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The Science & Business of BiopharmaceuticalsBioPharmINTERNATIONAL

September 2019

Volume 32 Number 9

PUTTING BIOPHARMA STABILITY TESTING UNDER

THE MICROSCOPE

www.biopharminternational.com

UPSTREAM PROCESSINGCELL-CULTURE ADVANCES

TEST BIOREACTOR PERFORMANCE MODELS

CELL AND GENE THERAPIESSEEKING SOLUTIONS FOR LARGE-SCALE GMP VIRAL

VECTOR MANUFACTURING

BIOSIMILARSMETHODS

ACCELERATE BIOSIMILAR ANALYSIS



THE MICROSCOPE

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The Science & Business of Biopharmaceuticals

BioPharmINTERNATIONAL

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September 2019

Volume 32 Number 9


THE MICROSCOPE


UPSTREAM PROCESSING

CELL-CULTURE ADVANCES

TEST BIOREACTOR

PERFORMANCE MODELS

CELL AND GENE THERAPIES

SEEKING SOLUTIONS FOR

LARGE-SCALE GMP VIRAL

VECTOR MANUFACTURING

BIOSIMILARS

METHODS

ACCELERATE

BIOSIMILAR ANALYSIS



THE MICROSCOPE


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INTERNATIONAL

BioPharmThe Science & Business of Biopharmaceuticals

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authors, and review manuscripts submitted for publication.

EDITORIAL

Editorial Director Rita Peters [email protected]

Senior Editor Agnes M. Shanley [email protected]

Managing Editor Susan Haigney [email protected]

European Editor Felicity Thomas [email protected]

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Table of Contents

4 BioPharm International September 2019 www.biopharminternational.com

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

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FEATURES

UPSTREAM PROCESSINGCell-Culture Advances Test Bioreactor Performance ModelsCynthia A. ChallenerThe evolution of cell-culture technology is driving the need for

improvements in modeling solutions. . .22

CELL AND GENE THERAPIESSeeking Solutions for Large-Scale GMP Viral Vector ManufacturingCynthia A. ChallenerInnovation in manufacturing technologies must occur to ensure the availability of

gene and cell therapies. . . . . . . . . . . . .26

QUALITYBest Practices for Studying Stability in BiologicsSusan HaigneyIndustry experts discuss the challenges and regulations of setting up a CGMP-

compliant stability testing program. . .30

BIOSIMILARSMethods Accelerate Biosimilar AnalysisMario DiPaola and Indu JaveriEffective application of mass-spectrometry tools can optimize biosimilar analysis, reducing

development time and cost. . . . . . . . .34

ANALYTICAL METHODSImproving Oligonucleotide AnalysisAnjali AlvingOligonucleotides, which are classified as both small molecules and biomolecules, pose unique analytical challenges. High-resolution mass spectrometry is becoming a method of choice for their

development. . . . . . . . . . . . . . . . . . . . .40

OPERATIONSContainer Closure Integrity Testing of Finished Sterile Injectable ProductDerek DuncanAs regulatory guidance has changed, so too has CCIT testing. In this article, possible CCIT strategy approaches are

outlined. . . . . . . . . . . . . . . . . . . . . . . . .44

DOWNSTREAM PROCESSINGWhat’s New in Manufacturing: Process ChromatographyLauren LavelleThe latest advances in process chromatography include pre-packed chromatography columns, process characterization kits, fast protein liquid chromatography systems,and mixed-

mode chromatography resins. . . . . . . .47

COLUMNS AND DEPARTMENTS

FROM THE EDITOR

The editors welcome

technical article contributions

from biopharma industry experts.

Rita Peters. . . . . . . . . . . . . . . . . . . . . . . . .6

REGULATORY BEAT

Industry and regulators seek global

system that reduces regional differences.

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

PERSPECTIVES ON OUTSOURCING

CDMOs are adding facilities and services

to their portfolios in anticipation of the

biologics industry’s continued growth.

Susan Haigney . . . . . . . . . . . . . . . . . . . .12

NEW TECHNOLOGY SHOWCASE . . . .49

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

ASK THE EXPERT

Providing regulators with a holistic

approach to addressing deficiencies is

the best response to an inspection.

Siegfried Schmitt . . . . . . . . . . . . . . . . . .50

COVER STORY

16 Putting Biopharma Stability Testing Under the MicroscopeStability testing for biologics is more complex than for small-molecule drugs, so companies should be aware of the potentially serious issues that can be costly and jeopardize drug development.

Cover Design by Maria ReyesImages: kkolosov/Stock.Adobe.com

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



http://www.biopharminternational.com/

The Foundations for

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6 BioPharm International www.biopharminternational.com September 2019

From the Editor

Rita Peters is the

editorial director of

BioPharm International.

The editors

welcome

technical article

contributions

from biopharma

industry experts.

Plan Now to Share Your Expertise in 2020

September—the traditional back-to-school time—has arrived, and the annual

holiday shopping season is sure to follow quickly. Personally, I don’t like rush-

ing the holidays and shop at the last minute. The publishing profession, how-

ever, forces me to plan and work well ahead of the calendar.

While we have yet to enter the last quarter of 2019, the editorial team is look-

ing ahead to 2020, planning topics, special issues, and features that BioPharm

International will cover next year. It is also prime time to remind readers of the

opportunities to share knowledge about bioprocessing by contributing a technical

article or peer-review paper to the publication.

Through peer-reviewed papers, technical articles, technology reports, regulatory

and business columns, and expert commentary, BioPharm International publishes

objective information related to process and formulation development, manufactur-

ing, analytics, drug delivery, and business development topics in print magazines,

digital publications, ebooks, and online at www.BioPharmInternational.com.

Ways to contributePeer-review papers are a vital part of BioPharm International’s coverage of scientific

and technical advances in biopharmaceutical development and manufacturing. Four

types of peer-review papers are considered: standard data-driven, novel research;

topical literature or patent review; technical case studies/technical application notes;

and science-based opinion papers. Manuscripts for peer-review papers are accepted

and reviewed on an ongoing basis; papers are published in the order in which they

are accepted by the editorial advisory board.

BioPharm International editors also welcome technical articles that are not peer-

reviewed from experts at bio/pharmaceutical companies, regulatory authorities,

industry suppliers, and consultants. The magazine’s editorial calendar lists the top-

ics scheduled for the monthly print issues, supplements, ebooks, or online at www.

BioPharmInternational.com. All submissions are reviewed and edited by the editorial

team; final publication is determined by the editors.

Ideas for contributions should be discussed with the editors four months prior to

the publication date. The editors will review an abstract (250 words) describing the

article focus and other details. If the topic is suitable, a word count—typically 1800–

2000 words—and deadline are assigned. Final articles, figures, and signed license

agreements are due approximately two months prior to publication.

The article/paper must be objective and cannot promote a company or its prod-

ucts. It must be original and submitted to BioPharm International on an exclusive

basis. And, all authors must sign a license agreement that provides BioPharm

International permission to publish the original article and its associated figures/

tables in print and online.

Share your knowledgeThe editors also interview industry experts from biopharma companies, contract ser-

vice providers, industry suppliers, regulatory authorities, and consulting groups for

technical articles on drug development and manufacturing topics. And, similar to

contributed articles, responses to questions must be objective and non-promotional.

To be considered for an interview, consult the editorial calendar for scheduled topics

and contact the editors approximately four months prior to the publication date.

Learn moreBioPharm International’s 2020 editorial calendar, which lists topics scheduled

for publication next year, plus sample articles, and an online form to submit an

article idea to the editors are available on the Submission Guidelines page on

www.BioPharmInternational.com. X


http://www.BioPharmInternational.com





November 21st 2019

The Oitavos

Lisbon/Cascais, Portugal

Eppendorf® and the Eppendorf Brand Design are registered trademarks of Eppendorf AG, Germany. All rights reserved, including graphics and images. Copyright © 2019 by Eppendorf AG.

Poster Session with Poster Prize / Abstract Submission and Registration on www.stemcellday.de

Bioprocessing Technologies in Stem Cell ResearchChaired by Paula Alves, PhD CEO of iBET (Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal)

Sessions

> Challenges in Bioprocess Engineering and

Scale-Up of Stem Cell Culture

> Process Controls and Analytics

> Successful Translation and Commercialization

Early-Morning-Workshop

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Lisbon 2019

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Regulatory Beat

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Regulatory officials in the United States,

Europe, and other regions are collabo-

rating with manufacturers to advance a

new framework for managing chemistry, manu-

facturing, and controls (CMC) changes more

efficiently across the product lifecycle. The

process is outlined in the Q12 guideline devel-

oped by an expert working group formed under

the International Council for Harmonization

(ICH) (1). ICH is expected to approve a revised

core Q12 guideline at the November 2019 ICH

meeting in Singapore, along with more detailed

annexes and a global training plan for industry

and regulators.

Support for a harmonized approach for

authorizing new manufacturing and testing

methods on approved drugs and biologics

reflects general agreement on a risk-based sys-

tem for categorizing and managing changes,

despite continuing differences among authori-

ties over specifics for reduced oversight of cer-

tain variations. In the works formally since

2014, the Q12 guideline is expected to provide

flexible oversight of post-approval changes with

reduced regulatory reporting for manufacturers

able to demonstrate enhanced knowledge about

product, manufacturing process, and

analytical procedures, as provided by

a company’s product quality system

(PQS).

A key feature of Q12 outlines how

manufacturers should define those

established conditions (ECs) that are

considered necessary to assure prod-

uct quality. Thus, a change to an EC

would require a regulatory submis-

sion, explained Chikako Torigoe, biol-

ogist in the office of the director of

FDA’s Center for Biologics Evaluation

and Research (CBER). At the same

time, those parameters that have less

risk of impacting product quality or process

consistency would not be considered ECs and

could be managed by a manufacturer’s internal

PQS and implemented without prior approval,

Torigoe noted at the July 2019 CMC Forum

sponsored by CASSS (2).

Manufacturers may reduce ECs requiring

regulatory approval through development and

submission of a post-approval change man-

agement protocol (PACMP) that describes

anticipated changes to a product or products.

The PACMP would provide the basis for agree-

ment between the applicant and regulatory

authority about information required to sup-

port certain changes. PACMPs could involve

multiple products, as with establishing a new

process to improve product sterility assurance,

upgrades to a vial wash room, or introduc-

ing a new product to a manufacturing facil-

ity. FDA is launching a pilot program to gain

experience in assessing proposed ECs, with

the aim of reviewing nine submissions of new

drugs, generics, and biotech products to see

how much time and effort is involved in identi-

fying ECs at time of approval.

An approach similar to Q12 already has been

established in Japan, pointed out Tomonori

Nakagawa, API project manager at Otsuka

More Predictable Post-Approval Change Policy on Horizon Industry and regulators seek global system that reduces regional differences.

Jill Wechsler

is BioPharm International’s

Washington editor,

[email protected].

It can take four to six years

to add a new site or comply

with a new standard for a

product that is approved

and distributed globally.




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Regulatory BeatRegulatory Beat

Pharmaceutical Co., at the CASSS

Forum. This initiative by Japan’s

Pharmaceuticals and Medical

Devices Agency (PMDA) has pro-

duced a more efficient program for

overseeing a robust change man-

agement system, he noted, based

on a demonstration of product and

process knowledge. And Health

Canada is ready to implement ICH

Q12, reported senior regulatory sci-

entist Anthony Ridgway at Health

Canada’s Biologics & Genetic

Therapies Directorate. That agency

has issued guidance on categories,

conditions, and data expected for

different reporting categories, and

policies already are aligned to pro-

vide flexibility in defining ECs

and in justifying reduced report-

ing. Health Canada encourages

adoption of risk-based categoriza-

tion of post-approval changes that

require only notification or listing

in an annual report, Ridgway said,

adding that more work is needed

to better define and align with

ICH recommendations for PACMPs.

Kavita Vyas, policy lead in the

Office of Policy for Pharmaceutical

Quality in the Center for Drug

Evaluation and Research (CDER),

described ongoing FDA efforts to

prepare to meet Q12 challenges.

An agency group is examining

case studies related to established

conditions and anticipates gaining

useful learnings from the upcom-

ing pilot project. Alexey Khrenov,

senior staff fellow at CBER’s Office

of Tissues and Advanced Therapies

(OTAT), noted that training is

being developed to familiarize

reviewers with Q12 approaches.

Greater regulatory f lexibi l ity

should benefit manufacturers, he

commented, although the specif-

ics for defining ECs and for assess-

ing PACMP and product lifecycle

management (PLCM) documents

remains uncertain.

ENCOURAGING IMPROVEMENTDespite the challenges, manufac-

turers hope to move forward with

Q12 as more products are tested,

produced, and dist r ibuted in

multiple regions, creating com-

plex supply chains that trigger

diverse testing requirements and

inspection processes. It can take

four to six years to add a new site

or comply with a new standard

for a product that is approved

and distributed globally, noted

Nakagawa. Similarly, consultant

Moheb Nasr, formerly with FDA

and Amgen, described how the

current system discourages firms

from adopting more efficient lab-

oratory test methods and modern

production systems.

Many manufacturers look to

advance strategies for managing

CMC changes under the firm’s

PQS, as supported by a series of

earlier ICH quality guidelines (Q8–

11). Leslie Bloom, executive direc-

tor of regulatory CMC at Pfizer,

described efforts to develop PLCM

plans to manage ECs over the

product lifecycle even before Q12

is finalized. And ongoing efforts

aim to provide more justification

for a range of risks related to ECs

and non-ECs, especially in process

and analytical methods.

K imberly Wolfram, director

of global regulatory affairs CMC

at Biogen, described herself as a

“realist” in anticipating the bene-

fits from Q12 after years of effort.

While she hopes for less confu-

sion in regional adoption of ECs

and guidelines, she and others

acknowledge challenges in gain-

ing global alignment on change

management protocols based on

risk assessment to enhance access

to therapies.

REFERENCES 1. ICH, Q12, Technical and Regulatory

Considerations for Pharmaceutical

Product Lifecycle Management, Step

2 (ICH, Nov. 16, 2017), www.ich.org/

fileadmin/Public_Web_Site/ICH_

Products/Guidelines/Quality/Q12/

Q12_Draft_Guideline_

Step2_2017_1116.pdf.

2. CASSS, “The Future of Post-approval

Changes is Coming–Are You Ready

for ICH Q12?” Conference

(Gaithersburg, MD, July 2019). X

FDA Publishes Guidance on Rare Pediatric Disease Priority Review

On July 30, 2019, FDA issued draft guidance that answers

questions regarding priority review vouchers for certain

rare pediatric disease treatments that meet criteria of

the Food, Drug, & Cosmetic Act (FD&C Act). As part

of the FD&C Act, FDA may give special incentives to

companies for the development of treatments for rare

pediatric diseases. This draft guidance revises a previous

draft guidance and clarifies the qualifications and process

for requesting priority review vouchers.

Specifically, the guidance gives detailed answers to

questions regarding the definition of a rare pediatric disease,

eligibility requirements, a sponsor’s responsibilities after

approval of an application, designation information requests,

the submission process, marketing applications, and use and

transfer of a rare pediatric disease priority review voucher. The

guidance also discusses drug-drug combinations, previously

approved drugs, and orphan drug designation questions.

—The Editors of BioPharm International



http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q12/Q12_Draft_Guideline_Step2_2017_1116.pdf

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Perspectives on Outsourcing

Do

n F

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The growth of the biologics indus-

try has created expansion in the con-

tract development and manufacturing

(CDMO) space. Liza M. Rivera, senior direc-

tor, global marketing, at Fujifilm Diosynth

Biotechnologies (FDB), says biologics have seen

success especially in the treatment of cancer

and autoimmune diseases. The following high-

lights some of the latest news from CDMOs in

the biomanufacturing space.

NEW FACILITIES AND SERVICESCDMOs are adding facilities and services to

their portfolios in anticipation of the industry’s

continued growth. Fujifilm is one of the com-

panies making significant investments in their

biomanufacturing offerings. “In recent years,

we have prioritized the expansion of our pro-

duction facilities, principally 2000-L medium-

sized tanks, in response to the rapid increase in

production demand for biologics,” Rivera says.

“As outlined in our medium-term manage-

ment plan, VISION2019, with fiscal year ending

in March 2020, we set our sights on enhancing

the growth of our healthcare business. To meet

this goal, we have actively made capital invest-

ments in our bio-CDMO business, achieving

double-digit growth in sales, exceeding market

growth. Going forward, we intend to continue

to promote growth along these lines, expanding

the bio-CDMO business as the growth driver for

the healthcare area,” says Rivera.

