14th Symposium on the

Practical Applications of

Mass Spectrometry in the

Biotechnology Industry

(Mass Spec 2017)

Symposium Co-chairs:

Michael Boyne, BioTechLogic, Inc.

Eef Dirksen, Synthon Biopharmaceuticals B.V.

September 19-22, 2017

Boston Park Plaza Hotel

Boston, MA USA

Organized by

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Table of Contents

Welcome Letter ................................................................................................. 3

Student Travel Grants ....................................................................................... 4

Program, Exhibitor and Media Partners ........................................................... 5

Scientific Program Summary ............................................................................ 8

Oral Abstracts ................................................................................................... 17

Roundtable Discussion Topics .......................................................................... 37

Technical Seminar Abstracts ............................................................................ 41

List of Posters for Thursday ............................................................................. 45

List of Posters for Friday ................................................................................. 53

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Welcome to the 14th Symposium on the Practical Applications of

Mass Spectrometry in the Biotechnology Industry We are pleased to welcome you to the 14th Symposium on the Practical Application of Mass

Spectrometry in the Biotechnology Industry. The focus of this Symposium is the application of mass

spectrometry (MS) 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, DNA, viral vectors), applications for conventional pharmaceuticals

will also be discussed. The Symposium will be held over a period of two and a half days devoted to

practical concerns in the use of MS within the biotechnology and pharmaceutical industries.

The success of this symposium will depend not only on our experienced and knowledgeable speakers

and 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 and Roundtable sessions that

have been designed to stimulate the exchange of ideas and information.

We would like to thank the speakers who are giving generously of their time and resources, and you

for your attendance, which will make this a successful endeavor.

We gratefully acknowledge the generosity of our sponsors and exhibitors. Thank you to 908 Devices,

AbbVie, Inc., Agilent Technologies, American Laboratory/labcompare, American Pharmaceutical

Review, The Analytical Scientist, Biogen, BioProcess International, Bruker Daltonics, Inc., Charles

River Laboratories, Inc., CMP Scientific, Corp., Eli Lilly and Company, Eurofins Lancaster

Laboratories, Inc., Genetic Engineering & Biotechnology News, Genedata Inc., Genentech, a

Member of the Roche Group, Genovis AB, International Pharmaceutical Quality, LCGC, New

England Biolabs, The Pathologist, Pfizer, Inc., Pharmaceutical Outsourcing, Promega Corporation,

Protein Metrics, Inc., ProZyme, Roche Diagnostics GmbH, Sanofi, separationsNOW.com, SCIEX,

Technology Networks, Thermo Fisher Scientific and Waters Corporation. We are also thankful for

the expert assistance and support of Anna Lingel, 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 2018!

THE ORGANIZING COMMITTEE:

Greg Adams, FUJIFILM Diosynth

Biotechnologies

Michael Boyne*, BioTechLogic, Inc.

Steven Cohen, SAC Analytical Consultants

Terry Cyr, Health Canada

Eef Dirksen*, Synthon Biopharmaceuticals B.V.

Anders Lund, Sanofi

Yelena Lyubarskaya, Sanofi

Jun Park

David Passmore, Tenaya Analytical Consulting

Richard Rogers, Just. Biotherapeutics

Jason Rouse, Pfizer, Inc.

Arjen Scholten, Janssen Infections Diseases and

Vaccines

John Valliere-Douglass, Seattle Genetics, Inc.

Christopher Yu, Genentech, a Member of the

Roche Group

*Symposium Co-chair

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CASSS Mass Spec Student Travel Grants

CASSS is pleased to provide a limited number of student travel grants for PhD students and post-

doctoral fellows who present applicable posters at the 14th Symposium on the Practical

Applications of Mass Spectrometry in the Biotechnology Industry (Mass Spec 2017). PhD

students or post-doctoral fellows conducting research at academic institutions throughout the

world are eligible.

This symposium offers insight to current topics and issues under discussion within the biotech

and biopharmaceutical industries, and as such, provides an opportunity to bridge between

industry, academia, and regulatory agencies. The presentations and workshops are focused on the

application of mass spectrometry to advance drug discovery and development in the

biotechnology industry. Topics will include the utility of MS as an alternative to conventional

assays such as peptide mapping, ion exchange, capillary electrophoresis, as well as for the

analysis of process-related impurities, such as host cell proteins; introduction of MS in the QC

laboratory, validation/transfer/compliance strategy, system suitability/assay acceptance criteria;

expectations from regulatory agencies for MS based assays for product characterization, release

and stability; technical challenges regarding quantitation, ionization, higher order structure

analysis, etc.; applications of MS for high throughput screening of cell-lines, raw materials, in-

process samples; and benchmarking of new technology.

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:

A Novel Method for On-line Native MS Analysis of Monoclonal Antibody Charge Variants

Applying Volatile Buffers in SCX Chromatography

Florian Füssl, NIBRT, Co. Dublin, Ireland

Glycomic and Glycoproteomic Analysis of Biopharmaceuticals in a High Throughput and

Highly Sensitive Manner

Guinevere Kammeijer, Leiden University Medical Center, Leiden, Netherlands

Native Mass Spectrometry for the Revelation of Highly Complex Glycosylation Patterns in

Biopharmaceuticals

Therese Wohlschlager, University of Salzburg, Salzburg, Austria

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The Organizing Committee gratefully acknowledges the following

program partners for their generous support of this Symposium

Strategic Program Partners

Platinum Program Partners

AbbVie, Inc.

Biogen

Gold Program Partners

Eli Lilly and Company

Pfizer, Inc.

Platinum Program Partners

Bruker Daltonics, Inc.

Genedata Inc.

Gold Program Partners

Agilent Technologies

Genentech, a Member of the Roche Group

Roche Diagnostics GmbH

SCIEX

Bronze Program Partner

Sanofi

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Exhibitor Partners

908 Devices

Agilent Technologies

Bruker Daltonics, Inc.

Charles River Laboratories International, Inc.

CMP Scientific, Corp.

Eurofins Lancaster Laboratories, Inc.

Genedata Inc.

Genovis AB

New England Biolabs

Promega Corporation

Protein Metrics Inc.

ProZyme

SCIEX

Thermo Fisher Scientific

Waters Corporation

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The Organizing Committee gratefully acknowledges the following

media for their promotional consideration of Mass Spec 2017

Media Program Partners

American Laboratory/Labcompare

American Pharmaceutical Review

The Analytical Scientist

BioProcess International

Genetic Engineering & Biotechnology News

International Pharmaceutical Quality

LCGC

The Pathologist

Pharmaceutical Outsourcing

separationsNOW.com

Technology Networks

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Mass Spec 2017

Scientific Program Summary

Tuesday, September 19, 2017

08:00 – 08:30 Breakfast (Full Day Short Course Attendees ONLY) in the Berkeley

Room

08:00 – 13:30 Short Course Registration ONLY in the Ballroom Foyer

Short Course in the Clarnedon Room

Short Course Instructor: Anders Lund, Sanofi, Framingham, MA USA

08:30 – 12:00 Fundamentals of Mass Spectrometry in the Analysis of Protein

Therapeutics

Anders Lund, Sanofi, Framingham, MA USA

12:00 – 13:00 Lunch (Full Day Short Course Attendees ONLY) in the Berkeley Room

13:00 – 17:00 Applications of Mass Spectrometry to Characterize Protein

Therapeutics

Anders Lund, Sanofi, Framingham, MA USA

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Wednesday, September 20, 2017

User Group Meetings (Must Preregister Online)

08:30 – 11:30 Thermo Fisher Scientific User Group in the Clarendon Room

08:30 – 11:30 Waters Corporation User Group in the Arlington Room

09:30 – 12:00 Protein Metrics Inc. User Group in the Berkeley Room

Mass Spec 2017 Scientific Program

12:00 – 17:15 Registration for Mass Spec 2017 in the Ballroom Foyer

13:00 – 13:15 Welcome and Introductory Comments in the Georgian Room

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

Keynote I Session in the Georgian Room

Session Chair: Jason Rouse, Pfizer, Inc., Andover, MA USA

13:15 – 14:15 Mass Spectrometry Imaging in Surgical Pathology and Drug

Development

Nathalie Y.R. Agar, Harvard Medical School, Boston, MA USA

14:15 – 14:45 Networking Break – Visit the Exhibits and Posters in the Terrace Room

Process and Product Characterization Session in the Georgian Room

Session Chairs: David Passmore, Tenaya Analytical Consulting, Redwood City, CA USA

and John Valliere-Douglass, Seattle Genetics, Inc., Bothell, WA USA

14:45 – 15:15 A Mass Spectrometry Approach for the Screening of Cell Culture

Raw Materials and Spent Media

Rachel Chen, Biogen, Cambridge, MA USA

15:15 – 15:45 ELISA Reagent Coverage Evaluation by Affinity Purification

Tandem Mass Spectrometry

Scott Henry, Seattle Genetics, Inc., Bothell, WA USA

15:45 – 16:15 A Platform Disulfide Mapping Method for Characterization of

Therapeutic Proteins with Various Formats

Bing Zhang, Genentech, a Member of the Roche Group, South San

Francisco, CA USA

10

Wednesday, September 20, 2017, continued

16:15 – 17:15 Roundtable Discussion Session I in Arlington/Berkeley/Clarendon

Rooms

17:15 – 18:45 Welcome Reception in Grand Ballroom B

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Thursday, September 21, 2017

07:30 – 08:30 Breakfast in the Terrace Room

07:30 – 08:30 Job Board Live! Breakfast in Arlington Rooms

08:00 – 17:30 Registration in the Ballroom Foyer

Bioanalytical Session in the Georgian Room

Session Chair: Eef Dirksen, Synthon Biopharmaceuticals B.V., Nijmegen, Netherlands

08:30 – 09:00 Paving the Way for Assessing in vivo Dynamics of Multiple Quality

Attributes for Protein Therapeutics

Haihong Zhou, Merck &Co., Inc., Kenilworth, NY USA

09:00 – 09:30 Characterizing the Immune Response to Novel Vaccine Candidates in

a Human Whole Blood Assay Using High-dimensional Single Cell

Proteomics

Michael Cohen, Sanofi Pasteur, Toronto, ON Canada

09:30 – 10:00 Immunocapture-LC/MS Detection of Anti-drug Antibodies

David Roos, Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT USA

10:00 – 10:30 Networking Break - Visit the Exhibits and Posters in the Terrace Room

Emerging Product Areas Session in the Georgian Room

Session Chair:

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

10:30 – 11:00 Glycomics Characterization of Influenza Hemagglutinin Glycoprotein

Antigens

John Cipollo, CBER, FDA, Silver Spring, MD USA

11:00 – 11:30 Quantification of Membrane Proteins in Genetically Engineered

Crops by Liquid Chromatography Coupled with Tandem Mass

Spectrometry

Lindsey Schacherer, DuPont Pioneer, Johnston, IA USA

11:30 – 12:00 Assessment of Monoclonal Antibody-Fc Receptor Interactions by

Online SEC-Native Mass Spectrometry

Hongxia (Jessica) Wang, Regeneron Pharmaceuticals, Tarrytown, NY

USA

12:00 – 12:15 Lunch for Technical Seminar Attendees – Please take lunch and return

to the Georgian Room for the “Lunch and Learn”

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Thursday, September 21, 2017, continued

12:15 – 12:45 Technical Seminar: Lunch and Learn

An Adapted Top-down LC-MS Approach for Screening Lipid-modified Proteins

Andrew James, Sanofi Pasteur, Toronto ON Canada

Sponsored by Genedata Inc. Georgian Room

12:45 – 12:50 Mini Break for Technical Seminar Attendees

12:50 – 13:20 Technical Seminar: Lunch and Learn

Applications of Bruker Technology in Support of Biotherapeutics

Kathleen Cornelius, Pfizer, Inc., St. Louis, MO USA

Sponsored by Bruker Daltonics, Inc. Georgian Room

13:20 – 15:20 Poster Session I - Visit the Posters in the Terrace Room

Young Scientist Session in the Georgian Room

Session Chair: Steve Cohen, SAC Analytical Consultants, Hopkinton, MA USA

15:20 – 15:40 Native Size Exclusion Chromatography Coupled with Multi-Angle

and Dynamic Light Scattering and Mass Spectrometry for Heightened

Characterization of High Molecular Mass Species in Monoclonal

Antibodies

Ying Zhang, Pfizer, Inc., Andover, MA USA

15:40 – 16:00 Glycomic and Glycoproteomic Analysis of Biopharmaceuticals in a

High Throughput and Highly Sensitive Manner

Guinevere Kammeijer, Leiden University Medical Center, Leiden,

Netherlands

16:00 – 16:20 A Novel Method for On-line Native MS Analysis of Monoclonal

Antibody Charge Variants Applying Volatile Buffers in SCX

Chromatography

Florian Füssl, NIBRT, Co. Dublin, Ireland

16:20 – 17:20 Roundtable Discussion Session II in Arlington/Berkeley/Clarendon

Rooms

17:20 – 18:20 Exhibitor Reception - Visit the Exhibits in the Terrace Room

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Friday, September 22, 2017

07:30 – 08:30 Breakfast in the Terrace Room

08:00 – 16:30 Registration in the Ballroom Foyer

Keynote II Session in the Georgian Room

Session Chair: Michael Boyne, BioTechLogic, Inc., Glenview, IL USA

08:30 – 09:30 Charge Detection Mass Spectrometry: Weighing Heavier Things

Martin Jarrold, Indiana University, Bloomington, IN USA

09:30 – 10:00 Networking Break - Visit the Exhibits and Posters in the Terrace Room

Viral Capsid Proteins Session in the Georgian Room

Session Chairs: Yelena Lyubarskaya, Sanofi, Westborough, MA USA

and Arjen Scholten, Janssen Infectious Diseases and Vaccines, Leiden, Netherlands

10:00 – 10:30 A Mass Spectrometry Based “ELISA” Assay for Adenovirus Vaccine

Jonathan Knibbe, Janssen Infectious Diseases and Vaccines, Leiden,

Netherlands

10:30 – 11:00 Pushing the Boundaries of Adenovirus Characterization:

Development and Application of LC-MS Methods for Comprehensive

Understanding of Adenovirus Capsid

Thomas Powers, Pfizer, Inc., Chesterfield, MO USA

11:00 – 11:30 Human and Baculovirus-Insect Manufacturing Platforms Generate

Chemically and Functionally Distinct AAV Vectors

Nicole Paulk, Stanford University, Stanford, CA USA

11:30 – 11:45 Lunch for Technical Seminar Attendees – Please take lunch and return

to the Georgian Room for the “Lunch and Learn”

11:45 – 12:15 Technical Seminar: Lunch and Learn

Innovations for Biopharmaceutical Characterization: Speed Up the Routine and Simplify

the Challenging

Sean McCarthy, SCIEX, Framingham, MA USA

Sponsored by SCIEX Georgian Room

12:15 – 12:20 Mini Break for Technical Seminar Attendees

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Friday, September 22, 2017, continued

12:20 – 12:50 Technical Seminar: Lunch and Learn

Advances in the Characterization of Protein Therapeutics by LC/MS

Steve Madden, Agilent Technologies, Santa Clara, CA USA

Sponsored by Agilent Technologies Georgian Room

12:50 – 14:20 Poster Session II - Visit the Posters in the Terrace Room

CMC Strategy and the Use of Mass Spec Session in the Georgian Room

Session Chairs: Anders Lund, Sanofi, Framingham MA USA

and Richard Rogers, Just. Biotherapeutics, Seattle, WA USA

14:20 – 14:50 Monitoring Biologics CQAs for Quality Control Using MAM

Michael Olma, Werthenstein BioPharma GmbH, Schachen, Switzerland

14:50 – 15:20 Analysis of Biopharmaceutical Attributes in a GMP Setting

Yan Jiang, Sanofi, Framingham, MA USA

15:20 – 15:50 Considerations for Mass Spectrometry Based Protein Therapeutic

Control Method

Sarah Rogstad, CDER, FDA, Silver Spring, MD USA

15:50 – 16:20 Panel Discussion

16:20 – 16:30 Poster Award Announcement in the Georgian Room

16:30 – 16:45 Closing Comments in the Georgian Room

Michael Boyne, BioTechLogic, Inc., Glenview, IL USA

15

NOTES:

16

NOTES:

17

Oral Abstracts

Mass Spectrometry Imaging in Surgical Pathology and Drug Development

Nathalie Y.R. Agar

Department of Neurosurgery and Department of Radiology, Brigham and Women's Hospital,

Boston, MA USA, Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard

Medical School, Boston, MA USA

Mass spectrometry provides multiple options for the direct characterization of tissue to support

surgical decision-making, and provides significant insight in the development of drugs targeting

tumors of the central nervous system. Using an array of mass spectrometry platforms, we rapidly

analyze specific tumor markers such as metabolites, fatty acids, lipids, and proteins from surgical

tissue for surgical guidance and rapid diagnosis. Using similar clinical protocols, we visualize

drug and metabolites penetration in brain tumor tissue in pre-clinical animal models and clinical

trial specimens and correlate with tumor heterogeneity and response to support drug

development.

NOTES:

18

A Mass Spectrometry Approach for the Screening of Cell Culture Raw Materials and

Spent Media

Rachel Chen, Zoran Sosic, Li Zang

Biogen, Cambridge, MA USA

During the modern bio-manufacturing, it is extremely important to ensure the consistent product

quality of the biological therapeutics, through a systematic control of impacting factors,

including cell culture process. Chemically defined media (CDM) is widely used in

pharmaceutical industry for mammalian cell culture. It contains various categories of

components such as amino acids, vitamins and inorganic salts that can be subject to multiple

types of stress including heat, light, humidity, etc. Yet, currently there is a lack of analytical

methods that can monitor the quality of chemically defined media with sufficient sensitivity and

wide scope of detection of its different components. On the other hand, cell culture media taken

from a bioreactor (a.k.a. “spent media”) contains important information about the consumption

of nutrients and accumulation of metabolites during the cell culture process. To this end, we have

developed an LC-MS based platform and applied it for the routine monitoring of quality of

chemically defined media, as well as for the analysis of cell culture spent media. This method

employs hydrophilic interaction chromatography (HILIC) separation coupled with electrospray

ionization mass spectrometry (ESI-MS) detection using an LTQ-Orbitrap instrument. It has

demonstrated capability of separating and detecting over 100 media components and cell culture

metabolites. The HILIC-MS platform has been used to identify several impurities from

chemically defined media that causes cell growth or product quality issues during cell culture

development. Application of the HILIC-MS method to the optimization of cell culture media has

yielded valuable knowledge regarding the correlation between certain nutrients and metabolites

and product quality attributes of recombinant monoclonal antibodies. The HILIC-MS method,

along with other spectroscopic methods, constitutes an important analytical platform for

thorough characterization of cell culture raw materials and cell culture process.

NOTES:

19

ELISA Reagent Coverage Evaluation by Affinity Purification Tandem Mass Spectrometry

Scott Henry

Seattle Genetics Inc., Bothell, WA USA

Downstream processing must adequately remove host cell proteins (HCPs) from recombinant

therapeutics to ensure patient safety, product quality, and regulatory compliance. HCP levels are

typically monitored using a polyclonal ELISA reagent that has sufficiently broad

immunoreactivity (coverage) against the HCPs expressed by the production cell line. Gel-based

methods that rely on optical comparison are utilized to evaluate coverage, and do not provide

protein-specific definition of immunoreactivity. It is challenging to provide assurance that a

given reagent can monitor all HCPs that persist downstream since ELISA reagents to do not

provide 100% immunoreactivity. We describe protein-specific determination of ELISA reagent

coverage by proteomic analysis following affinity purification using a polyclonal anti-HCP

reagent (AP-MS). The resulting HCP identifications enable a highly focused assessment of

ELISA reagent suitability relative to known downstream HCP impurities. The utility of this

approach is illustrated by coverage evaluation against HCPs in a monoclonal antibody after

Protein A purification, and against the HCPs present in a null cell harvest sample. Going

forward, we envision using the AP-MS approach to help reconcile the results of ELISA and MS-

based studies of HCP clearance. The AP-MS method also further enables a framework for HCP

impurity analysis driven by protein-centric characterization of process impurities, in contrast to

classical approaches that focus on evaluation of the total host cell proteome.

NOTES:

20

A Platform Disulfide Mapping Method for Characterization of Therapeutic Proteins with

Various Formats

Bing Zhang, Yonghua Zhang

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

Disulfide bond linkage is one of the many characteristics of therapeutic proteins that are critical

to their biological functions and stability. Peptide mapping by liquid chromatography-mass

spectrometry (LC-MS) analysis of enzymatically digested therapeutic protein under non-

reducing condition is a powerful method for disulfide linkage characterization to ensure product

quality. However, the existing disulfide methods suffer from varied digestion efficiency and

artifacts of disulfide scrambling.

In this study, eight non-reducing peptide mapping methods in literature and in-house were

compiled, evaluated and harmonized to a single well-defined platform method. Each of the

sample preparation steps was assessed thoroughly using three representative testing molecules

(an IgG1, an IgG4 and a Fab). The expected disulfide linkages for the molecules tested were all

identified with high digestion efficiency and minimized disulfide scrambling (total <0.5%).

Further incubation time course study showed that the observed low levels of scrambled

disulfides were induced during the incubation. The final method was demonstrated to be robust

with acceptable intermediate precision, and is applicable to a broad spectrum of molecules with

various formats (IgG1, IgG2, IgG4, Fab, fusion protein, bispecific). Information on other

cysteine-related variants such as free thiols, trisulfides and thiolether can also be derived from

the very same peptide map, which facilitates the cysteine related variants characterization.

NOTES:

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Paving the Way for Assessing in vivo Dynamics of Multiple Quality Attributes for Protein

Therapeutics

Haihong Zhou1, Yi Wang1, Richard Rogers2, Douglas Richardson2, Bhumit Patel1, Daniela

Tomazela3, Richard Wong1, Dong Hun Lee1, Sejal Patel1, Maribel Beaumont3, Yan-Hui Liu2,

Ayesha Sitlani1, David Pollard1, Shuangping Shi1

1Merck Co. & Inc., Kenilworth, NJ USA, 2Just. Biotherapeutics, Seattle, WA USA, 3Merck Co. &

Inc., Palo Alto, CA USA

During the bioprocessing of recombinant therapeutic proteins and antibodies, product variants

can form during cellular expression, purification and storage. These chemical conversions may

also occur in vivo. Therefore, a thorough understanding of the metabolism and clearance of

protein therapeutics can aid in the assessment of efficacy and safety, and help define criticality of

the quality attributes, thus directing manufacturing process development. Here we present a

general strategy on how to identify and quantify changes in multiple quality attributes of protein

therapeutics in vivo. To demonstrate proof of concept, we developed an affinity purification

procedure to pull down a therapeutic IgG1 monoclonal antibody from cynomolgus monkey

serum. Pharmacokinetic profiles of multiple quality attributes were obtained using peptide

mapping with high resolution mass spectrometric detection. Attention was given to optimizing

the affinity purification and digestion steps to ensure reproducible recovery of the quality

attributes and minimizing sample processing induced artifacts. For the detection of unknown

metabolites, several commercially available New Peak Detection algorithms were employed.

Their ability to detect unknown changes in the molecule was evaluated. Our results demonstrate

that it is feasible to recover and quantify individual protein variants from biological fluids. This

approach will have great utility in understanding the potential impact of product quality

attributes on characteristics and biological activity of the protein therapeutics.

NOTES:

22

Characterizing the Immune Response to Novel Vaccine Candidates in a Human Whole

Blood Assay Using High-Dimensional Single Cell Proteomics

Andrew James1, Michael Cohen1, Derek Wilson2, Roger Brookes1

1Sanofi Pasteur, Toronto, ON, Canada, 2York University, Toronto, ON, Canada

Monitoring the immuno-modulatory effects of vaccine formulations is critical for novel vaccine

development. While animal models have been effective, increasing evidence suggests differences

when translating to the human immune response. Hence, the development of new methods to

study complex and heterogeneous immune cell populations in a human-based system is of

critical importance.

In this study, we will utilize fresh human whole blood as a model to screen vaccine formulations.

The immune response will be characterized by high-throughput single cell analysis using mass

cytometry (CyTof Helios). Specifically, this deep proteomic profiling will provide crucial

biological information regarding immunophenotyping and the expression of key cytokines and

signaling cascades at a single-cell resolution.

We have conducted a proof of concept analysis on human peripheral blood mononuclear cells to

optimize the mass cytometry protocol and devise a panel of target antibodies. Preliminary results

revealed optimal immunophenotpying of naive, effector, and memory T-cells, as well as other

immune cells including B cells, monocytes, and NK cells. Furthermore, we have established an

efficient method for the fresh human whole blood assay by stimulating samples with a range of

vaccine formulations and measuring the baseline immune response by ELISA for interferon

gamma production. We are currently optimizing the mass cytometry protocol for use with the

human whole blood assay. This unique approach will produce high-dimensional data on

adjuvant-modulated, antigen-specific immune responses in a clinically relevant human model.

NOTES:

23

Immunocapture-LC/MS Detection of Anti-Drug Antibodies

David Roos

Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT USA

Therapeutic proteins and peptides have potential to elicit immune responses resulting in anti-

drug antibodies (ADA) that can pose problems for both patient safety and product efficacy.

During drug development, immunogenicity is usually examined by risk-based approach along

with specific strategies for developing “fit-for-purpose” bioanalytical approaches. ELISA is the

most widely used platform for ADA analysis. In this talk, we present a complementary,

immunocaputre-LC/MS based approach that can be used for detection and isotyping of ADA.

Briefly, ADA and/or drug-ADA complex is captured by biotinylated drug or an anti-drug capture

Ab, immobilized on streptavidin magnetic beads, and separated from human plasma by a

magnet. ADA is then released from the beads and subjected to trypsin digestion followed by

LC/MS detection of specific universal peptides for each ADA isotype. Detailed method

development along with two case studies is presented.

NOTES:

24

Glycomics Characterization of Influenza Hemagglutinin Glycoprotein Antigens

John F. Cipollo

CBER, FDA, Silver Spring, MD USA

Influenza hemagglutinin (HA) recognizes host cell surface sialyl N-glycans to facilitate virus

invasion of the host cell. It is also the major antigen present in seasonal influenza vaccine. HA

glycosylation can impact the host immune response in several ways including: 1) alteration of its

receptor site specificity; 2) masking antigenic sites and; 3) impacting interactions with the lectin

based host innate immune system elements, thus, influencing the response. In the vaccine

context HA glycosylation can vary with the cell substrate used to generate virus or subunit

protein for vaccine production. Differences in glycosylation can impact vaccine efficacy and

safety. To investigate glycosylation dependent structure-function relationships, we have

developed a glycomics workflow, which includes: 1) Analysis of released and permethylated -

glycans; 2) Glycopeptide analysis by nano-LC/MSE; 3) percent site occupancy determination; 4)

molecular modeling of glycosylation at the HA surface to investigate interactions with antigenic

sites and with host lectin based immune factors. We have also developed in-house

glycoinformatics tools to aid in our analyses. This work-flow will be discussed as well as our

major findings regarding structure function relationship involving HA glycosylation in H3N2,

H1N1 and H1N7 derived HA glycoproteins. Specific examples will describe how: increasing the

number of N-glycosylation sites impact interaction with lung surfactant SP-D; changes in glycan

subclass effect interactions with DC-SIGN; differences in HA peptide structure may alter N-

glycan subclass and therefore interaction with host immune response.

NOTES:

25

Quantification of Membrane Proteins in Genetically Engineered Crops by Liquid

Chromatography Coupled with Tandem Mass Spectrometry

Lindsey Schacherer

DuPont Pioneer, Johnston, IA USA

The detection and quantification of target proteins in genetically modified crops has been

accomplished primarily by immunoassays such as enzyme-linked immunosorbent assays

(ELISA). However, ELISAs require high quality antibodies that may be difficult and time-

consuming to produce and ELISAs may not work for membrane proteins. Liquid

chromatography coupled with tandem mass spectrometry (LC-MS/MS) has been used for many

years to quantify pharmaceuticals and pesticides, among other small molecules. In recent years,

LC-MS/MS has also been applied to quantify proteins in transgenic crops in multiplexed and

high throughput assays. We developed and validated LC-MS/MS assays to quantify two

membrane proteins, zmARGOS8 and gmDGAT1, expressed in maize leaf and soybean seed,

respectively. gmDGAT1 has nine transmembrane domains making solubilization and the

production of antibodies challenging. With LC-MS/MS, target proteins are quantified via peptide

surrogates and therefore denaturing conditions may be used to extract the proteins. An extraction

buffer containing CHAPS and Triton X-100 was determined to be suitable for both zmARGOS8

and gmDGAT1. Both methods exhibited high recovery and good reproducibility. Recombinant

proteins that had been expressed and purified from baculovirus or bush bean for zmARGOS8

and gmDGAT1, respectively, were used to prepare standard curves with good linearity and

accuracy. This study demonstrates the suitability of LC-MS/MS for quantification of membrane

proteins in transgenic crops.

NOTES:

26

Assessment of Monoclonal Antibody-Fc Receptor Interactions by Online SEC-Native Mass

Spectrometry

Hongxia (Jessica) Wang, Haibo Qiu, Ning Li, Thomas Daly

Regeneron Pharmaceuticals, Tarrytown, NY USA

In therapeutic proteins, post-translational modifications (PTMs) may affect drug stability, safety

and efficacy. For example, if PTMs are present in the Fc region, these PTMs may affect mAb

interactions with Fc receptors, which could alter antibody-dependent cell-mediated cytotoxicity,

complement dependent cytotoxicity and neonatal Fc receptor (FcRn)-mediated recycling

processes. FcRn plays an important role in regulating the serum half-lives of IgG antibodies.

Therefore, PTMs impacting the binding affinity of an antibody to FcRn could shorten the serum

half-life of the therapeutic antibody. Traditionally, PTMs are identified at the peptide level

through enzymatic digestion followed by peptide mapping. However, the PTMs impacted

specific protein-protein interactions are usually lost as a result of the denaturation. Recent

advances in native mass spectrometry (MS) have enabled the analysis of intact proteins and

protein-protein complexes at their near native states. In this study, we have developed an online

size exclusion chromatography (SEC)-native MS approach to investigate common PTMs in

mAbs and their effect on mAb-FcRn interaction using an Orbitrap mass spectrometer. A non-

covalent complex of an IgG1 antibody and human FcRn was selected as the model system. Our

preliminary result indicated that high levels of oxidation at HC Met258 and Met434 significantly

altered the binding of IgG1 to FcRn. The effect of other modifications such as glycation and

deamidation at the mAb-FcRn binding interface will also be investigated. Moreover, the

potential conformational alterations surrounding the binding site can be elucidated by Hydrogen

Deuterium Exchange MS. The binding affinity of the modified antibody to FcRn will be further

assessed by Surface Plasmon Resonance-Biacore assay. The use of native MS and associated

methods may therefore be a useful approach to predict the impact of specific PTMs within

specific domains of an antibody on FcRn binding and pharmacokinetics.

NOTES:

27

Native Size Exclusion Chromatography Coupled with Multi-Angle and Dynamic Light

Scattering and Mass Spectrometry for Heightened Characterization of High Molecular

Mass Species in Monoclonal Antibodies

Ying Zhang, Cliff Entrican, Yin Luo, Jason Rouse, Matt Thompson

Pfizer, Inc., Andover, MA USA

Recombinant protein therapeutics may contain small amounts of high molecular mass species

(HMMS) or aggregates, which are closely monitored due to potential product safety and quality

concerns. Size exclusion high-performance liquid chromatography (SE-HPLC/UV) coupled

with in-line multi-angle light-scattering (MALS) is the standard method for determining and

monitoring HMMS in most protein therapeutics. MALS provides a first principle determination

of the weight-averaged molar mass for a wide size range of HMMS under native conditions.

Characterization of the molecular attributes of HMMS at low abundance in a native, aqueous

environment can be challenging, even with mass spectrometry (MS) approaches. A recent

publication (Haberger et al. mAbs 2016 8:331) demonstrated in-depth characterization of HMMS

using SE-HPLC/UV and native MS. However, because the SE-HPLC mobile phase contains a

volatile salt, high-mass sensitivity is limited and very low levels of HMMS may not be detected.

Consequently, we improved the sensitivity of this approach by introducing an acidic, organic

solvent prior to sample ionization. We also applied in-line dynamic light-scattering (DLS) to

determine the hydrodynamic radius of the HMMS. Therefore, the combination of information

from all three in-line detectors affords a highly detailed, comprehensive understanding of low

abundance HMMS in protein therapeutics. In particular, we obtained size information using

MALS, shape information using DLS, and the specific molecular attributes of the dissociable

and non-dissociable species using MS.

NOTES:

28

Glycomic and Glycoproteomic Analysis of Biopharmaceuticals in a High Throughput and

Highly Sensitive Manner

Guinevere Kammeijer, David Falck, Noortje de Haan, Agnes Hipgrave Ederveen, Manfred

Wuhrer

Leiden University Medical Center, Leiden, Netherlands

The characterization of therapeutic proteins and their glycosylation is important for

biopharmaceutical industry as glycosylation influences the efficacy of the therapeutics as well

their safety. Therefore, there is a need to define the glycosylation profile of therapeutic proteins

in a fast, but accurate and precise manner. The characterization of the glycosylation can be

achieved either on intact, glycopeptide or on released glycan level and is dependent on the

criteria set by the industry and regulatory offices.

This study explores the consequence of using different analytical platforms (LC-ESI-MS, CE-

ESI-MS and MALDI-TOF-MS) for identifying the glycosylation profile of two NIST

monoclonal antibodies (mAbs) standards at the glycopeptide level. A total of 40 different glycan

species could be identified on the standards. G0F, G1F and G2F were the three most abundant

glycans for all used platforms. Notably, the N-acetylneuraminic acid appeared to be absent

while N-glycolylneuraminic acid was present. Furthermore, terminal alpha-1,3-linked galactose

was present, which could be expected as the mAbs were derived from murine cell lines. Overall,

all platforms revealed similar results whereas CE-ESI-MS appeared to be the most sensitive

platform, enabling the identification of 40 structures while LC-ESI-MS and MALDI-TOF-MS

could both identify up to 32 glycopeptides.

Furthermore, we set-up a complete workflow for the analysis of glycans on erythropoietin

(EPO), executable in 24 hours. It includes protein purification from cell culture supernatant,

glycan release, stabilization of the sialic acids, clean-up of the samples and the analysis of the

samples using MALDI-TOF-MS. The high-throughput platform for the analysis of glycans from

EPO revealed high recovery and an unbiased representation of the glycosylation profiles, that

were comparable to profiles obtained by well-established platforms such as CGE-LIF and

HILIC-FLD.

NOTES:

29

A Novel Method for On-line Native MS Analysis of Monoclonal Antibody Charge Variants

Applying Volatile Buffers in SCX Chromatography

Florian Füssl

NIBRT, Co. Dublin, Ireland

Monoclonal antibodies are the fastest growing class of pharmaceuticals due to their high target

specificity and excellent safety profiles. Numerous novel monoclonal antibodies are currently

under development and patents of already established mAbs are continuously expiring, therefore

opening the market for the release of biosimilar versions. A strong demand therefore exists for

effective methods for the thorough characterization of mAbs. Charge variant analysis is routinely

used for mAb characterization as it is a comparatively easy and effective method for the

separation of protein variants differing in their surface charge. One drawback however, is the

necessity of the presence of salt in the eluents when using either salt-based or pH-based elution

modes, which makes hyphenation of CEX to mass spectrometry (MS) problematic. In order to

overcome this we developed a SCX-based separation system applying a pH gradient using MS

friendly volatile elution buffers. By the application of individual gradients, we were able to

chromatographically resolve charge variants of several commercially available monoclonal

antibodies within short chromatographic run time. We furthermore tested MS compatibility by

on-line hyphenation to a Thermo ScientificTM Q-ExactiveTM Plus high-resolution mass

spectrometer with BioPharma option and were able to obtain native MS spectra for all

monoclonal antibodies analysed. Deconvolution and data analysis yielded in the identification of

lysine cleavage and deamidation to be among the main modifications chromatographically

resolved. In addition, major glycoforms were identified for most charge variants with high mass

accuracy. To the best of our knowledge, this work represents the first method employing native

MS analysis on-line after pH gradient based charge variant separation of intact monoclonal

antibodies. We also demonstrated that this method is applicable to a majority of therapeutic

mAb’s available which can make it a universal highly powerful analytical tool in antibody

characterization in the future.

NOTES:

30

Charge Detection Mass Spectrometry: Weighing Heavier Things

Martin Jarrold

Indiana University, Bloomington, IN USA

Charge Detection Mass Spectrometry (CDMS) is a single molecule method where the mass to

charge ratio (m/z) and charge (z) are measured for individual ions. Multiplying m/z and z gives

the mass for each ion; the masses are then binned into a histogram to give a true mass spectrum.

CDMS allows accurate mass measurements to be performed for high mass and heterogeneous

samples, samples which often confound convention mass spectrometry methods where only the

m/z ratio is measured.

I will describe how CDMS measurements are performed, and the recent technical developments

that have brought CDMS into the mainstream. Examples of applications of CDMS to the

analysis of protein complexes, protein aggregates, virus composition, and lipoproteins will be

provided.

NOTES:

31

A Mass Spectrometry based “ELISA” Assay for Adenovirus

Jonathan Knibbe, Annemiek Verwilligen, Arjen Scholten, Marta Germano

Janssen Infectious Diseases and Vaccines, Leiden, Netherlands

At Janssen, we are developing a tetravalent HIV vaccine based on the AdVac® platform. This

vaccine contains four different live, harmless, adenoviruses. Each is modified to encode for a

mosaic protein. These so-called transgenes consist of a variety of HIV antigens to protect against

a multitude of HIV subtypes and clades. Upon vaccination, adenoviruses infect cells, which then

express the mosaic antigens to elicit a broad, protective immune response against HIV. In

vitro potency testing is therefore expected to include a quantitative determination of transgene

protein expression after infection of an appropriate cell substrate. This is traditionally a cell

based assay with an ELISA read-out. In the case of our candidate HIV vaccine, four selective

antibodies are needed for quantification of each of the mosaic proteins. As these proteins have

sequences with over 90% homology, generation of such antibodies is challenging.

We developed a mass spectrometry method which allows for quantification of the expression of

the four mosaic antigens in a single analysis. The advantage of mass spectrometry is that

detection is based on monitoring the mass, retention time and fragmentation behavior of unique

transgene specific peptides (proteotypic peptides). While selective detection by antibody binding

may be impossible in the case of highly homologous proteins, for MS detection a difference of

one amino acid is enough. Quantification of the proteotypic peptides, which each represent one

of the four mosaic proteins, allowed us to determine their expression levels simultaneously. This

MS-based “ELISA” assay can be performed directly in cell lysate, without prior enrichment or

use of immune reagents. Method qualification is currently ongoing to amend the method for

quality control testing. The “MS-ELISA” is a promising new assay platform, which can

potentially be widely applied in the field of biologics and vaccines manufacturing and testing;

even in a GMP environment.

NOTES:

32

Pushing the Boundaries of Viral Vector Capsid Characterization: Development and

Application of LC-MS Methods for Comprehensive Understanding of Adeno-Associated

Virus and Adenovirus Capsids

Thomas Powers1, Elise Mullins1, Jason Rouse2, Olga Friese1, Herb Runnels1, Larry Thompson1

1Pfizer, Inc., Chesterfield, MO USA, 2Pfizer, Inc., Andover, MA USA

The advent of novel biotherapeutic modalities, including those that rely on viral vectors,

necessitates the development of new analytical strategies and methods for heightened product

characterization. Methods for two such vehicles, adeno-associated virus (AAV) and adenovirus,

have been developed to elucidate capsid protein composition and structural complexity. AAV

consists of three capsid proteins with related sequences that assemble to form a 5 MDa

icosahedral capsid, while the adenovirus capsid is composed of 11 primary proteins that form a

150 MDa capsid. Mass analysis of denatured, intact AAV capsid proteins confirmed one primary

structure with three N-terminal forms, and revealed post-translational modifications. Proteolytic

mapping was then used to verify these observations and enable site-specific assignment of

modifications. Application of these two characterization methods has resulted in the

identification of modifications unreported in literature, facilitated a more thorough understanding

of AAV drug products, and supported purification process changes to improve the quality and

consistency of AAV material. A comparison of multiple serotypes has strengthened our

understanding of AAV structure in general, as well as the quality attribute differences across

serotypes.

Methods were further optimized to account for the increased structural complexity of adenovirus

capsids. In addition to confirming capsid protein primary structure and modifications, mass

analysis of denatured, intact adenovirus proteins revealed sites of proteolytic truncations, the

presence of empty capsids in purified material, and was useful in characterizing an impurity in

accelerated stability samples. In addition to the impurity, an acidic shift was also observed in

accelerated stability samples, necessitating the optimization of the proteolytic mapping method

to minimize method-induced artifacts while resulting in complete digestion and sequence

coverage. When applied to stability samples, the proteolytic digestion method identified a single

site of deamidation where the rate of deamidation mirrored the rate of the acidic shift in the

anion exchange profile.

NOTES:

33

Human and Baculovirus-Insect Manufacturing Platforms Generate Chemically and

Functionally Distinct AAV Vectors

Nicole Paulk

Stanford University, Stanford, CA USA

Despite encouraging outcomes in pre-clinical and early adeno-associated virus (AAV) clinical

trials, vector efficacies in several recent gene therapy trials were discordant. Mechanisms

underlying this remain unknown. Transitions to new manufacturing platforms using baculovirus-

infected insect cells appeared to correlate with low-efficacy trials. We hypothesized that the two-

leading recombinant AAV manufacturing platforms, baculovirus-infected insect and transiently-

transfected human cell systems, result in different capsid post-translational modifications

(PTMs), affecting function.

We utilized multiple analytical approaches including deep proteomic profiling with high-

resolution and high-mass-accuracy mass spectrometry (nLC-ESI-MS/MS), two-dimensional

isoelectric focusing in combination with enzymatic modification removal, transmission electron

microscopy, structural modeling, and comparative functional transduction assessments in mice.

Our data indicate that rAAV capsids are differentially post-translationally modified in human

and insect cell preparations. Modifications included O-linked glycosylation, acetylation,

phosphorylation, and methylation. PTMs were seen on every serotype of AAV tested to

date including those serotypes (AAVs 1 and 5) which had poor-performing recent human trials.

In addition, capsids purified from media supernatant had different PTMs than those from cell

lysates. When compared head-to-head for functional transduction in vivo, we noted significant

differences in expression from insect and human-produced vectors in 72 mice treated

intravenously or intramuscularly. Significant sexually dimorphic functional transduction

differences were also seen in gender-controlled experiments where male mice experienced

greater expression than females.

Our results demonstrate that AAV capsids are differentially post-translationally modified when

produced by human and insect manufacturing platforms. These findings were reproducible

across numerous rAAV vendors including commercial producers, leading academic core

facilities, and individual lab preparations. Collectively, these PTMs may have profound

implications for capsid folding, viral replication, receptor binding, intracellular trafficking,

expression kinetics, functional activity, stability, half-life regulation, immunogenicity, and more.

Our findings may inform future directions for resource investments in GMP manufacturing

facilities currently being assembled.

NOTES:

34

Monitoring Biologics CQAs for quality control using MAM

Michael Olma1, Yi Wang2, Douglas Richardson2, Bhumit Patel2, Richard Rogers3, Yu Zou1,

Hans-Martin Müller1

1Werthenstein BioPharma GmbH, Schachen, Switzerland, 2Merck & Co. Inc, Kenilworth, NJ

USA, 3Just. Biotherapeutics, Seattle, WA USA

The use of monoclonal antibodies as therapeutic drugs requires a detailed understanding of the

proteins from discovery through delivery to clinics. In-depth characterization and monitoring of

critical quality attributes (CQAs) of these biologics, especially during manufacturing and release,

is essential to meet the strict regulatory requirements for patient safety. Currently, a panel of

different analytical approaches is applied in a quality control (QC) lab during drug release and

stability testing. In this study, the Multi-Attribute Method (MAM) using reduced peptide

mapping liquid chromatography mass spectrometry (LC/MS) was evaluated to monitor a large

number of molecular CQAs and process residuals in parallel. Our results suggest a path to use

MAM in a QC environment to potentially streamline and accelerate QC analysis for the release

and stability of biotherapeutics.

NOTES:

35

Analysis of Biopharmaceutical Attributes in a GMP Setting

Yan Jiang

Sanofi, Framingham, MA USA

Economic pressures continue to drive biopharmaceuticals to faster development and more

streamlined analysis. We have developed a platform method that allows direct monitoring of

multiple quality attributes of therapeutic antibodies in a single assay. The process includes a

platform method which automates the purification of inline samples (protein A purification) and

digestion. The analysis of protein digests on a validated (GMP) LC/UV/MS system identifies the

protein primary sequence by LC/UV peptide mapping and quantitatively measures various

targeted post-translational modifications and process-induced impurities by LC/MS(SIR).

Quality attributes including peptide map, N-glycosylation, oxidation, deamidation,

isomerization, truncation, and N- and C-terminal modifications can be monitored simultaneously

in a 40-min injection. The assay queue includes system suitability and an independent assay

control. This method has been applied to multiple monoclonal antibody products with good

selectivity and sensitivity and we have successfully used it for clone selection to help with cell

line development. A cost-effective and high-throughput platform approach like this can

potentially improve analytics efficiency and enhance the ability to make timely decisions for

process development and product disposition.

NOTES:

36

Considerations for Mass Spectrometry Based Protein Therapeutic Control Methodology

Sarah Rogstad

CDER, FDA, Silver Spring, MD USA

Mass spectrometry (MS) is an essential tool for the characterization of protein therapeutics.

Recent advances in high resolution accurate mass (HRAM) instrumentation have allowed for the

elucidation of many closely related species within these heterogeneous products. Modern

instruments also allow for the detection of degradation products, modifications and/or impurities

based on mass deviations. Currently, MS is primarily used for the characterization of these

products, but there has been a recent push in the field toward the use of MS-based methods for

quality control (QC) purposes.

Traditionally, several barriers have stood in the way of MS being used in the QC lab. MS has

been thought to be too complex for non-specialists to make quick pass/fail decisions.

Furthermore, MS instrumentation is expensive and is generally not thought to be sufficiently

high-throughput for the QC environment. Recent advances in HRAM instrumentation have

caused some to reconsider MS for QC purposes. The capabilities of HRAM instruments to easily

distinguish between highly related substances can allow for improved identification and

quantification of impurities. Such knowledge is valuable for establishing process controls that

could minimize impurities and improve the assurance of safety. Several issues should be

considered when assessing the possibility of MS-based QC assays including system suitability,

method validation and comparisons with traditional methods.\

Another major concern is whether or not the method is fit for purpose. For example, MS-based

methods are information rich, which is ideal for characterization purposes; however, when

converted into a control environment, such broad data coverage may not be necessary. Relatedly,

these broader datasets, which may cover a wider variety of critical quality attributes when

compared to traditional methods may allow for method consolidation. Deciding which data to

use is a complicated issue, which will be product specific and should be fully understood and

well characterized.

NOTES:

37

Roundtable Discussion Topics

Session I – Wednesday, September 20, 2017 – 16:15-17:15

Session II – Thursday, September 21, 2017 – 16:20-17:20

There are 15 roundtable topics. The Roundtable sessions will be a truly interactive workshop to

connect and discuss real issues with your peers. These sessions were designed to be informal (but

structured) discussions on topics which are of interest to participants, but were not able to be

incorporated into the other sessions within the program. To create useful discussion, we are

going to try and limit each topic to 10 attendees. Seating will be on a first come, first serve

basis. These discussions will include a facilitator, whose role is to help assist the discussion and

ensure a lively exchange, and a scribe, whose role is to make general, anonymous notes about the

discussion that will be posted on the Mass Spec 2017 website and mobile app. The Roundtable

Sessions are scheduled for Wednesday, September 20, 16:15-17:15 (Session I) and Thursday,

September 21, 16:20-17:20 (Session II). Listed below is a quick view of the Roundtable topics,

facilitators, and scribes. Please view abstracts in the mobile app or on the Mass Spec meeting

webpage (www.casss.org/page/MS1715).

Table 1 Host Cell Proteins: Sample Preparation, Methods, Instruments, Software,

Validation

SESSION I FACILITATOR: Ying Zhang, Pfizer, Inc.

SESSION I SCRIBE: Jacky Smith, Pfizer, Inc.

SESSION II FACILITATOR: Stephanie Wohlrab, Roche Diagnostics GmbH

SESSION II SCRIBE: Jacky Smith, Pfizer, Inc.

Table 2 Instrumentation: What is Required Re. Resolution, Mass Accuracy,

Sensitivity, System Suitability for Methods [MS, LC/MS, LC/MSn]

SESSION I FACILITATOR: Jonathan Josephs, Thermo Fisher Scientific

SESSION I SCRIBE: David Ripley, Pfizer, Inc.

SESSION II FACILITATOR: Greg Staples, Agilent Technologies

SESSION II SCRIBE: Jonathan Knibbe, Janssen Infectious Diseases and

Vaccines

Table 3 Product Characterization Strategies: Biosimilars, Viral Capsids, Gene

Therapy, mAbs, ADCs

SESSION I FACILITATOR: Ramsey Saleem, Amgen Inc.

SESSION I SCRIBE: Thomas Powers, Pfizer, Inc.

SESSION II FACILITATOR: Promod Mehndiratta, Janssen R&D LLC

SESSION II SCRIBE: Thomas Powers, Pfizer, Inc.

38

Table 4 Multi Attribute Methods: Early Development, Hot Spot Analysis, CQA

Assessment, Process Development, Release, Stability

SESSION I FACILITATOR: Kristin Boggio, Pfizer, Inc.

SESSION I SCRIBE: Promod Mehndiratta, Janssen R&D LLC

SESSION II FACILITATOR: Yan Jiang, Sanofi

SESSION II SCRIBE: David Ripley, Pfizer, Inc.

Table 5 Multi Attribute Methods: Low Versus High Resolution MS, Relative

Quantitation, Choice of Output, Explanations for Small Differences

SESSION I FACILITATOR: Haihong Zhou, Merck & Co., Inc.

SESSION I SCRIBE: Yelena Lyubarskaya, Sanofi

SESSION II FACILITATOR: Kristin Boggio, Pfizer, Inc.

SESSION II SCRIBE: Michael Boyne, BioTechLogic, Inc.

Table 6 Biotransformation and Bioanalysis: Analytical Strategies, Experiences with

Protein Therapeutics, Correlation with LBA Formats

FACILITATOR I & II: Steve Cohen, SAC Analytical Consultants

SCRIBE I & II: Jason Rouse, Pfizer, Inc.

Table 7 Glycosylation/Released Glycan Analysis: New Enzymes, Labels, LC/MS

Methods and Software

SESSION I FACILITATOR: Yihong Zhang, Bristol-Meyers Squibb Company

SESSION I SCRIBE: Eef Dirksen, Synthon Biopharmaceuticals B.V.

SESSION II FACILITATOR: Yihong Zhang, Bristol-Meyers Squibb Company

SESSION II SCRIBE: Yelena Lyubarkaya, Sanofi

Table 8 Applications of Native MS: Can it be Used for Relative Quantitation?

SESSION I FACILITATOR: Aaron Bailey, Thermo Fisher Scientific

SESSION I SCRIBE: David Passmore, Tenaya Analytical Consulting

SESSION II FACILITATOR: Hao Zhang, Amgen Inc.

SESSION II SCRIBE: David Passmore, Tenaya Analytical Consulting

39

Table 9 Regulatory Submissions: Effective Ways to Include MS Methods/Results in

INDs

SESSION I FACILITATOR: Scott Henry, Seattle Genetics, Inc.

SESSION I SCRIBE: Heather DeGruttola, Pfizer, Inc.

SESSION II FACILITATOR: Scott Henry, Seattle Genetics, Inc.

SESSION II SCRIBE: Christopher Yu, Genentech, a Member of the Roche Group

Table 10 Top-Down Sequencing: Is it Coming to Biotech Soon?

FACILITATOR I & II: Guillaume Tremintin, Bruker Daltonics, Inc.

SCRIBE I & II: Arjen Scholten, Janssen Infectious Diseases and Vaccines

Table 11 MS Deconvolution Software: Latest Software and Developments

SESSION I FACILITATOR: Rose Lawler, Protein Metrics, Inc.

SESSION I SCRIBE: Anders Lund, Sanofi

SESSION II FACILITATOR: Joe Shambaugh, Genedata Inc.

SESSION II SCRIBE: Anders Lund, Sanofi

Table 12 Sequence Variant Analysis: New Developments, Strategies and Methods

SESSION I FACILITATOR: Sreekanth Suravajjala, Amgen Inc.

SESSION I SCRIBE: Jay Jones, Seattle Genetics, Inc.

SESSION II FACILITATOR: Eric Carlson, Protein Metrics, Inc.

SESSION II SCRIBE: Heather DeGruttola, Pfizer, Inc.

Table 13 de novo Sequencing: Applications in a Discovery Setting

FACILITATOR I & II: St John Skilton, Protein Metrics, Inc.

SCRIBE I & II: Richard Rogers, Just. Biotherapeutics

Table 14 Peptide Mapping for Sequence Determination Versus Confirmation: Should

This be in an IND? Should This be Combined with Protein MS/MS?

SESSION I FACILITATOR: Bing Zhang, Genentech, a Member of the Roche

Group

SESSION I SCRIBE: Shibu Philip, Pfizer, Inc.

SESSION II FACILITATOR: Jessica Wang, Regeneron Pharmaceuticals

SESSION II SCRIBE: Shibu Philip, Pfizer, Inc.

40

Table 15 Higher Order Structure Analysis by MS: is this a Rare or Routine

Biopharma Application?

SESSION I FACILITATOR: Sean McCarthy, SCIEX

SESSION I SCRIBE: John Valliere-Douglass, Seattle Genetics, Inc.

SESSION II FACILITATOR: Sarah Rogstad, CDER, FDA

SESSION II SCRIBE: John Valliere-Douglass, Seattle Genetics, Inc.

41

Technical Seminars

Genedata. Inc. Technical Seminar: Lunch and Learn

Thursday, September 21

12:15 – 12:45

Georgian Ballroom

An Adapted Top-down LC-MS Approach for Screening Lipid-modified Proteins

Michael York1,2, Andrew James1,2, Manon Fradin1, Shaolong Zhu1,2, Bruce Carpick1

1Sanofi Pasteur, Toronto ON Canada, 2York University, Toronto, ON Canada

In vaccine development, liquid chromatography-mass spectrometry (LC-MS) is typically applied

to characterization and identity workflows, particularly for recombinant protein candidates.

Intact mass analysis by MS is particularly useful for establishing product identity, for both

therapeutic proteins and recombinant vaccine antigens. However, when the protein of interest

undergoes post-translational modification (PTM) during expression, intact mass analysis of the

heterogeneous product is more complex, and confirming identity becomes difficult. Often,

alternate MS workflows, such as a peptide mapping, are required to establish identity.

This work focuses on the development of an intact mass LC-MS method for the analysis of lipid

modified proteins. The method was originally evaluated for detection of the different protein

‘lipoforms’. By applying in-source fragmentation within the workflow, we were able to

determine protein sequence identity and further characterize the lipid profiles of individual

lipoprotein populations.

Lipid modifications are made on the N-terminal Cysteine of selected proteins during bacterial

expression. This creates a heterogeneously modified Cys residue that is more susceptible to in-

source fragmentation during MS experiments. The rest of protein remains intact, and can be

identified in a sequence specific manner using mass deconvolution. Meanwhile, the lipidated

Cys fragment can be detected and further analyzed to characterize and confirm specific PTM

structures. As this method evolves, we will apply principals of multiple attribute monitoring to

design an assay with readouts for protein identity, detection of protein lipoforms, lipid

identification, and protein purity.

NOTES:

42

Bruker Daltonics, Inc. Technical Seminar: Lunch and Learn

Thursday, September 21

12:50-13:20

Georgian Ballroom

Applications of Bruker Technology in Support of Biotherapeutics Characterization and

Development

Kathleen Cornelius1, Paul W. Brown1, Jason C. Rouse2, Olga V. Friese1

1Pfizer, Inc., St. Louis, MO USA, 2Pfizer, Inc. Andover, MA USA

Recent improvements in mass spectrometry (MS) instrumentation have assisted the

Biotherapeutics MS group at Pfizer in performing in-depth characterization and comparability

studies with more confidence and speed, as well as mitigating current challenges presented by

new modalities. The increased resolution of the Bruker Daltonics MaXis II electrospray

ionization quadrupole time-of-flight instrument has allowed for baseline resolution of trisulfides

allowing for more accurate quantitation at the intact protein level. Similarly, the increased

sensitivity and dynamic range of the MaXis II has allowed for detection of low-level amino acid

misincorporations leading to more accurate quantitation at both the subunit and intact protein

levels. For LC/MS – subunit analysis, the MaXis II provides an accurate monoisotopic mass for

the 50 kDa heavy chain, which facilitates rapid identification of low level isoforms and/or

misincorporations in mAbs (without IdeS digestion). Lastly, upgrades to the Bruker

ultrafleXtreme MALDI-ToF/ToF instrument has allowed for the routine mass analysis of large

heterogeneous vaccine molecules >150 kDa. Additionally, the enhanced sensitivity, resolution

and mass accuracy of the ultrafleXtreme have allowed us to perform time course studies to

monitor the reaction kinetics for protein conjugation. Bruker Daltonics instrumentation has been

successfully applied to biotherapeutics characterization, and not just for standard antibody

projects but for the more difficult-to-analyze cases as well.

NOTES:

43

SCIEX Technical Seminar: Lunch and Learn

Friday, September 22

11:45-12:15

Georgian Ballroom

Innovations for Biopharmaceutical Characterization: Speed Up the Routine and Simplify

the Challenging

Sean McCarthy

SCIEX, Framingham, MA USA

Biotherapeutics are complex molecules which require in-depth characterization to accurately

identify product critical quality attributes to ensure patient safety and efficacy. SCIEX

innovation can help you speed up routine tasks and simplify your most complex characterization

challenges. Now you can achieve insights faster and with greater confidence than you ever

thought possible. In this seminar, we will discuss:

▪ A streamlined solution for collecting and processing data for routine intact mass and

peptide mapping workflows which brings the power of mass spectrometry for routine

biopharmaceutical characterization to every user, regardless of their level of MS

expertise.

▪ An advanced, high-resolution strategy for resolving challenging critical quality attributes

such as deamidation, glycosylation, and drug-linker modifications at the intact, subunit,

and peptide levels by CESI-MS.

NOTES:

44

Agilent Technologies Technical Seminar: Lunch and Learn

Friday, September 22

12:20-12:50

Georgian Ballroom

Advances in the Characterization of Protein Therapeutics by LC/MS

Steve Madden

Agilent Technologies, Santa Clara, CA USA

Agilent has recently introduced several new and enhanced workflows that reduce the time and

complexity of characterizing protein therapeutics such as monoclonal antibodies (mAbs).

Sample preparation using the innovative AssayMAP Bravo automated protein platform allows

unique capabilities like on-cartridge deglycosylation. Precise separation and detection with

InfinityLab HPLC and the AdvanceBio LC/Q-TOF ensure you see more with every injection.

MassHunter BioConfirm makes quick work of processing large amounts of data.

Intact protein analysis provides a deconvoluted mass spectrum with the glycoforms peaks clearly

labeled. A table of relative quantitative values can easily be turned into a report or exported to a

spreadsheet program. The results from multiple data files can be quickly compared either in a

table or mirror plot spectrum to understand lot-to-lot variation or evaluate biosimilars.

For peptide mapping of protein digests, users can perform sequence confirmation with

confidence. A new peptide mapping algorithm uses MS/MS data to narrow down the list of

possible peptides—saving time vs. manual inspection. Results are displayed in the new

Sequence Coverage Map that allows the user to review multiple samples (e.g. different digest

enzymes) at the same time. Fast disulfide bond mapping can be used to untangle the scrambling

of disulfide bonds in a monoclonal antibody.

Finally, Agilent has added a workflow for released glycan profiling. Glycans are an especially

challenging PTM to characterize given the diversity in their composition and the need to

chemically tag them to get adequate sensitivity. The new Released Glycans workflow allows for

easy setup of the analysis and accommodates many commercial and custom tags. An extensively

curated database of glycans is provided which can be easily extended for custom glycans using

MassHunter tools. Identification is done using Agilent’s proprietary Find-by-Formula algorithm,

then results can be printed in the desired report format.

NOTES:

45

List of Posters

Process and Product Characterization

P-100-T

Native Ion Exchange Chromatography Coupled to Orbitrap Mass Spectrometry

Dramatically Improves Dynamic Range of Intact Mass Analysis

Aaron Bailey1, Guanghui Han2, Jennifer Sutton1, Paul Gazis1, Wendy Sandoval2, Jonathan

Josephs1 1Thermo Fisher Scientific, San Jose, CA USA, 2Genentech, a Member of the Roche Group, South

San Francisco, CA USA

P-101-T

Assessment of the Multi-Attribute Method Across Multiple Analysts, Instruments,

Laboratories, and Software Platforms

Kristin Boggio1, Carly Daniels2, Andrew Dawdy2, Anastasiya Manuilov1, Wenqin Ni1, Himakshi

Patel1, Thomas Powers2, David Ripley1, Joshua Woods2, Ying Zhang1, Matt Thompson1, Olga

Friese2, Keith Johnson1, Justin Sperry2, Jason Rouse1 1Pfizer, Inc., Andover, MA USA, 2Pfizer, Inc., Chesterfield, MO USA

P-102-T

Charge Heterogeneity Analysis of Intact Monoclonal Antibodies using CESI-MS

Esme Candish1, Olga Friese2, Elaine Stephens3, Marshall Bern4, St John Skilton5, Jason Rouse3,

Bryan Fonslow6 1SCIEX, Brea, CA USA, 2Pfizer, Inc., Chesterfield, MO USA, 3Pfizer, Inc., Andover, MA

USA, 4Protein Metrics Inc., San Jose, CA USA, 5Protein Metrics Inc., San Carlos, CA

USA, 6SCIEX, San Diego, CA USA

P-103-T

Structural Characterization of Protein Therapeutics by Mass Spectrometry

Robert Carney, Zsuzsa Lakos, Elena Dremina, John Snyder

Eurofins Lancaster Laboratories, Inc., Lancaster, PA USA

P-104-T

Evaluation of Fused-Core Particle Pore Size on Subunit Analysis of Antibodies and

Antibody Drug Conjugates by LC-MS

Ken Chanthamontri, Benjamin Cutak, Kevin Ray

MilliporeSigma, St. Louis, MO USA

46

P-105-T

Mass Spectrometric Identification and Quantitation of the Misincorporation of a Non-

Natural Amino Acid as a Result of Fermentation Conditions

Sharon Chen, Michelle Emrick, Mitchell Tai, Robert Mallett, Jeff Meyer, Karen De Jongh

Bristol-Myers Squibb Company, Seattle, WA USA

P-106-T

Determination of Amino Acid Concentration in Cell Culture Media using a Single

Quadrupole MS

Scott Corley, Christopher Warner

Boehringer Ingelheim Pharmaceuticals, Fremont, CA USA

P-107-T

Monitoring Multiple Critical Quality Attributes of mAbs at Subunit and Peptide Level

Using a Compliant Ready High-Resolution LCMS PlatformJing Fang, Nilini Ranbaduge,

Henry Shion, Min Du, Ying Qing Yu, Scott Berger, Weibin Chen

Waters Corporation, Milford, MA USA

P-108-T

Comprehensive Characterization of the NIST mAb Reference Standard using a QTof Mass

Spectrometer

Jing Fang, Nilini Ranbaduge, William Alley, Henry Shion, Ying Qing Yu

Waters Corporation, Milford, MA USA

P-109-T

Unveiling the Mysteries in the Mixed-Mode SEC Profile of a Monoclonal Antibody

Jinmei Fu1, James Chesko2, Brent Meengs2, Richard Ludwig1, Pauline Bariola2, Jeff Meyer2, Li

Tao1 1Bristol-Myers Squibb Company, Pennington, NJ USA, 2Bristol-Myers Squibb Company, Seattle,

WA USA

P-110-T

High-Throughput Multicomponent Profiling of Cell Culture Medium by LC/MS/MS for

Advance Process Monitoring of Biopharmaceutical Products

Gurmil Gendeh

Shimadzu Scientific Instruments, Inc., Saratoga, CA USA

P-111-T

Microfluidic Device for Integrated Imaged cIEF and Direct Electrospray Ionization-Mass

Spectrometry for Analysis of Intact Biopharmaceuticals

Erik Gentalen, Barry Clerkson

Intabio, Inc., Portola Valley, CA USA

47

P-112-T

Profiling and Heightened Characterization of O-linked Glycans from Therapeutic

Glycoproteins

Andrew Hanneman1, Elaine Sun1, Mario DiPaola1, Marshall Bern2 1Charles River Laboratories International, Inc., Woburn, MA USA, 3Protein Metrics Inc., San

Jose, CA USA

P-113-T

HDX-MS in Drug Development: Tackling Higher Order Challenges

Nikolai Hentze1, Markus Hollmann1, Medha Tomlinson2, Christopher Chumsae2, Carsten Jahn1 1AbbVie Deutschland GmbH & Co KG, Ludwigshafen, Germany, 2AbbVie Bioresearch Center,

Worcester, MA USA

P-114-T

Optimization of Mass Spectrometry for Quantifying Methionine Oxidation and Asparagine

Deamidation

Neil Hershey, Richard Ludwig, Li Tao, Tapan Das

Bristol-Myers Squibb Company, Pennington, NJ USA

P-115-T

A Recombinant Asp-Specific Protease for Bottom-up Mass Spectrometry Workflows

Chris Hosfield, Jim Hartnett, Ethan Strauss, Sergei Saveliev, Mike Rosenblatt, Marjeta Urh

Promega Corporation, Madison, WI USA

P-116-T

Characterization of Discolored Samples from an Monoclonal Antibody Formulation

Development

Yunping Huang1, Richard Ludwig1, Li Tao1, Ya Fu2, Anil Wagh2, Ming Zeng2, Anthony Leone2,

Reb Russell1 1Bristol-Myers Squibb Company, Pennington, NJ USA, 2Bristol-Myers Squibb Company, New

Brunswick, NJ USA

P-118-T

Sequence Variants Analysis of mAbs by LC-MS

Haitao Jiang, Yan-Hui Liu

Merck & Co. Inc., Kenilworth, NJ USA

48

P-119-T

Routine Monitoring of Host Cell Proteins and Non-Targeted Detection of New Features as

Part of Mass Spectrometry-based Quality Control

Jonathan Josephs, Michael Blank, Stephane Houel

Thermo Fisher Scientific, San Jose, CA USA

P-120-T

Automated Data Processing for Quality Monitoring of Biotherapeutics by Multi-attribute

Methods (MAMs)

Maurizio Bronzetti1, Joe Shambaugh1, David Bush1, Aude Tartiere2, Nick DeGraan-Weber1,

Cassandra Wigmore3, Peter Haberl4 1Genedata Inc., Lexington, MA USA, 2Genedata Inc., San Francisco, CA USA, 3Genedata AG,

Basel, Switzerland, 4Genedata GmbH, Munich, Germany

P-121-T

An Adapted Top-down LC-MS Approach for Screening Lipid-modified Proteins

Andrew James

Sanofi Pasteur, Toronto, ON Canada

P-122-T

Rapid Multi Attribute Methodology Based on Intact LC-MS Profiling in Support of

Upstream Process Development and Optimization

Mally Lev, Carolina Lanter

GlaxoSmithKline, King of Prussia, PA USA

P-123-T

Sequence Variant Identification by LC-MS/MS to Support Cell Line and Upstream Process

Development

Renpeng Liu, Lintao Wang, Alexandru Lazar

ImmunoGen, Inc, Waltham, MA USA

P-124-T

Determination of the Complex Disulfide Connectivity in a Binding Protein of Insulin-like

Growth Factor-1

Chen Li1, Serah Liu1, Shiaw-Lin Wu1, Paul A. Salinas2, Justin Prien2, Chris Barton2 1BioAnalytix Inc., Cambridge, MA USA, 2Shire Pharmaceuticals, Lexington, MA USA

P-125-T

HDX-MS Higher Order Structure Profiling of Somavaratan, a Novel Long-Circulating

rhGH Therapeutic

Shiaw-Lin Wu1, Jennifer S. Chadwick1, Yaping Sun1, Serah Liu1, Peter Li1, Chen Li1, Eric

Mueller2, Xiao Liu2, Gary Koe2, Patrick Murphy2 1BioAnalytix Inc., Cambridge, MA USA, 2Versartis Inc., Menlo Park, CA USA

49

P-126-T

LC-MS Based HCP Monitoring During Biologic Downstream Process Development

Chen Li1, Wanlu Qu1, Serah Liu1, Shiaw-Lin Wu1, Fengqiang Wang2, Yan-Hui Liu2, Douglas

Richardson2, Daisy Richardson2 1BioAnalytix Inc., Cambridge, MA USA, 2Merck & Co. Inc., Kenilworth, NJ USA

P-127-T

Advances in IgG2 Disulfide Isoform Characterization using MALDI and In-source Decay

Fragmentation

Anja Resemann1, Lily Liu-Shin2, Fang Wang2,3, Adam Fung2,4, Guillaume Tremintin5, Detlev

Suckau1, Gayathri Ratnaswamy2 1Bruker Daltonik, Bremen, Germany, 2Agensys, Inc., an affiliate of Astellas, Santa Monica, CA

USA, 3SCIEX, Brea, CA USA, 4Omeros Corporation, Seattle, WA USA 5Bruker Daltonics,

Billerica, MA USA

P-128-T

Highly Confident Peptide Mapping of Protein Digests Using Mass Spectrometry

Stephen Madden, David Wong, Linfeng Wu

Agilent Technologies, Santa Clara, CA USA

P-129-T

Automated Sequencing of Polyethylene Glycol Conjugation Sites on a Protein by High-

Resolution Mass Spectrometry

Matthew Maust

Protea Biosciences, Morgantown, WV USA

P-130-T

Workflow for Simultaneous Biotherapeutic Peptide Mapping and Host Cell Protein

Analysis Utilizing In-silico Peptide Monitoring

Matthew Maust

Protea Biosciences, Morgantown, WV USA

P-131-T

Optimizing Parameters to Get the Max Out of the MAM

Trina Mouchahoir1, John Schiel2 1NIST/IBBR, Rockville, MD USA, 2NIST/IBBR, Gaithersburg, MD USA

P-132-T

N-Glycan Analysis for a Monoclonal Antibody using RapiFluor® Labeling Method

Sheila Mugabe, Tasneem Bahrainwala, Krishnan Sampathkumar

MacroGenics, Inc., Rockville, MD USA

50

P-133-T

Site-specific O-glycan Analysis using a Novel O-glycan Protease

Philip Onigman1, Maria Nordgren2, Rolf Lood2, Fredrik Leo2, Fredrik Olsson2 1Genovis Inc, Cambridge, MA USA, 2Genovis AB, Lund, Sweden

P-134-T

NanoLC/MS: Improving Sensitivity for Quantification of Low Abundance Proteins

Michaela Owens

DuPont Pioneer, Johnston, IA USA

P-135-T

Implementation of a Dual Work Flow HRMS Platform for the Analysis of Antibody Drug

Conjugates and Small Molecules in a Walk-Up Environment

Michael Peddicord, Jonathan Marshall, Scott Miller

Bristol-Myers Squibb Company, New Brunswick, NJ USA

P-136-T

An Integrated Subunit LC/MS Analysis for Fast Core Fucosylation Assessment of mAb

Products

Nilini Ranbaduge, Henry Shion, Ying Qing Yu, Weibin Chen

Waters Corporation, Milford, MA USA

P-137-T

Comparison of Common Fluorescent Labels for Liquid Chromatography Analysis of

Released N-linked Glycans

John Yan, Andres Guerrero, Steven Mast, Emily Dale, Ted Haxo, Aled Jones

ProZyme, Hayward, CA USA

P-138-T

A Multi-Attribute Mass Spectrometry Method for Adenovirus Based Vaccines

Arjen Scholten, Carol de Ram, Jonathan Knibbe, Harold Backus, Annemiek Verwilligen

Janssen Infectious Diseases and Vaccines, Leiden, Netherlands

P-139-T

Vendor-Neutral Data Processing for Host Cell Proteins by Mass Spectrometry

St John Skilton1, Eric Carlson2, Ilker Sen1 1Protein Metrics Inc., San Carlos, CA USA, 2Protein Metrics Inc., San Jose, CA, USA

P-140-T

Ensuring Late Stage Readiness for a Monoclonal Antibody by Mitigating the Impact of

Host Cell Proteins and Misincorporations

Jacquelynn Smith1, Jose Gomes2, Ying Zhang2, Russ Shpritzer2, Dan Bereznyakov2, Phoebe

Baldus1, Olga Friese1, Gary Madsen1, Thomas Powers1, Paul Brown1, Jason Rouse2 1Pfizer, Inc., Chesterfield, MO USA, 2Pfizer, Inc., Andover, MA USA

51

P-141-T

TopN LC-MS/MS for Adjuvant Saponin Identification

Natalie Thompson1, Johan Bankefors2, Susanna MacMillar2, Ernest Maynard1, Karin Lovgren-

Bengtsson2, Oleg Borisov1 1Novavax, Inc., Gaithersburg, MD USA, 2Novavax AB, Uppsala, Sweden

P-142-T

Monitoring Critical Quality Attributes of an IgG4 Antibody During Process Development

with Genedata Expressionist

Larry Wang1, James Pollock2, Vijay Janakiraman1, Santosh Yadav1, Philip Savickas1, Marlis

Zeiler3, Arnd Brandenburg4 1Merck & Co., Inc., West Point, PA USA, 2Merck &Co., Inc., Kenilworth, NJ USA, 3Genedata

GmbH, Munich, Germany, 4Genedata AG, Basel, Switzerland

P-143-T

Using in vitro Models to Predict the in vivo Degradation of Therapeutic Monoclonal

Antibodies

Na Yang, Qing (Mike) Tang, Michael Lewis

Johnson and Johnson, Malvern, PA USA

P-144-T

MS Software Evaluation for Automated MS Data Analysis

Holly Yip

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

P-145-T

Orthogonal Technologies for the Epitope Mapping of Antibody Therapeutics

Edward J. Hsieh, Grigori Ermakov, Maribel Beaumont

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

52

NOTES:

53

Bioanalytical

P-202-F

Accurate Quantitation of Peptide-Conjugates with Improved Half-Life from Plasma using

Automated Affinity Capture Mass Spectrometry

Phillip Chu, Xinxin Gao, Douglas Leipold, Rami Hannoush, John Tran

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

P-203-F

Sample Preparation Method for Accurate Analysis of Non-enzymatic PTMs in

Biotherapeutic Proteins with Peptide Mapping

Kevin Cook, Sergei Saveliev, Chris Hosfield, Mike Rosenblatt, Marjeta Urh

Promega Corporation, Madison, WI USA

P-204-F

Intact Protein RP-LC-MS Method Development and Optimization Using a Well

Characterized Standard Protein Mix

Benjamin Cutak, Kevin Ray

MilliporeSigma, St. Louis, MO USA

P-205-F

Unraveling CES1c-mediated Cleavage of the Linker-drug in SYD985 (an anti-HER2 ADC)

in Wild-type Mice

Eef Dirksen, Myrthe Rouwette, Ruud Ubink

Synthon Biopharmaceuticals B.V., Nijmegen, Netherlands

P-206-F

Effects of Stationary Phase and Mobile Phase Modifiers on Reversed-Phase Polypeptide

Selectivity

Cory Muraco1, Ken Weber2 1MilliporeSigma, Bellefonte, PA USA, 2MilliporeSigma, St. Louis, MO USA

P-207-F

Automated Intact Mass Analysis for the Characterization of Antibodies

Peter Haberl1, David Bush2, Aude Tartiere3, Nick DeGraan-Weber2, Maurizio Bronzetti2,

Cassandra Wigmore5, Joe Shambaugh2 1Genedata GmbH, Munich, Germany, 2Genedata Inc., Lexington, MA USA, 3Genedata Inc., San

Francisco, CA USA, 5Genedata AG, Basel, Switzerland

54

P-208-F

Detecting Trisulfides in IgG1 and IgG2 Monoclonal Antibodies with Ultrahigh-Resolution

Mass Spectrometry

Mellisa Ly, Andrew Saati, Lisa Marzilli, Jason Rouse

Pfizer, Inc., Andover, MA USA

P-209-F

Analysis of Sialylated N-Glycans Using an Optimized HILIC-Fluorescence-MS Method

Jennifer Nguyen, Qi Wang, Matthew Lauber

Waters Corporation, Milford, MA USA

P-210-F

Use of Mass Spectrometry as a Process Analytical Technology for the Rapid Quantitation

of Underivatized Amino Acids in Crude Bioreactor Media

David Powers, Cyrus Agarabi

CDER, FDA, Silver Spring, MD USA

P-211-F

Robust Quantitation of a Low-concentration Biologic Using LC-MS

Annie He1, Dongdong Wang1, Wanlu Qu1, Jennifer S. Chadwick1, Shiaw-Lin Wu1, Nazila

Salamat-Miller2, Paul A. Salinas2 1BioAnalytix Inc., Cambridge, MA USA, 2Shire Pharmaceuticals, Lexington, MA USA

P-212-F

An Integrated Workflow for Automated Antibody Characterization

Shuai Wu

Agilent Technologies, Santa Clara, CA USA

P-213-F

Amino Acids and Metabolites/derivatives Analysis Without Derivatization using a Novel

Mixed-mode Column

Itaru Yazawa

Imtakt Corpoation, Kyoto, Japan

55

CMC Strategy and the Use of Mass Spec

P-214-F

Method Development of Multi-Attribute Monitoring by Peptide Mapping

Lu Dai

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

P-215-F

Adding Mass Detection to LC/UV-Based Workflows for Routine Analysis and Monitoring

of Biologics in Development and Quality Control Laboratories

Brooke Koshel, Ximo Zhang, Robert Birdsall, Ying Qing Yu

Waters Corporation, Milford, MA USA

P-216-F

Automated Workflow for Host Cell Protein Monitoring by Mass Spectrometry: From Raw

Data to Final Report

Joe Shambaugh1, David Bush1, Aude Tartiere2, Nick DeGraan-Weber1, Maurizio Bronzetti2,

Cassandra Wigmore5, Peter Haberl6 1Genedata Inc., Lexington, MA USA, 2Genedata Inc., San Francisco, CA USA, 5Genedata AG,

Basel, Switzerland, 6Genedata GmbH, Munich, Germany

P-217-F

The use of Mass Spectrometry in FDA Applications for Peptide Therapeutics

Xiaoshi Wang, Sarah Rogstad, Eric Pang, Xiaohui Jiang

CDER, FDA, Silver Spring, MD USA

56

Emerging Product Areas

P-218-F

Middle-Down LC-MS/MS Characterization on Glycation at Two Lys Residues in a Sucrose

Formulated Biotherapeutic

Difei Qiu, Scott Miller, Wenkui Lan, Wei Ding

Bristol-Myers Squibb Company, New Brunswick, NJ USA

57

Novel Technologies

P-219-F

A Novel Data Acquisition Mode for Identification, Quantification and Monitoring of Low-

Abundance Host Cell Proteins During Monoclonal Antibody Bioprocessing

Catalin Doneanu

Waters Corporation, Milford, MA USA

P-220-F

Monitoring Glycosylation Profile and Protein Titer in Cell Culture Samples using ZipChip

CE-MS

Peng Feng, Yan Wang, Zoran Sosic, Li Zang

Biogen, Cambridge, MA USA

P-221-F

Orthogonal, High Resolution Polar Biomolecule Analysis by CESI-MS

Edna Betgovargez1, Esme Candish2, Bryan Fonslow3 1SCIEX, Brea, CA USA, 2SCIEX, Framingham, MA USA, 3SCIEX, San Diego, CA USA

P-222-F

Examining the Structural Influence of Site Specific Phosphorylation by Ion Mobility-Mass

Spectrometry

Rebecca Glaskin1, Dawn Stickle2, Caroline Chu3 1Agilent Technologies, Lexington, MA USA, 2Agilent Technologies, Odessa, FL USA, 3Agilent

Technologies, Santa Clara, CA USA

P-223-F

Sub-unit and Middle-down Analyses of Monoclonal Antibody on an Orbitrap Fusion

Lumos Tribrid Mass Spectrometer

Stephane Houel, Romain Huguet, Jennifer Sutton, Seema Sharma, Aaron Bailey, Vlad

Zabrouskov, Jonathan Josephs

Thermo Fisher Scientific, San Jose, CA USA

P-224-F

The Separation of Intact Phosphorylated Proteins by Capillary Electrospray Ionization

(CESI)

Stephen Lock1, Edna Betgovargez2 1SCIEX, Pudsey, United Kingdom, 2SCIEX, Brea, CA USA

58

P-225-F

Characterization of Monoclonal Antibodies and Antibody Drug Conjugates using

Microchip Zone Electrophoresis-MS Technology

Erin Redman

908 Devices, Carrboro, NC USA

P-226-F

Integrated SEC-MS Analysis of Monoclonal Antibody (mAb) and Antibody Drug

Conjugates (ADCs) under Native Conditions.

Henry Shion1, Dale Cooper-Shepherd2, Laetitia Denbigh2, Maria Basanta-Sanchez3, Barbara

Sullivan4, Ying Qing Yu1, Weibin Chen1 1Waters Corporation, Milford, MA USA, 2Waters Corporation, Wilmslow, United Kingdom, 3Waters Corporation, Pleasanton, CA USA, 4Waters Corporation, Beverly, MA USA

P-227-F

LC-MS with Post-Column Reduction for Comprehensive Characterization of Disulfide

Bond Connectivity

Tian Wang

Amgen Inc., Thousand Oaks, CA USA

P-228-F

Enterprise Mass Spectrometry Software Solution Enabling Characterization of

Biotherapeutics from Discovery and Development to Production and Quality Control

Cassandra Wigmore1, Joe Shambaugh2, David Bush2, Nick DeGraan-Weber2, Aude Tartiere3,

Maurizio Bronzetti3, Peter Haberl4 1Genedata AG, Basel, Switzerland, 2Genedata Inc., Lexington, MA USA, 3Genedata Inc., San

Francisco, CA USA, 4Genedata GmbH, Munich, Germany

P-229-F

Native Mass Spectrometry for the Revelation of Highly Complex Glycosylation Patterns in

Biopharmaceuticals

Therese Wohlschlager1, Kai Scheffler2, Ines C. Forstenlehner3, Stefan Senn1, Eugen Damoc4,

Johann Holzmann3, Christian G. Huber1 1University of Salzburg, Salzburg, Austria, 2Thermo Fisher Scientific, Dreieich,

Germany, 3Sandoz GmbH, Kundl, Austria, 4Thermo Fisher Scientific, Bremen, Germany

P-230-F

Comprehensive Characterization on Monoclonal Antibody using a Newly Developed Q-

TOF/MS Instrument

David Wong

Agilent Technologies, Santa Clara, CA USA

59

P-231-F

Optimizing Electron Transfer Dissociation Conditions for Hydrogen/Deuterium Exchange

Mass Spectrometry and Its Application to the Study of Protein Conformation

Terry Zhang, Stephane Houel, Jonathan Josephs

Thermo Fisher Scientific, San Jose, CA USA

P-232-F

Identification of Structural Perturbations Caused by Methionine Oxidation on mAb by

Carboxyl Group Protein Footprinting Method

Hao Zhang, Alla Polozova

Amgen Inc., Cambridge, MA USA

60

Young Scientist

P-234-F

A Generic HPLC-UV-HRMS Method for Absolute Quantification of Oxidation in

Monoclonal Antibodies and Fc-Fusion Proteins

Christof Regl1, Therese Wohlschlager1, Johann Holzmann2, Christian G. Huber1 1University of Salzburg, Salzburg, Austria, 2Sandoz GmbH, Kundl, Austria

61

Late Breaking

LB-01-F

LC/MS/MS Bioanalysis of Therapeutic Antibodies Based on Nano-surface and Molecular-

Orientation Limited (nSMOL) Proteolysis

Tairo Ogura1, Gurmil Gendeh1, Toshiya Matsubara2, Noriko Iwamoto2, Nozomi Maeshima2,

Deepti Bhandarkar3, Masateru Oguri2, Rashi Kochhar3, Takashi Shimada2, Ichiro Hirano2 1Shimadzu Scientific Instruments, Columbia, MD USA, 2Shimadzu Corporation, Kyoto, Japan, 3Shimadzu Analytical India Pvt. Ltd., Mumbai, India

62

NOTES: