Volume 29 Number 1 Pharm -...

PharmThe Science & Business of Biopharmaceuticals

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January 2016

Volume 29 Number 1

FRAMING BIOPHARMA

SUCCESS IN 2016

DOWNSTREAM

PROCESSING

GOING SMALL TO

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PEER-REVIEWED

PRECIPITATION AS

AN ALTERNATIVE TO

CHROMATOGRAPHY IN THE

INSULIN MANUFACTURING

PROCESS

REGULATIONS

POLITICS AND PRICING

WILL CHALLENGE

MANUFACTURERS IN 2016

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INTERNATIONAL

BioPharmThe Science & Business of Biopharmaceuticals

EDITORIALEditorial Director Rita Peters [email protected] Editor Agnes Shanley [email protected] Editor Susan Haigney [email protected] Editor Randi Hernandez [email protected] Science Editor Adeline Siew, PhD [email protected] Manager Caroline Hroncich [email protected] Director Dan Ward [email protected] 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]/Mid-West Sales Manager Steve Hermer [email protected] Coast Sales Manager Scott Vail [email protected] Sales Manager Chris Lawson [email protected] Sales Manager Wayne Blow [email protected] Data and List Information Ronda Hughes [email protected] 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]

UBm LIfE SCIENCES

Tom Ehardt, EVP & Senior Managing Director, Life Sciences Tom Mahon, Senior VP, Finance Georgiann DeCenzo, EVP & Managing Director, UBM Medica Mike Alic, EVP, Strategy & Business Development Dave Esola, VP & Managing Director, Pharm/Science Group Johanna Morse, VP & Managing Director, CBI/IVT Becky Turner Chapman, VP & Managing Director, Veterinary Group Joy Puzzo, VP, Marketing & Audience Development Francis Heid, VP, Media Operations Jamie Scott Durling, Director, Human Resources

UBm AmERICAS

Simon Foster, Chief Executive Officer Brian Field, Chief Operating Officer Michael Bernstein, Head of Legal

UBm PLC

Tim Cobbold, Chief Executive Officer Andrew Crow, Group Operations Director Marina Wyatt, Chief Financial Officer Dame Helen Alexander, Chairman

© 2016 Advanstar Communications Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical including by photocopy, recording, or information storage and retrieval without permission in writing from the publisher. Authorization to photocopy items for internal/educational or personal use, or the internal/educational or personal use of specific clients is granted by Advanstar Communications Inc. for libraries and other users registered with the Copyright Clearance Center, 222 Rosewood Dr. Danvers, MA 01923, 978-750-8400 fax 978-646-8700 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].

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

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

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

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

EDITORIAL 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

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4 BioPharm International www.biopharminternational.com January 2016

Contents

BioPharmINTERNATIONAL

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.

COLUMNS AND DEPARTMENTS

6 From the Editor Infrastructure and payer decisions will determine drug choices. Rita Peters

7 Investment Outlook

47 Troubleshooting Miniature bioreactors can predict the cell culture kinetics in scaled-up reactors. Mohd Helmi Sani and Frank Baganz

50 Analytical Best Practices This article defines the concept, justification, and method of removal of out-of-trend points in stability modelling and shelf-life prediction. Thomas A. Little

56 Product Spotlight

56 New Technology Showcase

57 Biologics News Pipeline

57 Ad Index

58 Ask the Expert Siegfried Schmitt, principal consultant, PAREXEL, discusses how to streamline the document management process during market expansion.

Cover: Nastco/Andrey Machikhin/Martin McCarthy/

Mina De La O/Getty Images; Dan Ward

BIG PICTURE FOR 2016

Framing Biopharma Success in 2016Rita C. Peters

Corporate restructurings, regulatory

initiatives, and biosimilars will shape

biopharma development in 2016. 8

mAbs to Watch in 2016Randi HernandezWhich monoclonal antibodies will

gain US regulatory approval in 2016? 12

Politics and Pricing Will Challenge Manufacturers in 2016Jill WechslerThe bio/pharmaceutical industry will face increased scrutiny of product quality and

cost drivers. 14

Outsourcing Outlook for 2016Susan HaigneyIndustry experts discuss what the

outsourcing market holds for 2016. 16

BIOREACTORS

Tools for Continuous Bioprocess DevelopmentRajeev J. Ram

Could perfusion microbioreactors bring

more agility to biomanufacturing? 18

DOWNSTREAM PROCESSING

Going Small to Achieve Success on the Commercial ScaleCynthia A. Challener

Scale-down modeling is instrumental in supporting the development of downstream

biopharma manufacturing processes. 26

PEER-REVIEWED

Precipitation as an Alternative to Chromatography in the Insulin Manufacturing ProcessMadhavan Buddha, Shailabh Rauniyar, Shabandri Qais, Dinesh Goudar, Sai Srikar Kandukuri, Siddharth Mahajan, Sinash Siddik, and Partha Hazra

The authors evaluated two precipitation strategies as alternatives to the conventionally

used chromatographic process. 30

SERIALIZATION

Serialization: Getting Past the Quick FixAgnes Shanley

Traceability and transparency will remain elusive if manufacturers continue to approach serialization projects on a

case-by-case basis. 36

HEALTH-BASED

EXPOSURE LIMITS

EMA Guideline on Setting Health-Based Exposure LimitsAndrew Teasdale, Bruce D. Naumann, Gretchen Allison, Wendy Luo, Courtney M. Callis, Bryan K. Shipp, Laura Rutter, and Christopher Seaman

The results of an industry workgroup’s examination of EMA’s guide on shared

facilities are presented. 41

Volume 29 Number 1 January 2016

fEATURES


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From the Editor

Infrastructure and

payer decisions

will determine

drug choices in

emerging and

developed regions.

Emerging Nations Close the Medicine Use Gap

Biopharma industry headlines in 2015 reflected the positive (more drug

approvals and the first US-approved biosimilar); the negative (inflated

drug prices, Martin Shkreli, and tax inversion-driven mergers); and the

uncertain (multiple mega mergers and FDA initiatives).

In this issue, the editors look at key industry trends and expectations for the

year ahead, including these predictions for future global drug demand.

Improved patient access to chronic disease treatment and breakthrough

drug therapies will help reduce the “medicine use gap” between emerging and

developed markets, driving global spending on drugs to grow at a 4–7% com-

pound annual rate over the next five years. Regional economic conditions and

healthcare infrastructure, however, will dictate the types of drugs prescribed,

number of medicine doses, and ultimately, the volume of pharmaceutical sales.

By 2020, total patient spending on medicines will be $1.4 trillion, reports

the IMS Institute for Healthcare Informatics (1); however, the global spend-

ing increase from 2015 to 2020, estimated to be 29–32%, is below the 35%

increase of the prior five years.

Global medicine use will reach 4.5 trillion doses in 2020, the study predicts,

up 24% from 2015. Emerging markets will account for most of the increase,

led by India, China, Brazil, Indonesia, and Africa. Generic drugs, non-original

branded drugs, and over-the-counter products will account for 88% of total

medicine used in emerging markets, the report says. More than 90% of the

prescription drugs filled in the United States in 2020 will be generic drugs, up

from the present level of 88%.

Brand spending in developed markets is expected to reach up to $590 bil-

lion, a 34% increase in spending over 2015 on an invoice price basis, thanks to

new product launches, and price increases in the US—which may be offset by

discounts and rebates. Invoice price growth, which does not reflect discounts

and rebates received by payers, is expected to continue at historic levels during

the next five years; however, competition and payer resistance will keep net

price increases to 5–7% annually.

Spending growth will be curbed by patent expiries, which will result in $178

billion in reduced spending on branded products, including $41 billion on

biologics, as biosimilars become more widely adopted.

The rise of specialty medicinesGlobal spending on specialty medicines used to treat chronic, rare, or genetic

diseases is expected to reach 28% of the total spending by 2020. The report

estimates that more than 225 medicines will be introduced by 2020, with

one-third focused on treating cancer; other development areas are hepatitis C,

autoimmune disorders, heart disease, and rare diseases. Adoption of specialty

medicines will be most prevalent in established markets.

“During the next five years, we expect to see a surge of innovative medi-

cines emerging from R&D pipelines, as well as technology-enabled advances

that will deliver measurable improvements to health outcomes,” said Murray

Aitken, IMS Health senior vice-president and executive director of the IMS

Institute for Healthcare Informatics, in a press statement. “With unprec-

edented treatment options, greater availability of low-cost drugs and better

use of evidence to inform decision making, stakeholders around the world can

expect to get more ‘bang for their medicine buck’ in 2020 than ever before.”

Reference1. IMS Health, Global Medicines in Use in 2020 (Parsippany, NJ, November 2015).

Rita Peters is the editorial director of

BioPharm International.


Investment Outlook

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Shire Acquires Baxalta in $32 Billion DealShire and Baxalta have announced a $32 billion agreement

under which Shire will combine with Baxalta, creating a

company with the top rare diseases platform in revenue and

pipeline depth, the companies reported in a Jan. 11, 2016 press

statement. The new company is projected to achieve annual

revenues of $20 billion by 2020.

Shire reports that the combined companies will have

products in growing franchises in hematology; immunology;

neuroscience; lysosomal storage diseases; gastrointestinal/

endocrine; hereditary angioedema; and oncology; as well as a

late-stage ophthalmics pipeline.

Baxalta shareholders will hold approximately 34%

ownership in the combined company. The parties expect the

transaction to close mid-2016.

Boehringer Ingelheim to Establish Biopharmaceutical Production Facility in ViennaBoehringer Ingelheim will make a significant investment in

biopharmaceutical production at its Vienna site. The company

will establish a new large-scale biopharmaceutical production

facility for active ingredients manufactured using cell cultures.

With the roughly half billion-euro investment, Boehringer

Ingelheim will also create more than 400 new jobs in the

Austrian capital.

“This is a decision for Europe as a pharma location,” said

Professor Andreas Barner, PhD, chairman of the board of

managing directors at Boehringer Ingelheim. “We took a close

look at various international options as part of the investment

decision, also considering the research environment at

potential sites. The clincher for Vienna was ultimately the

company’s desire to additionally secure the market supply

of biopharmaceutical products and to balance the risk by

establishing a further independent facility.”

In Vienna, the company has already produced

pharmaceutical active ingredients using microorganisms;

over the next few years, cell-culture technology will also be

transferred to the plant. According to the company, the new

production plant will go into operation by 2021. Boehringer

Ingelheim has already been operating two large-scale facilities

for the market launch and cell-culture-based manufacture of

biopharmaceuticals in Biberach, Germany.

GSK Acquires Bristol-Myers Squibb R&D HIV AssetsGlaxoSmithKline (GSK) announced its global HIV business,

ViiV Healthcare, has reached two separate agreements with

Bristol-Myers Squibb (BMS) to acquire its late-stage HIV R&D

assets and its portfolio of preclinical and discovery-stage HIV

research assets.

Under the terms agreed in the two transactions, ViiV

Healthcare will acquire late-stage assets. Including fostemsavir

(BMS-663068), an attachment inhibitor, currently in Phase

III development for heavily treatment-experienced patients.

Fostemsavir has received a Breakthrough Therapy Designation

from FDA, and the company expects to file for regulatory

approval in 2018. Another Late-stage asset is a maturation

inhibitor (BMS-955176), currently in Phase IIb development for

both treatment-naive and treatment experienced patients. A

back-up maturation inhibitor candidate (BMS-986173) is also

included in the purchase.

Assets in preclinical and discovery phases of development

include a novel biologic (BMS-986197) with a triple mechanism

of action, a further maturation inhibitor, an allosteric integrase

inhibitor, and a capsid inhibitor. A number of BMS drug

discovery employees will also be offered the opportunity to

transfer to ViiV Healthcare.

According to GSK, the late-stage asset purchase comprises

an upfront payment of $317 million, followed by development

and first commercial-sale milestones of up to $518 million,

and tiered royalties on sales. The purchase of preclinical

and discovery-stage research assets comprises an upfront

payment of $33 million, followed by development and first

commercial-sales milestones of up to $587 million, and further

consideration contingent on future sales performance. The

two transactions are anticipated to complete independently

during the first half of 2016, subject to necessary approvals,

anti-trust, and regulatory clearances.

Takeda Acquires US Biologics Manufacturing FacilityTakeda Pharmaceutical Company Limited announced the

acquisition of a Brooklyn Park, Minnesota-based biologics

manufacturing facility from Baxalta Inc. Takeda is a Japan-

based pharmaceutical company focused on research and

development. It is the largest pharmaceutical company in

Japan.

Takeda intends to use the facility primarily for the

manufacture of Entyvio (vedolizumab) and other biologic

products. FDA approved Entyvio in May 2014 for the

treatment of ulcerative colitis and moderate-to-severe

Crohn’s disease. Terms of the transaction were not

disclosed.

January 2016 www.biopharminternational.com BioPharm International 7



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Dan W

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The US biopharmaceutical indus-

try turned a new page in 2015

when FDA approved Zarxio (fil-

grastim-sndz), the first biosimi-

lar approved for use in US markets, in

March 2015. Biotechnology stocks con-

tinued to outperform the Standards

& Poor’s 500 Index and NASDAQ

Composite Index. Venture capital invest-

ment and initial public offerings were

also near record levels (1). If the intro-

duction of generic alternatives is a sign

of maturity in the biologic-based drug

segment, the biopharma companies

may want to examine lessons learned by

small-molecule based pharma companies

as the market transitions.

In the past 20 years, biopharma

companies enjoyed success as block-

buster drugs dominated the market,

made adjustments as the patent cliff

approached, and in some cases, aban-

doned exist ing R&D programs to

seek alternate, more profitable routes.

Counterfeit drugs and drug shortages

disrupted the delivery of safe, effective

therapies to patients. Oversized, ineffi-

cient research, development, and manu-

facturing engines were downsized, or

transferred to outsourcing providers.

In 2015, FDA’s Center for Drug

Eva luat ion and Research (CDER)

approved 45 new molecular entity

and new biologic l icense applica-

tion approvals (2), the second highest

annual total. The Center for Biologics

Eva luat ion and Research (CBER)

approved more than a dozen new bio-

logics, vaccines, blood products, and

diagnostics. These numbers, and types

Framing Biopharma Success in 2016

Rita C. Peters

The Big Picture for 2016


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of drugs approved, indicate that

the drug-development market may

be headed in a new direction, rais-

ing questions about the ability of

the current R&D and manufactur-

ing infrastructure to adapt to the

changing market. While approv-

als are up, FDA cautions that the

number of new drug applica-

tions has been flat, indicating the

potential of a slowdown ahead.

BREAKTHROUGHS, BIOSIMILARS, AND BOTTLENECKSMany of the new molecular enti-

ties—41% of the approvals in

2014 and 47% of the approvals in

2015—were for rare diseases that

target smaller patient populations

and, therefore, require smaller

quantities of drug doses required

compared to blockbuster drugs.

This shift in production require-

ments—combined with lower

profit margins for former block-

buster brand drugs now prescribed

as lower-cost generic drugs—has

implications for drug development

and manufacturing processes.

A market research study esti-

mates that the global biosimilars/

follow-on-biologics market will be

$26.5 billion by 2020, up from

$2.55 billion in 2014. Although

the compound annual growth rate

is expected to be of 49.5% from

2015 to 2020, the growth is slower

than anticipated due to a lack of

regulatory guidelines in China

and the United States and a lack of

economies of scale for small-scale

manufacturers (3).

FDA’s expedited pathways for

drug approval have proved suc-

cessful; 60% of the 2015 approvals

were designated in one or more of

the expedited pathways (fast track,

breakthrough, priority review, and

accelerated approval). While this

faster route to market of needed

therapies is good news, FDA’s

inspection process, and the inabil-

ity of drug manufacturers to accel-

erate production timeframes, are

potential roadblocks. John Jenkins,

director of CDER’s Office on New

Drugs, reported at the FDA/CMS

Summit in December 2015 that

manufacturing and inspections—

not clinical development—are often

the “rate-limited steps” for expe-

dited approval.

INVESTING IN INFRASTRUCTUREAttractive credit terms in recent

years prompted some biopharma

manufacturers to invest in facili-

ties and equipment. Encouraging

compa n ie s to ma ke cap it a l

improvements to avoid future

quality and manufacturing prob-

lems, however, can be a tough

financial and regulatory argu-

ment. In a 2015 survey of 100

executives from top biopharma

companies (4), less than half

said they planned to increase

investments in late-stage R&D.

Instead, marketing/distribution

(79%), drug discovery/early-stage

R&D (70%), clinical trials (60%),

technology acquisit ion (59%),

and patenting (50%) were higher

investment priorities.

To encourage manufacturers to

adopt new production technolo-

gies, FDA issued a draft guidance

document (5) in December 2015

that provides a framework for drug

manufacturers and FDA to discuss

manufacturing design and devel-

opment issues early in the process.

The goal is to educate FDA staff

about the new technologies in

advance of chemistry, manufac-

turing, and controls (CMC) sub-

mission, thereby expediting the

approval process.


The view from the biopharma trenches

The big decisions about company acquisitions, mergers, and business development

may be made in the boardrooms of bio/pharma companies, but the frontline

professionals—those involved in formulation, development, and manufacturing

functions—can offer interesting observations about the impact those business

strategies have on the day-to-day tasks of bringing a drug to market.

More than 450 biopharma professionals from around the world participated in the

2015 BioPharm International annual employment survey (1). Respondents expressed

opinions about job security (74.6% feel secure in their current position), seeking new

opportunities (59.2% would like to leave their job), and increasing dissatisfaction

with salary levels. The respondents also expressed opinions about trends in the

industry, how changes impact their daily work, and future business prospects.

Almost 40% of those responded said business at their company increased in

2015, similar to data reported in the previous two years; 27.5% reported downsizing.

One-quarter of the respondents reported that their company had been through a

merger or acquisition in the past two years, down slightly from the 2014 survey.

Overall, respondents expressed a positive outlook for the bio/pharmaceutical

industry for the next year; however, fewer respondents said business would improve

(55.4% in 2015 compared with 62.2% in 2014). When looking at a longer-term horizon

(five years), nearly two-thirds of respondents (65.4%) predicted that business will

improve; however, 13.5% expect business to improve overseas, but not domestically.

More than half of the respondents (52%) predicted that their company’s business will

improve in 2016; more than one-third (34%) expected no significant change.

Reference

1. 2015 BioPharm International Employment Survey.



QUALITY WARNING SIGNSFDA plans to use quality metrics

to develop risk-based inspection

scheduling of drug manufactur-

ers; to improve its ability to predict

and mitigate drug shortages; and

“encourage the pharmaceutical

industry to implement state-of-the-

art, innovative quality manage-

ment systems for pharmaceutical

manufacturing” (6). Industry feed-

back to the d ra f t g u idance

reflected concerns about how FDA

will use the reported data to guide

further regulatory action, what

players in the supply chain were

responsible for reporting to FDA,

how the data would be interpreted

to assess overall product quality,

and factors used to determine the

success of a quality program. A key

issue was the perceived burden and

additional resources needed to per-

form quality testing.

THE MERGERS, ACQUISITIONS AND COLLABORATIONS PICTUREIn 2015, biopharma companies

and contact service organizations

actively repositioned their business

profiles though mergers, acquisi-

tions, and collaboration agree-

ments, as well as divestitures of

specific product lines or business

units. Senior executives of large

biotechnology and pharmaceutical

companies surveyed by ReedSmith

(3) plan to stay on the investment

track; 94% plan to initiate an acqui-

sition during the next 12 months;

more than one-third plan to

divest assets. The search for exter-

nal opportunities was a priority;

more than half of the respondents

said they are planning peer-to-peer

research partnerships; 85% report

that they plan to hire a contract

research organization.

More respondents (74%) said

major pharmaceutical produc-

ers would target companies with

strong drug discovery or early-stage

R&D potential versus companies

involved in late-stage R&D (69%).

RIGHT-SIZING R&D EFFORTS FOR IMPROVED RETURNSFor the past six years, Deloitte

and GlobalData analysts exam-

ined R&D results for the top 12

publicly held, research-based life-

science companies (measured by

R&D spending in 2008–2009); in

2015, four mid- to large-cap com-

panies were added to the study.

Analysis of the pharma industry’s

performance in generating return

on investment in new drug devel-

opment revealed a continuing pat-

tern of declining returns, from

10.1% in 2010 to 4.2% in 2015.

While the cost to develop an asset

increased 33%, from $1.188 billion

in 2010 to $1.576 billion in 2015,

the average peak sales per asset

dropped 50%, from $816 million

in 2010 to $416 million in 2015.

The numbers, the report authors

say, “do not add up for life-sci-

ences R&D to generate an appro-

priate return” (7).

There was some good news; the

mid- to large-cap companies out-

performed the top 12 publicly held

companies, indicating that different

R&D business models could pro-

duce better results.

Despite the decline in return on

investment, the companies in the

study continued to increase invest-

ment in R&D as a proportion of

cash generated, from 25.5% in

2004 to 29.4% in 2014. Investors,

however, seek returns. Companies

are more likely to return cash to

shareholders via dividends and

share buybacks than they are to

invest in acquisitions, product

licenses, and internal R&D, the

study concludes.

At larger R&D companies, legacy

infrastructure, which may be dif-

ficult to improve, and excess over-

head are major contributors to R&D

costs. The smaller companies in the

study did not have the large infra-

structure, but risk becoming too

complex and lose their R&D pro-

ductivity advantage as they grow.

Both groups must challenge addi-

tional investments at each phase

and evaluate returns for each asset,

the study reports.

The authors suggest that focusing

R&D efforts on stable therapy areas

and specialty therapeutics will add

to scientific, regulatory, and com-

mercial value propositions. Agility,

flexibility, and a focus on science

will allow external sources of inno-

vation to be optimized. Reducing

development complexity by stream-

lining functions and addressing

unproductive infrastructure can

improve returns.

FRAMING THE 2016 AGENDAEarly indications are for mergers

and acquisitions activity to con-

tinue in 2016, and prospects for

new drug approvals also are strong.

FDA has a busy agenda of legislative

approvals ahead, complicated by

an election year. Among these dis-

tractions, the industry must drive

ahead to seek shareholder value and

affordable, effective patient thera-

pies on the same route.

REFERENCES 1. Z. Tracer, Health Care in 2016: Eight

Charts You Need to Follow the Sector,

online, www.bloomberg.com/news/

articles/2016-01-08/the-eight-charts-

you-need-to-understand-health-care-

in-2016, accessed Jan. 10, 2016.

2. FDA, Novel New Drugs Summary

2015, online, www.fda.gov/Drugs/

DevelopmentApprovalProcess/

DrugInnovation/ucm474696.

htm, accessed, Jan. 5, 2016.

3. R. Deshmukh, World Biosimilars/Follow-

on-Biologics Market - Opportunities

and Forecasts, 2014–2020 (2015),

www.alliedmarketresearch.com/

pharma/global-biosimilars-market

4. ReedSmith, Life Lines: Life Sciences

M&A and the Rise of Personalized

Medicine (London, 2015).

5. FDA, Advancement of Emerging

Technology Applications to Modernize

the Pharmaceutical Manufacturing Base

Guidance for Industry, Draft Guidance

(Rockville, MD, December 2015).

6. FDA, Request for Quality Metrics

Guidance for Industry, Draft Guidance

(Rockville, MD, July 2015).

7. Deloitte Centre for Health

Solutions, Measuring the Return

from Pharmaceutical Innovation

2015 (London, 2015). ◆




Of the 45 new molecular enti-

ties and new therapeutic bio-

logical products approved by

FDA in 2015, nine products

were monoclonal antibodies (mAbs).

And there may be many more of these

types of proteins on the horizon—as

many as 14 are projected to hit the mar-

ket in 2016, according to data compiled

by EvaluatePharma, in collaboration with

BioPharm International (*).

MABS IN DEVELOPMENTIxekizumab—Eli Lilly’s ixekizumab is an

interleukin-17 drug that works by target-

ing the IL-17 ligand. This mAb is being

developed for the treatment of psoriasis

and psoriatic arthritis, and may directly

compete with Novartis’ Cosentyx

(secuk inumab) and Ast raZeneca/

Valeant’s brodalumab. Brodalumab,

however, will work a bit differently—it

is an antibody that binds to the IL-17

receptor, whereas secukinumab and

ixekizumab focus on the IL-17 ligand.

In clinical trials, ixekizumab demon-

strated superiority over Enbrel (etaner-

cept) for clearing skin plaques.

Brodalumab—Valeant and AstraZeneca’s

IL-17 therapy, brodalumab got some bad

press in May 2015 when Amgen dropped

out of a co-development project with

AstraZeneca for the drug after clinical-trial

results suggested that the drug was associ-

ated with suicidal ideation in patients. In

September 2015, however, Valeant stepped

in and agreed to be responsible for the

commercialization and development of

the project. Brodalumab was shown in

clinical trials to clear the scaly skin patches

associated with psoriasis more efficiently

than Johnson & Johnson’s (J&J) Stelara

(ustekinumab).

Obiltoxaximab—Elusys Therapeutics’

mAb, obiltoxaximab, which will go by the

trade name Anthim, differs significantly

from the rest of the mAb pack. The inves-

tigational agent for the treatment of inha-

lational anthrax infection is supported by

the Biomedical Advanced Research and

Development Authority (BARDA). Even

before the product’s approval by FDA,

BARDA granted Elusys its first delivery

procurement order for Anthim so that the

agency could add Anthim to its Strategic

National Stockpile (SNS) as a countermea-

sure against a potential bioterrorist attack.

Elusys has already received more than

$220 million in grants from government

agencies to develop the mAb. The drug,

meant to be used as both a treatment after

anthrax exposure and as an anthrax pro-

phylactic, is being investigated as an intra-

venous treatment as well as an emergency

injectable.

Atezolizumab—This PD-L1 checkpoint

inhibitor is being investigated for the treat-

ment of various solid tumors, including

indications for non-small cell lung can-

cer (NSCLC), bladder cancer, renal cell

carcinoma, and breast cancer. In clinical

trials, treatment with atezolizumab cor-

responded to an average 7.7 month-exten-

sion of life when compared with those

who received docetaxel chemotherapy;

this represents a statistically significant

survival benefit. If approved, atezolizumab

will likely compete with Merck’s Keytruda

(pembrolizumab), Bristol-Myers Squibb’s

Opdivo (nivolumab), and AstraZeneca’s

durvalumab, which is being investigated in

Phase II and III trials.

Reslizumab—Teva is gunning for FDA

approval in 2016 for its biologic resli-

zumab (trade name Cinquil), which tar-

gets IL-5. Although the company will first

seek an approval for the medication for

the treatment of eosinophilic asthma,

it is also researching the drug’s efficacy

in the treatment of eosinophilic esopha-

gitis. Reslizumab is in the same class as

AstraZeneca’s Nucala (mepolizumab).

According to Teva, a decision from FDA on

reslizumab is expected by March 2016.

Dupilumab—Sanofi and Regeneron’s

dupilumab is currently in late-stage devel-

opment for asthma and eczema/atopic der-

matitis. The drug is promising because of

mAbs to Watch in 2016Randi Hernandez

The Big Picture for 2016: Emerging Therapeutics

*Editor’s note: The forecasted

approval dates are drawn directly

from company-disclosed information

(e.g., press releases, company presen-

tations, etc.). As such, there may be

products that are filed or in Phase III

that are not included in these reports,

due to the lack of information regard-

ing approval/launch dates from

either of these sources. Similarly,

the original data were requested

by and provided to BioPharm

International by EvaluatePharma

in August 2015; therefore, some

of the original submissions have

been excluded and/or modified by

BioPharm International to reflect

regulatory changes. As examples,

although daratumumab, elotu-

zumab, and idarucizumab were orig-

inally included in EvaluatePharma’s

list as mAbs that would gain regula-

tory approval in 2016, these three

molecules actually gained US FDA

approval in 2015.



its ability to inhibit the biological

effects of both IL-4 and IL-13. It will

compete with AstraZeneca’s Nucala

(mepolizumab), which was approved

by FDA in November 2015, although

Nucala works by another mechanism

(through IL-5). Teva’s IL-5 inhibitor

Cinquil (reslizumab, currently in

Phase III trials), Roche’s IL-13 inhibi-

tor lebrikizumab (in Phase II trials),

and AstraZeneca’s IL-13 inhibitor

tralokinumab (in Phase III trials for

asthma) may also compete for market

share in this crowded uncontrolled

asthma mAb market.

Farletuzumab—Farletuzumab is

an investigational humanized IgG1

antibody targeting tumor cell surface

protein folate receptor alpha (FRA),

which is typically overexpressed

in epithelial tissue-derived can-

cers. Farletuzumab works by block-

ing the function of FRA. The mAb,

which is a radiotherapeutic that is

bound to a cytotoxic radioisotope,

received Orphan Drug designation in

the United States, European Union,

and in Switzerland for its potential

to treat platinum-sensitive ovarian

cancer. The investigational agent is

being developed by Eisai subsidiary

Morphotek and the Targeted Alpha

Therapy Group (TAT Group) at the

University of Gothenburg in Sweden.

Ibalizumab—The medication ibali-

zumab is believed to be a promising

treatment for HIV, as it represents

a treatment alternative for patients

who have become resistant to tradi-

tional antiretroviral therapies (ARTs).

Known as a viral-entry inhibitor, the

anti-CD4 human mAb of murine ori-

gin works by blocking HIV-1 entry in

vitro and has demonstrated in clini-

cal trials to reduce HIV viral load.

EvaluatePharma cites Roche,

Biogen, and The Aaron Diamond

AIDS Research Center as co-develop-

ers of the drug.

Inotuzumab ozogamicin—Pfizer’s

drug candidate for the treatment

of acute lymphoblastic leukemia

(ALL) is the only antibody-drug

conjugate on the potential approval

list for 2016. The drug received a

Breakthrough Therapy designation

from FDA in October 2015 based on

the results of the drug’s performance

in clinical trials compared with

long-term chemotherapy for patients

with relapsed or refractory CD22-

positive ALL. Current treatments

for ALL include Gleevec (imatinib)

and Sprycel (dasatinib), but these

are both examples of treatments for

patients whose leukemia cells have

the Philadelphia chromosome.

Ocrelizumab—Roche’s ocrel i-

zumab is a mAb being developed

to treat relapsing multiple sclerosis

(MS) and primary progressive mul-

tiple sclerosis (PPMS). In clinical tri-

als, ocrelizumab was shown to be

superior to interferon beta-1a, a

widely used treatment for relapsing

forms of the disease. There are cur-

rently no approved treatments for

the progressive form of the disease.

Ocrelizumab is designed to selec-

tively target CD20-positive B cells

that are thought to contribute to

myelin and axonal damage. Roche’s

Chief Medical Officer and Head of

Global Product Development Sandra

Horning, MD, stated in a press release

that ocrelizumab is the “first investi-

gational medicine to slow a clinically

meaningful and statistically signifi-

cant effect on the progression of dis-

ease in primary-progressive MS.”

Sarilumab—This IL-6-receptor

inhibitor from Sanofi and Regeneron

works by blocking the binding of

IL-6 to its receptor and interrupting

the cytokine-mediated inflamma-

tory signaling cascade. The drug is

being tested in clinical trials as a first-

and second-line therapy in combi-

nation with methotrexate or other

disease-modifying antirheumatic

drugs (DMARDs) and also as a mono-

therapy. Sarilumab is likely to com-

pete with first-line therapies such as

Bristol-Myers Squibb’s Orencia (abata-

cept), Genentech’s IL-6 inhibitor

Actemra (tocilizumab), and AbbVie’s

tumor necrosis factor (TNF)-blocker

Humira (adalimumab). The company

said it will have clinical trial results

comparing sarilumab’s efficacy to

adalimumab by 2016.

Tildrakizumab—This mAb from

Merck is in Phase III clinical trials

for the treatment of plaque psoria-

sis. Potential competitors that tar-

get IL-23 include J&J’s Stelara

(ustekinumab) and Janssen’s gusel-

kumab, which is currently in Phase

II trials.

Tremelimumab—Tremelimumab

is an immune checkpoint inhibi-

tor that prevents the blocking of the

action of cytotoxic T-lymphocytes,

allowing the immune system to more

effectively destroy cancer cells. The

fully humanized mAb binds to the

protein CTLA-4 on the surface of

activated T-lymphocytes, turning off

inhibitory immune signals.

In the ARCTIC, MYSTIC, and

NEPTUNE trials, MedImmune is

testing the drug’s efficacy in com-

bination with investigational agent

durvalumab for the first-line treat-

ment of metastatic non-small cell

lung cancer and head and neck can-

cers. FDA has also granted Fast Track

designation to tremelimumab as a

monotherapy for the treatment of

malignant mesothelioma, an aggres-

sive, rare form of cancer that affects

the lining of the lungs and abdomen.

Daclizumab—Daclizumab was orig-

inally approved by FDA in 1997 to

prevent organ rejection after trans-

plantation. The mAb’s new indica-

tion for multiple sclerosis would

be billed under the trade name

Zinbryta. According to co-developers

Biogen and AbbVie, it demonstrated

“superior outcomes” in clinical tri-

als when compared with Biogen’s

Avonex (interferon beta-1a).

Unlike investigational agent

ocrelizumab, which binds to CD20,

daclizumab binds to IL-2 subunit

CD25, modulating the activity of

IL-2 against abnormally activated

T-cells that attack myelin. Zinbryta

is believed to modulate the function

on IL-2 without disrupting general

immune system responses. ◆

The Big Picture for 2016: Emerging Therapeutics



The escalating attack on rising

expenditures for prescription

drugs and biologics is prompting

more proposals from Congress

and political candidates to strengthen

oversight of biopharmaceutical devel-

opment, manufacturing, and market-

ing. While Democrats have been most

vocal in challenging industry practices,

Republicans have joined the campaign,

citing the need to manage medical costs

as part of proposals for revising the

Affordable Care Act. All parties acknowl-

edge the importance of supporting bio-

medical and genomic discoveries able

to cure lethal conditions such as cancer

and hepatitis, but also want to ensure

that innovative therapies are available to

patients who stand to benefit.

The debate thus is moving beyond

industry “bad actors” and drug reim-

bursement issues to encompass a broad

range of policies affecting pharma opera-

tions. The Justice Department, for exam-

ple, is assessing competitive issues related

to pricing strategies of products marketed

by Eli Lilly, Merck, Mylan, and others.

Pharma mergers and acquisitions will face

even more scrutiny of potential impact

on competition and pricing, as well as

jobs, operations, and R&D. The Trans-

Pacific Partnership agreement and other

trade pacts may lose support among those

who consider these deals overly protective

of pharma intellectual property rights at

home and abroad.

The spreading epidemic of opioid

abuse and deaths from overdoses also will

increase scrutiny of manufacturer R&D

and marketing activities. State and local

governments are filing lawsuits that tar-

get industry practices seen to encourage

excessive opioid prescribing and dispens-

ing. FDA is encouraging development

of abuse-resistant formulations and new

easy-to-use opioid antidotes as it seeks to

provide patient access to needed painkill-

ers less likely to cause harm.

CONGRESSIONAL CONCERNSOne result of the anti-pharma rhetoric

has been to delay approval of the 21st

Century Cures legislation, as questions

multiply about the value of developing

new medicines that may be too costly

for many individuals to access. Such con-

cerns slowed efforts in the Senate to craft

a companion “Cures” bill and may dim

prospects for provisions in the House-

approved measure that provide added

incentives for drug research and testing.

Congressional committees will exam-

ine drug access issues more closely in

early 2016, as Republican leaders in the

House and Senate respond to calls for

reform, as well as pressure from De

mocrats to tackle rising costs. The

House Oversight & Government Reform

Committee has opened an investigation

into prescription drug pricing and expects

further hearings this year. The Senate

Special Committee on Aging set the pace

with a hearing in December 2015 focused

on price increases by Valeant and Turing

Pharmaceuticals. And the House Judiciary

Committee will continue to explore drug

pricing as part of its examination of fac-

tors affecting competition in the US

healthcare system, as seen at a November

2015 hearing on the role of pharmacy

benefit managers.

The Obama administration will fur-

ther examine strategies for manag-

ing outlays on drugs by public health

programs, as discussed at a high-profile

pharmaceutical forum in November

2015. A wide range of experts addressed

whether value-based purchasing and

other approaches can improve patient

access to medicines while maintaining

Politics and Pricing Will Challenge Manufacturers in 2016

Jill Wechsler

Jill Wechsler is BioPharm International’s

Washington editor, Chevy Chase, MD,

301.656.4634, [email protected].

The Big Picture for 2016: Regulations



incentives for biomedical innova-

tion. The White House has backed

calls for Medicare to have more lee-

way to negotiate drug prices, and

states are looking more closely at

options for revising Medicaid drug

reimbursement policy.

REGULATORY PRESSURESThese developments have entan-

gled FDA in cost and competition

issues much more than it desires.

At the November 2015 Senate hear-

ing to weigh the confirmation of

Robert Califf as the next FDA com-

missioner, Committee members

repeatedly pressed the nominee on

how the regulatory process could

improve access to medicines. Califf’s

main response was to promise to do

“a good job” to move new generic

drugs through the approval process

and to eliminate the generic-drug

application backlog. Similarly, FDA

approval of more biosimilars would

enhance patient access to less expen-

sive biotech therapies, a process

that would be facilitated by agency

clarification of controversial issues

related to biosimilar development,

such as product naming and inter-

changeability.

Califf also acknowledged the need

to streamline regulation of combi-

nation products, a topic of contin-

ued importance to biopharma and

medical-device manufacturers. And

he mentioned FDA efforts to set

standards for legitimate drug com-

pounding, which could produce

alternatives to costly medicines, as

proposed for a compounded version

of Turing’s costly antiparasitic treat-

ment. FDA aims to do more to track

and prevent drug shortages, with an

eye to avoiding monopoly situations

leading to price increases, as seen in

allegations involving manufacturers

of saline solutions and other com-

mon generic injectables.

Some of these issues may be

addressed through FDA initiatives to

improve drug and biotech product

quality. The agency hopes to final-

ize a program for collecting quality

metrics data that can help dem-

onstrate the state of operations at

pharma manufacturing facilities

and refine its risk-based method

for scheduling site inspections.

The larger benefit will be to reduce

drug recalls and quality-related

shortages and to reward manufac-

turers able to document high-per-

formance systems.

FDA also is stepping up scrutiny

of the accuracy of clinical and man-

ufacturing information submitted

to FDA by manufacturers in appli-

cations and reports, as part of its

increased focus on data integrity.

The agency wants to ensure that

information filed in all agency sub-

missions is complete and truthful,

particularly in numerous documents

related to production quality and

adherence to GMPs. FDA regards

data integrity as basic to any qual-

ity system, commented Doug

Stearns, director of the Office of

Enforcement and Import Operations

in FDA’s Office of Regulatory Affairs

(ORA), at the November 2015 FDA

Inspections Summit sponsored by

FDA News. False or altered data often

are hard to fix, particularly cases

involving overseas operations and

where errors arise more from neg-

ligence or carelessness than from

criminal activity. The agency still

will take enforcement action against

fraud, Stearns commented, but

noted that FDA seeks to rely on com-

panies to have “appropriate quality

systems” to address such problems

when they emerge.

In addition to ensuring indus-

try compliance with standards and

rules, FDA’s larger goal is to support

development and appropriate testing

of innovative therapies. The agency

is working on policies to advance

research on cellular and gene thera-

pies, which involve complex process-

ing and quality control issues.

In vitro diagnostics are in the

news, as drug and medical-device

makers look for clearer FDA stan-

dards for co-development and

appropriate use of these products.

The agency also is working with

genomic testing operators to develop

rational approaches for enhanc-

ing consumer access to personal

genomic information.

Many of these issues will be

on the table as FDA and industry

negotiate the next version of the

Prescription Drug User Fee Act

(PDUFA VI); the program has to be

reauthorized in 2017 to keep indus-

try payments flowing to support

FDA oversight of drugs and biologics,

as well as fees for medical devices,

generic drugs, and biosimilars. All

stakeholders look to continue ini-

tiatives for integrating patient per-

spectives into drug development and

regulatory decision-making, while

also enhancing FDA information

technology and its workforce and

ensuring the long-term stability of

fee programs. But hopes for quickly

enacting a “clean” user-fee reautho-

rization bill will continue to fade as

the political rhetoric heats up. ◆

The Big Picture for 2016: Regulations

Regulatory Topics to

Watch in 2016

In 2016, the US presidency, one-third

of the Senate seats, and all seats

in the House of Representatives

are up for election. In this active

political environment, the following

crucial issues affecting the bio/

pharma industry will be part of the

national discussion:

• Intellectual property protection

and competition

• Drug pricing and affordability

• International trade and regulations

• New FDA commissioner

• Alleviating drug shortages

• Improving product quality

• Data integrity oversight

• Development of innovative therapies

• Prescription Drug User Fee

Act reauthorization



To find out what the outsourcing

industry may look like in 2016,

BioPharm International spoke with

Elliott Berger, vice-president of

global marketing and strategy at Catalent

Pharma Solutions; Saharsh Rao Davuluri,

president of contract research, Neuland

Labs; Michael Lehmann, president, global

PDS and executive vice-president global

sales & marketing, Patheon; Paul Dupont,

vice-president marketing and business

development, Ropack Pharma Solutions;

Mark Rogers, senior vice-president, Life

Science Services, SGS North America; and

Peter Soelkner, managing director Vetter

Pharma International GmbH.

MARKET CONSOLIDATIONBioPharm: The contract services and man-

ufacturing market has experienced con-

solidation in the past year. What are the

implications of consolidation for drug

owners in terms of services available and

the cost of those services?

Berger (Catalent): Ongoing consolida-

tion enables major pharmaceutical com-

panies to optimize their supply networks

and source a greater range of services and

technologies from fewer partners. With a

more rationalized approach to sourcing, it

is easier for buyers to work with long-term

strategic partners to create joint quality

and operational scorecards against which

to measure suppliers, and this gives flex-

ibility to monitor and improve global sup-

ply chains.

Consolidation of allied services into

larger organizations creates partners that

are, on the whole, more flexible to clientsÕ

demands and have an attitude of expan-

sion and investment in new technologies,

global capacity, and world-class quality

systems to match the future demands of

sponsors. This may include co-creating

large custom manufacturing suites that

can accommodate new launches or tech

transfers with specific complex require-

ments from pharmaceutical clients who

look to free-up internal capacity; or the

placement of products which have been

launched or acquired into the companyÕs

portfolio and where no in-house capacity

currently exists.

As ever in the pharma industry, new

medicines are becoming increasingly more

complex, and the ability of contract service

companies to create customized solutions

is key to ongoing success. Products are

becoming more ÔglobalÕ, and the ability of

service providers to offer a global supply,

with the oversight of multiple regulatory

agencies, has never been more prescient.

Lehmann (Patheon): ItÕs true that the

contract development and manufactur-

ing industry has experienced significant

consolidation in recent years. The good

news is that there are solid benefits for

pharmaceutical companies that come from

outsourcing partners who have achieved

strategic consolidations. The increased

bandwidth, capacity, and capabilities,

together with an expanded regional and/or

global presence, are valuable to biopharma

companies. Improved economies of scale

and a simplified supply chain are also

important benefits.

Soelkner (Vetter): Consolidation has a

variety of implications for drug owners. It

reduces the number of different supplier

options for individual services. Also drug

owners want to simplify their service pro-

vider network and reduce the number of

providers, essentially creating a one-stop-

shop-approach whenever possible. As a

consequence, they expect their partners to

be strategic, not tactical. In practice, this

means that drug companies would rather

look at outsourcing opportunities for a

pipeline of several products, not just one

drug. This includes looking for attractive

cost models, which the provider can offer

to successfully contribute to developing,

Outsourcing Outlook for 2016

Susan Haigney

The Big Picture for 2016: Outsourcing

The Market Consolidates for Better Service



commercializing, and supplying this

drug pipeline.

Davuluri (Neuland Labs): As a result

of consolidation, vertically integrated

[contract manufacturing organiza-

tions] CMOs offer a Ôone-stopÕ solu-

tion to drug owners. Although this

idea has been promoted for quite

some time, consolidation could

potentially increase this focus. These

integrated CMOs are pitching their

services rather carefully to drug own-

ers. As a pure play API CMO, we tend

to focus on the advantages of being

API specialists with a lot of depth

in handling API-specific [chemistry,

manufacturing, and controls] CMC

issues. Integrated CMOs are also

separating their proposals for APIs

and drug products in order to avoid

coming across as a company who

insists that they do everything (or

nothing) and demonstrate skills that

are comparable to a standalone spe-

cialist who focuses purely on APIs

or finished products. Integrated

CMOs offer a potential cost savings

that drug owners can realize if they

choose the one-stop solution.

Dupont (Ropack): As consolidation

continues, benefits of long-term part-

nership between sponsors and [con-

tract research organizations] CROs/

CMOs become more apparent. Drug

development is becoming more com-

plex, and more venture capital fund-

ing and Big Pharma acquisition of

smaller and mid-size pharmaceuti-

cal companies continues to fuel new

product development. Both sponsors

and venture capitalists require that

candidates identify the drug candi-

dateÕs value point as quickly as pos-

sible. Yes, consolidation effectively

reduces the individual options avail-

able to sponsors. However, consoli-

dation provides sponsors with more

single-source options [and] helps

CROs and CMOs keep up with tech-

nology and develop more compre-

hensive service offerings.

Rogers (SGS): Like M&A activities in

any market, the consequential reduc-

tion in competition is not generally

an advantage to the consumer, and

the contract services and manu-

facturing market is no different.

However, such consolidation may

also make it easier for the drug own-

ers to find a Ôone-stop shopÕ, which

alleviates the need for the coordi-

nation and management of several

different providers but may also

increase the compliance and opera-

tional risks associated with a single-

source supplier.

MORE CONSOLIDATION AND OTHER CHANGES TO COMEBioPharm: Do you expect more con-

solidation or other changes in 2016?

Soelkner (Vetter): We expect con-

solidation to continue in 2016 and

beyond. Regularly, there is news

within the industry regarding a

merger or acquisition between large

and small companies, or at times

the consolidation of two equal large-

market participants. We believe this

will continue. But we do not see large

changes that can disrupt the indus-

try, but rather, the continuation of

existing challenges such as the ever-

increasing complexity of develop-

ment projects, greater expectations

for a high degree of flexibility at the

service providers pertaining to tim-

ing and batch size, as well as ever-

increasing regulatory demands.

Lehmann (Patheon): We know that

our clients are looking for partners

who can work differently with them

to deliver in a rapidly changing phar-

maceutical landscape .... The expand-

ing demand for sterile development

and manufacturing, biologics capac-

ity, and solutions for poorly soluble

compounds drive some of the con-

solidation efforts in the industry.

Berger (Catalent): On a global level,

the pharma industry is still very frag-

mented and continues to consolidate,

so we would expect the pharma ser-

vices sector to do the same. There

is increased demand for specialized

outsourced services, including global

solutions as well as customized and

full service options. At Catalent,

we have seen a marked increase in

the demand for complex products

requiring drug delivery expertise as

well as specialty handling services

including scheduled storage, cold

chain logistics and handling of DEA-

licensed drugs and potent and cyto-

toxic compounds.

Davuluri (Neuland Labs): API CMOs

will look at further modernizing

technology. New drug applications

are now required to include [qual-

ity-by-design] QbD data for any pro-

cesses where there could be control

or quality issues. Techniques such as

QbD can be effectively implemented

into development only if certain

infrastructure for lab scale, pilot, ana-

lytical, safety, and computer software

is in place. In addition, CMOs will

have to look at using modern tech-

niques in R&D and manufacturing

such as usage of parallel chemistry,

new chromatographic technologies,

flow chemistry, etc. This requires an

investment not just in infrastruc-

ture, but also in technologists with

the appropriate training and, most

importantly, a commitment from

management to make these technol-

ogies commercially viable long term.

Dupont (Ropack): Consolidation

within the drug-development mar-

ket continues to heat up. Ideally,

drug owners prefer to develop their

candidates under one roof and ben-

efit from a continuum of quality

throughout the development pro-

cess. This places greater pressure on

CMOs to offer additional services

supported by capital investments in

infrastructure, in equipment, and

in the recruitment of qualified per-

sonnel, while maintaining costing

models acceptable to sponsors and

clients. I believe that for these rea-

sons, CROs and CMOs will require

additional resources and revenue

to support the growing demand for

drug development and clinical mate-

rials. Meeting demand for compre-

hensive development capabilities can

be attained in the near-term only

through the M&A process. ◆

The Big Picture for 2016: Outsourcing



Ole

g P

rikho

dko

/Gett

y Im

ag

es

As biopharmaceutical man-

ufactur ing matures, it i s

natural to consider whether

concepts and trends used in

other manufacturing sectors, such as

automotive and electronics, could be

applied to bioprocessing.

In semiconductor manufacturing,

for example, a thorough understand-

ing of process variation allows com-

panies to manufacture circuits with

billions of transistors at high yields.

These variations are translated into a

set of design rules, which help ensure

that designs will be manufactured suc-

cessfully and meet safety and other

regulatory requirements.

The ability to codify manufacturing

into robust design rules has had a tre-

mendous impact on the semiconduc-

tor industry, and enabled it to move

from a simple vertically integrated

model where manufacturers designed

and manufactured product, to a model

in which a few global suppliers out-

source all manufacturing.

Would this structure be desirable

for biopharmaceutical manufactur-

ing? Although their respective regu-

latory environments are radical ly

different, the two industries share

some common elements: use of con-

tract manufacturers, for instance,

and a manufacturer focus on discov-

ery and validation. A key difference is

that biopharmaceuticals are manufac-

tured using living organisms, which

have yet to be fully characterized,

designed, and produced with preci-

sion genetic modifications. Therefore,

Tools for Continuous Bioprocess Development

Rajeev J. Ram

Could perfusion microbioreactors

bring more agility to

biomanufacturing?

Rajeev J. Ram is professor of

electrical engineering at MIT.

Bioreactors


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the equivalent design rules and

computer-a ided design tools

that are used in the electron-

ics industry cannot be applied

and the manufacturing process

must be developed, empirically,

for each new product. Bioprocess

development requires interaction

between manufacturer and cre-

ator because biopharmaceutical

product characteristics and qual-

ity are affected by the manufac-

turing process.

R e l i a b l e a n d s t a n d a r d -

ized scale-down models would

allow large-scale manufacturing

and product development to be

decoupled. If the large-scale bio-

reactor is well characterized, a

small-scale system can be used to

replicate all of its operating con-

ditions and potential variations.

Proper scale down requires good

simulation of large-scale bioreac-

tor conditions. The small-scale

reactor needn’t have the same

shape or geometry.

The industry needs technolo-

gies that can enable upstream

processes to move quickly and

seamlessly between partners,

whether internal or external.

Currently, cultural challenges

and established ways of working

pose challenges in moving to this

model. For example:

• Upstream process performance

(as measured by doubling time,

viability, and product quality)

is sensitive to a large number

of parameters (e.g., pH, gas con-

centration, shear, nutrient feed

rates, temperature, etc.).

• Bioreactors themselves, which

control this large number of

parameters, are large, complex

to operate, and have not been

standardized.

• Standardization is hampered

by use of various platforms and

lack of design rules.

For instance, in cell culture,

the typical biopharmaceutical

company uses at least a dozen

different platforms, including

shallow-well microtiter plates;

deep-well microtiter plates; shake

f lasks; benchtop bioreac tors

of various impeller and sparger

designs with various culture vol-

umes and larger bioreactors of var-

ious impeller and sparger designs

with various culture volumes

ranging from 100 L–15,000 L.

Generally, production teams

choose the platform that affords

them the greatest control of

the various process parameters.

Without design rules for pro-

cesses, this inevitably results

in choosing platforms that are

“familiar,” even if they might

not be the best choice for the

given process. In addition, dis-

covery teams tend to select plat-

forms that are easiest to use and

that offer the possibility of high-

throughput development.

As a result, biopharmaceutical

process development and scale

up are rarely linear. Fundamental

understanding of the cellular and

biochemical processes involved

is often sacrificed because it isn’t

always clear whether or not the

metabolism of the production

cells remains the same across var-

ious platforms.

In extreme cases, product qual-

ity can be affected. Consider the

case of Myozyme (alglucosidase

alfa), a lysosomal glycogen-spe-

cific enzyme indicated for use in

patients with Pompe disease. In

2008, FDA rejected Genzyme’s

application to produce Myozyme

in a 2000-liter-scale facility under

the same approval authorization

given for its 160-liter-scale plant.

The FDA ruling stated that the

glycosylat ion of the products

manufactured at each scale dif-

fered, and, thus, the 2000-liter

product required a new biologic

license application (1).

Why Is proCess Transfer harD?Scale up and scale down are well-

known barriers to biologics produc-

tion. Because volume and surface

area scale differently with length,

even in similar benchtop bioreac-

tors, the physical and chemical

environment seen by the cells will

be different from what is seen on

the industrial scale. The physical

and chemical environment of the

cells can strongly affect the cells’

physiology and productivity and,

hence, must remain constant or

above critical values during scaling.

First, the gas transfer rate of O2

and CO2 must be suffciently high

so that the dissolved oxygen (DO)

level remains above the oxygen

uptake rate of the cells, and waste

gases such as carbon dioxide are

effciently removed.

Bioreactors

Process Understanding

In the near future, biopharmaceutical companies will be looking into building

cellular function models that will help them understand the effects of feed rate,

and physical and chemical stresses on the productivity and growth of their

cell lines. Having predictive models of the impact of manufacturing conditions

on industrially relevant cell lines would greatly accelerate upstream process

optimization. Often, the overexpression of a recombinant protein is rate-limited

by an enzyme whose kinetics are not well understood. Understanding the rate-

limiting steps affecting the productivity of the cells will greatly reduce the effort

needed to find the optimal processing conditions of the recombinant cell line.

The large data sets required to form a complete cellular function model require

a high-throughput platform that can run at a lower cost than bench-scale

bioreactors but with the same set of instrumentations. —Rajeev J. Ram



AL

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Secondly, the maximum shear

rate seen by the cells must remain

the same or below the critical value

that affects productivity during

translation. This is especially impor-

tant for mammalian cells such as

Chinese hamster ovary (CHO) cells

due to their shear sensitivity.

The circulation time is also

an important parameter, since it

affects the frequency at which

the cells see the high shear. The

repeated deformat ion of the

endoplasmic reticulum can be

detrimental to protein glycosyl-

ation. Bioreactors with differ-

ent chamber volumes will have

different circulation times, and

hence, some benchtop bioreac-

tors are equipped with a circula-

tion line that allows the physical

env i ronment of the ce l l s to

mimic the circulation time seen

in large industrial-scale bioreac-

tors. When designing scale-down

models of bioreactors, the energy

dissipation rate has to be main-

tained constant so that the trans-

fer of internal energy to the cell

remains constant.

Decades of research have estab-

lished these guidelines for pro-

cess transfer from one platform

to another. While not complete,

they provide a set of recommen-

dations that are grounded in an

understanding of cellular pro-

cesses and biochemistry. Many

important principles of process

transfer are rarely followed, how-

ever, for the following reasons:

• Microtiter plates and shake

f lasks are incapable of repro-

ducing more than one (gas

transfer) of these parameters

(e.g., shear, circulation time,

and energy dissipation rate)

relative to a production biore-

actor.

• Most companies strive to keep

only a subset of these param-

eters constant as they scale

upstream processes. A presenta-

tion by Biogen illustrated using

either the kLa or the power dis-

sipation per unit volume as

scale-down parameters (2).

• Maintaining geometric similar-

ity over-constrains the problem

so that all of the relevant param-

eters cannot be held constant

across scale. A paper from ETH

Zurich (funded by Novartis)

demonstrated that geometric

similarity fundamentally does

not allow for scale invariance

(from 3 L to 15,000 L) of kLa,

shear stress, and mixing time

simultaneously (3).

In response to the increasing

need for parallelization and minia-

turization of controlled and moni-

tored bioreactors, commercial and

academic research groups have

developed microbioreactors with

working volume below 1 L to:

• De l ive r h i gh - t h roughput ,

easy-to-use platforms that can

replace the microtiter plates

and shake flasks used by dis-

covery teams

• Achieve the same gas-transfer

rates, shear, circulation, and

energy dissipation of 15,000-L

product ion reactors in the

high-throughput platform

• P rov ide backward compat-

ibility to all existing in-line

and final assays/instruments

performed by process develop-

ment teams.

sTaTe of The arT for sCale-DoWn Approaches to miniaturization

vary greatly, due to the many

different technologies currently

b e i ng deve lop e d . Howeve r,

advances in microbioreactors are

occurring, as the following exam-

ples demonstrate:

• pH and DO monitor ing are

increasingly being integrated

at microscale (4,5), as dispos-

able and non-invasive optical

pH and DO sensors are vali-

dated against the electrochemi-

cal probes used by large-scale

bioreactors.

• Optical DO sensors have been

implemented in shake flasks

as a first step to allowing the

monitoring of conditions in

these containers (6, 7).

• pH and feed control are being

implemented in shake flasks

with integrated syringe pumps

for l iqu id i nje c t ions a nd

pH probes (8), as shown by

Weuster-Botz et al.

Bioreactors

Figure 1: The fgure depicts the schematic for a disposable 1-mL polycarbonate

microbioreactor. The disposable device supports four input feed streams as well as a

perfusion output and waste output.



Bioreactors

• Equipment is being minia -

tur ized for control l ing DO

and pH (9, 10) by integrat-

ing optical pH and DO sen-

sors with 24-well plates and

sparging oxygen and carbon

d iox ide th rough a per me-

able membrane at the bottom

of the well, as seen with Pall

Corporation’s Micro-24 (M24)

reactor. The reactor has been

demonst rated in microbia l

cultivations (10), and has also

been shown to work for chi-

nese hamster ovary (CHO) cell

cultures as well (11).

• Microf luidics is being used

in well plates to improve the

mic rot ite r plate format in

appl icat ions such as m2p -

labs’ BioLector Pro. This tool

e nable s automate d l iqu id

inject ions to be per formed

using pneumatic valves in the

microfluidic section under the

well plate.

• Microbioreactors are also being

designed with optical density

(OD), conductiv ity, and pH

sensors (12). SimCell’s solution

for high-throughput minia-

turization is to design 700-μL

microbioreactors in the form

of cassettes agitated by a rota-

tor. The SimCell model uses

a robotic arm to transfer the

cassette from the rotator to a

sensing platform to measure

pH, DO, and OD. These online

measu rement s a re supple -

mented by sample removal for

off ine measurements of glu-

cose, viability, titer, and prod-

uct quality (13). The device

can use up to 324 cultures

simultaneously, allowing it to

be used for process optimiza-

t ion, screening, and media

optimization (14, 15).

• Mo s t r e c e nt l y, S a r t o r i u s

Sted im’s TAP ambr 15 and

ambr 250 have been gaining

tract ion for scale down. In

addition to optical pH and DO

sensors, the system combines

miniatur i zed st i r red tanks

with a pipett ing robot and

automated feed pumps, greatly

improving automation of the

fermentation process. By uti-

lizing a biosafety cabinet, the

system ensures that sterility is

maintained during automated

pipetting operations required

for feeding, inoculation, and

sampling. Both microbial and

mammalian processes have

been studied in the systems

and have shown reasonable

correlation with bench-scale

stirred tanks (16, 17). These

studies have also shown how

certain parameters, such as

agitation and power dissipa-

t ion, do not sca le l inearly

with bioreactor size, especially

when the st ir red-tank form

factor is maintained (18).

Nearly a l l of these micro -

bioreactors are capable of both

batch and fed-batch operation.

For the needs of conventional

biomanufacturing, these culti-

vat ion modes a re su f f ic ient .

However, cont inuous culture

operation, in all its forms—che-

mostat, turbidostat, and perfu-

sion— can suppor t metabol ic

flux analysis for increased pro-

cess understanding (see sidebar).

Continuous perfusion holds the

promise of improved product

quality and consistency because

of steady-state operat ion and

because the secreted molecule

does not have to be held until

the end for harvest.

Rea l i z ing such cont inuous

perfusion operation can be chal-

lenging for microbioreactors,

however. It requires long-term

cultures, and places greater stress

on sterile interfaces for the con-

tinuous in-flow and out-flow of

medium and cells.

For continuous perfusion pro-

cesses to be eff icient, the cell

density must be high, up to 100

million cells/mL, to convert the

nutr ient-r ich media into pro-

tein product, ef f ic iently (19).

Such high cell densities can be

demanding for gas t ranspor t

and fluid addition. In addition,

removal with cell retention is a

challenge. In fact, operation in

continuous perfusion culture mode

has not been reported for any of

the microbioreactors systems

described in previous passages.

sCale-DoWn for ConTInuous BIoproCessIngFor continuous perfusion bio-

processing, a lack of scale-down

technology is a major barrier to

entry (20). Because typical per-

fusion process experiments con-

sume 1–3 working volumes per

day and can run for 20–60 days,

culture media costs alone con-

tribute to prohibitively expen-

sive experimental campaigns. For

example, perfusion process devel-

opment experiments (21) using

a 4-L working-volume Wave bag

and alternating tangential f low

(ATF) or tangential flow filtration

(TFF) cell-retention filter, con-

Figure 2: An image of the instrument

that provides the fuidic interfaces and

the optical interfaces for the gas, pH,

and, dissolved oxygen (DO) samples.


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sumed between 55 L and 717 L,

with a median of 105 L. With a

typical medium cost of approxi-

mately $50/L, media costs alone

contribute to $5,000 per data

point. The high cost associated

with running perfusion experi-

ments is mainly due to the work-

ing volumes available for current

perfusion technology.

Hollow-fiber bioreactors are

currently used for small-scale

perfusion processing. These con-

sist of semipermeable tubing that

allows nutrients to diffuse to cul-

tured cells and removes waste

products. FiberCell Systems, for

example, provides disposable per-

fusion cartridges with volumes

as low as 2.5 mL. Typical usage

of the FiberCell system involves

seeding cells in the space out-

side of the fibers and pumping

fresh media through the fibers to

maintain a proper environment

for cell growth (22). Since cells

are typically seeded in the hous-

ing of the FiberCell system, it is

difficult to measure parameters of

the culture environment such as

dissolved oxygen and lack of mix-

ing makes cell sampling difficult.

External hollow-f iber systems

such as the ATF and TFF couple

hollow-fiber filters to a conven-

tional bioreactor. These systems

circulate cells between the con-

vent ional bioreactor and the

hollow fibers to allow for more

environmental control represen-

tative of large-scale production.

The tangential flow geometry of

these filters, in particular, the

alternating tangential flow of ATF

systems, helps to prevent fouling

of the hollow fiber filters and pro-

long filter life in high cell den-

sity applications. However, these

systems are currently unable to

operate at scales as small as the

FiberCell System. For example,

both Pall Corporation’s iCELLis,

used for adherent perfusion cul-

ture, and Repligen Corporation’s

ATF, used for suspension culture,

have minimum working volumes

of 1 L. Therefore, there is a real

need for small-volume perfusion

bioreactors capable of supporting

the high cell densities desired for

continuous perfusion processes.

T he p r i m a r y t e c h no lo g y

hurdle for existing microbiore-

actors is the development of

h igh- t h roughput mea ns for

continuous f luid addition and

harvesting with cell retention

capable of supplying up to 100

working volumes over 30 days.

This is diff icult for well-plate

technologies due to the close

proximity of growth chambers,

which constrains external fluid

connections. In addit ion, for

robotic pipetting architectures,

fluid removal with cell retention

cannot be accomplished with

standard pipette tips.

Microf luidic technology has

been applied to continuous cul-

ture over the past decade. This

technology has been success-

ful primarily for the culture of

attachment-based cells and tis-

sue culture (23–25). The large

surface area to volume ratio of

microf luidic devices facilitates

efficient diffusion of nutrients

while providing mechanical sup-

port for the cells. Unlike hollow-

fiber-type perfusion bioreactors,

microfluidics allows for the inte-

gration of various sensors and

also facilitates the precise control

of environment. There are a few

early examples of continuous sus-

pension culture using microflu-

idics (26, 27); these examples do

not incorporate control of pH and

dissolved gases, which is typically

required in a bioreactor.

A few commercial microf lu-

idic perfusion systems without

active mixing are currently avail-

able. The Mil l ipore Cel lASIC

traps cells in a closed chamber

and uses microfluidic channels

smaller than the size of the cell

to deliver media and reagents.

The small 2.8-mm diameter cul-

ture chambers can be observed

using microscopy and can be

used for screening and gene-

expression experiments (28).

neW aDvanCeMenTs In The fIelD of MICroBIoreaCTorsRecently, a microfluidics-based

bioreactor system was introduced

by Pharyx—a recent start-up out

of MIT—for continuous and per-

fusion processes (Figures 1 and 2).

This microbioreactor’s smaller

1–2 mL volume enables long-

term continuous experiments

with neglibile media usage. In

addition, all pumps and control

valves are integrated on-chip as

disposable elements, greatly sim-

plifying setup and operation.

Mixing is performed by moving

silicone diaphragms to push the

fluid between mixing chambers.

The relatively large surface area

of the fluid also allows for bub-

ble-free gas transfer through the

silicone diaphragms, with kLas

greater than 30 h^-1. Bubble-free

gas transfer enables online opti-

cal density measurements as well,

which provides new functionality,

such as automated cell bleeding

and on-line cell density control.

The form factor of the microbiore-

actor also enables integration of a

perfusion filter membrane directly

inside the growth chamber, where

constant mixing of the culture

provides passive filter cleaning,

prolonging the lifetime of the per-

fusion filter.

The original experiments with

a similar microfluidic bioreactor

focused on the demonstration of

various cell-culture modes and

on the development of metabolic

models (29). Due to the ability

to switch between multiple input

streams and accurately measure

the optical density, pH, and oxy-

gen, a variety of functions were

Bioreactors



Bioreactors

possible. Multiple experiments in

chemostat and turbidostat modes

with dif ferent media compo-

sitions were demonstrated in a

single device. These experiments

proved that modulation of input

sources was possible, high-per-

formance liquid chromatography

(HPLC) sample collection times

could be fast enough to look at

dynamics, and control of oxygen

during continuous culture could

be implemented. Additionally,

operation for three weeks without

evaporation was demonstrated,

all while maintaining sterility.

Modrirez et al. have used micro-

fluidic perfusion devices to study

therapeutic protein production

using Pichia pastoris (30). A perfu-

sion filter (polyethersulfone) with

a 1-cm diameter was incorporated

underneath the growth chamber

to allow for fluid flow-through

while maintaining all of the cells

inside the growth chamber and

enabling the switching of induc-

tion media. The ratio of the filter

surface area to bioreactor volume

was 0.758 – a factor of three greater

than in previously reported surface

area measurements in high-per-

formance, bench-scale perfusion

bioreactors (21). A combination of

perfusion flow and cell density con-

trol enabled process optimization of

various parameters, such as inducer

concentration and perfusion flow

rate. Cultures were also shown to be

stable, producing consistent levels

of both hGH and interferon alfa 2b

for more than 10 days.

In-line cell density and precise

flow-rate control enabled Han et

al. to study genetic switching of

Saccharomyces cerevisiae during

exponential growth in a steady-

state turbidostat (31). Cells were

genetically modified to produce

different recombinant proteins

in response to different chemi-

cal inducers and it was shown

that by testing the synthetic cir-

cuits in turbidostatic steady state,

circuit induction increased over

8x and the standard deviation

of activation decreased by more

than 50% when compared to

growth in test tubes.

W h i le cont i nuou s m ic ro -

bioreactors are early in their

development and commercial

deployment, the preliminary data

with microbial cell lines demon-

strate the capabilities for high

gas-transfer rates—supporting the

high cell densities required for

scale-down models of continuous

biomanufacturing, the ability to

maintain sterile conditions with

continuous in-flow and out-flow,

and the ability to support high

perfusion rates over a wide range

of operating conditions.

A mature perfusion microbio-

reactor platform has the poten-

tial to fill a crucial need in the

evolv i ng l a nd scape of b io -

manufacturing. As with earlier

microbioreactors for batch bio-

processing, perfusion microbio-

reactors could lower the cost for

bioprocess development. More

importantly, such devices can

fulfill multiplexing performance

and ease-of-use demands in early-

stage development and discov-

ery. Providing development and

discovery teams for robust scale-

down models in biomanufactur-

ing has the potential to help the

industry advance tremendously.

referenCes 1. G. Mack, Nature Biotech.

26, pp. 592 (2008).

2. V. Janakiraman et al., “Application

of Multivariate Analysis Tools

and Design of Experiments (DOE)

to Model the Design Space for

Characterization of a Mammalian Cell

Culture Process,” presentation from

the AIChE Annual Meeting (2012).

3. M. Soos et al., “Characterizing

Heterogeneity of Environmental

Conditions in Various Bioreactor

Scales Used for Cell Cultivation,”

presentation at the AIChE

Annual Meeting (2012).

4. H.R. Kermis et al., Biotech. Prog.

18 (5), pp.1047–1053 (2002).

5. M.A. Hanson et al., Biotech. 97

(4), pp. 833–841 (2007).

6. A. Gupta and G. Rao, Biotech.

Bioeng. 84 (3), pp. 351–358 (2003).

7. C. Wittmann et al., Biotech. Letters

25 (5), pp. 377–80 (2003).

8. D. Weuster-Botz, J. Altenbach-

Rehm, and M. Arnold, Biochem.

Eng. J. 7 (2), pp. 163–170 (2001).

9. A. Chen et al., Biotech. Bioeng.

102 (1), pp. 148–160 (2009).

10. K. Isett et al., Biotech. 98 (5),

pp. 1017–1028 (2007).

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(1104), pp. 149–165 (2013).

12. A. Buchenauer et al., Biosensors

Bioelectronics 24 (5), pp.

1411–1416 (2009).

13. R. Legmann et al., Biotech. Bioeng.

104 (6), pp. 1107–1120 (2009).

14. A.P. Russo et al., “Multi-Parameter

Process Optimization Using

the SimCell System,” in the

Proceedings of the 21st Annual

Meeting of the European Society

for Animal Cell Technology, N.

Jenkins, N. Barron, and P. Alves,

Eds. (Springer, Dublin, Ireland,

5th ed., 2009), pp. 515–518.

15. Z. Xiao et al., Methods Mol. Biol.

(1104), pp. 117–137 (2014).

16. V. Janakiraman et al.,

Biotechnol. Prog. doi: 10.1002/

btpr.2162 (2015).

17. M. Tai et al., Biotechol. Prog.

(5), pp. 1388–1395 (2015).

18. A.W. Nienow et al., Biochem. Eng.

J. (76), pp. 25–36 (2013).

19. M.S. Croughan, K.B. Konstantinov,

and C. Cooney, Biotech. Bioeng.

112 (4), pp. 648–651 (2015).

20. E.S., Langer and R.A.

Rader, BioProcess. J. (13),

pp. 43–49 (2014).

21. M.F. Clincke et al., Biotechnol.

Prog. 29 (3), pp. 754–767.

22. W.G. Whitford and J.J.S. Cadwell,

Bioproc. Int., (10), pp. 54–63 (2009).

23. M.J. Powers et al., Biotechnol.

Bioeng. 78, pp. 257–269 (2002).

24. S. Ostrovidov et al., Biomed.

Microdev. 6, pp. 279–287 (2004).

25. M. Reichen et al., PLoS ONE

7 (12):e52246 (2012).

26. F.K. Balagadde et al., Science

309, pp. 137–140 (2005).

27. References 5–9 as originally

cited in reference 28.

28. P. Lee, T. Gaige, and P. Hung,

Methods Cell Biol. (102),

pp. 77–103 (2011).

29. K.S. Lee et al., Lab on a Chip

(11), pp. 1730–1739 (2011).

30. N.J. Mozdzierz at al., Lab on a Chip

(15), pp. 2918–2922 (2015).

31. N. Han et al., “Microfluidics for

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2014, pp. 312–314 (2014). ◆



mst

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es

Biopharmaceutical manufactur-

ing involves a series of complex

unit operations linked together

to provide high-purity, biologic

actives with specified physicochemi-

cal and pharmacokinetic properties.

The development of commercial-scale

processes that consistent ly pro -

vide high-quality product that meets

those specifications requires extensive

examination of numerous process vari-

ables and potential process variations.

Conducting the required number of

studies on the commercial scale is not

practical; scale-down models are there-

fore employed to determine optimum

conditions for downstream separation

processes, including chromatography

and viral clearance steps. Qualification

that such models provide results truly

representative of commercial-scale pro-

cesses becomes necessary as molecules

move closer to commercialization.

whaT counTs as a scale-Down moDel?A scale-down model is a bench-scale

process designed to predict the results

that will be obtained when the process

is run at commercial scale. Unlike ini-

tial processes that are used during the

discovery and early development phases

to produce material for characteriza-

tion, efficacy, safety, and other studies,

scale-down models are intended to aid

in optimization and characterization of

the process that will be implemented

for manufacture of large quantities of

a biologic once the product has been

approved.

going small to achieve success on the commercial scale

Cynthia A. Challener

Scale-down modeling is

instrumental in supporting the development

of downstream biopharma

manufacturing processes.

Cynthia A. Challener, PhD, is

a contributing editor to

BioPharm International.

Downstream processing



While process simulations are an

ideal surrogate, given the complex-

ity and inaccuracy of many theo-

retical models, there often remains

no substitute for a physical scaled-

down model, according to Adam

J. Meizinger, a process engi-

neer in Genzyme’s Purification,

Manufac tur ing Sc ience, and

Technology Laboratory.

Scale-down models are used in

the development of nearly every

unit operation in the overall bio-

pharmaceutical manufacturing

process, including both upstream

and downstream steps, and sepa-

ration and filtrations steps, such

as chromatography and viral fil-

tration processes. A scaled-down

chromatography process, for

instance, is designed considering

the residence time, flow rate, and

column bed height of the large-

scale process. Additional param-

eters that are considered include

buffer preparation and feed stream.

“The ideal model behaves identi-

cally to the scaled-up process such

that the results obtained in the lab

exactly predict the results that will

be obtained in the manufacturing

plant,” says Paul Jorjorian, director

of global technology transfer for

Patheon. Because they are mod-

els, however, such a situation does

not occur. The goal, therefore, is to

mimic the commercial-scale pro-

cess as closely as possible.

The impacT of QbD anD DoeThe qual ity-by-design (QbD)

approach to process development

requires characterization of the

impacts of different process param-

eters on product critical quality

attributes (CQAs), which in turn

necessitates considerable experi-

mentation, according to Meizinger.

“While the consideration of up-

front risk assessments, access to

general scientific understanding,

and the use of design-of-exper-

iment (DoE) methodology mini-

mizes the required number of runs

and maximizes the informational

output, it is impractical to conduct

such characterization experiments

at full scale. Qualified scaled-

down models are instrumental

for conducting such characteriza-

tion work, as well as for viral clear-

ance studies, continuous process

improvement, and for conducting

impromptu investigations in sup-

port of large-scale operations, such

as when deviations occur,” he adds.

Dealing wiTh imperfecTionWhile ideal scaled-down models

correlate exactly with their large-

scale counterpart processes, gener-

ally a perfect match is not achieved

between the bench and commer-

cial scales, and therefore, model

processes are not perfectly scalable.

The causes, according to Jorjorian,

may not relate directly to the pro-

cess, but can be attributed to small,

nuanced differences that still have

an impact on the results at differ-

ent scales. “The issues are often

unavoidable and thus must be

addressed by developing correction

factors and/or transfer functions

that enable the accurate prediction

of the behavior of manufacturing

processes,” explains Jorjorian. “It is

important, however, to minimize

the need for correction factors and

transfer functions, and for those

that are required, provide thor-

ough justification and clearly dem-

onstrate their suitability,” he adds.

Another aspect of scale-down

models that can create issues when

predicting large-scale behavior is

the fact that models are qualified

at the center point of the process

design space. “This approach can

lead to discrepancies in predicted

and actual results when the model

is applied to the entire design

space,” Jorjorian observes. He adds

that timing and planning of mod-

eling runs is also important and

can negatively influence results if

not done effectively. For example,

it is important to use the same feed

used for production runs in scale-

down modeling runs; therefore,

modeling runs must be scheduled

when the feed is available. In many

cases, that isn’t possible, and the

feed may need to be frozen, which

introduces a new variable that can

impact the reliability of the model.

Specifically for chromatography

operations, some key consider-

ations in developing scaled-down

models are minimizing the dif-

ferences in the extra-column

volume, which varies consider-

ably with scale, contending with

increasing wall effects inherent in

small-diameter chromatography

columns, and accounting for dif-

ferences in detector-specific path

lengths and the associated non-lin-

earity of detector output, according

to Meizinger. He also notes that

some steps (e.g., gas overlays for

intermediate holds) may be chal-

lenging to implement as part of

sca led-down model develop -

ment, and mixing dynamics and

heat transfer often remain differ-


while process

simulations are an

ideal surrogate, given

the complexity and

inaccuracy of many

theoretical models,

there often remains

no substitute for a

physical scaled-down

model.



ent between scales. “The impact

of these differences must be care-

fully assessed for their resulting

impact on scaled-down model per-

formance,” he says.

Scale-down modeling of mem-

brane chromatography processes

can also be difficult, according to

Jorjorian. The reason often relates

to the designs used for small- and

large-scale membranes; they are

often different, with large-scale

membranes having added flutes or

pleats or other features that affect

their performance such that it is

difficult to develop bench-scale

processes that can reliably predict

production-scale outcomes.

QualificaTion aT laTer sTagesThere is nothing new about the

use of bench-scale process mod-

els during the development of

biopharmaceutical manufactur-

ing processes. What has become

an important trend in process

development is the need to dem-

onstrate that scaled-down models

truly represent their large-scale

counterparts. “It has only recently

become standard practice to dem-

onstrate that scaled-down models

provide results that are statisti-

cally equivalent to those obtained

from commercial-scale versions

of the same processes,” states

Meizinger.

Often during earlier stages of

development, even through Phase

I clinical trials, scale-down models

of downstream biopharmaceuti-

cal processes are generally not for-

mal, qualified models. “For these

models, the basis for use of each

model and the rationale for why

it is expected to be representa-

tive of the process at large scale

are documented,” notes Jorjorian.

As a molecule progresses toward

commercialization, however, for-

mal, qualified models are required.

The ability of the model to pre-

dict results at large-scale must be

demonstrated by performing at

least triplicate runs. The use of any

correction factors and/or transfer

functions must also be justified

and their application demonstrated

to provide robust results.

Qualification of most models

for chromatographic separations

is fairly straightforward, accord-

ing to Jorjorian. “There is a lot of

documentation and analyses to

complete, so in essence, qualifica-

tion of scale-down models is often

an exercise in coordination, rather

than presenting a substantial

technical challenge,” he observes.

Meizinger agrees that the use of

statistical equivalence for test-

ing quality attributes and process

performance indicators between

scales is generally not an issue.

“Demonstration of both individual

unit and linked unit output (recov-

ery, product quality attributes,

product purity, chromatographic

profiles/transition analysis, etc.)

comparability between small and

large scales remains both practical

and accepted,” he states.

moving To high-ThroughpuT moDelsQualification of scale-down mod-

els can be challenging, however,

when very small column sizes

are involved. There is significant

interest in using high-throughput

approaches for scale-down mod-

eling to speed up development

times. There are, however, techni-

cal issues that arise due to the dif-

ferent behaviors observed in very

large and very small columns,

such as the wall effects mentioned

previously.

“It is definitely more challeng-

ing to establish robust scale-down

models using high-throughput

methodologies because typically

transfer functions and correction

factors are required. As a result,

it is more difficult to adequately

demonstrate the scalability of

the models to the level required

for qua l i f icat ion,” Jor jor ian

says. He adds that there is a lot

of effort being devoted to over-

coming these issues to enable

high-throughput modeling of

downstream bioprocesses.

A few groups are also investi-

gating the application of first

principles calculations to the scale-

down modeling of both upstream

and downstream unit operations,

and particularly for chromatog-

raphy, for which there is signifi-

cant understanding of separation

behaviors and existing mathemati-

cal models. “This approach to

scale-down modeling is in the ear-

liest phases and largely limited to

large pharmaceutical companies

and university research groups

with the resources and computing

power required,” Jorjorian says.

exisTing Technology is highly valuable“Despite the limitations of exist-

ing scale-down modeling meth-

ods, the use of sca led-down

models is recommended wherever

possible, including for process

characterization, viral clearance

studies, continuous improvement,

manufacturing-related investiga-

tions, evaluation of changes in

raw materials, and so on, bar-

ring only validation efforts and

clinical trial/commercial manu-

facturing requirements for per-

formance of large-scale runs,”

asserts Meizinger. “Today, scaled-

down models serve as efficient

and cost-effective representations

of full-scale processes. While the

inability to demonstrate equiva-

lence between scaled-down and

large-scale operations is often

inevitable for some metrics, fur-

ther understanding of these scale-

related differences, particularly

through the development/refine-

ment of associated engineering

models, promises to continuously

improve the accuracy and util-

ity of scaled-down models,” he

states. ◆



How will you respond when the FDA asks:“Are you efectively monitoring your contract lab?”

Thursday, February 11, 20168 a.m. PST | 10 a.m. CST | 11 a.m. EST

Attend our

www.EurofinsLancasterLabs.com/Webinars

Presented by

EVENT OVERVIEW

In response to FDA draft guidance regarding the Quality

Metrics Program issued in July, 2015, the bio/pharma-

ceutical industry faces increasingly stringent regulatory

requirements in order to safeguard product quality and

patient safety. Moreover, suppliers and contractors are

now being viewed as an extension of the pharmaceutical

and biopharmaceutical manufacturer’s own facility rather

than an independent resource, forging a unique, strategic

relationship between vendors and sponsors.

While critical to meeting the recent regulations set forth

by FDA, the process of maintaining control over suppliers

requires effective organization, risk management, imple-

mentation of a strong quality culture, and quality over-

sight, which can lead to an extensive amount of time and

resources if not managed appropriately.

Join us for this informative webinar to learn how to foster

your relationships with contract partners to develop a qual-

ity surveillance program that will meet FDA scrutiny.

KEY TOPICS INCLUDE

• Critical aspects of an effective quality agreement

• Essential elements of a comprehensive quality process

• Best practices for a successful audit process

• Strategies for obtaining qualitative and quantitative

metrics reporting

WHO SHOULD ATTEND

Scientists, Managers, Directors and Quality Assurance/

Control Validation personnel in a bio/pharmaceutical

manufacturing company who are responsible for managing

contract testing services.

PRESENTERS

Arthur PezzicaSenior Director,

Quality Compliance

Eurofins Lancaster Laboratories

For questions, contact Kristen Moore

at [email protected]

Hosted by

Brittany CloudSenior Specialist,

Quality Compliance

Eurofins Lancaster Laboratories

Susan SchnieppFellow,

Regulatory Compliance

Associates



Mo d e r n d o w n s t r e a m

process ing of b iolog-

ics has elucidated sev-

e ra l spec i f ic prote i n

precipitation techniques that could

considerably reduce the number of

purification steps within a process.

Some of the specific precipitants (2)

used for these techniques include

polyelectrolytes, af f inity l igands,

metal ions, and protein-binding dyes.

The authors evaluated two pre-

c ipitat ion st rateg ies— one using

polyelectrolyte polyvinyl sulfonic

acid (PVS) and the other using zinc

chloride—as alternatives to the con-

ventionally used chromatographic

process. The authors also compared

the benefits of these strategies with

ion-exchange chromatography as

they relate to product purity, reduc-

tion of process-related impurities,

and cost of operation.

materiaLs and metHodsCell-free supernatant used for pre-

cipitation trials was generated in the

laboratory by a fermentation process

using a P. pastoris strain engineered

to secrete a human insulin precursor.

ABSTRACTRecombinant human insulin production using Pichia pastoris typically involves the expression of an insulin precursor as a single chain that is subsequently secreted into fermentation media. The first step in the

downstream process after cell separation typically involves capture by ion-exchange chromatography. In the capture step, the protein is concentrated by ion-exchange chromatography, which also results in the removal of a

significant proportion of the process-related impurities, such as pigments and host-cell proteins (HCPs) (1). The capture step requires an armamentarium of materials, including chromatographic resin media, various buffers, and

highly skilled personnel to handle the process. Although chromatography is a widely used step in this process, there exists a strong impetus to evaluate alternative process technologies from the perspective of process economics.

Precipitation as an alternative to Chromatography

in the insulin manufacturing Process

Madhavan Buddha, Shailabh Rauniyar, Shabandri Qais, Dinesh Goudar, Sai Srikar Kandukuri, Siddharth Mahajan, Sinash Siddik, and Partha Hazra

Madhavan Buddha, is senior scientific

manager; Shailabh Rauniyar is senior scientist;

Shabandri Qais is principal scientist; Dinesh Goudar is principal scientist; Sai Srikar Kandukuri is senior scientist; Siddharth

Mahajan is principal scientist; Sinash Siddik is senior scientist; and Partha Hazra is chief

scientific manager; all of whom are in the

research and development department at

Biocon research Limited in Bangalore, india.

PEER-REVIEWED

article submitted: may 29, 2015.

article accepted: aug. 19, 2015.

MA

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Peer-reviewed



Polyvinyl sulfonic acid (PVS), zinc chlo-

ride, phenol, sodium hydroxide, ortho-

phosphoric acid used for the precipitation

trials were procured from MilliporeSigma.

The standard Pierce BCA Protein Assay

Kit (Thermo Fisher Scientific) was used

to analyze the total protein content in

the samples. Sodium dodecyl sulfate–

polyacrylamide gel electrophoresis (SDS–

PAGE) was performed using Thermo Fisher

Scientific’s Novex NuPAGE 12% Bis–Tris

precast gel (1mm*10 well).

A Synergy HT microplate reader (BioTek)

was used to measure the optical density

of samples. Agilent’s 1260 Infinity HPLC–

Chip/MS was used to check the purity

profile and specific protein content of the

samples.

HPLC anaLytiCaL metHodA high-performance liquid chromatogra-

phy (HPLC) method—operable at alkaline

pH—was developed to analyze post-pre-

cipitation samples, because the polyvinyl

sulfonate/insulin complex precipitated at

acidic pH. A Zorbax C18 column from

Agilent (4.6*150mm) was used for purifi-

cation. Buffer A consisted of 100mM tris

and 15mM magnesium chloride and buffer

B consisted of 100% acetonitrile (HPLC

grade). An in-house qualified insulin stan-

dard with concentration of 4 mg/mL was

used to calculate the product content in

post-precipitation samples.

All precipitation samples were analyzed

using a 30-minute reverse-phase HPLC

method, and the HPLC column was main-

tained at 25°C. Elution was done with a

gradient formed by mixing buffers A and

B as follows: 85–60% B (0–15 minutes);

60–20% B (15–20 minutes); 20% B (18–

23 minutes); 20–85 % B (23–25 minutes);

85% B (25–30 minutes). The flow rate was

maintained at 1 mL/min, and the column

effluent was monitored at 215 nm.

BCa assay for Protein estimationThe authors followed the standard proto-

col described for the Pierce BCA Protein

Assay Kit (3) to establish a protein standard

curve. The protein samples were diluted to

approximately 1.5 g/L before performing

the assay. The BCA reagent (4), mixed with

the protein sample (total volume being

200 µl), was pipetted into a 96-well cell-

culture plate and absorbance was recorded

at 562 nm using a Synergy HT Multi-

Detection Microplate Reader from BioTek.

sds–PaGeThe authors followed the standard proto-

col for the NuPAGE kit (5). MES running

buffer (2-(N-morpholino)ethanesulfonic

acid), de-staining solution, sample buf-

fer (5X), staining dye were prepared as per

the procedures mentioned in the proto-

col. The samples were diluted to 1 g/L of

product concentration. Five volumes of

sample were mixed with one volume of

sample buffer, and the mixture was incu-

bated at 85 °C for 15 minutes. Next, 25 µl

of the sample was loaded into respective

wells of the precast gel. The gel was run

at 175V for 60 minutes. Post-completion,

the gel was rinsed in destaining buffer for

10 minutes followed by staining for three

hours. The gel was destained for nine

hours and examined for protein bands.

exPerimentaL ProCedure PVS-based precipitation

PVS concentrat ions of 0.10%, 0.25%,

0.50%, and 1% v/v were screened at pH

2.5, 3.5, and 4.5 to determine the opti-

mal conditions for precipitation. PVS was

added to clarified cell-free supernatant

under mixing and was allowed to mix

for approximately 8–10 minutes. The pH

was adjusted using concentrated ortho-

phosphoric acid. The mixture was further

mixed for 15 minutes and centrifuged at

7000 g for 25 minutes. The centrifuged

supernatant was analyzed for product

loss. The entire process was carried out

at approximately 20 –25 °C. The pellet

obtained was dissolved in 1M tris buffer

and checked for various process attributes

such as product purity, specific protein

content, and pigment content.

Zinc-based precipitation

Phenol concentration was screened at con-

stant zinc chloride and pH to determine

the optimal concentration required for

the precipitation process. The clarif ied

cell-free supernatant was treated with con-

centrations of 0.125%, 0.5%, and 0.75%

v/v of 100% phenol and allowed to mix for AL

L F

IGU

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Peer-reviewed



10 minutes. This was followed by addition

of 5% v/v of 4% w/v zinc chloride solu-

tion and mixed for 5–7 minutes. The pH

of the precipitation mixture was adjusted

to 4.5 by slow addition of 1N hydrochloric

acid. Post-pH adjustment, the precipitation

mixture was allowed to settle at 2–8 °C for

10–12 hours. The supernatant was decanted,

and the remaining slurry was centrifuged

at 6000 g for 25 minutes at 23+2 °C. The

obtained pellet was dissolved in 1M tris

to measure various process attributes such

as product purity, specific protein content,

and pigment content.

resuLts and disCussion The development of a precipitation pro-

cess for a protein to maximize yield and

product purity requires the screening

and optimization of various factors, such

as the nature and concentration of the

precipitant, the pH, the temperature, the

ionic strength, and the dielectric constant

of the medium. The precipitation of an

insulin precursor in the cell-free superna-

tant (CFS) stage strongly depends on the

buffer composition and various other fac-

tors that are present in the supernatant.

Polyelectrolyte-mediated precipitation

Polyelectrolytes are essentially charged

polymers that can either be anionic or

cationic in nature. Polyelectrolytes dis-

sociate in aqueous medium, which leads

to the formation of protein polyelectro-

lyte complexes (6). In addition to electro-

static forces, complex formation is also

influenced by hydrogen bonds and hydro-

phobic forces. This phenomenon—polyelec-

trolyte-mediated precipitation—has been

used in the protein purification process.

The earlier work by McDonald et al. on

antibody precipitation (7) demonstrated

the successful replacement of the chro-

matography step with polyelectrolyte pre-

cipitation. Such precipitation is able to

remove host-cell proteins, DNA, and other

impurities, and results in a product with

a purity that matches that of a product

obtained with the chromatographic pro-

cess. The precipitation of antibodies has

no influence on the activities of the anti-

bodies, which enables the iterative use of

the antibodies in additional protein puri-

fication processes. These antibody results

led the authors to evaluate a similar pre-

cipitation strategy for the purification pro-

cess of a human insulin precursor.

Various factors affecting polyelectro-

lyte precipitation of an insulin precursor

include: conductivity of the medium, pH

of the medium, and polyelectrolyte con-

centration. In the case of an insulin pre-

cursor, preliminary experiments revealed

that precipitation did not occur above the

conductivity of 25 mS/cm. However, there

exists a f lexibility of using higher con-

centration of polyelectrolytes to counter

the effects of higher conductivity of the

medium. The effect of pH (Figure 1) can

be explained by the charge neutralization

of the proteins as the pH increases toward

the isoelectric point (pI). Based on the

results (see Figure 1), PVS concentration

of 0.5% v/v and pH range of 2.5–3.5 were

found to be optimal for protein recovery

by precipitation. At higher concentrations

of polyelectrolytes, the electrostatic repul-

sion effect between the excess of free poly-

electrolyte and polyelectrolyte bound to

the protein increases the solubility of the

insulin precursor.

Zinc phenol-mediated precipitation

One of the fundamental methods of pre-

Peer-reviewed

Figure 1: Effect of concentration of polyvinyl sulfonic acid (PVS)

and pH on product recovery.

100

% R

eco

very

90

80

70

60

31.47 33.06

9.59

0.1% PVS 0.25% PVS

pH 2.5 pH 3.5 pH 4.5

0.5% PVS 1% PVS

68.23

75.6

93.7990.35 92.08

89

4.554.542.7

50

40

30

20

10

0



cipitating insulin precursor molecules

involves the use of metal ions. Zn+2 is the

most preferred metal ion because it is spe-

cific to insulin-like molecules (8). Zinc

induces the hexamerization of an insulin

precursor (9) and thus, stabilizes the mol-

ecule. Precipitation using zinc ions was

also evaluated as a substitute for the chro-

matography step. Hexamerization of insu-

lin precursor requires 0.3 moles of Zn+2

for every mole of insulin precursor. In

the current study, 5% v/v of 4% zinc chlo-

ride solution is added for precipitation,

which is in excess of molar requirement

for hexamerization of an insulin precur-

sor. In the authors’ unpublished study, it

was found that a molar excess of insulin

precursor is required to ensure complete

precipitation of an insulin precursor.

In the case of zinc phenol-mediated pre-

cipitation (precipitant concentration being

mainly phenol), the pH of the medium

and the protein concentrat ion before

addition of precipitants play a key role in

recovery of insulin precursor. Based on

the results (see Table I), 0.5% v/v of phenol

was found to be optimal for precipitation

of an insulin precursor. A higher-than-opti-

mal amount of phenol has shown to incur

higher product losses due to an increase in

solubility of the insulin precursor.

Product quality

assessment after precipitation

The product quality obtained through the

precipitation method was comparable to

the quality of a product purified through

traditional chromatography routes.

Certain parameters were monitored

to assess the quality of medications pro-

duced using precipitation methods com-

pared with products obtained through

chromatography. Host-cell proteins (HCPs),

host-cell pigments, and product purity

(as measured by HPLC) were studied. The

SDS–PAGE (see Figure 2) results show a

comparable band pattern for the chroma-

tography route and the polyelectrolyte

precipitation technique. The optical den-

sities at 600 nm, 450 nm, and 652 nm

and the specif ic protein contents were

recorded for the three strategies (the two

precipitation strategies in addition to tra-

ditional chromatography, as compared

in Table II). Reductions in the color of the

solution at a constant product concen-

tration indicate the removal of different

pigments. The optical densities and spe-

cific protein contents of the CFS at various

stages are tabulated in Table II. Reduction in

optical density is indicative of the removal

of the host-cell pigments by the respective

strategies. Specific protein is a measure of

the amount of insulin precursor relative to

the total protein content in the sample.

The product purities of the three strate-

Peer-reviewed

Phenol (%v/v) Zinc (%v/v) Recovery (%)

0.125 5 43.8

0.5 5 83.4

0.75 5 50.9

v/v: volume by volume

Table I: Effect of phenol concentration on

recovery of an insulin precursor with a constant

zinc concentration.

Figure 2: Sodium dodecyl sulfate–polyacrylamide gel

electrophoresis (SDS–PAGE) for precipitation feed and precipitate

samples.

Lane 1: Cell-free supernatant (CFS)

Lane 2: Clarifed CFS

Lane 3: Polyelectrolyte precipitate re-dissolved

Lane 4: Zinc-based precipitate re-dissolved

Lane 5: Ion-exchange elution pool

Lane 6: Human insulin drug substance re-dissolved

Lane 7: Protein ladder

1 2 3 4 5 6 7



Attributes

Optical density Specific protein based

on BCA Assay (%)

HPLC product

purity (%)450nm 600nm 652nm

Cell-free supernatant (insulin precursor, before clarification) (n=3)

0.30±0.05* 0.083±0.01 0.073±0.01 27.00±1.00 84.16±1

Polyelectrolyte precipitate redissolved (strategy 2) (n=3) 0.073±0.01 0.049±0.01 0.048±0.01 68.00±1.00 92.18±0.5

Zinc-based pellet redissolved (strategy 3) (n=3) 0.072±0.01 0.043±0.01 0.04±0.01 68.58±1.00 89.30±0.5

Cation-exchange (CIEX) elution pool (strategy 1) (n=3) 0.048±0.01 0.041±0.01 0.04±0.01 86.90±1.00 89.72±0.5

*The values represent mean ± standard deviation.

PVS precipitation Zinc phenol-precipitation Ion-exchange chromatography

Raw material Quantity required (g or mL)

Price($)

Raw materialQuantity required (g or mL)

Price ($)

Raw material Quantity

required (g or ml)

Price($)

1Polyvinyl sulfonic acid (PVS)

10 mL 2.80 Zinc chloride 4 g 0.34Ion-exchange

resin*166 mL 7.51

2Orthophosphoric acid

1 mL 0.02 Phenol 10 mL 0.62Sodium chloride

55 g 1.24

1N hydrochloric

acid 1 mL 0.01

Glacial acetic acid

4.5 mL 0.13

Sodium hydroxide

27 g 1.16

Total 2.82 0.97 10.04

*Resin reusability is considered in all cost calculations.

Table II: Optical densities and specific protein contents at various stages of the precipitation process. HPLC=high-

performance liquid chromatography.

Table III: Major raw material cost comparison of polyvinyl sulfonic acid (PVS)-based precipitation and zinc phenol-

mediated with ion-exchange chromatography to process 10 grams of insulin precursor.

gies measured by HPLC were comparable

(see Table II). Considering the purity of the

CFS under study, all the strategies gave a

significant increase in overall purity. This

could be connected to the increase in spe-

cific protein content as well after employ-

ing various strategies. Enrichment of the

specific protein content was accomplished

by precipitation strategies, although chro-

matography is a superior technique.

Removal of pigments prior to subse-

quent purification steps helps to improve

purification efforts. Whether these pig-

ments are associated with the insulin

precursor is still in question, but there

are instances where these pigments have

affected the chromatographic purification

of proteins (10). In a separate unrelated

study, these pigments—which are under-

stood to be alcohol-oxidase crystalloids—

were shown to interact with hydrophobic

proteins (such as human growth hormone),

resulting in altered charge and polarity

characteristics of the molecule (11). These

pigments considerably affect overall pro-

cess economics, leading to a lower bind-

ing capacity of target proteins, reduced

lifespan of the chromatographic media,

reduced yields, and lower product purity.

Cost considerations of precipitation

Use of a precipitation strategy is econom-

ical compared with the use of an ion-

exchange chromatography method. The

costs of the major raw materials used to

process 10 g of an insulin precursor using

PVS-mediated precipitation, zinc phenol-

mediated precipitation, and ion-exchange

chromatography are compared in Table III.

Even though ion-exchange resins can be

reused for 80–100 cycles, the cost of pro-

cessing by ion-exchange chromatography

Peer-reviewed



is 3.5 times higher than it is with the PVS-

based precipitation process and 10 times

higher than it is in zinc phenol-mediated

precipitation. Hence, precipitation pro-

vides a significant cost advantage in terms

of raw materials.

ConCLusion Precipitation offers several advantages

over chromatography. Precipitat ion is

cheaper, facilitates high-throughput pro-

cessing, and leads to higher concentration

of proteins. Chromatographic processes

are limited by the binding capacity of

the chromatographic media, a higher

cost of goods, and a larger volume of elu-

tion pools. In the present context, con-

sidering the example of enzyme reaction

after recovery of an insulin precursor

through various strategies, the authors

conclude that use of a precipitation step

offers the flexibility of working at higher

concentrations and at the buffer com-

positions suited for an enzyme reaction

that is associated with higher yield and

purity. Chromatography limits the choice

of eluents, which may be composed of

chemicals that could affect subsequent

processing steps.

The current study did not evaluate a

hybrid approach, which would include the

use of chromatography and precipitation

for purification. A hybrid approach that

employed a precipitation step, however,

may be a desirable approach in the future

for successive chromatography steps.

P ur i f icat ion of biolog ics t y pica l ly

involves several chromatography steps.

Thus, precipitation strategies discussed

here reduce the number of chromatog-

raphy steps required in a purif ication

process by serving to replace capture chro-

matography. A hybrid strategy could also

offer significant cost advantages by reduc-

ing the number of chromatographic steps

involved in the purification process.

In terms of infrastructure requirement,

precipitation involves the use of stirred

tanks for mixing and the solid-liquid sepa-

ration by either using centrifugation or fil-

tration. In contrast, the chromatographic

processes require a complex infrastructure

of chromatographic systems integrated to

buffer tanks, which significantly limits

the throughput of the process.

reCommendationsResults obtained in the current study are

promising in terms of cost reduction of

capture step of purification process with-

out compromising on the quality and func-

tionality of the product. Purification by

precipitation offers the opportunity for cost

reduction in capture steps without compro-

mising the quality and functionality of the

product. Therefore, precipitation is a promis-

ing method that could serve as an alternative

to expensive chromatography techniques.

referenCes 1. R.L. Fahrner et al., Biotech. Gen. Eng.

Rev. 18 (1), pp. 301–327 (2001).

2. M.Q. Niederauer and C.E. Glatz, “Selective

Precipitation,” in Bioseparation Advances

in Biochemical Engineering/Biotechnology,

G.T. Tsao, Ed. (Springer-Verlag, Berlin,

1st ed., 1992), 47, pp. 159–188.

3. T. Adilakshami and R.O. Laine, J. Biol.

Chem. 277, pp. 4147–4151 (2002).

4. P.K. Smith et al., Anal. Biochem. 150,

pp. 76–85 (1985).

5. I.M. Szalo et al., Clin. Diagn. Lab.

Immunol. 11 (3), pp. 532–537 (2004).

6. M. Braia et al., J. Chrom. 873

(2), pp. 139–143 (2008).

7. P. McDonald et al., Biotech. Bioeng.

102, pp. 1141–1151 (2009).

8. M. Sahyun, J. Biol. Chem. 138,

pp. 487–490 (1941).

9. G.D. Smith et al., Proc. Natl. Acad. Sci.

USA 81, pp. 7093–7097 (1984).

10. S.A. Minyasab et al., “A method of purifying

human growth hormone and purified growth

hormone thereof,” US Patent Application

WO2010134084 A1, Nov. 2010.

11. L.M. Damasceno et al., Protein Expr.

Purif. 37 (1), pp. 18–26 (2004). ◆

Peer-reviewed



M_a_y_

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As traffic in counterfeit and adul-

terated pharmaceuticals grows

(see Sidebar), the idea of a fully

traceable pharmaceutical supply

chain is taking shape. The concept has

changed drastically since the past decade,

when the first radio-frequency identifica-

tion (RFID)-based serialization systems

were piloted, and Florida set the first pedi-

gree requirements.

Today, 2-D matrix barcoding is the

technology of choice, and 40 countries

around the world have set requirements

for lot- and unit sales-level serialization

and traceability, each of which offers

specific challenges (Figure 1).

In the United States, the Drug

Supply Chain Security Act set a dead-

line of January 2015 for lot-level

traceability and verification. Unit seri-

alization requirements will take effect

in November 2017, and 2023 will be the

deadline for having serialization-based

track-and-trace systems in place. In the

European Union, the Falsified Medicines

Act has set a deadline of the end of 2018

for unit serialization and end-to-end ver-

ification and authentication.

Pharmaceutical serialization, aggre-

gation, and traceability pose huge

technical challenges, requiring the

easy transfer of, and access to, massive

amounts of data. These data will need

to flow from manufacturers—some of

whom have merged with or acquired

other companies—but also from their

contract manufacturing and packaging

partners. Data will also have to connect

to wholesale distributors, dispensers,

pharmacies, and clinics.

Serialization: Getting Past the Quick Fix

Agnes Shanley

Traceability and transparency

will remain elusive if

manufacturers continue to approach

serialization projects on a case-by-case basis.

Serialization



Over the past few years, the

pharmaceutical industry has made

progress in developing serializa-

tion strategies and investing in

the information technology (IT)

required. In UPS’ 2015 Pain in

the Chain Survey (1), 67% of 421

respondents viewed their invest-

ment in serialization IT as success-

ful in improving product security

(Figure 2).

However, challenges remain.

Even at the manufacturing level,

where IT connections will be

required between manufactur-

ers and contract manufacturing

and packaging organizations,

companies are at different levels

of achievement. Generally, Big

Pharma companies were ready for

serialization before the deadlines,

notes Darryl Brown, vice-president

of global strategic marketing at

Systech International. For smaller

firms and contract manufactur-

ing organizations (CMOs), readi-

ness varies. “Some companies have

robust programs in place, but oth-

ers have been working tactically, to

meet the bar set in different parts

of the world,” says Shabbir Dahod,

CEO of TraceLink, which offers a

cloud-based IT solution that won

a CPhI Award in 2015 for innova-

tion in supply chain and logistics

management.

“With less than two years to

meet the US deadline, both manu-

facturers and CMOs realize that

they have to move fast,” says

Steve Peterson, project manager at

CRB Consulting in St. Louis, Mo.

However, he says, over the past six

months, lead time for line-level

serialization equipment has gone

from 12–14 weeks to 10–16 weeks.

IF CMOS haven’t Started yet, they May already be tOO lateGenerally, it can take the smallest

pharmaceutical CMOs from one

to two years to develop a strategy

and foundation for serialization,

medium-sized CMOs from two

to three years, and large CMOs

more than three years, Vivian

McCain, vice-president of third-

party operations, Americas, at

Teva Pharmaceuticals, and pro-

gram owner, CMO Serialization

Program, told attendees at the

First Serialization Roundtable for

CMOs, held in Philadelphia, PA

on October 8, 2015 (2). For mid-

to-large CMOs that haven’t started

laying the groundwork, it may

already be too late, McCain said.

There are many complex ques-

tions to decide, he explained,

such as who should “own” label-

ing artwork issues or IT connec-

tivity projects, or how quality

assurance and exception manage-

ment should be handled.

Teva Pharmaceuticals began

developing its internal serialization

strategy four years ago, focusing on

its global network of 20 sites. This

year, the company began expand-

ing the program, funding a CMO

Serialization

A new standard aims to improve connectivity

The global market in counterfeit pharmaceuticals has been seeing double-

digit growth, according to Charlie Gifford, executive director of the Open

Serialization Communications Standard (Open-SCS) working group. Open-

SCS, launched in 2015 by the Open Platform Communication Foundation,

involves the participation of pharma companies that include Abbott, Teva

Pharmaceutical Industries Ltd., and Mylan Pharmaceutical, and vendors

including SAP AG, OptelVision Inc., Systech International, Werum IT Solutions

GmbH, and Antares Vision Srl. Its mission is a standard that would help

promote open communications and nonproprietary data interfaces.

The explosive growth in demand for generic drugs, and the fact that contract

manufacturing organizations now manufacture these drugs, has resulted in

huge opportunities for counterfeiters, Gifford says. “This issue of counterfeiting

is really a World Health Organization-type problem, and we’re seeing nothing

short of a pandemic,” he notes, “but it is only being addressed by countries and

individual manufacturers.”

The problem is that regulators in different countries, typically, don’t know

anything about manufacturing or supply-chain systems, or aggregating

information, Gifford says. In addition, he says, most regulatory agencies

are not actively participating in standards efforts, often out of concern for

the appearance of potential conflicts of interest. As a result, serialization

requirements can be difficult to meet, given typical plant floor realities.

Global Standards One (GS-1) has standardized communications protocols,

and current serialization data standards include its Electronic Product Code

Information Services (EPCIS) with pieces of various manufacturing process

control standards such as ISA 95, S88, and Make 2Pack, says Gifford.

Open-SCS would develop a comprehensive standard that would help

streamline and simplify data integration, says Gifford, reducing costs. The

group studied offerings from 10 leading packaging serialization equipment

vendors and found that architectures and data syntax varied greatly, potentially

increaasing the cost of serialization and traceability. Gifford notes two

challenges: vendor’s preference for proprietary systems and companies’

reluctance to subsidize employees’ participation in standards development.

Nevertheless, the group plans to launch new phases of the project this year.

For more information, visit www.opcfoundation.org/open-scs-working-group/



support program to help oversee

partner serialization efforts in its

external network of more than 700

CMOs. The program focuses on

issues that include IT connectiv-

ity, quality assurance, tools, and

applications. A CMO serialization-

management dashboard allows the

company to track each CMO’s seri-

alization readiness and progress

and assess the risk of noncompli-

ance. Teva is developing risk pro-

files for each partner to assess the

potential business impact of serial-

ization noncompliance.

To ensure flexibility, it’s impor-

tant to have an IT solution that’s

loosely coupled to the enterprise

system, says TraceLink’s Dahod.

However, Dahod sees non-standard

data exchange and integration

approaches as one of the big-

gest potential obstacles that the

industry faces in meeting upcom-

ing serialization and traceability

regulations.

Global Standards One (GS-1),

the nonprofit organization that

maintains standards for supply

chains across different industries,

and its Electronic Product Code

Information Services (EPCIS) stan-

dard have played a key role in help-

ing drive a common data exchange

framework between packaging

sites, enterprise serialization sys-

tems, and network trade partners,

he says.

He notes the potential of newer

standards as well. “The Open

Ser ia l izat ion Communicat ion

Standard (Open-SCS) Working

Group could extend these data

exchange harmonization efforts

even deeper in the serialization

process,” he says. “Particularly

between the packaging line and

packaging site level, where the

sheer diversity of line-management

systems and equipment manu-

facturers have made readiness for

serialization a great challenge.”

Jean-Pierre Allard, global serializa-

tion program manager for Optel

Vision, has called it “the standard

that CMOs need” (3).

Foundations for the Open-

SCS Working Group were laid

in February 2015, and executive

director Charlie Gifford sees a real

need for this work. “With trace-

ability, ultimately, we need to val-

idate the bottle that reaches the

patient,” Gifford says, “and the

current process—which includes

serializing at the bottle level,

aggregating and serializing in all

forms, and then de-aggregating to

the pharmacy level—is still vulner-

able to criminal interference.”

The idea behind Open-SCS is to

focus on the three or four data and

communication layers (line and

equipment, distribution centers

and warehouses, plant and ware-

house operations management)

between the plant and the corpo-

rate enterprise resource planning

(ERP) system. For serialization,

such a standard would help phar-

maceutical companies and CMOs

improve the aggregation of seri-

alization master data between

plants, and optimize plant-floor

strategies depending on whether

their manufacturing departments

assign a serialization number, or

whether a government regulatory

agency is providing numbers to

apply to product.

Given the number and variety

of regulations, and differences

between different vendors’ prod-

ucts, the challenges are great. “If

you walk into any CMO’s packag-

ing line today, every single piece of

equipment there is from a different

vendor. Manufacturing Execution

System (MES) and ERP vendors sell

proprietary systems. By the time

you get up to the top level to cre-

ate a report, it’s a mapping night-

mare,” Gifford says. As he explains,

line-equipment vendors tend to

promote proprietary solutions; at

Serialization

Figure 1. Different global requirements for serialization challenge pharma companies today. Respondents to UPS’ 2015 Pain

in the Chain survey (1) singled out the most challenging national regulations, as shown here (1).

International regulations that cause the most pain (global)

European Union GoodDistribution Practice

39%

16%

19%

7%

European Union Medical DevicesDirectives and Requirements

Brazil Serialization China Medical DeviceGood Supply Practice

Korea Medical Device andin-vitro Diagnostics Practice

U.S. Drug Supply ChainSecurity Act

Q. When thinking about regulatory compliance issues, what are the international regulations that cause you most pain?

39%

16%

24%

16%

All

fig

ure

s co

urt

esy

of U

PS


ON-DEMAND WEBCAST Originally aired December 8, 2015

Register for free at

www.biopharminternational.com/bp/UNit

EVENT OVERVIEW:

Formulation scientists are expected to do a lot more with less—

more constructs, higher concentrations, less volume. Novel

antibody structures and ADCs present a diferent challenge for

development. Platform formulations that were used for monoclonal

antibodies don’t always work and degradation can show up from a

number of biophysical and chemical pathways. Developing assays

that can characterize and predict the manufacturability, developa-

bility, and long-term stability of these complex biologics isn’t easy.

Obtaining information about their stability, whether it be thermally

driven structural changes, aggregation kinetics, viscosity, and more

can help make sense of what’s going to happen to the protein. In

this webinar, case studies from literature will be presented and a

powerful tool—the Unit—which can perform all of these measure-

ments with a lot less sample, will be presented and discussed.

Key Learning Objectives

n Learn the benefts of measuring Tm

and Tagg

simultaneously

n Learn how small sample sizes enable faster screening of

biologics

n Learn why full spectrum fuorescence and static light scattering

are critical for understanding protein unfolding and aggregation

PRESENTERS:

DAN LUND, PhD

Field Applications Scientist

Unchained Labs

MODERATOR:

SARA BARSCHDORF

Multimedia EditorBioPharm International

Who Should Attend

n Pre-formulation and formulation

scientists

n Biologic characterization scientists

n Structural biologists

Presented by

Way more biologic stability, a lot less protein

For questions contact Sara Barschdorf at [email protected]

Hosted by



Serialization

the MES level, they are looking at

the batch record, and, at the ERP

level, the batch record is different

from the master batch record. Also,

not all countries are standardized

on GS-1, so five different countries

may have five different require-

ments for reporting out of the

plant, Gifford says.

EPCIS addresses some, but not

all of these issues, so many proj-

ects become one-offs using custom

solutions. “This type of work does

not scale. What works in one plant

may not be able to accommodate

change, and work in five plants

around the world five years from

now,” says Gifford.

Another major problem with

one-offs is lack of transparency,

says Systech’s Brown. “Temporary

solutions will get you through the

checkbox for serialization, but if

there’s a problem down the line,

there will be no way to diagnose

it and retain the data, so it will

then be very difficult in situations

where, say, you need to recall mil-

lions of dollars worth of product.”

Supply Chain Wizard’s manag-

ing partner Burak Tiftikci sug-

gests that manufacturers “design

for tomorrow.” As he noted dur-

ing the October CMO conference,

“You can always dumb down the

richness of data to meet a specific

requirement” (4). He noted, how-

ever, that it can be “exponentially

more challenging” to work the

other way around.

A prerequisite to successful

serialization is having a strong

IT system that can handle serial

number mapping. Peterson adds,

“you need to be able to handle

high volumes of data, and to have

a disaster recovery and backup sit-

uation in place.”

A l l of the major ERP ven-

dors have introduced platforms

designed to facilitate serializa-

tion, although consultants note

that some may still be unproven.

In September 2015, SAP intro-

duced Adva nced Trac k a nd

Trace for Pharma (ATTP), which

replaces its prev ious Auto-ID

Infrastructure (AII) and object

event repository (OER) programs

as a way to generate serial num-

bers, send codes to packaging

lines, and then track them. That

same month, TraceLink intro-

duced an SAP Migration Kit to

allow users of SAP serialization

products to take that functional-

ity into the cloud.

POInt-OF-uSe COnneCtIOnS ChallenGe PharMaWell beyond the manufacturing

and CMO portion of the supply

chain, a major challenge is work-

ing with pharmacies, clinics, and

hospitals at the point of service,

Gifford says. “Today, between

20–50% of the cost of serialization

is due to paper pushing,” he says.

“We’re going into a rudimentary

IT world and telling them to put in

yet more systems.”

Concerns that can play out at

the point of service were recently

seen in Brazil, whose regula-

tory agency, ANVISA, postponed

a December 2015 deadline for

its traceability pilot phase. The

requirements cal led for drug

license holders to report to the

government, and pharmacies

feared that access to patient data

might give suppliers too much

information about stock levels and

trends, and allow them to control

pricing and supply (5). The gov-

ernment changed its requirements

so this information could not be

shared with distributors.

Even so, the challenges of data

integration at that level can be for-

midable, and standards can help.

This was shown in Roche’s recent

pilot project, which focused on

special products and medicines

for cancer, and involved collabo-

ration with the systems integra-

tor SPI and TraceLink. The project,

which won the 2015 GS-1 Brazil

Automation Award, used EPCIS to

Figure 2. Most life-sciences companies see investments in serialization as

enhancing product security, according to UPS’ 2015 Pain in the Chain survey (1).

Product Security

IT investment: bar coding,

serialization, etc.

Cooperation with law

enforcement

Visible authentication: visible

holographs, security inks, etc.

67%

41%

38%

Q.You indicated that you've been successful at addressing product security. What strategies

have made you successful?

Contin. on page 46



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An industry workgroup com-

prised of toxicologists and

manufacturing equipment

c lea n ing subjec t mat te r

experts was formed to examine the

European Medicines Agency (EMA)

shared facilities guideline, specifically

the provision for alternative approaches

to establishing limits and how to prac-

tically implement any proposed alter-

natives (1). This paper describes the

outcomes of these evaluations, includ-

ing alternatives for estimating health-

based exposure limits using available

assessments and a strategy to priori-

tize existing commercial products for

health-based exposure limit evaluation.

It provides a screening tool to identify

products that require calculation of a

health-based cleaning limit, and those

where cleaning to existing cleaning limits

(e.g., based on 1/1000th the minimum

therapeutic dose) may continue. The pre-

sumption is that all newly introduced

products will have formally documented

health-based exposure limits. EMA was

consulted during development of this

paper, which is intended to support imple-

mentation of the European Union GMP

Chapter 3 and 5 revisions for prevention

of cross-contamination (2).

EMA published a guideline on setting

health-based exposure limits for use in

risk identification in the manufacture of

different medicinal products in shared

facilities in November 2014 (1). The

European Commission (EC) also revised

related General GMP Chapters 3 and 5

in January 2015 by updating sections

on prevention of cross-contamination;

EMA Guideline on Setting Health-Based Exposure Limits

Andrew Teasdale, Bruce D. Naumann, Gretchen

Allison, Wendy Luo, Courtney M. Callis, Bryan

K. Shipp, Laura Rutter, and Christopher Seaman

The results of an industry

workgroup’s examination of

EMA’s guide on shared

facilities are presented.

Andrew Teasdale is a principal scientist

at AstraZeneca, andrew.teasdale@

astrazeneca.com; Bruce D. Naumann

is executive director, Toxicology, Merck &

Co.; Gretchen Allison is senior director,

Global Quality Operations, Pfizer Inc.;

Wendy Luo is associate director, Drug

Safety Evaluation, Bristol-Myers Squibb;

Courtney M. Callis is a research scientist,

Health/Safety/Environmental, Lilly

Research Laboratories; Bryan K. Shipp

is director, Risk Management, Pfizer

Inc.; Laura Rutter is SM manager,

Analytical Services, GlaxoSmithKline; and

Christopher Seaman is senior manager,

Occupational Toxicology, GlaxoSmithKline.

Health-Based Exposure Limits



these revisions became effective

on March 1, 2015 (2). The EMA

guide effective date was June

1, 2015 for new products and

Dec. 1, 2015 for existing prod-

ucts. These timelines represent

a significant challenge for the

industry.

The EMA guide implementation

timelines are aggressive consider-

ing the current number of exist-

ing products. The implications of

the EMA guide are that current

cleaning limits would need re-

evaluation in relation to newly

derived health-based cleaning

limits. If revisions are necessary,

then cleaning validation activi-

ties may need to be re-started for

impacted manufacturing equip-

ment. Cleaning sampling and ana-

lytical test method detection levels

may need to be re-evaluated, and

potentially lower detection limits

established/validated. Because re-

evaluation may consume signifi-

cant resources, efforts need to be

prioritized according to impact on

patient safety.

The EMA guide uses permitted

daily exposure (PDE) values as a

basis for establishing appropriate

limits but states that PDE values

are considered synonymous with

the acceptable daily exposure

(ADE) values. Both represent a dose

that is not expected to cause an

adverse effect in an individual,

even with a lifetime of exposure.

The EMA guide also states that

“the use of other approaches to

determine health-based exposure

limits could be considered accept-

able if adequately and scientifically

justified.”

This paper describes the poten-

tial to adopt scientifically jus-

t i f ied and hea lth-protec t ive

options for estimating ADE val-

ues from available assessments. It

also provides examples on how to

prioritize existing products and

evaluate currently used clean-

ing limits to identify those prod-

ucts that require calculation of

an ADE- or PDE-based cleaning

limit, and those where cleaning

to existing cleaning limits (e.g.,

1/1000th the minimum thera-

peutic dose) already provides suf-

ficient patient protection. The

presumption is that all newly

introduced products will have

documented health-based expo-

sure limits.

ALTERnATIvE APPROACHES fOR SETTInG HEALTH-BASED ExPOSuRE LIMITS: ESTIMATIOn Of ADE vALuES fOR SMALL MOLECuLESThe goal of applying alternative

approaches should be to dem-

onstrate whether or not current

cleaning practices provide a suf-

ficient margin of safety for patient

health. The approach proposed in

this document provides a ratio-

nale for estimation of ADE values

using existing potency and toxi-

cological evaluations performed

for worker safety purposes, fol-

lowed by an evaluation of facil-

ity attributes and product mix.

For example, many pharmaceuti-

cal organizations establish occu-

pational exposure limits (OELs)

for their compounds or use a

control banding approach (occu-

pational exposure bands [OEBs])

to categor i ze compounds of

increasing severity based on their

inherent pharmacological and tox-

icological properties and/or limited

dataset. ADE values can be esti-

mated using OELs or OEBs, and

if an adequate margin of safety is

identified between cleaning limits

based on the estimated ADE value

and maximum possible carryover

based on existing cleaning limits,

a formal comprehensive ADE value

monograph may not be required.

Adequate documentation could

initially include a brief summary

or spreadsheet reflecting the sci-

entific rationale, and formal ADE

values would only be needed for a

subset of compounds where facil-

ity attributes and product mix do

not afford an adequate margin of

safety. The evaluation approach

should be documented for each site

and included in plans available for

review by regulatory inspectors.

Identifying the Most Hazardous Drugs

Prior to issuance of the EMA

guideline on setting health-based

limits, categories of drugs were

identified that have the greatest

concern (e.g., “certain hormones,

certain cytotoxics,” etc.); however,

specific criteria were never pro-

vided. The International Society

for Pharmaceutical Engineering

(ISPE) Risk-MaPP baseline guide

outlines example characteristics of

compounds of traditional concern

that could benefit from further

review (3):

• Genotoxic (specificallymuta-

genic) compounds that are

known to be, or highly likely to

be, carcinogenic to humans.

• Compounds that can produce

reproductive and/or develop-

mental effects at low dosages.*

• Compounds that can produce

serious target organ toxicity,

anaphylaxis, or other signifi-

cant adverse effects at low dos-

ages.*


If effective protein

degradation and

inactivation could not

be demonstrated in

the cleaning studies,

cleaning limit should

be based on PDE.



* e.g., clinical doses <1–10 mg/

day or dosages in animal studies

<0.1–1 mg/kg/day

The threshold of toxicological

concern (TTC) concept may also

be useful to estimate ADE values

and applies to products contain-

ing mutagenic active ingredients

(ADE=1 µg/day), those that are

potent or toxic (ADE=10 µg/

day), or those that have no

prior evidence of these prop-

er t ies (ADE =100 µg/day) (4).

In pr inc iple, each of these

approaches can be used to identify

hazardous drugs for risk assess-

ments and, if scientifically justi-

fied, can be used for the derivation

of health-based cleaning limits.

Identification of a compound as a

hazardous drug should not auto-

matically lead to strict control

solutions, because it addresses only

hazard. A risk assessment should

be completed before conclusions

can be made about risk and the

need to modify controls.

Alternative Approach 1: If I Have an OEL

ADE or PDE value derivation and

OEL derivation share the same sci-

entific basis (e.g., the same method

to define the point-of-departure

[PoD]), with some differences in

the respective adjustment factors

related to the different exposure

scenarios or regulatory context.

By comparing the respective stan-

dard adjustment factors, it is pos-

sible to define the systematic

difference between the two calcu-

lations. If this systematic differ-

ence is adequately evaluated and

the OEL derivation is scientifically

justified, estimation of the ADE

from the available OEL could meet

expectations to derive health-

based cleaning limits, at least on

an interim basis to support priori-

tization and assessment of existing

cleaning limits.

The EMA guide acknowledges

that “good quality human clini-

cal trial data is highly relevant”

to the assessment for ensur-

ing patient safety. Following the

publicat ion by Fourman and

Mullen in 1993, many firms have

used a minimum therapeutic

dose approach (e.g., 1/1000th of

the minimum therapeutic dose)

to calculate cleaning limits (5).

The existing OEL monograph, if

available, can be used as a screen-

ing tool to determine if this

approach is health-protective. OEL

monographs typically include a

potency and toxicological evalu-

ation of relevant nonclinical and

clinical data. In the workplace,

compound-related effects (includ-

ing intended pharmacology and

unwanted toxicity) are consid-

ered adverse. Many products have

a favourable therapeutic index,

which means that the pharmaco-

logical activity is the most sensi-

tive effect. However, EMA doesn’t

address derivation of an ADE from

clinical data. Because the OEL is

frequently derived from the mini-

mum therapeutic dose, it is rea-

sonable to deduce that the ADE

value based on 1/1000th the

minimum therapeutic dose is suf-

ficiently conservative for most

compounds. ISPE Risk-MaPP out-

lines science-based adjustment

factors that support this assump-

tion. For example, a lower com-

posite adjustment factor (i.e., 10

to account for intra-species differ-

ences and 3 to extrapolate to a no-

effect level) can be scientifically

justified if using the minimum

therapeutic dose as the PoD for a

product with a reasonably robust

dataset (e.g., late- to commercial-

stage compound). Therefore, when

the human dose information is

used as the PoD for OEL deriva-

tion, the minimum therapeutic

dose cleaning limit approach may

be considered health-protective,

and no additional value would be

derived by proceeding to a formal

ADE derivation.

The exception would be com-

pounds (e.g., classical oncology

compounds) with an unfavour-

able therapeutic index that may

cause unwanted or even serious

adverse health effects at expo-

sures near or below the indicated

minimum therapeutic dose. A

mutagenic carcinogen adminis-

tered at a dose of 750 mg/day, for

example, would result in a limit

of 750 µg/day using the 1/1000th

approach, which is almost three

orders of magnitude higher than

the TTC level of 1 µg/day using

Dolan et al. (or 1.5 µg/day under

ICH M7) (4, 6). In these cases, a

review by a toxicologist is rec-

ommended. When the OEL is

calculated using a nonclinical

PoD (e.g., repeat-dose toxicity or

reproductive toxicity in animals),

or involves route-to-route extrap-

olation, then further review by a

toxicologist is also recommended

to determine an appropriate ADE

value.

Example of an Estimated ADE Value

Calculation Using Data from the OEL

Monograph for Compound A.

The following example illustrates

the calculation of an OEL for

Compound A using the minimum

therapeutic dose of 2.5 mg/day as

the PoD. All relevant nonclinical,

clinical, and post-marketing data

were evaluated. The details are

provided to show the similarity to

ADE derivation while highlighting


The threshold of

toxicological concern

(TTC) concept may

also be useful to

estimate ADE values.




that assessment factors used might

differ.

The OEL incorporates a compos-

ite adjustment factor (AF) of 30,

derived as follows (see Equation 1—

UF is uncertainty factor; LOAEL

is lowest observed adverse effect

level; NOAEL is no observed

adverse effect level).

For the calculation of the OEL,

a bioavailability correction fac-

tor of 0.3 is included in the

numerator to account for oral

bioavailability in humans (30%).

Therefore, an ADE est imated

from the OEL is conservative (i.e.,

approximately three-fold lower

than needed) if the oral route

of exposure is applicable for the

cross-contamination scenario (see

Equation 2—TWA is time weighted

average).

By comparison, 1/1000th the

minimum therapeutic dose is

2.5 µg/day (2.5 mg/day/1000),

which is 10 times lower than the

estimated ADE value. Therefore,

the existing cleaning limit is

acceptable, and development of a

formal ADE value is not a high pri-

ority at this time.

Alternative approach 2: What if I don’t

have an OEL but I do have an OEB?

Many companies assign new com-

pounds to an OEB, or equivalent

designation by airborne concentra-

tion ranges, to provide guidance on

safe handling before sufficient data

are available to establish a numerical

OEL. The OEB is assigned based on

the compounds’ inherent pharma-

cological and toxicological charac-

teristics, including the intended use,

mechanism-of-action, dose-response,

and safety pharmacology studies.

One option is to use the OEBs to esti-

mate ADEs for the most hazardous

subset of compounds (i.e., those with

the most restrictive OEBs).

Example OEB assignment crite-

ria are provided in Table I corre-

sponding to ADE values of 10 and

1 µg/day corresponding to respec-

tive OEBs of ≥ 1 <10 µg/m3 and

<1 µg/m3 because it is assumed

that 10 m3 are breathed during the

workday. These are the same values

that would be derived using the

TTC concept outlined in Dolan et

al. (4). Application of these default

values presumes that sufficient

compound-specific data are not

available to estimate an ADE (5).

The examples focus on “potent” or

“high containment” compounds,

but in practice, the principle

would still hold across less restric-

tive bands. As with application

of the OEL, a bioavailability cor-

rection could be considered if the

compound is anticipated to have

extremely low bioavailablity via a

relevant route.

• IntraspeciesDifferences(UFH) 10 DefaultAdjustment

• InterspeciesDifferences(UFA) 1 Human

• LOAELtoNOAEL(UFL) 3 Minimaltherapeutictono

therapeuticeffect

• Sub-chronictoChronic(UFs) 1 Experiencewithlong-termuse

• DatabaseCompleteness(UFD) 1 Completedatabase

UFC=(UFH)(UFA)(UFs)(UFL)(UFD)=(10)(1)(1)(3)(1)=30

Assumptions:

LOAEL=2.5mg/day

Bio-availabilityCorrectionFactor=0.3(30%oralbioavailabilityinhumans)

CompositeUncertaintyFactor(UFC)=30

ModifyingFactor(MF)=1(nomodifyingfactor)

V=Volumeofairbreathedin8hours(10m3) [Eq.1]

Table I. Example occupational exposure band (OEB) assignment criteria and corresponding estimated acceptable daily

exposures (ADEs).

Control band associated with airborne concentration range

≥1<10 µg/m3 <1 µg/m3

Estimated ADE (lower end of band x 10 m3)

10 µg/day <1 µg/day

Compound characteristics

High pharmacological potency (0.1–1 mg/day). Effects in humans may be serious and/or slowly reversible, but are not life-threatening and easily managed medically.

Very high pharmacological potency (< 0.1 mg/day). Acute exposures at very low doses may be incapacitating, life-threatening, and require medical intervention. Immediate and heroic medical intervention may be needed. Chronic effects may be irreversible, disabling, or life-shortening.

OEL=( 2 . 5 m g / d a y ) ( 0 . 3 )

(30)(1)x10m3/day

OEL=2.5µg/m3(8-hrTWA)

EstimatedADEvalue(dailysystemic

dose)=OELxV=2.5µg/m3x10m3/

day=25µg/day [Eq.2]



How can consistent screening to

evaluate the effectiveness of existing

cleaning limits using the OEL/OEB be

implemented?

Figure 1 provides an example deci-

sion process for systematic deter-

mination of whether traditional

cleaning limit calculations are

adequate using the OEL and OEB.

The decision tree shows that use of

OEL and application of 1/1000th

the minimum therapeutic dose

approach could be acceptable

under the following conditions:

• For existing commercial prod-

ucts where ex ist ing c lean-

ing limits are based on the

1/1000th approach

• IfanOELmonographincluding

toxicological evaluation of rel-

evant nonclinical and clinical

data is available

• Ifthepharmacologicalactivityis

the most sensitive (critical) effect.

LARGE MOLECuLE COnSIDERATIOnSThe EMA guide states that “deter-

mination of health-based expo-

sure limits using PDE limits of the

active and intact product may not


Figure 1. Example decision tree for evaluating effectiveness of existing cleaning limits using the occupational exposure

limits (OEL) or occupational exposure bands (OEB). PDE is permitted daily exposure. ADE is acceptable daily exposure. EHS

is environmental health and safety.

Start Cleaning ValidationScreen Tool

ADE or PDE ValueAvailable?

Yes

No

No

No

No

No

No

No

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Value Used inCurrent Cleaning

Limit?

Screen Complete. MaintainCurrent Cleaning Practice.Document Assessment and

Conclusions.

Calculate New Health -based Cleaning LimitUsing the ADE or PDE

Value

CurrentCleaning Limit

Based on 1/1000th

Minimum TherapeuticDose?

New Health - basedCleaning Limit > CurrentCleaning Limit or Within

One Order -of-Magnitude?

New Health - basedCleaning Limit > CurrentCleaning Limit or Within

One Order -of-Magnitude?

Screen Complete. Maintain CurrentCleaning Practice. Document

Alternative-based Approach Assessmentand Conclutions. No ADE Value

Required at this Time.

Screen Complete. Maintain CurrentCleaning Practice. Document

Alternative-based Approach Assessmentand Conclutions.

Screen Complete. Make AnyNecessary Change to the Current

Cleaning Limits. DocumentAssessment and Conclusions

Toxicologist to Prioritizeand Develop ADE Value

For Review and Approval

Notify ToxiCologist toPrioritize Development of

ADE Value forReanalysis.

Communicate Potentialfor Change to Current

Cleaning LimitRejected if Not in Alignment With the MinimumTherapeutic Dose Used for the OEL / OEB

EHS OEL/OEBBased on Clinical

Data?

Minimum Therapeutic Dose

“Cytotoxics”“Certain Antibiotics”“Highly Sensitizing Products”“Certain Hormones”

Product Classof Concern?

Stop Screen and ConsultToxicologist

Calculate New Health -based Cleaning LimitUsing the “Estimated

ADE Value”

CanToxicologist

ProvideAppropriate

“Estimate ADEValue”?

Review RiskAssessment /

Segregation AssessmentNo

Fig

ure

s 1 is

co

urt

esy

of th

e a

uth

or.




be required” for macromolecules

(proteins and peptides) (1). When

macromolecules are exposed to pH

extremes and/or heat, they rapidly

degrade and denature and are con-

sidered pharmacologically inactive.

Only breakdown products such as

smaller peptide fragments or amino-

acid derivatives formed due to pro-

tein hydrolysis would be present after

cleaning under conditions that are

shown to effectively degrade and

inactivate protein-based products.

In these scenarios, health-based

exposure limits of the active products

are no longer relevant. An experi-

mental approach and analytical

methods for assessing degradation

and inactivation of the API during

cleaning, and an approach for set-

ting safety-based acceptance limits

for inactivated product and process

residuals are described in separate

industry publications (7, 8). However,

if effective protein degradation and

inactivation could not be demon-

strated in the cleaning studies, clean-

ing limit should be based on PDE.

COnCLuSIOnThe proposed screening tool and

methods for estimating ADE values

are appropriate for implementation

of the EMA guide with the goal of

ensuring health-protective clean-

ing limits. It is valuable to note that

compound-specific hazard is not

the only concern as the hazard of

all compounds in the matrix must

be considered in order to calculate

the worst-case cleaning limit for all

Product A-to-Product B changeover

permutations. Hence, implemen-

tation of worst-case permutations

affords an additional level of safety

for many limits. Further assurance

is provided on completion of the

cleaning; when the equipment is

dry, a visual inspection is typically

performed for those surfaces that

can be visually inspected.

REfEREnCES 1. EMA, Guideline on Setting Health-

Based Exposure Limits for Use in Risk

Identification in the Manufacture of

Different Medicinal Products in

Shared Facilities (EMA, 20 Nov.

2014), www.ema.europa.eu/docs/

en_GB/document_library/Scientific_

guideline/2014/11/WC500177735.

pdf.

2. EU, EudraLex—Volume 4 Good

Manufacturing Practice (GMP)

Guidelines, Chapters 3 (Premises and

Equipment) and 5 (Production),

Revisions (January 2015), http://ec.

europa.eu/health/files/eudralex/

vol-4/chapter_5.pdf and http://ec.

europa.eu/health/files/eudralex/

vol-4/chapter_3.pdf.

3. ISPE, Volume 7: ISPE Baseline Guide:

Risk-Based Manufacture of

Pharmaceutical Products (Risk-MaPP)

(ISPE, September 2010).

4. D.G. Dolan et al. Regul. Toxicol.

Pharmacol. 43, 1–9 (2005).

5. G.L. Fourman and M.V. Mullen,

Pharm. Technol., April, 54–59 (1993).

6. ICH, M7 Assessment and Control of

DNA Reactive (Mutagenic) Impurities

in Pharmaceutical to Limit Potential

Carcinogenic Risk (ICH M7), Step 4

version (ICH, 23 June 2014, Adopted

by EMA September 2014), www.ich.

org/fileadmin/Public_Web_Site/

ICH_Products/Guidelines/

Multidisciplinary/M7/M7_Step_4.

pdf.

7. A. Mott et al., Journal of Validation

Technology 19 (4) (2013).

8. R. Sharnez et al., Journal of Validation

Technology 17 (4) (2012). ◆

Serialization—Contin. from page 40

share information among Roche,

its distributors, and clinics. The

work was accomplished within

three months. Integrating supply-

chain links was the biggest chal-

lenge, says Rafael Schirmer, SPI’s

director, who managed systems

integration for the project.

The pi lot used TraceLink’s

cloud-based system, so no instal-

lation or local software config-

uration was required, he says.

Connect ing with pharmacies

and distributors proved difficult

initially, Schirmer says, because

many of them did not want to

invest in the systems required.

Roche set up portals for them

to me e t r ep or t i ng r e qu i r e -

ments, and SPI configured web

boxes to enable them to access

TraceLink’s tool.

The project will soon move into

its next phase, but Brazil’s man-

date is clear, Schirmer says: All sup-

ply-chain members will be jointly

responsible for the traceability of

medicines.

Creating a supply chain that is

traceable, end to end, may seem

like a holy grail, but Open-SCS’

Gifford believes that standards will

make it easier. As he notes, they’ve

worked in other industries such as

semiconductors, where Sematech,

a consortium of the top 10 manu-

facturers, set a foundation in the

1990s. “Today, at AMD, Intel or

Samsung, at any plant anywhere

in the world, they’re all using the

same interfaces,” he says.

At this point, pharma is making

the connections required for trans-

parency and traceability. However,

as experts caution, only a strategic,

long-term approach will yield the

best results.

REfEREnCES 1. UPS, Eighth UPS Pain in the Chain

Survey Snapshot, www.ups.com/

media/en/UPS-PITC-Executive-

Summary-North-America.pdf

2. V. McCain, “Establishing CMO

Serialization Support Strategy,”

presentation at the First Serialization

Roundtable for CMOs (Philadelphia,

PA, Oct. 8, 2015).

3. J-P. Allard, “Serialization Solutions

Through Innovations,” presentation at

the First Serialization Roundtable for

CMOs (Philadelphia, PA, Oct. 8, 2015).

4. B. Tiftikci, “Serialization: Internal and

External IT Connectivity,” presentation

at the First Serialization Roundtable for

CMOs (Philadelphia, PA, Oct. 8, 2015).

5. P. Taylor, “Brazil’s Drug Traceability Pilot

Phase ‘Suspended,’” Oct. 7, 2015, www.

securingindustry.com/pharmaceuticals/

brazil-s-drug-traceability-pilot-phase-

suspended-/s40/a2548/#.Vo_

MCZMrI_U, accessed Jan. 8, 2015. ◆



Sve

ta D

em

ido

ff/G

ett

y Im

ag

es

Troubleshooting

There is a growing need to accelerate bio-

process development for mammalian cell

culture. Major pharmaceutical and bio-

tech firms are facing challenges to reduce pro-

cess development costs and cultivation times

(1). The conventional method for mammalian

cell-line development usually involves a series

of shake flasks for screening the cell-line prior

to large-scale cultivation. The shortcomings of

this method include long development times,

laborious operation, and limited experimental

throughput, which result in slow bioprocess

development of mammalian cell cultures.

Various scale-down miniature bioreactors

have been designed to speed up the biopro-

cess development of mammalian cell cultures.

Generally, it is accepted practice to perform the

small-scale experiment in a high throughput

and highly parallel manner. Current technol-

ogy endeavours to enable high-throughput pro-

cess development include the use of microtiter

plates, miniature stirred-tank bioreactors, and

microbioreactors (2, 3, 4).

Miniature stirred-tank bioreactors (MBRs)—

based on the conventional stirred-tank reactor

(STR)—enable a rapid and scalable experimental

process development. Experiments are usually

carried out in 4–16 parallel reactors running

simultaneously at scales of 10 mL to 500 mL. The

main advantages of these reactors are reduced

cultivation times and costs, and the ability to

conduct continuous monitoring and real-time

visualization of key process parameters in each

single bioreactor (3, 4). Moreover,

the capacity of miniature stirred

bioreactors for inline monitoring

and control of pH, dissolved oxygen

(DO), and temperature could make

these reactors an excellent alter-

native to shake-flask systems for

early-stage mammalian cell-culture

bioprocess development.

Scale translation of miniature bioreactors

to benchtop reactors remains a crucial issue

for mammalian cell-culture processes. Mixing

directly affects the heat transfer, gas dispersion,

and blending of different media components

in the reactor. Furthermore, poor mixing in

bioreactors can result in pH, nutrient, and tem-

perature gradients, as well as poor control of

operating parameters (5).

Mixing in mammalian cell-culture reactors is

achieved using either marine or pitched-blade

impellers to minimize shear damage. These

impeller designs will create low shear stress and

gently mix the culture (6). Detailed engineer-

ing characterization, such as mixing time, is

imperative to understand the performance of a

bioreactor. By characterizing the system during

the typical operating range, suitable parameters

for scale translation can be identified. In this

study, mixing time has been determined in a

prototype version of a commercial MBR system

and used as a criterion for translation to bench-

scale stirred bioreactors.

Materials and Methods Cell lines and media

All experiments in this work were carried out

using a Chinese hamster ovary (CHO) cell line

expressing an IgG antibody. The medium used

was of an animal-free origin and was chemi-

cally defined.

Miniature bioreactors

All MBR work was carried out with a 0.5-L min-

iature bioreactor system (HEL Ltd, UK) run in

parallel with a working volume of 0.35 L. The

operating parameters of pH, DO, and temper-

ature were controlled at 7.1, 30%, and 37°C,

respectively. Agitation was provided either by a

single three-blade marine impeller (direct driven)

or a four-bladed marine impeller (magnetic-bot-

tom-driven) with an agitation rate set to match

Mixing Time as a Criterion for Scale Translation of Cell-Culture Processes The authors conclude that miniature bioreactors can adequately predict the cell culture kinetics in scaled-up reactors using equal mixing times.

Mohd Helmi Sani is research

engineer in the department

of Biotechnology and Medical

engineering at the Universiti

teknologi Malaysia; and

Frank Baganz is senior lecturer

in the department of Biochemical

engineering at the University

College london.



the mixing time in the 5L-STR.

Aeration in the bioreactor vessel

was achieved either by a horseshoe

sparger or singular hole sparger.

Bench-scale bioreactors

The experiments were carried out in

5-L stirred-tank bioreactors (Biostat

B-DCU, Sartorius) run in paral-

lel with a working volume of 3.5 L

using the same operating parame-

ters as given for the MBRs. Agitation

was provided by a single three-blade

segment marine impeller. Aeration

in the bioreactor vessel was achieved

via a horseshoe-type sparger.

In all experiments at both scales,

the mode of operation was batch

phase from day zero until day six,

followed by fed batch from day

seven onwards. The cultures were

bolus-fed once a day to maintain

the glucose concentration at 2 gL-1.

Mixing time

Mixing time was measured in

the HEL-BioXplore miniature

bioreactor using the pH tracer

method (6). The experiment was

conducted using two types of

impellers; direct driven and mag-

netic driven with the horseshoe

and singular hole sparger. The

mixing time in the 5L-STR as a

function of agitation rate was

previously determined by inves-

tigator Silk (6).

Cell number and viability

The cell cultures were sampled

daily and cell concentration and

viability were analyzed using an

automated cell-counting device,

VI-Cell XR (Beckman Coulter),

which automates the trypan blue

dye exclusion method.

IgG HPLC assay

The quantification of IgG pro-

duced was determined through

the use of a high-performance liq-

uid chromatography (HPLC) sys-

tem (Agilent Technologies) with a

Protein-G affinity column.

resUlts and disCUssionMixing time is a useful parameter

to measure the mixing efficiency

in a reactor and homogeneity of a

fluid when agitated by an impel-

ler. These mixing time values vary

depending on the impeller and/or

sparger designs and geometry of

the reactor. Because the MBR exhib-

its a similar geometry as a conven-

tional STR, an empirical correlation

proposed by Nienow (7) was applied

to compare experimental and pre-

dicted values for the different MBR

configurations (Equation 1).

Tm= 5.9 (ε

Tg)−0.33 D

T0.67

Di

DT

−0.33

Tm = mixing time (s)

εTg = Total energy dissipation rate

in gassed bioreactor (Wm−3)

Di = impeller diameter (m)

DT = tank diameter (m)

[Eq. 1]

In agreement with other stud-

ies (Xing et al., [6]), Figure 1 shows

that mixing time is inversely pro-

portional to agitation rate. The

mixing time achieved ranges from

5–14 seconds and 4–11 seconds for

experimental and predicted values,

respectively. Although both experi-

mental data sets show the same

trend as the calculated values, the

measured values are either slightly

higher (magnetic driven) or lower

(direct driven) compared with the-

oretical predictions. These devia-

tions may be due to differences in

average energy dissipation rate that

were not measured. Nevertheless, the

results suggest that the Nienow (7)

correlation can be used to predict the

mixing times in the MBR with good

accuracy considering the experimen-

tal variations.

Matched mixing time between

miniature bioreactors and bench-

stirred tank reactors

In this case study, a typical fed-

batch CHO cell culture process was

chosen and mixing time was used

as a scale-translation criterion. The

troubleshooting

Figure 1: Comparison of experimental mixing times with Nienow correlation. A

dashed line represents the direct-driven impeller, the dotted line represents the

magnetic-driven impeller, and the solid line represents the Nienow correlation.

Mix

ing

tim

e (

s)

Agitation (rpm)



troubleshooting

operating conditions for each reac-

tor system with regard to agitation

speed, aeration rate, and feed addi-

tion were adjusted accordingly.

The CHO cell-growth profile and

percentage viability for the two

types of reactors, miniature biore-

actors, and bench 5-L stirred-tank

bioreactors are depicted in Figure 2.

Both cultures had a prolonged expo-

nential phase but reached the peak

viable cell concentration (VCC)

at different days (Figure 2A). The

parallel MBR cultures exhibited a

slower exponential growth between

70–168 hours of cultivation com-

pared with the benchtop cultures.

The peak viable cell concentrations

for the two reactors, however, are

almost identical (Table I).

The benchtop culture showed

a considerably longer stationary

phase compared with the MBR and

achieved higher cell viability on day

14. Final percentage viability of the

MBR was at 60%, while the viability

in the 5-L stirred-tank reactor was

at 80% after >300 hours (Figure 2B).

The higher viability observed in

the stirred-tank reactor on day 14

might be due to the better monitor-

ing and control of gas sparging in

the larger reactor. Besides that, the

feed addition, which was performed

daily from day seven, enabled pro-

longed cell viability and antibody

production until the harvest day.

Figure 3 shows the final anti-

body production for both bioreac-

tor cultures. Based on the HPLC

analyses, the MBR culture reached

a final IgG titer of 0.69 gL-1, which

is 17% lower than that in the 5 L

STR with 0.83 gL-1. The derived

growth parameters for both sys-

tems, however, show excellent

agreement in terms of specific

production rate (qP) and generally

good comparability with regard

to the cumulative integral viable

cell concentration (CiVC) (Table I).

Figure 2: Chinese hamster ovary (CHO) growth kinetics in fed-batch cultures for

two reactors: a 5-L benchtop (solid line) and a miniature bioreactor (MBR) (dashed

line). Graph (A) represents viable cell concentration; graph (B) shows cell viability

for each type of bioreactor. The arrows (↓) indicate the points in the process in

which feed is added.

0

5

10

0 200 400

VC

C

(x1

06 m

L-1)

Time (hours)

50.0

100.0

0 200 400

Cell v

iab

ilit

y (%

)

Time (hours)

(B)

(A)

Contin. on page 55



Analytical Best Practices

Imag

e: P

AS

IEK

A/S

cie

nce P

hoto

Lib

rary

/Gett

y Im

ag

es

Out-of-Trend Identification and Removal in Stability Modelling and Regression AnalysisThis article defines the concept, justification, and method of removal of out-of-trend points in stability modelling and shelf-life prediction.

Outliers in regression modelling may

cause incorrect and invalid results

to occur when predicting stability. A

clearly defined out-of-trend (OOT) protocol is

needed to correctly and consistently identify

and remove outliers from expiry and stability

modelling and prediction where technically

warranted. OOT is a point (measurement) in a

regression analysis that has statistically greater

error at a defined risk factor from a regression

line or multiple-factor regression model than

other determinations; it is a time-dependent

result that falls outside a predicted statistical

interval (see Figure 1). Simply put, an OOT event

is an outlier in a regression analysis. This article

provides an overview of OOT from literature,

guidance, and best analytical practice and the

procedure to be used during stability modelling

and analysis.

OOT points are considered to be non-repre-

sentative of the test sample and are due to ana-

lytical, transcription, or other sources of error.

Failure to remove OOT point(s) if they exist will

produce calculated rates of change that will not

be representative of the drug product nor drug

substance. The following are indications that

OOTs are present in the stability analysis:

• Pointsdonotlineupontheregressionline

• Confidenceintervalsofthefitareexcessively

wide

• Root-mean-squarederror(RMSE)oftheresid-

uals has excessively expanded well beyond

the characterized analytical error

• Expiryfromonetimepointtoanotherhasa

large amount of difference

• R2has a large amount of changewith and

without the OOT time point.

“OOT stabi l ity data can be

describedasaresultorsequenceof

results that are within specification

limits but are unexpected, given

the typical analytical and sampling variation

and a measured characteristic’s normal change

over time (e.g., an increase in degradation prod-

uct on stability)” (1).

Regression analysis is normally used

to determine change over time and associ-

ated 95% confidence limits relative to rates of

change and expiry. InternationalCouncil on

Harmonization (ICH)Q1A(R2) Stability Testing

of New Drug Substances and Product (2) states,

“the nature of any degradation relationship will

determine whether the data should be trans-

formed for linear regression analysis. Usually

the relationship can be represented by a linear,

quadratic, or cubic function on an arithmetic

or logarithmic scale. Statistical methods should

be employed to test the goodness of fit of the

data on all batches and combined batches

(where appropriate) to the assumed degradation

line or curve.”

OOT evaluation and elimination should be

used for the following applications and predic-

tion:

• Shelf-lifeestimation

• Storageevaluation

• Impurityformationandtrending

Failure to remove OOT

point(s) if they exist will

produce calculated rates

of change that will not be

representative of the drug

product nor drug substance.

Thomas A. Little, PhD, is president

of Thomas A. Little Consulting, 12401

North Wildflower Lane, UT 84003

USA, [email protected].



Analytical Best PracticesA

ll F

igure

s a

re c

ourt

esy o

f auth

ors

.

• In-processmonitoring and pre-

diction

• Trackingandtrendingoflotper-

formance.

LIKELY ROOT CAUSES FOR OOTThe following are typical possible

sources and mechanisms for OOT

events that may occur during sta-

bility evaluation:

• Sample selection and sample

handling errors

• Dilutionandsample-preparation

errors

• Samplematerialsandplateerrors

• Temperature, reaction time, and

pH effects errors

• Vendorandlotvariationoncrit-

ical reagents

• Flow rates and process time

errors

• Analysterrors

• Instrument variation and cali-

bration error

• Nonstandard test procedures

and not following the method

standard operating procedure

(SOP)

• Drift in standards or reference

materials

• Stability of test samples or criti-

cal reagents

• Calibration or compensation

errors

• Interactionandcompositeerrors

• Expiryofbulkmaterials.

HISTORICAL APPROACHES TO OOTThe following are typical histori-

cal approaches to OOT identifi-

cation and removal, though they

are not recommended approaches.

They are not considered to be sta-

tistically sound procedures for

OOT identification and removal.

Using procedures that are not sta-

tistically sound may remove time

points from a stability analysis

that cannot be defended nor jus-

tified upon review. There is no

statistical basis for the following

definitions of OOT and these do

not take into account process nor

method variation:

Figure 1: Out of trend (OOT) illustration and infuence of OOT.

Figure 2: Closed loop out of trend (OOT) identifcation and resolution.

No OOT

0 5 10 15 20 25Time (Months)

Linear Fit

Linear Fit

Summary of Fit

RSquare

RSquare Adj

Root Mean Square Error

Mean of Response

Observations (or Sum Wgts)

RSquare

RSquare Adj


Mean of Response


0.974613

0.970987

0.245665

19.05007

9

0.043636

-0.09299

3.63802

20.35897

9

Summary of Fit

Linear Fit

Linear Fit

0 5 10 15 20 25Time (Months)

Pro

tein

Co

nce

ntr

ati

on

ug

/mL

Protein Concentration ug/mL =

21.129718 - 0.1733043*Time (Months)

Protein Concentration ug/mL =

19.297252 + 0.0884766*Time

(Months)

Pro

tein

Co

nce

ntr

ati

on

ug

/mL23

22

21

20

19

18

17

16

30

25

20

15

10

OOT

OOTDetermination

OOTIdentifcation

OOTVerifcation

New TimePoint Data

StabilityPrediction




• The difference between consec-

utive results is outside of half

the difference between the prior

result and the specification

• The result isoutside±5%of ini-

tial result

• Theresultisoutside±3%ofpre-

vious result

• The result is outside±5%of the

mean of all previous results.

CLOSED-LOOP APPROACH TO OOT IDENTIFICATION AND REMOVAL

A best-practice approach to OOT

determination and removal is to

see it as a part of a closed-loop con-

trol system during stability moni-

toring and expiry prediction (see

Figure 2). The five steps to a closed

loop system for OOT are:

• Additionofnewtimepointsand

data

• OOTidentification

• OOT determination and point

removal where warranted

• OOTverificationandevaluation

of OOT influence

• Stability and performance pre-

diction.

Addition of New Time Points and

Data, Closed Loop Step 1

As each new time point is added

to the stability analysis, the time

point should be checked for OOT

potential. If they are within the

criteria for OOT identification,

then rates of change, expiry, etc.

are determined. OOT identifica-

tion, determination, and verifica-

tion are used if new time points

appear to be suspect.

OOT Identification,

Closed Loop Step 2

Some CMC teams recommend

the percent change from point-

to-point as well as from the ini-

tial time point to indicate an OOT

(3). For example,more than a 5%

change from baseline may be con-

sidered a possible OOT event (4).

Analytically, there are four

methods to identify a point as

OOT: visually, outlier boxplot

of the residuals, multivariate

Jackknife distances, and control

chart of the residuals (see Figures

3–6). Jackknife distances are the

most sensitive in identification of

OOT points in a regression anal-

ysis as they include and remove

each time point in the analysis

to evaluate their influence in the

model. Once an OOT has been

identified, the next step is to test

it to determine if the OOT will be

removed. Root cause as to why

the point is OOT is a secondary

investigation once the point has

been determined to be OOT. Once

the point is determined to be a

possible OOT, the point is tested

Figure 3: Visual analysis.

Figure 4: Outlier box plot of residuals.

Protein Concentration ug/mL By Time (Months)

Time (Months)

Pro

tein

Co

nce

ntr

ati

on

ug

/mL

25

24

23

22

21

20

19

18

17

160 5 10 15 20 25

2

1

0

Time (Months)

Co

un

t

Residuals Protein Concentration ug/mL

-3 -2 -1 0 1 2 3 4 5




statistically to determine if it is or

is not OOT.

OOT Determination,

Closed Loop Step 3

The following is the recommended

procedure for OOT outlier determi-

nation (see Figures 7 and 8):

• Exclude andhide the suspected

OOT point in the data analysis.

• Fit a linear regression linewith

the potential OOT time point

excluded.

• Savethepredictedresponse(con-

centration) from the linear fit.

• Calculate the difference (Delta)

at each time point.

• Calculateazscoreforeachtime

point (see Equation 1).

z score= (Measurement-predicted)/

s tde v (r e s idu a l s w i t h p o i nt

removed)

[Eq.1]

Once the z score for the OOT

has been calculated, it can be com-

pared with a risk threshold. A

k-sigma of 2.576 (or 99% risk) is

used to set the limit for OOT detec-

tion. Abs(z)> 2.576 (99%) isOOT,

so in this example, z is -27.238;

therefore, it is OOT. A z score with

a limit is the best method of OOT

detection. The key difference with

this procedure and other z score

procedures written in literature is

the z score is evaluated with the

point removed. This correctly

scales the residual error so that the

influence of the OOT point is not

included in the residual error and

the OOT time point can be cor-

rectly evaluated based on the other

measurements error.

OOT Verification

To verify the influence of the OOT,

the following measures are recom-

mended:

• ChangeinR2

• ChangeinRMSE

• Changeinexpirycalculation.

Comparingwithorwithout the

OOT time point will verify the

influence of the time point and

confirm the need for removal. If

the change in the three identi-

fied measures is trivial, then the

OOT has not been verified and

its removal is not warranted.

Differences inR2,RMSE,or expiry

of 3% or less are generally not

practically important to drug-sub-

stance or drug-product expiry or

stability evaluation. Verification

is performed by including, then

removing, the OOT point in the

stability evaluation and then mea-

suring the change in the key per-

formance metrics of the fit and the

prediction.Also,RMSEerrorcanbe

compared to the repeatability of the

analytical method to determine if

the residual error is primarily due

to analytical measurement error.

A control chart of the residuals

with the OOT time point excluded

will be a secondary confirmation

of OOT identification and removal

as an outlier (see Figure 9).Residual

Figure 7: Out of trend (OOT) determination.

1

Time(Months)

PredictedConcentration

Concentration z Score OOTDelta

2

3

4

5

6

0

3

6

9

12

18

0.02

0.0197

0.0195

0.0193

0.0177

0.0188

0.0199264151

0.0197320755

0.0195377358

0.0193433962

0.0191490566

0.0187603774

0.000074

-0.000032

-0.000038

-0.000043

0.000040

1.3832 OK

OK

OK

OK

OOT

OK

-0.6029

-0.7093

-0.8157

-27.2382

0.7448

Figure 5: Multivariate Jackknife distances.

Figure 6: Control chart of residuals.

Row Number

Jackknife Distances

Dis

tan

ce

50

0 1 2 3 4 5 6 7 8 9

30

10

-10

UCL = 3.67

6

A

A

B

B

C

C

1

UCL = 3.51

Avg = 0.00

LCL = -3.51

Sample

Resi

du

als

Pro

tein

Co

nce

ntr

ati

on

ug

/mL 4

2

1 2 3 4 5 6 7 8 9 10 11 12

0

-2

-4




points from the regression fit are

plotted onto a control chart, points

that are within the control limit

may be due to random or analyti-

cal method variation, points out-

side of the limits confirm points

are likely to beOOT. Remember

to exclude the OOT point when

building the chart so it does not

influence the limits.

Stability Prediction

Once the OOT point has been

removed and verified, stability

prediction can then be performed

per ICH Q1E (5). OOT points

should always be included in the

plot to indicate the measurement

but tagged as OOT to indicate

the point was included in graph

but not included in the analysis.

Including it in the plots will pro-

vide full disclosure as to all obser-

vations at all time points. Once a

point has been determined to be

OOT, it does not render the analy-

sis as additional data are added to

the stability prediction.

Single Factor, Single Batch,

and Multiple-Factor/Multiple-

Batch OOT

Thetechniqueforidentifyingand

removing an OOT point described

in this paper works equallywell

for single -batch/single -fac tor

versus multiple-batch/multiple-

factor stability modelling and

expiry prediction. The example

provided was for a single-batch,

single-factor analysis. For the mul-

tiple-factor and/ormultiple-batch

condition, the same approach

would be used. The model would

be fit with the OOT removed and

then the model would be saved, z

scores would be calculated for all

time points, and then OOT would

be determined based on a 2.576

(99%) threshold.

CONCLUSIONHaving a well-defined and sta-

tistically valid OOT protocol is a

powerful addition to any stabil-

ity program and needs a clearly

definedSOPtoconsistentlyapply

the logic to day-to-day stability

evaluation. Including an OOT

protocol to stability testing and

data analysis will produce more

stat ist ica l ly rel iable stabi l ity

determination and expiry predic-

tion. OOT determination based

on the protocol described in this

paper opens the door for the

automation of OOT determina-

tion and removal from any stabil-

Figure 8: Stability analysis with out of trend (OOT) removed.

RSquare

RSquare Adj


Mean of Response


0.986058

0.981411

6.143e–5

0.01946

5

Summary of Fit

Linear Fit

Concentration = 0.0199264 - 6.478e–5*Time

(Months)

Linear Fit

Time(Months)

Bivariate Fit of Concentration By Time (Months)

Co

nce

ntr

ati

on

OOT

0

0.021

0.02

0.019

0.018

0.017

5 10 15 20

Figure 9: Control chart of residuals.

Individual Measurement of Delta

8

Sample

UCL = 0.67

Avg = -0.00

LCL = -0.67

Delt

a

6

4

2

0

1 2 3 4 5 6 7 8 9 10 1211

1

-2

A

ABBC




ity analysis and may be the best

approach to systematically eval-

uate all time points in stability

analysis.

REFERENCES 1. PhRMA CMC Statistics and Stability

Expert Teams, “A Review of the

Potential Regulatory Issue and

Various Approaches,”

Pharmaceutical Technology 27

(2003).

2. ICH, Q1A(R2) Stability Testing Of

New Drug Substances (ICH,

February 2003).

3. T-J, Torbovska, “Method for

Identification of Out-of-Trend

Stability Results,” Pharmaceutical

Technology 37 (2003).

4. PhRMA CMC Statistics and Stability

Expert Teams, “Identification of

Out-of-Trend Stability Results, Part

II,” Pharmaceutical Technology (Oct

2, 2005).

5. ICH, Q1E Evaluation for Stability

Data (ICH, February 2003). ◆

CONCLUSIONThis study has demonstrated the

potential of miniature bioreac-

tors for scale translation using

equal mixing t ime as a cr ite -

rion. The mixing times obtained

for experimental and predicted

values in the miniature biore-

actors were comparable, which

suggested that the correlation

can be applied to predict mix-

ing t imes at this scale. Scale

comparison cultivations for fed-

batch CHO cell cultures were

carried out using miniature bio-

reactors and compared with a

standard 5-L stirred-tank reac-

tor at matched mixing time. The

results show that theMBR gave

comparable CHO cel l growth

and product kinetics compared

to that at the 5-L scale. The

results of this investigation sug-

gest that MBRs can be used as

sca le -down models of bench-

scale bioreactors.

ACKNOWLEDGEMENTSMohd Helmi Sani thankful ly

acknowledges the funding by the

Ministry of Higher Education,

M a l a y s i a a n d Un i v e r s i t i

TeknologiMalaysia.

REFERENCES 1. H. Zhang et al., Cur. Pharm. Biotech.

11 (1), pp. 103–112 (2010).

2. M.A. Hanson and G.Rao,

“Miniaturization of bioreactors,” in

Encyclopedia of Industrial

Biotechnology: Bioprocess,

Bioseparation and Cell Technology,

M.C. Flickinger, Ed. (John Wiley and

Sons, 2010).

3. J. Betts and F. Baganz, Microbial

Cell Factories, 5 (1), pp. 21–35

(2006).

4. R. Bareither and D. Pollard, Biotech.

Prog., 27 (1), pp. 2–14 (2011).

5. Z. Xing et al., Biotech. Bioeng. 103

(4), pp. 733–746 (2009).

6. N.J. Silk, “High Throughput

Approaches to Mammalian Cell

Culture Process Development,”

(EngD thesis), University College

London (2014).

7. A. Nienow, App. Mech. Rev. 51 (1), pp.

3–32 (1998). ◆

Figure 3: Antibody production in Chinese hamster ovaries (CHO) growth kinetics

in fed-batch culture for two reactors: a 5-L stirred-tank reactor (solid line) and a

miniature bioreactor (dashed line).

0.00

0.50

1.00

0 200 400

An

tib

od

y ti

ter

(gL

-1)

Time (hours)

Type of reactor MBR STR

Peak viable cell concentration

(x 106 cell mL-1)

9.56 10.04

Cumulative integrated viable cell

concentration (x 108 cell d-1 mL-1)

4.05 4.83

Max. IgG antibody titer (gL-1) 0.69 0.83

Specifc IgG production rate

(pg cell-1 d-1)

10.2 9.7

Table I: Derived growth and product parameters of fed-batch Chinese hamster

ovary (CHO) cell culture in a 0.5-L miniature bioreactor (MBR) and a 5-L stirred-

tank bioreactor (STR) with equivalent mixing time.

Troubleshooting—Contin. from page 49



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BIOLOGICS NEWS PIPELINE

IN THE PIPELINE

UK Pharmaceutical Companies Collaborate

on CAR-T Cell Immuno-oncology Therapies

Cell Therapy Catapult, University of Birmingham, and

Cancer Research Technology announced the launch

of a collaboration to develop a new immuno-oncology

cellular therapy created modifying the genes of T-cells

to target solid tumors.

The project is aimed at translating an academic

discovery program into a commercially viable cell

therapy. It is funded by Cancer Research United

Kingdom (UK) and was developed by Steven Lee,

PhD, and Professor Roy Bicknell, both of whom are

from the University of Birmingham. The collaborating

partners have launched a new company—Chimeric

Therapeutics Limited—to manage all future intellec-

tual property rights of any of the resulting discoveries.

The project is based on a new generation chimeric

antigen receptor T-cell (CAR-T) immuno-oncology ther-

apy for solid tumors. This involves directing the CAR-T

cell toward a new, highly specific marker of tumor angio-

genesis, CLEC14a. This therapy will act as a vasculature

disruptive agent, compromising oxygen supply to the

tumors and inhibiting growth. The technology is cur-

rently undergoing the final stages of preclinical develop-

ment will enter into clinical trials in the near future.

According to an announcement from the Cell Therapy

Catapult, the company will assist in accelerating the

translation of the academic discoveries around CAR-T

immunotherapies for solid tumors and the CLEC14a tar-

get toward a commercially available cell therapy.

Ionis Pharmaceuticals Receives

Orphan Drug Designation for HTTRxFDA has granted Ionis Pharmaceutical’s orphan drug

designation for IONIS-HTTRx, for the treatment of

patients with Huntington’s disease (HD). IONIS-

HTTRx is the first therapy to enter clinical develop-

ment that is designed to directly target the cause of

the disease by reducing the production of the protein

responsible for HD.

IONIS-HTTRx is a Gen. 2.0+ antisense drug in devel-

opment for the treatment of Huntington’s disease. The

drug is designed to reduce the production of all forms

of the HTT protein, which is the protein responsible

for HD. HD is referred to as a triplet repeat disorder

and is one of a large family of genetic diseases in which

certain gene sequences are mistakenly repeated. In HD,

the gene that encodes for the HTT protein contains

a trinucleotide sequence that is repeated in the gene

more than 36 times. The resulting HTT protein is toxic

and gradually damages neurons in the brain.

The drug has also been granted orphan drug des-

ignation by the European Medicines Agency for the

treatment of patients with HD.

Merck KGaA, Pfizer, and Syndax

Collaborate on Ovarian Cancer Treatment

Merck KGaA Darmstadt, Germany; Pfizer; and Syndax

announced a collaboration to evaluate the combina-

tion of avelumab and entinostat for heavily pretreated,

recurring ovarian cancer patients.

Avelumab is an investigational fully human anti-

PD-L1 IgG1 monoclonal antibody developed by Merck

KGaA and Pfizer. In November 2014, the companies

announced a collaboration to develop the antibody,

which is thought to potentially enable the activation

of T-cells and the adaptive immune system. Avelumab

is currently under clinical investigation across a broad

range of tumor types.

Syndax’s entinostat is an investigational oral

small molecule that targets immune regulatory cells

(myeloid-derived suppressor cells and regulatory

T-cells). According to Syndax, the delivery of entino-

stat in combination with hormone therapy can result

in improvements in overall survival in advanced HR+

breast cancer patients.

Merck KGaA, Pfizer, and Syndax have entered into

an exclusive agreement to study the combination of

these two investigational agents in ovarian cancer.

Syndax will be responsible for conducting Phase Ib/II

clinical trials in ovarian cancer.

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Ask the Expert

Q: Our company has been growing and

expanding into new markets in recent

years. Therefore, we need to revise the contents

of our quality system documentation, such as

standard operating procedures (SOPs), to reflect

the changed circumstances. However, we find it

difficult to change SOPs that require approval by

more than one department. Do you have any sug-

gestions for improving this process?

A: Market expansion provides a good oppor-

tunity to review your documentation man-

agement process, ensuring timely and efficient

revisions of documents, such as SOPs. The best

approach for changing SOPs that require approval

by more than one department is to restrict the

contents of SOPs. Process documentation should

be limited to only those steps included within the

remit and under the control of just one group or

department.

The modern approach to a pharmaceutical qual-

ity system (PQS) recommends ‘identification of the

pharmaceutical quality system processes, as well as

their sequences, linkages, and interdependencies.

Process maps and flow charts can be useful tools

to facilitate depicting pharmaceutical quality sys-

tem processes in a visual manner’ (1). This guide-

line has been adopted by the European Union,

Japan, and the United States, as well as many other

countries. These processes—such as change man-

agement, material management, or sample man-

agement—often involve several departments or

groups in an organization (see Figure 1).

Covering steps 3 and 4 in Figure 1 in one SOP

would require the collaboration of departments

A and C, plus their respective approvals. While

this approach is compliant with the applicable

regulations, it is discouraged for various reasons,

including:

• Proposed changes to the process (e.g., timing)

can be different among departments and thus

can lead to conflicting situations.

• There is a potential risk that one department

will impose upon another how to perform a

task. This can potentially lead to a suboptimal

or defective process.

• The approval process may differ among the

involved parties, leading to frustrating delays

and/or complications in achieving timely revi-

sions of the procedures or instructions.

These concerns point to the importance of

restricting the contents of SOPs. In the example

above, one would therefore separate the cur-

rent SOP for steps three and four into separate

SOPs—and the process-flow approach needn’t or

shouldn’t stop here. Preparing the detailed flow

for the activities within the SOP before writing any

text will drive a logical and sequential descriptive

procedure. All too often, this recommendation is

not followed, and the sequence is reversed (i.e., the

process flow follows the creation of the SOP’s text).

This rarely results in a well-written and easy-to-fol-

low SOP. An additional benefit of the process flow-

based approach is that it is also aligned with other

quality system approaches, such as International

Organization for Standardization (ISO) 9001:2015

Quality Management Systems–Requirements (2).

REFERENCES 1. ICH, Q10 Pharmaceutical Quality System (ICH, April 2009).

2. ISO, 9001:2015 Quality Management Systems–

Requirements (ISO, Sept. 15, 2015), www.iso.org.◆ Fa

na

tic S

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es

Fig

ure

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f th

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uth

or

Siegfried Schmitt,principal consultant,

PAREXEL

Managing Market Expansion’s Effect on ProceduresSiegfried Schmitt discusses how to streamline thedocument management process during market expansion.

Figure 1: Quality system processes.

Sample Management Process version 12

In Process Control Samples

Step 1

Dep

art

men

t A

Dep

art

men

t B

Dep

art

men

t C

Step 4

Step 2

Step 3


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