Table of Contents · Applied Photophysics Ltd. BioTools, Inc. ExSAR™ Corporation JASCO, Inc. SGS M-Scan, Inc. Waters Corporation Wyatt Technology Corporation Media Partners BioPharm

1

Table of Contents

Program, Exhibitor and Media Partners ............................................................ 2

Acknowledgements ........................................................................................... 4

Student Travel Grants ....................................................................................... 5

Scientific Final Program Summary ................................................................... 6

Plenary and Session Abstracts .......................................................................... 13

Technical Seminar I (Monday, September 26) Abstract .................................. 50

Technical Seminar II (Tuesday, September 27) Abstract ................................. 51

Poster Abstracts ................................................................................................ 52

2

The Organizing Committee gratefully acknowledges the Corporate Program Partners for

their generous support of the 1st Symposium on Higher Order Structure of Protein

Therapeutics

Platinum Program Partner

Genentech, a Member of the Roche Group

Gold Program Partners

Amgen, Inc.

Biogen Idec Inc.

Eli Lilly and Company

MedImmune

Novo Nordisk A/S

Waters Corporation

Bronze Program Partner

GlaxoSmithKline Pharmaceuticals

3

Exhibitor Partners

Applied Photophysics Ltd.

BioTools, Inc.

ExSAR™ Corporation

JASCO, Inc.

SGS M-Scan, Inc.

Waters Corporation

Wyatt Technology Corporation

Media Partners

BioPharm International

Genetic Engineering & Biotechnology News

Technology Networks.com

4

Acknowledgements

Symposium Co-Chairs: Wasfi Al-Azzam, GlaxoSmithKline Pharmaceuticals

John Dougherty, Eli Lilly and Company

Scientific Program Committee: Katherine Bowers, Fujifilm Diosynth Biotechnologies

John Carpenter, School of Pharmacy, University of Colorado at Denver

Mary Cromwell, Genentech, a Member of the Roche Group

Daron Freedberg, CBER, FDA

Wolfgang Friess, University of Erlangen

Parvez Haris, De Montfort University

Wim Jiskoot, LACDR, Leiden University

Tom Laue, University of New Hampshire

Aston Liu, GlaxoSmithKline Pharmaceuticals

Radhika Nagarkar, KBI Biopharma Inc.

Linda Narhi, Amgen Inc.

Tom Patapoff, Genentech, a Member of the Roche Group

Satish Singh, Pfizer, Inc.

Bonnie Wallace, Birbeck College, University of London

Ziping Wei, MedImmune

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

CASSS Staff: Karen A. Bertani, CMP, Symposium Manager

Stephanie L. Flores, CAE, Executive Director

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 1st International Symposium on Higher Order Structure of Protein

Therapeutics (HOS 2011). PhD students or post-doctoral fellows conducting research at academia

throughout the world are eligible.

This year‟s winners include:

Jennifer D'Antonio

North Carolina State University, Raleigh, NC USA

Comprehensive Analysis of Protein Therapeutics Secondary Structure Comparability by Fourier

Transform Infrared Spectroscopy

Lin Guo

University of Pennsylvania, Philadelphia, PA USA

Islet Amyloid Polypeptide Aggregation: Can We Probe the Difference that One Sidechain Makes?

Trushar Patel

University of Manitoba, Winnipeg, Canada

Towards a Structure-function Analysis of Wnt Proteins

6

1st International Symposium on Higher Order Structure of

Protein Therapeutics Scientific Program Summary

MONDAY, SEPTEMBER 26, 2011

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

07:30 – 08:45 Continental Breakfast in the Plaza Ballroom Foyer

08:45 – 09:00 Welcome and Introductory Comments

John Dougherty, Eli Lilly and Company

Wasfi Al-Azzam, GlaxoSmithKline Pharmaceuticals

Fundamentals of Higher Order Structure

Plenary Session in Plaza Ballroom I

Session Chairs: Katherine Bowers, Fujifilm Diosynth Biotechnologies and Wim Jiskoot, Leiden

University

09:00 – 09:25 Protein Engineering Functional Switches

Philip Bryan, University of Maryland, Rockville, MD USA

09:25 – 09:50 Non-Native Aggregate Assembly and Structure: A Multi-Scale Problem

Christopher Roberts, University of Delaware, Newark, DE USA

09:50 – 10:15 Chemical Protein Glycosylation: A New Approach to Protein Stabilization

Kai Griebenow, University of Puerto Rico, San Juan, Puerto Rico

10:15 – 10:45 AM Break – Visit the Exhibits and Posters in Plaza Ballroom II/III

10:45 – 11:10 Using Vibrational, Optical and NMR Spectroscopy to Explore Unfolded and

Misfolded States of Peptides

Reinhard Schweitzer-Stenner, Drexel University, Philadelphia, PA USA

11:10 – 11:35 Conformational Changes and Aggregate Structure: Can We Connect the

Dots for IgG Molecules? Alla Polozova, MedImmune, Gaithersburg, MD USA

11:35 – 12:00 Advantages and Limitations of Hydrogen/Deuterium Exchange with Mass

Spec Detection (H/DX-MS) in Conducting Higher Order Structural

Comparability Studies on Protein Therapeutics Steven A. Berkowitz, Biogen Idec Inc., Cambridge, MA USA

7

MONDAY, SEPTEMBER 26 continued…

12:00 – 12:25 Capabilities and Limitations of Methods used for Higher Order Structure

Characterization of Therapeutic Proteins

Ingo Lindner, Roche Diagnostics GmbH, Penzberg, Germany

12:30 – 13:00 Discussion – Questions and Answers

13:00 – 14:45 Hosted Lunch Break – in Plaza Ballroom II/III

13:30 – 14:30 Technical Seminar

Shaping Innovation for Higher Order Structure by Mass Spectrometry: Incorporating Ion

Mobility and Hydrogen Deuterium Exchange

Sponsored by Waters Corporation Plaza Ballroom I

Quality Approaches for Validation

Plenary Session in the Plaza Ballroom I

Session Chairs: Daron Freedberg, CBER, FDA and Linda Narhi, Amgen, Inc.

14:45 – 15:10 Higher Order Structure of Protein Products – Who Needs It?

Emily Shacter, CDER, FDA, Bethesda, MD USA

15:10 – 15:35 Qualification of CD and FTIR Spectroscopic Methods and their Applications

Cynthia Huimin Li, Amgen Inc., Thousand Oaks, CA USA

15:35 – 16:00 Circular Dichroism Spectroscopy for Higher Ordered Structures:

Validation, Calibration, Analyses, and the Protein Circular Dichroism Data

Bank (PCDDB)

Bonnie Wallace, Birbeck College, University of London, London, United

Kingdom


17:00 – 18:30 Exhibitor and Poster Reception in Plaza Ballroom II/III

18:30 Adjourn Day One

8

TUESDAY, SEPTEMBER 27, 2011


07:30 – 08:30 Continental Breakfast in Plaza Ballroom II/III

08:30 – 08:45 Announcements by John Dougherty, Eli Lilly and Company

Higher Order Structure Analysis: Challenges in Early Phase Development


Session Chairs: Tom Patapoff, Genentech, a Member of the Roche Group and Ziping Wei, MedImmune

08:45 – 09:10 Regulatory Considerations and Expectations on Higher Order Structure of

Therapeutic Proteins during the Different Phases of Development

Birgit Schmauser, BfArM, Bonn, Germany

09:10 – 09:35 Higher Order Structure Analysis in Protein Therapeutics Development

Yijia Jiang, Amgen, Inc., Thousand Oaks, CA USA

09:35 – 10:00 Pre-candidate Selection Screening for Developability

Emma Harding, GlaxoSmithKline R & D, Stevenage, United Kingdom



Higher Order Structure Analysis: Challenges in Commercial Phase Development


Session Chairs: Mary Cromwell, Genentech, a Member of the Roche Group and Bonnie Wallace,

Birbeck College, University of London

11:00 – 11:25 Higher Order Structure Information Useful for Biological License

Applications (BLAs)

Patrick Swann, CDER, FDA, Bethesda, MD USA

11:25 – 11:50 Challenges in Developing a Semi-Quantitative Visible Particulate Method for

Routine Commercial Testing George Saddic, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA USA

11:50 – 12:15 Sensitivity of Spectroscopic and Chromatographic Assays in Detecting HOS

Variants in Therapeutic Monoclonal Antibodies Andrew Kosky, Genentech, a Member of the Roche Group, South San Francisco,

CA USA


12:45 – 14:30 Lunch Break – Participants on their own

9

Tuesday, September 27 continued

Higher Order Structure Analysis: Challenges in Lifecycle Management

Plenary Session in the Plaza Ballroom I

Session Chairs: Satish Singh, Pfizer, Inc. and Radhika Nagarkar, KBI Biopharma Inc.

14:30 – 14:55 Regulatory Expectations for Post-marketing Changes for Biologics

Supported by Higher Order Structure Analysis Ravi Harapanhalli, PAREXEL Consulting, Bethesda, MD USA

14:55 – 15:20 Higher Order Structure Characterization of a Fusion Protein to Support

Process and Container Closure Changes

Angela Blake-Haskins, Human Genome Sciences, Inc., Rockville, MD USA

15:20 – 15:45 New Methods for Therapeutic Protein Higher Order Structure

Characterization

Henryk Mach, Merck Research Laboratories, West Point, PA USA


16:15 – 17:15 Poster Session One in Plaza Ballroom II/III

17:15 – 18:15 Technical Seminar

Protein Characterization with Light Scattering

Sponsored by Wyatt Technology Corporation Plaza Ballroom I

18:15 – 18:45 Mini-Break in Plaza Ballroom II/III

Young Scientists


Session Chairs: Wasfi Al-Azzam, GlaxoSmithKline Pharmaceuticals and John Dougherty, Eli Lilly and

Company

18:45 – 19:00 Islet Amyloid Polypeptide Aggregation: Can We Probe the Difference that

One Sidechain Makes?

Lin Guo, University of Pennsylvania, Philadelphia, PA USA

19:00- – 19:15 Towards a Structure-function Analysis of Wnt Proteins

Trushar Patel, University of Manitoba, Winnipeg, Canada

19:15 – 19:30 Comprehensive Analysis of Protein Therapeutics Secondary Structure

Comparability by Fourier Transform Infrared Spectroscopy

Jennifer D'Antonio, North Carolina State University, Raleigh, NC USA

10

Tuesday, September 27 continued


19:45 Adjourn Day Two

11

WEDNESDAY, SEPTEMBER 28, 2011


07:30 – 08:30 Continental Breakfast in Plaza Ballroom II/III

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

Biological Implications


Session Chairs: Claire Holland, GlaxoSmithKline Pharmaceuticals and Jamie Moore, Genentech, a

Member of the Roche Group

08:45 – 09:10 An Immunological Appreciation of Structure

Amy Rosenberg, CDER, FDA, Bethesda, MD USA

09:10 – 09:35 Higher-order Structural Consequences of Product Degradation in Interferon

Alpha

Mary Alice Hefford, Health Canada, Ottawa, Ontario, Canada

09:35 – 10:00 HOS: Challenges in Linking Form with Function

Valerie Quarmby, Genentech, a Member of the Roche Group, South San

Francisco, CA USA



New and Emerging Technologies


Session Chairs: Thomas Laue, University of New Hampshire and Aston Liu, GlaxoSmithKline

Pharmaceuticals

11:00 – 11:25 Cutting Edge Vibrational Spectroscopy for Protein Therapeutics

Rina Dukor, BioTools, Inc., Jupiter, FL USA

11:25 – 11:50 Fluorescent Dye-based Methods to Detect Changes in Higher Order

Structures Wim Jiskoot, LACDR, Leiden University, Leiden, The Netherlands

11:50 – 12:15 Protein Electrostatics, Charge Heterogeneity and Protein Rheology

Devendra Kalonia, University of Connecticut, Storrs, CT USA

12:15 – 12:40 Structures of Dynamic Protein Complexes: Prospects for 3D Electron

Microscopy at Atomic Resolution

Sriram Subramaniam, National Cancer Institute, NIH, Bethesda, MD USA

12

Wednesday, September 28, 2011 continued

12:45 – 13:15 Discussion - Questions and Answers

13:15 – 13:30 Closing Remarks by Wasfi Al-Azzam, GlaxoSmithKline Pharmaceuticals

13

Fundamentals of Higher Order Structure Session Abstract

Higher order structures in protein therapeutics, such as aggregates, particulates and amyloids, are critical

areas of study and concern for both academic and industrial researchers. What are protein higher order

structures, what drives the formation of these species from a protein folding, stability and

thermodynamic perspective and what are the biophysical tools that are currently utilized to study these

forms of protein therapeutics? The Fundamentals of Higher Order Structure session of HOS 2011 will

start to address some of these questions. Diverse speakers from industry and academia will present the

foundations of protein folding, stability and the formation of higher order structures and expound on the

biophysical methods that are being used to better understand the mechanism of formation and the

structure of these species. This opening session will provide the audience with a strong foundation

regarding protein therapeutic higher order structures that will built upon as the symposium progresses

into more in-depth discussions of current and emerging biophysical methods, quality and validation

activities and the biological implications of the presence of higher order structures in protein

therapeutics.

Session Questions:

1) What are protein higher order structures (definition)?

2) From a protein folding, stability and thermodynamic perspective, what are the driving forces for

the formation of these species?

3) What biophysical tools are currently available to study protein higher order structures?

4) What are the challenges from an analytical perspective in studying protein higher order

structures?

NOTES:

14

Plenary Session Abstracts

Protein Engineering Functional Switches

Philip Bryan

University of Maryland, Rockville, MD USA

Understanding the propensity of a protein to fold into a completely different structure as a result of

minor perturbation is central to understanding both protein folding in general and more specifically how

new protein structures and functions evolve. Our approach was to create two proteins which 1) are

stably folded into two different folds, 2) have two different functions, and 3) are very similar in

sequence. In this simplified sequence space we explore the mutational path from one fold to another. We

show that an IgG-binding, 4β+ fold can be transformed into an albumin-binding, 3- fold via a

mutational pathway in which neither function nor native structure is lost. On one side of the switch

point, the 4β/1 fold is >95% populated. A single mutation switches the conformation to the 3 fold

which is >95% populated. Basic principles for the design and characterization of proteins which switch

conformation will be presented.

NOTES:

15

Non-Native Aggregate Assembly and Structure: A Multi-Scale Problem

Christopher Roberts

University of Delaware, Newark, DE USA

Non-native aggregation is well known to involve some degree of disruption to the native tertiary and/or

secondary structure of proteins, but the extent of structural change that is needed to promote aggregation

is often not well known or discernable experimentally. Furthermore, initially small aggregates may grow

via different mechanisms, leading to different types of 'higher-order' structure or morphology spanning

multiple length scales. This presentation focuses on experimental examples for therapeutic and model

proteins, to examine changes in protein structure that span from small sequences to higher-order / larger

length-scale structures and morphology, and how this is related to monomer-monomer, aggregate-

monomer, and aggregate-aggregate interactions in solution, as well as thermodynamic driving forces for

creation or suppression of large aggregates and particles.

NOTES:

16

Chemical Protein Glycosylation: A New Approach to Protein Stabilization

Kai Griebenow

University of Puerto Rico, San Juan, Puerto Rico

Protein pharmaceuticals have outstanding potential in the cure and prevention of diseases and conditions

and have already substantially expanded the field of molecular pharmacology. Unfortunately, proteins

(and peptides) frequently display substantial chemical and physical instabilities hampering their success

as drugs. Detrimental stresses encountered during manufacturing, storage, delivery, and other

pharmaceutically relevant processes, frequently alter the chemical composition and the three-

dimensional structure of proteins thus negatively impacting their therapeutic efficacy and giving rise to

potential safety hazards for patients (e.g., immune reactions triggered by protein aggregates). This has

prompted an intense search for novel strategies to stabilize pharmaceutical proteins. Due to the well

known effect of glycans in increasing the overall stability of glycoproteins, rational manipulation of the

glycosylation parameters through glycoengineering could become a promising approach to improve both

the in vitro and in vivo stability of protein-based pharmaceuticals. The intent of this presentation is to

survey the different physicochemical instabilities displayed by proteins during their pharmaceutical

employment, how these can be prevented by glycosylation and to discuss the currently proposed

biophysical models by which glycans induce these stabilization effects.

NOTES:

17

Using Vibrational, Optical and NMR Spectroscopy to Explore Unfolded and Misfolded States of

Peptides

Reinhard Schweitzer-Stenner1; Andrew Hagarman

1, 2; Thomas Measey

1, 3; Daniel Verbaro

1; Siobhan

Toal1

1Drexel University, Philadelphia, PA USA;

2Department of Biochemistry, Duke University, Durham, NC

USA; 3Department of Chemistry, University of Pennsylvann, Philadelphia, PA USA

Over the last ten years multiple lines of evidence have been gathered in support of the notion that amino

acid residues in unfolded peptides and proteins do not sample the entire sterically allowed region of the

Ramachandran plot with nearly identical probabilities, as suggested by the classical random coil model.

The degree of disorder of a peptide or protein depends on the amino acid composition. We have started a

program aimed at obtaining the conformational distributions of all twenty amino scid residues in short

unfolded peptides. In this context we used GxG peptides (x:representing all 20 amino acids) as host-

guest system to determine the intrinsic conformational propensities of amino acid residues in aqueous

solution. To this end we measured and analyzed the amide I' profiles of the respective IR, polarized

Raman and vibrational circular dichroism spectra as well as seven J-coupling constant which all depend

on the dihhedral angles of the central x-residue. We found that alanine has a very high propensity of

polyproline II (pPII), the respective mole fraction is 0.78. Residues with (partially) hydrophobic side

chains exhibit a more balanced population of pPPII and β-strand. Branched aliphatic and aromatic

residues exhibit a slight preference for β-strand. Residues with side chains capable to donate or accept

hydrogen bonds (S, C, T, N and D) exhibit a disprotortional fraction of turns. The turns propensity is

particularly high for D. We investigated a limited number of peptides with non-glycine neighbours

(AAA,VVV,ADA). The results provided evidence for significant nearest neighbor interactions.

Moreover, we investigated the structural properties of various poly-alanines. We found that they

generally maintain an extended, pPII rich structure. However, if alanines were doped with e.g. positively

charged residues like lysine self-aggregation into fibrils and colloid like structures occur which can be

analyzed by vibrational spectroscopy.

NOTES:

18

Conformational Changes and Aggregate Structure: Can We Connect the Dots for IgG Molecules?

Alla Polozova

Analytical Biochemistry, MedImmune, Gaithersburg, MD USA

Aggregation is one of the major degradation pathways of therapeutic proteins. Understanding root

causes of aggregation is very important for successful development of safe and efficacious products.

Multiple factors, including conformational and colloidal stability of proteins, can play a role in

aggregation. Analysis of aggregate structure can shed light on the aggregation pathways. This

presentation will explore the connection between aggregate structure and aggregation pathways. Case

studies with IgG molecules will be discussed.

NOTES:

19

Advantages and Limitations of Hydrogen/Deuterium Exchange with Mass Spec Detection (H/DX-

MS) in Conducting Higher Order Structural Comparability Studies on Protein Therapeutics

Steven A. Berkowitz

Biogen Idec Inc., Cambridge MA USA

Two of the most important attributes of a protein are its higher order structure and its associated

structural dynamics. The analytical tools available to study and evaluate these two critical attribute areas

of protein therapeutics in a practical and routine manner for the purpose of assessing the biophysical

comparability in a biopharmaceutical setting are greatly lacking in sensitivity and spatial resolutions in

the case of higher order structure and non-existent in the case of structural dynamics. H/DX-MS offers

significant hope to improve this situation. Recent developments in instrumental hardware, along with

computer software now offer opportunities (for this nearly two decade old technique) to make

significant contributions in helping to build a much more capable biophysical toolbox to support

biopharmaceutical comparability studies. This talk will discuss these potential capabilities of H/DX-MS,

its present limitations, and future opportunities for overcoming these limitations.

NOTES:

20

Capabilities and Limitations of Methods used for Higher Order Structure Characterization 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. Protein drugs derived by recombinant DNA technology require an

extensive and stringent characterization of identity, purity and stability, as molecular modifications may

result in undesirable adverse biological effects. Especially the integrity of the higher order structure of

therapeutic proteins is an important issue since conformational changes in regions of the protein that are

involved in target binding could affect binding efficacy and can be an safety issue if the structural

change causes adverse effects.

Characterization of the higher order structure of proteins covers the determination of characteristics of

secondary structure like the α-helical and β-sheet part up to the integrity of the disulfide linkage

determining the quaternary structure important in particular in monoclonal antibodies.

Various methods are used to characterize the higher order structure of therapeutic proteins and it is

difficult to say which approach is most suitable. While the covalent structure is usually determined by

mass spec peptide mapping, the higher order structure is typically assessed by classical spectroscopic

methods like FT-IR, fluorescence or circular dichroism. These methods deliver a good overall value

within their range of sensitivity but are not site specific. Additional information can be assessed by

indirect structural measurements with chromatographic methods or hydrogen-deuterium exchange

experiments.

The presentation presents the results of several studies that were performed to show the capabilities and

limitations of different methods used for higher order structure analytics and discuss the suitability of

different methods for the characterization of the higher order structure of therapeutic proteins.

NOTES:

21

Quality Approaches for Validation Session Abstract

HOS analysis is an important part of biotherapuetic development. From the initial determination of the

folded structure of a protein to comparability assessments to demonstrate consistency of HOS across

product lots, these methods are included in every regulatory filing. How are these analyses qualified as

characterization methods, including demonstration of the fit for purpose, sensitivity, etc? This session

will focus on the qualification of HOS analytical methods from industrial, academic and regulatory

viewpoints, including case studies and examples of some approaches that have been successfully

employed, as well as regulatory expectations.

NOTES:

22

Higher Order Structure of Protein Products – Who Needs It?

Emily Shacter

CDER, FDA, Bethesda, MD USA

Proteins are defined by their amino acid sequence and folding. The secondary, tertiary, and quaternary

structure of proteins are all part of their higher order structure. The bioactivities, biodistribution,

pharmacokinetics, pharmacodynamics, and clinical safety and efficacy profiles of proteins all depend on

whether or not they have been synthesized and folded correctly, undergone post-translational in the cell,

and withstood exposure to chemicals and external stresses during purification and storage. The FDA‟s

regulatory expectations for evaluation of protein higher order structure are evolving as improved

analytical tools are becoming available. This talk will address the FDA‟s regulatory experience and

expectations with respect to the evaluation of higher order structure in protein products.

NOTES:

23

Qualification of CD and FTIR Spectroscopic Methods and their Applications

Cynthia Huimin Li

Amgen Inc., Thousand Oaks, CA USA

Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy are widely used to study

protein secondary and tertiary structure by the biopharmaceutical industry to study the effects of

manufacturing, formulation, and storage conditions on protein conformation and stability, and these

results are often included in regulatory filings. A comparison of two or more spectra is often required to

confirm that the protein‟s structure has been maintained. Traditionally, such comparisons have been

qualitative in nature, based on visually inspecting the overlaid spectra. In this presentation, we will

demonstrate the qualification of the CD and FTIR methods that established the precision and assessed

the sensitivity of the methods by the use of a numerical spectral comparison approach and that is the

methods are suitable for protein structural characterization in numerous biopharmaceutical applications.

NOTES:

24

Circular Dichroism Spectroscopy for Higher Ordered Structures: Validation, Calibration,

Analyses and the Protein Circular Dichroism Data Bank (PCDDB)

B.A. Wallace

Birkbeck College, University of London, London, United Kingdom

Circular dichroism (CD) spectroscopy is a widely used and established method for determining

protein secondary structure, monitoring tertiary structure, detecting conformational changes associated

with different conditions including ligand binding, examining stability and macromolecular interactions,

and is a designated method for the characterisation of proteins produced for use in human

pharmaceutical applications. A new extension of the method, synchrotron radiation circular dichroism

(SRCD) spectroscopy, has also been shown to be sensitive to higher order (quarternary) structure.

This talk will discuss methods for calibration and good practice in CD spectroscopic data

collection. It will present user-friendly tools available for processing, comparing, archiving and

analysing CD spectra, including the popular DichroWeb secondary structure analysis server, the new

Protein Circular Dichroism Data Bank (PCDDB) repository enabling access to published CD spectral

data and metadata, and a new program for identifying spectral nearest neighbours, DichroMatch. It will

also discuss the soon-to-be-released common IUPAC-approved JCAMP format for CD data that has

been agreed by representatives of all CD instrument manufacturers and SRCD beamline scientists.

Finally, the new ValiDichro software for evaluating spectral quality will be presented. Validichro

includes >20 test criteria for CD spectra developed in conjunction with an international panel of users,

instrument manufacturers and beamline specialists. It is freely available both as a standalone facility for

QA testing spectra and in conjunction with depositions to the PCDDB. It also produces a date-stamped

report that can be downloaded for documentation, regulatory and publication purposes.

These tools form a comprehensive resource for the calibration, analysis, validation, comparisons

and archiving of CD spectra, which should prove valuable in higher order structure characterisations of

proteins.

(Supported by grants from the UK Biotechology and Biological Sciences Research Council and the

International Union of Pure and Applied Chemistry)

References:

Wallace and Janes (2009) Modern Techniques in Circular Dichroism and Synchrotron Radiation

Circular Dichroism Spectroscopy. IOS Press.

Wallace, B.A. (2009) Protein characterisation by synchrotron radiation circular dichroism spectroscopy.

Quarterly Reviews of Biophysics 42:317-370.

Whitmore, Woollett, Miles, Janes, Wallace (2010) The protein circular dichroism data bank, a web-

based site for access to circular dichroism spectroscopic data. Structure 18:1267-1269.

Whitmore, Woollett, Miles, Klose, Janes,Wallace (2011) PCDDB: The protein circular dichroism data

bank, a repository for circular dichroism spectral and metadata. Nucleic Acids Research 39:D480-D486.

Whitmore and Wallace (2008) Protein secondary structure analyses from circular dichroism

spectroscopy: methods and reference databases. Biopolymers 89:392-400.

NOTES:

25

Higher Order Structure Analysis: Challenges in Early Phase

Development Session Abstract

This session will focus on the applications and challenges of higher order structure analysis during early

stage development of biotherapeutic products. Higher order structure analysis can be applied to evaluate

the stability of product candidates and develop optimized formulation. It is also an essential component

in understanding the relationship between higher order structure, the quality of therapeutic products, and

their impact on safety and efficacy. The understanding of these relationships plays an important role in

product and process understanding during early product development.

NOTES:

26

Regulatory Considerations and Expectations on Higher Order Structure of Therapeutic Proteins

During the Different Phases of Development

Birgit Schmauser

BfArM, Bonn, Germany

Regulatory expectations concerning the data to be submitted in support of Higher Order Structure (HOS)

remain to be defined, especially for early phase of development. HOS is essential for the biological

activity of a therapeutic protein. It is of fundamental importance among the various attributes describing

its quality due to the fact that it is correlated to efficacy. Consequently, from the earliest stage of

(pre)clinical development the use of suitable assays to ascertain the candidate therapeutic protein´s HOS

will support that the intended biological activity is displayed. Due to the inherent variability of

biological assays, techniques to confirm the correct HOS deserve increasing attention with ongoing

clinical development to bridge the gap between the desired consistency in HOS and its limited

predictability from biological activity assays per se. As such HOS analyses may similarly serve to

evaluate how the correct three-dimensional structure is retained during various storage conditions and

against various stress factors in order to build up knowledge on degradation pathways of the protein.

Analyses of HOS complement assays on biological activity. As such they should gain growing relevance

in assuring consistency in correct three-dimensional folding of therapeutic proteins during clinical

development. Regulatory expectations on analyses of HOS depend on the phases of clinical

development. Case studies will be presented to illustrate regulatory considerations.

NOTES:

27

Higher Order Structure Analysis in Protein Therapeutics Development

Yijia Jiang

Product and Process Development, Amgen Inc., Thousand Oaks, CA USA

Protein therapeutics is significantly different from small molecule pharmaceuticals due to the presence

of higher order structures such as secondary, tertiary and quaternary structures. The stresses encountered

during manufacture, transportation and storage processes of protein therapeutics can impact their high

order structure and stability, which may also affect their biological activity and patience‟s safety.

Therefore, understanding HOS changes can provide critical insight into manufacturing processes,

formulation development and characterization. In this presentation, we‟ll provide examples on how

manufacture and storage processes affects the higher order structure of protein therapeutics, the

commonly used techniques for characterizing HOS changes and the effect of the HOS changes.

NOTES:

28

Pre-candidate Selection Screening for Developability

Emma Harding

GlaxoSmithKline R & D, Stevenage, United Kingdom

The presentation will give an insight into how biophysical characterisation and understanding of product

attributes are applied during the discovery phase of drug development. This is used for screening novel

candidate molecules with respect to their „developability‟ including both processing and formulation, in

order that the right molecule is selected to go forward. An introduction to the Biopharm Process

Research department will be given to illustrate how this is carried out at GSK and the proposed benefits

this leads to in development and scale-up. This will focus on product characterisation with case studies

chosen to illustrate examples of the impact of higher order structure on the selection process.

NOTES:

29

Higher Order Structure Analysis: Challenges in Commercial Phase

Development Session Abstract

At the time a Sponsor is preparing for licensure, a significant amount of physicochemical and biological

characterization is performed to critically understand the product. The necessity for characterization of

higher order structures as part of this, the tools used for the assessment, and qualification of those tools

has generated much discussion recently in workshops. Regulatory agency and industry perspectives

regarding the analysis of higher order structure in support of dossiers will be presented.

Session Questions:

1) What kind of higher order structure information is useful to have in a license application for a

biological product? Does higher order structure information need to correlate with biological

function?

2) At the time of product licensure, are there preferred techniques to be employed for analysis of

higher order structure?

3) Are there biophysical methodologies that should NOT be included in license applications? If

so, why?

4) What are the sensitivity, specificity, and reproducibility requirements for techniques that

assess higher order structure used in license applications?

NOTES:

30

Higher Order Structure Information Useful for Biological License Applications (BLAs)

Patrick Swann


This talk will summarize regulatory guidance applicable to HOS information as found in BLAs. Results

of a survey of the type of data provided in BLAs as well as reviewer‟s questions arising from review of

HOS data will be presented. The survey is intended to provide an overview of the current opportunities

and challenges when incorporating HOS information as part of the license application process

NOTES:

31

Challenges in Developing a Semi-Quantitative Visible Particulate Method for Routine

Commercial Testing

George Saddic

GlaxoSmithKline Pharmaceuticals, King of Prussia, PA USA

Sometimes formulation components and sheer introduced by the secondary manufacturing process cause

particle formation. For many QC scientists in biopharmaceuticals, the term particulates equates to sub-

visible testing (particles ≤ 25 µm) and general appearance is solely used to monitor visible particles in

final drug formulations. By virtue of the subjectivity caused by visual acuity, general appearance may

not be enough as results are often inconsistent and inconclusive with respect to identity and quantity of

particles. With the advent of new instrumentation which combines flow cytometry and microscopy, full

characterization is possible. As a post-marketing commitment for Arzerra, GSK was required to develop

and institute a new method for visible particles. This presentation will discuss the challenges and

strategy used in developing a semi-quantitative method for visible particulates using a FlowCAM

Particle Imaging System (Fluid Imaging Technologies, Yarmouth, Maine) and how GSK implemented

its use for commercial DS and DP release testing.

NOTES:

32

Sensitivity of Spectroscopic and Chromatographic Assays in Detecting HOS Variants in

Monoclonal Antibodies

Andrew Kosky

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

We have compared commonly used spectroscopic methods (e.g. circular dichroism (CD) and Fourier-

transform infrared spectroscopy (FTIR)) and conventional chromatographic and functional assays to

determine which types of methods are most sensitive to higher order structural changes in monoclonal

antibodies. Our results demonstrate that commonly used spectroscopic techniques are often less

sensitive than conventional purity and potency assays to the types of structural changes that impact

protein function (in vitro) and overall product quality. We also found that spectra for different

monoclonal antibodies and variants of the same monoclonal antibody are essentially identical. Hence,

biological activity of the monoclonal antibody can be affected without detectable changes in CD or

FTIR spectrum. The spectroscopic methods can provide a direct assessment of the types of secondary

structure present in a monoclonal antibody, but they are not suitable as purity assays. Detailed

characterization data on the primary structure, the disulfide structure, and post-translational

modifications in conjunction with results from chromatographic and functional assays are of greater

value to understanding the overall structure of monoclonal antibodies than the spectroscopic assays.

NOTES:

33

Higher Order Structure Analysis: Challenges in Lifecycle Management

Session Abstract

A biologic product, even after approval, continues to evolve over its lifecycle. Changes ranging from

minor (e.g. filter material, fill volume, fill equipment, lyophilization cycle) to significant (e.g. cell line,

media, drug substance scale, purification process, formulation and concentration, container/closure etc,

lyophilization equipment / scale change) are likely to occur. Maintenance of HOS becomes a vital

parameter to ensure that the quality of the product does not deteriorate as a consequence of the change.

This session will focus on post-approval changes and the utility of HOS analysis in guiding and

enabling, as well as in support of regulatory filing of these changes.

Questions to be addressed will include:

1. What value does HOS analysis add for lifecycle management?

How can the methods be shown to be relevant or fit-for-purpose?

Qualitative or quantitative criteria?

How are “significant change” and “acceptance criteria” defined?

How can changes be correlated to clinical relevance?

2. Are specific techniques used based upon type of change being considered?

What are the limitations of techniques available?

3. Should these techniques be used during stability studies also and applied at both drug substance

and drug product levels?

What is the relevance of changes in accelerated testing?

NOTES:

34

Regulatory Expectations for Post-marketing Changes for Biologics Supported by Higher Order

Structure Analysis

Ravi Harapanhalli

PAREXEL Consulting, Bethesda, MD USA

In the recent years, FDA has recommended a Quality-by-Design (QbD) approach to drug and biologic

development. QbD requires a thorough understanding of a product‟s critical quality attributes and their

functional linkage to the critical process parameters. Understandably, probing a biologic product‟s

critical quality attributes requires a thorough knowledge of structure-activity relationship including the

significance of characterizing higher order structure that may be critical to a product‟s desired biologic

properties. Product characterization focused on higher order structure also helps in developing a robust

comparability program for supporting significant CMC changes that may occur pre- and post-approval.