In March 2019, Fujifilm acquired a large-scale

biologics manufacturing facility in Hillerød,

Denmark from Biogen as part of their goal

of expanding its global business. The facility

allows for the support of high-volume produc-

tion of biologics that requires large-scale culture

tanks and is equipped with six 15,000-L bio-

reactors. The facility also houses

an assembly, labeling, and packag-

ing facility; quality control laboratories; and

warehouses. “Most importantly, the nearly 800

women and men of the Hillerød facility will

contribute their world-class cGMP [current

good manufacturing practice] manufacturing

capabilities to FDB’s existing leadership bio-

CDMO, immediately strengthening our over-

arching goal to advance tomorrow’s medicines

and guarantee distribution of their lifesaving

properties to all who need them,” Rivera says.

HALIX, which specializes in clinical and

commercial proteins and viral products, com-

pleted its new cGMP manufacturing facility in

Leiden Bio Science Park in the Netherlands in

August. The 6700-sq-m facility will be used for

the development and production of biopharma-

ceutical drug substances. The facility contains a

manufacturing line for viral vaccines and viral

vectors in addition to a separate protein manu-

facturing area with a capacity of up to 1000-L

single-use bioreactors. Lab space is also avail-

able for process development, analytical devel-

opment, and quality control. All cleanroom

areas have a unidirectional process flow and are

designed to allow commercial manufacturing of

biopharmaceutical products (1).

“This new facility offers our current and

future clients’ capacity and flexibility for future

expansion and will allow for cGMP manufac-

turing solutions for viral products, proteins,

gene therapy, and client-specific new technolo-

gies,” said Roland Hecht, HALIX chief customer

officer, in a company press release.

Sartorius Stedim Biotech (SSB), a supplier

of biopharmaceutical manufacturing prod-

ucts and services, is now offering GMP mam-

malian cell bank manufacture, the company

announced on Aug. 8, 2019. The services will be

offered through its subsidiary, Sartorius Stedim

BioOutsource, a contract testing organization

based in Glasgow, United Kingdom, and in

Biologics Continue to Grow and Create Outsourcing OpportunitiesCDMOs are adding facilities and services to their portfolios in anticipation of the biologics industry’s continued growth.

Susan Haigney



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adaptable partner. By joining forces with you, we provide a highly fl exible, expert

service covering contract development and manufacturing services for mAbs and viral

vectors, product characterization, biosafety testing and toxicology testing services.

So whatever direction your project needs to take, we’ll help guide you there.

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US and Canada.

MilliporeSigma, the vibrant M and BioReliance

are trademarks of Merck KGaA, Darmstadt,

Germany or its affi liates. All other trademarks

are the property of their respective owners.

Detailed information on trademarks is available

via publicly accessible resources.

© 2019 Merck KGaA, Darmstadt, Germany

and/or its affi liates. All Rights Reserved.


http://www.merckmillipore.com/IN/en

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https://www.bioreliance.com/


Perspectives on Outsourcing

Cambridge, MA, in the United

States (2).

The services will feature the

manufacture of GMP master and

working cell banks for mamma-

lian suspension cells, which will

be conducted in a 260-sq.-m. GMP

cleanroom dedicated to mam-

malian suspension cell lines. The

facility enables closed-system

manufacture of GMP-compliant

cell banks, from vial thaw to auto-

mated filling, as a qualified broth

technology platform. The company

expects this platform to maximize

process reliability and assurance of

sterility.

SSB offers its cell bank manu-

facturing in a package along with

cell bank characterization services,

making the company a single-

source provider from vial thaw to

released cell banks.

With these new services, the

company can offer combined

cell-line development, cell bank

manufacturing, and cell bank

characterization and provide bio-

manufacturing solutions from

DNA to released GMP cell bank

within a 10-month timeline.

MERGERS AND ACQUISITIONSIn August 2019, Eurofins Genomics

completed its acquisit ion of

Blue Heron Biotech, a Bothell,

WA-based gene synthesis company.

The deal bolsters Blue Heron’s pro-

duction capabilities while expand-

ing Eurof ins Genomics’ gene

portfolio into cloning and com-

plex gene constructs, according to

the company. The acquisition also

strengthens Eurofins Genomics’

product portfolio in the synthetic

biology market (3).

The companies share similar

main product segments includ-

ing oligonucleotides, sequenc-

ing, and synthetic genes. Eurofins

Genomics’ product of fer ings

include oligonucleotide synthesis,

Sanger sequencing, next-gener-

ation sequencing, and gene syn-

thesis. With the acquisition, the

company can now provide holistic

solutions to customers, including

regulatory coverage for its prod-

ucts—ISO 13485, ISO 9001, CLIA,

CAP, good laboratory practice, and

FDA compliance—for the manufac-

turing of cGMP oligonucleotides

used in analyte-specific reagent

and in-vitro diagnostic products for

the clinical industry.

REFERENCES 1. Halix, “New cGMP facility starting

operational production in Q4-2019,”

Press Release, Aug. 9, 2019.

2. Sartorius, “Sartorius Stedim Biotech

Launches New Services for Mammalian

Cell Bank Manufacturing,” Press

Release, Aug. 8, 2019.

3. Eurofins, “Eurofins Genomics US

Expands Gene Synthesis Capabilities

with Acquisition of Blue Heron Biotech,”

Press Release, Aug. 5, 2019. ◆

Call for Peer-Review Papers

BioPharm International accepts four types of peer-review papers

that are considered: standard data-driven, novel research; topical

literature or patent review; technical case studies/technical

application notes; and science-based opinion papers.

Manuscripts for peer-review papers are accepted on an ongoing

basis. Publication priority is given to papers in the order they are

accepted for publication.

Submitted papers are initially screened by the editors, then

submitted for formal review by a member of the editorial advisory

board, who will review the article for technical interest and content

in a double-blind review process. Article acceptance is conditioned

on the reviewer’s approval. Once accepted for publication, a paper

typically is published within three to five months.

Peer-review papers are published in the print and digital editions

of BioPharm International, and on www.BioPharmInternational.com.

Links to the online versions of peer-review papers also are featured

in e-newsletters distributed to the publication’s audience.

To learn more about the peer-review submission process, click the Submission Guidelines link on

www.BioPharmInternational.com.

B

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A risk-based approach based on a pharmacological and toxico-logical evaluation is becoming perceived in the industy as a sci-

ence-based approach that ensures safety of pharmaceuticals. Since the publication of International Council for Harmonization (ICH) Q9 Quality Risk Management, the application of this approach to the quality management of pharmaceuticals has been considered essential, and its application has also been extended to the cleaning validation of pharmaceutical manufactur-ing equipment. Limits such as 1/1000, 1/10,000, and 10 ppm were convention-

ally used in cleaning validation, but these limits can not be scientif ically justif ied and are arbitrary. In September 2010, the International Society for Pharmaceutical Engineering (ISPE) published a new base-line guide called Risk-Based Manufacture of Pharmaceutical Products (Risk-MaPP). Risk-MaPP provides a scientific and risk-based approach, based on ICH Q9 Quality Risk Management principles, to manage the risk of cross-contamination to achieve and maintain an appropriate balance between product quality and operator safety (1). The basic concept of Risk-MaPP requires a consistent and science-based approach

Takashi Kaminagayoshi, is director and head of

manufacturing operations;

Kosuke Takenaka, Tetsuya Ohta, Tomohiro Doi, and

Makoto Sadamitsu are

principal scientists; Shunsuke Omori is scientist; and Shinji Tsuji and Yoshiaki Miko are

associate directors; all are at

Biopharmaceuticals Process

and Product Development,

Pharmaceutical Sciences,

Takeda Pharmaceutical.

Osamu Shirokizawa is director

and senior consultant, Life

Scientia, and Andrew Walsh

is president, Center for

Pharmaceutical Cleaning

Innovation.

PEER-REVIEWED

Submitted: Dec. 8, 2017 Accepted: Apr. 11, 2018.

ABSTRACTCurrently, risk management based on a scientific approach is becoming required in the establishment of cleaning validation limits for pharmaceutical manufacturing equipment, as the acceptable daily exposure (ADE), which is set based on pharmacological and toxicological evaluation, is increasingly applied. At the early stage of development, ADE values may be set using the threshold of toxicological concern (TTC) approach due to lack of human data on toxicity. However, TTC values are estimates, so their application requires careful consideration. Especially in biopharmaceuticals (mainly proteins), whether or not the target product item is inactivated and degraded after cleaning is an important issue in evaluating the cleaning process. Therefore, a study was conducted by carrying out “CIP (clean in place) + SIP (steam in place)” and “CI (caustic immersion: alkaline treatment over a certain period of time)”, which are usually processes used in the cleaning of antibody drug manufacturing equipment. The inactivation and degradation of antibody drug was evaluated from the molecular structure and physiological activity point of view, using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and surface plasmon resonance. This study was successful in establishing a reliable and effective method for evaluating cleaning processes based on risk.

TAKASHI KAMINAGAYOSHI, KOSUKE TAKENAKA, TETSUYA OHTA, TOMOHIRO DOI, SHUNSUKE OMORI, MAKOTO SADAMITSU, SHINJI TSUJI, YOSHIAKI MIKO,

OSAMU SHIROKIZAWA, AND ANDREW WALSH

Study on an Inactivation Evaluation Method of Cleaning Processes for

Biopharmaceuticals

Peer-Reviewed






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Stability Testing

Putting Biopharma Stability Testing Under the Microscope

Stability testing for biologics is more complex than for small-molecule drugs, so companies should be aware of the potentially serious

issues that can be costly and jeopardize drug development.

FELICITY THOMAS

Stability testing is an essential part of drug develop-

ment and approval processes for both small- and

large-molecule drugs, and enables regulators and

companies to discern an appropriate shelf-life of a prod-

uct so that drug efficacy is ensured and patient safety is

not at risk. The global pharmaceutical stability testing

market has been predicted to experience robust growth,

partly as a result of the continuous rise of biologics (1).

However, stability testing of biologics is a much more

complex affair than that for small-molecule drugs.

“Unlike small-molecule drugs that tend to have lim-

ited structural conformations, biologics are significantly

larger and, therefore, more complex with greater degrees

of conformational freedom,” explains Phil Kuhlman,

laboratory manager—biologics at RSSL. “In addition,

tertiary and quaternary structure is maintained by multi-

ple forces, which may be disrupted leading to conforma-

tional changes. These changes lead to reduced potency,

aggregation, and increased risk profile to the patient due

to enhanced immunogenicity. It is not that small mole-

cules don’t degrade, more that large molecules have more

options for reaching a state of reduced potency.”

A STABILITY-INDICATING PROFILEAs a result of the complexity of biologics giving rise to

potentially multiple degradation pathways, a sole sta-

bility indicating assay is not sufficient to evaluate all

critical quality attributes, emphasizes Alex Perieteanu,

director, biopharmaceutical services at SGS Agriculture,

Food and Life. “Manufacturers are, therefore, required to

propose a stability-indicating profile that provides assur-

ance that changes in the product potency, identity, or

purity are detectable,” he says.

However, Perieteanu continues, there is not a one-

size-fits-all approach for biologics. “Most people think

about shelf-life stability and lot commitment when the

topic of stability testing comes up,” he adds. “Although

these are a good proportion of what is required in evalu-

ations, there are also other types of stability studies that

are critical and required.” kko

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Stability Testing

Naming a few, Perieteanu states

some studies of note include in-

use stability, photostability, ship-

ping studies, and forced degradation.

These studies are intended to sub-

ject the drug product to conditions

it will encounter, whether during

manufacture, shipment, storage, or

in the clinic. “Of important consid-

eration,” he says, “are the types of

conditions, and stability-indicating

methodologies that form the stabil-

ity-indicating profile.”

AVAILABLE TOOLS AND GENERAL PRACTICESAlthough many of the same tools used

in stability testing of small-molecule

drugs, such as osmolality, pH, appear-

ance, and mass, find use in biolog-

ics, the more complex large molecules

also require additional analytical tools

to help in the detection of confor-

mational changes that can occur and

which often lead to aggregation, states

Kuhlman. Additional tools such as

size-exclusion chromatography (SEC)

using multi-angled laser light scatter-

ing (MALS) detection, or analytical

ultracentrifugation (AUC), or field-

flow fractionation (FFF) can be used

to aid in biologics testing.

“Larger molecules also undergo

post-translational modifications or

exist as closely related isomers, both

of which can lead to slight variances

in isoelectric potentials,” Kuhlman

continues. “The separation of these

isoelectr ic var iants by capil lar y

electrophoresis is often used when

characterizing and monitoring the

proportions of these populations of

molecules.”

“F irs t and foremost , ” asser ts

Perieteanu, “the analytics employed

should be validated and should be

shown to be stability indicating. In

general, these methodologies should

be able to detect changes in the prod-

ucts potency, identity, and purity.”

Potency is particularly product spe-

cific, he explains, and can be depen-

dent upon the known mechanism of

action. “Methodologies such as cell-

based functional assays, enzyme assays,

or binding assays are critical (and

commonly employed) as they provide

practical in-vitro solutions to monitor

potency,” Perieteanu says.

For biological products, ‘abso-

lute’ purity is difficult to determine,

Perieteanu continues. As a result of

the difficulties encountered, it is com-

mon for many methods to be used

so that multiple quality attributes

relating to purity can be assessed.

“Techniques such as SEC are often

used to detect soluble aggregation and

fragmentation,” he states. “Sodium

dodecyl sulfate-polyacrylamide gel

electrophoresis/capillary gel electro-

phoresis (SDS-PAGE/CGE) can

differentiate between covalent aggre-

gates, fragments, and in some cases

glycosylation. Hydrophobic interac-

tion chromatography, ion exchange

chromatography, or isoelectric focus-

ing can look at charge distribution as

an indication of deamidation/oxida-

tion events, and peptide mapping can

be used to provide a higher resolu-

tion understanding of such changes.

In cases of (relatively) small biolog-

ics, traditional reverse-phase high-

performance liquid chromatography

(HPLC) with high pore size can be

utilized to monitor degradation prod-

ucts as well.”

Further to the key purity and

potency tests, Perieteanu highlights

that other product characteristics,

including appearance, visible and sub-

visible particulates, pH, moisture lev-

els, reconstitution time, sterility, or

alternative, are commonly assessed.

“Additives or excipients may degrade

and adversely affect the quality of the

drug product, and as such may also

need to be monitored,” he notes.

PRESENCE OF PARTICULATES AND AGGREGATE FORMATIONA notable challenge affecting the

stability of biologics is the potential

presence of particulates. This issue is

of particular concern for injectables

as particulates can induce an immune

response within the patient, which is

extremely undesirable as it can lead

to adverse reactions and reduced drug

efficacy, explains Perieteanu.

Adding to Per ie teanu’s com-

ments further, Kuhlman confirms

that special considerations on the

container material for biologics are

necessary. “The extraction of leach-

able compounds leads to contami-

nation of small molecules, whereas

within high concentration biologic

samples, extraction of leachable

compounds can lead to a catalytic

formation of large-scale aggregates,”

he says. “This stability question

will be further complicated by the

route of administration, for example

the use of intravenous saline bags

as a transport medium for a drug.

Compatibility testing would need to

be performed as part of the stability

study to ensure that no adverse reac-

tions occur.”

As a result of the complex nature

of biologics and the further com-

plications that can occur due to the

route of administration, Perieteanu

adds that particulates are not an

uncommon occurrence and, as such,

significant formulation development

is required to prevent particulate

development. “A comprehensive test

paradigm is often employed during

development and stability to monitor

particulates and aggregate formation,”

he reveals.

“When a protein undergoes con-

formational changes, hydrophobic

amino acids, usually held in the core

of the folded protein structure, are

exposed and may coalesce with other

protein molecules in a similar state,”

asserts Kuhlman. “This process can

be catalytic, leading to the formation

of aggregates, or can be promoted by

extractables and leachables from con-

tainers, such as silicone used to coat

the vial stopper.”



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Stability Testing

Formation of aggregates is gen-

erally triggered by a change in the

local (atomic) environment of a

molecule, ultimately causing desta-

bilization of the native structure

of the molecule, adds Perieteanu.

“Destabilized structures are often

unfavorable energetically, and as

such a ‘stabilizing’ cascade of events

occur that ultimately culminate in

the formation of visible particulates,

which are energetically stable, but

unwanted,” he says. “The testing

panel employed (stability indicat-

ing profile) often focuses on detec-

tion various stages of these cascade

events.”

These aggregates can then cause

mechanical issues when using the

syringe for administration, notes

Kuhlman, and may, therefore, exhibit

an increased immunological r isk

profile. “Protein aggregates tend

to have increased immunogenicity,

which ultimately reduces the effi-

cacy of the biologic,” he explains.

“Circulating patient-derived anti-

bodies to the drug leads to increased

c learance and thereby reduced

potency and can ultimately lead

to the need for that patient to be

switched to a different medication.”

NOT ALL SMOOTH SAILINGPotential ly the most significant

issue that can arise for a company

developing a biological product is

finding out the product is not stable

under the respective stability con-

ditions, states Perieteanu. “Finding

this out during preclinical/clinical

phases may trigger a requirement to

manufacture multiple costly batches,

additional formulation studies, or

worse, may jeopardize the study,” he

cautions.

Performing a suitable formula-

tion study will help to prevent com-

mencement of a stability program

too early and finding that the bio-

logic will not last the duration of

the study, adds Kuhlman. “However,

it is also important to note that the

information gained during the sta-

bility study is only as good as the

assays being performed,” he says.

“If the CQAs have not been fully

defined using an appropriate forced-

degradation study, then the amount

of useful data may be reduced or

a critical attribute may be missed,

risking patient safety further down

the development path. Detecting

this kind of issue later in the devel-

opment cycle could also lead to the

need to repeat the study.”

Despite stability normally being

established or understood after for-

mulation development and around

the time prec linical batches are

being prepared, there are many fac-

tors that can change in the tran-

sition from bench-scale to larger

batch sizes, warns Perieteanu. “If

the process is not robust, there is

an increased risk of product insta-

bility,” he adds. “Most common

product-related failure modes in

our experience are covered under

aggrega t ion and ox ida t ion , o r

deamidation events.”

Dealing with particulates or aggregates in biologics

Particulates or aggregates can have a detrimental

effect on the efficacy and safety of a biological drug

in a number of ways. Aggregation, for example, can

lead to, or be a consequence of, misfolded proteins

in an inactive state, explains Alex Perieteanu, director,

biopharmaceutical services at SGS Agriculture, Food and

Life. “This effectively reduces the amount of soluble,

active molecule able to perform its intended function,”

he says. “Alternatively, even if the molecule remains

active, an immune response can result in antibody-

mediated neutralization of the protein’s activity or in its

bioavailability.”

Cer tain methods can be employed to identify

particulates or aggregates in a biological formulation.

Perieteanu states that for insoluble aggregates, it is

possible to simply use visual appearance for detection

when larger particles have formed. “Light obscuration

is employed to determine particle counts in the >2 μm,

>5 μm, >10 μm, >25 μm range, and to ensure that

requirements for injectables, or ophthalmics are met per

United States Pharmacopeia (USP ) <787>, USP, <788>, or

USP <789>,” he adds.

In instances where light obscuration is not feasible

due to the extensive formation of bubbles or color, it is

more suitable to use microscopic evaluation of sub-visible

particles, although, this technique is more labor intensive,

Perietenau notes. “Additionally, microflow imaging can

look at a similar range of particulate sizes to get more

detailed understanding of morphology and distribution,”

he says.

If the particulates or aggregates are soluble, it is

common practice to use techniques such as sodium

dodecyl sulfate-polyacrylamide gel electrophoresis/

capillary gel electrophoresis (SDS-PAGE/CGE), size-

exclusion chromatography (SEC) using multi-angled light

scattering (MALS), and analytical ultracentrifugation,

Perieteanu asserts.

—Felicity Thomas



www.biopharminternational.com September 2019 BioPharm International 21

Stability Testing

“There is a need to ensure that sta-

bility studies run smoothly, other-

wise the overall timelines for a drug’s

development will be perturbed,”

asserts Kuhlman. As there is a pos-

sibility that an unexpected result is

detected during stability tests—such

as unexpected development of a new

species in an assay—then there is a

regulatory requirement to be able to

investigate these occurrences, identify

the new product, and evaluate the

significance it may have in the final

drug product, he explains.

“Product-related impurities may

have little effect on the potency of the

drug,” Kuhlman adds. “Alternatively,

they may be immunogenic leading

to the need for formulation changes.

Either way, being able to rapidly

respond with a suitable investigation

is essential.”

Highlighting the example of the

FDA, Perieteanu specifies that if any

quality defects in a distributed drug

product that may potentially risk

patient safety are identified, submis-

sion of a field alert report is required.

“This includes reporting of any sig-

nificant chemical, physical, or other

change or deterioration in the distrib-

uted drug product,” he says.

However, Perieteanu underlines

that there are a number of critical

aspects that can facilitate a rapid reso-

lution of quality defects in a product.

“First, have a strong understanding of

analytical performance characteristics,”

he confirms. “Second, have a good

understanding of the mechanism of

degradation, the stability indicating

profile, and how various degradation

pathways manifest themselves. Third,

armed with the information in the

first two points, an experienced team

of technical and quality investigators

can investigate product failures within

a GMP quality system.”

BEST PRACTICESAn understanding of a molecule’s

d eg r ada t i on pa thway i s c r i t i -

cal, asserts Perieteanu, and can be

achieved ear ly on using limited

degradation studies. “These stud-

ies can almost immediately shed

light on any potential stability con-

cerns prior to them becoming a risk

to patient,” he says. “Furthermore,

t h e s t u d i e s w i l l p r o v i d e a n

understanding of how stabi l i t y

issues manifest themselves, enabling

r a p i d r e s o l u t i o n w h e n i s s u e s

potentially arise.”

For Kuhlman, an essential pre-

requisite for any company look-

ing to enter stability studies is the

performance of a suitable forced

degradation study to identify the

appropriate CQAs. “Having suitable

assays both in terms of what needs

to be detected as well as sensitivity

is essential to generate the best out-

come of a stability study,” he stresses.

“Considering the length of time of a

typical stability study (three years)

and the associated costs, every effort

should be made to optimize the

success rate.”

One way of increasing experience

level and reducing costs incurred is

to use an outsourcing partner. “The

primary benefits of outsourcing

come from access to facilities and

infrastructure, experts, and capac-

ity, at a comparatively low cost,”

s u m m a r i z e s Pe r i e t e a n u . “ T h e

right contract organizations have

an economy of scale that many

sponsors cannot achieve internally.

Additionally, outsourced partners

often have experienced experts who

have worked on stability programs

for dozens of molecular entities.

It ’s critical that the entire panel of

stability tests be performed within

their respective test window. Thus,

expert capacity is paramount.”

REFERENCE1. Frost & Sullivan, “Global

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frost.com, Jan. 13, 2017. ◆

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Upstream Processing

Cell-Culture Advances Test Bioreactor Performance Models

The evolution of cell-culture technology is driving the need for improvements in modeling solutions.

CYNTHIA A. CHALLENER

Modeling and simulation are recognized to provide

assistance in predicting bioreactor performance,

both for initial laboratory runs and when scaling to

the pilot plant and commercial manufacturing. “Bioreactor

performance has consistently improved in biomanufacturing

due to a greater understanding of the engineering and biol-

ogy of cellular systems. To continue to drive efficiency and

reduce cost of manufacturing while maintaining or improv-

ing quality, reducing bioreactor variation will be required,

and predictive models can enable these variation reductions,”

asserts Tom Mistretta, director of data sciences at Amgen.

The continual development of higher-producing cell lines,

the switch to chemically defined media, and other advances

in cell-culture technology are, however, challenging develop-

ers of modeling and simulation software.

TRADITIONAL, RELIABLE METHODTypically, bench-top bioreactors are operated in the perfor-

mance design space of larger bioreactors to model scale-up

performance, according to Parrish M. Galliher, chief tech-

nical officer for upstream and founder of Xcellerex, a GE

Healthcare Life Sciences business. “Successful modeling

of bioreactors can reduce costs associated with engineer-

ing testing and engineering runs, accelerating pathways

through preclinical and early clinical phases,” adds Mark

T. Smith, staff engineer for single-use technologies in the

BioProduction Division of Thermo Fisher Scientific.

In particular, the inexpensive computing power and excel-

lent computational talent entering the biopharma industry,

combined with improvements in computational fluid dynam-

ics (CFD) and metabolic modeling software, are promising

to open new doors for more complete cell culture models,

according to Smith. “I predict that as modeling becomes

more robust, it may be possible to entirely eliminate tradi-

tionally performed engineering runs, which in fact is already

being done to some extent because in many cases companies

and engineers simply ‘get to know’ their bioreactor networks

empirically and heuristically, without the need for complex

physical modeling in silico,” he asserts.

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CYNTHIA A. CHALLENER, PhD, is a contributing editor to





Upstream Processing

The key to successful modeling for

the prediction of bioreactor perfor-

mance, stresses Galliher, is to first calcu-

late the performance design space of the

bioreactor at all relevant scales. “Once

these design spaces are understood, the

scientist can operate any scale bioreac-

tor within the design space of any other

bioreactor scale and model any bioreac-

tor of any scale using a bioreactor of any

other scale,” Galliher says. He adds that

this approach has been used effectively

for decades.

The process of modeling has, in fact,

advanced due to the increased capability

to aggregate various sources of data (sen-

sor measurements, batch record entries,

product quality measurements, main-

tenance records, etc.) needed to model

bioreactor performance, according to

Mistretta. “The industry has become

more efficient due to the significant

improvements in technology for central-

ized access to various data sources via

data lakes,” he observes. In addition, the

information systems technology used has

been validated and demonstrated to be

cost effective, secure, compliant, and scal-

able with the volume of data generated

from manufacturing processes.

CONTINUOUS CONSIDERATIONSIn the past, continuous processing in

perfusion mode was generally limited to

cell-culture processes involving sensitive

products that degrade under conven-

tional batch conditions. Today, there is

increasing use of perfusion cell culture

to realize other cost, efficiency, and pro-

ductivity advantages.

Some of the challenges with perfu-

sion processes include sensor insta-

bility and fouling over the course of

the culture, which impacts the abil-

ity to effectively monitor and control

these processes, according to Galliher.

Perfusion mode can also create addi-

tional risks of contamination due to

the continuous removal of depleted

media and introduction of fresh

media, and these processes involve a

significant increase in the media vol-

umes required, which has logistical and

cost implications for a manufacturing

facility, adds Mistretta.

Overall, perfusion mode generally

places higher performance demands

on the reactor, according to Smith.

He points to the higher cell densi-

ties in perfusion mode, which result in

increased oxygen demand in a higher

viscosity solution with potentially more

cell-free biopolymers and lipids than a

fed-batch culture. “Viscosity and solu-

tion property changes can fundamen-

tally change mass-transfer mechanisms

by impacting bubble size, bubble coales-

cence, mixing times, shear rates, and

other parameters,” he explains.

As a result, in-silico modeling of sys-

tems must be adjusted for such param-

eters in a meaningful way. There is,

however, a limited understanding in

how to model such high cell densities

due to the relative newness of these

operating regimes, according to Smith.

Computational models of perfusion

processes additionally require consider-

ation of filter effects such as fouling and

sieving, Mistretta notes.

SHEAR ISSUES IN SINGLE-USE BIOREACTORSThe shift to single-use technologies

has also introduced a wider diversity

of bioreactor designs, particularly with

respect to spargers, baffles, and impeller

configurations. “Whereas stainless-steel

reactors could be made with relatively

similar configurations within and across

bioreactor networks, having multiple

vendors represented in single-use bio-

reactor networks increases the level of

data and modeling required to success-

fully predict behavior,” Smith explains.

In addition, Smith notes there is

a need for improved mass trans-

fer performance with reduced shear

in single-use bioreactors. “As cultures

become more intensified, the need

for oxygen transfer increases and is

generally accommodated by increas-

ing the oxygen gas passing through

the micro-sparger. In many cases, the

increased flow through the micro-

sparger leads to shear-associated cell

damage, likely related to increasing

shear at the gas-liquid interface,” he

explains.

To address this problem, oxygen

delivery must be achieved through

efficient spargers with lower gas-liq-

uid shear rates. “While some vendors

already offer such sparger technolo-

gies, the industry has been slow to

update to these sparger technologies,

likely due to the revalidation burden

of changes to established bioprocesses,”

Smith comments.

Mistretta agrees that variability of

single-use systems does impact cell cul-

ture, but Amgen has been able to rap-

idly detect and determine the root cause

for this cell culture variation through

the accessibility of process data, material

lot data, and process performance mod-

els. “These models also support data-

driven decision making for impacted

lots, and we continue to develop moni-

toring strategies through predictive

models, near-real-time monitoring, and

material-control strategies to mitigate

unintended variation in performance,”

he states.

More advanced use of hybrid models

such as CFD combined with first prin-

ciples kinetic models are also effective

to assess the impact of bioreactor design

on performance, according to Mistretta.

“Use of CFD models to determine kLa

[mass transfer coefficients], mixing time,

and shear effect have become pretty

standard applications for technology

transfer, scale-up, process optimiza-

tion, and troubleshooting activities at

Amgen,” he says.

In addition, Amgen has demon-

strated the utility of finite element

models for studying operational risk

factors for single-use bioreactor sys-

tems. For instance, models were devel-

oped to predict how material science

aspects of the single-use bioreactors

impact pressurization and other oper-

ational risks, according to Mistretta.




Upstream Processing

Amgen has also observed more

collaboration with single-use bioreac-

tor suppliers with respect to exchang-

ing data and developing models that

enable more reliable operations.

ULTIMATE GOAL:MODELING COMPLETE BIOREACTOR PERFORMANCEModeling of the performance of the

whole bioreactor including predicting

full cell-culture performance seems to

be the holy grail, but according to Smith,

broad application of complete models

has yet to be achieved due to to the

complexities of the variables required for

accuracy, including the oxygen transfer

rate, oxygen uptake rate, carbon diox-

ide evolution, carbon dioxide stripping,

metabolism, cell-line traits, etc.

In addition, Smith notes that

improvements in understanding of cell

biology have led to dramatic changes

in what a “typical” cell line is capable

of producing, with improvements com-

ing from cell-line traits such as gene

promoter strength, gene integration site,

integration stability, modified metabolic

pathways, etc. Furthermore, aspects of

bioprocess engineering have shifted,

for example from natural to chemically

defined media and from batch to fed-

batch (and toward continuous) modes.

“In short,” Smith concludes, “model-

ing the ‘complete’ bioreactor cell-culture

performance for universal application

may be not yet be in sight.” He does

comment that there appears to be a push

among large biopharma toward the use

of complete models for the prediction

of bioreactor performance in their given

networks. However, in any published

literature regarding these efforts, there

is rarely sufficient detail provided to

enable easy reproduction of the models

used, and almost never is source code

made public for direct application. “In

this sense, trade secrecy and intellectual

property concerns may be stymying the

potential rapid industry-wide advances

in performance prediction,” Smith

observes.

“Even simpler aspects of bioreactor

performance are generally still poorly

modeled.” He points to the modeling

of mass transfer coefficients, which dic-

tate whether a reactor can provide suf-

ficient oxygen to maintain viable cell

mass, as an example. “In the past three

years, there have been several hundred

publications on CFD-based prediction

of kLa in bioreactors. While this quan-

tity suggests great interest in the area,

they frequently describe the resulting

models as ‘adequate,’ ‘sufficient,’ or simi-

larly hesitant verbiage, suggesting in fact

there is still space to improve in our

performance prediction as an industry,”

Smith explains.

Potentially because of this remaining

gap in the predictive power of CFD,

many of the product development/

pilot-scale models used today, accord-

ing to Smith, are driven primarily by

empirical data leveraging fairly tra-

ditional correlations. “This approach

seems to be able to provide a similar

predictive power to that of CFD with-

out the additional computational effort

and resources,” he comments.

ROLE OF PROCESS ANALYTICAL TECHNOLOGYUseful models must be robustly vali-

dated with real data. The greater the

amount of good data incorporated into

a model, the more insight the model

can provide. Traditional off-line sam-

pling once or twice daily of key parame-

ters (e.g., viable cell density, glucose, key

amino acids, titer, etc.) will not provide

the robust data set needed to robustly

predict performance on a moment-by-

moment basis, according to Smith.

That is where process analytical

technology (PAT) comes in. “Relatively

recent application of PAT, such as

Raman spectroscopy and biomass

probes for inline sensing, are now pro-

viding more complete data on cell-cul-

ture performance and enabling better,

more timely control of reactor automa-

tion,” he observes. Advances in sensor

technologies have also contributed to

enhanced real-time performance pre-

dictions of bioreactor performance and

enable approaches such as model pre-

dictive control-based models, according

to Mistretta. It is, in fact, improvement

of automation control strategies within

the existing capabilities of the bioreac-

tor—not sampling and sensing alone—

that is essential to improving bioreactor

performance, stresses Smith.

Further advances in PAT are still

needed, however, to enable them to

really contribute to model develop-

ment, according to Galliher. As an

example, he notes that while infrared

sensors have been touted as being able

to measure relevant bioreactor param-

eters and generate predictive models,

they require large computing power

and algorithms to cancel out interfer-

ence from cells, gas bubbles, and media

components, all of which change over

the course of the run.

POTENTIAL FOR ARTIFICIAL INTELLIGENCE?The availability of data (particularly

genomic and proteomic data on cel-

lular systems) and the capability to

efficiently capture, store, and contex-

tualize the process data needed for

modeling have been the most notable

advances in predictive bioreactor per-

formance, according to Mistretta. The

decreasing cost of the infrastructure

needed to run computational models

has, he adds, also made it faster to pro-

totype and deploy predictive models

for bioreactors.

One area of rapid development

involves the potential application of

artificial intelligence (AI) and machine

learning (ML) to bioreactor perfor-

mance modeling. Bioengineers are

beginning to discuss how to apply

machine learning and digital-twin

algorithms to the modeling of bioreac-

tor performance, coupled with CFD

modeling of multiphase (liquid and

gas) solutions, according to Galliher.

He does note, though, that these

approaches are in conceptual stage.




Upstream Processing

In fact, the capability to design, develop, and implement

machine learning algorithms more efficiently has also allowed

modeling to be done in a more systematic way, according to

Mistretta. “Machine learning models based on the ability to

leverage larger datasets have enabled automated data-driven

models of bioreactor performance and offer the potential to

help determine fitted parameters in first principles models of

bioreactor performance,” he remarks.

One possible challenge to leveraging AI/ML could be the

need for massive data sets in order to avoid overfitting issues.

“Considering that biopharma companies are generally trying

to minimize the number of culture runs for any given product

and that even processes for blockbuster products may only be

run 12–20 times per year, there is a relative dearth of informa-

tion for most cell-line cultures with respect to typical ML

data sets,” Smith explains. While each run produces thousands

of data points (e.g., second-by-second pH, dissolved oxygen,

viable cell density, etc.), these variables are inextricably linked

to the chronology of the culture and could lead to further bias,

he adds.

Smith stresses, though, that although AI and ML

pose unique challenges to bioengineers, it is possible that

experts well-versed in how and when to apply AI may

develop creative solutions for deconvoluting and dealing

with these types of challenges in the near future, to success-

fully enable the utilization of these technologies within the

bioprocess industry.

EXPECT MORE ADVANCES SOONToday, most of the measures of bioreactor performance are at a

macro level in commercial facilities, according to Mistretta. To

get to highly accurate predictions of bioreactor outputs, addi-

tional measurements and a framework for extensible model-

ing that describes the physiology, chemistry, and mechanics

of a bioprocess must be developed, he says. “Genomics and

metabolomics-level understanding of bioreactor performance

supplemented by real-time advanced sensors to measure

post-translational modifications are necessary, and we predict

to see these types of advances in the next three to five years,”

Mistretta asserts. ◆

Samsung Demonstrates Commerical-Scale ATF Perfusion

Samsung BioLogics has demonstrated the viability of using

alternating tangential flow (ATF) technology in a perfusion

reactor at commercial scale at its in Songdo, South Korea site.

The system allows for a 10-fold improvement in cell culture

densities while retaining cell viabilities of over 98% at the

seed stage, enabling inoculation within 15,000-L bioreactors

at higher cell densities; peak cell densities can be achieved

within shorter culture durations, the company reported in a

press statement (1). Samsung worked with Repligen to design

the stainless-steel-housed system. Validation, including ATF

system water and media tests as well as autoclave cycle

development and sterility performance testing, was completed

within six months, the company reported.

Reference1. Samsung, “Samsung BioLogics Implements Large Scale N-1

Perfusion for Commercial Application,” Press Release, Aug. 12, 2019.

—The editors of BioPharm International

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Cell and Gene Therapies

Seeking Solutions for Large-Scale GMP Viral Vector Manufacturing

Innovation in manufacturing technologies must occur to ensure the availability of gene and cell therapies.

CYNTHIA A. CHALLENER

The rapid advance of gene and modified cell therapies

and growing interest in viral vaccine therapies are creat-

ing significant demand for large-scale viral-vector man-

ufacturing capabilities. Biopharma companies and contract

manufacturers alike face a host of challenges as they work

to meet this crucial market need, from a limited availability

of technology to a lack of standardization to complex and

evolving regulatory pathways.

MANY SIGNIFICANT CHALLENGESThere are numerous challenges to sourcing effective large-

scale manufacturing solutions for viral vector production.

Based on conversations with customers over the past several

years, Univercells has identified the major hurdles in large-

scale virus manufacture, which include expensive manufactur-

ing facilities, lack of expertise, limitations in the number of

scalable manufacturing technologies available in the market,

and the high cost of good manufacturing practice (GMP)-

grade reagents including transfection mix, plasmids, and

bovine serum, according to Thomas Theelen, business devel-

opment manager at the company.

“Equipment and facility setup using the technology that is

available, most of which has been adapted from other thera-

peutic areas, is very expensive and often does not support prod-

uct manufacture through different maturity stages including

process development, clinical trials, and commercialization. In

addition, the level of expertise around developing large-scale

manufacturing processes for gene therapies is limited, and the

use of flatware for cell culture and sub-optimal downstream

processing protocols result in low yields; both factors also influ-

ence the overall cost of goods sold (COGS),” Theelen explains.

“Existing manufacturing processes are often complex with

hard-to-control unit operations and unfavorable COGS pro-

files,” agrees Xin Swanson, head of commercial development

for viral vector gene therapy, Lonza Pharma & Biotech. As an

example, she points to the widely used transient transfection

process for associated adeno virus and lentivirus (AAV and LV,

respectively) production, which mainly uses adherent cells and

lacks scalability and lot-to-lot consistency due to variability in

vecto

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CYNTHIA A. CHALLENER, PHD, is a contributing editor to




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transfection efficiencies. In addition, she

notes that many downstream processes

for early-stage clinical production often

employ gradient centrifugation steps that

lack the ability to be furthered scaled.

“Overall the volumetric productivity

of virus particles per cell from culture

systems and complete virus recovery

rates through downstream processing

are sub-optimal among existing pro-

duction systems, both of which nega-

tively impacts COGS. As a result,

the lack of standardized production

platforms that support industrialized

scale processes is the leading challenge,”

Swanson concludes.

Further complicating the situation is a

lack of standardized and advanced analyt-

ical methods for vector characterization

and release testing (including in-process

samples), which makes it difficult to have

well-understood manufacturing processes

before incorporating process improve-

ment steps, according to Swanson. Other

challenges include a lack of automation

in key steps of the manufacturing process;

immature supply chains, which creates

risk and leads to a lack of standardization/

innovation of critical raw materials; and

rapidly advancing regulatory pathways

that are complex to manage among dif-

ferent jurisdictions.

INCREASING URGENCYMany of these challenges have existed

for several years, with some develop-

ments occurring along the way. For

instance, Theelen observes that novel

scalable bioreactor systems, the imple-

mentation of continuous bioprocess-

ing, and the introduction of new resins

for viral vector purification are hav-

ing an impact. “Several companies are

developing suspension-based processes

and implementing stable producer cell

lines,” he notes. However, he adds that

even though stable cell lines have the

potential to significantly reduce COGS,

developing robust processes using

these stable cell lines is still a challenge.

The same is true for suspension-based

processes, which currently require sig-

nificant investments in process develop-

ment and extend the time to market.

What has largely changed, according

to Swanson, is the timeline for over-

coming these challenges. “It is critical

to meet the clinical and commercial

manufacturing needs for these curative

therapies, and the need is becoming

more urgent due to the rapid advance

of clinical progress. The lack of manu-

facturing scalability has created a vector

shortage, and the collection of suffi-

cient vector chemistry, manufacturing,

and controls (CMC) information has

become a bottleneck during the product

development lifecycle,” she asserts.

Theelen adds that to ensure that

more gene and other next-generation

therapies reach patients, reducing

COGS is necessary to increase avail-

ability, facilitate reimbursement, allevi-

ate the burden on healthcare budgets,

and ensure that innovator companies

can offer their products at an afford-

able price while maintaining sustainable

gross margins. “Scalable and reliable

technologies for cost-effective cell and

gene therapy manufacture will reduce

the reliance of biopharmaceutical com-

panies on hard-to-acquire expertise

and dependence on contract develop-

ment and manufacturing organizations

(CDMOs),” he says.

Additionally, Theelen notes that

developing technologies that facilitate

process and product development will

shorten the time-to-market and reduce

development costs, while increasing the

number of technology candidates will

intensify competition and finally drive

down materials and equipment costs.

INNOVATION IS A PRIORITYTechnology innovation is key to

addressing the challenges associ-

ated with large-scale GMP viral vec-

tor manufacturing, agrees Tania Pereira

Chilima, NevoLine product manager at

Univercells. “Solutions that are tailored

for gene therapy manufacture and com-

bine a low capital investment, scalability,

ease-of-operation, and robustness while

maintaining product quality are needed,”

she comments.

It is also important that these tech-

nologies also are able to accommodate

the manufacture of gene therapy prod-

ucts with low and high annual demands

in order to ease the development and

commercialization of personalized gene

therapies and viral vector-based vaccines.

Lowering the capital investment is also

necessary for reducing the entry barriers

for gene therapy start-up companies.

Swanson adds that all of the major

challenges must be tackled concurrently

in order to address the manufactur-

ing challenges. “The optimal goal is to

deliver products that will meet target

product profiles with defined quality

attributes while realizing the need to

increase process productivity and reduce

COGS,” she says.

ROLE OF CDMOsCDMOs have a vital role to play when it

comes to manufacturing innovation for

viral vector production. “Given the fast

pace of clinical development timelines

and the unconventional demand curve

of curative therapies, CDMOs offer

competitive advantages not only from a

manufacturing technology advancement

perspective, but also from a cost perspec-

tive with respect to optimizing capital

expenditures and better managing oper-

ating expenses,” Swanson states.

Pereira Chilima agrees that CDMOs

are a major gateway for the adoption

Complicated

processes and

sub-optimal

yields impact the

COGS for large-

scale viral vector

manufacturing.FOR PERSONAL, NON-COMMERCIAL USE




of new technologies. “Many estab-

lished gene-therapy developers rely on

CDMOs throughout all stages of prod-

uct development and even once they

reach the commercialization stage. The

expertise of CDMOs in large-scale pro-

duction helps cell therapy developers

evaluate different avenues for cell therapy

manufacture and select the technologies

to be used once they decide to internalize

product production,” she explains.

CDMOs are also in the unique

position of developing and providing

manufacturing platform processes that

will allow drug developers to focus on

product innovation and significantly

shorten the gene-to-product develop-

ment timeline, according to Swanson.

ACHIEVING SOME PROGRESSOne of the most important devel-

opments highlighted by Swanson has

been the transitioning of cell-culture

processes from an adherent format to a

suspensions format via cell-line adapta-

tion, which is enabling better scalability.

Development of stable producer cell lines

is also reducing process variability and

enabling the adoption of intensified pro-

cessing solutions. A better understanding

of the important qualities of critical raw

materials, such as animal serum, plasmids,

non-chemically-defined media, and the

extractables/leachables associated with

single-use technologies has also helped to

address some manufacturing challenges.

Incorporating design-of-experiment

study principles and use of improved bio-

process control and data analysis software

are allowing process development/opti-

mization to be conducted at the mul-

tiparameter level, while new analytical

methods, such as more accurate viral vec-

tor quantitation techniques, provide a

better understanding of impurity profiles

and allow for better process monitor-

ing. In downstream processing, density-

gradient centrifugation is being replaced

with chromatography methods for more

consistent and efficient operations.

Collaboration between the different

stakeholders in the industry is neces-

sary to build on preexisting expertise

and capitalize on academic innovation

to help guide technology developers on

the gaps that must be addressed in the

industry, according to Pereira Chilima.

Adds Swanson, “Col laborat ion

between members of the value chain,

ranging from innovators, manufactur-

ers, and reagent and equipment sup-

pliers to regulators, as well as close

collaboration between academic and

industry partners, is critical in driving

solution development.”

Pereira Chilima also notes that

involving the regulatory bodies and

payers early on is needed to reduce

regulatory barriers and ensure adequate

reimbursement.

These types of collaborative efforts

are ongoing, she says. Good examples

include the efforts made by the Cell

and Gene Therapy Catapult and Cobra

Biologics to investigate continuous man-

ufacturing. Univercells is also actively dis-

cussing promising collaborations with

established gene therapy companies

with the aim of combing its expertise to

develop manufacturing solutions for end-

to-end viral vector production for gene

therapy applications.

PLATFORM DEVELOPMENT Lonza has invested substantial internal

research and development efforts in plat-

form development, particularly in the

area of AAV and LV production. Specific

focus areas include scalable suspension-

based transient transfection processes

and stable producer cell-line technolo-

gies, according to Swanson. Concurrently,

the company is investing in the develop-

ment of novel analytical methods and

new media formulations with the aim to

provide turn-key solutions.

Separately, Lonza has invested heav-

ily in a manufacturing capacity expan-

sion with the opening of its dedicated,

300,000-sq.-ft. cell- and gene-therapy

facility in Houston in 2018. The facil-

ity uses an extensive modular design

concept and employs single-use tech-

nologies to allow clients quick access to

scaled-up production capacity based on

project needs.

Univercells, meanwhile, has devel-

oped the NevoLine platform, a modular

and automated manufacturing system

delivering affordable viral products such

as vaccines and gene therapy vectors.

This design of the NevoLine platform

relies on a modular approach, allow-

ing for a variety of configurations that

provide tailored solutions for a range of

products, according to Pereira Chilima.

Viral-vector-specific NevoLine plat-

forms can be designed for enabling

AAV or LV manufacture for small- and

large-scale applications.

At the core of the NevoLine platform

lies the scale-X fixed-bed bioreactor

coupled with in-line product concentra-

tion to intensify upstream processing and

deliver large product quantities within a

reduced footprint. “Intensification of each

unit steps allows for drastic footprint

reduction, enabling the entire process to

be placed in self-contained modules such

as isolators or biosafety cabinets,” Pereira

Chilima explains. “The platform aims to

minimize the capital investment for gene

therapy manufacturers while delivering

the low COGS and flexibility required

to support the development and com-

mercialization of gene therapy products,

eventually ensuring their availability to

patients,” she says. ◆

Collaborative

efforts between

innovators,

academia,

regulators, and

suppliers are

essential to

drive solutions.




Quality

Best Practices for Studying Stability in Biologics

Industry experts discuss the challenges and regulations of setting up a CGMP-compliant stability testing program.

SUSAN HAIGNEY

BioPharm International spoke with Bhroma Patel, head

of product stability, and Baldev Jogi, lead scientist,

both at Lonza; and Will Hatcher, senior manager, QC,

and Rekha Patel, director, biologics analytical solutions, both

at Catalent, about what steps companies need to take when

setting up a current good manufacturing practice (CGMP)-

compliant stability testing program for biologics.

BEST PRACTICES IN STABILITYBioPharm: What are the first steps for setting up a CGMP-

compliant stability program for biologics?

Hatcher (Catalent): The first step in setting up a CGMP-

compliant stability program is to determine the analytical assays

that are stability-indicating and to verify that the method qualifi-

cation/validation has been performed appropriately to prove that

these assays are indeed stability-indicating. After the analytical

assays are determined, the next step is to set up the stability strat-

egy, which will include long-term stability at the determined stor-

age condition, short-term stability (accelerated and stressed), and

possibly photostability or freeze/thaw studies. It is very important

to be conservative with regards to timepoints within a stabil-

ity study. If the appropriate amount of data [are] not gathered

at the appropriate conditions, then it is possible that an entire

study would need to be repeated, which becomes extremely time-

consuming and costly, and could be detrimental to a regulatory

filing timeline.

Bhroma Patel and Jogi (Lonza): An amendment

agreement is required between the clients and outsourcing

company to see what stages of work [are] required from start

to end. This will include objective, activities (e.g., timepoints,

temperatures, and intended storage temperature), test meth-

ods, and delivery of reports. In addition to this, methods and

equipment require validation and specifications put in place

prior to starting the stability study.

BioPharm: What challenges should companies be aware of

when setting up a CGMP stability program for biologics?

Bhroma Patel and Jogi (Lonza): The challenges com-

panies must be aware of when setting up the GMP stability

programs are that regulatory requirements, such as the ICH

[International Council for Harmonization] guideline, are fol-

lowed throughout the stability program, from pilot stability

studies to drug product studies. Companies may require large Orl

an

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Quality

facilities for laboratory capacity, storage, as

well as stability chambers under different

climatic conditions and relative humidity

and freezers. In addition, companies are

required to develop, validate, and evaluate

testing methods and equipment to per-

form stability studies.

Rek ha Pa te l (C a t a len t ) :

Companies should ensure that they have

redundancies in power, water, and air han-

dling systems as well as appropriate alarm

notification systems, to safeguard against

possible impacts to the study. To ensure

integrity of the study, sample traceabil-

ity and inventory systems should also be

in place. Having a sufficient volume of

materials should not be overlooked. Study

coordinators should ensure that they have

enough materials to progress through

the study as well as any investigations or

extensions. If special studies are needed,

such as freeze/thaw, in-use, forced degra-

dation studies, photostability, etc., proper

material amounts should be ensured as

volume is often a challenge.

Study coordinators should ensure that

methods are proven to be stability-indi-

cating during qualification/validation, are

appropriately set up beforehand, and that

orthogonal methods are available where

appropriate. Analytical methods may

change going from early to late phase (e.g.,

moving from ELISA [enzyme-linked

immunosorbent assay]-based to cell-

based), and investigators should plan for

any bridging activities that may be needed.

Appropriate planning also includes a thor-

ough understanding of the study matrix

and any special handling required. The

timing of the study should be laid out

to meet any critical regulatory filings. If

planning to file globally, consider testing

conditions specific to different regions in

the same study, as this will provide suf-

ficient data without the time and cost

needed to conduct a separate study.

REGULATORY EXPECTATIONSBioPharm: What are the specific

regulatory requirements for a CGMP-

compliant stability program?

Bhroma Patel and Jogi (Lonza):

The specific regulatory requirement

for stability programs for biologics are

defined in ICH guidelines with ref-

erence to ICH Q1A (R2), Stability

Testing of New Drug Substances and

Products, ICH Q5C, Stability Testing for

New Dosage Forms, and EMA/CHMP/

BWP/534898/2008 rev. 1 corrigendum,

Guideline on the Requirements for Quality

Documentation Concerning Biological

Investigational Medicinal Products in

Clinical Trials. For recommendations

on how to establish shelf life or retest

period based on stability studies, ICH

Q1E, Evaluation of Stability Data, is

followed. In total there are six ICH

basic guidelines for stability studies,

Q1A to Q1F.

Hatcher (Catalent): The regula-

tory requirements for CGMP stabil-

ity programs mainly come from ICH

guidelines, specifically: Q7 11.5, Q1A

(R2), Q1B, Q1C, Q1D, Q1E, Q5C.

There are also regulations from the

FDA (21 Code of Federal Regulations

211.166) and European Medicines

Agency (EMA) (Guideline 3AB5a)

that govern what should be contained

in a stability strategy. In addition to

these regulations, FDA provides guid-

ance that can help determine an appro-

priate stability study strategy.

With this bolus of information, it can

be challenging to determine a clear sta-

bility strategy. However, as long as the

assays performed are stability-indicating

and the stability strategy includes long-

term at storage condition, accelerated

and stress stability studies, in most cases

this can be enough for an initial filing.

As a product moves through the clini-

cal lifecycle, photostability and freeze/

thaw studies will need to be performed.

Additionally, depending on the proper-

ties of the product and the results of the

stability studies, additional studies may

be warranted.

BioPharm: Can you provide an

example of how a company can ensure

their stability program is following reg-

ulations?


Companies can ensure [the] stability pro-

gram is following regulations by [creating]

standard operating procedures (SOPs) to

help set up stability studies and protocol

in compliance with regulatory expecta-

tions.

[A] stability program should be

described in the protocol to support

the shelf life and storage condition and

include:

• Objective/scope of the study (e.g.,

the stability study results may form

part of submissions to the regulatory

authorities to support the use of the

product in toxicological studies and

clinical trials).

• Storage conditions (e.g., intended,

accelerated, and stress storage

conditions)

• Sampling plan (e.g., samples to be

tested at 0, 3, 6, 9, 12, 18, 24, and 36

months)

• Stability indicating parameters for

testing of product characteristics,

identity, potency, purity, and safety,

which have been developed and

validated

• Stability test methods (e.g., capillary

electrophorsis sodium dodecyl sulfate

[CE SDS], image capillary isoelectric

focusing [icIEF], gel permeation

chromatography [GPC], ELISA),

which have been qualified for usage

• Acceptance criteria (e.g., limits for the

test results)

• Reference standard to compare the

sample against

• Approval process (e.g., approved by

quality assurance [QA])

• Stability chambers are serviced,

inspected, calibrated, and qualified

regularly

• Out of specification and out of trend

SOPs

• QA regulated

• Evaluation of the acquired data to

provide a shelf life

• Trained operators, which includes

GMP training and data integrity

training annual.




Quality

BioPharm: Can you provide any

examples of mistakes companies have

made when preparing stability stud-

ies for investigational new drug (IND)

submissions?


As a CDMO [contract development and

manufacturing organization], the biggest

issue we encounter is often around data.

For example, companies provide insuf-

ficient data and information to support

stability of drug substance or informa-

tion is erroneous due to lack of time to

prepare for IND submission. Failure to

follow FDA guidance or to summit all

the information required from the IND

submission checklist is another potential

oversight.

Hatcher (Catalent): The main mis-

take that companies make when setting

up their stability strategy is not perform-

ing accelerated, stressed, photostability,

or freeze/thaw stability studies. Often, a

process will change throughout the clini-

cal development of a product, and with

these changes comes the need to repeat

stability studies. I have seen a trend where

companies will not repeat the accelerated

or stressed stability; however, they will

repeat standard stability at the storage

condition for the product. This is a very

risky strategy, as regulators will want data

to show that the change in the process

did not impact the stability of the prod-

uct, including stressed and accelerated

stability studies.

OUTSOURCING STABILITYBioPharm: What are the benefits of

outsourcing CGMP stability studies for

biologics?


Companies have access to regula-

tory experts and can meet the most

up-to-date regulatory requirements.

Companies can meet their needs and

requirements (planning and designing

stability studies). [There is] cost sav-

ing, as large amount of capital is not

required on resources, such as on analyt-

ical equipment and stability chambers.

[They can] gain scientific and analytical

knowledge for data generation, inter-

pretation, and reporting. [Outsourcing]

enables companies to focus internal

resources on new drug discovery and

development.

Hatcher (Catalent): The main

benefit of outsourcing CGMP stability

studies for biologics is that CMOs are

exposed to many different strategies

from sponsors and, therefore, have a

good handle on trends in the industry.

This level of expertise can be very ben-

eficial for the sponsor and will ensure

that the stability program is compliant

and up to date with trends in the indus-

try. Another benefit is the analytical

expertise that CMOs possess because

the analysts in a QC [quality control]

CMO laboratory are familiar with sev-

eral types of analytical methods, which

means CMOs are well placed to help

the sponsor troubleshoot methods to

make improvements when there are

issues.

Outsource facilities also typically have

significant storage space and analytical

instrumentation. This helps the sponsor

know that there will not be equipment

or space constraints that could negatively

impact their stability program.

BioPharm: What are the challenges

of outsourcing CGMP stability studies

for biologics?

Hatcher (Catalent): The main chal-

lenge from a CDMO perspective is when

a sponsor changes the stability strategy

late in the game. This can be detrimental

to the timeline and ultimately could delay

approval. Another challenge is balancing

cost-savings versus performing stabil-

ity using a conservative approach. There

are times when a sponsor is focused on

cost-savings and then limits the stability

program, only to have to come back later

and perform the study again. This is why

it is very important to perform the study

in a conservative manner, to avoid having

to repeat.


Finding a trusted outsourcing partner

with the right level of expertise and

process knowledge, as well as the right

assets and technology is key. Without

this, delivery may be late or below expec-

tations, leading to a delay in progress-

ing drug production to the next level.

Confidentiality and security are essential

to avoid breaches of proprietary informa-

tion in a multi-customer facility.

Lastly, a strong track record of qual-

ity compliance and experience working

with regulatory bodies in different juris-

dictions is also paramount.

BioPharm: How can a sponsor

company ensure their outsourcing facil-

ity is following CGMPs?


Companies should conduct site audits

to verify outsourcing best practices

and standards of GMP. Also, to check

validated systems and processes, and

that staff are experienced and properly

trained. Checking the FDA, MHRA

[Medicines and Healthcare products

Regulatory Agency], etc., history of

an outsourcing partner as well as the

QA procedures in place ensures that an

established culture of regulatory com-

pliance and high standards exists.

Hatcher and Patel (Catalent):

When outsourcing stability studies, it

is important to ensure that the contract

provider can meet regulatory require-

ments. One way in which our company

helps reassure clients about CGMP

compliance is through on-site customer

audits, where auditors can dig into the

procedures and processes and verify that

appropriate regulations are followed.

Having the outsourcing facility provide

raw data to the client is also an excellent

way to provide transparency and confi-

dence in CGMP compliance.

When working with an outsourc-

ing facility, on-site audits should be

performed prior to setting down

any studies. In addition, the sponsor

should ensure that the stability cham-

bers to be used for the study are on

site at the provider location. Lastly,

the site’s regulatory history should be

reviewed to confirm that the site has

received approval from all relevant

regulatory agencies. X



LIVE WEBCAST: Monday, September 16, 2019 at 9am EDT | 2pm BST | 3pm CEST

Register for this free webcast at: http://www.biopharminternational.com/bp_p/bioreactorsAll attendees will receive a free executive summary of the webcast!

EVENT OVERVIEWStirred-tank bioreactors are gaining more and more importance in the industrial production of pharmaceutical products, biofuels and food. When compared with 2D-cultivation systems, bioreactors are easy to handle, and multiple bioreactors can be controlled in parallel without a considerable increase of workload. In stirred-tank bioreactors cells can be cultivated as free cells in solution, cell aggregates or on microcarriers in batch, fed-batch or continuous cultivation modes.

8LI�ƼVWX�TEVX�SJ�XLMW�[IFGEWX�[MPP�JIEXYVI�E�VIZMI[�SJ�XLI�FEWMG�TVMRGMTPIW� of stirred-tank bioreactors. Ankita Desai of Eppendorf North America will explain factors that must be considered before and during the process, how to set up perfect conditions for cells, and how the WLETI�SJ�XLI�MQTIPPIV�MRƽYIRGIW�XLI�WXIQ�GIPP�GYPXYVI�

Within the second part, Arie Reijerkerk and Farbod Famili from Ncardia, a stem cell drug discovery and development company, will provide insight into how the advent of human-induced pluripotent stem cell (hiPSC) technology has substantially expanded the availability of human cells for drug discovery. The availability of these cells helps researchers to uncover the potential for better translation to the clinic and reduction of late-stage drug attrition.

Ncardia will present a case study of large-scale manufacturing development and their hiPSC-based drug screening platform, DiscoverHIT, including results from the generation of disease- relevant human stem cell-derived models and the development of predictive, phenotypic assays to enable high-throughput screening.

KEY LEARNING OBJECTIVES• �-)��*0/�/# ��#�'' )" .��)�� ) �/.�

of stirred-tank bioreactors

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• ��$ )/$./.�$)�./ (�� ''�

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• �-0"��$.�*1 -4�.�$ )/$./.

Presenters

Ankita Desai

Bioprocess Field Application Specialist

Eppendorf, North America

Dr. Arie Reijerkerk

Head of Services and Innovation

Ncardia

Dr. Farbod Famili

Project Leader

Assay Development and High Through-put Screening

Ncardia

Jennifer Markarian

Manufacturing Editor

BioPharm International

Presented bySponsored bySponsored by

From Small-Scale Stem Cell Production to Large-Scale hiPSC-Based Drug Development: The Flexibility of Stirred-Tank Bioreactors


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https://www.eppendorf.com/IN-en/



Biosimilars

Methods Accelerate Biosimilar Analysis

Effective application of mass-spectrometry tools can optimize biosimilar analysis, reducing development time and cost.

MARIO DIPAOLA AND INDU JAVERI

The backbone of biosimilar development is the ana-

lytical characterization performed on the innova-

tor—or originator or reference—product, initially

to define innovator product attributes and subsequently

on the biosimilar in development along with the innova-

tor product in order to demonstrate similarity. In May

2019, FDA published a draft guidance on the analytical

assessment and other quality-related considerations for

biosimilars, clarifying regulatory requirements and expec-

tations for demonstration of biosimilarity in support of a

marketing application (1).

As the foundation in the development of biosimilars is

the analytical strategy, this paper reviews effective analyti-

cal methodologies available to perform in-depth character-

ization of both innovator and biosimilar products. Focus is

placed mainly on high-resolution mass spectrometry (HR-

MS) for primary structural analysis and characterization

of post-translational modifications and various biophysical

techniques for higher-order structural analysis, while con-

sidering the FDA draft guidance.

US BIOSIMILAR APPROVAL ROADBLOCKSAs of Aug. 1, 2019, FDA has approved 21 biosimilars; of

these, only five are currently commercially available. Of the

approved drugs, five are biosimilars of trastuzumab; three

are biosimilars each of infliximab and adalimumab; two are

biosimilars each of filgrastim, pegfilgrastim, etanercept, and

bevacizumab, respectively; and one is a biosimilar each of

rituximab and epoetin.

Currently, the development of biosimilars is lengthy,

taking up to seven years to gain approval; the effort is

relatively expensive, costing between $150–$200 million

per biosimilar product. Identifying analytical method-

Bill

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MARIO DIPAOLA is head of operation, and INDU JAVERI is president and CEO, both with CuriRx, Inc., Wilmington, MA.



Register for this free webcast at: http://www.chromatographyonline.com/lcgc_p/data_system

Event Overview

Bioanalytical studies performed with LC-MS are a fundamental part of the drug-development process; ĉŠŖƓĶŖƙìŎŎƺ�ĖƳŠŎƳĶŖĬ�ƃĖĬƙŎìƓŠƃƺ�ĬƙĶďĖŎĶŖĖƊ�ìŖď�ĶŖƓĖŖƊĶǟĖď�

demands on data integrity mean that traceability, accuracy, and integrity of any associated data is vital to ensure the safety of therapeutic products.

In this webcast, you will learn how seamless integration of chromatography, mass spectrometry (both Quadrupole and High Resolution), chromatography data system (CDS) software and your laboratory information management system (LIMS) create a compliant, secure, scalable, and reliable solution for bioanalysis studies whilst increasing

ŎìĈŠƃìƓŠƃƺ�ĖǜĉĶĖŖĉƺ�ìŖď�ƓĲƃŠƙĬĲƀƙƓɐ

Key Learning Objectives

• DĶŖď�ŠƙƓ�ĲŠƴ�ì�ƊŠīƓƴìƃĖ�ƴŠƃŋǠŠƴ�ďĖƊĶĬŖĖď�ƓŠ�ŕĖĖƓ�ƓĲĖ�

needs of bioanalysis can streamline your processes

• Learn how a CDS can expand the potential for quadrupole and high-resolution mass spectrometry within your laboratory

• Discover how the use of a single-software platform ĉìŖ�ĶŖĉƃĖìƊĖ�ƀƃŠďƙĉƓĶƳĶƓƺ�ìŖď�ŠƀƓĶŕĶǃĖ�ĖǜĉĶĖŖĉƺ� using compliance tools, networking capabilities,

data processing, and more

For questions or concerns, email

[email protected].

Solving Problems When Using FT-IR

LIVE WEBCAST: Thursday, Sept. 26, 2019 at 9am EDT | 2pm BST | 3pm CEST

Sponsored by

Presented by

Expanded Capabilities for

Bioanalysis Leveraging a

Chromatography Data System

¡ƓƃĖìŕŎĶŖĖ�ÑŠƙƃ�d!ɥl¡�ËŠƃŋǠŠƴƊ

Presenters

Peter Zipfell

Product Marketing Manager

ªĲĖƃŕŠ�DĶƊĲĖƃ�¡ĉĶĖŖƓĶǟĉ

Kaylynn Chiarello-Ebner

Managing Editor

Special Projects

Who Should Attend:

• Laboratory Technicians / Operators

• Laboratory Managers /

Supervisors / Directors

• Quality Assurance Managers

• Pharmaceutical Quality Control

Analytical Scientists


http://www.chromatographyonline.com/lcgc_p/data_system



https://lcgc.com/


Biosimilars

ologies that provide the necessary

characterization and product com-

parability data in a shorter time can

reduce the overall product develop-

ment cycle time and, consequently,

development costs. Various analyti-

cal approaches can fit the goal of

reducing development time and cost

for biosimilars.

MASS SPECTROMETRY TOOLSMass spectrometry has been a key

technology for the characteriza-

tion of the originators’ products and

to perform comparability studies

between innovator product and bio-

similar molecules. The new genera-

tion of mass spectrometers, including

time-of-flight (ToF) instruments,

as well as hybrid systems such as

quadrupole (Q)–ToF, ion trap–ToF

or ToF–ToF, and orbital ion trap

mass spectrometers offer high mass

resolution and mass accuracy. Using

this new generation of equipment

introduced in the past five years, it

is possible to extract several product

attributes by simply determining the

intact mass of the molecule. The data

provided in Table I and illustrated in

Figures 1 and 2 demonstrate prod-

uct characteristics that can be quickly

derived from the determined intact

mass profile by a Q-ToF HR-MS.

From the mass profiles presented in

the figures, it can be concluded that

both innovator and biosimilar prod-

ucts contain similar proteoforms; of

these, the most abundant is the G0F

glycoform. While similar species are

Table I. Summary of proteoforms identified based on intact mass analysis data.

Intact mass analysis by liquid chromatography/mass spectrometry

(electrospray ionization-quadropole time of flight)

Peak#

ProteoformsTheoretical

average mass (Da)

Observed average masses (Da) for

innovator product

Observed average masses (Da) for

biosimilar product

1 unsubstituted 146637.7 146637.7 146636.8

2 G0F-GlcNAc 147879.9 147879.2 147878.8

3 G0F 148083.1 148083.1 148083.0

4 G0F+1 Lys 148210.2 148209.1 148209.0

5 G1F 148244.3 148243.6 148244.0

6 G0F + 2 Lys 148339.4 148338.4 148340.1

7 G1F + 1 Lys 148372.4 148372.3 148371.2

8 G2F 148406.3 148406.1 148405.8

Amgen Wins Enbrel Patent Case, Sandoz to Appeal

The United States District Court for the District of New

Jersey ruled in Amgen’s favor on the validity of two

patents that describe and claim Enbrel (etanercept), the

company’s anti-inflammatory biologic, and methods for

making it, Amgen announced in an Aug. 9, 2019 press

release (1).

Amgen af f i l iates Immunex Corp. and Amgen

Manufacturing, Ltd, along with the owner and licensor

of the two patents, Hoffmann-La Roche Inc., brought

the patent infringement action in Federal Court against

Sandoz Inc., Sandoz International GmbH, and Sandoz

GmbH (together, Sandoz). Before trial, Sandoz, a division

of Novartis, acknowledged that its biosimilar etanercept

infringes seven patent claims in US Patent Nos. 8,063,182,

“Human TNF receptor fusion protein,”and 8,163,522,

“Human TNF receptor”. Trial proceeded only on Sandoz’s

challenges to the validity of those claims. The Court found

that Sandoz had not met its burden to prove all seven

asserted claims invalid.

Immunex/Amgen and Sandoz have entered into an

agreement with respect to a preliminary injunction

regarding Sandoz’s etanercept as set out in the Court’s

order of June 7, 2018.

Responding to the ruling, Sandoz has said it will

appeal to the US Court of Appeals for the Federal Circuit,

according to an Aug. 9, 2019 press release issued by

the company (2).

Sandoz is the first biosimilar company to receive FDA

approval for a biosimilar etanercept, Erelzi (etanercept-

szzs), which has been approved for nearly three years,

the company stated. However, the company has been

unable to launch the medicine because of the ongoing

patent litigation from Amgen. FDA approved Erelzi on

Aug. 30, 2016 for all indications included in Enbrel’s

product label.

References1. Amgen, “Amgen Wins Patent Case on Enbrel (etanercept),”

Press Release, Aug. 9, 2019. 2. Novartis, “Sandoz Will Appeal District Court of New Jersey

Ruling in Biosimilar Erelzi (etanercept-szzs) US Patent Case,” Press Release, Aug. 9, 2019.

—The editors of BioPharm International




Biosimilars

present in both the innovator prod-

uct and the biosimilar product, close

inspection of peak intensities shows

that the innovator product has greater

enrichement of the G0F-GlcNAc

species relative to the biosimilar. In

turn, the biosimilar product displays

a higher level of the G2F glycoform

relative to the innovator molecule.

Intact mass analysis by HR-MS

can provide an abundance of informa-

tion about the integrity of the mol-

ecule and glycoforms comprising the

protein product or the presence of

oxidation, truncation, or other poten-

tial product modification. For more

detailed and accurate quantification

of these modifications and to identify

the sites where these modifications

occur, however, it is necessary to per-

form peptide mapping by liquid chro-

matography (LC)-MS/MS.

Such a quantitative approach,

involving large-scale, targeted search

of peptide mapping data has been

referred previously as a multi-attri-

bute method (MAM) (2). Figure 3

shows the typical output from the

peptide mapping of the innovator and

biosimilar products of a monoclonal

antibody as analyzed with a Q-ToF

mass spectrometer. A snippet of the

extracted data from the peptide maps

is summarized in Table II.

Based on the peptide mapping data,

summarized in Table II, the methio-

nine residue at position 256 in the

AA253-AA278 peptide is partially

oxidized; from the abundance val-

ues, it is calculated that 14.9% of this

methionine is in the oxidized form.

Additionally, the asparigyl residue at

position 319 is partially deamidated

with approximately 14.1% having con-

verted to the aspartic/isoaspartic form.

Furthermore, from the relative mea-

sured abundances, it can be quickly

determined that, of the glycoforms as

summarized in Table II, 97.7% cor-

respond to G0F, 1.3% to G1F, and

1.1% to G2F. It is quite clear from

this example that multiple product

attributes can be readily extracted and

quantified from the intact mass analy-

sis and peptide mapping.

CHARACTERIZING HIGHER-ORDER STRUCTUREHigh-resolution tandem mass spec-

trometry has shown to be quite use-

Figure 1. Intact mass profile of innovator product.

140441.2 143045.5 146638.2

148082.9

150678.1 152135.5 153813.8 156601.0 159730.3

146638.2

148082.9

148243.7

12

3

4

5

6 7 8

142767.8 145674.8

148082.8

150818.5 154086.7 156598.1 159171.2

146636.7147880.0

148082.8

148243.9

149681.1

12

3

5

4 6 7 8

Figure 2. Intact mass profile of biosimilar product.

FIG

UR

ES

CO

UR

TE

SY

OF

TH

E A

UT

HO

RS




Biosimilars

6 16 26 36 46 56 66 76 86 96

A(2

16-2

24)

AspN

A(1-29)

A(405-416)

A(152-215)B

(185-1

94)

B(1

51-1

66)

B(1

95-2

14)

A(6

2-7

2)

A(4

34-4

50)

A(4

17-4

50)

A(2

53-2

68)o

x

A(3

80-4

02)

D

A

(26

9-2

83

)

A(4

03-4

16)

A(2

84-3

15)

gly

co

syla

tio

n

A(2

53-2

68)

A(3

80-4

02)

A(2

74-2

83)

B(7

0-8

1)

B(8

2-1

21)

A(3

60-3

79)

B(1

7-6

9)

A(3

0-5

8)

A(1

09-1

51)

A

(225-2

52)B(1

85-2

14)

B(1

-16)

A(4

17-4

33)

A(7

3-8

9)

A(3

16-3

59)

B(1

70-1

84)

A(1

22-1

50

B(1

7-8

1)

Figure 3. Mirror image of peptide maps of reduced/alkylated and endo-Lys C digested monoclonal antibody product lots

(Upper: Innovator product; Lower: Biosimilar product).

ful for the characterization of primary

structure and both enzymatic and non-

enzymatic post translational modi-

fications of biologics. Until recently,

higher-order structures, including sec-

ondary and tertiary structures, were

mostly evaluated by a combination

of biophysical techniques, includ-

ing intrinsic tryptophan fluores-

cence, as well extrinsic fluorescence,

far- and near-UV circular dichroism,

Fourier-transform infrared spectros-

copy (FTIR), differential scanning

calorimetry (DSC), among others.

Unfortunately, these methods are low

resolution and fail to provide structural

details that distinguish among subtle

differences in higher-order structures.

Table II. Partial summary of peptide mapping data (innovator product lot).

Peptide Peptide sequence ModificationPredicted

mass (Da)

Measured

mass (Da)

Mass

deltaAbundance

A(152-214)DYFPEPVTVSWNSGALTSGVHTFPAVLQSS

GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK

6712.3072 6712.3190 1.76 1186377

A(218-222) KVEPK 599.3643 599.3643 -0.01 3115089

A(227-250) THTCPPCPAPELLGGPSVFLFPPK 2618.3025 2618.3039 0.51 8279074

A(253-278) DTLMISRTPEVTCVVVDVSHEDPEVK 2954.4365 2954.4395 1.00 7197104

A(253-278) DTLMISRTPEVTCVVVDVSHEDPEVK Oxidation 2970.4314 2970.4333 0.62 1259040

A(279-292) FNWYVDGVEVHNAK 1676.7947 1676.7948 0.03 3030949

A(293-321) TKPREEQYNSTYRVVSVLTVLHQDWLNGK 3459.7899 3459.7939 0.65 11536

A(293-321) TKPREEQYNSTYRVVSVLTVLHQDWLNGKG0F

Deamidation4905.3078 4905.3180 2.08 231089

A(293-321) TKPREEQYNSTYRVVSVLTVLHQDWLNGK G0F 4904.3238 4904.3264 0.54 1381426






Biosimilars

It is possible to use multidimen-

sional nuclear magnetic resonance

(MD–NMR) or X-ray crystallography

to obtain detailed analysis of both sec-

ondary, tertiary, and—as applicable—

quaternary structures, but these latter

methods are very time consuming and

quite expensive to run and certainly

not suitable to be easily applied for

the analysis of multiple lots.

Mass spectrometry has also been

found to be quite useful in provid-

ing detailed higher-order structural

information in a very timely man-

ner. These methods, which include

native ion-mobility mass spectrom-

etry (nIM–MS), collision activated

ion-mobility mass spectrometry, and

hydrogen-deuterium exchange mass

spectrometry (HDX–MS), are sub-

stantially less time consuming than

MD-NMR or X-ray crystallography

and can be easily applied across mul-

tiple lots for comparability studies.

Furthermore, the output from these

MS techniques is substantially more

informative than the low-resolution

biophysical methodologies.

In a publication by Upton, et al.,

the authors, using the combination

of IM–MS, HDX–MS, and colli-

sion-activated IM–MS, were able to

detect some minute differences in

higher- order structures among lots

of innovator Herceptin (3). While

nIM-MS and, more so, collision-

activated IM-MS have gained trac-

tion in the past few years in their

applicability for analysis of higher

structures of biopharmaceutical mol-

ecules, HDX–MS has been avail-

able for quite some time and has

been used in several comparabil-

ity studies between innovator and

biosimilar products (4). With the

introduction of more advanced soft-

ware to analyze HDX–MS data,

such as Deuteros (5), it is expected

that HDX–MS will become a more

widely used tool in the analysis of

biosimilars for demonstrating com-

parability to their respective innova-

tor products.

It is worth noting that IM has

also been used for top-down analy-

sis of proteins, even when coupled

with medium-resolution mass spec-

trometers, yielding two-dimensional

spectra. By means of two new data

analysis programs, IMTBX (IM

Toolbox) and Grppr (Grouper), the

two-dimensional spectra have been

easily and automatically processed to

obtain not only full protein sequence

but also post-translational modifica-

tions (6).

CONCLUSIONThe biosimilar industry in the United

States has been evolving more slowly

than anticipated and drug competi-

tion that would have resulted in lower

costs for biologics has yet be real-

ized. One of the reasons for this slow

start in biosimilars commercialization

is partially due to patent litigation;

however, development time and over-

all costs for biosimilars have certainly

played a central role in the failure

of biosimilars to meet commercial

expectations.

Shor tening the deve lopment

time by using more efficient ana-

lytical technologies, as discussed in

this paper, not only will speed up

the introduction of these follow-

on biologics but will also decrease

the development costs. Shortening

the development cycle and reduc-

ing development costs should entice

more biopharmaceutical as well as

start-up companies to enter the bio-

similar space, resulting in increased

competition that should translate into

reduced costs for these biologics on

behalf of the consumers.

REFERENCES1. FDA, Draft Guidance to Industry;

Development of Therapeutic

Protein Biosimilars: Comparative

Analytical Assessment and Other

Quality-Related Considerations

(Rockville, MD, May 2019).

2. R. S Rogers, et al., Mabs, (5),

881-890 (2015).

3. R. Upton, et. al., Chem Sci., 10 (9),

2811-2820 (2019).

4. R.E. Iacob, et. al., LCGC, 35(6),

382-390 (2017).

5. A.M. Lau, et.al., Bioinformatics,

2019, 1-3 (Jan. 14, 2019).

6. D.M. Avtonomov, et. al, Anal.

Chem., 2018, 90(3), 2369-2375. ◆

Turning to Plant Cell Culture for Sustainability

In an effort to develop a sustainable means to manufacture a

therapeutically important molecule, Agenus Inc., a Lexington,

MA-based immuno-oncology company, is developing

a method to produce its proprietary QS-21 Stimulon, an

adjuvant used in the manufacture of vaccines, from a plant

cell-culture-based process. Currently, the molecule is derived

from a saponin extract of the Chilean soap bark tree and

purified into an extract. Earlier in 2019, however, the Bill &

Melinda Gates Foundation awarded the company a grant of

approximately $1 million to develop an alternative, plant cell

culture-based manufacturing process to ensure a continuous

future supply of the adjuvant, which boosts the immune

respons of vaccines through multiple mechanisms.

Jennifer Buell, PhD, chief operating officer at Agenus,

discussed the company’s work on developing this new

process with BioPharm International. To read the full article,

go to www.biopharminternational.com/agenus.

—Feliza Mirasol


http://www.biopharminternational.com/agenus



Analytical Methods

Improving Oligonucleotide AnalysisOligonucleotides, which are classified as both small molecules and

biomolecules, pose unique analytical challenges. High-resolution mass spectrometry is becoming a method of choice for their development.

ANJALI ALVING

Oligonucleotides (polymeric sequences of RNA and

DNA) are becoming increasingly vital research tools

due to their ability to inhibit genes or to function as

aptamers to interact with protein targets. Over the next six

years, demand for oligonucleotides in areas that include gene

therapies is expected to grow by 20.6% per year to reach

US$3697 million by 2026 (1).

One of the ways in which oligonucleotides work is by

targeting RNA function at the cellular level, where specific

malfunctioning genes can be targeted, manipulated, and/or

modulated (2). Robust, accurate analytical characterization

of oligonucleotides is necessary in order to confirm their

identity, and to determine purity, quality, and strength. A

wide range of oligonucleotides are available for clinical and

research use, with specific tools ranging in size, chemical

modifications, and degree of conjugation.

The diversity of oligonucleotides has made their analysis

challenging, necessitating the development of new ana-

lytical techniques that can address the varied tasks in a

high-throughput environment. This article highlights mass

spectrometry (MS) techniques that are improving the analy-

sis of oligonucleotides and enabling high-throughput analysis.

Oligonucleotides are commonly produced through syn-

thetic solid-phase chemical synthesis, and precise analytical

characterization is required to confirm identity, determine

safety, and to identify and quantify contaminants. Numerous

impurities must be identified and removed, and post-syn-

thesis processing must be monitored. Due to the complexity

and diversity of oligonucleotides in clinical development,

for example, differences in identity, structure, biological

potency, and physiochemical properties, a range of analyti-

cal approaches has been developed. These methods include

liquid chromatography–mass spectrometry (LC–MS),

high-performance liquid chromatography (HPLC), nuclear

magnetic resonance (NMR), Fourier transform infrared

spectroscopy (FTIR), and polyacrylamide gel electrophoresis

(PAGE).

Oligonucleotide analysis is an essential part of drug devel-

opment, registration, and quality control (QC), so methods

that are capable of separating and identifying impurities must

be established.

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ANJALI ALVING, [email protected], is a senior scientist for pharmaceuticals and biopharmaceuticals at Bruker Daltonics.




Covering the science and business

of biopharmaceuticals• Quality/Analytics • Upstream Processing • Downstream Processing

• Peer-Reviewed Technical Notes • Product Spotlight • Perspectives on Outsourcing

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Analytical Methods

The limited accuracy and speed of

electrophoretic techniques such as

PAGE have led the industry towards

HPLC for the separation of oligonu-

cleotides for analysis. However, oli-

gonucleotides with similar molecular

weights present a challenge for HPLC,

particularly those with higher molecu-

lar weights, so development of robust

MS methods to characterize and quan-

titate oligonucleotides and synthesis

contaminants has become a key focus.

HPLC-MS is superior due to its com-

bination of high mass accuracy, high

throughput, reproducibility, robustness,

sensitivity and precision that the other

analytical techniques lack.

Determining molecular weight and

confirming the nucleotide sequence

of an oligonucleotide are fundamental

criteria for establishing the molecule’s

identity, which is a regulatory require-

ment. Oligonucleotide synthesis is

a complex process that requires more

than 100 sequential chemical reactions

to make a single 25-base sequence (3),

and the quality of each synthesized oli-

gonucleotide must be evaluated prior to

use to ensure that the correct sequence

was made and that purity meets regu-

latory standards.

Numerous methods can be applied

to obtain this information, and devel-

opments in high-resolution MS in

particular have provided a viable alter-

native to other methods such as PAGE,

offering significantly better accuracy

for the determination of both mass and

sequence of oligonucleotides. PAGE

is a subjective method of analysis and

can only give average mass informa-

tion whereas MS is a high resolution

technique and high resolution accurate

mass (HRAM) mass spectrometers can

provide monoisotopic mass information

of the oligonucleotides.

Oligonucleotides are considered

neither small nor large molecules,

leading to many regulatory challenges

and a lack of guidance. The variety in

their structure (i.e., whether they are

single or double-stranded), molecular

weight, molecular size, and number of

negative charges all affect the molec-

ular interaction with targeted tissue.

These factors have led to disagreement

between regulatory agencies, such

as the European Medicine Agency

(EMA) and the US FDA, in how to

classify these species. Specifically, FDA

considers oligonucleotides as small

molecules, but the EMA uses a cen-

tralized procedure required for drug

products manufactured using biotech-

nology processes (4). Consequently,

there is no official FDA guidance for

QC expectations. However, the agency

has issued some guidance on the anal-

ysis of oligonucleotides with respect to

identity, purity, quality, and strength,

as part of the chemistry, manufactur-

ing, and controls (CMC) regulatory

process (5).

In addition to their regulatory ambi-

guities, oligonucleotides can be chal-

lenging to use in gene therapies

because their diverse modes of action

can complicate their delivery in vivo.

For one thing, oligonucleotide delivery

is a two-fold process. First, the oligo-

nucleotide must be transported to the

tissue of therapeutic interest with mini-

mized exposure to other tissues. Then

it must then be delivered to the correct

intracellular compartment in order to

function. In order for an oligonucle-

otide to downregulate gene expression,

it must penetrate into the targeted cells.

Systemic oligonucleotide delivery has

been accomplished in rodent models,

but their action becomes localized to

the liver (6), and high oligonucleotide

accumulation is also observed in the kid-

ney and other organs such as the spleen,

heart, pancreas, and the brain, which

show far lower concentrations.

Single-stranded DNA and RNA oli-

gonucleotides also have other proper-

ties that complicate drug development.

For example, they can be degraded by

nucleases when introduced into biologi-

cal systems; other potential problems

include: poor uptake through cell mem-

branes, unfavorable bio-distribution

and pharmacokinetic properties, and

sub-optimal binding affinity for com-

plementary sequences (7). Fortunately,

challenging delivery issues are being

addressed (e.g., through approaches

such as chemical modification of the

oligonucleotide itself; implementation

of lipid or polymeric nanocarriers; and

linking oligonucleotides to receptor-

targeting agents such as carbohydrates,

peptides, or aptamers).

As a result, significant contributions

have been made at a therapeutic and

clinical level, applying tailored solutions

based on the characteristics of the mol-

ecules involved. Even with the most effi-

cient synthesis, however, it is crucial that

synthesized oligonucleotides be evalu-

ated for quality before they are used in

molecular biology applications.

ANALYZING WITH MSMS techniques, including electrospray

ionization (ESI) and matrix-assisted

laser desorption/ionization (MALDI),

are being used to obtain accurate

molecular weight, sequence confirma-

tion, and characterize impurities in

both high- and low-throughput modes.

These powerful technologies can be

applied across the biopharmaceutical

pipeline, from oligonucleotide devel-

opment through to QC.

High-resolution MS generates accu-

rate data to support characterization

requirements. A mass spectrum can

be a useful QC tool to help verify that

a custom oligonucleotide was syn-

thesized correctly and, more specifi-

cally, that it has the expected molecular

weight based upon the requested base

sequence. When considering intact

mass, low-resolution instrumentation

can only be used to obtain the average

molecular weight. In contrast, high-res-

olution mass spectrometry enables the

determination of accurate mass. This

method is based on obtaining negative

ion spectra of the oligonucleotide fol-

lowed by interpretation of the spectra.

This is typically done using deconvolu-

tion software algorithms where all the




Analytical Methods

multiply-charged species are recalcu-

lated into the singly-charged form.

The accuracy of these measurements

is typically less than 5 ppm, and, as

such, the mass can be used to help

establish the empirical formula of the

molecule, which is in turn used to pos-

tulate or confirm structure (8).

In addition to determining accu-

rate molecular weight, MS provides

information about the sequence and/or

structure of the biomolecules. When

comparing the use of MALDI and ESI

for the analysis of oligonucleotides, it

is important to consider the type of

mass analyzer used because that will

determine the limits of resolution and

mass range. Analyses of LC-separated

and ionized samples can be accom-

plished through the use of different

mass analyzers. These can be divided

into two main groups based on their

working methods: beam type analyzers

that continually scan ions (e.g., time-

of-flight [TOF] and quadrupole) and

trap-based analyzers that capture ions

of interest for a specific time to acquire

mass spectrum.

A quadrupole mass analyzer acts

as a variable mass filter that separates

ionized species using only electri-

cal fields generated by a direct current

and superimposed radio-frequency

potential. Ions are then introduced in

a path parallel to that of a quadrupole

rod and only ions of a particular mass

and charge can pass through, with all

non-confirming ions filtered out. The

majority of quadrupole TOF (QTOF)

MS systems have tandem capabilities,

so that precursor ions that have been

separated by mass-to-charge ratio in

the first stage (MS1) can be selected

and then separated and detected in

the second stage (MS2) as fragments

(product ions) in the QTOF. Precursor

ions are selected in the quadrupole and

sent to the collision cell for fragmenta-

tion, and the generated product ions are

then detected by TOF MS.

MALDI-TOF offers high sensitiv-

ity for oligonucleotides, is relatively

easy to use, and is well-suited to a

high-throughput laboratory environ-

ment. The method is also reasonably

tolerant of the presence of salts, buffers,

and other additives. Accurate measure-

ment by MALDI-TOF is limited to

oligonucleotides less than 50 bases in

length. Longer oligonucleotides tend

to ionize poorly or “fly” in a MALDI–

TOF instrument.

Alternatively, ESI technology

maintains high mass accuracy, reso-

lution, and sensitivity over a range

of lengths (20–120 bases), but has

a reduced throughput compared to

MALDI–TOF. MALDI–TOF is ame-

nable to high throughput since the

sample/matrix mixture is spotted on

a metal grid which is then analyzed

in the instrument, with data typically

acquired in a fraction of a second. In

ESI–MS, the sample is introduced

via a syringe or injected by an HPLC

system, and data acquisition takes

place in a matter of several minutes,

depending on the complexity of the

samples.

This requires closer attention to be

given to sample cleanliness, which is

important in ESI as the instrument

is more susceptible to contamination.

Generally, ESI is most effective in syn-

theses involving more than 50 bases.

THE FUTURE OF ANALYSISOligonucleotides continue to present

a growing opportunity, as well as an

analytical challenge, to biopharmaceu-

tical developers. MS techniques such

as ESI-QTOF and MALDI-TOF

can be used for ultra-high-resolution

analysis of oligonucleotides to achieve

sensitive detection with enhanced

speed and data quality. Current data

analysis software such as Bruker

Daltronics’ BioPharma Compass

3.1 is available that can work with

both high resolution ESI-QTOF and

MALDI-TOF data.

Advances in these technolo-

gies promise to facilitate the ongo-

ing development of more efficient

oligonucleotide therapies, improving

methods of delivery as well as the

QC pipeline. Oligonucleotides show

low stability against nuclease in vivo,

but progress in the development of

chemically modified nucleic acids and

technology has allowed the develop-

ment of a number of stable and effec-

tive candidate products.

In 2016 Nusinersen (Spinraza), an

antisense oligonucleotide drug for

spinal muscular atrophy, was approved

as an oligonucleotide therapeutic for

infants (9). Current techniques and

studies are paving the way for future

analysis of polymeric sequences of

nucleotides. Coupled with novel data-

analytical software, MS is supporting

the development of new oligonucle-

otide therapeutics.

REFERENCES1. Zion Market Research, “Oligonucleotide

Synthesis Market Expected to Reach USD 3,697 Million By 2026, Globally,” Press Release, January 2019.

2. B2B Labs, “Oligonucleotides: Opportunities, Pipeline and Challenges,” accessed Dec. 12, 2016, www.b2blabs.com/2016/06/oligonucleotides-opportunities-pipeline-challenges/

3. Integrated DNA Technologies, “Technical Report: Mass Spectrometry Analysis of Oligonucleotide Syntheses,” 2017, www.sfvideo.blob.core.windows.net/sitefinity/docs/default-source/technical-report/mass-spec-of-oligos.pdf?sfvrsn=ca483407_6

4. B2B Labs, Oligonucleotides: Opportunities, Pipeline and Challenges, June 2016. http://b2blabs.com/2016/06/oligonucleotides-opportunities-pipeline-challenges/

5. FDA, “CMC Regulatory Considerations for Oligonucleotide Drug Products: FDA Perspective,” pqri.gov, 2017. www.://pqri.org/wp-content/uploads/2017/02/3-SapruPQRI-FDA-Conference-Oligo-2017-Presentation.pdf

6. D. Argyle et al., “Molecular/Targeted Therapy of Cancer,” in Small Animal

Clinical Oncology (Fourth Edition), ed. Stephen J.. Withrow, David M. Vail, (Withrow & MacEwen, 2007).

7. X. Shen, D. R Corey, Nucleic

Acids Research, 46(4) (2018).8. R. Houghton, “Oligonucleotides: The

Next Big Challenge for Analytical Science,” Chromatography

Today, March 2011).9. C. Stein and D. Castanotto, Molecular

Therapy, 25 (5) (2017). X



http://b2blabs.com/2016/06/oligonucleotidesopportunities-pipeline-challenges/

http://www.sfvideo.com/

http://b2blabs.com/2016/06/oligonucleotidesopportunities-pipeline-challenges/

http://pqri.org/wp-content/uploads/2017/02/3-SapruPQRI-FDA-Conference-Oligo-2017-Presentation.pdf


Operations

Container Closure Integrity Testing of Finished Sterile Injectable Product

As regulatory guidance has changed, so too has CCIT testing. In this article, possible CCIT strategy approaches are outlined.

DEREK DUNCAN

Container closure integrity (CCI) plays an important

role in maintaining the sterility and stability of sterile

injectable products. The defects that cause a sterile

vial to leak are not necessarily detectable by a visual inspec-

tion process. Examples of such defects are those that are

hidden by the crimp, microscopic cracks and scratches in

the glass, or temporary defects such as stopper pop-up that

result in temporary container leakage.

New regulatory guidance has triggered changes in industry

best practices in the area of CCI testing (CCIT). This article

summarizes the current state of container closure integrity testing

in the pharmaceutical and biopharmaceutical industries and out-

lines possible approaches for developing a CCIT strategy.

REGULATORY ENVIRONMENT FOR CCIHistorically, good CCI has been linked to the maintenance of

sterility. A container that loses, or does not have, good closure

integrity is at risk for microbial contamination. However, the con-

text of CCI has become broader over the years.

An increasing number of formulations have significant

sensitivity to oxygen and need to be packaged under an inert

atmosphere. Freeze-dried product requires protection against

water vapor and is often packaged at a partial vacuum to help

with reconstitution and/or seating of the stopper. In these cases,

good CCI is necessary not only for the maintenance of sterility

but also to maintain critical headspace gas conditions.

Note that, quite generally, a container that is gas-tight will also

be tight against microbial ingress. Therefore, the requirement to

maintain headspace gas conditions imposes higher standards on

CCI than the requirement to maintain sterility.

In light of the importance of CCI for product sterility and sta-

bility, regulatory guidance has placed an increasing emphasis on

CCI concepts. The current United States Pharmacopeia (USP)

<1207> chapter titled Package Integrity Evaluation—Sterile

Products was implemented in late 2016 and represents the most

thorough guidance document to date on CCI concepts for sterile

injectable product (1).

The chapter gives an overview on CCIT technolo-

gies and approaches for CCI control over the product life-

idam

be

er

- S

tock

.Ad

ob

e.c

om

DEREK DUNCAN, PHD is director of Lighthouse Instruments, Amsterdam, The Netherlands.




Operations

cycle. Traditional CCIT methods, such

as microbial challenge tests or blue dye

ingress tests, are described as methods

associated with probabilistic outcomes

having some uncertainty in the results

which, in turn, makes such methods dif-

ficult to quantitatively validate for the

detection of critical leaks (1). The chap-

ter also makes clear that CCIT should

be performed throughout the product

lifecycle. Deterministic CCIT methods

based on non-destructive analytical mea-

surements can be used to generate sci-

ence-based CCI data that, coupled with

a risk-based approach, enable informed

decisions about a CCIT strategy in

commercial manufacturing.

A draft revision of the European

Union’s Annex 1 requirements for sterile

product manufacturing was released at

the end of 2017 (2). CCIT was a popular

discussion topic for the revision, and the

draft text contains new requirements for

CCIT in manufacturing. Other world

regulatory bodies, Russia and South

Korea for example, have also been putting

increasing emphasis on CCI control for

finished sterile products. It is clear from

these developments that regulators are

wanting to see improved industry prac-

tices in the area of CCIT.

CCI TEST METHODSUSP <1207> provides an overview of

CCIT technologies and categorizes them

as being deterministic or probabilistic (see

Table I). The chapter emphasizes that

this overview of CCIT technologies is

not exhaustive but is a summary of tech-

nologies that have been implemented for

CCIT in the pharmaceutical industry

and that are described by a body of peer-

reviewed literature.

It is important to distinguish between

CCI technologies and CCI test methods.

Once a leak testing technology has been

chosen as the basis for a test method, the

chapter emphasizes the need to perform

method development studies generating

data that demonstrate detection of a criti-

cal leak for a specific product container

configuration using defined test method

parameters (1): “After a methodology has

been selected for use, the test equipment

operation and performance is qualified.

Test method parameters are optimized

during method development and con-

firmed during validation. Thus, a final leak

test method is specific to a particular con-

tainer-closure or product-package system.”

Another point emphasized in the

chapter is that “no one test is appropri-

ate for all packages or for all leak test-

ing applications.” The chapter and its

three sub-sections describe a framework

in which appropriate CCI test method-

ologies are chosen, optimized per product

configuration, and a robust validation of

the method for detecting a critical leak

is performed. In selecting a methodology,

“deterministic leak test methods are pre-

ferred over probabilistic methods when

other key method selection criteria per-

mit.”

Package integrity data are generated

over the product lifecycle and serves as

input for an ongoing database of CCI

data (the package integrity profile), which

then serves as a risk management tool

to ensure that CCI of finished product

meets the product quality requirements.

The framework described in the chapter

is currently driving changes in industry

best practices for CCI testing, including:

• Implementation of a ‘toolbox’ of CCI

test methods optimized and chosen

on a per product configuration basis

rather than the application of a single

legacy test method in a one-size-fits-

all approach

• Generation of science-based CCI data

in robust method validation studies,

which demonstrate the detection of

a critical leak represented by various

types of positive controls.

STATISTICAL SAMPLINGA big topic of current discussion is

how much CCIT is required, especially

for commercial batches of finished

sterile product. Despite the general

consensus that CCI is a critical quality

parameter for finished sterile product,

the industry has historically expended

much more effort on testing for par-

ticle contamination than for CCI.

Visual inspection to detect particulate

contamination has been a requirement

for many years with 100% inspection of

finished parenteral product being done

manually or by automated inspection

platforms. In the context of risk to the

patient, a loss of CCI would, in general,

be assessed as being just as critical as par-

ticle contamination.

The current EU Annex 1 guidelines

require 100% leak testing for certain types

of product containers. “Containers closed

by fusion, e.g., glass or plastic ampoules,

should be subject to 100% integrity test-

ing” (3). This requirement is a result of the

fact that the inherent failure rate of the

sealing process for these types of contain-

ers cannot be sufficiently controlled.

The ongoing draft revision of the

EU Annex 1 guidelines again states the

requirement of 100% integrity testing

for fused containers and adds the follow-

ing requirements for all other types of

containers. “Samples of other containers

should be checked for integrity utilizing

validated methods and in accordance with

Table I. Overview of container closure integrity testing (CCIT) technologies.

Deterministic Probablistic

Electrical conductivity and capacitance (high-voltage leak detection)

Bubble emission

Laser-based gas headspace analysis Microbial challenge, immersion exposure

Mass extraction Tracer gas detection, sniffer mode

Pressure decay Tracer liquid (blue dye ingress)

Tracer gas detection, vacuum mode

Vacuum decay

Source: Adapted from USP 40 <1207.2>




Operations

QRM, the frequency of testing should be

based on the knowledge and experience

of the container and closure systems being

used. A statistically valid sampling plan

should be utilized. It should be noted that

visual inspection alone is not considered

as an acceptable integrity test method”

(2). If finalized in this form, these CCIT

requirements will require the evolution of

best practices for CCIT in the manufac-

turing environment.

Currently, a small percentage of the

industry performs statistical CCIT of fin-

ished commercial product. Most compa-

nies point to the 100% visual inspection

process to justify meeting current CCIT

guidance, such as the following from the

FDA (4). “A container closure system that

permits penetration of microorganisms is

unsuitable for a sterile product. Any dam-

aged or defective units should be detected,

and removed, during inspection of the

final sealed product.” The language of the

draft EU Annex 1 revision makes clear

that visual inspection is not considered an

acceptable integrity test method; in other

words, the CCI test methods that enable

the testing of larger amounts of samples

will need to be implemented.

To demonstrate statistical confidence

in the process requires the generation of

statistical CCI data. However, an argu-

ment could be made that a better place

to do this in the product lifecycle is in

process development and scale-up rather

than in manufacturing. The guidance pro-

vided in USP <1207> to collect package

integrity data throughout the product life-

cycle so that a package integrity profile

database is built up implies an approach

in which a significant amount of CCI

data are generated outside of the manu-

facturing environment. The generation of

robust CCI data providing knowledge of

the container and closure system (which

then gives guidance to a CCIT strategy

in manufacturing) is also implied in the

text of the draft revised EU Annex 1, “the

frequency of testing should be based on

the knowledge and experience of the con-

tainer and closure systems being used.”

Figure 1 outlines a possible approach

to generating CCI data that enables the

design of an appropriate CCI testing pro-

gram in manufacturing.

After validation of the fundamental clo-

sure system, data need to be generated to

understand if the process introduces risk to

CCI. To gain statistical confidence in the

process, it would be necessary to perform

testing on statistical sample sets. This in

turn will require the use of non-destructive

deterministic test methods because the

probabilistic legacy test methods (blue dye

and microbial ingress testing) have limited

throughput capability. Testing could be

done on either a pilot scale or with test

and engineering batches from the manu-

facturing environment. Once a baseline

failure rate has been established, process

controls could be implemented to improve

the process, if necessary.

Product from the improved process

would be tested to quantify the residual

risk to CCI after which a decision could

be made for an appropriate testing strat-

egy in manufacturing. Packages and pro-

cesses having a high inherent failure rate

that is difficult to control would require a

heavier inspection process and vice versa.

In this way, the decision for an inspection

process design is driven by science-based

statistically relevant data.

SUMMARYThe current environment for CCIT of

sterile injectable product is evolving. New

regulatory guidance recognizes CCI as a

quality parameter that is critical for the

maintenance of both the sterility and the

stability of finished sterile product. New

concepts introduced in the regulatory

guidance are changing industry best prac-

tices and include the following:

• Generate science-based CCI data

throughout the product lifecycle to

build up a package integrity profile

database that can be used as input for

risk management.

• When possible, use deterministic CCI

test methods that have been validated

to detect a critical leak.

• There is no one-size-fits-all CCI test;

a toolbox of CCIT technologies that

can be optimized on a per-product

package configuration is necessary for

a robust CCIT program.

Because industry best practices will

be evolving as the impact of new guid-

ance becomes clearer, a certain amount

of uncertainty in CCIT best practices is

to be expected in the near term. However,

a general approach that includes the

implementation of validated deterministic

CCIT methods and the increased genera-

tion of science-based CCI data to enable

informed risk assessments will help pre-

pare the industry for the future.

REFERENCES1. USP, USP 40 <1207> “Sterile

Product Packaging—Integrity Evaluation” (US Pharmacopeial Convention, Rockville, MD, 2017).

2. EC, EU GMP Annex 1 Revision:

Manufacture of Sterile Medicinal Products

(Draft) (Brussels, December 20, 2017). 3. EC, EudraLex, Volume 4, EU Guidelines to

Good Manufacturing Practice-Medicinal

Products for Human and Veterinary

Use, Annex 1, Manufacture of Sterile Medicinal Products (Brussels, 2009).

4. FDA, Guidance for Industry: Sterile Drug

Products Produced by Aseptic Processing—

Current Good Manufacturing Practice

(FDA, Rockville, MD, September 2004).◆

Figure 1. Schematic of possible approach to generating container closure integrity (CCI) data.

Fig

ure

is

cou

rte

sy o

f th

e a

uth

or

Understand base-

line failure rate

Quantify

residual risk

CCI testing

strategy

Implement / improve

process controls



Downstream Processing


sin

hyu

- S

tock

.Ad

ob

e.c

om

What’s New in Manufacturing: Process Chromatography

Advances in process chromatography are necessary for

accuracy and productivity in biologics manufactur-

ing. The following products promise to improve and

enhance the reliability of process chromatography.

PRE-PACKED CHROMATOGRAPHY COLUMNSThe ReadyToProcess chromatography column with 600 mm

inner diameter is the latest addition to the ReadyToProcess

chromatography column line from GE Healthcare. The pre-

packed, ready-to-use column offers enhanced flexibility and

productivity in clinical and commercial manufacturing by

eliminating the need for column preparation, packing, and

validation procedures, according to the company.

Introduced to the market in July 2019, the

ReadyToProcess 600 mm inner diameter chromatography

column comes equipped with bed heights of 100–250 mm.

The column is compatible with a variety of chromatography

resins including those used for monoclonal antibody (mAb)

purification, viral vectors, plasmids, recombinant proteins,

and more.

Validation columns from GE Healthcare involve pre-

packed scale down Tricorn 10/200 columns for process

development and process validation. The columns can be run

on chromatographic systems, including the company’s own

ÄKTA chromatography systems, and are prepacked to save

time, improve reproducibility, and eliminate the need for

packing expertise, the company says.

The columns come equipped with a bed height of 20

cm and an inner diameter of 10 mm. MabSelect PrismA,

MabSelect SuRe, MabSelect SuRe LX, Capto S ImpAct,

Capto Q, Capto SP ImpRes, and Capto Adhere prepacked

resins are available for use with these columns.

The latest advances in process chromatography include pre-packed chromatography columns, process characterization kits, fast protein liquid

chromatography systems,and mixed-mode chromatography resins.

LAUREN LAVELLE




Downstream Processing

CHROMATOGRAPHY PROCESS CHARACTERIZATION KIT GE Healthcare also introduced

Process Characterization Kits to its

catalog in July 2019. The kits provide

access to knowledge from resin devel-

opment projects, enable risk assess-

ments and process characterization

of resin variability, and interplay with

process parameter impact on pro-

cess performance and critical quality

attributes. According to the company,

this aids in the understanding of pro-

cess parameters and raw material

attributes, which are key to develop-

ing successful control strategies and

productive processes.

The kits come with three lots of a

given resin with different ligand den-

sities (high, center, and low) and are

available for Capto S ImpAct, Capto

SP ImpRes, Capto adhere, Capto

adhere Impres, Capto MMC, Capto

MMC ImpRes, Capto Phenyl, Capto

Phenyl ImpRes, and Capto Butyl

ImpRes resins.

SOON-TO-BE-RELEASED CHROMATOGRAPHY PRODUCTS ÄKTA go is GE Healthcare’s most

recent addition to the ÄKTA chro-

m a t o g r a p h y s y s t e m s p o r t f o -

l io. Deve loped for automated

chromatography descended from

fast protein liquid chromatography

(FPLC) technology, the system sup-

ports routine lab-scale protein purifica-

tion for affinity, size exclusion, and ion

exchange chromatography.

With a flow rate range of 0.01 to 25

mL/min, an operating pressure of 0 to

5 MPa (50 bar, 725 psi), pressure, ultra-

violet, conductivity, and pH monitors,

and dimensions of 335 × 482 × 464

mm without accessories, the ÄKTA

go is designed to simplify method cre-

ation, achieve desired purity with ease,

and make the most of bench space in

crowded laboratories. The company

plans to introduce the system to the

market in October 2019.

Chromatography

products promise

to improve and

enhance the

reliability of process

chromatography.

Similarly, Repligen will introduce

a new proprietary method of pack-

ing ceramic hydroxyapatite resin

in its OPUS pre-packed columns

in September 2019. The columns

will be ≤45 cm in diameter with a

range of configurable bed heights

(5–30cm) and standard OPUS pre-

packed column lead times will apply,

according to the company.

PROCESS CHROMATOGRAPHY RESINSOriginally introduced in 2016, the

next generation of Pall Biotech’s

CMM HyperCel mixed-mode resin

is designed for large-scale drug man-

ufacturing. The resin works to sepa-

rate proteins with similar isoelectric

points and hydrophobicity in broad

conditions while using a pH from

five to eight and conductivity up to

50 ms/cm.

The product is made up of a cel-

lulosic matrix on which amino ben-

zoic acid provides the ionic charge and

hydrophobicity attached to it. It aids in

process and product-related impurity

removal and is particularly suitable for

new engineered modality of mono-

clonal antibodies (mAbs) that are dif-

ficult to purify or recombinant proteins.

The resin is also able to work from

low to high conductivity and avoid the

dilution needs required with cation

exchange conditions.

The rebooted resin has an applied

flow rate allowing three-to-four-

minute residence time, a binding

capacity of 100 mg/mL immuno-

globulin G (IgG) at pH 5, 12 ms/cm,

and > 60 mg/ml mAb at 25 ms/cm

efficient ion exchange at high con-

ductivity with no need for dilution

or defiltration, the company states.

The Eshmuno CPS Resin from

MilliporeSigma is a cation exchange

resin that combines high dynamic

binding capacity and separation

efficiency in high-salt purification

processes. According to the com-

pany, the resin’s salt tolerance allows

for direct loading of high conduc-

tivity feed streams, which reduces

costs, time, and manufacturing. It

also features easy process develop-

ment capabilities with bind and

elute conditions and selection of

process parameters.

The resin is composed of a base

matr ix , hydrophi l ic polyv iny le-

ther polymer, and a sulfonate cat-

ion exchanger. It also has a capture

and intermediate polishing step of

recombinant proteins, enzymes, anti-

body fragments, growth factors, etc.

The product has a linear flow rate up

to 500 cm/h (<3.0 bar net pressure)

and a binding capacity of approxi-

mately 160 mg lysozyme/mL of gel.

GE Healthcare’s high resolution

Capto HiRes Q and Capto HiRes S

ion exchange chromatography col-

umns are designed for high reso-

lution separation for protein and

nucleic acid purification or analysis.

Prepacked in a Tricorn high-perfor-

mance column, the resins are based

on high-flow agarose with a particle

size of 8 um.

The resins come with bed heights

of 50 mm (5/50 column) and 100

mm (10/100 column); column inner

diameters of 5 mm (5/50 column)

and 10 mm (10/100 column); max-

imum pressure over a packed bed

of 4.0 MPa; and an ionic capacity/

ml packed resin of ~0.23 mmolCl-/

mL resin for the Capto HiResQ and

~0.12 mmol H+/mL resin for the

Capto HiRes S. X



September 2019 www.biopharminternational.com BioPharm International 49

New Technology Showcase

Ask the Expert — Contin. from page 50

INTEGRATED BIOLOGICS DEVELOPMENT SERVICESCatalent Biologics has the

capabilities and experience in

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Tel.+1 888.765.8846, [email protected], www.catalent.com

OSMOTECH® PRO MULTI-SAMPLE MICRO-OSMOMETERThe OsmoTECH PRO Multi-Sample Micro-Osmometer

from Advanced Instrument tests osmolality–an essential

parameter for process control and QC. Designed to meet

these testing pharmacopeia guidelines, OsmoTECH

PRO utilizes freezing point depression–the gold

standard method for testing osmolality. Designed

for user-friendliness, OsmoTECH PRO has an intuitive touchscreen

interface, a 20-position turntable (that requires only a small sample

of 30μL), and arrives factory-calibrated so your lab can start testing

quickly. Built with the most data-management and security features

of any osmometer currently on the market, the OsmoTECH PRO

also helps support 21 CFR Part 11 and EU Annex 11 compliance.

Advanced Instruments, www.aicompanies.com

VALPLUS™ CONFIDENCE THROUGH VALIDATIONSaint-Gobain Life Sciences

ValPlus™ provides pharmaceutical

manufacturers increased confidence

in the cleanliness of the tubing

products they use in critical applications. Available with a variety of

Saint-Gobain tubing brands such as C-Flex® and Sani-Tech®, ValPlus

is certified to USP <788> for particulate, USP <85> for bacterial

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Saint-Gobain Life Sciences, www.biopharm.saint-gobain.com/valplus

AMBR® 15 CELL CULTURE GENERATION 2ambr® 15 cell culture from

Sartorius Stedim Biotech

is a high throughput,

automated bioreactor

system for 24 or 48 parallel

cultivations at the 10–15

mL microbioreactor scale.

Offering new functionality,

a high level of flexibility and improved performance the

Generation 2 system is ideally suited for applications including:

clone selection, media and feed development, early stage process

optimization, and screening under perfusion mimic conditions.

Sartorius Stedim Biotech, www.sartorius.com

• Your personnel training and personnel following

procedures is correct

• Your contamination control strategy for all points of

risk of contamination from gloved hands is appro-

priate and sufficient.

If not, you should be able to immediately identify

corrective action and preventive action (CAPA) mea-

sures to reduce the risks to an acceptable level.

The CCS will bring all these assessments together

and provide a holistic view of your approach to con-

tamination control. The CCS is not only needed for

responding to the inspection observations, it is an

essential document for any pharmaceutical operation

and, in particular, for sterile manufacturing ones.

REFERENCES 1. EC, Annex 1, Manufacture of Sterile Medicinal Products,

December 2017. Link: https://ec.europa.eu/health/sites/

health/files/files/gmp/2017_12_pc_annex1_consultation_

document.pdf

2. PHSS, Control Strategy in Manufacture of Sterile

Pharmaceutical/Drug Products, Whitepaper, www.phhs.co.uk.

3. CDC, Morbidity and Mortality Weekly Report, 51 (RR-16), (Oct.

25, 2002). Link: www.cdc.gov/mmwr/PDF/rr/rr5116.pdf ◆

Ad Index

Company Page

AGILENT TECHNOLOGIES 19

CATALENT PHARMA SOLUTIONS 52

CPHI WORLDWIDE 51

EPPENDORF 7, 33

IRVINE SCIENTIFIC 15

KNAUER GMBH 21

MASTER CONTROL SYSTEMS 9

MILLIPORE SIGMA 13

PDA 11

PENDOTECH 25

PHRMA 2

READING SCIENTIFIC SERVICES LTD 27

SARTORIUS STEDIM BIOTECH 5

THERMO FISHER SCIENTIFIC 17, 35

YOURWAY TRANSPORT COVER TIP



http://www.catalent.com

http://www.aicompanies.com

http://www.biopharm.saint-gobain.com/valplus

http://www.sartorius.com


https://ec.europa.eu/health/sites/health/files/files/gmp/2017_12_pc_annex1_consultation_document.pdf

https://www.fda.gov/drugs/development-approval-process-drugs/pharmaceutical-quality-resources

https://www.cdc.gov/mmwr/PDF/rr/rr5116.pdf


Ask the Expert

Contin. on page 49 Fa

na

tic S

tud

io/G

ett

y I

ma

ge

s

Siegfried Schmitt, PhD, vice-president, technical,

Parexel Consulting.

Q: A recent inspection of our facility resulted

in several observations, including insuf-

ficient hand sanitization by personnel in the

sterile filling area, deficient gowning in the

microbiology laboratory, and use of wooden

pallets in the cold storage area. Before conclud-

ing, the inspectors told us that they want us to

address these deficiencies holistically. Can you

give some advice on how to do this?

A: It is correct that all too often, compa-

nies address each and every inspection

observation individually, rather than address

fundamental flaws or gaps in the systems, pro-

cesses, or organizations. The regulators want

the industry to find the true root causes for

their compliance lapses. This is why your

inspectors want to see the issues they identified

addressed holistically. For example, they want

you to clearly identify the functional relation-

ship between the parts that lead up to a compli-

ant operation and the whole.

Looking at the examples you cite, it seems

that you would be best served by preparing a

contamination control strategy (CCS). Control

strategies are an increasing requirement in the

European Union good manufacturing prac-

tice regulations (EU GMPs) and expected to

be referenced in the next revision of EU GMP

Annex 1 (1). The draft text, which is likely to be

adopted in the final version, reads:

‘Quality assurance is particularly impor-

tant, and manufacture of sterile products must

strictly follow carefully established and vali-

dated methods of manufacture and control. A

contamination control strategy should be imple-

mented across the facility in order to assess the

effectiveness of all the control and monitoring

measures employed. This assessment should

lead to corrective and preventative actions being

taken as necessary. The strategy should consider

all aspects of contamination control and its life

cycle with ongoing and periodic review and

update of the strategy as appropriate.’

The regulators want

the industry to find the

true root causes for their

compliance lapses.In your case, the CCS should consider all the

integral elements of sterile product manufactur-

ing, including quality risk management (QRM)

principles and supporting risk assessments for

contamination control and monitoring (detect-

ability of contamination event) (2). How should

or could you develop this CCS? First, you need a

complete process flow description of all materi-

als and personnel. This will allow you to iden-

tify points of critical risk of contamination and

points of inherent risk of contamination.

If we take the examples provided by your

inspectors, you will need to identify where

there is a risk for contamination (e.g., the oper-

ators) and how you control this risk of con-

tamination (e.g., gowning and barriers, such

as isolators and disinfection). Next, you need

to determine whether these control measures

(individually and as a whole) are sufficient

to reduce the risk to an acceptable level. For

example, is hand sanitization performed often

and sufficiently, and is the procedure following

industry best practices (3)? Are your personnel

suitably trained and are they following the pre-

scribed procedure? Have you collected sufficient

data (e.g., from swabs and dabs) that prove the

efficiency of the sanitizing regime? Once you

have performed this risk assessment, you will

have sufficient information and data to under-

stand, whether:

• Your hand sanitizing procedures are adequate

and effective

Developing an Effective Contamination Control StrategyProviding regulators with a holistic approach to addressing deficiencies is the best response to an inspection.

Siegfffriiieddd SSSchhhmiitittt PPPhDhDhD



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