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13th Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology Industry

(Mass Spec 2016)

Symposium Co-Chairs:

Eef Dirksen, Synthon Biopharmaceuticals B.V. Christopher Yu, Genentech, a Member of the

Roche Group

September 27-30, 2016

The Westin San Diego Hotel San Diego, CA USA

Organized by

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Table of Contents

Welcome Letter ................................................................................................. 3 Student Travel Grants ....................................................................................... 4 Program, Exhibitor and Media Partners ........................................................... 5 Scientific Program Summary ............................................................................ 8 Plenary and Session Abstracts .......................................................................... 16 Round Robin Table Topics ............................................................................... 40 Technical Seminar Abstracts ............................................................................ 42 Poster Abstracts ................................................................................................ 48

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Welcome to the 13th Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology Industry We are pleased to welcome you to the 13th Symposium on the Practical Application of Mass Spectrometry in the Biotechnology Industry. The focus of this Symposium is the application of mass spectrometry for product characterization, process monitoring, formulation development and release testing in the pharmaceutical industry. Since mass spectrometry is a critical technology for a wide array of applications, the meeting will provide scientists in the industry an opportunity to share their data and learn from their colleagues. The symposium will feature a diversified selection of speakers from academia, industry, and regulatory agencies, who will discuss applications of state-of-the-art mass spectrometry to proteins, glycans, nucleic acids, viral vectors and other molecules of therapeutic interest. The success of this symposium will depend not only on our experienced and knowledgeable speakers and workshop leaders but also on the interactions and open discussion that take place among the attendees. We encourage you to participate wholeheartedly in the discussion sections that have been designed to stimulate the exchange of ideas and information. We would like to thank the speakers who are giving generously of their time and resources, and you for your attendance, which will make this a successful endeavor. We gratefully acknowledge the generosity of our sponsors and exhibitors. Thank you to 908 Devices, AbbVie, Inc., Agilent Technologies, American Laboratory/labcompare, American Pharmaceutical Review, Analyst, Analytical Methods, Analytical Scientist, Biogen, Biognosys, BioProcess International, Bruker Daltonics, Inc., ChemComm, Chemical Science, ChemSocRev, CMP Scientific, Eurofins Lancaster Laboratories, Inc., Genetic Engineering & Biotechnology News, Genedata AG, Genentech, a Member of the Roche Group, Genovis AB, Green Chemistry, Integrative Biology, IPQ, LCGC, MedChemComm, The Medicine Maker, Molecular BioSystems, New England Biolabs, The Pathologist, Pfizer, Inc., Protein Metrics, ProZyme, Roche Diagnostics GmbH, Royal Society of Chemistry, separationsNOW.com, SCIEX, Technology Networks, Thermo Fisher Scientific and Waters Corporation. We are also thankful for the expert assistance and support of Renee Olson and CASSS, as well as the audiovisual expertise of Michael Johnstone from MJ Audio-Visual Productions. We hope you enjoy the conference, build new contacts and return for new information in 2017! THE ORGANIZING COMMITTEE: Greg Adams, FUJIFILM Diosynth Biotechnologies Alain Balland, Boehringer Ingelheim Pharma GmbH & Co. KG Michael Boyne, BioTechLogic, Inc. Patrick Bulau, Roche Diagnostics GmbH James Carroll, Pfizer, Inc. Steven Cohen, Northeastern University Eef Dirksen, Synthon Biopharmaceuticals B.V. (Co-chair)

Vivian Lindo, MedImmune, A member of the AstraZeneca Group Anders Lund, Genzyme, a Sanofi company Yelena Lyubarskaya, Codiak Biosciences Jun Park, CDER, FDA David Passmore, Bristol-Myers Squibb Company Jason Rouse, Pfizer, Inc. John Valliere-Douglass, Seattle Genetics, Inc. Christopher Yu, Genentech, a Member of the Roche Group (Co-chair)

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CASSS Mass Spec Student Travel Grants

CASSS is pleased to provide a limited number of student travel grants for PhD students and post-docs who present applicable posters at the 13th Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology Industry (Mass Spec 2016). PhD students or post-doctoral fellows conducting research at academic institutions throughout the world are eligible.

This symposium offers insight to current topics and issues under discussion within the biotech and biopharmaceutical industries, and as such, provides an opportunity to bridge between industry, academia, and regulatory agencies. The presentations and workshops are focused on the application of mass spectrometry to advance drug discovery and development in the biotechnology industry. Applications will highlight uses of MS in various areas of product development including lead selection & optimization, high throughput screening, identification of PTMs, process development and in-process testing, drug product characterization, higher-order structure, and adoption of innovative MS technologies. Participants will have an excellent opportunity to meet, network and participate in exchanging knowledge for mutual education with other MS practitioners.

Requirements are: -Present a poster on a MS topic -Proof of studentship/post-doc status -Recommendation from the supervisor/advisor

This year’s grant winners include:

Application of Quantitative Liquid Chromatography – Mass Spectrometry Techniques to Facilitate Enhanced Bioprocessing of Therapeutic Proteins Amy Farrell, NIBRT, Ireland Quantification of Serum Vitamin D Binding Protein by Novel LC-MS/MS Methodology Indicates Similar Concentrations in Blacks and Whites Clark Henderson, University of Washington, USA Monitoring the Site-Specific Glycosylation, Site Occupancy and other PTMs in Plant Bioengineered Recombinant Therapeutical Glycoproteins John Muchena, University of California, Davis, USA

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

Strategic Program Partners

Platinum Program Partners AbbVie, Inc.

Biogen

Gold Program Partner Pfizer, Inc.

Platinum Program Partners

Bruker Daltonics, Inc.

Genedata AG

Gold Program Partners

Agilent Technologies Genentech, a Member of the Roche Group

Roche Diagnostics GmbH SCIEX

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Exhibitor Partners

908 Devices

Agilent Technologies Biognosys

Bruker Daltonics, Inc. CMP Scientific

Eurofins Lancaster Laboratories, Inc. Genedata AG Genovis AB

New England Biolabs Protein Metrics Inc.

ProZyme SCIEX

Thermo Fisher Scientific Waters Corporation

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The Scientific Organizing Committee gratefully acknowledges the following media for their promotional consideration of Mass Spec 2016:

Media Program Partners

American Laboratory/labcompare American Pharmaceutical Review

Analyst Analytical Methods

The Analytical Scientist BioProcess International

ChemComm Chemical Science

ChemSocRev Genetic Engineering & Biotechnology News

Green Chemistry Integrative Biology

IPQ LCGC

MedChemComm The Medicine Maker

Molecular BioSystems The Pathologist

Royal Society of Chemistry separationsNOW.com Technology Networks

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Mass Spec 2016

Scientific Program Summary

Tuesday, September 27, 2016 08:30 – 09:30 Breakfast (Full Day and Morning Short Course Attendees ONLY) 08:30 – 13:30 Short Course Registration ONLY in the Pearl Room

Short Course in the Pearl Room Short Course Facilitator: Anders Lund, Genzyme, a Sanofi company, Framingham, MA USA

09:30 – 12:30 Fundamentals of Mass Spectrometry in the Analysis of Protein

Therapeutics Anders Lund, Genzyme, a Sanofi company, Framingham, MA USA

12:30 – 13:30 Lunch (Full Day Short Course Attendees ONLY) on the Pool Deck 13:30 – 17:00 Applications of Mass Spectrometry to Characterize Protein

Therapeutics Anders Lund, Genzyme, a Sanofi company, Framingham, MA USA

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Wednesday, September 28, 2016 07:30 – 18:00 Registration in the Ballroom Foyer 07:30 – 08:30 Breakfast in Emerald Ballroom 08:30 – 08:45 Welcome and Introductory Comments in the Crystal Ballroom

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

Keynote I Session in Crystal Ballroom

Session Chair: Michael Boyne, BioTechLogic, Inc., Glenview, IL USA 08:45 – 09:45 Development and Applications of Ultraviolet Photodissociation Mass

Spectrometry for Biological Problems Jennifer Brodbelt, University of Texas at Austin, Austin, TX USA

09:45 – 10:15 Break – Visit the Exhibits and Posters in Emerald Ballroom

ABC’s and XYZ’s of Sequencing Session in Crystal Ballroom Session Chairs: James Carroll, Pfizer, Inc. St. Louis, MO USA

and Christopher Yu, Genentech, a Member of the Roche Group, South San Francisco, CA USA 10:15 – 10:45 Mass Spectrometry-Based Sequencing to Rescue an Antibody From a

Lost Hybridoma Clone Jason Hogan, Bristol-Myers Squibb Company, Redwood City, CA USA

10:45 – 11:15 Traditional de novo Sequencing Methodologies Applied to Biosimilar

Product Development Jason Rouse, Pfizer, Inc., Andover, MA USA 11:15 – 11:45 Multiple Ion Activation Techniques for Improved Top-down and

Middle-down Antibody Characterization Luca Fornelli, Northwestern University, Evanston, IL USA 11:45 – 12:00 Lunch for Technical Seminar Attendees – Please take lunch and return

to Crystal Ballroom for the “Lunch and Learn”

12:00 – 12:30 Technical Seminar: Lunch and Learn MS-based In-depth Characterization of a Therapeutic Monoclonal Antibody for Identification and Monitoring of CQAs Miriam Böhm, Sanofi-Aventis Deutschland GmbH, Frankfurt am Main, Germany Sponsored by Genedata AG Crystal Ballroom

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Wednesday, September 28, 2016, continued 12:30 – 12:35 Mini Break for Technical Seminar Attendees 12:35 – 13:05 Technical Seminar: Lunch and Learn Overcoming the Challenges of Intact Biotherapeutics Characterization with Ultra-High Resolution Mass Spectrometers Keith A. Johnson, Pfizer, Inc., Andover, MA USA Sponsored by Bruker Daltonics, Inc. Crystal Ballroom 13:05 – 13:20 Break - Visit the Exhibits and Posters in Emerald Ballroom

CE-MS Session in Crystal Ballroom Session Chairs: Steven Cohen, Northeastern University, Boston, MA USA

and Steffen Kiessig, F. Hoffmann-La Roche Ltd., Basel, Switzerland 13:20 – 14:00 Microfluidic Capillary Electrophoresis-Electrospray Devices for

Analysis of Biopharmaceutical Materials J. Michael Ramsey, University of North Carolina, Chapel Hill, Chapel Hill, NC USA

14:00 – 14:30 Cutting-Edge Mass Spectrometry for mAbs & Related Product

Structural Characterization Elsa Wagner-Rousset, Centre d'Immunologie Pierre Fabre, Saint Julien

en Genevois, France 14:30 – 15:00 CE-MS Analysis of A Single Dose Pharmacokinetic Study of Two Fc-

Fusion Protein Constructs Mei Han, Amgen Inc., South San Francisco, CA USA 15:00 – 17:00 Poster Session I - Visit the Exhibits and Posters in Emerald Ballroom 17:00 – 18:00 Round Robin Table Discussions in Crystal and Emerald Ballroom 18:00 – 19:30 Welcome Reception at the Pool Deck

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Thursday, September 29, 2016 08:00 – 17:00 Registration in the Ballroom Foyer 07:30 – 08:30 Breakfast in Emerald Ballroom 07:30 – 08:30 Job Board Live Breakfast in Emerald Ballroom

Novel Technologies, Product and Process Characterization Session in Crystal Ballroom Session Chairs: David Passmore, Bristol-Myers Squibb Company, Redwood City, CA USA and

Jason Rouse, Pfizer, Inc., Andover, MA USA 08:30 – 09:00 Measuring the Quality of Immunization-induced Immune Responses

using Hydrogen/Deuterium Exchange Mass Spectrometry Lee Frego, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT USA

09:00 – 09:30 Performance of a Chip-based CE-MS Interface for Biotherapeutic Characterization: Evaluation and Applications Justin Sperry, Pfizer, Inc., Chesterfield, MO USA

09:30 – 10:00 Ion Mobility-Mass Spectrometry and Collision Induced Unfolding

Rapidly Detect Subtle Differences in Antibody Glycoforms Yuwei Tian, University of Michigan, Ann Arbor, MI USA

10:00 – 10:30 Break - Visit the Exhibits and Posters in Emerald Ballroom 10:30 – 11:00 LC-MS Characterization of Adeno-Associated Virus Capsid Proteins Xiaoying Jin, Genzyme, a Sanofi company, Framingham, MA USA 11:00 – 11:30 Simultaneous Monitoring of Multi-Attributes for Product Attribute

Control (PAC) by Mass Spec Based Method Yi Wang, Merck & Co., Inc., Kenilworth, NJ USA 11:30 – 12:00 Investigating Therapeutic Protein Structure and Binding Interactions

with Diethylpyrocarbonate Labeling and Mass Spectrometry Robert Vaughan, Indiana University, Bloomington, IN USA

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Thursday, September 29, 2016, continued 12:00 – 12:15 Lunch for Technical Seminar Attendees – Please take lunch and return

to Crystal Ballroom for the “Lunch and Learn”

12:15 – 12:45 Technical Seminar: Lunch and Learn Developments in CE/MS Analysis of Proteins Michael D. Knierman, Eli Lilly and Company, Indianapolis, IN USA Sponsored by Agilent Technologies Crystal Ballroom 12:45 – 12:50 Mini Break for Technical Seminar Attendees 12:50 – 13:20 Technical Seminar: Lunch and Learn The Simpler and Faster Solution for Routine Biopharmaceutical Characterization Sean McCarthy, SCIEX, Brea, CA USA Sponsored by SCIEX Crystal Ballroom 13:20 – 15:20 Poster Session II - Visit the Exhibits and Posters in Emerald Ballroom

Young Scientist Session in Crystal Ballroom Session Chairs:

Alain Balland, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany 15:20 – 15:40 Rapid Characterization of Biotherapeutic Proteins by Size-exclusion

Chromatography Coupled to Native Mass Spectrometry Anna-Katharina Heidenreich, Roche Diagnostics GmbH, Penzberg,

Germany 15:40 – 16:00 Application of Quantitative Liquid Chromatography – Mass

Spectrometry Techniques to Facilitate Enhanced Bioprocessing of Therapeutic Proteins Amy Farrell, NIBRT, Dublin, Ireland

16:00 – 16:20 LC-MS/MS Approach to Amino Acid Misincorporation Detection for

Monoclonal Antibodies Benjamin Moore, Genentech, a Member of the Roche Group, South San

Francisco, CA USA 16:20 – 17:30 Exhibitor Reception - Visit the Exhibits in Emerald Ballroom

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Friday, September 30, 2016 08:00 – 14:00 Registration in the Ballroom Foyer 07:30 – 08:30 Breakfast in Emerald Ballroom

Regulatory Session in Crystal Ballroom Session Chair: John Valliere-Douglass, Seattle Genetics, Inc., Bothell, WA USA

and Anders Lund, Genzyme, a Sanofi Company, Framingham, MA USA 08:30 – 09:00 Bioanalytical Strategies for Next-Generation ADCs using Hybrid

Immunoaffinity LC-MS/MS Violet Lee, Genentech, a Member of the Roche Group, South San Francisco, CA USA

09:00 – 09:30 Influencing Early-stage Process Development with Peptide Mapping

Catherine Eakin, Seattle Genetics, Inc., Bothell, WA USA 09:30 – 10:00 Break - Visit the Exhibits and Posters in Emerald Ballroom 10:00 – 10:30 Mass Spectrometry: What is it Good For? Terry Cyr, Health Canada, Ottawa, ON Canada 10:30 – 11:00 Opportunities and Challenges in Applying LC-MS Multi-Attribute

Method (MAM) to Biopharmaceutical Release Li Zang, Biogen, Cambridge, MA USA 11:00 – 11:30 Panel Discussion 11:30 – 11:45 Lunch for Technical Seminar Attendees – Please take lunch and return

to Crystal Ballroom for the “Lunch and Learn”

11:45 – 12:15 Technical Seminar: Lunch and Learn Streamlining Workflows for Sequence Variant Analysis in Biotherapeutic Characterization Michelle English, Pfizer, Inc., Andover, MA USA Sponsored by Protein Metrics Crystal Ballroom

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Friday, September 30, 2016, continued 12:15 – 12:20 Mini Break for Technical Seminar Attendees 12:20 – 12:50 Technical Seminar: Lunch and Learn Deploying MS in Late Development and QC: Defining Fit for Purpose Scott J. Berger, Min Du, Waters Corporation, Milford, MA USA Sponsored by Waters Corporation Crystal Ballroom 12:50 – 13:05 Break - Visit the Exhibits and Posters in Emerald Ballroom

Bioanalytical Session in Crystal Ballroom Session Chairs: Eef Dirksen, Synthon Biopharmaceuticals B.V., Nijmegen, Netherlands and Vivian Lindo, MedImmune, A member of the Astra Zeneca Group, Cambridge, UK

13:05 – 13:35 Bioanalytical Strategies for the Quantitation of in vivo Site Specific

Modifications of Therapeutic Antibodies in Early Discovery Cyno Studies using IP-LC-MS

Celia D’Arienzo, Bristol-Myers Squibb Company, Princeton, NJ USA 13:35 – 14:05 Characterizing the Molecular Integrity of Antibody-Drug Conjugates:

Applying Preclinical Learnings to the Clinic Brooke Rock, Amgen, Inc., South San Francisco, CA USA 14:05 – 14:35 LC-MS Based Platform for Drug Assay and its Applications to

Biotherapeutics: Fc-Fusion Proteins, Full Length Antibodies, and Antibody Variants

Hetal Sarvaiya, Merck & Co., Inc., Palo Alto, CA USA 14:35 – 14:40 Poster Award Announcement in Crystal Ballroom 14:40 – 14:50 Closing Comments in Crystal Ballroom

Eef Dirksen, Synthon Biopharmaceuticals B.V., Nijmegen, Netherlands

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Development and Applications of Ultraviolet Photodissociation Mass Spectrometry for Biological Problems

Jennifer Brodbelt

University of Texas at Austin, Austin, TX USA

Recent advances in instrumentation and experimental design have propelled mass spectrometry to the forefront for protein identification, biomarker discovery, and quantitative proteomics. Conventional bottom-up proteome analysis is based on the ability to sequence the constituent peptides of an enzymatically digested protein mixture. This limits the ability to determine sequence truncations, single nucleotide polymorphisms, and the combinatorial nature of post-translational modifications. The top-down approach, an alternative that involves the MS/MS analysis of intact proteins, provides both intact protein and fragment mass measurements and yields unparalleled maps of sequence and modifications. Activation and dissociation of intact proteins remains one of the major hurdles which has limited the widespread adoption of the top-down approach for proteomic studies. We have recently implemented ultraviolet photodissociation (UVPD) on a high performance mass spectrometer (the Orbitrap platform). UVPD affords broader sequence coverage via more extensive backbone fragmentation than can be obtained using any other MS/MS method, and ion activation/dissociation can be accomplished using a single 5 nanosecond laser pulse. This translates to an MS/MS technology that can characterize intact proteins in detail, including mapping post-translational modifications (PTMs) such as phosphorylation that play critical roles in cell signaling and metabolic regulation. In addition to using UVPD-MS to sequence and characterize proteins in high throughput LCMS applications, there has also been growing interest in employing top-down approaches to directly interrogate native-like protein structures, primarily using MS/MS methods to disassemble the complexes, sequence the proteins, and draw conclusions about protein conformation based on fragmentation behavior. Ultraviolet photodissociation (UVPD) in particular has provided unsurpassed levels of sequence coverage for native-like proteins and protein complexes.

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Mass Spectrometry-Based Sequencing to Rescue an Antibody From a Lost Hybridoma Clone

Jason Hogan

Bristol-Myers Squibb Company, Redwood City, CA USA

The large sequence diversity of monoclonal antibodies, resulting from the combination of different germline genes and somatic mutations, leads to incorporation of different amino acids relative to the translated germline genes. Many of these mutations occur in the complementary determining regions (CDRs), creating diverse antibody libraries for antigen recognition. Challenges are presented when the underlying DNA sequence of an antibody is unavailable, but it is critical to be able to express additional material for follow-up studies. An equipment malfunction resulted in the loss of multiple antibody producing hybridoma cell lines that had not been sequenced. Sequencing attempts of the remaining cellular material was unsuccessful and the only remaining material was a small amount of purified antibody from the lost hybridomas. A mass spectrometry-based workflow was utilized to determine the amino acid sequence of the antibodies from the hybridoma clones and identify all of the amino acid mutations. Multiple enzyme digestions were utilized to create a diverse set of peptides. These peptides were analyzed by LC-MS/MS and searched against a custom database of germline genes. The identified peptides were subsequently mapped to a target antibody sequence and the location and identity of all amino acid mutations were determined. Expression of the identified amino acid sequence resulted in an antibody with similar binding kinetics to the original antibody material. This mass spectrometry-based workflow allows both the heavy and light chain amino acid sequences to be identified simultaneously in a single experiment and can be used to assist DNA sequencing for amino acid sequence identification.

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Traditional de novo Sequencing Methodologies Applied to Biosimilar Product Development

Jason Rouse, Heather DeGruttola, Matthew Thompson, Chee-Keng Ng Pfizer, Inc., Andover, MA USA

Biosimilars, which are approved versions of originator biologic medicines, have the potential to significantly improve the lives of people around the world by expanding treatment options to patients. Because it remains highly challenging to produce an identical copy of any biologic medicine, a biosimilar must show no clinically meaningful differences from the originator’s reference product. Early biosimilar product development involves reverse-engineering the structural and functional quality attributes (QAs) of the originator biologic, as well as assessing the lot-to-lot variability for each QA. Consequently, these QAs are used to guide the design, development and optimization of a modernized manufacturing process for the proposed biosimilar. Multiple state-of-the-art analytical tools and robust methods are used at every process and product development decision point to establish that the nascent biosimilar has a “QA profile” that matches the originator protein molecule with a high degree of similarity. In principle, when both reverse engineering and process development are successful, then the structure and function of the biosimilar should be highly similar to the reference product via totality of evidence.

One of the first analytical activities in biosimilar product development is reverse-engineering the primary structure of the reference product, which for monoclonal antibodies (mAbs), involves elucidation of the correct full-length amino acid sequences for the light and heavy chain polypeptides. Regulatory agencies expect that the encoded amino acid sequence for the proposed biosimilar will be identical to that of the reference product. However, a reliable database with manually annotated, verified therapeutic mAb amino acid sequences remains elusive. For many therapeutic mAbs, the CAS Registry in combination with original patents, journal articles and/or Swiss-Prot is a dependable solution for generating a preliminary in silico full-length sequence. This preliminary sequence is confirmed experimentally with LC-MS(/MS) methodologies using a reference product sample, which then allows cell line development activities to commence. In this presentation, we will share our initial experiences and challenges in rapidly confirming preliminary mAb sequences to start cell line development on time. As biosimilars development strategy evolved, a more comprehensive de novo sequencing approach became necessary, which involved amino acid-by-amino acid validation with 100% sequence coverage (including Leu/Ile differentiation) via peptide mapping with multiple proteases, LC-MS/MS, Edman degradation, and nanoESI-MS/MS.

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Multiple Ion Activation Techniques for Improved Top-down and Middle-down Antibody Characterization

Luca Fornelli, Kristina Srzentić, Philip Compton, Neil Kelleher

Northwestern University, Evanston, IL USA

Top-down (TD) and middle-down (MD) mass spectrometry techniques offer important advantages for the characterization of biotherapeutics with complete molecular specificity. Particularly for antibodies TD/MD allow for reduced sample manipulation and therefore minimized introduction of artifactual modifications of the sample. This aspect is potentially crucial when evaluating the quality of production processes, storage conditions or emerging biosimilars. However, reaching the protein sequence coverage required to confidently verify the complete primary sequence of the antibody or map post-translational modifications can be challenging in TD/MD. Here, trihybrid Orbitraps (OT) combine high resolution detection of product ions with four different ion activation techniques. Specifically, we fragment either an intact, 150 kDa IgG or large antibody fragments (25-50 kDa, produced by rapid digestion with highly-selective proteases) via interaction with neutral molecules (higher-energy collisional dissociation, HCD), electrons (electron transfer dissociation, ETD) or high-energy photons (213 nm ultraviolet photodissociation, UVPD). Additionally, we applied combinations of these techniques (EThcD). Interestingly, these ion activation methods showed high degree of complementarity enabling >75% overall sequence coverage as read out in ProSight Lite (http://prosightlite.northwestern.edu/). Besides crude numbers, a particular focus was the correct assignment of product ions and also the development of strategies aimed at improving data quality.

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Microfluidic Capillary Electrophoresis-Electrospray Devices for Analysis of Biopharmaceutical Materials

J. Michael Ramsey

University of North Carolina, Chapel Hill, NC USA

We have pioneered the development of a sensitive, stable, and efficient microchip electrospray interface that enables the integration of MS detection with rapid and highly efficient microfluidic separation methods. The separative performance of these devices is near the theoretical diffusional limit for cationic species. This performance is achieved through the use of novel surface modification strategies that result in highly homogeneous surface characteristics. These devices yield electrospray ionization (ESI) performance commensurate with commercial nanoESI emitters without sacrificing the quality of microfluidic separations. Compared to CE-MS performed using fused silica capillaries, microchip CE-MS can achieve greater separation efficiency in shorter analysis times as the integrated injection and ESI functional elements greatly reduce extra-column band broadening. Microchip CE-ESI-MS has been used for challenging applications such as the characterization of intact biopharmaceuticals and antibody-drug conjugates, where achieving optimal separation efficiency is crucial for the success of the analysis. Moreover, peptide mapping can also be performed rapidly with high coverage. Most of these separations are completed in less than three minutes and most samples require minimal sample preparation.

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Cutting-Edge Mass Spectrometry for mAbs & Related Product Structural Characterization

Elsa Wagner-Rousset

Centre d'Immunologie Pierre Fabre, Saint Julien en Genevois, France

Monoclonal antibodies (mAbs) are highly complex tetrameric glycoproteins that must be extensively analytically and structurally characterized to become drug candidates. This is also true for biosimilar and biobetter antibodies and for IgG-related products such as antibody drug conjugates (ADCs). These immunoconjugates are based on highly cytotoxic Small Molecular Drugs (SMDs) covalently attached via conditionally stable linkers to mAbs and are among the most promising next generation empowered biologics for cancer treatment. ADCs are more complex than naked mAbs, as the heterogeneity of the conjugates adds to the inherent microvariability of the biomolecules. The development and optimization of ADCs rely on improving their analytical and bioanalytical characterization by assessing several critical quality attributes (CQAs), namely the distribution and position of the drug, the amount of naked antibody, the average drug to antibody ratio (DAR), and the residual drug-linker and related product proportions (SMDs). Progresses of multi-level (top, middle, bottom, small molecular drugs) state-of-the art mass spectrometry methods (Native MS, Ion Mobility-MS, CESI-MS, 2D-LC-MS, Extended Bottom Up, Top Down Sequencing) combined with chromatographic and electrophoretic techniques will be presented. They will be illustrated by extensive structural characterization of reference FDA and EMA approved therapeutic mAbs and ADCs.

• Wagner-Rousset E. et al. Fast workflow to rank critical versus non-critical charge variants of therapeutic antibodies. Journal of Chromatography A, 2016 in press.

• Beck A. et al Cutting-Edge MS methods for multi-level ADC structural characterization. Expert Review of Proteomics, 2016.

• Resenman A, Beck A et al. Full Validation of Therapeutic Antibody Sequences by Middle-Up Mass Measurements and Middle-Down Protein Sequencing. mAbs, 2016.

• Gahoual R, Beck A et al. Independent highly sensitive characterization of asparagine deamidation and aspartic acid isomerization characterization by sheathless CZE-ESI-MS/MS. J Mass Spec 2016.

• François YN, Beck A et al. Characterization of cetuximab Fc/2 dimers by off-line CZE-MS. Anal Chem Acta 2016.

• Gahoual R. Cutting Edge capillary electrophoresis characterization of monoclonal antibodies and related products. J. Chrom. B 2016.

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CE-MS Analysis of A Single Dose Pharmacokinetic Study of Two Fc-Fusion Protein Constructs

Mei Han

Amgen Inc., South San Francisco, CA USA

Abstract not available at the time of print.

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Measuring the Quality of Immunization-induced Immune Responses using Hydrogen/Deuterium Exchange Mass Spectrometry

Lee Frego

Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT USA

To determine the effectiveness of immunization strategies used in therapeutic antibody or vaccine development, it is critical to assess the quality of immunization-induced polyclonal antibody responses. Here we developed a workflow that uses sensitive methods to quantitatively and qualitatively assess immune responses against foreign antigens with regard to antibody binding affinity and epitope diversity. The application of such detailed assessments throughout an immunization campaign can significantly reduce the resources required to generate highly specific antibodies. Our workflow consists of two steps: 1) the use of Surface Plasmon Resonance (SPR) to quantify antigen-specific antibodies and evaluate their apparent binding affinities, and 2) the recovery of serum IgGs using an automated small-scale purification system, followed by the determination of their epitope diversity using Hydrogen Deuterium Exchange (HDX) coupled with mass spectrometry. We demonstrated that these methods were sensitive enough to detect antigen-specific IgGs in the ng/μl range, and that they provided information for differentiating the antibody responses of the various immunized animals that could not be obtained by conventional methods. We also showed that this workflow can guide the selection of an animal that produces high-affinity antibodies with a desired epitope coverage profile, resulting in the generation of potential therapeutic monoclonal antibody clones with desirable functional profiles. We postulate that this workflow will be an important tool in the development of effective vaccines to combat the highly sophisticated evasion mechanisms of pathogens.

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Performance of a Chip-based CE-MS Interface for Biotherapeutic Characterization: Evaluation and Applications

Justin Sperry

Pfizer, Inc., Chesterfield, MO USA

The diversity of biotherapeutics continues to broaden through the use of novel protein platforms, and for bioconjugates through the use of innovative conjugation chemistries. These facets, which often result in increased product heterogeneity, further emphasize the importance of thorough structural characterization to enable rapid process and product development. In-depth structural elucidation of complex biotherapeutics relies heavily on a variety of routine and non-routine mass spectrometry-based assays coupled with highly efficient separation techniques. This presentation will highlight the evaluation of a new chip-based capillary electrophoresis (CE) interface for the mass spectrometry characterization of biotherapeutic charge variants. A focus of the work will encompass that of intact and proteolytic mapping of monoclonal antibodies and antibody-drug conjugates (ADCs). The seamless integration of electrophoretic separations with nanoelectrospray ionization mass spectrometry affords excellent speed and resolution, coupled with high quality mass information for definitive species identification.

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Ion Mobility-Mass Spectrometry and Collision Induced Unfolding Rapidly Detect Subtle Differences in Antibody Glycoforms

Yuwei Tian, Brandon Ruotolo University of Michigan, Ann Arbor, MI USA

Glycosylation is one of the most intensively studied monoclonal antibody (mAb) post-translational modifications, having a significant impact on effector function, immunogenicity, plasmatic clearance, and resistance towards proteases. Many tools used to characterize mAb glycosylation, such as liquid chromatography coupled to bottom-up style mass spectrometry (LC-MS), allow for the detailed analysis of glycostructure and modification site; however, such data typically requires hours to acquire and is blind to changes in protein stability, as well as higher-order structure. Recently, we introduced an ion mobility-mass spectrometry (IM-MS) approach for intact mAb analysis, which involves the collision induced unfolding (CIU) of selected antibody ions. Collision energy is added to the antibody ions in a step-wise fashion, and 'fingerprint plots' are created that track the amount of unfolding undergone as a function of the energy imparted to the ions prior to IM separation. We have used these fingerprints to rapidly distinguish between antibody isoforms, possessing different numbers and/or patterns of disulfide bonds and general levels of glycosylation. Here, we present our latest data probing the information content accessible by rapid CIU analyses of both intact antibodies and antibody Fc Fragments modified with assorted heterogeneous or homogeneous N-linked glycans.

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LC-MS Characterization of Adeno-Associated Virus Capsid Proteins

Xiaoying Jin, Lin Liu, Catherine O'Riordan, Eric Pastor, Anders Lund, Karen Lee, Kate Zhang Genzyme, a Sanofi Company, Framingham, MA USA

Recombinant AAVs are popular vectors for gene therapy due to their nonpathogenic nature and ability of long term expression of therapeutic genes. As viral capsid proteins (VPs) are critical for viral infectivity, complete characterization of the viral capsid proteins including their sequence and post-translation modifications is required for gene therapy research and development. Conventional SDS-PAGE followed by in-gel digestion and LC/MSMS was used in the characterization of VPs, however, due to the limited recovery of peptides from the gel, the exact N-termini of the viral capsid proteins from various AAVs have not been confirmed by mass spectrometry in the literature. In this study, a direct LC/MS intact protein analysis method to characterize all VP isoforms in recombinant AAVs without prior SDS-PAGE separation was developed. Both the N- and C-termini of the VPs of selected AAV serotypes have been identified based on accurate mass measurement. This method can be used to confirm AAV serotype identity or monitor VP heterogeneity to support the recombinant AAV gene therapy development. Complete sequence coverage of AAV2 was achieved through multiple enzymatic digestions and LC/MS/MS peptide mapping. LC/MS/MS has confirmed the N, and C-termini of VPs and identified acetylation on the N-termini of VP1 and VP3, consistent with LC/MS intact protein analysis data.

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Simultaneous Monitoring of Multi-Attributes for Product Attribute Control (PAC) by Mass Spec Based Method

Yi Wang1, Richardson Douglas1, Richard Rogers2, Alex Fridman1, Bhumit Patel1, Mohammed Shameem1, Huijuan Li1

1Merck & Co. Inc, Kenilworth, NJ, USA, 2Just. Biotherapeutics, Seattle, WA USA

Therapeutic monoclonal antibodies are complex and heterogeneous biologics with complex mechanisms of action. In-depth characterization and monitoring of the product quality attributes (PQAs) of these biotherapeutics is essential to ensure that the protein drug products meet desired quality during manufacturing process and for use in clinical trials. To monitor the product attributes, a panel of different analytical approaches is needed. In this study, a Mass Spec based Multi-Attribute Method (MAM) was evaluated to simultaneously monitor most of the product quality at molecular level. Our results demonstrated the ability of MAM to potentially replace several methods currently used in quality control to release therapeutic molecules.

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Investigating Therapeutic Protein Structure and Binding Interactions with Diethylpyrocarbonate Labeling and Mass Spectrometry

Eric Graban1, Robert Vaughan2, John Hale3, Richard Vachet4 1QuarryBio, LLC, Tallahassee, FL USA, 2Indiana University, Bloomington, IN USA, 3QuarryBio, LLC, Klamath Falls, OR USA, 4University of Massachusetts, Amherst, Amherst, MA USA

Current technologies to study the structure of protein therapeutics are technically challenging (x-ray crystallograpy, NMR, HDX) or have limited resolution (CD, intrinsic fluorescence). Methods that can monitor changes in higher order structure that are relatively fast and straightforward are needed. We describe a method that uses covalent labeling with diethylpyrocarbonate (DEPC) and mass spectrometry (MS) to determine the site of protein-protein interactions. The method can also identify three dimensional structural changes to protein therapeutics upon exposure to perturbing conditions (e.g. thermal stress). Using Rituximab and human growth hormone (hGH) as test systems, we have identified specific protein regions that are impacted by thermal stress conditions. We also demonstrate that DEPC labeling can effectively identify the binding site of the CD20 antigen on Rituximab. DEPC labels Ser, Thr, Lys, Tyr, His and Cys side chains, covering up to 30% of the residues in the average protein, thus providing excellent structural resolution. In addition, DEPC labeling overcomes some significant shortcomings of methods such as hydrogen-deuterium exchange MS, as labeling is covalent and not subject to back-exchange. The DEPC label also greatly simplifies identification of modified residues, thereby increasing confidence in the resulting data. These advantages allow highly sensitive and reproducible data to be generated without the need for highly specialized equipment or training. Potential applications include protein binding site determinations and developabilty studies to support early stage biologic candidate selection.

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Rapid Characterization of Biotherapeutic Proteins by Size-exclusion Chromatography Coupled to Native Mass Spectrometry

Anna-Katharina Heidenreich

Roche Diagnostics GmbH, Penzberg, Germany

High-molecular weight aggregates such as antibody dimers and other side products derived from incorrect light or heavy chain association typically represent critical product-related impurities for bispecific antibody formats. In this study, an approach employing ultra-pressure liquid chromatography size-exclusion separation combined with native electrospray ionization mass spectrometry for the simultaneous formation, identification and quantification of size variants in recombinant antibodies was developed. Samples exposed to storage and elevated temperature(s) enabled the identification of various bispecific antibody size variants. This test system hence allowed us to study the variants formed during formulation and bioprocess development, and can thus be transferred to quality control units for routine in-process control and release analytics. In addition, native SEC-UV/MS not only facilitates the detailed analysis of low abundant and non-covalent size variants during process characterization/validation studies, but is also essential for the SEC-UV method validation prior to admission to the market

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Application of Quantitative Liquid Chromatography – Mass Spectrometry Techniques to Facilitate Enhanced Bioprocessing of Therapeutic Proteins

Amy Farrell1, Kai Scheffler2, Jenny Ho3, Peter Mowlds3, Patrick Pankert3, Andrew Williamson3, Ken Cook3, Jonathan Bones1

1NIBRT, Dublin, Ireland, 2Thermo Fisher Scientific, Dreieich, Germany, 3Thermo Fisher Scientific, Hemel Hempstead, UK

Chinese Hamster Ovary (CHO) cells lines are the dominant expression system used in the biopharmaceutical industry for the production of therapeutic proteins. Despite numerous improvements to CHO bioprocessing based on empirical observations, there is a still a very poor fundamental understanding of the molecular mechanisms governing biotherapeutic protein production from these important expression systems. Following serum free suspension batch culture, quantitative proteomics was performed on a Chinese Hamster Ovary (CHO) cell line that expressed an anti-Interleukin-8 IgG1 monoclonal antibody (mAb), to elucidate the cellular response to altered culture conditions of pH, temperature and dissolved oxygen. A multiplexing workflow using Tandem Mass Tags (TMT) and multi-dimensional liquid chromatography- mass spectrometry (LC-MS3) incorporating synchronous precursor selection (SPS) was applied for identification and quantification of proteins differentially expressed in response to culture conditions. In addition the multiplexing workflow was also utilised for the analysis of naïve CHO K1 cells following their exposure to spent culture media from the various production runs. Furthermore, complete characterisation of the expressed mAb was carried out using advanced LC-MS methods including high resolution middle down MS; MS determination of sequence-level modifications, intact protein analysis of critical quality attributes, stable isotope based quantitative glycan analysis, hydrogen deuterium exchange MS for structural comparability analysis and surface plasmon resonance spectroscopy for determination of changes to the functional integrity of the expressed mAb. Combined, the findings herein provide a holistic insight into the effect of upstream parameters on the quality of therapeutic proteins and facilitate a greater understanding of the molecular mechanisms governing biopharmaceutical production systems, thereby creating a hypothesis for improved future cell line development using various engineering strategies.

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LC-MS/MS Approach to Amino Acid Misincorporation Detection for Monoclonal Antibodies

Benjamin Moore

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

In recent years, peptide map LC-MS/MS has proven to be an extremely sensitive and versatile technique for general protein characterization and for the identification and quantification of sequence variants. Peptide map LC-MS/MS can be used to detect genetic mutations by careful examination of the MS and MS/MS data, searching for single amino acid substitutions. However, amino acid misincorporation typically results in a larger number of peptides containing the same type of amino acid substitution. This difference in biological amino acid substitution pattern potentially allows for an optimized data analysis approach for misincorporation detection, thus leading to higher throughput and better sensitivity compared to more traditional approaches. In this presentation, the detection of amino acid misincorporation by peptide map LC-MS/MS will be discussed. In addition to the 20 canonical amino acids, unusual amino acids (norleucine, methylnorleucine, and hydroxynorvaline) found in protein produced in E. coli are also detected and quantified in a high-throughput approach for multiple monoclonal antibodies. Electron transfer dissociation (ETD) is also used to provide side chain structural elucidation of these unusual amino acids.

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Bioanalytical Strategies for Next-Generation ADCs using Hybrid Immunoaffinity LC-MS/MS

Violet Lee, Surinder Kaur, Ola Saad Genentech, a Member of the Roche Group, South San Francisco, CA USA

Antibody-drug conjugates (ADCs) are a class of cancer therapeutics designed with the goal of minimizing severe side effects of conventional chemotherapy by targeted delivery of drugs to the tumor. ADCs are monoclonal antibodies conjugated with a cytotoxic drug to form a complex molecule containing characteristics of large-molecule biologics and small-molecule drugs. The complexity of ADCs propels the need to establish bioanalytical approaches that address both classes of molecules in assessing and understanding their pharmacokinetics (PK). The pharmacokinetics of an ADC can be assessed through a combination of mass spectrometry-based assays and immunoassays: total antibody, antibody-conjugated drug (acDrug) and unconjugated drug quantitation. To measure acDrug, we developed a hybrid immunoaffinity LC-MS/MS assay for disulfide-linked payloads to assess the PK of various new THIOMABTM antibody drug conjugates (TDCs). Given the same linker-drug, drug release kinetics was impacted by conjugation site, antibody tertiary structure, and surrounding pKa. The development of the acDrug assay for disulfide linked TDCs was naturally extended into developing a multiplexed assay which combines total antibody quantitation, with the potential of providing average drug-to-antibody ratio (DAR), a measurement of TDC stability. As the ADC landscape continues to evolve, next-generation ADCs require us to continually assess and adjust our bioanalytical strategies as needed.

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Influencing Early-stage Process Development with Peptide Mapping

Catherine Eakin

Seattle Genetics, Inc., Bothell, WA USA

Product quality concerns that can impact molecule stability and efficacy include post-translational modifications such as deamidation, oxidation, molecule clipping, etc. Peptide mapping with mass spectrometry detection has proven itself as a powerful tool for identifying and monitoring these product variants. However, the lengthy sample preparation, data collection and analysis times associated with peptide mapping have often limited the direct influence the data has on process development decisions. In this talk, I will present a case study of a monoclonal antibody with deamidation in a complementarity-determining region (CDR) region where peptide mapping was used to both inform and influence early-stage process development. With process decisions gated based on the observation of low or no change in deamidation a faster peptide map looking specifically at deamidation levels was implemented. Data from the peptide map was used to support several process decisions including cell culture duration, hold times during purification, and formulation development. Enabling peptide map data to be on the leading edge of process decisions directly resulted in improved product quality.

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Mass Spectrometry: What is it Good For? Terry Cyr Health Canada, Ottawa, ON Canada Advanced separation methods coupled to high resolution mass spectrometers can generate information-rich data from the analysis of complex biological products. The regulatory research community uses innovative software tools to interrogate these data and provide key information about products’ attributes such as: structure, stability, purity, and bioavailability. There is an increasing effort by some groups to expand the role of mass spectrometry (MS) into the characterization and regulation of biological medicinal products. This effort is especially focussed on recombinant biosimilar proteins, but also has been applied to more complex mixtures such as vaccines and cell based therapies. Data and conclusions from MS analyses are gradually filtering into regulatory presentations, product submissions and inevitably will be used as part of lot release testing. In this presentation, our efforts to use MS for the identification of influenza strains in multivalent vaccines and the simultaneous quantitation of all major antigens will be used for illustration. We compared the Hi3 method for the quantitative analysis of influenza vaccine antigens to methods requiring labeled isotopes. The data illustrate some key issues and highlight how MS analyses can constructively contribute to enhancing our understanding of biologics. NOTES:

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Opportunities and Challenges in Applying LC-MS Multi-Attribute Method (MAM) to Biopharmaceutical Release

Li Zang

Biogen, Cambridge, MA USA

The multi-attribute method (MAM) based on LC-MS peptide mapping has demonstrated its capability in quantitating primary sequence modifications and detecting clipped forms as well as other impurities in protein biopharmaceuticals. The quality attributes monitored using the MAM are potentially linked to the purity, impurity, N-glycan and charge heterogeneity profiles of the biopharmaceuticals. This offers an opportunity to replace multiple conventional release tests for these quality attributes using the MAM. Results of an IgG4 molecule analysis from MAM appeared to be highly correlated with the % acidic species from ion exchange chromatography (IEC) as well as the % terminal sialylation and galactosylation by N-glycan analysis; therefore, the MAM could potentially replace the IEC and N-glycan release assays for the IgG4. However, significant challenges need to be overcome before a LC-MS MAM method can be implemented in a GMP environment. Major challenges lie in the instrument and method variability, which makes accurate and consistent quantitation of low-level signals or detection of trace-level new impurity peak extremely hard; data processing automation and reliability as well as limited options of GMP-compliant data acquisition and processing software packages. Implementation of system suitability control, development of robust sample preparation and optimized LC-MS conditions, as well as use of GMP-compliant software packages such as Chromeleon may provide an opportunity to address some of the challenges. In addition, the correlation between MAM method and other conventional analytical methods need to be examined for each specific biopharmaceutical. Randomly distributed low-level modifications such as glycation may pose a challenge for reliable quantitation by the MAM; however those can be directly monitored using traditional separation method like IEC.

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Bioanalytical Strategies for the Quantitation of in vivo Site Specific Modifications of Therapeutic Antibodies in Early Discovery Cyno Studies using IP-LC-MS

Celia D'Arienzo, John Mehl, Bogdan Sleczka, Eugene Ciccimaro, Christian Caporuscio, Ekaterina Deyanova, Richard Huang, Timothy Olah

Bristol-Myers Squibb Company, Princeton, NJ USA

During drug discovery, therapeutic antibodies under consideration are evaluated for chemical stability in formulation buffer, typically undergoing stressed conditions including extremes of heat and pH. The “stressed” compound then undergoes full characterization by intact mass spectrometry to reveal site specific modifications to the protein sequence. Isomerization of aspartic acid and deamidation of asparagine are two common amino acid modifications that are of particular concern, especially if located within the complementarity-determining region (CDR) regions of the therapeutic antibodies since potency and binding affinity can be affected. Although the nature of these modifications is not enzymatic, there is the possibility of additional in vivo modification to these sites when dosed which can impact pharmacological and toxicological properties. With the information obtained from intact mass characterization, we have developed bioanalytical assays to quantify the extent of in vivo isomerization and deamidation of human monoclonal antibodies (mAbs) dosed in Discovery cyno studies employing immunoprecipitation (IP) and LC-MS-based detection providing selective enrichment, separation, and detection of naïve and modified forms of proteolytic peptides. In this presentation, the bioanalytical strategies and assays in three case studies are discussed. These case studies represent site specific aspartic acid isomerization of a bispecific fusion protein, a site specific asparagine deamidation within the light chain of a monoclonal antibody, and a site specific asparagine deamidation on the Fc portion of a second monoclonal antibody.

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Characterizing the Molecular Integrity of Antibody-Drug Conjugates: Applying Preclinical Learnings to the Clinic

Brooke Rock

Amgen, Inc., South San Francisco, CA USA

Characterizing the mechanisms of ADC instability and release of free cytotoxin are germane in the design of the next generation of ADCs. The metabolism and disposition of antibody drug conjugates (ADCs, especially in regards to cytotoxins that are conjugated to an antibody with a linker that requires cellular catabolism for release (e.g. non cleavable) is not fully understood. This presentation will discuss novel mass spectrometry assays that lead to the understanding of the mechanism behind release of the cytotoxin moiety, which is important in both the efficacy of the ADC as well as toxicity profile.

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LC-MS Based Platform for Drug Assay and its Applications to Biotherapeutics: Fc-Fusion Proteins, Full Length Antibodies, and Antibody Variants Hetal Sarvaiya, Daniela Tomazela, Maribel Beaumont

Merck & Co., Inc., Palo Alto, CA USA

The past few years have seen an increase in the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the quantification of protein drugs in biological matrices for early discovery and development of biotherapeutics. LC-MS/MS assays are orthogonal to traditional immunoassay and offer many complementing analysis characteristics based on the unique selectivity, the mass-based precision determination, and ease of multiplexing.

Multiple reaction monitoring (MRM)-MS approach measures one or more selected peptides that are unique to the target protein and serves as quantitative stoichiometric surrogate for protein concentrations in the sample. To develop an LC-MS/MS assay with good selectivity and sensitivity, the selection of surrogate peptides with prominent MS responses and unique amino acid sequences as well as immuno-affinity reagent for target enrichment is critical. Known concentrations of isotopically labeled 13C/15N (heavy) synthetic peptides are spiked in the sample and protein concentrations are derived from ratios of the peak areas measured for each heavy and light peptide.

Drug exposure is a very important readout for any in-vivo pharmacokinetics (PK) or efficacy study. Several factors can influence the PK properties and PK linearity of a mAb such as drug-target binding, complex formation, concentration and distribution of target antigen etc. Hence, it becomes crucial to consider the specificity of the assay for measuring bound, free, or total antigen along with the drug in order to compare PK behavior of different mAbs.

Here at Merck Palo Alto- we have developed several specific methods for measuring antibody based therapeutics, antibody variants, as well as fusion proteins from in-vivo studies. We use intact mass measurements to determine the capture specificity as well as any in-vivo modifications or degradation of the drug routinely along with the drug assay. Furthermore, we have utilized this platform to understand drug-target engagement by developing total antigen as well as drug bound antigen assay.

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Round Robin Table Discussions

Wednesday, September 28, 2016 17:00 – 18:00

There are 15 roundtable topics. The plan is for these to be active discussions, not presentations or lectures. To create useful discussion we are going to try and limit each topic to 10 attendees. Seating will be on a first come, first serve basis. These discussions will include a facilitator, whose role is to help assist the discussion and ensure a lively exchange, and a scribe, whose role is to make general, anonymous notes about the discussion that will be posted on the Mass Spec 2016 website. Listed below is a quick view of the Roundtable Topics, Facilitators, and Scribes:

Table 1 Multi-attribute Methods (MAM)

Rich Rogers, Just. Biotherapeutics Catherine Eakin, Seattle Genetics, Inc.

Table 2 Native Mass Spectrometry and its use in Biopharmaceutical Protein

Characterization David Passmore, Bristol-Myers Squibb Company Olga Friese, Pfizer, Inc. Table 3 Ion Mobility MS and its use in Biopharmaceutical Protein Characterization Scott Berger, Waters Corporation James Carroll, Pfizer, Inc. Table 4 Sample Preparation – Consistency, Efficiency, Robustness: How to Optimize Trina Formolo, NIST

Jason Hogan, Bristol-Myers Squibb Company Table 5 Best Practices for Quantifying Proteoforms in Peptide Maps

John Valliere-Douglass, Seattle Genetics, Inc. Benjamin Moore, Genentech, a Member of the Roche Group Table 6 Higher-order Structure Analysis by MS Vivian Lindo, MedImmune, A member of the AstraZeneca Group Carly Daniels, Pfizer, Inc.

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Round Robin Table Discussions Table 7 The Do's and Don’ts for MS Data Deconvolution and Quantitation Based on

Intact Protein Data Aaron Bailey, Thermo Fisher Scientific Jason Rouse, Pfizer, Inc. Table 8 Qualification/Validation of MS-based Characterization Methods for use in a

Regulated Environment Alain Balland, Boehringer Ingelheim Pharma GmbH & Co. KG John Schiel, NIST

Table 9 Best Practices for Host Cell Protein Analysis using MS Ying Zhang, Pfizer, Inc. Sean McCarthy, SCIEX

Table 10 Quantitation Differences Across Instrument Platforms Anders Lund, Genzyme, a Sanofi company StJohn Skilton, Protein Metrics Inc

Table 11 Maximizing the Benefit of Stable Isotope-labeled Internal Standards Steven Cohen, Northeastern University Wenzhou Li, Amgen, Inc. Table 12 Mass Calibration and Trending of Mass Spectrometer Stability Rama Tummala, Agilent Technologies Ewald van den Bremer, Genmab Table 13 Capillary Electrophoresis-MS Abigail Turner, NIST Xiaoying Jin, Genzyme, a Sanofi company Table 14 Best Practices for Di- and Trisulfide Detection via MS Jason Wood, Bruker Daltonics, Inc. Li Zang, Biogen

Table 15 What Should be Reported Considering with the Sensitivity of the Machines These Days?

Michael Boyne, BioTechLogic, Inc. Sarah Rogstad, CDER, FDA

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Technical Seminars Genedata AG Technical Seminar: Lunch and Learn Wednesday, September 28 12:00 – 12:30 Crystal Ballroom MS-based In-depth Characterization of a Therapeutic Monoclonal Antibody for Identification and Monitoring of CQAs

Miriam Böhm, Udo Roth, Annette Pieper

Sanofi-Aventis Deutschland GmbH, Frankfurt am Main, Germany The high complexity of biotherapeutic drugs such as monoclonal antibodies requires the identification of critical quality attributes (CQAs) which are then monitored in stability studies within the development process. Typical CQAs include protein deamidation, oxidation, and clipping within the protein backbone. Mass spectrometry is the state-of-the-art technology for the precise determination of these modifications, allowing site-specific, quantitative detection based on changes in molecular mass. This talk will focus on the application of different LC-MS-based technologies, including both bottom-up and middle-down approaches, and downstream data processing and investigation to ensure reproducible and reliable results. As a result of this case study, one Asn deamidation site in a CDR region and an oxidation site in the Fc domain were identified. Additionally, a halfmer formation was observed to a minor extent. The deamidation site was rated a high priority CQA. An LC-MS-based assay for monitoring its progression was established and is presented in this talk. NOTES:

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Bruker Daltonics, Inc. Technical Seminar: Lunch and Learn Wednesday, September 28 Crystal Ballroom 12:35 – 13:05 Overcoming the Challenges of Intact Biotherapeutics Characterization with Ultra-High Resolution Mass Spectrometers Keith A. Johnson, David Ripley, Andrew Saati, Lisa A. Marzilli, and Jason C. Rouse Pfizer, Inc., Andover, MA USA The maXis II and the Impact II, state-of-the-art, ultrahigh-resolution electrospray ionization hybrid quadrupole time-of-flight (ESI-QTOF) tandem mass spectrometers, were installed recently in our Analytical R&D (ARD) laboratories. ARD, a BioTherapeutics Pharmaceutical Sciences organization, is responsible for the structural and functional characterization of biotherapeutic product candidates at all stages of development. With these newly installed mass spectrometers, we analyzed a panel of protein standards and an in-house monoclonal antibody (mAb) via on- and off-line MS in an effort to benchmark routine mass accuracy levels for intact proteins. Additionally, we are establishing points of reference for instrument sensitivity, exploring the spectral accuracy of intact protein isotopic distributions, and verifying routine spectral deconvolution strategies for efficient data analysis by comparing the performance of these instruments with our previously-installed maXis instruments. Two case studies will be presented. First, a proof-of-concept study will demonstrate how ultrahigh-resolution can be used to detect deamidation in a monoclonal antibody. Next trisulfide detection in an intact mAb will be shown, and our method for estimating the level of trisulfides in the intact mAb will be discussed. The performance of the Bruker instruments, coupled with on-line techniques and accurate mass determinations, will advance our ability to rapidly verify product attributes for therapeutic proteins at the intact level, such as amino acid sequence fidelity and N-glycosylation distributions. For detailed product characterization, the Bruker instruments will provide enhanced capability to resolve and identify new protein isoforms with increased confidence. NOTES:

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Agilent Technologies Technical Seminar: Lunch and Learn Thursday, September 29 12:15 – 12:45 Crystal Ballroom

Developments in CE/MS Analysis of Proteins Michael D. Knierman Eli Lilly and Company, Indianapolis, IN USA CE/MS is gaining popularity mainly driven in improvement in mass spectrometry’s sensitivity. My talk will cover the implementation of a nanosheath CE/MS interface. The interface was placed on an Agilent 6550 and a Thermo orbitrap velos pro mass spectrometer using a commercial Agilent CE system. The primary aim was to develop a universal method for large protein separation online with mass spectrometry. Several problems were observed and solved such as bubble formation and contamination of the sheath liquid. These problems hampered its routine use in the laboratory. Improvements in operation and stability that yielded a robust platform for routine intact protein analysis will be described along with examples of protein separations using the nanosheath CE/MS. Using this improved interface a divert mechanism is enabled that allows selective deletion of a peak in the total ion electropherogram and diversion away from the MS source during capillary flushing. In summary, CE-MS has become a routine tool in my lab for solving critical problems in protein characterization. NOTES:

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SCIEX Technical Seminar: Lunch and Learn Thursday, September 29 12:50 – 13:20 Crystal Ballroom The Simpler and Faster Solution for Routine Biopharmaceutical Characterization Sean McCarthy SCIEX, Brea, CA USA Biotherapeutics are complex molecules which require in-depth characterization to accurately identify product critical quality attributes to ensure patient safety and efficacy. As new molecules move through development, there is an increasing need to use mass spectrometry for routine characterization workflows, such as intact mass and peptide mapping analyses, in downstream labs. Historically, implementation of mass spectrometry in downstream labs has been challenging due to complex software and instrumentation, which are not well-suited for non-expert users. In this seminar, we will discuss a streamlined solution for collecting and processing data for routine intact mass and peptide mapping workflows which brings the power of mass spectrometry for routine biopharmaceutical characterization to every user, regardless of their level of MS expertise. NOTES:

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Protein Metrics Inc. Technical Seminar: Lunch and Learn Friday, September 30 11:45 – 12:15 Crystal Ballroom

Streamlining Workflows for Sequence Variant Analysis in Biotherapeutic Characterization

Michelle English1, Paul Brown3, Jason Rouse1, Lisa Marzilli1, Eric Carlson2, Ilker Sen2, StJohn Skilton2, Chris Becker2

1 Pfizer, Andover, MA USA, 2 Protein Metrics Inc., San Carlos, CA USA, 3 Pfizer, Chesterfield, MO USA

Sequence variants (SVs) typically are low-level protein isoforms that contain unintended substitutions in the amino acid sequence as a result of genetic mutations, mistranslation events or misincorporations due to amino acid depletion. In order to select a production cell line without genetic SVs or amino acid misincorporations, mass spectrometric SV analysis is performed alongside genetic sequencing methods. However, SV analysis via mass spectrometry (MS) remains one of the most challenging assays for the characterization of therapeutic proteins even though automated data analysis tools are utilized. This is because SVs exist at very low abundances (e.g. a fraction of one percent), and consequently analysts often need to manually interrogate mass spectra with poor signal-to-noise to confirm each SV identification and rule out false positives. Additionally, there are SV masses which are easily confused with sample preparation artefacts or PTMs, and then monoisotopic masses are often miscalled causing further errors in identification. In this talk, we will demonstrate a streamlined SV methodology based on Orbitrap LC-MS/MS analysis of cell clone samples digested with 1-2 enzymes. The subsequent data analysis is conducted using Byonic™ to search a protein fasta file with all possible single-base mutation sequence variants and other modifications enabled. Byologic® software is used for inspection, validation, label-free quantification and report generation.

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Waters Corporation Technical Seminar: Lunch and Learn Friday, September 30 12:20 – 12:50 Crystal Ballroom Deploying MS in Late Development and QC: Defining Fit for Purpose Scott J. Berger, Jing Fang, Brooke Koshel, Robert Birdsall, Min Du, Weibin Chen, Ying Qing Yu Waters Corporation, Milford, MA USA The proposal of “Multi-Attribute Method (MAM)” based LCMS peptide mapping methods for semi-targeted monitoring of biotherapeutic protein attributes has been greeted with both excitement for their potential to streamline development processes through the reduction of redundant assays, and concerns over their appropriateness for deployment into regulated environments. The challenges of deploying and operating High-Resolution-MS (HRMS) methodologies in regulated environments must be weighed against more established and routine nominal mass detection approaches that may require more rigorous evaluation in establishing their suitability for MAM based analyses. This lunch seminar will explore the capabilities of HRMS and nominal mass detection for attribute monitoring workflows, and discuss suitable platforms for deploying these analyses in early development, regulated development and for QC lot-release. NOTES:

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Poster Abstract

Bioanalysis P-101-T Quantitative Proteomic Analysis of Acetaminophen Toxicity in Liver Micro Tissues using Data Independent Acquisition (DIA) Based Hyper Reaction Monitoring Mass Spectrometry (HRM-MS) Florian Marty1, Roland Bruderer1, Yue Xuan2, Fadi Abdi3, Chris Balagtas4, Oliver M. Bernhardt1, Tejas Gandhi1, Lukas Reiter1 1Biognosys AG, Zurich, Switzerland, 2Thermo Fisher Scientific, Bremen, Germany, 3Biognosys AG, Cambridge, MA USA, 4Biognosys AG, Zurich, Germany

P-102-W Assessing the Elimination of Therapeutic IgG1 Glycoform Variants in Humans using LC-MS/MS Qing Tang Janssen Pharmaceutical R&D, LLC, Malvern, PA USA

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Data Analysis and Software P-103-T Revolutionizing MS Data Processing, Analysis and Management in Late-phase Biopharmaceutical Development Nicolette de Wijs-Rot Merck, Sharpe & Dohme, Oss, Netherlands

P-104-W Enterprise Mass Spectrometry Software Solution Enabling Characterization of Biotherapeutics from Discovery and Development to Production and Quality Control Lem del Rosario1, Maurizio Bronzetti1, Joe Shambaugh1, Aude Tartiere1, Arnd Brandenburg2 1Genedata Inc., Lexington, MA USA, 2Genedata AG, Basel, Switzerland

P-105-T Electronic Annotation of LC/MS Peaks with Complementary Therapeutic Protein Modifications Jin Li1, William Haskins2 1Genentech, a Member of the Roche Group, South San Francisco, CA USA, 2Consultant, South San Francisco, CA USA

P-106-W 21st Century Intact Mass Deconvolution, Prettiness, and Automated Reporting Marshall Bern1, Yong Kil1, K. Ilker Sen1, Paul Dominic Olinares2, Alexander Ivanov3, St John Skilton1, Chris Becker1 1Protein Metrics Inc., San Carlos, CA USA, 2Rockefeller University, New York, NY USA, 3Northeastern University, Boston, MA USA

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Drug Product Characterization P-107-T LC-MS Method Development for Antibody Drug Conjugates Using a Non-Toxic ADC Mimic Brian Gau, Kevin Ray, Pegah Jalili, Benjamin Cutak MilliporeSigma, St. Louis, MO USA

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Higher-order Structure P-109-T Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) Analysis Revealed High Structure Similarity between IgG4 Wild Type mAb and IgG4 S228P Mutant mAb Qinqin Ji, Yuan Hu, Jeffrey Chapman, Brent Williams, Corey Strickland, Giovanna Scapin, Zhuyan Guo, Brad Sherborne, Huijuan Li, Mohammed Shameem, Yan-Hui Liu Merck & Co., Inc., Kenilworth, NJ USA

P-110-W Antibody Aggregate Interface Mapping by Hydrogen Deuterium Exchange Mass Spectrometry using a Customized Automation Instrument Qian Zhang, Hui Xiao, Shailin Patel, Yu Huang, Yunlong Zhao, Xiaobin Xu, Chen Li, Ning Li, Tom Daly Regeneron Pharmaceuticals, Tarrytown, NY USA

P-111-T Evaluation of Different Hydrogen/Deuterium Exchange Data Processing Software by Looking at Conformational Changes Induced by mAb Aggregation Terry Zhang1, Shanhu Lin2, Stephane Houel1, Jennifer Sutton1, Xiaodong Liu2, Jonathan Josephs1 1Thermo Fisher Scientific, San Jose, CA USA, 2Thermo Fisher Scientific, Sunnyvale, CA USA

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Innovative Mass Spec Technologies P-112-W Chip-Based CE-MS for Biotherapeutic Characterization and Analysis Carly Daniels, Justin Sperry, Josh Woods, Andrew Dawdy, Jason Starkey, Nathan Lacher Pfizer, Inc., Chesterfield, MO USA

P-113-T High Selectivity MRM (HS-MRM) Assay for Quantification of Host Cell Protein in Biotherapeutics Catalin Doneanu, Jing Fang, Yun Alelyunas, Ying Qing Yu, Mark Wrona, Weibin Chen Waters Corporation, Milford, MA, USA

P-114-T Echo-MS: A Versatile Platform for Ultrafast Mass Spectrometry Analysis in Drug Discovery Lucien Ghislain1, Martin Bachman2, Ian Sinclair2, Jonathan WIngfield2, Dan Addison2, Eric Hall1, Rick Stearns1, Lars Majlof1, Rich Ellson1, Rhys Jones3, Steve Pringle3, Sammy Datwani1 1Labcyte Inc, Sunnyvale, CA USA, 2AstraZeneca, Macclesfield, UK, 3Waters Corporation, Wilmslow, UK

P-115-W Microchip Electrophoresis with MS Detection for High-throughput Analytical Applications in Biopharmaceutical Development Seth Madren1, Li Zang2 1Biogen, Research Triangle Park, NC USA, 2Biogen, Cambridge, MA USA

P-116-W PALMS - Parallel Targeted/Untargeted Mass Spectrometry Platform Alexey V. Melnik1, Ricardo Da Silva1, Embriette Hyde2, Fernando Vergas1, Amina Bouslimani1, Ivan Protsyuk4, Theodore Alexandrov1,4, Rob Knight2,3,, Pieter C. Dorrestein1. 1Collaborative Mass Spectrometry Innovation Center, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA USA, 2Department of Pediatrics, University of California, San Diego, La Jolla, CA USA, 3Department of Computer Science & Engineering, University of California, San Diego, La Jolla, CA USA, 4European Molecular Biology Laboratory, Heidelberg, Germany

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Mass Spec in a Regulated Environment P-117-T Deploying MS in Late Development and QC: Defining Fit for Purpose Scott Berger, Jing Fang, Brooke Koshel, Robert Birdsall, Min Du, Weibin Chen, Ying Qing Yu Waters Corporation, Milford, MA USA

P-118-W NISTmAb Reference Material 8671: A Research Tool Grounded in Quality Measurements John Schiel NIST, Rockville, MD USA

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Post-translational Modifications P-119-T One-pot Consolidated Glycomics and Glycoproteomics Experiment using Permethylation of Glycoproteins and High-resolution Tandem MS Analysis Parastoo Azadi Complex Carbohydrate Research Center, Athens, GA USA

P-120-W Synergy Between CESI-MS and CE-LIF Analyses of Released, APTS-labeled N-glycans Edna Betgovargez1, Stephen Lock2, Bryan Fonslow1, Boglarka Donczo3, Marton Szigeti3, Andras Guttman3 1SCIEX, Brea, CA USA, 2SCIEX, Pudsey, UK, 3University of Debrecen, Debrecen, Hungary

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Process Characterization P-121-T Proteomic Identification of Bioprocess Markers for Cell Stress and Cell Death during Perfusion Culture Simone Albrecht1, Christian Kaisermayer2, Anna Lindeberg2, Jonathan Bones1 1NIBRT, Dublin, Ireland, 2BioMarin International Limited, Cork, Ireland

P-122-W Automated Data Analysis Enabling Mass Spectrometry–Based Assays Maurizio Bronzetti1, Joe Shambaugh1, Aude Tartiere1, Lem del Rosario1, Arnd Brandenburg2 1Genedata Inc., Lexington, MA USA, 2Genedata AG, Basel, Switzerland

P-123-T LC-MS/MS Peptide Mapping as a Half Antibody Charge Assay to Support Process Development Kasia Catherman1, Hongbin Liu1, Milady Ninonuevo1, Charlene Li1, Gihani Wijewickrama2 1Genentech, a Member of the Roche Group, South San Francisco, CA USA, 2Eurofins Lancaster Laboratories, South San Francisco, CA USA

P-124-W Mass Spectrometry as a Powerful Tool Box for Host Cell Protein Analysis Heiner Falkenberg, Anke Schnabel, Gerhard Koerting, Anja Dommermuth, Anna Lendzian, Thomas Flad, Roland Moussa Protagen Protein Services GmbH, Dortmund, Germany

P-125-T Evaluation of High Temperature Short Time (Htst) Treatment on Industrial Cell Culture Media by Untargeted LC-MS/MS Analysis Patrick Floris1, Christian Kaisermayer2, Anna Lindeberg2, Jonathan Bones1 1NIBRT, Dublin, Ireland, 2BioMarin International Limited, Cork, Ireland

P-126-W Misincorporation in Biotherapeutics: Demonstration of Process and Product Consistency by Quantitative Mass Spectrometric Methods Olga Friese1, Jason Rouse2, Kathleen Cornelius3, Kristin Boggio2, Lisa Marzilli2, Matthew Thompson2 1Pfizer, Inc., Chesterfield, MO USA, 2Pfizer, Inc., Andover, MA USA, 3Pfizer, Inc., St. Louis, MO USA P-127-T Identification of Compound Signatures of Process Variability and Development of a Targeted LC-QqQ-MS Method for Subsequent Detection and Quantification Nicola McGillicuddy, Jonathan Bones NIBRT, Dublin, Ireland

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P-128-W Multi-Attribute LC-MS/MS Peptide Mapping for High Throughput Cell Culture Testing without Purification Support Matthew Traylor Shire Pharmaceuticals, Lexington, MA USA

P-129-T Optimization of Orthogonal HCP Analysis by LC/MS/MS Facilitates Bioprocess Understanding Ying Zhang1, Michelle English2, Joseph Martin1, Phoebe Baldus1, Justin Sperry1, Jason Starkey1, Brian Korniski1, Bruno Figueroa2, Ned Mozier1, James Carroll1, Jason Rouse2, Olga Friese1 1Pfizer, Inc., Chesterfield, MO USA, 2Pfizer, Inc., Andover, MA USA

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Process Characterization P-130-W A Streamlined Analytical Platform with GU Scientific Library Searching for Glycan Comparability Assessment of Innovator and Biosimilar Infliximab William Alley, Ying Qing Yu, Min Du Waters Corporation, Milford, MA USA

P-131-T Integrated Characterization of a Lysine-linked Antibody-drug Conjugate by Native Intact Mass Analysis and Peptide Mapping Performed on a Hybrid Quadrupole-Orbitrap Mass Spectrometer with High Mass Range Aaron Bailey1, Stephane Houel1, Eugen Damoc2, Kai Scheffler3, Jonathan Josephs1 1Thermo Fisher Scientific, San Jose, CA USA, 2Thermo Fisher Scientific, Bremen, Germany, 3Thermo Fisher Scientific, Dreieich, Germany

P-132-W Versatile Data Processing Software Leverages Orbitrap Data for Intact and Sub-unit Mass Analysis of Protein Biotherapeutics Aaron Bailey1, Stephane Houel1, Jennifer Sutton1, Eugen Damoc2, Kai Scheffler3, Jonathan Josephs1 1Thermo Fisher Scientific, San Jose, CA USA, 2Thermo Fisher Scientific, Bremen, Germany, 3Thermo Fisher Scientific, Dreieich, Germany

P-133-T Comparative mAb Charge Heterogeneity Assays by CESI-MS and CZE/cIEF-UV Analyses Edna Betgovargez1, Stephen Lock2, Bryan Fonslow1, Ying Zhang3, Olga Friese3, Steven K. Cook3, John Yates III4, Jason Rouse5 1SCIEX, Brea, CA USA, 2SCIEX, Pudsey, UK, 3Pfizer, Inc., Chesterfield, MO USA, 4The Scripps Research Institute, La Jolla, CA USA, 5Pfizer, Inc., Andover, MA USA

P-134-W Application of a Mass Spectrometry Based Quality Control Method for Characterization and Attribute Monitoring in Antibody Drug Conjugates Michael Blank, Stephane Houel, Aaron Bailey, Jonathan Josephs Thermo Fisher Scientific, San Jose, CA USA

P-135-T Poly(D,L-lactide) Nanofibers with Poly(ethylene glycol) Additive as Cyclosporine A Delivery System Controlled by HPLC-MS/MS Zuzana Bosakova1, Zuzana Hampejsova1, Jakub Sirc2 1Charles University in Prague, Prague, Czech Republic, 2Academy of Sciences of the Czech Republic, Prague, Czech Republic

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P-136-W Intact Mass Analysis of Antibody-drug Conjugates (ADCs) Arnd Brandenburg1, Aude Tartiere2, Maurizio Bronzetti2, Lem del Rosario2, Joe Shambaugh2 1Genedata AG, Basel, Switzerland, 2Genedata Inc., Lexington, MA USA

P-137-T Online Desalting of LC Fractions Containing Monoclonal Antibodies using AdvanceBio Desalting – RP Cartridges Suresh Babu C.V.1, Ning Tang2 1Agilent Technologies, Bangalore, India, 2Agilent Technologies, Santa Clara, CA USA

P-138-W A Single Software Suite for a Multi-Attribute Method Comparison of Infliximab Eric Carlson1, Chris Becker1, St John Skilton1, K. Ilker Sen1, Michael Ford2, Sergei Saveliev3, Karthik Pisupati4, Anna Schwendeman4 1Protein Metrics Inc., San Carlos, CA, USA, 2MS Bioworks, Ann Arbor, MI USA, 3Promega, Madison, WI USA, 4University of Michigan, Ann Arbor, MI USA

P-139-T NIST Interlaboratory Study on Assessing Variability of Glycosylation Analysis Methods in Monoclonal Antibodies M. Lorna De Leoz, David Duewer, Stephen Stein NIST, Gaithersburg, MD USA

P-140-W Whole Molecule LC-MS Analysis for HT Monitoring of N->S Substitution in an Antibody: Challenges in Method Performances Assessment Bertaccini Diego, Celine Duhot Merck Serono S.A., Corsier-sur-Vevey, Switzerland

P-141-T Advancing Attribute Control of Antibodies and Its Derivatives using High Resolution Analytics Jing Fang, Henry Shion, William Alley, Ying Qing Yu, Weibin Chen Waters Corporation, Milford, MA USA

P-142-W Optimization of LC-MS/MS Peptide Mapping for Comparability Assessment of NISTmAb Reference Material 8671 Trina Formolo, John Schiel NIST, Rockville, MD USA

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P-144-W Application of Electron-Transfer Dissociation Mass Spectrometry to the Characterization of Antibody-Drug Conjugates Zhiqi Hao1, Qiuting Hong2, Michael Kim1, William Haskins1, Yan Chen1

1Genentech, a Member of the Roche Group, South San Francisco, CA USA, 2Eurofins Lancaster Laboratories, Inc., Lancaster, PA USA

P-145-T Peptide Mapping of Lysine Linked Antibody-drug Conjugate using HCD and ETD Fragmentation with an Orbitrap Fusion Tribrid Mass Spectrometer Stephane Houel, Terry Zhang, Aaron Bailey, Romain Huguet, Jennifer Sutton, Jonathan Josephs Thermo Fisher Scientific, San Jose, CA USA

P-146-W CESI-MS and its Application to Intact Protein Analysis Stephen Lock SCIEX, Pudsey, UK

P-147-T Characterization of mAb N-terminal Heterogeneity Caused by Improper Signal Peptide Cleavage Andy Mahan Janssen Pharmaceutical R&D, LLC, Spring House, PA USA

P-148-W Rapid Characterization of Low Level Amino Acid Variants During Clone Screening for a Biosimilar Monoclonal Antibody Irina Perdivara, Margo Wilson, Clara Smith, Greg Adams FUJIFILM Diosynth Biotechnologies, Morrisville, NC USA

P-149-T Power of Tandem Mass Spectrometry for Structural Characterization of N-Linked Oligosaccharides Nalaka Rannulu, Promod Mehndiratta Janssen Pharmaceutical R&D, LLC, Malvern, PA, USA

P-150-W Rapid Proteolysis of IgG for Bioanalysis Workflows Michael Rosenblatt, Sergei Saveliev, Marjeta Urh Promega, Madison, WI USA

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P-151-T A Mass Spectrometry Toolkit for Rapid and Efficient Characterization of a Bispecific Antibody Felicia Schnur, Adam Catherman, Matthew Kalo Genentech, a Member of the Roche Group, South San Francisco, CA USA

P-152-W Developing Novel Integrated LC-MS Workflows for Oligonucleotide Characterization, High Throughput Mass Confirmation and Impurity Profiling Henry Shion, Robert Birdsall, Joe Fredette, Ying Qing Yu Waters Corporation, Milford, MA USA

P-153-T Two-Dimensional Time-Resolved LC-MS Deconvolution for Intact Mass Analysis of Biological Drugs Aude Tartiere1, Joe Shambaugh1, Maurizio Bronzetti1, Lem del Rosario1, Arnd Brandenburg2 1Genedata Inc., Lexington, MA USA, 2Genedata AG, Basel, Switzerland

P-154-W Evaluation of a Rapid Method for Deamidation Profiling of Monoclonal Antibodies Suitable for Early Molecular Selection in Drug Discovery Guillaume Tremintin1, Lisa Marzilli2, Heather DeGruttola2, Elaine Stephens2, Michelle English2, Keith Johnson2, Anja Wiechmann3, Jason Rouse2, Wolfgang Jabs3 1Bruker Daltonics, Inc., Fremont, CA USA, 2Pfizer, Inc., Andover MA USA, 5Bruker Daltonik, Bremen, Germany

P-155-W Middle-down Approach for Monitoring Monoclonal Antibody Variants and Deglycosylation Shanhu Lin1, Zoltan Szabo1, Yury Agroskin1, Terry Zhang2, Jonathan Josephs2, Xiaodong Liu1 1Thermo Fisher Scientific, Sunnyvale, CA USA, 2Thermo Fisher Scientific, San Jose, CA USA

P-156-W Characterization of the Acidic Species During Monoclonal Antibody Process Development and Optimization Yunping Huang1, Richard Ludwig1, Li Tao1, Bai Kelvin1, Zhijun Tan2, Jie Chen2, Zhiqiang Chen2, Zheng Jian Li2, Tapan Das1 1Bristol-Myers Squibb Company, Pennington, NJ USA, 2Bristol-Myers Squibb Company, Devens, MA USA

P-157-T Characterization of Antibody Drug Conjugates by Liquid Chromatography Mass Spectrometry Aarti Jashnani, David Passmore, Chetana Rao, Srikanth Kotapati, Patrick Holder, Mary Huber, Ganapathy Sarma, Adam Salles Bristol-Myers Squibb Company, Redwood City, CA USA

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P-158-W Development of a 5-Minute Deglycosylation Method and Instant Labeling Dye for High-throughput N-Glycan Analysis by Mass Spectrometry Michael Kimzey, Aled Jones, John Yan, Vaishali Sharma, Andres Guerrero, Alexander Gyenes, Justin Hyche, Emily Dale, Ted Haxo, Sergey Vlasenko ProZyme, Hayward, CA USA

P-159-T Aminopyrene-based Multi-positively Charged Labels for CE/LIF and CE/MS Analysis of Glycans Jana Krenkova1, Marcela Liskova1, Gyula Vigh2, Frantisek Foret1 1Institute of Analytical Chemistry, v. v. i., Brno, Czech Republic, 2Texas A&M University, College Station, TX USA

P-160-W Automated False Positive Removal and Result Archiving for MS based Sequence Variant Analysis Wenzhou Li, Robert J. Duff, Jette Wypych Amgen, Inc., Thousand Oaks, CA USA

P-161-T Determination of Disulfide Connectivity of a Glycosylated Insulin-Like Protein by Top-Down LC-MS with ETD Analysis Serah Liu1, Chen Li1, Billy Wu1, Richardson Douglas2, Yan-Hui Liu2, Huijuan Li2 1BioAnalytix, Inc., Cambridge, MA USA, 2Merck & Co., Inc., Kenilworth, NJ USA

P-162-W CESI-MS and it Application to Neuropeptide Analysis Stephen Lock SCIEX, Pudsey, UK

P-163-T Multilevel Characterization of Monoclonal Antibodies using Microfluidic CE-MS Technology Erin Redman, Joshua Guerrette, Scott Mellors 908 Devices, Inc., Carrboro, NC USA

P-164-W Automated Peptide Mapping for Quantitative Comparison of Biotherapeutics Joe Shambaugh1, Aude Tartiere1, Lem del Rosario1, Maurizio Bronzetti1, Arnd Brandenburg2 1Genedata Inc., Lexington, MA USA, 2Genedata AG, Basel, Switzerland

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P-165-T Performance and Comparison of High Resolution Mass Spectrometry Systems for Product Variant Quantitative Measurement Chi Yan Tam, Melissa Alvarez, Armando Cordoba, Christopher Yu Genentech, a Member of the Roche Group, South San Francisco CA USA

P-166-W Common Post-translational Modification Analysis of Therapeutic Antibodies Hsien-Yu Tsai, Yen-ling Huang, Yuan-hsin Lu, Fang-ciao Hsu, I-Ling Kuo Institute of Biologics, Development Center for Biotechnology, Taipei City, Taiwan

P-167-T In Control of Controlled Fab Arm Exchange: Native H-L Pairing is Retained in the Formation of Stable Bispecific IgG1 Ewald Van den Bremer1, Ramon van den Boogaard1, Aran Labrijn1, Patrick Priem1, Kai Scheffler2, Janine Schuurman1, Paul Parren1, Rob de Jong1 1Genmab, Utrecht, Netherland, 2Thermo Fisher Scientific, Dreieich, Germany

P-168-W Determining Sources of Artificial Oxidation of Therapeutic Proteins in Peptide Mapping Aria Veach, Izydor Apostol, Da Ren Amgen, Inc., Thousand Oaks, CA USA

P-169-T Discovery and Characterization of a C-terminal Extension Variant of an IgG1 Monoclonal Antibody during Early Cell Line Selection Yan Wang, Chongfeng Xu, Zoran Sosic, Li Zang Biogen, Cambridge, MA USA

P-170-W Monitoring the Critical Quality Attributes of Antibody Drug Conjugates (ADCs) as Part of Biosimilar Development: Case Studies of Ado-trastuzumab Emtansine Ying Qing Yu, Henry Shion, Min Du, Weibin Chen Waters Corporation, Milford, MA USA

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R&D Applications P-171-T Automated Antibody de novo Sequencing K. Ilker Sen, Wilfred Tang, Marshall Bern, Yong Kil, Chris Becker Protein Metrics Inc., San Carlos, CA USA

P-172-W Comparison of a 2D LC-MS/MS Method and an Anti-CHOP Antibody-based Enrichment Method for Identification of Host Cell Proteins using High-resolution ESI-mass Spectrometric Detection Yu Zhou, Lin Chen, Jeffrey Lemontt, Sushmita Mimi Roy BioMarin Pharmaceuticals, Novato, CA USA

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Young Scientist P-174-W Quantification of Serum Vitamin D Binding Protein by Novel LC-MS/MS Methodology Indicates Similar Concentrations in Blacks and Whites Clark Henderson University of Washington Medical Center, Seattle, WA USA

P-175-T Determination Of Six Porphyrins In Human Urine Samples By Liquid Chromatography-Mass Spectrometry Emre Ispir1, Muhittin A. Serdar2, İsmail Kurt3 1Balıkesir Military Hospital, Balıkesir, Turkey, 2Acıbadem University, İstanbul, Turkey, 3Gulhane Military Medical Academy, Ankara, Turkey

P-176-W Monitoring the Site-Specific Glycosylation, Site Occupancy and other PTMs in Plant Bioengineered Recombinant Therapeutical Glycoproteins Muchena J. Kailemia1, Kalimuthu Karuppanan2, Yanhong Li1, Wanghui Wei1, My Phu3, Abhaya M. Dandekar3, Xi Chen1, Somen Nandi4, Karen McDonald2, Carlito Lebrilla1 1Department of Chemistry, University of California, Davis, CA USA, 2Chemical Engineering & Materials Science, University of California, Davis, Davis, CA USA, 3Department of Plant Sciences, University of California, Davis, Davis, CA USA, 4Department of Molecular & Cellular Biology, University of California, Davis, Davis, CA USA P-177-T Application of Quantitative Liquid Chromatography – Mass Spectrometry Techniques to Facilitate Enhanced Bioprocessing of Therapeutic Proteins Amy Farrell1, Kai Scheffler2, Jenny Ho3, Peter Mowlds3, Patrick Pankert3, Andrew Williamson3, Ken Cook3, Jonathan Bones1 1NIBRT, Dublin, Ireland, 2Thermo Fisher Scientific, Dreieich, Germany, 3Thermo Fisher Scientific, Hemel Hempstead, UK

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Late Breaking LB-01-W Advantage of Premixed Mobile Phases for Consistent Chromatography of Peptides using EASY-nLC 1200 Haibo Wang, Subhra Bhattacharya, Stephen C. Roemer Thermo Fisher Scientific, San Jose, CA USA LB-02-T Complete Characterization of Biotherapeutic Proteins by Automated Data Processing on High Resolution Accurate Mass Spectrometry Milla Neffling1, Sean McCarthy2, Xu Guo, Doug3, Simmons3, Vanaja Raguvaran3, Kelli Jonakin2 1SCIEX, Warrington, UK, 2SCIEX, Framingham, MA, USA, 3SCIEX, Concord, ON Canada

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