKee concludes. The

strong value proposition that RMMs

offer, however, extends to the devel-

opment of assays targeting other

adventitious contaminants includ-

ing viruses, mycoplasmas, residual

DNA, and residual proteins. Assays

that can be performed near the bio-

reactor location and in real time are

definitely the assays for the future.

REFERENCES 1. International Council for Harmonisation,

Q5A (R1) Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin (September 1999).

2. FDA, “FDA issues final rule on sterility testing of biological products,” Press Release, May 3, 2012.

3. K. Williams, BioPharm International, 28 (7) 28-33 (2015).



Photo

by M

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/Gett

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Forced degradation studies are

performed by means of various

stressing agents such as pH,

temperature, light, chemical

agents (e.g., oxidizing, deamidating

agents, etc.), and mechanical stress to

speed up the chemical degradation,

physical degradation, or instability

of a molecule. Currently, there are no

industry guidelines available defin-

ing how to perform forced degrada-

tion studies for biopharmaceuticals.

The guidelines only provide useful

definitions, general comments, and

a rough concept about degradation

studies (1–5). Strict guidelines with

specific ranges or exact conditions

for forced degradation studies are not

necessarily possible, as every mole-

cule is different, and certain freedoms

for selecting stress conditions for bio-

pharmaceuticals are inherent (4, 5).

Hence, conditions should be carefully

selected on a case-by-case basis (3).

Regulatory guidance documents

specify the following expectations on

forced degradation:

t 5IFNBOVGBDUVSFSTIPVMEQSPQPTFB

stability-indicating profile that pro-

vides assurance that changes in the

identity, purity, and potency of the

product can be detected (2, 3).

t 3FTVMUT G SPN GPSDFE EFHSBEBUJPO

studies will form an integral part of

the information provided to regula-

tory authorities (4, 5).

Furthermore, studies exposing the

biopharmaceuticals to stress condi-

Forced Degradation Studies for Biopharmaceuticals

Anette Skammelsen

Schmidt

The author addresses

critical issues to consider prior to

performing forced degradation studies and

provides best practice

recommendations for these types

of studies.

Anette Skammelsen Schmidt, PhD,

is senior research scientist,

API analytical development, at

Novo Nordisk A/S, Denmark.

Analytical Testing



Company DescriptionTosoh Bioscience LLC is a major supplier of chromatography

products worldwide, particularly to the pharmaceutical,

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provide bulk chromatographic resins for the purification

of biopharmaceutical drugs in commercial manufacturing

processes, and we manufacture analytical HPLC columns.

Tosoh’s portfolio of more than 500 TSKgel® and

TOYOPEARL® products encompasses all common modes of

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that it is both the ligand and base bead.

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Major Product Innovationsr $B1VSF)" IZESPYZBQBUJUFNFEJBGPSUIFQVSJGJDBUJPO

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tions may be useful in determin-

ing whether accidental exposures

to conditions other than those

proposed (e.g., during transpor-

tation) generate changes in the

molecule. Stress studies are also

useful for evaluating which spe-

cific test parameters may be the

best indicators of product sta-

bility and should be monitored

under proposed storage condi-

tions (3).

THE PURPOSE OF FORCED DEGRADATIONForced deg radat ion st udy i s

defined as an intentional break-

dow n of a molec u le to a n

appropriate extent by means of

various stressing agents (includ-

ing mechanical stress) to speed

up the chemical and physical

degradation and instability of a

biopharmaceutical. A forced deg-

radation study can give a range of

information regarding the likely

degradation products of a specific

biological drug. This informa-

tion can be useful for many pur-

poses, and can help to establish

the degradation pathways and the

intrinsic stability of the molecule.

Challenging the analytical proce-

dures helps validate the method’s

stability-indicating power (4, 5).

Prior to performing a forced

degradation study, the goal of the

study needs to be defined. Several

purposes might be addressed

in one study. When relevant, a

forced degradation study can be

performed at different develop-

ment stages. Figure 1 shows exam-

ples of the various reasons that

forced degradation studies are per-

formed.

Degradation products for bio-

pharmaceuticals may be either

product-related substances or

product-related impurit ies, as

some degradation products may

retain biological activity (1–3). An

example of this is illustrated in

Figure 2 and describes a situation

in which oxidation is not associ-

ated with a decrease in activity.

DEGRADATION PATHWAYS OF BIOPHARMACEUTICALSBiopharmaceuticals can usually

degrade in many different path-

ways following different kinet-

ics. The extent of stress needs

to provide a measurable change

and confirm the most relevant

degradation pathways. Too much

stress, however, might form sec-

ondary degradation products not

seen in formal stability studies,

and the level of stress might not

ref lect actual potential stress-

ors. An extent of degradation of

approximately 5–20% is assumed

to be suitable for most purposes

and for most analytical meth-

ods. An adequate level of stress

should be carefully selected on a

case-by-case basis (3).

The selection of the degradation

pathways to be investigated dur-

ing forced degradation should be

based on known and anticipated

degradation pathways—as well as

prior knowledge from similar mol-

ecules, if such knowledge exists.

The degradation pathways are typ-

ically either physical (e.g., aggrega-

tion) or chemical (e.g., oxidation)

in nature.

Aggregation can be noncovalent

in nature, such as an association of

monomers that are dissociable at

the right conditions (e.g., solvent,

temperature). These noncovalent

aggregates are mainly formed by

denaturation and unfolding of the

molecule, or by an interaction with

interfaces such as liquid-air, liquid-

solid, or even liquid-liquid. These

associations are typically a result of

mechanical stress such as shaking,

stirring, rotation, pumping; freeze-

thaw cycles; heating; or exposure

to acidic pH.

Analytical Testing

Figure 1: The purpose of forced degradation studies (FDS).

Figure 2: Determined oxidized forms (A) and activity (B) of drug substance (DS)

and drug product (DP) after exposure to 0.003% H2O2 at 22 °C for 0, 2, 4, 6, and

24 hours.

Identify likely

degradation

productsSupport process

development

Information to

process robustness

studies

Validation of

analytical methods,

including stability

indicating power

Impurity / variant

characterization Support

formulation

development

Support formal

stability studies

Provide samples for

analytical development

FDS

Establish and

understand

degradation

pathways

Determine

intrinsic stability

of molecule

60

50

40

30

20

10

0

Oxid

ized

fo

rm

s (

%)

0 10 20 30

Time (hours)

0 10 20 30

Time (hours)

A B

DS

DP 2 mg

DP 1 mg

DS

DP 2 mg

DP 1 mg

Activ

ity


Aggregation can also be cova-

lent in nature, such as chemical

bonding between the molecules,

and is non-dissociable during buf-

fer change. These chemical bonds

are often formed by rearranged

disulfide bridges or other altered

intramolecular bridges. They are

typically a result of reactions of the

amino acid residues with trace met-

als (copper or iron) or an incom-

plete reduction of the protein.

Side chains of methionine,

cysteine, histidine, tryptophan,

or tyrosine residues are suscep-

tible to oxidation, where methio-

nine is the most reactive residue.

Oxidation can alter the physico-

chemical properties of a protein,

such as folding and subunit asso-

ciations. The oxidation is mainly

due to exposure to atmospheric

O2 under condit ions of l ight,

heat, moisture, agitation, or to

exposure to oxidizing agents.

Deamidation is a hydrolytic con-

version of asparagine or gluta-

mine to a free carboxylic acid

residue and is typically due to

changes in pH, ionic strength,

temperature, and humidity in

the case of lyophilized proteins.

The overall effect of a chemical

modification of a single amino

acid residue depends on its posi-

tion in the protein and on the

specific role the residue has in

the functionality and active site

of the protein.

Photolysis by exposure to light

involves a free radical mecha-

nism that affects many functional

groups (e.g., carbonyl groups). The

free radicals can result in oxida-

tion, aggregation, or peptide bond

cleavage. The photolysis is due

to exposure to photo-irradiation,

which is typically in the form of

ultraviolet irradiation.

Hydrolysis (f ragmentat ion)

is a cleavage of peptide bonds

between amino acid residues

releasing smaller peptide chains.

The peptide bonds of Asp-Pro

and Asp-Gly are the most suscep-

tible to hydrolysis. Hydrolysis is

mainly a result from exposure to

acidic or alkaline pH.

Disu l f ide br idge exchange

might cause incorrect paired

disulfide bridges, which affects

the tertiary structure of a protein.

Such incorrect disulfide bridges

might be a result of partial cleav-

ing and reformation of disulfide

bonds as a result from denatur-

ing/reducing conditions (expo-

sure to reagents such as GnHCl,

u rea, and 1,4 -Dith iothre itol

[DTT]) and oxidation of cysteine

residues such as oxidation by Cu

(II) or Fe (II) ions.

Severa l biopharmaceut ica ls

contain ligands or conjugates.

Such bound moieties (e.g., acyla-

tion and conjugation) might be

lost due to chemical or physical

stress on the molecule.

SELECTION OF MATERIALS FOR A FORCED DEGRADATION STUDYWhen performing forced degra-

dation studies, it is important

to use a single batch of material.

Forced degradation studies usu-

ally require a large amount of

material. However, the material

could be non-GMP, a test batch,

or even an out-of-specification

batch (if such is available), as long

as the choice of batch is justified.

A l l re levant sample t y pes

should be included in the forced

degradation study. Drug prod-

uct at both high- and low-dose

levels can be included for drug

product-specific methods. If the

molecule is modif ied (e.g., by

acylation, glycosylation, or con-

jugation), the inclusion of the

intermediate is highly recom-

mended to aid understanding of

Analytical Testing

Table I: Examples of selected analytical methods for evaluation of degradation

pathways.

PathwayRP–HPLC

SE–HPLC

IE–HPLC

Peptide mapping

SDS–PAGE

Activity*

Aggregation - xxx - x x x

Fragmentation/clips

xx x x x xxx x

Oxidation xxx - - xxx - x

Deamidation x - xxx xx x x

Disulfide bridge exchange

x x - x - x

*Effect depends on location and extent of change in molecule

- indicates no effect, x indicates small effect, xx indicates significant effect, xxx indicates severe effect

RP–HPLC: reversed-phase high-performance liquid chromatography

SE–HPLC: size-exclusion high-performance liquid chromatography

IE–HPLC: ion-exchange high-performance liquid chromatography

SDS–PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis

Due to the complexity

of biopharmaceuticals,

there is no single

stability-indicating

method that can

profile all its stability

characteristics.


Analytical Testing

the changes seen in the underly-

ing structure of the molecule.

Solution/buffer blanks and con-

trols (excipients) are included for

evaluation of peak profile regard-

ing occurrence of new peaks

as a result of stress conditions.

Always include reference samples

in each experiment.

SELECTION OF ANALYTICAL METHODS FOR FORCED DEGRADATION STUDIESDue to the complexity of bio-

pharmaceuticals, there is no sin-

gle stability-indicating method

that can profile all its stability

characteristics (2, 3). The nature

of biopharmaceuticals will dic-

tate which test methods to use.

In general, methods that are

used in stability studies should

be included in forced degrada-

tion studies, as well as methods

that determine identity, purity,

content, and methods for moni-

toring impurities. The methods

should provide reliable data—as

measured by a satisfactory selec-

tivity between the main peak

and impur it ies—an adequate

intermediate precision, and be

able to detect the change i f/

when it occurs. Examples of ana-

lytical methods to evaluate deg-

radation pathways are shown in

Table I.

Examples of common methods

to employ for analysis of biophar-

maceuticals during forced degra-

dation are appearance (i.e., color,

clarity, particulate matter); activ-

ity measurement; sodium dodecyl

sulfate polyacrylamide gel electro-

phoresis (SDS–PAGE); microchip

gel electrophoresis; size-exclusion

high-performance liquid chro-

matography (SE–HPLC) (e.g., for

protein content and aggregates);

reversed-phase high-performance

liquid chromatography (RP–HPLC)

(e.g., for purity and specific impu-

rities); isoelectric focusing (IEF)/

imaged capillary isoelectric focus-

ing (iCE)/ion-exchange HPLC (IE–

HPLC) (e.g., for deamidated forms);

peptide mapping; and physico-

chemical analysis (e.g., differen-

tial scanning calorimetry [DSC],

circular dichroism [CD], and fluo-

rescence). Additional analysis can

be employed based on the results

obtained by the initially selected

analytical methods.

SUITABLE CONDITIONS FOR FORCED DEGRADATION STUDIESAll molecules can be degraded

by some chemical or physical

means. Figure 3 shows examples

of common st ress condit ions

known to induce different deg-

radation pathways for biophar-

maceuticals. The conditions used

in forced degradation have to be

harsher than conditions used in

accelerated studies. If the condi-

tions result in no change, longer

exposure time is recommended,

rather than the use of a more

ext reme temperat u re. W hen

selecting the relevant stress con-

dit ions, the fol lowing points

must be considered:

t8FSFBMMEFHSBEBUJPOQBUIXBZT

covered?

Figure 3: Examples of common stress conditions, including light for forced

degradation studies.

DTT: 1,4-Dithiothereitol; ICH: International Council for Harmonization

Oxidation

8 e.g., 0.003% H2O

2 overnight 25 ºC, or

atmospheric O2

8 pH

8 e.g., overnight at 5 ºC and 25 ºC

8 Elevated temperature

8 e.g., 1-2 weeks or even up to 4 weeks

8 Depends on normal storage temperature

(and accelerated studies)

8 Normal storage (e.g., 25 ºC), then

stressed study (e.g., 50 ºC to 60 ºC)

8 Mechanical stress

8 e.g., rotation, shaking, agitation,

freeze-thaw

8 Freeze-thaw (e.g., 5-10-15 cycles)

8 Reduction

8 e.g., 0.01 M DTT overnight 25 ºC

Light

8 Exposure - ICH Q1B conditions

8 ≥ 1.2 million lux hours and ≥ 200 W

hours per m2 (25 ºC); ICH Confirmatory

conditions

8 Dark control (25 ºC); wrapped in

aluminium foil and placed next to

exposed sample

8 Exposure - stressed conditions

8 ≥ 2.4 million lux hours and ≥ 400 W

hours per m2 (25 ºC); stress conditions

8 Dark control (25 ºC); wrapped in

aluminium foil and placed next to

exposed sample

8 Depending on the biologics, even

harsher conditions can be employed

Figure 4: (A) Determined high-molecular weight proteins (HMWP) and (B) total

protein content after storage at elevated temperature (5 °C, 25 °C, and 37 °C)

at one and two weeks. The chromatogram (C) from the high-performance liquid

chromatography illustrates the increasing HMWP peak at high temperature.

45

40

35

30

25

20

15

10

5

0

1.2

1.0

0.8

0.6

0.4

0.2

0.0

8.00 10.00 12.00 14.00 16.00 18.00 20.00

A

C

B

T=0 (as is)

1 week

2 weeks

T=0 (as is)

1 week

2 weeks

5 25 37

Temperature (ºC)

5 25 37

Temperature (ºC)

HMWP

Mo

no

mer

Retention time (minutes)

37ºC 2 weeks

25ºC 2 weeks

5ºC 2 weeks

T = 0 (as it)

HM

WP

(%

)

Ab

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12

10

8

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new methods and character-

ization) were used?

The total protein content should

be measured for all samples (as

shown in Figure 4) to evaluate

the presence of insoluble aggre-

gates. As the determined total

protein content is constant under

the applied conditions, insoluble

aggregates are not formed under

these conditions. Conditions of

high temperature and long peri-

ods of time, however, lead to high

amount of high-molecular weight

proteins (HMWP). Reference

samples have to be placed next

to forced degradation samples in

order to evaluate the cause for an

observed effect. All samples from a

specific study need to be analyzed

in the same analytical series to

exclude the effect of possible ana-

lytical variation.

FORCED DEGRADATION DURING THE DEVELOPMENT PHASESForced degradation studies can be

performed in early development

or late development depending on

the purpose of the study and the

amount of material available. The

health authorities expect forced

degradation studies to be carried

out during development Phase

III at the latest, but no guide or

specific requirements exist about

when to perform forced degrada-

tion studies.

A forced degradation study will

provide knowledge about the deg-

radation pathways of the molecule.

By performing such studies early

in development, this knowledge

about the molecule will be avail-

able for optimal process and for-

mulation development.

The degraded samples can aid

the development of stability-indi-

cating analytical methods by dem-

onstrating if the current methods

are sufficient to evaluate stabil-

ity (e.g., use oxidized samples to

develop method for determination

of oxidized forms) and by identify-

ing which test parameters are the

best indicators of stability.

Degraded samples are also use-

ful during analytical validation,

as they can be spiked in valida-

tion samples. However, a limited

amount of material is usually

available at the early stage of

development and the analytical

package might be incomplete.

During development, the process

steps and the formulation might

change. Additionally, the analyti-

cal methods might change due to

further optimization of the ana-

lytical conditions. Hence, forced

degradation studies most likely

need to be repeated or extended

at a later stage of development. In

conclusion, a limited forced degra-

dation study should be performed

as early as possible during develop-

ment, and a more comprehensive

forced degradation study during

Phase III should be performed.

GENERAL EVALUATION OF FORCED DEGRADATION STUDIESResults f rom forced degrada-

t ion st ud ies shou ld be pre -

sented graphically and should

include compare plots for chro-

matographic methods. A result

matrix is an excellent way to

show results, as such a matrix

will be able to indicate which

forced degradation conditions

resulted in changes for which

degradation pathway. Statistical

and kinetic tools should be used

for evaluation of data when pos-

sible to aid the understanding

of the degradation kinetics. A

forced degradation study reveals

the most important degrada-

tion pathways. Such pathways

can, for example, be indicators

of aggregation or the formation

of specific impurities that could

cause concern. The forced deg-

radat ion study a lso indicates

which analytical methods are

most concerning and whether

these methods are able to detect

the change that occurs. In sum-

mary, degradation pathway stud-

ies can help investigators predict

whether an analytical package

is suff icient for a molecule or

whether a need exists for the

development of other analytical

methods.

REFERENCES 1. ICH, Q6A, Specifications: New

Chemical Drug Substances and

Products, Step 4 version (1999).

2. ICH, Q6B, Specifications Test

Procedures and Acceptance Criteria

for Biotechnological/Biological


3. ICH, Q5C, Stability Testing of

Biotechnological/Biological


4. ICH, Q1A(R2), Stability Testing

of New Drug Substances and


5. EMEA, Guideline On Stability Testing:

Stability Testing of Existing Active

Substances and Related Finished

Products (London, October 2003).

Analytical Testing

Forced degradation studies can be performed

in early development or late development

depending on the purpose of the study and the

amount of material available.


This article reviews the current

dynamics in the RNA thera-

peutics/vaccines market as well

as differences between small-

interfering RNA (siRNA), RNA interfer-

ence (RNAi), microRNA (miRNA), and

messenger RNA (mRNA). In addition,

the authors outline the general produc-

tion processes for these platforms, the

challenges encountered during process

development and production, and the

strategies to overcome them.

MARKET OVERVIEW RNA-based therapeutics target the treat-

ment of diseases such as diabetes, cancer,

tuberculosis, and some cardiovascular

conditions. There is currently a great

deal of money being put into this rela-

tively new class of therapeutics and vac-

cines, which is projected to grow 12%

in 2016 and reach $1.2 billion by 2020

(1). The 2015 research and develop-

ment (R&D) biotech pipeline is shown

in Figure 1. There are more than 700

nucleic acid-based therapeutics (DNA

and RNA) in the pipeline and more than

60% of the nucleic acid-based therapeu-

tic pipeline is in preclinical development.

It is interesting to note that 35% of such

pipeline is focused on oncology (2, 3).

Several companies (approximately 160)

and many academic institutes (approxi-

mately 65) are developing RNA-based

therapeutics. Table I provides a non-

comprehensive list of a few (4). Two

companies have marketed R NA-

based therapies: NeXstar and Ionis

Pharmaceuticals. There are 12 mRNA

vacc ines in development, seven

of which are being developed by

Curevac (Germany). Based on current

outlook, the RNA therapeutics market

seems more promising than the mar-

ket for DNA therapeutics.

From a partnership perspective, Ionis

Pharmaceuticals has entered into a global

collaboration with Janssen Biotech, Inc.

to discover and develop antisense drugs

to treat autoimmune disorders of the gas-

trointestinal tract (5), and Merck & Co.

(MSD) has bet $100 million on Moderna’s

mRNA technology (6). Moderna also has

previously announced collaborations

ABSTRACTIn 2014, the monoclonal antibodies market had the highest growth rate (19%)

for the number of new molecules in the pipeline. DNA and RNA therapeutics were not far behind, achieving 12% year-over-year growth. Industry analytics data

suggest that the RNA-based therapeutics market will reach $1.2 billion by 2020.

Bioprocessing Technology Trends of

RNA-Based Therapeutics and Vaccines

Elina Gousseinov, Mikhail Kozlov,Claire Scanlan, Aaron Hammons, Ling Bei, Youssef Benchek, Karim Pirani, and Ruta

Waghmare are all from MilliporeSigma.

Priyabrata Pattnaik works in the

Biomanufacturing Sciences and Training

Centre at Merck Pte Ltd., Singapore.

PEER-REVIEWED

Article submitted: Nov. 9, 2015.

Article accepted: April 5, 2016.

LA

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ett

y I

ma

ge

sPeer-Reviewed

Claire Scanlan, Priyabrata Pattnaik, Ruta Waghmare, Elina Gousseinov,

Mikhail Kozlov, Aaron Hammons, Ling Bei, Youssef Benchek, and Karim Pirani



Our CompanyAs a premier contract discovery, development, and

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WuXi Biologics, a wholly owned

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Free Trade Zone, Shanghai, China 200131

[email protected]

www.wuxibiologics.com


with Alexion, AstraZeneca, and the Defense

Advanced Research Projects Agency (DARPA)

totalling $450 million. Moderna has raised $625

million in equity funding (7, 8).

