The Organizing Committee Gratefully Acknowledges the Symposium

2nd Symposium on Higher Order Structure of Protein Therapeutics

February 11 – 13, 2013 Hyatt Regency Baltimore on the Inner Harbor Baltimore, Maryland USA

Organized by

Table of Contents

Program Partners ...... 3

Exhibitor and Media Partners...... 4

Acknowledgements ...... 5

Student Travel Grants ...... 6

Scientific Final Program Summary ...... 7

Plenary and Session Abstracts ...... 16

Technical Seminar I (Monday, February 11) Abstract ...... 55

Technical Seminar II (Tuesday, February 12) Abstract...... 56

Technical Seminar III (Tuesday, February 12) Abstract ...... 57

Poster Abstracts ...... 58

2 The Organizing Committee gratefully acknowledges the Corporate Program Partners for their generous support of the 2nd Symposium on Higher Order Structure of Protein Therapeutics Sustaining Gold Program Partners AbbVie Inc. Agilent Technologies Sustaining Silver Program Partner Pfizer, Inc. Platinum Program Partners Biogen Idec Genentech, a Member of the Roche Group Gold Program Partner Amgen Inc. Silver Program Partners Bristol-Myers Squibb Company Eli Lilly and Company Bronze Program Partner Fujifilm Diosynth Biotechnologies

Exhibitor Partners Agilent Technologies Applied Photophysics Ltd. BioTools, Inc. ExSAR™ Corporation JASCO KBI Biopharma Malvern Instruments, Inc. NanoSight, LLC Pall Life Sciences TA Instruments Waters Corporation Wyatt Technology Corporation Leading Media Partner Technology Networks Limited

Acknowledgements

Symposium Co-Chairs: Wasfi Al-Azzam, GlaxoSmithKline Pharmaceuticals Steven Cohen, Waters Corporation

Scientific Program Committee: Steven Berkowitz, Biogen Idec Katherine Bowers, Fujifilm Diosynth Biotechnologies John Carpenter, School of Pharmacy, University of Colorado at Denver Guodong Chen, Bristol-Myers Squibb Company Otmar Hainzl, Sandoz Biopharmaceuticals Wim Jiskoot, LACDR, Leiden University Tom Laue, University of New Hampshire Aston Liu, GlaxoSmithKline Pharmaceuticals Jamie Moore, Genentech, a Member of the Roche Group Radhika Nagarkar, KBI Biopharma Inc. Linda Narhi, Amgen Inc. Jason Rouse, Pfizer, Inc. Satish Singh, Pfizer, Inc. William Weiss, Eli Lilly and Company

Audio-Visual: Michael Johnstone, MJ Audio-Visual Productions

CASSS Staff: Karen Bertani, Symposium Manager Stephanie Flores, Executive Director Linda Mansouria, Manager of Meetings and Events Renee Olson, Manager of Meetings and Events Mikaela Sanford, Project Coordinator Catherine Stewart, Finance Manager

5 CASSS Higher Order Structure 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 2nd International Symposium on Higher Order Structure of Protein Therapeutics (HOS 2013). PhD students or post-doctoral fellows conducting research at academia throughout the world are eligible.

This year’s winner is:

P-04 Hai Yue Washington University, St. Louis, University City, MO USA Development of Far-UV and Near-UV Circular Dichroism Method for the Characterization and Comarability of Protein Higher Order Structure

2nd International Symposium on Higher Order Structure of Protein Therapeutics Scientific Program Summary

MONDAY, FEBRUARY 11, 2013

07:00 – 17:00 Registration in the Constellation Ballroom Foyer

07:00 – 08:30 Continental Breakfast in Constellation Ballroom A

08:00 – 08:15 CASSS Welcome and Introductory Comments in Constellation Ballroom B Steven Cohen, Waters Corporation

Higher Order Structure Welcome and Introductory Comments in Constellation Ballroom B Wasfi Al-Azzam, GlaxoSmithKline Pharmaceuticals

Keynote Session in Constellation Ballroom B Session Chairs: Wasfi Al-Azzam, GlaxoSmithKline Pharmaceuticals and Linda Narhi, Amgen Inc.

08:15 – 09:00 The Evolving Role of Analyzing Higher Order Structure for Biotechnology Products and its Scientific and Regulatory Implications Anthony Mire-Sluis, Amgen Inc., Thousand Oaks, CA USA

Higher Order Structure Introduction and Fundamentals Plenary Session in Constellation Ballroom B Session Chairs: Norma Allewell, University of Maryland, College Park and Jason Rouse, Pfizer, Inc.

09:10 – 09:30 Effects of Silicone Oil and Silicone-coated Glass on Protein Aggregation John Carpenter, School of Pharmacy, University of Colorado, Denver, Aurora, CO USA

09:30 – 09:50 Structures of Amyloid Precursor Protein in a Membrane Environment David Thirumalai, University of Maryland, College Park, MD USA

09:50 – 10:10 Structural Principles of Insulin Analog Design Michael Weiss, Case Western University of Medicine, Cleveland, OH USA

10:10 – 10:30 Chaperoning Antibody Folding and Assembly Johannes Buchner, Technical University Munich, Munich, Germany

10:30 – 11:00 AM Break – Visit the Exhibits and Posters in Constellation Ballroom A

7 Monday, February 11 continued…

11:00 – 11:20 The Present State and Future Outlook for Characterizing the Higher Order Structure of Protein Drugs in the Biopharmaceuticals Industry Steven Berkowitz, Biogen Idec, Cambridge, MA USA

11:20 – 11:45 Discussion – Questions and Answers

Hydrogen/Deuterium Exchange Mass Spectrometry in Higher Order Structures Plenary Session in Constellation Ballroom B Session Chairs: Guodong Chen, Bristol-Myers Squibb and Steven Cohen, Waters Corporation

11:45 – 12:05 Hydrogen/Deuterium Exchange Mass Spectrometry: A Valuable Tool for Protein Higher Order Structure Characterization Roxana Iacob, Northeastern University, Boston, MA USA

12:05 – 12:25 Applications of Hydrogen/Deuterium Exchange Mass Spectrometry in Comparability Studies and Epitope Mapping Jingjie Mo, Bristol-Myers Squibb Company, Princeton, NJ USA

12:25 – 12:45 Hydrogen/Deuterium Exchange with Mass Spectrometry Detection for Higher Order Structure Evaluation of Therapeutic Proteins Ingo Lindner, Roche Diagnostics GmbH, Penzberg, Germany

12:45 – 13:05 Impact of Chemical Degradation on the Conformational Dynamics of Monoclonal Antibody Ping Hu, GlaxoSmithKline, King of Prussia, PA USA

13:05 – 13:30 Discussion – Questions and Answers

13:30 – 15:00 Lunch Break – Participants on their own

HOS in Protein Therapeutics Discovery and Early Candidate Selection Plenary Session in Constellation Ballroom B Session Chairs: Yijia Jiang, Amgen Inc. and Jamie Moore, Genentech, a Member of the Roche Group

15:00 – 15:20 Integrated Early Developability Assessment: Monitoring Higher Order Structure Attributes for Candidate Selection Alla Polozova, MedImmune, Gaithersburg, MD USA

15:20 – 15:40 Molecule Assessment and Early Development Cynthia Li, Amgen Inc., Thousand Oaks, CA USA

Monday, February 11 continued…

15:40 – 16:00 Understanding Sequence/Structure Impact on Pharmaceutical Development of a Monoclonal Antibody Vikas Sharma, Genentech, a Member of the Roche Group, South San Francisco, CA USA

16:00 – 16:30 Discussion – Questions and Answers

16:30 – 17:00 PM Break – Visit the Exhibits and Posters in Constellation Ballroom A

17:00 – 18:00 Technical Seminar

Advances in the Integrated Measurements of Protein Size and High Order Structure

Sponsored by Malvern Instruments, Inc. Constellation Ballroom B

Young Scientists Plenary Session in Constellation Ballroom B Session Chairs: Wasfi Al-Azzam, GlaxoSmithKline and Steven Cohen, Waters Corporation

18:15 – 18:30 Development of Far-UV and Near-UV Circular Dichroism Method for the Characterization and Comparability of Protein Higher Order Structure Hai Yue, Washington University, St. Louis, University City, MO USA

18:30 – 20:00 Exhibitor and Poster Session One in Constellation Ballroom A

20:00 Adjourn Day One

TUESDAY, FEBRUARY 12, 2013

07:30 – 17:30 Registration in the Constellation Ballroom Foyer

07:30 – 08:45 Continental Breakfast in Constellation Ballroom A

Process Development: HOS Studies for Formulations and Stability Plenary Session in Constellation Ballroom B Session Chairs: Katherine Bowers, Fujifilm Diosynth Biotechnologies and Anthony Cannon, Grifols, Inc.

