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7:00 am Registration and Morning Coffee

Mechanistic Perspectives On Aggregation

8:30 Chairperson’s Opening Remarks

David Litzinger, Ph.D., Director, Pharmaceutical Sciences, Amylin Pharmaceuticals, Inc.



Yatin GokarnGood Proteins, Bad Environment: The Role of Ions in Governing Stability, Solubility, and Viscosity of Proteins

Yatin Gokarn, Ph.D., Associate Director, Late-stage Pharmaceutical and Process Development, Genentech

9:10 The Role of Physical Stress on Aggregate Formation during Processing and Storage of Biotherapeutic Proteins

Mark Pollo, Associate Senior Biophysical Chemist, Bioproduct Research and Development, Eli Lilly & Co.

A major challenge for production and delivery of proteins is to preserve the physical and chemical stability after exposure to varying processing and storage conditions. We have performed studies utilizing biophysical methods to monitor changes in higher-order structures and formation of non-native species for an antibody exposed to physical stresses, including agitation and freeze-thaw. The formation of different aggregate types resulting from different physical stresses and outline rational, evidenced based controls for minimizing protein perturbation.

9:40 Proximity Energies and Protein Aggregation

Thomas M. Laue, Ph.D., Professor, Biochemistry and Molecular Biology; Director, Biomolecular Interaction Technologies Center (BITC), University of New Hampshire

The weak, short-range electrostatic and electrodynamic forces between molecules dominate solution behavior, including solubility. These forces become significant at high concentrations. The potential energies that result in these forces are called proximity energies. Thinking about high concentration solutions using a framework of proximity energies clarifies a number of solution phenomena, and can lead to formulation changes that will optimize solubility. Proximity energies will be described, along with solvent and solute properties that influence them.

10:10 Coffee Break, Poster and Exhibit Viewing

11:10 Solution Factors Affecting Aggregation in a High Concentration Antibody Solution

Devendra (Davy) S. Kalonia, Ph.D., Professor of Pharmaceutics, University of Connecticut

This talk will focus on the importance of solution properties of a monoclonal antibody at high concentrations. The ionic strength affect can both reduce or enhance aggregation depending on the protein-protein interactions in solution. The solution storage modulus or elastic behavior correlates with the extent of aggregation at various pH conditions.

11:40 A Case Study of Process Stress Induced Aggregation

Carl “Charlie” Hitscherich, Jr., Ph.D., Associate Director, Protein Pharmaceutical Development, Biogen Idec

When optimizing processing conditions for unit operations downstream the traditional purification unit operations one must closely monitor the impact that processing conditions can have on aggregation.  This talk will provide several bench scale and manufacturing scale examples of where process stress leads to increased aggregate levels in the final drug product.  The talk will also discuss the approach that Biogen Idec takes to minimize increases in aggregation levels during scale up and processing

12:10-12:40 pm Luncheon Presentation I

Sponsored by

Current Approaches to Characterization of Aggregates and Sub-Visible Particles in Protein Formulations
Alan C. Herman, Ph.D., Vice President of Product Development & Chief Scientific Officer, Althea Technologies, Inc.
Identifying the presence and nature of multimeric product forms in protein-based therapeutics remains a significant challenge.  Traditionally, the method of choice for measuring soluble multimers has been analytical ultracentrifugation; a labor intensive technique requiring a skilled operator and tedious data interpretation.  The method of choice for sub-visible particle quantification has been light obscuration which provides little or no data on particle morphology and identification.  Recent advances in field flow fractionation, static light scattering, and micro-flow imaging open new possibilities for more rapid and accurate assessment.

12:40-1:10 Luncheon Presentation II
            Sponsored by
Enzo Life Sciences Assay Designs
Rapid Assessment of Aggregation in Protein-Based Pharmaceuticals
Wayne F. Patton, Ph.D., Chief Scientific Officer, Enzo Life Sciences
A homogenous fluorescence-based assay is described for investigating the impact of adverse conditions that a protein product might be exposed to during manufacture, storage, shipping, freeze/thaw cycles, oxygen exposure, light, and physical stress. The assay facilitates assessment of the impact that these conditions might have on drug safety and quality and can be performed using a simple fluorimeter, microplate reader or RT-PCR instrument.  Sub-visible particles are detectable without a requirement for sample separation, solvent exchange or dilution. The assay has been benchmarked using a wide range of practical applications.


Tools And Methods For Analysis

1:30 Chairperson’s Remarks

Joel Richard, Ph.D., Senior Director, Head of Drug Product Development, Pharmaceutical Development, Ipsen

1:35 Biophysical Techniques to Explore Protein Aggregation or Aggregation Propensity

Min Huang, Ph.D., Principal Scientist, Pharmaceutical Research and Development, Global Biologics, Pfizer, Inc.

Protein aggregation poses a considerable challenge in the manufacturing and delivery of biopharmaceuticals. This talk will present some case studies employing some new biophysical analytical techniques to characterize protein aggregation as well as their aggregation propensities. These techniques could potentially be useful orthogonal tools to understand and characterize protein aggregation.

2:05 Analysis of Subvisible Particles in Protein Therapeutics: Methods and Applications

Shawn Cao, Ph.D., Principal Scientist, Process and Product Development, Amgen, Inc.

The subvisible particles that might be present in protein therapeutics have been identified by the regulatory agencies as a potential safety issue. Analytical methods are needed for the monitoring and control of these subvisible particles, and to study the mechanism of particle formation. The methods available to subvisible particle analysis, their strengths and weaknesses, and some case studies showing how these techniques can be applied to address particle characterization during the product lifecycle will be discussed in this presentation.

2:35 DLS Characterization of High Concentration Protein Formulations in Shelf Life StudiesSponsored by
Malvern small


Kevin Mattison, Ph.D., Senior Bioanalytical Scientist,
Research & Development, Malvern Instruments Ltd.

Dynamic light scattering (DLS) is a common technique for detecting protein aggregation. While historically delegated to dilute solutions, technological advances have moved DLS instrumentation into the realm of high concentration measurements. The ability to measure at high concentration however, does not negate the possibility of physical effects such as multiple scattering, restricted diffusion, and particle interactions, all of which can lead to erroneous interpretation of DLS results. This presentation highlights approaches to addressing high concentration effects.

3:05 Networking Refreshment Break, Poster and Exhibit Viewing

3:50 Problem Solving Break-Out Sessions

Table 12: The Physical Properties of mAbs That Lead to Aggregation and How to Control Them

Moderator: Thomas M. Laue, Ph.D., Professor, Biochemistry and Molecular Biology; Director, Biomolecular Interaction Technologies Center (BITC), University of New Hampshire

Discussion topics include:
• Which physical properties are most important?
• How can they be measured?
• How do solvent properties (e.g. excipients) affect the protein properties?

Table 13: Sub-visible Particles in Therapeutic Protein Formulations: Analytical and Safety Aspects

Moderators: Henryk Mach, Senior Investigator, Bioprocess Analytical and Formulation Sciences, Merck and Co.
Shawn Cao, Ph.D., Principal Scientist, Process and Product Development, Amgen Inc.

Discussion topics include:
• New analytical techniques: differentiation between protein and foreign particles
• Identification of degradation/aggregation mechanisms
• Correlation between size, structure, antigen spacing and immunogenicity
• Is USP <788> adequate and appropriate for protein therapeutics?
• How to analyze sub-µm particles in protein therapeutics?
• What will particle analysis look like in 10 years from now?

Table 14: Aggregation and Immunogenicity:  Can We Identify/Characterize  Aggregates That Are "Provocative" to the Immune System and How Do we Tackle Them?

Moderator: Joel Richard, Ph.D., Senior Director, Head of Drug Product Development, Pharmaceutical Development, Ipsen

Discussion topics include:
• What are the most "provocative " aggregates to the immune system?
• How do we identify and characterize them?
• Can we set "smart" specifications on aggregate content, and what is an acceptable aggregate level as regards immunogenicity?
• Are there advantages with PEGylation and other post-translational modifications? What is the effect of preservatives on aggregation in multi-dose formulations, and do we expect these formulations to be more immunogenic?

Table 15: New Technologies in Use or Under Investigation by Scientists

Moderator: Mark Pollo, Associate Senior Biophysical Chemist, Bioproduct Research and Development, Eli Lilly & Co.

Discussion topics include:
• Characterization of solutions (soluble species)
• Suspensions (particulates)
• Solids (dosages)

Table 16: Addressing Issues of Observed Protein Aggregation: Risk Associated with Product Quality and Immune Responses

Moderator: Greg Walsh, Senior Scientist, Technology Development, Genzyme

Discussion topics include:
• Are all aggregates (native vs. denatured, small vs. large, and sub-visible particles) considered to have comparable immunogenic risk?
• What does the presence of protein aggregates in therapeutic protein product indicate about states of process and product quality?
• What are the most relevant stress models currently employed to determine robustness with respect to protein aggregation and sub-visible particles?

Table 17: Peptide and Protein Modifications to Improve Pharmaceutical Properties

Moderator:  David Litzinger, Ph.D., Director, Pharmaceutical Sciences, Amylin Pharmaceuticals, Inc.

Discussion topics include:
• What aspects should be considered when making an analog, mutant, or conjugate?  What are the desired changes?  What are potential undesirable consequences?
• What should guide choices for modifications (what site, what modification?)?
• At what stage in discovery, development or product life-cycle should this be explored?
• What screening assays should be done to show improved pharmaceutical properties, including aggregation?

4:50 Networking Cocktail Reception in the Exhibit Hall

6:00 End of Day


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Program Navigation

Phage and Yeast Display of Antibodies and Proteins Engineering Antibodies Antibody Optimization Difficult to Express Proteins Pre-Clinical/Clinical Development Revival of Bispecific Antibodies Immunogenicity of Therapeutic Biologics Protein Aggregation in Biopharmaceutical Products Biotherapeutic Targets

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