RNA interference (RNAi) and RNA anti-

sense technologies appear to be dominating

the market. RNAi is a gene-silencing technol-

ogy in which RNA molecules inhibit gene

expression by targeting and destroying spe-

cific mRNA molecules. RNA antisense tech-

nology involves synthesizing an RNA strand

that binds to a specific mRNA or to a splic-

ing site on a pre-mRNA molecule to prevent

translation. The major challenges associated

with the commercialization of these RNA-

based therapies are toxicity and drug delivery.

RNA-BASED THERAPEUTICSWith the advent of RNA-based therapeu-

tics and their potential in treating a variety

of chronic diseases, it is important to note

the number of enabled technologies used to

exploit the RNA mechanism/pathway, some

of which are discussed in the following.

RNAi

RNAi technologies work by “silencing” or

turning off a gene through the use of its own

DNA sequence (Figure 2). The process is initi-

ated by double-stranded RNA (dsRNA) that

expresses either as a small or short hairpin

RNA (shRNA) or as a microRNA (miRNA) tran-

script. Using this silencing mechanism, RNAi

is commonly used to gain a better under-

standing of gene function, which can then

be used to generate additional targeted thera-

peutics (9). Small interfering RNA (siRNA) and

miRNA are the core elements of RNAi tech-

nology based therapeutics.

siRNA

RNAi utilizes a “dicer” enzyme to cut dsRNA

into 21 oligonucleotide segments, called

siRNA. These siRNAs can then bind to a

specific family of proteins called Argonaute

proteins, of which there are two classes: Ago

and Piwi. Ago proteins bind to siRNAs or

miRNAs, while Piwi proteins bind to Piwi-

interacting RNA (piRNA) and are used to

silence mobile genetic elements. The siRNA,

miRNA, or piRNA complex bound to the

Argonaute protein is called the RNA-induced

silencing complex (RISC). Once bound to the

Argonaute protein, one strand of the dsRNA

is removed and the remaining strand binds

to and directs the degradation of the comple-

mentary RNA target sequence, which then

leads to the loss of protein expression (10). AL

L F

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Peer-Reviewed

Figure 1: R&D biotech pipeline expansion.

9765

2209

727

2331

427

1104

15631488

976

295

1952

377

660

733

2009

8490

20152014

487

917

Monoclonal antibody

Bioengineered vaccine

Cell therapy

DNA & RNA therapeutics

Gene therapy

Antibody-drug conjugates

Other biotechnology product

Recombinant product

Figure 2: An insight into the RNAi pathway. Small hairpin RNA

(shRNA) is a class of double-stranded RNA (dsRNA). The dsRNA is

cleaved or degraded by a “dicer” enzyme into oligonucleotide segments

called small interfering RNA (siRNA), which then enter a cell to form

the RNA-inducing silencing complex (RISC). The siRNA strands then

separate or unwind to form the activated RISC complex, which can then

target messenger RNA (mRNA), bind to it, and cleave it.

Dicer

dsRNA cleavage

Target mRNA

Unwound siRNA

Target mRNA cleavage

RISC Complex

RISC Assembly

siRNA

or shRNA

dsRNA


It has been reported that synthetic siRNA

is able to knock down targets in various dis-

eases in vivo, including hepatitis B, human

papilloma virus, ovarian cancer, bone cancer,

hypercholesterolemia, and liver cirrhosis. Only

a few molecules of siRNA per cell are required

to produce effective gene silencing (11). siR-

NAs are most commonly delivered into cells

using microinjection or a transfection agent.

Many companies now offer siRNA-delivering

reagents to simplify this process (12).

miRNA

miRNA do not code for proteins, as they

belong to specific class of non-coding RNAs.

miRNA are 19–25 nucleotides in length and

are encoded within introns (i.e., the portions

of the gene sequence that are not expressed in

the protein) (13). miRNA acts as a guide strand

for the RISC complex to its mRNA target in

vertebrates. Approximately 30% of genes in the

human genome are regulated by miRNA (14).

Though siRNA silencing requires exact

match between target and small interfering

RNA, miRNA are non-specific and can exert

action through imperfect base pairing. In

addition, miRNA triggers translation inhibi-

tion (i.e., prevents the RNA from synthesiz-

ing protein from amino acids), while siRNA

triggers mRNA degradation.

mRNA

mRNA, which codes for protein, is an essen-

tial component of the central dogma of life

(DNAmRNAprotein). mRNA is tran-

scribed from a DNA template. mRNA takes

the genetic code from DNA to the ribosome

where the mRNA is translated to protein.

There has been a significant increase in

mRNA-based therapies in large part due to

the many advantages that mRNA has over

DNA in relation to gene expression and

transfer. While RNAi and antisense RNA

technologies are used primarily for gene

Peer-Reviewed

Table I: Biopharmaceutical companies developing RNA-based therapeutics and vaccines.

siRNA=small interfering RNA, miRNA=microRNA, and mRNA=messenger RNA.

siRNA miRNA mRNA

Kyowa Hakko Kirin Andes Biotechnologies CureVac

Silence Therapeutics Mirna Therapeutics Biontech RNA Pharmaceuticals

Debiopharm miRagen Therapeutics Boehringer Ingelheim

Marina Biotech Marina Biotech Johnson & Johnson

Ipsen Moderna Therapeutics Ludwig Institute for Cancer Research

Alnylam Pharmaceuticals Alnylam Pharmaceuticals BioNTech

Sanofi Pasteur Sanofi Pasteur Sanofi Pasteur

Tekmira Pharmaceuticals Tekmira Pharmaceuticals

NanoCarrier Regulus Therapeutics

Dicerna Pharmaceuticals Biogen Idec

BioCancell Therapeutics GlaxoSmithKline

Samyang Group AstraZeneca

Silenseed Ionis Pharmaceuticals

siRNAsense Les Laboratoires Servier

Reference Biolabs Celsion

Avena Therapeutics Rosetta Genomics

Lipella Pharmaceuticals Santaris Pharma

Arrowhead Research Shire

InteRNA Technologies

Alexion Pharmaceuticals

t2cure

Rigontec

Microlin Bio


silencing, mRNA technologies are often used

in vaccines or gene therapy (15). In both

cases, after injection into the human body,

mRNA is translated to protein, which can

ultimately replace a missing protein (ther-

apeutic) or induce an immune response

(preventive approach). The production of

synthetic mRNA for therapeutic use is rela-

tively straightforward, and the challenges

associated with its stability and delivery have

been tackled through scientific advances in

recent years (16).

RNA-BASED VACCINESConceptually, mRNA-based vaccines are a

simple approach to inducing an immuno-

logical response by delivering the coding

genetic element as a translation-ready mol-

ecule. Upon direct vaccination with mRNA

molecules, dendritic cells (antigen-present-

ing cells) take-up, process, and encode the

target antigen, which in turn induces an

immune response. Typically, mRNA vaccines

are produced by in-vitro synthesis through

an enzymatic process. Such a synthetic pro-

cess can be tightly controlled, resulting in

a quality and predictable product profile.

mRNA can be easily tailored to offer a spe-

cific immunogenic profile and pharmacoki-

netics (17). mRNA’s stability and antigenic

properties can be easily manipulated by

changing codon or modifying base pairs.

Ongoing clinical trials show that mRNA

can be delivered as naked mRNA; immobi-

lized on particles or in liposome nanopar-

ticle; or transfected in dendritic cells in vitro

resulting in a discernible immune response

and protective efficacy. mRNA can also act

as an adjuvant and mRNA also has been

explored to stimulate the innate immune

system through toll-like receptors (18). RNA-

based vaccines are comparatively simple

to produce and can be developed, manu-

factured, and administered in a short time

period, therefore, they are suitable for pan-

demic situations. Thermostability of mRNA

vaccines can also significantly contribute to

their low cost, as they do not require cold-

chain distribution.

MANUFACTURING RNA-BASED BIOPHARMACEUTICALSAs more experimental RNA drugs move

through the clinic and into large-scale tri-

als, the demand for efficient and cost-effec-

tive manufacturing strategies will grow (19).

RNA-based biopharmaceuticals are inher-

ently susceptible to endonucleases, so spe-

cial handling is required for production

and purification. Degradation of product

during manufacturing adds heterogeneity

and chemical instability to the product.

Therefore, the manufacturing and purifi-

cation methods used in RNA-based thera-

peutics differ from that of DNA and other

proteins (20).

mRNA purification (post-chemical syn-

thesis) includes concentration precipitation,

extraction, and chromatographic methods

(including high-performance liquid chroma-

tography) (19). The purpose of the upstream

concentration and diafiltration step is to

concentrate (if lower titer) and change the

buffer to the necessary pH and conductiv-

ity for the first chromatography step. The

objective of the final concentration and dia-

filtration step is to de-salt and achieve the

necessary final concentration prior to ster-

ile filtration. A 5-kD membrane cut-off is

generally used for concentration and diafil-

tration in mRNA processes. Because siRNA

are smaller than mRNA, a 1-kD membrane

cut-off is used for adequate retention of the

siRNA product (21).

CHROMATOGRAPHIC PURIFICATION STEPSSince the breakthrough discoveries of cat-

alytic RNAs in the early 1980s and RNA

interference in the late 1990s, more than

50 RNA or RNA-derived therapeutics have

reached clinical testing. In RNA purifica-

tion, despite the different techniques such

as arginine-affinity, ion-pairing reversed-

phase, or pellicular anion exchange, the

traditional ion-exchange (IEX) media—

especially anion exchange (AEX)—remains

the most popular technique used in both

pure RNA and RNA packaged for delivery

(22, 23, 24, 25).

Sm and Sm-like proteins, which can form

heteromeric complexes or bind to vari-

ous RNAs, were proven to contain ancient

RNA-binding motifs (Sm domain) with

oligo(U) specificity (26). Fractogel TMAE

(MilliporeSigma), a strong anion-exchange

resin, was used for the purification of small

nuclear ribonucleoproteins (snRNP). The

snRNP molecule was eluted with Tris/HCl

Peer-Reviewed


and 300 mM NaCl. Ribonucleoprotein and

uncoupled RNA were separated from free pro-

tein, and the sample was immediately used

for negatively stained electron microscopy.

Both AEX and reversed-phase (RP) tech-

nologies are widely used in the RNA puri-

fication process. Quarternary amine (Q)

and Dimethylaminoethyl (DMAE) chemis-

try are among the choices for AEX (27, 28,

29, 30). One study proved that a few AEX

resins can be used for RNA purification

with optimized experimental conditions to

achieve high dynamic binding capacity. In

this study, among the 18 AEX medias that

were screened, only four resins—Q Sepharose

FF (GE Healthcare), POROS 50HQ (Applied

Biosystems), Q Ceramic HyperD F (Pall), and

Fractogel DEAE (MilliporeSigma)—showed

baseline separations of RNA and plasmid

DNA (31). After optimized loading and elut-

ing conditions, Fractogel DEAE had a wider

range of operation, higher dynamic binding

capacity, and complete separation of RNA

in the breakthrough from plasmid in the

elute. The high recovery, robustness, and

reproducibility also met the requirement for

large-scale manufacturing. These binding

and elution conditions can be utilized as a

starting point for optimal experimental con-

ditions in RNA purification.

Overall, many biochromatography res-

ins are suitable for RNA purification similar

to use in other biomolecule separations. In

many cases, the Fractogel resins showcased

superior capacity and efficiency, largely due

to the “tentacular” structure whereby func-

tional groups are located at the end of long

arms grafted to the bead surface, which cir-

cumvent the steric hindrance caused by large

biomolecules (32).

FORMULATION AND DELIVERYThe most challenging aspect of RNA-based

therapeutics is its delivery to target cells.

Several methods have been explored and

tested in clinical trials. Some of the most

promising approaches are explained in the

following passages.

Polymer conjugation/

chemical modification

Native RNA and RNA-based therapies are

vulnerable to degradation from the many

r ibonucleases found within the cel l .

Chemical modification is one method for

hardening the RNA against such enzymatic

attacks. Modifications to the molecule can

also increase its target affinity, decrease its

undesired immunogenicity, and improve its

overall efficacy. Hardening strategies include

modifications to the backbone, sugar, or base

of the RNA molecule.

Conjugation of the RNA therapeutic is

a strategy that is increasingly being used

for improved delivery and uptake. Alnylam

Pharmaceuticals has adopted a method of

conjugating an amino sugar derivative of

galactose, N-Acetylgalactosamine (GalNac)

to improve the delivery of siRNA therapies

to the liver. The GalNac-conjugated siRNA

is taken up by asialoglycoprotein receptors

in the liver resulting in a fivefold increase in

efficacy versus the parent molecule (33).

Arrowhead Research is developing a com-

peting conjugation strategy. Arrowhead’s

delivery technology, termed Dynamic

Polyconjugates (DPCs), is a siRNA bound to

an endosomolytic polymer backbone via a

disulfide bond. The endosomolytic polymer

enables the quick and efficient release of the

siRNA from the endosome. Arrowhead’s most

recent strategy includes attaching cholesterol

to the siRNA and GalNac to the endosomo-

lytic polymer, ensuring they are both deliv-

ered to the hepatocytes. The co-injection

therapy was shown to increase the efficacy

of siRNA-cholesterol 500-fold with a 90%

knockdown (34).

Encapsulation

The dominant and most-studied strategy for

the delivery of RNA-based therapeutics is

lipid-based delivery systems. One successful

platform is the use of stable nucleic acid lipid

particles (SNALPs), which are lipid particles

formed from a fusogenic lipid, cationic lipid,

and PEG-lipid mixture. The SNALP deliv-

ery system has been developed and champi-

oned by Tekmira Pharma; the company now

refers to it as LNP technology. According to

Tekmira, the LNP “encapsulates siRNAs (also

mRNA) with high efficiency in uniform lipid

nanoparticles that are effective in delivering

RNAi therapeutics to disease sites in numer-

ous preclinical models” (35).

Another promising lipid delivery tech-

nology is the proprietary Smarticles deliv-

ery platform developed by Novosom and

Peer-Reviewed


now owned by Marina Biotech. Similar to

SNALPs, the Smarticles technology can

change their surface charge to facilitate both

stability and endosomal release. Smarticles

are capable of encapsulating both single-

and double-stranded nucleic acid therapies.

Smarticles are comprised of cationic, anionic,

and neutral lipids. The negatively charged

Smarticles avoid the often seen toxic effects

of positively charged lipids at physiologi-

cal pH but convert to a positive charge in

the acidic environment of the endosome,

facilitating its release. Other interesting

encapsulation techniques involve PLGA

nanoparticles (36, 37).

CONCLUSIONRNA-based therapeutics are a relatively

new class of therapies that has bright pros-

pects in the treatment and prevention of

difficult-to-treat chronic and rare diseases.

RNAi work by interfering with the tran-

scription process, and thereby inhibit pro-

tein translation. Though such a therapeutic

approach is highly selective and targeted,

special care is required during the produc-

tion of these therapies and vaccines because

of their susceptibility to ubiquitous RNAse-

induced degradation. Large-scale manufac-

turing of new class of therapeutics would

require bioprocessing components, chemi-

cals, and tools free from RNAse. Technology

and tool providers need to consider making

such products available to enable large-scale

production of RNA-based therapeutics. The

surmounting challenges related to poten-

tial toxicity and drug delivery need to be

addressed before such products can be com-

mercialized. However, new technologies are

emerging to overcome some of these chal-

lenges, and the future of RNA-based thera-

peutics is very promising.

REFERENCES 1. Allied Market Research, “RNA Therapeutics

Market is Expected to Reach $1.2 Billion,

Globally, by 2020,” Press Release, www.

prnewswire.com/news-releases/rna-therapeutics-

market-is-expected-to-reach-12-billion-globally-by-

2020---allied-market-research-274471461.html,

accessed May 24, 2016.

2. Personal communication, Donia Slimani, EMD

Millipore (now MilliporeSigma).

3. EvaluatePharma, World Preview 2015,

Outlook to 2020 (8th Edition, June 2015).

www.evaluategroup.com/public/reports/

EvaluatePharma-World-Preview-2015.aspx,


4. E. Gousseinov et al., Genetic Engineering &

Biotechnology News (Sept. 15, 2015), www.

genengnews.com/insight-and-intelligence/rna-

based-therapeutics-and-vaccines/77900520/,


5. Ionis Pharmaceuticals (n.d.), www.ionispharma.

com/, accessed May 24, 2016.

6. D. Garde, “Merck Bets $100M on Moderna and

its Pioneering RNA Tech,” www.fiercebiotech.com/

partnering/merck-bets-100m-on-moderna-and-its-

pioneering-rna-tech, accessed May 24, 2016.

7. Moderna Messenger Therapeutics, “Our

Core ‘Expression’ Platform: Messenger RNA

Therapeutics,” www.modernatx.com/mrna-

expression-platform, accessed May 24, 2016.

8. B. Fidler, “With Massive Venture Round, Moderna

Has $450M Reasons to Stay Private,” www.

xconomy.com/boston/2015/01/05/with-massive-

venture-round-moderna-has-450m-reasons-to-

stay-private/2/, accessed May 24, 2016.

9. UMass Medical School, “How RNAi Works,” www.

umassmed.edu/rti/biology/how-rnai-works,


10. J. Höck and G. Meister, Genome Biol. 9 (2):210.

doi:10.1186/gb-2008-9-2-210. Feb. 26, 2008).

11. Gene Link, “What is RNAi and siRNA?”, www.

genelink.com/sirna/RNAiwhatis.asp, accessed

May 24, 2016.

12. M. Gujrati and Z.R. Lu, “Targeted Delivery of

Therapeutic siRNA,” in Gene Therapy of Cancer:

Translational Approaches from Preclinical Studies to

Clinical Implementation, E.C. Lattime and S.L. Gerson,

Eds. (Academic Press, 3rd ed., 2013), pp. 47–65.

13. Sigma-Aldrich, “miRNA (microRNA) Introduction,”

www.sigmaaldrich.com/life-science/functional-

genomics-and-rnai/mirna/learning-center/mirna-

introduction.html, accessed May 24, 2016.

14. Qiagen, “MicroRNA–Why Study It and How,” www.

sabiosciences.com/pathwaymagazine/pathways7/

microrna.php, accessed May 24, 2016.

15. R. Scott McIvor, Mol. Therapy 19 (5), pp. 822–

823 (2011).

16. U. Sahin, K. Karikó, Ö Türeci, Nat. Rev. Drug

Discov. 13 (10), pp. 759 –780 (October 2014).

17. T. Kramps and L. Probst, Wiley Interdisp. Rev.

RNA 4 (6), pp. 737–749 (July 25, 2013).

18. S. Pascolo, Handb. Exp. Pharmacol. 183, pp.

221–235 (2008).

Peer-Reviewed

Contin. on page 48

ON-DEMAND WEBCAST Aired June 21, 2016

Register for free at www.biopharminternational.com/bp/development

Functional Comparability Studies for

BiosimilarDevelopment

EVENT OVERVIEW:

Regulatory authorities require applicants for biosimilar

therapies to demonstrate that the proposed product is

biosimilar to the reference product using analytical and

other studies. Comparative analytical testing that evaluates

factors including structure and function can be used to

make decisions about the scope of subsequent studies and

could result in a shortened clinical development process.

This webcast will review key factors in developing,

optimizing and implementing comprehensive func-

tional comparability studies to provide data needed to

advance the development process. Experts will discuss

the regulatory requirements and guidelines for functional

comparability studies and the types of studies that should

be conducted; review approaches to understand multiple

mechanisms of action and assay development; and the

qualification of assays used in these studies.

Key learning objectives

Review the regulatory requirements for comparability studies

Learn which functional comparability studies should be conducted

Understand the role of reference products for comparability studies

Learn about development & qualification of assays for comparability studies

For questions contact Kristen Moore at [email protected]

Who should attend

Laboratory managers

Product development scientists

Process development leaders

Quality control professionals

Sponsored by

Presenters

Hoss A Dowlat, PhD

Vice President,

Regulatory Affairs, Global

Strategy

PharmaBio Consulting

Abhi Saharia, PhD

Director, Cell-based

Assays and Biologics

DiscoverX

Nicolas Fourrier, PhD

Director Biomarker

and Biopharmaceutical

Testing

SGS Life Sciences

Moderator

Rita Peters

Editorial Director

BioPharm International

Presented by


The International Organization

for Standard izat ion ( ISO)

published the long-awaited

revisions to its standards for

classification and monitoring of air

cleanliness in cleanrooms on Dec. 15,

2015. ISO 14644-1:2015 “Cleanrooms

and associated controlled environments

Part 1: Classification of air cleanliness

by particle concentration” (1) replaces

ISO 14644-1:1999, and ISO 14644-

2:2015 “Cleanrooms and associated con-

trolled environments Part 2: Monitoring

to provide evidence of cleanroom per-

formance related to air cleanliness by

particle concentration” (2) replaces ISO

14644-2:2000.

The 2015 editions are the result of a

systematic review and include changes

made in response to requests by users and

experts in the cleanroom community. In

particular, the requests for reviewing Part 1

were related to “the basis for the number of

sampling locations and, most importantly,

the whole statistical basis of classification

of cleanliness using the Student T-test for

one to nine sampling locations,” notes

Gordon Farquharson, convenor of the ISO

TC209 working group 1, which performed

the review and revisions. Because Part 2 is

closely aligned with Part 1, the committee

reviewed both parts together.

CLASSIFICATION BY PARTICLE CONCENTRATIONThe addition of “by particle concen-

tration” to the title of the standard is

a long-overdue clarif ication, com-

ments Karen Ginsbury, CEO at PCI

Pharmaceutical Consulting. “For years,

I have heard cleanroom contractors and

practitioners alike wrongly describe ISO

14644-1 and 2 as cleanroom ‘validation’

or ‘qualification’ standards.” She notes

that the standards only address airborne

particles, not other factors crucial to

cleanroom qualification, such as smoke

tests to determine airflow patterns.

The introduction of Part 1 explains,

“This part of ISO 14644 specifies classes

of air cleanliness in terms of the num-

ber of particles expressed as a concentra-

tion in air volume. It also specifies the

standard method of testing to deter-

mine cleanliness class, including selec-

tion of sampling locations” (1).

SAMPLING CHANGESThe primary changes to Part 1 involve the

number of samples and the selection of

sampling locations. “The number of sam-

ples will increase from what was required

previously,” explains Marsha Stabler

Hardiman, senior consultant at ValSource.

“The minimum number of samples is now

determined from a lookup table (instead

of an equation), and that number is set to

be statistically significant.”

According to ISO, the new method

for selecting the sites and number of

sampling collections uses a more con-

sistent statistical approach based “where

samples are drawn randomly without

replacement from a finite population.

The new approach allows each location

to be treated independently with at least

a 95% level of confidence that at least

90% of the cleanroom or clean zone

areas will comply with the maximum

particle concentration limit for the target

class of air cleanliness. No assumptions

are made regarding the distribution of

the actual particle counts over the area

of the cleanroom or clean zone; while in

ISO 14644-1:1999 an underlying assump-

tion was that the particle counts follow

the same normal distribution across the

room” (1). The sampling locations are

to be chosen representatively, meaning

that “features such as cleanroom or clean

Revised ISO Cleanroom Standards Improve Air Cleanliness Classification

Jennifer Markarian

Revised versions of ISO 14644

Parts 1 and 2 introduce changes to

sampling procedures

and monitoring plans for

cleanrooms and clean zones.

Cleanroom Standards


zone layout, equipment disposition,

and airflow systems should be con-

sidered when selecting sampling

locations” (1).

This representative selection of

sample locations is a big change from

the previous random selection, says

Hardiman. “A company now has to

have a rationale and justification for

sample site location to ensure that

the sample locations selected are rep-

resentative of the characteristics of

that section. Companies will have to

look at the new number of sample

locations and then determine where

the representative sample locations

will be collected. If contracting out

the classification activities, you

should make sure that your contrac-

tor is now using your new, represen-

tative sample locations.”

CLASSIFICATION OF MACRO AND NANO-PARTICLESThe revision makes a change

regarding large (≥ 5 μm) particles,

which are required to be measured

for some classifications in the EU

Annex 1 GMP guidelines (3) and

others. “The experts working on

the revision of ISO 14644-1 were

of the opinion that particles ≥ 5

μm diameter should not be used

to classify ISO class 5 and cleaner

environments because of the

uncertainty associated with par-

ticle collection efficiency and

accuracy of counting low con-

centrations,” says Farquharson.

“In order that the European

Union (EU), the Pharmaceutical

Inspec t ion Convent ion and

Ph a r m a c e ut i c a l I n s p e c t io n

Co-operat ion Scheme, World

Health Organization, and Chinese

GMPs are not left without a clas-

sification tool for their Grades A (at

rest and operational) and B (at rest),

ISO 14644-1:2015 provides a mech-

anism of extrapolating the macro-

particle descriptor for class limits of

20 and 29 particles ≥ 5 μm.”

The new document does not

address nano-scale particles, which

were formerly defined as ultrafine

particles in ISO 14644-1:1999, but

will address these under a new

Part 12, notes Farquharson, who

explains, “These particles are mea-

sured using a different particle

counter, and industries such as

semi-conductor monitor for con-

centration of these very small air-

borne particles at critical control

points. These particles are not gen-

erally of interest to the pharmaceu-

tical and life sciences industries.”

MONITORING AND TESTINGISO 14644-2:2015 now requires

monitoring to provide evidence of

cleanroom performance, explains

Fa rqu ha r son. T he s t a nda rd

addresses airborne particle concen-

tration, airflow, and device pres-

sure difference. New topics include

monitoring of critical parameters

and setting action and alert alarms.

The revised standard now allows

companies to use risk management

to set their periodic classification

testing schedules, notes Hardiman.

“In the past, the retesting was pre-

scribed in a table and the timing was

based on the ISO class of the clean-

room or clean zone. Now, compa-

nies can put more emphasis on the

day-to-day data that they generate in

their own facilities to help determine

the appropriate testing and frequen-

cies needed for continued cleanroom

compliance. If a company is generat-

ing great data and the risk to contin-

ued cleanroom compliance is low,

then they can set a longer periodic

classification frequency,” explains

Hardiman. She notes that risk-based

sample site selection is crucial for

environmental monitoring. “The key

is understanding your unique prod-

ucts and processes and selecting sites

that best address the risks that your

products and processes present. It is

important to be able to identify all of

the potential contamination sources

in each cleanroom and to select

environmental monitoring sample

locations in close proximity to these

sources. It is also very important to

understand the people, material, and

waste flows,” concludes Hardiman.

Pharmaceutical cleanrooms typi-

cally already have monitoring plans,

which are required by Annex 1 of

the EU GMPs, says Ginsbury (3). She

notes that users should, however,

check with their cleanroom contrac-

tors to determine whether a contrac-

tor is qualified and familiar with

the revisions, because the regulators

reference the ISO standards and they

must be followed, says Ginsbury. “I

would recommend having the dis-

cussion now so that, by 2017, your

contractors and in-house staff are

fully up to speed and following the

new standard,” she notes. “Even if

you use contractors, the responsi-

bility for review and approval of

their results and compliance with

regulatory standards lies with you.

ISO may not require 5 μm particles

and may let you use risk assessment

to determine frequency of classifi-

cation. However, EU Annex 1 still

requires measuring 5 μm particles

as part of classification, and current

industry practice (EU [3] and FDA

[4]) is to perform cleanroom classi-

fication twice a year for aseptic core.

We haven’t heard the regulators

on this one but I wouldn’t rush to

reduce that frequency based on risk

assessment,” cautions Ginsbury.

REFERENCES 1. ISO, ISO 14644-1:2015, Cleanrooms

and associated controlled

environments – Part 1: Classification

of air cleanliness by particle

concentration (Geneva, 2015).

2. ISO, ISO 14644-2:2015, Cleanrooms

and associated controlled

environments – Part 2: Monitoring

to provide evidence of cleanroom

performance related to air cleanliness by

particle concentration (Geneva, 2015).

3. EC, EudraLex Volume 4: Good

manufacturing practice Guidelines,

“Annex 1, Manufacture of Sterile

Medicinal Products,” (Brussels, 2008).

4. FDA, Guidance for Industry:

Sterile Drug Products Produced

by Aseptic Processing—Current

Good Manufacturing Practice

(Rockville, MD, 2004).

Cleanroom Standards


Biopharmaceutical manufactur-

ing is a complex process involv-

ing many unit operations,

precise amounts of materials, and

numerous variables. These elements must

be addressed properly to ensure consistent

product quality and drug product yield.

While a strong focus on quality, safety,

and drug efficacy is absolutely essential,

biopharmaceutical manufacturers are

also committed to finding ways to reduce

costs and improve manufacturing efficien-

cies. One production step with significant

costs (and potential risk to quality) is the

buffer and cell-culture materials prepara-

tion process: it is labor-intensive, requires

investment in storage and environmental

resources, and involves repeated quality

assurance (QA) testing as bulk materials

are subdivided for individual process runs.

New innovations in raw materials

packaging technologies can directly

impact this process—streamlining oper-

ations, mitigating risks, and contribut-

ing to operational excellence (OpEx). In

this article, the author examines new

methods of raw materials packaging

and how they may lead to manufac-

turing efficiencies, elimination of raw

material yield losses, reduction in QA

testing time and costs, and lower costs

in weigh and dispense production.

TRADITIONAL RAW MATERIAL DELIVERY METHODSUpstream biopharmaceutical processes

consume various raw materials, includ-

ing cell-culture media, carbohydrates,

amino acids, and buffers, which are typ-

ically supplied in powder form. The bio-

reactors and medium preparation tanks

that use these materials often oper-

ate around the clock. This operation

includes both large-scale reactors with

10,000 L capacity that run con-

t i nu o u s l y f o r a ny w h e r e f r o m

15–35 days, to newer generation single-

use technologies, with multiple 2000-L

bioreactors operating in overlapping

sequences to achieve similar or greater

productivity.

In general, two kinds of packaging

systems are in use today: Traditional

steel 100 kg drums with one or two plas-

tic liners, and smaller cardboard boxes

with plastic liners holding 50 kg. Both

bulk-packaging systems are part of stan-

dard practices that most raw materials

suppliers have established for their sup-

ply chain systems. The end user (i.e.,

the biopharmaceutical producer) typi-

cally orders, receives, and stores enough

salts, buffers, and other cell-culture

powdered materials to last several weeks

or months. The material consumption

rates can be substantial: a typical buf-

fer media preparation/usage cycle may

consume between 150 and 400 kg of

dry products per run, and uses multiple

raw materials per cycle. These materials

are then subdivided by the biopharma-

ceutical producer and used in smaller

amounts depending on the processes

they are running. In essence, this bulk

material packaging and delivery meth-

odology satisfies the raw material sup-

plier’s operational requirements, without

full consideration of how that material is

used by the biopharmaceutical producer.

OPERATIONAL INEFFICIENCIES AND RISKSFollowing the more traditional method

described above, the biopharmaceuti-

cal manufacturer must follow multiple

processing steps to properly manage and

use these bulk raw materials.

t #VML NBUFSJBMT BSF SFDFJWFE BOE

inventoried in storage areas that

must have the appropriate temper-

Raw Materials Packaging Innovations for Biopharmaceutical Manufacturing

Nandu Deorkar

Recent trends in raw

materials packaging

may impact manufacturing,

quality, and cost of

biopharma-ceuticals.

Nandu Deorkar, PhD, MBA, is

vice-president of R&D for Avantor

Performance Materials. He has

more than 25 years of experience in

materials technology research and

development, and has worked on

various aspects of chemical/polymer

R&D, drug development, formulation,

drug delivery technologies, process

development, and technology transfer.

Packaging Trends


ature and humidity controls

to maintain the mater ia l’s

integrity.

t5IF DPOUBJOFSTPVUFSQBDLBHJOH

is cleaned and sanitized. A sam-

ple is taken to independently

confirm via lab analysis that the

product’s quality, purity, and

characterization match what was

ordered. Once this is confirmed

(and analysis can take multi-

ple days), the bulk material is

cleared for use in the producer’s

dispensing operation.

t 0ODF DMFBSFE UIF DPOUFOUT PG

the drum are subdivided by

hand according to manufactur-

ing requirements. For example,

if 45 kg are initially required,

that quantity is removed and the

remainder is put back into stor-

age. For a 2000-L bioreactor, a

manufacturer may need to sub-

divide a 100 kg drum of material

between two and five times.

t 5IJT TVCEJWJTJPO TUFQ JT UJNF

consuming and risks cross-con-

taminating the remaining bulk

material. Some biopharmaceu-

tical producers conduct multi-

ple lab analyses of these drums,

each time a new quantity of

material is removed, to confirm

that no issues have occurred.

Much of this activity precedes,

and is not directly related to, the

value-added process of protein

manufacturing. It is time and

effort spent on materials manage-

ment and warehousing activities,

particularly the sub-dividing step,

to supply the bioreactors with the

precise amount of material needed

for production.

COMPLICATIONS DUE TO MATERIAL CAKING/CLUMPINGVarious raw materials, such as salts,

buffers, amino acids, and carbohy-

drates, have an intrinsic propensity

to form clumps or cake due to their

crystal structure and surface mois-

ture content. The presence of avail-

able surface moisture catalyzes the

process of caking when free mois-

ture migrates onto the surface of

the crystals and dissolves a small

portion, forming a salt bridge. This

caking, or clumping (Figure 1), is

a common problem with packag-

ing, storing, and sub-dividing pow-

dered materials in bulk containers.

Changes in ambient temperature

or humidity are the principal fac-

tors driving this process; the num-

ber of temperature-change cycles

will increase the strength of the

cake. Severe cases of caking can

result in complete solidification of

the entire package. Caked materi-

als must be completely broken up

in order to measure out the precise

amounts needed for bioreactor pro-

cesses. This is a time-consuming

manual activity with an open con-

tainer, which is at risk for cross-

contamination and absorption of

additional moisture, potentially

extending the problem. This prac-

tice also creates a potential safety

risk, as operators work to manu-

ally break up clumps while the

container is open, and can lead to

material loss.

MATCHING PACKAGING SIZE TO PROCESS NEEDSOver the past few years, pharma-

ceutical and biopharmaceutical

manufacturers have worked with

bulk material suppliers to modify

packaging approaches. The goal

was to enhance operational excel-

lence and reduce wasted time

and impact on process and prod-

uct quality. The initial focus was

on bulk material container sizes,

which typically held many more

materials than were needed, and

forced on-site storage and subdi-

viding steps. While continuing

to offer the standard 50 kg and

100 kg containers, suppliers began

to provide smaller, more manage-

able container sizes, such as 12 kg

and 25 kg.

This approach reduced the

length of time containers were

kept in storage and the number of

times a container was subdivided.

Subdividing was still required, how-

ever, because the exact amounts

of sugars, buffers, salts, and other

powdered bulk materials varied

greatly by manufacturer, based on

the specific process, protein, and

bioreactor equipment being used.

Subdividing also adds a measure of

uncertainty to the amount of raw

materials dispensed in a particu-

lar process step. Biopharmaceutical

production uses extremely tight

control on process parameters to

protect the final drug’s safety and

quality, and finding ways to strictly

control the amount of raw mate-

rial that goes into bioreactors can

enhance operational excellence and

process yield.

NEW PACKAGING FOR PRE-WEIGHED, FREE-FLOWING MATERIALSInnovative chemicals suppliers

have begun offering single-use,

pre-weighed product bags that pro-

vide biopharmaceutical produc-

ers with an easy-to-use method

for dispensing salts, buffers, and

other cell-culture materials directly

into their media or buffer prepara-

tion tanks, in the exact amounts

they specify for a given process.

The packaging options that are

now used essentially complete the

evolution from 50 kg and 100 kg

bulk product drums, to individual

direct dispense bags. The packag-

ing is constructed of transparent

polymers, the same materials that

have already been used to line the

traditional bulk drums; therefore,

biopharmaceutical manufactur-

ers do not have to re-validate the

material as safe for use.

The packaging’s design offers

biopharmaceutical manufactur-

ers more choices with regard to

packaging size. Manufacturers can

order the materials in a wide range

of smaller, precise quantities (e.g.,

from 250 g to 100 kg). The size,

shape, sealing, and seams of these

Packaging Trends


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bags are designed so that when they

are inverted, they dispense virtually

all the pre-weighed material into

the bioreactor. An important con-

sideration here is that pre-weighed

amounts should be within a 1%

tolerance of the amount of mate-

rial required. This is crucial, espe-

cially when the direct dispense bags

are used in single-use bioreactors.

If a biopharmaceutical manufac-

turer has determined that an exact

amount of glucose or sodium citrate

is needed to achieve maximum

yield on a process, the ability of

the packaging to freely deliver that

amount to very tight tolerances

must be assured.

To help ensure a free-flowing

dispensing system, the bags also

incorporate design features to

eliminate clumping, including

outer and inner layers with spe-

cial desiccant materials installed

between the two layers (Figure 2).

The inner layer uses a vapor per-

meable material already in use in

other container-lining applica-

tions. Any moisture that develops

within the bag passes through this

material and is controlled by the

desiccant, to maintain the cor-

rect moisture levels and reduce

clumping to an absolute minimum

(Figure 3).

DIRECT DISPENSE BAGS SIMPLIFY SAMPLING AND TESTINGTraditional large-volume bulk

container packaging also necessi-

tates the time-consuming process

of sampling and testing to verify

the material properties of a newly

delivered drum of product. Direct

dispense bag systems use trans-

parent polymers that are compat-

ible with non-destructive identity

testing tools, such as contact-free

Raman spectroscopy. With Raman

testing, there is no need to open

the bag and take a physical sample

to verify the product, the closed

bag can be scanned and verified

upon delivery, saving multiple

testing steps. The packaging is also

tamper-evident to ensure validity

and supply chain security.

In addition to near Raman test-

ing, some suppliers also will pro-

vide a tailgate sample along with

the bags. Biopharmaceutical pro-

ducers that are required to conduct

full analyses of all materials used in

their processes don’t need to open

the bag to obtain a product sample.

The tailgate sample process must be

validated to ensure that the material

in the tailgate sample is equivalent

to the materials packed in the bag.

DIRECT DISPENSE SYSTEMS: TIME AND COST SAVINGSExpanding the use of these direct

dispense systems can help advance

operational excellence initiatives

and reduce costs within the bio-

pharmaceutical industry’s supply

chain. By adopting flexible manu-

facturing technology, such as the

direct dispense systems, biophar-

maceutical manufacturers may

be able to reduce their operating

costs in certain areas by up to 40%.

There are multiple savings associ-

ated with the use of these systems:

t -BCPS&MJNJOBUFT UIF UJNF BOE

cost of personnel who need to

weigh, subdivide, and dispense

materials from bulk containers.

The savings can be significant.

Depending on the process, each

1000 L run of a cell-culture reac-

tor may require approximately

1000 to 2000 kg of more than

50 raw materials for the produc-

tion process. The media/buf-

fer preparation activities could

require 30–50 labor hours for

dispensing and adding the mate-

rials to the reactors.

t 'BDJMJUJFT 6TF PG EJSFDU EJT-

pense systems can eliminate the

need for dedicated raw material

preparation areas, drum storage

and handling equipment, and

environmental (temperature

and humidity) control equip-

ment. In some cases, these sys-

tems can reduce f loor space

needs by 40–70%.

t 5FTU JOH WB M JEBU JOH 6TF PG

Raman testing and tailgate sam-

ples greatly simplifies the testing/

validating step. Products do not

have to be re-validated each time

material is sub-divided from a

bulk container. In addition, the

primary packaging material typ-

Packaging Trends

Figure 1. A common problem with packaging, storing and sub-dividing powdered

materials in bulk containers is material caking or clumping.


Packaging Trends

ically used for these bags is the

same as standard drum liners, so

contact material does not have to

be re-validated.

t 2VBMJUZ1SFXFJHIFEEJSFDUEJT-

pense systems eliminate the need

to clean the weighing and dis-

pensing area for another opera-

tion, saving time and eliminating

risk of cross-contamination and

employee exposure.

t .BUFSJBMTUBCJMJUZBOEFGGJDJFOUVTF

Anti-clumping packaging design

improves raw material yields by

avoiding material non-confor-

mities and inaccurate ingredi-

ent measurements from clumped

materials.

t 4BGFUZ "OUJDMVNQJOH QBDLBH-

ing leads to sound environmen-

tal, health, and safety practices,

as employees no longer need to

engage in the potentially unsafe

practice of breaking up clumps

that can form in large drums.

t 3BX NBUFSJBMT TBWJOHT 1SF

weighed amounts in direct dis-

pense bags that match specific

biopharmaceut ica l process

requirements eliminate the need

to buy and store material in bulk,

reducing overages, out-of-date

materials, and disposal costs.

ADVANCES IN BULK MATERIAL PACKAGING FOCUS ON IMPROVING OVERALL OPEXBy evolving raw material packag-

ing and delivery options to align

more closely with the operational

requirements of biopharmaceutical

producers, raw materials suppli-

ers are helping to eliminate inef-

ficiencies and drive down costs

within the overall supply chain

and production environment. The

success of these bag-based, pre-

weighed, free-flowing direct dis-

pense systems is also encouraging

the development of streamlined

systems for other types of materi-

als beyond bulk powders. Some

raw-materials suppliers are inves-

tigating new packaging methods

to deliver ready-made liquid solu-

tions to customers, which would

eliminate the biopharmaceutical

manufacturing step of taking solid

materials and creating solutions.

While the transportation and

storage considerations for liquid

solutions are more complex than

solids, there are opportunities to

apply innovations to raw-materi-

als packaging designs to improve

the efficiency, productivity, safety,

and quality of biopharmaceutical

manufacturing.

Figure 2. To help ensure a free-flowing dispensing system and eliminate

clumping, some bags have outer and inner layers with special desiccant materials

installed between the two layers. The inner layer uses a vapor permeable material;

any moisture that develops within the bag passes through this material and is

controlled by the desiccant, to maintain the correct moisture levels.

INNER TYVEK®

LAYER

4” TC FERRULE

BLANKING CAP

4” TC CLAMP

4” TC CASKET

(EPDM)

2-OFF DESI-PAK

ACTIVATED CLAY

DESICCANT

(4 UNITS)

DESICCANT PACKS

FITTED BETWEEN

TYVEK® LAYER AND

OUTER PE LAYER

Figure 3. With new developments in raw material packaging, even after four

weeks at 40 °C and 90% relative humidity, the material in this 10 kg bag remains

free-flowing.


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Troubleshooting

Single-use technologies (SUT) have made sig-

nificant inroads in biopharmaceutical and

vaccine manufacturing. Greater adoption in

the years to come can be anticipated, given their

broad use in pre-clinical and clinical manufactur-

ing and expanding use for approved therapeutics.

With increasing regulatory oversight of SUT pro-

cesses, it’s worthwhile to review basic concepts of

design and qualification that apply to single-use

components and systems (SUS).

EQUIPMENT DESIGN REGULATIONS AND GUIDANCEWhile drug and vaccine manufacturers are sub-

ject to regulatory review and inspection of how

equipment is used, that is not the case for the

manufacturers of process equipment, including

SUT components and systems, even when sold for

use under good manufacturing practice (GMP).

Suitability of design and qualification for use must

be determined by the therapeutic manufacturer.

The US Code of Federal Regulations (21 CFR 211.63),

Section 211.63 states, “Equipment used in the

manufacture, processing, packing, or holding of a

drug product shall be of appropriate design, ade-

quate size, and suitably located to facilitate opera-

tions for its intended use and for its cleaning and

maintenance” (1). While this is the responsibility

of the user, equipment manufacturers need to

understand and design to user requirements for

what may be considered appropriate,

adequate, or suitable.

US 21 CFR does provide some

information for process equipment

designers: Section 211.65, paragraph

(a) states, “Equipment shall be con-

structed so that surfaces that contact

components, in-process materials, or

drug products shall not be reactive,

additive, or absorptive so as to alter

the safety, identity, strength, qual-

ity, or purity of the drug product

beyond the official or other established require-

ments” (1). While not specifying those require-

ments, this requirement highlights the need for

SUT equipment designers to consider operational

performance criteria, as well as potential chemical

interactions and equipment-derived impurities or

particulate contaminants that may be introduced

into applicable processes and potentially impact

intermediates or final dosages.

Much of SUT use occurs in therapeutic pro-

tein API production. International Council

on Harmonization (ICH) Q7, European

Medicines Agency (EMA) Q7, and FDA Q7A

Good Manufacturing Practice Guidance for Active

Pharmaceutical Ingredients incorporate essen-

tially the same design requirement statement:

“Equipment should be constructed so that surfaces

that contact raw materials, intermediates, or APIs

do not alter the quality of the intermediates and

APIs beyond the official or other established speci-

fications” (2–4).

Fortunately, established SUT manufacturers

have significant experience working with drug and

vaccine companies and have learned to adopt SUT

equipment designs to meet user needs in GMP-

regulated processes. Adoption of established good

engineering practices for design and deployment

of SUTs is crucial and requires close collaboration

between users, component designers and system

engineers having experience in polymer materials,

device manufacturing, human interface engineer-

ing, and other related practices (5).

SUT DESIGN CONSIDERATIONSDesign considerations for SUT manufacturers can

be divided into six categories:

t 1IZTJDPDIFNJDP QSPQFSUJFT DPWFS TFMFDUJPO

and performance of the materials of construc-

tion and finished component under antici-

pated use conditions, with reasonable safety

factors. These properties can include pressure

requirements (e.g., operating, burst, creep, pres-

Design and Qualification of Single-Use Systems The author provides a review of the concepts of design and qualification that apply to single-use systems.

Jerold M. Martin is an independent

consultant and Chairman Emeritus,

BPSA, [email protected].


TVSFESPQ NBYNJO UFNQFSBUVSF

requirements (e.g., operating,

thermal sterilization, melt, cryo-

genic, impact of temperature on

pressure performance), mechani-

cal properties (e.g., flexibility,

rigidity, and tensile strength),

optical properties (e.g., clarity or

opacity), cleanliness (e.g., surface

or embedded particles), biocom-

patibility, material oxidation and

radiation stability, gas barrier

properties, polymer additives,

transmissible spongiform enceph-

alopathy-bovine spongiform

encephalopathy (TSE–BSE) status,

and other raw material supplier

data, etc.

t 'MVJE DPOUBDU QSPQFSUJFT UIBU

should be considered in product

design include chemical compat-

ibility (e.g., polymer solubility,

swelling, embrittlement, etc.),

chemical reactivity, extractables

under appropriate solvents and

extraction conditions, particle

TIFEEJOH BOEQSPUFJOCJOEJOH

adsorption to contact surfaces

(along with any other formulation

ingredients).

t 'PSN GJU BOE GVODUJPO SFMBUFT

to the “fitness for purpose” of

the physical design of the

component(s) and assemblies.

It includes handling and other

ergonomic properties, accessibil-

ity for installation and removal,

microbial barrier and physical

and fitment (leak) integrity prop-

erties, sterilizability, drainability,

incorporation into an automated

platform as the disposable fluid

path, etc.

t .BOVGBDUVSBCJMJUZ FOTVSFT UIBU

the SUT production equipment

and environment are suitable to

effectively produce the compo-

nent or system as designed and

maintain quality specifications

with a high degree of assurance.

Manufacturability should also

include operator training and in-

process and final product testing

(as needed) for quality factors such

as integrity (i.e., retention prop-

erties of filters, leak absence of

containers, connector and hose fit-

ments, microbial barrier, etc.) bio-

burden, endotoxins, and particle

contamination or generation.

t 1BDLBHJOHBOETIJQNFOUDPOUBJO-

ers must be designed to effectively

protect the SUT component or

assembly during transport from

the manufacturing or assembly

site, as well as ease and security of

unpacking and installation at the

user site.

t %PDVNFOUBUJPO TIPVME BMTPCF

considered as part of product

design, to include support data

for performance claims, design

and production validation data

reports, operation guides, and

other information that may be

requested by users or from regu-

latory authorities.

QUALIFICATIONQualification is the action of proving

that any equipment works correctly

(as designed) and can be expected

to perform as intended. For the user,

qualification includes confirming

that the equipment is the right tool

for the job. While the term valida-

tion is sometimes applied to incor-

porate the concept of qualification,

validation means verifying (and doc-

umenting) that the equipment con-

sistently functions within a specified

range of operations to produce an

intended result. Qualification studies

are, therefore, done with representa-

tive samples prior to use. Validation

is conducted on process equipment

(or scaled-down models) under actual

use conditions.

While the word qualification is not

specifically mentioned in 21 CFR 211,

the interpretation of these regula-

tions by both industry and regulatory

agencies has introduced terms such

as design qualification (DQ) (i.e., is

the design suitable for user require-

ments?), installation qualification

(IQ) (i.e., is the equipment installed

properly?), operational qualification

(OQ) (i.e., does it operate according

to the manufacturer’s specifications?),

and performance qualification (PQ)

(i.e., does it consistently perform

to meet the user’s requirements?).

Additional terms that combine these

concepts include factory acceptance

qualification (FAQ) or test (FAT) (e.g.,

DQ, IQ, OQ) and site acceptance

qualification (SAQ) or test (SAT) (e.g.,

IQ, OQ, PQ).

The first step of qualification

should be DQ. For SUT, this occurs

in two phases: confirmation by the

equipment supplier that the equip-

ment meets its’ design and opera-

tion criteria, and evaluation by the

user that the component design and

performance is suitable for use in the

intended application. To the degree

that the equipment supplier can

anticipate the user’s requirements,

some portion of the manufacturer’s

qualification data can also serve as

the user’s evaluation and be incor-

porated into the user’s documenta-

tion to be presented to regulatory

authorities. This is of course provided

UIBU UIFNBOVGBDUVSFSTVQQMJFSQSP-

cedures and data are suitable for GMP

use, the user review of the data is

documented, and a user audit is con-

ducted to confirm data validity.

Conformance of the SUT design

with the manufacturer’s claims

and user’s process requirements

should be demonstrated and docu-

mented. According to European

Union GMP Annex 15, Section

3, “Qualification activities should

consider all stages from initial

development of the user require-

ments specification (URS) through

to the end of use of the equip-

ment, facility, utility or system.”

Once the URS is developed, “The

next element in the qualification

of equipment, facilities, utilities,

or systems is DQ where the com-

pliance of the design with GMP

should be demonstrated and doc-

umented. The requirements of

Troubleshooting

Contin. on page 48

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Company DescriptionDistek, Inc., headquartered in North Brunswick, NJ, is

a leading manufacturer of laboratory testing instruments

for the pharmaceutical and biotechnology industry, as well

as an experienced provider of validation and qualification

services.

Distek’s robust product portfolio includes water

bath and bathless dissolution, dissolution media heating,

degassing, dispensing and disposal, in-situ fiber optic UV,

bathless tablet disintegration, content uniformity, digital

video monitoring, programmable automated sampling,

and now a single-use bioreactor system for mammalian cell

culture applications.

Founded in 1976, Distek has an excellent reputation

for innovation, product reliability, and customer

support, and we believe that these, as well as continual

improvement, are essential to our current and future

growth and crucial to ongoing success.

For technological advancements that offer reliable

and consistent performance, the pharmaceutical industry

trusts Distek.

Markets ServedDistek’s core market includes brand name and generic

pharmaceutical drug manufacturers, CROs, CMOs,

Nutraceuticals, government agencies, and universities.

To ensure consistent quality and to meet the international

standards of our world market, Distek has been ISO

certified since 2002.

Services & InnovationsDistek does not simply design ‘me too’ products. Distek

instruments are considered to be highly innovative, which

is exemplified in the product portfolio. Distek engineers

technically modern and user-friendly instruments that

have provided many innovative features to customers,

worldwide. Distek holds several instrumentation and

accessory patents and has been ISO certified since 2002.

Distek’s robust product portfolio includes water

bath and bathless dissolution, dissolution media heating,

degassing, dispensing and disposal, in-situ fiber optic UV,

bathless tablet disintegration, content uniformity, digital

video monitoring, programmable automated sampling,

and now a single-use bioreactor system for mammalian cell

culture applications.

Major Product InnovationsYou don’t need to make a large capital investment to

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cabinet, probes, motor, heating jacket and recipes. Since

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process. All materials are USP Class VI, animal derivative

free and utilized in existing single-use products.

Distek, Inc.

Distek, Inc.121 North Center Drive

North Brunswick, NJ 08902

Phone: 732.422.7585

Fax: 732.422.7310

www.distekinc.com


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Troubleshooting

the user requirements specifica-

tion should be verified during the

design qualification” (6).

SUPPLIER QUALITY SYSTEM AND QUALIFICATION PRACTICESSUT and SUS are unique from tra-

ditional stainless equipment in that

the user depends on the supplier’s

quality system. Review of the suppli-

er’s quality system should be part of

supplier qualification. Furthermore,

users cannot normally perform

incoming testing prior to using SUSs

intended for implementation in pro-

duction, so are also more dependent

on suppliers’ product qualification

practices.

Supplier qualification of SUT

products can be summarized in

product qualification and process

validation reports suitable for user

documentation and submittal to

regulatory authorities. Applicable

test methods for pre-use qualifica-

tion are detailed in Bio-Process

Systems Alliance (BPSA’s) Quality Test

Reference Matrices document, along

with supplemental BPSA guides

with specific recommendations on

qualification of extractables, steril-

ization, and particulates testing (7).

Additional tests for SUS qualification

have been described by users (8–12).

The United States Pharmacopeial

Convention (USP) has published a

draft standard on extractables test-

ing for plastic process equipment

(13). A BPSA guide on SUS leak test-

ing is also under development. In

addition, BPSA provides an indus-

try consensus-generated Quality

Agreement Template that can be

used to document accountabili-

ties (7), and audits should be used

to review these under the supplier’s

quality management system.

Also important for SUS users is

to qualify the SUT equipment sup-

plier’s reliability and delivery his-

tory. Potential supplier performance

can be assessed by the experience

of integrators and users who have

previously worked with them.

Attendance at industry meetings,

participation on scientific asso-

ciation SUS committees, or trade

associations such as BPSA pro-

vide important access to suppli-

ers, integrators, and users to aid in

EFUFSNJOJOH BQQSPQSJBUF TVQQMJFS

integrator qualification practices.

REFERENCES 1. FDA, US Code of Federal Regulations (21

CFR 211.63), Section 211.63, Current

Good Manufacturing Practice for Finished

Pharmaceuticals (FDA, April 2014).

2. ICH, Q7 Good Manufacturing Practice

Guide forActive Pharmaceutical Ingredients

(ICH, November 2000).

3. EMA, Note for Guidance on Good

Manufacturing Practice for Active

Pharmaceutical Ingredients (CPMP/

ICH/4106/00), November 2000.

4. FDA, Guidance for Industry, Q7A Good

Manufacturing Practice Guidance for Active

Pharmaceutical Ingredients, August 2001

5. M. Botterill and B. Rawlings, BioProcess

Int. (December 2008).

6. EMA, EU Guidelines for Good

Manufacturing Practice for Medicinal

Products for Human and Veterinary Use,

Annex 15: Qualification and Validation,

February 2014.

7. BPSA, Component Quality Test Matrices,

Gamma Irradiation and Sterilization,

Extractables and Leachables, Particulates,

Quality Agreement Template, www.

bpsalliance.org

8. PDA, Technical Report No. 66,Application

of Single-Use Systems in Pharmaceutical

Manufacturing (PDA, 2014).

9. D. M. Stephenson, J. Val. Technol.

(February 2003).

10. M. A. Petrich, Amer. Pharm. Rev. 16 (7)

(November/December 2013).

11. W. Ding, BioPharm Int. 28 (9) (September

2015) pp. 32–39.

12. D. Riedman and J. Martin, BioProcess Int.

9 (S2) (2011) pp. 28-35.

13. USP, <661.3> (draft): Plastic Systems

Used for Manufacturing Pharmaceutical

Products, USP—Pharmacopoeal Forum

42(3) (USP, March 2016).

19. R. Martins, J.A. Queiroz, and F. Souza, J. Chrom. A 1355,

(August 2014).

20. T. Schlake et al., RNA Biol. 9 (11), pp. 1319–1330 (Nov. 1, 2012).

21. Personal communication with BSN team, EMD Millipore (now

MilliporeSigma).

22. R. Martins, J.A. Queiroz, and F. Sousa, Anal. Bioanal. Chem.

405 (27), pp. 8849–8858 (November 2013).

23. P.M. Swiderski et al., Anal. Biochem. 216, pp. 83–88 (1994).

24. J.R. Thayer et al., Chrom. Today, (March 2011), www.

chromatographytoday.com/article_read/985/, accessed

May 24, 2016.

25. F. Wincott et al., Nucl. Acids. Res. 23 (14), pp. 2677 –2684 (1995).

26. T. Achsel, H. Stark, and R. Luhrmann, Proc. Natl. Acad. Sci.

U.S.A. 98 (7), pp. 3685–3689 (March 27, 2001).

27. C. Miller et al., Mol. Syst. Biol. 7 (458), doi: 10.1038/

msb.2010.112 (Jan. 4, 2011).

28. L.E. Easton et al., RNA 16 (3), pp. 647–653 (2010).

29. W.J. Issa et al., “Ion Exchange Purification of mRNA,” US

patent WO2014144767 A1, Sept. 18, 2014.

30. W.H. Pan et al., Mol. Ther. 9 (4), pp. 596–606 (April 2004).

31. A. Eon-Duval et al., J. Chrom. B Analyt. Technol. Biomed. Life

Sci. 804 (2), pp. 327–335 (2004).

32. I. Theodossiou, M. SØndergaard, and O.R. Thomas,

Bioseparation 10 (1–3), pp. 31–44 (2001).

33. J.K. Nair et al., J. Am. Chem. Soc. 136 (49), pp. 16958–

16961 (Dec. 10, 2014).

34. S.C. Wong et al., Nucleic Acid Ther. 22 (6), pp. 380–390

(December 2012).

35. Arbutus Biopharma, “Tekmira Presents Recent Advances in

mRNA Delivery at Scientific Symposium,” Press Release, Feb.

25, 2014, http://investor.tekmirapharm.com/releasedetail.

cfm?ReleaseID=827952, accessed May 24, 2016.

36. P. Pantazis et al., “Preparation of siRNA-Encapsulated PLGA

Nanoparticles for Sustained Release of siRNA and Evaluation of

Encapsulation Efficiency,” in Nanoparticles in Biology and Medicine:

Methods and Protocols, Methods in Molecular Biology, M. Soloviei,

Ed. (Springer Science+Business Media, 2012), pp. 311–319.

37. M. Chen et al., ACS Nano 6 (6), pp. 4835–4844 (2012).

Peer-Reviewed—Contin. from page 36

Contin. from page 45


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BD BIOSCIENCES 5

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EUROFINS LANCASTER LABORATORIES 10–11

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POLYTHERICS LTD 15

SGS LIFE SCIENCE SERVICES 37

TOSOH BIOSCIENCE 25, 51

UNIVERSITY OF NEBRASKA-LINCOLN 2

WUXI APP TEC 31, 52

PRODUCT SPOTLIGHT


BIOLOGICS NEWS PIPELINE

IN THE PIPELINE

Using a New CRISPR Effector to Edit RNA

A naturally occurring CRISPR (clustered regularly

interspaced short palindromic repeats) system that spe-

cifically can be used to modify the RNA of an organ-

ism is the newest development in the technology’s

evolution. A new study, published in Science, identifies

C2c2, a bacterial protein that can be used as a tool

to cleave single-stranded RNA sequences at desired

locations. As an RNA-guided RNAse, C2c2 can be har-

nessed to defend against viral intruders and turn off

gene expression of certain conditions, according to

researchers.

Typically, gene silencing is a gene expression manip-

ulation performed by small interfering RNA (siRNA),

but the study researchers attest that the C2c2 complex

is even more efficient when it comes to editing RNA.

Specifically, C2c2 can be used to add to or delete infor-

mation from existing RNA sequences, and can also be

used to tag RNA to learn more information about the

function of certain sequences. In addition, C2c2 can

be programmed to knock out certain messenger RNA

(mRNA), disrupting the DNA-->mRNA-->protein tran-

scription process. C2c2 requires only a single RNA to

function, and is “genetically encodable,” the researchers

said in a Broad Institute/Harvard press release accompa-

nying the study. As an RNA-targeting immune system

mechanism, they say C2c2 is a promising tool for future

RNA manipulation.

The authors concluded that the CRISPR-C2c2 complex

is probably not the only system that can be programmed

to alter RNA sequence, and other, patentable editing tools

may be on the horizon. They wrote, “It is likely that other,

broadly analogous Class 2 RNA-targeting immune systems

exist, and further characterization of the diverse members

of Class 2 systems will provide a deeper understanding of

bacterial immunity and provide a rich starting point for

the development of programmable molecular tools for in

vivo RNA manipulation.”

Although the research on CRISPR-C2c2 was conducted

by various teams at MIT, the Broad Institute, Harvard

University, the National Institutes of Health, Rutgers

University-New Brunswick, and the Skolkovo Institute

of Science and Technology, the Broad Institute’s Feng

Zhang is at the helm of the research and serves as one of

the paper’s senior authors. Zhang is notorious for being

one of the founders of the CRISPR-Cas9 complex and its

applications for editing DNA, however, there is currently

an ongoing legal battle between the Broad Institute and

the University of California (involving work by researcher

Jennifer Doudna and by French researcher Emmanuelle

Charpentier) for the patent rights to the technology.

Study: HIV-1 Neutralizing Antibodies

in Infants May Impact Vaccine Development

A new article in Cell that studied HIV-neutralizing antibod-

ies in an infant may have important implications in the

development of an HIV vaccine. The novel study exam-

ined broadly neutralizing antibodies (bnAbs) in a Nairobi

infant prior to receiving antiretrovirals and approximately

one-year post infection. Researchers isolated and character-

ized infant HIV-1 neutralizing antibodies in order to fur-

ther understand their impact on the HIV virus.

Multiple studies have been done on HIV-1 bnAbs in

adults, but little is known about infant bnAbs contributing

to broad plasma responses. While a subset of adults with

HIV-1 develop bnAbs, these antibodies exhibit high levels

of somatic hypermutation (SHM) and only neutralize the

disease after years of affinity maturation. By comparison,

infant’s bnAbs develop broad responses early, and “HIV-1-

specific neutralization breadth can develop without pro-

longed affinity maturation and extensive SHM.” Therefore,

infants may have the ability to neutralize the disease more

quickly than adults.

“Most studies of adults have looked at responses many

years after infection and these have suggested that broad

and potent HIV neutralizing antibodies take years to

develop and that they require extensive hypermutation to

be effective,” study author Julie Overbaugh, PhD, member

of the Human Biology Division at the Fred Hutchinson

Cancer Research Center, told BioPharm International. “This

is the first example of broadly neutralizing antibodies

detected this early in infection. These infant antibodies

have much less hypermutation and yet, they are nonethe-

less broad and potent.”

Overbaugh notes the infant studied had a polyclonal

response to the virus, meaning, the infant’s antibodies

targeted multiple epitopes, making it more difficult for the

virus to survive. This presents an interesting comparison

to studies done on adult antibodies, which target only one

dominant epitope, Overbaugh says.

The challenge is now finding ways to harness these

unique attributes of infant bnAbs for practical applications

such as vaccine development. The goal, researchers say,

is to better understand infant bnAbs that develop early

in natural infection in order to develop vaccines that can

elicit neutralizing antibodies more quickly.

“Infants mount a more rapid potent antibody

response to HIV than adults—understanding how they

do this is important, both in terms of how they do it

more rapidly and whether there are fundamental dif-

ferences in the pathway,” said Overbaugh. “Adults take

a long time to develop these responses [naturally], lon-

ger than is possible to imagine for a vaccine regimen.”

www.tosohbioscience.com

Tosoh Bioscience and TOYOPEARL are registered trademarks of Tosoh Corporation.

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4785

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an ADC drug’s global IND filing

stable cell lines developed

for GMP Manufacturing

cell-based assays developed

including 15 qualified for GMP

Volume 29 Number 7 BioPharm · 2018-09-11 · Publisher Mike Tracey [email protected] National...

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Transcript of Volume 29 Number 7 BioPharm · 2018-09-11 · Publisher Mike Tracey [email protected] National...