08:30 – 08:50 Similarity of Higher Order Structure: Moving Towards Best Practices for Spectral Comparability Mark Manning, Legacy BioDesign, LLC, Johnstown, CO USA

08:50 – 09:10 High-throughput Biophysical Analysis of Protein Stability Applied to Both Formulation Development and Comparability Assessments David Volkin, University of Kansas, Lawrence, KS USA

09:10 – 09:30 Doing More with Less – High Throughput Development and its Applications in Protein Therapeutic Formulation Development Feng He, Amgen Inc., Seattle, WA USA

09:30 – 09:50 Assessment of Particles in Prefilled Syringes of Protein-based Products: Case Studies Jun Park, CDER, FDA, Bethesda, MD USA

09:50 – 10:20 Discussion – Questions and Answers

10:20 – 10:50 AM Break – Visit the Exhibits and Posters in Constellation Ballroom A

HOS in Development: Characterization and Comparability Plenary Session in Constellation Ballroom B Session Chairs: Linda Narhi, Amgen Inc. and Leonard Olszewski, GlaxoSmithKline

10:50 – 11:10 Assessing Higher Order Structure and Comparability of Protein Therapeutics – A Regulator’s Perspective Evie Struble, CBER, FDA, Rockville, MD USA

11:10 – 11:30 Protein Quaternary Structure Characterization: The Effect of Protein Self- association on Analytical Method Development Qin Zou, Pfizer, Inc., Chesterfield, MO USA

10 Tuesday, February 12 continued…

11:30 – 11:50 Higher Order Structure Characterization for MA and Beyond John Gabrielson, Amgen, Inc., Longmont, CO USA

11:50 – 12:10 Mechanistic Complexity of Subvisible Particle Formation: Links to Protein Aggregation are Highly Specific Robert Simler, Genzyme, A Sanofi Company, Framingham, MA USA

12:10 – 12:40 Discussion – Questions and Answers

12:45 - 14:30 Hosted Lunch Break – in Constellation Ballroom A

13:15 – 14:15 Technical Seminar – Lunch and Learn

Seeing the Forest and the Trees: Exploring HDX and Microcalorimetry as Techniques for the Analysis of Biotherapeutic Higher Order Structure

Sponsored by Waters Corporation Constellation Ballroom B

14:30 – 15:15 Poster Session Two in Constellation Ballroom A

HOS of Biosimilars Plenary Session in Constellation Ballroom B Session Chairs: Steven Berkowitz, Biogen Idec and Radhika Nagarkar, KBI Biopharma

15:15 - 15:35 Characterization of Higher Order Structure for Biosimilars Maria-Teresa Gutierrez-Lugo, CDER, FDA, Bethesda, MD USA

15:35 – 15:55 Biophysical and HOS Characterization of Biosimilars: Providing a Comprehensive Method and Technology Portfolio for an Expanding Field in the Biotech Industry Otmar Hainzl, Sandoz Biopharmaceuticals, Oberhaching, Germany

15:55 – 16:15 Fingerprinting the Higher Order Structure of a Biosimilar and its Innovator Drug at High Resolution in Order to Assess Comparability Yves Aubin, Health Canada, Ottawa, ON, Canada

16:15 – 16:35 Development of Antibody Arrays for Novel and Biosimilar mAb Higher Order Structure (3-D) Comparability Xing Wang, Array Bridge, Inc., St. Louis, MO USA

16:35 – 17:05 Discussion – Questions and Answers

11 Tuesday, February 12 continued…

17:15 – 18:15 Technical Seminar

Characterizing Biomolecular Interactions by CG-MALS

Sponsored by Wyatt Technology Corporation Constellation Ballroom B

18:15 Adjourn Day Two

WEDNESDAY, FEBRUARY 13, 2013

07:30 – 17:00 Registration in the Constellation Ballroom Foyer

07:30 – 08:45 Continental Breakfast in Constellation Ballroom A

08:30 – 08:45 Announcements by Wasfi Al-Azzam, GlaxoSmithKline

Biological Consequences of HOS Plenary Session in Constellation Ballroom B Session Chairs: Wasfi Al-Azzam, GlaxoSmithKline and Yiqing Feng, Janssen Research & Development, LLC

08:45 – 09:05 Tying Higher Order Structure with in-vitro Activity Through a Forced Degradation Study Laura Duitch, GlaxoSmithKline, King of Prussia, PA USA

09:05 – 09:25 Immunogenicity of Protein Aggregates Ted Randolph, Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO USA

10:15 – 10:45 Discussion – Questions and Answers

10:45 – 11:15 AM Break – Visit the Exhibits and Posters in Constellation Ballroom A

Higher Order Structure Emerging and Novel Technologies Plenary Session in Constellation Ballroom B Session Chairs: Thomas Laue, University of New Hampshire and William Weiss, Eli Lilly and Company

11:15 – 11:35 High-resolution NMR Analysis of Protein Therapeutics: An Inter-laboratory Round Robin Study John Marino, National Institute of Standards and Technology, Rockville, MD USA

11:35 – 11:55 Mapping Molecular Interfaces Michael Brenowitz, Albert Einstein College of Medicine, Bronx, NY USA

11:55 – 12:15 Brownian Dynamics Simulation of High-concentration Protein Solutions John Tsavalas, University of New Hampshire, Durham, NH USA

13 Wednesday, February 13 continued…

12:15 – 12:45 Discussion - Questions and Answers

12:45 – 13:00 Closing Remarks and Invitation to HOS 2014 Steven Cohen, Waters Corporation

13:00 Adjournment

14 NOTES:

Keynote Abstract

The Evolving Role of Analyzing Higher Order Structure for Biotechnology Products and Its Scientific and Regulatory Implications

Anthony Mire-Sluis

Amgen Inc., Thousand Oaks, CA USA

The evolution of technology to probe higher order structure of proteins is becoming an ever increasingly powerful tool in the characterization 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. Ever complicated assay readout profiles require the time and expertise to understand the exact nature of what is being reported as well as efforts to ensure that the methodology itself is appropriately qualified or, in some cases when used for lot release, validated. It is also important to understand the limitations of higher order structure methods, such as the power to detect differences between protein moieties, as well as what the readout of an assay is actually telling you regarding the structure of the protein. How one goes about assessing what is background noise or simply assay variability versus something that is 'real' 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. The advent of multiple technologies to test the same attribute (e.g. protein particles) has introduced even more complexity since these orthogonal methods may or may not provide the same answers. 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:

Higher Order Structure Introduction and Fundamentals Session Abstract

“HOS Introduction and Fundamentals” is the first session of the Higher Order Structure 2013 conference for protein therapeutics. Distinguished speakers from academia and industry will make presentations toward introducing the major themes of the conference, in addition to conveying the fundamental elements and significance of protein higher-order structure. Topics will include secondary and tertiary structure/sub-structures; aggregation, amyloids, and intrinsically disordered proteins; impact of protein HOS on molecular interactions, binding and function; protein folding pathways and molecular chaperones; protein behavior versus overall HOS in terms of particular amino acids, modifications, charge, hydrophobicity, etc.; and HOS comparability studies for industry. The intent, content and positioning of this session is especially important to the conference proceedings given that many attendees may be new to the biophysical sciences and the fact that the biophysical sciences involves complex theories and phenomena, as well as a multitude of approaches, techniques and methods in the determination of molecular size distributions; secondary, tertiary and quaternary structure; conformational stability; intermolecular interactions; molecular-level images; etc.

NOTES:

Plenary Session Abstracts

Effects of Silicone Oil and Silicone-coated Glass on Protein Aggregation

John F. Carpenter

University of Colorado Center for Pharmaceutical Biotechnology, Aurora, CO USA

Silicone oil is used to lubricate prefilled syringes and cartridges, to facilitate smooth plunger movement. As a result, there can be oil microdroplets sloughed off into the formulation, as well as the silicone- water interface at the barrel wall. With “baked-on” silicone, the sloughing of droplets into solution is mitigated but there is still the silicone-coated glass of the barrel.

In studies with silicone oil microdroplets, we have found that monoclonal antibodies readily adsorb to the oil-water interface. The magnitude of adsorption depends on the individual antibody molecule. Similarly, the degree of perturbation of the adsorbed protein’s tertiary structure – as assessed with fluorescence quenching spectroscopy – varies among the different antibodies studied. Also, at least for one antibody, the ionic strength of the solution affects the adsorption-induced structural perturbation.

In studies of protein aggregation, it was found that a combination of silicone oil microdroplets and agitation act synergistically to cause loss of monomeric protein to aggregates; as measured by reduction in monomer level using size exclusion chromatography. Employing a neutral pH, at which the model antibody studied, had reduced colloidal stability, greatly enhanced protein aggregation compared to that noted at pH 5. Also, addition of polysorbate to the formulation inhibited the loss of monomer, which can be attributed, at least in part, to the surfactant’s capacity to reduce protein adsorption to the silicone- water and the air-water interfaces.

It is difficult to study formation of subvisible particles – a critical product quality attribute – in formulations containing silicone oil microdroplets. To address this issue and to investigate a surface mimicking baked-on silicone oil, we prepared siliconized glass beads. With this system, we also found that adsorption of the model monoclonal antibody to the silicone-water interface caused perturbation of tertiary structure. Furthermore, the combination of siliconized beads and agitation was synergistic in causing formation of protein microparticles. Interestingly, polysorbate was effective at inhibiting the gross loss of monomer to insoluble aggregates, but subvisible particles were still formed, albeit to reduced level.

Taken together, the results of these studies, document that proteins readily adsorb to silicone-water interfaces, often with a resulting perturbation of tertiary structure. And combination of agitation and a silicone-water interface can foster rapid aggregation and particle formation by proteins.

NOTES:

18 Structures of Amyloid Precursor Protein in a Membrane Environment

David Thirumalai

University of Maryland, College Park, MD USA

Aggregation of Amyloid  (A) peptide has been linked to the neurodegenerative Alzheimer’s disease and implicated in other amyloid diseases including cerebral amyloid angiopathy. A peptide is generated by cleavage of the amyloid precursor protein (APP) by transmembrane proteases. It is crucial to determine the structures of -amyloid peptides in a membrane to provide a molecular basis for the cleavage mechanism. I will report the structures of amyloid -peptide (A1-40 and A1-42) as well as the 672-726 fragment of APP (referred to as A1-55) in a membrane environment determined by replica exchange molecular dynamics simulation. A1-40 is found to have two helical domains A (13-22) and B(30-35) and a type I -turn at 23-27. The peptide is localized at the interface between membrane and solvent. The dominant simulated tertiary structure of A1-40 is similar to the simulated A1-42 structure. However, there are differences in the overall conformational ensemble, as characterized by the two- dimensional free energy surfaces. The fragment of APP (A1-55) is observed to have a long transmembrane helix. The position of the transmembrane region and ensemble of membrane structures will be elucidated. The conformational transition between the transmembrane A1-55 structure, prior to cleavage, and the A1-40 structure, following cleavage, will be proposed.

