Cambridge Healthtech Institute’s 10th Annual
Protein Aggregation and Emerging Analytical Tools
January 17-18, 2019
The popular 10th Annual Protein Aggregation and Emerging Analytical Tools conference covers latest trends, challenges and solutions in understanding, characterization and mitigation of problems generated by protein aggregation in biopharmaceuticals.
This conference will feature in-depth case studies, new and unpublished data and interactive discussions on immunogenicity of aggregates, mechanisms of aggregation, new tools for detection and quantitation of aggregates, and how the data is used in
regulatory filings. It will also discuss mechanistic understanding of protein aggregation and present case studies on prevention of particle formation by engineering and formulation approaches, aggregation in ADCs, bispecifics, impact of aggregation
on production, aggregates as a factor for immunogenicity, and approaches for improvement of biophysical properties of protein solutions.
Final Agenda
5:45 - 8:45 Recommended Dinner Short Courses*
SC3: Protein Aggregation: Mechanism, Characterization and Consequences
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*Separate registration required
THURSDAY, JANUARY 17
7:45 am Registration and Morning Coffee (Sapphire West Foyer)
8:10 Organizer’s Welcome Remarks
Nandini Kashyap, Conference Director, Cambridge Healthtech Institute
8:15 Chairperson’s Opening Remarks
Thomas Laue, PhD, Professor Emeritus, Molecular, Cellular and Biomedical Sciences, University of New Hampshire
KEYNOTE PRESENTATION
8:20 Chemical Protein Modifications and Immunogenicity Risks
Christian Schöneich, PhD, Takeru Higuchi Distinguished Professor and Chair, Department of Pharmaceutical Chemistry, The University of Kansas
Chemical modifications can play an important role in the immunogenicity of proteins. We have designed experiments to test whether specific chemical protein modifications induced by light are immunogenic. Peptides derived from a light-exposed humanized
monoclonal antibody were fractionated, and these fractions injected into transgenic mice designed to tolerate native human IgG. Specific peptide fractions showed immunogenic responses, and chemical modifications present in these fractions were characterized
by HPLC-MS/MS analysis.
FEATURED PRESENTATION
9:00 Protein Polymorphism, Heterogeneity and the Immunogenicity of Biotherapeutics
Roy Jefferis, PhD, MRCP, FRCPath, DSc, Emeritus Professor, Institute
of Immunology & Immunotherapy, University of Birmingham
Administration of biotherapeutic drug may be considered: 1) to introduce/supplement a deficit in a natural (self) protein/glycoprotein (P/GP); 2) to manipulate/eliminate the activity of a self-molecule/cell. Clinical experience shows that a proportion
of patients produce an anti-therapeutic antibody drug (ATA) immune response. This may be due to: 1) absence of the natural molecule or exposure to an unmatched polymorphic variant; 2) exposure to a molecule lacking structural fidelity with a self
P/GP.
9:30 Next Steps in Biophysical Characterization and Screening: RPC/IEX-MALS and HT-SLS
Jeff Ahlgren, PhD, Senior Application Scientist, Wyatt Technology
SEC-MALS and high-throughput DLS (HT-DLS) are widely implemented across biopharma to characterize molar mass, aggregation, oligomerization and fragmentation, and to screen candidates and formulations for aggregation and stability. Recent extensions of
light scattering will be presented: a light-scattering plate reader that measures both dynamic and static light scattering to determine size, molar mass, kD, A2, thermal stability and viscosity; and the use of multi-angle light scattering with reversed-phase
and ion-exchange chromatography.
10:00 Coffee Break in the Exhibit Hall with Poster Viewing (Sapphire Ballroom)
11:00 Use of an In Vivo Model and In Vitro Cell Based Assays to Assess the Potential Immunogenicity Risk of Critical Quality Attributes of Biotherapeutics
Cathie Xiang, MS, Senior Associate Scientist, Attribute
Science, Amgen
Although some critical quality attributes, e.g. hydroxylysine are commonly modifications found in biotherapeutics, there is little to no information available on their safety impact. This uncertainty has led to high severity risk scores being assigned
to them and the subsequent need for process control. A comprehensive evaluation of the potential immunogenicity risk of some critical quality attributes were conducted using both in vivo models and in vitro cell based assays.
11:30 IgG Charge
Thomas Laue, PhD, Professor Emeritus, Molecular, Cellular and Biomedical
Sciences, University of New Hampshire
Charge is a fundamental property of practical and biological importance. ZDHH has been measured in formulation (pH 5) and physiological (PBS) solvent for three different IL-13-specific mAbs. For each mAb, ZDHH has been measured for four IgG subclasses,
as well as their Fc and F(ab’)2 fragments. Also, the distribution of ZDHH has been determined for human poly-IgG in PBS. The results illustrate how little is known about protein charge.
12:00 pm Session Break
12:10 Luncheon
Presentation I: A Stunning Combo for Quick and Thorough
Assessments of Protein Quality
Dina Finan, PhD, Marketing Manager, Analytics, Marketing, Unchained Labs
There are many reasons to assess the quality of protein samples prior to downstream analysis, such as comparing batches of purified material, changing formulation conditions, or checking the integrity of thawed or stressed samples. With Stunner,
you can now perform painless quality checks by determining the concentration, hydrodynamic size, and polydispersity at the same time with just 2 µL of sample, enabling you to move on to the next steps in your workflow with confidence.
12:40 Luncheon Presentation II (Sponsorship Opportunity Available)
1:10 Ice Cream Break in the Exhibit Hall with Poster Viewing (Sapphire Ballroom)
2:15 Chairperson’s Remarks
Christian Schöneich, PhD, Takeru Higuchi Distinguished Professor and Chair, Department of Pharmaceutical Chemistry, The University of Kansas
2:20 Investigating the Mechanism of Protein Aggregation and Subvisible Particle Formation Mediated by Solid-Liquid Interfaces
Cavan Kalonia, PhD, Scientist, Late Stage Formulation Sciences,
MedImmune
Physical degradation and aggregation of proteins at solid-liquid interfaces can negatively impact the manufacturability, shelf-life stability, and administration of protein therapeutics. Despite the critical impact of solid-liquid interfaces on protein
stability, the mechanisms of interfacial degradations remain poorly understood and highly speculative in the pharmaceutical literature. In this work, we implement and develop state of the art metrology and modeling tools to investigate protein
interfacial degradation at pharmaceutically relevant surfaces.
2:50 Mechanism, Consequence and Control of Protein Opalescence
Wei Wang, PhD, Senior Scientist, Biologics Development, Bayer U.S. LLC
Protein opalescence is a commonly-observed phenomenon. It is often accompanied by phase separation, especially at high protein concentrations. Both protein opalescence and phase separation are undesirable physical properties in the development of
a successful protein pharmaceutical product. This presentation discusses the mechanism of protein opalescence, its potential consequences, and various means of controlling protein opalescence.
3:20 Elucidating Sites and Mechanisms of Protein
Aggregation for Improved Candidate Progression
Belinda Pastrana, CEO, Protein Dynamic Solutions
Therapeutic mAb candidates were compared for developability based on extent and mechanism of stress-induced aggregation. A label-free, array-based platform was used to determine and compare regions prone to aggregation, assess domain stability and
to pinpoint deamidation of asparagine & glutamine residues. Stabilizing effects of excipients were compared at varying concentrations.
3:35 Networking Refreshment Break (Sapphire & Aqua West Foyer)
4:00 Formulation and Container Closure System Strategies for Biopharmaceuticals with Higher Stability
Susumu Uchiyama, PhD, Professor, Department of Biotechnology, Graduate School of Engineering, Osaka University
We have identified causes of protein aggregation in biopharmaceuticals and attempted to optimize formulation and container closure system to reduce the protein aggregates. Secondary virial coefficient can be effective parameter for the prediction
of aggregation tendency. Meanwhile, appropriate selection of barrel material is necessary for biopharmaceuticals with better quality. Silicon oil free polymer-based syringe is most suitable for biopharmaceuticals. All together formulation and
container closure system strategies will be introduced.
4:30 Aggregation Mechanisms and Molecular Profiling of Therapeutic Antibodies
Peter M. Ihnat, PhD, Principal Scientist, Biologics Preformulation
and Drug Delivery, AbbVie Bioresearch Center
Isothermal chemical denaturation was used to calculate the free energies of unfolding as a function of concentration and determine the mechanisms of oligomerization for a series of IgG1 antibodies. Most of the IgG1s favored the native state mechanism
of association which was sensitive to pH. The mechanisms were correlated with thermal analysis, aggregation kinetics and structural attributes to illustrate screening and risk assessment of IgG1 candidates.
5:00 Novel Biopharmaceutical Compositions to Reduce the Rate of Aggregation
Jan Jezek, PhD, CSO, Research & Development, Arecor, Ltd.
Despite considerable progress in candidate screening and formulation approaches, protein aggregation during manufacturing, storage and use remains one of the key challenges of biopharmaceutical development, particularly for a number of new modalities
such as bispecific antibodies. This talk will show on several case studies how novel and unconventional formulations can significantly decrease the rate of aggregation alongside other degradation pathways and enable development of competitive
patient-friendly products.
5:30 Close of Day
FRIDAY, JANUARY 18
8:00 am Registration (Sapphire West Foyer)
8:00 BuzZ Sessions with Continental Breakfast (Sapphire Foyer)
Protein therapeutics is a fast-growing global market. As the science improves, so does the complexity of the R&D organization. Ensuring product quality plus speed to market requires insights from stakeholders working across the stages of protein
science R&D. Join experts representing this PepTalk pipeline, peers, and colleagues for an interactive roundtable discussion. Topics include highlights from the week’s presentations, new technologies and strategies, challenges, and future
trends.
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Moderator: Thomas Laue, PhD, Professor Emeritus, Molecular, Cellular and Biomedical Sciences, University of New Hampshire
9:00 Chairperson’s Remarks
Jan Jezek, PhD, CSO, Research & Development, Arecor, Ltd.
9:05 Novel Analytical Approaches for Mechanistic Understanding of Protein Aggregation
Ulla Elofsson, PhD, Associate Professor, Senior Scientist,
RISE Research Institutes of Sweden
The use of scattering techniques (electrons, neutrons) to investigate aggregation mechanisms at high resolution in space and time will be explored. Predictive methods are built on this knowledge in combination with stability data generated by traditional
(long term stability studies) and other techniques such as DLS and AF4. As an example, we will present methods to study surface induced protein aggregation.
9:35 Water Proton NMR for in situ Detection of Protein Aggregation
Yihua Bruce Yu, PhD, Professor, Department of Pharmaceutical
Sciences, University of Maryland School of Pharmacy
The water proton (1H2O) NMR signal is sensitive to protein aggregation. Compared with conventional analytical techniques, 1H2O NMR can be performed on protein solutions inside sealed containers and thereby is applicable to both drug substance and
drug products. 1H2O NMR can detect both small (nanometer sized) and large (micrometer) aggregates. 1H2O NMR can be implemented using benchtop NMR spectrometers; data collection and analysis takes 1-2 min per sample.
10:05 Development Strategy of Fibril-Prone Peptide Therapeutics: Aggregation Kinetics, Predictive Methods, and Detection Methods
Jingtao Zhang, PhD, Principal Scientist, Pharmaceutical Sciences,
Merck Research Laboratories
Peptide aggregation such as fibrillation presents significant challenges for DS and DP development of peptide therapeutics. Different development criteria and control strategy are required for fibril development in contrast to protein aggregation.
The unique nature of fibril also presents significant challenges in the analytical development, especially in aggregation measurement. Approaches to close gaps in these areas will be shared in the presentation, which includes the investigation
on the aggregation kinetics of a fibril-prone peptide, the projection of physical stability shelf-life, and the development of highly sensitive characterization methods for fibrils.
10:35 Networking Coffee Break (Sapphire & Aqua West Foyer)
11:00 Stress-Induced Aggregation of Mouse IgG2c Depends on Antibody Nature and Sub-Micron Aggregates are Detectable by Cell-Surface Low Affinity Mouse Fcγ Receptors
Dana I. Filoti, PhD, Senior Scientist II, NBE Analytical R&D,
AbbVie Bioresearch Center
Proteinaceous aggregates have been linked to the incidence of immunogenic responses but specific factors responsible haven’t been identified because the physiological mechanisms are not well understood. Where biophysical characterization of
stressed IgG solutions showed little to no differences, using FACS we show significantly more binding to Fcγ receptors expressed on the surface of CHO cells compared to unstressed IgG solutions with solutions containing higher amounts of
sub-micron sized aggregates.
11:30 Investigation of Oxidation Potential of Protein Formulation Excipients and Processes Using Dansyl-Methionine
Lin M. Luis, Senior Research Associate, Late Stage Pharmaceutical Development, Genentech
Proteins and excipients are continually exposed to internal and external oxidants. The external factors and processes that give rise to these oxidants include light, metals, cavitation, etc. We have results showing dansyl-methionine is a good protein
surrogate that is capable of picking up, irreversibly, very low amount of oxidation due to peroxides from formulation excipients and processes.
12:00 pm Conference Wrap-Up
Thomas Laue, PhD, Professor Emeritus, Molecular, Cellular and Biomedical Sciences, University of New Hampshire
12:30 Close of Conference