2016 Archived Content

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Cambridge Healthtech Institute’s 7th Annual
Protein Aggregation and Emerging Analytical Tools
Mechanism, Prediction, Screening, Immunogenicity and Formulation Challenges
January 21-22, 2016

The popular Protein Aggregation and Emerging Analytical Tools conference covers latest trends, challenges and solutions in understanding, characterization and mitigation of protein aggregation. It features in-depth case studies, new and unpublished data and interactive discussions on mechanisms of aggregation, new tools for detection and quantitation of aggregates, and how the data is used in regulatory filings. It also discusses mechanistic understanding of protein aggregation and present case studies on prevention of particle formation by engineering and formulation approaches, impact of aggregation on production, aggregates as a factor for immunogenicity, and approaches for improvement of biophysical properties of protein solutions. We are looking for cutting-edge research findings and unpublished data to be featured at this forum.

We invite you to present a poster and join colleagues from around the world in this discussion of the key challenges and solutions in protein aggregation in biotherapeutics.

Final Agenda

THURSDAY, JANUARY 21

7:45 am Conference Registration and Morning Coffee

8:15 Chairperson’s Opening Remarks

Jan Jezek, Ph.D., CSO, Research & Development, Arecor Ltd.

KEYNOTE PRESENTATION

8:20 Aggregation of Antibodies

Dimiter_Dimitrov Dimiter S. Dimitrov, Ph.D., Senior Investigator, Protein Interaction Group, FNLCR, NCI, National Institute of Health

Aggregation of antibodies in several formats including full-size, domains and antibody-drug conjugates will be reviewed. Examples of computational analyses and experimental data will be discussed for several antibodies including some generated in our group. These results could help decrease or eliminate aggregation in newly identified antibodies and antibody based fusion proteins.

9:00 The Mechanistic Understanding Protein Aggregation

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

The creation of antibody-drug conjugates, bispecific and multi-specific biotherapeutics raises concerns about whether there needs to be a fundamental shift in how these molecules are formulated. The simple answer is no - the underlying principles of macromolecular interaction remain unchanged. The energetics of the pathway leading to a protein-protein complex will be outlined, with special attention paid to the role of desolvation energies. Attention will be paid to how the properties of both the protein and the solvent impact the pathway energetics.

9:30 The Impact of Process Impurities on mAb Degradation

Sreedhara Alavattam, Ph.D., Senior Scientist, Senior Group Leader, Late Stage Pharmaceutical Development, Genentech Inc.

The talk will focus on the impact of metal ion impurities during mAb formulation development. Metal impurities, such as Cu2+, can bind to mAbs and undergo hydrolysis or oxidation, leading to fragmentation of the molecule. To better understand Cu2+-mediated mAb fragmentation, hinge cleavage products and their rates of formation were studied as a function of pH with and without Cu2+. Results suggest that a charge may contribute to stabilization of a specific molecular structure, leading to the possible formation of a Cu2+ binding pocket that causes increased susceptibility of the hinge to degradation. Additionally, mAb interactions with Zn2+ led to the formation of high molecular weight species (HMWS) all the way from dimers to hexamers. These HMWS were dissociable upon dilution but were dependent on both mAb and Zn2+ concentration.

10:00 Coffee Break in the Exhibit Hall with Poster Viewing

11:00 Poster Presentation: Morphology of Alpa-1 Proteinase Inhibitor Aggregates Monitored Using Hydrogen Deuterium Exchange and Covalent Labeling Mass Spectrometry

Jainik P. Panchal, Department of Industrial and Physical Pharmacy, Purdue University

11:30 Separation of Protein from Non-Proteinaceous Particles in Biopharmaceutical Formulations with MVAS by Microflow Imaging MFI

Akhunzada_Zahir_web Zahir Akhunzada, Ph.D., PPD Consultant, Analytical & Bioanalytical Development, Bristol-Myers Squibb

The presence of sub-visible particles (SVPs) is a major challenge in the development of therapeutic protein formulations. Distinction between proteinaceous and non-proteinaceous SVPs is vital in monitoring the formulation stability. The current compendial method based on light obscuration (LO) has limitations in analyzing translucent particles, requires large analysis volume and therefore demands urgent need for an unambiguous method to characterize SVPs. A number of attempts have been made to characterize SVPs, albeit with limited success. This presentation reveals a method that successfully characterizes and distinguishes, both potentially proteinaceous and non-proteinaceous SVPs in protein formulations by using Microflow Imaging (MFI) in conjunction with the MVAS (MFI View Analysis Suite) software.

12:00 pm Session Break

12:15 Luncheon Presentation (Sponsorship Opportunity Available) or Enjoy Lunch on Your Own

1:15 Ice Cream Break in the Exhibit Hall with Poster Viewing

2:00 Chairperson’s Remarks

Marisa K. Joubert, Ph.D., Senior Scientist, Process Development, Amgen, Inc.

2:05 Novel Chromatography-Based Approaches to Investigate Protein-Protein Interactions and Its Application in Formulation Screening Workflows

Sanket_Patke Sanket Patke, Ph.D., Research Investigator, Drug Product Science and Technology, Pharmaceutical Development, Bristol-Myers Squibb

Protein-protein interactions influence colloidal stability parameters such as solubility and aggregation. Measurement of osmotic second-virial coefficients, B22, provides one method to quantify protein interactions at the molecular level. Self-interaction chromatography is a novel method of measuring B22 with modest material and time requirements. Here we discuss the application of this approach to investigate protein–protein interactions in mAbs. This approach can potentially be used as a tool during formulation screening.

2:35 Innovative Approaches to Controlling Aggregation of Therapeutic Proteins

Jan_Jezek Jan Jezek, Ph.D., CSO, Research & Development, Arecor Ltd.

Aggregation of therapeutic proteins remains one of the key challenges during their manufacture and storage. Formulation is a powerful tool to minimise aggregation. The talk will outline innovative approaches to addressing protein aggregation to simplify production and enable products to be used outside the cold chain. This will be demonstrated on several data driven case studies using relevant therapeutic proteins, describing the specific formulation features employed to achieve superior stability.

3:05 Molar Mass, Size and Interactions: Light Scattering Tools for Essential Biophysical Characterization

Hassouneh_Wafa Wafa Hassouneh, Ph.D., Applications Scientist, Customer Service, Wyatt Technology

Biophysical techniques based on static and dynamic light scattering address many of the key analytical challenges in biotherapeutic R&D, from early candidate selection through scale-up, formulation, characterization and comparability studies. This seminar will review light scattering technology and instrumentation, then present select examples illustrating how Wyatt’s light scattering solutions facilitate rapid and effective development of biologics including mAbs, ADCs, PEGylated and other proteins.

3:35 Refreshment Break in the Exhibit Hall with Poster Viewing

4:15 Characterization of High Concentration Monoclonal Antibodies by Combined DLS and Raman Spectroscopy

Geetha_Thiagarajan Geetha Thiagarajan, Ph.D., Associate Principal Scientist, Sterile Product and Analytical Development, Merck

The study highlights applications of Zetasizer Helix, which allows concurrent measurement of dynamic light scattering and Raman spectroscopy for high-concentration solutions (>50mg/ml), providing size and secondary/tertiary structural information. Distinct structural changes involving disulfides, aromatics, hydrogen bonding, and secondary structure were identified on thermally stressed mAbs, whereas DLS captured changes for photostressed mAb. Helix-measured unfolding temperatures correspond with DSC, while providing additional structural perturbations. Minimal sample volume and manipulations makes Zetasizer Helix a useful analytical tool for exploring stress/formulation induced-changes in therapeutic proteins.

4:45 Assessing Biotherapeutics Stability Using Raman Spectroscopy

Marinella_Sandros Marinella Sandros, Ph.D., Assistant Professor, Department of Nanoscience, University of North Carolina at Greensboro

Self-administered protein-based therapeutics need to be developed at high concentrations. However, there is an increase in tendency for proteins to aggregate at these levels (>100 mg/m L). Our group has investigated the potential of Raman as a non-invasive and label-free tool to assess protein stability. Results from this study identified specific signature bands that can be used to highlight individual amino acid residues that are responsible for structural changes in proteins.

5:15 Site Directed Spin Labeling for Detection of Protein Aggregation: an Emerging Analytical Tool

Lawrence_Berliner Lawrence Berliner, Ph.D., Professor of Chemistry and Biochemistry, University of Denver; Emeritus, Ohio State University

The site-directed spin labeling technique utilizes cysteine substitutions in proteins, has been shown to be a boon to protein structure determination where other methods fail. It is highly suited for membrane proteins where crystallography is not feasible. The advantages are no requirement for optical transparency, molecular weight limits are not an issue and solid state matter is applicable. It can be applied to detection of protein aggregates and particulates.

5:45 Close of Day

6:00-7:00 Reception in the Tiki Pavilion

FRIDAY, JANUARY 22

8:00 am Conference Registration and Morning Coffee

8:30 Chairperson’s Remarks

Joël Richard, Ph.D., Vice President, Peptides, CMC & Engineering, Ipsen

8:35 Predicting the Risk of Extractables and Leachables (E&L) Interacting with Therapeutic Proteins

Kim_Li Kim Li, Ph.D., DABT, MPH, Senior Manager, Environment, Health, Safety and Sustainability, Amgen, Inc.

Therapeutic proteins can be subject to chemical modifications which may lead to product quality and safety concerns. Extractables and leachables (E&L) arising from process- and product-contact surfaces present the risk of interacting with the protein products. This presentation will describe the mechanisms of such interactions and the use of an in silico software that classifies E&L structures into reactive functional groups for risk prediction.

9:05 Extractables & Leachables in Liquid Formulations of Proteins: Impact on Stability, Aggregation, Potency and Immunogenicity of Drug Product

Joel_Richard Joël Richard, Ph.D., Vice President, Peptides, CMC & Engineering, Ipsen

Extractables and leachables are impurities that can contaminate liquid formulations of biologics, leaching from the surface of glass walls or being extracted from the rubber stoppers and plungers. They can interact with and degrade proteins, modifying their higher order structure, forming mixed micelles, or composite protein aggregates. These impurities may have a strong impact on stability, quality attributes and immunogenicity profile of the protein drug products.

9:35 Oxidation of Methionine in Aggregated Antibodies does not Increase the Potential Risk of Immunogenicity

Marisa_Joubert Marisa K. Joubert, Ph.D., Senior Scientist, Process Development, Amgen, Inc.

This talk will give an update on the aggregate attributes of monoclonal antibodies (mAbs) that may cause an immune response. The potential impact of Met oxidation of both aggregated and monomeric antibodies was investigated in a population of human peripheral blood mononuclear cells (PBMC) from healthy and disease state individuals (50+ donors tested) in vitro. Met oxidation was found not to increase the potential risk of immunogenicity of both aggregated and monomeric antibodies.

10:05 Coffee Break with a Poster Pavilion

PepTalk is proud to support and recognize the protein scientists of tomorrow during the Poster Pavilion. This time has been set aside to view the Student Fellowship posters and interact with presenters one on one. This opportunity gives job seekers the chance to share their expertise with future/potential employers or develop contacts to further their research.

11:00 Investigation of Reversible Self-Association during Early Stage Development of a Low Concentration Antibody-Drug Conjugate

Elizabeth Bartlett, Scientist II, Analytical & Pharmaceutical Sciences, ImmunoGen, Inc.

Reversible self-association is often present in high concentration antibody products, but may also occur in lower concentration preparations. In the case of antibody-drug conjugates (ADCs), a novel class of molecules for the treatment of cancers, this property can present substantial challenges to successful formulations. In this study, a multi-technique approach was used to identify and investigate the effects of various excipients on reversible self-association in a low concentration ADC.

11:30 Characterizing Changes in Protein Quality Attributes to Assess Leachable Risks from Single-Use Bioprocess Containers

Nina_Xiao Nina Xiao, Senior Research Associate, Late Stage Pharmaceutical Development, Genentech, Inc.

Application of single-use bioprocess containers for the manufacturing of biologics have increased significantly over the years. This study examines two monoclonal antibodies in a small-scale stressed model to detect and assess the presence of leachables by monitoring protein quality attributes. The results from this study demonstrate that the stress model can inform a risk assessment of leachables on protein quality attributes during routine manufacturing. Leachable characterization will also be discussed.

IT’S A WRAP:
PEPTALK 2016 CLOSING PLENARY PANEL DISCUSSION
Friday, January 22, 12:00 pm

Protein therapeutics is one of the fastest-growing global markets, driven by increasing adoption of protein- over non-protein drugs, growing funding for protein engineering and reduced drug discovery timelines and costs. As the science improves, so does the complexity of the R&D organization: it really does “take a village” to bring nextgeneration therapies to market and patients who need them. Ensuring product quality plus speed to market requires collective insights from experts working across the stages of protein science R&D – as embodied by panelists representing each PepTalk Pipeline topic.

They discuss: