CE in Biotechnology & Pharmaceutical

Industries: 13th Symposium on the Practical Applications for the Analysis of

Proteins, Nucleotides and Small Molecules

(CE Pharm 2011)

Symposium Chair:

Margaret Ruesch, Pfizer, Inc. Cari Sänger - van de Griend, Kantisto BV

October 9-13, 2011

The Ritz Carlton, Amelia Island Amelia Island, FL

Organized by

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Table of Contents CE Pharm Award ................................................................................................................. 4 Student Travel Grants .......................................................................................................... 5 Corporate, Exhibitor and Media Program Partners .............................................................. 6 Scientific Final Program Summary ...................................................................................... 8 Session Abstracts ............................................................................................................... 17 Workshop Session I (Monday, October 10) Description ................................................... 41 Workshop Session II (Tuesday, October 11) Description ................................................. 42 Workshop Session III (Wednesday, October 12) Description ........................................... 43 Technical Seminar (Monday, October 10) Abstract .......................................................... 44 Technical Seminar (Tuesday, October 11) Abstract .......................................................... 44 Technical Seminar (Wednesday, October 12) Abstract ..................................................... 46 Poster Abstracts ................................................................................................................. 48

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Welcome to CE in the Biotechnology and Pharmaceutical Industries: 13th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules We are pleased to welcome you to Capillary Electrophoresis in the Biotechnology and Pharmaceutical Industries: 13th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules, 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 CE analysis of protein, nucleotide and small molecule pharmaceuticals. The presentations and workshops will be devoted completely to practical concerns to 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. The symposium will feature presentations from leading experts within the industries and the regulatory agencies from around world. 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, improving sensitivity and technique, new technology, and QbD. Three workshops will focus on Best Practices, CE-MS and CE Methods in Monographs. 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 discussion 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, Analis, Beckman Coulter, Inc., BioPharm International, Calipers Life Sciences, Genentech, a Member of the Roche Group, LCGC North America, Novartis Pharma AG, Pfizer, Inc., Picometrics, Inc., ProteinSimple, Royal Society of Chemistry and Technology Networks. 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 ORGANIZING COMMITTEE François de l'Escaille, Analis Gamze Belin, Novartis Pharma AG Stefan Christians, Paul-Ehrlich-Institut Chantal Felten, Alpine Analytical Academy Franka Kálmán, University of Applied Sciences Western Switzerland Sarah Kennett, CDER, FDA Mark Lies, Beckman Coulter, Inc. SungAe Suhr Park, Amgen, Inc. Meg Ruesch, Pfizer, Inc. (Co-chair) Oscar Salas-Solano, Seattle Genetic, Inc. Cari Sänger - van de Griend, Kantisto BV (Co-chair) Dieter Schmalzing, Genentech, A Member of the Roche Group

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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 - 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 on your evaluation.

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CASSS CE Pharm Student Travel Grants

CASSS is pleased to provide a limited number of student travel grants for PhD students and post-docs who present applicable posters at CE Pharm 2011. PhD students or post-doctoral fellows conducting research in academia or industry throughout the world are eligible. Why you should apply: This symposium gives an insight into the current topics and issues under discussion within the pharmaceutical and biotech industry and, as such, gives attendees the opportunity to bridge 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 This year’s recipients include: Soo Hyun Park KIST, Seoul, South Korea Capillary Electrophoretic Mobility Shift Assay for Binding of DNA with NFAT3, a Transcription Factor from H9c2 Cardiac Myoblast Cells Ting Shi University of Illinois at Urbana-Champaign, Urbana, IL USA Detecting and Quantifying the Storage of D-Serine and Other Signaling Molecules in Astrocytic Synaptic-like Vesicles Using Two Different CE Platforms Jelynn A. Stinson Wright State University, Dayton, OH USA Conductivity Studies for Improved Sequence Coverage in Peptide Mapping of Lysozyme

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The Organizing Committee gratefully acknowledges the following program partners for their generous support of this Symposium:

Sustaining Program Partners

Platinum Agilent Technologies

Silver

Pfizer, Inc.

Platinum Program Partners

Beckman Coulter, Inc. Genentech, A Member of the Roche Group

Silver Program Partners

Novartis Pharma AG

Exhibitors

Agilent Technologies Beckman Coulter, Inc. Caliper Life Sciences

Picometrics ProteinSimple

Media Program Partners

BioPharm International

Genetic Engineering & Biotechnology News LCGC North America

Royal Society of Chemistry Technology Networks Limited

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CE Pharm 2011 Scientific Final Program Summary

Sunday, October 9, 2011

08:30 – 09:00 Registration and Breakfast (for course attendees ONLY) in Amelia Room Foyer 09:00 – 15:00

Short Course in Amelia Room Application of Capillary Electrophoresis to the Analysis of Protein Therapeutics

Short Course Facilitators: Chantal Felten, Alpine Analytical Academy, Whistler, BC Canada David A. Michels, Genentech, A Member of the Roche Group, South San Francisco, CA USA

and Oscar Salas-Solano, Seattle Genetics, Bothell, WA USA 10:30 – 11:00 Break in the Amelia Room 12:30 – 13:30 Hosted Lunch (for course attendees ONLY) in Amelia Room

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Monday, October 10, 2011 07:30 – 17:00 Registration in the Talbot Registration Desk 07:30 – 08:30 Breakfast in the Talbot D&E 08:30 – 08:45 Welcome and Introductory Comments in Talbot A-C

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

Best Practices Session I – Experience from Long Term Use in Talbot A-C Session Chair: SungAe Suhr Park, Amgen Inc., Thousand Oaks, CA USA

08:45 – 09:10 Intercompany Study to Evaluate the Robustness of Capillary Isoelectric

Focusing Technology for the Analysis of Monoclonal Antibodies Oscar Salas-Solano, Seattle Genetics, Bothell, WA USA

09:10 – 09:35 Capillary Electrophoresis in the Certification and Control of Port and Douro Wine Ana Oliviera, Instituto dos Vinhos do Douro e do Porto, I.P., Porto, Portugal

09:35 – 10:00 Short End Analysis - Uses and Limitations David Perrett, Queen Mary University of London, London, UK 10:00 – 10:25 Comparability Strategy for Capillary Electrophoreses Instrumentation Anu Bansal, Genentech, A Member of the Roche Group, South San Francisco, CA

USA 10:25 – 10:40 Discussion 10:40 – 11:10 Break – Visit the Exhibits and Posters in Talbot D&E 11:10 – 12:00 CE Pharm Partner Showcase 12:00 – 12:15 Break – Visit the Exhibits and Posters in Talbot D&E 12:15 – 13:15 Technical Seminar/Lunch and Learn

Sponsored by Agilent Technologies Talbot A-C 13:15 – 13:30 Break – Visit the Exhibits and Posters in Talbot D&E

Best Practices Session II – Experience from Long Term Use in Talbot A-C Session Chair: Gamze Belin, Novartis Pharma AG, Basel, Switzerland

13:30 – 13:55 Modified Pressure Injections and DTT as a Reductant in cSDS to Overcome

Analytical Challenges Posed by Particular Therapeutic Monoclonal Antibodies Tara Enda, ImClone Systems, a wholly-owned subsidiary of Eli Lilly, Branchburg, NJ USA

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Monday, October 10, 2011 continued

13:55 – 14:20 Technical Challenges in the Analysis of PEGylated Proteins by Capillary

Electrophoresis Yingchen Chen, Bristol-Myers Squibb, Pennington, NJ USA

14:20 – 14:45 Capillary Electrophoresis Cation and Protein Analyses for Industrial

Biosolutions in Grain Processing Applications Don Cannon, Genencor, A Danisco Division, Cedar Rapids, IA USA 14:45 – 15:00 Discussion 15:00 – 15:30 Break – Visit the Exhibits and Posters in Talbot D&E 15:30 – 16:30

Workshop I: Where to Start when you Encounter a Problem with your Capillary Electrophoresis System? in Talbot A-C

Workshop Facilitators: Chantal Felten, Alpine Analytical Academy, Vancouver, BC Canada and

François de l’Escaille, Analis, Namur, Belgium

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Tuesday, October 11, 2011 07:30 – 08:30 Breakfast in the Talbot D&E 08:00 – 17:00 Registration in the Talbot Registration Desk

Purity/Impurity Session in Talbot A-C Session Chair: Meg Ruesch, Pfizer, Inc., Chesterfield, MO USA

08:30 – 08:55 Characterization of CE-SDS Light and Heavy Chain Heterogeneities in

Monoclonal Antibodies Marc Hassel, Novartis Pharma AG, Basel, Switzerland 08:55 – 09:20 Deamidation Specific CZE Methods for Characterization and Quality

Control of Biopharmaceuticals Frank Moffatt, Solvias AG, Kaiseraugst, Switzerland 09:20 – 09:45 Characterization of Antibody Charge Variants Isolated by Free Flow

Isoelectric Focusing Brian Hosken, Genentech, A Member of the Roche Group, South San Francisco, CA USA

09:45 – 10:00 Discussion 10:00 – 10:30 Break – Visit the Exhibits and Posters in Talbot D&E

Transitioning to CE Session in Talbot A-C Session Chair: Mark Lies, Beckman Coulter, Inc., Brea CA USA

10:30 – 10:55 Making the Switch to CE in Biopharmaceutical Development; Lessons

Learned Melody Gossage, Eli Lilly & Company, Indianapolis, IN USA

10:55 – 11:20 Practical Approach of Amino Acids Testing or How to Make Life Easier Malgorzata Jaworska, National Medicines Institute, Warsaw, Poland

11:20 – 11:35 Discussion 11:35 – 11:50 Break – Visit the Exhibits and Posters in Talbot D&E

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Tuesday, October 11, 2011 continued

11:50 – 12:50 Technical Seminar/Lunch and Learn Unleashing the Power of your Mass Spectrometer – an Introduction to CESI-MS Ed Horton Beckman Coulter, Inc., Brea, CA, USA The Efficient and Sensitive Characterization of Intact Proteins of Pharmaceutical Interest by CESI-MS Govert W. Somsen Utrecht University, Utrecht, The Netherlands Sponsored by Beckman Coulter Talbot A-C 12:50 – 14:20 Poster Session – Visit and Discuss with Posters Presenters and Exhibitors in

Talbot D&E

CE/MS Session in Talbot A-C Session Chair: Franka Kálmán, University of Applied Sciences Western Switzerland, Sion, Switzerland

14:20 – 14:45 The Need for Novel Capillary Coatings – Optimization of CE-MS Methods

for (Glyco)peptide Analysis Carolin Huhn, Forschungszentrum Jülich, Jülich, Germany 14:45 – 15:10 Targeted High-throughput Glycan Profiling using Multiplexed CGE-LIF

and Protein Enrichment L. Renee Ruhaak, University of California, Davis, Davis, CA USA

15:10 – 15:35 Capillary Electrophoresis – Mass Spectrometry Methods for the Analysis of

Therapeutic Antibodies Colin Whitmore, Genentech, A Member of the Roche Group, South San

Francisco, CA USA 15:35 – 16:00 Comprehensive Glycoform Profiling of Biopharmaceuticals by Low Flow

CESI-MS Govert Somsen, Utrecht University, Utrecht, The Netherlands 16:00 – 16:15 Discussion 16:15 – 16:45 Break – Visit the Exhibits and Posters in Talbot D&E

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Tuesday, October 11, 2011 continued 16:45 – 18:15

Workshop II: CE-MS Function / New Developments and Applications in Talbot A-C

Workshop Facilitators: Franka Kálmán, University of Applied Sciences Western Switzerland, Sion, Switzerland and SungAe Suhr Park, Amgen Inc., Thousand Oaks, CA USA

Workshop Presenters:

Martin Greiner, Agilent Technologies, Waldbronn, Germany; Ed Horton, Beckman Coulter Inc., Brea, CA, USA; Carolin Huhn, Forschungszentrum Jülich, Jülich, Germany; Govert Somsen, Utrecht

University, Utrecht, The Netherlands

18:15 – 19:30 Exhibit and Poster Reception in the Talbot D&E

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Wednesday, October 12, 2011 07:30 – 08:30 Breakfast in the Talbot D&E 07:30 – 16:00 Registration in the Talbot Registration Desk 7:30 – 08:30 Sunrise Technical Seminar LabChip GXII for High Throughput Analysis of Protein Quality in Bioprocess Development Bahram Fathollahi Caliper Life Sciences, Alameda, CA USA Sponsored by Calipers Life Sciences Talbot A-C 08:30 – 08:45 CE Pharm Award Presentation

Regulatory Session in Talbot A-C Session Chair: Dieter Schmalzing, Genentech, A Member of the Roche Group, South San Francisco, CA

USA 08:45 – 09:10 The European Pharmacopeia, a Tool to Promote the use of Capillary

Electrophoresis? Guy Rautmann, EDQM, Strasbourg, France

09:10 – 09:35 Uses of Capillary Electrophoresis for Pharmaceutical Quality Control in

Japan Kumiko Sakai-Kato, National Institute of Health Sciences, Tokyo, Japan 09:35 – 10:05 Break – Visit the Exhibits and Posters in Talbot D&E 10:05 – 10:30 Requirements for Capillary Electrophoresis Test Procedures in Compendial

Monographs Stefan Christians, Paul-Ehrlich-Institut, Langen, Germany 10:30 – 10:55 Use of Capillary Electrophoresis as a Compendial Test for

Biopharmaceutical Products – Recent Developments at USP Tina Morris, US Pharmacopeia, Rockville, MD USA 10:55 – 11:20 Discussion 11:20 – 12:20 Workshop III: Capillary Electrophoresis Methods in Monographs Panel Discussion in Talbot A-C

Workshop Facilitators: Stefan Christians, Paul-Ehrlich-Institut, Langen, Germany and François de

l’Escaille, Analis, Namur, Belgium

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Wednesday, October 12, 2011 continued 12:20 – 12:35 Break – Visit the Exhibits and Posters in Talbot D&E 12:35 – 13:35 Technical Seminar Lunch/Learn Get There, Faster with iCE 1Susan Darling; 2Jiaqi Wu ProteinSimple, Santa Clara, CA USA1, ProteinSimple, Toronto, ON Canada2 Sponsored by ProteinSimple Talbot A-C 13:35 – 13:50 Break – Visit the Exhibits and Posters in Talbot D&E

CE Analysis of Antibody Drug Conjugates Session in Talbot A-C Session Chair: Oscar Salas-Solano, Seattle Genetics, Bothell, WA USA

13:50 – 14:15 Antibody-Drug Conjugate Characterization and Quality Assurance

Sarah Kennett, CDER, FDA, Bethesda, MD USA 14:15 – 14:40 Capillary Electrophoresis; a Practical Tool for Characterization of

Antibody-Drug-Conjugates (ADC) Darren Allender, Seattle Genetics, Bothell, WA USA

14:40 – 15:05 Applications of CE Methods for the Characterization of Antibody Drug

Conjugates Yan Chen, Genentech, A Member of the Roche Group, South San Francisco, CA USA

15:05 – 15:30 Use of Capillary Electrophoretic Techniques in the Analysis of Bioconjugates

Jeffery Schneiderheinze, Pfizer, Inc., St. Louis, MO USA 15:30 – 15:45 Discussion 15:45 – 16:00 Closing Comments in Talbot A-C

Meg Ruesch, Pfizer, Inc., Chesterfield, MO USA

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Thursday, October 13, 2011 8:30 – 14:00 Registration in the Amelia Room Foyer 8:30 – 9:00 Breakfast (for course attendees ONLY) in the Amelia Room 9:00 – 15:00

Short Course in Amelia Room Method Development, Optimization and Good Working Practice in Capillary Electrophoresis Short Course Facilitators: François de l'Escaille, Analis, Namur, Belgium and Cari Sänger - van de

Griend, Kantisto BV, Baarn, The Netherlands 10:30 – 11:00 AM Break in the Amelia Room 12:30 – 13:30 Hosted Lunch (for course attendees ONLY)

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Oral Abstracts

Intercompany Study to Evaluate the Robustness of Capillary Isoelectric Focusing Technology for the Analysis of Monoclonal Antibodies 1Oscar Salas-Solano; 1Darren Allender; 2Kunnel Babu; 3SungAe Suhr Park, 4Xinfeng Zhang, 4Li Zhang; 5Zoran Sosic; 5Boris Boumajny; 6Ming Zeng; 6Kuang-Chuan Cheng; 7Angelia Reed-Bogan; 7Stacey Cummins-Bitz; 8David A. Michels; 8Monica Parker; 9Paulina Bonasia; 10Mingfang Hong; 11Steven Cook, 11Margaret Ruesch, 11Brian Nunnally; 12David Lamb; 13Dora Bolyan; 14Steffen Kiessig Seattle Genetics, Bothell, WA USA1, Bristol-Myers Squibb, Pennington, NJ USA2 , Amgen, Inc., Thousand Oaks, CA USA3, Amgen, Inc., Fremont, CA USA4, Biogen Idec Inc., Cambridge, MA USA5, Bristol-Myers Squibb, Bloomsbury, NJ USA6 ,Eli Lilly and Company, Indianapolis, IN USA7, Genentech, a Member of the Roche Group, South San Francisco, CA USA8, Genzyme, a Sanofi Company, Framingham, MA USA9, Johnson and Johnson Company, Radnor, PA USA10, Pfizer, Inc., Chesterfield, MO USA11, Sanofi Pasteur, Swiftwater, PA USA 12, Solvias AG, Basel, Switzerland13, F. Hoffmann-La Roche Ltd, Basel, Switzerland14 Interlaboratory comparisons are essential to bringing emerging technologies into biopharmaceutical industry practice and regulatory acceptance. As a result, an international team including 12 laboratories from 10 independent biopharmaceutical companies in the United States and Switzerland was formed to evaluate the precision and robustness of capillary isoelectric focusing (CIEF) and imaged capillary isoelectric focusing (iCIEF) to determine the charge heterogeneity of monoclonal antibodies. The different laboratories determined the apparent pI and the relative distribution of the charge isoforms of a representative monoclonal antibody (rMAb) sample using the same CIEF and iCIEF method. Statistical evaluation of the data was performed to determine within and between-laboratory consistencies and outlying information. The apparent pI and the percent peak area data of the various rMAb charge variants showed very good precision across different laboratories, with different analysts using different lots of ampholytes and multiple instruments. Taken together, these results validate the appropriate use of CIEF and icIEF in the biopharmaceutical industry in support of regulatory submissions. NOTES:

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Capillary Electrophoresis in the Certification and Control of Port and Douro Wines Ana Oliveira Instituto Vinhos Douro e Porto, Porto, Portugal The Mission of the Port and Douro Wine Institute (IVDP, I.P.) is that of promoting the certification, quality control, and quantity of Port Wines, along with regulating the production process, the defense and protection of the Douro and Port denominations of origin, and also the geographical indication (GI) of the Douro Region. The IVDP, I.P. Laboratory has been accredited since 1994, in accordance with the EN ISO/IEC 17025, for various tastings of licorous wines and brandies. The laboratory provides analytical services both for the Port Wine and the National Wine-Growing sectors in general. This laboratory is equipped with state of the art analysis resources, one of which is the capillary electrophoresis (CE) used for the analysis of sulphates in wine. It is worth mentioning in this regard the collaborative trial organized under the sponsorship of OIV (International Organization for Vine and Wine), which was designed to assess the qualitative performance of the method and to enable the adoption of the OIV type II method. The trial was carried out through seven international laboratories from five European countries. These laboratories received eight coded duplicate wine samples and one spiked sample with concentrations from 0.4 g/l to 2.4 g/l of sulphates (expressed in potassium sulphate). The coefficients of variation values for sulphates ranged from 2% to 8% within CVr laboratories and from 6% to 14% among CVR laboratories. The results of the interlaboratory trial served to confirm that the analytical method is robust and reliable and can be used as a standard method for analyzing sulphates. This method could be a complement to the OIV type II method for acid analysis. NOTES:

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Short End Analysis - Uses and Limitations David Perrett Queen Mary University of London, London, UK Most commercial CE systems can operate in a ‘reversed’ mode where the usual inlet (anode) becomes the cathode and samples are injected by reversing the applied pressures. The detection window is then usually only a few centimetres from the point of injection. This permits much faster analysis of samples. We have investigated the utility and performance of operating in this short end mode. We have employed short end injection for both pharmaceutical and clinical analyses. Fully validated assays using short end analyses using a Agilent CE system have been developed for tetracaine and lidocaine and used for pharmacokinetic studies. The peaks of interest typically migrated at ca. 1 minute. There was no difference in linearity, sensitivity and reproducibility between short and long end injections. The data was also comparable to that generated by HPLC. For urine and blood screening studies, both complex samples, there is some loss of peak capacity and resolution for small molecular weight species but this more than countered by the throughput improvements achieved. The analysis of proteins in serum/plasma and other biofluids by short end injection is also possible. NOTES:

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Comparability Strategy for Capillary Electrophoreses Instrumentation Anu Bansal; Alice Yee; Helen Kumagai; Thomas Chan Genentech, A Member of the Roche Group, South San Francisco, CA USA Instrumentation changes due to discontinuation of instrument model, continuous technological improvements, or assay transfers to testing sites with different analytical platforms are routine for state-of-the-art quality control laboratories. These changes require a thorough technical impact assessment to the validated state of the method and a subsequent experimental comparability exercise, if warranted, prior to implementation. Inadequate comparability assessment can lead to a critical difference with the original assay validation and performance that might reveal itself over time during routine testing, e.g. unexpected out of trend results, out of specifications results. Furthermore, an established approach for instrument comparability can minimize the resources and time involved in identifying and qualifying a new instrument for routine QC use. An overview of the critical parameters in determining instrument comparability with case studies will be discussed in this presentation. NOTES:

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Modified Pressure Injections and DTT as a Reductant in cSDS to Overcome Analytical Challenges Posed by Particular Therapeutic Monoclonal Antibodies. Tara Enda; Gordon Freckleton; Tim Blanc; Zhengang Wang; Qinwei Zhou ImClone Systems, a Wholly-owned Subsidiary of Eli Lily, Branchburg, NJ USA Capillary SDS (cSDS) is now a well established separation technique that has evolved to replace Coomassie SDS slab gels for the analysis of therapeutic monoclonal antibodies. Our lab generally tests monoclonal antibody products with matrices composed of various strengths of ionic additives. In addition, we have found several antibodies that have needed special sample preparation conditions including high SDS concentrations. Some sample matrices and some sample preparation buffers have rendered electrokinetic injection impractical. We have found that a modified pressure injection, in place of electrokinetic injection, results in reliable sample introduction and consistent repeatable data that can be achieved over a multitude of sample matrices and sample preparation buffers. Our first attempt of pressure injecting reduced samples did not resolve a peak in front of the heavy chain that is typically seen in electrokinetic sample injection. By adding a water plug injection prior to the sample injection we were able to successfully resolve the small peak seen before the heavy chain peak. Sample introduction via pressure injection also enabled the use of higher SDS concentrations in the sample buffer without adversely affecting the amount of sample introduced into the capillary. This can be especially advantageous when analyzing antibodies that may not reduce completely under the standard sample handling procedures. The standard cSDS separation condition uses β-mercaptoethanol in place of DTT to reduce proteins, despite DTT being a much more efficient reducing reagent than β-mercaptoethanol at pH 9.0. After screening several buffers, we found using a borate buffer in combination with DTT followed by alkylation with Iodoacetic Acid shifts the migration time of the DTT peak to well before the sample peak migration times. This modified cSDS procedure has enabled us to fully reduce one particular IgG that had not been fully reduced under the standard sample preparation conditions. NOTES:

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Technical Challenges in the Analysis of PEGylated Proteins by Capillary Electrophoresis Yingchen Chen; Jinping Liu; Reb Russell; Michael Grace Bristol-Myers Squibb, Pennington, NJ USA Protein modification through covalent coupling of polyethylene glycol (PEG) to therapeutic agents has now become a standard technique in biopharmaceutical industry. The covalent attachment of PEG (PEGylation) to biomolecule improves their pharmacological properties by reducing their renal clearance (increased circulation half-life), toxicity and immunogenicity. Several methods for analysis and characterization of PEGylated proteins have been reported in the past, including chromatography, capillary electrophoresis, and mass spectrometry. However, the nature of the PEGylation and polydispersity of the PEG molecule itself present some unique analytical challenges. This presentation will detail some of the method development strategies and challenges for the analysis of PEGylated proteins by capillary electrophoresis. NOTES:

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Capillary Electrophoresis Cation and Protein Analyses for Industrial Biosolutions in Grain Processing Applications Don Cannon Genencor, a Danisco Division, Cedar Rapids, IA USA Biotechnology solutions continue to enhance and expand industrial grain processing applications. Advancements toward a comprehensive bio-refinery concept require analytical methods capable of sensitive, high-throughput determinations in complex matrices. Specifically tasked for enzyme treatment innovations in grain processing applications, our applied innovation center utilizes many of the common modalities of capillary electrophoresis (CE), including CE-SDS and cIEF for protein analysis, CE-LIF for carbohydrate profiling, and dynamic coating CE with indirect UV detection for small molecule ion determinations. This presentation will focus on practical, customer-centric applications of CE for both wet-mill and dry-mill grain processing industries. CE cation analyses have the required sensitivity (low ppm) and throughput ability to profile numerous process streams from a multitude of customer manufacturing sites. These ion audits have allowed further site-specific optimization of a critical alpha-amylase enzyme treatment step in the breakdown of starch granules. Quantitative and qualitative data from these CE cation analyses will be presented along with cIEF characterization of general ion dependencies of alpha-amylase enzymes. For wet-mill processing of starch slurries with up to 35% dissolved solids, both free and total cation levels are used for optimizing alpha-amylase activity. To measure total cation levels, acid treatment is used lower the pH (pH<1); resulting in unbinding associated cations. With no more than a subsequent 2x dilution of the supernatant, total cation profiles have been reproducibly measured by CE without apparent degradation of the poly-ion dynamic capillary coatings. For dry-grind corn-based ethanol fermentations, cation profile data have led to changing flow patterns of re-circulated process streams for sufficient cation presence in this early enzyme step. These unique applications for CE cation analyses, combined with protein cIEF insights, exemplify that ability of CE to contribute to expanding biotechnology for new, complex industrial applications. NOTES:

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Characterization of CE-SDS Light and Heavy Chain Heterogeneities in Monoclonal Antibodies Marc Hassel Novartis Pharma AG, Basel, Switzerland The capillary gel electrophoresis after denaturation with sodium dodecyl sulfate (CE-SDS) as a size based separation technique is more and more replacing the formerly well established SDS-PAGE in quality control of monoclonal antibodies. Because of the higher resolution of CE-SDS, heterogeneities of the light and the heavy chain which are not observed in SDS-PAGE, become now visible as shoulders or even distinct peaks that need to be identified. As in CE-SDS a very low amount of protein is injected, the identification by fraction collection or hyphenation of the CE with a second technique such as MS, is very difficult if not impossible. In this presentation alternative approaches to characterize light and heavy chain heterogeneities detected by CE-SDS are discussed. NOTES:

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Deamidation Specific CZE Methods for Characterization and Quality Control of Biopharmaceuticals Frank Moffatt; Maria Anna Schwarz Solvias AG, Kaiseraugst, Switzerland Capillary isoelectric focusing (cIEF) is commonly used to show charge heterogeneity of intact monoclonal antibodies (mAbs). Usually between 3-6 isoform groups (highly complex mixture) are visible. To improve resolution of the charged variants, digestion of mAbs using papain and following cIEF analysis of the formed Fab and Fc fragments has been investigated. In this study we will present data of various commercial and development mAbs to demonstrate the use of cIEF following papain digestion. In all investigated mAbs, additional variants were visible that were masked using conventional cIEF with no digestion. The results show the potential of cIEF with papain digestion as a general tool for quality control and characterization of mAbs but also for first assumptions in which part of the mAb deamidation is progressed. For a more detailed (quantity) and fast localization of all deamidation sites a deamidation specific digestion coupled to a CZE or HPLC separation will be shown. In this investigation Asn or Gln deamidation and Asp isomerization of mAbs proteins under stressed conditions were characterized and quantified by a new and highly specific analysis technique. Protein deamidation without isomerization provides new cutting sites for a site specific endopeptidase. Therewith a new peptide fragment is detected in the CZE separation profile. If isoAsp is formed an additional signal can be observed with a typical ionic mobility shift in the CZE separation. By this approach both deamidation and isomerization of each deamidation site can be quantified in a simple way. NOTES:

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Characterization of Antibody Charge Variants Isolated by Free Flow Isoelectric Focusing Brian Hosken Genentech, A Member of the Roche Group, South San Francisco, CA USA Capillary isoelectric focusing (cIEF) is widely used in the biopharmaceutical industry to measure the charge distribution of therapeutic antibodies. However, the implementation of this technology has created a new analytical challenge. The nanoliter volumes that enable rapid, automated and high resolution separations also prevent capillary methods from being used for variant isolation and identification. Therefore, a semi-preparative IEF separation is needed to fractionate the charge variants for downstream characterization. We developed a free flow isoelectric focusing method to isolate the product related variants observed in a cIEF charge profile. The fractionated isoforms were purified from the separation matrix and then characterized using physiochemical and biological assays. Acidic and basic variants were identified using this approach. NOTES:

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Making the Switch to CE in Biopharmaceutical Development; Lessons Learned Melody Gossage; Kevin Overstreet; Tewelde Tesfai Eli Lilly & Company, Indianapolis, IN USA Capillary electrophoresis (CE) based methods have become a common part of analytical biopharmaceutical development for analysis of monoclonal antibodies (mAbs). Although these CE methods offer many well know advantages over their traditional counterparts there have been growing pains and implementation challenges along the way. Over the past 10 years Lilly has progressed from CE methods being considered only when typical, routine methods were not feasible to being a front line technique. Bioproduct Research and Development at Lilly now considers CE one of our platform technologies. Some of the challenges, pitfalls, and successes of implementation from acceptance of a new technology to routine testing in a QC laboratory will be presented. NOTES:

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Practical Approach of Amino Acids Testing or How to Make Life Easier Malgorzata Jaworska National Medicines Institute, Warsaw, Poland α-Amino acids, their derivatives and peptides are widely used in medicine, mostly in products for parenteral and enteral nutrition. Some of the amino acids and their derivatives are also indicated for another purposes: supportive treatment during clinical nutrition, non-nutritional indications, auxiliaries. Medicinal products containing amino acids are manufactured in different pharmaceutical forms, based on over a dozen active substances. Currently, the quality control of these preparations commonly used chromatographic tests, since the separation of each of groups of chemicals usually requires a separate analytical procedure. Amino acid analysis with liquid chromatography is still considered a significant challenge due to the diversity of analytes, with most of them not having a chromophore, and some very similar in their chemical structure. The procedure often requires derivatization and multi-step gradient systems. The ionic nature of amino acids and their derivatives makes them also suitable for testing by capillary electrophoresis (CE) that offers several advantages over HPLC. Thanks to these, CE is increasingly being used in pharmaceutical analysis, both for the determination of active substances and related impurities. The presentation gives an overview of the CE methods developed in our laboratory and proposed to quality control of drugs containing amino acids and their derivatives. Systematic optimization of electrophoresis conditions for each group of preparations allowed the selective determination of active ingredients in the medicinal product, as well as impurities. Validation of the methods and result comparison with the reference method confirmed the suitability of capillary electrophoresis as a tool in pharmaceutical analysis. It should be pointed out that, in many cases the application of this technique made it possible to shorten the run time and simplify the analytical procedure, for example by replacing several HPLC methods by one CE method. NOTES:

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The Need for Novel Capillary Coatings – Optimization of CE-MS Methods for (Glyco)peptide Analysis Martin Pattky; Carolin Huhn Forschungszentrum Jülich, Jülich, Germany Capillary coatings are widely used for the capillary electrophoretic analysis of proteins and peptides in order to reduce the degree of analyte adsorption onto the capillary surface. However, beside separation efficiency, basic separation parameters including speed of analysis, resolution of analyte signals and robustness can be optimized to a large extent by the choice of convenient coating materials. Using a set of neutral and cationic adsorbed coatings for capillary electrophoresis – mass spectrometry, we here show the strong need for the development of further coating materials. We compare adsorbed coatings, one newly synthesized cationic coating, a commercial cationic (polybrene) as well as a commercial neutral coating (based on polyacrylamide, called LN®). All three coatings showed very high plate numbers up to the range of 500 000 in the analysis of tryptic peptides, indicating that analyte adsorption can be neglected. However, large differences are visible regarding resolution of analyte pairs, induced by differences in the electroosmotic flow velocity. Speed of analysis was highest for polybrene and lowest for the neutral coating. Lowest robustness, mainly with regard to spray stability, was found for the neutral coating, it was higher for the cationic coatings providing significant flow towards the CE-MS sheath-liquid interface. Highest resolution was achieved for the LN coating, however at cost of highest analysis times and lowest robustness. Using cationic polybrene coating gives rise to extremely fast separations at the cost of resolution. Our novel cationic coating with an intermediate EOF velocity offers a compromise on resolution and speed of analysis. The method was applied to tryptic digests of (glyco)proteins and small intact glycoproteins. NOTES:

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Targeted High-throughput Glycan Profiling using Multiplexed CGE-LIF and Protein Enrichment L. Renee Ruhaak1, 2; Cynthia Williams1; Carolin Huhn3; Manfred Wuhrer2; Carlito B. Lebrilla1 1University of California Davis, Davis, CA USA; 2LUMC, Leiden, The Netherlands; 3Forschungszentrum Julich, Julich , Germany Methods to analyze N-glycosylation patterns of biofluids, tissues and individual proteins in larger sample sets have recently become available, and the potential of glycans as biomarkers for diseases such as cancer, diabetes and Alzheimer is now widely recognized. As the range of protein concentrations in complex biological samples is very high, protein N-glycosylation patterns from such samples is dominated by glycans from high-abundant proteins. One strategy for large scale glycan analysis is based on CGE-LIF analysis of APTS-labeled glycans using a DNA sequencer. We recently optimized the derivatization method as well as sample purification procedure to allow fast sample preparation. Noticeably, the commonly used, but toxic reducing agent NaCNBH3 is replaced by 2-picoline borane, which is non-toxic and yields superior results with respect to the robustness of the labeling. Moreover, a HILIC-based SPE material was used for clean-up. The method has now been applied to several sample sets. The use of a hexapeptide library for the depletion of high-abundant proteins and enrichment of low-abundant proteins from complex biological samples has been described in proteomics analyses. We here apply the developed CGE-LIF method for the evaluation of this method for N-glycan profiling and discuss the applicability of the hexapeptide library for glycan-based biomarker discovery. NOTES:

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Capillary Electrophoresis – Mass Spectrometry Methods for the Analysis of Therapeutic Antibodies Colin Whitmore; Lynn Gennaro Genentech, a Member of the Roche Group, South San Francisco, CA USA Capillary electrophoresis is a powerful separation method for the analysis of therapeutic antibodies, but the identification of unknown peaks can be cumbersome. The use of online mass spectrometry detection provides for identification of biomolecules, but coupling mass spectrometry to capillary electrophoresis poses a number of challenges. We have developed two methods that handle these challenges in order to deliver useful CE-MS data. First, LC-MS separates tryptic peptides of therapeutic antibodies with excellent resolution and very good sequence coverage. For example, the sequence coverage of the therapeutic antibody analyzed in this work is 97%. There were ten undetected peptides, all three or fewer amino acids in length; among the ten is one complementarity determining region peptide. These small peptides elute in the salt front of the LC separation, and so are not subject to MS analysis. Here, CE-MS is presented as a complimentary method to LC-MS for the detection of small peptides. Data was gathered using two distinct electrospray CE-MS interface designs: a traditional sheath flow interface, and a novel sheathless interface. All small peptides were detected with both interfaces. The sheathless CE-MS gave 100% sequence coverage, while the sheath system was more robust in our hands. Second, capillary isoelectric focusing (CIEF) is finding routine use in the characterization of antibodies, as its pI based focusing reveals variants not resolved by other separations. Unfortunately, most reagents used with CIEF are incompatible with mass spectrometry. We have developed an online CIEF-MS method using a sheath liquid interface. The sheath liquid provides the CIEF catholyte during focusing, then is switched to an acid for gradient mobilization and ionization. We use volatile reagents where possible, and maximize the antibody to ampholyte ratio. Using this method, we demonstrate the resolution and identification of antibody variants. NOTES:

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Comprehensive Glycoform Profiling of Biopharmaceuticals by Low Flow CESI-MS Govert W. Somsen; Rob Haselberg; Gerhardus J de Jong Utrecht University, Utrecht, The Netherlands Sheathless integrated capillary electrophoresis electrospray ionization (CESI) offers attractive possibilities for mass spectrometric (MS) detection of intact proteins. It provides the selectivity and sensitivity required to distinguish structurally related species. This presentation outlines the performance of CESI-MS for glycoform analysis of biopharmaceuticals at the intact protein level. It will be shown that CESI provides very favorable signal-to-noise ratios, yielding protein detection limits in the sub-nM range and allowing detailed glycoform profiling. The ultra low flow rates applied are demonstrated to circumvent protein ionization suppression and to permit efficient protein separation. The usefulness of CESI-MS for biopharmaceutical applications will be illustrated by the assessment of the glycoform heterogeneity of recombinant human interferon-β (rhIFN), recombinant human erythropoietin (rhEPO) and monoclonal antibody (mAb) products. For rhIFN, 20 different glycoforms spanning two orders of magnitude in concentration could be reliably assigned in a single run, which encompasses a significant improvement over current characterization methods. CESI-MS of BRP rhEPO revealed the presence of more than 250 isoforms within an analysis time of less than 45 min. It will be shown that CESI-MS provides semi-quantitative glycoform profiles that allow the specific distinction of EPO products, including biosimilars. CESI-MS was also applied to the direct analysis (no sample preparation) of mAb formulations (IgG, 150 kDa) containing MS-interfering surfactants, revealing various isoforms. NOTES:

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The European Pharmacopeia, a Tool to Promote the use of Capillary Electrophoresis? Guy Rautmann EDQM, Strasbourg, France An overview of the European Regulatory bodies involved in the legislation governing medicinal products will be given. Particular emphasis will be made on the role and responsibilities of the European Directorate for the Quality of Medicines and HealthCare (EDQM) of the Council of Europe. Capillary electrophoresis (CE) competes with liquid chromatography (LC) for the quality control of biopharmaceuticals. Perceived once as a promising alternative, CE is still not widely used as an analytical tool and this is also reflected in the compendia. Some possible reasons for this will be presented from a regulatory perspective and illustrated by examples. NOTES:

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Uses of Capillary Electrophoresis for Pharmaceutical Quality Control in Japan Kumiko Sakai-Kato National Institute of Health Sciences, Tokyo, Japan Capillary electrophoresis is an analytical method which targets range from small molecules to macromolecules including proteins and nucleic acids. In Japan, capillary electrophoresis procedures are described in JP (Japanese Pharmacopoeia) General Information and the pharmacopoeial texts can now be used as interchangeable in the ICH regions as the result of Q4B process. In this presentation, we will report a survey how capillary electrophoresis is used for the pharmaceutical quality control in pharmaceutical companies in Japan. Although the main target products are proteins or peptides, the survey shows that the capillary electrophoresis is used in many test assays reflecting the various separation modes of the method. NOTES:

