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The Organizing Committee gratefully acknowledges the pharmaceutical corporate sponsors for their generous support of the academic speaker travel grants:

Platinum Program Partner Waters Corporation

Diamond Program Partner Genentech, Inc.

Gold Program Partners Amgen, Inc. Biogen Idec

Eli Lilly and Company Genzyme Corporation

MedImmune Novartis Biologics

Silver Program Partners Bristol-Myers Squibb Company

Centocor, Inc. Roche Diagnostics GmbH Bronze Program Partner

Biologics Consulting Group, Inc. Friends of CASSS Program Partner

Novo Nordisk A/S

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Exhibitor Partners 551 Linus Pauling Group

AAI Pharma AAPS

Agilent Technologies ARTEL

Beckman Coulter, Inc. BioQuality

BioReliance Corporation Blue Stream Laboratories

Brightwell Technologies, Inc. Convergent Bioscience

Fluid Imaging Technologies, Inc. Lancaster Laboratories, Inc.

M-Scan Inc. MDS Analytical Technologies

NanoImaging Services Northstar BioProducts

Postnova Analytics PPD

ProZyme, Inc. Solvias, Inc.

Tandem Labs Waters Corporation WindRose Analytica

Wyatt Technology Corporation YMC America, Inc.

Media Partners BioProcess International

International Scientific Communications, Inc. LC-GC North America

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Acknowledgements Symposium Co-Chairs Barry Cherney, CDER, FDA Philip Krause, CBER, FDA Wassim Nashabeh, Genentech, Inc.

Permanent Committee Robert Cunico, Bay Bioanalytical Laboratory, Inc. John Dougherty, Eli Lilly and Company John Frenz, GlobeImmune, Inc. Christopher Joneckis, CBER, FDA William Hancock, The Barnett Institute, Northeastern University Michael Kunitani, Genentech, Inc. Thomas Layloff, Management Sciences for Health Robert Sitrin, (Chair), Merck Research Laboratories

Workshop Committee Co-Chairs Roman Drews, CBER, FDA Elizabeth Fowler, Xcellerex, Inc. Mark Schenerman, MedImmune Patrick Swann, CDER, FDA

Special thanks to all the Workshop Session Co-Leaders

Program Committee Sid Advant, Tunnell Consulting Robert Baffi, BioMarin Pharmaceutical Narendra Bam, GlaxoSmithKline Pharmaceuticals Laura Bass, Pfizer Scott Buckel, Xencor Kathleen Francissen, Genentech, Inc. John Hennessey, NovaDigm Therapeutics, Inc. Peter Johnson, 3M Drug Delivery Systems Chulani Karunatilake, Amgen, Inc. Anthony Lubiniecki, Centocor R & D, Inc.

Stacey Ma, Genentech, Inc. Robert Mattaliano, Genzyme Corporation Rohin Mhatre, Biogen Idec Inc. Inger Mollerup, Novo Nordisk A/S Edwin Moore, Baxter Healthcare Corporation Nadine Ritter, Biologics Consulting Group, Inc. Reb Russell, Bristol-Myers Squibb Company Zahra Shahrokh, Shire HGT Philip Wyatt, Wyatt Technology Corporation Yuan Xu, Novartis Biologics

Collaborating Organizations American Association of Pharmaceutical Scientists (AAPS), Biotec Section American Chemical Society, Division of Analytical Chemistry (ACS, DAC) American Society for Gene Therapy (ASGT) Association of Biomolecular Resource Facilities (ABRF) Central New England Chromatography Council (CNECC)

Fédération of Internationale Pharmaceutique (FIP), Special Interest Group Pharmaceutical Biotechnology International Association for Biologicals (IABs) International Society for Cellular Therapy (ISCT) National Institute for Biological Standards and Control (NIBSC) PDA, West Coast Chapter United States Pharmacopeia (USP)

Audio-Visual: Michael Johnstone, MJ Film/Video Productions CASSS Staff: Karen A. Bertani, CMP, Symposium Manager Stephanie Flores, CAE, Executive Director Erin Kelch, CMP, Senior Program Manager Kristina Peterson, Project Coordinator

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General Information Name Badges Please wear your name badge in order to gain admittance to the meetings, poster sessions, exhibit hall and social functions. A name badge with a red circle represents a one-day registration, which does not include the cost of the Museum of Modern Art. Tickets to the Museum of Modern Art may be purchased at the registration desk at a cost of $125.00. Business Center The Intercontinental San Francisco Hotel has a Business Center that is located on the Lobby Level. The Business Center is open 24 hours a day, seven days a week and is accessible with your room key. Photographic Equipment The use of cameras is not permitted during the lecture program, workshops or poster sessions. Cameras are permitted on the exhibit floor. However, permission from the vendors involved must be obtained before photographs can be taken. Poster Sessions All posters will be set up the entire three days of the Symposium in the Pacific Terrace Foyer on the Fourth Floor. Posters in the P-100-M series will be presented on Monday from 15:15 – 16:15. Posters in the P-200-W series will be presented on Wednesday from 14:00 – 15:00. Poster abstracts can be found beginning on page 70 in this book. Registration Registration will be set up in the Grand Ballroom Foyer on the Third Floor from Monday, January 12 through Wednesday, January 14. Social Program Welcome Reception Museum of Modern Art (off-property) Monday, January 12, 19:30 – 22:30 Hosted Luncheon Grand Ballroom Foyer, Third Floor Tuesday, January 13, 12:15 – 14:00 Exhibitor Reception Grand Ballroom Foyer, Third Floor Tuesday, January 13, 18:30 – 20:00 If you have special dietary needs, please notify the Symposium Manager Monday morning so that an alternate menu may be prepared. Also, please note that Museum of Modern Art tickets for non-registered guests or one-day registrants may be purchased at the Symposium registration desk for $125.00 Technical Seminars Four technical seminars will be held on Tuesday, January 13 from 12:45 – 13:45. Two technical seminars will be held on Tuesday, January 13 from 17:30 – 18:30. Please refer to the program summary for more details. Technical Seminar abstracts may be found beginning on page 60.

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Meeting Room Layout PETER – YOU NEED TO TWEAK THE ROOM LAYOUT PDF – CALL ME! KAREN

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WCBP 2009 Scientific Program Summary

MONDAY, JANUARY 12, 2009 07:15 – 17:30 Registration in the Grand Ballroom Foyer 07:15 – 08:15 Continental Breakfast in the Grand Ballroom Foyer 08:15 – 08:30 Welcome and Introductory Comments Wassim Nashabeh, Genentech, Inc.

Regulation of Biopharmaceutical Products: Government Perspectives Plenary Session in the Grand Ballroom

Session Chairs: Barry Cherney, CDER, FDA, Philip Krause, CBER, FDA and Gene Murano, Genentech, Inc. 08:30 – 08:55 Center for Drug Evaluation and Research: Biotechnology Products and Agency Initiatives

Steven Kozlowski, CDER, FDA, Bethesda, MD USA 08:55 – 09:20 Center for Biologics Evaluation and Research: Update on Current Activities and

Future Directions Carolyn Wilson, CBER, FDA, Rockville, MD USA

09:20 – 09:45 Overview of Activities in the European Union - Present and Future Tasks

John Purves, European Medicines Agency, London, United Kingdom 09:45 – 10:10 Current Topics in Japan on Evaluation and Control of Biotechnology Products Takao Hayakawa, Pharmaceuticals and Medical Devices Agency, Tokyo, Japan 10:15 – 10:45 AM Break Visit the Exhibits in the Grand Ballroom Foyer (Third Floor) Visit the Posters in the Pacific Terrace Foyer (Fourth Floor) 10:45 – 11:15 Panel Discussion – Questions and Answers

Critical Quality Attributes and Specifications Plenary Session in the Grand Ballroom

Session Chairs: Timothy Lee, CBER, FDA and Mark Schenerman, MedImmune 11:15 – 11:35 Critical Quality Attributes for Monoclonal Antibodies Reed Harris, Genentech, Inc., South San Francisco, CA USA 11:35 – 11:55 Critical Quality Attributes for Live Viral Vaccines Mark Galinski, MedImmune, Mountain View, CA USA 11:55 – 12:15 Critical Quality Attributes for Blood Products Timothy Lee, CBER, FDA, Rockville, MD USA

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Monday, January 12 continued 12:15 – 12:30 Panel Discussion – Questions and Answers 12:30 – 14:00 Lunch Break - Participants on their own

Particulate Analysis Plenary Session in the Grand Ballroom

Session Chairs: Barry Cherney, CDER, FDA and Stacey Ma, Genentech, Inc. 14:00 – 14:20 Particulates in Parenteral Biotech Products – Requirements & Methods

Hanns-Christian Mahler, F. Hoffmann-La Roche Ltd., Basel, Switzerland

14:20 – 14:40 Aggregation of Therapeutic Proteins: Causes, Control, Consequences and Challenges John Carpenter, University of Colorado Denver, Denver, CO USA 14:40 – 15:00 Subvisible Particulates in Protein Therapeutics: Why the Immune System Cares Amy Rosenberg, CDER, FDA, Bethesda, MD USA 15:00 – 15:15 Particulate Analysis Panel Discussion – Questions and Answers 15:15 – 16:15 Poster Session One in the Pacific Terrace Foyer (Fourth Floor) 16:15 – 17:45 Workshop Session 1

• Critical Quality Attributes and Specifications Grand Ballroom A Laurie Graham, CDER, FDA; Chulani Karunatilake, Amgen, Inc.; Timothy Lee, CBER, FDA

• Particulates and Aggregates Grand Ballroom B Barry Cherney, CDER, FDA; Steven Rubin, CBER, FDA; Ziping Wei, MedImmune

• FDA Moves Forward with Exempting Phase 1 Drugs from cGMPs Grand Ballroom C Sid Advant, Tunnell Consulting; Patricia Hughes, CDER, FDA; Laurie Norwood, CBER, FDA; John Purves, EMEA

• Heparin: A Case for Continuous Improvement in Pharmacopeia Monograph Development Union Square Room

Ali Al-Hakim, CDER, FDA; Todd Wielgos, Baxter Healthcare Corporation; Wes Workman, Pfizer 19:30 – 22:30 Welcome Reception at the San Francisco Museum of Modern Art Sponsored by Genentech, Inc. and Waters Corporation

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TUESDAY, JANUARY 13, 2009 07:30 – 17:00 Registration in the Grand Ballroom Foyer 07:30 – 08:30 Continental Breakfast in the Grand Ballroom Foyer 07:30 – 08:15 Sunrise Summary: CMC Forum July 2008: Quality by Design – The Next Phase: Approach for Filing the

QbD Data and Potential Regulatory Implications Update Session in the Grand Ballroom

Forum Chairs: Sid Advant, Tunnell Consulting; John Dougherty, Eli Lilly and Company; Steven Kozlowski, CDER, FDA; Rohin Mhatre, Biogen Idec Inc.

08:30 – 08:40 Announcements by Wassim Nashabeh, Genentech, Inc. The Development and Approval Process for Device-Based Combination Products for Biopharmaceuticals

Plenary Session in the Grand Ballroom Session Chairs: Peter Johnson, 3M Drug Delivery Systems and Zahra Shahrokh, Shire HGT

08:40 – 09:00 Combination Products: An Overview

Kathy Lee, CDER, FDA, Bethesda, MD USA 09:00 – 09:20 Combination Products Aren't Drugs or Devices: A Regulatory Perspective

Aric Kaiser, CDRH, FDA, Rockville, MD USA 09:20 – 09:40 Are Recent Trends in Combination Product Review and Approval Improving Patient Outcomes? James Collins, Eli Lilly and Company, Indianapolis, IN USA 09:40 – 09:55 Panel Discussion – Questions and Answers 10:00 – 10:30 AM Break Visit the Exhibits in the Grand Ballroom Foyer (Third Floor) Visit the Posters in the Pacific Terrace Foyer (Fourth Floor)

10:30 – 12:00 Workshop Session 2 • Quality by Design for Analytical Methods Grand Ballroom A

Christof Finkler, Hoffmann-La Roche AG, Rajesh Gupta, CBER, FDA; Kathy Lee, CDER, FDA; Dieter Schmalzing, Genentech, Inc.

• Regulatory Risk Management and Improving Application Quality Grand Ballroom B John Dobbins, Eli Lilly and Company; Mahmood Farshid, CBER, FDA; Chana Fuchs, CDER, FDA; Joseph Kutza, MedImmune

• Changes to Cell Substrates Grand Ballroom C Roman Drews, CBER, FDA; Subramanian Muthukkumar, CDER, FDA; Helena Yusuf-Makagiansar, Biogen Idec

• Upcoming Regulations and Guidances from FDA: A Case Study for Biopharmaceuticals and the Implication of Immunogenicity for Combination Products Union Square Room LeeAnn Chambers, Eli Lilly and Company; Suzanne Kiani, Genentech, Inc.; Susan Kirshner, CDER, FDA; Steven Oh, CBER, FDA

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Tuesday, January 13 continued 12:00 – 13:45 Hosted Lunch in the Grand Ballroom Foyer 12:30 – 13:30 Technical Seminars

• Antibody Analysis: From Characterization to Manufacturing Sponsored by Agilent Technologies Grand Ballroom A

• Sedimentation Velocity Analytical Ultracentrifugation for Protein Aggregate Quantification: Managing Method Performance Expectations Sponsored by Beckman Coulter, Inc. Grand Ballroom B

• High-Performance, Routine Characterization of Protein Biopharmaceuticals with a Novel LC/MS System Solution Sponsored by Waters Corporation Grand Ballroom C

• ICIEF in Quality: Guidance on Method Validation and Maintenance Through the Use of QbD, DOE and Risk Management Principles Sponsored by Convergent Bioscience Union Square Room

State of the Art Analytical Technologies for Protein Characterization

Plenary Session in the Grand Ballroom Session Chairs: Emily Shacter, CDER, FDA and Robert Garnick, Genentech, Inc.

13:45 – 14:05 Innovative Mass Spectrometry Technology for the Study of Cell Signaling Donald Hunt, University of Virginia, Charlottesville, VA USA 14:05 – 14:25 The Transparent Structural Details within Therapeutic Glycoproteins Vernon Reinhold, University of New Hampshire, Durham, NH USA 14:25 – 14:45 Emerging Mass Spectrometry-Based Methods to Characterize Higher Order Structure of Biopharmaceuticals Igor Kaltashov, University of Massachusetts, Amherst, MA USA 14:45 – 15:00 Panel Discussion – Questions and Answers 15:00 – 15:30 PM Break Visit the Exhibits in the Grand Ballroom Foyer (Third Floor) Visit the Posters in the Pacific Terrace Foyer (Fourth Floor)

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Tuesday, January 13 continued 15:30 – 17:00 Workshop Session 3

• New and Emerging Analytical Techniques Grand Ballroom A Vladimir Chizhikov, CBER, FDA; Reb Russell, Bristol-Myers Squibb Company; Emily Shacter, CDER, FDA

• CMC Strategies for Rapid Progression of Programs from Phase 1 to Phase 3 Grand Ballroom B

Leslie Bloom, Pfizer; Brigitte Brake, BfArM; Michelle Frazier-Jessen, MedImmune; Denise Gavin, CBER, FDA; Susan Kirshner, CDER, FDA

• Update on Revisions to the EMEA Guideline on Production and Quality Control of Monoclonal Antibodies and Related Substances Grand Ballroom C Kowid Ho, AFSSAPS; Barbara Rellahan, CDER, FDA; Mark Schenerman, MedImmune

• Critical Quality and Manufacturing Issues Related to Antibody-Based Conjugates Union Square Room

Milan Blake, CBER, FDA; Rajesh Krishnamurthy, ImmunoGen, Inc., Jun Park, CDER, FDA 17:15 – 18:15 Technical Seminars

• Characterization of Nanoparticles Using Cryo-Electron Microscopy Sponsored by NanoImaging Services, Inc. Grand Ballroom A

• The Detection and Measurement of Particles by MALS Combined with Fractionation Techniques: Large Aggregates, Virus-Like Particles, Liposomes and Gold Colloids Sponsored by Wyatt Technology Corporation Grand Ballroom B

18:15 – 20:00 Exhibitor and Poster Reception Visit the Exhibits in the Grand Ballroom Foyer (Third Floor) Visit the Posters in the Pacific Terrace Foyer (Fourth Floor)

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WEDNESDAY, JANUARY 14, 2009 07:30 – 17:00 Registration in the Grand Ballroom Foyer 07:30 – 08:30 Continental Breakfast in the Grand Ballroom Foyer 07:30 – 08:15 Sunrise Summary: Overview of the Extractables and Leachables Safety Information Exchange (ELSIE)

Initiative Update Session in the Grand Ballroom Moderator: Jon Cammack, Baxter Healthcare Corporation

08:30 – 08:40 Announcements by Wassim Nashabeh, Genentech, Inc. 08:40 – 09:00 Acknowledgments Robert Sitrin, Merck Research Laboratories

Comparability Plenary Session in the Grand Ballroom

Session Chairs: Patrick Swann, CDER, FDA and Yuan Xu, Novartis Pharmaceuticals 09:00 – 09:20 Comparability During Clinical Development: One of the Challenges of Cell Line Changes Kathleen Francissen, Genentech, Inc., South San Francisco, CA USA 09:20 – 09:40 Comparability Assessment in the Development of Follow-On Protein Products: Case Studies from Omnitrope Development Andreas Premstaller, Sandoz GmbH, Kundl, Austria 09:40 – 10:00 Characterisation Studies to Support Comparability of a Complex Glycoprotein Post Manufacturing Changes Brian Fitzpatrick, Wyeth Biotech, Dublin, Ireland 10:00 – 10:20 Comparability of Insulins Produced by Second Generation Processes Lene Hørlyck, Novo Nordisk A/S, Gentofte, Denmark 10:20 – 10:40 Panel Discussion – Questions and Answers 10:40 – 11:10 AM Break Visit the Exhibits in the Grand Ballroom Foyer (Third Floor) Visit the Posters in the Pacific Terrace Foyer (Fourth Floor)

Current Topics in Vaccine Development Plenary Session in the Grand Ballroom

Session Chairs: Robin Levis, CBER, FDA and Robert Sitrin, Merck Research Laboratories 11:10 – 11:30 Evaluating Novel Cell Substrates for Use in Vaccine Manufacture Philip Krause, CBER, FDA, Rockville, MD USA

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Wednesday, January 14 continued 11:30 – 11:50 Data Mining Builds Process Understanding for Vaccine Manufacturing Julia McNeill, Merck & Co., West Point, PA USA 11:50 – 12:10 New Perspectives on Vaccine Development, Stockpiling and Manufacturing Infrastructure Building Robin Robinson, US Department of Health and Human Services, Washington, DC USA

12:10 – 12:25 Panel Discussion – Questions and Answers 12:30 – 13:30 Lunch – Participants on their own 13:30 – 14:30 Poster Session Two in the Pacific Terrace Foyer (Fourth Floor) 14:30 – 16:00 Workshop Session 4

• Comparability: Evolving Concepts and Applications Grand Ballroom A Mahmood Farshid, CBER, FDA; Brendan Hughes, Wyeth Biotech; Anthony Lubiniecki, Centocor R&D, Inc.; Patrick Swann, CDER, FDA

• Biophysical Techniques Grand Ballroom B Daron Freedberg, CBER, FDA; Sarah Kennett, CDER, FDA; Thomas Patapoff, Genentech, Inc.

• New and Emerging Product and Process Technologies Grand Ballroom C Kimberly Benton, CBER, FDA; Larry Couture, Beckman Research Institute of City of Hope; Anthony Ridgway, Health Canada

• Well Characterized Vaccine Products Union Square Room John Frenz, GlobeImmune, Inc.; Robin Levis, CBER, FDA

16:00 – 16:15 Break

New and Emerging Biopharmaceutical Products Plenary Session in the Grand Ballroom

Session Chairs: Edwin Moore, Baxter Healthcare Corporation and Carolyn Wilson, CBER, FDA 16:15 – 16:35 Cellular and Gene Therapy Products - CBER Update Kimberly Benton, CBER, FDA, Rockville, MD USA 16:35 – 16:55 Manufacturing and Characterization Challenges for Human Stem Cell Products Larry Couture, Beckman Research Institute of City of Hope, Duarte, CA USA 16:55 – 17:15 Strategies for Characterization of Human CD34+ Stem Cells Richard Johnson, Baxter Healthcare Corporation, Round Lake, IL USA 17:15 – 17:30 Panel Discussion – Questions and Answers 17:30 – 17:45 Closing Remarks by Elizabeth Fowler, Xcellerex, Inc.

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NOTES:

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Plenary Session Abstracts Center for Drug Evaluation and Research: Biotechnology Products and Agency Initiatives Steven Kozlowski CDER, FDA, Silver Spring, MD USA Ensuring pharmaceutical product quality is of critical importance. Knowledge gained during the pharmaceutical development program is important for an enhanced understanding of product quality and provides a basis for risk management. Quality by Design (QbD), Pharmaceutical cGMPs for the 21st Century, and Process Analytical Technology are initiatives being applied to small molecule pharmaceutical development and manufacturing. Although the principles of these initiatives are applicable to biopharmaceuticals, there may be some unique considerations for these products. A number of approaches may facilitate the application of these initiatives to biotechnology products. The FDA critical path initiative may also provide opportunities for enhancing the development of biopharmaceuticals. NOTES:

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Center for Biologics Evaluation and Research: Update on Current Activities and Future Directions Carolyn A. Wilson CBER, FDA, Bethesda, MD USA The Center for Biologics Evaluation and Research (CBER) is responsible for regulating blood and blood products, blood screening test kits for infectious agents, prophylactic and therapeutic vaccines, tissues, novel therapeutic products including cellular and gene therapy, cancer vaccines, tissue-engineered, xenotransplantation and certain combination products. CBER proactively engages in research, stakeholder workshops, and professional, scientific, and global organizations to generate, be aware of, and apply the most advanced science available for regulatory decision-making and policy development, facilitating development of novel, safe and effective products. Areas of emerging public health needs include preparedness for acts of terrorism and pandemic influenza. In addition, CBER is committed to fostering development of new or improved treatments for diseases that do not have adequate treatment options. We accomplish this by fostering application of novel technologies that enable improved therapeutic strategies, establishing new approaches to product manufacture and characterization, and by application of genomics to engender an era of effective, personalized medicine. CBER is working to identify novel means to improve late stage product development, such as use of modeling and adaptive clinical trial design. To improve product safety, CBER epidemiologists are applying new analytic approaches to large clinical databases to post-marketing surveillance for low frequency adverse events in patients receiving licensed biologics. As a community, we are at the nexus of improved computational, scientific, and clinical knowledge. CBER is profoundly committed to our ongoing mission to apply a unique combination of scientific and regulatory expertise to improve the public health and ensure product safety. NOTES:

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Overview of Activities in the European Union - Present and Future Tasks John Purves European Medicines Agency, London, United Kingdom This presentation will provide an introduction, looking at the environmental and legislative changes in the Europe Union. This will start with a reflection of the EMEA mission statement and be followed by a brief examination of future challenges and environmental changes in both a European and an International basis, taking into account the evolution of legislation and science. The second part of the presentation will deal with the new tools for the review of medicinal products and address the centralised procedure and how changes to the legislation have influenced consideration of “Conditional” marketing authorisations, “Accelerated” review, risk management systems, Paediatric Investigational Plans (PIPs), Small and Medium Size Enterprises (SMEs) and suggestions on how to further promote the benefits of early contact with the Regulators. The thirds section will provide an overview of current work on biosimilar and advanced therapy medicinal products, recent developments in vaccines and experience with the provision of Scientific Opinions (SO) and reference to more recent guidelines which have been developed. These guidelines cover monoclonal antibodies, allergen products, products from transgenic plants and investigational medicinal products. Finally, the fourth section will look forward to the opportunities available to both the Regulators and the Industry, taking into account the evolution of the science, the regulatory procedures, transparency and the global market. NOTES:

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Current Topics in Japan on Evaluation and Control of Biotechnology Products Takao Hayakawa Pharmaceuticals and Medical Devices Agency, Tokyo, Japan This presentation will focus on updating the key regulatory activities in Japan in 2008. Two new guidelines regarding cells/tissue-based products issued by MHLW in 2008 will be outlined, which describe the basic technical elements to ensure the quality and safety of pharmaceuticals and medical devices derived from processing of autologous and allogeneic human cells/tissue, respectively. These guidelines also describe differences with respect to data requirements and evaluation between marketing authorization submission and submission for verification of conducting investigational clinical trial. The latter is to evaluate if there is any quality and safety problem that might pose an obstacle to start a clinical trial. On the other hand, both guidelines emphasize that when conducting or evaluating tests on individual product, it is necessary to take flexible approaches case-by-case in line with the purpose of the guideline, based on the type, characteristics and intended clinical use of a product in question. Reflection of scientific progress and accumulation of experience in relevant field is always encouraged. Recently, MHLW study group has started to develop another new guideline which specially addresses potential quality and safety issues on products derived from human stem cells including embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). A draft guidance on subsequent-entry protein products issued by MHLW for public comment on September 17, 2008 will also be introduced. The guideline applies to recombinant proteins and polypeptides, their derivatives, and products of which they are components, (e.g., conjugates). These proteins and polypeptides are produced from recombinant cell-culture expression systems and can be highly purified and characterized using an appropriate set of analytical procedures. There are several key suggestions in the draft guidance to encourage developing subsequent-entry protein products in a scientific, efficient, effective and economical way. On the other hand, there may be several other key issues to be further clarified to make the guidance clearer and more applicable for both applicants and reviewers. NOTES:

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Critical Quality Attributes for Monoclonal Antibodies Reed Harris Protein Analytical Chemistry Department, Genentech, Inc., South San Francisco, CA USA Critical quality attributes (CQAs) are defined as “A physical, chemical, biological or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality.” Interpreting this definition for actual CMC strategies has been challenging. We are developing CQAs for our antibody products based on a combination of: 1) what is the potential impact to patients if this characteristic or impurity is present at a high or low level, and 2) how certain are we that we’ve answered question #1 correctly? Impact is a 2–20 point scale that considers a combination of potency, pharmacokinetics, immunogenicity and adverse events risks, while uncertainty is a 1–7 point scale that acknowledges whether our knowledge derives from studies of this attribute for this molecule, or if we’re relying on information from related molecules or literature. The impact and uncertainty values are multiplied to generate an overall severity score, with scores above a threshold value considered CQAs. Levels of each form or impurity are not considered when CQAs are evaluated. Several antibody CQA examples will be reviewed. CQAs are reviewed during the pre-IND period, prior to process characterization study initiation, and when a final commercial control strategy is being developed. CQAs will generally be controlled by testing (specifications) or by process control (validation), and some quality attributes may need to be included in comparability assessments. NOTES:

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Critical Quality Attributes for Live Viral Vaccines Mark Galinski Vaccine Analytical Sciences, MedImmune, Mountain View, CA USA Currently licensed live viral vaccines (LVVs) are complex biologic products that differ from other biologics (and pharmaceutics) in that they are infectious and express the relevant viral antigen(s) following administration of the drug product. Infectivity is a requirement of the active pharmaceutical ingredient (API) and is a critical attribute that assures (1) amplification of the relevant viral antigen(s) and (2) presentation of the antigen(s) in a “natural fashion” that induces adaptive immunity and subsequent protection from disease. Manufacturing of LVV drug substance requires several intermediate products (Master Virus Seed and Working Stock Seed), a well characterized cell substrate for virus growth (Master Cell Bank, Manufacturers Working Cell Bank), and well controlled raw materials (culture media, excipients). A general overview of LVV manufacturing and identification of Critical Quality Attributes (CQAs) that impart the desired quality, potency, safety, and efficacy of these products will be discussed. NOTES:

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Critical Quality Attributes for Blood Products Tim Lee CBER, FDA, Rockville, MD USA Currently licensed blood products are derived either from natural sources or recombinant DNA technology. Plasma-derived products have been traditionally manufactured from poorly defined starting materials using processes that lack robustness. In addition, some of the impurities in these relatively low-purity products are often not inert, which in turn affect the efficacy and safety of the intended product. Their recombinant analogs, while more pure and better characterized, are still complex and heterogeneous molecules that differ from their naturally occurring counterparts in gross and subtle ways. The least of which is the nature and extent of post-translational modifications, such as glycosylation, proteolytic processing and gamma-carboxylation. An overview of the manufacture of blood products, their critical quality attributes and control strategies, which include in-process controls, specifications for drug substance and drug product, and stability testing, will be discussed. NOTES:

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Particulates in Parenteral Biotech Products - Requirements & Methods Hanns-Christian Mahler F. Hoffmann La Roche Ltd., Basel, Switzerland This presentation will briefly discuss regulatory requirements for particulate in parenteral drug products (incl. biotech), methods in use to detect such particulates as well as upcoming methods. Additionally, attempts to identify particulates will be discussed. Gaps of current practice including knowledge of the relevance of sub-visible and visible particulates in clinical practice, a common understanding or definition of requirements and expectation and harmonization of methodologies will also be discussed. NOTES:

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Aggregation of Therapeutic Proteins: Causes, Control, Consequences and Challenges John Carpenter Department of Pharmaceutical Sciences, University of Colorado Denver, Denver, CO USA Causes: Proteins usually aggregate from partially unfolded molecules. Even in a formulation that greatly stabilizes the native state, significant aggregation can occur; especially over pharmaceutically-relevant time scales. Furthermore, exposure to interfaces (e.g., air-liquid and solid-liquid) can induce aggregation. Sometimes protein particles (visible and subvisible) are generated due to heterogeneous nucleation by foreign micro- and nanoparticles shed from filling pumps and the product closure/container. Control: Protein aggregation can be slowed by maximizing the stability of the native conformation (“conformational stability) and the energetics of intermolecular repulsion (“colloidal stability”). This also may help to reduce interface-induced aggregation, but often a non-ionic surfactant is also required. Particle formation caused by foreign materials shed from filling pumps and containers/closures is a difficult problem to solve. One must assess and optimize formulation compatibility with pumps and container/closure, and additional formulation studies are needed if these systems are changed for a given product. Consequences: Even at relatively low levels (e.g., 1% of the protein molecules) aggregates may cause adverse effects in patients such as immunogenicity and anaphylaxis. Challenges: Size exclusion chromatography (SEC) is used to quantify aggregate levels and sizes, but the results can be inaccurate. Corroborating SEC results with analytical ultracentrifugation (AUC) is useful, but AUC has its own challenges. Also, subvisible particles often are not analyzed properly in protein products. The actual amount of protein in the particles may not be sufficient (e.g., 0.1%) to allow their detection as loss of native protein. USP guidelines for particle analysis, which were developed for small molecule parenteral drug products, are followed for protein particle analysis. These guidelines require quantitation of particles > 10 micron. But, examples exist where there were hundreds of thousands of particles < 10 micron, yet the protein sample still passed the USP test. NOTES:

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Subvisible Particulates in Protein Therapeutics: Why the Immune System Cares Amy Rosenberg CDER, FDA, Bethesda, MD USA Subvisible particles in the size range of 0.1-10um have not been routinely assessed in therapeutic protein products. Recently, several papers have illustrated the presence of large numbers of these particles in several protein therapeutics, with the demonstration that they potentially contain thousands to millions of natively conformed protein molecules. In fact, large MW arrays of natively conformed proteins have been shown to be efficient inducers of immune responses, a fact that is being exploited to generate vaccines using particulate microspheres encapsulating or displaying microbial proteins or peptides. The underlying reason for efficient immune responses to particles of such size may well relate to the finding that many viruses, bacteria, yeast, and molds span this size range of subvisible particles, suggesting that the immune system views such particles as threats. This seminar will examine the basic immunology of responses to particulate antigens and how this may bear on a regulatory approach to subvisible particles in protein therapeutics. NOTES:

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Combination Products: An Overview Kathy Lee CDER, FDA, Bethesda, MD USA What are Combination Products and how are they regulated by the FDA? What is the Office of Combination Products and what do they do? Which Center takes the review lead? What are some of the CMC challenges? This talk will attempt to answer all of these questions and provide an introduction to the complex world of Combination Products. NOTES:

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Combination Products Aren't Drugs or Devices: A Regulatory Perspective Aric Kaiser CDRH, FDA, Rockville, MD USA In the past, all medical products could relatively easily be regulated as drugs, biologics or devices. As new medical technologies have been devised, it has become necessary to define a fourth type of medical product– combination products. As their name suggests, these are medical products that consist of a combination of at least two of the other types of medical products. These products are regulated by the Center determined to have lead regulatory jurisdiction, but receive consultative input from the Center(s) assigned the secondary regulatory jurisdiction. For “traditional” medical products, a single set of regulations governs the review and regulation of the product. In the case of combination products; however, the regulations associated with each Center are available for use and are applied as necessary to insure the most appropriate control of the potential risk to the treated population and/or user. The applicable regulations range from those associated with non-clinical studies (Good Laboratory Practices) to conduct of clinical trials (informed consent, clinical trial design and reporting, institutional review boards and investigator selection) to manufacturing (cGMPs and QSR), and are dependent on the lead Center. The work necessary to bring a combination product to market incorporates a complicated regulatory process involving interactions with at least two Centers within FDA. Because of differences in legal/regulatory requirements between the various Centers, the non-clinical and clinical evaluation of a combination product can be a more difficult, but not insurmountable task. This presentation will attempt to outline some of the similarities and differences between traditional and combination products, as well some of the similarities and differences between the Centers and how these impact the evaluation and regulation of combination products. NOTES:

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Convergence of Biologics with Devices Challenges and Opportunities William Van Antwerp Medtronic USA, Inc., Valencia, CA USA Abstract was not available at time of printing. NOTES:

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Are Recent Trends in Combination Product Review and Approval Improving Patient Outcomes? James Collins Eli Lilly and Company, Indianapolis, IN USA Over the last three years, Eli Lilly and Company submitted six pen delivery devices to US and EU regulatory authorities for approval. These submissions provide a unique opportunity to evaluate the global regulatory review process for combination products. All six devices were developed using the same device development quality system, utilized established ISO 11608 standards for testing, provided a very similar data set to regulatory authorities, and were for use with previously approved therapeutics. Given the existing combination product regulations, CDRH reviewed two products as 510k's and CDER reviewed four products as NDA supplements. In Europe, three products were self certified and three products were reviewed as variations to previous EU submissions. Even though the product technologies, device requirements, and data provided was very similar, the time to approval and number of question cycles was significantly higher for CDER with input from OSE than any other reviewing body. Given the similarities in these delivery devices, it is our hypothesis that the difference in approval time and question cycles can be directly correlated to the reviewing body. Each delivery device submitted provided benefits to the end user, and the delays in approval withheld the benefits from the market. Were patient outcomes improved based on the delays? NOTES:

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Innovative Mass Spectrometry Technology for the Study of Cell Signaling Donald Hunt Departments of Chemistry and Pathology, University of Virginia, Charlottesville, VA USA This lecture will focus on several different aspects of cell signaling and the mass spectrometry technologies developed to study them. By using electron transfer dissociation (ETD)-tandem mass spectrometry it is now possible to analyze intact proteins on a chromatographic times scale (1 protein/2-5 sec). Proteins are converted to gas-phase, positive ions by electrospray ionization and then allowed to react with fluoranthene radical anions. Electron transfer to the multiply charged protein promotes random fragmentation of amide bonds along the protein backbone. Multiply charged fragment ions are then de-protonated in a second ion/ion reaction with the carboxylate anion of benzoic acid. The m/z values for the resulting singly, doubly, and triply charged ions are used to read a sequence of 15-60 amino acids at both the N and C termini of the protein. This information, along with the measured mass of the intact protein, is used to identify unknown proteins, to confirm the amino acide sequence of a known protein, to detect post-translational modifications, and to determine the presence of possible splice variants. Applications of this technology to the study of class I and class II antigen processing pathways, labile post-translational modifications (phosphorylation and O-GlcNAcylation), and comlex patterns of post-translational modifications on histone proteins that regulate gene expression, stem cell differentiation, reprogramming of DNA in the egg, and changes of phenotype that do not involve altering the sequence of DNA (epigenics) will be discussed. NOTES:

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The Transparent Structural Details within Therapeutic Glycoproteins Vernon Reinhold The Glycomics Center in Partnership with Glycan Connections, LLC, University of New Hampshire, Durham, NH USA As a consequence of genomic expression, cell milieu, and temporal factors, molecular glycosylation represents an unsurpassed chemical fingerprint of tissues, cells, and molecular structure. In this situation, differential mass profiles of normal and diseased tissue samples provide an important opportunity to untoward function, physiological evaluation, and QC of pharmaceuticals. In implementing a glycosylation evaluation, it must be recognized that each connecting monomer may introduce a new topology (branching), institute alternative stereochemistry (equatorial or axial hydroxyl groups), and most probable, establish a different structural isomer through a multiplicity of linkage positions. These exquisite details of glycan structure have long been appreciated as providing the structural basis for major but yet discrete biological responses. These would include blood group substances, plant lectins, and the specific attachments of viruses, bacteria, and parasites. Unfortunately, many of these critical details of glycan function are transparent to mass analysis, with the corollary if you can’t detect a structural modification it’s not present. Correlation of glycan mass profiles with gene modulation, physiological change, and differing expression vehicles, provides a powerful approach to the growing appreciation of glycan function. But in using such strategies a representative glycome profile remains fundamental, as do all components that comprise each ion. We will make the case in this presentation that a comprehensive structural evaluation is possible by reaching beyond the usual tandem MS with the application of ion trap MS, (MSn), e.g., digging into the isomeric components of glycan structure, where the details of linkage and branching reside. To resolve this structural information glycans are chemically or enzymatically released, methylated, mass profiled and each ion disassembled by MSn. In numerous samples types, including metastatic, developmentally regulated, and perceived normal tissues multiple isomers were identified as pendant non-reducing termini of di-, and trisaccharide fragments, probably transparent to a tandem MS approach, but distinctively not to sequential ion trap MSn detection. Similar evaluations have proceeded on stem cell lines, and therapeutic glycoproteins with particularly unique results. Although numerous analytical strategies have been introduced to identify selected components of carbohydrate structure, it has been the continued focus of this and previous efforts to only build upon protocols that can be integrated into a high throughput strategy consistent with automation. Such protocols could bring an important analytical focus to carbohydrate sequencing that is greatly lacking. NOTES:

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Emerging Mass Spectrometry-Based Methods to Characterize Higher Order Structure of Biopharmaceuticals Igor Kaltashov University of Massachusetts, Amherst, MA USA Unlike small molecule drugs, the conformational properties of protein biopharmaceuticals in solution are influenced by a variety of factors that are not solely defined by their covalent chemical structure. Since the conformation (or higher order structure) of a protein is a major modulator of its biological activity, the ability to detect changes in both the higher order structure and conformational dynamics of a protein, induced by an array of extrinsic factors, is of central importance in producing, purifying, and formulating a commercial biopharmaceutical with consistent therapeutic properties. This presentation focuses on evaluating two complementary mass spectrometry (MS) based techniques (analysis of ionic charge state distribution and hydrogen/deuterium exchange) as potent tools for monitoring conformational changes in protein biopharmaceuticals. The utility of these approaches is demonstrated by detecting and characterizing conformational changes in the biopharmaceutical product interferon β-1a (IFN-β1a). The protein degradation process is modeled by inducing a single chemical modification of IFN-β1a (alkylation of its only free cysteine residue with N-ethylmaleimide), which causes significant reduction in its antiviral activity. Analysis of IFN-β1a ionic charge state distributions unequivocally reveals a significant decrease of conformational stability in the degraded protein, while hydrogen/deuterium exchange measurements provide a clear indication that the higher order structure is affected well beyond the covalent modification site. Importantly, neither technique requires that the location or indeed the nature of the chemical modification be known prior to or elucidated in the process of the analysis. In contrast, application of the standard armamentarium of biophysical tools, which are commonly employed for quality control of protein pharmaceuticals, met with very limited success in detection and characterization of conformational changes in the modified IFN-β1a. Detailed analysis of the conformational consequences of IFN-β1a degradation also suggests the mechanism of the protein inactivation, as the observed conformational changes affect its binding interface with one of the cognate receptors. This mechanism of protein inactivation is verified by direct MS characterization of IFN-β1a interaction with ectodomains of its two cognate receptors. The presentation will also discuss the potential impact of MS-based experimental strategies on the studies targeting conformation and interactions of highly heterogeneous biopharmaceuticals, such as PEGylated proteins heparin. Several examples will be presented to highlight the role MS can and should play in the biopharmaceutical industry beyond the presently assigned task of primary structure analysis. NOTES:

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Comparability During Clinical Development: One of the Challenges of Cell Line Changes Kathleen Francissen Genentech, Inc., South San Francisco, CA USA During the development of biologics (from preclinical stages up through initiation of Phase III) manufacturing processes are often changed. It is not unusual to change cell lines during clinical development in order to improve productivity. Cell line screening involves assessment of protein production and certain product quality attributes, such as aggregation, as well as sequence variants. Based on historical data at Genentech, approximately 15% of CHO cell lines contain some level of sequence variants during the screening process. Current evidence suggests that some of the errors occur at the DNA level. Sequence variants may be detected at levels well below 1% or as high as 50% as measured at the protein level. These variants are generally detected using reversed-phase HPLC-UV peptide mapping and mass spectrometry to attain sufficient sensitivity, though other analytical methods may prove useful. Because of the extremely high number of potential variants in any recombinant protein product, it is not possible to guarantee their detection or predict the best mode of quantification during screening. However, the presence of unintended sequence variants in the protein product should be avoided, or at least minimized. NOTES:

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Comparability Assessment in the Development of Follow-On Protein Products: Case Studies from Omnitrope Development Andreas Premstaller Sandoz GmbH, Kundl, Austria The assessment of comparability plays a crucial role in the development of follow-on protein products. Comparability exercises include not only the comparison to the reference product, but may also be warranted to justify process changes during development or to demonstrate safety and efficacy of novel drug product formulations. Depending on the nature of the change and the depth of product- and process-understanding, physicochemical and biological data can be sufficient to demonstrate comparability. In other cases, preclinical and even clinical data may be required. The decision which levels of comparison are required is driven by a scientific assessment in the individual case. An array of orthogonal analytical techniques was developed and applied for the comprehensive physicochemical and biological characterization of the active substance somatropin in Omnitrope. The development of any follow-on protein product necessitates a comprehensive analysis and comparison of the profile of product-related variants between candidate and reference product. Critical quality attributes need to be identified and controlled. Specifically, the absence of any unwanted product-related impurities should be demonstrated. Differences in product-related variants must be assessed regarding their impact on safety and efficacy of the product, and the extend of this assessment will again depend on the understanding of the product. The development of a liquid formulation poses similar challenges; the justification of the product profile at the end of shelf life will be addressed in the talk. NOTES:

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Characterisation Studies to Support Comparability of a Complex Glycoprotein Post Manufacturing Changes Brian Fitzpatrick Wyeth Biotech, Dublin, Ireland The demonstration of Comparability of a biotechnology-derived product involves a detailed characterisation of biological and physiochemical properties of the molecule. This talk will highlight the analytical studies undertaken to demonstrate the comparability of a complex glycoprotein post-manufacturing changes. The presentation will identify the key challenges undertaken within the analytical studies to demonstrate comparability of the product profile. The talk will highlight how the combined use of a variety of orthogonal analytical approaches and biological techniques were necessary to confirm that the isoform distribution and glycan composition of the molecule were consistent with historical product profile. NOTES:

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Comparability of Insulins Produced by Second Generation Processes Lene Hørlyck Novo Nordisk A/S, Gentofte, Denmark The Comparability Guidance documents have been implemented for some years with significant impact in the pharmaceutical industry. In particular, for minor changes in approved manufacturing processes the concept has greatly decreased the implementation time without compromising the quality of the product. For major changes to approved processes the comparability mindset can be applied as well, provided that a high degree of process and product expertise is available in-house. Case studies will be presented describing implementation of second-generation processes using the comparability mindset. Emphasis will be on the appropriate design of the analytical programme for the quality comparability studies and the subsequent evaluation of data. In addition, the criteria for conclusion of comparability will be discussed, as well as the consequences to be mitigated if this conclusion cannot easily be reached. NOTES:

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Evaluating Novel Cell Substrates for Use in Vaccine Manufacture Philip Krause CBER, FDA, Rockville, MD USA The need to develop vaccines against new pathogens, as well as the interest in using new technologies to develop more effective vaccines, has led to renewed interest in use of novel cell substrates for vaccine manufacture. New technologies are being developed to better address concerns about novel cell substrates that are related to the potential for carry-over of undesirable attributes, including oncogenicity and adventitious agents. Degenerate PCR, followed by high-throughput sequencing was used to evaluate cell lines for the potential presence of adventitious agents. This and similar techniques have the potential to detect all viral sequences present in a cell. Scientific pitfalls associated with the increasing availability of newer, more sensitive techniques such as high throughput sequencing will be discussed. NOTES:

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Data Mining Builds Process Understanding for Vaccine Manufacturing Julia McNeill Merck & Co., Inc., West Point, PA USA Vaccine bulk manufacturing is a complex biological process composed of many steps carried out over months or even years. A critical quality attribute of the product is potency measured with biological assays. Hundreds of raw material characteristics and process variables are monitored for every lot. Identifying the root cause of potency changes is extremely challenging because of the large number of candidate variables and the inherent variation of the measurement system. Recently, two key vaccine bulk products were studied to better understand and control potency. Initially the traditional methods of univariate statistical process control and least squares linear regression were used. These basic approaches left many questions unresolved, so a number of more advanced techniques were applied. The primary challenges of building process understanding are pinpointing the timing of changes in potency, and identifying root cause variables with the strongest correlation to potency. Random forests and partial least squares regression proved extremely valuable for generating hypotheses about causes of potency shifts. These data mining methods have set a new standard for building vaccine process understanding within Merck. Their effectiveness will be illustrated with a case study. NOTES:

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New Perspectives on Vaccine Development, Stockpiling, and Manufacturing Infrastructure Building Robin Robinson Biomedical Advanced Research and Development Authority, U.S. Department of Health and Human Services, Washington, DC USA Addressing public health emergencies caused by man-made threats and emerging naturally-occurring diseases requires public-private sector partnerships at global, national, State, and local levels. Forging a path forward for U.S. pandemic influenza preparedness to provide pandemic vaccines requires a multipartite approach utilizing vaccine development, pre-pandemic vaccine stockpiling, and increased domestic and global influenza vaccine manufacturing surge capacity. Shortening timelines for availability of pandemic influenza vaccines requires changes in government regulations on influenza virus permitting, testing, and shipping and generation and calibration of vaccine potency assay reagents. Lastly, policy discussion and decision on when pre-pandemic vaccination should occur – during interpandemic phase or at the onset of a pandemic- require risk-benefit analyses and development of suitably sized clinical studies to support widespread usage of pre-pandemic vaccines. The U.S. pandemic influenza vaccine rubric provides a model for addressing other public health emergencies caused by chemical, biological, radiological, and nuclear threats as well as emerging infectious diseases. NOTES:

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Cellular and Gene Therapy Products – CBER Update Kimberly Benton CBER, FDA, Rockville, MD USA Cellular and gene therapies are a novel and rapidly evolving class of products that promise to be the new generation of biological products. CBER places a high priority on scientific and regulatory activities that promote the development of safe and effective products, while working to avoid an unnecessary regulatory burden. The scientific and regulatory issues raised by this diverse group of products are extraordinarily complex, multifaceted, and subject to rapid change, as technology progresses. The unique nature of the products and their manufacturing processes necessitates development of specialized assays for individual products and the establishment of appropriate release criteria for the parameters of identity, purity, and potency. CBER’s Office of Cellular, Tissue, and Gene Therapies has responsibility for the regulation of cell therapy, gene therapy, therapeutic vaccines, and related products. Regulatory challenges for these products and summary of recent OCTGT outreach activities such as workshops, advisory committees, guidance for industry will be discussed. NOTES:

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Manufacturing and Characterization Challenges for Human Stem Cell Products Larry Couture Beckman Research Institute of City of Hope, Duarte, CA USA Human pluripotent cells such as human embryonic stem cells (hESCs) represent an important source of healthy tissues for cell therapies in regenerative medicine. Manufacture of these cellular products will likely require the creation of large master cell banks of the undifferentiated parental hESC lines as well as cell banks, or single-patient lots, of the differentiated derivatives. Large scale production of undifferentiated hESC cell banks presents challenges in establishing reproducibility, in maintaining genetic stability and in retaining an undifferentiated state of the cells. Current techniques for the propagation of hESCs were developed by laboratory researchers for small scale culture primarily to maintain hESCs in a predominately undifferentiated state. These processes typically require the cells to be grown as colonies on mouse or human feeder cells; expansion often involves labor-intensive mechanical separation of each colony. These protocols demand a high degree of operator training and skill and are not consistent with robust cGMP manufacturing processes. In addition, hESCs can be unstable in culture. Differentiation state and genetic stability are susceptible to modest changes in cell propagation techniques and culture conditions. Efforts to develop scalable cell culture methods for feeder-free, single cell adherent or suspension cultures are needed. Product characterization also poses challenges. Traditional cell product characterization methods may be insufficient to assess the genetic stability, differentiation state, purity, and residual tumorgenicity of hESC-derived cell products. While the ultimate measure of residual contamination by undifferentiated pluripotent cells in a final product is the formation of teratomas in animal models, this method is insensitive, time consuming and costly, and is not feasible as an in-process or patient-specific differentiated final product test method. Surrogate characterization methods with a correlation to tumorgenicity are needed. Methods such as epigenetic fingerprinting, x-chromosome inactivation or other genetic and epigenetic characterization methods may provide these alternatives. This presentation will discuss the unique challenges of stem cell product manufacture and characterization of hESC and genetically engineered neural stem cells. NOTES:

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Strategies for Charactization of Human CD34+ Stem Cells Richard Johnson Baxter Healthcare Corporation, Round Lake, IL USA Human adult CD34+ stem cells have been employed for oncology applications for many years. Recently, investigators have been exploring the application of this heterogeneous population of cells for other potential settings, including cardiovascular disease. This presentation will discuss the Isolex approach to purification of mobilized peripheral blood derived stem cells and the various methods that have been used to characterize these unique cells. This will include both in vitro techniques as well as an in vivo test system, along with a brief discussion of clinical parameters. NOTES:

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WYATT TECHNOLOGY B/W AD

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Workshop Descriptions

Workshop Session 1 Monday, January 12, 2009

16:15 – 17:45

Critical Quality Attributes and Specifications Laurie Graham, CDER, FDA; Chulani Karunatilake, Amgen, Inc.; Timothy Lee, CBER, FDA Workshop Description Grand Ballroom A Critical Quality Attributes (CQA) are those properties that impart the desired product quality, safety and efficacy. Due to the large number of attributes associated with biotechnology products, determining the criticality of an attribute can be challenging, especially during early development when limited data is available. A life-cycle approach, applied to CQAs, may involve risk based evaluations to define/refine the list of CQAs during product development and after licensure. Once identified, CQAs can focus the development and characterization of the manufacturing process onto those attributes that have the highest probability of impacting product safety and/or efficacy. The identification of CQAs, therefore, allows for an evaluation of the manufacturing process with regard to product risks, and should drive the design of product control strategies. During the workshop we will discuss current practices in areas described above with the aim of identifying best practices, issues and difficulties, with particular emphasis on the relationship between CQAs and product specifications. Audience participation is encouraged via sharing examples of what has worked and difficulties encountered during this process. NOTES:

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Particulates and Aggregates Barry Cherney, CDER, FDA; Steven Rubin, CBER, FDA; Ziping Wei, MedImmune Workshop Description Grand Ballroom B Large particles and aggregates of therapeutic proteins, fractionated blood products and other biopharmaceutical products can form during the manufacturing process and potentially impact efficacy and safety (including immunogenicity) of products. Particles greater than 10 µm in size are typically monitored by light obstruction test and visible appearance tests, while the presence of sub-visible particles between 0.1 and 10 µm is not routinely monitored or controlled. This workshop will focus on the analytical methods that are useful in quantifying these particulates, the risks associated with product quality, and approaches to the design of suitable control strategies that mitigate the identified risk to product quality. The following questions will be discussed:

1. What are the risks to product quality? 2. What analytical methods can be used to monitor particles?

A. What are the methods for visible and sub-visible particles? B. How can protein particles be identified? C. What are the challenges for using light obscuration methods with protein therapeutics? How well

do they correlate with other methods? D. How are acceptable limits for particles established?

3. What are the mechanisms of action for particle formation? A. Is there a connection between soluble aggregates and particles? B. Are there any common precursors for particle formation?

4. What control strategies are available to prevent and minimize particle formation? NOTES:

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FDA Moves Forward with Exempting Phase 1 Drugs from cGMPs Sid Advant, Tunnell Consulting; Patricia Hughes, CDER, FDA; Laurie Norwood, CBER, FDA Workshop Description Grand Ballroom C Adherence to CGMPs in the manufacture of clinical supplies is intended to assure safety, quality and supply of investigational products from development to commercial manufacture. To facilitate product development, FDA published a final rule in the Federal Register amending the CGMP regulations for human drugs, including biological products, to exempt most investigational “Phase 1” drugs from complying with the CGMP regulations (21 CFR210/211) on July 15, 2008. This exemption became effective on September 15, 2008. Phase 1 investigational drugs remain subject to the statutory CGMP requirements of the FD&C Act 501(a)(2)(B) and the statutory provisions in the Act continue to allow the agency to inspect manufacturing and testing sites and take appropriate actions during manufacturing of phase 1 materials. The exemption to the CGMP regulations does not apply to an investigational drug for use in a phase 1 study once the investigational drug has been made available for use by or for the sponsor in a phase 2 or phase 3 study or the drug has been lawfully marketed. An FDA Guidance on “CGMPs for Phase 1 Investigational Drugs” was published in July 2008 to provide guidance to industry on attaining compliance with CGMP during manufacturing of clinical supplies for phase 1 studies. This workshop will explore how the change in CGMP regulations and the recently published FDA guidance will facilitate the manufacturing of clinical materials in the US and globally. Specifically:

• How do the new regulations and the new FDA guidance on “CGMPs for Phase 1 Investigational Drugs” reduce the regulatory burden and facilitate the development of new drugs?

• How do the new regulations and the new guidance affect existing manufacturing facilities? • How do the current CGMP expectation for investigational drugs differ between the US and Europe? How

will the differences affect product development? NOTES:

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Heparin: A Case for Continuous Improvement in Pharmacopeia Monograph Development? Ali Al-Hakim, CDER, FDA; Todd Wielgos, Baxter Healthcare Corporation; Wes Workman, Pfizer Workshop Description Union Square Room In January 2008, an increase in the number of adverse reactions associated with the administration of heparin was observed. In February 2008 a voluntary drug recall was initiated, by Baxter, the major supplier of Heparin in the United States. Shortly thereafter, reports of similar adverse reactions began to come in from different countries threatening the worldwide heparin supply. An intense effort was undertaken by the affected parties to develop biological and analytical methods to determine the cause of the adverse events. Capillary Electrophoresis (CE) and Nuclear Magnetic Resonance (NMR) methods were developed and appeared on the Food and Drug Administration (FDA) web site in March. These methods were capable of screening API Heparin and were successful in reducing contaminated heparin from the world supply and the number of adverse reactions showed an immediate decline. The contaminant was soon determined to be oversulfated chondroitin sulfate (OSCS), a heparin like compound that could not be detected by the pharmacopeia methods in place at the time of the contamination. The source of the contamination has not been identified, though it was found in the crude heparin used to produce Heparin API, but it was theorized that it was a byproduct of attempting to increase the potency of heparin by sulfation. This workshop will consider whether the pharmacopeias should have an active program of continuous improvement to keep monographs current with respect to modern analytical technology. Furthermore, could such a continuous improvement program have anticipated the heparin contamination? And could it prevent other future contamination issues with other product monographs? NOTES:

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Workshop Session 2 Tuesday, January 13, 2009

10:45 – 12:15

Quality by Design for Analytical Methods Christof Finkler, Hoffmann-La Roche AG; Rajesh Gupta, CBER, FDA; Kathy Lee, CDER, FDA; Dieter Schmalzing, Genentech, Inc. Workshop Description Grand Ballroom A Quality by Design (QBD) is a progressive regulatory strategy originally designed for biotechnological product development and manufacturing processes. For manufacturing processes, implementation of QBD involves scientifically designing of product and process performance characteristics to meet specific objectives, not just empirically deriving from performance of test batches. Recently a similar philosophy or thinking has been considered in design and evaluation of analytical methods. This workshop aims at extending QBD concept to analytical methods (QBDA) that are employed for quality control testing. Specifically, the workshop will explore how QBDA could transform the established regulatory practices for quality control test methods. It will investigate how this transformation might enable continuous analytical innovation and dynamic specification life-cycle management. Case studies will be used to guide the discussion. The workshop will also explore if QBDA could ultimately lead to a paradigm shift how both industry and regulatory agencies approach method validation. NOTES:

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Regulatory Risk Management and Improving Application Quality John Dobbins, Eli Lilly and Company; Mahmood Farshid, CBER, FDA; Chana Fuchs, CDER, FDA; Joseph Kutza, MedImmune Workshop Description Grand Ballroom B The product lifecycle covers not only a wide range of development and testing but also a wide range of regulatory requirements. Keeping current with regulatory and scientific developments, maintaining a good relationship with regulators and updating regulatory submissions with new information gathered during product development is critical to meeting clinical trial requirements and efficient assembly of a marketing application. CMC issues that commonly invite questions from regulators or that can lead to a clinical hold will be discussed along with approaches to improving submission quality that may reduce the number of comments and questions from regulators. Participants are encouraged to bring their own examples and ideas for discussion. NOTES:

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Changes to Cell Substrates Roman Drews, CBER, FDA; Subramanian Muthukkumar, CDER, FDA; Helena Yusuf-Makagiansar, Biogen Idec Workshop Description Grand Ballroom C Occasionally there is a need to implement changes to cell substrates or its growth or storage conditions. For example, increases in product demand may prompt a change to a more productive cell line, while the need to improve the manufacturing process’s consistency, yield and/or quality attributes can necessitate the change to a more suitable parental cell line. Safety concerns may warrant changes to the cell banking or growth procedures, such as the removal of serum or other animal-derived component which may require re-cloning of the original cell line or generation of a new cell line all together. While these changes may be warranted, they also carry a high risk of having an impact on product quality attributes and possibly product safety. This workshop is aimed at providing an interactive discussion between pharmaceutical companies and regulatory agencies on what constitutes cell substrate or growth/storage changes, the regulatory ramification of specific changes and the type of data that would be required to support product comparability. Participants are encouraged to bring examples of changes related to cell substrate that have been made to an existing process and the approaches that were used to perform revalidation or demonstrate comparability. NOTES:

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Upcoming Regulations and Guidances from FDA: A Case Study for Biopharmaceuticals and the Implication of Immunogenicity for Combination Products LeeAnn Chambers, Eli Lilly and Company; Suzanne Kiani, Genentech, Inc.; Susan Kirshner, CDER, FDA; Steven Oh, CBER, FDA Workshop Description Union Square Room This workshop will build on highlighted topics from the plenary session and address specific topics that speak to development, regulation and issues with combination products. The workshop will cover upcoming rules and guidance documents that are being drafted and reviewed by the Office of Combination Products and question the audience on how they are preparing for them. We will then cover an industry case study that is illustrative of combination products for biopharmaceuticals. Our FDA representative will address immunogenicity testing for combination products. NOTES:

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Workshop Session 3 Tuesday, January 13, 2009

15:45 – 17:15

New and Emerging Analytical Techniques Vladimir Chizhikov, CBER, FDA; Reb Russell, Bristol-Myers Squibb Company; Emily Shacter, CDER, FDA Workshop Description Grand Ballroom A Analytical technologies continue to advance at a rapid pace. Industry and regulatory authorities are continuously engaged in the evaluation and implementation of these technologies in support of medicines. One area that has challenges is the determination of protein higher order structure and conformation. During this workshop, methods for protein secondary and tertiary structure will be discussed and compared, with an eye on identifying which are most discriminating and quantitative. The relationship between protein structure and biological activity as well as exposure of antigenic epitopes will be discussed. As with all emerging methods, we will address questions around when and if a new technology can be implemented in the regulatory setting and whether it will ever migrate to a release test in a quality control laboratory setting. NOTES:

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CMC Strategies for Rapid Progression of Programs from Phase 1 to Phase 3 Leslie Bloom, Pfizer; Brigitte Brake, BfArM; Michelle Frazier-Jessen, MedImmune; Denise Gavin, CBER, FDA; Susan Kirshner, CDER, FDA Workshop Description Grand Ballroom B This workshop is a continuation of last year’s First in Man workshop and is intended to generate discussion around industry experience in rapidly moving programs from FIH to Phase 3. The goal is to share both technical and regulatory challenges/successes/strategies in the following areas and to highlight differences is strategies based on type of product.

Comparability is the major place where we develop information as we progress through drug development from the initial toxicology supplies through Phase 3. The extent of the analysis and available information expands throughout the course of development. Multiple Scenarios:

• Phases 1, 2 and 3 with tight timelines: highly coordinated progression as fast as possible without lags in time between trials driven by CMC.

• From Phase 1 directly to Pivotal Study: usually for unmet medical needs (often Orphan Indications), common in Oncology setting.

Comparability Analysis to Support Rapid Progression from FIH to Phase 3:

• Are all elements of the investigational filings equally impacted by comparability analysis or are there specific focus areas to build the comparability story to support rapid progression?

• How can process modifications be developed to enhance the probability of demonstrating comparability?

• Use of platform manufacturing processes, DOE and QbD principles throughout development, production of additional small pilot lots.

• Maintenance of reference standards and retain samples to support comparability, analytical method development and specification setting.

• Use of development data to minimize time delays in comparability programs. When can development data be used instead of clinical lot data?

• What role does stability testing play in timing of comparability analysis and investigational filings? Analytical Method Development: Implementing New Assays, Improving Assays and Modifying Acceptance Criteria without Introducing Timeline Delays:

• Methods are always scientifically sound and fit for purpose. Validation of many methods may not occur until either the pivotal study or submission of license application.

• Implementation of cell based bioassays; challenges faced, timing, success stories. • Impurity profiling: how much and when, refer to reference standards and retain samples? • Rely on contractors with method expertise and experience • Determining the potency of product related substances, expectations based on levels present.

o Testing variants in a range of assays. • Introduction of a process specific HCP assay

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Specifications: • Limits established early for safety. Characterization proceeds from report results through acceptance

criteria. Leverage preclinical and historical data. • Use standard specifications for certain classes of molecules throughout development. • When are additional lots helpful, required? • Specification refinement in later phases. How much data do you evaluate before you tighten acceptance

criteria? How do you loosen inappropriate acceptance criteria? Regulatory Filings:

• Substantial amendments o What constitutes a substantial amendment? How do you reduce the need for substantial

amendments? How do you manage shelf life extension in the EU? Worldwide? • IND amendments

o What are the best methods for companies to manage timing and feedback on IND amendments? NOTES:

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Update on Revisions to the EMEA Guideline on Production and Quality Control of Monoclonal Antibodies and Related Substances Kowid Ho, AFSSAPS; Barbara Rellahan, CDER, FDA; Mark Schenerman, MedImmune Workshop Description Grand Ballroom C The previously approved EMEA Guideline for Monoclonal Antibodies entered a revision process in 2007. After numerous comments were received from the industry on the initial draft, a meeting was requested between the Biologics Working Party and industry trade groups. The newly revised guideline has now been submitted for CHMP approval. The workshop will describe the revision process from both the regulatory and industry perspectives culminating in the soon to be issued Guideline. In addition, some next steps and follow-up items will be discussed including platform manufacturing processes and evaluation of glycosylation in relation to effector function. NOTES:

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Critical Quality and Manufacturing Issues Related to Antibody-Based conjugates Milan Blake, CBER, FDA; Rajesh Krishnamurthy, ImmunoGen, Inc.; Jun Park, CDER, FDA Workshop Description Union Square Room Antibody-based conjugates (AC) are a promising class of molecules for the treatment of various types of cancers that is attracting a lot of interest. ACs are designed to deliver a cytotoxic drug (small molecule) through a targeting moiety. Ideally, an antibody-drug conjugate remains non-toxic during circulation in vivo until it reaches its target site and then is functional only upon binding to the target. Although such antibody-based conjugates are simple in principle, significant chemistry, manufacturing, and control (CMC)-related challenges have been presented in the regulatory review and approval of these complicated products. These include identifying potential critical quality attributes (CQAs) for the conjugates including those for antibody, the cytotoxic component, and the linker. The goal of this workshop is to highlight and discuss quality and manufacturing issues unique to this class of molecules. Some examples of topics that would be discussed include:

a. Designation of the different components of the conjugate and the practical implications of these designations.

b. Release & stability methods for the conjugate and its components, potential CQAs, methods to monitor

heterogeneity. c. Comparability exercise following changes in the manufacturing process of the conjugate or its

components. d. Creation, characterization, and change of reference standard. e. Risk factors for immunogenicity e.g. contribution of linker towards immunogenicity.

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SOLVIAS B/W AD

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Workshop Session 4 Wednesday, January 14, 2009

15:00 – 16:30

Comparability: Evolving Concepts and Applications Mahmood Farshid, CBER, FDA; Brendan Hughes, Wyeth Biotech; Anthony Lubiniecki, Centocor R&D, Inc.; Patrick Swann, CDER, FDA Workshop Description Grand Ballroom A Comparability is a perennial topic for WCBP plenary sessions and workshops. While ICH Q5e concepts are well established, each comparability assessment brings its own particular challenges and it is not unusual for regulatory agencies to require results from additional studies. In this workshop, we would like to explore how a systematic approach to product development as described by ICH Q8 (R1) could facilitate manufacturing changes and the assessment of comparability. For example, enhanced knowledge of the manufacturing process (including the ability to link material attributes and process parameters to critical quality attributes) can facilitate the risk assessment of the impact of a planned manufacturing change on the quality, safety, and efficacy of the drug product. In addition, when sensitive analytical techniques detect differences, the risk assessment will then turn to how these differences relate to safety and efficacy. Addressing the impact of observed analytical differences requires consideration of the products intended use in clinical setting, dosage form, route of administration (i.e. Quality Target Product Profile). On the other hand, ICH Q8 (R1) concepts do not include nonclinical/clinical considerations as described in ICH Q5e (e.g. complexity of the product structure-activity relationships; strength of the association of quality attributes and safety/efficacy; or the product’s mode(s) of action). This session will provide an overview of the comparability concepts and challenges for biotechnology products.

• How can QbD approaches facilitate the demonstration of comparability? • What are common problems in comparability assessments? • How is comparability assessed in different regions and at different stages of development? • Are there recently developed tools/protocols that have improved comparability assessments?

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Biophysical Techniques Daron Freedberg, CBER, FDA; Sarah Kennett, CDER, FDA; Thomas Patapoff, Genentech, Inc. Workshop Description Grand Ballroom B There have been several WCBP workshops and an immense amount of discussion about the use of biophysical methods to study protein-protein interactions and aggregation. This workshop is NOT another one of them. The intent of this workshop is to explore old, new, and emerging biophysical methods that can help elucidate many of the three dimensional aspects of proteins that can impact the solution properties, stability and other characteristics important to a pharmaceutical product. These qualities include but are not limited to the charge and hydrophobicity distribution on the surface of the protein, dynamic structural fluctuations, binding of ions and surfactants, and denaturation or phase separation induced by low or high temperatures. Techniques used to investigate these qualities are an alphabet soup containing FRET, NMR, FTIR, CD, MS, CE, ITC, DSC but also including such things as small molecule probes, in silico analysis, protein net charge analysis, and others. Discussion by the audience on the merits and limitations of each method presented is encouraged especially with respect to their use in protein characterization, control system, lot release, and forensic/investigations. Questions to stimulate discussion on the topic:

1. Can you cite (potential) examples where using one or more of these techniques helped answer a question in either research or development?

2. Can any of these techniques help troubleshoot a manufacturing or stability issue either by determining

the source of the problem or ruling certain sources out? 3. Are there times when the results of some of these methodologies should be included in a formal

regulatory filing? Why? 4. Are there times when the results of some of these methodologies should NOT be included in a formal

regulatory filing? Why? 5. If a biotech company only had the resources to buy two instruments, what two should they purchase? 6. What techniques require dedicated personnel to maintain and operate the instrument and interpret the

results? Does the benefit of having the instrument outweigh the cost? NOTES:

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New and Emerging Product and Process Technologies Kimberly Benton, CBER, FDA; Larry Couture, Beckman Research Institute of City of Hope; Anthony Ridgway, Health Canada Workshop Description Grand Ballroom C Gene therapy and stem cell technologies represent new product opportunities that have emerged from the rapidly developing scientific fields of genomics and cell and developmental biology. Cellular and gene therapy products are complex biological therapeutics requiring unique and often innovative strategies for controlling product consistency, safety, and therapeutic efficacy. The inability to subject these products to purification protocols that will remove or inactivate pathogens, typically used for traditional biotechnology products, such as recombinant proteins or monoclonal antibodies, introduce additional challenges into the manufacturing schema. Stem cell-derived therapies introduce additional challenges since the manufacturing process must reproducibly result in a product with the correct cell phenotype in order to ensure both the safety and the efficacy of the product. The key characteristics that can be used to ensure product identity, purity, and potency may not always be obvious. These and other unique aspects of product control for these products, including regulatory as well as technical and analytical methods, will be presented and discussed. NOTES:

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Well Characterized Vaccine Products John Frenz, GlobeImmune, Inc.; Robin Levis, CBER, FDA Workshop Description Union Square Room This session will focus on issues relevant to the development of assays for vaccine products. While some issues discussed will be specific for vaccines, others will be relevant for all well characterized biological products. The primary purpose of most quality assays is to assure the safety and efficacy of the final product. This requires manufacturers to determine what vaccine characteristics are relevant and critical for safety and efficacy. Since vaccines can range from relatively simple highly purified polysaccharides or proteins, to relatively complex assemblies of virus-like particles or to highly complex inactivated cell-based vaccines, addressing these challenges constantly presses the limits of available technologies. Some of the potential areas of discussion in this workshop include:

• How can challenges be addressed for potency assays for combination vaccines and adjuvanted vaccines (for which animal assays may be most relevant)?

• What are the critical product attributes that need to be included in a comparability protocol? • What is the role of process validation in interpreting the results of quality assays? • What information is needed for the introduction of assays based on new technologies? • What are the challenges associated with the rapid development of assays to support products used for

emergency response? NOTES:

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Technical Seminar Abstracts

Tuesday, January 13, 2009 12:45 – 13:45

In Grand Ballroom A Sponsored by Agilent Technologies Antibody Analysis: From Characterization to Manufacturing Taegen Clary and Christine Miller Agilent Technologies, Santa Clara, CA USA This talk will highlight a variety of analytical techniques and new technologies for antibodies. In biologic manufacturing, monitoring and control are critical to obtaining high yields and high purity. The Agilent Bio-Monolith HPLC columns offer rapid solutions for calculating IgG fermentation titers, monitoring preparative and process scale purifications, monitoring the fermentation and purification of macro bio-molecules such as viral particles, among other applications. The Bio-Monolith family consists of strong cation exchange, weak and strong anion exchange and Protein A phases. The short monolith discs used in these columns enables the rapid separation of macro biomolecules, providing crucial data needed to make crucial next-step downstream decisions. The primary characterization of a monoclonal antibody using an Agilent HPLC-Chip system coupled with an Agilent accurate mass Q-TOF MS will also be discussed. The results obtained from these studies show that the nanoflow methodology is sensitive, robust and capable of identifying heterogeneity in the antibody sample. The mass assignment of large fragments generated either by chemical or enzymatic cleavage was easily interpreted due to the high mass accuracy. The data shows that this method can be used for the routine analysis of biopharmaceuticals with the use of minimal sample. The analysis of this data using BioConfirm software for confirmation of the protein, including the use of a new large molecule feature extraction algorithm, will be shown. NOTES:

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Tuesday, January 13, 2009 12:45 – 13:45

In Grand Ballroom B Sponsored by Beckman Coulter, Inc. Sedimentation Velocity Analytical Ultracentrifugation for Protein Aggregate Quantification: Managing Method Performance Expectations John Gabrielson Amgen, Inc., Thousand Oaks, CA USA Sedimentation velocity analytical ultracentrifugation (SV-AUC) is routinely applied in biopharmaceutical development for measuring protein aggregate content and size distribution. SV-AUC allows quantification of protein aggregates directly in product formulation and eliminates complications of traditional size separation techniques (e.g. resin-protein interactions, mobile phase effects, etc.). However, SV-AUC exhibits poor precision compared to conventional size separation methods such as SEC. Additionally, factors such as sub-optimal experimental practice and solution non-ideality can lead to inaccurate aggregate quantification. Improving method performance is critical to ensure the appropriate application of SV-AUC in protein biopharmaceutical development. This presentation will focus on approaches to improve SV-AUC performance, including how to evaluate and select optimal centerpieces and alignment of the centerpieces to the center of rotation. Furthermore, a strategy is proposed to enable accurate aggregate quantification in non-ideal formulation conditions. NOTES:

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Tuesday, January 13, 2009 12:45 – 13:45

In Grand Ballroom C Sponsored by Waters Corporation High-Performance, Routine Characterization of Protein Biopharmaceuticals with a Novel LC/MS System Solution Weibin Chen; Alistair Wallace Waters Corporation, Milford, MA USA; Waters Corporation, Manchester, United Kingdom LC/MS has been an enabling cornerstone technology for implementing the WCBP paradigm for biotherapeutic drug development, including peptide mapping and intact mass analysis. While there is no question of the power of this technology to generate in-depth characterization data, the complexity of high-end mass spectrometry combined with labor-intensive data analysis has minimized this technique’s impact throughout the development of a biopharmaceutical protein. It is well-documented that broader implementation of LC/MS leads to faster development times, greatly increased process understanding, and safer, more effective drugs.

Showing real-world examples with antibodies and other recombinant proteins, Waters introduces an LC/MS system solution designed for both high-performance and routine characterization of protein therapeutics. In creating a new approach to the biopharmaceutical LC/MS workflow, we leverage market leading UPLC separations, an MS designed to deliver unsurpassed benchtop performance with any analyst accessibility, a broad range of MS consumables, and powerful informatics tools that automatically analyze, annotate, and compare LC/MS peptide mapping and intact mass analysis data sets. This comprehensive data set enables the thorough understanding of protein drugs, the characterization of underlying variation, and the demonstrated control of this variation during production as required by regulatory authorities. As the power and robustness of LC/MS technologies have evolved, so have the challenges to develop them for every analytical task - and every user. Biopharmaceutical scientists will learn how to easily and rapidly obtain detailed information from LC/MS peptide maps and intact protein mass analysis with this system solution. NOTES:

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Tuesday, January 13, 2009 12:45 – 13:45

In the Union Square Room Sponsored by Convergent Bioscience PRESENTATION: Imaged Capillary Isoelectric Focusing (ICIEF) Method from Development to Validation Zara Safarian Genentech, Inc., South San Francisco, CA USA WORKSHOP: ICIEF in Quality: Guidance on Method Validation and Maintenance Through the Use of QbD, DoE and Risk Management Principles Chantal Felten; Jiaqi Wu Cardiome Pharma, Vancouver, BC Canada; Convergent Bioscience, Toronto, ON Canada The method lifecycle can roughly be divided into four phases: development, initial validation, maintenance and replacement. The seminar will give an overview on the principles of QBD, DOE and risk assessment with the goal of achieving proactive method lifecycle management instead of reactive method lifecycle management. Underlying the application of all above mentioned principles is a sound scientific understanding of the critical method parameters and how they influence the separation. Over the last few years, the iCE280 system has seen many applications within the biopharmaceutical industry, allowing Convergent to present a short review on critical method parameters for method development and validation on the iCE280 system. This technical seminar is designed in a workshop format. First, a scientist from a major biopharmaceutical company will present an example of cIEF method development and validation in the QC environment. Then, an expert in quality guidelines and validation will outline the principles of QbD, DoE and risk management, and how to potentially apply these principles to cIEF method development and validation. And to conclude, risk assessment for critical method parameters in cIEF methods will be briefly reviewed by a Convergent scientist. We encourage and value the participation of the audience. QbD, DoE and risk management are still new concepts with respect to analytical method validation and we would like to hear your thoughts. NOTES:

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Tuesday, January 13, 2009 17:30 – 18:30

In Grand Ballroom A Sponsored by NanoImaging Services, Inc. Characterization of Nanoparticles Using Cryo-Electron Microscopy Bridget Carragher NanoImaging Services, Inc., San Diego, CA USA Cryo-transmission electron microscopy (cryoEM) provides a method for directly observing hydrated specimens in their native state, avoiding the artifacts often associated with alternative preparation methods like negative staining or freeze fracture. CryoEM imaging provides direct visualization of nanoscale particles in solution and is ideally suited for characterization of protein macromolecules as well as lipid vesicles. For particles with a regular size and shape (e.g. virus like particles) the method has the potential to provide 3D structural information to sub-nanometer resolution, where secondary structure begins to be resolved. It thus provides a complement to high resolution X-ray structures of individual proteins and allows for the interpretation of very large complexes to near atomic resolution. CryoEM can also routinely provide very specific and accurate size and morphological analysis of the sample population and the ability to visualize aggregates within the sample. These data can be used to help interpret, and accurately calibrate, other bulk sample measurements, for example dynamic light-scattering. In this technical seminar we will provide an overview of electron microscopy and the variety of methods within this area that can be used for biological specimen characterization. The technology used for cryoEM will be described in detail, including sample preparation, image acquisition and data analysis methods. We will describe and contrast two methods used to provide 3D reconstructions of macromolecules, viz. electron tomography and single particle reconstruction. We will demonstrate the use of cryoEM for the characterization of a variety of hydrated specimens, including protein macromolecules and lipid vesicles in solution. NOTES:

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Tuesday, January 13, 2009 17:30 – 18:30

In Grand Ballroom B Sponsored by Wyatt Technology Corporation The Detection and Measurement of Particles by MALS Combined with Fractionation Techniques: Large Aggregates, Virus-Like Particles, Liposomes and Gold Colloids Philip Wyatt* Wyatt Technology Corporation, Santa Barbara, CA USA The ability to detect and measure particulates is an important requirement for many elements of pharmaceutical formulations. On the one hand, the deleterious effects of large aggregates has been stressed throughout these meetings, so their presence and populations must be detected and measured in order to understand their source and to develop techniques to prevent or minimize their appearance. New means for drug delivery using virus-like particles as well as structural elements such as liposomes need careful monitoring to insure uniformity of size and concentration for their accurate subsequent delivery as therapeutics. Finally, the importance of gold sols to a variety of new cancer therapies requires an a priori knowledge of their size distributions. Each of the aforementioned classes of particles may be well described and classified by means of their chromatographic separation followed by a multiangle light scattering measurement. Each class, however, brings with it a set of measurement problems. *Dr. Philip Wyatt is the 2009 Recipient of the American Physical Society Prize for the Industrial Application of Physics. NOTES:

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CASSS CALENDAR (in color)

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CONVERGENT BIOSCIENCE COLOR AD

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Poster Abstracts

Poster Session One Monday, January 12, 2009

15:15 – 16:15 Pacific Terrace Foyer (Fourth Floor)

P-101-M Lot to Lot Variation of Carrier Ampholytes Used in Isoelectric Focusing (IEF) Jiaqi Wu Convergent Bioscience Ltd., Toronto, ON Canada At Convergent Bioscience, the final testing for our whole-column detection capillary isoelectric focusing (cIEF) instruments, the iCE280, includes running a standard sample in Pharmalytes pH 3-10 (by GE Healthcare). The data collected from the final testing is a good record of variation of the Pharmalytes pH 3-10. In this presentation, data obtained using different lots of Pharmalytes pH 3-10 over a three year period (2005 – 2008) are analyzed. First, e-grams of the standard sample obtained from different instruments, different column and different years, but in the same lot of Pharmalytes are shown to confirm the reproducibility of the method used in the testing. Then, the e-grams obtained in different lots of the Pharmalytes in the three years will be presented and discussed. Knowledge about the variation between different lots of carrier ampholytes is important in cIEF method development and validation since it partly determines the design space in the cIEF methods. P-102-M Chemical Interference in Capillary Isoelectric Focusing (cIEF) That Distorts the pH Gradients Created by Carrier Ampholytes Jiaqi Wu; Tiemin Huang Convergent Bioscience Ltd., Toronto, ON Canada Isoelectric focusing (IEF) is a special electrophoretic technology that is performed in a pH gradient created by carrier ampholytes. The pH gradient can be distorted mainly by two kinds of chemical compounds; salts (ionic compounds) and amphoteric compounds. All protein samples analyzed by cIEF contain salts in their matrices. Salts in the IEF media can squeeze the pH gradient. As for the amphoteric compounds, they will be focused as a component of the used carrier ampholytes; thus, they distort the gradient at the position where they are focused. Many protein samples contain some amphoteric compounds such as histidine and arginine to stabilize the proteins. Protein samples themselves also act as the components of the used carrier ampholytes and distort the pH gradient. In this presentation, several strategies will be discussed to minimize the effect of these chemical interference. For salts, as long as their concentration is lower than 15 mM, their effect can be compensated for by two internal pI markers. For the distortion caused by the samples themselves, the two pI marker method shows

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much better reproducibility than multiple pI markers. When sample peaks are adjacent to the locations where the interfering amphoteric compounds are focused, the calibrated pI values of the sample peaks will be changed by the different sample concentrations. This effect may be minimized by selecting correct pI markers. Convergent Bioscience provides a full range of pI markers for this purpose. Two mAb samples containing histidine are used as the examples in this presentation. NOTES:

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P-103-M Assessment of Protein Concentration Using a Calibration-Free Method Kevin Lindquist; Åsa Sporrong; Anna Sylvan; Anita Larsson; Robert Karlsson; Ewa Pol GE Healthcare, Uppsala, Sweden Monitoring protein concentration while at the same time assuring its continued ability to interact specifically with a particular binding partner is important throughout biotherapeutic development. This starts with the selection of potent candidates, progresses through the optimization of purification, formulation and storage, and finally requires lot-to-lot consistency assessment and product stability control. The method discussed here, Calibration-Free Concentration Analysis (CFCA), is a progression of Biacore™-based concentration methodology, which provides highly sensitive and robust determination of the active protein concentration without the need for a standard curve. CFCA is a label-free, ligand-binding assay, which relies on changes in binding rates with varying flow rates, when the transport rate of the molecules to the detection area is partially limited by diffusion. In this poster we will describe the general approach of CFCA, but we will also dive into the detailed methodology and explore how different experimental conditions like selection of flow rates and protein immobilization levels affect assay robustness, reproducibility, and dynamic range. Detailed insights will be provided into the practical considerations and experimental design required to successfully employ this novel methodology to monitor the concentration and activity of biologics. To verify the protocol we developed, the total concentrations of eight commercial antibodies were determined and compared using two capture reagents that target the Fc part of the antibody: an anti-Fc antibody and protein A. The concentrations obtained were compared against each other, as well as with the concentration values declared by the antibody manufacturers. For all eight antibodies, the concentrations were in good agreement whether measured against the anti-Fc antibody or protein A. Significantly, however, the CFCA-derived concentrations varied in their degree of agreement with those specified by the manufacturers, with the maximum difference being as much as 50% of the declared value. P-104-M pH Gradient Ion Exchange Chromatography for the Charge Heterogeneity Profiling of Recombinant Monoclonal Antibodies Tony Moreno; Dell Farnan Genentech, Inc., South San Francisco, CA USA Ion-exchange chromatography is widely used for profiling the charge heterogeneity of monoclonal antibodies. The development of ion-exchange methods is generally very time consuming, in particular, the identification of both the optimal pH value for the mobile phase and the ionic strength gradient for each mobile phase pH value. An alternative approach is to use pH gradients to elute the antibodies instead of the more conventional ionic strength gradient. Experimental data from ionic strength based elution and a pH gradient elution method will be discussed for a series of monoclonal antibodies. In general, there was good agreement between the ionic strength elution method and the platform pH gradient method. There were some subtle differences in the elution profiles, as would be expected, when considering the different elution mechanisms. The impacts of operational parameters, such as flow rate, column length, sample loading, and gradient volume on the elution profiles

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observed when using the pH gradient will be discussed. In our studies it is seen that the use of a pH gradient as an elution mechanism in ion-exchange chromatography give consistent data to ionic strength elution with the key advantage that the method could be used for multiple products without change. NOTES:

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P-105-M Protein Sequencing Using High Resolution LC-MS/MS Shankaramma Shivaprasad; Robert Duff; Jon Kauffman Lancaster Laboratories, Inc., Lancaster, PA USA Purpose: This study demonstrates a systematic approach for peptide sequencing and PTM characterization using high-resolution and high mass accuracy tandem mass spectrometry. Tandem mass spectrometry (MS/MS) has become a valuable tool for characterization of biomolecules owing to its reliability, speed and sensitivity. In this study, we demonstrate a MS/MS method that provides extensive sequence information over the whole length of a protein chain in a single series of experiments. Method: Ovalbumin, a major protein in avian egg-white which belongs to the serpin super-family, was used for the analysis. The method involves enzymatic protein digestion, liquid chromatography (LC) separation of resulting peptides and data dependent MS/MS analysis. Analysis was carried out on high resolution LTQ XLTM Orbitrap (Thermo Electron Corp., Waltham, MA) hybrid mass spectrometer in conjunction with an AccelaTM high-speed chromatography system. Mass spectral data was acquired using high mass accuracy full scan and normal accuracy data dependent product ion spectra. All the data was analyzed using BioworksTM software to produce amino acid sequence information. Results and Conclusions: Confirmation of the sequence was accomplished by LC-MS/MS analysis combined with data processing and sequence similarity database searching tools. The method demonstrates the advantage of fast chromatography combined with high resolution, high mass accuracy mass spectrometry to confirm sequence identity of proteins. The method is simple, rapid and useful approach for the characterization of complex proteins. The method can be extended for characterization of unknown proteins sharing identical peptides with related database sequences. P-106-M Characterization of Synthetic Oligonucleotides by High Resolution LTQ-Orbitrap Mass Spectrometry: Method Development and Qualification Robert Duff; Jon Kauffman Lancaster Laboratories, Inc., Lancaster, PA USA Purpose: To develop and qualify a high-resolution mass LC/MS/MS method for the characterization of a RNA oligonucleotide (5’-AUCCGUAGCUAACGUCAUGG) and the homologous DNA oligonucleotide (5’-ATCCGTAGCTAACGTCATGG) in order to compare the quality of a commercial source. Methods: This method utilized a hybrid linear ion trap, the Thermo Scientific LTQ XL™ Orbitrap mass spectrometer (LC-ESI-MS/MS) as used in conjunction with an Accela™ high-speed chromatographic system. Development began with U-HPLC separation using reverse phase C18 columns (Phenomenex Luna (250 x 2.0 mm, flow 0.2 mL/min) and Supelcosil LC-18S column (250 x 4.6 mm, flow 0.8 mL/min)). The oligoribonucleotide was eluted using various mobile phases and conditions. Two mobile phase compositions were primarily examined: 1) triethylammonium bicarbonate (TEAB), pH 7.0 in acetonitrile and 2) 1,1,1,3,3,3-hexafluoro-2-propanol/triethylamine (pH 7.0) in methanol. Sequence analysis was carried out using through MSn analyses number. ESI mass spectra were acquired in negative ion mode.

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Results: Confirmation of sequence was accomplished. The crude synthetic mixtures were examined for impurities and artifacts of the synthesis. Acceptable intraday and interday assay precision (<5% RSD) and accuracy (<5% diff.) of the purified oligonucleotides were observed over an acceptable linear range (nM). Conclusion: The advantage using an LTQ-Orbitrap to confirm the sequence identity of the oligonucleotides is its ability to provide high-resolution mass data with the LC-MS analysis. Both oligonucleotides were sequenced for identity. Finally, the method was robust with respect to linearity, accuracy and its ability to separate then identify the failed synthetic sequences (n-1, n-2 etc. oligomers) and product-related impurities. NOTES:

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P-107-M A Characterization Strategy for Recombinant Polyclonal Antibody Products Torben Frandsen Symphogen A/S, Lyngby, Denmark Recombinant polyclonal antibody products represent a unique new class of antibody products where target specific mixtures of antibodies are manufactured as a single batch from a polyclonal cell bank. Recombinant polyclonal antibody products consist of a defined number of antibodies targeting a single or multiple antigens. Symphogen’s lead candidate the Sym001 product combines 25 different RhD specific antibodies and is presently in phase II clinical trials for the treatment of two different indications. Development of a viable characterization strategy for recombinant polyclonal antibody products has been a challenge. In accordance with ICH Q6B recombinant polyclonal antibody products are characterized thoroughly. Due to the inherent properties of recombinant polyclonal antibodies, i.e., a one batch manufacturing process of a defined number of antibodies, some of the applied release and characterization assays will provide information about the average characteristics for all of the antibodies in a recombinant polyclonal antibody product. In addition, a number of assays are being performed to characterize the constant region of recombinant polyclonal antibody product, which is identical for all of the antibodies. Finally, some analytical methodologies are being developed to address the specific identity of recombinant polyclonal antibody product in terms of polyclonality. This poster presentation will include examples of specific assays which form part of the overall structural characterization strategy and furthermore will focus on the analysis of the polyclonality of recombinant polyclonal antibody products. P-108-M Automating Amino Acid Analysis Using Robotic Sample Preparation Instrumentation Thomas Wheat1; Paula Hong1; Hillary Hewitson1; Diane Diehl1; Jennifer Goode2

1Waters Corporation, Milford, MA USA; 2Tecan US, Research Triangle Park, NC USA Amino acids play an important role in many biological and physiological processes. Their importance has led to the use of amino acid analysis across a wide number of applications and research endeavors. These include protein structure characterization, determining the nutritional value of foods and feeds, monitoring cell cultures, and clinical research applications involving metabolic pathways. In all cases, the measurements must be accurate and precise. The analytical process must have the highest throughput consistent with reliable results. We have developed two robust protocols that include both the derivatization of amino acids and chromatographic analysis. The UPLC® Amino Acid Analysis Solution has been applied across the range of applications, including protein characterization and the analysis of cell culture media. For physiological amino acids, the MassTrak™ Amino Acid Solution provides the same characterization for research use only. Both methods provide faster and more robust analyses than traditional methods. The limiting step in the analysis process is now in sample handling. A robotic sample preparation instrument has been optimized for the amino acid derivatization. Results are compared to the established manual process for reproducibility, accuracy and precision. The specific variables requiring optimization are described in detail. This automation can reduce the possibility of human error when preparing large number of samples. These results will demonstrate how a robotic sample derivatization combined with a robust analytical solution can lead to higher throughput with accurate and precise amino acid analysis.

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P-109-M Analysis of PEGs by ESI-IMS-TOF MS Coupled with Ion-Molecule Reactions Asish Chakraborty; Weibin Chen; John Gebler Waters Corporation, Milford, MA USA Poly (ethylene glycols) (PEGs) and its derivatives are widely used in the biopharmaceutical industry for the delivery of therapeutic drugs. Mass spectrometric analysis of high molecular weight PEGs is very challenging due to the great complexity and diversity of the materials. The use of gas phase ion-molecule reactions for the analysis of PEG and PEGylated proteins has precedent within the biopharmaceutical industry today (e.g. Anal. Chem. 2008, 80, 2408-2418). This presentation describes an improved method to accurately measure the average molecular weight of large poly(ethylene glycols) (PEGs) using ion-mobility time-of-flight mass spectrometry coupled with gas-phase ion–molecule reactions. The method is developed based on a simple and flexible modification to a commercial MS instrument with off-the-shelf components, thus enabling a gas-phase ion–molecule reaction to be effectively coupled to the high-performance tandem mass spectrometer without sacrificing any high performance attributes of the original instrumentation. During these studies, ion–molecule reactions are carried out by introducing gaseous superbases to the trap cell of the commercial ion mobility Q-TOF mass spectrometer, and therefore dramatically change the charge states of multiply charged PEG ions generated by electrospray ionization. As a result, spectral congestion is significantly reduced, and the average molecular weights and molecular weight distributions for the synthetic polymers (5-40 kDa) are accurately determined. PEGs are also analyzed based on the chain lengths using ion mobility separations, providing enhanced specificity to the characterization of the materials. Our results demonstrated that this method has great potential to rapidly characterize PEG materials to the extent unachievable by other methods. It is conceivable that this configuration can be readily applied to many challenging analytical tasks in pharmaceutical industry. P-110-M Developing Methods for Protein Separations on a New Reversed-Phase Column Hillary Hewitson; Thomas Wheat; Diane Diehl Waters Corporation, Milford, MA USA The growing need for fast and accurate characterization of large biological molecules has required ever increasing sensitivity and resolution in the separations of these molecules. With the application of Waters® ACQUITY UPLC and the development of a new column for the reversed-phase separation of proteins, users can leverage these technologies for increased confidence and efficiency of their processes. The development of this material will be described, including the influence of the base particle, pore size, bonded phase on the separation. The impact of design choices was measured based on the behavior of protein probes representing a range of properties as well as some representative applications. The comparisons encompass large and small, acidic and basic, hydrophobic and hydrophilic proteins, as well as, monoclonal antibodies and their subunits. The measurement probes include peak shape, peak area, resolution, and carryover. The same set of proteins were also used to evaluate the influence of mobile phase constituents, solvent choice, separation temperature, gradient slope, and flow rate in developing a method for separation. We will show that simple modifications can provide useful alterations in the selectivity that can be adapted to particular samples. Whether it is for identification and quantitation of target proteins and their possible modification, or monitoring trace impurities, we will show that the Waters BEH300 C4 column provides a suitable material for reversed-phase protein separations.

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P-111-M Concentration Gradient Static Light Scattering for Characterization of Protein Self Association and Protein-Protein Interaction Inhibition Kinetics Amy Hanlon; Michelle Chen Wyatt Technology Corporation, Santa Barbara, CA USA Concentration Gradient Static Light Scattering can be used to measure both equilibrium and time-dependent variables of protein-protein interactions. Sample preparation and delivery, as well as data acquisition and analysis, was completely automated using the Wyatt Calypso System. P-112-M Disulfide Mapping of Recombinant Therapeutic Proteins Using On-Line LC-MS with Electron Transfer Dissociation (ETD) Shiaw-Lin Wu Northeastern University, Boston, MA USA The disulfide map of recombinant therapeutic proteins such as monoclonal antibodies and plasma proteins are fully characterized by the new LC-MS method with ETD. Disulfide-linked peptides were identified by CID and ETD fragmentation, and the disulfide-dissociated (or partially dissociated) peptides were characterized in the subsequent MS3 step. The on-line LC-MS approach is successfully demonstrated in the characterization of disulfide linkages of a recombinan monoclonal antibody (16 disulfides) and tissue plasminogen activator (17 disulfides with one unparied cysteine attaching to either a glutathione or cysteine). The characterization of disulfide-dissociated or partially dissociated peptides in the MS3 step is important to assign the disulfide linkages, particularly, for intertwined disulfide bridges and the disulfide scrambling if exist. The disulfide-dissociated peptides are shown to be obtained either directly from the ETD fragmentation of the precursors (disulfide-linked peptides) or indirectly from the charge-reduced species in the ETD fragmentation. The simultaneous observation of disulfide-linked and disulfide-dissociated peptides provided not only facile interpretation with high confidence but also simplified the conventional approach, which often requires two separate experiments (i.e. with and without chemical reduction). The on-line LC-MS with ETD methodology represents a powerful approach to aid in the characterization of correct folding of therapeutic proteins. P-113-M Improving Process Development Using Displacement Chromatography to Enhance Impurity Detection Barry Haymore; Richard Goodin; Charles Little; C. Patrick McAtee Sachem, Inc., Austin, TX USA Although generally seen as a preparative purification method, displacement chromatography is also a powerful enrichment technique which enables facile detection and quantization of trace components in a sample. Minor components with low concentrations are enriched in the transition zones located between the major components in the displacement train. This allows the detection and measurement of impurities and low-level components not seen by other methods.

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Using new high-purity, high affinity displacers, these transition zones are narrower and much better defined. This leads to further enrichment (10-400X) of minor and trace components and simultaneous depletion of major components. This combination allows much easier identification and quantization of the minor components and impurities compared to other analysis methods. The analyses of enriched fractions from the transition zones can be carried out by any traditional method of analysis This enhanced ability to more fully characterize product streams can be important during process optimization of downstream processes as well as when modifying upstream conditions to aid downstream processing. In cases to be presented, new impurities were found in some proteins using this technique that were not detectable in the analysis of the original sample. This also allowed for improved accuracy for biocomparability studies. The concentrating effect of displacement chromatography also offers the ability of isolating and purifying sufficient amounts of low-level components to allow for chemical and structural characterization of impurities and product isoforms. By accurately identifying trace components one can more efficiently scale up processes and understand transfer issues more completely. NOTES:

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P-114-M Development of a New Peptide Mapping Method Hongji Liu; Bi Xu; Manas Ray; Zahra Shahrokh Shire HGT, Cambridge, MA USA A new peptide mapping method has been developed for therapeutic protein identification and stability test. The uniqueness of the new method is, unlike a standard peptide mapping method employing an acidic mobile phase with TFA as an additive, a basic mobile phase using ammonia as the additive was used for separation. While the same high-resolution peptide separation is maintained, the new method exhibited significantly different selectivity. This provides possibility to separate peptides difficult to be separated with the acidic mobile phase. For example, a deamidated peptide is usually difficult to be separated from its native peptide due to the minimal difference in the hydrophobicity between the converted amide group and the original carboxyl group at an acidic condition (each group is present in the mobile phase with a non-ionic status). However, the hydrophobicity difference becomes much more significant in a basic mobile phase (the carboxylic group is charged while the amide group is not), which ensures baseline separation between the deamidated and the native peptides. Another example is the separation between the regular C-terminal peptide of a protein substance and its truncated C-terminal peptide with 3 of its amino acid residues (PHA) being cleaved at C terminus. The advantages of using the new method include a broader pH usage range, an increase in tryptic sequence coverage and an increase in peptide detection sensitivity. Due to the use of a high-pH tolerant RP column, no significant difference in the column life-time was observed between the two methods. Peptide mapping reproducibility was also similar under both conditions. No detectable on-column deamidation was observed at the used pH (9-10) and and temperature (30-40 °C). In this poster, new method optimization and repeatability results will be reported. P-115-M High Performance Routine Characterization of Proteins and Peptides by LC/MS Weibin Chen; Asish Chakroborty; John Gebler Waters Corporation, Milford, MA USA All too often high performance combined with routine analysis methods result in a compromise in system performance. This work will illustrate how one can obtain high-performance in a routine manner without compromises. This LC/MS system and methods will be demonstrated with intact proteins and peptide maps from antibodies and recombinant proteins. The value to a work-flow of an optimized LC, columns, MS, and software for data analysis will also be presented.

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P-116-M HPLC Without the C: Flow Injection Analysis (FIA) for High Throughput Characterization of Protein Aggregates Using In-Line Laser Light Scattering (LLS) and UV Detection Robert Cunico1; David Fulchiron1; Vicki Gruhn1; Lien Vonguyen1; Rajiv Nayar2; Mitra Mosharraf2

1Bay Bioanalytical Laboratory, Inc., Hercules, CA USA; 2HTD Biosystems, Inc., Hercules, CA USA Measurement and characterization of protein aggregates continues to be a challenging analytical problem even after 20 years of technical advances. Many techniques have been used to analyze aggregates including SEC-HPLC, Field Flow Fractionation, (FFF), AUC, light and electron microscopy, mass spectrometry and even neutron scattering. No single method has emerged as the definitive way to characterize aggregates. In 1987, Welinder et al. used FIA to measure column recovery. The FIA method can provide a way to characterize protein aggregates using conventional HPLC equipment with on-line detectors such as UV (FIA-UV) and (FIA-UV-LLS). Suitability of the FIA method was demonstrated using Ovalbumin, Thyroglobulin and IgM. Relative Standard Deviations (RSD) for peak area are typically less than 1%. For example, Thyroglobulin was dissolved in mobile phase (PBS) at 1 mg/mL and filtered through a 0.22 micron filter. Unfiltered and filtered samples were compared on the FIA-UV-LLS system. The average UV peak areas were not statistically different, p>0.2 (215.7K for unfiltered versus 215.2K for filtered), however average LLS areas were statistically very different, p<<0.001 (381.2K for unfiltered, RSD 0.8% versus 252.3 K for filtered, RSD 0.4%). This technique can be particularly useful in process development, formulation development, and stability analysis. FIA-UV- LLS offers advantages in speed, simplicity, and sensitivity without any investment in new equipment. The sensitivity to large particles and aggregates dictates that impeccable laboratory techniques and proper controls be practiced. P-117-M Use of “Zero” Length Cross-Linking for Evaluation of Aggregation in Manufactured rhPDGF-BB Vladimir Kery; Jim Bonomo; Ken Shockley; Ashley Thompson; Leo Snel Biomimetic Therapeutics, Franknlin, TN USA PURPOSE: Recombinant human PDGF-BB (PDGF) expressed in yeast in its native form is a homodimer inter-connected by two disulfide bonds between Cys43-Cys52 and Cys52-Cys43. This protein is physically and chemically stable even at elevated temperatures. Nonetheless, about 1% of PDGF is in the form of dimer-dimer, and during production of recombinant protein therapeutics there is potential for formation of high molecular weight aggregates. To aid in the evaluation of such aggregates “zero” length cross-linking method was used to prepare rhPDGF-BB aggregates with defined sizes resembling those occurring in manufactured preparations of rhPDGF-BB from yeast. The cross-linked species were then used for calibration of the sizing methods such as SDS-PAGE and high performance size exclusion chromatography (HPSEC). METHODS:

1. To prepare high molecular weight aggregate forms of PDGF and the size/shape of oligomeric PDGF a “zero-length” cross-linking method was applied using EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride).

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2. A series of multimers were created by forming amide cross-links between amino and carboxyl residues. 3. To verify the suitability of the aggregate preparations, samples were analyzed by size exclusion

chromatography (SEC) and gel electrophoresis (SDS-PAGE). 4. Size estimates from SEC and SDS-PAGE data were determined using calibration curves prepared with

protein standards. RESULTS:

1. Different ratios of cross-linked products were formed with up to 35% of double dimer and 14% of quadruple dimer depending upon conditions in the cross-linking reaction.

2. Although the molecular weight of PDGF-BB dimer is 24.6 kDa the native PDGF homodimer runs at the apparent size of 16.4 kDa on SEC due to its compact native structure.

3. Double dimer and quadruple dimer eluted at 31.5 kDa and 55.4 kDa respectively. 4. Multiple well-defined aggregates were distinguished in the reducing SDS-PAGE analysis

CONCLUSIONS: “Zero-length” cross-linking method proved to be a useful tool to investigate higher oligomeric structures of manufactured rhPDGF-BB NOTES:

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P-118-M Particle Characterization by Flow Microscopy David Thomas Brightwell Technologies, Inc., Ottawa, ON Canada Protein-based parenteral formulations often present unique challenges to traditional counting/sizing techniques in terms of characterizing particles in the sub-visible and visible size range. Protein aggregates are highly heterogeneous, irregular in shape, fragile, and often unstable. They may also possess refractive indices close to that of the buffer in which they are contained, and therefore pose a particular challenge with respect to detection. In addition to this, it is not uncommon for high concentration formulations to be viscous, turbid, and opalescent, thereby creating additional analytical challenges. Recent developments in Flow Microscopy technologies have been shown to provide improved sensitivity, sample attribute flexibility, and characterization potential. MFI Flow Microscopy is based upon analyzing images of particles which are captured in succession as a sample stream passes through a flow-cell centered in the field-of-view of a magnification system having a well-characterized and extended depth-of-field. Size, shape, and intensity of each individual particle are measured, as well as the count and concentration of the entire particle population. Maintaining a correlation between each particle’s morphological parameters and its image, aids in the classification of unique particle species and the development of filters that can then be used to isolate the sub-populations for further diagnostics. This paper will describe the MFI Flow Microscopy technology and compare results to those obtained by light obscuration. Studies will be included that demonstrate how Flow Microscopy may be used to characterize protein formulations for the purposes of studying aggregation formation and isolating particle sub-populations of interest (e.g. air bubbles, silicone oil droplets, protein aggregates, foreign particles). Results will also be presented that demonstrate the suitability of Flow Microscopy for analyzing challenging samples such as high concentration protein formulations. P-119-M A Multi-Tiered Analytical Approach for the Analysis and Quantitation of High Molecular Weight Aggregates in a Recombinant Therapeutic Glycoprotein Heather Hughes1; Charles Morgan2; Elizabeth Brunyak1; Kristen Barranco1; Emily Cohen3; Tim Edmunds1; Karen Lee1

1Genzyme Corporation, Framingham, MA USA; 2Genentech, Inc., South San Francisco, CA USA; 3Yale University, New Haven, CT USA In this study, we have performed a systematic evaluation of aggregation in a high molecular weight, recombinant glycoprotein (rGP) using sedimentation velocity analytical ultracentrifugation (AUC-SV), size exclusion chromatography (SEC) and circular dichroism (CD). The results of this study showed that the formation and molecular weight distribution of rGP aggregated species were dependent upon the formulation conditions, as well as the storage or stress conditions used to induce aggregation. The utility of CD as a probe for non-native, aggregated species was affirmed, as this method was sensitive to low levels (≤ 4%) of aggregated rGP. An extensive evaluation of AUC-SV variability was performed using nine levels of spiked rGP aggregate that were analyzed on six occasions. Based on our data, the Precision of the AUC-SV results

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increased with increasing levels of aggregate, with a mean RSD of 37.2%. The Limit of Quantitation (LOQ) for the AUC-SV method, which was based on a Precision criterion of RSD < 20%, was determined to be ≥ 3% aggregated rGP. The Precision and LOQ of the SEC method, determined using the same rGP sample set, was found to be 3.8% and ≥ 0.23%, respectively. In general, there was good agreement between the levels of aggregated rGP determined using the AUC-SV and SEC methods, with a slight positive bias noted for the AUC-SV results. These studies emphasize the value of applying multiple, well-characterized analytical tools in the evaluation of therapeutic protein aggregation. NOTES:

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P-120-M Aggregation Index: A Convenient UV Method for Monitoring Aggregates in Protein Formulations Rajiv Nayar; Jackie Campos; Mitra Mosharraf HTD Biosystems Inc., Hercules, CA USA Various techniques such as dynamic light scattering, size-exclusion chromatography, analytical ultra-centrifugation, and field-flow fractionation to characterize protein aggregation have been employed to characterize protein aggregates. A major challenge has been to obtain a reproducible quantitative value for protein aggregation that can be utilized to monitor aggregation during process development, formulation development, and stability analysis conveniently and reproducibly. This paper formulizes a simple UV method for detection of protein aggregates as a result of light scattering at wavelengths above 320 nm. Protein aggregation was monitored using three different proteins, htPA, tau-IFN, and ovalbumin. These proteins were treated under various stresses or storage conditions such as nebulization (htPA), storage at various temperatures (tau-IFN), and thermal stress (ovalbumin). A UV diode-array spectrometer is employed to analyze the protein solutions with various levels of aggregates. The amount of aggregates was calculated using a simple equation that assigns an Aggregation Index (A.I) value to a protein formulation. Aggregation Index, A.I. = 100 X (Abs adj340 nm/(Abs 280-Abs adj340)), where Abs adj340 is the extrapolated light scattering contribution at 280 nm. The results showed that there was a direct correlation between the A.I values and the 90 oC laser light scattering. Increased A.I values also inversely correlated with percent protein monomer in the case of nebulized htPA. These studies suggest that by utilizing a simple UV measurement, aggregation in protein formulations can be measured in a convenient reproducible fashion that can be utilized in process development and characterization studies. This technique is sensitive, non-destructive, amenable to analysis at high protein concentrations, and requires minimal sample volumes. This assay could also be used in a QC environment if validated for a specific protein drug product. P-121-M Structural Characterization of Human Monoclonal Antibody Aggregates by Vibrational Spectroscopies Tiansheng Li HTL Biosolutions Inc., Newbury Park, CA USA Aggregation of human monoclonal antibody often occurs during formulation, process and manufacturing development. Currently, there is a lack of understanding of the structures of human antibody aggregates, due to the large molecular sizes of aggregates. In this poster, we shall discuss the application of vibrational spectroscopies including FT-IR, Raman and Raman Optical Activity (ROA), to characterize the structures of various types of monoclonal antibody aggregates formed under different stresses. Two different classes of human monoclonal antibodies, IgG1 and IgG2, have been subjected to this structural investigation. The common stresses leading to antibody aggregation, mis-folding or unfolding include exposure to acidic pHs, heat and shear force. The effect of different types of stresses on the structure and aggregate formation of human monoclonal antibodies has been investigated by employing vibrational spectroscopic techniques.

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P-122-M Using Eclipse-MALS to Study Temperature Effects on Monoclonal Antibody Aggregation Michelle Chen Wyatt Technology Corporation, Santa Barbara, CA USA Size exclusion chromatography (SEC) system equipped with a UV absorption detector has been the primary tool to determine the amount of aggregation present in pharmaceutical protein products, such as monoclonal antibodies. Multi-angle light scattering (MALS) is often added to such systems to enhance the characterization of the peaks of interest. However, the SEC column is often the subject of concern, as it can remove large aggregates or alter the aggregation amount in the sample. Therefore, an orthogonal technique is called for to confirm the amount of aggregates measured from the SEC analysis. Analytical ultracentrifugation (AUC) is one such orthogonal technique used currently. This poster demonstrates the use of field flow fractionation (FFF) combined with MALS as a useful tool to study temperature effects on monoclonal antibody aggregation. We found that FFF has a similar accuracy as AUC in quantifying small amounts of dimer. However, the FFF-MALS method is able to detect and characterize large antibody aggregates that were often missed by an SEC analysis. P-123-M Conformance Thresholds and Peptide Map Qualifications: When is a Peak Really a Peak? Adam Harder; Steven Cockrill; Heather Connelly Amgen,Inc., Longmont, CO USA Increased regulatory scrutiny around defining the level at which a previously unseen peak in a peptide map triggers an internal investigation have led to a proposal for a new qualification strategy. Here we have coined the term “Conformance Threshold” to describe the signal intensity for any given peptide map method at which a new peak reaches the level that initiates an internal investigation. This threshold is set using the fundamental chromatographic noise associated with the method. The new qualification design allows for semi-dynamic assignment of the conformance threshold and acceptance criteria in order to account for the particular nuances of individual methods. P-124-M Predicting Shelf Life Stability of Monoclonal Antibodies Osigwe Esue; Thomas Patapoff Genentech, Inc., South San Francisco, CA USA Biologics undergo modifications through physical and chemical reactions under various conditions such as solution pH, temperature and ionic conditions that ultimately affect shelf life. These modifications are manifest as product qualities such as size, charge variants as well as activity that could result in safety and efficacy concerns during human clinical trials. Using the Arrhenius equation, this study models size and charge variants of monoclonal antibodies under stressed conditions (elevated temperatures) and use the model to predict product stability at real time storage conditions (lower temperatures).

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P-125-M Detection of Heparin Contamination using Anion Exchange Chromatography Weibin Chen; Catalin Doneanu; John Gebler Waters Corporation, Milford, MA USA Heparin is a blood thinning drug that is primarily used to prevent the development of blood clots. Heparin and low-molecular-weight heparin (LMWH) belong to the group of linear polysaccharides called gly¬cosaminoglycan (GAG) and they are widely used as anticoagulant drugs during surgery and kidney dialysis. Recent incidents, including severe allergic reactions and several deaths have been attributed to heparin adulteration, resulting in a massive recall of heparin drugs by the manufacturer [1]. Oversulfated chondroitin sulfate (OSCS) is the heparin contaminant responsible for the adverse clinical events [2]. Because heparin is a drug commonly used in clinics, these adverse events have created a worldwide crisis and a call for an analytical method that can readily monitor the purity of heparin API before formulation of the drug. Here we present a simple method to separate and quantify OSCS in the presence of heparin. The method uses anion exchange chromatography to achieve complete separation between heparin and OSCS on a 4.0 x 250 mm Spherisorb 5 µm SAX column, followed by UV detection at 202 nm to quantify the concentrations of heparin and OSCS. The reproducibility of the chromatographic separation indicated very good retention time RSD (relative standard deviations) values for both heparin (0.08%) and OSCS (0.05%). The dynamic range of the method was linear over two orders of magnitude for heparin concentrations ranging from 0.1 mg/mL to 5 mg/mL. The results dem¬onstrate that this method not only generates reproducible, fast separations (10 minutes) but also is able to detect OSCS at a concentration of less than 1% of overall content. This sensitive testing method can be used for monitoring heparin quality and OSCS adulteration in order to protect patient health. References: 1. Chem. & Engineering News, 2008, 86, p46. 2. Nature Biotechnology, 2008, 26, p669. P-126-M A Systematic Approach to the Evaluation of Regulatory Risk Associated with Process and Product Changes Throughout Development Vandana Chauhan; Vassia Tegoulia; Julia Edwards; Jennifer Ferrence; Jenifer Lundberg Genentech, Inc., South San Francisco, CA USA A risk assessment tool for the evaluation of regulatory risk associated with process and product changes has been developed. The use of risk assessment tools such as Risk Ranking and Filtering (RRF) provides a systematic approach to the evaluation of regulatory risk. Three factors are considered by the RRF tool: (1) phase of development, (2) type of molecule (i.e., platform knowledge), and (3) the potential impact of the change on the identity, strength and purity of the molecule as it relates to safety and efficacy. This approach to regulatory risk can be utilized throughout the product lifecycle and may be leveraged in regulatory submissions from IND to BLA. ICH Q9 Quality Risk Management, as well as, ICH Q5E Comparability of Biotechnological/Biological

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Products Subject to Changes in their Manufacturing Process and EMEA/CHMP/BMWP/101695/2006 Guideline on Comparability of Biotechnology Derived Medicinal Products After a Change in Manufacturing Process provided the foundation for the proposed risk factors and principles. NOTES:

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P-127-M Characterization and Comparability of Trastuzumab-DM1 Fred Jacobson; Yaning Wang; Aditya Wakankar; Javier Riverra; Chien Lee; Charity Bechtel; Yan Chen Genentech, Inc., South San Francisco, CA USA In spite of the high degree of molecular heterogeneity, lysine-linked antibody drug conjugates (ADCs) can be characterized using the same analytical techniques as used for non-conjugated monoclonal antibodies. T-DM1 is an ADC comprised of the her2 targeting monoclonal antibody, trastuzumab, linked to the anti-microtubule drug, DM1, through a heterobifunctional compound SMCC. T-DM1 has been manufactured at increasing scales to meet the needs of Phase I, II, and III clinical development. Product comparability has been demonstrated using a mix of techniques including chromatography (SEC and reversed-phase), mass spectrometry, capillary electrophoresis (CE-SDS and CE-IEF), peptide mapping, ELISA, and cell-based potency. Data will be presented demonstrating that the manufacturing process is highly reproducible across scales and that the product is stable during storage. The cell cytotoxicity assay for T-DM1 uses a breast cancer cell-line (BT474) that over-expresses the her2/neu receptor, and binding to this protein is also the basis of the ELISA. The excellent correlation between these two methods, including samples that have been stressed through high temperature storage, will be shown. This data suggests that the two potency assays for this product yield similar information about this product. P-128-M A DOE Approach to Setting System Suitability Criteria for HPLC Assays Clara Smith; Heather Ragonese-Baldwin; Greg Adams Diosynth Biotechnology, a part of Schering-Plough, Cary, NC USA System suitability criteria for HPLC assays are integral for ensuring that the complete testing system is suitable for the intended application. Criteria is typically set using a combination of development data and assay qualification data and may involve the use of theoretical plates, resolution, peak tailing, injector precision, capacity factor, or other calculations. This work illustrates the use of designed experimentation (DOE) in the form of robustness testing to aid in determination of system suitability criteria. An example using a monoclonal antibody is presented in which a standard system suitability criteria, theoretical plate number, was eliminated following analysis of DOE data. P-129-M Extractables/Leachables from Integrated Single-Use Systems in Biopharmaceutical Manufacturing Weibing Ding Pall Life Sciences, Port Washington, NY USA Leachables study has become an integral part of characterization of biopharmaceutical products. To successfully complete leachables evaluation and satisfy the regulatory requirements, the drug product manufacturer can rely on the full extractables data from all process contact surfaces, including single-use system, and design an efficient leachables test. Single-use systems are often applied right before the final filling

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line. Therefore there are concerns about chemical compounds that may migrate from the contact surfaces of single-use system into the finished biopharmaceutical products. To facilitate leachables study, it is imperative to develop a suitable method to study extractables from single-use systems. This poster presents a new set of results of the systematic study of extractables from single-use systems into water and ethanol via novel concepts, practical design, and analytical detection using advanced techniques. In order to tackle the fairly complex systems, we studied the components first, which included filters, sterile connectors, tubing and biocontainers, and then examined the full single-use systems comprised of filter, tubing, sterile connectors and biocontainers. This approach greatly simplified the identification of the extractable compounds from the integrated systems. The test design was based on actual biopharmaceutical manufacturing process conditions using a worst-case scenario. The complete extractables results were obtained using validated analytical methods, including quantitative non-volatile residue measurement and FTIR for qualitative assessment, headspace GC/MS for volatile compounds and direct-injection GC/MS for semi-volatile compounds, derivatization GC/MS for fatty acids, HPLC/UV/MS and LC/MS/MS for non-volatile and heat-sensitive compounds, and ICP/MS for inorganic elements. The poster provides a well-designed method for extractables/leachables study on single-use systems. The results are beneficial to characterization of biopharmaceuticals in contact with single-use systems. NOTES:

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P-130-M A Homogeneous Apoptosis Bioassay for Quantifying the Biological Activity of an Anti-CD22 Immunotoxin Hyun Jun Kim; Xu-Rong Jiang; Tianmeng Shao; Robert Strouse MedImmune, Gaithersburg, MD USA An anti-CD22 immunotoxin composed of the Fv portion of a humanized monoclonal antibody fused to a truncated form of Pseudomonas exotoxin A is being developed to target CD22-bearing tumor cells. This immunotoxin binds to the cell surface of CD22-bearing cells, is endocytosed, and the exotoxin domain is translocated to the cytosol. Once in the cytosol, it catalyzes the adenosine diphosphate (ADP) ribosylation of elongation factor 2, causing the inhibition of protein translation and cell death. Here we describe the development of a homogeneous bioassay for quantifying the bioactivity of an anti-CD22 immunotoxin. The bioassay measures the ability of the immunotoxin to induce apoptosis in a CD22-expressing human B-cell Burkitt’s lymphoma cell line (Daudi). Once bound to CD22 and internalized, anti-CD22 immunotoxin induces Daudi cells to undergo apoptosis via Caspase 3/7 activation. This caspase activation can be measured by a commercially available luminescent substrate. The amount of luminescence is proportional to the amount of apoptosis, and is quantified in a luminometer. Anti-CD22 immunotoxin test sample causes a dose-dependent induction of apoptosis of Daudi cells that is measured relative to a reference standard. This bioassay offers advantages over conventional anti-proliferation assays that include, reduction of assay time to under 1 day; and a homogeneous format that is simple to perform and amenable for use in a regulated environment. Assay performance data will be presented that includes accuracy, precision and specificity. P-131-M Efficacy-Indicating Bioassay for Arylsulfatase B Using the Natural Substrate of the Enzyme Ginger Chen; Charles Hague; Jeff Lemontt; William Prince; Erno Pungor BioMarin Pharmaceutical, Novato, CA USA An assay has been developed to measure the intracellular enzymatic activity of recombinant human Arylsulfatase B (rhASB) on its natural substrate, Deramatan Sulfate (DS) when the enzyme is taken up into cultured ASB-deficient human fibroblasts. The enzyme ASB is a lysosomal exohydrolase, cleaving sulfate from N-acetylgalactosamine-4-sulfate (GalNAc-4S) from the nonreducing terminal of glycosaminoglycan (GAG) structures. ASB-deficient cells accumulate DS, which may be partially hydrolyzed by other lysosomal hydrolases, with the reactions stopping if a GalNAc-4S residue is reached on the nonreducing end of the oligosaccharide. When rhASB is added to the culture medium, the enzyme is taken up, translocates to the lysosomes through the Cation Independent Mannose-6-Phosphate receptor, and intracellular DS is depleted, demonstrating that the uptake of rhASB is able to restore lysosomal function in an in vitro cell based assay. The accumulation and depletion of DS is measured by monitoring a characteristic disaccharide digestion product of DS by Laser Induced Fluorescence-Capillary Zone Electrophoresis (LIF-CZE). This assay measures depletion of DS independently of its molecular size or processing state: in this approach all DS-like substances accumulating in the absence of rhASB activity are considered natural substrates of the enzyme.

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P-132-M MALDI-TOF Peptide Mass Fingerprinting for Distinguishing Therapeutic Proteins – Ready for a QC Lab? Mary Zhu; Lin Gan; Min Zhu; Edward Hoff; Viswanatham Katta Genentech, Inc., South San Francisco, CA USA Growth in the number of therapeutic monoclonal antibodies (MAb) in the clinical pipeline created a need for an identity assay that ensures specificity, yet simple and robust enough for implementation in QC environment. Peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is widely used in proteomics arena for protein identification. There are several challenges in applying PMF method to MAbs as they are multi-chain proteins and have high sequence homology between them. It is also perceived to be difficult to implement an essentially a qualitative mass spectrometric method in QC environment. We simplified various aspects of the method - digestion, sample spotting, mass spectrometer parameters, submission of spectrum to search engine, and interpretation of search results. Here we demonstrate the robustness of the method (R&D lab) using five different antibody products. Though our focus is on clinical products, the assay was designed to distinguish them from marketed products as well. During the method transfer to the QC lab, we encountered several challenges for this new MS based assay. Many technical issues were considered in design of the test procedure as a simple platform assay to be practiced by people with less technical background in the MS field. Use of a simpler spectrometer (TOF only instrument) brought its own challenges in terms of mass accuracy and resolution. An internal calibration step became necessary to ensure high mass accuracy which is essential to the success of the assay. Though it is not necessary in principle, use of a reference material side-by-side ensured the test performed as expected. Separate acceptance criteria became necessary for non-antibody products. The simplicity and robustness of the assay was demonstrated by the applicability to >30 protein products. Each product could be uniquely identified and distinguished from the other therapeutic proteins reproducibly. P-133-M Detection and Identification of a Sequence Variant of a Recombinant Monoclonal Antibody During Stable Cell Line Development Charlene Li; Boyan Zhang; Kathleen Francissen Genentech, Inc., South San Francisco, CA USA In the early stages of cell line development for recombinant humanized monoclonal antibody expression, several stably transfected clones are evaluated for cell growth characteristics and product quality attributes before the final selection of a production clone. The integrity of the primary structure is one of the important quality attributes in this evaluation process. Identification of a sequence variant at a high level results in the rejection of the cell line. During the LC-MS analysis of reduced material of a recombinant humanized monoclonal antibody (referred to as rhuMAb) produced for an animal toxicity study, a strong signal with the mass of the predicted light chain plus 16 Da was detected in addition to the signals for the light chain and heavy chain of rhuMAb. The extra light chain species was isolated by a reversed phase HPLC method and characterized by utilizing tryptic and chymotryptic peptide mapping with LC-MS/MS characterizations. This species was identified as an amino acid substitution with a proline residue replaced by a leucine residue in the light chain of rhuMAb, instead of a commonly considered oxidation species with a mass shift of +16 Da. The analytical techniques developed for detecting and identifying the sequence variant will be presented.

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Poster Abstracts

Poster Session Two Wednesday, January 14, 2009

14:00 – 15:00 Pacific Terrace Foyer (Fourth Floor)

P-201-W The Linearity and Resolving Power of High pH Range Carrier Ampholytes Tiemin Huang; Jiaqi Wu Convergent Bioscience Ltd., Toronto, ON Canada It is known that the resolving power of basic pH range carrier ampholytes is relative low due to the current synthesis methodologies of the carrier ampholytes. In this presentation, the pH gradient linearity and resolving power of high pH range carrier ampholytes (Pharmalyte 8-10.5, Servalyt 9-11) were investigated by a set of well characterized pI marker using whole-column detection cIEF instrument, iCE280. The peak pattern of different pI markers were observed when different brand name carrier ampholytes were used. Separation resolution can be improved by using mixtures of carrier ampholytes of different brand names. P-202-W Risk Assessment for Critical Method Parameters in cIEF Method Jiaqi Wu Convergent Bioscience Ltd., Toronto, ON Canada In order to achieve proactive method lifecycle management instead of reactive method lifecycle management, risk assessment for method parameters is important during method development and validation. The assessment requires scientific understanding of the method parameters and how they influence the results. In past four years, Convergent Bioscience’s Fast IEF Analyzer, iCE280, has seen its many applications within the biopharmaceutical industry. In this poster, critical method parameters for method development and validation on the iCE280 Analyzer will be reviewed. They include carrier ampholytes, pI markers and instrument’s resolution and sensitivity.

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P-203-W Identification of Low Level Serine to Asparagine Misincorporation in a Monoclonal Antibody Produced by Chinese Hamster Ovary (CHO) Cell Expression Christopher Yu; Victor Ling Genentech, Inc., South San Francisco, CA USA Very low levels of serine (Ser) to asparagine (Asn) variants were discovered in a monoclonal antibody molecule produced by CHO expression. Initial identification was made during routine sequence variant analysis of trypsin digest facilitated with MASCOT error tolerant search. A single Ser in the variable region of the heavy chain was found to be substituted with Asn. The same substitution was apparent in samples from multiple clones, as indicated by the presence of a mass 27 Da higher than expected and approximately 2 minutes earlier in elution. Quantitation by peak area relative to that of the normal peptide mass in extracted ion chromatograms indicated approximately 0.1% level of substitution. Subsequent experiments with high resolution mass spectrometry confirmed the identity and presence of the Ser to Asn substitution within 2 ppm accuracy. A peptide with the corresponding Ser to Asn substitution was synthesized and used in spiking experiments that further confirmed the identity of the variant peptide. We hypothesized that this variant was the result of amino acid misincorporation during translation, and that the misincorporation might not be limited to a single Ser position. Upon closer examination, Ser to Asn substitution was indeed revealed in multiple tryptic peptides containing Ser residues. The levels of substitution were found in a range of 0.01% to 0.1% by relative peak areas in extracted ion chromatograms. Tandem mass spectrometric analysis identified at least six Ser residues where substitution occurs. These results support the conclusion that amino acid misincorporation during translation can be an alternate source of protein sequence variant in monoclonal antibodies produced by CHO expression. P-204-W Multi-Dimensional Chromatography for Monitoring Charge Heterogeneity of Monoclonal Antibodies in Cell Culture Medium Prajakta Chaudhari; Methal Albarghouthi; John Hope; Ziping Wei; Patricia Cash MedImmune, Gaithersburg, MD USA Charge heterogeneity of monoclonal antibodies is an important product quality attribute. It is typically caused by modifications, such as C-terminal lysine truncation, deamidation, and sialylation, occurring during cell culture processes. Therefore, monitoring the charge heterogeneity of antibodies during the production processes is helpful to ensure the quality of the antibody product. A multi-dimensional chromatography system was built as a potential process analytical technology (PAT) tool to monitor charge heterogeneity of monoclonal antibodies produced by cell culture processes. The system employs a single HPLC instrument equipped with a quaternary pump and three chromatographic dimensions: protein A affinity chromatography, size exclusion chromatography (SEC), and ion exchange chromatography (IEC). The system is automated, provides fast sample analysis (~ 1 hour), and is capable of providing rapid evaluation of antibody charge heterogeneity. The integration and automation of the multi-dimensional separation eliminate the need for sample handling, resulting in significant reduction of labor and time needed to perform the analysis off-line. The multi-dimensional separation was successfully applied to monitor changes in charge heterogeneity of an antibody during the cell culture process. The analysis conditions are applicable to several monoclonal antibodies with similar isoelectric points. The multi-dimensional chromatography system may be a useful PAT tool for monitoring antibody production processes to ensure that the desired charge isoform profile is maintained.

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P-205-W Mass Spectrometry Tools for Comparison of Protein Batches Beth Gillece-Castro; Barry Dyson; Keith Richardson; Marc Gorenstein; Daniel Golick; Diane Diehl; Thomas Wheat Waters Corporation, Milford, MA USA Qualitative and quantitative batch comparisons are used during the development of biopharmaceutical development. Optimization of cell culture, purification, formulation and stability require detection of modified amino acids. Defining the site and level of modification as it relates to changes in biopharmaceutical manufacture allow appropriate decisions to be made regarding final conditions which produce the most stable, safe and efficacious drug. Site-specific quantitation of degraded residues such as oxidation of methionine and tryptophan is most often performed by peptide mapping. Highly resolving chromatography, accurate mass LC-MS, and software tools have been combined to more efficiently correlate peptide maps with protein structure. Optimized chromatography still requires confirmation of peak identity and purity so it is useful to couple the separation to the exact mass measurements possible with an oa-ToF mass spectrometer. Peptides can be identified based on molecular weight, and co-elutions can be detected. This additional information links the chromatographic pattern to the structure of the protein and in this way the protein coverage of peptide maps can be readily assessed. Additionally, the amount of trace degradation or contamination can be assessed. When a batch of a 97kDa protein was compared to a batch treated to forced oxidation the methionine-containing and tryptophan-containing peptides were examined for changes in abundance. The speed and ease with which the comparison was made demonstrates the utility of the Biopharmalynx software package. P-206-W An Ion Mobility ESI-TOF MS for the Top-Down Analysis of Antibody Fragments Asish Chakraborty; Weibin Chen; John Gebler Waters Corporation, Milford, MA USA Top- or middle-down MS methodology directly fragments intact proteins or their components to obtain information for protein characterization. However, direct fragmentation of large proteins and their components tends to generate many different types of fragment ions, making the spectra interpretation and sequence deduction very difficult. The hybrid ion mobility/time-of-flight mass spectrometer possesses a unique ion-mobility separation (IMS) function and is capable of separating fragment ions by size, shape, and charge prior to mass spectrometric detection. In this presentation, we have demonstrated the utility of IMS separation coupling with CID in the top-down sequencing of antibody fragments for the characterization of a recombinant IgG1 monoclonal antibody. During these studies, intact antibodies were cleaved into 3 fragments using a limited proteolysis method with endoproteinase Lys-C followed by reduction with DTT to produce the light chain (LC), Fc half (Fc/2) fragment and F determinant (Fd). These fragments were separated using a reversed-phase LC in-line with a quadrupole ion-mobility/time-of-flight mass spectrometer. The fragmentation was done with a number of selected charge states. Fragments coming out the collision cell were subsequently transferred into the ion-mobility cell for ion

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separation based on the charges, sizes and masses prior to time-of-flight mass measurement. Spectral information that contains fragment ions of similar charge states were then generated from the drift scope by selecting different regions of m/z vs. drift time plots. This ion-mobility separation and post data processing lead to the simplification of fragmentation data, thus revealing low intensity peaks that would have been masked by high mass signals in a spectrum where no mobility separation is used. The post-fragmentation IMS separation thus allowed a segment of antibody sequence to be easily deduced.This technique has the potential to be used in identifying unknowns and sites of post-translational modifications associated with an antibody. NOTES:

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P-207-W An Automated LC/MS Data Analysis of Interferon: A Case Study Using Biopharmalynx Application Manager Joomi Ahn; Ying Qing Yu; Beth Gillece-Castro; John Gebler Waters Corporation, Milford, MA USA Interferon (IFN) α proteins are a group of genetically related cytokins with a wide spectrum of biological activities including antiviral, antiproliferative, and immunomodulatory properties. The goal of the study is to characterize and compare the recombinant Interferon α-2b proteins harvested from different cell growth conditions. Intact Interferon protein and the enzymatically digested protein were analyzed using Liquid Chromatography-Time of Flight Mass Spectrometry (LC/TOF-MS). LC/TOF-MS has been extensively used for biotherapeutic drug development. Conventionally, LC/TOF-MS data generated for these studies are interpreted manually or being processed with a limited automation to confirm protein masses and peptide maps. They are often time-consuming processes and offer limited information due to the high complexity nature of the data. BiopharmaLynx, a new application manager, significantly reduces data analysis time for LC/TOF-MS peptide mapping and intact protein analyses. It automatically annotates the peaks from LC/TOF-MS data. We demonstrated that BiopharmaLynx enables the automatic comparison of two batches of Interferon drug. Both the Top down Intact Protein analysis and the Peptide Mapping analysis lead to the same conclusion that is the two batch of Interferon only differs at N-terminus; the analyte batch has N-acetylation. P-208-W Online A2:Rapid Characterization of the Second Virial Coefficient Amy Hanlon; Michelle Chen Wyatt Technology Corporation, Santa Barbara, CA USA Measurement of weak protein-protein interactions is essential in a wide array of pharmaceutical and biotechnology applications, such as therapeutic protein formulation, protein mixture purification, and protein crystallization. Protein self-association can be quantified by the second virial coefficient, A2. A2 values are dependent on both the protein and its environment: Temperature, salinity, pH, chemical excipients, etc., can all induce dramatic shifts in A2. Positive A2 corresponds to repulsive intermolecular interaction, whereas negative A2 corresponds to attractive intermolecular interaction. Rapid determination of the buffer conditions yielding an optimal A2, such as a negative A2 for crystallization, is highly desirable. Wyatt Technology has developed an accurate, automated, and high-throughput method for measuring A2-Online A2. In Online A2, only a single stock solution is needed. In addition to online dialysis, the online A2 method has the added benefit of flushing the light scattering flow cell between each injection, helpful for researchers working with “sticky” proteins.

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P-209-W Refractive Index, Quantitation of High Concentration Protein Solution Without Sample Dilution Joseph Molon; Marisol Burt; Damiano Migani; Joe Siemiatkoski; Li-Ping Hsieh; Jeremy Pariseau; Elena Belitsky; Pete Bryngelson Biogen Idec, Cambridge, MA USA The determination of protein concentration is an essential test for release, stability and in-process samples. In QC the current preferred method of measuring drug substance and drug product concentration is UV-Vis spectrophotometry, which requires sample dilution if concentrations are greater than approximately 1 g/L. This apparently simple step becomes very difficult and require additional training when the sample concentration is very high and when the solution is viscous. It is the major reason for anomalous (AR) and out of specifications (OOS) results. A new method based on Refractive Index technology was developed that allows for measurement of protein concentration without dilution. The technique is widely used in food and beverage industries. Here we present the extension of this technology to the biopharmaceutical industry for the accurate and precise determination of protein concentrations in well defined matrices. The assay can be performed using Digital Handheld Refractometer. The instrument is reliable, easy to validate and operate in a GMP environment. It was shown that bovine serum albumin samples have a linear refractive index (nD) response over the range 20-220 g/L with R2 values > 0.999. A range of buffer excipients were systematically tested and shown to have minimal effects on the response of nD to protein concentration. High concentration samples of recombinant antibody (up to 180 g/L) were also examined and indicate that each protein formulation has a different nD response requiring a different calibration for each program similar to individual extinction coefficients currently used for A280 measurements. Method comparability study demonstrated that the RI based method and a conventional A280 method give comparable results. The study also showed that the RI method is more precise. Overall, the refractive index method is simpler and less time consuming than a conventional method allowing for significantly higher precision and minimal analyst interference. P-210-W CryoTEM as a Tool for Characterization of Liposomes in Solution Bridget Carragher; Clint Potter NanoImaging Services, Inc., San Diego, CA USA Direct determination of particle morphology (size distribution and shape), and assembly (lamellarity) of therapeutic lipid particles is critical to liposome characterization during pharmaceutical and biotechnology process development and formulation. CryoTEM (Transmission Electron Microscopy) is a method that allows for the direct observation of native fully hydrated biological structures including proteins and liposomes. The particles are preserved in their native buffer and the method does not require the use of heavy metal stains and thus the image contrast is directly representative of the particles morphology and free of artifacts. This allows for the direct visualization and analytical characterization of the morphology, assembly, and aggregation of nanoparticles in their solution state. The three-dimensional structure of the individual particles and their interactions can also be reconstructed from these cryoTEM images using tomographic data collection methods.

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In this poster, we show the results obtained by using cryoTEM to examine a liposome preparation composed of a bilayer-forming phospholipid and cholesterol and compare these results to those obtained using negative staining and freeze fracture methods. NOTES:

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P-211-W XPS Analysis of Protein Adsorption on Bioprocessing Materials Rebecca A. Zangmeister NIST, Gaithersburg, MD USA Protein therapeutics are adversely affected by protein aggregation. Aggregation can decrease the safety and efficacy of the therapeutic biologic protein product. Currently, the causes of protein aggregation and ways to avoid it are under investigation. The adsorption of protein therapeutics to bioprocessing materials used in production, storage and delivery may create nucleation sites for further protein aggregation. X-ray photoelectron spectroscopy was used to analyze protein adsorption on bioprocessing materials to screen for those with increased probability of contributing to aggregation of the protein therapeutic. The standard overlayer model and protein labeling were used to estimate the thickness of adsorbed protein layers. P-212-W N- and C-Terminal Sequencing of Proteins by Mass Spectrometry (MALDI-ISD) – Can Edman Sequencing be Replaced? Martin Blueggel1; Sonja Bailey1; Susanne Mette1; Rick Garretson2; Andreas Wattenberg1

1Protagen AG, Dortmund, Germany; 2Protagen Inc., Chester, NJ USA According to ICH Q6B N- and C-terminal primary structure sequencing is required for structural characterization of proteins. Today Edman degradation is the method of choice. However performance i.e. C-terminal sequencing is limited and no technical improvement has been achieved in the last decade. Conversely mass spectrometry performance has increased dramatically in sensitivity, accuracy and ease of use. Alternatives to Edman sequencing, such as MALDI-ISD have recently been developed. (Suckau, Resemann, Anal. Chem. 2003, 75, 5817ff). MALDI in-source decay (ISD) spectra are acquired on a MALDI-TOF mass spectrometer in reflector mode. The decay of an intact protein in the ion source of the MS provides fragments like c-ions from the N- and y-ions from the C-terminus. Reflector mode analysis of ISD spectra increased mass accuracy and resolution to facilitate a precise determination of sequence tags. Here we present our very positive experience on the analysis of more than 50 different proteins over the last five years among those therapeutic antibodies, N-terminal blocked, modified proteins and our recent results on the ABRF ESRG 2009 study. Additionally we compare pro and cons of both methods.

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P-213-W Single Drop Microextraction for the Analysis of Basic Drugs in Urine by Capillary Electrophoresis Jungyeon Kim; Kihwan Choi; Jihye Kim; Doo Soo Chung Seoul National University, Seoul, South Korea Basic drug compounds were preconcentrated using single droplet microextraction (SDME) and analyzed with capillary electrophoresis (CE). A small drop of an acidic aqueous run buffer covered with a thin layer of organic liquid was formed at the tip of a capillary by simple manipulation of the liquid handling functions of a commercial CE instrument. The basic analytes in a basic aqueous donor phase were concentrated into the acidic acceptor drop through the organic layer by the driving force of the pH difference between the two aqueous phases. Since the acceptor phase volume inside the 2-layer drop was in the nanoliter range, the enrichment factor, the equilibrium value of which is given by the volume ratio between the donor and acceptor phases, was greatly enhanced. From 5-min SDME with the agitation of the donor phase by a small stirrer retrofit to the CE instrument, enrichment factors were more than thousand-fold. In addition to preconcentration, sample cleanup is another advantage of SDME. Basic analytes in the urine were successfully analyzed with SDME/CE. P-214-W UPLC Method Development for a Mixed Protein Product Chad Marten1; Samuel Molesworth1; Susan Park2; Enona Gopinath2

1Hospira, Inc., McPherson, KS USA; 2Tercica, Inc., Brisbane, CA USA Monitoring the heterogeneity profile for lot release and stability studies typically requires several orthogonal techniques to address different modes of degradation. For small proteins, some type of chromatographic method is often the primary stability indicating assay. In the unique case of a product with two active ingredients that are well-characterized proteins, the number of assays needed for routine testing can be overwhelming. In this study, an ultra-performance liquid chromatography (UPLC) purity method was developed for a mixture of two small model proteins, insulin-like growth factor 1 (rhIGF-1) and human growth hormone (hGH). The primary degradation pathway of rhIGF-1 at accelerated conditions is by diketopiperazine formation and cleavage, resulting rhIGF-1 that is missing the two N-terminal residues. This variant is usually detected by a neutral reversed-phase HPLC (RP-HPLC) method that has a run time of 72 minutes. The primary degradation products of hGH at pH 6.0 are deamidated hGH and the cyclic imide at Asp130, which can be quantitated by ion exchange chromatography (IEC). Oxidized species of hGH and N-terminal clipped hGH are detectable by RP-HPLC. Both hGH methods typically run for 40 to 60 minutes. The UPLC method was developed to analyze mixtures of rhIGF-1 and hGH in a single run with enough resolution to establish it as the primary purity method for both proteins. The results show that the two proteins are easily separated from each other in a single 60 minute run. Product related variants normally detected by RP-HPLC and IEC are also apparent due to mixed mode separation. The primary degradation products for rhIGF-1 and hGH are identified in the UPLC chromatogram using stressed samples, as well as isolated variants. These studies show that UPLC is capable of significantly reducing the total analysis time of rhIGF-1/hGH mixtures, while providing resolution of the primary product-related degradation products.

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P-215-W A New Developed Hydrophilic Polymer-Based Ion Exchange Chromatography Column for Separation of Various Biological Molecules Noriko Shoji1; Akiko Matsui1; Masakatsu Omote1; Naohiro Kuriyama1; Ernest Sobkow2

1YMC Co., Ltd, Kyoto, Japan; 2YMC America, Inc., Allentown, PA USA YMC has developed new porous and non-porous ion exchange columns featuring QA and SP chemistries bonded to a hydrophilic polymer spherical bead. The columns are applicable for analysis and lab scale purification of MAb's and other proteins as well as DNA species. Separations in the poster show analytical and small scale preparative applications on various biological molecules. Current development plans call for expansion of the product line beyond the initial offering. P-216-W Transferability of Pipette Calibrations – A Prerequisite for Method Validation and Method Transfer A. Bjoern Carle; George Rodrigues ARTEL, Westbrook, ME USA High confidence in the amounts of liquid aliquots delivered or transferred is crucial in almost every laboratory setting. Establishing this confidence is dependent on two main variables: knowledge and proof of correctly functioning equipment and appropriately trained and skilled operators of such equipment. Frequent calibration of instruments not only ensures their correct functionality, while recurring operator training ensures that instrument users are following the same standardized procedures. Both quality requirements are important elements of ISO 17025. Calibration of an instrument means that its performance is traceable to the internationally recognized standards as set forth and maintained by the individual country’s National Standards and Measurement Agency (e.g., NIST in the United States), and that this traceability is then extended to the individual laboratory in which the instrument is used. Such extension of traceability requires an evaluation of measurement uncertainties at each link in the traceability chain. This poster will address different means of transferring this traceability to individual laboratories and/or instruments. The suitability of various calibration transfer standards pertaining to pipettes and liquid delivery devices will be discussed. The challenges faced when comparing “calibrated” pipettes between different laboratory sites or even different countries will be highlighted. A discussion and evaluation of different calibration methods and their uncertainties will be presented. Method transfers commonly occur from research laboratories to development sites, and then finally to production sites. It is imperative that all facilities operate under the same performance uncertainties, regardless whether they are located in the same building, the same city, or on different continents.

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P-217-W Single-Batch Production of Recombinant Human Polyclonal Antibody Soeren Rasmussen Symphogen A/S, Lyngby, Denmark Symphogen has developed an expression platform, Sympress, for production of target-specific recombinant polyclonal antibodies. The Sympress platform has been used for manufacturing of Sym001, a recombinant human polyclonal antibody comprising 25 different antibodies against the RhD erythrocyte antigen. The Sympress technology as used in Sym001 is based on generation of 25 individual polyclonal antibody library (PALS) cell lines. The PALS are generated using site-specific integration technology in a CHO Flp-In cell line to ensure insertion of each of the antibody encoding genes into the same genetic locus in the host cell. The characterization of the individual polyclonal antibody library stock (PALS) cell lines constituting Sym001, the cell banking approach, and the single-batch production strategy will be presented. Analytical methods examining the batch-to-batch consistency have been developed and examples at both the protein and mRNA level will be given in the poster presentation. P-218-W Manufacture of Sym002 Recombinant Human Polyclonal Antibody for Prophylaxis or Therapy of Smallpox Andrea Porchia Symphogen A/S, Copenhagen, Denmark Symphogen´s proprietary antibody technology provides methods for the generation of fully human antigen-specific, recombinant polyclonal antibody preparations representing a new class of therapeutic antibodies expected to have important clinical advantages compared to blood-derived immunoglobulin-based products and monoclonal antibodies. Sym002 is a recombinant polyclonal antibody product, consisting of 26 vaccinia virus-specific antibodies reacting against different viral proteins. Preclinical proof-of-concept has been established for Sym002, which has demonstrated superior neutralizing activity over commercially available VIG, both in vitro and in vivo. A manufacturing process as well as a series of release and characterization assays has been developed for the Sym002 product based on the knowledge, information, and process that was developed previously for Symphogen´s Sym001 product. This poster will focus on the up-scaling of the manufacturing process for Sym002 polyclonal antibody as well as on the strategy to release and characterize recombinant polyclonal antibody products.

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P-219-W Challenges in Measuring Comparability of Aggregation and Conformation for Biosimilars (and often innovator products too!) John Philo; Tsutomu Arakawa Alliance Protein Laboratories, Thousand Oaks, CA USA There is much current interest in analytical methods for demonstrating comparability of biosimilars to the reference product. Heightened concerns about potential differences in immunogenicity make it particularly important to show comparability of product molecular conformation as well as aggregate content and distribution. However key biophysical tools such as analytical ultracentrifugation, light scattering, and circular dichroism (CD) may not be applicable to the final product due to interference from excipients. In such cases some biosimilar developers have tried to re-create the innovator’s bulk drug substance by purifying the active ingredient out of the reference final product. While this approach may give material suitable for some analytical methods, we show that for erythropoietin (EPO) this creates a drug substance surrogate that is damaged and not equivalent with respect to aggregation. Further, in this case subjecting two different final products to the same re-purification protocol does not produce equivalent changes and thus would give a potentially invalid comparison. Some specifics about excipient interference with AUC, light scattering, CD, and/or SEC will be discussed, with examples. Polysorbate 20 or 80 can be particularly troublesome when present at levels above ~0.3% and may cause false peaks in SEC, sedimentation velocity, or DLS. Further, because the detergent-associated impurities in product samples typically differ from those present in the formulation buffers it is often not possible to correct for the detergent effects by running control samples or buffer blanks. For CD the UV absorbance of simple salts such as citrate can cause problems. The strong UV absorbance of bacteriostatic agents present in multi-dose formulations can pose severe challenges for CD and AUC using absorbance detection. Further, for AUC the presence of sugars or polyols at levels above ~2% can cause significant interference with aggregate detection due to unwanted density and viscosity gradients across the measuring cell. P-220-W Validation of a RP-HPLC Assay for Residual Solvent-Detergent in Hemofil M A. Maximilian Fernandez; Tina Badiei; Wentong Guo Baxter Healthcare Corporation, Thousand Oaks, CA USA Virus inactivation is a critical step in the production of biopharmaceuticals. The most dangerous viruses, such as HIV and the hepatitis B virus, are lipid-enveloped, so they can be inactivated by the solvent-detergent procedure. For Baxter’s monoclonal antibody-purified, solvent detergent-treated antihemophilic factor (Hemofil M), a combination of tri-n-butyl phosphate (TnBP) and octoxynol 9 is used to inactivate lipid-enveloped viruses in solution. To ensure that solvent-detergents have been effectively removed after final processing, residual concentrations are measured in the final drug product. A reversed-phase (RP) HPLC assay with refractive index detection (RID) to simultaneously quantify TnBP and octoxynol 9 replaces two tests that were performed separately: a capillary GC method for TnBP and an ion chromatography method for octoxynol 9. It is a QC-friendly assay that relies on just one sample preparation procedure and robust HPLC methods of quantitation. It is specific, sensitive, and accurate at levels of 18 ng TnBP per AHF International Unit and 50 ng octoxynol 9 per AHF International Unit. In this poster, the successful validation of this RP-HPLC assay according to ICH guidelines is presented.

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P-221-W From Selection of Antibodies to Immunogenicity Studies: The Use of Label-Free Techniques in Biotherapeutic Development Kevin Lindquist; Tanja Jarhede; Eva Pol; Pär Säfsten; Gary Franklin; Anita Larsson; Robert Karlsson GE Healthcare, Uppsala, Sweden Therapeutic antibodies represent a rapidly growing area, with close to twenty approved and more than a hundred in development. Once a therapeutic target is validated, antibody discovery proceeds through multiple stages. Expression, characterization, selection, formulation, production and quality control of antibodies are followed by clinical studies, where safety and efficacy are investigated. In this poster we demonstrate how Biacore™ analysis, providing product characterization based on label-free, real-time protein interaction data, can be integrated across the discovery and development process. In addition to the elimination of labeling requirements, the main advantage of this approach in pre-clinical development is that high quality data on antibody kinetics and affinity, epitope specificity and concentration can be obtained from one system. The same approach is rapidly gaining importance in clinical studies, where early detection of unwanted immune responses against therapeutics is desired. Here the real-time analysis allows detection of low/medium affinity anti-drug antibodies (ADA) with rapid kinetics, which may be of clinical relevance and are otherwise difficult to measure. Recent advances in Biacore technology also permit detection of unwanted immune response even in the presence of excess drug. P-222-W Thermodynamic Stability of Monoclonal Antibodies: Exploring the Phenomenon of Cold Denaturation Kristi Lazar; Tom Patapoff; Vikas Sharma Genentech, Inc., South San Francisco, CA USA Monoclonal antibodies (MAbs) are exposed to a variety of solution environments and stresses during processing. Various stresses can lead to the formation of non-native conformations, which contribute to physical instability. The susceptibility of a MAb towards unfolding can be probed through estimation of the free energy of unfolding, Delta_Gu. While readily obtained for single-domain proteins exhibiting reversible unfolding, it is often challenging to obtain Delta_Gu values for multi-domain proteins. We used guanidine hydrochloride (GuHCl) induced denaturation to obtain the unfolding curves (and subsequently Delta_Gu values) of two MAbs, MAb1 and MAb2 (differing only in the CDR sequences), and investigated the temperature-dependence of Delta_Gu to explore cold denaturation of these MAbs. Fluorescence and CD spectroscopy were used to follow the unfolding of MAb1 and MAb2 and the respective Fc domain with GuHCl from 0-50oC at pH 6.3. DSC was used to monitor the thermal denaturation. Unlike thermal denaturation, chemical denaturation was reversible for both MAbs. GuHCl induced unfolding curves of both MAbs and the Fc showed the presence of at least one intermediate. The results show that MAbs exhibit a temperature of maximal thermodynamic stability around ambient temperature and have a potential to undergo cold denaturation at sub-zero temperatures.

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P-223-W Comparability Studies of the Conformation of Interferon alpha-2 during Formulation and Deformulation Using the NMR Fingerprint Assay. Naim Panjwani2; Derek Hodgson1; Genevieve Gingras1; Simon Sauve1; Yves Aubin1

1Health Canada, Ottawa, ON, Canada; 2University of Waterloo, Waterloo, ON, Canada Development of analytical techniques for the characterisation of recombinant protein therapeutics are sometimes complicated by the presence of excipients in a given product. These may interfere with the method and may require their removal. In the latter case, a validation of the method may be required to correlate results in the absence of excipients with the formulated product. This paper reports the results of our investigation on the effects of various formulations on the conformation of IFN-alpha2a and IFN-alpha2b using the recently published NMR fingerprinting assay. Samples of labelled interferon were produced in our laboratory and their activity was inferred by comparing their NMR spectra with spectra recorded for the corresponding EDQM (European Directorate for Quality Medicine) reference standards. The proteins were then mixed with appropriate excipients to reproduce formulations used in innovator products of Roferon-A® and Intron® A and de-formulated by passage on a weak cation-exchanger. The conformation of IFN-alpha2 was monitored by 2D-NMR spectroscopy after the formulation and de-formulation procedures. By monitoring the conformation of the protein (active pharmaceutical ingredient) in the absence/presence of excipients, we show that the NMR fingerprinting assay is more than a new method for product analysis, it can also be used as a tool for method validation. P-224-W Approaches for Improving Peptide Isolation and Purification Jo-Ann Jablonski; Thomas Wheat; Diane Diehl Waters Corporation, Milford, MA USA Peptides play important roles in the physiology of organisms. Pure peptides are required to effectively study biochemical interactions in organisms as well as to understand structure-activity relationships in the development of peptide therapeutics. Many types of impurities complicate the isolation and purification of peptides. Failure, damaged, and deleted sequences, in addition to those sequences modified by incomplete peptide cleavage and deprotection require purification techniques that efficiently isolate the peptide product. Hydrophobic peptides that exhibit limited solubility are among the most difficult samples to purify. Solvents that dissolve crude peptide mixtures often jeopardize the chromatographic separation. Focused gradients and temperature control both move impurities away from the product of interest and increase sample solubility in the mobile phase. Optimized at-column dilution steps increase mass loading on the column and prevent high pressure problems associated with sample solubility. In this study, we illustrate the implementation of these three separation optimization techniques applied to Waters Peptide Separation Technology Columns to improve the isolation of peptides with large numbers of nonpolar amino acid residues.

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P-225-W High-Throughput Characterization of Biomolecules with Dynamic Light Scattering Sigrid Kuebler; Michelle Chen Wyatt Technology Corporation, Santa Barbara, CA USA Dynamic light scattering (DLS) is an established technique for determining the size of submicron particles, such as proteins and other biomolecules, liposomes, nanoparticles and compound aggregates. Typical applications are determining biomolecule size and size distribution, protein and RNA unfolding, thermal stability, aggregation and solubility behavior. With the DynaPro Plate Reader, a multitude of sample conditions, such as buffer compositions, concentrations, time and temperature stability can be screened automatically and directly in disposable microwell plates. P-226-W Characterizing MW and Stoichiometry of Protein Conjugates and Their Aggregates Michelle Chen Wyatt Technology Corporation, Santa Barbara, CA USA Characterizing aggregation of protein conjugates is a challenging task as the aggregates can vary not only in molar mass but also in stoichiometry and conformation. Traditional techniques using calibration or assumptions on conformation, like gel electrophoresis and size exclusion chromatography (SEC), often give erroneous results. We will explain the use of hyphenated detection system, multi-angle light scattering (MALS), UV absorbance, and differential refractive index (DRI) detectors, following SEC or field flow fractionation (FFF). The combined MALS, UV, and DRI detection can readily elucidate, for each data slice, the respective molar masses of conjugate, protein, and modifier of various protein conjugates and their aggregates. Examples will be discussed to demonstrate that this method is fast, simple, accurate, robust, and comprehensive. P-227-W Development of a Homogeneous Cell-Based Potency Assay for an Auristatin-Containing Antibody-Drug Conjugate Joyce Penelope Pasion ; Xu-Rong Jiang; Victoria Bushman; Tianmeng Shao; Robert Strouse MedImmune, Gaithersburg, MD USA An antibody-drug conjugate (ADC) comprising a potent anti-mitotic agent (auristatin) conjugated to a monoclonal antibody is being developed to target EphA2 expressing cancer cells. This ADC binds to the EphA2 receptors on the cell surface and is then internalized into the cell. Once inside the cell, the ADC is cleaved, releasing the auristatin moiety that inhibits tubulin polymerization and causes cell death. Here we will describe the development of a homogeneous cytotoxicity assay that quantifies the biological activity of this ADC. This bioassay measures ADC-induced cytotoxicity of the prostate cancer epithelial cell line PC3. After incubating PC3 cells in the presence of various concentrations of ADC reference standard or test sample, a single-step commercial reagent is added to the assay wells to detect ATP. The number of viable PC3 cells is proportional to the amount of ATP present in metabolically active cells. Based on the signals from the reference standard and

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test sample, the relative cytotoxicity of the test sample is determined. This bioassay offers a simple, homogeneous and robust assay format that is suitable for use in a quality control environment. Data on specificity, linearity and range, precision and accuracy are presented. NOTES:

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P-228-W Analysis and Characterization of Subdomains in Antibody IgG2 Subclass by Reversed-Phase Liquid Chromatography and Mass Spectrometry. Boxu Yan; Tamer Eris; Zac Yates; Robert hong; Weichun Wang; Gerd Kleemann; Jennifer Liu Amgen, Inc., Seattle, WA USA It has been known for decades that papain cleaves an IgG1 molecule to release Fab and Fc, which can be used for further analysis and characterization; however, papain was found unable to release such subdomains from an IgG2. Here we present a combinatory strategy, for the first time, to analyze and characterize the light chain, single chain Fc and Fab portion of the heavy chain of Ig G2 molecules released by papain cleavage under mild reducing conditions. These subdomains and their counterparts with specific modifications were well separated on reversed-phase high performance liquid chromatography (RP-HPLC) and characterized by online reversed-phase liquid chromatography time of flight mass spectrometry (LC-TOF MS). This strategy is also proven suitable for IgG1 molecules as well. This procedure not only provides a simplified way for characterization of antibody biomolecules by facilitating the detection of low level modifications at subunits with a higher sensitivity than peptide mapping, but also presented a new strategy as an identification assay to distinguish each IgG2 molecule on RP-HPLC, where highly conserved Fc domains remain at a constant retention time (RT) unique to the subisotype it belongs, and varying RTs in the light chain and Fab portion of the heavy chain distinguish the monoclonal antibody against other molecules of the same isotype based on the underlining characteristics for each antibody. P-229-W Photostability of a Recombinant Monoclonal Antibody Hongbin Liu; Patricia Molina; Yilma Adem; Osi Esue Genentech, Inc., South San Francisco, CA USA Evaluation of photostability of new biopharmaceutical drug substances and products is an integral part of drug development process. ICH has established guidelines for photostability studies. A recombinant monoclonal antibody (MAb) is subjected to light stress and the resulting changes were assessed by various functional and analytical methods. Compared with control samples, light stressed MAb showed different characteristics profile, including: 1) reduced potency (antigen binding assay); 2) increased aggregation (size exclusion chromatography and CE-SDS analysis); 3) different charge profile with decreased main peak and increased acidic region (imaged capillary IEF assay); and 4) presence of potential oxidation products in both the intact and reduced antibody (LC/MS analysis). A number of oxidation sites were identified by LC/MS/MS analysis of tryptic peptide map, including two methionine residues in the Fc region that were oxidized to an extent of 30 – 40%. Two tryptophan residues within the CDR regions were also identified to be oxidized to a smaller extent.

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P-230-W Identification of an Unusual High Molecular Weight Monoclonal Antibody Variant Connie Lu; Hongbin Liu; Dandan Liu; Paul Motchnik Genentech, Inc., South San Francisco, CA USA Aggregates are a common high molecular weight species observed in recombinant monoclonal antibody (MAb) preparations. Size Exclusion Chromatography (SEC) is the most commonly used method to separate and quantify antibody high molecular weight species. MAb X is an IgG1 subtype humanized MAb that is recombinantly produced in CHO cells. SEC analysis of MAb X resolved a peak, named Peak 1, which elutes between monomer and dimer peaks. All the MAb X lots produced from different clones and production scales have 0.2-0.3% of SEC Peak 1. In these same lots, peak 1 was not detected upon non-reduced Capillary Electrophoresis-sodium Dodecyl Sulfate (NR CE-SDS) with fluorescence detection analysis of MAb X (performed on either Beckman PA800 or Agilent 2100 Bioanalyzer). However, non-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (NR SDS PAGE) analysis of MAb X shows two minor bands right above the monomer band that are not commonly seen in other IgG1 molecules. To investigate this unusual high molecular weight species, the SEC Peak 1 fraction was collected from three different lots of MAb X bulk drug substances. The results of electrospray ionization time of flight (ESI-TOF) mass spectrometry, Bioanalyzer, and SDS PAGE analyses of the non-reduced and reduced SEC Peak 1 fractions demonstrate that the Peak 1 contains the MAb X with one or two additional light chains. The results of peptide map LC/MS analyses suggest that the extra light chain is linked to the MAb X light chain through disulfide bonds, which indicates that some MAb X heavy chains are linked to light chain dimers instead of a single light chain. Details of the peptide mapping that enabled identification of the disulfide bonding will be presented. P-231-W Characterization of a Humanized IgG4 Monoclonal Antibody’s Charge Heterogeneity Daniel McDonald; Jennifer Wang; Victor Ling Genentech, Inc., South San Francisco, CA USA Characterization of a monoclonal antibody’s charge heterogeneity distribution is important in understanding the quality attributes of that drug substance. Charge heterogeneity analysis becomes a powerful tool over the life of a product. It is frequently used throughout product development, such as during comparability assessment, and the charge profile typically becomes part of a marketed product’s control system. This poster describes how a CHO (Chinese hamster ovary cell) derived antibody’s charge variants were characterized, during a NS0 (a mouse myeloma cell line) to CHO cell line change. A novel approach to isolate and characterize the N-terminal glutamine variant, which rapidly converts to the cyclized pGlu variant, using a typical fractionation technique, is illustrated. The results of the charge heterogeneity analysis as part of the comparability assessment support the change from NS0 to CHO.

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P-232-W Characterization of a Monoclonal Antibody Using Peptide Mapping with UPLC-MSE Hongwei Xie; Martin Gilar; John Gebler Waters Corporation, Milford, MA USA Monoclonal antibodies are a class of recombinant proteins. They are subject to a variety of enzymatic or chemical modifications during expression, purification, and long-term storage. Effective control and monitoring these variations require a sensitive and reproducible strategy to characterize such product and process related modifications. We have applied an online Ultra Performance Liquid Chromatography –Data Independent Acquisition Mass Spectrometry (UPLC-MSE) approach to map digests of Herceptin monoclonal antibody. High (>97%) sequence coverage and detailed characterization of the antibody are presented. Tryptic digests of the antibody were separated on a 2.1x150mm, BEH300Å 1.7μm C18 ACQUITY™ PST column, then, eluted and fragmented in a SYNAPT™ MS™ instrument. Both precursor and fragment data of LC-eluted peptides were acquired in a parallel and unbiased mode. The collected data were processed by IdentityE for peptide sequencing and determining the type, site and stoichiometry of covalent modifications existed in the antibody. More than six N-deamination sites and one M-oxidation site from heavy chain and three N-deamination sites from light chain were identified from the antibody. The stoichiometry of these modifications was inferred from the relative ratio of the deconvoluated MS signal intensities between modified and unmodified peptides. Synthetic modified peptides were applied to further confirm the deamidations in “PENNY” peptide of heavy chain, distinguish aspartic and isoaspartic isoforms of N-deamidations, and exclude potential M-oxidation artifact produced in MS ion source. P-233-W Optimal Method Development for Separation of Monoclonal Antibodies Using ProPac Weak Cation Exchange (ProPac WCX) Columns: Effect of Fe3+ Complexes on Column Performance Srinivasa Rao; Chris Pohl Dionex Corporation, Sunnyvale, CA USA Charge heterogeneity is generally exhibited by monoclonal antibodies (MAbs) that can result from various reasons including oxidation, aspartic isomerization, asparagine deamidation, lysine truncations and glycan modifications. Manufacturing and subsequent stability testing procedures of MAbs involve routine analysis and monitoring of the impurities resulting from these situations. ProPac weak cation exchange (WCX) columns are widely used to characterize MAb heterogeneity. ProPac WCX columns are packed with non-porous particles which are well suited for high resolution analytical separations of both acidic and basic monoclonal antibody variants. Using ProPac WCX columns, we have separated MAbs on MES (pH 5.6), ACES (pH 6.7), and Tris (pH 7.3) and compared the influence of these conditions. Further, we studied the influence of varying pH, gradient conditions and temperature and discussed optimal conditions for separation of monoclonal antibodies.

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We have explored the problems associated with metal poisoning of ProPac WCX columns when stainless steel HPLC systems were used. Metal components in such HPLC systems corrode and form a metal complex when salts and low pH eluents are in contact with them. Such metal complexes from the corrosion will leach on to the column and inhibit its performance. We have tested this phenomenon with a laboratory experiment employing Fe3+ conjugates on the separation of MAb using ProPac WCX columns on inert HPLC systems. The loss of resolution and capacity were completely reversible after washing the column with oxalate solution suggesting that it was due to the metal binding on cation exchange sites. Earlier, we have shown that SST HPLC systems have a seasoning effect. These observations provide evidence that inert systems are ideal for MAb separations by cation-exchange chromatography. P-234-W Characterization of Antibodies by High Resolution Protein Analytical Methods Martin Blueggel1; Susanne Mette1; Andreas Wattenberg1; Richard Garretson2

1Protagen AG, Dortmund, Germany; 2Protagen Inc, Chester, NJ USA In recent years the development of biopharmaceuticals has increased enormously and also in the next years an immense growth is expected. The production of recombinant antibodies is a challenging field and changes in the protein structure or posttranslational modifications can occur with and without influence on the function of the protein and on the therapeutic efficacy. Currently modifications of therapeutic antibodies with high interests are glycosylation, deamidation/isomerisation or C-terminal Lysine cleavage although a full physicochemical characterisation program is required following ICH Q6B. Total characterization of therapeutic antibodies on the protein sequence can be achieved by employing a combination of classical protein analysis technologies and techniques with high resolution techniques like 2D PAGE and mass spectrometry (MALDI-MS, MALDI-MS/MS, MALDI-ISD, LC-MS and LC-MS/MS) supported by bioinformatics for detailed interpretation. We have developed methods for the analysis of therapeutic antibodies and these modifications. We show the analysis of the full primary sequence by high-confidence peptide mapping experiments, the glycan profiling by MALDI MS and MS/MS and the analysis of protein deamidation by isotope pattern simulation. Additionally we utilize high resolution 2D PAGE for the analysis of a HCP Reference, impurity analysis and process control in downstream processing. The present workflow delivers a comprehensive high quality data set for QC on biotherapeutics. NOTES:

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CMC FORUM EUROPE 2009 COLOR AD – SIDE ONE

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CMC FORUM EUROPE 2009 COLOR AD – SIDE TWO