Biophysical analysis is now playing a significant role in the research and early development for a new generation of complex protein therapeutics. As protein engineers and analytical scientists increase their reliance on biophysical analysis, they are driving a move to instruments with higher throughput and resolution – and working to quantify analytical results previously used only for qualitative assessments. The PEGS Biophysical Analysis meeting brings together an international audience of protein scientists and analytical specialists to explore the latest technologies and techniques used for problem solving in this dynamic field.

Final Agenda

Recommended Short Course(s)*

SC8: Introduction to Biophysical Analysis for Biotherapeutics: Discovery & Development Applications

Christine P. Chan, PhD, Principal Scientist, Global Manufacturing Science & Technology, Sanofi

*Separate registration required.

WEDNESDAY, MAY 2

7:30 am Registration(Commonwealth Hall) and Morning Coffee (Harbor Level)

METHODS AND INSTRUMENTS
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8:30 Chairperson’s Remarks

George Bou-Assaf, PhD, Scientist, Technical Development, Biogen

8:40 Integrating Biophysical Analyses Earlier in the Discovery Process to Improve Final Lead Selection

Jennifer F. Nemeth, PhD, SCPM, Director, Biophysics, Structural Characterization, Biologics Discovery Sciences, Janssen Research & Development

Biophysical characterization has a critical role in drug discovery from target identification, to early hit lead screening, to intensive structural characterization at the pre-NME stage. Historically, biophysics has been applied heavily in the late discovery phases as a drug candidate approaches the NME declaration. I will examine what assays are having the greatest impact in our workflows across the discovery space, and where we are looking to make in-roads into new functional areas.

9:10 Alternatives to SEC for Measuring Aggregates and Fragments

David Hayes, PhD, Principal Scientist, Boehringer Ingelheim Pharmaceuticals, Inc.

It is known that some aggregates cause immunogenicity and therefore it is important for analytical scientists to use multiple techniques to robustly monitor and control aggregate levels. Analytical Ultracentrifugation (AUC) is the gold standard technique orthogonal to SEC for aggregate quantification. This talk will outline best practices for using AUC and also discuss limitations of AUC in terms of precision and repeatability illustrated with examples using synthetic data.

9:40 KEYNOTE PRESENTATION: Capturing, Identifying and Visualizing Preaggregate Transients Using Chaperonin-Based Biolayer Interferometry Platforms

Mark T. Fisher, PhD, Professor, Biochemistry and Molecular Biology, University of Kansas Medical Center

We can detect the presence of transient preaggregates within protein solutions using Chaperonin (GroEL) Biolayer interferometry (BLI) biosensors. ATP binding to GroEL Biosensors can specifically release captured proteins from the biosensor into microvolume aliquots. These released proteins are easily visualized by electron microscopy and evaluated using mass spectroscopy analysis. In some instances, we can even obtain low resolution 3D structures of released proteins released into these microvolumes using Electron Tomography.

10:10 Coffee Break in the Exhibit Hall with Poster Viewing (Commonwealth Hall)

10:55 AUC for the Biophysical Characterization of Gene Therapy Products

George Bou-Assaf, PhD, Scientist, Technical Development, Biogen

During production of AAV-based therapeutics, some viral particles remain empty while others incorporate a partial gene of interest. Both species constitute impurities. Hence, accurate separation and quantitation from the main product, the full particle, is indispensable for the characterization of AAV-based therapeutics. We developed two AUC-based methods for the quantitation of aggregates and empty versus full particle ratios in gene therapy products to enable process development and drug product characterization.

11:25 Screening and Miniaturization Approaches for Continuous Improvement in Biologics Development

Dana Filoti, PhD, Senior Scientist, Biologics Preformulation DPD, AbbVie Bioresearch Center

11:55 Evaluation of Factors Contributing to the Presence of Subvisible Particles in Dose Preparation and Administration Volumes for Biotherapeutics

Mark Pollo, Principal Scientist, Pfizer, Inc.

Particulate matter in drug products is a major concern in development of biopharmaceuticals and extensive work is performed in drug product development to minimize the level of particulates during manufacturing. Sources of particulates in dose preparation vary based on the formulation, manufacturing process and packaging. Studies were conducted to systematically investigate intrinsic and extraneous sources of particulate matter arising from materials and procedures associated with dose preparation and administration. 

12:25 pm High Throughput, Low Volume Subvisible Particle Screening

Robert Hart, CEO, Halo Labs

Halo labs will present a subvisible particle screening tool, the HORIZON, with detailed explanation of its Backgrounded Membrane Imaging (BMI) technology. A comparative analysis between HORIZON and flow imaging will be presented and key performance indicators including sample volume, throughput, dynamic range, instrument repeatability will be evaluated.

12:40 CLIPS-Based Protein Mimicry: Extending Epitope Mapping Technology with Paratope Mapping

Steffen van der Wal, PhD, Peptide Chemist, Pepscan

Most therapeutic antibodies recognize conformational epitopes. To address the 3D spatial conformation of complex epitopes, Pepscan has perfected its array technology through the addition of CLIPS chemistry resulting in CLIPS Precision Epitope Mapping. To comprehensively characterize Ab-Ag interactions also paratope mapping has been added to the platform technology.

12:55 Luncheon Presentation I: A Quick Protein Quality Ccheck That Will Improve Biotherapeutic Candidate Purification and Characterization Workflows

Peter Fung, PhD, Senior Manager, Product Marketing, NanoTemper Technologies

Starting with material of questionable quality for protein purification and characterization leads to irreproducible or ambiguous results. Transitioning between upstream and downstream workflows can be a challenge for bioprocessing researchers, particularly when the quality of the sample material is not known. We present a new platform that swiftly identifies sample quality and relative functionality in minutes complementing and guiding bioprocessing workflows—making go/no go decisions easy and quick—saving time, effort and producing more consistent results.

1:25 Luncheon Presentation II: Computational Approaches for Pptimizing the Developability of Biotherapeutics

Nels Thorsteinson, Scientific Services Manager, Biologics, Chemical Computing Group

mAb candidates identified from high-throughput screening or binding affinity optimization often present liabilities for developability, such as aggregation-prone regions or poor solution behavior. In this work, we optimized an integrin α11 binding mAb for developability using homology modeling and rational design where reducing hydrophobic surface patches improved HIC behavior. A retrospective data analysis demonstrates that 3D descriptors and multi-parameter models can screen candidates and enrich libraries with favorable developability properties for a range of biotherapeutics.

1:55 Session Break

VISCOSITY MEASUREMENT
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2:10 Chairperson’s Remarks

Deniz B. Temel, PhD, Senior Research Investigator, Bristol-Myers Squibb

2:15 Predicting Viscosity of Therapeutic Antibodies: A Systematic Study Involving Small-Scale in vitro and in silico Methods

Hubert Kettenberger, PhD, Senior Principal Scientist, Protein Engineering, Roche Innovation Center Munich, Germany

Subcutaneous or intravitreal administration of antibodies often requires a high protein concentration as well as a low viscosity. Viscosity measurement is low-throughput, cumbersome and demands comparatively high sample amounts. Using a set of approximately 30 therapeutic antibodies, we aim at identifying small-scale in vitro and in silico methods to predict viscosity.

2:45 Emerging Automated Viscosity Measurements with Small Sample Size: An Evaluation of Multiple Methods and Technologies

Deniz B. Temel, PhD, Senior Research Investigator, Bristol-Myers Squibb

It is crucial to optimize the usage of available samples and time for candidate differentiation, especially in terms of developability. There are many emerging methods and technologies with the promise to measure biophysical properties in a high throughput fashion with small sample consumption. In this talk, a number of instruments and methods, based on various physical principals, are evaluated and compared to measure (or predict) viscosity of biologics at high concentrations.

3:15 High-Throughput Biophysical Screening for Development and Formulation of Biologics

John Champagne, Northeast Regional Manager, Wyatt Technology

A smorgasbord of biophysical screening capabilities for development and formulation of biologics: light scattering does it in plates.

3:45 Refreshment Break in the Exhibit Hall with Poster Viewing (Commonwealth Hall)

4:45 Problem-Solving Breakout Discussions (Commonwealth Hall)

Detecting the Elusive Preaggregation State

Mark T. Fisher, PhD, Professor, Biochemistry and Molecular Biology, University of Kansas Medical Center

• Experimental setup and specific release of enriched preaggregates
• 3 D electron microscopy of proteins isolated in microvolume quantities
• Capturing and evaluating bacterial/viral protein toxin following acid transitions
• Rapid identification of modified or mutant proteins within mixtures

Analytical Characterization for Novel Biologics

Zhi (Jay) Guo, PhD, Senior Scientist, Global Protein Sciences, AbbVie Bioresearch Center

• Bispecific Biologics Design
• Screening platform for novel biologics
• Novel biologics with various payloads
• Useful analytical methods for novel biologics

Is What You See What You Get? Analytical Issues in Late Research and Early Development

David Hayes, Ph.D., Principal Scientist, Boehringer Ingelheim Pharmaceuticals, Inc.

• How pure should the tox lot be? Can too much purity cause problems later on? Can too little purity lead to mischaracterization?
• Can sample preparation cause aggregation or fragmentation?
• Which comes first, method validation or degradation pathway definition?
• How should we deal with a “new” aggregate peak?
• Limits of detection: is characterization of minor variants (below 1%) an efficient use of development resources?

Use of Biophysics in Antibody Discovery

Jennifer F. Nemeth, Ph.D., SCPM, Director, Biophysics, Structural Characterization, Biologics Discovery Sciences, Janssen Research & Development (Confirmed)

• What analytical techniques are most impactful in the funnels stage?
• What analytical techniques are most impactful when determining manufacturability?
• What are the current bottlenecks in delivering analytical data?
• What new techniques or assays are showing significant impact?

Challenges and Opportunities in the Use of Surfactants in Protein Drug Products

Andrea Allmendinger, PhD, Senior Scientist, Late-Stage Pharmaceutical and Processing Development, Biologics, F.Hoffmann-La Roche, Switzerland

• Polysorbates: (enzymatic) degradation, particle formation and mitigation strategy
• Comparison of Poloxamer 188 and Polysorbates, interchangeability in protein formulations
• Potential future alternative surfactants, testing and approval

5:45 Networking Reception in the Exhibit Hall with Poster Viewing (Commonwealth Hall)

7:00 End of Day

THURSDAY, MAY 3

8:00 am Registration(Commonwealth Hall) and Morning Coffee (Harbor Level)

ADVANCES IN SPECTROMETRIC METHODS
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8:30 Chairperson’s Remarks

Guodong Chen, PhD, Research Fellow, Bioanalytical and Discovery Analytical Sciences, Bristol-Myers Squibb

8:35 Development and Implementation of a Component-Based Hydrogen-Deuterium Exchange MS System

Kristopher Truncali, Scientist, Biophysics, Structural Characterization, Biologics Discovery Sciences, Janssen Research & Development

Hydrogen-Deuterium Exchange (HDX) mass spectrometry can provide critical insights into protein binding interactions. However, the technique’s complexity has raised confusion around its routine utility and application. Here, we present an advanced HDX platform for early biophysical characterization of protein therapeutics. The workflow employs a LEAP H/D-X PAL, Thermo Orbitrap Fusion Lumos, Vanquish UHPLC, HDExaminer software, and in-house built automation software. The platform setup, applications, and limitations will be discussed.

9:05 Novel Mass Spectrometry-Based Strategies to Study Biomolecular Structure, Dynamics and Interactions in Complex Systems

Igor Kaltashov, PhD, Associate Professor, Chemistry, University of Massachusetts, Amherst

LC/MS is now routinely used in characterization of biopharmaceuticals, with reversed phase HPLC being the preferred separation method. In this presentation, we will discuss the advantages of using MS and MS/MS in combination with non-denaturing separation methods (ion exchange and size exclusion) to characterize complex therapeutic proteins. We will also present examples of using native LC/MS for characterization of other extremely heterogeneous macromolecular therapeutics, such as heparin and heparin-based medicines.

9:35 The QC Watchdog: Forensic ID of Any Particle

Greg Manley, PhD, Product Manager, Unchained Labs

Particles muck up the quality of a drug and can shut down its production. Unchained Labs has the only tool out there that identifies particles by their shape and then forensically identifies them by their chemical and elemental fingerprints. In minutes, researchers know the culprit with-out a doubt, can track it back to the source, and fix their workflow or manufacturing process.

10:05 Coffee Break in the Exhibit Hall with Poster Viewing (Commonwealth Hall)

CHARACTERIZATION OF HIGHER ORDER STRUCTURE
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11:05 Higher Order Structure Determination: Strategy and Case Study

Guodong Chen, PhD, Research Fellow, Bioanalytical and Discovery Analytical Sciences, Bristol-Myers Squibb

The higher order structure (HOS) of biologics plays an important role in stability, biophysical attributes, biological potency, and may have impact on safety and efficacy of biologics. Determining the HOS of biologics is a critical aspect of biologics discovery and development in the biopharmaceutical industry. This presentation will describe HOS determination strategy and methodology including case studies.

11:35 Novel Benchtop Method of Oxidative Footprinting for HOS Determination

Michael Brenowitz, PhD, Professor, Biochemistry and Molecular Pharmacology, Albert Einstein College of Medicine

Controlled oxidation is an insightful method of protein structure determination well-suited to mapping protein-protein interfaces such as those between antibodies and epitopes. We present a robust method of quantitative hydroxyl radical generation based on Fenton chemistry that is readily implemented on the benchtop using common laboratory tools and scalable to robotic sample handling. Example maps of protein surface accessibility and binding interfaces will be presented.

12:05 pm The Role of Biophysical Tools in Understanding Comparability and Biosimilarity

William Weiss, PhD, Principal Research Scientist and Group Leader, Biophysical Characterization, Eli Lilly and Company

Higher order structure (HOS) characterization is an important component of the broader analytical data package required to provide a comprehensive understanding of comparability/biosimilarity. This presentation will include an overview of established and emerging biophysical techniques for HOS characterization as well as discussion of important considerations for establishing comparability/biosimilarity of processes/products including selecting HOS techniques, developing and controlling methods based on these techniques, and comparing the resulting data.

12:35 End of Biophysical Analysis of Biotherapeutics