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Requirements for Capillary Electrophoresis Test Procedures in Compendial Monographs Stefan Christians Paul-Ehrlich-Institut, Langen, Germany The emerging of capillary electrophoresis (CE) in the field of analytical sciences allowed to combine the high resolution power of electrophoretic techniques with the reliability and quantifiability of HPLC techniques. Thus it is not surprising that the literature on CE is exploding. Surprising it is instead that up to now relatively few CE methods have made their way into pharmacopoeias. What are the advantages of CE-test procedures in monographs? Currently in many monographs for therapeutic proteins rather antiquated gel electrophoresis test procedures are required. These are often not very reliable and prone to subjectivities in quantification. If such procedures are required even sophisticated tests like CE methods have to be validated against the less reliable test. Requirements of test procedures to qualify as compendial method: The main requirements are the robustness and the reliability of the test procedure. It should be robust enough that the vast majority of manufacturers of the relevant product but also the regulatory test laboratories should be able to perform the test and come to very similar results. Validation of CE methods before the implementation: Basically the analytical test procedure is used to ensure that products meet the quality requirements of the monograph. Test procedures aimed to be implemented in monographs usually have to be tested also in inter-laboratory studies. Proprietary elements in compendial test methods: There are some excellent and reliable test kits on the market which would be very useful for the assessment of the quality especially of therapeutic proteins. From an analytical point of view these test kits may be appropriate as compendial test procedures. Serious challenges, however, may be the permanent availability of the test kits and the limited information on the properties of fundamental elements. NOTES:

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Use of Capillary Electrophoresis as a Compendial Test for Biopharmaceutical Products – Recent Developments at USP Tina Morris US Pharmacopeia, Rockville, MD USA USP has recently made significant progress in updating procedural guidance for capillary electrophoresis as it applies to the analysis of biotechnology –derived products, as well as the ancillary materials used in their production. Capillary Electrophoresis was first captured in the USP-NF in Chapter <727> Capillary Electrophoresis. This Chapter has recently been omitted and CE general procedural information is now primarily found in the harmonized Chapter <1053> Capillary Electrophoresis. Beyond that, USP has been very actively working on analyte-specific compendial procedures: specifically in the area of glycoprotein and glycan analysis. The presentation will focus on the development of several specific compendial EC procedures, but also discuss the general approaches and challenges that apply to establishing a compendial test. NOTES:

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Antibody-Drug Conjugate Characterization and Quality Assurance Sarah Kennett CDER, FDA, Bethesda, MD USA Antibody-drug conjugates (ADCs) have been developed as a mechanism for targeting delivery of highly toxic drugs to specific cells. Regulatory submissions for ADCs have seen a significant increase in the last 3-4 years, and, with the promise of advances in ADC scientific knowledge and technologies and the recent U.S. licensure of Adcetris™ (brentuximab vedotin), it is expected that the interest in developing ADCs will continue to grow. Analytical assay development will be a crucial aspect of ADC product development, and it is imperative that the assays used to evaluate therapeutic products are suitable. ADC characterization, release, and stability analysis will require testing of the naked antibody, the small molecule drug and linker components, and the resulting conjugate. Unique challenges to analysis are presented by ADCs due to the conjugation reactions, and analysis of different ADCs will require different analytical tools. This presentation is intended to provide a monoclonal antibody reviewer's perspective on the current expectations regarding characterization and quality assurance of ADCs. NOTES:

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Capillary Electrophoresis; a Practical Tool for Characterization of Antibody-Drug Conjugates (ADC) Darren Allender; Oscar Salas- Solano Seattle Genetics, Bothell, WA USA Antibody-drug conjugates (ADCs) are monoclonal antibodies linked to cell-killing agents and are considered effective delivery systems for anti tumor cytotoxic drugs. The analysis and physicochemical characterization of ADCs is a regulatory expectation to ensure the safety, potency, quality control and stability of these therapeutic proteins. Charge heterogeneity is a critical quality attribute (CQA) which can impact pharmacokinetics (PK), potency, and can be used to define both the purity and potential degradation pathways of the molecule. Several analytical techniques are commonly used to monitor and characterize charge heterogeneity including cation exchange chromatography (CEX) and capillary isoelectric focusing (cIEF). CEX is a widely used method for naked antibodies due to its ease of use and preparative capabilities. However, CEX is not necessarily an appropriate assay for ADCs as it may not provide adequate resolution of minor species, probably due to interactions of the ADC with the stationary phase of the separation column. Imaged Capillary Isoelectric Focusing (icIEF) is an emerging technology that separates charged species primarily based on differences in isoelectric point. Due to the difference in the mechanism of separation, icIEF is an attractive complementary technique and offers an alternative to CEX. However, characterization of the charged variants present in the icIEF profile can be challenging as the method is not preparative. Here we describe the use of Aglient Technologies OFFGEL 3100 Fractionator as an alternative to preparatively collect charged variant fractions of both naked antibody intermediate and ADC for subsequent characterization using several analytical techniques. NOTES:

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Applications of CE Methods for the Characterization of Antibody Drug Conjugates Yan Chen Genentech, A Member of the Roche Group, South San Francisco, CA USA Antibody-drug conjugates (ADC) are being explored as anti-cancer therapeutics which allows targeted delivery of the cytotoxic drug moiety to tumors. The primary sites used for protein-directed conjugation are the amino groups of lysine residues or the sulfhydryl groups of the inter-chain cysteine residues. The conjugation technology, regardless of the site and process used for linkage, often results in an ADC molecule that is heterogeneous with respect to both the distribution and loading of cytotoxic drug species on the mAb. For any given ADC, the chemical properties of the cytotoxin and linker, combined with selection of linkage site, will dramatically affect the physicochemical attributes, and the selection of analytical methods to assess these attributes will depend on this architecture. Assays used for the parent mAb may not work for its corresponding ADC, or assays used for one type of ADC, may not be applicable to an ADC with a different architecture. Depending on the ADC, the same assay may provide different information. During the past decade, capillary electrophoresis (CE) methods emerged as a promising, effective, and economical approach for the analysis of therapeutic proteins. The application of various CE-based methods for the characterization of ADCs will be presented. Examples will be provided to demonstrate that the applicability of a particular CE method to ADC analysis depends on the nature of the drug-linker, the mode of attachment, and the resulting complexity of the product. NOTES:

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Use of Capillary Electrophoretic Techniques in the Analysis of Bioconjugates Jeffrey Schneiderheinze Pfizer, Inc., St. Louis, MO USA Bioconjugation is a term used to define the process by which a pharmaceutically active compound is covalently linked to a larger, usually inert molecule such as poly(ethylene) glycol (PEG), antibodies, enzymes or nucleic acids. The purpose of this conjugation is usually to bestow beneficial properties upon the pharmaceutically active component (typically a small molecule or peptide) such as increased aqueous solubility, increased resistance to proteases or a more beneficial PK/PD profile. The nature of the conjugated molecule, however, adds additional complexity to the analytical characterization, especially when attempting to analyze and quantify product-related degradants and impurities. This presentation will discuss will discuss specific challenges associated with antibody-peptide conjugates. Specifically, challenges associated with analysis of the peptide modification and degradation will be discussed as well as analytical methods and technology that were utilized to overcome these challenges. NOTES:

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Workshop Descriptions

Workshop Session I

Monday, October 10, 2011 15:30 – 16:30

Where to Start When You Encounter a Problem with your Capillary Electrophoresis System? Workshop Facilitators: Chantal Felten, Alpine Analytical Academy, Vancouver, BC Canada François de l’Escaille, Analis, Namur, Belgium Troubleshooting is the process of diagnosing the source of a problem/failure in a process. The basic theory of troubleshooting is that you start with the most general (and often most obvious) possible problems, and then narrow it down to more specific issues. The most beneficial approach to troubleshooting should follow a logical process of elimination to identify the true source of a problem. John W. Dolan (* LC-GC Europe July 2011) presented in his article “Troubleshooting Basics (Part 1)” the following six guiding rules for troubleshooting LC systems: • The Rule of One • The Rule of Two • The Divide and Conquer Rule • The Module Substitution Rule • The Put It Back Rule • The Documentation Rule Although LC and CE are different technologies, these general rules of troubleshooting still apply. During the workshop, the rules shown above will be discussed and adjusted to fit Capillary Electrophoresis. Based on the rules the Workshop Participants shall troubleshoot existing failures, define the underlying cause and offer solutions. The target outcome of the workshop is to develop a structural approach for troubleshooting Capillary Electrophoresis problems, based on the common method concerns, such as instrument/hardware maintenance, sample preparation errors and training issues. This structural approach should be beneficial for in-house resolution of problems/failures as well as for communication with manufacturers support team. NOTES:  

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Workshop Session II Tuesday, October 11, 2011

16:45 – 18:15

CE-MS Function / New Developments and Applications Workshop Facilitators: Franka Kálmán, University of Applied Sciences Western Switzerland, Sion, Switzerland SungAe Suhr Park, Amgen Inc., Thousand Oaks, CA USA Workshop Presenters: Martin Greiner, Agilent Technologies, Waldbronn, Germany Carolin Huhn, Forschungszentrum Jülich, Jülich, Germany Ed Horton, Beckman Coulter Inc., Brea, CA, USA Govert Somsen, Utrecht University, Utrecht, The Netherlands In the workshop the function and latest developments in interfacing Capillary Electrophoresis with Mass Spectrometry will be presented by G. Somsen & C. Huhn, for many years experts in the field. Technical solutions to interface CE with the MS will be explained as the conventional sheath flow way of interfacing CE with MS ("triple tube" approach) by M. Greiner from Agilent Technologies and the sheath less CE-MS interface presented by M. Lies from Beckman Coulter. Interfaces “to touch” will be available. The different technical solutions will be compared in terms of application possibilities, easiness of use, strength, sensitivity, reproducibility and robustness. The experts will provide practical advises how to run CE-MS in the daily routine in an efficient and economic way, also addressing sample preparation issues. Typical and novel applications of CE-MS for biotherapeutics, biosimilars e.g. glycoproteins but also from other application areas will be covered by the presenters. Workshop participants who are involved in these fields are invited to share their experiences with the others in the workshop. NOTES:

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Workshop Session III Wednesday, October 12, 2011

11:20 – 12:20

Panel Discussion – Capillary Electrophoresis Methods in Monographs Workshop Facilitators: Stefan Christians, Paul-Ehrlich-Institut, Langen, Germany François de l’Escaille, Analis, Namur, Belgium  “The international pharmacopeias such as USP, EP, and JP, being responsible for the quality of drugs are in a continuous process of revision of their monographs. Despite the fact that a drug’s production might have changed and a different impurity profile has to be expected, the development of new analytical methods is mirrored in the pharmacopeias….” Holzgrabe U. (2008). The need for CE methods in pharmacopeial monograghs. Capillary Electrophoresis Methods for Phamaceutical Analysis, Volume 9, pp. 245 – 258, Elsevier Inc. Today capillary electrophoresis is more and more used in pharmaceutical and biopharmaceutical environment. Despite this fact there are only a few monographs available. The aim of this workshop is to discuss the reasons of this situation and how to overcome them. There may be different reasons for this. One major hindrance may be that the analytical power of CE-methods are still not sufficiently enough recognized among the decision-makers in compendial groups. Furtheron the fear that results from CE-methods might be not comparable to existing gel electrophoresis techniques or HPLC techniques. Another reason may be that existing methods, in use in the quality control of pharmaceutical companies do use some proprietary or patented elements such as sieving gels and capillary coatings. The aim of this workshop is to start a discussion between regulatory, industry and vendors how to coop with the introduction of new monographs and how to fill in the different elements needed for a monograph. NOTES:

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Technical Seminar Abstracts

Technical Seminar

Monday, October 10, 2011 12:15 – 13:15

Talbot A-C Sponsored by Agilent Technologies TBD Abstract not avaliable at the time of print.

Technical Seminar Tuesday, October 11, 2011

11:50 -12:50 Talbot A-C Sponsored by Beckman Coulter, Inc. Unleashing the Power of your Mass Spectrometer – an Introduction to CESI-MS Ed Horton Beckman Coulter, Inc., Brea, CA, USA Both mass spectrometry (MS) and capillary electrophoresis (CE) have become indispensable technologies for the analysis, characterization, and identification of therapeutic proteins. In this workshop we present a new front-end separation-and ionization technology called CESI, which fully integrates the high efficiency and ultra-low flow characteristics of capillary electrophoresis (CE) with electrospray ionization (ESI) in a single dynamic process within the same device. Reduction of flow with ESI serves to significantly improve ionization efficiency as well as reduce ion suppression. We will provide you with an upfront view of how this process works, as well as highlight its commercial embodiment as the CESI 8000 High Performance Separation – ESI Module. Our intent of this workshop is to demonstrate how CESI-MS has the potential to extend the reach of your mass spectrometer by preserving intact protein structure, increasing sensitivity and performance, and as we like to say, allow you to “See what you’ve been missing.”

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The Efficient and Sensitive Characterization of Intact Proteins of Pharmaceutical Interest by CESI-MS Govert W.Somsen, Rob Haselberg, Gerhardus J. de Jong Utrecht University, Utrecht, The Netherlands In biopharmaceutical analysis, there is a strong demand for methodologies that permit quality assessment of intact proteins. Mass spectrometry (MS) preceded by integrated capillary electrophoresis electrospray ionization (CESI) offers attractive possibilities for protein analysis. It provides the high selectivity and sensitive detection required to distinguish structurally related species. This presentation outlines the performance of CESI-MS for intact protein analysis. It will be shown that CESI presents a significant enhancement in signal-to-noise ratio, yielding protein detection limits in the sub-nM range. Use of dedicated capillary coatings ensures high protein resolution while maintaining full MS compatibility. The usefulness of CESI-MS for pharmaceutical protein applications will be demonstrated by the analysis of drug-protein conjugates, therapeutic glycoproteins, and monoclonal antibodies (mAbs). CESI-MS is shown to provide quantitative drug-protein and glycoform profiles, allowing detemination of conjugate stochiometries and specific assignment of more than 250 isoforms per protein in a single run. It is concluded that CESI represents a novel versatile tool for biopharmaceutical analysis. NOTES:

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Technical Seminar Wednesday, October 12, 2011

07:30 – 08:30 Talbot A-C Sponsored by Caliper Life Sciences LabChip GXII for High Throughput Analysis of Protein Quality in Bioprocess Development Bahram Fathollahi Caliper Life Sciences, Alameda, CA USA Protein therapeutics are typically large complex molecules that are often heterogeneous in molecular structures. The control and characterization of the product quality throughout the development process has been a major focus of the biotechnology industry. Design of Experiment (DOE) studies to test the effect of cell culture changes on post translational modifications of protein therapeutics or to monitor its purification process produces large number of samples that can greatly exceed the capacity of modern analytical laboratories. With increased samples comes the demand for high throughput analytical platform with high precision, automation, and ease-of-use. Although capillary electrophoresis SDS (CE-SDS) is now widely used in modern analytical laboratories in place of conventional SDS-PAGE for the analysis of monoclonal antibodies, there are still clear needs for further improvements in performance, reliability, and throughput. In the past several years, microfluidic-based assays for screening protein product quality are also finding wide use because they address the limitations of SDS-PAGE and CE-SDS. Caliper Life Sciences has developed an automated high-throughput integrated platform (LabChip GXII) that performs separation and quantification of denatured proteins and profiling of N-glycans with analysis time of 40 seconds per sample. In this presentation we will describe the use of Microchip-CE platform for a number of characterization assays including purity assessment of monoclonal antibodies under reducing and non-reducing conditions and glycan profiling. In addition, we will describe the development of a high throughput Microchip-CZE method for determination of protein charge heterogeneity. NOTES:

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Technical Seminar Wednesday, October 12, 2011

12:35 – 13:35 Talbot A-C Sponsored by ProteinSimple Get There, Faster with iCE Susan Darling; Jiaqi Wu 1ProteinSimple, Santa Clara, CA, 2ProteinSimple, Toronto, ON, Canada Proteins are complex, no way around it. They behave differently, come in a variety of shapes and sizes and some are linked to other molecules making analysis a challenge. For over 10 years iCE technology has helped scientists analyze difficult proteins and samples accelerating the development of biologics. Please join us for lunch and our latest advances in simplifying complex protein analytics. NOTES:

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Poster Abstracts

P-101 CE-SDS-LIF:FQ Labeled Antibodies. Comparative Study using a 488nm Laser and a Light Emitting Diode Audrey Boutonnet1; Pierre Naccache1; Jacques Fabre1; Bernard Feurer1; François Couderc2 1Picometrics, Toulouse, France; 2Université Paul Sabatier IMRCP, Toulouse, France Laser induced fluorescence (LIF) detection is routinely used as a powerful tool with Capillary Electrophoresis for the characterization and quantification of MAb’s. The use of 3-(2-furoyl)-quinoline-2 carboxaldehyde (FQ) as a labeling reagent of proteins, improves the sensitivity, sample preparation and the robustness of the quantification (Michels DA, Brady LJ, Guo A, Balland A. Anal Chem. 2007 Aug 1;79(15):5963-71. 2007 Jun 26). In fact, purification of labeled product is not necessary since FQ is a fluorogenic reagent which becomes fluorescent only upon reaction with a primary amine. A comparative study was performed with a 7100 Agilent Technologies CE and two light sources Fluorescence detectors, the Zetalif Discovery with a 488nm solid state laser and a new Zetalif detector with a Light Emitting Diode (LED). This recently developed detector employs an original optical arrangement to optimize the light beam and meet the usual LIF performance. We observed an extremely low detected concentration (LOD) with the 488nm solid state laser and blue LED. The LOD for minor species were determined from spiked contaminant of known concentration at 0.5%, 1%, 2%, 5%. P-102 Comparison Between Three Fluorescent Derivatization Methods of Proteins by CE-SDS with a Light-Emitting-Diode Induced Fluorescence (L(ed)IF) Detector Pierre Naccache1; Audrey Boutonnet1; Arnaud Morin1; Bernard Feurer1; François Couderc2 1Picometrics, Toulouse, France; 2Université Paul Sabatier IMRCP, Toulouse, France CE-SDS is an important separation technique in biopharmaceutical manufacturing and CE-SDS-LIF has become a popular method for characterization and quantification of MAb’s. Dyes such as 5-TAMRA.SE or FQ are widely used for labeling MAb’s for fluorescence detection and many different CE methods have been described in the literature. In this work, the Hunt & Nashabeh’s procedure (Anal Chem. 1999, 71, 2390-2397) was chosen using 5-TAMRA.SE, 3-(2-furoyl)-quinoline-2 carboxaldehyde (FQ) and Naphthalenedialdehyde (NDA) as derivatizing agents. The derivatives were detected at the wavelengths using appropriate Light-Emitting-Diodes for excitation and the sensitivity obtained from each derivative was compared.

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For this work, a Picometrics detector with a Light-Emitting-Diode (LED) was integrated into a Capillary Electrophoresis (CE) system from Agilent Technologies. The high divergent light beam of the LED was focused on the capillary using an original patented optical arrangement. CE L(ed)IF provides an extremely powerful and flexible tool for the analysis of therapeutic monoclonal antibodies in pharmaceutical quality control analysis. NOTES:

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P-103 Sensitive and Reliable Capillary Electrophoresis of Trace Metal Ions in a Saline Sample Kihwan Choi; Sunyoung Cho; Asif Riaz; Jihye Kim; Doo Soo Chung Seoul National University, Seoul, South Korea For the determination of trace metal ions with CE, chromophoric complexation is widely used since most metal ions are neither naturally absorbing UV/Vis nor fluorescent. However, for high-conductivity samples such as most physiological samples containing salts of high concentration, the performance of CE suffers from destacking of peaks. By applying transient isotachophoresis (TITP) using the salt anion (chloride) as a leading electrolyte (LE), we not only overcame the destacking problem but also enhanced the detection sensitivity further. With a dye, 4-(2-pyridylazo) resorcinol (PAR), the metal ions such as Ni2+, Fe2+, and Zn2+ in a saline sample were converted into anionic complexes having strong absorbance near 500 nm. A large volume of the metal-PAR complex sample solution injected into a capillary was stacked isotachophoretically and separated using PAR in the sample as a terminating electrolyte (TE). However, 4-(2-thiazolylazo) resorcinol (TAR) was used for metal ions including Cd2+, the mobility of TAR was higher than those of metal-TAR complexes and TAR could not be used as a TE. Thus, a long plug of the saline sample of metal ions, but devoid of TAR, was injected instead of pretreating the sample with TAR. Then a zone of isotachophoretically concentrated TAR was formed at the rear of the sample matrix and swept the metal cations as metal-TAR complexes, yielding highly efficient metal-TAR peaks. We also resolved the annoying problem of migration time changes in TITP due to uncontrolled concentration of LE. Calibration using multiple internal standards enabled us to predict the migration times to an accuracy of over 99.9% without conductivity normalizing or desalting of the sample. Our method of combining the dye complexation and TITP provided detection limits for trace metal ions in the low ppb range with absorbance detection, and was successfully applied to urine samples without pretreatment or desalting. NOTES:

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P-104 Applications of Electrophoretic Techniques for the Characterization of Therapeutic Biomolecules Suresh Babu Cugati Vishweshwaraiah1; Ravindra Gudihal1; Tobias Preckel2; Andreas Ruefer2; Christian Wenz2; Martin Greiner2 1Agilent Technologies, Bangalore, India; 2Agilent Technologies, Waldbronn, Germany The characterization of therapeutic proteins such as monoclonal antibody (mAb) during different stages of manufacturing is crucial for timely and successful product release. Regulatory agencies require a variety of analytical technologies for comprehensive and efficient protein analysis. Electrophoresis-based techniques and liquid chromatography (LC) either standalone or coupled to mass spectrometry (MS) are at the forefront for the in-depth analysis of protein purity, isoforms, stability, aggregation, posttranslational modifications, PEGylation, etc. In this presentation, a combination of various electrophoretic techniques such as liquid-phase isoelectric focusing, microfluidic and capillary-based electrophoresis (CE) and combinations of those with mass spectrometry techniques will be discussed. We present a workflow based approach to the analysis of therapeutic proteins. In successive steps critical parameters like purity, accurate mass, aggregation, peptide sequence, glycopeptide and glycan analysis are analyzed. In brief, the workflow involved proteolytic digestion of mAb for peptide mapping, N-Glycanase and chemical labeling reaction for mAb glycan analysis, liquid-phase isoelectric focusing for enrichment of charge variants followed by a very detailed analysis using state of the art methods such as CE-MS and LC-MS. For the analysis of glycans, we use combinations of CE-MS and LC-MS to highlight the sweet spots of these techniques. CE-MS is found to be more useful in analysis of highly sialylated glycans (charged glycans) while nano LC-MS seems to be better adapted for analysis of neutral glycans. These two techniques can be used to get complementary data to profile all the glycans present in a given protein. In addition, microfluidic electrophoresis was used as a QC tool in initial screening for product purity, analysis of papain digestion fragments of mAb, protein PEGylation products, etc. The described workflow involves multiple platforms, provides an end to end solution for comprehensive protein characterization and aims at reducing the total product development time. NOTES:

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P-105 Fully Automated Capillary-Based Western Analysis System Using On-Line Stacking as an Easy Tool for the Enhancement of Sensitivity and Resolution Irina Kazakova; Uyen Nguyen; Jessica Dermody; Francisco Ramirez; Tom Yang; Robert Gavin; Annegret Boge ProteinSimple, Santa Clara, CA USA Size-base characterization of proteins has been predominately performed by either Western analysis or by capillary electrophoresis (CE). Each technique has advantages – Western blotting exploits high sensitivity as well as specificity of antibody binding and CE offers high resolution and reproducibility. The reliability of the results obtained from either of these methods is often challenged by the ability to pre-concentrate or stack enough protein sample before the separation step. Simon, a new size-based separation platform that runs Simple Western assays, combines for the first time the advantages of both Western blotting and CE into a single automated workflow. The work presented illustrates the dependency of stacking efficiency upon separation lengths of matrices, protein identity and running conditions that results in more reproducible and reliable results. Assay optimization with the Simple Western is easy to perform and enables the user to achieve optimal detection and resolution of their target proteins. Stacking conditions can be optimized in a Simple Western assay to gain as much as a 10-fold increase in signal detection. Assay conditions for Simple Western Assays run on Simon have been optimized for the best resolution and linear, wide dynamic range of detection. P-106 Size and Charge Based Analysis of ERK1/2 and 4-EBP1 Using Automated Capillary-Based Systems for Nanoscale Protein Analysis Uyen Nguyen; Jessica Dermody; Francisco Ramirez; Irina Kazakova; Tom Yang; Bob Gavin; Annegret Boge ProteinSimple, Santa Clara, CA USA Aberrant expression and signaling in the EGF signaling cascade is a common occurrence in a variety of cancers including breast cancer. Understanding how EGF signaling impacts disease progression is key to the development of novel therapeutics. Analysis of ERK1/2 and 4E-BP1 expression in cancer samples frequently employs Western blot analysis. However, in order to fully understand signaling events, changes in signaling proteins are evaluated utilizing size-based as well as charge-based separation techniques, each of which is followed by immunoassay detection. The Simon Simple Western System performs size-based separation of proteins in nanoscale volumes. NanoPro systems utilize charge-based separation in capillaries via isoelectric focusing (cIEF). Using Capillary Electrophoresis (CE) modes coupled to an automated workflow, Simon and NanoPro

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instruments, eliminate the need for manual processing of multiple steps. Proteins from a single sample preparation were analyzed on two platforms: either in a native conformation using charge-based separation (NanoPro) or in a denatured state followed by size-based separation (Simon). Utilizing both platforms allowed for detailed analysis of post-translational modifications and protein abundance within a prepared sample. As a validation of synergy between the platforms, we investigated key members of the EGFR signaling cascade in different phosphorylation states. We showed that the same cellular lysate preparation can be used for charge or size-based separation. Advantages of the Simple Western assay over Western blot assays include ease of use, minimal user intervention, automatic analysis, and excellent reproducibility. Simon and Simple Westerns allow for the first time to run a fully automated Western workflow, resulting in significant time savings and improved reproducibility. NanoPro delivers -rich data sets with exquisite sensitivity that allows an in-depth look at post-translational modifications of small protein samples. In combination, data from both platforms correlate very well with each other as well as with literature while providing synergistic information. NOTES:

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P-107 Comparison of Western Blot Analysis with a Fully Automated Capillary Electrophoresis System for Size-based Analysis of the AKT Signaling Cascade Francisco Ramirez; Uyen Nguyen; Anna Johnson; Irina Kazakova; Jessica Dermody; Tom Yang; Robert Gavin; Annegret Boge ProteinSimple, Santa Clara, CA USA Protein kinase AKT is a central player for a variety of cell stimuli affecting downstream events such as the inhibition of apoptosis, regulation of glycogen synthesis, and cell cycle regulation. Many of these events are routinely assessed by Western blot analysis which exploits high sensitivity of enzyme amplification as well as specificity of antibody binding. The process itself, however, is very labor intensive and has not significantly changed since it was first introduced. Simon, a new system from ProteinSimple, runs Simple Western assays. Simple Westerns are a complete reinvention of the Western blot. We present data comparing analysis of key targets of the AKT signaling cascade via Western blot and Simple Western on Simon, highlighting work flow, biological response, sensitivity, and resolution. Conclusions Simple Western assays run on Simon are a fully automated reinvention of the Western blot including all the manual steps associated with the process. Simple Westerns generate highly reproducible, quantitative, reliable and sensitive assessment of key targets in the AKT signaling cascade. Simple Westerns streamline the total time to results to about 3-5 hours in a walk-away mode. P-108 Characterization of a Charge Heterogeneity Profile for a Complex Glycoprotein Alan Akiyama; Chi-Ting Huang Acceleron Pharma, Inc., Cambridge, MA USA Receptor extracellular domain-Fc fusion protein biotherapeutics represent a growing class of biotherapeutics that have been developed to modulate the growth of red blood cells, bone, muscle, fat and the vasculature at Acceleron Pharma. These highly glycosylated Fc-fusion proteins contain multiple sources of charge heterogeneity ranging from variations in the protein backbone to differences in glycosylation and sialylation. In addition, cell culture and harvesting conditions can have an effect on the glycan isoforms during processing and can be detected by capillary isoelectric focusing. In order to characterize the changes in the Fc-fusion protein that result in cIEF profile changes, an Acceleron Fc-fusion protein was fractionated using the Agilent OFFGEL 3100 system and the resulting fractions were characterized by MS.

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P-109 Optimized Gel Buffer for CE-SDS Analysis of Complex Glycoproteins Heather Jean; Jennifer Manfra; Kannappan Ragavan Pfizer, Inc., Andover, MA USA Complex glycoproteins, when analyzed by CE-SDS kits, often result in change in the mobility causing inaccurate molecular weight determinations when compared to low level glycosylated protein standards. This may be due to the inability of SDS to bind complex glycoproteins in the sample buffer at a typical SDS:protein ratio of 1.4:1g. This less optimum SDS:protein binding ratio decreases the charge to mass ratio required for optimum separation (retention times), which result in inaccurate molecular weight determinations. Shifted retention times and the resultant inaccurate molecular weight determination can lead to comigration of process related impurities causing the quantitation of impurities difficult for releasing products. To minimize these retention time shifts, several gel buffers were formulated containing increased SDS and polymer concentrations. An optimal gel buffer was chosen and then tested on several glycoproteins to determine if a more representative molecular weight determination could be achieved. P-110 A High-Throughput Platform for Preparation of APTS-Labeled N-Glycans: Improving the Accuracy, Reproducibility and Time-to-Results of N-Glycan Profiling Michael Kimzey1; Shiva Pourkaveh1; Steven Mast1; Bopha Sun1; Abdel Minalla1; Ted Haxo1; Craig Nishida1; Barry Karger2; Andras Guttman2; Zoltan Szabo2; Jeff Chapman3; Jo Wegstein1 1ProZyme, Inc., Hayward, CA USA; 2Northeastern University, The Barnett Institute, Boston, MA USA; 3Beckman Coulter, Inc., Brea, CA USA Analysis of APTS-labeled glycan conjugates using capillary electrophoresis (CE) is a sensitive, robust, and fast approach to characterize N-linked sugar structures. However, this method has not been widely implemented in screening applications, due in part to the lack of commercially available tools for automated sample preparation and analysis. Here we present an overview of a glycoanalysis platform comprising rapid sample preparation coupled with CE-based glycan analysis including: 1) an automatable format with an optional purification module to allow direct screening of monoclonal antibody (MAb), cell-culture samples; 2) quantitative deglycosylation and separation of N-glycans; 3) complete glycan labeling for laser-induced fluorescent (LIF) detection; 4) efficient glycan sample cleanup and desalting to reduce excess reagent peaks; and 5) N-glycan profiling and glucose unit (GU) value-based structural prediction with a high degree of accuracy. The platform enables the generation of up to 96, high-quality results overnight. The GlykoPrep® N-Glycan Sample Preparation protocol features optional purification of MAbs, enzymatic deglycosylation, APTS labeling, cleanup, and standardization of N-glycans optimized for downstream CE-LIF analysis. Using proprietary reagents, the optimized labeling reagents provide complete derivatization with only one-hour incubation time, without degradation of important labile groups, such as sialic acid and core or outer-arm fucose. After labeling, the remaining free APTS is

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efficiently (>99.9 %) removed and the sample eluted in water, enabling the potential for mass spectrometry confirmation of atypical peaks. Spike-in, lower and upper internal mobility standards are used to normalize glycan migration times further reducing variability between runs; GU assignment based on normalized glycan migration times showed significant improvement in precision compared to GU assignment without use of the internal mobility standards. It is envisioned that this unique glycoanalysis solution will be important for the standardization and expansion of N-glycan profiling in such applications as clone selection and cell-culture optimization. NOTES:

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P-111 Measuring Charge Heterogeneity Profiles of Antibody-Maytansinoid Conjugates Pui-King Amy Leung; Megan Ellis; Karan Shah; Joyce Lin; Xuan Chen; Michael Fleming; Alex Lazar; Godfrey Amphlett ImmunoGen, Inc, Waltham, MA USA A number of antibody-maytansinoid conjugates (AMCs) are in clinical testing that consist of a highly potent maytansinoid cancer cell killing agent attached to the antibody component via a linker that forms an amide bond with a lysine ε-amino group. Each maytansinoid so linked eliminates a basic site on the antibody. Thus, the isoelectric point (pI) of the antibody decreases each time a lysine residue is modified. Different antibody molecules in a conjugate lot are modified to different, but defined and consistent, extents. This defined distribution of conjugated species is expected to have a corresponding distribution of differently charged species, with AMCs having lower pI values likely corresponding to species with increasing levels of conjugation. In several AMCs, the various charged species observed by cIEF are consistent with the different numbers of maytansinoid molecules attached to the antibody, resulting in profiles similar to those observed by mass spectrometry. Charge heterogeneity of AMCs can be complicated by the charge heterogeneity profile of the unconjugated antibody (due to the presence of variants such as C-terminal Lys cleavage, N-terminal pyro-Glu and deamidation). For several AMCs the peaks of differently charged isoforms are only partially resolved. One complication is that attachment of the hydrophobic maytansinoid molecules, combined with the hydrophobic properties of the antibody, can result in precipitation of the conjugated species during analysis. The presentation will provide examples of AMCs with different behaviors during charge heterogeneity measurement. P-113 Heterogeneity of Therapeutic Albumin Highlighted by Capillary Electrophoresis Coupled to Mass Spectrometry. Anne-Lise Marie1; Cédric Przybylski2; Florence Gonnet2; Régis Daniel2; Rémi Urbain3; Sylvie Jorieux3; Myriam Taverna1 1University of Paris, Châtenay-Malabry, France; 2University of Evry-Val d’Essonne, Evry, France; 3LFB, Les Ulis, France Human Serum Albumin (HSA) is the most abundant plasma protein and an important circulating carrier of endogenous and exogenous ligands in the blood. Endogenous HSA contributes to maintain the osmotic pressure; as a very abundant and important circulating antioxidant, it provides a permanent preventive protection. Clinically, HSA is used to restore and maintain blood volume in hypovolemic situation when colloid administration is appropriate. Due to its bioprocess, storage conditions and natural sources, therapeutic albumin, obtained by fractionation of donors’ plasma, is heterogeneous. It has been shown that HSA undergoes many chemical or physical alterations (aggregation, dimerization,

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oxidation, cysteinylation), which could highly impact on the quality of the HSA preparation and thereby on therapeutic activity (elimination rate, antioxidant properties, stability…). Recently, the LPNSS in collaboration with the LFB has developed an analytical method based on capillary electrophoresis and a semi-permanent polyethylene oxide coating allowing for the separation and quantification of 9 different forms of HSA (including native and oxidized forms). In the present work, three therapeutic albumins, including Vialebex marketed by LFB, have been compared. Vialebex was shown to contain the highest proportion of native form compared with competitive albumin preparations. In contrast, competitors’ preparations contained more oxidized forms than any other form and a higher proportion than Vialebex. To definitely identify the different forms found in the therapeutic albumin and the differences already pointed out between different therapeutic albumins, a coupling of capillary electrophoresis with mass spectrometry (CE-MS) was carried out. This coupling was applied to identify each different form of HSA existing in the pharmaceutical preparations (native, oxidized, cysteinylated, nitrosylated, glycated, truncated forms) and to determine more precisely the differences in composition between the albumin preparations. Key words: human serum albumin, capillary electrophoresis, mass spectrometry NOTES:

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P-115 Aglycosylated Antibody Effects on PK Half-Life: A Case Study of huIgG1 and huIgG2 Limary Medina Abbott Bioresearch Center, Worcester, MA USA Antibody glycosylation is a key post-translational modification that has been shown to contribute to Fc effector function including complement activation, FcRn interaction and in in-vitro/vivo stability. However, the effects of glycosylation on serum half-life remain controversial. Several observations suggest there is little difference between point mutated (N297A) and glycosylated IgG1 antibodies with respect to FcRn binding and PK phenotypes (1). Other studies indicate that various oligosaccharides or glycan subtypes directly affect the serum half-life of IgGs (2). Purification expression systems such as HEK-293 and CHO influence antibody glycosylation states. In observation, relative shorter PK half-life has been found using HEK293-derived huIgG1 in contrast to the same antibody produced in CHO. Understanding the effects of cell culture conditions, origin of production and glycosylation on in-vivo stability becomes essential. Since lead antibodies are generated in HEK-293 during early rat/cyno PK studies, we need to investigate the impact of aglycosylated antibody, as it may reduce PK half-life. Additionally, we need to find out if controlling HEK-293 culture conditions would reduce aglycosylated antibody and minimize glycosylation heterogeneity. Here we present a comparative study of huIgG1 and huIgG2 derived from HEK293 and CHO. Various analytical tools were utilized during characterization to obtain information on aglycosylated antibody level, oligosaccharide profiles, antibody heterogeneity and aglycosylated peptides. In addition, we studied the impact on rat PK, using antibody mixtures at various deglycolsylation percentages. Our results confirm that aglycosylated and deglycosylated IgG1 and IgG2 are associated with shorter rat PK half-life and higher clearance. Due to the importance of these findings, the quantitation of aglycosylated antibody differences is therefore imperative prior to antibody PK studies. 1) PNAS, December 23, 2008, Vol. 105, No. 51, p. 20167–20172 2) Current Opinion in Immunology 2008, 20:471–478 P-116 Identification of CE-SDS Peaks by Different Spiking Approaches and Mass Spectrometry Bernd Moritz; Andrea Heyne; Anja Bathke; Joerg Hoernschemeyer 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). In order to enable 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.

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Peak identification for reduced and non-reduced CE-SDS separations of IgG1 antibodies has been performed by different approaches. Partial reduction of IgG1 material by DTT resulted in possible combinations of light and heavy chains, which matched well with the peak profile of typical CE-SDS separations. Spiking with N-Glycosidase F treated mAb allowed identification of the deglycosylated IgG1 monomer and the deglycosylated heavy chain. Fab/c and Fab fragments were obtained by fractionation of SE-HPLC separations or Papain digestion. By spiking experiments CE-SDS peaks of the Fab/c, Fab and HC parts thereof could be identified. These fragments were also characterized by mass spectrometry (MS) and were compared with the apparent CE-SDS masses. By this approach a high number of CE-SDS peaks were successfully identified in the electrophero-gram and annotated with the underlying molecular structures. NOTES:

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P-117 Rapid Sample Preparation of Biologics to Support High-throughput/High-resolution Glycan Analysis by Capillary Electrophoresis Andras Guttman1; Zoltan Szabo1; Barry Karger1; Jeff Chapman2; Michael Kimzey3; Ted Haxo3; Jo Wegstein3 1Northeastern University, The Barnett Institute, Boston, MA USA; 2Beckman Coulter, Inc., Brea, CA USA; 3ProZyme, Inc, Hayward, CA USA Structural characterization of the glycan moieties of biologics, especially recombinant monoclonal antibody (rMAb) therapeutics, is critical during clone selection, cell-culture optimization and product characterization. High-throughput screening methods are required in order to return results promptly to allow multiple iterations for selection of optimal candidates. This poster gives an overview of a state-of-the-art screening protocol with rapid sample preparation coupled with capillary electrophoresis (CE)-based glycan analysis that includes: automatable sample preparation with optional purification modules to allow direct screening of cell-culture samples; glycan labeling for laser-induced fluorescent (LIF) detection; clean-up and desalting to reduce excess reagent peaks; and oligosaccharide profiling, carbohydrate sequencing and glucose unit (GU) value-based structural prediction. Glycans released from subnanomolar amounts of rMAbs are quickly and accurately prepared and analyzed, e.g., rapid (<7 min) CE separation of twelve, key, IgG glycans can be obtained along with 96-well format operation for convenient overnight processing. Particular attention is paid to full separation of corefucosylated and afucosylated forms, as the presence or absence of this modification is of high importance in regulating the effector function of rMAbs. The excellent relative migration time reproducibility of the optimized CE separation method (RSD <0.09%) facilitates high-fidelity peak assignments for the individual components in the glycan pools, and consequently allows high-precision, structural predictions using GU-value databases. Exoglycosidase, array-based, sequence verification of the predicted glycans is also presented. P-118 Use of Quality by Design in Developing a Capillary Electrophoresis Method to Replace Traditional SDS-PAGE for Product Purity Determination Emily Parker; Claire Davies Genzyme, a Sanofi Company, Framingham, MA USA Traditional SDS-PAGE with Coomassie staining has known limitations such as low throughput, low sensitivity and poor resolution. Capillary electrophoresis offers improvements in these areas and is therefore being investigated as an alternative to SDS-PAGE. As the use of CE has grown, several types of CE-SDS have emerged. Microchip CE-SDS has more recently begun to gain popularity as a higher throughput method than standard CE-SDS. This presentation will focus on standard CE-SDS (Beckman PA 800) and Microchip CE-SDS (Caliper GXII) as potential alternatives for the current SDS-PAGE release method.

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Using Quality by Design (QbD), a gap analysis was performed and critical quality attributes were identified and assessed. Side by side experiments were performed using standard CE-SDS and Microchip CE-SDS to directly compare the two techniques in terms of their robustness, sensitivity, resolution, and reproducibility. NOTES:

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P-119 Development of a High Throughput Capillary Electrophoresis-based Method to Rapidly Evaluate the PK Properties of Candidate Molecules Early in Discovery. Reema Piparia; Ivan Correia Abbott Bioresearch Center, Worcester, MA USA Identification of high throughput technologies that facilitate rapid screening of a large pool of candidate molecules in serum with good pharmacokinetic (PK) properties are highly desirable. The LabChip GXII is a high throughput capillary electrophoresis (CE) based system that will analyze a protein sample in 40 seconds directly from a 96 well plate. The GXII has a sizing range from 14 to 200 kDa and a 4 log linear range of detection (50 pg/mL to 100 ng/mL) for pre-labeled proteins. Labeling of the protein is carried out using the Pico Protein dye provided by the manufacturer, followed by quenching and buffer exchange. In this study we used the LabChip GXII to analyze the PK properties of a pre-labeled monoclonal antibody (mAb) in rat and compared it to the PK of the unlabeled molecule. Following IV administration, the non-labeled mAb displayed low clearance (0.2 mL/h/kg), small volumes of distribution (106 mL/kg) and a half-life of 15 days. Analysis on the GXII revealed that the pre-labeled mAb showed comparable PK properties with clearance of 0.16 mL/h/kg, volumes of distribution of 112.5 mL/kg and half-life of approximately 20 days. Furthermore, the CE profile illustrates that the mAb was highly resistant to fragmentation and aggregation when in circulation. In summary, this data demonstrates that labeling with the fluorescent dye did not alter the clearance of this particular mAb, pharmacokinetic parameters were comparable for both labeled and unlabeled molecules. In a follow up experiment, mAb dimer was created by photo-induced cross-linking of unmodified proteins (PICUP) and labeled with the Pico Protein dye. The labeled dimer was found to have similar PK profile to labeled monomer. This high throughput method could be used to quickly assess the PK properties of multiple antibodies and evaluate PK properties of mAb aggregates or fragments. P-120 Detecting and Quantifying the Storage of D-Serine and Other Signaling Molecules in Astrocytic Synaptic-like Vesicles Using Two Different CE Platforms Ting Shi1; Magalie Martineau2; Ann M. Knolhoff1; Jean-Pierre Mothet3; Jonathan V. Sweedler1 1University of Illinois at Urbana-Champaign, Urbana, IL USA; 2University of Münster, Münster, Germany; 3Universite Aix-Marseille, Marseille, France Major receptors in our brains are perhaps the most promising targets for pharmaceutical intervention related to mental illnesses. The N-methyl-D-aspartate receptor (NMDAr) is important in learning and memory and excess stimulation of the NMDAr is responsible for excitotoxicity in neurodegenerative disorders, such as Parkinson’s and Alzheimer’s disease. Thus, interest in the NMDAr is increasing in the pharmaceutical industry. Nowadays, D-serine (D-Ser) is recognized as a key brain messenger that gates

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the function of the NMDAr; then, D-Ser and D-Ser related molecules can be developed as novel therapeutic agents for these challenging central nervous system diseases. Elucidation of the molecular mechanisms of the release and uptake of D-Ser will contribute to the rationale development of novel therapeutics related to this signaling pathway. Here, a specific basic question is addressed; does D-Ser released from glia or neurons originate from a cytosolic pool or from secretory organelles. We answer this question by testing whether D-Ser and other amino acids are found within immunopurified astrocytic vesicles. Capillary electrophoresis with laser induced fluorescence (CE-LIF) was used to analyze the purified synaptic-like vesicles from astrocytes. Cyclodextrin-modified micellar electrokinetic chromatography (MEKC) was employed in chiral separation and the separation buffer components were optimized to achieve baseline resolution of serine enantiomers. D-Ser, along with other signaling molecules, was detected and quantified using three methods on two CE platforms, CE-LIF and capillary electrophoresis-mass spectrometry (CE-MS). One chiral and two non-chiral separation methods together greatly enhance the reliability of our quantification. Our results agree with the hypothesis of D-Ser vesicular release; the intracellular concentration of both D-Ser and glutamate is estimated to be around 20 mM, within the expected range of vesicular concentration for a signaling molecule. NOTES:

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P-121 Capillary Electrophoretic Mobility Shift Assay for Binding of DNA with NFAT3, a Transcription Factor from H9c2 Cardiac Myoblast cells Soo Hyun Park1, 2; Eunmi Ban1; Young Sook Yoo1; Hyunjung Lee1; Doo Soo Chung2; Eun Joo Song1 1KIST, Seoul, South Korea; 2Seoul National University, Seoul, South Korea Nuclear factor of activated T-cells (NFAT) is a transcription factor involved in the development of cardiac and skeletal muscle and the nervous system. NFAT is activated by calcium signal pathway and translocated into the nucleus. The quantification of binding between NFAT and NFAT-specific DNA gives important information about cardiac hypertrophy. We investigated the binding of NFAT3 in nuclear extracts from H9c2 cells to its specific DNA by capillary electrophoretic mobility shift assay (CEMSA). The binding reaction time required for stable formation of the DNA-NFAT3 complex was 3 h and the separation of the complex and free DNA was achieved within 10 min by CE. The formation of NFAT3-DNA complex was confirmed by the competitive reaction. Comparison of the ratios of complex/free DNA peak area for 1 μM endothelin-1 (ET-1) treated cells and control cells showed the NFAT3 translocation into the nucleus promoted by ET-1. The binding constant between NFAT3 and DNA was estimated to be 7.7 × 109 M–1 at 4°C. [This research is funded by the Korea MEST NRF grant (20110002143) and the Korea Institute of Science and Technology (2E22260)] P-122 Performance Characteristics of Commercially Available Gels for Protein Analysis by Capillary Gel Electrophoresis with UV Detection Christian Wenz; Martin Greiner Agilent Technologies, Waldbronn, Germany Capillary gel electrophoresis is a widely used tool for the size-based analysis of proteins. Due to several advantages with regard to automation, reproducibility and resolution it has replaced the classical technique sodium dodecyl sulfate-polyacrylamide gel electrophoresis in many labs, especially in the biotechnology industry. Capillary gel electrophoresis it is now routinely used in the quality control environment to assess purity and integrity of therapeutic proteins including monoclonal antibody. Commonly commercially available dextran-based separation matrices are used for these separations. Here, two separation matrices for protein characterization by capillary gel electrophoresis with UV detection, the SDS Gel Buffer from Beckman Coulter and the recently introduced Protigels from Advanced Analytical, were compared in detail on the Agilent 7100 Capillary Electrophoresis system. Two different sample sets were analyzed: first, a protein size standard and BSA as a test protein for molecular weight determination; second, a reduced antibody standard for quantification of light chain, non-glycosylated heavy chain and heavy chain. To get an estimate for batch-to-batch variability, all experiments were performed with two different gel and capillary batches, respectively. Across these experiments, the gels from both suppliers showed a similar performance. Furthermore, impurity detection experiments were done with a low molecular weight protein spiked into a non-reduced antibody standard sample. With the gels of both suppliers it was possible to detect the low molecular weight protein down to a level of 0.1 %.

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P-123 Development of a Simplified Nanospray Sheath Flow CE-MS Interface for Novel Approaches to Proteomic Analysis Roza Wojcik; Norman Dovichi University of Notre Dame, South Bend, IN USA Nanospray CE-MS interface developed in our lab retains versatility of a traditional sheath flow interface but is simpler in operation, does not result in significant sample dilution and operates in nanospray regime. Fast peptide separations with ~2 minute separation time window allow for comprehensive protein identification in low complexity samples with low attomole detection limits while coupled with LTQ Obitrap Velos mass spectrometer. The interface retains CE separation efficiency, producing sub second peak widths and over 200 thousand theoretical plates for amino acid and peptide analysis. The interface is also capable of supporting two dimensional CE separations. Diagonal two dimensional CE incorporating online alkaline phosphatase microreactor is a fully automated targetted approach for phosphopeptide analysis. The proof of principle fo this technique, coupled with LIF and MS detection was demonstrated on peptide and protein standards. P-124 Evaluation of the dPC Fractionator for Peak Identification in the iCE IEF Analyzer Jiaqi Wu ProteinSimple, Toronto, Canada ProteinSimple’s iCE IEF Analyzer is the best tool for protein charge heterogeneity characterization and has been widely used as the platform method in innovative biotech pharmaceutical companies, including all the largest in the world. The iCE has higher resolution and throughput compared to ion exchange chromatography; however, due to its narrow capillary column, collecting fractions of the separated peaks from the column for further peak identification is impossible. One option for peak identification is using off line fractionators that fractionate protein samples based on isoelectric point (pI). Part of the collected fractions can be re-injected into the iCE for confirmation. The balance of the fractions will be analyzed by mass spectrometers (MS) for identification after the confirmation. ProteinSimple’s dPC fractionator is a fraction collector based on sample pIs. However, the fraction amounts obtained from the current format of the dPC are too small for the iCE application, as the quantity collected of each fraction is limited by the IPG gel plug volume on the dPC chips, which is about 1 uL. In order to use the dPC for iCE peak identification, its chips are modified from pH gradient format into single pH format. In this way, the fraction volume on the chip is increased by 40 times; in

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addition, the catholyte and anolyte can also collect fractions. The fraction amounts in the modified dPC are big enough for re-injection into the iCE and MS. NOTES:

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P-125 Enhancing Peak Pattern Stability and Reproducibility in Capillary Isoelectric Focusing (cIEF) by Plugging the Capillary Column during the Isoelectric Focusing Jiaqi Wu ProteinSimple, Toronto, Canada In isoelectric focusing (IEF), at the end of the focusing process all components in a sample are focused and stop at their pI points. In order to perform IEF in a capillary column (cIEF), two forces within the column that interfere with the focusing process have to be eliminated: electroosmotic flow (EOF) and hydrodynamic flow. In the commercial cIEF instruments, the EOF is substantially reduced by column coatings and the hydrodynamic flow is eliminated by placing the both ends of the column at the same level. In ProteinSimple’s iCE IEF Analyzer, the hydrodynamic flow within the separation column is eliminated by using a specially structured, constant fluid level waste vial at the outlet of the column and a balancing vial at the column inlet in the autosampler that has the same fluid level as the waste vial. This design constantly provides equal fluid levels at both ends of the column regardless of the waste volume deposited into the waste vial. However, some high concentration additives can generate an unbalancing force within the column during IEF for some unknown reasons. One example is high concentration urea. When these high concentration additives are used, the peak pattern sometimes is pushed back or forth within the separation column making the peak pattern unstable, which can reduce the separation resolution and reproducibility. We determined that this problem can be solved by plugging one end of the column during the IEF process. This plugging is accomplished by a switch valve in the iCE, which is incorporated in ProteinSimple’s new model iCE3 instrument. In this presentation, we will compare peak pattern stability in the iCE3 and the old iCE when 8 M urea is used as the additive. NOTES:

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P-126 Capillary Isoelectric Focusing (cIEF) Analysis of Monoclonal Antibodies under a Denatured and Reduced Condition Jiaqi Wu ProteinSimple, Toronto, Canada Capillary isoelectric focusing (cIEF) is the best tool for protein charge heterogeneity characterization. Common sources of charge heterogeneity for monoclonal antibodies include heavy chain C-terminal lysine heterogeneity, deamidation, and sialyation associated with the glycosylation sites on the antibodies. Under the denatured and reduced condition, disulfide bonds in the antibodies can be reduced and the monoclonal antibodies are broken into heavy chains and light chains. The heavy chain and light chain are expected to have different pI values, thus, they can be separated from each other by IEF. In this way, the contributions of the heavy chain and light chain to the entire antibodies’ charge heterogeneity can be observed by IEF analysis. In this presentation, monoclonal antibodies are analyzed by cIEF under 8 M urea (denatured) and DTT condition. In the example shown in the presentation, the heavy chain and light chain are well separated and the charge heterogeneity of both is characterized. P-127 SDS-PEG Binding Affected the Linearity Range of the CE-SDS Method for Pegylated Proteins Jian Zhang PPD, Inc., Middleton, WI USA During qualification of a CE-SDS method wtih electrokinetic injection for a pegylated protein (12 kD protein with a 40 kD covalently linked PEG), it is observed that the normalized peak area of the pegylated protein plateaus above approximately 0.7 mg/mL. It is known that the SDS binds to the PEG and affects the charge and mobility of the pegylated protein molecule. Since the PEG presents as the majority of the pegylated protein molecule, it is likely to have dominated the charge profile of the molecule and consumed all SDS in the sample matrix. With the SDS being limited, as the protein concentration increases, the charge per protein molecule (and therefore the mobility of the protein) decreases. Therefore, the injection amount under electrokinetic injection mode remains constant. By increasing the SDS concentration in the final sample matrix from 0.5% to 1.5%, the linearity range was expanded to 1.5 mg/mL. Based on the concentration where the linearity curve turns plateau, it was estimated that the SDS binds to the PEG at 4-6 PEG units per SDS ratio. NOTES:

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P-128 Evaluation and Improvement of SDS Capillary Gel Electrophoresis Method for Early Phase Formulation Study of Monoclonal Antibody Pipelines Lin Zhu; Tim Blanc; Tara Enda; Joseph Liu; Joel Goldstein; Qinwei Zhou ImClone Systems, A wholly owned subsidiary of Eli Lilly, Branchburg, NJ USA Sodium Dodecyl Sulfate - Capillary Gel Electrophoresis (SDS-CGE) has been widely used to replace the traditional SDS-PAGE method for quantitative analysis of purity for monoclonal antibodies in the biopharmaceutical industry. An optimized electrokinetic sample-injection technique as an important step of the SDS-CGE method is recommended for monoclonal antibody analysis using the Beckman PA 800, as a result of a series of collaborative studies in which various pharmaceutical companies and regulatory authorities participated. However, when applied to formulation study samples, this method demonstrates an intrinsic problem associated with electrokinetic injection diminishing the reliability of the SDS-CGE method. High variance of the sample injection amount was found to be dependent not only on the concentration of the IgG sample, but also on the ionic strength and conductivity of the sample solution. This issue is most acute since most formulation study samples are formulated with different pH buffers and different excipients. In this presentation, a hydrodynamic sample injection method has been used to support various formulation studies. The results demonstrate a variety of advantages over the electrokinetic injection method that includes no loss of resolution or sensitivity. Additional improvements in SDS buffer preparation and sample treatment under non-reduced or reduced conditions has also demonstrates the method to be simpler, faster, reliable and reproducible. Currently, this method has been used to support early formulation studies for pH selection, excipients selection, thermo and photo stress studies and comparability studies. The potential to become a fully automated SDS-CGE method will be discussed. P-129 Conductivity Studies for Improved Sequence Coverage in Peptide Mapping of Lysozyme Jelynn A. Stinson Wright State University, Dayton, OH USA In this study, a capillary zone electrophoresis approach is used for peptide mapping of lysozyme. To determine the effects of conductivity on peptide identification and protein sequence coverage, ionic strength of running and sample buffers, as well as sample plug length is varied. All studies are carried out in an ammonium acetate buffer system. To measure the effects of conductivity variation upon improving mapping outcomes, the capillary electrophoresis instrument is equipped with a UV detector for monitoring peptide migration, and coupled to a high resolution time-of-flight mass spectrometer through electrospray ionization for determining peptide identification at a given moment of elution. States of optimum conductivity are accompanied by improved resolution and separation in the capillary electropherogram. This provides the highest peak production rate and consequently results in higher sequence coverage for lysozyme.

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P-130 Separation of Fucosylated, non-Fucosylated, and Complex Carbohydrates Associated with Monoclonal Antibodies using Capillary Electrophoresis Sushma Rampal1; Lynn Gennaro2; Mark Lies1

1Beckman Coulter Inc., Brea, CA USA, 2Genentech, A Member of the Roche Group, South San Francisco, CA USA In order to gain a comprehensive understanding of therapeutic Monoclonal Antibody (MAb) function, it is necessary to critically characterize glycosylation associated with them. Carbohydrates are known to play an important role in the structure, function, and clearance of MAbs and have been shown to be responsible for invoking immune responses in humans. Changes in carbohydrate composition or concentration can significantly impact the overall efficacy of therapeutic MAbs and can also lead to side effects. Because of their link to antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC), accurate analysis of oligosaccharide fucosylation, sialylation, and antennary structure is critical for a complete understanding of MAb microheterogeneity. Capillary electrophoresis (CE) technology has been successfully used to separate major IgG N-linked oligosaccharides G0, G1, and G2 structures from one another. The basis for this separation relies on electrophoresis of oligosaccharides labeled with amino pyrene tri-sulfonic acid (APTS). The complexity of glycans associated with many molecules calls for high resolution separation in order to assess heterogeneity among carbohydrate isomers and co-migrating carbohydrate species. Since CE is already an established technology for automated and quantitative analysis of N-linked oligosaccharides, we set out to develop methodology by which fucosylated, afucosylated, sialylated and complex antennary oligosaccharides can be differentiated from one another. Optimization of chemistry and CE methods enabled separation of these species in a simple, reproducible assay environment. P-131 A Platform cIEF Method for Monoclonal Antibody Charge Heterogeneity Analysis Scott Mack; Ingrid D. Cruzado-Park; Mark Lies; Chitra K. Ratnayake Beckman Coulter, Inc., Brea, CA USA The use of cIEF for the characterization of therapeutic monoclonal antibodies (MAbs) has been increasingly adopted in recent years. The pI determination adds a critical dimension to establishing identity, purity, post-translational modification and stability of therapeutic MAb preparations. Most MAbs have charge isoforms with a pI in the basic range of the pH gradient (7-10). The analysis of basic compounds presents the greatest challenge in cIEF due to the inadequate focusing and buffering nature of ampholytes that comprise the basic region. These obstacles can be overcome by the incorporation of cathodic and anodic stabilizers in the cIEF sample. The optimization of stabilizer solution volumes, focusing times and ampholyte concentration was critical for the development of a single universal cIEF method with high reproducibility in the basic pH range. This poster describes the universal method and summarizes the results of an inter-day reproducibility study carried out with three unique MAbs during six days using two instruments and two lots of neutral-coated capillaries. The results obtained demonstrate the high reproducibility and robustness of this universal cIEF method.

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P-132 Analysis of Fentanyl and Its Metabolite, Norfentanyl by Capillary Electrophoresis Coupled to Mass Spectrometry with the CESI 8000 and Opti-MS Technology John C. Hudson; Mark Lies Beckman Coulter Inc., Brea, CA, USA A sheathless electrospray interface for capillary electrophoresis-mass spectrometry (CE-MS), currently in development, was evaluated in a forensic application requiring high sensitivity. Fentanyl, a potent analgesic and recreational drug, is commonly administered at low dosages, 25 micrograms/patch, resulting in very low therapeutic levels of 0.3 to 1.2 ng/mL of serum after 24 hours (1). There is a need in forensic casework to detect and quantify low levels of fentanyl after extended periods of time between administration of the drug and collection of the sample for analysis. These challenging types of cases include Drug Facilitated Sexual Assault (DFSA) and Driving Under the Influence of Drugs (DUID) where there may be a substantial delay in the assault being reported or samples from an impaired driver being obtained. In this work, fentanyl along with its main metabolite, norfentanyl, were analyzed in extracts of spiked serum. Samples were externally prepared along with blind controls and analyzed in duplicate in an automated process. The results for both fentanyl and norfentanyl showed excellent linearity (R >0.995) over a range of 0.1 to 50 ng/mL of serum with LOD and LOQ values of 0.1 ng/mL. Analysis of controls prepared by an external agency, were within acceptable precision over the whole concentration range. The results confirm that the interface is capable of providing the necessary ionization efficiency, robustness and precision for a challenging, qualitative and quantitative forensic drug analysis. Very low sample volumes (6 nL in this work) are required compared to existing technology such as LC-MS, making CE-MS using this sheathless interface, virtually a non-destructive sampling technique. This is significant in forensic cases because analysis can then be done on extracts of very small samples of the matrix of interest (50 to 100 microliters of serum). Reference: 1. Baselt, R.C. Disposition of Toxic Drugs and Chemicals in Man, pp. 616-619, 8th Edition, Biomedical Publications, Foster City, California, 94404 (2008). The HSPS interface is for Laboratory Use Only; not for use in diagnostic procedures. NOTES:

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P-133 Characteristics and Potential of CESI-MS for the Analysis of Complex Peptide Mixture Jean-Marc Busnel1,2;Jeff D. Chapman1; Ed Horton1; Bart Schoenmaker2; Anton A. Heemskerk2; Andre M. Deelder2; Oleg A. Mayboroda2

Beckman Coulter Inc. Brea, CA, USA, Leiden University Medical Center, Leiden, The Netherlands In this presentation we present a new front-end separation-and ionization technology called CESI, which fully integrates the high efficiency and ultra-low flow characteristics of capillary electrophoresis (CE) with electrospray ionization (ESI) in a single dynamic process within the same device. Reduction of flow with ESI into the low nanoflow region serves to significantly improve ionization efficiency as well as reduce ion suppression of co-migrating analytes. With CESI we are able to generate stable and robust electrospray with bulk flows of electrolyte at or below 10 nl/min, that when combined with the orthogonal selectivity provided by CE allows for an increase in proteome coverage. We demonstrate that this technique is truly complimentary to nanoLC-ESI-MS taking advantage of an increased compatibility with small and hydrophilic peptides but also with rather large peptides, whose behavior is close to the one of small proteins. We will highlight examples of this complimentary coverage as well as achievements of ultrahigh sensitivity, detecting peptides at picomolar concentrations, from minute samples. P-134 Assessing Biosimilars: Applying the European Pharmacopoeia Capillary Zone Electrophoresis Method for the Separation of Recombinant Human Erythropoietin (rhEPO) Marcia Santos; Mark Lies Beckman Coulter, Inc., Brea, CA USA Erythropoietin (EPO) is a naturally occurring hormone that stimulates red blood cell production and release from bone marrow and was one of the first therapeutic recombinant glycoproteins commercialized for the treatment of anemia. EPO is known to have complex N- and O-linked glycosylation patterns, and can exist as numerous protein isoforms that play a critical role in the bioavailability, activity, potency and stability of rhEPO. Proper characterization of rhEPO is extremely important in order to ensure the comparability or efficacy of a biologic preparation. The European Pharmacopoeia1 (EuPh) first defined a method to qualitatively determine EPO isoform distribution using capillary electrophoresis in 1999. Subsequently, collaborative studies among pharmaceutical companies, government agencies and academic institutions have refined this method and also developed a suitability standard. We present a method for the analysis of isoform distribution in rhEPO using the PA 800 plus Pharmaceutical Analysis System and describe, sample preparation, instrument setup and data analysis. Note: For Research Use Only; not for use in diagnostic procedures.

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P-135 High Resolution Intact and Glycoprotein Analysis by CESI Scott Mack; Kimberly Kwong; Ed Horton; Chitra Ratnayake Beckman Coulter, Inc., Brea, CA, USA The analysis of intact proteins by Electrospray Ionization - Mass Spectrometry (ESI-MS) provides the most direct route for the identification and characterization of these highly complex molecules. With the potential presence of multiple isoforms, it is also widely accepted that the presence of a liquid-phase separation technique upstream from the MS detection step would greatly contribute to a complete deciphering of the structure of these molecular entities. Capillary electrophoresis (CE), compatible with the preservation of delicate protein structure, post-translational and other modifications, while simplifying sample complexity through high resolution separation capabilities, appears as a very valuable alternative prior to introduction into the mass spectrometer. To achieve the greatest advantage of coupling CE with ESI-MS for intact protein analysis, a CESI interface currently in development, was implemented for the analysis of a model glycoprotein, Ribonuclease B. The efficient coupling between these liquid and gas phase separation systems allowed the rapid, sensitive and high resolution detection of the five known Ribonuclease B glycoforms. Further deconvolution of the resulting Time of Flight (ToF) mass spectra by the Maximum Entropy Algorithm identified differences in the attached high mannose structure as the source of the electrophoretic heterogeneity. Produced on multiple sheathless sprayers, the data confirm that these separations are highly repeatable and reproducible. The High Sensitivity Porous Sprayer interface is for Laboratory Use Only; not for use in diagnostic procedures. P-136 50 Ways to Improve your Method Inspired by, and Messing Around with, Paul Simon’s Excellent Text Cari Sänger – van de Griend Kantisto BV, Baarn, The Netherlands The problem is all inside your head She said to me The answer is easy if you Take it logically I'd like to help you in your struggle To be free There must be fifty ways To improve your method

She said it's really not my habit To intrude Furthermore, I hope my meaning Won't be lost or misconstrued But I'll repeat myself At the risk of being crude There must be fifty ways To improve your method

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P-137 Comparison of HPLC Methods with Orthogonal Capillary Electrophoresis Methods used for Registration of New Active Pharmaceutical Ingredients Gamze Belin, Christoph Meyer, Joerg Herren Novartis Pharma AG, Basel, Switzerland Most commonly used analytical methods to test for the purity of active pharmaceutical compounds are based upon reversed-phase liquid chromatography. International Committee on Harmonization guideline Q2, validation of analytical procedures, however, requests that orthogonal analytical purity testing methods with a different separation mechanism are applied to confirm the validity (specificity) of the first choice liquid chromatography method and to confirm the obtained impurity profile of active pharmaceutical ingredients. Also, in earlier phase drug development it must be ensured that all impurities are detected and properly quantified to assure patient safety in clinical trials. Here, capillary electrophoresis methods are discussed in view of their relevance for the detection of drug substance impurities which may not be detected using other separation techniques. We describe the analytical method development for the quantitative detection of ionic drug substance synthesis by-products that were only separated by applying buffer additives which yielded complexes with different frictional force properties respectively different hydrodynamic radii than the complex formed with the active ingredient. Further, the detection of an UV inactive synthesis educts is addressed making use of the indirect detection mode. Also, demands on the applicability of capillary electrophoresis in the quality control laboratory environment are highlighted: Robustness and ruggedness are essential for the daily routine high-throughput analysis of marketed drug products. NOTES: