CE in the Biotechnology & Pharmaceutical Industries · 2018. 4. 2. · 1 CE in the Biotechnology & Pharmaceutical Industries: 17th Symposium on the Practical Applications for the

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CE in the Biotechnology &

Pharmaceutical Industries:

17th Symposium on the Practical

Applications for the Analysis of

Proteins, Nucleotides and Small

Molecules

(CE Pharm 2015)

Symposium Co-Chairs:

Tim Blanc, Eli Lilly & Company

Henry Luo, Regeneron Pharmaceuticals, Inc.

September 20-24, 2015

New York Marriott at the Brooklyn Bridge

Brooklyn, NY

Organized by

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Table of Contents

Welcome Letter .......................................................................................................... 3

CE Pharm Award ....................................................................................................... 4

Student Travel Grants ................................................................................................ 5

Program Partners, Exhibitors and Media Partners ..................................................... 6

Scientific Final Program Summary ............................................................................ 8

Session Abstracts ..................................................................................................... 15

Workshop I Description ........................................................................................... 36

Workshop II Description .......................................................................................... 37

Technical Seminar Abstract ..................................................................................... 38

Poster Abstracts ....................................................................................................... 42

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Welcome to CE Pharm 2015: CE in the Biotechnology and Pharmaceutical Industries: 17th

Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small

Molecules

We are pleased to welcome you to CE Pharm 2015, a symposium devoted to the practical concerns that

will strengthen the use of CE within the biotechnology and pharmaceutical industries. The goal of this

symposium is to provide a forum for the discussion of recent developments in the analysis and

characterization of protein therapeutics, nucleotides and small molecules by CE and related techniques.

The symposium will feature presentations from leading experts within industry and regulatory agencies

from around the world. Applications will highlight the use of CE in various areas of product development

including high-throughput screening, process development, product characterization, formulation studies,

validated lot release and stability testing. Attendees will have the opportunity to discuss the use of CE

with regulatory agencies. In addition, CE troubleshooting approaches will be presented and

instrumentation companies will show advances in CE instruments, sensitivity and reagents. The

symposium will allow for open discussions aimed at improving and increasing the use of CE for analysis

of proteins, small molecules, carbohydrates, metabolites, and other molecules, with a focus on validation

and qualification, new technology and QbD. Workshops will focus on best practices and troubleshooting

techniques.

The success of this symposium will depend not only on the outstanding cast of experienced and

knowledgeable speakers and workshop leaders, but also on the interactions and open discussions that take

place among the attendees. We encourage you to participate whole-heartedly in the discussion sections

that have been designed to stimulate exchange of ideas and information.

We would like to thank the speakers who are generously giving their time and resources and also you for

your attendance, which will make this endeavor a success.

We gratefully acknowledge the generosity of our exhibitors and program partners: Agilent Technologies,

Amgen Inc., The Analytical Scientist, Biogen, Bio Tech International, BioProcess International, CMP

Scientific, Genentech, a Member of the Roche Group, Genetic Engineering & Biotechnology News,

International Pharmaceutical Quality, LCGC North America, MedChemComm, The Medicine Maker,

The Pathologist, Pfizer, Inc., ProteinSimple, ProZyme, Inc., Regeneron Pharmacuticals, Inc., RSC

Advances, seperationsNOW.com, SCIEX, Technology Networks and Thermo Scientific.

We are thankful for the expert assistance of CASSS and the audiovisual expertise of Michael Johnstone

from MJ Audio-Visual Productions. Their experience and guidance in the preparation of this symposium

have been invaluable.

THE SCIENTIFIC ORGANIZING COMMITTEE

Tim Blanc, Eli Lilly & Company (Co-chair)

François de l'Escaille, ANALIS s.a./n.v.

Göran Hübner, Boehringer Ingelheim Pharma

GmbH & Co. KG

Sarah Kennett, CDER, FDA

Steffen Kiessig, F. Hoffmann-La Roche Ltd.

C. Mark Lies, SCIEX

Jiann-Kae Henry Luo, Regeneron Pharmaceuticals,

Inc. (Co-chair)

David A. Michels, Genentech, a Member of the

Roche Group

SungAe Suhr Park, Amgen Inc.

Meg Ruesch, Pfizer, Inc.

Richard Rustandi, Merck & Co., Inc.

Oscar Salas-Solano, Seattle Genetics, Inc.

Cari Sänger - van de Griend, Kantisto BV

Zoran Sosic, Biogen

Hermann Wätzig, Technical University

Braunschweig

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CE Pharm Award History and Qualifications

Objective: Recognize and award an individual for sustained and significant contribution to the practical

application of CE to the analysis of biotechnology and pharmaceutical products.

Qualification for Award:

a. Advocate for CE from biotechnology and pharmaceutical industry b. Technical advancement or considered as a leader in developing or implementing

various CE applications, such as:

New CE Application for R&D

CE Method Qualification

CE Method Validation

CE Method Transfer

c. Technical reputation, in terms of number of presentations, publications and

patents

d. Dedication to CE Pharm meeting as speaker, tutor, poster presenter or committee

member

e. Mentor, advisor and advocate of industrial-based CE practitioners in other

industrial applications such as food chemistry, forensics and clinical.

Past Recipients of the "CE Pharm Award" include:

2006 - Norberto Guzman – Johnson & Johnson

2007 - Kevin Altria – GlaxoSmithKline

2008 - Anthony Chen and Wassim Nashabeh – Genentech, Inc.

2009 - Stacey Ma – Genentech, Inc.

2010 - SungAe Suhr Park – Amgen Inc.

2011 - Oscar Salas-Solano – Seattle Genetics, Inc.

2012 - Franka Kálmán – University of Applied Sciences Western Switzerland

2013 - András Guttman – Northeastern University

2015 - Michel Girard – Health Canada

2016 - Winner will be announced Wednesday at 8:30 AM.

Do you think we are missing someone influential? Add your suggestion to the list.

Suggestions for next year’s award can be submitted with your post-meeting evaluation.

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CASSS CE Pharm Student Travel Grants

CASSS is pleased to provide a limited number of student travel grants for students who present

applicable posters at CE Pharm 2015. PhD students or post-doctoral fellows conducting research

in academia or industry throughout the world are eligible.

Why you should apply:

This symposium gives insight into the current topics and issues under discussion within the

pharmaceutical and biotech industry and, as such, gives attendees the opportunity to bridge

between industry, academia and regulatory agencies. The presentations and workshops will be

devoted to practical concerns that strengthen the use of CE within the biotechnology and

pharmaceutical industries. Applications will highlight uses of CE in various areas of product

development, including high-throughput screening, formulation studies, process development,

product characterization and validated lot release and stability testing. As a participant, you will

have an excellent opportunity to meet, network and participate in exchanging knowledge for

mutual education with other CE practitioners.

Requirements are:

- Present a poster on a CE topic

- Proof of studentship/post-doc status

- Recommendation from the supervisor/advisor

CASSS has awarded student travel grants to the following individuals:

P-104

CE-SDS-CZE-MS Using an In-capillary Approach to Eliminate SDS Interferences

for Antibody Analysis

Cristina Montealegre, Aalen University, Germany

P-149

Affinity Capillary Electrophoresis to Investigate the Interactions of Pentosan Polysulfate

Sodium and Related Substances with Proteins

Mona Mozafari, Technical University Braunschweig, Germany

P-151

Hyphenation of 2D heart cutting CIEF/CE-MS

Daniel Sydes, University of Tuebingen, Germany

P-127

Fast Separation and Analysis of Reduced Monoclonal Antibodies with Capillary Zone

Electrophoresis Coupled to Mass Spectrometry

Yimeng Zhao, University of Notre Dame, USA

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The Organizing Committee gratefully acknowledges the following program

partners for their generous support of CE Pharm 2015:

Sustaining Program Partners

Platinum

Biogen

Silver

Pfizer, Inc.

Platinum Program Partner

SCIEX

Gold Program Partners

Agilent Technologies

Regeneron Pharmaceuticals, Inc.

Silver Program Partner

Genentech, a Member of the Roche Group

Bronze Program Partner

Amgen Inc.

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Exhibitors

Agilent Technologies

CMP Scientific

ProteinSimple

ProZyme, Inc.

SCIEX

Thermo Scientific

Media Program Partners

The Analytical Scientist

Bio Tech International

BioProcess International

Genetic Engineering & Biotechnology News

International Pharmaceutical Quality

LCGC North America

MedChemComm

The Medicine Maker

The Pathologist

RSC Advances

separationsNOW.com

Technology Networks

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CE Pharm 2015

Scientific Final Program Summary

Sunday, September 20, 2015

08:30 – 09:00 Breakfast (for course attendees ONLY) in the Park Slope Room

08:30 – 13:30 Registration (for course attendees ONLY) in the Ballroom Foyer

09:00 – 17:00

Short Course in Park Slope Room

Application of Capillary Electrophoresis to the Analysis of Protein Therapeutics

Short Course Facilitators: Chantal Felten, Alpine Analytical Academy, Whistler, BC Canada

and David A. Michels, Genentech, a Member of the Roche Group, South San Francisco, CA USA

10:30 – 11:00 Break in the Park Slope Room

12:30 – 13:30 Hosted Lunch (for course attendees ONLY) in the Park Slope Room


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Monday, September 21, 2015

07:30 – 18:00 Registration in the Ballroom Coatroom

07:30 – 08:30 Breakfast in Ballroom A-C

08:30 – 08:45 Welcome and Introductory Comments in Ballroom D

Tim Blanc, Eli Lilly & Company, Branchburg, NJ USA

Keynote I Session in Ballroom D

Session Chair: Tim Blanc, Eli Lilly & Company, Branchburg, NJ USA

08:45 – 09:30 Defining Product Quality using Analytical Methods

Reed Harris, Genentech, a Member of the Roche Group, South San

Francisco, CA USA

09:30 – 09:45 Discussion

09:45 – 10:15 Break – Visit the Exhibits and Posters in Ballroom A-C

Protein Analysis and Emerging Therapeutics in Ballroom D

Session Chair: Oscar Salas-Solano, Seattle Genetics, Inc., Bothell, WA USA

10:15 – 10:40 Applications of Capillary Electrophoresis in the Discovery and Early-

Development of Antibody Based Drugs

David Passmore, Bristol-Myers Squibb Company, Redwood City, CA USA

10:40 – 11:05 Charge Variant Characterization of Bispecific Antibodies and Their

Halves

Feng Yang, Genentech, a Member of the Roche Group, South San

Francisco, CA USA

11:05 – 11:30 Approaches for Analysis of Unconjugated Components in GBS

Glycoconjugate Vaccine: From Chromatography to Capillary

Electrophoresis

Luca Simeone, GlaxoSmithKline, Siena, Italy

11:30 – 11:55 Characterization of Complex Glycosylated Therapeutic Proteins by

Capillary Isoelectric Focusing (cIEF)

Georg Hölzl, Sandoz GmbH, Kundl, Austria


12:10 – 12:25 Lunch for Technical Seminar Attendees – Please take lunch and return

to Ballroom D for the “Lunch and Learn”

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Monday, September 21, 2015 continued

12:25 – 13:25 Technical Seminar/Lunch and Learn

Application of icIEF in Charge Heterogeneity Analysis, Formulation Development and

Automation: Case Studies

Ya Fu, Bristol-Myers Squibb Company, New Brunswick, NJ USA

Sponsored by ProteinSimple Ballroom D

13:25 – 15:10 Poster Session - Visit the Exhibits and Posters in Ballroom A-C

Method Development Session in Ballroom D

Session Chair: C. Mark Lies, SCIEX Brea, CA USA

15:10 – 15:35 Optimization of a CZE Method for Charge Variant Analysis in

Monoclonal Antibodies and the Comparison of this Method to iCIEF

and HPIEX

Tijmen Verweij, Merck Sharp & Dohme, Oss, The Netherlands

15:35 – 16:00 The Use of Capillary Electrophoresis fro Protein Analysis in Vaccine

Characterization

Jerome Thiebaud, Sanofi Pasteur, Marcy l’Etoile, France

16:00 – 16:25 Common Fragmentations in mAbs Detected by CE-SDS

Umesh Kishnani, Merck & Co., Inc., West Point, PA USA


16:40 – 17:10 CE Pharm Partner Showcase

17:10 – 18:10 Technical Seminar

Novel Fluorescent Labels for High Throughput N-glycan Analysis

Shaheer Khan, Thermo Scientific, South San Francisco, CA USA

Sponsored by Thermo Scientific Ballroom D

18:10 – 19:15 Exhibition Reception – Visit the Exhibitors in Ballroom A-C

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Tuesday, September 22, 2015


07:30 – 08:30 Breakfast in the Ballroom A-C

Keynote II Session in Ballroom D

Session Chair: Hermann Wätzig, Technical University Braunschweig, Braunschweig, Germany

08:30 – 09:15 Seeing while CE-ing

Michael Breadmore, University of Tasmania, Hobart, Australia



Deep Dive into CE Session in Ballroom D

Session Chairs: François de L’Escaille, Analis R&D Diagnostics , Namur, Belgium

and Richard Rustandi, Merck & Co., Inc., West Point, PA USA

10:00 – 10:25 Rapid Analysis of RNA in Virus like Particle Vaccine by Capillary

Zone Electrophoresis in Uncoated Capillary

Yan He, Pfizer, Inc., Chesterfield, MO USA

10:25 – 10:50 The Characterization and Analysis of PEG-erythropoietin by CE and

CE-MS

Maria Schwarz, Solvias AG, Kaiseraugst, Switzerland

10:50 – 11:15 A CZE Method for the Identification of Therapeutic Antibodies and

Quality Control of Infusion Bags at the Hospital

Claire Smadja, University of Paris Sud, Châtenay-Malabry, France


11:30 – 11:45 Lunch for Technical Seminar Attendees – Please take lunch and return

to Ballroom D for the “Lunch and Learn”

11:45 – 12:45 Technical Seminar/Lunch and Learn

Fully Automated Sample Preparation for Ultrafast N-glycosylation Analysis of Antibody

Therapeutics

András Guttman, SCIEX, Brea, CA USA and Horváth Csaba Laboratory of Bioseparation

Sciences, University of Debrecen, Hungary

Sponsored by SCIEX Ballroom D

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Tuesday, September 22, 2015 continued


Novel Technologies Session in Ballroom D

Session Chair: Zoran Sosic, Biogen, Cambridge, MA USA

13:00 – 13:25 Characterization of Charge Variants Separated by Free-Flow

Electrophoresis Seth Madren, Biogen, Research Triangle Park, NC USA

13:25 – 13:50 Intact Mass Analysis of Monoclonal Antibodies by Capillary

Electrophoresis – Mass Spectrometry Mei Han, Amgen Inc., South San Francisco, CA USA

13:50 – 14:15 High-Throughput Michrochip Electrophoresis (MCE) for the

Analysis of Antibody Based Biotherapeutics

Friedericke Winkhaus, Roche Diagnostics GmbH, Penzberg, Germany


14:30 – 15:00 Break - Visit the Exhibits and Posters in Ballroom A-C

Regulatory Session in Ballroom D

Session Chairs: Sarah Kennett, CDER, FDA

and Meg Ruesch, Pfizer, Inc.

15:00 – 15:25 Compendial CE Analytical Procedures for Therapeutic Proteins

Edith Chang, US Pharmacopeia, Rockville, MD USA

15:25 – 15:50 Mind the Gap: Analytical Method Management from Development

Through Transfer

Joel Welch, CDER, FDA, Silver Spring, MD USA

15:50 – 16:15 The NISTmAb: A Platform for Open Innovation

John Schiel, IBBR/NIST, Rockville, MD USA


16:45 – 17:45

Workshop I: Root Cause Analysis of Invalid Assays in Ballroom D

Workshop Facilitators: Sarah Kennett, CDER, FDA, Silver Spring, MD USA,

Cari Sänger -van de Griend, Kantisto BV, Baarn, The Netherlands

and Hermann Wätzig, Technical University Braunschweig, Braunschweig, Germany

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Wednesday, September 23, 2015


07:30 – 08:30 Breakfast in the Ballroom A-C

08:30 – 08:45 CE Pharm Award Presentation

08:45 – 09:45

Workshop II: Troubleshooting in Ballroom D

Workshop Facilitators: Tim Blanc, Eli Lilly & Company, Branchburg, NJ USA

Cari Sänger - van de Griend, Kantisto BV, Baarn, The Netherlands

and Bernd Moritz, F. Hoffmann-La Roche Ltd. Basel Switzerland


Method Lifecycle Management Session in Ballroom D

Session Chairs: Henry Luo, Regeneron Pharmaceuticals, Tarrytown, NY USA

and David Michels, Genentech, a Member of the Rocher Group, South San Francisco, CA USA

10:15 – 10:40 Developing an iCIEF Method to Replace Slab Gel for a Commercial

Product Charge Variant Analysis

Kun Lu, Regeneron Pharmaceuticals, Inc., Rensselaer, NY USA

10:40 – 11:05 Characterization of Glycation in Monoclonal Antibodies by Reduced

Capillary Electrophoresis Sodium Dodecyl Sulfate

Matthew Myers, Merck & Co., Inc., Kenilworth, NJ USA

11:05 – 11:30 Implementing New Instrument Models in the Global

Roche/Genentech Network: Challenges, Risks & Opportunities

David Fischer, Genentech, a Member of the Roche Group, South San

Francisco, CA USA


11:45 – 12:00 Closing Comments in Ballroom D

Henry Luo, Regeneron Pharmaceuticals, Inc., Tarrytown, NY USA

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Thursday, September 24, 2015

08:30 – 09:00 Breakfast (for course attendees ONLY) in the Park Slope Room

08:30 – 13:30 Registration (for course attendees ONLY) in the Ballroom Foyer

09:00 – 17:00

Short Course in Park Slope Room

Method Development, Optimization and Good Working Practice in Capillary

Electrophoresis

Short Course Facilitators: François de l'Escaille, Analis s.a./n.v., Namur, Belgium

and Cari Sänger - van de Griend, Kantisto BV, Baarn, The Netherlands


12:30 – 13:30 Hosted Lunch (for course attendees ONLY) in the Park Slope Room


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Defining Product Quality using Analytical Methods

Reed Harris

Genentech, a Member of the Roche Group, South San Francisco, CA USA

The assessment of product quality during clinical development requires some definitions and

acceptance criteria. Early stage specifications use a common set of assays, with acceptance

criteria based on precedents from related products or by comparison of the clinical batches to the

quality of the material used in toxicology studies. Specification acceptance criteria are

augmented by the use of QC profile assessments and alert limits.

Critical quality attributes are identified by assessing patient impacts such as bioactivity,

pharmacokinetic impact, immunogenicity risk and safety risk, each with quantitative or

qualitative criteria, combined with an uncertainty assessment regarding the impact determination.

This assessment enables identification of special tests that may need to be added, and the

uncertainty aspect encourages further studies to better understand the patient impacts of product

variability and impurities.

Clinical comparability is assessed using defined rules, generally without quantitative criteria

until post-approval. A historical review of the Genentech clinical material quality enabled

defined product quality target ranges for new development programs.

NOTES:

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Applications of Capillary Electrophoresis in the Discovery and Early-Development of

Antibody Based Drugs

David Passmore, Aarti Jashnani, Jennifer Juliano

Bristol-Myers Squibb Company, Redwood City, CA USA

Capillary electrophoresis (CE) is a proven technology for characterization of recombinant

proteins, and the technology is routinely applied in the biotechnology industry as an analytical

control for the production of biologic drugs. This presentation demonstrates the utility of CE in

the discovery and early-development of therapeutic monoclonal antibodies (mAbs) and antibody

drug conjugates (ADC). Various modes of CE are discussed including: capillary laser induced

fluorescence (CE-LIF), capillary SDS gel (CE-SDS), and imaged capillary electrophoresis (iCE).

cLIF is an effective method to determine the glycosylation pattern of mAbs; here, we

demonstrate its use to characterize the glycan profile of mAbs with enhanced Fc effector

functions. CE-SDS is generally applied in our laboratory to assess mAb fragmentation and to

determine the % non-glycosylated mAb; we demonstrate batch variation with respect to these

attributes. Both iCE and CD-SDS methods are used to support forced degradation studies to

confirm the physicochemical stability of antibody lead candidates. CE plays an important role in

the characterization of antibody drug conjugates and is a critical analytical tool in development

of novel drug conjugation technologies. CE is applied in our laboratory to develop antibody

expression technologies, monitor product quality, inform lead candidate selection, and to support

ADC development.

NOTES:

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Charge Variant Characterization of Bispecific Antibodies and Their Halves

Feng Yang, Anh Nguyen Dang, Nicole Liu, Renee Yang, Will McELory, David Fischer, Yun

Tang, David Michels


Bispecific antibody (bsAb) recognizes two distinct targets, and has attracted substantial

therapeutic interest in recent years.1-4 One efficient way to generate a bsAb is through knob-into-

hole technology, whereby complementary mutations are made in the CH3 domain of each heavy

chain to form knobs and holes. 5,6 However, there are many challenges to characterize product-

related variants of bsAbs, including the charge variant analysis of half antibodies of bsAbs.

Antibody charge variants have gained considerable attention due to their potential influence on

stability and biological activity. Compared to regular monoclonal antibodies, greater charge

heterogeneity for our CHO-based bsAbs has been observed. Therefore, there is great need to

develop a charge variant assay for half antibodies to support their cell culture process

development and clone selection, which eventually can improve product quality of the final

assembled bsAb products. Due to unique features of half antibodies compared to full antibodies,

many issues were found to develop such an assay.

In this work, we developed a high throughput and robust image capillary isoelectric focusing

method for the half antibodies, with good resolution and auto-sampler stability. This method has

been applied to monitor charge variants of half antibodies from different bsAbs to support

process development and clone selection.

(1) Lameris, R.; de Bruin, R. C.; Schneiders, F. L.; van Bergen En Henegouwen, P. M.; Verheul,

H. M.; de Gruijl, T. D.; van der Vliet, H. J. Critical reviews in oncology/hematology 2014, 92,

153-165.

(2) Spiess, C.; Merchant, M.; Huang, A.; Zheng, Z.; Yang, N. Y.; Peng, J.; Ellerman, D.; Shatz,

W.; Reilly, D.; Yansura, D. G.; Scheer, J. M. Nature biotechnology 2013, 31, 753-758.

(3) Chan, A. C.; Carter, P. J. Nature reviews. Immunology 2010, 10, 301-316.

(4) Byrne, H.; Conroy, P. J.; Whisstock, J. C.; O'Kennedy, R. J. Trends in biotechnology 2013,

31, 621-632.

(5) Merchant, A. M.; Zhu, Z.; Yuan, J. Q.; Goddard, A.; Adams, C. W.; Presta, L. G.; Carter, P.

Nature biotechnology 1998, 16, 677-681.

(6) Klein, C.; Sustmann, C.; Thomas, M.; Stubenrauch, K.; Croasdale, R.; Schanzer, J.;

Brinkmann, U.; Kettenberger, H.; Regula, J. T.; Schaefer, W. mAbs 2012, 4, 653-663.

NOTES:

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Approaches for Analysis of Unconjugated Components in GBS Glycoconjugate Vaccine:

From Chromatography to Capillary Electrophoresis

Luca Simeone, Mila Toppazzini, Stefano Ricci, Marcello Morelli, Barbara Fabbri, Galasso Rosy,

Sara Giannini, Cristiana Campa

GlaxoSmithKline, Siena, Italy

Group B Streptococcus (GBS) is a bacterium responsible for serious disease in infants, pregnant

women, elderly and immunosuppressed adults. Fetal exposure to GBS in utero may result in

stillbirth, neonatal pneumonia or sepsis and is strongly associated with prematurity. A candidate

vaccine for maternal immunization against GBS has been obtained by coupling purified capsular

polysaccharide antigens of GBS with an immunogenic carrier protein and it is currently in

clinical Phase II. The availability of fast and reliable approaches for the analysis of

glycoconjugates and their intermediates is of primary importance for conjugation process

monitoring and purity testing. In addition to glycoconjugates, protein and polysaccharides are

among the analytes to be characterized and separation techniques with specialized selectivity are

required.

This work reports the evolution of analytical approaches for the quantitative determination of

unconjugated components in GBS glycoconjugates. Capillary Electrophoresis, which initially

has been considered a complementary technique to HPAEC-PAD (High Performance Anion

Exchange Chromatography with Pulsed Amperometric Detection) and SEC-HPLC approaches,

has become the method of choice, fully satisfying the requirements of the Analytical Target

Profile. The electrophoretic MEKC (micellar electrokinetic chromatography) method is able to

separate and quantify unconjugated carrier protein and GBS free polysaccharidic antigen in a

single analysis. MEKC method was validated following the ICH guidelines: accuracy,

repeatability, intermediate precision, limit of quantification, linearity, range, specificity and

robustness were evaluated. Validation results were then compared to the liquid chromatographic

methods showing better results for MEKC approach.

NOTES:

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Characterization of Complex Glycosylated Therapeutic Proteins by Capillary Isoelectric

Focusing (cIEF)

Georg Hölzl1, Matthias Habeler2, Wolfgang Gutleben1

1Sandoz GmbH, Kundl, Austria, 2Medical University of Innsbruck, Innsbruck, Austria

Isoelectric focusing (IEF) is a high-resolution electrophoretic technique for separation and

analysis of proteins in a pH gradient. In the past years IEF has become an indispensable

bioanalytical measurement method in research, development and manufacturing quality control

of biopharmaceuticals [1]. Consequently, the method has become of significant importance when

determining the similarity of an innovator biopharmaceutical product to a biosimilar candidate.

In this presentation, a capillary isoelectric focusing (cIEF) method for charge isoform

distribution analysis of a highly complex glycosylated therapeutic protein is presented. Standard

practice charge heterogeneity analysis by ion exchange chromatography (IEC) or capillary zone

electrophoresis (CZE) fails to adequately resolve subtle differences in isoelectric point (pI). As a

result charged variants originating from a proteinaceous charge heterogeneity or extensive

diverse glycosylation such as sialylation may be superimposed in the chromatographic or

electrophoretic separation. To meet this challenge, a cIEF method was established and

experimentally optimized for charge heterogeneity profiling and comparison of biosimilar

candidate and originator. Several commercially available broad-range carrier ampholytes were

evaluated with regard to pH gradient linearity, baseline noise and resolution power of protein

isoforms. With the optimized methodology in summary approximately 35 different isoforms

could be separated. This clearly demonstrates the extraordinary performance of cIEF over

orthogonal methods for the characterization of charge heterogeneity of biopharmaceuticals.

[1] Zhao, Shuai Sherry; Chen, David D. Y.; Electrophoresis 2014, 35, 96–108 Applications of

capillary electrophoresis in characterizing recombinant protein therapeutics

NOTES:

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Optimization of a CZE Method for Charge Variant Analysis in Monoclonal Antibodies and

the Comparison of this Method to iCIEF and HPIEX

Tijmen Verweij, Joop Waterval

Merck, Sharp & Dohme, Oss, The Netherlands

Capillary zone electrophoresis (CZE) is an upcoming technique in the pharmaceutical industry

for the determination of charge variants of monoclonal antibodies (mAbs). Previous studies

showed that one of the main strengths of this technique is the high resolution that can be

achieved.

A CZE method was optimized for eight different mAbs with isoelectric points (pIs) varying from

6.5 to 9.2. The separation buffer contained 6-aminocaproic acid (EACA), triethylenetetramine

(TETA) and hydroxypropyl methylcellulose (HPMC) as proposed by Yan He (Pfizer) in an

earlier publication.

A single platform method for charge variant determination of mAbs could not be created.

However, many parameters could be fixed, leaving only the TETA concentration and the

capillary length as specific parameters per mAb. It was observed that the pI value of the mAb

dictates the optimal TETA concentration and separation length.

A comparison between the optimized CZE method, an ion exchange chromatography (IEX)

method and an imaged capillary isoelectric focusing (icIEF) method was made, showing that the

CZE method is equal in resolution and precision compared to IEX and better compared to the

icIEF method.

Finally a full co-qualification of the CZE method for one of the mAbs was performed, according

to the ICH guidelines, by three different departments in three different countries. Even though

two of the departments had no prior experience with this CZE method, the qualification was

successful.

NOTES:

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The Use of Capillary Electrophoresis for Protein Analysis in Vaccine Characterization

Jerome Thiebaud, Aurelie Deliot, Afifa Bouadam, Jean François Cotte, Philippe Talaga, Olivier

Adam

Sanofi Pasteur, Lyon, France

Capillary electrophoresis (CE), using fused silica capillaries was introduced by Jorgenson and

Lukas in 19811. High voltages are used to separate molecules based on differences in charge and

size. Various separation methods can be used in CE, depending on the type of capillary and the

background electrolyte, as well as various detection devices, including UV, fluorescence or MS.

The variety of separation methods, i.e. mainly capillary zone electrophoresis (CZE), capillary

isoelectric focusing (cIEF), capillary gel electrophoresis (CGE), micellar electrokinectic

chromatography (MEKC), the small sample size and its separation performance make CE a

preferred method for the analysis of proteins in complex biological mixtures.

One of the most important aspects of vaccine manufacturing is to develop simple and robust

methods for process and product characterization. In this context, capillary electrophoresis can

be used throughout the production process, whether for quantification of proteins by CGE and

MEKC or for the characterization of glycoprotein and glycoconjugate vaccine.

1 Jorgenson, JW., Lukas, KD., Zone electrophoresis in Open Tubular Glass Capillaries Anal.

Chem. 59 (1981), 1298-1302.

NOTES:

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Common Fragmentations in mAbs Detected by CE-SDS

Umesh Kishnani, Yan An

Merck & Co., Inc., West Point, PA USA

CE-SDS is a widely used technique for mAb purity testing. Advantage of non-reduced CE-SDS

over SEC (size exclusion chromatography) is that the fragments in CE-SDS profile are well

separated, whereas the fragments in SEC are generally not separated from each other. In our

approach, the light induced fragmentation of six mAbs (including three IgG1, one IgG2, and two

IgG2) were analyzed by non-reduced CE-SDS. Denatured SEC was employed in fractionation of

the peaks which are corresponding to the fragment peaks in the non-reduced CE-SDS

electropherograms. The collected fractions were further characterized by LC-MS. Our results

showed that common mAb fragmentations include, but not limited to, the covalent bond

cleavage (mostly peptide bond cleavage and disulfide bond cleavage) resulted from deamidation,

isomerization, oxidation and non-enzymatic hydrolysis.

NOTES:

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Seeing While CE-ing

Michael Breadmore

University of Tasmania, Hobart, Tasmania, Australia

The poor sensitivity of capillary electrophoresis is often cited as one of its biggest disadvantages.

When compared to liquid chromatography, the concentration detection limits are often 100-1,000

times worse. A number of strategies have been developed over the past two decades for on-line

enrichment. This presentation will provide an introduction to the most common approaches for

on-line concentration including stacking, sweeping and extraction, and various combinations of

these, for a range of target analytes including small molecules such as inorganic ions and

pharmaceuticals through to oligosaccharides. Theoretical and practical insight into these

approaches will be discussed and limitations and problems will addressed.

NOTES:

24

Rapid Analysis of RNA in Virus Like Particle Vaccine by Capillary Zone Electrophoresis

in Uncoated Capillary

Yan He, Vicky Hou, Helen Sato, Michael Jones, Margaret Ruesch

Pfizer, Inc., Chesterfield, MO USA

A capillary zone electrophoresis (CZE) method for quantitation of total RNA in virus like

particle (VLP) vaccine is presented. RNA of different sizes released from VLP has similar

charge-to-mass ratio, and is separated as one peak with CZE. The separation is carried out in

short (10 cm effective length) uncoated capillary. Zwitterionic buffer (HEPES) and low

concentration of HPMC (0.05%) is used to form dynamic coating to suppress electroosmotic

flow, and enable fast separation. Internal standard is used to reduce variability of quantitation.

Under optimal conditions, rapid separation of RNA and internal standard is achieved within 3

min with moderate field strength of 400 V/cm. The method has been validated to quantify RNA

in different VLPs. Limit of quantitation is 25 µg/ml. Linearity (R2>0.99) is from 25 to 130

µg/ml. Intermediate precision is 9.4%.

NOTES:

25

The Characterization and Analysis of PEG-erythropoietin by CE and CE-MS

Maria Anna Schwarz, Alena Ferenc, Angelina Rafai

Solvias AG, Kaiseraugst, Switzerland

Owing to the changed molecular properties the analytical handling of PEGylated proteins is

different when compared to the non-PEGylated form. Especially the increased size heterogeneity

of the modified protein introduced by PEGylation is demanding in terms of finding useful

analytical methods in order to prove structural identity, post-translation and process related

modifications beside the need to analyze the PEGylation degree and PEG-site.

Using classical CZE analysis of the intact molecule the overall PEGylation degree can be easily

analyzed, however, protein related variants are difficult to access. In order to localize the

PEGylation site or to analyze charge variants the complexity of the molecule has to be reduced

by digestion of the protein backbone with suitable enzymes depending on the PEGylation site

and the amino acid sequence of the protein followed by CZE-UV analysis of the peptide map.

The present study describes a new approach for the characterization of erythropoietin PEGylated

randomly at 5 – 8 different PEGylation sites applying enzymatic Lys-C digests. The evaluation is

based on the comparison of the resulting CE-UV profiles of PEGylated and non-PEGylated

erythropoietin. Thereby, PEGylated peptides may be attributed to broad signals at higher

migration times resulting from different q/rh ratios. Furthermore, PNGase F/Sialidase A and Lys-

C treated test samples analyzed by HPLC and CE-MS are involved in the study in order to

confirm the identity of PEGylated and non-PEGylated peptides containing also N-/O-glycans. By

evaluation of changed A% of the considered PEGylated/non PEGylated peptides the PEGylation

degree can be determined as well as an estimation in terms of present charge variants (e.g.

deamidation) can be made.

NOTES:

26

A CZE Method for the Identification of Therapeutic Antibodies and Quality Control of

Infusion Bags at the Hospital

Claire Smadja1, Emmanuel Jaccoulet2, Myriam Taverna1

1University of Paris Sud, Châtenay-Malabry, France, 2Hôpital Européen Georges Pompidou,

Paris, France

Monoclonal antibodies (mAbs) are widely used in cancer therapy. Their compounding is

performed in a production unit of the hospital. Identification of these drugs, individually

prepared in bags for infusion and just on line before patient administration is of paramount

importance to avoid medication errors. Analytical methods for these specific QC have to be fast

and simple to facilitate their implementation at the hospital. At this stage an extensive

characterization of the infusion bags is not required as for final batches. The most important rely

on the identification and accurate quantification of the mAbs. Several techniques including

capillary electrophoresis are now available to control or characterize mAbs glycosylation, charge

heterogeneity or to detect undesired degraded forms in final products. None of these methods can

really fulfill the requirements for a quality control process at the hospital. Therefore, a new

method based on capillary zone electrophoresis combined to a cationic coating (hexadimethrine

bromide) was developed for the discrimination of the most widely used compounded mAbs for

cancer therapy in France (bevacizumab, cetuximab, rituximab and trastuzumab). This work

describes the extensive optimization of the BGE composition and coating protocol, we

performed to achieve a full resolution of these four mAbs and a very reproducible method. This

was quite challenging considering the high structural and physico-chemical similarities of these

mAbs and their tendency to adsorb to surfaces. The separation profile was greatly improved with

the addition of perchlorate in the BGE. The specificity of the method was assessed by the

expected excipients analysis of the four commercial mAbs. Finally, we have demonstrated, by a

blind test, that this method allowed an accurate mAbs identification.

NOTES:

27

Characterization of Charge Variants Separated by Free-Flow Electrophoresis

Seth Madren1, Gerhard Weber2, Robert Wildgruber2

1Biogen, Research Triangle Park, NC USA, 2FFE Service, Feldkirchen, Munich, Germany

Monoclonal antibodies (mAbs) are an important class of therapeutic proteins; however

heterogeneity introduced by post-translational modifications can be difficult to control.

Identifying these modifications is critical in determining their impact on the safety and efficacy

of the final drug product. Many of these modifications create charge variants with different

isoelectric points (pI). Imaging capillary isoelectric focusing (icIEF) can be used to determine

and monitor shifts in the charge variant profile. However, icIEF is not preparative and can not be

used to isolate the charge variants for further characterization. Free-flow electrophoresis (FFE) is

a continuous separation method that uses an electric field to separate analytes as pressure drives

the sample through a separation region. A pH gradient can be formed in the separation region to

focus the charge variants based on their pI values. The charge variants can then be collected as

they exit the separation region for additional characterization. Our collaborators at FFE service

have used this technique to fractionate the charge variants of several monoclonal antibodies.

These fractions have been characterized by icIEF, capillary gel electrophoresis, and mass

spectrometry. The icIEF analysis confirmed the successful fractionation of isoforms with a Δ pI

< 0.1. The mass spectrometry and capillary gel electrophoresis data was used to identify the post-

translational modifications responsible for the different charge variants. Additionally, the binding

affinities of the isoforms were characterized to determine the relative potency of the different

isoforms.

NOTES:

28

Intact Mass Analysis of Monoclonal Antibodies by Capillary Electrophoresis – Mass

Spectrometry

Mei Han

Amgen Inc., South San Francisco, CA USA

Abstract not available at time of print.

NOTES:

29

High-Throughput Michrochip Electrophoresis (MCE) for the Analysis of Antibody Based

Biotherapeutics

Friederike Winkhaus

Roche Diagnostics GmbH, Penzberg, Germany

These days, traditional monoclonal antibodies are more and more replaced by novel complex

antibody formats. To ensure product quality, a well-controlled manufacturing processes as well

as thorough analytical characterization are required. Quality by Design (QbD) studies and

Design of Experiments (DoE) approaches to understand the relation between critical process

parameters and product quality as well as continuous process automation increase the number of

samples that need to be processed. Conventional analytical methods like CE-SDS, CZE or IEC

are very precise and reproducible but only have a low sample throughput. However, Microchip

Capillary Electrophoresis (MCE) provides an automated high-throughput platform to monitor

antibody quality. Here, we demonstrate the characterization of classical and novel formats of

antibody based biotherapeutics using Perkin Elmer’s LabChip GXII. The MCE assay shows a

good linear range, high sensitivity and provides a resolution superior to CE-SDS for many

aspects. Moreover, the assay can be used to identify and to monitor specific antibody fragments

and modifications during bioprocessing. With the use of DoE studies and by optimizing assay

conditions we aim to better understand critical assay parameter and to overcome current assay

limitations.

NOTES:

30

Compendial CE Analytical Procedures for Therapeutic Proteins

Edith Chang

US Pharmacopeia, Rockville, MD USA

The main advantages of using capillary electrophoresis (CE) as an analytical tool are enhanced

separation efficiency and shorter analysis times compared to traditional liquid chromatography

method such as HPLC. USP has recently developed several compendial applications of CE for

analysis of therapeutic proteins. A validated procedure based on capillary electrophoresis with

laser induced fluorescence detection (CE-LIF) for analysis of glycosylation through profiling of

released N-linked oligosaccharides (or N-glycans) is described in both <212> Oligosaccharide

Analysis and General Chapters <129> Analytical Procedures for Recombinant Therapeutic

Monoclonal Antibodies and. This procedure is used to analyze mostly neutral bi-antennary

glycan chains with low levels of sialylated structures. Additionally, chapter <212> also

describes the usage of two references standards (RS), Oligosaccharide System Suitability

Mixture A RS (glycan species released from human polyclonal IgG) and Oligosaccharide System

Suitability Mixture B RS (glycan species released from bovine RNase B) to assess the system

suitability of this CE-LIF procedure. USP Monoclonal IgG System Suitability RS has been

developed to assess the system suitability of a CE-SDS procedure. Information about these

compendial procedures and representative data are presented.

NOTES:

31

Mind the Gap: Analytical Method Management from Development Through Transfer

Joel Welch

CDER, FDA, Silver Spring, MD USA

The regulatory and scientific expectations for the validation of a given analytical method for a

biotechnology product are well-understood. However, the management of an analytical method

from early clinical development through its introduction to routine commercial manufacturing

requires more than development of a robust procedure. It requires deliberate consideration of the

impact of method changes throughout development and culminates in an appropriate method

transfer. This talk will describe considerations and potential missteps in managing method

changes during the development lifecycle and the expectations for designing appropriate method

transfer activities.

NOTES:

32

The NISTmAb: A Platform for Open Innovation

John Schiel

IBBR/NIST, Rockville, MD USA

The road to developing quality biotherapeutic proteins is a rapidly and ever changing landscape.

Now more than ever, analytical and biophysical characterization plays pivotal yet evolving roles

from R&D to commercialization. The ultimate pursuit is technological innovation, followed by

practical implementation, of broadly informative methodology. The NISTmAb, an IgG1k class-

specific biopharmaceutical reference material, is a means to fuel milestone technologies and

bridge their widespread industry utilization. Global conclusions drawn from the NISTmAb

crowdsourcing collaboration “State-of-the-Art and Emerging Technologies for Therapeutic

Monoclonal Antibody Characterization” will highlight the importance of open innovation

focused around common materials. The RM is expected to more firmly underpin fit-for-purpose

method selection and facilitate the development of originator and follow-on biologics. The

NISTmAb is intended for a variety of uses including, but not necessarily limited to: establishing

method or instrument performance and variability, comparing changing analytical methods,

assisting in method qualification, etc. Unique aspects of RM lifecycle management, fit-for-

purpose characterization, and ongoing research expanding the biopharma RM program will be

discussed.

NOTES:

33

Developing an iCIEF Method to Replace Slab Gel for a Commercial Product Charge

Variant Analysis

Kun Lu 1,2, Linsey Lipari1, Nicole Nall1, Paul Biogwarfe1, Ron Wang1, Jeffrey Schneiderheinze1

1Regeneron Pharmaceuticals, Inc., Rensselaer, NY USA; 2Regeneron Pharmaceuticals, Inc.,

Tarrytown, NY USA

Imaged Capillary Isoelectric Focusing (iCIEF) method has been extensively used in

biopharmaceutical industry as a tool for characterization, product release, formulation

development, stability monitoring and identification. Comparing to the traditional labor-intensive

IEF slab gel method and even standard cIEF, iCIEF offers many advantages including

quantitation, reproducibility, robustness and speed. A specific example is provided here to

demonstrate the activities related to the development and robustness study of an iCIEF assay for

the analysis of charge variants of a commercialized recombinant Fc-fusion protein. Conditions to

stabilize the charge variants and improve the method reproducibility have been optimized for this

heavily sialylated Fc-fusion protein. The iCIEF method shows sensitivity to urea concentration.

To overcome this problem, an approach to prepare well-controlled urea solution is included in

the method. After optimization, acceptable linearity, range, precision, accuracy and robustness

have been obtained. The method does not show sensitivity to sample formulation matrix and is

suitable for the analysis of drug substance, formulated drug substance and drug product. In

addition, the method is stability indicating as a distinct shift in pI from the basic to the neutral

and acidic region when samples were exposed to light.

NOTES:

34

Characterization of Glycation in Monoclonal Antibodies by Reduced Capillary

Electrophoresis Sodium Dodecyl Sulfate

Matthew Myers

Merck & Co., Inc., Kenilworth, NJ USA

Presented here is the identification and characterization of glycation (non-enzymatic addition of

a sugar) on monoclonal antibodies by reduced Capillary Electrophoresis Sodium Dodecyl Sulfate

(CE-SDS). Initially after switching apertures on the Beckman Coulter PA 800plus the

appearance of a shoulder which was previously unresolved on the light chain in reduced CE-SDS

was observed for multiple monoclonal antibodies. Through CE-SDS investigations, review of the

literature, and orthogonal Liquid Chromatography Mass Spectrometry (LC-MS) studies this

shoulder was identified to be glycation. Glycation is a nonspecific process that can occur at

multiple locations and changes a protein’s secondary and tertiary structure. Based on location of

glycation there is also concern that it may interfere with a proteins functional properties and thus

should be treated as a critical quality attribute until shown otherwise. For this reason Reduced

Peptide Mapping data identifying the multiple sites where glycation can occur on both the light

chain and heavy chain are included here. Finally, a strategy for integration and data collection in

our development labs is presented.

NOTES:

35

Implementing New Instrument Models in the Global Roche/Genentech Network:

Challenges, Risks & Opportunities

David Fischer, David A. Michels

Genetech, a Member of the Roche Group, South San Francisco, CA USA

In biopharmaceutical analysis, there is an ever increasing demand for technology that is able to

provide better, faster and cheaper business solutions. Analytical instrumentation must also be

reliable, robust, and capable of providing precise and accurate information regarding product

quality.

Through the constant evolution of technology, analytical instrument manufacturers are able to

introduce new features which increase functionality while improving performance. Typically,

these upgrades come in the form of a new instrument model which brings welcomed, and often

necessary, updates. However, new instrument models are distinctly different from those being

replaced and require additional considerations for network implementation.

The challenges of implementing the new CE instrumentation within the Roche/Genentech global

network will be discussed. Implications ranging from instrument training, platform support,

regulatory compliance, and implementation strategy will be discussed.

NOTES:

36

Workshops

Workshop I: Root Cause Analysis of Invalid Assays

Tuesday, September 22

16:45 – 17:45

Ballroom D

Facilitators:

Sarah Kennett, CDER, FDA, Silver Spring, MD USA,

Cari Sänger-van de Griend, Kantisto BV, Baarn, The Netherlands

Hermann Wätzig, Technical University Braunschweig, Germany

Scribe:


No-one wins when a test has a high rate of invalid analytical results. Reanalysis is costly;

including material costs and the laboratory analyst time, not to mention potential delays.

Everyone wins with appropriate reliable test methods. This is a statement anyone can agree with,

only how do we translate it to and apply it to the real life laboratory?

At different recent CASSS meetings, there were discussions about the invalid rates of certain

tests among which CE-SDS. Analytical scientists are usually pressed to get the methods up and

running as quickly as possible. We are given little time and limited resources to invest in

comprehensive method optimization and performance robustness studies. However, there are no

winners when a method has a high invalid rate!

What is an invalid assay and how do we track that? To start the discussion, we need to establish

common terminology and similar metrics. In the next step we will focus on the CE-SDS method

for monoclonal antibodies. This is a broadly used method that some claim to be working

perfectly and others state invalid rates as high as 40 %. So after we established a common

terminology and similar metrics to assure the data from a wide number of labs are similarly

collected and mutually interpretable, we want to apply this to CE-SDS and collect data as well as

discuss root causes for invalid analyses. All in order to improve successful performance and

reduce costs and delays.

NOTES:

37

Workshop II: Troubleshooting


08:45 – 09:45

Ballroom D

Facilitators:

Tim Blanc, Eli Lilly and Company, Branchburg, NJ USA

Cari Sänger-van de Griend, Kantisto BV, Baarn, The Netherlands

Bernd Moritz, F. Hoffmann – La Roche Ltd., Basel, Switzerland

Scribe:


As a community of Capillary Electrophoresis, sharing the expertise among the industry is one of

the primary objectives of the CEPharm Meeting and also the focus in our annual troubleshooting

workshop. Each laboratory makes unique distinctions about common problems and devise

unique and cleaver solutions to such problems within their organization. Some have

affectionately coined this acquired information as “Tribal Knowledge.” Internally, it may be

viewed as too trivial to publish, even though it is critical to the performance of important

methods. The goal of this workshop is to share and harness such tribal knowledge across our CE

community.

While lively and informative discussions are the goal, a picture (or Electropherogram) can

provide a much higher level of clarity to the discussion. This year we have invited attendees to

submit electropherograms representative of their troubleshooting issues. The hope is that the

electropherograms will bring a new level of clarity to questions that focus discussion and send

attendees home with solutions.

NOTES:

38

Technical Seminar Abstracts

ProteinSimple Lunch and Learn Seminar

Monday, September 21

12:25 – 13:25

Ballroom D

Application of icIEF in Charge Heterogeneity Analysis, Formulation Development and

Automation: Case Studies

Ya Fu

Bristol-Myers Squibb Company, New Brunswick, NJ USA

Imaged capillary isoelectric focusing (icIEF) has been widely used at BMS and throughout the

industry for characterization of the charge heterogeneity in protein-based biopharmaceutical

products. Current trends in biopharmaceutical development are driving the demand for higher

throughput analytical platform to identify the relationship between critical process parameters

and product quality. The new iCE3 analyzer enables onboard automated sample preparation

feature, which can elevate preparative artifacts, reduce operator to operator variability, and

enable high-throughput compatibility.

In this study we compared the results of automated sample preparation to manual sample

preparation at multiple aspects, including preparation time, within-run precision, between-run

precision and linearity, therefore demonstrated the advantage of auto sample preparation. We

further evaluate the advantages of using automated sample preparation on supporting

formulation development and characterizing charge heterogeneity of therapeutic monoclonal

antibodies (mAbs) and fusion proteins.

NOTES:

39

Thermo Scientific Seminar

Monday, September 21

17:10 – 18:10

Ballroom D


Shaheer Khan

Thermo Fisher Scientific, South San Francisco, CA USA

Glycosylation is one of the key critical quality attributes of mAb based biotherapeutics.

Glycosylation changes can impact biological drug’s safety, efficacy, clearance and

immunogenicity, making it necessary to accurately detect changes. Glycan profiling begins at

cell line development and continues through process development. Current glycan analysis

methods involve laborious multistep sample preparation that takes anywhere from a day to

multiple days for 96 samples, followed by single channel LC or CE separation.

Here, we report the development of a high throughput glycan analysis method utilizing a 24

capillary polymer filled array with laser induced fluorescence detection. For glycan labeling

along with conventional APTS dye, two new rapidly reacting fluorescent dyes were developed.

Glycan cleavage, dye labeling and excess dye removal steps were streamlined for automation.

We also eliminated the toxic sodium cynaoborohydride chemistry and vacuum centrifugation

steps. N-glycans from 96 samples were analyzed with <3 hours of hands on time and CE analysis

were completed in 7-9 hours. Some of the IgG glycans that were unresolved when labeled with

APTS were fully baseline resolved when labeled with our proprietary dyes. These novel dyes on

a simple streamlined workflow in combination with multi capillary CE instrument offer a high

resolution high throughput glycan analysis platform for rapid separation and quantitation of

antibody glycans.

NOTES:

40

SCIEX Lunch and Learn Seminar


11:45 – 12:45

Ballroom D

Fully Automated Sample Preparation for Ultrafast N-glycosylation Analysis of Antibody

Therapeutics

András Guttman1,2, Marton Szigeti2, Clarence Lew1

1SCIEX, Brea, CA USA, 2Horváth Csaba Laboratory of Bioseparation Sciences, University of

Debrecen, Hungary

There is a growing demand in the biopharmaceutical industry for high throughput and, large

scale N-glycosylation profiling of therapeutic antibodies in all phases of product development.

This is especially important during clone selection where hundreds of samples, with limited

amounts, should be analyzed in a short period of time for their glycosylation to ensure the

biological activity of the product to be developed. Our group has recently introduced a magnetic

bead based protocol for N-glycosylation analysis of glycoproteins to alleviate the hard-to-

automate centrifugation and vacuum-centrifugation steps of the currently used protocols. Glycan

release, fluorophore labeling and clean-up were all optimized with a magnetic bead based

process with excellent yield, and high repeatability. In this presentation we demonstrate the next

level of this work by fully automating all steps of the optimized magnetic bead based protocol

from endoglycosidase digestion, through fluorophore labeling and clean-up with high throughput

sample processing in 96 well plate format in <60 minutes, using an automated laboratory

workstation. Capillary electrophoresis analysis of the fluorophore labeled glycans was also

optimized for rapid (<3 min) separation to accommodate the high throughput processing of the

automated sample preparation workflow.

NOTES:

41

Agilent Ad Here

42

Poster Abstracts

Novel Technologies

P-100

Unification of Charge Heterogeneity, Purity, and Molecular Weight Analyses of mAbs into

a Single Analysis using CESI-MS

Bryan Fonslow1, Olga V. Friese2, K. Steven Cook2

1SCIEX, Brea, CA USA, 2Pfizer, Inc., Chesterfield, MO USA

Charge heterogeneity, purity, and molecular weight analyses are powerful CE-based methods for

mAb characterization. Combining similar CE separations of intact mAbs with mass

spectrometric (MS) detection could allow for unification of these three methods into one.

Additionally, the MS detection would facilitate identification of unknown CE peaks and may

also provide more accurate and sensitive purity and molecular weight measurements than with

optical detection alone. The integration of capillary electrophoresis (CE) and electrospray

ionization (ESI) into one process (CESI) provides these possibilities while also lowering the

sample mass analysis requirements which can be particularly useful in the mass-limited

development phase. We describe the use of a single CESI-MS analysis that provides charge

heterogeneity, purity, and molecular weight information. For such, the analyses of representative

IgG1, IgG2, and IgG4 molecules were performed at the intact and reduced levels using both

CESI- and CE-based methods. The CESI-MS results are compared to existing industry-accepted

CE-based charge heterogeneity, purity, and molecular weight analyses. Notably, charge

heterogeneity separations by CESI-MS using a CZE-based separation mechanism showed similar

profiles to a cIEF-based method. With the MS-based detection, molecular changes that cause

charge heterogeneity, such as deamidation, were correlated to CE migration shifts. Other peaks

within the charge heterogeneity separation could also be attributed to molecular weight

impurities within the samples. Collectively, the results demonstrate the advantages of using MS

as the detector for a charge heterogeneity analysis since it also provides molecular weight and

purity information.

NOTES:

43

NOTES:

44

P-101

Comprehensive Characterization of a Representative Antibody-drug Conjugate-like

Molecule by CESI-MS

Bryan Fonslow1, Lubica Supekova2, Chan Hyuk Kim3

1SCIEX, Brea, CA USA, 2The Scripps Research Institute, La Jolla, CA USA, 3California Institute

for Biomedical Research, La Jolla, CA USA

Antibody-drug conjugates (ADCs) are an important new class of biotherapeutics where a drug

molecule is covalently attached to a therapeutic monoclonal antibody (mAb) via a linker

molecule. Thus, ADCs present additional characterization challenges to mAbs including

determination of localization and stoichiometry of the drug-linker conjugation to the mAb and

the drug-antibody ratio (DAR). The integration of capillary electrophoresis (CE) and

electrospray ionization (ESI) into one process (termed CESI) provides the capabilities to improve

the separations, sensitivity, speed, and comprehensiveness of ADC characterization by mass

spectrometry while also lowering the mass requirements, particularly useful in the mass-limited

development phase and for safety and cost reasons. We describe the trifunctional use of CESI-

MS for the comprehensive characterization of a representative ADC-like molecule using small

molecule, peptide mapping, and intact protein analysis. In this case, the ADC-like molecule is a

fragment antigen binding (Fab) domain with lysine-conjugated polyethylene glycol–fluorescein

isothiocyanate (PEG-FITC). First, the representative NHS-PEG-FITC drug-linker was infused

using CESI-MS to characterize the fragmentation pattern of the FITC and FITC-PEG molecules.

Small molecule MS/MS information was used for peptide mapping data analysis. Second,

peptide mapping was performed to localize the FITC-PEG on primary amines among the

peptides and estimate their stoichiometries through relative quantification. Both small and large

peptides containing the FITC-PEG-modified peptides were identified and quantified. Third, the

Fab and Fab-PEG-FITC molecules were both analyzed at the intact level to generate intact MS

spectra for DAR calculation and stoichiometry measurements. Collectively, the trifunctional

analysis demonstrates the capabilities and benefits of CESI-MS in ADC development and

characterization.

P-102


Shaheer Khan, James Stray, Jenkuei Liu, Bharti Solanki-Nand, Brian Evans

Thermo Fisher Scientific, South San Francisco, CA USA

Glycosylation is one of the key critical quality attributes of mAb based biotherapeutics.

Glycosylation changes can impact biological drug’s safety, efficacy, clearance and

immunogenicity, making it necessary to accurately detect changes. Glycan profiling begins at

cell line development and continues through process development. Current glycan analysis

45

methods involve laborious multistep sample preparation that takes anywhere from a day to

multiple days for 96 samples, followed by single channel LC or CE separation.

Here, we report the development of a high throughput glycan analysis method utilizing a 24

capillary polymer filled array with laser induced fluorescence detection. For glycan labeling

along with conventional APTS dye, two new rapidly reacting fluorescent dyes were developed.

Glycan cleavage, dye labeling and excess dye removal steps were streamlined for automation.

We also eliminated the toxic sodium cynaoborohydride chemistry and vacuum centrifugation

steps. N-glycans from 96 samples were analyzed with <3 hours of hands on time and CE analysis

were completed in 7-9 hours. Some of the IgG glycans that were unresolved when labeled with

APTS were fully baseline resolved when labeled with our proprietary dyes. These novel dyes on

a simple streamlined workflow in combination with multi capillary CE instrument offer a high

resolution high throughput glycan analysis platform for rapid separation and quantitation of

antibody glycans.

NOTES:

46

P-103

Detection of ADP-Ribosylation in PARP-1 and Bacterial Toxins Using a Capillary-based

Western System

John Loughney, Richard R. Rustandi, Melissa Hamm, Sha Ha


Both poly and mono ADP-ribosylation are common post-translational protein modifications. For

example, poly ADP-ribosylation is involved in DNA repair mechanisms through the poly (ADP-

ribose) polymerase (PARP) family of enzymes. While mono ADP-ribosylation has been known

to trigger cell death exhibited by many bacterial toxins. Because of the wide role of ADP-

ribosylation, the detection and analysis are very important for further understanding of the PARP

family of enzymes and the molecular mechanisms leading to cell toxicity in the presence of

bacterial enzymes. Here we describe a novel technique utilizing a capillary electrophoresis (CE)-

based Western technology to detect and analyze ADP-ribosylated proteins. The method is based

on a nano-volume size separation that is automated, quantitative, offers great sensitivity, and is

high-throughput for potential use in PARP drug screening inhibitor assays.

Reference:

Rustandi, R. R., Hamm, M., Loughney, J. W., Ha, S. Electrophoresis (2015)

P-104

CE-SDS-CZE-MS using an In-capillary Approach to Eliminate SDS Interferences for

Antibody Analysis

Cristina Montealegre, Laura Sánchez Hernández, Christian Neusüß

Aalen University, Aalen, Germany

CE hyphenation to ESI-MS detection is increasingly applied for sensitive detection and

identification purposes. Unfortunately, many CE techniques and methods established in research

and industry are not compatible to ESI-MS since essential components of the background

electrolyte interfere in ES ionization. CE-SDS is one of the most commonly used analytical

method for quality control and purity assessment in pharmaceutical industries due to its high

separation efficiency and selectivity. However, its incompatible coupling with MS detection does

not allow obtaining essential information of proteins as unequivocal identity, glycosilation,

degradation, or possible impurities.

First, the usefulness of an in-capillary strategy for the removal of SDS interferences before ESI-

MS was demonstrated. Based on the interaction of SDS with a cationic surfactant, successful

results for the detection by ESI-MS of mAb in the presence of SDS were reached.

Using the potential of this in-capillary removal of SDS, the setup of a complete 2D-CE system

will be described by a mechanical valve as interface. In this way, direct MS analysis of protein

47

samples coming from CE-SDS can be performed. First results of the complete CE-SDS-CZE-MS

system will be presented and the possibilities of this new 2D-CE system for on-line coupling

SDS/MS methods will be discussed.

NOTES:

48

P-105

Automated Capillary Western Dot Blot Method for the Identity of a 15-Valent

Pneumococcal Conjugate Vaccine

Melissa Hamm, Sha Ha, Richard Rustandi


Simple Western™ is a new technology that allows for the separation, blotting, and detection of

proteins similar to a traditional western except in a capillary format. Traditionally, identity

assays for biological products are performed using either an ELISA or a manual dot blot western.

Both techniques are usually very tedious, labor-intensive, complicated for multivalent vaccines

and can be difficult to transfer to other laboratories. An advantage this capillary western

technique has over traditional manual dot blot western method is the speed and the automation of

electrophoresis separation, blotting and detection steps which performed in 96 capillaries. This

study describe details the development of an automated identity assay for a 15-valent

pneumococcal conjugate vaccine (PCV15-CRM197) using capillary western technology.

Keywords: Pneumococcal Conjugate Vaccine, Dot Blot, Capillary Western, Simple Western,

CRM197, polysaccharide.

Reference:

Hamm, H., Ha, S., Rustandi, R. R. Anal. Biochem. (2015) 478, 33-39

P-106

Applications of an Automated and Quantitative CE-based Size and Charge Western Blot

for Therapeutic Proteins and Vaccines

Richard Rustandi, Catherine Lancaster, Melissa Hamm, John Loughney


Capillary Electrophoresis (CE) is a versatile and indispensable analytical tool that can be applied

to characterize proteins. In recent years, labor intensive SDS-PAGE and IEF slab gels have been

replaced with CE-SDS (CGE) and CE-IEF methods, respectively, in the biopharmaceutical

industry. These two CE based methods are now an industry standard and are an expectation of

the regulatory agencies for biologics characterization. Another important and traditional slab gel

technique is the western blot, which detects proteins using immuno-specific reagents after SDS-

PAGE separation. This technique is widely used across industrial and academic laboratories, but

it is very laborious, manual, time consuming and only semi-quantitative. Here, we describe the

applications of a relatively new CE-based western blot technology which is automated, fast and

quantitative. We have used this technology for both charge- and size-based CE westerns to

analyze biotherapeutic and vaccine products. The size-based capillary western can be used for

fast antibody screening, clone selection, product titer, identity, and degradation while the charge-

49

based capillary western can be used to study product charge heterogeneity. Examples using this

technology for monoclonal antibody (mAb), Enbrel, CRM197, and Clostridium difficile (C.

difficile) vaccine proteins are presented here to demonstrate the utility of the capillary western

techniques.

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P-107

An Integrated Solution for High-throughput, User-friendly Glycoanalysis Using Rapid

Separation by CE

Zoltan Szabo, Michael Kimzey, Shirley NgJustin Hyche, Aled Jones, Ted Haxo, Sergey

Vlasenko

ProZyme, Inc., Hayward, CA USA

Glycan characterization is becoming necessary in the earliest stages of biotherapeutic cell line

development, to the point where cell culture screening often requires glycan profiling. This

entails significantly increased throughput for sample preparation, analytical instrumentation, data

processing, and expertise in glycan characterization. Unfortunately, these factors can result in a

bottleneck to results. To address these unmet needs, a glycan analysis solution is presented that

provides relative N-glycan quantification for 96 cell culture samples in 8 hours. These results are

obtained in an accessible format for technicians without prior glycan experience. At the front

end, simple and automatable sample preparation uses InstantDye chemistry to label released

glycans from Protein A purified cell culture samples. For N-glycan separation and detection, we

introduce a novel capillary electrophoresis system that has taken a cue from the speed and

ruggedness/ reproducibility of DNA analysis, with a total run time of 2 minutes per sample. At

the back end, peaks are integrated and glycan assignments are made using smart and

customizable analytical software. The quality of data from this system satisfies requirements for

screening and is similar to technologies such as UHPLC-FLR and CE-LIF. We anticipate that

this glycan analysis system is poised for future applications in glycomics and diagnostics.

P-108

Novel iZE Separation of Monoclonal Antibody Isoforms by Free Flow Electrophoresis

Christopher Timm, Robert Wildgruber, Katarina Herzig, Gerhard Weber

FFE Service GmbH, Feldkirchen, Germany

A lot of clinically and scientifically relevant proteins, like monoclonal antibodies (mAB),

undergo posttranslational modifications (PTM) stemming for example from glycosylation or

mRNA splicing. These protein isoforms can differ from each other in their activity and therefore

have to be closely studied and possibly separated from each other ahead of clinical application.

The matrix-free separation of these proteins by Free Flow Electrophoresis (FFE) with its fast

separation, high sample-throughput and -recovery make the FFE an ideal tool for semi-

preparative separation of protein isoforms.

In a different approach we demonstrated the separation of mAB isoforms by Continuous

Horizontal Isoelectric Focusing FFE (CHIEF-FFE). Here the protein isoforms were separated in

an electrical field, by using an ampholyte containing buffer, to establish a continuous pH

51

gradient. This approach results in a resolution of 0.02 ∆pI between single fractions and was

therefore very well suited for isolation of single isoforms.

In a novel approach mAB isoforms were separated by FFE in interval zone electrophoresis (iZE)

mode. The buffers used for interval zone only contain well defined chemicals (one acid, one base

and mannitol) and no polymers or ampholytes to form a stepwise pH gradient. Thus, this

technique is compatible for direct clinical and pre-clinical applications or crystallography.

NOTES:

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P-109

Quantitative Separation of Protein Isoforms by Free Flow Electrophoresis

Robert Wildgruber1, Christopher Timm1, Gerhard Weber1, Katarina Herzig1, Seth Madren2

1FFE Service GmbH, Feldkirchen, Germany, 2Biogen, Research Triangle Park, NC USA

Protein isoforms are defined as variants of a single polypeptide which generally alter its function.

More than 90% of naturally occurring isoforms arise from post translational modifications

(PTMs) and less than 10% from mRNA splice variations. In many cases, PTMs change the

biological activity of proteins. Therefore their properties have to be closely studied before being

administered as a drug.

Here, we introduced Continuous Horizontal Isolelectric Focussing Free Flow Electrophoresis

(CHIEF-FFE) to isolate up to 100 milligram amounts of individual protein isoforms of mABs

under native conditions that enable further biological studies. Resulting fractions were well

suited for direct use in further studies such as enzyme- and/or immune-assays.

In contrast to competing technologies like capillary electrophoresis (CE) and imaged capillary

isoelectric focusing (iCIEF) we are able to separate semi-preparative amounts of protein in a

very fast workflow.

Variable lengths of electromigration and a variable span of pH, generated by using homemade

ampholytes, gives us total control over protein separation. Zooming into ultra-flat pH gradients

resulted in a resolution of 0.02 ΔpI between single fractions. Combined with rapid UV detection

we were hereby able to separate and characterize multiple isoforms of different samples in

quantitative amounts.

P-110

Determination of Impurities in Small Molecules by Capillary Electrophoresis at an

Electrolyte pH where the Major Component is at Neutral Charge

Robert Weinberger

CE Technologies, Inc., Chappaqua, NY USA

Impurity analysis by CE is often hampered by the general detection problem. Most stacking

techniques enrich both the major component along with the impurities yielding little advantage.

A large chemical company manufactures 99.95% pure phenol and the 0.05% impurities are of

comprised of multiple components. If the sample concentration could be increased beyond the

usual 1 mg/mL, then perhaps the minor components could be visualized. By operating at a pH

where phenol is neutral, the sample concentration can be increased to 10 mg/mL without the

consequences of antistacking and electromigration dispersion. Separation of phenol and its

impurities is by MEKC. The peak height of the major component can be an astounding 2

53

absorbance units full scale. The method is robust, has simple sample preparation and exceeds

HPLC in speed and sensitivity (1). Four HPLCs were replaced with a single CE instrument.

The method was also applied to a zwitterionic drug substance (2) and most recently to a chiral

separation of ibuprofen using mixed cyclodextrins. This lecture will focus of the basis of the

technique, its limitations, describe pH effects at the surface of micelles and introduce the latest

data employing cyclodextrins.

Sayler, K., Weinberger, R, “Separation of Phenols as Neutral Compounds by Micellar

Electrokinetic Chromatography”, J. Chromatogr. 1014(2003)179.

Allen, L., Weinberger, J., Weinberger, R. Determination of Impurities in the Drug 5-

Aminosalicylic Acid by Micellar Electrokinetic Capillary Chromatography Using an Electrolyte

pH that Approaches the pI of the Parent Compound. J. Chromatogr. 1053(2004)217.

NOTES:

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P-111

Automated Sample Preparation for CE-SDS-LIF

Martin Weiss, Rolf Ketterer, Patrick Heim, Sonja Gisin, Jan Stracke, Bernd Moritz

F. Hoffmann-La Roche Ltd., Basel, Switzerland

CE-SDS-NGS emerged as a powerful and well accepted methodology in the biopharmaceutical

industry to support purity evaluation of recombinant humanized monoclonal antibodies

(rhmAbs) and new formats derived thereof. In order to enable highly sensitive LIF (laser induced

fluorescence-) detection the samples are labeled by a fluorescent dye. After denaturation with

SDS and optional reduction by DTT the electrophoretical separation is performed within a fused

silica capillary.

Automation of the time-consuming, multi-step sample preparation was assessed. For this a

Hamilton Microlab Star roboter was used. Sample preparation was adapted from a standard

manual protocol and optimized for this purpose. Prepared samples can be automatically

transferred into PCR tubings that directly fit into the autosampler of the CE system. The obtained

results are comparable with those obtained with manually prepared samples. This work

demonstrates that automated sample preparation for CE-SDS-LIF is valuable to reduce hands-on

time and costs for routine CE-SDS LIF analytics for protein pharmaceuticals.

NOTES:

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NOTES:

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Protein Analysis and

Emerging Therapeutics

P-112

Top-down and Middle-Down CE-MS for Deep Characterization of Biopharmaceuticals

with Glycan Heterogeneity: Identification of Interferon-β1 and Monoclonal Antibody

Proteoforms

David Bush1,2, Arseniy M. Belov1, Alexander R. Ivanov1, Li Zang3, Barry L. Karger1

1Barnett Institute, Northeastern University, Boston, MA USA, 2Genedata USA, Inc.,

Framingham, MA USA, 3Biogen, Cambridge, MA USA

Biopharmaceutical proteins typically consist of heterogeneous collections of proteoforms,

accurate knowledge of which is critical for assessing the safety and stability of a drug. Intact

separations of highly heterogeneous biopharmaceuticals is challenging due to the limited ability

of current separation techniques in resolving proteoforms with small structural changes. A new

sheathless CE-MS interface facilitates high resolution proteoform separation coupled to high

resolution mass spectrometry for thorough characterization of biopharmaceutical proteins.

A Sciex Separations CESI 8000 was coupled to a Thermo Fisher Scientific Orbitrap ELITE mass

spectrometer equipped with ETD. An etched porous silica capillary for sheathless CESI was

coated with polyethylenimine which was cross-linked to improve column durability.

Recombinant human interferon-β1 (Avonex) and an mAb were used in this study. Proteins was

buffer exchanged into H2O and, for MS2, were reduced with TCEP

Interferon-β1 (Avonex) is a 166 AA, glycoprotein (N80) with a disulfide bond (C31-C141) and

deamidation (N25). In order to identify constituents of Avonex that could not be assigned by the

glycan composition alone, TCEP was introduced into the sample buffer to reduce the disulfide

bond and facilitate top-down MS. Numerous c-ions confirmed the loss of the N-terminal

methionine, which contradicts an assignment for the same peak previously reported in the

literature as a mixed glycan which was unlikely to occur in the expression system used.

Sequence coverage for the unmodified and methionine-loss was 76% and 80% with both ETD

and HCD. More interesting is that the methionine loss is far more abundant with the deamidated

forms than the non-deamidated forms; this correlation would be otherwise lost with proteolysis.

Furthermore, by optimizing separation and injection conditions, we were able to resolve non-

reduced forms with glycan isomers.

NOTES:

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P-114

Detection and Quantitation of Low Abundance Proteins using Capillary Gel

Electrophoresis with Laser Induced Fluorescence Detection

Sharon Chen, Holly Craven, Karen De Jongh

Bristol-Myers Squibb Company, Seattle, WA USA

SDS-PAGE gels have traditionally been used to assay protein drug substance purity. However,

the manual nature of slab gels can result in method artifacts, as well as, poor detection and

quantitation of impurities. Capillary electrophoresis (CE) is a method demonstrated to have

increased reliability and reproducibility over slab gels. In recent literature reports, CE with laser-

induced fluorescence detection (CE-LIF) has been shown to be a more accurate and reproducible

protein quantitation method for the detection of lower abundance impurities than silver stained

SDS-PAGE. In these reports, the proteins were labeled with a fluorophore and detected using

specialized instrument settings and/or custom filters specific to the fluorophore. Here we present

a CE-LIF acquisition scheme that operates within the excitation and emission wavelength limits

of the default LIF carbohydrate analysis filters of the Beckman PA800 Plus using a

commercially available fluorophore that was previously unreported for this use. As a case study,

the scheme was demonstrated on forced degraded stability samples with minor modified forms.

The minor modified forms were identified as protein fragments and dimers by mass

spectrometry, were above the limit-of-detection of silver stain SDS-PAGE, and were below the

limit-of-detection of both Coomassie stained SDS-PAGE and CE-UV. In addition to being a

method that uses a commercially available fluorophore that operates with the instrument

manufacturer’s filters without unnecessary customization, this CE- LIF scheme provides an

alternative to silver stain SDS-PAGE for the identification of lower abundance impurities.

P-115

Precise Handling of Ultra-small Sample Volumes in Conventional Capillary

Electrophoresis Instruments

Sami El Deeb, Mona Mozafari, Markus Nachbar

Technical University Braunschweig, Braunschweig, Germany

Capillary electrophoresis is an appropriate and important technique for analysing

pharmaceuticals and biopharmaceuticals. In addition to low requirements in sample purity, only

a small volume of the sample is required to perform the analysis.

Most of the conventional CE instruments need approximately 50 µL of the sample in the

injection vial to perform the analysis, even though the injected sample volume in capillary

electrophoresis is only in the nanoliter range.

Since many proteins which are important from an analytical point of view undergo complicated

manufacturing processes and are produced in microgram quantities, it is important to reduce the

59

required sample volume and apply high concentrations of valuable samples. This approach

would be very advantageous from an economic point of view.

In order to fully profit from the low injection volumes, smaller vial volumes are required.

Therefore our experiments were performed using silicone oil as a filler of the vial dead volume

and the results were compared to that performed without this filling. As study example affinity

capillary electrophoresis was used to investigate the interactions of two proteins, namely HSA

and vitronectin, with three ligands namely enoxaparin sodium, unfractionated heparin and

pentosan polysulfate sodium (PPS). Mobility shift precision results over 12 hours analysis

showed that the employment of the filling with silicone oil has no noticeable effect on any of the

protein-ligand interactions (RSDs% for mobility ratios and peak areas were better than 0.8% and

1.5%, respectively). Accordingly, the employed silicone oil is suitable as a water immiscible and

inert liquid for filling the dead volume of sample vials. Using a commercial instrument and an

autosampler the required sample volume is reduced down to 10 µL, and almost this complete

volume can be subsequently injected during all experiments. Therefore the application of this

successful approach can be strongly recommended.

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P-116

Challenges of Demonstrating Biosimilarity

Eva Ennemann, Fabian Milz, Susanne Hensel, Martin Blüggel

Protagen Protein Services GmbH, Dortmund, Germany

The development and production of recombinant biopharmaceuticals and their biosimilars is a

challenging field due to their structural complexity. While demonstrating batch to batch

comparability is a demanding analytical task, demonstrating biosimilarity is even more

challenging as it is already performed during early development with limited knowledge of the

structural features of the biosimilar candidate and limited molecule specific method experience.

Additionally analytical methods in biosimilar development need to be applied for different

aspects like throughput (e.g. clone screening), initial and final comparability (qualified to be

sensitive to pick up difference), but also for release testing aspects (robust, validated).

The demands for analytical methods like CE are three-fold. Firstly, the depth of analytical

characterization is constantly increasing, necessitating new analytical approaches. Secondly, the

throughput of analytical methods needs to be increased, to support the fast development

timelines. Thirdly, orthogonal methods need to be provided for each quality attribute to ensure

the best possible comparability assessment.

For characterization of a mAb biosimilar candidate and the respective originator protein

modifications such as glycosylations, C-terminal lysine modifications, deamidations, oxidaton or

disulfide linkages are of special interest. By determination of the protein sequence, the

characteristic charge profile, the specific isoelectric point or the size distribution of the intact and

the reduced protein important structural and physicochemical information are made available as

a fingerprint. Here we present an analytical concept for a biosimilar development including

detailed side-by-side characterization of mAb biosimilar candidates and the corresponding

originator batches using qualified capillary electrophoresis and mass spectrometry assays for

biosimilar comparability.

P-117

Evaluation of a Shoulder Peak Detected in CE-SDS Testing of mAb A under Non-Reducing

Condition

Ya Fu1, Hangtian Song2, Jinping Liu3, Li Tao3, Ming Zeng1, Tapan Das3

1Bristol-Myers Squibb Company, New Brunswick, NJ USA, 2Bristol-Myers Squibb Company,

Bloomsbury, NJ USA, 3Bristol-Myers Squibb Company, Hopewell, NJ USA

Size variants of monoclonal antibodies (mAbs) are commonly monitored by reducing and non-

reducing CE-SDS methods. In the testing of a specific mAb samples (mAb A) by CE-SDS, a

minor shoulder peak under non-reducing condition was observed. The shoulder peak appeared

between the main IgG peak and partially separated NG-IgG (nonglycosylated IgG) peak. A

61

distinct shoulder peak was not observed in the reference material of the reported experiment.

Review of CE-SDS results generated from BMS and contract laboratories revealed that the

detection of this shoulder peak was found to be in random drug substance lots. In order to

identify the size variant of mAbs, three different methods were explored, which include (1)

enzyme digestion of mAb A samples; (2) reducing CE-SDS method; (3) Intact MS analysis. All

three methods provided consistent results in the identification of the fragments observed in CE-

SDS. The shoulder peak was found to be a partial de-glycosylated IgG.

NOTES:

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P-118

Enabling Rapid, High Throughput Analysis of Size-Based Purity Using SDS-Microfluidic

Capillary Electrophoresis

Emily Garguilo, Brian Schmidt, Tom Robinson

AbbVie, Inc., Redwood City, CA USA

Fragmentation is generally considered to be a critical quality attribute for therapeutic proteins

that must be monitored and controlled. Capillary gel electrophoresis with sodium dodecyl sulfate

(CE-SDS) has become a widely adopted method within the biopharmaceutical industry for

quantitative analysis of size-based purity. In recent years, newer microfluidic “capillary on a

chip” technologies have emerged as potential higher throughput alternatives to traditional

capillary electrophoresis.

We have successfully implemented microfluidic capillary electrophoresis (SDS-MCE) for use in

evaluating size-based purity and impurities of biopharmaceutical proteins, including monoclonal

antibodies. The 96-well plate based sample preparation strategy and rapid separation time of 41

seconds per sample, greatly improve the efficiency and throughput of the SDS-MCE method

compared to traditional CE-SDS. The high efficiency of the method offers benefits in

applications where rapid screening of large numbers of samples or conditions is desired. This

efficiency also enables use of sample replicates as a strategy for increasing precision of sample

results as is often desired in Quality Control. The SDS-MCE method was qualified according to

ICH recommendations for validation of analytical methods and successfully implemented for

GMP lot release and stability monitoring. Selected qualification and GMP testing results

demonstrating SDS-MCE method performance are presented here.

The high throughput and robust performance of the SDS-MCE method make it an ideal platform

assay for fragment analysis that can be used seamlessly across the development spectrum from

R&D to Quality Control.

Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial

support for this research were provided by AbbVie. AbbVie participated in the interpretation of

data, review, and approval of the publication.

P-119

The NISTmAb: A Platform for Open Innovation

John Schiel

NIST, Rockville, MD USA

The road to developing quality biotherapeutic proteins is a rapidly and ever changing landscape.

Now more than ever, analytical and biophysical characterization plays pivotal yet evolving roles

from R&D to commercialization. The ultimate pursuit is technological innovation, followed by

63

practical implementation, of broadly informative methodology. The NISTmAb, an IgG1k class-

specific biopharmaceutical reference material, is a means to fuel milestone technologies and

bridge their widespread industry utilization. Global conclusions drawn from the NISTmAb

crowdsourcing collaboration “State-of-the-Art and Emerging Technologies for Therapeutic

Monoclonal Antibody Characterization” will highlight the importance of open innovation

focused around common materials. The RM is expected to more firmly underpin regulatory

decisions and facilitate the development of originator and follow-on biologics. The NISTmAb is

intended for a variety of uses including, but not necessarily limited to: establishing method or

instrument performance and variability, comparing changing analytical methods, assisting in

method qualification, etc. Unique aspects of RM lifecycle management, fit-for-purpose

characterization, and ongoing research expanding the biopharma RM program will be discussed.

NOTES:

64

P-120

Fractionation of Antibody Drug Conjugates Utilizing Free-Flow Electrophoresis and

Protein A Purification

Aron Lee1, Jeffrey Zhang1, Meena Narsimhan2, Bradley Calvin2, William Haskins1, Yan Chen1

1Genentech, a Member of the Roche Group, South San Francisco, CA USA, 2Aseda Sciences,

West Lafayette, IN USA

Antibody drug conjugates (ADCs) have developed into a promising modality of antibody

therapeutics. However, manufacture of the antibody precursor produces charge heterogeneity

that can impact pharmacokinetics and potency. For ADCs with cysteines conjugated to drugs

that do not contain a charge, the overall charge is not expected to be changed. However, the

ADC charge variants still need to be isolated for characterization of potential unexpected low

level modifications and assessment of biological potency. Many conjugated drugs are highly

hydrophobic, which means ion exchange is not an effective method for charge variant isolation

due to nonspecific interactions with the column matrix. Free flow electrophoresis (FFE) is an

effective alternative because it is a liquid based phase separation which enables high recoveries

of sample components and good resolution.

FFE isolation can introduce excipients, such as hydroxypropylmethylcellulose (HPMC), which

might interfere with downstream analysis. Since HPMCs form oligomers of varying sizes,

simple filtration is not an option for its removal. Protein A capture can effectively remove the

excipients, but might introduce aggregation due to the low pH elution conditions. The current

work presents results from FFE separation of charge variants for an ADC conjugated to a potent

antimitotic agent and development of a protein A capture condition that minimizes aggregation.

P-121

Using Non-Denaturing icIEF to Estimate Drug-to-Antibody Ratio and Percent

Unconjugated Antibody in Antibody-Drug Conjugates Containing Charged Drug-Linkers

Lily Liu-Shin

Agensys, Inc., Santa Monica, CA USA

Imaged capillary isoelectric focusing (icIEF) is a widely-used analytical tool for characterizing

charge variant distributions of monoclonal antibodies (mAbs) and antibody-drug conjugates

(ADCs). Charge variant distributions of ADCs may be quite complex in that the chemical

properties of the antibody intermediate are inherited, and are further influenced by the drug-

linker chemotype and conjugation technology. One of the advantages of using a non-denaturing

icIEF method is the ability to directly compare the biochemical changes occurring pre- and post-

conjugation of the intact molecule. In the case of ADCs conjugated with charged drug-linkers

(DL), the icIEF profile contains several additional peaks (in comparison to the mAb) that

correspond to various drug-loaded species. We demonstrate the correlation between ADC drug-

65

loading distribution and the resulting icIEF profile, and provide a complimentary method for

estimating drug-to-antibody ratio (DAR) and amount of unconjugated antibody.

NOTES:

66

P-122

Advantages of CESI-MS for Peptide Quantitation: A Comparison to Analytical flow LC-

MS

Stephen Lock1, Anton Heemskerk1, Bryan Fonslow2, Edna Betgovargez2

1SCIEX, Pudsey, United Kingdom, 2SCIEX, Brea, CA USA

Standard reverse phase liquid chromatography separates constituents based on their differential

interactions with the column stationary phase and an organic mobile phase. CESI-MS is a

different separation technique and is the integration of CE and ESI into one dynamic process and

provides highly efficient peptide and protein separations based on their size and charge. The

ionization is at the ultra-low nanoflow regime (25-100 nL/min) and uses an open capillary which

eliminates some of the challenges of stationary phase based separations such as lack of retention

of small polar peptides as well as over retention of larger hydrophobic peptides.

The purpose of this work is to compare CESI-MS to high flow LC-MS analysis. In this

comparison the MS method remains the same with the exception of the source conditions which

had to be optimized for the different flow rates used by CESI-MS and HPLC-MS. The runtimes

were adjusted so that throughput was reasonable and injection to injection analysis would be

below 20 minutes for each final method. Sensitivity, linearity and reproducibility of both

methods are compared using each of the 5 peptides present in series of calibration standards.

P-123

A New High Resolution Approach to the Detection of Insulin Degradation Products

Stephen Lock

SCIEX, Pudsey, United Kingdom

Despite recent developments in liquid chromatography column and hardware technology

analysis of larger intact peptides, for example insulin and its derivatives, by liquid

chromatography mass spectrometry (LCMS) remains challenging. These larger peptides often

exhibit properties which adversely affect their chromatography. In some instances they are polar

eluting in the solvent front, or overly retained causing peak broadening, tailing or problems with

carryover.

In this presentation, we will present an alternative workflow used to overcome some of the

challenges of stationary phase based separations. We will compare capillary electrospray

ionization (CESI-MS, which has a revolutionary design where the CE flows directly into the MS

source at low nL/min) with reverse phase LC-MS in the analysis of a sample containing intact

insulin which had been heated to induce degradation. We will use the same accurate mass MS

method and sample to compare both techniques and assess their ability to detect degradation

products of insulin with the aim to identify both cleavage and deamination products.

67

NOTES:

68

P-127

Fast Separation and Analysis of Reduced Monoclonal Antibodies with Capillary Zone

Electrophoresis Coupled to Mass Spectrometry

Yimeng Zhao1, Liangliang Sun1, Michael D. Knierman2, Norman J. Dovichi1

1University of Notre Dame, Notre Dame, IN USA, 2Eli Lilly and Company, Indianapolis, IN USA

Capillary zone electrophoresis (CZE) separates ions by their mass to charge ratio rather than

hydrophobicity, which is more efficient for proteins. CZE also has an advantage over liquid

chromatography in separation speed. These two features of CZE make it a potential high

throughput screening method for quality control of monoclonal antibodies. In this work, capillary

zone electrophoresis-electrospray ionization-mass spectrometry (CZE-ESI-MS) was used for

analysis of reduced antibodies. We first developed a simple protocol to condition commercial

linear-polyacrylamide coated capillaries for use in top-down proteomics. We then suspended

reduced antibodies in a solution of 35% acetic acid, 50% acetonitrile in water. Heavy and light

chains were baseline resolved within 10 minutes and with 3 to 30 µg/mL detection limits using a

0.1% aqueous formic acid background electrolyte. Quintuplicate runs of a two-antibody mixture

produced relative standard deviations of ~1% in migration time and 10% in peak amplitudes.

Resolution was further improved for the two-antibody mixture by using 5% acetic acid as the

background electrolyte, highlighting the potential of capillary electrophoresis-mass spectrometry

for analysis of antibody mixtures. This system provides fast separation, good reproducibility, and

reasonable sensitivity. Furthermore, the separation of a two-antibody mixture highlighted the

possibilities for CZE-MS to characterizing mAb mixture and even polyclonal antibodies.

This work was funded by the National Institutes of Health (R01GM096767) and Eli Lilly Inc.

through the Lilly Research Award Program.

NOTES:

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70

QbD and Method Development

P-128

Method Development and Characterization of Fusion Protein using Imaged Capillary

Isoelectric focusing (iCIEF)

Ying-Chen Chen, Anulfo Valdez, Girija Krishnamurthy, John Devincentis, Reb Russell

Bristol-Myers Squibb Company, Bloomsbury, NJ USA

This study has been used to evaluate the use of iCIEF technology for the analysis the charge

heterogeneity of a fusion protein. The fusion protein consists of ligand-binding domain and

constant region of IgG heavy chain (Fc). As compared to traditional slab gels, the Imaged

capillary isoelectric focusing (iCIEF) method provides greater sensitivity and allows the

generation of highly quantitative data of the charge variants.

The scope of the presentation is to show the development of the methodology to characterize

charge variants in a protein. The development and optimization, of the iCIEF are highlighted in

this presentation. Enzymatic treatment was also used to characterize the separation profiles

related to lysine variants and glycoforms of fusion protein. The results of iCIEF method

demonstrate its application in characterization, of charge variants in the fusion protein.

P-129

Evaluating the Effect of Capillary Temperature on Structure Specific Glycan Migration

Behavior

András Guttman1,2, Marton Szigeti1,2, Marta Kerekgyarto2 and Gabor Jarvas1,2

1MTA-PE Translational Glycomics Research Group, University of Pannonia, Veszprem,

Hungary, 2Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen,

Debrecen, Hungary

Temperature dependent differential electromigration of helical (maltooligosaccharides) and

branched (biantennary IgG glycans) carbohydrates were studied in capillary electrophoresis. The

activation energy concept was applied to understand the irregularities in their electrophoretic

mobilities. In addition to the regularly used lithium acetate buffer system, background

electrolytes containing low and high molecular weight hydrophilic modifiers of ethylene glycol,

linear polyacrylamide and polyethylene oxide were also investigated and the same phenomena

were observed. APTS labeled helical and branched glycans were separated by capillary

electrophoresis with laser-induced fluorescence detection in the temperature range of 20-50°C.

The activation energy values were derived from the slopes of the Arrhenius plots. Temperature

71

dependent glucose unit (GU) shifts were observed for the different shape carbohydrates both in

additive-free and additive-containing background electrolytes, emphasizing the importance of

temperature control during glycosylation analysis by capillary electrophoresis. Based on the

experimental data, a novel high resolution separation matrix was designed enabling ultrafast (<5

min) analysis of monoclonal antibody born fucosylated and afucosylated biantennary glycans of

recent biopharmaceutical interest.

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P-130

Band Confirmation for SDS-PAGE/Capillary Gel Electrophoresis Crossover using the

Simple Western Platform

Heather Jean, Martha Jackson, Poonam Aggarwal, Mary Denton, Jeff Smith

Pfizer, Inc., Andover, MA USA

As part of therapeutic protein characterization activities related to SDS-PAGE/capillary gel

electrophoresis (CGE) cross over, protein band confirmation is performed. This is often

achieved by N-terminal sequencing, which is time consuming and requires running SDS-PAGE,

transfer of bands to PVDF, excising bands of interest and then sequencing each excised band.

Frequently, trace level bands are not detectable during the initial round of sequencing and

multiple subsequent rounds of isolation and analysis are required. An alternative to N-terminal

sequencing is Western blotting, which uses antibodies directed against epitopes on the proteins

(bands) being confirmed. Traditional Western blotting is also resource intensive with SDS-

PAGE analysis, transfer, wash steps and detection. The Simple Western instrument

(ProteinSimple), employs a capillary electrophoresis format with Western blotting reagents to

provide rapid analysis. This platform can analyze up to 25 samples simultaneously and provides

semi-quantitative results. The potential use of the Simple Western platform for SDS-PAGE band

confirmation as part of the SDS-PAGE/CGE characterization activities for a late-stage

monoclonal antibody was evaluated. This study presents results from optimization activities

related to sample preparation and resolution on the Simple Western platform for reducing and

non-reducing SDS-PAGE band confirmation activities.

P-131

Comparison of Cleanup Methods for N-linked Oligosaccharide Analysis

Bindiya Juneja, Simon Yeung, Kathir Muthusamy, William Matousek, Jiann-Kae Luo, Erica

Pyles

Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA

To date, more than thirty monoclonal antibodies (mAbs) have been approved for the treatment of

various diseases such as rheumatoid arthritis, breast cancer, and multiple sclerosis. Glycan

characterization of mAbs is valuable due to its vital role in function, protein stability, and

efficacy. Detailed characterization of mAbs as well as routine, well-controlled release assays for

each manufactured lot is required for regulatory filings. The techniques most commonly used for

glycan characterization include hydrophilic-interaction liquid chromatography (HILIC) and

capillary electrophoresis (CE). For CE-based analysis, N-linked oligosaccharides are released

from mAbs using Peptide N Glycanase F (PNGase F), then labeled with 8-aminopyrene-1,3,6-

trisulfonate (APTS) dye by reductive amination. APTS-labeled glycans are electrophoretically

separated and then quantitated using CE coupled to laser induced fluorescence detection.

Presence of free APTS in the sample can interfere with glycan analysis, and also lead to poor

73

signal to noise ratio. There are a few APTS cleanup methods available such as rapid magnetic

beads based sample cleanup, and cleanup module from commercial resources. Magnetic beads-

based method utilizes differential binding affinity of carboxyl-coated magnetic microparticles

(COOH-beads) between labeled glycan and free APTS in solution. Prozyme kit cleanup modules

include cleanup spin columns, which selectively capture labeled glycans. These two clean-up

methods for glycan analysis were compared and our results indicate that both cleanup methods

are reliable, fast, and generate reproducible results.

NOTES:

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P-132

Assessment of Disulfide Related Fragmentation and Modification Processes; Analytical

Capabilities of CE-SDS and Other Methods

Bernd Moritz

F. Hoffmann - La Roche Ltd., Basel, Switzerland

Disulfide related fragmentation & modification processes, their effect on potency & clearance

and their detectability by CE-SDS and other analytical methods are reviewed. Focus is on IgG1,

IgG2 and IgG4 antibodies which are the main mAb classes for therapeutic use. Literature

information about reductive cleavage of inter- and intra-chain disulfide bonds, processes in the

hinge region, trisulfides, cysteinylation and disulfide scrambling is summarized.

There are several methods that can detect disulfide induced fragmentation and modification

processes. In case of inter-chain disulfide cleavage and hinge cleavage CE-SDS is the method of

choice. Other disulfide related modifications are characterized by methods like IEC.

Influence of disulfide related modifications on potency depends on the mode of action, i.e.

monovalent/bivalent binding of antigens and requirement of Fc effector functions. Clearance is

influenced by FcRn binding and size. Small fragments like the light chain are rapidly cleared by

the kidney. Clearance of larger fragments like Fab/c depends on FcRn functionality that can be

influenced by disulfide related modifications. In vivo occurrence in endogenous antibodies or in

vivo repair was observed for many of these modifications.

Available literature information supports CQA assessment for disulfide related fragmentation &

modification processes.

P-133

Faster and Easier Charge Heterogeneity Analysis with iCE3

Alpana Prasad, Scott Mack

ProteinSimple, San Jose, CA USA

Three major usability enhancements are now available for the iCE3 system for improved charge

heterogeneity analysis. A new HT cIEF cartridge increases resolution while reducing run times

by eliminating the need for methylcellulose. A redesigned electrode arm assembly minimizes

cathodic drift allowing robust analysis of 100 samples in a batch. The pI calibration and data

export processes have been combined into a single automated procedure through the

development of enhanced software features. These updates offer greater speed and improved

ease of use. In this poster, we present the results of these improvements on a model iCE method

for analysis of a basic IgG1 mAb. The new HT cartridge reduces analysis time to 10 minutes,

while still providing a highly resolved peak profile comparable to the original FC cartridge. An

75

intermediate precision study demonstrated a %CV of less than 10% for peak all major peak

clusters (>5% percent composition).

NOTES:

76

P-134

Enriching the Analytical Toolbox for Viral Vaccines with Capillary Electrophoresis

Lars Geurink1, Ewoud van Tricht1, Cari Sänger-van de Griend2

1Janssen Infectious Diseases and Vaccines, Leiden, The Netherlands, 2Kantisto BV, Baarn, The

Netherlands

A wide variety of analytical and biological methods are required to monitor the safety, efficacy

and quality throughout the production process of viral vaccines. Nevertheless, extension of the

toolbox is desired as better characterization is still needed.

Capillary electrophoresis (CE) is currently relatively little used to assess viral vaccines. Typical

characteristics for CE are fast separations, low sample volumes, qualitative analysis with good

precision and accuracy and a wide range of applications. Additionally, this analytical technique

has the advantage to be able to analyse under native conditions and the potential to directly inject

a wide range of sample matrices.

Within Janssen Infectious Diseases and Vaccines a workgroup was established to investigate the

potential of the capillary electrophoresis techniques. Different modes of capillary electrophoresis

such as Capillary Zone Electrophoresis (CZE), Capillary Gel Electrophoresis (CGE), Capillary

IsoElectric Focussing (cIEF) and CE-MS, are being investigated. Applications include

quantification of viral proteins content, host cell DNA content, virus particle content, and antigen

expression after cell infection.

This poster presents our implementation strategy and discusses the requirements for a successful

implementation programme for new technology.

P-135

Improved Method for Quantification of Virus Particles in Complex Matrices using

Capillary Electrophoresis

Ewoud van Tricht1, Lars Geurink1, Cari Sänger-van de Griend2

1Janssen Infectious Diseases and Vaccines, Leiden, The Netherlands, 2Kantisto BV, Baarn, The

Netherlands

Analytical and biological methods are required to monitor the quantities (in virus particles per

ml) of viruses throughout the production process, to ensure the safety, efficacy and quality of the

vaccines. HPLC-based techniques (e.g. size exclusion or ion exchange) have been typically used

to quantify virus particles but showed to be unsuitable for samples containing cell lysate or high

salt concentrations. Carry-over and recovery issues were observed resulting in unrepeatable and

inaccurate results. There is a distinct need for a single method that can quantify viruses

throughout the entire production process with high accuracy and precision, and a limited time to

result. Capillary electrophoresis (CE) was selected to overcome the issues as observed for the

77

HPLC-based techniques, since literature described the application of CE for the analysis of

viruses, proteins and complex biomolecules, even in complex matrices. The main challenge

during development was to prevent adsorption of the virus and matrix components to the

capillary wall. A capillary electrophoresis method was developed suitable for the accurate and

precise analysis of all samples from the full process containing either cell lysate and cell debris,

or high salt concentrations. The method was validated and will be implemented in the quality

control department to determine the quantity of virus particles.

NOTES:

78

P-136

Computer-Aided Design of Experiment (DoE) for Imaging Capillary Isoelectric Focusing

(icIEF) Assay Development, Optimization, and Re-qualification: Case of a Problematic

Protein

Richard Smart, Peter Bryngelson

Biogen, Cambridge, MA USA

Imaging Capillary Isoelectric Focusing (icIEF) has been performed at Biogen for many years

utilizing iCE technology from Protein Simple®. Biogen’s Analytical Development group is now

looking to apply Quality by Design (QbD) and Computer Aided Design of Experiments (DoE)

for icIEF assay development and qualification. This innovative approach uses Stat-Ease Design-

Expert® software and a 96-well plate coupled with automated sample preparation. This approach

is fast, efficient, and enables AD to meet development timelines for multiple products.

An IgG protein currently under development has seen a recent increase in assay failures due to

significant inconsistency in measured relative percent of high pI Isoforms. The source of the

issue was suspected to be aggregation caused by precipitation during the isoelectric focusing

step. In order to solve the assay issue, an experiment was designed using Design-Expert® to

evaluate various parameters such as urea content, narrow and wide range ampholyte content, and

protein load on the icIEF profile. It was determined that the addition of urea to the sample matrix

and an optimization of the narrow range ampholytes resulted in better separation of charged

isoforms and greater assay robustness. Therefore, the assay was optimized and re-qualified

according to ICH guidelines using QbD and DoE principles. The approach decreases the time

necessary to develop and qualify icIEF assay conditions as well as troubleshoot similar issues in

the future while improving assay quality.

P-137

Optimized Microelectrophoretic Characterization of the NISTmAb Reference Material

Abigail Turner1, 2, John E. Schiel2

1University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD

USA, 2NIST, Rockville, MD USA

The National Institute of Standards and Technology is developing a monoclonal antibody (mAb)

reference material and accompanying characterization data whose purpose is to facilitate and

harmonize analytical characterization of therapeutic mAbs. Therapeutic mAbs comprise the

fastest-growing class of biologic drugs, including novel proteins as well as biosimilar and bio-

better follow-on biologics. In particular, the growing market for biosimilars has inspired a call

for improved analytical characterization of therapeutic mAbs which may reduce the burden of

animal studies and clinical trials needed for regulatory approval. The NIST mAb is intended to

fulfill the need for a highly characterized, well-defined and widely available reference protein.

79

Assurance of material quality requires robust microelectrophoretic methods for defining size and

charge heterogeneity in the NIST mAb reference material. We present optimized methods for

analysis of size variants by capillary SDS gel electrophoresis (cSDS) and of charge variants by

capillary isoelectric focusing (cIEF), focusing on resolution of known product variants produced

under stressed conditions. This work is part of an ongoing effort to create a comprehensive

analytical profile of NIST mAb purity and stability which are essential to its utility as a reference

material.

NOTES:

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P-138

cIEF Method Development For a Bispecific Antibody

Eric Waltimyer, Shelly Ji

Janssen Pharmaceutical R&D, LLC, Malvern, PA USA

Bispecific antibodies are believed to have great therapeutic potential in various disease

indications and are increasing in development within the biologics industry. The advantage of

bispecific antibodies is that they bind to two different epitopes either on the same, or on different

targets which may improve the antibodies' specificity and efficacy in inactivating the disease

targets. One of the bispecific antibody technologies is the DuoBody® platform (Labrijn et al.,

Proc Natl Acad Sci U S A. 2013 Mar 26; 110(13): 5145–5150) which is composed of heavy and

light chains from each parent antibody.

In this presentation, we describe the development of a single capillary isoelectric focusing (cIEF)

method for monitoring the charge heterogeneity of the DuoBody®, and both parental antibodies.

The presentation will outline the study design and optimization of the cIEF parameters, and

discuss the challenges during method development.

P-139

Development of a CZE Method to Monitor Charge Heterogeneity in a Protein with a Low

pI

Rebekah Ward, Prachu Bhoskar, Michael Jankowski, Neil Steinmeyer

Pfizer, Inc., Andover, MA USA

Charge heterogeneity of a protein therapeutic is a common quality attribute monitored as part of

a robust characterization, release, and stability testing package. Ion-exchange chromatography

(IEX) and imaged capillary electrophoresis (iCE) are the typical techniques employed when

developing a charge-based separation. In the case of a protein with a low pI and containing

multiple N-linked glycosylation sites with varying sialic acid content, the above mentioned

techniques fail to reproducibly resolve the charge isoforms. As such, a capillary zone

electrophoresis (CZE) method was developed for this particular protein. The method was found

to have optimal separation of the charge isoforms and was deemed appropriate to reproducibly

monitor the charge heterogeneity. In this study, multiple background electrolytes (BGE) were

evaluated along with various separation conditions. A BGE solution of boric acid, pH adjusted

with sodium hydroxide to pH 8, was found to yield an optimal resolution of the various acidic

species. As anticipated for this molecule, the heterogeneity in the acidic region was found to be

mainly due to sialylation. This was confirmed enzymatically by de-glycosylation and de-

sialylation of the protein prior to analysis by CZE. A small subset of parameters was evaluated

for robustness of this method. These parameters include pH and concentration of the BGE,

81

protein concentration, and injection duration. This CZE method was determined to be a robust

and reproducible method for separating charge isoforms for a protein with a low pI.

NOTES:

82

Regulatory and QC

P-141

Recombinant DNA Sequencing using GenomeLab GeXP Genetic Analysis System

Jane Luo1, Doni Clark2, Handy Yowanto1

1SCIEX, Brea CA, USA, 2Beckman Coulter, Inc., Brea, CA USA

Within the process of manufacturing biologics utilizing recombinant DNA technology, living

cells producing recombinant proteins could potentially undergo mutations that alter the

properties of a protein with adverse impact to the intended products. Therefore, it is critical to

verify the fidelity of the nucleotide sequence encoding the expression product in the Master Cell

Bank. For many pharmaceutical companies, verification of coding sequence of the recombinant

DNA insert in expression vector is also conducted at the level of Manufacturers Working Cell

Bank (MWCB) and End of Production cells. The GenomeLab GeXP Genetic Analysis System

performs Sanger sequencing with high quality results using unique DNA sequencing reagents

including linear polyacrylamide gel (LPA), coated capillaries and near-infrared dyes with low

background and high signal to noise ratio. In addition, the online thermal denaturation further

ensures more uniform sequencing results across the sample plates. In this poster, we present

results obtained with the GeXP system in sequencing various recombinant DNA in expression

vectors. The dITP utilized in the GeXP Dye Terminator Cycle Sequencing chemistry reduces

GC compression, making it ideal for routine sequencing analysis for many recombinant DNA

sequences. In cases of difficult templates containing repeat regions or a polymerase hardstop, an

alternatively available dGTP chemistry successfully enables accurate reads through these

sequences. The GenomeLab GeXP Genetic Analysis System is therefore a valuable tool for

verifying coding sequences in the production process of biologics.

P-142

CE Based Sequencing Analysis as a Tool for Investigation of Sterility Positives with Fast

Turnaround Time

Jane Luo1, Kai Uchiumi2, Manfred Souquet3, Handy Yowanto1

1SCIEX, Brea, CA USA, 2SCIEX, Tokyo, Japan, 3SCIEX, Darmstadt, Germany

During the development and manufacturing processes of biologics, it is critical to monitor the

sterility of pharmaceutical ingredients, water for pharmaceutical use, the manufacturing

environment, intermediates and finished products. In addition, Master Cell Bank, each seed lot

and cells used in each production run should be tested for adventitious agents including

mycoplasma, bacteria, fungi, viruses, and virus-like particles. When microbial growth is

83

observed, an investigation needs to be conducted and FDA guideline requires sterility test

isolates be identified to the species level. It has been shown that molecular sequencing is superior

in identifying microbes to the species level, when compared against conventional microbiology

staining and culture methods. In this poster, we describe a process for sequencing 16s ribosomal

RNA gene of microorganism using the GenomeLab GeXP Genetic Analysis System that offers

fast turnaround time (8 hrs) and high microbial identification accuracy. Upon isolation of nucleic

acid from each microbial sample, the gene target is amplified using a polymerase chain reaction

(PCR), followed by sequencing reaction utilizing the Dye Terminator Cycle Sequencing (DTCS)

on the GenomeLab GeXP Genetic Analysis system. Results are compared against the NCBI

microbial library to identify the contaminants.

NOTES:

84

Troubleshooting, Method Lifecycle

Management, Validation

P-143

Leading Peak Artifacts in Gel Electrophoresis of Monoclonal Antibodies

Megan Ely1, Douglas Johnson2, Paula Lei2, Michelle Brown1, Qiang Qin1, Roujian Zhang1,

1MedImmune, A member of the AstraZeneca Group, Frederick, MD USA 2MedImmune, A

member of the AstraZeneca Group, Gaithersburg, MD USA

One source of poor reproducibility in gel electrophoresis of non-reduced monoclonal antibodies

has been identified and can be prevented by adjusting the composition of the sample buffer.

Atypically large pre-peaks in front of the intact antibody that are not reproducible between

sample preparation replicates are indicative of incomplete protein denaturation1. A study of

sample preparation factors found these pre-peak artifacts correlate with the amount of dodecyl

sulfate (DS) added to the reaction (either the volume of sample buffer used or the amount of DS

added to the sample buffer) rather than the heating conditions, amount of alkylating agent added

used, or other factors tested.

Supplementing the sample buffer with DS may improve the robustness of this test method by

reducing both the frequency and size of these artifact peaks. However, the benefits of increasing

the DS concentration must be balanced against the risk of losing sensitivity in fluorescent

detection systems if too much DS is added.

References:

1. Brody T (1997) Multistep denaturation and hierarchy of disulfide bond cleavage of a

monoclonal antibody. Anal Biochem 247:247-256

P-144

Bringing iCIEF Technologies into QC Network: The Challenges Moving Forward

Sarafina Lou Sai Ho, Connie Lu, Sherry Guo


A marketed product was approved by the U.S. Food and Drug Administration in June 2000 for

use to reduce mortality associated with acute myocardial infarction (AMI). There is no charge

heterogeneity method in the control system since approval. Expectation evolves over a product’s

lifecycle and the original approved control system become outdated.

85

An image Capillary Isoelectric Focusing (iCIEF) method was developed and validated for

meeting new Health Authority expectations and keeping the control system in modern and

compliant state. The results of iCIEF method validation will be presented.

Although the new iCIEF method is validated to determine the charge variants of this highly

complex protein, there are still challenges to be overcome in order to be implemented as a robust

QC testing method.

NOTES:

86

P-145

Challenges of Implementing CE Methods for Marketed Products During Control System

Lifecycle Management

Connie Lu


Capillary electrophoresis (CE) is a technique used to separate charged molecules within a small

bore capillary filled with an electrolyte based on their size to charge ratio. CE technology offers

a lot of advantages over traditional sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE), isoelectric focusing gel electrophoresis (IEF), and high performance liquid

chromatography (HPLC) methods, such as short analysis time, high resolution, and small sample

and solvent volume. Therefore, CE technology has been used in a wide range of applications in

biopharmaceutical industry. During marketed products control system lifecycle management, we

try to replace SDS-PAGE with CE-SDS, IEF gel with imaged capillary IEF (iCIEF), or add

additional CE method to the control system. This presentation will discuss challenges that we

encountered during method development, validation, and implementation of CE methods for QC

testing in the global network. Solutions to overcome the challenges will also be presented.

P-148

Identification of an Unexpected Peak in Non-Reduced CE-SDS of a Monoclonal Antibody

Qing Zhu, Jia Chen, Paula Kong


The use of Capillary Electrophoresis Sodium-Dodecyl Sulfate (CE-SDS) with LIF detection is an

important tool to monitor and quantify size heterogeneity of therapeutic proteins at Genentech.

Highly reproducible profiles are essential to ensure the detection of size-related product variants

accurately. An unexpected peak was observed at the trailing edge of the main peak as compared

to the typical profile in non-reduced CE-SDS for a monoclonal antibody (mAb) using

Fluorogenic FQ Dye. This peak was observed randomly among different test sessions and

analysts. We investigated multiple steps and factors during CE-SDS sample preparation to

understand the root cause of the unexpected peak. The results showed that the unexpected peak

resulted from the sample preparation mixing step, which caused mAb aggregation. Therefore, the

assay test procedure was modified to eliminate this assay artifact. This poster presents the details

and conclusion of this unexpected peak investigation.

NOTES:

87

NOTES:

88

Young Scientist

P-149

Affinity Capillary Electrophoresis to Investigate the Interactions of Pentosan Polysulfate

Sodium and Related Substances with Proteins

Mona Mozafari, Sami El Deeb, Hermann Wätzig


Pentosan polysulfate (PPS) is highly sulfated polysaccharide derived from beechwood

hemicelluloses by sulfate esterification. Beside the well known antithrombotic activity, other

properties such as anti-inflammatory, anti-angiogenesis and inhibition of cell adhesion are still

under investigation for this substance as well as for heparins [1, 2]. Accordingly, it is important

to know how strong PPS interacts with some important proteins in comparison to heparin and

other heparinoids.

For this purpose affinity capillary electrophoresis methods have been developed due to its

numerous advantages such as short analysis duration and minute consumption of samples. In

this study we used some model proteins namely HSA, BSA, myoglobin, ovalbumin, beta-

lactoglobulin and two other important proteins namely vitronectin and P-selectin, which are

involved in coagulation cascade.

The interactions were calculated using mobility ratios (R) of the EOF-marker, acetanilide and the

proteins to avoid effects from the migration time shifts, which are not related to interactions [3-

4]. Among the investigated proteins myoglobin, ovalbumin and beta lactoglobulin, showed no

significant interactions with the heparinoids as expected.

HSA and BSA showed stronger interactions with PPS than heparin and other heparinoids. In

case of HSA, the shape of the peak obtained by electrophoresis was changed and splitted into

two peaks after interaction with PPS. The higher the concentration of PPS, the more distinct this

effect was. P-selectin showed very strong interaction with the capillary wall so that the use of

coated capillaries and a special rinsing procedure were necessary. As well, p-selectin showed

strong interactions with PPS and heparinoids, which was probably dependant on the presence of

Ca2+ ions. With an appropriate ACE method we could demonstrate that heparinoids exhibit

strong affinity to some of these important proteins.

References

1. H. Abdel-Haq et al. J. of Chrom. A, 2012, 1257, 125-130.

2. M. Degenhardt et al. Arch. Pharm. Pharm. Med. Chem., 2001, 334, 27-29.

3. S. Redweik et al., Electrophoresis 2012, 33, 3316-3322.

4. H.A. Alhazmi et al. J. Pharm. Biomed. Anal. 2015, 107, 311-317.

89

P-150

Precise Handling of Ultra-small Sample Volumes in Conventional Capillary

Electrophoresis Instruments

Mona Mozafari, Markus Nachbar, Sami El Deeb


Capillary electrophoresis is an appropriate and important technique for analysing

pharmaceuticals and biopharmaceuticals [1-4]. In addition to low requirements in sample purity,

only a small volume of the sample is required to perform the analysis.

Most of the conventional CE instruments need approximately 50 μL of the sample in the

injection vial to perform the analysis, even though the injected sample volume in capillary

electrophoresis is only in the nanoliter range [5].

Since many proteins which are important from an analytical point of view undergo complicated

manufacturing processes and are produced in microgram quantities, it is important to reduce the

required sample volume and apply high concentrations of valuable samples. This approach

would be very advantageous from an economic point of view [6].

In order to fully profit from the low injection volumes, smaller vial volumes are required.

Therefore our experiments were performed using silicone oil as a filler of the vial dead volume

and the results were compared to that performed without this filling. As study example affinity

capillary electrophoresis was used to investigate the interactions of two proteins, namely HSA

and vitronectin, with three ligands namely enoxaparin sodium, unfractionated heparin and

pentosan polysulfate sodium (PPS). Mobility shift precision results over 12 hours analysis

showed that the employment of the filling with silicone oil has no noticeable effect on any of the

protein-ligand interactions (RSDs% for mobility ratios and peak areas were better than 0.8% and

1.5%, respectively). Accordingly, the employed silicone oil is suitable as a water immiscible and

inert liquid for filling the dead volume of sample vials. Using a commercial instrument and an

autosampler the required sample volume is reduced down to 10 μL, and almost this complete

volume can be subsequently injected during all experiments. Therefore the application of this

successful approach can be strongly recommended.

References

1. S. El Deeb et al. TrAC, 2013, 48, 112-131.

2. H. Wätzig et al. Electrophoresis, 2007, 28, 2324-2328.

3. S. El Deeb et al. Electrophoresis, 2014, 35, 170-189.

4. H.A. Alhazmi et al. J. Pharm. Biomed. Anal., 2015, 107, 311-317.

5. A. Beutner et al. Microchim Acta, 2015, 182, 351-359.

6. R. Walter et al. Anal. Chem., 2002, 74, 3575-3578.

NOTES:

90

P-151

Hyphenation of 2D Heart Cutting CIEF/CE-MS

Daniel Sydes1, Pablo A. Kler2, Daniel Lutz3, Peter Zipfl4, Carolin Huhn1

1University of Tuebingen - IPTC, Tuebingen, Germany, 2CIMEC, Universidad Nacional del

Litoral, Santa Fe, Argentina, 3CalvaSens GmbH, Aalen, Germany, 4University of Applied

Sciences, Aalen, Germany

The hyphenation of capillary isoelectric focusing (CIEF) with mass spectrometry is a very

promising tool for the characterization of proteins and peptides especially for R&D and quality

control of monoclonal antibodies. However, the CIEF-MS coupling is still challenging due to the

high number of MS incompatible carrier ampholytes. We seek to establish a 2D separation

approach for CIEF/CE-MS to separate analytes from carrier ampholytes in the second CE

dimension before MS detection. A microfluidic chip device with a common intersection for both

separation dimensions (thus forming the well-defined injection plug for the CE separation) is

used as interface for the hyphenation. First 2D experiments using myoglobin as a model analyte

show that the CIEF pH gradient can be sampled sequentially (heart cutting) and each fraction

injected via the microfluidic chip interface into the second CE separation dimension and

identified via MS. Positioning of the pH gradient in the first dimension to have the analyte of

interest focused in the common intersection was realized via different plug lengths of anolyte and

catholyte, respectively. The optimization of the sample transfer regarding carryover and peak

broadening was achieved by monitoring and therefore controlling of the IEF focused protein

plug via intermediate on-chip LED-LIF detection within the common intersection of the

interface. The second dimension separation of the protein from the accompanying ampholyte

fraction was further optimized via a combination of computer simulations and experimental data

to understand the transition from CIEF to CE conditions.

P-152

On-chip Intermediate Fluorescence, Conductivity and Potential Detection for 2D

Electrophoretic separations

Daniel Sydes1, Pablo A. Kler2, Hans Meyer3, Daniel Lutz4, Peter Zipfl5, Carolin Huhn1

1University of Tuebingen - IPTC, Tuebingen, Germany, 2CIMEC, Universidad Nacional del

Litoral, Santa Fe, Argentina, 3J&M Analytik AG, Essingen, Germany, 4CalvaSens GmbH, Aalen,

Germany, 5University of Applied Sciences, Aalen, Germany

Regarding two dimensional electrophoretic separations the transfer of the analyte from the first

to the second separation dimension is one of the most challenging aspects by means of

minimizing band broadening, transfer timing and ensuring orthogonally of both separation

dimensions. We introduced a modular hybrid 2D electrophoretic separation approach using a

microfluidic chip interface with on-chip contactless capacitively coupled conductivity detection

(C4D) and mass spectrometry for identification. Following this work we here present first proof

91

of principle on-chip C4D, fluorescence and intermediate potential detection for 2D

electrophoretic separations.

Intermediate C4D detection was implemented for the simultaneous ITP-C4D/CE-MS analysis of

the 20 proteinogenic amino acids in a non-aqueous electrolyte system using DMSO.

Spatially resolved on-chip fluorescence detection was realized via a linear optical fiber array and

a push broom imager for spectrally and spatially resolved detection of model proteins in an

intersection of the chip with is shared by both dimensions of the setup and thus forms the

injection plug for the second dimension. With the on-chip fluorescence detection, the presence of

the band of interest for CIEF/CE-MS analysis can be monitored for sample transfer.

On-chip potential measurements were conducted via a passivated electrode inside the chip

interface to monitor and thus control potential variations at the cross section of the interface

during electrophoretic separations.

NOTES:

92

Late Breaking

LB-01

Development of High Resolution icIEF Platform Methods for Monoclonal Antibodies

Min Liu, Jiann-Kae Luo, Erica Pyles

Regeneron Pharmaceuticals, Inc., Tarrytown, NY USA

Post-translational modifications within proteins, including glycosylation, phosphorylation, and

deamidation can lead to charge heterogeneity. To ensure reproducible quality of the therapeutic

protein, it is necessary to characterize and quantify the global charge variant profile of the

protein. Imaged capillary isoelectric focusing (icIEF) is a widely used technique to assess protein

charge heterogeneity. Typically, a broad pI range (pI 3-10) platform method is used during

monoclonal antibody development. This method provides suitable resolution of charge variant

heterogeneity in early stage development. In late stage development, a higher resolution method

may be desired to better monitor charge variants of monoclonal antibodies. One way to improve

resolution is by adding narrow range ampholytes into the broad range ampholyte (pI 3-10 range).

This maximizes capillary separation capacity. Several narrow range ampholytes were titrated

separately into a broad range ampholyte and evaluated for separation efficiency. Here we

present our findings and recommendations for high resolution icIEF separation conditions for

acidic (pI 6-7), neutral (pI 7-8) and basic (pI 8-9) monoclonal antibodies.

LB-02

Development of a High-Throughput Microchip CE-SDS (MCE-SDS) Assay to Support

Process Validation for a Therapeutic Fab Molecule

Thomas P. Niedringhaus, Zherylynn Vinyard


Over the past few years, there have been several promising molecules entering late-stage clinical

trials at Genentech. These late-stage projects require extensive characterization of the cell

culture, purification, and formulation process prior to commercialization. To meet the analytical

demands of the large sample volumes generated throughout these Process Characterization and

Process Validation (PC/PV) studies, a set of high-throughput analytical assays were developed.

One such assay is an automated microchip CE-SDS (MCE-SDS) assay that utilizes automation

to prepare samples and can analyze 46 samples under both non-reduced and reduced conditions

in less than 3 hours. This assay has been successfully employed for several late-stage mAb

93

molecules and was presented in detail during the 2014 CE in the Biotechnology and

Pharmaceutical Industries conference. Recently, however, a Fab molecule entering PC/PV

studies proved to have unique process conditions that required divergence from the established

MCE-SDS assay. This poster highlights the challenges faced when analyzing the Fab molecule

with the current MCE-SDS assay and outlines the development of a next-generation automated

MCE-SDS assay that is compatible with low molecular weight proteins.

NOTES:

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LB-03

An Iterative Approach to Develop a Challenging mAb-specific CE-SDS LIF Method

Brian Roper, Leslie Welch, Patricia Molina, Thomas Niedringhaus

Genentech, a Member of the Roche Group, South San Francisco, CA, USA

Genentech has a long history of developing and validating CE-SDS LIF methods to monitor and

quantify the size heterogeneity of therapeutic proteins in an effort to support clinical process

development and enable product release. Currently, we have a collection of generic or platform

CE-SDS methods that serve as the starting point to evaluate therapeutic proteins. While these

methods are suitable for a majority of the diverse molecules in our pipeline, there are instances

that require deviation from the platform CE-SDS method and a product-specific method is

needed.

One such example of a unique therapeutic protein in Genentech’s pipeline is mAb 1. After the

initial platform CE-SDS LIF assessment, we observed a large discrepancy between the high-

molecular weight species (HMWS) data collected by CE-SDS LIF and data generated from an

orthogonal size-based analytical method (i.e. SEC). In addition, we observed variable HMWS

results among our other generic CE-SDS methods, including a CE-SDS UV method and a

microchip CE-SDS LIF method. We performed numerous experiments to examine the different

factors that affect the HMWS response in the CE-SDS LIF assay and we eliminated the sample

preparation induced HMWS peak observed in the platform CE-SDS LIF method. Ultimately, a

product-specific non-reduced CE-SDS method was developed and additional experiments were

performed to produce a method that incorporates both reducing and non-reducing conditions in

one sample preparation procedure. This poster presents the details of this work.

LB-04

Assessing Platform Electrophoresis-Based Assays for Routine Testing of Unique Bispecific

Molecules in Quality Control Labs

Laura Yee, Cheryl Lovato, Wenni Gao, Thomas Niedringhaus, David Fischer

Genentech, a Member of the Roche Group, South San Francisco, CA, USA

At Genentech, the Platform Method Control (PMC) is one approach used to streamline method

assessment and validation of molecules during clinical development. The PMC approach

reduces the time spent on new molecules by utilizing a systematic one-time comprehensive

method validation to establish method acceptance criteria with a representative antibody. This

process allows us to leverage existing knowledge of the platform methods while still providing

flexibility for phase appropriate GMP work. This approach works well with the majority of

95

Genentech’s generic antibodies, but with the addition of bispecific molecules this approach will

need to be reexamined. This poster presents an overview of both the CE-SDS and iCIEF PMC

assessments performed for two exciting bispecific molecules.

NOTES:

Documents

CE in the Biotechnology & Pharmaceutical Industries · 2018. 4. 2. · 1 CE in the Biotechnology & Pharmaceutical Industries: 17th Symposium on the Practical Applications for the