NOTES:

19 Structural Principles of Insulin Analog Design

Michael A. Weiss

Case Western Reserve University School of Medicine, Cleveland, OH USA

The emerging pandemic of Type 2 diabetes mellitus (DM) and progressive increase in global incidence of Type 1 DM has renewed interest in the molecular engineering of insulin to enhance the safety and efficacy of insulin replacement therapy. Over the past decade the successful clinical use of first- generation insulin analogs has shown that human insulin (although the products of stringent evolutionary selective pressure for in vivo physiological function) is not optimal as a diabetes drug. Unmet clinical needs pertain to pharmacokinetics, pharmacodynamics, compatibility with computer- controlled pumps (algorithm-based closed-loop systems), formulation strength, and thermal stability; frontier goals envisage the engineering of glucose-responsive “smart” analog formulations, tissue- specific signaling and post-receptor partial agonist activity. Whereas insulin analog design has traditionally been based on crystal structured of classical zinc hexamers as first reported by D. C. Hodgkin and coworkers more than 40 years ago, a new perspective is informed by co-crystal structures of model hormone-receptor complexes (Menting, J. G., et al., How insulin engages its primary binding site on the insulin receptor surface. Nature 493, 241-5 (2013)). This presentation will focus on design criteria for next-generation insulin analogs and their potential therapeutic role in the treatment of DM. An emerging principle is that the foundation of rational design is provided not by a single structure but instead by the conformational “lifecycle” of a protein, i.e., its repertoire of structures in successive higher-order protein-protein assemblies.

NOTES:

20 Chaperoning Antibody Folding and Assembly

Johannes Buchner

Technical University Munich, Germany

Antibodies are hetero-oligomeric proteins consisting of structurally highly similar heavy and light chains. It is well established that unassembled heavy chains are actively retained in the endoplasmic reticulum (ER) until they associate with light chains. Our mechanistic analysis of this critical quality control step revealed that, unlike all other antibody domains studied, the CH1 domain of the heavy chain is an intrinsically disordered protein in isolation. It adopts the typical immunoglobulin fold only upon interaction with its native partner, the CL domain. The molecular chaperone BiP modulates CH1/CL association by binding unfolded states of the CH1 domain and thus preventing secretion of incompletely assembled IgG. We identified conserved BiP binding sites within the CH1 domain and determined critical features of these BiP binding sequences. The chaperone-assisted assembly of protein complexes in the ER may be a general means of controlling the secretion of oligomeric proteins.

NOTES:

21 The Present State and Future Outlook for Characterizing the Higher Order Structure (HOS) of Protein Drugs in the Biopharmaceuticals Industry

Steven A. Berkowitz

Biogen Idec, Cambridge, MA USA

This talk will initially focus its attention on the present capabilities and limitations of the most commonly used biophysical tools employed in the research and process development areas of the biopharmaceutical industry to characterize the HOS of protein drugs. These capabilities and limitations will then be contrasted against a discussion concerning the needs of the biopharmaceutical industry in assessing the HOS of protein drugs. In so doing particular attention well be given to the task of assessing comparability and understanding the impact of drug related impurities (drug variant forms) on drug product quality. Finally this talk will then turn its focus to those less commonly used biophysical tools that offer improved capabilities to better satisfy the needs of industry, as well as regulatory agencies, in order to help achieve a positive and efficient drug approval.

NOTES:

Hydrogen/Deuterium Exchange Mass Spectrometry in Higher Order Structures Session Abstract

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as one of the most powerful techniques that provide conformational information for proteins not amenable to classical structural tools. This session will cover the fundamental principles, instrumentation development, data analysis and practical applications of HDX-MS in higher order structure characterization of protein therapeutics. A thorough coverage of approaches toward HDX-MS method development for comparability studies and epitope mapping experiments will provide a good starting point for understanding practical issues encountered during implementation and applications of HDX-MS in the biopharmaceutical industry. Future directions in HDX-MS will also be highlighted.

NOTES:

Plenary Session Abstracts

Hydrogen / Deuterium Exchange Mass Spectrometry: A Valuable Tool for Protein Higher-order Structure Characterization

Roxana E. Iacob and John R. Engen

Northeastern University, Boston, MA USA

The higher-order structure of proteins is responsible for their uniqueness and dictates their function. In order to characterize higher-order structure, a wide array of orthogonal analytical techniques is needed. Higher-order structure can be interrogated with mass spectrometry and in particular by hydrogen exchange mass spectrometry (HX MS). Although HX MS was introduced more than 20 years ago, it has only recently become a widely used tool in the structural characterization of protein therapeutics and has opened new frontiers in the characterization of biopharmaceuticals.

HX MS monitors deuteration of protein backbone amide hydrogens. Deuteration is a function of solvent exposure and hydrogen bonding thereby indirectly providing information about higher-order structure and dynamics. For example, details of local conformational and dynamic changes upon binding to a partner can be obtained with micrograms of material and proteins that will not crystallize can also be studied by HX MS in many buffer and solution conditions. In this presentation, we will give an overview of HX MS, focusing on its strengths and weaknesses in characterizing protein therapeutics. Improvements in the technology, including automation, software, and dedicated instrumentation will be described, including why the technique is now amenable for comparability studies of protein biopharmaceuticals.

NOTES:

24 Applications of Hydrogen/Deuterium Exchange Mass Spectrometry in Comparability Studies and Epitope Mapping

Jingjie Mo

Bristol-Myers Squibb Company, Princeton, NJ USA

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) probes protein conformation and conformational dynamics in solution by monitoring the rate and extent of deuterium exchange of backbone amide hydrogen and the conformation of the protein. As a sensitive analytical tool, MS can precisely measure the mass increases of the protein upon HDX. A global view of protein conformation can be obtained by measuring the molecular weight of a protein and following its rate of deuterium incorporation as a function of incubation time in deuterated buffer (global HDX). When this technique is paired with enzymatic digestion, structural features at the peptide level can be resolved, enabling differentiation of surface exposed peptides from those folded inside (peptide HDX). In this presentation, applications of HDX-MS in comparability studies at both global and peptide levels will be described. In addition, applications of HDX-MS in epitope mapping will also be presented for studies on mechanisms of actions in early drug discovery.

NOTES:

25 Hydrogen/Deuterium Exchange with Mass Spectrometry Detection for Higher Order Structure Evaluation of Therapeutic Proteins

Ingo Lindner

Roche Diagnostics GmbH, Penzberg, Germany

Therapeutic applications with proteins and especially with monoclonal antibodies (mAb) have been rapidly growing in recent years. The function, efficacy, and safety of therapeutic proteins are tied to their three-dimensional structure. The analysis and verification of this higher order structure is critical in demonstrating manufacturing consistency and in analyzing the absence or presence of structural changes in response due to changes in production or stability. It is, therefore, essential to have reliable and high sensitive biophysical tools capable of interrogating protein structure and conformation. The analytical tools available to study and evaluate this critical attributes, which are in general the classical spectroscopic methods like FT-IR, fluorescence or circular dichroism, are lacking sensitivity and spatial resolution. Hydrogen/deuterium exchange (HDX-MS) offers significant hope to improve this. The use of HDX-MS for protein biotherapeutics can include epitope mapping, binding, protein-drug interaction studies, aggregation studies, effect of mutation on conformation and localization of conformational changes as a result of post-translational modifications. Due to recent developments in robotic automation of sample handling and improvements in chromatographic separation and mass spec detection along with the development of appropriate computer software, HDX-MS has now become a technology that is capable of making significant contributions in biopharmaceutical characterization by providing more spacial resolved information compared to the classical spectroscopic methods. It has also the potential to be used as a tool to support biopharmaceutical comparability studies.

This talk will present several examples of HDX analysis applied to mAb samples to elucidate the influence of posttranslational modifications on the protein structure as well studies of aggregation behavior of these molecules. It will discuss the potential capabilities of HDX-MS and its present limitations on hardware and software.

NOTES:

26 Impact of Chemical Degradation on the Conformational Dynamics of Monoclonal Antibody

Aming Zhang, Ping Hu, Paul MacGregor, Aston Liu, Leonard Olszewski

GlaxoSmithKline, King of Prussia, PA USA

Conformation plays a critical role in the stability of protein biopharmaceutical and its biological functions. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a powerful technique that has recently been successfully used to probe the conformation of biopharmaceutical products. In this study, we investigated the impact of various chemical modifications (methionine oxidation, aspartic acid isomerization and asparagine deamidation) on the conformational dynamics of IgG1 monoclonal antibodies using HDX-MS.

Two oxidized IgG1 proteins that have common glycosylation profile and Fc region sequence showed a similar increase of deuterium incorporation into the heavy chain residues adjacent to the CH2 domain oxidation site. Although heavily oxidized, only minor change in the conformation of CH3 domain was detected in either protein. Another glycosylated IgG1 protein with three oxidized methionines in the CH3 domain showed slight increase of conformational change in this domain, but the major conformational change remained in the CH2 domain. On the other hand, oxidation in aglycosylated IgG1 was found to not only induce a dramatic change in the conformation across its CH2 domain, but also in the CH2-CH3 domain interface. IgG1 conformational changes caused by isomerization and deamidation will also be discussed in this presentation.

NOTES:

HOS in Protein Therapeutics Discovery and Early Candidate Selection Session Abstract

An emerging trend in the development of biopharmaceuticals is to conduct molecule developability studies to inform the candidate selection process. The goal of these studies is to raise the probability of success of achieving the target product profile and to ease development. In addition, to evaluating key properties such as PK and binding affinity, other molecule properties that affect development and delivery are also characterized. The higher order structure of the candidate molecule can be one such characteristic. This session will include case studies from early development programs describing the use of various higher order structure techniques to support candidate selection.

NOTES:

Plenary Session Abstracts

Integrated Early Developability Assessment: Monitoring Higher Order Structure Attributes for Candidate Selection

Alla Polozova

MedImmune, Gaithersburg, MD USA

The increasing demand for next generation protein therapeutics with enhanced efficacy and safety, reduced immunogenicity and improved delivery has resulted in an expanding cohort of more extensively engineered novel molecules being considered as candidates for development. Recent advances in our understanding of the relationship between protein structure and stability or function relationships, combined with advances in biophysical methods, are enabling more comprehensive and accurate developability and manufacturability screening during early development stages. This presentation focuses on the application of various biophysical methods, such as Differential Scanning Calorimetry, Circular Dichroism, Dynamic Light Scattering and Analytical Ultracentrifugation, in monitoring higher order structure properties for developability screening. The suitability of several biophysical tools is discussed in the context of stage-related requirements and constraints, such as predictability, speed, and limited material and resource availability. Case studies will be presented to demonstrate the value of such early developability assessment in identifying risk associated with in-process (low pH) stability, storage stability and high concentration solution properties.

NOTES:

29 Molecule Assessment and Early Development

Cynthia Li, Xichdao Nguyen, Jenny Li, Jie We, Linda Narhi and Yijia Jiang

Amgen Inc., Thousand Oaks, CA USA

The development of a successful protein therapeutic is dependent on the product quality and stability during processing, storage and delivery, optimal production yield and solution property in addition to its biological activity. Selecting the best candidate early is very important during the drug product development life cycle. It can save time and resources by minimizing the issues that may be encountered later during the development life cycle. Molecule assessment is a body of work that considers product quality, stability and solution property when designing and selecting lead protein therapeutic candidates. It is used to select the lead molecule(s) from multiple candidates and for identifying potential issues for further development. Molecule assessment is also an important step of Quality by Design (QbD). In this presentation, we’ll show a few of case studies to demonstrate how molecule assessment helped the candidate selection at an early stage.

NOTES:

30 Understanding Sequence/Structure Impact on Pharmaceutical Development of a Monoclonal Antibody

Vikas K. Sharma

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

Monoclonal antibodies, with differences largely only in the CDR sequence, exhibit vastly different behavior during various steps of development including purification, formulation, and processing. A case study is presented on a monoclonal antibody, mAb 1, which behaves atypically during the purification steps, readily forms insoluble particles under formulation conditions and exhibits high viscosity. We hypothesized that such atypical behavior is a result of unusual sequence/structural attributes of this mAb in the variable domain compared to a well-behaved mAb and investigated the origin of such atypical behavior through sequence mutations. Various mutants were generated to alter the charge, hydrophobicity, and CDR loop length of the mAb and the mutants were then evaluated for their purification behavior, solubility, and viscosity properties. We show that certain molecular attributes have a rather dramatic affect on the behavior of this mAb under various test systems. The outcome of this investigation will be presented and the impact of various molecular attributes on mAb behavior will be discussed.

NOTES:

Young Scientists Session Abstract

Development of Far-UV and Near-UV Circular Dichroism Method for the Characterization and Comparability of Protein Higher Order Structure

Hai Yue

Washington University in St. Louis, University City , MO

Circular Dichroism (CD) spectroscopy is widely used in academia and industry to study protein secondary and tertiary structure. In the biopharmaceutical industry particularly, CD spectroscopy is routinely used to evaluate the effects of manufacturing, formulation and storage conditions on protein stability and conformation. The results are usually required by regulatory agencies. Therefore, establishing CD spectral comparability quantitatively and the relationship between protein structural change and CD spectral change are important and challenging since traditionally, such comparisons have been qualitative in nature and based mainly on visual inspection. To avoid possible inaccuracies generated from the inherently subjective visual assessment, and in compliance with the recent requests from regulatory agencies of demonstrating the suitability of the CD spectroscopic method for the purpose of comparing spectra, in this study, we use three different algorisms: spectral correlation coefficient (SCC), area of overlap (AO) and modified area overlap (mAO) to determine spectral comparability of a protein as a function of changes in pH or temperature, and their ability to do so and sensibility are compared. The result is a framework for quantitative determination of whether any two spectra differ significantly.

NOTES:

Process Development: HOS Studies for Formulations and Stability Session Abstract

The formulation of stable protein therapeutics can be complicated due to the changes in higher order structures that may impact the long-term shelf life stability, safety, and deliverability of a product. Understanding the propensity of a protein candidate to form or change higher order structures early in the formulation development process is paramount, as well as developing and understanding of how this propensity will affect long-term storage stability. The formation of higher order structure during protein formulation development and stability can be probed by a wide array of biophysical methods. With the development of high throughput methodologies, the amount and diversity of biophysical data that can be collected for a protein therapeutic leads to potential analytical challenges in interpreting and compiling vast data sets. In this series of talks, the audience will hear from distinguished speakers whose research interests include both high throughput biophysical techniques and the development of methodology for analyzing large biophysical data sets for formulation development and stability.

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Plenary Session Abstracts

Similarity of Higher Order Structure: Moving Towards Best Practices

Mark Cornell Manning1,2; Brian M. Murphy1,2; Robert W. Payne1,2;Wasfi Al-Azzam3

1Legacy BioDesign, LLC, Johnstown, CO USA; 2Department of Chemistry, Colorado State University, Fort Collins, CO USA; 3GlaxoSmithKline, King of Prussia, PA USA

Maintaining higher order structure (HOS) of therapeutic proteins is essential for retention of potency and reducing the likelihood of aggregation and immunogenicity. While one can characterize the HOS of proteins using a variety of biophysical methods, there remains a central question about how one demonstrates comparability and similarity in a non-biased, quantitative fashion. To this end, we have been systematically evaluating a number of algorithms that can measure the degree of similarity of spectra obtained from biophysical methods that have been shown to indicate the extent of organized secondary and tertiary structure. The goal of these studies is to identify the optimal method(s) in an attempt to delineate the best practice for demonstrating similarity at the HOS level. Examples will be given from our analysis of infrared and circular dichroism spectra. These approaches can be used to examine not only the effects of solution conditions on HOS, but they are also applicable to assessment of comparability between highly similar biologics from different sources or between different batches of a recombinant protein.

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34 High-throughput Biophysical Analysis of Protein Stability Applied to Both Formulation Development and Comparability Assessments

David Volkin

University of Kansas, Lawrence, KS USA

This presentation will examine the challenges and opportunities of using high-throughput biophysical techniques to assess protein stability as part of formulation development and comparability assessments. The utility and limitations of various analytical methods to detect and monitor conformational changes and aggregate formation will be presented by use of illustrative case studies with large, multidomain proteins including IgG1 and IgG2 mAbs, an albumin-fusion protein, and a pentameric plasma glycoprotein. Utilization of data visualization techniques to analyze these large biophysical stability data sets has demonstrated utility for formulation development, and potential application in comparability studies to examine the conformational stability of protein therapeutics.

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35 Doing More with Less - High Throughput Development and Its Applications in Protein Therapeutic Formulation Development

Feng He

Amgen Inc., Seattle, WA USA

Recent development trends in the biotechnology industry have emphasized the development and application of high throughput methodologies. High throughput methods are especially powerful because of the low resource demand and rapid turnaround speed. In this presentation, I will summarize the development of a few high throughput tools applied to large molecule drug assessment. While these methods can be utilized throughout the development of biological entities, this presentation specifically focuses on the formulation aspects. Examples will be provided to illustrate the strategies of applying high throughput methodology to the screening of drug formulation.

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36 Assessment of Particles in Prefilled Syringes of Protein-based Products: Case Studies

Jun Park

CDER, FDA, Bethesda, MD USA

Abstract was not available at the time of printing.

NOTES:

HOS in Development: Characterization and Comparability Session Abstract

As protein therapeutics move through the product lifecycle HOS analysis is a key part of comparability studies, and process understanding. These techniques can also play a role in support of NC investigations. This includes secondary and tertiary structure characterization, as well as analysis of the amount, size, and morphology of protein aggregate. This session will feature talks on the strategy and application of HOS analysis from late stage commercial development through support of marketed products.

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Plenary Session Abstracts

Assessing Higher Order Structure and Comparability of Protein Therapeutics – A Regulator’s Perspective

Evie Struble

CBER, FDA, Rockville, MD USA

Therapeutic proteins are characterized by a complex higher order structure which is highly dependent upon the conditions used for production, purification, handling and storage. To ensure that marketed biotherapeutics continue to remain safe and effective throughout their lifecycle, adequate product characterization, appropriate in-process and release specifications, as well as tight control of manufacturing process are necessary. When changes to the manufacturing process are instituted, the comparability of the product pre- and post-change should be assured. In this talk, a discussion of current and emerging methods that can be used for characterization of protein therapeutics during the production process, upon release and to demonstrate comparability, as well as pertinent regulatory guidances will be presented.

Disclaimer: This presentation reflects the views of the author and should not be construed to represent FDA’s views or policies.

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39 Protein Quarternary Structure Characterization: The Effect of Protein Self-association on Analytical Method Development

Qin Zou

Pfizer, Inc., Chesterfield, MO USA

Size exclusion chromatography (SEC) is the method of choice for routine analysis of protein aggregates across the biopharmaceutical industry. SEC has also been used historically for native protein-protein association to gain insight about the protein quaternary structure and the thermodynamics and kinetics of the association. However, weak protein association may not be detected due to significant sample dilution and protein-column interaction. On the other hand, stronger association may also complicate the SEC method validation due to the changes as a function of sample loads. Orthogonal methods, such as analytical ultracentrifugation (AUC), should facilitate further characterization of the protein association at the concentration and buffer conditions that are difficult to implement in the SEC method. Implementation of orthogonal confirmation during SEC method development will help better understand the performance of the method and ensure its suitability for intended use. Several case studies are provided to demonstrate the use of AUC in supporting the SEC analysis regarding protein-protein association.

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40 Higher Order Structure Characterization for MA and Beyond

John Gabrielson1; Kelly Arthur1; Yijia Jiang2; Brent Kendrick1

1Analytical Sciences, Amgen Inc., Longmont, CO USA; 2Process & Product Development, Amgen Inc., Thousand Oaks, CA USA

Characterization of the higher order structure of therapeutic protein products is a requirement for marketing authorization of these products in many jurisdictions. Spectroscopic, calorimetric, and separation methods are used to characterize higher order structure, thermal stability, and association states of protein products and to verify that these attributes are preserved following changes to manufacturing processes. This presentation provides important considerations for selecting appropriate methods, and applying quantitative analysis approaches, to ensure a robust assessment of higher order structure is performed to enable product approval and post-marketing changes.

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41 Mechanistic Complexity of Subvisible Particle Formation: Links to Protein Aggregation are Highly Specific

Robert Simler

Genzyme, A Sanofi Company, Framingham, MA USA

Little is known about the mechanistic features of the protein aggregation pathway which lead to subvisible particles (0.1-100 μm in size). Specifically, the relationship between soluble aggregates (those that are less than 0.1 μm in size) and subvisible particle formation is largely unknown. This talk will focus on agitation experiments coupled with flow microscopy and size-exclusion chromatography, performed on a series of proteins to investigate this relationship. Additionally, statistical analysis of morphological parameters describing the shape of subvisible particles in agitated and non-agitated samples is used to characterize subvisible particle populations and identify protein specific characteristics of the subvisible particle formation. Collectively, these results suggest/illustrate the complexity of elucidating an aggregation mechanism that encompasses both soluble aggregates and subvisible particles.

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HOS of Biosimilars Session Abstract

In assessing the comparability of biosimilars to their innovator counterpart drug product molecule, the assessment of their higher order structure (HOS) is one of the first major steps that need to be undertaken and established. How this should best be done and to what level is not a straight forward template process. In this session discussions concerning the how and what that should be done with what analytical biophysical tools to meet and achieve regulatory guidance acceptance will be the key theme of this meeting. In addition, challenges facing the biosimilar manufacturer, such as establishing appropriate reference innovator material to make these comparability studies, especially when these innovator drugs are imbedded in complex formulation matrices will be addressed.

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Plenary Session Abstracts

Characterization of Higher Order Structure for Biosimilars

Maria-Teresa Gutierrez-Lugo

CDER, FDA, Bethesda, MD USA

Analytical similarity involves a comprehensive comparative evaluation of physicochemical and functional properties, including higher order structure (HOS) between the proposed biosimilar product and the reference product. HOS impacts the product’s functional activity and conformational stability, thus differences in HOS may impact product performance. This presentation will provide a regulatory perspective on the evaluation of HOS for biosimilars and the challenges related to an assessment of HOS similarity using current methodologies.

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44 Biophysical and HOS Characterization of Biosimilars: Providing a Comprehensive Method and Technology Portfolio for an Expanding Field in the Biotech Industry

Otmar Hainzl

Sandoz Biopharmaceuticals, Oberhaching, Germany

Targeted technical development of follow-on biologics (biosimilars) requires continuous comparison with the originator product and monitoring of the product development process. For this, a thorough assessment of dozens of parameters of the product is performed, and in addition to classical physicochemical methods, biophysical characterization gives insight into the structural organization of the protein, which may allow predictions of biological function and activity. As HOS analysis involves both ensemble methods which show the overall (population average) status of a protein sample, as well as methods which can detect subtle differences between populations within a sample, it delivers comprehensive information on the protein. In order to gain a sensitive methodology that is able to deliver clear and reliable results, these methods need to be both well understood and qualified. With established HOS methods at hand, a so-called “technical proof of similarity” exercise comprises a major milestone in biosimilar development, where many analytical results are used to demonstrate the required high similarity to the originator product. Examples from “proof-of-similarity” and comparability exercises demonstrate how precisely biopharmaceuticals can be analyzed and compared.

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45 Fingerprinting the Higher Order Structure of a Biosimilar and its Innovator Drug at High Resolution in Order to Assess Comparability

Yves Aubin

Health Canada, Ottawa, ON, Canada

The current perception of nuclear magnetic resonance (NMR) spectroscopy applied to proteins is most often characterized with the words complicated, time-consuming and expensive, to name only these. In this paper, we will show through a case study that a simple NMR method can provide detailed information on the higher order structure of protein therapeutics.

Filgrastim is the generic name for recombinant methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF). It is produced in Escherichia coli (E. coli) in a non-glycosylated form. Filgrastim is marketed under the brand name Neupogen® by Amgen. Since this product has lost patent protection, many subsequent entry versions have been approved or are in the process of filing for market authorization throughout the world, including Canada. In order to be authorized as a subsequent entry product, the sponsors must demonstrate similarity with an approved product in Canada via an appropriate comparability exercise. Here we show that the NMR fingerprint assay can be used to assess the three-dimensional structure of the active ingredient in the formulated product from two different sources as well as a comparator, the approved product Neupogen®.

Recombinant metHuG-CSF was prepared in E. coli and isotopically enriched with 13C and 15N isotopes. Samples were analysed by NMR to study the effects of varying the pH, the concentration of excipients (sorbitol and polysorbate-80), the ionic strengths with several salts, and co-solutes. Spectra of mutants have been recorded to assess the sensitivity of the method to small structural changes. Finally, NMR fingerprints were recorded for Neupogen®, purchased at a local pharmacy, and a chemical reference standard from the European Directorate for Quality Medicine (EDQM).

The NMR fingerprint assay applied to Filgrastim provided residue specific information of the structure of the active ingredient of a product. In addition to current methods, the ability to assess the conformation with a high degree of resolution can greatly facilitate the comparability exercise.

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46 Development of Antibody Arrays for Novel and Biosimilar mAb Higher Order Structure (3-D) Comparability

Xing Wang

Array Bridge, Inc., St. Louis, MO USA

For biologics, the three dimensional structure (Higher Order Structure) of the molecule is closely related to its efficacy and safety including immunogenicity. Recent guidelines from FDA on biosimilars gave specific instructions on the analysis of protein tertiary structure. This presentation described a new approach to measure biosimilar molecule conformational changes systematically and at molecular level. A large number of different antibodies were developed toward a single biosimilar molecule to cover the whole protein sequence, and interrogate its conformational changes and compare the information with that of the innovator molecule. Case studies will be presented to demonstrate that this sensitive and robust technology can be applied to biosimilar as well as novel monoclonal antibody cell line selection, process and formulation development, and many other aspects of biologics development.

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Biological Consequences of HOS Session Abstract

This session will focus on exploring the potential impacts of protein therapeutic higher order structure on the product safety and potency. Safety related issues such as possible immunogenicity will be discussed. In addition, product activity related topics such as binding and cell based potency will be discussed with relation to product higher order structure. In vivo and In vitro case studies in addition to risk assessment of potential impact of protein therapeutic higher order structure on safety and potency will be discussed from various subject matters expert.

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Plenary Session Abstracts

Tying Higher Order Structure with in-vitro Activity Through a Forced Degradation Study

Laura Duitch

GlaxoSmithKline, King of Prussia, PA USA

Higher order structures have been known as part of the critical product attributes for protein therapeutics. Maintaining intact higher order structures is a key to maintaining biological activities of proteins. Here we presented a case study to correlate higher order structures to in vitro activity of an IgG molecule through a forced degradation study. The IgG molecule was stressed at high storage temperature (50C), acidic and basic pH’s (pH 3 and 10) through an extended time period. Secondary and tertiary structures of stressed samples at different time points were analyzed via FTIR and Near UV CD. Amount of high MW aggregates, acidic and basic variants were measured with SEC and CIEF respectively. A Biacore based assay was used to measure antigen binding activity. In vitro activity of the samples was also evaluated by a cell based assay. An incubation time dependent structural degradation was observed with acidic pH stressed samples, corresponding to a gradual decrease in antigen binding and in vitro activity. Meanwhile, no significant structural changes and activity losses were observed with either heat stressed or basic pH treated samples.

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49 Immunogenicity of Protein Aggregates

Theodore W. Randolph

Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO USA

Immunogenicity in therapeutic proteins is a concern due to the effects of anti-drug antibodies (ADAs) on clinical efficacy of the therapy. Little is known about the mechanism(s) underlying immune responses to therapeutic proteins, but the presence of particulate matter has been suggested as a potential contributing factor. I will discuss some recent murine studies of protein immunogenicity, as well as historical data (including human clinical data) suggesting that the presence of protein aggregates may greatly enhance immunogenicity.

NOTES:

Higher Order Structure Emerging and Novel Technologies Session Abstract

Understanding and controlling next-generation protein therapeutics will require next-generation technologies. This session will highlight cutting-edge tools and techniques for the analysis of higher order structure, including both experimental and computational approaches. In addition to a detailed treatment of the current state of ground-breaking research, speakers are encouraged to provide forward- looking statements highlighting key opportunities for innovation in the coming years.

NOTES:

Plenary Session Abstracts

High-resolution NMR Analysis of Protein Therapeutics: An Inter-laboratory Round Robin Study

John P. Marino

Institute for Biotechnology and Bioscience Research, National Institute of Standards and Technology, Rockville, MD USA

In contrast to small molecule therapeutics, protein biologics are many kDa in size and require higher- order folding of the primary sequence (i.e., tertiary structure) for therapeutic function. While correct folding of a protein biologic is critical for drug efficacy, misfolding may impact drug safety by eliciting unwanted immune and/or other off-target responses. High-resolution NMR uniquely provides a relatively simple, robust spectroscopic approach for obtaining higher-order structural 'fingerprints' of the bioactive form(s) of protein therapeutics at atomic resolution in solution. Such 'fingerprints' of the structure(s) of protein therapeutics can be used as a tool for establishing consistency in drug manufacturing, for detecting drug product variations inherent to or resulting from modifications in the manufacturing process, and for comparing a biosimilar to an innovator reference product. In this presentation, progress will be described on an inter-laboratory round robin study involving laboratories at the FDA, NIST, Health Canada and MPA-Sweden, on the comparability of NMR spectral 'fingerprints' that can be obtained using standardized NMR experimental techniques on representative recombinant protein biologics.

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52 Mapping Molecular Interfaces

Michael Brenowitz; Joerg Schlatterer; Keiji Takamoto; Micheal Leser; Steve Almo

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY USA

Michelle Khine1, 3; Matt Law2, 3

Departments of Biomedical Engineering1, Chemistry2 and Chemical Engineering & Materials Science3, University of California at Irvine, Irvine, CA USA

Molecular interfaces mediate cellular interactions ranging from simple dimerization to the assembly of large nucleo-protein complexes. While the determination of atomic resolution structures is the gold standard for molecular interface maps, the structures of many biologically interesting complexes are not known. In addition, structural analyses often report only the end state of an assembly reaction, i.e. the complex. Chemical or enzymatic mapping (aka ‘footprinting’) approaches can provide insight into the structure of molecular interfaces with as fine as single residue resolution. With appropriate protocols, footprinting can follow thermodynamic or kinetic transitions from their initial to final states and thus directly connect structure with function. A footprinting probe of proven value is the hydroxyl radical that can map the solvent accessible surface of nucleic acids and proteins. We are developing a new method of hydroxyl radical generation for footprinting that utilizes an Fe(II) containing solid surface to catalyze the Fenton reaction. This surface is compatible with standard robotic sample handling systems and thus will enable high-throughput analyses of proteins and nucleic acids. We present the development and validation of this approach in the context of the assembly of complexes that mediate immune responses including those between an antibody and its antigen.

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53 Brownian Dynamics Simulation of High-Concentration Protein Solutions

John Tsavalas1; Robert Carrier2

1Materials Science Program, University of New Hampshire, Durham, NH USA; 2Research Computing Center, University of New Hampshire, Durham, NH USA

The behavior of biomolecules in solution at high concentration, primarily in terms of their interaction potential and the resultant impact on solution viscosity, has become an increasingly important topic over the last decade. Humanized monoclonal antibodies (mAbs) serve as a great example with the focus shifting from oral delivery of low concentration solutions to subcutaneous delivery of more highly concentrated solutions (faster delivery time, less compliance issues, and patient convenience). The movement toward higher concentration solutions (> 100 mg/mL) inherently leads to the case where molecular crowding and intermolecular collisions become more probable as the protein-protein separation distance is markedly reduced. Non-idealities in the solution behavior present themselves as a result of these increased collisions and molecular interactions including protein aggregation, phase separation, and non-linear rheological behavior. Aggregation can lead to increased immunogenicity, which may result in loss of drug effectiveness or, even worse, anaphylactic shock. Most literature appropriately ascribes this solution behavior at higher concentration to the reduced separation distance between the biomolecules, their charge and response to pH, and the ionic strength of the solution. The general finding seems to be that it is the nature of the charges, both the net and the partial due to dipoles, on the protein that most influence the interactions and resultant rheological profile of the solution.

In this work, a dynamic model is presented that can evaluate and predict this behavior of proteins in concentrated solutions as a function of their charge, charge distribution, and resultant interaction potential. In particular, the viscosity response to the stability of the proteins in solution is discussed as a dynamic output during the simulation. Emphasis is made for the impact of the significant difference between the effective hydrodynamic radius of the proteins and the “hard” radius. The larger effective radius is in part due to the high aspect ratio of a single mAb, but is exacerbated by weak clustering of multiple mAbs in solution. The modeling of an anisotropic charge distribution (including charge-charge, and dipole-dipole induced interactions) is hypothesized to help explain some of the tendency towards these higher aspect ratio entities and the non-ideal rheological behavior observed in experiment.

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54 NOTES:

Technical Seminar Abstract

Monday, February 11, 2013 17:00 – 18:00

Sponsored by Waters Corporation

Seeing the Forest and the Trees: Exploring HDX and Microcalorimetry as Techniques for the Analysis of Biotherapeutic Higher Order Structure

Michael Eggertson1; Dile Holton2

1Waters Corporation, Milford, MA USA; 2TA Instruments – A Waters LLC, New Castle, DE USA

This lunch session will cover two fundamental techniques (HDX and microcalorimetry) that enable biopharmaceutical organizations to answer the multitude of higher order structure questions about their biotherapeutic, and the molecules with which they interact. As the general HOS meeting will deftly discuss many applications of these technologies, we have taken the opportunity to invite two scientists, key to developing our HDX and microcalorimetry solutions, to address some of the fundamental scientific concepts underpinning the products and solutions that we have developed in partnership with the biopharmaceutical industry.

ABOUT: Michael Eggertson, the key developer of the Waters DynamX 2.0 HDX informatics platform, will describe the data acquisition and processing strategies that have evolved to address the fundamental data complexity issues inherent to HDX MS analysis, and how proper assessments of measurement error can facilitate more confident data interpretation.

Dile Holton, has been a lead developer and supporting scientist for biopharmaceutical and life science applications of microcalorimetry at TA Instruments for more than five years. He will describe how two techniques (DSC and ITC) enable scientists to optimize biotherapeutic drug candidate selections, using acquired knowledge of the thermodymanics underlying their structure, and the molecular binding forces driving their interactions.

NOTES:

Technical Seminar Abstract

Tuesday, February 12, 2013 17:15 – 18:15

Sponsored by Wyatt Technology Corporation

Characterizing Biomolecular Interactions by CG-MALS

Daniel Some

Wyatt Technology Corporation, Santa Barbara, CA USA

Dr. Daniel Some of Wyatt Technology will present a technical seminar regarding the characterization of protein self-association and other biomolecular interactions by Composition-Gradient Multi-Angle static Light Scattering (CG-MALS) and Wyatt’s Calypso® system. CG-MALS is a scientifically rigorous, label-free, immobilization-free biophysical technique that quantifies a wide range of phenomena related to biomolecular binding, stoichiometry, dissociation and aggregation. In the context of drug quality and safety, it is a powerful tool for investigating the reversible and irreversible quaternary structure of biotherapeutics.

ABOUT: As principal scientist at Wyatt Technology Corporation, Dr. Some currently investigates techniques for characterization of macromolecular interactions, developing both hardware and software for new instruments and applications. His prior record of experimental research and applied R&D encompasses semiconductor diode lasers, photoluminescence spectroscopy of semiconductors, ultrafast THz spectroscopy, ultrafast laser amplifiers, laser-coupled scanning tunneling microscopy and atomic force microscopy, and laser scattering for defect detection on patterned semiconductor wafers.

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Poster Abstracts

P-01

Analysis of Local Dynamics of Human Insulin and a Rapid-acting Insulin Analog by Hydrogen Deuterium Exchange Mass Spectrometry

Joomi Ahn1; Shiori Nakazawa2, 3; Noritaka Hashii3; Kenji Hirose4; Nana Kawasaki2, 3

1Waters Corporation, Milford, MA USA; 2Hokkaido University, Sapporo, Japan; 3National Institute of Health Science, Tokyo, Japan; 4Nihon Waters, Tokyo, Japan

Human insulin, used by diabetics to regulate blood sugar, was first introduced as a recombinant therapeutic drug nearly 30 years ago. It is, however, very challenging to adequately characterize the higher-order structures of biotherapeutic products and to monitor the changes in oligomeric stability. Human insulin and insulin lispro have identical primary structure, except for the transposition of a pair of amino acids. Lispro is one of the rapid-acting insulin analogs, which has higher tendency to dissociate than human insulin. In this study, we present an analytical workflow to allow us to detect the difference in the oligomeric dynamics using a Hydrogen Deuterium Exchange Mass Spectrometry (HDX MS). The HDX analysis on Insulin and Lispro peptides was conducted to identify the location where different deuterium uptakes were observed between human insulin and lispro. The detected areas were illustrated in various display formats to help understand their flexibility associated with rapid dissociation of insulin oligomers.

Drug products, human insulin (Humulin R) and lispro (Humalog), were reduced and digested online by pepsin. Deuterium labeling, quenching, and injection to on-line pepsin digestion were prepared using a robotic sample manager. Labeling experiments in 0, 0.5, 5, 10, 60, and 180 min interval were duplicated for both samples. The peptic digests were separated on a UPLC system dedicated for HDX experiments at 0 °C. Q-TOF mass spectrometer was used to measure the deuterium incorporation of identified peptides. The amount of deuterium was determined by automated HDX data processing software, DynamX 2.0. The HDX results were compared in uptake curves, heat maps, and 3D-structures, which were enhanced features of the software.

We obtained 98% of sequence coverage for both human insulin and lispro. The coverage map of the peptic digests demonstrated that the efficient digestion was achieved with a high redundancy score resulted by multiple overlapping peptides. From peptide HDX determination, two regions were revealed distinctive different values in deuterium uptakes between human insulin and lispro; the N terminus of chain A, and a region adjacent to the C terminus of chain B. We attributed this localized behavior to the relation of hexamerization and dimerization, respectively. Furthermore, characteristic profiles that showed different deuteration margins between two insulins were determined, which was also consistent with their involvement in hexamer and dimer formation.

59 NOTES:

60 P-02

Ferric Ions Perturb Tertiary Structure of a Monoclonal Antibody Leading to Loss of Storage Stability

Shuxia Zhou1; Sandeep Kumar2; Qian Wang2; Jianxin Guo2; Satish Singh2, 2; Christian Schoeneich3

1Johnson & Johnson, Radnor, PA USA; 2Pfizer, Inc., Chesterfield, MO USA; 3University of Kansas, Lawrence, KS USA

Trace amounts of metal ions can degrade biopharmaceutical drug product leading to potency loss. We report the impact of ferric ions on the conformation and aggregation of an IgG monoclonal antibody. The experiments show that the mAb secondary structure is not impacted but the microenvironment around aromatic residues is impacted, leading to a small but immediate increase in protein aggregation followed by a dose-dependent second phase of slower aggregate growth. A decrease in the first melting points as well as the hydrophobic exposure thermal transition temperatures and a change in chemical denaturation unfolding process indicate that the ferric ions destabilize the protein less stable domain. A homology-based model of the full length IgG indicates that ferric ions binding residue clusters including aromatic residues and aggregation prone regions (APRs) are spatially proximal in its complementarity determining regions (CDRs). We propose that ferric ions bind to the available chelating sites near aromatic residues in a specific manner, leading to minor structural perturbations. This binding also destabilizes the protein less-stable domain, resulting in enhanced aggregation over time. The molecular level observations help us rationalize the experimental results and are relevant to rational design and formulation of biotherapeutic drug candidates for improved molecular integrity.

P-03

Higher Order Protein Structure Comparability Assessment using ECHOS-NMR

Carlos Amezcua; Christina Szabo

Baxter Healthcare Corporation, Round Lake, IL USA

An NMR method for quantitative assessment of higher order structure (HOS) comparability of protein- based biopharmaceutical products, named ECHOS-NMR (Easy Comparability of Higher Order Structure by NMR), has been developed. ECHOS-NMR uses the correlation coefficient derived from linear regression analysis of binned NMR spectra to estimate the similarity between samples. The method was successfully applied to 1D- and 2D-NMR spectra of non-glycosylated proteins spanning a molecular weight range of 6-67 KDa. It will facilitate and increase the use of NMR as an orthogonal analytical technique in comparability exercises for product development and submission to regulatory agencies.

61 NOTES:

62 P-04

Development of Far-UV and Near-UV Circular Dichroism Method for the Characterization and Comparability of Protein Higher Order Structure

Hai Yue

Washington University in St. Louis, University City, MO USA

P-05

Developing an Automated Workflow for Disulfide Linkage Analysis of Therapeutic Proteins

Henry Shion; Scott Berger; Stephane Houel; Weibin Chen

Waters Corporation, Milford, MA USA

Disulfide bond formation is critical for facilitating the three-dimensional folded structure and for maintaining the proper function of therapeutic proteins. Confirming the presence of expected disulfide linkages and demonstrating the absence of mispaired disulfides have become a standard element of a biotherapeutic characterization package. In this study, non-reduced peptide maps were generated and processed for several proteins using an integrated workflow containing on-line UPLC/MSE QTof MS detection coupled with an informatics platform for automated data processing and interpretation. Disulfide containing peptides were identified by accurate precursor ion masses and confirmed by the presence of peptide fragment ions. Both expected and unexpected (scrambled) disulfide linkages could be allocated by the automated workflow and presented using a customized reporting template. The ability to deploy a holistic system, combining high performance LC-MSE acquisition and automated disulfide map processing, should significantly reduce the burden of this complex characterization task, enabling higher sample throughput or reallocation of analytical resources to other equally important biotherapeutic structural questions.

63 NOTES:

64 P-06

Development of Antibody Arrays for Monoclonal Antibody Higher Order Structure Analysis

Xing Wang

Array Bridge, Inc., St. Louis, MO USA

Protein Higher Order Structure is important to its safety and efficacy but difficult to define. A novel technology is developed using antibody arrays to analyze monoclonal antibody therapeutics Higher Order Structure. This technology provides a sensitive, systematic and high-throughput approach for mAb Higher Order Structure comparability analysis, generating valuable information for cell line selection, process development and formulation development. Examples will be presented to demonstrate the applications of the antibody array in biosimilar as well as novel mAb development and its complementary value to the bioassays and other analytical technologies.

P-07

The Use of Temperature Dependent Intrinsic Fluorescence for the Analysis of In-process Stability of a Protein

Yamuna Dasarathy1; John Welsh2; Stephanie Hyde2; John Woodgate2

1Pall Life Sciences, Port Washington, NY USA; 2Pall Europe Limited, Portsmouth, United Kingdom

The manufacturability of proteins is heavily influenced by their stability to the processing conditions required to purify and formulate them. It would therefore be very useful to have a fast, efficient method for screening a wide range of manufacturing conditions to discover the optimal conditions for each step and, more importantly, the conditions to avoid. To demonstrate this approach a study was undertaken using in-process samples of a monoclonal antibody from a conventional purification development. Each stage of the process was assessed using a DOE approach to map the design space and to find the optimal processing conditions. In parallel, a stability assessment of the antibody at each stage was carried out using the Optim®1000 instrument to determine the overall manufacturability of the product as impacted by the Concentration of the protein in solution, the buffer in which it is stored (pH and/or conductivity as appropriate) and the stage of the process.

65 NOTES:

66 P-08

Evaluating the Impact of Oxidation on Glucocerebrosidase Higher Order Structure

John J. Thomas1; Cedric E. Bobst2; Gang Yan1; Muthu Meiyappan1; Paul Salinas1; Donald Gillies1; Igor A. Kaltashov2; Phil Savickas1; John Amari1; Mark Hardy1

1Shire Human Genetic Therapies, Lexington, MA USA; 2University of Massachusetts, Amherst, MA USA

Characterizing and monitoring the structural effects of protein oxidation requires several orthogonal techniques. Here we present three methodologies, size-exclusion chromatography (SEC), hydrogen- deuterium exchange mass spectrometry (HDX-MS) and in-silico molecular dynamic simulations (MDS) to characterize glucocerebrosidase (GCB), a therapeutic enzyme for the treatment of Gaucher disease. GCB is a monomeric glycoprotein that includes two disulfide bonds and three free cysteines. This work demonstrates the structural effects of GCB oxidation and the ability to monitor the monomeric purity of this therapeutic product.

SEC was performed using a phosphate buffer mobile phase (pH 6.1) and UV detection on GCB stored at 4-8 °C/4months (temperature-stressed) and -65°C with no added oxidants. HDX experiments were carried out using forced oxidized GCB generated from hydrogen peroxide exposure (0.1 %, 25° C, 3 hours). Over a time course, deuterium-exchanged peptides were separated by reversed-phase LC and analyzed on a quadrupole time-of-flight mass spectrometer. MDS were performed in 1 nanosecond steps at a temperature of 300 K using BioLuminate 1.0 software.

SEC analysis of GCB shows two low-level shoulder peaks with apparent molecular weights corresponding to monomeric GCB. Peptide mapping indicates temperature stress increased the level of one shoulder peak with an elevated level of oxidation on one prominent cysteine, whereas an earlier- eluting shoulder peak was highly oxidized on multiple sites. The elution times of the oxidized species suggest reduced column interaction. HDX-MS analysis on oxidatively-stressed GCB shows structural destabilization remote to many of the oxidation sites. Molecular modeling of oxidized GCB reveals subtle changes in the secondary structure with the exception of some loop regions. The combined data from these methodologies suggest oxidation-induced conformational variations lead to changes in chromatographic analysis of GCB.

P-09

Biophysical Characterization of an Alphabody™ Drug Candidate

Richard Dixon1; Vivian Ring1; Jurgen Debaveye2; Thore Hettman2; Ignace Lasters2; Eric Lorent2; Abhinav Shukla1; Tim Kelly1; Prathima Acharya1

1KBI Biopharma, Durham, NC USA; 2Complix NV, Ghent, Belgium

The Alphabody™ is a novel small protein scaffold that incorporates naturally occurring protein structural elements with computer-aided rational design. Comprised of a single-chain, three-helix coiled-

67 coil structure, the Alphabody™ is a highly stable, protease-resistant structure with certain sequence positions dedicated for targeting binding interactions. Complix is developing Alphabodies™ as a platform technology for the discovery and development of high-affinity biopharmaceuticals against a diverse set of disease targets. KBI Biopharma has successfully cloned and expressed an Alphabody™ drug candidate in Pichia pastoris and developed a purification process to support preclinical development and product characterization. Dynamic light scattering (DLS), circular dichroism (CD), and differential scanning calorimetry (DSC) were used to characterize the secondary and tertiary structure of the Alphabody™ drug candidate. In addition, chemical and thermal denaturation studies were used to evaluate the stability of the folded state of the protein. Our results confirmed that the expected secondary structure elements are present in the Alphabody™ and the protein fold has remarkable thermodynamic stability.

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68 P-10

Characterization of a Recombinant Fusion-Protein Drug Substance by Analytical Ultracentrifugation

Christopher Sucato

Blue Stream Laboratories, Cambridge, MA USA

The extent of aggregation in protein biopharmaceuticals can have important consequences for the safety and efficacy of drug formulations. Characterization of aggregates or degradents in drug formulations is a challenging task, due to the reversible nature of some species, and limitations of instrumentation. Here we describe methods that were employed at Blue Stream Labs using sedimentation velocity analytical ultracentrifugation to characterize the size distribution of species in a complex sample comprised of a peptide-protein conjugate. Centrifugation of sample was performed using a Beckman XLA analytical ultracentrifuge with UV-detection. The resulting sedimentation profiles were analyzed with the continuous sedimentation coefficient-distribution method. The capabilities of the fitting model to accurately resolve the distribution profile were then evaluated. Work here is focused on crucial parameters including the confidence level (F-ratio) and the grid spacing. Data will be presented that demonstrate how the appropriate use of the algorithm can successfully quantify structural variants that are closely-spaced in mass, and that monomeric fusion variants can be reliably distinguished from higher order aggregates. This methodology is widely applicable to address challenging structural features of protein biopharmaceuticals.

P-11

Optimization of Sub-micron Size and Count Measurements using Nanoparticle Tracking Analysis (NTA)

Duncan Griffiths1; Sonja Capracotta1; Gary Linz1; Jonathan Smith2; Patrick Hole2

1NanoSight Inc., Costa Mesa, CA USA; 2NanoSight Ltd., Amesbury, United Kingdom

Nanoparticle Tracking Analysis (NTA) is the only automated method for counting protein aggregates as small as 30 nm and has gained increasing popularity for the characterization of these polydisperse distributions. As these measurements have become more important in understanding product stability, improvements in reproducibility and accuracy have been achieved through optimization of sampling statistics and analytical protocols. As a particle-by-particle analysis, the statistics required for optimum reproducibility and resolution need to be considered. The requirements for sufficient statistics depend on the requirements of the analysis and sample type. Analyzing increasing numbers of particles can be achieved by extending analysis times or by multiple sub-samplings. Data shows that a practical limit is reached beyond which increasing analysis time shows a diminished improvement in reproducibility. Multiple samplings show further improvement. Additionally, carrying out analyses under a constant slow flow rather than static provides a further significant improvement. Monodisperse materials such as

69 standards can be well characterized even with short run times. For polydisperse distributions as most aggregated protein samples are, guidelines have been developed to optimize the experimental design. When taking multiple measurements, the series of separate results can be used to estimate the error of parameters. The standard error can be used to estimate the ‘error’ on a particular parameter, allowing confidence intervals to be constructed and hypothesis tests to be performed. Repeat measurements should be taken to allow a sufficient number of particles to be analyzed and to enable estimates of the errors of certain parameters (mean, distribution, standard deviation, concentration) using standard errors. Fresh sample should be introduced between measurements, or sample can be continuously flowed through, to increase particle numbers. The number of repeats should be increased until desired precision is achieved.

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70 P-12

Fluorescence Spectroscopy Combined with Principal Component Analysis for Characterization of Unfolding in mAbs

Satyan Pai; Jessica Stark; Vikas Sharma

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

Fluorescence spectroscopy is used extensively in studying protein dynamics and conformation. In particular, intrinsic trytophan fluorescence of proteins is sensitive to changes in the surrounding environment (i.e. polarity, proximity of surrounding residues, solvent exposure) and can reveal information about protein conformation states. In this study, the thermal unfolding of six therapeutic monoclonal antibodies (including IgG1, IgG4, aglycosylated, glycosylated, and bispecific frameworks) in various pH and buffer systems was monitored using tryptophan fluorescence. Several data analysis techniques (such as monitoring emission wavelength at 362nm, intensity at lambda max, and global principal component analysis) were evaluated for their ability to extract protein conformational information from the fluorescence data. The structural changes seen in the fluorescence data were also confirmed using orthogonal techniques such as differential scanning calorimetry (DSC) and near-UV circular-dichroism (CD). The combination of intrinsic fluorescence with PCA provided a more direct means of measuring protein Tm values as compared to DSC, which requires significant data manipulation to determine the melting onset temperature. The shift in Tm values as a function of pH and formulation buffer species was successfully monitored and, in some cases, used to guide formulation screening studies. For one of the molecules analyzed, mAb D, a low-temperature conformational transition was detected by fluorescence whereas no such transition was observed in the DSC data. This transition is currently the topic of further investigation. This study showed how tryptophan fluorescence combined with principal component analysis may be used to monitor the thermal-induced onset of unfolding events in mAbs and guide formulation selection. The techniques developed are also conducive to high-throughput formulation screening formats, which are currently being evaluated.

P-13

Amide Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) of Therapeutic Protein at Various pHs

Kathleen Molner; Stephen Coales; Yoshitomo Hamuro

ExSAR™ Corporation, Monmouth Junction, NJ USA

Proteins are the fastest growing class of therapeutics. Additional analysis is necessary for their development and production compared with traditional small molecule drugs, due to their large size and complex structure. The protein construct and its formulation have to be optimized during development, and its structural integrity must be closely monitored during production. HDX-MS is the ideal analytical tool to monitor protein structural integrity in the development and production of whole protein

71 therapeutics. HDX-MS can rapidly determine the effects of mutation, chemical modification, formulation change and/or process change on protein folding/dynamics during development. HDX-MS can localize any changes to 5 – 15 amino acid residues long resolution. Human growth hormone (hGH) therapy has been applied in the treatment of growth hormone-deficient children to promote linear growth since 1984. The EC approved Sandoz' hGH as the first biosimilar in 2006 for the treatment of growth hormone deficiency. hGH is known to exhibit distinct conformations at acidic and neutral pH. While the native state is populated at neutral pH, an alternative less stable conformation is populated at acidic pH. Although the molecular conformations at acid and neutral pH share virtually identical extents of secondary structure, differences in the tertiary structure have been observed. The less stable acidic conformation is also implicated as the intermediate for undesirable aggregation. Here, HDX-MS experiments were conducted to detect the change in dynamic characteristics of hGH at four different pHs.

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72 P-14

Mapping of Discontinuous Conformational Epitopes by Amide Hydrogen/Deuterium Exchange Mass Spectrometry

Stephen Coales; Steven Tuske; Yoshitomo Hamuro

ExSAR™ Corporation, Monmouth Junction, NJ USA

Epitope mapping is a critical step for therapeutic antibody development for scientific, regulatory and intellectual property (IP) reasons. Recently, many pharmaceutical companies are including epitope information in their antibody patent applications. Hydrogen/deuterium exchange mass spectrometry technology (HDX-MS) is a widely-applicable analytical technique compatible with discontinuous conformational epitopes. HDX-MS epitope mapping works as follows: First, an antigen is deuterated in the presence and absence of its antibody. Second, the deuterated antigen is digested by immobilized pepsin (digestion). Third, the deuteration levels of each antigen peptide are determined by LCMS (LC- MS analysis). The antigen peptides which deuterated less in the presence of its antibody are the HDX- MS identified epitopes. Several real world examples of HDX-MS epitope mapping will be described in this presentation. In all cases, the epitopes identified by a medium-resolution and widely-applicable HDX-MS method are in good agreement with the epitopes identified by high-resolution yet difficult-to- obtain X-ray co-crystal structures.

P-15

Hydrogen/Deuterium Exchange Reveals the Conformational Changes of Human α1-Acid Glycoprotein upon Glycosylation

Richard Yu-Cheng Huang1, 2; Jeffrey Hudgens1, 2

1NIST, Gaithersburg, MD USA; 2IBBR, Rockville, MD USA

Human α1-Acid Glycoprotein (AGP) or orosomucoid, a 40 kDa acute phase glycoprotein, presents predominantly in blood. The glycosylation and serum concentration of AGP change in response to tissue injury, inflammation or infection, and these changes are highly correlated with hepatic synthesis. With its ability to bind basic, lipophilic, and acidic drugs, AGP has been served as a suitable drug carrier. It has been shown that the drug-binding activities of AGP depend strongly on its carbohydrate composition. The details of the effects of glycosylation on the AGP conformation, however, are unknown. Here we report the use of hydrogen/deuterium exchange (H/DX) coupled with mass spectrometry to elucidate the changes in conformation and dynamics of AGP upon different types and degree of glycosylation. Our peptide-level H/DX not only reveals the dynamics of AGP variants, but also demonstrates that glycosylation modulates dynamics of five peptide regions (residue 9-18, 44-48, 50-61, 80-89, and 114-127) surrounding the ligand-binding cavity of AGP, which must cause significant impact on its drug-binding activities and immunomodulatory function.

73 NOTES:

74 P-16

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals the Binding Interfaces Between Proliferating Cell Nuclear Antigen (PCNA) and SPIP

Richard Yu-Cheng Huang1, 2; Zhuo Li2; Zvi Kelman1, 2; Jeffrey Hudgens1, 2

1NIST, Gaithersburg, MD USA; 2IBBR, Rockville, MD USA

Proliferating Cell Nuclear Antigen (PCNA) is a 29 kDa protein that forms a stable trimers. PCNA encircles duplex DNA and upon binding to DNA polymerase increases its processivity. In addition to its interaction with the polymerase, PCNA was shown to bind, and regulate the activity of many other enzymes involved in nucleic acid metabolic process. We previously observed that a unique 9.8 kDa small PCNA interacting protein, SPIP, can interact with PCNA in vivo. Recently, we have shown that the interaction leads to PCNA dissociation. The detailed information of the interaction interfaces on these two members, however, is unknown. We have used hydrogen deuterium exchange mass spectrometry (H/DX-MS) coupled with enzymatic digestion to investigate the protein dynamics together with the interfaces between these two members in the complex. Our peptide-level H/DX results on the protein dynamics confirm the trimeric conformation of PCNA and further reveal the unstructured nature of SPIP. A comparison of apo-PCNA (PCNA alone) and holo-PCNA (PCNA-SPIP) provides information of the complex interfaces located on PCNA, whereas the comparison of apo-SPIP and holo- SPIP provides information of the complex interfaces located on SPIP. We conclude that peptide region 187-204 of PCNA, located outside of the ring structure, interacts with either peptide region 22-28 or region 55-58 of SPIP during the complex formation. Moreover, peptide region 104-110 of PCNA, located at the trimer interface, becomes more structurally flexible upon SPIP binding, indicating trimer dissociation of PCNA. The outcome reported in this study provides information for future mutagenesis, which aims to alter the PCNA activities by modulating the PCNA-SPIP interactions.

P-17

Analysis of cGMP/cAMP Binding to the PKGI Beta Regulatory Domain by HDX using High Resolution Orbitrap Mass Spectrometer

Jawad Pashmi1; Sheng Li2; Bryant Kou2; Darren Casteel2

1Thermo Fisher Scientific, San Jose, CA USA; 2University of California, San Diego, San Diego, CA

The type I cGMP-dependent Protein Kinases (PKGI) plays important roles in regulating the cardiovascular and nervous systems. Mammalian cells express two isoforms of PKGI (PKGI and PKGI) 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 PKGI regulatory domain (amino acids 4-352) when bound to saturating amounts of cGMP or cAMP (1 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

75 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 PKGI, these data provide insight into the mechanism of cyclic nucleotide specific activation of PKGI.

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76 P-18

Structure and Functional Analysis of Survivin Protein using HDX and High Resolution Mass Spectrometry

Rachel Altura1; Dennis Pantazatos1; Jawad Pashmi2

1Brown University, Providence, RI USA; 2Thermo Fisher 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-19

A Study of Apolipoprotein E4 Oligomerization by H/DX and Electron-transfer Dissociation

Richard Huang2; Jawad Pashmi1

1Thermo Fisher 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’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 involved in apoE oligomerization.

77 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.

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78 NOTES: