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9th Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology Industry

(Mass Spec 2012)

Symposium Co-Chairs:

Yelena Lyubarskaya, Biogen Idec Inc. David Passmore, Bristol-Myers Squibb

September 11-14, 2012

Catamaran Hotel San Diego, CA USA

Organized by

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Table of Contents

Student Travel Grants ....................................................................................... 4 Program, Exhibitor and Media Partners ........................................................... 5 Scientific Final Program Summary ................................................................... 8 Plenary and Session Abstracts .......................................................................... 15 Workshop Session I (Wednesday, September 12) Description ........................ 36 Technical Seminar I (Wednesday, September 12) Abstract ............................. 37 Technical Seminar II (Thursday, September 13) Abstract ............................... 38 Wednesday Poster Abstracts ............................................................................ 40 Thursday Poster Abstracts ............................................................................... 74

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Welcome to the 9th Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology Industry We are pleased to welcome you to 9th 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 used routinely 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. Although most of the applications may deal with biopharmaceuticals (proteins, nucleic acids, viral vectors), applications for conventional pharmaceuticals will also be discussed. 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 AB SCIEX, Agilent Technologies, Amgen, Inc., Analytical Methods, Beckman Coulter, Inc, Biogen Idec, Inc., Bristol-Myers Squibb, Bruker Daltonics, ExSAR Corporation, Genedata AG, Genentech, a Member of the Roche Group, Genetic Engineering & Biotechnology News, Genovis AB, LabX, LCGC North America, MicroLiter Analytical Supplies, Inc., Pfizer, Inc., Technology Networks Limited, 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 2013! THE ORGANIZING COMMITTEE: Greg Adams, FUJIFILM Diosynth Biotechnologies Alain Balland, Amgen, Inc. James Carroll, Pfizer, Inc. Steven Cohen, Waters Corporation Victor Ling, Genentech, a Member of the Roche Group Anders Lund, Genzyme, a Sanofi Company Yelena Lyubarskaya, Biogen Idec Inc. (Co-chair) Jun Park, CDER, FDA David Passmore, Bristol-Myers Squibb (Co-chair) Dietmar Reusch, Roche Diagnostics GmbH Jason Rouse, Pfizer, Inc. Hansjörg Toll, Sandoz GmbH

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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 9th Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology Industry (Mass Spec 2012). PhD students or post-doctoral fellows conducting research at academia throughout the world are eligible. Why you should apply: 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:

Cedric Bobst University of Massachusetts Amherst, Amherst, MA USA Screening Stressed Glycoprotein Therapeutics for Disulfide Scrambled Species L. Renee Ruhaak University of California, Davis, Davis CA USA Biomarkers for Cancer: MS--based Glycosylation Analysis of Serum and its Specific Proteins Natalie J. Thompson Utrecht University, Utrecht, The Netherlands Antibody Profiling by Native Mass Spectrometry Using Q‐TOF and Orbitrap Platforms

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

Sustaining Platinum Program Partner

Agilent Technologies

Sustaining Silver Program Partner

Pfizer, Inc.

Gold Program Partner

Biogen Idec Inc.

Silver Program Partner

Amgen, Inc. Genentech, a Member of the Roche Group

Bronze Program Partner

Waters Corporation

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Friend of CASSS Program Partner

Bristol-Myers Squibb

Exhibitor Partners

AB SCIEX

Agilent Technologies Beckman Coulter, Inc.

Bruker Daltonics ExSAR Corporation

Genedata AG Genovis AB

MicroLiter Analytical Supplies, Inc. Thermo Scientific

Waters Corporation

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Leading Media Program Partner

Technology Networks Limited

Media Program Partner

Analytical Methods

Genetic Engineering & Biotechnology News LabX

LCGC North America

Supporting Organizations

The Association of Biomolecular Resource Facilities Bay Area Mass Spectrometry Discussion Group

CACO Pharmaceutical & Bioscience Society Deutsche Gesellschaft für Massenspektrometrie

Greater Boston Mass Spec Discussion Group MASSEP

Midwest Mass Spectrometry Discussion Group

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Mass Spec 2012

Scientific Final Program Summary

Tuesday, September 11, 2012 12:00 – 17:00 Registration in the Aviary Ballroom Foyer

Short Course in Macaw Session Chair: Anders Lund, Genzyme, a Sanofi Company, Framingham, MA USA

13:00 – 17:00 Fundamentals of Mass Spectrometry in the Analysis of Protein

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

17:00 – 18:00 Welcome Reception – All Conference Attendees – Beach North

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Wednesday, September 12, 2012 07:30 – 17:30 Registration in the Aviary Ballroom Foyer 07:30 – 08:30 Breakfast in Aviary Ballroom Foyer 08:30 – 08:45 Welcome and Introductory Comments in Cockatoo/Macaw

David Passmore, Bristol-Myers Squibb, Sunnyvale, CA USA

Plenary Session in Cockatoo/Macaw Session Chair:

Victor Ling, Genentech, a Member of the Roche Group, South San Francisco, CA USA 08:45 – 09:45 Mass-Spectral Protein Footprinting: A Tool for Protein Engineering

Michael L. Gross, Washington University in St. Louis, St. Louis, MO USA

09:45 – 10:15 Break – Visit the Exhibits and Posters in Toucan and Aviary Ballroom Foyer

Product Impurities Session in Cockatoo/Macaw

Session Chair: Victor Ling, Genentech, a Member of the Roche Group, South San Francisco, CA USA

10:15 – 10:45 Next Generation Sequence Variant Analysis for Therapeutic Proteins

Maximiliane Hilger, Roche Diagnostics GmbH, Penzberg, Germany 10:45 – 11:15 Alanine to Serine Sequence Variants Observed in an IgG4

Monoclonal Antibody Jinmei Fu, Bristol-Myers Squibb, Princeton, NJ USA 11:15 – 11:45 Identification and Monitoring of E. coli Host Cell Protein

Katrin Bomans, Roche Diagnostics GmbH, Penzberg, Germany 11:45 – 12:15 Detection of Low Level Tyrosine to Phenylalanine Misincorporation

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

12:15 – 12:30 Break – Visit the Exhibits and Posters in Toucan and Aviary Ballroom

Foyer 12:30 – 13:30 Technical Seminar: Lunch and Learn Structural Conformation is a Critical Attribute for Model Airplanes and Biotherapeutics Weibin Chen, Waters Corporation Sponsored by Waters Corporation Cockatoo/Macaw

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Wednesday, September 12, 2012 (continued)

13:30 – 13:45 Break – Visit the Exhibits and Posters in Toucan and Aviary Ballroom

Foyer

Vaccines Session in Cockatoo/Macaw Session Chair: James A. Carroll, Pfizer, Inc., St. Louis, MO USA

13:45 – 14:15 Real High-Throughput Glycoanalysis Combining the High-

Performance Analysis-Systems CE-MS and xCGE-LIF Erdmann Rapp, Max Planck Institute Magdeburg, Magdeburg, Germany

14:15 – 14:45 Characterization of a Complex Prophylactic Vaccine with Protein and

Glycoconjugate Components Paul W. Brown, Pfizer, Inc., St. Louis, MO USA

14:45 – 15:15 Analysis of Influenza Virus and Vaccines using Isotope Dilution Mass

Spectrometry Tracie Williams, Centers for Disease Control and Prevention, Atlanta, GA

USA 15:15 – 16:45 Poster Session - Visit the Exhibits and Posters in Toucan and Aviary

Ballroom Foyer 16:45 – 17:45

Host Cell Protein Analysis Workshop in Cockatoo/Macaw Session Chair: Steve Cohen, Waters Corporation, Milford, MA USA

and Andrew Goetze, Amgen, Inc., Thousand Oaks, CA USA

Introduction to HCP Methods and How Mass Spectrometry Can Play an Important Role Andrew Goetze, Amgen, Inc., Thousand Oaks, CA USA

Identification & Quantification of Host Cell Proteins in Biotherapeutics using MSE and Applications in Process Matt Schenauer, Allergan, Inc., Irvine, CA USA A Comparison Approach to Host Cell Proteins Anders Lund, Genzyme, a Sanofi Company, Framingham, MA USA Using MS to Solve an ELISA Issue Christopher Yu, Genentech, a Member of the Roche Group, South San Francisco, CA USA

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Wednesday, September 12, 2012 (continued)

Host Cell Protein Analysis in Drug Product Using Mass Spectrometry Qingchun Zhang, Amgen, Inc., Thousand Oaks, CA USA

17:45 – 19:00 Exhibitor and Poster Reception

Visit the Exhibits and Posters in Toucan and Aviary Ballroom Foyer

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Thursday, September 13, 2012 07:30 – 18:00 Registration in the Aviary Ballroom Foyer 07:30 – 08:30 Breakfast in Aviary Ballroom Foyer

Plenary Session in Cockatoo/Macaw Session Chair: Alain Balland, Amgen, Inc., Seattle, WA USA

08:30 – 09:30 The Ever Increasing Power of Mass Spectrometry in the Analysis of

Biotechnology Products - Where Do We Draw the Line? Anthony Mire-Sluis, Amgen, Inc., Thousand Oaks, CA USA

09:30 – 10:00 Break – Visit the Exhibits and Posters in Toucan and Aviary Ballroom

Foyer Characterization of Biological Samples and Protein Complexes Session in Cockatoo/Macaw

Session Chair: Anders Lund, Genzyme, a Sanofi Company, Framingham, MA USA 10:00 – 10:30 The Rapid Integration of LC-MS-based Bioanalytical Methods to

Quantify Protein Therapeutics in Drug Discovery Timothy Olah, Bristol-Myers Squibb, Princeton, NJ USA

10:30 – 11:00 The Role of Mass Spectrometry in Antibody Drug Conjugate Drug Development: Trastuzumab Emtansine (T-DM1)

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

11:00 – 11:30 A Proteomics Approach to Measuring In-vitro Innate Immune

Response in Human Cells Da Ren, Amgen, Inc., Thousand Oaks, CA USA 11:30 – 12:00 Ion Mobility-MS for Native Proteins and Complexes Frank Sobott, University of Antwerp, Antwerp, Belgium 12:00 – 12:15 Break - Visit the Exhibits and Posters in Toucan and Aviary Ballroom

Foyer 12:15 – 13:15 Technical Seminar: Lunch and Learn Characterization of Therapeutic Proteins for Biologic Development Ning Tang, Agilent Technologies Mellisa Ly, Agilent Technologies Sponsored by Agilent Technologies Cockatoo/Macaw

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Thursday, September 13, 2012 (continued) 13:15 – 14:45 Poster Session - Visit the Exhibits and Posters in Toucan and Aviary

Ballroom Foyer

Regulatory Issues: Biosimilars Session in Cockatoo/Macaw Session Chair: Jun Park, CDER, FDA, Bethesda, MD USA

14:45 – 15:45 Role of Advanced Analytics in the Development of Biosimilar Protein

Products Emily Shacter, ThinkFDA LLC, Takoma Park, MD USA 15:45 – 16:00 Break – Visit the Exhibits and Posters in Toucan and Aviary Ballroom

Foyer 16:00 – 16:30 Targeted Development of Highly Similar Biosimilars

Uwe Demebauer, Sandoz – Hexal AG, Oberhaching, Germany 16:30 – 17:00 TBD

17:00 – 18:00 Roundtable Discussion – Biosimilars

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Friday, September 14, 2012 07:30 – 08:30 Breakfast in the Aviary Ballroom Foyer 08:00 – 13:00 Registration in the Aviary Ballroom Foyer 08:30 – 08:45 Poster Award Announcement in Cockatoo/Macaw

Plenary Session in Cockatoo/Macaw Session Chair: Jason Rouse, Pfizer, Inc., Andover, MA USA

08:45 – 09:45 Protein Quantitation using Stable Isotope Labeling in Mammals

John Yates, The Scripps Research Institute, La Jolla, CA USA

09:45 – 10:00 Break – Visit the Exhibits and Posters in Toucan and Aviary Ballroom Foyer

Product Characterization Session in Cockatoo/Macaw

Session Chair: Hansjoerg Toll, Sandoz GmbH, Kundl, Austria 10:00 – 10:30 Characterization of Currently Marketed Heparin Products: Analysis

of Heparin Digests by Mass Spectrometry David Keire, DPA, FDA, St. Louis, MO USA

10:30 – 11:00 Molecular Heterogeneity of Polysorbates and Its Implications Studied with LC-MS Oleg Borisov, Amgen, Inc, Thousand Oaks, CA USA

11:00 – 11:30 MS Methods for Top Down Characterization of Cysteinyl Linked

Antibody Drug Conjugates John Valliere-Douglass, Seattle Genetics, Bothell, WA USA

11:30 – 11:45 Break 11:45 – 12:15 Characterization of a Heavily Glycosylated Fusion Protein with

Unique Modifications Keith A. Johnson, Pfizer, Inc., Andover, MA USA

12:15 – 12:45 Comprehensive Mass Spectroscopic Characterization of Recombinant

Phenylalanine Ammonia Lyase Erno Pungor, BioMarin Pharmaceutical, Inc., Novato, CA USA

12:45 – 13:00 Closing Comments in Cockatoo/Macaw

Yelena Lyubarskaya, Biogen Idec Inc., Cambridge, MA USA

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Oral Abstracts

Mass-Spectral Protein Footprinting and Top-Down Sequencing: Tools for Biotechnology Michael L. Gross Washington University in St. Louis, St Louis, MO USA Our goal is a rapid, sensitive, and specific means of determining protein integrity, interactions and folding by using chemical footprinting coupled with MS. Driving this approach is the wide availability of mass spectrometers for analytical proteomics; these should also be applicable to footprinting. To this end, we are developing fast photochemical oxidation of proteins (FPOP) and hydrogen/deuterium exchange (e.g., PLIMSTEX) to interrogate protein interactions, interfaces, and dynamics of folding/unfolding. A significant advantage of H/DX is its simplicity and generality. FPOP is fast owing to the use of free radicals; radical generation occurs in low ns, and the radical reactions are complete in 1 microsec. Only a single conformation of the protein exists during footprinting. Besides OH radicals, other radicals such as I• and •OSO3

-can be used. FPOP can also be the probe in a classic, two-laser "pump-probe" experiment whereby a temperature-jump perturbation is produced as a pump by one laser and a second laser initiates FPOP as the probe. Using subsequent MS analysis, this experiment is capable of probing protein structural dynamics at the sub millisecond level with high sensitivity and more detailed structural resolution than any physical chemical method. Better understanding of folding will emerge from this approach than from those using usual physical probe methods (e.g., fluorescence). Top-down sequencing of proteins coupled with footprinting and sometime ion mobility can provide additional information to characterize proteins, particularly those of therapeutic utility. Collaborating with Justin Sperry and James Carroll at Pfizer, we have preliminary results on the efficacy of this combination. We propose that these MS-based methods will occupy a "middle ground" in protein science between the high resolution methods of X-ray crystallography and NMR and the low resolution methods of fluorescence, absorbance, CD, calorimetry, and SPR, because the MS methods can determined sites of interaction and folding. NOTES:

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Next Generation Sequence Variant Analysis for Therapeutic Proteins Maximiliane Hilger1; Christopher Yu2; Hans Rainer Voelger1; Michael Mølhøj1; Georg Drabner1; Wilma Lau1; Vincent Larraillet1; Melissa Alvarez2; Cornelia Wagner1; Victor Ling2; Joerg Thomas Regula1

1Roche Diagnostics GmbH, Penzberg, Germany; 2Genentech, a Member of the Roche Group, South San Francisco, CA USA Sequence variants (SVs) are protein species with unintended amino acid substitutions that occur in recombinantly expressed proteins as they do naturally in endogenous proteins. Due to its high sensitivity, mass spectrometry (MS) is the method of choice for sequence variant analysis (SVA) compared to sequencing techniques. Moreover all sources of SVs such as mutations, transcriptional and posttranslational errors can be covered analyzing SVs on the protein level. Typical MS-based SVA workflows involve peptide mapping using LC-MS/MS on a high-resolution mass spectrometer and identification of SVs by Mascot error tolerant search (ETS) followed by extracted ion chromatogram-based quantitation. Orthogonal data analysis by SIEVE features detection of all types of LC-MS differences between two samples including SVs not detected by Mascot ETS. High numbers of false positives by Mascot ETS and SIEVE, that both need to be evaluated manually, make SVA very time consuming. Therefore we aimed to automate SVA by developing a custom-made software tool that significantly reduces data analysis time especially for MS/MS validation and quantification. Linking expert knowledge to typical Mascot hits additionally helps us to quickly recognize false positives. Applying the newest SIEVE version (2.0) with optimized parameters allows us to significantly reduce the number of false positives in SIEVE. Although MS-based SVA is capable of detecting SVs down to sub percent levels, not all potential SVs can be detected down to that level. While inappropriate size and ionization may be compensated by alternative enzymatic digestion, dynamic range limitations of the current MS instruments make some peptides undetectable as low level variants. Here application of an optimized UPLC gradient improved sensitivity. Generally, tight control of the entire SVA workflow is required to ensure highest sensitivity, which led us to develop a system suitability test that needs to be passed at the beginning and end of each SVA. NOTES:

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Alanine to Serine Sequence Variants Observed in an IgG4 Monoclonal Antibody Jinmei Fu1, 2; Jacob Bongers1, 2; Richard Ludwig1, 2; Li Tao1, 2; Michael Grace1, 2; Reb Russell1, 2

1Bristol-Myers Squibb, East Syracuse, NY USA; 2Bristol-Myers Squibb, Princeton, NJ USA Low levels of sequence variants resulting from two mutated constructs were identified in an IgG4 monoclonal antibody by high performance liquid chromatography and tandem mass spectrometry. The levels of the identified sequence variants A183S and A152S, both in the light chain, have been determined to be 7.8-9.9% and 0.5-0.6 %, by extracted ion currents of the tryptic peptides L16 and L14, respectively. The A183S mutation was confirmed through tryptic map spiking experiments using synthetic peptide, SDYEK, which incorporated Ser at the position of native Ala in the tryptic peptide L16. Both mutations were also observed by endoproteinase Asp-N peptide mapping. The variant level of A183S was also quantified by UV absorption at 280 nm and native fluorescence of tyrosine residues on the tryptic peptides. The results from LC-MS, UV, and fluorescent detection are in close agreement with each other. The levels of the sequence variants are comparable among the antibody samples manufactured at different scales as well as locations, indicating variants’ levels are not affected by manufacture scale or locations. DNA sequencing on the master cell bank revealed the presence of mixed bases at position 183 encoding both wild and mutated populations, whereas bases encoding the minor sequence variant at position152 were not detected. The root cause for A152S mutation is still being investigated. NOTES:

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Identification and Monitoring of E. coli Host Cell Protein Katrin Bomans; Patrick Bulau Roche Diagnostics GmbH, Penzberg, Germany Recombinant antibodies are produced in nonhuman host cells derived from Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, several E. coli host cell proteins (ECP) were identified by HPLC fractionation, SDS-PAGE and ESI-MS peptide mapping. Identification of low abundant ECP from final drug substance samples was feasible via Anti-ECP column combined with sensitive analysis by LTQ Orbitrap–MS. By using this strategy we were able to develop two high throughput ECP quantification assays on a Cobas Integra 400 plus system. NOTES:

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Detection of Low Level Tyrosine to Phenylalanine Misincorporation Christopher Yu; Jennifer Wang; Betty Chan; David Michels; Melissa Alvarez; Amy Shen; Lauren Feeney; Michael Laird; Victor Ling Genentech, a Member of the Roche Group, South San Francisco, CA USA Translation errors including amino acid misincorporation during protein biosynthesis result in low level product variants. These variants can be discovered using rigorous product characterization efforts and various strategies can be evaluated to reduce and/or control these amino acid substitutions. Peptide map analysis by reversed phase high performance liquid chromatography and high resolution mass spectrometry was used to detect and identify possible variants in monoclonal antibody samples. Tyrosine to phenylalanine substitution variants were observed in multiple tryptic peptides containing tyrosine residues. The identification was facilitated with the use of Mascot error tolerant search, supported by high resolution MS1 data acquisition with ±10 ppm accuracy. MS/MS spectra were examined, with -16 Da mass shifts confirmed to occur at positions corresponding to expected tyrosine residues in multiple peptides. The retention times of peptides containing phenylalanine variants were observed to be significantly later than that of the peptides containing tyrosine, consistent with the more hydrophobic nature of phenylalanine residues. Integration of peak areas in the extracted ion chromatogram of the expected and variant peptide ions proved to be useful means of determining the relative level of misincorporation in each peptide. The phenylalanine variant level was found in a range from as high as 5% to below the estimated detection limit of 0.01%. Although the majority of tyrosine containing peptides showed phenylalanine containing variants within a given sample, variable levels of misincorporation were observed for different tryptic peptides. For relative ranking of misincorporation levels in samples with different cell culture media and feed conditions, we found it useful to compare the variant level of each peptide among samples. With this approach, we were able to demonstrate a good correlation between the observed misincorporation level and the tyrosine level in the production culture media during the last several days of the culture. NOTES:

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Real High-Throughput Glycoanalysis Combining the High-Performance Analysis-Systems CE-MS and xCGE-LIF Erdmann Rapp1, 2; René Hennig1, 2; Svenja-Catharina Bunz3; Thilo Muth1, 2; Christian Neusüß3; Udo Reichl1, 4

1Max Planck Institute Magdeburg, Magdeburg, Germany; 2glyXera GmbH, Magdeburg, Germany; 3Aalen University, Aalen, Germany; 4Otto-von-Guericke University, Magdeburg, Germany Glycomics is a rapidly emerging field that can be viewed as a complement to other “omics” approaches including proteomics and genomics. Hence, there is a dramatic dynamic increase in the demand for sophisticated databases and analytical tools in glycobiology respectively, glycobiotechnology. In order to enhance and improve the comparatively small existing glycoanalytical toolbox, fully automated highly sensitive, reliable, high-throughput and high-resolution analysis methods including automated data evaluation are required - i.e. high-performance methods. Especially one glycoanalysis approach, based on multiplexed capillary gelelectrophoresis with laser induced fluorescence detection (xCGE-LIF), shows high potential for high-performance analysis of glycoconjugates. The developed xCGE-LIF based high-performance glycoanalysis system (method, software and database), allows to evaluate the generated “normalized” electropherograms of glycomoieties (“fingerprints”) on three stages: (1) “simple” qualitative and quantitative pattern comparison ("fingerprint"-analysis), (2) identification of compounds in complex mixtures via database matching (“glycoprofiling”) and (3) extended structural analysis using exoglycosidase sequencing in combination with xCGE-LIF based glycoprofiling. The development of this innovative glycoanalysis system and its application to different fields with respect to sample preparation, separation and data analysis is presented [1,2,3]. In particular the build-up of the corresponding oligosaccharide-database is demonstrated [1,4]. Therefore, APTS-labeled glycans are identified by capillary electrophoresis-mass spectrometry (CE-MS). Online coupling is performed by ESI in the negative ion mode using a coaxial sheath-liquid interface in front of a quadrupole time-of-flight hybrid mass spectrometer (QTOF-MS). In this mode, for CE-MS the same elution order is achieved as for xCGE-LIF. Thus, this approach allows a fast database build-up by linking the identification of unknown glycans using the accurate mass of TOF-MS, with the “normalized” migration times of the peaks within xCGE-LIF “fingerprints.” This novel modular high-performance glycoanalysis system allows fully automated, highly sensitive, instrument-, lab- and operator-independent "real" high-throughput glycoanalysis. NOTES:

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Characterization of Complex Prophylactic Vaccines with Protein and Glycoconjugate Components Paul W. Brown; Deanna C. Schuchmann; Nathan A. Lacher; Robert L. Dufield; James A. Carroll; Jason C. Rouse Pfizer, Inc., St. Louis, MO USA In general, prophylactic vaccines are a very heterogeneous group of preventative medicines with an increasingly wide variety of proteins, polysaccharides and adjuvants used in their formulation. Modern mass spectrometric (MS) methods provide many advantages for the in-depth characterization of vaccine components relative to classical techniques. Three example applications of MS for multi-component vaccines are presented where MS enabled faster, more comprehensive product characterization at higher sensitivity. For another protein constituent in a vaccine, an uncommon post-translational modification, gluconoylation, was detected at low levels and identified from on-line mass analyses of the intact protein and proteolytic map. These modifications were localized to the N- terminus as determined via on-line tandem mass spectrometry. A protein component in a particular vaccine was characterized by both reversed phase LC-MS and peptide mapping. A host cell protein was detected at very low levels in drug substance via accurate mass analysis, and identified by a top-down proteomics approach in which all intact proteins were fragmented via collision-induced dissociation (CID). The CID mass spectrum was deconvoluted and interrogated using MASCOT. The top-down approach provided faster means of identification as compared to fraction collection followed by conventional Edman sequencing or proteolytic digestion and LC/MS/MS. The host cell protein subsequently was detected and confirmed at very trace levels in the routine peptide map. Lys-C proteolytic digestion of a protein-polysaccharide conjugate gave rise to the detection of low-level acetylated, miscleaved lysines in the peptide map. A mass spectrometry-based method was developed for the semi-quantitation of the acetylated peptides to support the development of the conjugation step in the manufacturing process. The key reaction intermediate responsible for acetylation also was identified by high resolution tandem mass spectrometry. With this information, the conditions for the conjugation chemistry were adjusted to minimize acetylation. NOTES:

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Analysis of Influenza Virus and Vaccines using Isotope Dilution Mass Spectrometry Tracie Williams Centers for Disease Control and Prevention, Atlanta, GA USA Influenza vaccination is the primary method for preventing influenza and its severe complications. Influenza viral strains undergo rapid mutations and may experience an abrupt change in the surface proteins hemagglutinin (HA) and neuraminidase (NA), which can result in a virus that is radically different from those previously circulating in human populations. This is the reason that seasonal vaccines must be annually updated. The mutations and their effects on the virus and its subsequent transmissibility to or between humans cannot be adequately foreseen. Thus, the strategy of predicting which strain of influenza may cause the next worldwide pandemic so that vaccines can be manufactured and stockpiled prior to the emergence of an influenza pandemic may not be successful. It is instead, imperative that the process of producing a vaccine that will protect the public in the face of an unpredicted virus strain be streamlined and as swift as possible. We have developed an isotope dilution mass spectrometry method to simultaneously quantify viral proteins of numerous influenza subtypes. This method can be used in many different phases of vaccine production—from analysis of seed strains to aid in the decision of which is the most efficient producer to accurately quantifying the amount of antigen in the final production lot. This method can be applied to allantoic fluid, purified virus preparations, and to trivalent inactivated influenza vaccines with improved speed, sensitivity, precision, and accuracy. This LC/MS/MS approach may substantially increase the reliability of methods used to quantify the amount of antigen in seasonal and pandemic influenza vaccines and reduce the time and effort to deliver influenza vaccines for public health use during the next influenza pandemic. NOTES:

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The Ever Increasing Power of Mass Spectrometry in the Analysis of Biotechnology Products - Where Do We Draw the Line? Anthony Mire-Sluis Amgen, Inc., Thousand Oaks, CA USA Mass Spectrometry is becoming an ever increasingly powerful tool in the characterization and lot release of biotechnology products. However, this increasing depth of analysis comes with a price - how much information is actually useful and relevant for safety and efficacy or lot to lot consistency. Ever complicated spectroscopic profiles requires the time and expertize to understand the exact nature of what is being reported as well as efforts to ensure that the methodology itself is appropriately qualified or validated. How one goes about assessing what is background noise versus something that is a 'real' peak and what to do with it remains a challenge. One has to ask the philosophical question of where to stop and how to justify not investigating minor species to a regulator, whose role is naturally conservative. In addition, the algorithms that are required to analyze complex readouts can lead to challenges during method development and qualification/validation from a regulatory and GMP perspective - decision making moves from the analyst to the 'technology'. NOTES:

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The Rapid Integration of LC-MS-based Bioanalytical Methods to Quantify Protein Therapeutics in Drug Discovery Timothy Olah Bristol-Myers Squibb, Princeton, NJ USA Because of the combination of selectivity and sensitivity, LC-MS-based methods are rapidly evolving as a viable technique for both the accurate quantitation of proteins in biological fluids and as a complementary technique to the more established ligand binding assay in support of early Drug Discovery. As an attribute of their technical expertise to rapidly develop multiple analyte methods, bioanalysts are refining the bioanalytical process for the simultaneous detection of multiple peptides that are generated from the enzymatic digestion of proteins as a strategy for quantifying and assessing proteins on a molecular level in biological fluids. This presentation describes the challenges in rapidly developing quantitative LC-MS-based methods for diverse types of therapeutic, target, and biomarker proteins in biological fluids for in vivo Discovery studies. This strategy of quantitative bioanalysis is based on the selected enrichment of proteins from biological fluids, enzymatic digestion of the sample or extract, and LC-MS-based detection of multiple specific peptides for the quantitation of the proteins. The proteins are either separated from endogenous proteins using different preparation techniques (Liquid Extraction, Immunocapture, Dried Blood Spot sampling) and/or digested with specific enzymes directly. The samples are injected onto LC-MS systems and selected peptides are detected on different platforms using both accurate mass detection on OrbiTrap or QTOF mass spectrometers and Selected Reaction Monitoring (SRM) on triple quadrupole mass spectrometers. The challenges in establishing sensitive methods that simultaneously determine multiple analytes with different physicochemical properties is discussed along with examples that supported the results of studies by increasing the level of detailed molecular information that was gained from these more complex analyses. NOTES:

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The Role of Mass Spectrometry in Antibody Drug Conjugate Drug Development: Trastuzumab Emtansine (T-DM1) Ola M. Saad; Keyang Xu; Luna Liu; Neelima Koppada; Surinder Kaur Genentech, a Member of the Roche Group, South San Francisco, CA USA Antibody drug conjugates (ADCs) are tumor specific antibodies used to deliver a cytotoxic drug to cancer cells. Upon internalization, ADCs can release the cytotoxic drugs inside target tumor cells, minimizing the exposure to non-target cells and thereby reducing potential side effects of the treatment. Analysis of ADCs in vivo has been challenging due to their complex and heterogeneous structures. Our bioanalytical strategies involve a number of mass spectrometry approaches for the quantitative and qualitative analysis of the fate of these biotherapeutics in vivo. Trastuzmab emtansine (T-DM1) is Genentech’s first ADC currently under evaluation in PhIII clinical studies and will be used as a case study here. It contains the recombinant humanized monoclonal antibody Trastuzumab linked to the cytotoxic drug maytansinoid, DM1. Due to the structural complexity of T-DM1, which presents unique analytical challenges, a number of bioanalytical assay methodologies were used. These incorporated strategies for both large molecules and small molecules to characterize the pharmacokinetics and biotransformation of T-DM1 in plasma. In addition to standard immunoassays to measure antibody concentrations, we used a validated LC-MS/MS PK assay to measure DM1 and several exploratory assays to measure small molecule catabolites of T-DM1. An affinity capture LC-MS method was also used to characterize intact molecular masses of the ADC in plasma to assess structural changes over time in vivo. The improved strategy, including the combination of methods and incorporating several mass spectrometry approaches, has provided a powerful tool for the development of the ADC therapeutic platform. NOTES:

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Ion Mobility-MS for Native Proteins and Complexes Frank Sobott University of Antwerp, Antwerpen, Belgium Recently native approaches in mass spectrometry combined with ion mobility have been applied to characterize the conformations of proteins and other biomolecules, as well as the composition, topology and subunit arrangement of large protein complexes. But what does a measured collision cross section really tell us about the conformational space of a protein or the subunit arrangement of a complex? How critical is the absence of bulk solvent in the gas phase, and how much do the measured size and the flexibility depend on the internal energy ('temperature') of the ions? We are using nano-ESI coupled with T-wave IM-MS (Waters Synapt G1 and G2) to assess the structure and conformational space of a couple of well understood proteins and complexes, and correlate the data with results from other biophysical methods such as size-exclusion chromatography, surface plasmon resonance and small-angle x-ray and neutron scattering. In direct comparison, closely related protein sequences (e.g. mutations, truncations or post-translational modifications) show subtle changes which are frequently reflected in the position and width of the drift time profile (i.e. “size” and “flexibility”). The global, structural effects of ligand binding can also become apparent using IM-MS in screening approaches. In addition, we can for example determine the threshold of unfolding or the 50% unfolding point as an indirect measure of the gas-phase stability of native biomolecular structures. Taken together these data highlight the importance of controlling the internal energy of ions carefully in the experiment, and raise some critical points regarding the measurement of absolute (rather than relative) sizes by IM-MS. They also demonstrate the level of information we can extract from careful, systematic measurements of collision cross sections of ions of biochemical and pharmaceutical interest. NOTES:

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A Proteomics Approach to Measuring In-vitro Innate Immune Response in Human Cell Da Ren Amgen Inc., Thousand Oaks, CA Immunogenicity is one of the major concerns for the development of protein therapeutics. To understand the immunogenic potential of therapeutic proteins and protein aggregates, we established an in vitro model to study the innate immune response by using LC/MS/MS based proteomics method followed by pathway analysis. Different forms of human IgG aggregates generated by heat, stirring, and pH stress have been tested in human acute monocytic cells, THP-1. The immunogenic response of human monocytes was measured by proteomics analysis over time (6 and 24 hours). Lipopolysaccharides (LPS) and a buffer sample were used as positive and negative controls and protein abundances in each sample were correlated to these two controls. The averaged correlation coefficients of the proteomes for each sample were plotted by the proximity to positive and negative controls to establish a two-dimensional immunogenic scale. Three proteins STAT1, ISG15 and MX1, which are part of the IFN/STAT signaling pathway of immune response, have been found to have strong correlation with the established immunogenic scale. Additionally, pathway analysis revealed several heat shock protein (HSP) family members known as chaperokines appeared with high scores in key node analysis. The model established in the study can be used for further immunogenic evaluation of therapeutic proteins. NOTES:

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Role of Advanced Analytics in the Development of Biosimilar Protein Product Emily Shacter ThinkFDA LLC, Takoma Park, MD USA The FDA’s draft guidance on the regulatory expectations for biosimilars delineates over-arching scientific considerations as well as specific analytical, nonclinical, and clinical approaches for the demonstration of similarity to a US-licensed reference product. It is clear from the guidance documents that the use of advanced protein analytics will be fundamental to the demonstration of similarity. This talk will provide insights into the FDA’s overall approach to the regulation of biosimilars and will discuss the tools that should be used to analyze and evaluate molecular similarity. NOTES:

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Targeted Development of Highly Similar Biosimilars Uwe Demelbauer1; Martin Schiestl2; Thomas Stangler2; Andreas Seidl1

1Sandoz Biopharmaceuticals, Hexal AG, Holzkirchen, Germany; 2Sandoz Biopharmaceuticals, Sandoz GmbH, Kundl, Austria Setting goal posts and the definition of the quality target profile are essential parts of biosimilar development. At Sandoz this definition is done utilizing the variability of the reference product (originator). The characterization of multiple batches of originator defines the target range of quality attributes (QA) for process development. These quality attributes are ranked with a risk based approach assessing impact of the QA on efficacy, PK/PD, immunogenicity and safety and the knowledge strength which is available (uncertainty). Together with the assessment of the structural functional relationship understanding this risk assessment of QAs drives the direction or prioritization of process development. Throughout the biosimilar development, for major project milestones, a similarity exercise is done for comparison between originator and biosimilar candidate. This detailed characterization covers the primary structure elucidation, including the analysis of the micro heterogeneity originating from post translational modifications, the secondary and tertiary structure of the protein, the behavior under stressed and non-stressed stability studies and a biological characterization. In all of these areas of analytics, mass spectrometry is used as a sensitive and selective analytical tool. The talk will present case studies throughout development process of a proposed biosimilar. The basic sequence determination with MS and MS/MS techniques with amino acid resolution, identification and quantitation of sequence variants or post translational modifications with ultra-exact mass traces, evaluation of higher order structure HDX experiments or the variant specific determination of PK profiles in pre-clinical studies. NOTES:

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Protein Quantitation using Stable Isotope Labeling in Mammals John Yates; Daniel McClatchy; Jeff Savas; Lujian Liao; Robin Park The Scripps Research Institute, La Jolla, CA USA A component to understanding biological processes involves identifying the proteins expressed in cells as well as their modifications and the dynamics of processes. Several major technologies, but especially mass spectrometry, have benefited from large-scale genome sequencing of organisms. The sequence data produced by these efforts can be used to interpret mass spectrometry data of proteins and thus enables rapid and large-scale analysis of protein data from experiments. Advances in multi-dimensional separations as well as mass spectrometry have improved the scale of experiments for protein identification. This has improved the analysis of protein complexes and more complicated protein mixtures. Quantitative mass spectrometry can be used to study biological processes. We developed methods to label rats and mice with 15N for use as internal standards for complex tissue samples 1. This allows the study of animal models of development and disease2-4. The advantages and disadvantages of the approach in the study of several different disease and injury models will be discussed. Keywords: Proteomics, Rats, Mice, Quantitative Mass Spectrometry, Stable Isotopes References 1. C. C. Wu, M. J. MacCoss, K. E. Howell et al., Anal Chem 76 (17), 4951 (2004). 2. D. B. McClatchy, M. Q. Dong, C. C. Wu et al., J Proteome Res 6 (5), 2005 (2007). 3. D. B. McClatchy, L. Liao, S. K. Park et al., Genome Res 17 (9), 1378 (2007). 4. D. B. McClatchy, L. Liao, S. K. Park et al., PLoS One 6 (1), e16039 (2011). NOTES:

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Characterization of Currently Marketed Heparin Products: Analysis of Heparin Digests by Mass Spectrometry David Keire1; Bo Wang1; Adam Brustkern1; Michael Boyne1; Lucinda Buhse1; Ali Al-Hakim2

1DPA, FDA, St. Louis, MO USA; 2CDER, FDA, Silver Spring, MD USA Intact heparin is a complex mixture of monosaccharide chains of varying length that contain micro-heterogeneity in sulfation pattern, acylation and epimerization along the sequence. Because of past issues with contaminated heparin products entering the US market, the FDA has studied mass spectrometry methods to assess the quality of the drug. Unfortunately, mass spectrometry of intact heparin mixtures are complicated by the loss of the labile sulfate groups upon ionization and variation in the protonation state of heparin acidic groups which leads to unresolved mass spectra even on modern high resolution mass spectrometers. However, disaccharide level enzymatic digests of heparin are sensitive to the composition of the drug and can be studied by liquid chromatography-mass spectrometry methods. Here we applied reverse phase ion-pairing high-pressure or ultra-high pressure liquid chromatography (RPIP-HPLC or RPIP-UHPLC) coupled with an ion trap or a quadrupole time-of-flight (Q-TOF) mass spectrometer to separate and quantify heparin digest mixtures. Digests of twenty lots of heparin sodium active pharmaceutical ingredients (APIs) or ten low molecular weight heparin (LMWH: dalteparin, tinzaparin or enoxaparin) lots were characterized representing multiple lots manufactured by different processes. These data establish the normal variation in the composition of heparin sodium or LMWHs as measured by this assay. These values are useful as possible product benchmarks and for surveillance of the quality of heparin products being imported into the US market. NOTES:

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Molecular Heterogeneity of Polysorbates and Its Implications Studied with LC-MS Oleg Borisov Amgen, Inc., Thousand Oaks, CA USA Polysorbates are widely used as surfactants in the agricultural, food, personal care, and pharmaceutical industries due to their cost effectiveness, biocompatibility, formulation flexibility, low toxicity, and good stabilizing and protecting properties. The polysorbates are often pictured as polyoxyethylated sorbitan monoesters of saturated and/or unsaturated fatty acids. In reality, polysorbates are complex mixtures of multiple components, as follows from the reactions involved in their production. In this talk, a novel application of liquid chromatography mass spectrometry (LC-MS) for the characterization of polysorbates, will be presented. This method takes advantage of accurate mass measurements and information on the identity of a fatty acid from “in-source” generated characteristic dioxolanylium ions. The method allowes to perform quick profiling of fatty acids in PS 20 and 80 which, combined with a computer-aided peak assignment algorithm, facilitated detailed characterization of their constituents. Use of the LC-MS method to detect and monitor degradation products of polysorbates and their pathways upon oxidative stress, will be presented and discussed. NOTES:

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MS Methods for Top Down Characterization of Cysteinyl Linked Antibody Drug Conjugates John Valliere-Douglass; William McFee; Jay Jones; Oscar Salas-Solano Seattle Genetics, Bothell, WA USA We present here a method for the rapid determination of the intact mass of noncovalently associated antibody heavy chains (HC) and light chains (LC) which result from the attachment of drug conjugates to interchain cysteine residues. We have employed a native SEC-MS method to maintain the intact bivalent structure of the ADC, which ordinarily would decompose as a consequence of denaturing chromatographic conditions typically used for liquid chromatographic-mass spectrometric (LCMS) analysis. The mass of the desalted ADC is subsequently determined using standard desolvation and ionization conditions. The native SEC-MS method has been found to be suitable for determining the intact mass of IgG1 mAbs conjugated with maleimidocaproyl-monomethyl Auristatin F (mcMMAF) and valine-citrulline-monomethyl Auristatin E (vcMMAE) at interchain cysteine residues. Additionally, the relative distribution of drug loaded forms obtained using our native SEC-MS method is in agreement with other orthogonal off-line methods such as hydrophobic interactions chromatography (HIC) which are used to quantitate the molar ratio of drugs on ADC’s. Finally, we have found that the native SEC-MS method is ideally suited for application in a process development environment for the routine, high-throughput characterization of ADC’s with drugs attached at interchain cysteine residues. NOTES:

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Characterization of a Heavily Glycosylated Fusion Protein with Unique Modifications Keith A. Johnson; Himakshi Patel; Lisa A. Marzilli; Jason C. Rouse Pfizer, Inc., Andover, MA USA Recombinant fusion proteins result from attaching the amino acid sequences of more than one protein together to form a single, chimeric protein. In the pharmaceutical industry, fusion proteins can be used to target more than one indication or, in the case of the receptor-Fc protein described here, to increase the half-life of the circulating biotherapeutic. Receptor-Fc proteins typically have molecular masses that are less than monoclonal antibodies (mAbs), but the extensive structural heterogeneity of receptor-Fc proteins makes their analytical analysis difficult. This complexity can be predicted from the amino acid sequence from the presence of N-glycosylation consensus sites and other known sequence motifs for additional posttranslational modifications. Some posttranslational modifications to the protein backbone (e.g. O-glycosylation) are not predictable based on the amino acid sequence alone. The routine high-resolution mass spectrometry methods for mAbs make a good starting point for receptor-Fc protein characterization, but many of these methods must be optimized, further developed, or in some cases replaced with new techniques. This presentation will demonstrate the adaptability of some mAb mass spectrometry-based methods to this receptor-Fc protein, as well as highlight other off-platform methods that were used for the successful characterization of the receptor-Fc biotherapeutic. This presentation will also illustrate how complex proteins still test the limits of state-of-the-art mass spectrometers. Finally, an assessment of the methods and their applicability to future complex biotherapeutics will be summarized. NOTES:

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Comprehensive Mass Spectroscopic Characterization of Recombinant Phenylalanine Ammonia Lyase Fangfei Yan1; Dan Wendt2; Phil Alferness2; William Hancock1; Shiaw-lin Wu1; Erno Pungor2

1The Barnett Institute, Northeastern University, Boston, MA USA; 2BioMarin Pharmaceutial Inc, Novato, CA USA Phenylketonuria (PKU) is an autosomal recessive disorder characterized by deficiency in the phenylalanine hydroxylase, an enzyme that converts phenylalanine to tyrosine. In PKU, phenylalanine can accumulate in the blood and body tissues causing neurological damage. A recombinant form of the phenylalanine ammonia lyase from Anabaena variabilis (rAvPAL), converting phenylalanine to trans-cinnamate is now in clinical development to treat PKU. The active site of rAvPAL contains an ASG sequence that is spontaneously converted into 4-methylidene-imidazole-5-one (MIO) ring through a double dehydration. The MIO in the active site can decompose under certain conditions ultimately leading to a cleavage in the peptide backbone of the 61 kDa intact protein, resulting in a 16 kDa and a 45 kDa protein fragment. As the MIO is not stable under denaturing conditions and /or acidic environment, traditional peptide mapping approaches could not confirm its presence on rAvPAL. Our studies using multiple and sequential proteolytic digestions coupled high resolution (FT-ICR) mass spectroscopy and with LC-MS/MS analysis identified the exact break-down sites and revealed that the decomposition of the MIO and the chain cleavage are results of formation of free radical peptides. Capillary Gel Electrophoresis analysis demonstrated that the peptides containing free radical moiety can also react with the intact rAvPAL protein to form non-reducible stable aggregates. Eliminating the double bond on the methylidene group by Michael-type additions stopped the decomposition of the MIO site and allowed for direct LC-MS/MS identification of the of the Michael addition products. These results were then confirmed with direct mass spectroscopic analysis of the whole protein. NOTES:

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Workshop Abstract

Host Cell Protein Analysis Wednesday, September 12 16:45-17:45 Cockatoo/Macaw Host cell protein (HCP) assays are an essential component in characterizing recombinant protein biopharmaceuticals as their presence in the final drug product may compromise patient safety due to an immunogenic response to the residual proteins. Removal of proteins that co-purify with the active substance can be difficult, and regulatory agencies are always concerned about the level of HCP in the final product. The major agencies have formulated guidelines, but there are still many questions concerning the actual levels and the identification of host cell proteins present in protein biotherapeutics. Common HCP assays include western blotting (WB), 1 or 2-D gels with silver staining and ELISA based methods. Each of these techniques provides at least semi-quantitative information on the presence of HCP, but detection, quantitation and/or identification of HCP present may lack sensitivity or specificity. For example, ELISA methods are sensitive to sub ppm levels, but provide little or no information regarding the amount or identity of individual components present. ELISA methods will also fail to detect any proteins that do not elicit an immune response in animal immunized with a host cell protein mixture. On the other hand, WB methods may not provide better than 50-100 ppm sensitivity, and can fail to detect proteins that are denatured with loss of the relevant epitope during sample preparation. Recent advances in mass spectrometric methods offer the promise of both identifying and quantifying HCP based on technology developed for proteomics. However, there are significant challenges to obtaining sufficiently sensitive, reproducible data based on mass spectrometry (MS) as their detection in the presence of an enormous excess of product protein is a daunting task. Moreover, as yet no MS based methods have been validated. This workshop will discuss those challenges and present several approaches to solving the problems posed in MS based HCP analysis. NOTES:

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Technical Seminar Abstracts

Waters Corporation Lunch and Learn Seminar Wednesday, September 12 12:30 – 13:30 Cockatoo/Macaw Structural Conformation is a Critical Attribute for Model Airplanes and Biotherapeutics Weibin Chen, Waters Corporation

A room of scientists can be given the same model airplane kit, assemble the pieces, and launch their planes. Some will fly long and true. Others will tumble and crash with varying degrees of success. Is this any different than commercializing a biotherapeutic protein?

The analysis of biotherapeutic primary structure (sequence, modifications) has become a routine task for the majority of analytical organizations tasked with their analysis. The tools developed for such analyses fail to address questions of Higher Order Structure(HOS), typically those relating to biotherapeutic protein folding, dynamics, and interactions. Yet, biotherapeutic HOS is increasingly scrutinized as a critical product attribute by regulators, as questions of process improvement, biosimilarity, and product interchangeability become more common. Difficult questions are now challenging development organizations:

• Why are bioassays or binding assays showing differences, when peptide maps show no significant alterations in primary structure?

• Do candidate biosimilars that have the same sequence and variant profiles still present safety and efficacy risks from differences in conformation or epitope recognition?

• Which product variants are structurally neutral, which create local perturbations in product structure, and which result in significant changes to global protein structural dynamics?

In our seminar, we will discuss the tools scientists have available to examine questions of biotherapeutic higher order structure and dynamics. We will focus on the ability of hydrogen deuterium exchange and ion mobility mass spectrometry to address these problems, and on the ability of software and informatics tools to simplify, and accelerate, studies of complex product attributes such as disulfide linkage analysis. NOTES:

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Agilent Technologies Lunch and Learn Seminar Thursday, September 13 12:15-13:15 Cockatoo/Macaw Characterization of Therapeutic Proteins for Biologic Development Ning Tang, Agilent Technologies Mellisa Ly, Agilent Technologies Protein biologics now represent a significant share of pharmaceutical sales and future growth potential, particularly in an era of increasing patent expirations. A range of analytical methods is required to determine the purity, identity and integrity of protein biologics at multiple points along the manufacturing process, from cell culture to downstream purification, product characterization and lot release. In this presentation, we will be showing the application of high resolution and high mass accuracy Q-TOF for intact mAb measurement, peptide mapping, and characterization of PTM. A new protein deconvolution algorithm, pMod, has been developed that employed automatic peak modeling technique. It decreased maximum entropy deconvolution artifacts and improved confidence in the final results. Another import aspect of protein characterization for glycoproteins is identify the glycan components and the sites of glycosylation. Glycosylation is important in biotherapeutics design and disease progression and detection. The unique properties of glycopeptides create analytical challenges because of their low abundance compared to peptides, the presence of heterogeneous mixtures of glycoforms at each site, and the suppression of their ionization by peptides that co-elute during LC/MS runs. A major breakthrough was the ability to couple HILIC enrichment with reversed-phase separation, two chromatographic phases which have been assumed to be incompatible. Automated, on-chip HILIC enrichment of glycopeptides was demonstrated on a microfluidic device coupled to a QTOF mass spectrometer. Advantageously, no sample handling was required and tedious off-line purification steps were avoided. Results fom haptoglobin and erythropoietin will be presented. NOTES:

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Wednesday Poster Abstracts

P-101 Isotopo Towards Quantitative Mass Isotopomers Distribution Analysis using Spectral Data Zeeshan Ahmed1; Saman Majeed1; Wolfgang Eisenreich2; Thomas Dandekar1

1University Wuerzburg, Wuerzburg, Germany; 2TU Munich, Munich, Germany Mass isotopomer distribution analysis (MIDA) is a technique towards the measurement of amalgamation of polymers by involving the process of quantification of relative abundances of molecular species with mass spectrometry. The objective of this research is to study metabolic isotope to quantify the fraction of metabolites of interest in the mixture typically by tracing isotopes. Estimating mass isotopomers distribution from spectral data is an extension of the quantitative mass spectrometric method to a multi component mixture analysis. Focusing on identification of the quantity of population of labelled isotopomers for resolving the exact rate of synthesized fractions present in the mixture and metabolic experimental data management, a new software application named “Isotopo” is developed. Isotopo is an application with the ability of performing quantitative mass spectrometry to readily mixtures of materials labelled with stable isotopes can be very important for both biomedicine and biochemistry. Most recent version of Isotopo is with the ability of processing experimental isotopomers data and as the result of successful experimental data processing, Isotopo estimates mass values and relative intensities. It predicts natural abundance values, relative isotopic abundance values and fractional molar abundance values of each fragment from labelled substance based experimental data elements. Using Isotopo it is also possible to process data sets with multiple data entries up to three actual intensity values against one mass to charge ratio values, estimate absolute enrichment, mean and standard deviation of both natural and relative isotopic abundances. Isotopo also provides the standardization of experimental data with a file based record keeping system for experimental data manipulation and management. The strength and effectiveness of Isotopo is validated using datasets based on observed values during GC-MS experimentation, consisting of the information obtained using different metabolites. Resultant outcomes are observed in both textual and visual (spectrometric visualization) formats, produced by Isotopo. NOTES:

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P-102 Hydrogen Deuterium Exchange Mass Spectrometry Study of Fragment Antibody Binding to a Highly Conserved Influenza Epitope Joomi Ahn1; Eveline Sneekes-Vriese3; Otto Diefenbach3; Alex Muck2; Ying Qing Yu1; Adrian C. Apetri3

1Waters Corporation, Milford, MA, USA; 2Waters Corporation, Manchester, UK; 3Crucell BV, Leiden, The Netherlands Recombinant antibodies with high-affinity binding directed against a conserved epitope on the major surface antigen are a powerful strategy in influenza vaccine development and future prevention against a wider range of virus strains. A hemagglutinin (HA) epitope recognized by the broadly neutralizing human antibody (CR6261) was previously identified by cocrystallization of Fab-CR6261 in complex with HA from the 1918 H1N1 influenza pandemic. Fab-CR6261 bound with HA 1918 H1N1 was prepared in a homotrimeric complex. In this study, we present hydrogen deuterium exchange mass spectrometry (HDX MS) as a rapid method to probe interactions between Fab-CR6261 and the HA 1918 H1N1 influenza. The HDX results were directly compared with the co-crystal structure to confirm the interaction domains. Fab-CR6261 bound with and without HA 1918 H1N1 were prepared. HDX MS experiments were conducted with a nanoLC system, which has online pepsin digestion capability and independent low temperature control. The eluent was directed into a standard ESI interfaced Q-Tof MS. Data independent LC-MS were acquired to identify peptic peptides from digestion of all proteins. Deuteration at peptide level was automatically measured using a HDX dedicated software. Differences in deuterium uptake between bound and unbound were displayed in comparative formats to locate the interaction regions. With HDX, we successfully identified all of the Fab regions involved in HA binding. High sequence coverage of both light and heavy chains (81% and 86%, respectively) was achieved. Four different regions in the Fab heavy chain revealed a significantly lower level of deuterium uptake when the antibody was bound to HA. Consistent with cocrystallization results, the four different loops of the Fab-CR6261 derived from HDX results contained all contact residues, where were previously identified in the co-crystal structure. These data demonstrate that the HDX MS is an efficient method for an epitope-paratope mapping analysis in addition to crystallography. P-103 High-throughput Sample Preparation Strategies for Protein Digests Jonathan Woodard; Nicolas Angell Amgen, Inc., Thousand Oaks, CA USA The recent adoption of high resolution separations and separation-free mass spectrometry approaches for protein digests, along with advances in instrumentation, has greatly increased the

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quality and throughput of peptide analysis. However, protein digest sample preparation is still somewhat time consuming. While rapid approaches have been reported, many lack ruggedness and reproducibility and are not suitable for applications in a regulated environment. Additionally, many of the approaches still do not achieve the desired throughput. We report on recent attempts to decrease the time and method complexity of protein digests by combining novel techniques and highly parallel sample processing with an end target of completing all of the necessary preparation steps, including digestion, in less than 2 minutes per sample. NOTES:

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P-104 Formic Acid Elution from an IgSelect Anti-human FC Column of a "Humanized" Therapeutic Antibody in Mouse Plasma Micheal Batt Lilly Biotechnology Center, San Diego, CA USA Recently a number of groups have reported using the IgSelect Anti-Human Fc column from GE Healthcare to isolate “humanized” therapeutic antibodies from mouse plasma samples. The column is easy to set up and can be used on a broad range of therapeutic proteins. The column has unique selectivity for Fc fragments of human IgG which makes it ideal for an affinity based separation of “humanized” therapeutic proteins from various non-human serums. Here we present an alternative procedure with the column set up on an AKTA FPLC using formic acid elution. High salt (1X Dulbecco’s PBS +100 mM NaCl pH 7.4) and low salt (0.1 M TRIS-HCl, pH 7.5) column rinses were done prior to the 0.2% formic acid elution. The eluted sample was concentrated via speed-vac and pH adjusted to 7 for further top-down and bottom-up analyses. In our example, an in-vivo collection from 4 mice was pooled (~ 35 µg of a “humanized” therapeutic antibody in ~ 500 µL of mouse plasma),diluted 1:1 with (1X Dulbecco’s PBS) and then loaded onto the column manually. After purification the sample was split into two portions. The first portion (~25 µg) was digested with trypsin for bottom-up analysis. The second portion (~10 µg) was used for reduced and non-reduced intact mass analysis. Using formic acid elution, we obtained tryptic digest results to identify minor chemical modifications and obtained greater than 98% sequence coverage. But more importantly, the reduced and non-reduced intact molecule mass spectrometry results were clean and would allow us to identify low level impurities, such as clips and minor chemical modifications if present. We believe that this elution method is simple with little hands-on sample manipulation. It allows us the option of performing “clean” intact molecule mass spectrometry when compared to the typical eluent choices. P-105 Multiplex Fragmentation and Ion Mobility Separations can Improve the Quality of Rapid LCMS Peptide Mapping Analyses for Biotherapeutics. Scott Berger; Asish Chakraborty; Henry Shion; St John Skilton; Weibin Chen Waters Corporation, Milford, MA USA LCMS Peptide mapping is the fundamental technique for defining biotherapeutic protein primary structure. Improvements in separations, mass detection, and informatics have shortened data acquisition to about an hour, and reduced data processing from days to hours. Mapping proves an invaluable tool for biotherapeutic characterization, but lacks the throughput needed to screen for biotherapeutic variants as part of many early (clone screening, QbD) and late (Formulations, Stability) development activities. Modern TOF analyzers have enabled the collection of rapid

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accurate mass peptide mapping data, but “accurate mass only” identifications are ambiguous for some peptide assignments, and constitute insufficient evidence for confident identification of new variants. Here, we investigated how multiplexed LCMSE fragmentation and High Definition (ion mobility) LCMSE (HDMSE) can promote more confident peptide assignments in rapid peptide mapping studies. Analytical-scale methods were developed on the SYNAPT HDMS QTof platform for rapid (5, 10 min gradient) and typical (90 min) peptide maps applied to a biotherapeutic mAb. In selected experiments, the ion mobility functionality was enabled, permitting additional gas-phase separation of peptide precursor ions, prior to the collision cell. Data processing was accomplished using BiopharmaLynx informatics. Results indicated that rapid mAb peptide maps yielded high coverage (>90%), and presented MSE fragmentation patterns that could validate peptide accurate mass assignments. Peptide coelutions (peptides eluting within 1/7th peak width at half height) were observable in these shorter analyses, resulting in chimeric MSE fragmentation spectra. This chimeracy did not preclude using fragment ions in a confirmatory role (e.g. quality metrics for a minimum number of accurate mass b/y ions needed to validate peptide accurate mass assignments). Parallel studies conducted with the ion mobility functionality enabled effectively eliminated fragment ion chimeracy and improved the ability for human verification of peptide assignments, but proved non-essential for automated rapid map data processing. NOTES:

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P-106 Screening Stressed Glycoprotein Therapeutics for Disulfide Scrambled Species Cedric Bobst1; Adriana Kita1; Igor Kaltashov1; Melanie Lin2; Paul Salinas2; Philip Savickas2

1University of Massachusetts Amherst, Amherst, MA USA; 2Shire, Lexington, MA USA Acid-β-glucocerebrosidase (GCase) is a lysosomal protein responsible for hydrolyzing the glycosphingolipid glucosylceramide into glucose and ceramide. GCase possesses four cysteines clustered at the N-terminus and proper connectivity of these residues by disulfide bonds is required for optimal enzymatic activity. We have developed a mass spectrometry (MS) based method tailored for the detection of non-native disulfides in GCase. Using a combination of proteolytic and glycolytic enzymes under carefully controlled reaction conditions we are able to screen samples to confirm the native disulfide structure as well as screen for the subpopulation of alternate forms. We evaluated a variety of proteolytic and chemical fragmentation techniques in our quest to introduce cleavages between each of the four N-terminal cysteine residues. Additionally, we enzymatically removed an N-terminal glycan that otherwise complicated gas phase fragmentation of the resulting disulfide linked peptides. Evaluating the specificity and yield of each reaction, we sought conditions that would minimize the introduction of disulfide scrambling during analysis. We conclude that the MS analysis of various disulfide containing fragments by collision induced dissociation is adequate to confirm the presence of native cystine formations. More importantly, the ability to screen for alternate forms is made possible with the ability to introduce backbone cleavages between each of the disulfide linked cysteine residues. Having successfully established such conditions, we are then able to analyze the resulting fragments by LC-MS/MS to elucidate their cystine connectivities. This method was applied to screen disulfide connectivity in stressed samples. We demonstrate that the method is well suited for detecting the presence of scrambled species. P-107 Rapid Structural Comparison of American and Foreign Sourced Rituximab Bo Wang; David Keire; Michael Boyne DPA, FDA, St. Louis, MO USA Liquid chromatography combined with mass spectrometry (LC-MS) is a powerful analytical tool that can provide fast, robust and sensitive structural characterization for both the quality assurance and comparison of protein therapeutics. Here, structural characterization and comparison of a monoclonal antibody drug, rituximab, from US and foreign sources was

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conducted. Rituximab is a chimeric monoclonal antibody directed against CD20 and has clinical indications for lymphomas, leukemia, and rheumatoid arthritis. The foreign product is not approved for marketing in the US, and thus, sensitive methods are needed to distinguish this version from the innovator product. Here, a specific enzymatic digestion and LC-MS method was applied to show US and foreign sourced products could be partially characterized and differentiated analytically. Based on the small number of lots tested, the foreign product shows more charge heterogeneity with differential modifications to the N and C termini as well as differences in abundance of the primary glycan structures. NOTES:

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P-108 Characterization of Antibodies and Antibody Drug Conjugates by a Novel High Resolution Quadrupole Time-of-Flight Mass Spectrometer Asish Chakraborty1; St John Skilton1; Steven Pringle2; Nick Tomczyk2; Gareth Booth2; Weibin Chen1

1Waters Corporation, Milford, MA USA; 2Waters MS Technologies, Manchester, UK Monoclonal antibodies (mAbs) and derivatives are the fastest growing class of biotherapeutics for the treatment of cancers and inflammatory diseases. mAbs are often combined with cytotoxic drugs to enhance their therapeutic efficacy. Alternatively, small anti-neoplastic molecules can be chemically conjugated to mAbs, used both as carriers and as targeting agents. Typically, several molecules of a cytotoxic-agent are covalently linked to a MAb, thus forming an antibody drug conjugates (ADCs) that differs in number of drugs linked and the sites of linkage. Therefore, ADCs have more complex and heterogeneous structures with different drug-to-antibody ratios (DARs) than the corresponding antibodies, so structural characterization can be challenging. Here, we present a high-sensitivity quadrupole/time-of-flight system with novel source ion-guide for characterization of several mAbs and couple of ADCs (trastuzumab conjugates). Two different chromatographic conditions (SEC and RP) were used to introduce samples to the ESI-MS. The use of MS-friendly SEC with sub-2 micron column packing materials directly coupled to ESI-MS provided a routine analysis method for profiling of antibodies and conjugates, degree of conjugations, sequence confirmation (isotope resolution), and MW measurement. High-resolution MS detection was able to istopically resolve light chain (25 kDa). Identities of either intact mAbs or trastuzumab conjugates, or the subunits were automatically confirmed by deconvoluting the mass spectra using maximum entropy. The deconvoluted mass spectrum of deglycosylated trastuzumab conjugate shows several peaks with same mass differences and with varying intensities. The masses of these peaks are assigned to naked mAb and to trastuzumab conjugates with varying number of covalently linked drug-molecules. The deconvoluted MS spectra of deglycosylated reduced conjugate show both light and heavy chains of trastuzumab are conjugated with drugs. Conjugation sites were determined by trypsin/AspN peptide mapping using UPLC/MSE approach. The UPLC-TOF MS approach presented here facilitates rapid characterization of naked antibodies and antibody drug conjugates. NOTES:

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P-109 Characterization of Therapeutic Antibodies and Biosimilars Using a Combinatorial Enzymatic Treatment Workflow and Mass Spectrometry Jiawei Chen; Kevin Ray Sigma-Aldrich Biotechnology, St. Louis, MO USA Therapeutic antibodies have played an important role in drug development in the past decade. The function of therapeutic antibodies is associated with the diversity and distribution of glycoforms, which often have to be characterized extensively during development and monitored throughout production. In addition to the conserved glycosylation site at Asn297 in the constant region, a significant number of antibodies contain a secondary glycosylation site in the variable region, complicating the characterization by intact mass spectrometry. We report the use of combinatorial enzymatic treatment of such therapeutic antibody, wherein three parallel treatments are implemented within one workflow. The first treatment utilizes FabRICATORTM, a cysteine protease that cleaves IgG below the hinge region, to generate scFc and Fd’ fragments, both glycosylated. The second treatment employs the combination of FabRICATORTM and IgGZEROTM, a specific endoglycosidase that hydrolyzes the conserved glycans attached to Asn297, to generate glycosylated Fd’ and deglycosylated scFc. The third treatment combines FabRICATORTM and PNGase F, a non-specific endoglycosidase that releases all N-glycans, to generate deglycosylated scFc and Fd’. The fragments resulting from each treatment are analyzed with RP-HPLC/MS. The glycoforms are identified for two glycosylation sites separately, and other modifications are revealed on the antibody subunit level as well. Moreover, we apply this strategy to a biosimilar candidate, and show the ability to quickly assess similarity of the molecule to the innovator’s drug. We propose that such strategies utilizing these new enzymes can be incorporated in the routine characterization workflow for therapeutic antibodies, including those with complex glycoform distributions. P-110 Profiling the Glycoforms of Highly-Glycosylated Biotherapeutic Proteins using a Quadrupole Time-of-Flight Mass Spectrometer and Chromatographic Separations Asish Chakraborty; Stephane Houel; Ying Qing Yu; Weibin Chen Waters Corporation, Milford, MA USA The work presented here provides a systematic study for the characterization of four therapeutic glycoproteins, r-hFSH (recombinant human follicle stimulating hormone), EPO, alpha1-antitrypsin (A1PI) and Etanercept. Both RP and SEC methods coupled to ESI-Q-TOF MS were developed and used to analyze intact proteins and subunits generated by chemical or enzymatic reactions. The objective is to understand how the MW of protein with increasing level of glycosylation affects the glycoprofile information acquired at intact mass level and to compare

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the glycan-profile at intact and released glycan levels. Alpha and beta chains of glycosylated r-hFSH were separated by RP and analyzed by ESI-MS. Deconvoluted mass spectra of deglycosylated r-hFSH showed two major peaks at ~11 kDa and ~13kDa corresponding to alpha-and beta-subunits respectively. Major peaks observed in glycosylated alpha and beta subunits correspond to the addition of di-antennary, tri-antennary, di-sialyted N-glycans to deglycosylated-subunits. Deconvoluted mass spectra for intact EPOs showed similar (26-32 kDa) glycoprofiles between two biosimilars and differ (35-38 kDa) from the 3rd-biosimilar and revealed a series of masses differing by 365 Da (HexNAcHex) and 291 Da (Sialic acid). Although most of the glycans were present in all the EPO-biosimilars, the relative quantities were different. The analysis of alpha-1-antitrypsin yielded glycoprofile of protein A1PI despite of 52 kDa MW, much higher than FSH and EPO. The data showed that major glycans of A1PI contained diantennary, disialylated and smaller amounts triantennary, trisialylated N-glycans. The success of the A1PI glycoprofile analysis leads to our next target, Etanercept, which is a dimeric fusion- protein containing six N-glycosylation sites and multiple O-glycans. The mass analysis of Etanercept are focused on the subunit (fragments) of the molecules generated from an enzymatic digestion process (IdeS). Released-glycans assays to correlate the glycoprofile information acquire at the subunit protein level and free glycan levels are also performed. NOTES:

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P-111 Tracking Host Cell Proteins Variations During Purification Process of a Biotherapeutic Protein Catalin E. Doneanu1; Vincent Monchois2; Weibin Chen1

1Waters Corporation, Milford, MA USA; 2R&D and Pilot Services, Novasep Process, Metz, France Detailed information on HCP composition and concentrations in the samples will help to design efficient purification schemes so the final products meet the HCP regulatory guidelines, therefore protecting patient safety. In addition, knowledge about the identity and concentration of individual HCP in the products during purification stages allows each HCP to be tracked, building greater confidence about the purification process. We have recently developed an efficient LC/MS assay that allows the identification and quantification of HCPs in biopharmaceuticals over five orders of magnitude in concentration. This analytical methodology was applied to the identification and quantification of HCPs present in preparations of recombinant human protein X expressed in eukaryotic cell and produced using an alternative purification process. At the end of each purification step, the samples were spiked with four protein standards, derived from a different organism than the host cell. 2D RP/RP LC separation provided enhanced dynamic range and the multiplexed data collection method allowed us to reproducibly detect low-level peptides. The data was searched against a NCBI database (containing ~ 25 K entries) for the cell line. Twenty four HCPs were identified after the first purification stage with a total HCP concentration of approx. 6,000 ppm, but only 4 of these proteins were detected in the final purified finished product with a total HCP concentration of 20 ppm. The results of the LC/MS assay (total HCPs concentrations expresses in ng HCPs/mg protein or ppm) were compared with with ELISA measurements for the same samples, and the results correlated very well. The results show that the assays provide detailed valuable information to understand the relative contributions of purification schemes to the nature and concentrations of HCP impurities in biopharmaceutical samples, and the assays has great potential to be used as generic methods for HCP analysis in the biopharmaceutical industry. P-112 Quantification of Intact mAb Trastuzumab Using LC-QTOF Caroline S. Chu; Carol H. Ball; Vadiraja B. Bhat; Alex Zhu; Yanan Yang Agilent Technologies, Santa Clara, CA USA Manufacturing recombinant proteins may require reproducible, quick, and robust methods for the characterization. LC-MS techniques are routinely used to monitor both the manufacturing process and resulting product. LC-MS analysis can also provide better information about biotransformation of therapeutic proteins. The LC-QTOF provides high mass accuracy to

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identify the mAb as well as any impurities in the process. However, LC-QTOF is not widely used for quantitative analysis of intact proteins applied to manufacturing processes or biotransformations of therapeutic proteins by LC-MS. Here we present a high resolution and accurate mass approach to quantify intact mAb’s using different LC-QTOF platforms and quantitative approaches. Different concentrations (1ng/mL-200ug/mL) of intact mAb, Trastuzumab, were prepared in both 0.1%FA in water and 0.1%FA+ 5% ACN, in water. For mAb quantitation, 5 uL of samples were injected on to Poroshell SB-300 C8 (5um) column and data acquired in MS scan mode using 7.5 min chromatographic gradient. LC-MS data was acquired on 3 different LC-QTOFs to compare the LOD and LOQ. Quantitative LC-MS as a technology is extensively used for small molecule pharmaceuticals, however for intact proteins, quantitative LC-MS techniques are not well established. In this study, LC-Q-TOF will be shown to be a great platform for quantitative analysis of large molecules. Q-TOFs with different resolving power ranging from 20,000 to 40,000 are used for data acquisition and several quantitative approaches, including quantitation of the different protein glycoforms. Trastuzumab elutes at 3.8 min using a 7.5 min LC gradient. The limit of detection (LOD) for Trastuzumab in a clean standard is 1 ng or 6.7 fmol on column based on m/z 2907. The calibration curve shows good linearity over three orders of dynamic range. The peak area relative standard deviations (RSDs) are less than 10% over the dynamic range using triplicates. NOTES:

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P-114 Microfluidic Chip-based Liquid-chromatography Mass Spectrometry for Quantitative Determination of Biomarker and Drug Metabolite in Human Urine Ming-Ren Fuh Soochow University, Taipei, Taiwan Microfluidic chip-based liquid-chromatography coupled to mass spectrometry (LC-MS) has played an important role in proteomic research for high throughput analysis. Up to date, the use of chip-based LC-MS for quantitative determination of small molecules in pharmaceutical and clinical applications has not been thoroughly investigated. This paper will discuss the utilization of chip-based LC-MS systems for quantitative determination of small molecules (including abused drugs/drug metabolites and disease/tumor-associated biomarkers) for bioanalytical application. An integrated chip-nanoLC device integrates a microfluidic switch, a chip column design having a pre-column (enrichment column) for sample enrichment prior to an analytical column for separation, as well as a nanospray emitter on a single polyimide chip was utilized in this study. The approach to minimize ion suppression of urine sample will be presented; furthermore, the stability and lifetime of chip will be evaluated. In addition, the applicability of microfluidic chip-based LC-MS for the analysis of 7-aminoflunitrazepam and 8-isoprostaglandin in human urine will also be presented. Reference 1. H.Y. Bai, S.L. Lin, Y.T. Chung, T.Y. Liu, S.A.Chan, M.R. Fuh, J. of Chromatogra. A, 1218 (2011) 2085 2011 2. H.Y. Bai, S.L. Lin, S.A. Chan, M.R. Fuh, Analyst, 135 (2010) 2737. P-115 Automated MAb Workflow: from Harvest Cell Culture to Intact Mass Analysis of Variants Gurmil Gendeh; Shanhua Lin; Zhiqi Hao; Wim Decrop; Remco Swart; Andreas Huhmer; Srinivasa Rao; Yury Agroskin; Chris Pohl Thermo Scientific, San Jose, CA USA Early in the development of recombinant monoclonal antibodies (MAbs), a large of number of harvest cell culture (HCC) samples must be screened for IgG titer, aggregations, and charge variants. Affinity chromatography is often used first to purify MAbs, with typical yields of more than 95%. Size-exclusion chromatography is followed to identify and quantify MAb aggregations. Ion-exchange chromatography is then used to characterize charge variants. Among the detection tools used for the analysis of therapeutic MAb, mass spectrometry (MS), especially high resolution and accurate mass MS, has become more and more important in providing

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accurate information on various protein properties, including intact molecular mass and glycosylation form distribution. In the present study, we automate a 2-Dimensional HPLC workflow using a biocompatible HPLC system. This system consists of a dual-gradient pump, a UV/VIS detector, a column oven, and an autosampler capable of both sample injection and fraction collection. First, the HCC is injected onto the protein A column and IgG fractions are collected by the autosampler. Subsequently, the IgG fractions are injected onto a SEC size-exclusion, or a strong cation ion-exchange column for further analysis. Variants separated on the SCX column were collected onto a 96-well plate and analyzed by a benchtop quadrupole Orbitrap mass spectrometer. Full MS spectra of intact or reduced MAb were analyzed using Protein Deconvolution 1.0 that utilizes the ReSpect algorithm for molecular mass determination. NOTES:

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P-116 Automated Antibody Verification for Lead Discovery Utilizing Automated MS Characterization Strategie Simone Greven1; Birge Dübel1; Heiner Apeler1; Wolfgang Jabs2; Catherine Evans3; Laura Main4; Carsten Baessman2

1Bayer Pharma AG, Wuppertal, Germany; 2Bruker Daltonik GmbH, Bremen, Germany; 3Bruker Daltonics GmbH, Fällanden, Switzerland; 4Bruker Daltonics Ltd, Coventry, UK Monoclonal antibodies play an increasing role as therapeutic agents in the pharmaceutical industry. Within the antibody lead discovery and optimization process the expression, purification and analytical characterization of large numbers of antibodies or antibody fragments is required. At Bayer Pharma Global Biologics Mass Spectrometric analyses are employed during lead discovery for screening & selection workflows and for QC of the final product. In both cases it is highly desirable to utilize automated MS characterization approaches to ensure consistent data quality and high throughput. We present here details of the implementation of such an automated MS characterization strategy for antibody verification. Using Biopharma Compass, an open access MS platform, which provides semi-automated sample sequence generation in combination with an automated sample acquisition, processing and report generation, has dramatically increased our productivity and sample throughput. Furthermore, with its ability to link both to our in-house Protein Database and also to export data into Excel, Biopharma Compass has enabled us to automatically integrate the acquired data and results into our Biologics Data Platform. Thus permitting further streamlining of the lead discovery and optimization process. P-117 Evaluation of Intact Mass Spectrometry for the Quantitative Analysis of Protein Therapeutics Ashley Gucinski; Michael Boyne DPA, FDA, St. Louis, MO USA Implementation of modern analytical techniques, such as intact mass spectrometry, may allow for more detailed quality assessments of protein therapeutics. The complexity of the protein therapeutic manufacturing process as well as the sensitivity of these drugs to different storage conditions can lead to the presence of several undesired products, including truncations, degradation products, byproducts, and differentially-modified protein variants that are difficult to detect by peptide mapping. Intact mass spectrometry can be used to identify the intact protein composition, inclusive of post-translational modifications (PTMs), but can also generate a chemical fingerprint of the different protein species present in a given sample. In this work, we systematically evaluated the influence of multiple charge states, multiple isotopes per charge

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state, and operating resolution on the suitability of intact mass spectrometry for quantitative analysis using insulin and somatotropin as model systems. Standard curves could be generated using absolute intensity data or using the relative ratio between the analyte and internal standard. This was explored using an insulin detemir standard curve with a 25 nM insulin regular internal standard and an insulin regular standard curve with a 2 µM insulin detemir internal standard. Standard curves were evaluated in terms of their linear dynamic range (LDR), % RSD, and % recovery. Improved linearity was observed in the presence of the 2 µM internal standard. T-tests were used to determine at what level of precision one protein variant could be quantified in the presence of another. For somatotropin, standard curves were generated at operating resolutions of 7,500, 30,000, and 100,000. While the largest LDR was observed at the highest resolution (25-2500 nM), the other operating resolutions also afforded adequate standard curves. These methods demonstrate the validity of quantitative intact mass spectrometry for the analysis of protein therapeutic drugs, providing a foundation for future comparative methods. NOTES:

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P-118 Analysis of Polyethylene Glycol (PEG) and PEGylated Therapeutic Protein Using LC/MS Ravindra Gudihal1; Sundaram M Palaniswamy1; Suresh Babu CV1; Umamaheshwari S2; Suneel Kumar M Basingi2; Ning Tang3

1Agilent Technologies, Bangalore, India; 2GangaGen Biotechnologies Pvt Ltd, Bangalore, India; 3Agilent Technologies Inc, Santa Clara, CA USA Covalent modifications involving the chemical attachment of polyethylene glycol (PEG) to the therapeutic protein is one of the important processes used in biopharmaceutical industry. This modification is known for improving the therapeutic value of protein and also increasing the half life of the protein drug. As PEG themselves are heterogeneous in nature, the PEGylation reaction often leads to product heterogenetity. Therefore, it is required to analyze these PEG reagents and PEGylated protein drugs before product release. Liquid chromatography/Mass spectrometry (LC/MS) technology is a powerful and sensitive technique for the characterization of such complex and heterogeneous products. In the present study the intact PEGylated therapeutic protein samples and also the PEG reagents were analyzed using HPLC-coupled to Q-TOF/MS system. The PEG and PEGylated proteins were separated and eluted from HPLC, while the charge stripping agent, triethylamine (TEA), was delivered through a syringe pump via tee junction. TEA was mixed with PEGylated sample before it went into the mass spectrometer and acted as a charge reducing reagent. The addition of amine improved the mass spectrum and was useful for interpreting the results. The results showed that the PEGylated therapeutic protein under study as well as PEG reagents were heterogeneous. The intact protein after PEGylation showed multiple peaks with each peak differing by ~44Da mass units which is the mass of the ethylene glycol. P-119 Extensive Identification of Naturally-Occurring Peptides in Human Milk by Chip-Based Mass Spectrometry Andres Guerrero University of California, Davis, Davis, CA USA Human milk contains active proteases (namely plasmin, trypsin, elastase,cathepsin D and kallikrei) as well as its corresponding anti-proteases. The presence of proteases and anti-proteases in milk suggests a balance of proteolytic degradation in the mammary gland. A large number of milk peptides produced by in vitro digestion have been found to be bioactive. Bioactivities of milk peptides include immunomodulation, opioid-like activity, antimicrobial action and probiotic action. These peptide fragments often exist within milk proteins that, when intact, are not bioactive.

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Pooled, mature term breast milk was analyzed for presence of naturally ocurring peptides by nano-LC Chip Quadrupole Time-of-Flight Tandem Mass Spectrometry (Chip-Q-TOF). Tandem-MS spectra were analyzed with both MS-GFDB and X!Tandem against a human milk protein library. For each tandem-MS analysis, the list of identified peptides was used as an exclusion list in a subsequent round of tandem analysis to prevent re-fragmentation of the same peptides and to allow further peptide discovery. Tandem analysis was repeated with this iterative exclusion lists strategy until few new peptides were identified. Overall, more than 600 unique naturally ocurring milk peptides from more than 30 proteins were identified, most were derived from β-casein. Phosphorylation was present in 22% of the peptides, moreover, new sites of phosphorilation were identified for the first time. Sixty peptides found had significant sequence overlap with known bioactive peptides with antimicrobial or immunomodulatory functions. As far as we know this is the most complete analysis of naturally-occurring peptides in the human milk up to the moment. NOTES:

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P-120 Bioinformatics Tools For Biologics: Automated LC/MS Peptide Mapping Processing

1Xu Guo, 1Mark M. Garner, 2Eric Johansen, 2Robert Deutschman, 1Steve Tate

1 AB SCIEX, Concord, ON, Canada; 2 AB SCIEX, Foster City, CA, USA

With the fast growing biopharmaceutical markets, Mass spectrometry has become an important analytical tool in the characterization of biologics and to address increasingly challenging regulatory requirements. In the development of novel biologics, intact protein and peptide mapping LC/MS workflows provide the complementary information on full sequence coverage, purity assessment, quantification, and product identifications. Compared to intact protein workflow, peptide mapping provides detailed structural information, and allows one to localize the sites of biological and non-biological modifications ( e.g. acetylation, oxidation , deamidation etc. ). However, manual data analysis for routine work such as assessing lot-to-lot variation is time consuming and low efficiency for the development cycle. In this work, we describe an automated LC/MS peptide mapping processing using a prototype software for biologics data. A Shimadzu Prominence XR system with an ACQUITY UPLC BEH300 C18 Column, 2.1 x 150 mm, 1.7 µm was used for separating antibody H tryptic digests peptide mixture. The samples were eluted in a linear gradient of 5 -80% acetonitrile and 0.1% formic acid in 80 min at the flow rate of 0.2 mL/min. AB SCIEX TripleTOF® 5600 system was used with Analyst® TF 1.5 Software for instrument control and data acquisition utilizing the power of Information Dependent Acquisition (IDA). PeakView® software was used for data review and a prototype software was used for peptide mapping analysis. Antibody H tryptic digest mixture were well separated under the LC condition selected. The AB SCIEX TripleTOF® 5600 system exhibited high resolution (~ 35,000 full width at half mass) and sub ppm mass accuracy across the entire m/z range we examined. The mass accuracy and processing algorithm ‘Enhance’ LC/MS Peak-Finding Filter enable peptide identification with high confidence and better sequence coverage. Furthermore the MS/MS spectra of corresponding matched peptides were examined. NOTES:

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P-121 A High Resolution Bench-Top Orbitrap LC-MS Workflow Solution for Comprehensive Intact Monoclonal Antibody Characterization Zhiqi Hao1; Yi Zhang1; David Horn1; Shiaw-Lin Wu2; Xiaoyue Jiang1; Jessica Wang1; Patrick Bennett1; Andreas Huhmer1

1Thermo Fisher Scientific, San Jose, CA USA; 2The Barnett Institute, Northeastern University, Boston, MA USA Monoclonal antibodies (mAbs) are increasingly being commercially developed for diagnostic testing and disease therapy. Although the primary structure of mAb’s are well defined, most therapeutic antibodies produced are heterogeneous, which makes characterization of these large antibodies analytically challenging. In this study, an automated high resolution LC-MS based workflow solution was developed and evaluated for robustness, accuracy and comprehensive characterization of an intact mAb using a bench-top Orbitrap mass spectrometer, the Q Exactive. Full MS spectra of the intact mAb and its substructure including Fab, light chain and Fab heavy chain were analyzed using Protein Deconvolution software for intact mass determination and relative quantification of glycoforms. The Q Exactive produced accurate and reproducible measurements for both molecular mass and relative abundance of glycoforms. Mass errors of less than 10 ppm for average mass and less than 3 ppm for monoisotopic mass were achieved for all measurements. This includes multiple instruments. CVs of relative abundance for five major glycoforms were less than 5%. Oxidized species of Fab, light chain and Fab heavy chain were identified with high confidence at the intact substructure level. In the same LC-MS run, top-down sequencing of intact light chain and Fab heavy chain was performed using the unique multiplexing HCD of the Q Exactive at 140K resolution. Analysis of these high resolution top-down MS/MS spectra using ProSightPC software generated extensive sequence coverage from more than 30% of backbone fragmentation and also mapped the internal disulfide linkage. Results from this study indicated that both precise mass measurement and highly confident structural information can be obtained for mAb’s using a single workflow. The fast chromatography, superior mass resolution and accuracy, and the high throughput MS/MS capabilities combined with easy, accurate data analysis of this workflow solution provides a high-confidence screening tool to accelerate biopharmaceutical product development. NOTES:

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P-122 Comparison of Two Analytical Methods for Glycosylation Profiling of Monoclonal Antibodies Kim Højlys-Larsen; Martin Ørgaard; Jette Wagtberg Sen Symphogen A/S, Lyngby, Denmark Antibodies are the largest group of glycoproteins produced recombinantly, where glycosylationhas a profound impact on antibody effector functions as e.g. antibody-dependant cellular cytotoxicity (ADCC). N-linked glycosylation is a posttranslational modification that occurs in the rough endoplasmatic reticulum, involves the attachment of high-mannose oligosaccharide structures to a tripeptide motif NXT/S (X not proline). In the Golgi apparatus the glycosylated antibody can be further modified, which result in microheterogeneity of the end-stage glycosylated antibody. This generates molecular subsets that have distinct physical and biochemical properties and thus functional diversity. Hence knowledge of antibodies glycosylation profile is indicatory for how well the production is performed and controlled and the glycosylation status is an important and integral part of regulatory requirements. The present study compares two approaches (LC-MS and LC-FLD) for the characterization of glycosylation profile of IgG’s. The LC-MS method measure IgG’s intact masses by Q-ToF after a short (10 min) desalting step. The method is characterized by a shot run time and automatic detection and deconvolution. Comparison with known reference masses estimates the glycosylation patterns and the combination of the different glycoforms. Thus this is a cheap, fast and robust method, with the drawback that only the major glycoforms is detected. The LC-FLD method analysis is enzymatic cleavage of the N-linked carbohydrates with N-glycanase followed by InstantAB labeling (Prozyme). The labeled glycans are separated on a UHPLC system equipped with an HILIC column, detected using fluorescence, and quantitated by integration. The labeled oligosaccharides are identified by comparison with a human IgG N-linked glycan library. Pros and cons of this method is the identification of all glycans and quantification, but this comes with high sample costs and long analysis time. We show results comparing the two methods with respect to dynamic range, linearity and sample loading. NOTES:

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P-123 Automated Screening of Protein Variants by LC-MS Ryan Hylands1; Malcolm J Saxton1; Wolfgang Jabs2; Joanne Waters1

1Novozymes Biopharma UK Ltd, Nottingham, UK; 2Bruker Daltonik GmbH, Bremen, Germany We describe an automated UPLC-MS based method for the analysis and quality confirmation of the intact mass for protein variants. The automation is implemented using Bruker Daltonics’ (BD) Biopharma Compass software which allows for data acquisition, processing and analysis to occur with minimal user intervention. Data acquisition is set up through Biopharma Compass ensuring all acquisition parameters are traceably linked to the samples analysed and any future processing that may occur. Post-acquisition samples are automatically processed to determine the intact mass of the protein variant. The difference in measured mass from the theoretical mass is automatically calculated indicating whether the measured mass is within the specified acceptance criteria. The intensity fraction of the major species is also displayed as a percentage, providing information on sample homogeneity. Once processing is complete, data is presented in a graphical interface which allows the identification of samples that have passed or failed the set acceptance criteria and also highlighting any samples which require further user investigation. Ultimately the various automation steps allow intact mass screening for numerous protein variants while saving significant resources and ensuring high quality data and interpretation occurs. P-124 Comparability Assessment of Innovator and Biosimilar Rituximab using UNIFI Biopharmaceutical System Solution Vera Ivleva; Ying Qing Yu; Scott Berger; Weibin Chen Waters Corporation, Milford, MA USA Today biopharmaceutical companies are challenged with the design of an efficient analytical strategy for detailed assessment of the structural comparability of biosimilar and innovator products. Extensive characterization assures that the biosmiliar product is safe and meets regulatory compliance criteria. Here we demonstrate how the newly developed comprehensive UNIFI solution addresses the challenges by integrating data acquisition, data processing, and

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data reporting into a seamless workflow. A fully integrated LC/UV/TOF-MS approach with UNIFI software was applied for structure characterization of Rituximab monoclonal antibody (mAb) allotropes and rapid comparison among innovator and the biosimilar products. Comparability analysis was performed at intact protein, reduced protein, and peptide mapping levels; the results were reported under the same method and data processing platform. The experiments were acquired using RPLC/UV/TOF-MS operated with UNIFI Scientific Information System. High level of heterogeneity was revealed in the Biosimilar 1 sample based on observed variable mass discrepancy and large chromatographic peak width of the intact protein compared to that of innovator product. The location of the modification was deduced from the reduced protein mass analysis and confirmed by tryptic peptide mapping data. This heterogeneity was due to pyroglutamination Gln, C-terminal Lys variation, and glycosylation levels, which were measured and compared across all samples included using the automated data processing in UNIFI. A consistent mass shift between the innovator drug and a Biosimilar 2 candidate was revealed at the intact protein level. Reduced protein data examination confined the mass alteration to the heavy chain. Tryptic digest failed to explain this mass discrepancy, so a mutation site was discovered by using chymotryptic protein digest. A single amino acid substitution was identified: Lys218 substituted for Arg218, which explains why tryptic digest analysis could not localize this mutation site. Higher level of G0F glycosylation was observed in Biosimilar 2 compared to the innovator mAb. NOTES:

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P-125 Fast and Extensive Mass Spectrometry Characterization of Therapeutic mAbs: The Panitumumab Case Study Wolfgang Jabs1; Anja Resemann1; Waltraud Evers1; Catherine Evans2; Laura Main3; Carsten Baessmann1; Detlev Suckau1; Elsa Wagner-Rousset4; Daniel Ayoub4; Alain Beck4

1Bruker Daltonik GmbH, Bremen, Germany; 2Bruker Daltonics GmbH, Fällanden, Switzerland; 3Bruker Daltonics Ltd, Coventry, UK; 4Centre d’Immunologie Pierre Fabre, Saint Julien en Genevois, France A number of fundamental discoveries and innovations in ESI and MALDI Mass Spectrometry instrument engineering and software have led to significant advancements in monoclonal antibody analytical characterization. These include techniques for identity testing such as intact mass and subunit analysis, and primary sequence coverage by Top Down Sequencing, peptide mass fingerprints and LC-peptide mapping. Furthermore, new approaches for probing various modifications including sequence variants, truncations, oxidation, deamidation and glycosylation, have been developed. Here we present an analytical case-study of panitumumab, a humanized IgG2 directed against EGFR, highlighting the use of these different approaches for rapid identity confirmation and modification analysis. Special emphasis will be put on the subunit and glycan analysis. P-126 Determination of Core Fucosylation in Glycopeptides of Monoclonal Antibodies Anja Resemann1; Ulrike Schweiger-Hufnagel1; Nannan Tao2; Daniel Kolarich3; Detlev Suckau1

1Bruker Daltonik GmbH, Bremen, Germany; 2Bruker Daltonics Inc., Fremont, CA USA; 3Max Planck Institute of Colloids and Interfaces, Berlin, Germany Fucosylation is one of the most common modifications of glycans and can affect glycoprotein confirmation and their interaction with other proteins. The α;-1,6 fucosylation of the chitobiose core of N-linked glycans plays an important role in many physiological processes. Therapeutic proteins such as IgG like antibodies carry N-linked glycans with different grades of core fucosylation mediating antibody-dependent cell-mediated cytotoxicity. We describe here a fast and reliable method to unambiguously determine core fucosylation of glycopeptides in tryptic digests measured by MALDI-TOF/TOF and ESI-IT. A bioinformatics software platform uses instrument specific fragmentation patterns to identify glycopeptides and localize the fucose within the glycans. We used the typical fragmentation patterns from N-linked glycopeptides in MALDI-TOF/TOF-MS and ESI-IT-MS analysis to fish for the glycopeptides in a large number of peptide MS/MS spectra, to determine the mass of the peptide and the glycan of every glycopeptide and to detect a fucosylation at the chitobiose core.We extended a commercially available software by editing lists of fragmentation patterns to distinguish different glycan

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structures. We found high glycan microheterogeneity at ASN 294 of the heavy chain of the murine IgG1 MOPC21 with core fucosylation of more than 50 % of the glycans. The results of the glycopeptide classification are displayed in a table sorted by absence and presence of fucosylation. The main glycan in MOPC21 is the GOF1 (GlucNAc4 Man3 Fuc1), the second abundant is the same structure without core fucose G0. Subsequently, the core fucosylation classification results were verified by glycan database searches and spectrum matches. NOTES:

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P-127 N-terminal Top-Down Protein Sequencing of the ABRF-PSRG2012 Study Samples by ETD-UHR-TOF Mass Spectrometry Guillaume Tremintin1; Stephanie Kaspar2; Wolfgang Jabs2; Ralf Hartmer2; Carsten Baessmann2; Christian Albers2

1Bruker Daltonics Inc., Fremont, CA USA; 2Bruker Daltonik GmbH, Bremen, Germany MS based top-down protein sequencing (TDS) techniques are becoming increasingly important mainly because relevant sequence information such as N-terminal sequence assignment is obtained very quickly and accurately. This meets the demand of many growing areas like identity confirmation of biopharmaceuticals. In this study, we applied a general analysis strategy for top-down sequencing (TDS) based on Electron Transfer Dissociation (ETD) mass spectrometry. Samples were obtained for the participation in the ABRF-PSRG research study from the PSRG and the identities were provided: BSA, Endostatin and protein A. Samples were directly subjected to offline nano-spray. Using a Bruker maXis ETD mass spectrometer the molecular weight of the intact proteins as well as the charge states for the ETD fragmentation were determined and the ETD fragment spectra acquired. Identification of N-terminal sequences was readily obtained using BioTools software applying a two-step approach: The first step consists of a TDS based Mascot database search. If the protein terminus cannot be obtained, most likely because the protein sequence is not included in the database, a BLAST protein homology search is performed as second step. For the BLAST search, different sequence tags of the proteins top-down spectrum are automatically generated. The retrieved sequences were aligned with the ETD fragment sequence tags. Misalignments indicate sequence variation such as methylation as in protein A. The three proteins were identified via Mascot database search. N-Terminal sequences could be determined directly for BSA and two Collagen alpha-1-(XVIII) fragments (Endostatin) up to the position of the first Cysteine-Cysteine bridge. As protein A could not be identified via Mascot database search, alternative BLAST searches were performed. ETD-TDS in combination with dedicated TDS software provided N-terminal sequences for all PSRG samples - even in cases of a ragged or modified N-term. Precision intact MW determination provided also information about C-term Lys-excision in Endostatin. P-128 Exploring Targeted Proteomic Approaches on a Q-TOF system Stephanie Kaspar; Wolfgang Jabs; Stuart Pengelley; Carsten Baessmann Bruker Daltonik GmbH, Bremen, Germany Mass spectrometry-based quantitative proteomics is a popular tool in biopharma drug development. Advances in speed of separation of proteomics samples and the quality of mass spectrometric detection enable validation of promising potential drugs and its variations in

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complex biological matrices using targeted approaches. These validation approaches heavily rely on methods that facilitate targeting and measuring the quantity of specific peptides and proteins in complex proteomic samples. Typically, SRM and MRM (single- and multi-reaction monitoring) approaches are applied. Nevertheless, those methods require detailed target knowledge and do not allow re-examining of acquired data. We describe here targeted proteomics approaches using the high resolution and accuracy achievable with an Ultrahigh Resolution (UHR) Q-TOF system. Different data acquisition methods are used for targeted quantitative proteomics, namely MS-based acquisition, broad band collision induced dissociation (CID), and middle band CID. Target protein quantification is performed in a complex E.coli background (500 ng) spiked with different amounts of a proteomic standard, which spans a concentration range of 6 decades (UPS-2, Sigma). Protein identification information used as targets for quantification is obtained out of the equimolar proteomic standard (UPS-1, Sigma) containing the same set of proteins as the dynamic range mixture. Quantification of the target peptides spiked into E.coli background based on pure MS data acquisition by application of high resolution extracted ion chromatograms reveals reliable results of targeted proteomics. In total 28 proteins were significantly regulated (p<0.05) demonstrating a high quantification efficiency from 500 fmol down to the amol range. Results of targeted proteomics approaches using broad band and middle band CID will not only provide quantitative but also qualitative data in single runs. Quantification efficiency with regard to the coverage of a high dynamic range of these methods will be compared to the pure MS-based acquisition. NOTES:

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P-129 Automated Termini Assignment of Biologics by Routine Top-Down Mass Spectrometry Anja Resemann1; Nannan Tao2; Rainer Paape1; Lars Vorwerg1; Detlev Suckau1

1Bruker Daltonik GmbH, Bremen, Germany; 2Bruker Daltonics Inc., Fremont, CA USA During recombinant production of proteins, fast and reliable QC methods are needed. Bottom-up approaches have been the method of choice since many years for identification of MS/MS spectra via database search, leading to more or less high sequence coverage of the target proteins. However, the N- and C-terminal sequence status is rarely covered by this method. Top-down approaches like In-Source-Decay (ISD) using MALDI-TOF/TOF instrumentation reveal N-terminal and also C-terminal sequence stretches of up to 80 amino acids. Sample preparation, spectrum acquisition and interpretation only take minutes. A method was developed for very fast and simple validation of the terminal sequences of proteins with MALDI-Top-Down-Sequencing (MALDI-TDS) using ISD. Samples of rich ISD spectra were analyzed with dedicated software (BioPharma Compass) that automates the interpretation of the TDS spectra based on suggested sequence candidates, an acquired ISD spectrum, yielding a detailed N- and C-terminal sequence assignment as pdf report. The ISD-spectra were automatically matched against a database of predicted protein sequences including possible modifications. The software calculated the a, c, y and z+2 ions for each protein sequence (up to the 116 kDa b-galactosidase) and matched the fragments against the spectrum. The results were presented in an in-depth report (PDF-document) that shows the annotated ISD spectrum together with the protein sequence for the best matching protein. For all protein sequences the information about a confirmed N- or C-terminus is presented. Created reports are collected in a common report directory and they include assignment of modifications in the range covered by the termini analysis (residues 1-80 from both sides). Using a batch process it was possible to match a series of spectra against a series of protein sequences. Terminal truncations, modifications such as acetylation or pyroglutamylation were detected in various investigated proteins as well as terminal sequence elongations. P-130 Top-Down Mass Spectrometric Analysis of Glycosylated and Phosphorylated Proteins Nannan Tao1; Anja Resemann2; Lars Vorwerg2; Detlev Suckau2

1Bruker Daltonics Inc., Fremont, CA USA; 2Bruker Daltonik GmbH, Bremen, Germany Top-down approaches allow for direct analysis of intact proteins using their mass and their fragments for identification and characterization. In contrast to bottom-up techniques, labile PTM&rsquo;s like phosphorylation remain stable in top-down analysis and enable direct modification site determination. MALDI-In-Source-Decay (MALDI-ISD) is a powerful Top-down-Sequencing (TDS) tool and typically provides long N- and C-terminal sequence tags of up

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to 80 amino acids and more, allowing for the detection of large PTM&rsquo;s like N-linked glycans even at central sites of proteins up to 15 kDa. TDS were applied to standard glycoproteins such as RNAse B, horseradish peroxidase, a- and b-caseins for positive as well as negative ionization mode MALDI-ISD was explored. Glycosylation: Using 1,5-diaminonaphtalene (DAN) as matrix, reduction of the SS-bridges occurred and ISD fragmentation of RNaseB was extended to residue 40 including the glycosylated Asn34. The most abundant high-mannose-type glycans Man5 and Man6 were detected. Horseradish peroxidase was prepared the same way and the N-linked glycan structure typical for plants was identified at Asn13. Additionally, the pyro-glutamate at the N-terminus of horseradish peroxidase was found. To our knowledge, this is the first time TDS analysis included the assignment of N-linked glycans. Phosphorylation: Negatively charged phosphate modifications in b-casein limited the N-terminal ISD fragment detection in positive ion mode, only 2 phosphorylation-sites were detected. Negative ion mode, however, enabled the detection of all 5 N-terminally phosphorylated serine residues. In a-S1-casein, Ser--41 was identified as non-phosphorylated in contrast to uniprot description, S46 and S48 were confirmed as being phosphorylated. a-S2-Casein carries 5 phosphorylated serine residues near the N-terminus, all of them were confirmed using negative mode ISD. As a result, we propose to use negative ion mode ISD in cases where phosphorylation or other negative charged residues effect the formation of fragment ions in TDS analyses. NOTES:

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P-131 Immuno-LC-MS a Possibility for Pharmacokinetic Measurements of Biologics Carsten Krantz; Wai Siang Law; Christian Miess; Gwenola Treton; Sherri Dudal; Peter Lloyd; Andrew Paul Warren Novartis Pharma AG, Basel, Switzerland Due to a combination of relatively high molecular weight analyte and the complex protein mixture contained in serum matrix, protein drug bioanalysis by LCMS has challenges to meet levels of sensitivity routinely achieved by ligand-binding assays. However, recent triple quadropole MS combined with sample pretreatment designed to enrich the analyte enables LCMS-based bioanalytical methods for proteins to be considered as viable bioanalytical options that can provide complementary input alongside, and in some cases instead of ligand-binding assays.. Here we present a case study of a bioanalytical PK assay for a human IgG drug with ng/mL sensitivity in cynomolgus monkey serum. This level of sensitivity was achieved by including an anti-human IgG enrichment step prior to the digestion step. The resulting PK profiles in both high- and low-dosed animals were comparable to profiles generated using a validated ligand-binding assay and provide further evidence that Immuno-LC-MS can be successfully deployed in routine PK analysis with high sensitivity and reproducibility NOTES:

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Thursday Poster Abstracts

P-201 High-throughput Critical Quality Attribute (CQA) Screening for Desired Monoclonal Antibody rDNA Clone by LC-MS Anita Krishnan; Himanshu Gadgil Intas Biopharmaceuticals Ltd, Ahmedabad, India Clone development is the first step towards the birth of a biopharmaceutical drug. Appropriate analytics can lead to the selection of a top performing clone. Using multiple criteria it is possible to assess the right clone with all the desired qualitative attributes and concomitantly with a yield that is commercially viable. Time and effort invested in this process would certainly result in gains at a later stage of product development cycle. So the major question remains - what is the most rapid and effective strategy for clone screening? Since every glycoprotein targets a different mechanistic pathway it is critical to screen for the right qualitative criteria (CQAs). Clones are initially assessed by the titres and on crossing the titre gate, qualitative analysis for CQAs are performed. Subsequently primary structure variants, mismatching charge variants and unusual glycan variants are assessed for their criticality. High-throughput methods that can effectively screen a series of minipools during clone selection and later during process optimization for CQAs, essentially lead to the selection of a variety of top-performing clones. Mass Spectrometry in combination with an LC workflow was able to effectively filter (a) misprocessed signal peptide - two amino acid 213Da difference in mAb(b) Fc variant allotype - 32Da in the conserved region of IgG1 (c) 52kDa clipped version of a fusion protein and more. We have also assessed the glycan peculiarities that were observed due to host cell line differences - a biobetter version. All these findings involved the least sample analysis time and resulted in valuable clone selection decisions. NOTES:

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P-202 Surface Modified BaTiO3 NPs for the Extraction and Analysis of Phospholipids and Hydrophobic Proteins in Escherichia coli in MALDI MS Suresh Kumar1; Hui-Fen Wu2

1SVNIT, Surat, India; 2National Sun Yat Sen University, Kaohsiung, Taiwan e describes that the dual role of HSA-tailored BaTiO3 NPs in MALDI MS for the analysis of phospholipids and hydrophobic proteins in E. coli. The surface modified BaTiO3 NPs were effectively acted as liquid matrix for the analysis of phospholipids (PS and PA) in MALDI-MS. HSA-tailored BaTiO3 NPs provided a good desorption/ionization efficiency for phospholipids with less fragmentation. This method provided good quantitative linearity for PS and PA (R2= 0.9905 - 0.9980). We also demonstrated that the applicability of HSA-tailored BaTiO3 NPs as extracting probes for the LLME of hydrophobic proteins in E. coli. HSA-tailored BaTiO3 NPs were effectively extracted and preconcentrated the hydrophobic proteins in E. coli. This tailored BaTiO3 NPs is uniquely suited for the analysis of phospholipids and hydrophobic proteins in biological samples with high sensitivity. P-203 Product Identity, MALDI-TOF Method Validation of Clinical Products by Bruker Systems Emily Liu; Connie Kong; Heidi Chae; Lin Gan; Ed Hoff Genentech, a Member of the Roche Group, South San Francisco, CA USA Peptide mass fingerprinting by MALDI-TOF-MS is used as an identity test for protein products at Genentech. Genentech has a database that is limited to its products to identify proteins by probability based matching of the observed set of peptide masses with theoretical ones. Method validation of peptide mass fingerprinting was completed for 39 clinical products using the Bruker MALDI-TOF-MS Systems. The Bruker MALDI-TOF-MS offers the advantages of a security controlled software and simplified sample preparation by eliminating the use of an internal standard mass calibrant. Two of the challenges encountered during the method validation were distinguishing two products with only one amino acid difference in sequence; and identifying a particular product due to its unique peptide overlapping with another peptide, but 1Da variation. This poster presents an overview of these challenges and their resolutions. NOTES:

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P-204 LC/MS of Glycopeptides for Determining Microheterogeneity Mellisa Ly; Gregory O. Staples; Ning Tang; Hongfeng Yin; Kevin Killeen Agilent Technologies, Santa Clara, CA USA The carbohydrate components of glycoproteins are of biological interest because they play important roles in development, disease, and homeostasis. However, there lacks a robust, rapid, and simple analytical toolbox for glycosite profiling. The unique properties of glycopeptides create analytical challenges because of their low abundance compared to peptides, the presence of heterogeneous mixtures of glycoforms at each site, and the suppression of their ionization by peptides that co-elute during LC/MS runs. Traditionally, glycopeptides are purified from protein digests by numerous techniques, often involving some type of tedious off-line solid-phase extraction. Here we present alternative analytical approaches for the study of glycosite microheterogeneity of glycopeptides using HPLC-chip/MS. A major breakthrough was the ability to couple HILIC enrichment with reversed-phase separation, two chromatographic phases which have been assumed to be incompatible. Automated, on-chip HILIC enrichment of glycopeptides was demonstrated on a microfluidic device coupled to a QTOF mass spectrometer. Advantageously, no sample handling was required andoff-line purification steps were avoided. The human proteins haptoglobin and erythropoietin were proteolyzed for analysis. The resultant separation of glycopeptides enriched by a HILIC trapping column followed by separation using a 150 mm reversed-phase analytical column provided very high resolution of glycoforms at each of the glycosites. Separation was primarily based on peptide sequence and secondarily by glycoform structure. Lower abundance glycopeptides were better identified using HILIC enrichment with reversed-phase separation, as compared to traditional analysis using reversed-phase. This HILIC enrichment coupled to reversed-phase separation should be a particularly well suited analytical tool for glycopeptide mapping, where the composition and abundance of glycans at a given site are determined. P-205 Combined Physical-Chemical and Biological Characterization Strategy For Effective Screening of Bispecific Antibodies Clones Patricia Molina; Domingos Ng; Blair Wilson; Phuong Tran; Amy Shen; Pamela Chan; Hongbin Liu Genentech, a Member of the Roche Group, South San Francisco, CA USA The main challenge for recombinant bispecific antibody production is the ability to produce sufficient amounts of BsAbs with high purity for therapeutic use. Recombinant BsAbs production has been explored in the last two decades and has rendered numerous BsAbs formats. At Genentech, a “knob-into-hole” bispecific IgG architecture has been developed. To evaluate

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different”knob-into-hole” formats, thousands of transient clones are generated and screened to determine which configuration might be the most promising to move into stable cell line development. As a result, a step-wise strategy for the effective screening of transient bispecific-producing clones has been devised. The strategy consists of the initial evaluation of the bispecific content and yield using two Enzyme-Linked ImmunoSorbent Assay (ELISA) formats to detect total IgG and the bispecific antibodies. Few of the selected clones are then scaled up, the BsAbs are purified from the media, and evaluated using RP-UPLC-MS analysis. Intact molecular mass measurements are used to assess the correct nature of bispecific. Disulfide bonds are chemically reduced and the resulting four species, two light chains (LCs) and two heavy chains (HCs), are also analyzed by RP-UPLC-MS. UV monitoring was used to determine the amount of LCs and HCs and their relative ratios present in each sample whereas online MS data provided their identities. Results from these experiments have demonstrated that the expression ratios of LC and HC from the same half molecules, and ratios between two halves, are key product quality indicators. There is a good correlation between the LC-MS and the ELISA analysis, i.e., the clones identified to have properly balanced expression ratios by LC-MS are also the clones that have high bispecific content from the ELISA data. This combined strategy provides relatively rapid and effective screening of BsAbs-producing transient clones as well as valuable physical-chemical and biological information. NOTES:

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P-206 A Study of Apolipoprotein E4 Oligomerization by H/DX and Electron-Transfer Dissociation Richard Huang2; Jawad Pashmi1

1ThermoFisher Scientific, San Jose, CA USA; 2NIST, Rockville, MD USA Apolipoprotein E, a 299 amino acid lipoprotein, plays a key role in the triglyceride and cholesterol metabolism. Of the three common isoforms viz, apoE2, 3 and 4 (which differ by single amino acid changes) only apoE4 is a risk factor for Alzheimer&rsquo;s disease and for cardiovascular diseases. Lipid-free ApoE self-associates to form oligomers such as dimers and tetramers at nM to µM concentrations. However, a monomeric form of apoE can be prepared by substituting 4-5 residues in the C-terminal domain. We have used hydrogen deuterium exchange (H/DX) coupled with enzymatic digestion and ETD fragmentation to investigate the differences between oligomeric and monomeric forms of the ApoE4 at the amino-acid level. The outcome, which we report here, provides an improved understanding of residues invovled in apoE oligomerization. A peptide-level H/DX comparison of wild-type ApoE proteins and their monomeric mutants allows us to locate the interfaces between the oligomers. With ETD fragmentation, we achieved residue-level resolution in which amino-acid residues involved in the oligomerization process, either directly or allosterically, can be visualized by comparing their extent of deuterium uptake. We found significant differences in deuterium uptake between the WT-apoE isoforms and their respective monomeric mutants in the regions 230-243 and 262-270. We then analyzed the peptic peptides undergoing H/DX at the amino-acid level for these two regions of ApoE4. The results indicate that Glu234, Gln 235 and Val236 in peptide 230-243 and amino acids Trp264 (Arg264 in the monomeric mutant) in the peptide 262-270 are highly involved in the oligomerization. We are now applying H/DX-ETD strategy to obtain, for the first time, a nearly complete picture of oligomer formation of ApoE4 at the amino-acid level. NOTES:

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P-207 Structure and Functional Analysis of Survivin Protein using HDX and High Resolution Mass Spectrometry Y. Cheng1, K Panagopoulos2, M. Holloway1, Y. K. Nguyen1, D. Pantazatos 2, R. Altura1, Virgil L. Woods3, Jr. Jawad Pashmi4.

1Department of Pediatrics and 2Department of Medicine, The Warren Alpert Medical School at Brown University and Rhode Island Hospital, Providence, RI USA, 3University of California San Diego, La Jolla, CA USA, 4ThermoFisher Scientific, San Jose, CA USA The smallest member of the Inhibitor of Apoptosis (IAP) family, Survivin (16.5kDa) is a master regulatory protein involved in key cellular functions that are dependent on its intracellular localization. In the nucleus, Survivin primarily associates with the chromosomal passenger complex (CPC) to regulate cell division, while in the cytoplasm Survivin promotes cell survival by actively inhibiting apoptotic pathways. Numerous studies have suggested that Survivin is highly upregulated and preferentially localized to the cytoplasm in breast, pancreatic, and colorectal cancers. In association with the CRM1/RanGTP nuclear export complex, Survivin is shuttled from the nucleus to the cytoplasm in order to exercise its cytoprotective and tumor-promoting functions. This study aims to further current efforts in the design of selective small molecule CRM1 inhibitors by identifying residues specific and essential to the Survivin-CRM1 association. Here, we apply Deuterium Exchange Mass Spectrometry (DXMS) first on isolated Survivin, to identify disordered and unstructured regions which may provide insight into biological function. We find a rapidly exchangeable N-terminal BIR domain (>90%) labeling at 10s) and a highly dynamic C-terminal region. These results indicate that Survivin exists predominantly as a monomer in solution and implicate functional involvement of these regions in protein-protein interactions. The results provide insight into regions of Survivin involved in protein-ligand interactions with its partner ligands. P-208 Analysis of cGMP/cAMP Binding to the PKGI Beta Regulatory Domain by HDX using High Resolution Orbitrap Mass Spectrometer Sheng Li 2; Jawad Pashmi1; Bryant Kou2; Darren Casteel2

1ThermoFisher Scientific, League City, TX USA; 2University of California, San Diego, San Diego, CA USA The type I cGMP-dependent Protein Kinases (PKGI) play important roles in regulating the cardiovascular and nervous systems. Mammalian cells express two isoforms of PKGI (PKGIα and PKGIβ) which are slice variants from a single gene. In this study we used the latest improvements in deuterium exchange mass spectrometry to compare the dynamics of the PKGIβ regulatory domain (amino acids 4-352) when bound to saturating amounts of cGMP or cAMP (1

81

mM). The improved experimental set up consisting of an automated H/D exchange platform from LEAP Technologies and Orbitrap Elite Mass Spectrometer from Thermo Scientific allowed us to achieve 100% sequence coverage of the regulatory domain, which we were unable to get in earlier studies (peptides comprising the leucine zipper domain were not previously detected). When we compared H/D exchange between the cGMP- and cAMP-bound proteins, we found that most regions of the protein showed equivalent decreases in H/D exchange when bound to either nucleotide compared to unbound protein. However, the C-helix in the second cGMP-binding pocket showed lower H/D exchange in the presence of cGMP compared to cAMP, indicating that cGMP was better at stabilizing the helix. Since stabilization of the C-helix is important for activation of full-length PKGIβ, these data provide insight into the mechanism of cyclic nucleotide specific activation of PKGIβ. NOTES:

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P-209 Characterization of a Monoclonal Antibody using a High Resolution and Accurate Measurement Mass Spectrometer with Multiple Fragmentation Techniques Jie Qian; Greg Kilby Thermo Fisher Scientific, Somerset, NJ USA Monoclonal antibodies (mAbs) have become a promising type of therapeutics for biopharmaceutical industry. Thorough characterization of mAbs is needed for research and production. Mass spectrometry (MS) with high resolution and accurate mass has become an essential analytical tool for mAb structural determination. A monoclonal IgG1 kappa antibody, composed of two heavy chains of 451 amino acids and two light chains of 213 amino acids, is characterized in this study. The mAb was enzymatically treated before liquid chromotography (LC)-MS analyses. Both intact proteins and peptides eluted from the column were analyzed using an Orbitrap mass spectrometer. Multiple fragmentation techniques were applied towards the amino acid sequencing.The top-down and bottom-up data was interpreted with a variety of software packages, providing an easy and fast workflow for investigating amino acid sequence and modification sites of the antibody. LC-MS spectra of intact or reduced mAb were analyzed using a piece of protein deconvolution software (Protein Deconvolution 2.0). For amino acid sequencing and modification identification of the protein, both top-down and bottom-up approaches were sophistically performed for a thorough study of the model protein. A complimentary combination of fragmentation techniques were applied including traditional collision induced dissociation (CID), high energy collision dissociation (HCD), electron transfer dissociation (ETD) and etc. Coverage by different fragmentation types were compared and reported. The full MS spectra from direct injections of the intact antibody followed by LC-MS analyses demonstrate a clean distribution of the whole charge envelope. Without deglycosylation, each charge state peak shows four major glysosylation species. The protein was further deglycosylated or deglycosylated and reduced for intact measurement.Results from this study shows that the HR/AM Orbitrap mass spectrometer equipped with multiple fragmentation techniques is the instrument of choice for a thorough mAb characterization. NOTES:

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P-210 The Medicinal Potential of Promising Marine Organisms: A Proteomic Approach Md. Azizur Rahman University of the Ryukyus, Nishihara, Japan In the sea, there are many resources and novel chemical entities with biological activities that may be useful for finding drugs with greater efficacy and specificity to treat a variety of human diseases. Although there are some reports on promising natural products to date, the technology on small-molecule-based methods to isolate protein sets using a proteomic approach, which is currently useful for pharmaceuticals, is lacking. The separation of proteins from marine organisms, especially calcifying marine organisms, which are part of one of the most biologically diverse and ecologically important ecosystems on the planet, is difficult due to contamination by skeletal tissues and the high sensitivity of tissues to handling. Here, we highlight some recent studies of marine organisms, paying particular attention to their discovery, medicinal activities, and mechanisms of action. In addition, we introduce a newly established highly effective protocol from sample preparation to protein purification for proteomic analysis using Mass Spectrometry to isolate candidate proteins for target identification, helping turn the current wealth of proteomic information into drug discovery and optimization. This information might be useful for medicinal chemists, biologists and biotechnologists for further studies utilizing the great resources of these promising marine organisms. P-211 Qualitative and Semi-quantitative Analysis of Composite Mixtures of antibodies by Native Mass Spectrometry Sara Rosati Univeristy of Utrecht, Utrecht, The Netherlands Monoclonal antibodies represent a fast growing class of therapeutic biomolecules gaining more and more interest among pharmaceutical companies. Besides the well-established single product formulations, more complex antibody products are lately studied as drug candidates. Co-expression of monoclonal antibodies can result in the formation of bispecific species. A unique characteristic of bispecific antibodies is their ability to target two different antigens simultaneously. Likewise, mixtures of antibodies can target different antigens involved in the same disease resulting in a synergistic effect with increased efficacy and potency, or in addition target different epitopes of the same antigen. A reliable technique suitable for the analysis of antibody mixtures of increasing complexity, is essential for the both drug development and quality control. Using native mass spectrometry, we are able to simultaneously identify and relatively quantify a mixture of monospecific and bispecific monoclonal antibodies co-expressed in a single cell platform. Moreover, to validate our quantitative data, the same antibody mixtures

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were analyzed by cation exchange chromatography. We conclude that native mass spectrometry has reached a high level of reliability, and it is ready to be used for the routine characterization of therapeutic antibodies. It presents a fast and robust approach for the qualitative and quantitative profiling of biopharmaceutical products. The exact mass measured for each antibody species reveals its identity, and simultaneously, from the ion signal intensity the relative abundance of each component can be determined. NOTES:

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P-212 Top-down Characterization of Monoclonal Antibody Therapeutics via a 12T SolariX Hybrid Qq-Fourier Transform Ion Cyclotron Resonance (FTICR) Mass Spectrometer Lisa Marzilli1; Christopher Thompson2; Katherine Kellersberger2; Daniel Haq1; Andrew Saati1; Michael Easterling2; Brian Stall2; Victor Fursey2; Jason Rouse1

1Pfizer, Inc., Andover, MA USA; 2Bruker Daltonics, Inc., Billerica, MA USA Therapeutic monoclonal antibodies (mAbs) are characterized methodically with top-down and bottom-up MS-based approaches, in concert with process and product development, to achieve complete structural understanding of the active mAb substance prior to commercialization. Ultrahigh-resolution (UHR) ESI-QTOF mass spectrometers have revolutionized antibody characterization by providing enhanced sensitivity/dynamic range, 30-60k resolution, and <2ppm mass accuracies for IdeS subunit/domain mapping, Lys-C peptide mapping, and released N-glycan mapping. Similarly, direct analysis of intact mAbs in bulk solution via offline nanoESI-QTOF MS is an effective method to clearly demonstrate that the correct 148kDa antibody was manufactured with the intended amino acid sequence, 4-chain architecture, and expected posttranslational modifications. However, in the analysis of intact mAbs, UHR ESI-QTOF instruments afford similar experimental resolving powers as older QTOFs resulting in average mass determinations with higher measurement errors (5-30ppm). FTICR MS is a definitive technique emerging for the characterization of intact mAbs; recently, baseline isotopic resolution was achieved for a representative intact IgG1k mAb using a customized 9.4T FTICR mass spectrometer with enhanced data collection/processing capabilities (Valeja et al., Anal. Chem., 2011). Accordingly, we investigated intact mAb analysis using a commercially available FTICR instrument. Using the same IgG1k mAb, we demonstrate that a Bruker Daltonics 12T SolariX equipped with a dynamically harmonized ICR cell (Nikolaev et al., J. Am. Soc. Mass Spectrom., 2011) can provide similar baseline isotopic resolution (RP=330k, magnitude mode) for the intact N-glycoforms, in addition to reliable, accurate monoisotopic mass determinations with low ppm errors, following external calibration and SNAPII deisotoping. For the major G0F/G0F N-glycoform, the observed mass, 147664.84 Da, compared well to the theoretical mass (monoisotopic), 147664.35 Da, yielding a 3.3ppm error. Minor N-glycoforms had mass errors <10ppm. With offline ESI, longer detection times (13.4 vs. 8.8s/scan; 200 scans) resulted in more refined isotopic profiles and more accurate masses, 147664.42 Da (0.5ppm error). NOTES:

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P-213 Biomarkers for Cancer: MS-based Glycosylation Analysis of Serum and its SpecificPproteins Renee Ruhaak1; Jincui Huang1; Qiuting Hong1; Carol Stroble1; Hai-Chi Pham2; Lauren Dimapasoc1; Suzanne Miyamoto2; Carlito Lebrilla1

1University of California Davis, Davis, CA USA; 2University of California Davis Cancer Center, Sacramento, CA USA Many of the clinically approved biomarkers for the detection of cancer are glycoproteins and several of the stains used to distinguish malignant tissue in histology are directed against carbohydrates. It is, therefore, not surprising that over the last decades, several successful studies have emerged on the development of candidate glycan biomarkers. So far, most studies have focused on whole biofluids and it seems likely that changes in specific glycoforms of cancer-related proteins will result in clinically more important biomarkers, with improved sensitivity and specificity. Here, we have developed methods to investigate protein-specific glycan profiles of proteins associated with cancer. First, we released N-glycans from captured IgG, and using nLC-PGC-chip-TOF-MS 35 N-glycans could be observed consistently and were quantified with good CV’s. This method has been applied to a cohort of lung cancer patients, and altered IgG glycans were observed. Subsequently, tryptic digests of IgG were analyzed using RP-LC-QQQ-MS, where stable transitions could be obtained for 14 (IgG1), 10 (IgG2 and 3) and 8 glycans (IgG4) with a CV of 6%. This method has been applied to a cohort of gastic cancer patients; changes in IgG glycosylation that are associated with gastric cancer will be presented. A similar method was used for the enrichment of fibronectin, a large (440kDa) protein that is associated with several types of cancer, from serum and ascites fluid. To obtain site-specific glycosylation patterns of fibronectin, the GLYCOFIND procedure where fibronectin was digested with pronase, was used. We will present results on the association of fibronectin glycosylation with ovarian- and lung cancer. Overall, we have developed methods for the analysis of site specific glycosylation profiles from individual cancer-related proteins were using MALDI-FTICR-MS(-MS), nLC-PGC-chip-Q-TOF-MS of N-glycans and pronase digests or RP-LC-QQQ-MS of tryptic digests. Using these techniques we now show, for the first time, site-specific glycan markers. NOTES:

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P-214 Extended Characterization Methods for Confirmation of Disulfide Bond Structure and PEGylation Site in a PEGylated Factor VIII Nicole Samuels; Raghu Chitta; Susmitha Sompalli; Joanne Severs Bayer Healthcare, Berkeley, CA USA Long-acting B-domain deleted Factor VIII variants (FVIII) have been engineered with a polyethylene glycol (PEG) polymer conjugated to a surface-exposed cysteine (Mei, B. et al.). The PEGylation process for the FVIII variants includes reduction with tris(2-carboxyethyl)phosphine (TCEP) prior to the conjugation of PEG-maleimide to the engineered cysteine. We present the detailed characterization of a representative PEGylated construct, a 60 kDa PEG FVIII variant. A procedure for the analysis of disulfide bonds under neutral conditions has been employed for PEGylated FVIII variants to prevent the induction of scrambling during sample preparation. An LC-MS method was developed for screening non-reduced tryptic digests of the PEGylated FVIII variant to confirm the presence of the expected disulfide bonds and the absence of scrambling. High mass Matrix-Assisted Laser Desorption (MALDI) mass spectrometry was employed to confirm PEGylation of the expected domain of the FVIII variant. Edman sequencing of the PEGylated peptide isolated from a tryptic digest of the FVIII variant provides supporting confirmation of the site-specific PEGylation of a surface-exposed cysteine introduced through site-directed mutagenesis of a BDD FVIII. P-215 Automated Acquisition and Data Analysis of Protein Conjugation by LC-MS Malcolm J Saxton1; Ryan Hylands1; Wolfgang Jabs2; Karl Nicholls1; Joanne Waters1

1Novozymes Biopharma UK Ltd, Nottingham, UK; 2Bruker Daltonik GmbH, Bremen, Germany We describe an automated UPLC-MS based method for the monitoring and analysis of the conjugation of small molecules and peptides to proteins. The automation is implemented using Bruker Daltonics (BD) Biopharma Compass software which allows for data acquisition, processing and analysis to occur with minimal user intervention. Data acquisition is implemented through Biopharma Compass ensuring all acquisition parameters, and any future processing that may occur, are traceably linked to the samples analysed and all data produced. Post-acquisition, samples are automatically processed to determine the intact mass of the produced protein conjugates. From this deconvoluted data

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conjugation ratios are automatically calculated for the various conjugation states produced, along with the percentage of correct conjugation state produced and an indication of the purity of the final conjugated product. Once processing is complete, data is presented in a graphical interface, allowing the user to easily determine the conjugation state of the sample and which samples may require further user intervention. The automation of acquisition, data processing and analysis allow for both a simple confirmation of correct sample conjugation and the analysis of complex conjugation time course experiments to be carried out with the minimum of user intervention required, saving significant resources while ensuring high quality data is produced and interpretation occurs. NOTES:

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P-216 Improving Intact Antibody Characterization by Orbitrap Mass Spectrometry Kai Scheffler1; Eugen Damoc2; Mathias Mueller2; Martin Zeller2; Thomas Moehring2

1Thermo FisherScientific, Dreieich, Germany; 2Thermo Fisher Scientific, Bremen, Germany The ultrahigh resolution of the Orbitrap mass detector is one of the most important prerequisites for intact protein characterization and top-down analysis. However, resolving power of large intact protein mass measurements requires a very low pressure in the Orbitrap mass analyzer to prevent fast transient decay caused by collisions with residual gas. We have modified a Thermo Fisher Scientific Orbitrap instrument for improved high molecular weight ion characterization. The modifications of the instrument relate to the implementation of a switching valve in the C-trap collision gas supply used to control respectively reduce the pressure in the Orbitrap mass analyzer as well as changes in the instrument control software. Reduction of the gas load in the C-trap/HCD collision cell allows for the reduction of the pressure in the Orbitrap mass analyzer and hence reduces the decay of transients of intact proteins and allows for the detection of more discrete beats in the transient of high molecular weight ions, thus increasing the upper mass limit of proteins isotopically resolved in online analysis up to ~50 kDa. To demonstrate the improved instrument performance resulting from the implemented modifications we have analyzed an intact monoclonal antibody in non-reduced and reduced condition by LC-MS. The intact antibody respectively the separated light and heavy chains were analyzed in Full MS experiments as intact protein species as well as with top-down experiments using different fragmentation techniques such as in-source CID (SID), CID, HCD and ETD. For data evaluation ProSight PC 2.0 and Protein Deconvolution 1.0 software packages were used. P-217 A Flexible Workflow-based Platform for Automating Mass Spectrometry Data Processing and Analysis to Standardize and Improve PTM Biopharmaceutical Characterization Joseph Shambaugh1; Jennifer Nemeth2; Steven Pomerantz2; Jessica Qi1; Jens Hoefkens1

1Genedata, Lexington, MA USA; 2Janssen R&D, Radnor, PA USA The ability to fully characterize post translational modifications (PTMs) is of significant importance along the research, development and manufacturing process of therapeutic biomolecules. Biopharmaceutical companies seek to establish scalable, automated processes using high-performance mass spectrometry to optimize efficiency and increase sample throughput. Establishing standardized data analysis processes that utilize minimal resources to maximize productivity and drive quick, reproducible decisions, is imperative to remain

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competitive. In an environment of rapid improvements in mass spec technology, it is equally important to adopt analytical methods that are platform-independent and promote utilization of available or future resources. Here we report on the development of activities and processes to enhance and streamline methods for the successful analysis of PTMs using Genedata Expressionist for Mass Spectrometry. We have worked together with our Pharmaceutical and Biotech partners to address the need to fully characterize biotherapeutics for PTMs. For example, together with scientists at Janssen's Biotechnology Center of Excellence, automation of data analysis was undertaken to address the analytical challenge of increasing the number of mAb candidates that are fully characterized for potential PTM liability prior to lead selection. The workflow incorporates label-free HPLC-MS experimental data for front-end data generation, with Expressionist for Mass Spectrometry for the processing of biologic data. When a biopharmaceutical is subjected to a stress study, it is exposed to thermal, pH, or light conditions known to induce structural changes such as oxidation and deamidation. The system was designed to facilitate the identification and delta change of defined PTMs in stability-stressed samples of biotherapeutics. This method also enables discovery of sites with unexpected liability. Similar workflows have also been applied by other customers to the assessment of batch-to-batch changes, measuring comparability, in biopharmaceutical production. NOTES:

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P-218 Identification of a Tryptophan Oxidation Antibody Variant Isolated by Size Exclusion Chromatography Alex Strahan; Louisette Basa; Boyan Zhang Genentech, a Member of the Roche Group, South San Francisco, CA USA Size exclusion chromatography (SEC) is routinely used to determine the size variant distribution of recombinant humanized monoclonal antibodies (rhuMAbs). During assessment of a rhuMAb using SEC, it was observed in the chromatographic profile that the main peak eluted later than expected. Further analysis uncovered the presence of a peak that would merge with the main peak if the mobile phase contained organic solvent (i.e., isopropanol). Enriched SEC fractions of the peak that merged with the main peak were prepared and then analyzed with intact/reduced mass determination and LC-MS/MS peptide mapping using two different enzymes. The data identified the peak as being a result of oxidation on a tryptophan residue in the CDR-H3 of the rhuMAb. This poster presents the techniques used for detecting and identifying the peak discovered in the SEC profile. P-219 Antibody Profiling by Native Mass Spectrometry Using Q-TOF and Orbitrap Platforms Natalie J. Thompson; Sara Rosati; Rebecca J. Rose; Esther van Duijn; Albert J.R. Heck Utrecht University, Utrecht, The Netherlands Advances in mass spectrometry technology, including the routine application of electrospray ionization and increases in sensitivity and mass resolution for multiple instrument platforms, have opened the field to the analysis of large intact protein complexes. Specifically, the study of monoclonal antibodies by mass spectrometry has led to the development of novel methods for profiling of antibody modifications and mixtures of antibodies. Here, native mass spectrometry on a Q-TOF platform has been evaluated for qualitative and semi-quantitative analysis of composite mixtures of antibodies representing biopharmaceutical products co-expressed from single cells. We show that by using automated peak fitting of the ion signals in the native mass spectra, we can quantify the relative abundance of each of the antibodies present in mixtures, with an average accuracy of 3%, comparable to a cation exchange chromatography based approach performed in parallel. Moreover, using native mass spectrometry we were able to identify, separate, and quantify nine antibodies present in a complex mixture of ten antibodies, whereas this complexity could not be unraveled by cation exchange chromatography. In preliminary work we now have also explored how native mass spectrometry can be coupled with Orbitrap platforms for the analysis of large molecules. Minor instrumental modifications were made on a research grade instrument not yet available allowing measurement of a range of protein assemblies with superior mass resolution and a sensitivity down to the detection of single

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ions. From the presented data, we conclude that native mass spectrometry presents a valuable alternative to existing analytical methods for qualitative and semi-quantitative profiling of biopharmaceutical products. It provides both the identity of each species in a mixture by mass determination and relative abundance through comparison of relative ion intensities. Native mass spectrometry is a particularly effective tool for characterization of heterogeneous biopharmaceutical products such as bispecific antibodies and antibodies mixtures. NOTES:

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P-220 Characterization of Tyrosine Sulfate Residues in Therapeutic Proteins by Liquid Chromatogrpahy Electrospray Ionization Tandem Mass Spectromety using Ion-paring Reagent Iva Turyan; Ruth Frenkel; Zoran Sosic; Yelena Lyubarskaya; Andy Weiskopf Biogen Idec Inc, Cambridge, MA USA Tyrosine sulfation is a common posttranslational modification in secreted and transmembrane proteins and a key modulator of extracellular protein-protein interactions. In contrast to protein phosphorylation, determination of protein tyrosine (Tyr) sulfation remains a challenge due to extreme lability of tyrosine sulfo&ndash;ester bonds. Quantitation of tyrosine sulfate residues using a combination of reversed-phase liquid chromatography and electrospray ionization mass spectrometry will be presented. Two different ion-pairing reagents were tested to achieve selective complexation of sulfated Tyr residues via non-covalent interactions: nonafluoropentanoic acid (NFPA) and trifluoroacetic acid (TFA). TFA was shown to provide the lowest degree of interference for tyrosine sulfate detection. Hypercarb HPLC columns were found to be particularly useful for the analysis of the polar tyrosine sulfates, which are difficult to retain on C18 columns. ESI conditions were further optimized to preserve the labile Tyr-O-SO3 linkages. As a result, sulfoester fragmentation was successfully minimized. Sulfated tyrosine was quantified through multiple reaction monitoring (MRM) as a sum of multiple transitions including the neutral loss of COOH (major signal), the loss of SO3 from sulfated tyrosine, and monosodiated tyrosine. Isotopically labeled tyrosine (d4-Tyr) was used as an internal standard. Tyr and d4-Tyr were monitored using the loss of COOH from the parent ions. This approach was shown to be ideally suited for highly sensitive analysis of Tyrosine sulfate residues in therapeutic proteins. P-221 Chip-based nano ESI-MS for Monoclonal Antibody Characterization on an Orbitrap Mass Spectrometer Lisa Vampola; Melissa Alvarez; Victor Ling Genentech, a Member of the Roche Group, South San Francisco, CA USA Characterization of monoclonal antibodies (MAbs) remains a challenging task due to their large size and heterogeneity. Peptide mapping with LC-MS/MS detection is typically employed to obtain detailed structural information such as post-translational modifications, degradation products and sequence variants. Our typical approach involves separating peptides by gradient elution on an analytical column and introducing analytes into an Orbitrap mass spectrometer via electrospray ionization (ESI). Due to the high flow rates used in ESI, contaminants present in the solvents often cause inefficient peptide ionization. In addition, previous studies indicate that

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background chemical noise can interfere with the assignment of peptide charge states during acquisition, thus preventing the peptide ion from being selected for fragmentation and subsequently detected. Here we report the development of a chip-based nanoESI-MS method for the characterization of MAbs by peptide mapping. This method couples the TriVersa NanoMate with a standard HPLC and converts LC gradients operating in microliter flow to nanoliter flow rates for analyte introduction into the MS. The MS signal response was optimized by adjusting the position of the chip, spray voltage and capillary temperature on the MS while maintaining stable spray. With the LC flow significantly reduced, the expectation is that the signals from the contaminants would become less prominent. Since this change affects the signal-to-noise ratio, the levels of chemical noise from nanoESI were evaluated and compared with regular ESI. The LC coupling + fraction collection feature on the NanoMate was utilized to collect LC-MS/MS data while simultaneously fractionating segments of the peptide map into a 96-well plate. Due to the low sample consumption of nano-ESI, in-depth analysis could be performed on specific fractions of interest that were either not selected for MS/MS or required further analysis using other MS modes. NOTES:

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P-222 Using Label-free On-line System of Particle Plasmon Resonance Immuno-biosensor with Liquid Chromatography Mass Spectrometry to Determine Proteins and Small Molecules Szi-Han Fang; Yu-Jane Huang; Chia-Yu Lee; Lai-Kwan Chau; Shau-Chun Wang Natl Chung Cheng University, Chia-Yi, Taiwan Unlike conventional immuno-biosensing methodologies using detection tags such as fluorophores or radioactive elements, which require chemical synthesis procedures to derive, label-free detection employing localized plasmon resonance on nano-particles of noble metals is a more straightforward means without tedious steps to synthesize derivatives. Label-free methods also posses less risk to change the binding linkage structures of immunoassays. Particle plasmon resonance (PPR) is therefore an adequate label-free spectroscopic detection scheme to adopt in microfluidic immuno-biosensor, especially when sample consumption amount is limited and critical. In this paper, on-line PPR immunoassay microfluidic system with liquid chromatography mass spectrometry (LC-MS) is developed to determine proteins and small molecules. Having been sealed in microchannel, one segment of stripped optical fiber of which functionalized gold nano-particles immobilized with antigen or antibody probes are covered on the cladding layer. When the incident light passes through the fiber intensity decrease is sensitive to the bindings with the molecular probes planted on the nano-particles. The standard curve of intensity attenuation versus detected molecule concentration can be used to develop assays to determine target molecules such as antibody proteins and antigen small molecules. Having been bound with biotion immobilized nano-particles on the cladding section of fibers in microfluidic device, strepavidin proteins are detached from the nano-particles and flush out off the microchannel via infusing solution for desorption, and finally to detect with LC-MS to confirm molecular weight. Similarly, other PPR assays of anti-ovalbumin with ovalbumin and amphetamine derivatives with their antibodies are developed and the target molecules are also washed with buffer to desorb and to confirm with LC-MS. P-223 Quantification and Qualification of Oligonucleotides by High Resolution/Accurate Mass Orbitrap MS Hongxia (Jessica) Wang; Kevin Cook; Patrick Bennett Thermo Scientific, San Jose, CA USA Qualitative and quantitative analysis of oligonucleotides in biological matrices are important aspects of the drug development process. There is a trend towards the use of high resolution mass spectrometry (HRMS) as an alternative to overcome the limitations of nominal resolution provided by triple quadrupole MS. A high throughput generic UHPLC-HR-MS assay is demonstrated for oligonucleotide analysis using Q-Exactive Orbitrap MS. The qualitative

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information provided during quantitation of analytes within one injection is a crucial advantage to accelerate drug development process by reducing instrument analysis time and sample consumption. A 20-mer synthetic oligonucleotide was quantified with 15-mer oligonucleotide as internal standard. Samples were analyzed on a Thermo Q-Exactive Orbitrap equipped with Thermo XRS UHPLC pump and Open Accela autosampler. Oligonucleotides were analyzed first by full scan mode at 70,000 resolution for the charge state distribution envelope. The most abundant ions, [M-9H] 9- for 20-mer and [M-7H] 7- for 15-mer, were then used for quantitation. A comparison of oligonucleotide quantitation using both selected ion monitoring (SIM) and targeted MS/MS was performed with 8-min gradient on Thermo Hypersil Gold C18 column(1.9µm, 2.1x50mm) to separate analyte with internal standard. SIM at 70,000 resolution effectively isolated the analyte of interest from interferences in human plasma spiked with synthetic oligonucleotide. To minimize the absorption, three different carrier solutions and sample vials were evaluated. Preliminary data for samples with human plasma as a carrier indicates good linearity with a calibration range of 1-5nM to10µM. SIM method (70,000 resolution) shows 5 fold more sensitivity than targeted MS/MS method (17,500 resolution). SIM at 140,000 resolution and targeted MS/MS provide higher selectivity for the assay with complex matrices. In summary, the 20-mer phosphodiester oligonucleotide is used as a model compound to demonstrate the extraction recovery and assay specificity while high confident identification of oligonucleotides is achieved with fragmentation accurate mass data. NOTES:

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P-224 An LC-MS Method for Determination of Residual, Free Small-Molecule Cytotoxin in Antibody Drug Conjugate Lab Isolator Surface Cleansing Verification Samples Leslie Welch Genentech, a Member of the Roche Group, South San Francisco, CA USA In the search for oncology therapeutics that are more potent and cause fewer side effects, numerous biotechnology companies are actively investigating a variety of monoclonal antibodies (mAbs) conjugated to various small-molecule cytotoxins whereby the cytotoxin is delivered only to those cancer cells and specific proliferative pathways targeted by the mAbs. The success of this approach has led directly to the establishment of antibody drug conjugate (ADC) laboratories in manufacturing facilities, and the necessity for post-manufacturing clean-up and monitoring of the free (i.e., unconjugated), small-molecule cytotoxins. A sensitive, quantitative assay using liquid chromatography coupled to mass spectrometry (LC‑MS) was developed to monitor the level of residual, free small-molecule cytotoxin on the surfaces of lab isolators in a conjugation laboratory. Swabbing the surfaces with an organic solvent, and then extracting the cotton swabs with the solvent was used to verify cleaning of the isolator. The level of cytotoxin in the extracted samples was determined by reversed‑phase high‑performance liquid chromatography (RP‑HPLC) and an in‑line mass spectrometer set to selectively monitor the representative ion‑of‑choice for the free cytotoxin. Concentrations of cytotoxin in the extracted samples were calculated from the average mean response factor obtained from cytotoxin standard curves of the doubly‑charged molecular ion. Average recoveries of cytotoxin spiked into samples ranged from 92 ‑ 97%. Injection repeatability was demonstrated by analyzing mid- and high-range control samples over several days (5% and 2%, respectively). The mass spectrometric method permitted quantitative analysis of residual, free cytotoxin remaining on the isolator surfaces at a level ten‑fold below the acceptable surface limit (ASL) of 5 ng/cm2. Results from the LC‑MS assay were compared to those from an orthogonal method, an ELISA, and found to be similar. P-225 Enhanced and Automated Peak Modeling Charge Deconvolution Method for Intact Protein Data Analysis using Mass Spectrometry Xiaoling Wu1; Linda DeNoyer2; Ning Tang1; Maithilee Samant1; Keith Waddell1

1Agilent Technologies, Santa Clara, CA USA; 2Spectrum Square Associates, Ithaca, NY USA In LC/MS data analysis, the maximum entropy deconvolution method has been widely used for transforming multiple-charged spectrum into zero-charged mass spectrum. With a deconvoluted spectrum, the complexity of the data is reduced significantly; however, the maximum entropy

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deconvolution method can introduce unwanted artifacts through extraneous signals. We present an advanced data processing methodology that can solve this problem by applying an automatic peak modeling technique, delivering the most probable deconvolution results that are virtually free of artifacts. This peak modeling deconvolution method results in improved signal-to-noise ratios and enhanced resolution of protein peaks for precise analysis. The benefits of this improved methodology are illustrated by the results in several proteins’ data. Protein samples including antibody samples were analyzed using Q-TOF mass spectrometers. Two different methods of charge state deconvolution were compared. Maximum Entropy Charge Deconvolution. A well-known and powerful method for electrospray mass spectra of intact proteins deconvolution, it can condense each series from a given protein into a single peak on a true molecular weight scale, determining the most probable zero-charge mass spectrum. Peak Modeling Charge Deconvolution. This advanced algorithm based on the maximum entropy result, automatically generate peak models without manual intervention and apply these models through fitting and validating procedures, providing a highly resolved zero-charge spectrum and a set of mass error assessments for each peak. The new peak modeling deconvolution produces a much cleaner result with almost no artifacts, it has an enhanced resolution and overlapped peaks are well resolved. This enabled differentiating small modifications from the main heterogenous glycoprotein profile with much greater clarity. Our examples showed the new peak modeling methodology is robust even for poor S/N data and severely overlapped peaks. A full comparison of the maximum entropy deconvolution and peak modeling deconvolution methods and their results will be presented in this poster. NOTES:

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P-226 Higher-Order Structural Characterization of an IgG2 Monoclonal Antibody by Disulfide Mapping and Hydrogen Deuterium Exchange Mass Spectrometry Joomi Ahn1; Stephane Houel1; Justin Sperry2; Ying Qing Yu1; Weibin Chen1

1Waters Corporation, Milford , MA USA; 2Pfizer Inc., Chesterfield, MO USA Recombinant human monoclonal antibodies (mAbs) are widely used in biotherapeutics research and development as potential therapies for various diseases. After antibody production several analytical techniques focused on primary and higher-order structural analyses are conducted to adequately characterize these complex therapeutics. Recent reports detailing the characterization of IgG2 mAbs revealed the presence of disulfide-linked structural isoforms, termed IgG2-A, IgG2-B, and IgG2-A/B. In this study, we present an analytical workflow to automatically map out the disulfide -linked isomers using data independent LC/MS acquisition and disulfide bond mapping informatics tools. Once the peptide map information is obtained, hydrogen deuterium exchange mass spectrometry (HDX MS) using on-line pepsin digestion and peptic peptide mapping was used to investigate the consistency of multiple IgG2 batches. The HDX MS experiments, which combined online pepsin digestion and automated peptide assignment, afforded high sequence coverage of both light and heavy chains (88% and 93% respectively). We observed interesting deuterium exchange kinetics for a peptide representing the C-terminus of the light chain (VTHQGLSSPVTKSFNRGEC, residue numbers 196 to 214). The major IgG2 isoforms present in this mAb are the IgG2-B and IgG2-A/B. Each of these isoforms contain an interchain disulfide bond between the light chain (at position 214) to the heavy chain hinge region. The deuterium exchange profile of this particular light chain peptide exhibited EX1-like behavior, as observed as having multiple deuterium distributions at long deuterium exchange times. We attribute this behavior to the presence of multiple disulfide isoforms present in the mAb. Furthermore, the same exact deuterium uptake profile was also observed for this peptide in the other batch of IgG2. HDX MS combined with disulfide bond mapping was found to be a powerful approach to assess the primary and higher-order structure IgG2 mAbs across multiple production batches. NOTES:

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P-227 HR/AM Targeted Peptide Quantification on Q Exactive: A Unique Combination of High Selectivity, High Sensitivity and High Throughput Yi Zhang1, 2; Zhiqi Hao1, 2; Markus Kellmann1, 2; Andreas Huhmer1, 2

1ThermoFisher Scientific, San Jose, CA USA; 2ThermoFisher Scientific, Bremen, Germany Quantitative proteomics enables the identification of large number of protein candidates, which display biologically interesting dynamics on a global scale in the early discovery phase. Targeted MS approach, in particular selected reaction monitoring (SRM), has become the preferred platform for quantitatively analyzing tens to hundreds of peptide candidates across large number of samples, either for understanding of signaling regulation or for verification and selection of potential clinical biomarkers. As hardware innovations dramatically increase sensitivity and scan speed of high resolution and accurate mass (HR/AM) mass spectrometers, HR/AM MS method provides an attractive alternative for targeted peptide analyses. In this study, the performance of Thermo Scientific Q Exactive mass spectrometer was evaluated for targeted protein quantification. The linear dynamic range and LOD/LOQ of 15 peptide targets were investigated in complex matrix background, using two HR/AM methods, multiplexed targeted selected ion monitoring (msx tSIM) and targeted HCD (tHCD). For msx tSIM method, the high resolution of 140,000 and high mass accuracy (<5ppm) ensure confident identification of target ion in complex matrix. Two unique features, parallel filling/detection and spectrum multiplexing, dramatically increase total scan speed and throughput. A LOD of 10amol and an excellent linear dynamic range of 4 orders were obtained for all peptide targets in a medium complex background of 10ng of E coli whole cell digest. For tHCD method, the high mass accuracy (< 5ppm) and high resolution of 17,500 ensure unmatched confident identification of fragment ions compared to triple quadrupole based SRM method. A LOD of 10-50amol and an excellent linear dynamic range of 3-4 orders were obtained for all peptide targetsl in a strong complex background of 500ng of E coli whole cell digest. P-228 Assessment of Stable Isotope Incorporation into Recombinant Proteins Xin Zhang; Quanzhou Luo; Izydor Apostol; Shun Luo; Matthew Jerums; Gang Huang; Xinzhao Jiang; Jessica Gastwirt; Nimesh Savjani; Ronald III. Keener; Jeffrey Lewis; Jette Wypych Amgen Inc., Thousand Oaks, CA USA Stable isotope labeling combined with mass spectrometry has been widely utilized in a diverse set of applications in the biochemistry and biomedical fields. In many of the quantitative approaches, the stable isotope labeled protein is used as relative reference protein or internal standard. When stable isotope labeled proteins are produced via metabolic labeling of the cell culture, a comprehensive quality check or precise measurement of the labeling pattern of the

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target protein is imperative. In this study, we present a mass spectrometry-based analytical tool developed for quantitatively tracing the incorporation of stable isotopes into the recombinant proteins (such as monoclonal antibodies and Fc fusion proteins expressed in different host systems) using combined total mass analysis, peptide mapping analysis, and amino acid analysis. This work also describes the strategies of metabolic labeling the cell culture to produce uniformly labeled recombinant proteins from both E-Coli and Chinese hamster ovary (CHO) cell line. In addition, we show that the analytical technique developed here is a powerful tool to provide valuable insights into studying cell metabolism and performing flux analysis during cell culture. NOTES:

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P-229 Py-GC/MS Study of Oak Wood Decayed by Hymenochaete Rubiginosa and Stereum Hirsutum Leila Karami Motaghi University of Hamburg, Hamburg, Germany The Quercus robur degraded by the white-rot fungi Hymenochaete rubiginosa and Stereum hirsutum was analyzed by analytical pyrolyis (Py-GC/MS). In the resulting pyrograms, the relative peak areas of the degradation product of lignin, belonging to the guaiacyl (G) and syringyl (S) type phenols, and carbohydrates were evaluated. The pyrograms of sound and decayed wood showed the same degradation products but with different quantities. The most essential fragment of lignin were: 2-methoxy-phenol, 4-methylguajacol, vinyl guaiacol, eugenol, syringol, isoeugenol (trans), 4-methylsyringol, vanillin, homovanillin, acetoguaiacone, 4-vinylsyringol, guaiacyl aceton, 4-(1-Propenyl)-2,6-dimethoxyphenol (trans), 4-(1-propenyl)-2,6-dimethoxyphenol (trans), syringaaldehyde, homosyringaaldehyde, acetosyringone, coniferyl alcohol (trans), coniferyl aldehyde, syringylacetone, sinapylalcohol (cis), sinapyl alcohol (trans), sinapaldehyde. The main compounds of carbohydrate origin are: propanal-2-one, hydroxyacetaldehyde, acetic acid, 1-hydroxy-2-propanone, 3-hydroxypropanal, furan-(2H)-3-one, butanedial, 2-furaldehyde, an isomer of 4-hydroxy-5,6-dihydro-(2H)-pyran-2-one, 2(5H)-furanone, 4-hydroxy-5,6-dihydro-pyran-(2H)-2-one, 1,5-anhydro-ß-D-xylofuranose, (2-hydroxymethyl-5-hydroxy-2,3-dihydro-(4H)-pyran-4-one), 1,5-anhydro-ß-D-xylofuranose, and levoglucosan. White rot caused a decrease of S/G ratio and, expectedly, also decreased the lignin/carbohydrate ratio. P-230 A Peptide Mapping Method with Mass Spectrometry Detection for Determination of Glycosylation Occupancy Jenny Sun; Zhuchun Wu; Mike Byrne Human Genome Sciences, Inc., a GSK Company, Rockville, MD USA This poster describes a peptide mapping method with mass spectrometry detection for determination of the N-linked glycosylation site occupancy of a glycoprotein that contains multiple N-linked glycosylation sites. The method involves initial removal of N-linked glycans by PNGase F digestion. The deglycosylated protein is then subjected to trypsin digestion, generating deglycosylated peptides, which are analyzed by reversed-phase HPLC and MS detection. The deglycosylated peptide has a mass of one additional Dalton compared with the unglycosylated peptide, due to the conversion of Asn to Asp at the glycosylation site by PNGase F digestion. Therefore, the occupancy of the glycosylation site can be determined by integration

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of the extracted-ion chromatogram of the deglycosylated and unglycosylated peptides obtained using an ESI-Tof mass spectrometer. NOTES:

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