12th Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology Industry (Mass Spec 2015)

Transcription

1 12th Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology Industry (Mass Spec 2015) Symposium Co-Chairs: Steven Cohen, Northeastern University Christopher Yu, Genentech, a Member of the Roche Group September 23-25, 2015 New York Marriott at Brooklyn Bridge Brooklyn, NY USA Organized by 1

2 Table of Contents Student Travel Grants... 4 Program, Exhibitor and Media Partners... 5 Scientific Final Program Summary... 8 Plenary and Session Abstracts Workshop Technical Seminar Abstracts Poster Abstracts

3 Welcome to the 12th Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology Industry We are pleased to welcome you to the 12th 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 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 AbbVie, Inc., Agilent Technologies, Analytical Scientist, Biogen, Bio Tech International, BioProcess International, Bruker Daltonics, Inc., CMP Scientific, Eurofins Lancaster Laboratories, Inc., Genetic Engineering & Biotechnology News, Genedata AG, Genentech, a Member of the Roche Group Genovis AB, IPQ, LCGC North America, The Medicine Maker, New England BioLabs Inc., The Pathologist, Pfizer, Inc., Protein Metrics, ProZyme, Inc., Roche Diagnostics GmbH, RSC Advances, separationsnow.com, SCIEX, Technology Networks, Thermo 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 2016! 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, (Co-chair) Eef Dirksen, Synthon Biopharmaceuticals B.V. Vivian Lindo, MedImmune, A member of the AstraZeneca Group Anders Lund, Genzyme, a Sanofi company Yelena Lyubarskaya, Biogen Jun Park, CDER, FDA David Passmore, Bristol-Myers Squibb Company Jason Rouse, Pfizer, Inc. Hansjörg Toll, Sandoz GmbH Christopher Yu, Genentech, a Member of the Roche Group (Co-chair) 3

4 CASSS Mass Spec Student Travel Grants CASSS is pleased to provide a limited number of student travel grants for PhD students and postdocs who present applicable posters at the 12th Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology Industry (Mass Spec 2015). PhD students or postdoctoral 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, higherorder 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: P-166-T Protein-protein Interactions: Protein Footprinting and Mass Spectrometry Map The Interface of Marburg Virus VP24 Interacting with Keap1 Jing Li, University of Washington in St. Louis, USA Thursday, September 24 Young Scientist Session High Resolution Accurate Mass LC-MS/MS based Characterization of Industrial Cell Culture Media to Identify Compound Signatures of Process Variability Brian Morrissey, NIBRT - The National Institute for Bioprocessing Research and Training, Ireland P-167-F Intact Monoclonal Antibody Analysis via Microchip CE-MS Erin A. Redman, University of North Carolina, Chapel Hill, USA P-168-T Comprehensive Characterization of Human Growth Hormone Degradants by Integration of a Top-down and Bottom Mass Spectrometry Approach Annie Yu Wang, The Barnett Institute, Northeastern University, USA 4

5 The Organizing Committee gratefully acknowledges the following program partners for their generous support of this Symposium: Sustaining Program Partners Platinum Program Partners AbbVie, Inc. Biogen Silver Program Partner Pfizer, Inc. Diamond Program Partner Agilent Technologies Platinum Program Partners Bruker Daltonics, Inc. Genedata AG Gold Program Partners Genentech, a Member of the Roche Group Roche Diagnostics GmbH SCIEX 5

6 Welcome Reception Bruker Daltonics, Inc. Exhibitor Partners Agilent Technologies Bruker Daltonics, Inc. CMP Scientific Eurofins Lancaster Laboratories, Inc. Genedata AG Genovis AB MRM Proteomics Inc. New England BioLabs Inc. Protein Metrics ProZyme, Inc. SCIEX Thermo Scientific Waters Corporation 6

7 Media Program Partners Analytical Scientist Bio Tech International BioProcess International Genetic Engineering & Biotechnology News IPQ LCGC North America The Medicine Maker The Pathologist RSC Advances separationsnow.com Technology Networks Supporting Organizations Chicago Mass Spectrometry Discussion Group Connecticut Mass Spectrometry Discussion Group Dutch Society for Mass Spectrometry German Society for Mass Spectrometry Greater Boston Mass Spectrometry Discussion Group MASSEP.org, A Separation Science Discussion Group Midwest Mass Spectrometry Discussion Group Pharmaceutical & Bioscience Society (PBSS) International Scripps Center for Metabolomics and Mass Spectrometry (SANDMAN) Washington-Baltimore Mass Spectrometry Discussion Group 7

8 Mass Spec 2015 Scientific Program Summary Tuesday, September 22, :30 09:30 Breakfast (Full Day and Morning Short Course Attendees ONLY) 08:30 13:30 Short Course Registration ONLY in the Park Slope Foyer Short Course in the Park Slope Room Short Course Facilitators: 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) 13:30 17:00 Applications of Mass Spectrometry to Characterize Protein Therapeutics Anders Lund, Genzyme, a Sanofi company, Framingham, MA USA 8

9 Wednesday, September 23, :00 17:00 Registration in the Ballroom Coatroom 13:00 13:15 Welcome and Introductory Comments in the Ballroom D Steve Cohen, Northeastern University, Boston, MA USA Plenary I Session in Ballroom D Session Chair: Jason Rouse, Pfizer, Inc. Andover, MA USA 13:15 14:15 Ultrahigh Resolution Mass Spectrometry: Extending the Size and Detail of Biomolecule Structure Analysis Alan Marshall, Florida State University, Tallahassee, FL USA 14:15 14:45 Break Visit the Exhibits and Posters in Ballroom A-C Process and Product Characterization Session in Ballroom D Session Chairs: Alain Balland, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany and Christopher Yu, Genentech, A Member of the Roche Group, South San Francisco, CA USA 14:45 15:15 Minimizing Trisulfides in Monoclonal Antibodies: Early Screening and Detection using Ultrahigh-resolution Mass Spectrometry to Ensure Product Quality and Consistency Lisa Marzilli, Pfizer, Inc., Andover, MA USA 15:15 15:45 Application of Mass Spectrometry for Process Understanding and Advanced Process Control Kazumi Kobayashi, Biogen, Cambridge, MA USA 15:45 16:15 Implementing a LC/MS Based Multi-attribute Method in GMP Environment Da Ren, Amgen Inc., Thousand Oaks, CA USA 16:15 16:30 The Application of High Resolution Top Down Mass Spectrometry to the Study of Degradation Reactions in Stability Samples of Protein Pharmaceutical Jeffrey Agar, Northeastern University, Boston, MA USA 16:30 16:40 Short Moving Break 9

10 Wednesday, September 23, 2015 continued 16:40 17:40 Technical Seminar Breakthrough Applications to Expand and Speed Biopharmaceutical Characterization Bryan Fonslow, SCIEX, Brea, CA USA Sponsored by SCIEX Ballroom D 17:45 19:30 Welcome Reception 501 Union Hosted by Bruker Daltonics, Inc. Please meet in lobby for transportation to reception 501 Union is a space of subtle distinction and quiet refinement. Erected in 1916, the building served in the restoration of classic cars through the glory years of the American automotive industry. It was brought back to life in Embedded in the stone and steel of this quiet landmark is the timeless integrity of a continually modern New York. 10

11 Thursday, September 24, :30 17:30 Registration in the Ballroom Foyer 07:30 08:30 Breakfast in Ballroom A-C 07:30 08:30 Technical Seminar Vendor-Neutral Software for the Comprehensive Characterization of Therapeutic Proteins Chris Becker, Protein Metrics Inc., San Carlos, CA USA Sponsored by Protein Metrics Inc. Ballroom D Plenary II Session in Ballroom A-C Session Chair: Steven Cohen, Northeastern University, Boston, MA USA 08:30 09:30 Innovative Instrumentation and Methods for Sequence Analysis of Antibodies and Posttranslationally-modified, Intact Proteins on a Chromatographic Time-scale Donald Hunt, University of Virginia, Charlottesville, VA USA 09:30 10:00 Break Visit the Exhibits and Posters in Ballroom A-C 10:00 11:00 Workshop I: Streamlining Workflows in the Mass Spectrometry Laboratory for Increased Productivity in Ballroom A-C Facilitator: Jason Rouse, Pfizer, Inc., Andover, MA USA Presenters: Melissa Alvarez, Genentech, a Member of the Roche Group, South San Francisco, CA USA Richard Rogers, Just Biotherapeutics, Seattle, WA USA Host Cell Proteins and Other Process Related Impurities Session in Ballroom D Session Chair: Patrick Bulau, Roche Diagnostics GmbH, Penzberg, Germany and Steven Cohen, Northeastern University, Boston, MA USA 11:00 11:30 Novel Approaches to Targeted Protein Analysis using LC-MRM in an HSV2 Vaccine Candidate Andrew James, Sanofi Pasteur Ltd., Toronto, ON Canada 11

12 Thursday, September 24, 2015 continued 11:30 12:00 An Accelerated LC-MS/MS Workflow for Assessment of the Host Cell Protein Composition during Biotherapeutic Process Development Don Walker, Genentech, A Member of the Roche Group, South San Francisco, CA USA 12:00 12:15 Lunch for Technical Seminar Attendees Please take lunch and return to Ballroom D for the Lunch and Learn 12:15 13:15 Technical Seminar: Lunch and Learn Sponsored by Agilent Technologies Ballroom D 13:15 13:30 Break - Visit the Exhibits and Posters in Ballroom A-C Young Scientist Session in Ballroom D Session Chair: James Carroll, Pfizer, Inc. St. Louis, MO USA 13:30 13:45 Impact of Sequence Variants on the HOS Analysis of Monoclonal Antibodies using an Optimized HDX-MS Method with Two Protease Column Approach Sasidhar Nirudodhi, Pfizer, Inc., Chesterfield, MO USA 13:45 14:00 High Resolution Accurate Mass LC-MS/MS Based Characterization of Industrial Cell Culture Media to Identify Compound Signatures of Process Variability Brian Morrissey, NIBRT, Dublin, Ireland 14:00 14:15 LC-MS Based Determination of Critical Attributes of a Novel Antibody-Drug Conjugate (ADC) Srikanth Kotapati, Bristol-Myers Squibb Company, Redwood City, CA USA 14:15 15:45 Poster Session I - Visit the Exhibits and Posters in Ballroom A-C Mass Spec Approaches for Quantitative Studies Session in Ballroom D Session Chair: Eef Dirksen, Synthon Biopharmaceuticals B.V., Nijmegen, The Netherlands and Vivian Lindo, MedImmune, A member of the AstraZeneca Group, Cambridge, United Kingdom 15:45 16:15 Data Analysis Strategies for Detecting and Characterizing Unknown Chemical and Posttranslational Modifications on Proteins John Valliere-Douglas, Seattle Genetics, Bothell, WA USA 12

13 Thursday, September 24, 2015 continued 16:15 16:45 Functional Assessment of Antibody Oxidation by Native Mass Spectrometry Markus Haberger, Roche Diagnostics GmbH, Penzberg, Germany 16:45 17:15 Understanding of Critical Quality Attributes of Biopharmaceuticals In Vivo Yinyin Li, Biogen, Cambridge, MA USA 17:15 18:15 Exhibitor Reception - Visit the Exhibits in Ballroom A-C 13

14 Friday, September 25, :30 17:00 Registration in the Ballroom Foyer 07:30 08:30 Breakfast in Ballroom A-C 07:30 08:30 Technical Seminar Routine and Advanced Comparability Studies for the Next Generation of Innovators and Biosimilars Asish Chakraborty, Waters Corporation, Milford, MA USA Sponsored by Waters Corporation Ballroom D Plenary III Session in Ballroom D Session Chair: Yelena Lyubarskaya, Biogen, Cambridge, MA USA 08:30 09:30 Rapid Production of Versatile Nanobody Repertoires Brian Chait, The Rockefeller University, New York, NY USA 09:30 10:00 Break Visit the Exhibits and Posters in Ballroom A-C Regulatory Session in Ballroom D Session Chair and Moderator: Michael Boyne, BioTechLogic, Inc., Glenview, IL USA 10:00 10:30 Characterization of Filgrastim using Intact and Top-down MS Johann Holzmann, Sandoz GmbH, Kundl, Austria 10:30 11:00 Applications of MS in Biosimilar Product Development and Similarity Assessment Jennifer Liu, Amgen Inc., Thousand Oaks, CA USA 11:00 11:30 The Use of Mass Spectrometry in FDA Applications for Biotherapeutics: A Retrospective Review Sarah Rogstad, CDER, FDA, Silver Spring, MD USA 11:30 12:30 Panel Discussion 12:30 12:45 Lunch for Technical Seminar Attendees Please take lunch and return to Ballroom D for the Lunch and Learn 14

15 Friday, September 25, 2015 continued 12:45 13:45 Technical Seminar: Lunch and Learn Unbiased In-depth Characterization of CEX-fractions from a Stressed Mab by MS Matthias Berg, Novartis AG, Basel, Switzerland Sponsored by Genedata AG Ballroom D 13:45 15:00 Poster Session II - Visit the Exhibits and Posters in Ballroom A-C Novel Technologies Session in the Ballroom D Session Chair: David Passmore, Bristol-Myers Squibb Company, Redwood City, CA USA 15:00 15:30 Mass Spectrometry Assisted Development of an Efficient In-silico Prediction Tool for Aspartate Degradation Sites Hans Rainer Voelger, Roche Diagnostics GmbH, Penzberg, Germany 15:30 16:00 Utility of Ion-mobility Mass Spectrometry in Antibody Drug Conjugates Characterization Richard Huang, Bristol-Myers Squibb Company, Princeton, NJ USA 16:00 16:30 In-depth, Quantitative (Host Cell) Protein Fingerprinting of Adenovirus Based Vaccines by Mass Spectrometry Annemiek Verwilligen, Janssen Infectious Diseases and Vaccines, Leiden, The Netherlands 16:30 16:45 Poster Award Announcement in Ballroom D 16:45 16:55 Closing Comments in Ballroom D Christopher Yu, Genentech, a Member of the Roche Group, South San Francisco, CA USA 15

16 Ultrahigh Resolution Mass Spectrometry: Extending the Size and Detail of Biomolecule Structure Analysis Alan Marshall Florida State University, Tallahassee, FL USA Most mass analysis relies on "nominal" mass accuracy (i.e., to within 1 Da). However, more and more applications are based on much more accurate mass measurement. High mass resolving power (m/ m50% > 500,000 over a wide mass range) offers two major advantages. First, it becomes possible to separate complex mixtures without prior chromatographic or gel separation. Second, elemental composition may be determined from accurate (to ~100 ppb) mass measurement alone for unknown molecules up to ~1,000 Da. Examples include protein posttranslational modifications and site-specific redox potentials, lipidomics, and mapping contact surfaces in protein assemblies for drug targets. Fourier transform ion cyclotron resonance mass spectrometry offers x higher mass resolution than other mass analyzers. [Supported by NSF DMR and CHE and the State of Florida. Noble, A. J.; Zhang, Q.; O'Donnell, J.; Hariri, H.; Bhattacharya, N.; Marshall, A. G.; Stagg, S. M." A Pseudoatomic Model of the COPII Cage Obtained from Cryo-EM and Mass Spectrometry," Nature SMB 2013, 20, Scotcher, J.; Bythell, B. J.; Marshall, A. G. "Unequivocal Determination of Site-Specific Protein Disulfide Bond Reduction Potentials by Top-down FT-ICR MS: Characterization of the N- and C-Terminal Redox-Active Sites in Human Thioredoxin 1," Anal. Chem. 2013, 85, Brownstein, N. C.; Guan, X.; Mao, Y.; Zhang, Q.; DiMaggio, P. A.; Xia, Qiangwei; Zhang, Lichao; Marshall, A. G.; Young, N. L. "Paired Single Residue-Transposed Lys-N and Lys-C Digestions for Label-Free Identification of N-Terminal and C-Terminal MS/MS Peptide Product Ions: Ultrahigh Resolution FT-ICR MS and MS/MS for Peptide De Novo Sequencing,"RCMS. 2015, 29, Guan, X.; Noble, K. A.; Tao, Y.; Roux, K. H.; Sathe, S. K.; Young, N. L.; Marshall, A. G. "Epitope Mapping of 7S Cashew Antigen in Complex with Antibody by Solution-Phase H/D Exchange Monitored by FT-ICR MS," JMS 2015, 50,

17 Minimizing Trisulfides in Monoclonal Antibodies: Early Screening and Detection using Ultrahigh-resolution Mass Spectrometry to Ensure Product Quality and Consistency Lisa Marzilli, Mellisa M. Ly, Andrew E. Saati, Jason C. Rouse Pfizer, Inc., Andover, MA USA A trisulfide bond (-S-S-S-) is a post-translational modification that has been identified in both recombinant and endogenous plasma-derived antibodies (Gu et al., 2010; Kshirsagar et al., 2012), as well as in several other classes of proteins (Nielsen et al., 2011). In monoclonal antibodies (mabs), trisulfide modifications have only been detected in place of the disulfide bonds between the light and heavy chain, or between the two heavy chains. While trisulfide bonds do not appear to impact mab target binding or effector functions, their presence in mabs interferes with the reduction step in antibody-drug conjugation (Cumnock et al., 2013). During early stage process development support, we discovered several mabs with a high level of trisulfide modification, as indicated by +32 and +64 Da mass additions to the intact antibody. A combination of methods and data will be presented that demonstrate the detection, confirmation, site localization, and relative quantitation of trisulfides using mass spectrometry. Implementation of our LC/MS-based platform intact antibody profiling method to rapidly screenfor trisulfide levels has become a vital component of our product quality assessment for final clone selection. Initial findings indicate the relative level of trisulfide may vary with both the cell clone and media. This early screening and characterization work contribute to immediate product understanding with the ultimate goal of minimizing the level of trisulfides in our recombinant antibodies for more robust conjugation processes and enhanced batch-to-batch consistency. 17

18 Application of Mass Spectrometry for Process Understanding and Advanced Process Control Kazumi Kobayashi Biogen, Cambridge, MA USA Under a Quality by Design (QbD) framework, a robust and flexible manufacturing control strategy can be derived using a systematic approach based on sound science and quality risk management. Testing, monitoring or controlling is often shifted earlier into the process and results may be used for real time release, parametric release, or feedback/feed-forward process controls. Mass spectrometry offers rapid and accurate analysis of multiple product quality attributes in a single assay. It is a powerful tool for monitoring product quality in process samples when combined with high-throughput sample preparation and data analysis. Depending on the correlation with the data obtained using the traditional test methods, mass spectrometry may potentially be used not only for process and product understanding but also for process control/real time release. In this talk, examples of mass spectrometry application for process understanding and advanced process control strategy development for monoclonal antibody products will be presented together with discussions on future applications. 18

19 Implementing a LC/MS Based Multi-attribute Method in GMP Environment Da Ren Amgen Inc., Thousand Oaks, CA USA The Quality by Design (QbD) paradigm requires deep understanding of product attributes in biopharmaceutical industry. An attribute specific, LC/MS based Multi-Attribute Method (MAM) has been developed to replace some non-attribute specific assays used for process and product characterization. This assay can automatically identify and quantify product attributes based on peptide mapping data generated by high mass accuracy, high resolution LC/MS systems. Recently, this Multi-Attribute Method has been implemented in GMP environment for clinical material disposition and stability testing. Qualification of the MAM system includes Installation Qualification (IQ) and Operational Qualification (OQ) of the LC system, mass spectrometer, and software. Controlling PC hardware, data repository, user roles and permissions were also carefully selected to ensure data integrity and security. Several issues related to system hardware stability have been resolved. Challenges and lessons learned during the implementation of Multi- Attribute Method in GMP environment will be discussed. 19

20 The Application of High Resolution Top Down Mass Spectrometry to the Study of Degradation Reactions in Stability Samples of Protein Pharmaceutical Jeffrey Agar, Jared Auclair, Joseph Salisbury, Yang Tang, Di Wu, Yu Wang, William Hancock Northeastern University, Boston, MA USA We have developed an LC/MS system utilizing the SolariX (XR) ESI-FTICR with mass resolution greater than 1 million and which allows the characterization of a variety of oxidation and other degradation reactions in the intact molecule as well as chain cleavage reactions. In the presentation we will show the potential of this powerful approach with the analysis of oxidized growth hormone, 2-chain and stability samples. We will also discuss the application of this technology to antibody therapeutics. 20

21 Innovative Instrumentation and Methods for Sequence Analysis of Antibodies and Posttranslationally-modified, Intact Proteins on a Chromatographic Time-scale Donald Hunt University of Virginia, Charlottesville, VA USA This lecture will focus on data generated with a new ion source that facilitates simultaneous generation of positively charged sample ions by electrospray ionization and negatively charged reagent ions for both electron transfer dissociation (ETD) and ion-ion proton transfer (IIPT) reactions on Orbitrap mass spectrometers. Implementation of multiple C-trap fills for enhanced sensitivity will be discussed and both parallel peak parking, and ion ejection strategies to facilitate protein separation and enhanced sequence coverage of intact proteins will be described. Use of IIPT/ETD facilitates near complete sequence coverage on many intact proteins and is ideally suited for locating multiple post-translational modifications on the same protein molecule. Sequence analysis of antibodies with an enzyme reactor that generates 3-10 KDa fragments in seconds will also be discussed. 21

22 Novel Approaches to Targeted Protein Analysis using LC-MRM in an HSV2 Vaccine Candidate Andrew James, Artur Pedyczak, Bruce Carpick Sanofi Pasteur Ltd., Toronto, ON Canada The capabilities of mass spectrometry are continuously increasing within the bio-pharmaceutical industry. Until recently, the primary focus of mass spectrometry in vaccine research and development has been as a characterization and identity tool, particularly for recombinant protein candidates. However, newer mass spectrometry workflows are being evaluated for both targeted analysis and quantitation of proteins in more complex vaccine candidates. This work focuses on the development of a targeted assay for two HSV-1 proteins: HELI and DNBI (UL5 and UL29 gene products), detected in an HSV-2 replication deficient, live-attenuated vaccine candidate. The HELI and DNBI proteins are expressed by a complementary cell line, AV529, used to produce the HSV529 candidate. After detection and characterization of the vaccine candidate for HELI and DNBI components using nanolc techniques; the development of a novel MRM assay using a high flow UPLC and a simplified chromatographic method was designed. Sensitivity of the MRM assay for HELI and DNBI was established at high flow, and linearity of response was shown in the range of the LLOQ. Our results demonstrate the unique sensitivity of MRM methods for detection of targeted proteins, and establish specificity for the HSV-1 proteins HELI and DNBI, in the candidate vaccine lots. 22

23 An Accelerated LC-MS/MS Workflow for Assessment of the Host Cell Protein Composition During Biotherapeutic Process Development Don Walker Genentech, a Member of the Roche Group, South San Francisco, CA USA Recent advances in LC-MS/MS methods for detection of residual host cell proteins (HCPs) in biologics continue to improve in detection sensitivity. However, lengthy analysis time and poor sample throughput remain as challenges to the technique and the realization of its full value in support of fast and high quality biopharmaceutical development. We demonstrate the development of a robust, high throughput 1D-UPLC-MS/MS method (1 hour analysis time) that utilizes improved UPLC column separation technology and fast MS/MS scanning speed at 20Hz to improve method speed and throughput. A separation method with greater peak capacity and sample loadability is critical for successful detection of HCP contaminants in the presence of drug product due to the inherent wide dynamic range. By combining data-dependent and dataindependent mass spectrometry approaches, our workflow utilizes upstream, partially purified inprocess chromatography pools to identify specific HCPs and track their clearance in downstream pools by MS2 peak area quantification. When appropriate, HCP levels in final pools can also be estimated by calibration against a spiked protein internal standard used as a universal response factor. The utility of this robust and high throughput workflow is further demonstrated in the analysis of complex mixtures of CHO host cell culture fluid (HCCF), specifically in the ability to routinely identify more than 1000 proteins in one hour of analysis time. Data dependent acquisition allows semi-quantitative, yet highly relevant studies of HCPs in the assessment of different cell strains and culture conditions and their impact on HCP composition and relative levels. Data independent acquisition of the samples can be used for MS2-based relative quantification between samples, with estimation of absolute levels obtainable using calibration with a universal response factor. These results complement the traditional immuno-based HCP characterization and help ensure the development of biopharmaceutical products with low and consistent levels of HCP impurities. 23

24 Impact of Sequence Variants on the HOS Analysis of Monoclonal Antibodies using an Optimized HDX-MS Method with Two Protease Column Approach Sasidhar Nirudodhi 1, Lin Tzihsuan 2, Justin Sperry 1, Jason Rouse 2, James A. Carroll 1 1 Pfizer, Inc., Chesterfield, MO USA, 2 Pfizer, Inc., Andover, MA USA The unintended amino acid changes in a protein sequence are defined as the sequence variants (SVs) and such errors can be caused during DNA replication, DNA-RNA transcription or RNAprotein translation. Presence of SVs in recombinant biotherapeutic mabs contribute to product variability. Advanced analytical techniques are needed to identify and characterize the product variability of mabs as it might lead to the potential impact on product safety. In contrast to the existing biophysical techniques that provide low resolution information on the global level, HDX-MS technique provides localized information with higher resolution in biotherapeutics. HDX MS was performed using selected batches of mabs observed to have increased levels of SVs and compared to batches with no detectable SVs in order to determine the impact on the higher order structure (HOS) of the mab. A custom digestion column packed with two separate immobilized proteases, pepsin and Type XII, was used for digestion (Novabioassays, Woburn, MA). A Waters HDX-MS platform with Synapt G2-Si system and Leap automation setup was used for automated labeling, quenching, on-line proteolytic digestion and LC/MS analysis. Preliminary deuterium uptake data showed that conservative amino acid substitutions did not induce conformational changes in the localized domain whereas non-conservative amino acid substitution resulted in measurable local conformational changes. These examples illustrate the potential impact of SVs on the mab on a case by case basis depending on the type of amino acid substitution. 24

25 High Resolution Accurate Mass LC-MS/MS Based Characterisation of Industrial Cell Culture Media to Identify Compound Signatures of Process Variability Brian Morrissey 1, Anna Lindeberg 2, Christian Kaisermayer 2, Sean Forrestell 3, Erik Fouts 3, Jonathan Bones 1 1 NIBRT, Dublin, Ireland, 2 BioMarin International Ltd., Cork, Ireland, 3 BioMarin Pharmaceutical Inc., Novato, CA USA Monitoring the variability of culture media used in biopharmaceutical production is essential to ensure a stable and efficient cell culture. For industrial scale production basal media are often supplemented with hydrolysates and other additives which are heterogeneous and create a significant source of process variability. Here, we describe a LC-MS/MS platform developed for the characterisation of production media and soy hydrolysates to identify compound signatures that correlate with production variability observed in an industrial scale perfusion based cell culture. A reverse phase positive ionisation LC-MS/MS platform and appropriate bioinformatics was applied to analyse 8 soy hydrolysates and 18 production media used in the production of a recombinant therapeutic enzyme. Following LC-MS/MS analysis and bioinformatic processing, principle component analysis (PCA) and hierarchical clustering analysis (HCA) was conducted to identify the presence of sample variability and differentially expressed features present in the media. Interestingly, where multiple production media samples were produced from the same parent soy hydrolysate, a grouping of the production media was observed in the PCA plots and thereby suggests that soy hydrolysate component is responsible for the majority of the variance in the media samples. In addition a number of outlier samples have been identified and the chemical identity of the compounds responsible for this variance is now being explored. 25

26 LC-MS Based Determination of Critical Attributes of a Novel Antibody-Drug Conjugate (ADC) Srikanth Kotapati, Lourdes Thevanayagam, Mei-Chen Sung, Janet Tam, David Passmore, Vangipuram S. Rangan Bristol-Myers Squibb Company, Redwood City, CA USA Antibody-drug conjugates (ADCs) are complex biomolecules composed of an antibody attached to a cytotoxic payload via a linker. Compared to conventional chemotherapeutics used to treat cancer, ADCs offer the distinct advantage of delivering the payload to specific tumor cells expressing the target thereby maximizing efficacy and minimizing side effects. It is of utmost importance that the payload remains conjugated to the antibody and not released during systemic circulation resulting in unwanted off-target toxicity. If the payload is cleaved from the ADC in serum, it results in a change in the drug to antibody ratio (DAR) further leading to changes in the ADC efficacy. Although the payload is not deconjugated from the ADC in plasma, it can undergo biotransformation to form an inactive payload. It is critical to determine the percent conversion of active to inactive ADC to understand the ADC exposure-response relationship. LC-MS based bioanalytical methods were developed to determine the critical attributes of a novel ADC with a microtubule polymerization inhibitor payload attached to the antibody via a cathepsin B cleavable linker. The conjugated payload in this case can also be potentially converted from active to inactive form in vivo. An LC-MS method was developed for the determination of DAR of the purified ADC by cleaving the payload from the ADC with Cathepsin B. In vitro serum stability studies of the ADC were performed and the percent payload deconjugated in serum was determined by LC-MS based MRM of the released payload. These methods were further applied to in vivo and pharmacokinetic studies of ADC, where the total payload was quantified after enzymatic cleavage with Cathepsin B. The kinetics for the conversion of conjugated payload from active to inactive form in vitro and in vivo was also determined similarly using the above LC-MS methods. 26

27 Data Analysis Strategies for Detecting and Characterizing Unknown Chemical and Posttranslational Modifications on Proteins John Valliere-Douglass Seattle Genetics, Inc., Bothell, WA USA Over the years, mass spectrometry data analysis software has been developed to meet the needs of researchers carrying out proteomics-type studies on highly complex samples. In certain key areas such as sequence variant analysis and host-cell protein detection, the application of proteomics-based data search software to biopharmaceutical development has lead to important insights that could be leveraged to make key process changes that improved product quality. However, in other areas relevant to biopharmaceutical development, such as the elucidation of unknown protein modifications and accurate, reliable MS1-based quantitation of molecular variants, MS data analysis software has been slower to evolve to fit the needs of the bench scientist. In this talk, we will describe a generic workflow using LCMS and proteomics-based data analysis software to identify and characterize unknown covalent adducts observed on lyophilized, heat-stressed mabs and antibody-drug conjugates (ADCs). While successful, this workflow was not straightforward so we will also highlight some of the key areas in which continuing evolution of MS data analysis software is needed to support the biopharmaceutical industries increasing reliance on mass spectrometry to characterize product quality. 27

28 Functional Assessment of Antibody Oxidation by Native Mass Spectrometry Markus Haberger Roche Diagnostics GmbH, Penzberg, Germany Oxidation of methionine (Met) residues is one of several chemical degradation pathways for recombinant IgG1 antibodies. Studies using several methodologies have indicated that Met oxidation in the constant IgG1 domains affects in vitro interaction with human neonatal Fc (hufcrn) receptor, which is important for antibody half-life. Here, a completely new approach to investigating the effect of oxidative stress conditions has been applied. Quantitative ultraperformance liquid chromatography mass spectrometry (MS) peptide mapping, classical surface plasmon resonance and the recently developed FcRn column chromatography were combined with the new fast-growing approach of native MS as a near native state protein complex analysis in solution. Optimized mass spectrometric voltage and pressure conditions were applied to stabilize antibody/hufcrn receptor complexes in the gas phase for subsequent native MS experiments with oxidized IgG1 material. This approach demonstrated a linear correlation between quantitative native MS and IgG-FcRn functional analysis. In our study, oxidation of the heavy chain Met-265 resulted in a stepwise reduction of mab3/hufcrn receptor complex formation. Remarkably, a quantitative effect of the heavy chain Met-265 oxidation on relative binding capacity was only detected for doubly oxidized IgG1, whereas IgG1 with only one oxidized heavy chain Met-265 was not found to significantly affect IgG1 binding to hufcrn. Thus, mono-oxidized IgG1 heavy chain Met-265 most likely does not represent a critical quality attribute for pharmacokinetics. 28

29 Understanding of Critical Quality Attributes of Biopharmaceuticals In Vivo Yinyin Li 1, Yu Huang 1, Emma Zhang 2, Peter Li 2, Billy Wu 2, Patrick Swann 1, Yelena Lyubarskaya 1 1 Biogen, Cambridge, MA USA, 2 BioAnalytix, Cambridge, MA USA Understanding of critical quality attributes (CQAs) of biologics is essential in biopharmaceutical development and production. Often, the CQA assignment is built upon prior knowledge and results derived from In Vitro studies, although the relevance of those may be incomplete. In this work, we are investigating the quality attributes of our biopharmaceuticals using preclinical and clinical samples. This approach will allow us to obtain dynamic In Vivo proteoform information. This information will enable more thorough understanding of the product quality attributes, will guide the development of appropriate process control strategy, optimize quality and yield of biopharmaceutical manufacturing, establish scientifically based specifications. 29

30 Rapid Production of Versatile Nanobody Repertoires Brian Chait The Rockefeller University, New York, NY USA Nanobodies are small ( 15 kda) single domain antibodies derived from the variable regions of camelid atypical immunoglobulins. Their strong and specific binding to antigens, extreme diversity, and small size are reminiscent of these properties in toxins. Nanobodies show great promise as high affinity reagents for research, diagnostics and therapeutics due to their high specificity, small size and straightforward bacterial expression. However, identification of repertoires with sufficiently high affinity has proven time consuming and difficult, significantly hampering nanobody implementation. Here, we present a rapid, straightforward approach that generates large repertoires of readily expressible recombinant nanobodies with high affinities and specificities against a given antigen. We demonstrate the efficacy of this approach through the production of large repertoires of nanobodies against two antigens, GFP and mcherry, with Kd values into the sub-nanomolar range. After mapping diverse binding epitopes on GFP, we were also able to design ultra-high affinity dimeric nanobodies with Kds down to ~30 pm. The approach presented is well-suited for the routine production of high affinity capture reagents for a multitude of biomedical applications. 30

31 Characterization of Filgrastim using Intact and Top-down MS Johann Holzmann, Hansjörg Toll Sandoz GmbH, Kundl, Austria In March of this year, the Food and Drug Administration (FDA) approved Sandoz s Zarxio as the first biosimilar in the United States (U.S.). Detailed characterization using a number of analytical techniques most prominently mass spectrometry (MS) - provided the foundation for approval by demonstrating the high similarity between Zarxio and the reference product Neupogen. With the continuous improvement in MS instrumentation and data analysis software, intact and top-down MS are becoming highly attractive methods for the characterization of protein drugs. Using a benchtop Exactive MS we identified several modifications in filgrastim with high sensitivity on the intact level and subsequently performed site assignment using the instruments all-ion fragmentation mode. In our presentation we will show some examples of successful top-down MS experiments such as the site assignment of methionine oxidized variants as low as 0.1%. 31

32 Applications of MS in Biosimilar Product Development and Similarity Assessment Jennifer Liu, Tamer Eris, Qingchun Zhang, Quanzhou Luo, Zhongqi Zhang Amgen Inc., Thousand Oaks, CA USA To ensure successful development of a biosimilar product candidate, it s important to establish a well-defined quality target product profile based on a comprehensive knowledge of the biological properties and a thorough physicochemical characterization of the reference product. In addition to demonstrating identical amino acid sequences to the reference product, the presence of any sequence variants due to cell line or manufacturing process should be carefully identified and evaluated for potential efficacy, safety, and immunogenicity concerns. Posttranslational modifications including glycosylation site occupancy and the composition of glycan forms which have varying degrees of biological relevancy, including impact to the pharmacokinetics and ability to illicit effector functions in IgG1, should be sufficiently resolved by chromatographic-based separation methods and characterized by mass spectrometry for identity. Chemical modifications for all product-related substances and impurities should be characterized and quantified using orthogonal methods, and the comparison to the reference product should take into consideration the stability profiles and material ages. Finally, characterization of the residual host proteins in both the proposed biosimilar product and the reference product is necessary to ensure no impact to safety and immunogenicity. State-of-art mass spectrometry-based analytical techniques play crucial roles during the development of biosimilar products and provide high confidence and quality data in the analytical similarity assessment at the time of regulatory submissions. The authors will present the approaches and applications using mass spectrometry in Amgen biosimilar product development and analytical similarity assessment. 32

33 The Use of Mass Spectrometry in FDA Applications for Biotherapeutics: A Retrospective Review Sarah Rogstad 1, Anneliese Faustino 1, David Keire 2, Michael Boyne 3, Jun Park 1 1 CDER, FDA, Silver Spring, MD USA, 2 CDER, FDA, St. Louis, MO USA, 3 BioTechLogic, Glenview, Il USA Protein based biotherapeutics comprise a large and promising market within the United States. Due to the complex nature of their production these products are often heterogeneous and require elaborate technical analysis. Thus, modern analytical techniques are necessary for their characterization. Mass spectrometry (MS) is an indispensable tool for the analysis of such products. A recent FDA data mining study (Read et al, 2011) surveyed the methods used for glycosylation analysis in monoclonal antibody drug applications and found that the use of MS was highly prevalent. This study inspired an all-inclusive analysis of MS use in protein biologics license applications (BLAs). The current study aims to monitor the usage and progress of MS in these BLAs. The BLAs for 67 approved protein biotherapeutics were analyzed for the use of MS. This analysis discerned a variety of MS techniques and instrumentation. Results were further interpreted based on the relationship of MS use over time and between drug types. MS data was included in all but five of the 67 BLAs examined, which covered submissions from 1989 to Primarily, MS was used to analyze amino acid sequence, molecular mass, glycosylation, and other protein modifications. The methods applied were largely based on intact mass and/or peptide mapping experiments. However, recently, more modern approaches have been developed and employed for novel applications (i.e., QC assays, higher order structure, antibody drug conjugates, bispecific antibodies, or biosimilars). Thus, increased use of high resolution instrumentation and methods in academia and industry has translated into increased presentation of MS data in BLAs. As the complexity of the drug being analyzed for quality increases, the greater the need for higher resolution and sensitivity in the techniques used to measure its critical quality attributes. Hence, the presence of modern MS approaches is predicted to increase in BLAs submitted to the agency. 33

34 Mass Spectrometry Assisted Development of an Efficient In-silico Prediction Tool for Asparagine and Aspartate Degradation Sites Hans Rainer Voelger, Hubert Kettenberger Roche Diagnostics GmbH, Penzberg, Germany Monoclonal antibodies and proteins containing antibody domains are an important class of biotherapeutics in many disease areas. Besides functional properties, the stability of therapeutic proteins is a key criterion for the development of a drug candidate. Chemical degradation events such as asparagine deamidation or aspartate isomerization may limit stability as well as binding affinity of these proteins. Purely sequence motif-based methods for elimination of deamidation or isomerization sites are prone to massive over-prediction and therefore prevent the efficient identification of true hotspots of the drug candidates. We will present our approach to establish a fast and robust filtering for deamidation and isomerization inert protein candidates. First, we studied the structural properties of a large, uniform dataset of Asn and Asp residues in the variable domains of antibodies. Then their structural parameters were correlated with the degradation propensities measured by mass spectrometry. From these results we derive an accurate in silico prediction method for the degradation propensity of both Asn and Asp residues in the complementarity-determining regions of monoclonal antibodies. 34

35 Utility of Ion-mobility Mass Spectrometry in Antibody Drug Conjugates Characterization Richard Huang 1, Ekaterina Deyanova 1, David Passmore 2, Vangipuram Rangan 2, Shrikant Deshpande 2, Adrienne Tymiak 1, Guodong Chen 1 1 Bristol-Myers Squibb Company, Princeton, NJ USA, 2 Bristol-Myers Squibb Company, Redwood City, CA USA Antibody drug conjugate (ADC) becomes an emerging therapeutics in pharmaceutical industry. Compared to traditional chemotherapeutic cancer treatments, the treatments of ADCs expand the therapeutic window owing to the specificity of mab binding to biological target and the more predictable stability of mab. Characterization of drug-to-antibody ratio (DAR) of ADC, therefore, becomes a key assessment in support of drug discovery and development processes. For DAR characterization, analytical strategies such as UV absorbance, intact protein MS measurement, and HILIC column separation have already been implemented and have showed good success. This presentation will discuss the current MS-based strategies for ADC s DAR measurement. The use of ion mobility mass spectrometry (IM-MS) as a way to provide orthogonal information in DAR characterization will also be presented. 35

36 In-depth, Quantitative (Host Cell) Protein Fingerprinting of Adenovirus Based Vaccines by Mass Spectrometry Annemiek Verwilligen, Dayar van der Steeg, Jonathan Knibbe, Arjen Scholten, Harold Backus, Johan Nijenhuis Janssen Infectious Diseases and Vaccines, Leiden, The Netherlands The recombinant viral vectors employed in Janssen s AdVac vaccine platform are harmless adenoviruses. These are modified to genetically encode proteins of viral origin, so-called transgenes, which are expressed in the human cell upon administration. Adenoviruses are nonenveloped and consist of 14 capsid (structural) proteins and DNA. In analytical terms, manufactured recombinant adenovirus based vaccines constitute a mixture of adenovirus proteins and a small portion of residual host cell proteins (HCPs). For batch release of such vaccines, protein identity and quantity of both protein constituents is required. Currently, such tests are laborious and time consuming and are biased towards the much higher abundant adenovirus proteins due to the large variation in dynamic range. To overcome these challenges, we have developed a fast all-in-one semi-quantitative mass spectrometry-based approach. The method combines filter assisted digestion, C18 RP-UPLC separation coupled to ESI-Q-TOF MS E data acquisition. This allowed us to confirm the presence of all 13 adenovirus capsid proteins with ample sequence information and at the same time identify a significant amount of HCPs in a single 3h analysis. Adding, relative quantitation based on signal intensity further refined the method to estimate the ratio of adenovirus protein to HCPs. The obtained method is currently employed in characterization to gain product knowledge on multiple properties of the adenovirus vaccine, such as virus identity confirmation, evaluation of HCP co-purification from the vaccine manufacturing process and assessment of batch-to-batch variation. We envision the method should be embedded in targeted quantification of frequently co-purifying HCPs to aid in the development of FACS and ELISA platforms. Because of the short execution time of only three hours and the deep coverage this assay exemplifies the importance of mass spectrometry in the characterization of complex (adeno)virus based vaccines. 36

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38 Workshop Workshop I: Streamlining Workflows in the Mass Spectrometry Laboratory for Increased Productivity Thursday, September 24 10:00 11:00 Ballroom D Facilitator: Jason Rouse, Pfizer, Inc., Andover, MA USA Presenters: Melissa Alvarez, Genentech, a Member of the Roche Group, South San Francisco, CA USA Richard Rogers, Just Biotherapeutics, Seattle, WA USA Current general business mantra continues to be do more with less, while sustaining quality, market share, and innovation. For biotech companies in particular, this climate demands that departments, laboratories and colleagues work smarter and continually find ways to improve efficiency by basically rethinking strategies, revising workflows, updating methods, and investing in new technologies. Essentially every mass spectrometry (MS) laboratory and colleague supporting drug discovery, product and process development, and marketed-product lifecycle management face similar daily challenges: an overwhelming number of samples for analysis, manual data analysis, tedious report writing, tight deadlines, increased meetings/presentations, unplanned investigations, and new departmental initiatives. Unfortunately, each of these challenges are in direct competition with having sufficient blocks of time to investigate, develop, and implement new technologies and time-saving workflows/practices. While tweaking a strategy or re-designing a workflow will bring new laboratory efficiencies, it is the novel ideas, methods, instruments and/or software that afford both efficiency and enhanced information, which truly advances productivity to the next level. The first half of the workshop features two short case-study presentations from leading scientists in the industry, covering automated data analysis software and cross-laboratory method/system harmonization, respectively. The second half of the workshop will be an open forum discussion, where conference attendees are highly encouraged to openly share their needs, concerns, experiences, best practices, and practical tricks and tips toward streamlining laboratory workflows for increased efficiency and enhanced information. As time permits, additional open forum topics for discussion include: software for reliable automated data analysis and report generation; strategies for data storage, access, management, and long-term backup; automated sample preparation for high-throughput MS/multi-attribute testing; and futuristic MS in QC, MS for upstream product attribute control, and integrated analytics/real-time release. The workshop is envisioned to be highly beneficial for both industry practitioners and MS vendor companies/technology partners alike, especially given that it is our shared goal to deliver safe and effective medicines to patients in the shortest time possible. 38