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CE in the Biotechnology &

Pharmaceutical Industries:

19th Symposium on the Practical

Applications for the Analysis of

Proteins, Nucleotides and Small

Molecules

(CE Pharm 2017)

Symposium Co-Chairs:

Steffen Kiessig, F. Hoffmann – La Roche Ltd.

David Michels, Genentech, a Member of the Roche Group

September 17-20, 2017

Boston Park Plaza Hotel

Boston, MA

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 ............................................................................ 9

Session Abstracts ..................................................................................................... 17

Troubleshooting Workshop ..................................................................................... 39

Roundtable Discussions ........................................................................................... 41

Technical Seminar Abstract ..................................................................................... 43

Poster Abstracts ....................................................................................................... 47

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Welcome to CE Pharm 2017: CE in the Biotechnology and Pharmaceutical Industries: 19th

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

Molecules

We are pleased to welcome you to CE Pharm 2017, 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.

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 the 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: 908 Devices, Agilent

Technologies, American Laboratory/Labcompare, American Pharmaceutical Review, Amgen Inc., The

Analytical Scientist, Biogen, Bioprocess International, CMP Scientific, Corp., Eli Lilly and Company,

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

Pharmaceutical Quality, LCGC, Merck & Co., Inc., The Pathologist, PerkinElmer, Pfizer, Inc.,

Pharmaceutical Outsourcing, ProteinSimple, a Bio-techne brand, ProZyme, Regeneron Pharmaceuticals,

Inc., seperationsNOW.com, Sanofi, SCIEX, Technology Networks and Thermo Fisher 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 and Company

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

Mei Han, Amgen Inc.

Göran Hübner, Boehringer Ingelheim Pharma

GmbH & Co. KG

Steffen Kiessig, F. Hoffmann-La Roche Ltd. (Co-chair)

Nathan Lacher, Pfizer, Inc.

C. Mark Lies, SCIEX

Henry Luo, Regeneron Pharmaceuticals

David A. Michels, Genentech, a Member of the Roche

Group (Co-chair)

SungAe Suhr Park, Samsung Bioepis

Richard Rustandi, Merck & Co., Inc.

Oscar Salas-Solano, Seattle Genetics, Inc.

Maria Schwarz, Solvias AG

Zoran Sosic, Biogen

Ewoud van Tricht, Janssen Infectious Diseases and

Vaccines

Hermann Wätzig, University of Braunschweig

University of Braunschweig

Joel Welch, CDER, FDA

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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

2014 - Michel Girard – Health Canada

2015 - Cari Sänger - van de Griend – Kantisto BV

2016 - Sarah Kennett – CDER, FDA

2017 - Winner will be announced Monday at 18:00

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 2017. 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:

Isomeric Separation of Positively Labeled N-glycans by CE-ESI-MS

Guinevere S.M. Kammeijer, Leiden University Medical Center, Leiden, Netherlands

Enhancing Microchip Electrophoresis with Embedded Fused Silica Capillary for Cellular

Analysis

Benjamin T. Mehl, St. Louis University, St. Louis, MO USA

Capillary Electrophoresis of Antiperspirant Ingredients: Characterization and

Quantification of Aluminum Chlorohydrates Oligomer

Nesrine Ouadah, IBMM University of Montpellier, Montpellier, France

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The Scientific Organizing Committee gratefully acknowledges the following

partners for their generous support of this Symposium:

Strategic Program Partners

Platinum

Biogen

Gold

Eli Lilly and Company

Pfizer, Inc.

Diamond Program Partners

Genentech, a Member of the Roche Group

ProteinSimple, a Bio-Techne brand

PerkinElmer

Platinum Program Partner

SCIEX

Gold Program Partner

Agilent Technologies

Silver Program Partner

Amgen Inc.

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Bronze Program Partner

Sanofi

Friend of CASSS Partner

Merck & Co, Inc.

Welcome Reception

Regeneron Pharmaceuticals

Exhibitors

908 Devices Inc.

Agilent Technologies

CMP Scientific, Corp.

PerkinElmer

ProteinSimple, a Bio-Techne brand

ProZyme

SCIEX

Thermo Fisher Scientific

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The Scientific Organizing Committee gratefully acknowledges the following

media for their promotional consideration of CE Pharm 2017:

Media Program Partners

American Laboratory/Labcompare

American Pharmaceutical Review

The Analytical Scientist

BioProcess International

Genetic Engineering & Biotechnology News

International Pharmaceutical Quality

LCGC

The Pathologist

Pharmaceutical Outsourcing

separationsNOW.com

Technology Networks

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CE Pharm 2017

Scientific Final Program Summary

Sunday, September 17, 2017

08:30 – 09:00 Breakfast (for course attendees ONLY) in Berkeley Room

08:30 – 13:00 Registration (for course attendees ONLY) in the Ballroom Foyer

09:00 – 17:00

Short Course in the Clarendon Room

Applications of Capillary Electrophoresis to the Analysis of Protein Therapeutics

Short Course Facilitators:

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

and Cari Sänger - van de Griend, Kantisto B.V., Baarn, The Netherlands

12:30 – 13:30 Hosted Lunch (for course attendees ONLY) in Berkeley Room

16:30 – 18:00 Registration CE Pharm 2017 in the Ballroom Foyer

17:00 – 18:30 Welcome Reception in the Statler Ballroom

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Monday, September 18, 2017

07:30 – 18:15 Registration in the Ballroom Foyer

07:30 – 08:30 Breakfast in the Terrace Room

08:30 – 08:45 Welcome and Introductory Comments in the Georgian Room

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

Keynote I Session in the Georgian Room

Session Chair:

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

08:45 – 09:30 Global Biologics Regulatory Trends: Opportunities and Challenges

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

09:30 – 09:45 Discussion

09:45 – 10:15 Break – Visit the Exhibits and Posters in the Terrace Room

Analysis of Product and Process Related Impurities Using CE Session in the Georgian Room

Session Chairs: Tim Blanc, Eli Lilly and Company, Branchburg, NJ USA

and C. Mark Lies, SCIEX, Brea, CA USA

10:15 – 10:40 Characterization of Highly Basic Species of High Concentration mAb

by CE and HPLC

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

10:40 – 11:05 Analytical Challenges Faced During the Development of a Capillary

Gel Electrophoresis Method for Nanobody® Molecules

Mark Anthony Haverick, Merck & Co., Inc., Kenilworth, NJ USA

11:05 – 11:30 Chiral Purity of Amino Acids Originated from Oligopeptides

Claudia Michael, Solvias AG, Basel, Switzerland


11:45 – 12:00 Lunch for Technical Seminar Attendees – Please take lunch and return

to the Georgian Room for the “Lunch and Learn”

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Monday, September 18, 2017 continued

12:00 – 13:00 Technical Seminar/Lunch and Learn

Fast Glycan Labeling & Analysis: Sensitive CE-LIF Detection with Automated Glycan

Identification

András Guttman, SCIEX, Brea, CA USA

Sponsored by SCIEX Georgian Room

13:00 – 13:15 Mini Break – Visit the Exhibits and Posters in the Terrace Room

Late Breaking Session in the Georgian Room

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

13:15 – 13:40 Isomeric Separation of Positively Labeled N-glycans by CE-ESI-MS

Guinevere Kammeijer, Leiden University Medical Center, Leiden,

Netherlands

13:40 – 14:05 Novel Approaches for CE-SDS Peak Characterization – Comparison

to Current Approaches and Combination with Orthogonal Methods

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

14:05 – 14:30 Integration of Imaged cIEF and MS with a Novel Microfluidic System

for Real-time Protein Analytics

Erik Gentalen, Intabio, Inc., Portola Valley, CA USA


14:45 – 16:15 Poster Session – Visit the Exhibits and Posters in the Terrace Room

16:15 – 17:00

CE Pharm Partner Showcase in the Georgian Room

Facilitators: Mei Han, Amgen Inc. South San Francisco, CA USA

and Hermann Wätzig, University of Braunschweig, Braunschweig, Germany

17:00 – 18:00

Troubleshooting Workshop in the Georgian Room

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

and Cari Sänger - van de Griend, Kantisto B.V., Baarn, Netherlands

18:00 – 18:15 Presentation of the CE Pharm Award

18:15 – 19:15 Exhibition Reception – Visit the Exhibitors in the Terrace Room

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Tuesday, September 19, 2017



07:30 – 08:30 1st Time Attendee Breakfast in Arlington Room

Keynote II Session in the Georgian Room

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

08:30 – 09:15 CE Enantioseparations and Application to the Determination of the

Stereoisomeric Purity of Drugs

Gerhard Scriba, Friedrich Schiller University Jena, Jena, Germany


09:30 – 10:00 Break – Visit the Exhibits and Posters in the Terrace Room

Deep Dive into CE Session in the Georgian Room

Session Chair: Maria Schwarz, Solvias AG, Kaiseraugst, Switzerland

10:00 – 10:25 Using SDS-Titrations Monitored by Differential Scanning

Calorimetry (DSC) to Develop a Robust Non-Reduced Capillary

Electrophoresis Sodium Dodecyl Sulfate (NR-CE-SDS) Method for an

Atypical IgG1 mAb

Patricia Molina, Genentech, a Member of the Roche Group, South San

Francisco, CA USA

10:25 – 10:50 Evaluation of High-Throughput Microchip Capillary Electrophoresis

Assays for QC Testing - An Intracompany Multi-Site Ring Trial

Friederike Winkhaus, Roche Diagnostics GmbH, Penzberg, Germany

10:50 – 11:15 Capillary Electrophoresis of Small Molecules – Challenges and

Applications

Roman Szucs, Pfizer Global R&D, Sandwich, United Kingdom


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

to the Georgian Room for the “Lunch and Learn”

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Tuesday, September 19, 2017 continued

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

Maurice, the Workhorse CE System for your Biologics

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

Chris Heger, ProteinSimple, a Bio-Techne brand, San Jose, CA USA

Sponsored by ProteinSimple, a Bio-Techne brand Georgian Room

12:45 – 14:15 Poster Session - Visit the Exhibits and Posters in the Terrace Room

Keynote III in the Georgian Room

Session Chair:

Ewoud van Tricht, Janssen Infectious Diseases and Vaccines, Leiden, Netherlands

14:15 – 15:00 AQbD and CE – Speeding up or Slowing Down?

Cari Sänger - van de Griend, Kantisto B.V., Baarn, Netherlands


15:15 – 16:15 Technical Seminar

A Tale of Three Transfers: The Good, the Bad, and the Ugly - Transfer of mAb Purity

Analysis to cGMP Organizations

Niomi Peckham, Alexion Pharmaceuticals, New Haven, CT USA

Sponsored by PerkinElmer Georgian Room

16:15 – 16:30 Mini Break

Vaccines Session in the Georgian Room

Session Chairs: Nathan Lacher, Pfizer, Inc., Chesterfield, MO USA

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

16:30 – 16:55 Novel CZE Method for the Quantifications of Intact Adenovirus

Particles – AQbD Implementation and Application

Ewoud van Tricht, Janssen Infectious Diseases and Vaccines, Leiden,

Netherlands

16:55 – 17:20 Charge-based Separation of Lipid Nanoparticles for mRNA Vaccine

by Imaged Capillary Isoelectric Focusing

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

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Tuesday, September 19, 2017 continued

17:20 – 17:45 Identification and Quantitation of Intact Virus-like Particles of

Human Papillomavirus (HPV-VLP) using Capillary Electrophoresis

Virginie Bettonville, University of Liège, Liège, Belgium


18:00 – 19:00 Roundtable Discussions in Arlington/Berkeley Rooms

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Wednesday, September 20, 2017



Regulatory Session in the Georgian Room

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

08:30 – 08:55 CE’ing is Believing: Capillary Electrophoresis in Biosimilar

Development

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

08:55 – 09:20 Evaluation of New Technology for the Quality Control Laboratories

Jeffrey M. Schneiderheinze, Regeneron Pharmaceuticals, Rensselaer, NY

USA

09:20 – 09:45 Development of Complementary Approaches Using Capillary

Electrophoresis for the Evaluation and Regulation of Vaccine Product

and Biologics

Simon Sauve, Health Canada, Ottawa, ON Canada


10:00 – 10:30 Break in the Terrace Room

Hyphenated Techniques Session in the Georgian Room

Session Chair: Mei Han, Amgen Inc., South San Francisco, CA USA

10:30 – 10:55 Capillary Electrophoresis-Mass Spectrometry Method Development

for Biomarkers of Indoleamine 2,3-Dioxygenase

Yunan Wang, Amgen Inc., South San Francisco, CA USA

10:55 – 11:20 Microchip Electrophoresis Coupled to MS for Process Monitoring

and Rapid Product Quality Determination for mAbs

Seth Madren, Biogen, Research Triangle Park, NC USA

11:20 – 11:45 High Resolution CZE-MS Peptide Mapping with Improved

Separation, Peptide Recovery, and PTM Analysis for mAbs and

ADCs

Oluwatosin Dada, Seattle Genetics, Inc., Bothell, WA USA

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Wednesday, September 20, 2017 continued


12:00 – 12:05 Closing Comments in the Georgian Room

David A. Michels, Genentech, a Member of the Roche Group, South San

Francisco, CA USA

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Oral Abstracts

Global Biologics Regulatory Trends: Opportunities and Challenges

Wassim Nashabeh

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

This presentation will overview the emerging trends in the regulatory environment of

pharmaceuticals, with particular focus on Biotherapeutics. Current challenges with divergent

regulatory requirements across the world and their impact on initial product registration as well

as post-approval changes for manufacturing and analytical changes will be discussed. Efforts

undergone by ICH, WHO and regional initiatives to bring greater convergence of requirements

and their relevance to enabling streamlined variation changes with focus on analytical

technologies will be discussed. The presentation will end with an open discussion on our

collective role in shaping the environment to enable fast and efficient adoption of new and

emerging technologies.

NOTES:

18

Characterization of Highly Basic Species of a High Concentration mAb by CE and HPLC

Yan He, Michael Jones

Pfizer, Inc., Chesterfield, MO USA

High concentration mAb formulations pose challenges during manufacturing, stability, and

during analytical method development. During the development of a high concentration mAb

formulation, highly basic species were observed by iCE. To characterize these species, an array

of CE and HPLC methods including iCE, CZE, CGE, SEC, RPLC, CEX, CEX-MS, and CEX-

MALLs were employed. In conclusion, the highly basic species were determined to be a

heterogeneous mixture of extremely large aggregates

NOTES:

19

Analytical Challenges Faced During the Development of a Capillary Gel Electrophoresis

Method for Nanobody® Molecules

Mark Haverick

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

Next generation protein based drugs such as bispecific mAbs and Nanobodies® pose several

analytical challenges. Capillary gel electrophoresis (CGE) is the major work horse for

monitoring size variants, mainly fragmentation, during the manufacture of protein therapeutics.

CGE has been well established for the use with monoclonal antibodies. Here, we discuss the

analytical challenges in developing a capillary gel electrophoresis method for Nanobodies®. In

particular, the use of the Beckman PA800 plus has been the method of choice routinely used for

release and stability. We discuss the impact of sample preparation, SDS gel, and running

conditions on the size based separation of a single chain protein fragment. The Nanobodies®

have been analyzed by orthogonal sized based methods such as SDS-PAGE, denaturing SEC and

LabChip® CE-SDS and the results will be discussed.

NOTES:

20

Chiral Purity of Amino Acids Originated from Oligopeptides

Claudia Michael, Maria A. Schwarz

Solvias AG, Kaiseraugst, Switzerland

The current investigations are focused on the development of a CE test method suitable for the

quantification of amino acids present in a therapeutic peptide. The main focus was laid on the

chiral separation of the individual amino acids. The analysis of undesired enantiomers as

impurities resulting from original raw material but also followed by racemization during

synthesis/analysis requires not only sufficient UV/fluorescence sensitivity, possible by

derivatization, essential are also the preservation of the chirality of the amino acids during

labelling. The history of method development is presented from the first thoughts considering

favorable hydrolyzation labelling reagents accessible for primary and secondary amino groups

and formation of chiral inclusion complex applying cyclodextrin up to a robust and selective test

method. Furthermore, the aspects pH, ionic strength and type of running buffer besides an

appropriated coating of capillary surface are discussed. The completed test method provides the

separation of all individual amino acids and separation of enantiomers in therapeutic peptides.

NOTES:

21

Isomeric Separation of Positively Labeled N-glycans by CE-ESI-MS

Guinevere Kammeijer, Noortje de Haan, Sander Wagt, Pablo Mohaupt, Manfred Wuhrer

Leiden University Medical Center, Leiden, Netherlands

Glycosylation is known for its important role in biomarker discovery as well for

biopharmaceutical characterization in the industry. Glycosylation may affect the efficacy as well

as half-life of biopharmaceuticals (e.g. sialylation). However due to a large variety in the glycan

moiety, retrieving information about linkage positions remains a challenging task as

fragmentation patterns in postive ionization mode (CID) are quite often not able to reveal any

differences between isomers. The hyphenation of capillary electrophoresis (CE) with mass

spectrometry (MS) via electrospray ionization (ESI) presents an attractive approach to

characterize glycans as it allows high separation efficiencies. Furthermore, by implementing a

dopant enriched nitrogen gas during the ionization process optimal sensitivity can be achieved in

combination with a sheathless interface.

For this study glycans were released with PNGase F from polyclonal immunoglobulin G. The

CE separation selectivity was improved by neutralizing the negative charge of the sialylated

glycans with a derivatization step. A cationic charge was implemented on the N-glycans by

introducing a label on the reducing end. CE-ESI-MS experiments were carried out in positive ion

mode with a commercially available neutrally coated capillary. By investigating a “zero-flow”

principle (i.e., without applied pressure but with applied capillary voltage) the method was

further developed where a special focus was set on the separation of galactose positional isomers

(e.g. either α3- or α6-arm).

The analysis of the labeled and stabilized PNGase F released glycans, showed that CE–ESI-MS

has great potential as an analysis platform for in-depth glycomic research. The “zero-flow”

approach led to higher peak capacities and further improved the separation efficiencies,

providing new possibilities for separation of isomeric glycan species. Further studies are ongoing

to improve the labelling efficacy and CE selectivity to allow its use for biopharmaceutical

analysis.

NOTES:

22

Novel Approaches for CE-SDS Peak Characterization - Comparison to Current

Approaches and Combination with Orthogonal Methods

Bernd Moritz2, Laura Sánchez-Hernández1, Christina Montealegre1, Steffen Kiessig2, Andrea

Bathke2, Anja Bathke2, Christian Neusüß1

1Aalen University, Aalen, Germany, 2F. Hoffmann-La Roche, Basel, Switzerland

CE-SDS-NGS emerged as a powerful and well accepted analytical technique to support purity

evaluation of recombinant humanized monoclonal antibodies in the biopharmaceutical industry.

In order to allow sensitive control of fragments labeling with a fluorescent dye and LIF detection

are performed.

Peak identification for reduced and non-reduced CE-SDS-NGS separations of

biopharmaceuticals can be performed by different MS assisted spiking and matching approaches

with fractions from other separation techniques like size exclusion chromatography. N-

glycosidase F treatment allows the identification of de-glycosylated forms. In case of antibodies

partial reduction identifies peaks that result from the sequential loss of light and heavy chains.

However, all these indirect characterization methods are often not suitable for all peaks and may

not detect peak heterogeneities due to comigrating species.

Direct coupling of CE-SDS to MS was considered to be a valuable tool for addressing these

problems. However, that was prevented by MS interfering SDS. Meanwhile, a novel online CE-

SDS/CZE/MS was developed and successfully applied to unknown peaks in CE-SDS-NGS

profiles. RP-HPLC/MS, SEC/MS and peptide mass fingerprinting of SDS-PAGE bands were

found to confirm the obtained results either as orthogonal methods or by providing

supplementary results.

Different approaches for CE-SDS-NGS peak characterization are compared and application

strategies are discussed. In conclusion, a combination of the new set of tools provides a novel in

depth insight into CE-SDS peak identities.

NOTES:

23

Integration of Imaged cIEF and MS with a Novel Microfluidic System for Real-Time

Protein Analytics

Erik Gentalen, Barry Clerkson

Intabio, Inc., Portola Valley, CA USA

The complexity of biologic drug production demands that multiple measurements be performed

to characterize proteins throughout development and manufacturing. Capillary isoelectric

focusing “cIEF” is routinely used as the analytical platform for initial isoform assessment

because it provides exquisite resolution, high precision and is robust and easily reproducible

across laboratories. Mass spectrometry (“MS”) provides precise molecular identification of an

isoform and how it has been modified. However, weeks of scale up, chromatographic method

development and sample isolation can separate the first detection of an isoform by cIEF from the

MS analysis required to identify the nature of the protein modification.

To address this issue, Intabio has developed a proprietary protein analytics platform, Blaze™ to

integrate cIEF with MS analysis, reducing the time delay and infrastructure needs required to

identify protein variants. Blaze utilizes microfluidic functionality to integrate (1) separation of

protein isoforms by isoelectric focusing, (2) UV imaging of protein isoforms for detection and

quantitation, and (3) MS sample preparation and delivery by electrospray into an adjacent mass

spectrometer to identify each isoform. We demonstrate the integration of these three functions

with NIST standards, recombinant proteins, and monoclonal antibody samples. We also

demonstrate that Blaze has the flexibility to run in 2 modes: as a stand-alone for cIEF only, or

connected to a mass spectrometer for cIEF-MS.

NOTES:

24

CE Enantioseparations and Application to the Determination of the Stereoisomeric Purity

of Drugs

Gerhard Scriba

Friedrich Schiller University Jena, Jena, Germany

The importance of the stereochemistry of pharmaceutical drugs is well recognized as

stereoisomers often differ in their pharmacological, toxicological and/or pharmacokinetic profile.

Consequently, powerful analytical techniques are required in drug development and quality

control allowing the accurate and sensitive determination of the stereoisomeric purity of

synthetic drugs, natural products or pharmaceutical formulations. Apart from HPLC, CE has

become an attractive alternative for this purpose.

In CE the chiral selector is added to the background electrolyte acting as a pseudostationary

phase, which is also mobile in contrast to chromatographic methods. Consequently, two

stereoselective principles contribute to stereoisomer separations, i.e. the formation of transient

diastereomeric complexes between analyte enantiomers and the chiral selector (also referred to

as the thermodynamic or chromatographic enantioselective mechanism) as well as the motility of

these complexes (electrophoretic enantioselective mechanism). Both principles can cooperate or

counteract each other.

The presentation will discuss the effects of analyte complexation and mobility of the analyte-

selector complexes on enantioseparations in CE. The application of design of experiments (DoE)

in method development for chiral drugs such as levomepromazine and dextromethorphan will be

addressed.

NOTES:

25

Using SDS-Titrations Monitored by Differential Scanning Calorimetry (DSC) to Develop a

Robust Non-Reduced Capillary Electrophoresis Sodium Dodecyl Sulfate (NR-CE-SDS)

Method for an Atypical IgG1 MAb

Patricia Molina

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

Capillary electrophoresis analysis under non-reducing and reducing conditions has been adopted

by the biopharmaceutical field to detect product-related variants (fragments and aggregates) and

process-related impurities (host-cell proteins). An IgG1 monoclonal antibody (mAb) was

analyzed using a platform non-reduced capillary electrophoresis sodium dodecyl method using

laser-induced fluorescence (NR-CE-SD-LIF) detection. Results from the analysis showed an

unusual amount of high molecular weight (HMW) forms of about 17%. The level of HMW

forms with an orthogonal method such as size-exclusion high performance liquid

chromatography (SE-HPLC) was only about 2%. Consequently, further evaluation and

additional development of the NR-CE-SDS method was required for this molecule. Examination

of the electropherogram for the molecule showed that the main specie contributing to the high

HMW forms was due to the presence of an unexpected peak that migrated right after the main

peak. This peak is not normally observed in an electropherogram for a typical IgG1 mAb.

Analysis of the molecule in the presence of SDS using Differential Scanning Calorimetry (DSC)

indicated that this unexpected peak might be due to incomplete denaturation of the protein.

Subsequently, we used DSC to monitor SDS-titrations as a simple “proof-of-concept” strategy to

optimize the SDS-protein complexation step of the NR-CE-SDS method. The optimal SDS

concentration and temperature to achieve proper denaturation of the molecule were determined

and resulted in a superior electropherogram profile, thus eliminating several iterative steps

usually needed for sample preparation optimization. Therefore, this simple screening strategy

allowed a better understanding of the behavior of the protein in the assay and significantly

accelerated the development time of an improved and robust NR-CE-SDS method specific for

this molecule.

NOTES:

26

Evaluation of High-Throughput Microchip Capillary Electrophoresis Assays for QC

Testing - An Intracompany Multi-Site Ring Trial

Friederike Winkhaus

Roche Diagnostics GmbH, Penzberg, Germany

Sodium Dodecyl Sulfate (SDS)-based separations play an important role in monitoring quality

attributes of therapeutic proteins. Capillary Electrophoresis (CE)-SDS is the current standard for

release testing and stability testing, however the instrument setup is complex which requires

extensive training and the assay has limited throughput. Alternatively, Microchip Capillary

Electrophoresis (MCE) offers fast separations on a user-friendly instrument as a high-throughput

substitute for CE-SDS. Here, we describe an intracompany multi-site ring trial to evaluate MCE

as an alternative method in the QC environment.

Three different MCE methods - using covalent as well as non-covalent labelling- have been

evaluated using the LabChip GXII and the LabChip GX Touch instruments from Perkin Elmer.

To evaluate assay reproducibility, selected therapeutic proteins as well as different reagent lots

were tested across different labs. In addition, parameters like sensitivity, sizing range and

stability indicating properties have been compared among the MCE assays and with our standard

CE-SDS. The results of the ring trial will be presented.

NOTES:

27

Capillary Electrophoresis of Small Molecules – Challenges and Applications

Roman Szucs

Pfizer Global R&D, Sandwich, United Kingdom

In this presentation, we will discuss some applications of capillary electrophoresis (CE) on small

molecules of pharmaceutical interest. We will explain where CE fits into the product

development workflow which begins from nomination of development candidate all the way to

commercialization. Using real life examples, we will highlight application areas where CE

offers clear advantage over other separation techniques as well as point out technical challenges

which prevent its wider implementation. In the final part, we will demonstrate some recent

developments in this technique and predict some potential future applications.

NOTES:

28

AQbD and CE – Speeding up or Slowing Down?

Cari Sänger – van de Griend1, Ewoud van Tricht2, Lars Geurink2

1Kantisto B.V., Baarn, Netherlands, 2Janssen Infectious Diseases and Vaccines, Leiden,

Netherlands

“Is Capillary Electrophoresis really robust and can it be use in QC?” “Isn’t QbD just good

science?” “Do we need more regulation than the usual guidelines, doesn’t AQbD take an awful

lot of time?” These are questions I often get asked in more or less polite/diplomatic versions.

So what actually is Analytical Quality by Design, why does everybody talk so much about it and

does it have any advantage for the CE user? Is AQbD applicable at all on a technique such as

capillary electrophoresis? How does the general flow of AQbD translate into CE method

development?

This lecture gives an overview of approaches to Method Development, zooming in on the

applicability for developing robust, sensitive and precise CE methods. With better developed

methods and a better understanding about the critical parameters and good working practices for

CE, method validation becomes just a step in the chain of events that encompasses Method Life

Cycle Managements. In this lecture, we will also look at how neat CE and Analytical Quality by

Design fit together and result in more and higher quality information with less effort.

NOTES:

29

Novel CZE Method for the Quantification of Intact Adenovirus Particles – AQbD

Implementation and Application

Ewoud van Tricht1, Lars Geurink1, Harold Backus1, Marta Germano1, Cari Sänger – van de

Griend2

1Janssen Infectious Diseases and Vaccines, Leiden, Netherlands, 2Kantisto B.V., Baarn,

Netherlands

During development of adenovirus-based vaccines, samples have to be analyzed in order to

either monitor the production process or control the quality and safety of the product. An

important quality attribute is the total concentration of intact adenoviruses, which currently is

determined by quantitative polymerase chain reaction (qPCR) or anion exchange-HPLC.

The objective of this study was to obtain a reliable, efficient and robust cost-saving method for

the quantification of intact adenovirus particles in upstream processing (USP) and downstream

processing (DSP) samples. An analytical quality by design (AQbD) method development

approach was embraced. The analytical target profile (ATP) compromised of precision < 10%

RSD on intact adenovirus particle concentration, accuracy (as spiked recovery) between 90 –

110% over a range of 0.5×1011–1.5×1011 adenovirus particles per ml (~80 – 250 pmol/l), with a

time-to-result of less than 1 day. Capillary electrophoresis (CE) was selected for method

development since this technique potentially met all requirements from the ATP. The critical

method parameters of CE were defined based on a criticality assessment and these parameters

were the main focus during method development. Full factorial design of experiments was used

for method optimization as part of the analytical quality by design (AQbD) method development

approach. The CE method was validated for the quantification of adenoviruses on five

representative samples from the manufacturing process in the range of 0.5×1011 –

1.5×1011 adenovirus particles per ml. The CE method showed intermediate precision of 7.8%

RSD on concentration and an accuracy (spiked recovery) of 95–110%. Risk assessments were

performed prior to robustness testing and transfer to control the critical method parameters and to

define the criteria of the system suitability test. CE was successfully implemented on four

different sites for in-process control testing for adenovirus vaccine manufacturing and proved

highly useful for process development support.

NOTES:

30

Charge-based Separation of Lipid Nanoparticles for mRNA Vaccine by Imaged Capillary

Isoelectric Focusing

John Loughney

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

Lipid nanoparticles (LNP) have been employed for drug delivery in small molecules, siRNA,

mRNA, pDNA for both therapeutic and vaccines. Typically, LNP contain at least four different

lipids, and one of them is an ionizable amino acid lipid. Characterization of LNP is challenging

because they are heterogeneous mixture of large polydisperse particles. Many different tools of

particle size and morphology analysis have been applied; however, there is only limited surface

charge LNP characterization. CZE has been done for many different large particles such as

liposomes, polymer, and viruses. However, capillary isoelectric focusing has not been used of

this type of LNP materials. Here we describe the development of imaged capillary isoelectric

focusing using the new technology named Maurice for LNP-based mRNA vaccine.

NOTES:

31

Identification and Quantitation of Intact Virus-like Particles of Human Papillomavirus

(HPV-VLP) using Capillary Electrophoresis

Virginie Bettonville, Jérôme T.J. Nicol, Tania Furst, Nicolas Thelen, Géraldine Piel, Marc Thiry,

Marianne Fillet, Nathalie Jacobs, Anne-Catherine Servais

University of Liège, Liège, Belgium

Human papillomaviruses (HPV) are small, non-enveloped, icosahedral and double-stranded

DNA viruses that are responsible for approximately 5% of all cancers. Because HPV in

vitro production leads to low virus titers, HPV studies have used virus-like particles (VLP) as

model. Capillary electrophoresis (CE) is a very interesting alternative technique compared to

those currently used in viral analysis, such as SDS-PAGE, Western blot or protein assay that are

destructive and semi-quantitative or non-specific. However, the analysis of viral particles by CE

represents a real challenge not only due to their large size but also to their propensity to

adsorption and aggregation. In this context, the use of a coated capillary and of a BGE containing

high salt concentration and low SDS concentration was found to be necessary in order to obtain

reproducible HPV-VLP analysis. Another major issue in virus analysis is the absence of

reference material. Therefore, strategies for the identification and quantitation of HPV-VLP have

been developed. HPV-VLP peak assignment was done by comparison with a production made

using a wild-type baculovirus and with Gardasil® after adjuvant dissolution as well as by affinity

CE using a conformational H16.V5 antibody. Regarding the quantitation purpose, HPV16-VLP

concentration was determined using ELISA with Gardasil® after adjuvant dissolution as

reference material and H16.V5 antibody. HPV16-VLP concentration was found to influence the

electrophoretic mobility of the particles until a plateau was reached for concentrations ≤ 50 µg

ml-1. The concentration dependence of the electrophoretic mobility could be explained by an

overlap of the electrical double layers of adjacent particles. Finally, the CE method was

successfully validated following the ICH Q2R1 guidelines.

NOTES:

32

CE’ing is Believing: Capillary Electrophoresis in Biosimilar Development

Joel Welch

CDER, FDA, Silver Spring, MD USA

The development of a biosimilar candidate requires not only consideration of a quality target

product profile common to all development programs, but also consideration of the outcome of

the thorough characterization of the reference product and its corresponding critical quality

attributes. For this reason, analytical methods, and in particular CE methods, play a unique and

pivotal role in guiding the development process. This talk will provide a description of the

current regulatory landscape for biosimilar development, as well as the unique role CE methods

play in leading process development, characterizing the reference product, and evaluating

functional assays necessary to explore its mechanism of action. Challenges associated with

qualifying these methods will also be described.

NOTES:

33

Evaluation of New Technology for the Quality Control Laboratories

Jeffery M. Schneiderheinze

Regeneron Pharmaceuticals, Rensselaer, NY USA

The testing demands placed on today’s Quality Control (QC) laboratories for biological products

requires the implementation of robust, reproducible and user-friendly technology. This allows

for the generation of quality analytical data while minimizing the number of invalid test results

and instrument-related investigations. While historical QC methodologies relied upon traditional

biochemical methods of protein analysis such as gel electrophoresis, the transition to capillary-

based methodologies has dramatically expanded the throughput and robustness of the QC labs.

Further, the instrumentation trends toward miniaturization and microscale types of

methodologies has further challenged the traditional analytical boundaries. However, before

new methodology can be implemented in the QC lab, it must be thoroughly vetted for the

aforementioned attributes (robust, reproducible and user-friendly). This presentation will discuss

several case studies for the implementation of new instrumentation and technology into the QC

laboratory.

NOTES:

34

Development of Complementary Approaches Using Capillary Electrophoresis for the

Evaluation and Regulation of Vaccine Product and Biologics

Simon Sauve

Health Canada, Ottawa, ON Canada

Part of the mandate of agencies such as Health Canada is to regulate biological drugs that are

manufactured or imported into their country. To accomplish this task, risk-based assessment

programs and regulatory research towards Vaccine product and Biologics are undertaken.

However, many of the current methods used for determining potency and batch-to-batch

consistency are based on animal methods and/or in vivo assays with high variability rates.

Failures due to test variability and delays caused by re-testing put additional pressure on the

availability of biologics being supplied to the public. In addition, there are the ethical questions

of using animal models that are of questionable relevance to the human immunological response

for the purposes of quality control testing.

With the advancements made in analytical techniques such as CE and HPLC, complimentary

approaches using bioassays for potency and quality assays based on biochemical and

physiochemical methods could provide for better consistency testing and quality control of

biologics as they have a significantly lower variability rate, require less time to perform, are

generally less expensive to conduct and yield results which are easier to interpret. Here, we will

provide concrete examples of complementary approaches that are being developed to address

such issues in Vaccines (Influenza, Bacterial combination) and Biologics (Interferon).

NOTES:

35

Capillary Electrophoresis-Mass Spectrometry Method Development for Biomarkers of

Indoleamine 2,3-Dioxygenase

Yunan Wang, Mei Han, Jing Man Wong, Dan Rock, Brooke Rock

Amgen Inc., South San Francisco, CA USA

Indoleamine 2,3-dioxygenase (IDO) modulates the T-cell response and the effectiveness of the

tumor therapy. In the tryptophan pathway, IDO catabolizes tryptophan into kynurenine. Previous

studies have correlated tryptophan/kynurenine ratio with poor clinical results in acute myeloid

leukemia and diffuse large B-cell lymphoma patients. (Corm S., Leuk Research, 2009; Ninomiya

S., Leuk Lymphoma, 2012). Tryptophan, kynurenine, and possibly other downstream

metabolites can potentially serve as biomarkers for IDO activity in T-cell response. The IDO

substrates selected as potential biomarkers in this project include tryptophan, kynurenine,

serotonin, nicotinamide, 2-picolinic acid, 3-hydroxyanthranilic acid, quinolinic acid, kynurenic

acid, xanthurenic acid, and 3-hydroxy-DL-kynurenine. Here, we propose a capillary

electrophoresis-mass spectrometry (CE-MS) method to quantify the potential biomarkers for

IDO. The CE-MS system consists the Agilent 7100 CE system coupled to Agilent 6230 time-of-

flight mass spectrometer (TOF-MS) through CMP Scientific CE-MS EMASS-II interface. With

preliminary conditions, the nine metabolites could be separated in 6 minutes. In the linearity

assay, nicotinamide, 3-hydroxyanthranilic acid, kynurenic acid, tryptophan, and 3-hydroxy-DL-

kynurenine have a linear range of 0.5-10 µM with R2>0.999. Xanthurenic acid, L-kynurenine,

and serotonin have a linear range of 0.5-10 µM with R2>0.99. 2-Picolinic acid, quinolinic acid,

have the linear range of 2-50 µM with R2>0.99.

NOTES:

36

Microchip Electrophoresis Coupled to MS for Process Monitoring and Rapid Product

Quality Determination for mAbs

Seth Madren, Linda Yi

Biogen, Research Triangle Park, NC USA

The bioreactors used to produce monoclonal antibodies (mAbs) is a complex system, contributed

by the inherent variability in both biological processes and the complex media required to

maintain high cell growth. Quality controls on the raw materials used to prepare the cell culture

media can reduce the process variability. An in-depth understanding of how variations in the cell

culture conditions impact the final product quality is critical in developing a control strategy, by

permitting corrective action when a deviation is detected. The complex nature of the cell culture

media requires a highly efficient separation technique to monitor a wide range of metabolites and

nutrients to thoroughly characterize how the bioreactor is performing. A fast separation with

minimal sample preparation facilitates rapid corrective action. Microchip electrophoresis can

perform highly efficient separations and is able to handle sample matrixes that are often

problematic for LC based separations. When coupled to MS, it can also rapidly provide confident

identification and characterization of both cell culture media and the mAb products. We have

developed methods on a commercially available ZipChip interface coupled to a Thermo orbitrap

mass spectrometer. It can monitor and quantitate amino acids, vitamins, salts and metabolites in

cell culture harvest in less than 7 minutes with minimal sample preparation. Several product

quality attributes of mAbs, such as deamidation, N-terminal variant, C-terminal variants, and Fc

N-glycosylation, can be characterized in less than 5 minutes after IdeS digestion performed

directly with cell culture harvest instead of purified mAb.

NOTES:

37

High Resolution CZE-MS Peptide Mapping with Improved Separation, Peptide Recovery,

and PTM Analysis for mAbs and ADCs

Oluwatosin Dada, Yimeng Zhao, Nomalie Jaya, Oscar Salas-Solano

Seattle Genetics, Inc., Bothell, WA USA

Peptide mapping with mass spectrometry (MS) detection is a powerful technique routinely used

for interrogating physicochemical properties of proteins. Peptide mapping benefits from an

efficient front-end separation to increase selectivity and reduce complexity prior to MS

detection. The most commonly used method for peptide mapping is based on reverse phase

liquid chromatography with mass spectrometry (RPLC-MS). Capillary zone electrophoresis with

mass spectrometry (CZE-MS) is an orthogonal technique with growing attention for peptide

mapping due to its high efficiency and sensitivity. However, that growth has been slow due to

poorer peptide resolution and method robustness compared to RPLC. Here we present results

from optimization of CZE-MS peptide mapping separation using mixed aqueous - aprotic dipolar

solvent as the background electrolyte (BGE) to improve separation performance. We also

demonstrate the utility of CZE-MS as an orthogonal and complementary technique to RPLC-MS

to support characterization of proteins.

NOTES:

38

NOTES:

39

Troubleshooting Workshop

Monday, September 26

17:00 – 18:00

Georgian Ballroom

Facilitators:

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

Cari Sänger-van de Griend, Kantisto B.V., Baarn, Netherlands

Scribe:

Nathan Lacher, Pfizer, Inc., Chesterfield, MO USA

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

Zoran Sosic, Biogen, Cambridge, MA USA

Analytical methods subject matter experts (SMEs) play an important business role by ensuring

the success of technologies in labs supporting characterization and GMP testing. As a

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

the primary objectives of the CE Pharm Meeting and is the focus in our annual troubleshooting

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

clever solutions to mitigate 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 again 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. Three years ago, CASSS and the CE Pharm Committee

began soliciting examples for this workshop and the response has been impressive. Reports from

the past workshops can be found at http://www.casss.org/page/CEPharmTroubleshoot. A report

of this year’s troubleshooting session will be published on this page as well.

NOTES:

40

NOTES:

41

Roundtable Discussions

Tuesday, September 19, 2017

18:00 – 19:00

There are 10 roundtable topics. The plan is for these to be active discussions, not presentations

or lectures. To create useful discussion, we are going to try and limit each topic to 10 attendees.

Seating will be on a first come, first serve basis. These discussions will include a facilitator,

whose role is to help assist the discussion and ensure a lively exchange, and a scribe, whose role

is to make general, anonymous notes about the discussion that will be posted on the CE Pharm

2017 website.

Listed below is a quick view of the Roundtable Topics, Facilitators and Scribes:

Table 1 What Are Opportunities and Challenges for Further Implementation of CE-

MS (including Chip-MS) in Development of Biopharmaceuticals?

Facilitator: Mei Han, Amgen Inc.

Scribe: Zoran Sosic, Biogen

Table 2 CE in Formulation Development Support and Stability Analytics

Facilitator: Ewoud van Tricht, Janssen Infectious Diseases and Vaccines

Scribe: Guinevere Kammeijer, Leiden University Medical Center

Table 3 Chip Based Separations vs Classical CE Separations: Advantages &

Disadvantages Comparing Platforms and Approaches

Facilitator: Nomalie Jaya, Seattle Genetics, Inc.

Scribe: Claudia Michael, Solvias AG

Table 4 Reduced and Non-reduced CE-SDS for Antibodies. When to Eliminate One

Versus the Other?

Facilitator: Henry Luo, Regeneron Pharmaceuticals

Scribe: Bernd Moritz, F. Hoffmann - La Roche Ltd.

Table 5 Applications of CE to Nucleic Acid Analysis

Facilitator: Richard Rustandi, Merck & Co, Inc.

Scribe: Kelsey Dent, Genentech, a Member of the Roche Group

Table 6 Instrument Quality, Reliability and Failure Rate

Facilitator: Hermann Wätzig, University of Braunschweig

Scribe: Friederike Winkhaus, Roche Diagnostics GmbH

42

Roundtable Discussions

Table 7 What Are the Most Challenging Separation/Identification Problems Now

(e.g. Subvisible Particles) and How Does CE Fit In?

Facilitator: Joshua Woods, Pfizer, Inc.

Scribe: Yunan Wang, Amgen Inc.

Table 8 Trends in Pharmaceutical Application of CE

Facilitator: Maria Schwarz, Solvias AG

Scribe: Nathan Lacher, Pfizer, Inc.

Table 9 Hot Topic (TBD)

Facilitator: David Michels, Genentech, a Member of the Roche Group

Scribe: Tim Blanc, Eli Lilly & Company

Table 10 Hot Topic (TBD)

Facilitator: Steffen Kiessig, F. Hoffmann - La Roche Ltd.

Scribe: Joel Welch, CDER, FDA

43

Technical Seminars Technical Seminar: Lunch and Learn

Monday, September 18

12:00 – 13:00

Georgian Ballroom

Fast Glycan Labeling & Analysis: Sensitive CE-LIF Detection with Automated Glycan

Identification

András Guttman

SCIEX, Brea, CA USA, University of Debrecen, Debrecen, Hungary

A Fast Glycan Labeling & Analysis technology has been developed for N-linked oligosaccharide

profiling of glycoproteins along with a gel-buffer system to ensure rapid and high-resolution

separation of the target molecules. Glycan release, fluorophore labeling and clean-up parameters

were all optimized resulting in 60 min sample preparation time using a novel magnetic bead

mediated protocol that assures excellent yield, high reproducibility and easy automation.

Optimization included rapid endoglycosidase digestion and fluorophore labeling time and

temperature as well as sample clean-up, supporting fast sample processing. The procedure does

not require any centrifugation and vacuum centrifugation steps, otherwise necessary for most

glycan sample preparation methods. Capillary electrophoresis analysis with laser induced

fluorescence detection (CE-LIF) of the fluorophore (APTS) labeled glycans was also optimized

to enable rapid and high-resolution separations of the labeled sugars just within a few minutes, in

order to accommodate both manual and automated sample processing. We also report on the

design and implementation of a co-injection based triple-internal standard mediated

glycoinformatics method, to alleviate the need of an accompanying run of the

maltooligosaccharide ladder for precise glucose unit (GU) calculation based structural

assignment. The importance of precise temperature control during CE-LIF analysis is also

emphasized. Worked examples will show comprehensive glycan composition analysis of some

of the most prescribed monoclonal antibody therapeutics and their biosimilar counterparts.

Quantitative assessment and automatic structural identification of glycans of CQA importance in

biologics development will also be discussed.

NOTES:

44

Technical Seminar: Lunch and Learn

Tuesday, September 19

11:45 – 12:45

Georgian Ballroom

Maurice, The Workhorse CE System for your Biologics

XiaoPing He1, Chris Heger2

1Pfizer, Inc., Chesterfield, MO USA, 2 ProteinSimple, a Bio-Techne brand, San Jose, CA USA

Years ago, iCE revolutionized how cIEF is used in Biologics development processes with the

introduction of imaged cIEF.

Now, Maurice further streamlines and extends the use of capillary electrophoresis to simplify

your protein profiling. On Maurice, we’ve augmented our icIEF technology with sensitive native

fluorescence detection capabilities and now offer in addition seamless switching to the newly

added CE-SDS mode to bring a lot more “iCE” to your biologics analysis.

At this technical seminar, you will be hearing about the evaluation of Maurice at Pfizer and

Fujifilm. XiaoPing He, M.Sc. (Senior Scientist, Pfizer) will discuss her experience with Maurice

in Size and cIEF mode during her validation of the platform. Chris Heger Ph.D., Manager,

Applications Science at ProteinSimple San Jose will discuss aspects of CE-SDS method

development on Maurice using real world examples from a collaboration with FujiFilm and give

an introduction on combining Maurice and our Simple Western technology in your bioprocess

workflow.

The seminar will be interactive to encourage discussion with speakers, the ProteinSimple team,

and to share your own experiences.

NOTES:

45

Technical Seminar

Tuesday, September 19

15:15-16:15

Georgian Ballroom

A Tale of Three Transfers: The Good, the Bad, and the Ugly - Transfer of mAb Purity

Analysis to cGMP Organizations

Niomi Peckham

Alexion Pharmaceuticals, New Haven, CT USA

Alexion’s focus on rare diseases often translates into accelerated development timelines, making

high throughput and flexible platforms a necessity. Protein analysis on the Caliper LabChip has

been adopted by Alexion for monoclonal antibodies from Pre-Clinical through Validated Phase

II/III release and stability applications. Contract manufacturing organizations (CMOs) have also

been utilized for manufacturing and analytical support to progress several clinical stage

molecules, resulting in repeated transfers of the LabChip assays to cGMP laboratories. Here, we

describe transfer of validated assays to three cGMP labs concurrently.

NOTES:

46

NOTES:

47

List of Posters

Analysis of Product and

Process Related Impurities Using CE

P-101

Non-Glycosylated Heavy Chain Analysis Crossover: Agilent Bioanalyzer 2100 to

PerkinElmer LabChip® GXII

Troy Adams, Jigna Patel, Mike Smith, Byron Dipaolo, Jennifer Dally

GlaxoSmithKline, King of Prussia, PA USA

P-102

Development of CE-SDS Method using Maurice for the Purity of Adeno-associated Virus

(AAV) Capsids

Wei-Chiang Chen

Biogen, Cambridge, MA USA

P-103

Comparison Between Maurice and iCE

K. Steven Cook, Xiaoping He


P-104

A High Throughput Sample Preparation and Analytical Method for N-Glycan Analysis on

Multi-capillary CE and UHPLC

Anahita Eckard, Johnie K. Young, Baburaj Kunnummal, Peter Bell

Thermo Fisher Scientific, South San Francisco, CA USA

P-105

A Method for Rapid and Complete Release of N-Glycans from Simple and Complex

Glycoproteins

Anahita Eckard, David Dupont, Johnie K. Young, Baburaj Kunnummal, Peter Bell


P-106

Characterization of a Protein Impurity by SDS Capillary Electrophoresis

Shiao-Yan Fang

Ambrx Inc., La Jolla, CA USA

48

P-107

Glycosimilarity Index for Biotherapeutics

András Guttman1, Beata Borza2, Marton Szigeti2, Akos Szekrenyes2, Laszlo Hajba3 1SCIEX, Brea, CA USA, 2University of Debrecen, Debrecen, Hungary, 3Universiy of Pannonia,

Veszprem, Hungary

P-108

icIEF Quantification of a Product-Related Impurity in an Antibody Candidate

Steve Kauffman, Jennifer Kyauk, Diya Ren

Gilead Sciences, Inc, Oceanside, CA USA

P-109

Analysis of Highly Sialylated and Low-Input Glycoprotein Samples on the GlycanAssure™

System

Shaheer Khan, Natalee Gautam, Jenkuei Liu, Bharti Solanki-Nand, Baburaj Kunnummal, Peter

Bell


P-110

Identification and Characterization of a Thermally Cleaved Fragment of Monoclonal

Antibody-A Detected by Sodium Dodecyl Sulfate-capillary Gel Electrophoresis

Kei Kubota

Daiichi-Sankyo Co., Ltd., Hiratsuka-shi, Kanagawa, Japan

P-111

Method Development and Qualification Approach to Support the Fast-to-FIH Timelines

for Biologics: A Case Study for CE-SDS Methods

Ruiqiong Li1, Madesh Belakavadi1, Amit Katiyar2, Tapan Das1 1Bristol-Myers Squibb Company, Pennington, NJ USA, 2Bristol-Myers Squibb Company,

Hopewell, NJ USA

P-112

Quantitative Analysis of Low Molecular Weight Species (LMW) Generated During Human

IgG4 Process Development

Mengxiao Lu, Nesredin Mussa, Barry Drew

Bristol-Myers Squibb Company, Devens, MA USA

P-113

From ELISA to ELLA Plug & Play Custom Immunoassay Development with Simple Plex

to Enable Rapid, Automated, High Performance Bioprocess Analysis

Gregory Marusov

ProteinSimple, a Bio-Techne brand, Wallingford, CT USA

49

P-114

Method Transferability of Capillary Isoelectric Focusing (cIEF) to Simple Western

Jiaqi Wu, Chris Heger, Annegret Boge

ProteinSimple, a Bio-Techne brand, San Jose, CA USA

P-115

Optimization of Cysteine Exchange for new THIOMAB™ Antibody Formats During

Charge Variant Analysis

Mary Montti1, Yayan Zhou2, Aron Lee1, Diana Liu1, Yushi Wang1, Zhiqi Hao1, Jeffrey Zhang1,

Michael Taejong Kim1, Christopher Cornell1, Yan Chen1, Fred Jacobson1 1Genentech, a Member of the Roche Group, South San Francisco, CA USA, 2Eurofins Lancaster

Laboratories, South San Francisco, CA USA

P-116

Challenges in the Determination of Amyloid Oligomeric Species by Two Electrophoretic

Techniques

Aurore Napp, Virginie Houbart, Alice Demelenne, Anne-Catherine Servais, Marianne Fillet

University of Liège, Liège, Belgium

P-117

Capillary Electrophoresis of Antiperspirant Ingredients: Characterization and

Quantification of Aluminum Chlorohydrates Oligomers

Nesrine Ouadah1, Fabien Brothier2, Jean-François Kuntz2, Claudine Moire2, Hervé Cottet1 1IBMM University of Montpellier, Montpellier, France, 2L'Oréal Research & Innovation,

Aulnay-sous-bois, France

P-118

Microfluidic Capillary Electrophoresis with Real-Time Calibration and Enhanced

Resolution for Protein Impurity Analysis

Zhiyong Peng1, White James1, Derek Troiano1, Megan Sierant1, Anubhav Tripathi2, Brian

Gerwe1 1PerkinElmer, Hopkinton, MA USA, 2Brown University, Providence, RI USA

P-119

A Capillary Based iCIEF Technology Approach to Replace Gel Based System in the

Quality Control Laboratories. Making a Transition from Gel in the QC Laboratories.

Anu Rambhadran

Regeneron Pharmaceuticals, Tarrytown, NY USA

P-120

Robust cIEF Analysis with Longer Neutral Capillary Run-Life while Maintaining High

Resolution and Reproducibility

Chitra Ratnayake1, Zaifang Zhu1, Ingrid Cruzado-Park1, David Neyer2 1SCIEX, Brea, CA USA, 2SCIEX, Redwood City, CA USA

50

P-121

Fast Glycan Labeling & Analysis: High-Resolution Separation and Glycan Identification in

Minutes

Matthew Salem

SCIEX, Redwood City, CA USA

P-122

A Tale of Two CE-SDS Platforms: A Head to Head CE-SDS Assessment of the AB Sciex

PA800 Plus and the ProteinSimple Maurice Using Multiple Biologic Compounds

Kristin Schultz-Kuszak, Frank Gorelik, Eric Meinke, Xiangyang Wang

MedImmune, A member of the AstraZeneca Group, Gaithersburg, MD USA

P-123

Development of a CGE‐SDS Method for Routine Fragmentation Monitoring in a High

Complex Fusion

Natascia Sciamanna

Merck Serono, Guidonia, Italy

P-124

IgG2s and their Idiosyncrasies: Charge Assay Challenge Accepted!

Pawankumar Suresh


P-125

Inter-instrument Evaluation applying CE-SDS for Protein Analysis

Hermann Wätzig, Julia Kahle, Kai Jorrit Maul

University Braunschweig, Braunschweig, Germany

P-126

Charge Isoform Heterogeneity Analysis by Capillary Isoelectric Focusing (cIEF)

Hio (Cara) Wong, Chuck Hague, Crystal Conlan

BioMarin Pharmaceutical Inc., Novato, CA USA

P-127

Evaluation of the Maurice for Fragment Characterization by Reducing and Non-reducing

CE-SDS

Joshua Woods


51

Deep Dive into CE

P-128

Use of a Total TCA Range in CGE (CE-SDS) Analysis of Quality Control Samples

Edith Binder

Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany

P-129

NISTmAb Characterization: Purity, Charge Heterogeneity and Glycan Analyses on a

Single Platform

Esme Candish, Chitra Ratnayake, Mervin Gutierrez, András Guttman

SCIEX, Brea, CA USA

P-130

Evaluation of Automated Wes System as an Analytical and Characterization Tool to

Support Monoclonal Antibody Drug Product Development

Ying-Chen Chen1, Jinyu Wang2, Anulfo Valdez1 1Bristol-Myers Squibb Company, Pennington, NJ USA, 2Rutgers University, New Brunswick, NJ

USA

P-131

Double Dipping the Chip -- Maurice as 1) An iCE3 Alternative for Existing Methods and 2)

A New Tool for Challenging Sample Types

Kelsey C. Dent, Pawankumar Suresh, David J. Fischer, David A. Michels


P-132

Evaluation of Protein Simple Maurice CE-SDS for Purity and Stability Indicating Methods

used in Process Development and Commercial Manufacturing

Abbie Esterman1, Tapan Das1, Amit Katiyar2 1Bristol-Myers Squibb Company, Pennington, NJ USA, 2Bristol-Myers Squibb Company,

Hopewell, NJ USA

P-133

The Power of Gu and the Importance of Temperature Control

András Guttman1, Marton Szigeti2, Jeff Chapman1 1SCIEX, Brea, CA USA, 2University of Debrecen, Debrecen, Hungary

52

P-134

Don't We Have a Machine for That? Automated Fluorescent Labeling for CE-SDS LIF in

QC

Koman Joe, Thomas Niedringhaus, Mary Han, Jian Zhang, Sean Mojabi, Yun Tang, Sarah Du

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

P-135

A New, Narrow-range – High-resolution cIEF Method for High pI Molecules

Filippo Marchioni, Brian Hosken, Tony Cano

AbbVie, Inc., South San Francisco, CA USA

P-136

Enhancing Microchip Electrophoresis with Embedded Fused Silica Capillary for Cellular

Analysis

Benjamin T. Mehl, R. Scott Martin

St. Louis University, St. Louis, MO USA

P-137

Utility of Maurice cIEF Native Fluorescence in cIEF Analysis of Protein Mixtures

Jiaqi Wu, Chris Heger, Annegret Boge

ProteinSimple, a Bio-Techne brand, San Jose, CA USA

P-138

Capillary Electrophoresis of Antiperspirant Ingredients: Study of their Interaction with

Proteins

Nesrine Ouadah1, Fabien Brothier2, Jean-François Kuntz2, Claudine Moire2, Hervé Cottet3 1IBMM University of Montpellier, Montpellier, France, 2L'Oréal Research & Innovation,

Aulnay-sous-bois, France

P-139

Analytical Characterization of Protein-Polymer Conjugates used for Long Acting Delivery

Therapeutics

Cinzia Stella


P-140

A Smart Microfluidic Pharmaceutical Analysis System

Anubhav Tripathi, Adam Snider, Richard Park, Alexandra Riccardi

Brown University, Providence, RI USA

P-141

Incorporation of a Strong Acid Catalyst Addresses Labeling Bias for Afucosylated N-

linked Glycans

Jonathan van Dyck

Seattle Genetics, Inc., Bothell, WA USA

53

P-142

Characterization of a Monoclonal Antibody Minor Variant Using CE-SDS and Peptide

Mapping

Qing Zhu


54

NOTES:

55

DoE/QbD/Life Cycle Management

P-143

Application of Analytical Quality by Design (AQbD) Approach in a Design of a Robust

mAb Non-Reduced CE-SDS Purity Method

Qian Guan, Sergey Voronov, Julia Ding, Nesredin Mussa

Bristol-Myers Squibb Company, Devens, MA USA

P-144

Quantitative Assessment and Automatic ID of Glycans of CQA Importance in Biologics

Development

András Guttman1, Marton Szigeti2, Jeff Chapman1 1SCIEX, Brea, CA USA, 2University of Debrecen, Debrecen, Hungary

P-146

The Journey to QC Readiness: A Search for a Robust Microchip CE-SDS Assay

Brian Roper¹, Zherylynn Vinyard¹, Timothy Holewinske¹, Stefanie Wohlrab², Ines Lavergne³,

Friederike Winkhaus², Thomas Niedringhaus¹

¹Genentech, a Member of the Roche Group, South San Francisco, CA USA, ²Roche Diagnostics

GmbH, Penzberg, Germany, ³F. Hoffmann-La Roche Ltd., Basel, Switzerland

56

NOTES:

57

Hyphenated Techniques

P-147

Charge Heterogeneity Analysis of Intact Monoclonal Antibodies using CESI-MS

Esme Candish1, Olga Friese2, Elaine Stephens3, Marshall Bern4, St John Skilton5, Jason Rouse3,

Bryan Fonslow6 1SCIEX, Brea, CA USA, 2Pfizer, Inc., Chesterfield, MO USA, 3Pfizer, Inc., Andover, MA

USA, 4Protein Metrics Inc., San Jose, CA USA, 5Protein Metrics Inc., San Carlos, CA

USA, 6SCIEX, San Diego, CA USA

P-148

Higher Order Structure of Intact Proteins by Capillary Electrophoresis Native Ion

Mobility Mass Spectrometry

Caroline Chu1, Pat Perkins1, Andy Gieschen2, Martin Greiner3 1Agilent Technologies, Santa Clara, CA USA, 2Agilent Technologies, San Diego, CA

USA, 3Agilent Technologies, Waldbronn, Germany

P-149

Orthogonal, High Resolution Polar Biomolecule Analysis by CESI-MS

Edna Betgovargez1, Esme Candish2, Bryan Fonslow3 1SCIEX, Brea, CA USA, 2SCIEX, Framingham, MA USA, 3SCIEX, San Diego, CA USA

P-150

Development and Applications of a Novel Fluorescent Dye for Glycan Labeling and

Analysis on Multi-capillary CE and CE-MS

Shaheer Khan1, Jenkuei Liu1, James Xia2, Baburaj Kunnummal1 1Thermo Fisher Scientific, South San Francisco, CA USA, 2CMP Scientific Corp., Brooklyn, NY

USA

P-151

The Study of Intact Casein as a Model System for the Separation of Intact Phosphorylated

Proteins by CESI-MS

Stephen Lock1, Edna Betgovargez2 1SCIEX, Pudsey, United Kingdom, 2SCIEX, Brea, CA USA

P-152

Characterization of Monoclonal Antibodies and Antibody Drug Conjugates using

Microchip Zone Electrophoresis-MS Technology

Erin Redman

908 Devices Inc., Carrboro, NC USA

58

P-153

An Integrated Platform for N-Glycan Analysis Using Rapid Sample Preparation and 2-

Minute Separation by Capillary Electrophoresis

John Yan, Aled Jones, Michael Kimzey, Andres Guerrero, Tom Rice, Justin Hyche, Emily Dale,

Ted Haxo, Sergey Vlasenko

ProZyme, Inc, Hayward, CA USA

P-154

Automation of Gly-X Sample Prep with InstantPC and InstantQ Dyes

Ted Haxo, Emily Dale, Adele Taylor

ProZyme, Inc, Hayward, CA USA

P-155

Monitoring Glycosylation Profile and Protein Titer in Cell Culture Samples using ZipChip

CE-MS

Zoran Sosic, Peng Feng, Yan Wang, Li Zang

Biogen, Cambridge, MA USA

59

Vaccines

P-156

Quantitation of CRM197 Using Imaged Capillary Isoelectric Focusing with Fluorescence

Detection and Capillary Western

Richard Rustandi, Sha Ha, John Loughney

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

60

Late Breaking

LB-01

Micellar Electrokinetic Chromatography for Measurement of Dopamine and D-serine

Secretion from Human Islet of Langerhans

Kimberly Evans, Michael G. Roper

Florida State University, Tallahassee, FL USA

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