State-of-the art bioanalytical methods of characterization are expected to probe the three properties of

therapeutic proteins, namely the post-translational modifications, three-dimensional structure, and

protein aggregation.

An assessment of product‟s charge variants (isoforms), glycoform profiling, and disulfide linkage

determination is expected. Spectroscopic techniques such as NMR and CD may be useful. Often, for

complex molecules, the physicochemical information may be extensive but unable to confirm the

higher-order structure. The Potency test, with appropriately established acceptance criteria, assesses

higher-order structure of a biologics and biotechnology drug substance. Suitably chosen, a Potency test

procedure can be stability-indicating. Activity-based procedures, such as enzyme assay, ligand binding,

and cell culture–based procedures can be stability-indicating.

The talk highlights the importance of HOS analysis at various stages of product development covering

discovery to late stage. Specifically, the regulatory aspects of the use of HOS analysis in guiding and

documenting late stage comparability will be discussed with a focus on the regulatory expectations and

extent of qualification/validation expected for characterization tests. Risks of comparability testing and

approaches to their derisking will also be discussed.

NOTES:

35

Higher Order Structure Characterization of a Fusion Protein to Support Process and Container

Closure Changes

Angela Blake-Haskins

Human Genome Sciences, Inc., Rockville, MD USA

Structural characterization of therapeutic proteins is an important component of biopharmaceutical

product development, in particular, as changes are implemented as part of process optimization. This

presentation describes the higher order structure characterization of albinterferon alfa-2b, a genetic

fusion protein consisting of human serum albumin (HSA) and interferon alpha-2b (rIFNa-2b),

performed to support changes in the manufacturing process and final container closure.

Analyses of albinterferon alfa-2b, HSA, rIFNa-2b, and mixtures of HSA and rIFNa-2b in native and

misfolded states were performed using qualified biophysical methods including DSC, UV, CD, and

fluorescence spectroscopies. Subvisible particle (≥ 1 µm) levels in product manufactured in vials and

prefilled cartridge were also investigated using light obscuration, flow imaging microscopy, and photon

correlation spectroscopy.

The biophysical data showed that albinterferon alfa 2b compared well to the sum of the individual HSA

and rIFNa-2b data and the mixture of HSA and rIFNa-2b. The biophysical methods were capable of

detecting thermally misfolded domains of HSA and rIFNa-2b in the presence of the complimentary

native protein when qualitative and quantitative analysis was employed. Silicone from prefilled

cartridges caused significant elevation of subvisible particle levels by light obscuration and flow

imaging microscopy in both placebo and active containing product. Chemically induced aggregates of

albinterferon alfa-2b were proven to be detectable in the presence of silicone oil by flow imaging

microscopy and photon correlation spectroscopy when spiked into product. No protein aggregates were

detectable in prefilled cartridges containing albinterferon alfa-2b product alone.

The product manufacturing process and container closure changes did not impact the higher order

structure of albinterferon alfa-2b. Structural characterization studies using a variety of qualified

biophysical methods were instrumental in supporting these process changes.

NOTES:

36

New Methods for Therapeutic Protein Higher Order Structure Characterization

Henryk Mach

Merck Research Laboratories, West Point, PA USA

Changes in the process or formulation of therapeutic proteins and vaccines may bring unexpected

changes in the propensity to aggregate despite favorable chemical and spectroscopic comparability.

Development of precise and efficient methods to characterize the higher order structure behavior during

production and administration often requires adaptations of the existing hardware. Examples include

applications such as atomic force microscopy and computer simulations to characterize morphology and

size distribution of aggregates, flow cytometry to selectively quantify proteinacious subvisible particles,

fluorescence spectroscopy of an extrinsic dye to measure thermal unfolding and phase separation

propensity, zeta potential to assess the effect of counterions on protein charge, HPLC backpressure to

estimate solution viscosity, as well as use of HP-SEC to assess the extent of binding of monoclonal

antibodies to subcutaneous tissue. The emphasis on throughput and sample size minimization by

applying 96-well plate formats and macro programming in data reduction facilitates timely and efficient

definition of QBD landscape.

NOTES:

37

Young Scientists Session Abstracts


Lin Guo; Feng Gai


Islet amyloid polypeptide (IAPP) aggregation in the extracellular matrix of β-cells is known to be

associated with the development of type II diabetes. However, a mechanistic understanding of how

IAPP aggregation causes islet β-cell degeneration and loss has not been established. For example, while

the rat and human IAPP1-19 peptides (i.e., rIAPP1-19 and hIAPP1-19) differ by only one residue, hIAPP1-19

is significantly more toxic. Herein we employ fluorescence correlation spectroscopy (FCS) to study the

aggregation properties of rIAPP1-19 and hIAPP1-19 in the presence of model membranes. Our results

show that at low peptide/lipid ratios, wherein both peptides are presumably in monomeric form, rIAPP1-

19 actually shows a larger effect on the lipid dynamics and membrane structures, whereas at medium

peptide/lipid ratios hIAPP1-19 is more readily to form oligomers, and at high peptide/lipid ratios they

show similar aggregation behaviors. Taken together these results indicate that FCS is a useful technique

to probe membrane-induced peptide aggregation and higher-structure formation and also provide new

insights into the mechanism of IAPP toxicity.

Reference P-20 for additional information.

NOTES:

38


Trushar Patel1; Suat Özbek

2; Joerg Stetefeld

1

1University of Manitoba, Winnipeg, Canada;

2University of Heidelberg, Heidelberg, Germany

Wnt, Dkk and Frizzled receptors belong to the Wnt signaling pathway that regulates several biological

functions. Wnts are characterized by a conserved pattern of 23 cysteine residues and play fundamental

role in embryonic development, cell migration, cell polarity, cell proliferation and cell fate specification

along with various forms of cancer. Out of all Wnts, Wnt3A is functionally one of the most prominent

members of the protein family. As a first step towards understanding of structure-function relationship

of Wnt proteins, there is a need of high amounts of purified protein that behaves as a monodisperse

protein in solution. Unfortunately, attempts of expression and purification of Wnts in E. coli have not

been successful so far due to their high cysteine content and extreme hydrophobicity. In the present

study, we report an expression of Wnt3A using the pGEV2 plasmid in BL21 (DE3) cells and its

purification using affinity chromatography. The purified Wnt3A however was not a monodisperse

preparation and large oligomeric species were observed. To formulate suitable buffer to obtain

monodisperse solution, the dynamic light scattering experiments were conducted in a range of buffer,

pH and ionic strength conditions with different additives. Intense DLS studies suggested the buffer with

pH of 6.5 to 7, presence of reducing agent and detergent helps solubilizing the Wnt3A. The long-term

objective of this project is to study high-resolution structure of Wnt3A that will enable us to understand

various molecular aspects of Wnt-receptor interactions and its role in various forms of cancer.


NOTES:

39



Jennifer D'Antonio1; Brian M. Murphy

2; Mark Cornell Manning

2; Wasfi Al-Azzam

3

1North Carolina State University, Raleigh, NC USA;

2Legacy BioDesign, LLC, Johnstown, CO USA;

3GlaxoSmithKline, King of Prussia, PA USA

Protein therapeutics require a native-like structure in order to maximize their potency and minimize

interference with the immune system and avoid adverse effects. Therefore, the assessment of higher

order structure (HOS), which involves the analysis of secondary and tertiary structure of protein drugs,

is essential to ensure a consistent supply of high quality therapeutic products and comply with regulatory

agencies. Comparability studies are routinely carried out by manufacturers to assess product similarities

that are produced by different sites or processes. Evaluation of the secondary structure of a protein is an

important HOS quality attribute of any protein. Fourier transform infrared (FTIR) spectroscopy is one of

the main technologies used to determine protein secondary structure composition. There are two

mathematical approaches commonly used to measure the similarity of FTIR spectra: spectral correlation

coefficient (SCC) and area of overlap (AO). However, there is no comprehensive study comparing these

approaches for quantitative comparison of FTIR spectra. In this work, an extensive study using four

model proteins will be presented using both SCC and AO to compare spectra. In addition, a modified

area of overlap (MAO) method will be described that displays an extended dynamic range relative to the

original AO method.


NOTES:

40

Biological Implications Session Abstract

This session will focus on exploring the potential impacts of protein therapeutic higher order structure

on the product safety and efficacy. Safety related issues such as possible immunogenicity and adverse

effect of the product 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.

NOTES:

41

An Immunological Appreciation of Structure

Amy Rosenberg


The immune system is poised to respond to infectious agents whose signature is that of a particulate

with patterned displays of proteins, lipids and sugars. From this concept, this seminar will explore the

relevance of higher order structure of proteins to immune responses to therapeutic proteins, beginning

with particles bearing highly ordered protein arrays to other, less well organized structures, to soluble

aggregates.

NOTES:

42

Higher-order Structural Consequences of Product Degradation in Interferon Alpha

Mary Alice Hefford

Health Canada, Ottawa, Ontario, Canada

Protein-based therapeutic products almost invariably contain small amounts of product-related

impurities (such as oxidized, deamidated or clipped variants of the active ingredient) and these

impurities are known to increase with time and storage of the drug product. Biologics manufacturers

invest considerable time and resources in identifying and quantifying such product-related impurities in

order to set release specifications and determine shelf-life but their effects on the higher order structure

of the drug substance are less studied. We have made several specific variants of interferon alpha 2a,

either by site-directed mutagenesis (to mimic product deamidation) or by enzymatic and/or chemical

modification (to produce “clipped” and “process” variants). These protein variants were assessed for

secondary and tertiary structure, folding stability and tendency to aggregate using circular dichroism

(CD), fluorescence spectroscopy, stability to extremes of pH or temperature and size exclusion

chromatography, respectively. Results were compared to those obtained for the EDQM reference

standard. The data show that impurities resulting from deamidation, protein “clipping” and other product

modifications can have subtly different conformations and, as a result, affect protein stability,

aggregation tendencies and promote product degradation by other pathways

NOTES:

43

HOS: Challenges in Linking Form with Function

Valerie Quarmby

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

Protein therapeutics may elicit an unwanted immune response in some subjects, and this can

compromise product safety or efficacy. Therefore, during biotherapeutic product & process development

it is important to use informative methods to monitor quality attributes which may have the potential to

enhance immunogenicity.

For protein therapeutics, amino acid sequence dictates conformational and higher order structure. The

biophysical properties of protein therapeutics can be assessed with a range of different methods.

However, such methods often provide ensemble average of structural information for a molecule; data

from these methods may therefore lack sufficient precision/resolution to provide actionable information.

This talk will review some of the challenges involved in linking higher order structure with

biotherapeutic safety & efficacy.

NOTES:

44

New and Emerging Technologies Session Abstract

New and better methods are required to detect and characterize protein higher order structure properties

including oligomerization and particulates formation. In addition, predictive tools are needed that can

forecast which proteins are most likely to suffer from aggregation problems at various conditions such

as in high concentration formulations. In this session novel and improved spectroscopic and microscopic

methods will be described for detecting and characterizing protein aggregates. In addition, emerging

methods will be presented for assessing a protein‟s secondary and tertiary structure and their propensity

to aggregate based on its electrostatic and spectroscopic features.

NOTES:

45

Cutting Edge Vibrational Spectroscopy for Protein Therapeutics

Rina Dukor1; Laurence Nafie

1,2

1BioTools, Inc., Jupiter, FL USA;

2Department of Chemistry, Syracuse University, Syracuse, NY USA

Biopharmaceutical industry is now over two decades old and produced over a dozen life-saving or

enhancing therapies. Protein-based therapeutics require structure characterization at all stages of

development – from R&D to formulation, manufacturing and QC. Although detailed structure is

required for developing new drug targets, an average conformation, a fold, or even more importantly a

conformational change is sufficient for the development of biopharmaceuticals. No other technique is

better poised to address this need than vibrational spectroscopy. Vibrational spectroscopy is not new to

protein structural studies but it has been plagued by „common knowledge‟ that such studies require high

protein concentrations and long collection times. Although there is truth to some of these claims – the

advantages outweigh them. The vibrational spectroscopy of proteins - consisting of four techniques -

FT-IR, VCD, Raman and ROA, allows comparison in all types of formulations – liquids, gels, sprays

and solids allowing analysis of API‟s in solution, injectable, or formulated tablets. It is fast, inexpensive

and provides detailed information on the type of fold or family, secondary structure and tertiary

structure. There is no limit on the size or type of protein – antibody, hormones, factors, glycoproteins

and membrane proteins – all can be analyzed by vibrational spectroscopy. Each technique, in turn, has

its own advantage and provides complementary data to others.

The use of FT-IR spectroscopy as a probe of formulations is now widespread throughout the

biopharmaceutical industry. More recently, Raman spectroscopy has been used for tertiary structure

studies such as tyr-tyr degradation and disulfide bond conformations. ROA has high specificity for

glycoproteins and enhanced sensitivity to structural perturbations. VCD was found effective in

discriminating between proteins and excipients in monitoring protein stability and changes in secondary

structure. In particular, glycine, a common excipient, possesses a strong absorption near the amide I

band of proteins in the IR, but shows no signature in the corresponding VCD since glycine is an achiral

molecule. Furthermore, VCD exhibits an increased sensitivity to fibril formation and can be used to

follow the long-term growth and maturation of protein fibrils. In this presentation, we will discuss

advances in all four forms of vibrational spectroscopy as applied to structural studies of proteins.

NOTES:

46

Fluorescent Dye-based Methods to Detect Changes in Higher Order Structures

Wim Jiskoot

LACDR, Leiden University, Leiden, The Netherlands

Protein aggregation is a major concern in the development of protein pharmaceuticals, because

aggregates affect product quality and may enhance immunogenicity. Detection and characterization of

protein aggregates is challenging because aggregates are heterogeneous with regard to their size, shape,

morphology, protein structure, reversibility and solubility. In this presentation I will highlight the use of

fluorescent dye-based detection methods for higher-order protein structures as sensitive and versatile

tools to detect protein aggregates and conformational variants. The usefulness of these dyes will be

illustrated with several recent examples of studies performed in my lab, showing that fluorescent dye-

based detection methods are a valuable addition to the conventional arsenal of characterization methods

for higher-order protein structures.

Further reading:

Hawe, A., Sutter, M., and Jiskoot, W. (2008) Extrinsic fluorescent dyes as tools for protein

characterization. Pharm. Res. 25: 1487-1499.

Hawe, A., Friess, W., Sutter, M., and Jiskoot, W. (2008) Online fluorescent dye detection method for the

characterization of IgG aggregation by size exclusion chromatography and asymmetrical flow field-flow

fractionation. Anal. Biochem. 378: 115-112.

Hawe, A., Kasper, J., Friess, W., and Jiskoot, W. (2009) Structural properties of monoclonal antibody

aggregates induced by freeze-thawing and thermal stress. Eur. J. Pharm. Sci. 38: 79-87.

Hawe, A., Filipe, V., and Jiskoot, W. (2010) Fluorescent molecular rotors as dyes to characterize

polysorbate-containing IgG formulations. Pharm. Res. 27: 314-326.

Hawe, A., Rispens, T., Herron, J.N., and Jiskoot, W. (2011) Probing Bis-ANS binding sites of different

affinity on aggregated IgG by steady state fluorescence, time resolved fluorescence and isothermal

titration calorimetry. J. Pharm. Sci. 100: 1294-1305.

Van Maarschalkerweerd, A., Wolbink, G., Stapel, S., Jiskoot, W., and Hawe, A. (2011) Comparison of

analytical methods to detect instability of etanercept during thermal stress testing. Eur. J. Pharm.

Biopharm. 78: 213-221.

NOTES:

47

Protein Electrostatics, Charge Heterogeneity and Protein Rheology

Devendra Kalonia

University of Connecticut, Storrs, CT USA

Current market trends for high dose therapeutic proteins require concentrated liquid formulations for

patient convenience, in home subcutaneous administration, and to cut manufacturing costs and to

improve product marketability. Protein-protein interactions play an important role in governing solution

viscosity and rheology. High net charge on a protein molecule results in a positive second virial

constant, repulsive interactions and low solution viscosity. On the other hand, at a low net charge, the

charge distribution on a protein can affect the overall dipole moment. A strong dipole moment at high

concentrations can result in strong attractive interactions. This talk will focus on the effect and nature of

various interactions; discuss a novel method for determining dipole moment at low protein volumes, and

the role of charge heterogeneity in governing protein solution rheology.

NOTES:

48

Structures of Dynamic Protein Complexes: Prospects for 3D Electron Microscopy at Atomic

Resolution

Sriram Subramaniam

National Cancer Institute, NIH, Bethesda, MD USA

Emerging methods in 3D biological electron microscopy provide powerful tools and great promise to

bridge a critical gap in imaging in the biomedical size spectrum. This comprises a size range of

considerable interest that includes cellular protein machines, giant protein and nucleic acid assemblies,

small subcellular organelles and bacteria. These objects are generally too large and/or too heterogeneous

to be investigated by high resolution X-ray and NMR methods; yet the level of detail afforded by

conventional light and electron microscopy is often not adequate to describe their structures at

resolutions high enough to be useful in understanding the chemical basis of biological function. The

long-term mission of our research program is to obtain an integrated molecular understanding of cellular

architecture by combining novel technologies for 3D biological imaging with advanced methods for

image segmentation and computational analysis. I will review our recent progress in imaging and

modeling dynamic biological systems, with particular emphasis on applications to signal transduction,

HIV/AIDS and cancer, and assess the prospects of describing dynamic protein complexes at near-atomic

resolution.

NOTES:

49

NOTES:

50

Technical Seminar Abstract

Monday, September 26, 2011

13:30 – 14:30

In Plaza Ballroom I Sponsored by Waters Corporation

Shaping Innovation for Higher Order Structure by Mass Spectrometry: Incorporating Ion

Mobility and Hydrogen Deuterium Exchange

St John Skilton

Waters Corporation, Milford, MA USA

The three-dimensional structure of protein biopharmaceuticals affects function, efficacy, and safety.

Thus, biophysical tools to interrogate conformational dynamics are important in demonstrating

manufacturing consistency and understanding structural changes that arise from sequence variants,

formulation or process changes. The biotech industry is deploying these tools with increasing regularity

to satisfy regulators, fill pipelines more efficiently, demonstrate batch or process comparability, and

protect intellectual property. Two mass spectrometry based analytical methodologies now effectively

achieve these diverse structural goals: Hydrogen-deuterium exchange MS (HDX-MS), and Ion Mobility-

MS.

The first commercial system dedicated to HDX/MS has been rapidly adopted by the biopharmaceutical

industry due to recent innovations in the underlying analytical methodologies. The use of UPLC enables

high resolution separations at low temperatures, MSE data acquisition enables unbiased comprehensive

component detection and quantitation, and the novel automated informatics tools (DynamX™) have

drastically slashed the time taken to perform HDX experiments from weeks to days. Recent applications

include epitope mapping, comparability studies for PEGylated species, and the localization of

conformational changes down to peptide and residue levels. HDX/MS measurements are quantitatively

reproducible using only picomoles of protein, to achieve high coverage peptide level structural

resolution.

Ion mobility is a complementary technique that separates on the basis of molecular shape and provides

orthogonal information to chromatographic and mass-to-charge based separations. This has meant the

ability to distinguish between IgG2 disulfide isomers in seconds, more comprehensive HDX/MS studies,

advances in glycoprotein characterization, yielding greater insights into structural biology of a number

of biotherapeutic classes. The combination of both techniques in SYNAPT™ HDMS systems has

revolutionized mass spectrometry, and has enabled more informative and robust biotherapeutic

characterization studies than were previously available to the biopharmaceutical industry. This seminar

will highlight some of this exciting work and highlight several workflows particularly relevant to the

characterization and comparability needs of the biopharmaceutical industry.

NOTES:

51

Technical Seminar Abstract

Tuesday, September 27, 2011

17:15 – 18:15

In Plaza Ballroom I Sponsored by Wyatt Technology Corporation

Protein Characterization with Light Scattering

John Champagne

Wyatt Technology Corporation, Santa Barbara, CA USA

Light scattering (LS), including classical and dynamic, has been widely employed to characterize

biotechnology-derived pharmaceuticals, such as proteins, DNA, polysaccharides, and viruses. Classical

LS, especially multi-angle light scattering (MALS), determines the absolute molecular weight of

pharmaceuticals in solution. Dynamic light scattering (DLS) directly measures diffusion coefficient and

thus derives the hydrodynamic radius. Both MALS and DLS can be used as stand-alone (batch)

instruments or online coupled with a separation system, such as liquid chromatography and field flow

fractionation (FFF). When used in conjunction with a separation system, light scattering detection

provides the absolute molecular weight, root-mean square radius, and hydrodynamic radius of individual

peaks of interest. In addition, it can be used for the assessment of size heterogeneity (aggregation and

fragmentation), molecular weight distribution, stoichiometry, and preliminary conformational analysis

of proteins. Electrophoretic mobility (zeta potential) has been used as a means of measuring electrostatic

interactions of proteins and colloidal particles and thus assessing their formulation stability. However,

reliable measurements of molecular charge of different protein formulations are challenging due to the

low detection sensitivity of the existing instrumentation. This challenge has been overcome by the

newly-developed Massively Phase Analysis Light Scattering (MP-PALS) technology.

In this technical seminar, we will discuss the latest advancements in MALS and DLS and their

applications for measuring protein oligomerization state and aggregation as well as protein-protein

interaction, pegylated proteins, and membrane proteins. We will also present how, for the first time,

electrophoretic mobility of protein formulations can be measured consistently and non-destructively at a

reasonable sample concentration and volume.

NOTES:

52

Poster Abstracts

P-01

Application of Higher Order Structural Characterization Techniques to Understand the

Functional Relationship of a Monoclonal Antibody and its Target Ligand

Kelly Arthur

Amgen Inc., Longmont, CO USA

Application of higher order structural characterization techniques to study the association of protein

therapeutics to their targets can provide an enhanced understanding of the therapeutic‟s mechanism of

action and critical quality attributes. In this study solution based biophysical characterization techniques

were applied to study the in vitro binding of a monoclonal antibody to its target ligand. The association

of native antibody to ligand, including elucidation of the binding stoichiometry, was investigated by

application of sedimentation velocity analytical ultracentrifugation (SV-AUC), size exclusion HPLC

coupled to static light scattering detection (SE-HPLC-SLS), and differential scanning calorimetry

(DSC). The same biophysical techniques were then applied to study a forcibly oxidized antibody sample

with known modifications to its primary structure and reduced in vitro potency compared to the native

antibody. Biophysical characterization identified possible structural mechanisms for the oxidized

molecule‟s observed decrease in potency, thereby providing an enhanced understanding of the

functional relationship between the monoclonal antibody and its target ligand.

P-02

Biophysical Characterization for Comparability of a Therapeutic Monoclonal Antibody

James Zobel; Lucy Liu; Qin Zou

Pfizer, Inc., Chesterfield, MO USA

Various drug substance lots of a monoclonal antibody in development were made at two different

manufacturing sites. The quality of the drug substance needs to be highly similar to ensure comparable

clinical outcome. Biophysical characterization was used as part of an analytical characterization package

to assess the product comparability. An array of diverse techniques was applied to evaluate the integrity

of higher order structure, size distribution and conformational stability. The results show that all the

drug substance lots are comparable to the reference standard material in all three categories.

53

NOTES:

54

P-03

Localized Conformation Analyses of Biopharmaceuticals By Hydrogen/Deuterium Exchange Mass

Spectrometry

Joomi Ahn1; Xiaojun Lu

2; St John Skilton

1; Ying Qing Yu

1; Jihong Wang

2

1Waters Corporation, Milford, MA USA;

2MedImmnune, Gaithersburg, MD USA

Hydrogen/deuterium exchange mass spectrometry (HDX MS) is an indispensible analytical method for

the study of local changes to protein conformation. Recent improvements in LC-MS systems have made

HDX MS a useful biophysical tool for the development and discovery of protein drugs. Amino acid

substitution on biopharmaceuticals is often explored to improve pharmacokinetics properties. Because

the higher order of protein structure is closely related to its function, the localized conformation due to

the mutation needs to be carefully monitored. Typically the three-dimensional structure is determined by

X-ray crystallography. However the localized conformational changes at the substitution sites may not

be easily detected in a crystal structure comparison. The HDX LC-MS workflow and its system were

deployed in this study to demonstrate the capability of detecting differences in deuterium uptakes at

peptide level. This information could be related to the subtle and distinct changes of conformation due to

local effect. Time consuming HDX data processing was improved by an innovative HDX software tool.

The software automatically calculated the deuterium uptakes and the HDX results were displayed in

convenient comparative views: uptake curves, a butterfly chart, and a difference plot. We report the

efficient comparability studies using a well-established HDX LC-MS method including an informatics

package.

P-04

Automated Analysis of Hydrogen/Deuterium Exchange Mass Spectrometry Data Using DynamX

Joomi Ahn; Michael Eggertson; Martha Stapels; Keith Fadgen; St John Skilton; Ying Qing Yu

Waters Corporation, Milford, MA USA

Recent improvements of hydrogen/deuterium exchange mass spectrometry (HDX MS) have been

proved to be useful as a biophysical analytical tool for the study of protein dynamics. Conventionally,

HDX data are interpreted manually or - at best - processed with semi-automated tools to determine the

deuterium uptake at peptide level. This is time-consuming because of the need to track hundreds of

peptides across multiple time-courses in comparative analyses. In order to improve the efficiency of data

processing, an innovative HDX software tool, DynamX was deployed in this study. The automated

software extracts information on peptic peptides using retention time, intensity, fragment ions, drift

time, and mass accuracy. DynamX tracks all peptides that are reproducibly found in replicates, ensuring

consistency in monitoring the deuterium exchange. The software also calculates the amount of

deuteration and displays the results in convenient comparative views. The data processing time is

significantly reduced from months for manual processing to hours for automated processing. Ion

mobility separation (IMS) is used in the HDX LC-MS workflow, providing additional, orthogonal

separations to chromatography and mass dimensions. Overlapping interfering ions are successfully

resolved by IMS and displayed in DynamX. We report the recent advances in informatics for automated

HDX data analysis of calmodulin, BSA, and phosphorylase b proteins.

55

NOTES:

56

P-05

Fast Detection of Proteins with Low Fluorescent Membrane in Stain Free Gel System

Xuemei Yang

Bio-Rad Laboratories, Hercules, CA USA

The Criterion Stain Free SDS-PA gel (Bio-Rad Laboratories) is a new type of gel that does not require

staining or destaining when used with the SF imager. This system is fast, reproducible and

environmentally friendly. Resolved proteins are visible on Criterion Stain Free gel imaging system

within 2.5~5 minutes after activated by UV light. The use of Low Fluorescence membrane can help to

obtain publication-quality blot images and analyzed results with just 0.5 seconds after transfer. The

activation of the membrane works well with downstream detection, and have similar sensitivity with

Western Blot ChemiDoc detection, and better quality than SyproRuby Stain.

P-06

A Systematic Approach to CD Method Development

Elizabeth Brunyak; Heather Hughes; Scott VanPatten; Karen Lee

Genzyme, A Sanofi Company, Framingham, MA USA

Circular dichroism (CD) is a key technique for investigating the solution structure of proteins and has

been used by Genzyme as a tool to support therapeutic protein development programs and to

characterize released product. This poster describes the development of CD method parameters with the

aim of maximizing both sensitivity and reproducibility. Individualized near and far UV CD parameters

were optimized for two of Genzyme‟s glycoproteins. Parameters such as protein concentration, response

time/scanning speed, data pitch, bandwidth, and number of accumulations were individually evaluated,

revised and incorporated into the final method if found to increase assay sensitivity and/or

reproducibility. Assay variability was then defined for each optimized method by overlaying the spectra

of replicate analyses of the same lot of each product.

P-07

Characterization of Biological Nanoparticles Using Temperature Controlled Field-Flow

Fractionation

Soheyl Tadjiki; Trevor Havard; Evelin Moldenhauer; Roland Welz

Postnova Analytics, Salt Lake City, UT USA

Asymmetrical Flow Field-Flow Fraction (AF4) is a powerful separation and characterization technique

for biological macromolecules, lipids, viruses and cell particulates. The separation in AF4 takes place in

an open channel and is based on diffusion coefficient or hydrodynamic diameter of sample components.

The AF4 channel is made of two parallel walls with a thickness of 0.0190-0.0500 cm. Sample

57

components are pushed toward the semi-permeable lower channel wall by the separation field (cross

flow) and transported along the channel at different flow velocities by a secondary flow (channel flow).

The AF4 channel can also be made of a semipermeable ultrafiltration hollow fiber where the cross flow

exits radially through the fiber wall and the channel flow is flowing axially along the fiber length.

Resolution, reproducibility and sample recovery of the planar and hollow fiber cartridges were studied

using a Bovine Serum Albumin standard for more than 100 consecutive injections. The average

retention time RSD of the monomer and dimer peaks were found to be below 1% in the hollow fiber

cartridge. The Planar cartridge had 60% more resolution and 3 times more sample recovery than the

hollow fiber cartridge.

The planar and hollow fiber cartridges were hyphenated with diode array UV/Vis , multi-angle light

scattering and dynamic light scattering detectors to characterize protein mixtures, a human plasma

sample and virus particles.

The results showed a baseline separation of HDL, LDL and VLDL for the plasma sample. The virus

particles exhibited a narrow size distribution with a mean hydrodynamic diameter of 84 nm.

NOTES:

58

P-08

Circular Dichroism Spectroscopy: Quantitation, Analyses and Validation

B.A. Wallace

Birbeck College, University of London, London, United Kingdom

Circular Dichroism (CD) spectroscopy is a widely used and established method for determining protein

secondary structure, detecting conformational changes associated with different conditions including

ligand binding, examining macromolecular interactions, and is a designated method for the

characterisation of proteins produced for use in human applications. A new extension of the method,

synchrotron radiation circular dichroism (SRCD) spectroscopy, has also been shown to be sensitive to

higher order (quarternary) structure.

This talk will discuss methods for calibration and good practice in CD data collection. It will present

user-friendly tools available for processing, comparing, archiving and analysing CD spectra, including

the popular DichroWeb secondary structure analysis server and the new Protein Circular Dichroism

Data Bank (PCDDB) facility for accessing, searching and depositing published CD data and metadata.

Finally, the new Validichro software for evaluating spectral quality will be presented. Validichro

includes >20 test criteria for CD spectra developed in conjunction with an international panel of users,

instrument manufacturers and SRCD beamline specialists. It is freely available both as a standalone

facility for testing spectra and in conjunction with depositions to the PCDDB. It produces a date-

stamped report that can be downloaded for documentation, regulatory and publication purposes.

(Supported by grants from the UK Biotechology and Biological Sciences Research Council and the

International Union of Pure and Applied Chemistry)

Wallace and Janes (2009) Modern Techniques in Circular Dichroism and Synchrotron Radiation

Circular Dichroism Spectroscopy. IOS Press.

Whitmore, Woollett, Miles, Janes, and Wallace (2010) Structure 18:1267-1269.

Whitmore, Woollett, Miles, Klose, Janes,and Wallace (2011) Nucleic Acids Research 39:D480-D486.

Whitmore and Wallace (2008) Biopolymers 89:392-400.

P-09

Plain and Mono-PEGylated Recombinant Human Insulin Exhibit Similar Stress-induced

Aggregation Profiles

Riccardo Torosantucci

LACDR, Leiden University, Leiden, The Netherlands

PEGylation has been suggested to improve the stability of insulin, but evidence for that is scarce. Here,

we compared the forced aggregation behavior of insulin and mono- PEGylated insulin. Therefore,

recombinant human insulin was conjugated on lysine B29 with 5-kDa PEG. PEG–insulin was purified

59

by size-exclusion chromatography (SEC) and characterized by mass spectrometry (MS). Next, insulin

and PEG–insulin were subjected to heating at 75°C, metal catalyzed oxidation, and glutaraldehyde

cross-linking. The products were characterized physicochemically by complementary analytical

methods. Mono-PEGylation of insulin was confirmed by SEC and MS. Under each of the applied stress

conditions, insulin and PEG–insulin showed comparable degradation profiles. All the stressed samples

showed submicron aggregates in the size range between 50 and 500 nm. Covalent aggregates and

conformational changes were found for both oxidized products. Insulin and its PEGylated counterpart

also exhibited similar characteristics when exposed to heat stress, that is, slightly changed secondary and

tertiary structures, covalent aggregates with partially intact epitopes, and separation of chain A from

chain B. Both glutaraldehyde-treated insulin and PEG–insulin contained covalent and non covalent

aggregates with intact epitopes, showed partially perturbed secondary structure, and substantial loss of

tertiary structure. From these results, we conclude that PEGylation does not protect insulin against

forced aggregation.

NOTES:

60

P-10

The Development of a Pharmaceutically Relevant Model Systems to Evaluate the Consequences of

Adsorption-Induced Structural Perturbations of Proteins

Flora Felsovalyi1; Paolo Mangiagalli

2; Christophe Bureau

2; Sanat Kumar

1; Scott Banta

1

1Dept. of Chemical Engineering, Columbia University, New York, NY USA;

2BD Medical -

Pharmaceutical Systems, Pont de Claix, France

The successful formulation of a biologically-active therapeutic agent is critically linked to maintaining

its stability throughout the drug‟s lifecycle. Interfacial effects, such as protein adsorption to solid

surfaces, may lead to instabilities. Drug storage and delivery provide ample opportunity for

protein/surface interactions to occur, especially in prefilled systems, where proteins contact devices

surfaces for extended time periods, sometimes under sub-optimal conditions. Surface adsorption may

lead to protein unfolding, activity loss and population of non-native, aggregate-prone states. Such events

may compromise the safety and efficacy of biologics.

A central paradigm that underpins our understanding of protein/surface interactions is that protein

adsorption leads to changes in secondary structure. Bound proteins tend to denature and the non-native,

adsorbed structures are likely stabilized by loss of alpha-helices and concomitant formation of

intermolecular beta-sheets. This research seeks to gain better understanding of the structural state of

adsorbed proteins following surface-induced perturbations, where irreversible conformational change

may lead to aggregation or other forms of instability. We employ a robust study design to examine the

kinetics of adsorption, desorption, and structural transitions of lysozyme on fumed silica nanoparticles

as a function of surface coverage, an important parameter when modeling pharmaceutically relevant

systems. We use circular dichroism (CD) spectroscopy to monitor structural transitions on the surface, in

situ. By applying a new approach to isolate adsorbed protein signal, the nature of structural changes the

surface elicits is assessed. The results show that despite significant adsorption-induced structural loss,

adsorption is reversible. We find evidence of a two-state model, involving exchange between a native-

like dissolved and highly perturbed adsorbed state. Adsorbed protein secondary structure is independent

of coverage, which sheds new light on the role of surface coverage. These findings hold promise to

facilitate better predictability of instabilities related to drug-container interactions.

P-11


Trushar Patel1; Suat Özbek

2; Joerg Stetefeld

1

1University of Manitoba, Winnipeg, Canada;

2University of Heidelberg, Heidelberg, Germany

Wnt, Dkk and Frizzled receptors belong to the Wnt signaling pathway that regulates several biological

functions. Wnts are characterized by a conserved pattern of 23 cysteine residues and play fundamental

role in embryonic development, cell migration, cell polarity, cell proliferation and cell fate specification

along with various forms of cancer. Out of all Wnts, Wnt3A is functionally one of the most prominent

members of the protein family. As a first step towards understanding of structure-function relationship

of Wnt proteins, there is a need of high amounts of purified protein that behaves as a monodisperse

protein in solution. Unfortunately, attempts of expression and purification of Wnts in E. coli have not

61

been successful so far due to their high cysteine content and extreme hydrophobicity. In the present

study, we report an expression of Wnt3A using the pGEV2 plasmid in BL21 (DE3) cells and its

purification using affinity chromatography. The purified Wnt3A however was not a monodisperse

preparation and large oligomeric species were observed. To formulate suitable buffer to obtain

monodisperse solution, the dynamic light scattering experiments were conducted in a range of buffer,

pH and ionic strength conditions with different additives. Intense DLS studies suggested the buffer with

pH of 6.5 to 7, presence of reducing agent and detergent helps solubilizing the Wnt3A. The long-term

objective of this project is to study high-resolution structure of Wnt3A that will enable us to understand

various molecular aspects of Wnt-receptor interactions and its role in various forms of cancer.

NOTES:

62

P-12

Characterizing the Electrophorectic Mobility and Effective Charge of IgG1 Samples by MP-

PALS, A Novel New Technique

John Champagne; Robert Collins


Electrical charge is a fundamental property of all macromolecules. In colloidal suspensions, the

formulation stability depends critically on the amount of charge developed at the interfaces between

particles and their solvent. For most biomolecules-like proteins-electrostatic interactions excercise a

profound influence on their conformation, function and efficacy. Practically speaking, electrophoretic

mobility has been the most popular and widely accepted proxy for molecular charge and interface

potential, also known as zeta potential. However, traditional commercial instruments have hit a wall

trying to characterize proteins and other nanoparticles less than 5nm. The invention of the new Mobius

instrument, utilizing Massively Parallel Phase Analysis Light Scattering (MP-PALS), shatters this

barrier and extends the measurable sample size down to 1nm. In this poster, we describe the theory of

the new Wyatt Mobius instrument utilizing MP-PALS and how it is used to characterize the

electrophoretic mobility and effective charge of antibodies. The results show how effectively this

technique is able to differentiate the charge of several IgG1 sanples and then use this information to

determine the formulation stability of the samples.

P-13

Charaterizing Protein-Protein Interactions by Composition-Gradient Multi-Angle Light

Scattering

Sophia Kenrick; John Champagne


Macromolecular interactions influence all phases of biopharmaceutical development from drug

discovery and target validation to the characterization of stable drug formulations at therapeutic doses.

Composition-gradient multi-angle light scattering (CG-MALS) is a powerful, label free technique for

quantifying reversible interactions and can be applied to many stages of the drug development process.

Analysis of CG-MALS data yields a second virial coefficient (A2) for non-specific interactions as well

as equilibrium association constants (Ka) and stoichiometry for specific self and hetero-association.

Because assays are performed in solution, complex interations can be observed without being influenced

by tags or surface immobilization. This not only provides for absolute characterization of interaction

strength and stoichiometry but also enables study of the effects of solvent composition, pH, and

molecular conformation on the interaction of interest. In this poster, we describe several applications of

CG-MALS characterizing protein-protein binding and other interactions automated by the Wyatt

Calypso system.

63

NOTES:

64

P-14

A Case Study of Kinetic Stabilization by Self-association: A Recombinant Therapeutic

Glycoprotein in which Dimerization Controls the Rate of Degradation

Jonathan Kingsbury; Karen Albee; Tim Edmunds; Karen Lee

Genzyme, A Sanofi Company, Framingham, MA USA

A recombinant human glycoprotein produced in CHO cells (gpD), is a stable non-covalent dimer. The

contribution of the quaternary structure to the overall structural stability of the molecule has not yet been

established, but is of critical importance to the complete understanding of the structure/function

relationship. Furthermore, accurate models of degradation/aggregation for this protein may predict

molecular qualities important for sample handling, comparability assessment, and assay development.

Equilibrium chemical denaturation studies supported by sedimentation analyses indicate that the

monomeric form of the protein is highly unstable and prone to aggregation when the self-association is

perturbed at neutral pH. Thermal denaturation studies confirm these findings and suggest that the

dimeric form is kinetically stabilized. These results suggest that self-association is the key rate-limiting

contributor to overall stability. Modeling of this defining property within the Lumry-Eyring framework

of protein stability leads to several theoretical consequences that are of practical interest to

biopharmaceutical development support activities. In addition, the concept of kinetic stabilization (rather

than the oft encountered thermodynamic alternative) is of general scientific significance. In that respect,

gpD appears to be a useful model system for investigating the role of higher order structure within this

theoretical framework.

P-15

High-throughput Molecular Microscopy as a Novel Tool for Quantitative Sub-micron Aggregate

Characterization of Protein Therapeutics

Anke Mulder; Joyce Sung; Clint Potter; Bridget Carragher

NanoImaging Services, Inc., La Jolla, CA USA

The objective of this study was to demonstrate the application of high-throughput molecular microscopy

to monitor sub-micron aggregates in protein therapeutics. The reproducibility and sensitivity of the

method were demonstrated using 100nm latex beads, and the method was validated using IgG and EPO

in a series of controlled stress experiments. IgG was subjected to temperature (heat, freeze-thaw)

stresses and aggregate formation was monitored over time. Images were acquired at multiple scales of

magnification (6,500 – 52,000x) using automated image collection software. Individual IgG monomers

(MW 150 kDa) and IgG aggregates were clearly visible in these images and monomers were counted

using automated particle selection and classification software. The loss of free protein and accumulation

of aggregates could be simultaneously assessed and quantitatively characterized. This study

demonstrates the potential of molecular microscopy for providing an orthogonal quantitative method for

characterizing protein aggregation in the sub-micron size range. The unique advantage of molecular

microscopy is that it provides a direct means of observing aggregates and discriminating them from

contaminates or impurities.

65

NOTES:

66

P-16

Dissecting Contributors to Particle Formation in a Protein Drug Product Sourced from

Alternative Drug Substance Processes

Ge Jiang; Monica Pallitto Goss; Merleen Gholdston; Jason Ko; Nancy Jiao; Neil Kitchen; Chris

Crowell; Tom McNerney

Amgen Inc., Thousand Oaks, CA USA

Drug substance (DS) materials of a mAb prepared from two alternative processes (A & B) were

analytically comparable by standard stability-indicating assays. However, particle formation was

observed in drug product (DP) sourced from Process B but not from Process A. The poster will focus on

the impact of the cell culture and purification processes (A vs. B), on this atypical particle formation.

The process was segregated to determine if the particle formation differences in DP were due to the cell

culture and/or purification process:

Study 1 (Process Swap): The cell culture and purification processes were combined in a full factorial

design. Process A purification led to particle-free DPs, regardless of cell culture conditions of either A

or B. DPs sourced from Process B purification formed particles over time and the kinetics differed

slightly between A and B cell culture conditions. Therefore, the effect from cell culture on particle

formation was less pronounced than purification process.

Study 2 (Post Column 2 Cross Over): The Column 2 pool from Process B was purified further

downstream with either A or B process conditions, and particles were observed in B conditions but not

in A conditions. Therefore, post Column 2 conditions of Process B, specifically the 3rd chromatography

and viral filtration, were identified to contribute to DP particle formation.

Study 3 (Post Column 2 characterizations): Further characterizations assessed the individual effects

from Column 3 and viral filtration operations in Process A or B by including or excluding the

corresponding step, changing the order of these steps, and evaluating the Column 3 resin and buffers.

The Column 3 resins showed the most critical impact on particle formation by removing particle prone

components/subspecies.

The findings highlighted the link between DS process and DP stability and the need for effective cross-

functional collaboration.

P-17

Epitope Mapping by Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS)

Stephen Coales; Jessica Lee; Kelly E; Yoshitomo Hamuro

ExSAR Corporation, Monmouth Junction, NJ USA

Amide hydrogen/deuterium exchange (HDX) coupled with proteolysis, HPLC separation, and mass

spectrometry (MS) was used to map antigen-antibody interactions. Briefly, (i) an antigen was deuterated

in solution by mixing with neutral deuterated buffer (on-exchange), (ii) the deuterated antigen was

67

loaded onto an antibody column, (iii) the antibody column was washed with neutral aqueous buffer (off-

exchange), (iv) the antigen was eluted from the antibody column by a cold acidic buffer, (v) the eluted

antigen was digested by acid stable protease(s), and (vi) the deuteration levels of each antigen peptide

were determined by LC-MS.

The binding of an antibody to its antigen should retard the exchange of amide hydrogen to deuterium

through solvent exclusion, and/or restriction of conformational fluctuation at the antigen-antibody

interface. If an amide is within the epitope, the amide should carry a significant amount of deuterium

after on-exchange in solution and off-exchange in the column reactions. On the other hand, if an amide

is not in the epitope, the amide should carry very little deuterium after on/off exchange reactions.

First, HDX-MS was used to map the epitopes of three antigen-antibody interactions. All of them were

validated by X-ray crystallographic data. These three epitopes identified were discontinuous

conformational epitopes. Second, HDX-MS was applied to map epitopes of more challenging

glycoprotein against two different antibodies. The success rate of HDX-MS epitope mapping is over

90%. HDX-MS technology is widely applicable for the epitope mapping of various antigen-antibody

interactions providing epitope resolution between 5-15 amino acids per region.

NOTES:

68

P-18

Amide Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) of Human Growth

Hormone (hGH) at Various pHs

Kathleen Molnar; Stephen Coales; Yoshitomo Hamuro

ExSAR Corporation, Monmouth Junction, NJ USA

Proteins, including monoclonal antibodies, are the fastest growing class of therapeutics. Compared with

traditional small molecule drugs, much additional analysis is necessary for their development and

production, 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 therapeutics. During development, HDX-MS analysis can rapidly determine

the effects of mutation, chemical modification, formulation change and/or process change on protein

folding/dynamics, and localize any changes at the sub-molecular level.

Since 1984, somatropin (recombinant DNA-derived hGH) therapy has been applied in the treatment of

growth hormone-deficient children to promote linear growth. The EC approved Sandoz' Omnitrope

(somatropin) 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 test dynamic characteristics of hGH at various pHs.

P-19



Jennifer D'Antonio1; Brian M. Murphy

2; Mark Cornell Manning

2; Wasfi Al-Azzam

3

1North Carolina State University, Raleigh, NC USA;

2Legacy BioDesign, LLC, Johnstown, CO USA;

3GlaxoSmithKline, King of Prussia, PA USA

Protein therapeutics require a native-like structure in order to maximize their potency and minimize

interference with the immune system and avoid adverse effects. Therefore, the assessment of higher

order structure (HOS), which involves the analysis of secondary and tertiary structure of protein drugs,

is essential to ensure a consistent supply of high quality therapeutic products and comply with regulatory

agencies. Comparability studies are routinely carried out by manufacturers to assess product similarities

that are produced by different sites or processes. Evaluation of the secondary structure of a protein is an

important HOS quality attribute of any protein. Fourier transform infrared (FTIR) spectroscopy is one of

the main technologies used to determine protein secondary structure composition. There are two

69

mathematical approaches commonly used to measure the similarity of FTIR spectra: spectral correlation

coefficient (SCC) and area of overlap (AO). However, there is no comprehensive study comparing these

approaches for quantitative comparison of FTIR spectra. In this work, an extensive study using four

model proteins will be presented using both SCC and AO to compare spectra. In addition, a modified

area of overlap (MAO) method will be described that displays an extended dynamic range relative to the

original AO method.

NOTES:

70

P-20


Lin Guo; Feng Gai


Islet amyloid polypeptide (IAPP) aggregation in the extracellular matrix of β-cells is known to be

associated with the development of type II diabetes. However, a mechanistic understanding of how

IAPP aggregation causes islet β-cell degeneration and loss has not been established. For example, while

the rat and human IAPP1-19 peptides (i.e., rIAPP1-19 and hIAPP1-19) differ by only one residue, hIAPP1-19

is significantly more toxic. Herein we employ fluorescence correlation spectroscopy (FCS) to study the

aggregation properties of rIAPP1-19 and hIAPP1-19 in the presence of model membranes. Our results

show that at low peptide/lipid ratios, wherein both peptides are presumably in monomeric form, rIAPP1-

19 actually shows a larger effect on the lipid dynamics and membrane structures, whereas at medium

peptide/lipid ratios hIAPP1-19 is more readily to form oligomers, and at high peptide/lipid ratios they

show similar aggregation behaviors. Taken together these results indicate that FCS is a useful technique

to probe membrane-induced peptide aggregation and higher-structure formation and also provide new

insights into the mechanism of IAPP toxicity.

P-21

Strategies for Comparing Far-UV Circular Dichroism Spectra of Similar Proteins: A Study of the

Conformation of Eight Mammalian Albumins

David Gregson; Lindsay Cole

Applied Photophysics Ltd., Leatherhead, United Kingdom

CD spectroscopy is a sensitive probe of protein secondary structure in solution and can be used to

compare the conformations of different proteins. When making such comparisons, it is important to take

account of differences in the spectra that could be caused by differences in protein concentration or cell

pathlength which, in practice, can be difficult to achieve. In this study, two methods of comparing far-

UV CD spectra are described, neither of which needs precise knowledge of the protein concentrations or

the cell pathlengths.

The first requires that each CD spectrum be normalised using the absolute CD values at each measured

point summed over the whole wavelength range and that difference spectra be calculated relative to a

designated reference standard, in this case human serum albumin (HSA), to establish if statistically

significant differences between the normalised CD spectra exist.

The second method described uses the G-factor, which is the ratio of the CD (as delta A) to the total

absorbance of the sample (A). The G-factor retains much of the conformational specificity of the CD

spectrum but as a ratio it is dimensionless with no dependence on the sample concentration or cell

pathlength. Comparing the G-factor spectra of sample serum albumins to a reference G-factor spectrum

(again HSA) will establish if there are significant differences between them and hence the protein

secondary structures.

71

A key aspect of this study is the assessment of statistical significance: the combination of a fixed-probe

robotic autosampler and the Chirascan-plus CD spectrometer enables us to make replicate measurements

easily and facilitates the determination of standard deviations in the measurements. The standard

deviations in the measurements are propagated in the derived parameters and provide a solid foundation

upon which to assess similarity.

NOTES:

72

P-22

Aggregates and Their Analysis---Some Perspectives and Lessons from Working with Over 250

Proteins

John Philo

Alliance Protein Laboratories, Thousand Oaks, CA USA

This poster will give examples of aggregate analysis and aggregation issues drawn from experience with

over 250 different therapeutic and vaccine products. A particular focus will be examples from SV-AUC

or SEC-MALS experiments which illustrate interesting phenomena, different aggregation mechanisms,

and things our clients often misunderstand or forget to investigate themselves.

P-23

Application of FTIR Spectroscopy in Structure Analysis of Protein Therapeutics: Case Studies

Lucy Liu

Pfizer BioTherapeutics, Andover, MA USA

The advent of modern Fourier transfer infrared (FTIR) spectrometer and attenuated total reflectance

(ATR) sample accessory made it possible to characterize the secondary structure of protein therapeutics

directly in their aqueous formulation as well as lyophlized cake. 1. FTIR in comparability studies: two

case studies are shown for protein secondary structure analysis. 2. FTIR in overcoming analytical

challenges of high concentration formulations: two areas of applications are presented – the first is the

determination of protein unfolding temperature for high concentration mAbs and the second is

monitoring protein secondary structure alteration of a high concentration protein therapeutic in the

presence of Ca2+ as a functional assay.

P-24

Unfolding and Aggregation of Epoetin Alfa Under the Influence of Tungsten

Otmar Hainzl1; Alexander Bepperling

1; Britta Deutel

2; Stephan Boehm

2; Robert Fischer

1; Andreas

Seidl1

1Hexal AG, Oberhaching, Germany;

2Sandoz GmbH, Kundl, Austria

In recent years, pre-filled syringes have become the preferred container-closure system for

biopharmaceutical proteins. During the manufacturing process, a tungsten pin is used to form the needle

channel and the fluid path at the tip of the glass barrel of the syringe. Mechanical stress and a chemically

challenging, highly reactive environment of heated glass and hot air rapidly wears off the tungsten pin

and residual tungsten may be deposited on the inner surface of the syringe barrel and dissolve from there

into the product. Heavy metals have been demonstrated to interact with proteins, and some including

tungsten can cause the formation of protein aggregates in model systems. Implications of tungsten on

product quality were also found in the context of biopharmaceutical manufacturing. However, the

activity of tungsten towards therapeutic proteins and the exact consequences on structure and

73

immunogenicity of the respective proteins are not known in detail. Further, as the chemical identity of

the tungsten forms which may arise during syringe manufacturing and subsequently may get in contact

with the therapeutic protein cannot be fully elucidated, we tested various forms of tungsten in

preparation of the present study. We found that several tungsten species (from a set of tungsten forms

that potentially may arise during the syringe manufacturing process) have significant influence on the

folding state of Epoetin alfa and some of them trigger oligomerization and aggregation of the protein.

Among those tungsten species, Na-polytungstate showed the most profound effects. Various methods of

biophysical characterization were applied to assess the mechanism of action of the interaction of Na-

polytungstate with Epoetin alfa, and the consequences on the structure and oligomeric state of the

therapeutic protein.

NOTES:

74

P-25

A Study of Protein Unfolding by Differential Scanning Calorimetry, Differential Scanning

Flourimetry and HPLC.

Brian Lang; Kenneth Cole

National Institute of Standards and Technology, Gaithersburg, MD USA

The study of protein unfolding is of great importance especially with regards to therapeutic molecules

since melting temperature, Tm, and transition enthalpy, ΔmH°, can used as a measure of the overall

stability of the protein. The gold standard for protein unfolding measurements is differential scanning

calorimetry (DSC), however DSC generally requires large amounts of sample in order to obtain

sufficient signal for analysis. Recently, a technique has been developed which uses a dye reporter to

monitor the unfolding process. The method, differential scanning fluorimetry (DSF), relies on specific

dyes having different fluorescent properties in hydrophilic versus hydrophobic environments. As the

protein unfolds, its hydrophobic regions are exposed allowing greater interaction of the reporter dye

with this region of the protein, thus changing the dyes fluorescence. While the DSF technique has been

shown to have a good correlation with Tm determined by DSC for immunoglobulins, there is little data

on how well the data correlates for other classes of proteins. In this study we determine the melting

temperature of the proteins using DSC under various conditions and compare it to studies using DSF

using different reported dyes. Furthermore we then have used HPLC to characterized the aggregation

products of the reactions. Some of the model proteins we have studied are BSA, lysozyme, lactose

dehydrogenase, and Rituxan. While there is generally correlation in the melting temperature between the

techniques, there are also many anomalies in the DSF method that are important to understand before

using DSF, and give insight on how the various dyes interact with the proteins and their aggregates.

P-26

A Combined Morphological Imaging and Raman Spectral Approach for the Characterization of

Aggregation in Protein-Based Biotherapeutics

E. Neil Lewis; Linda Kidder; Kenneth Haber

Malvern Instruments, Inc., Columbia, MD USA

Sub-visible particles in pharmaceutical formulations can be of extrinsic or intrinsic origin and USP

<788> requires that particles >10 and >25 microns be counted for quality control. The test is typically

carried out using light obscuration with no requirement for their identification as the approach is only

aimed at preventing physical blockage in the circulatory system. However, biopharmaceutical parenteral

products have an additional safety concern associated with a potential immunogenic response from self-

aggregation of the product within the formulation. The size, frequency, origin, stability or chemical

characteristics of these particulates are not well understood. We will describe a new technique that

combines digital imaging with morphologically-guided Raman microscopy for the chemical

characterization of the detected particles. The technique can provide novel information not only with

respect to the identity of the particulate material but also on their higher order (molecular) structural

characteristics which may help shed some additional light on the mechanism(s) of aggregation. The

potential benefits of this new approach will be discussed with regards to the current measurement gaps

and potential future measurement requirements.

NOTES:

75

P-27

Secondary Interactions Contributing to Non-ideal Size Exclusion Behavior of a Fusion Protein

Ruth Frenkel; Li Zang; Julie Wei; Andy Weiskopf

Biogen Idec, Cambridge, MA USA

Size exclusion chromatography (SEC) is the industry standard method for routine measurement of

aggregates in biopharmaceuticals. Column-to-column robustness is a crucial factor in selection of a

column during development of SEC methods for QC release testing. Analysis of a large fusion protein

on several commercial SEC columns revealed that some column resins resulted in deviations from a

purely size-exclusion separation mechanism, leading to potentially inaccurate molecular weight (MW)

assignment based solely on retention times. During method development, it was found that some batches

of columns resulted in the resolution of an “apparent dimer” peak prior to the monomer peak. However,

SEC coupled with light scattering analysis showed that the MW of this peak was almost the same as the

monomer MW. Further investigation revealed that resolution between this alternate monomer species

and the main peak monomer was dependent on the column temperature. Minor increases above ambient

temperature caused a greater increase in retention time of the main peak compared to that of the

alternate monomer, which indicates that the separation was operating under a mixed-mode mechanism.

During method qualification/validation activities, it was found that the temperature required to achieve a

consistent profile varied from batch-to-batch of the same column resin and from lab-to-lab. Various

compositions of mobile phase were explored in an effort to elucidate and suppress the secondary

interactions causing the mixed-mode separation. The optimization of an SEC mobile phase system for

this molecule will be presented.

NOTES:

76

NOTES:

Documents

Table of Contents · Applied Photophysics Ltd. BioTools, Inc. ExSAR™ Corporation JASCO, Inc. SGS M-Scan, Inc. Waters Corporation Wyatt Technology Corporation Media Partners BioPharm