As new product formats progress through development and into the regulatory process, the role of analytical characterization is taking on new meaning. Regulatory agencies are themselves learning about these new formats, and to support filings, industry
companies are being asked to provide ever more complex data across a wide range of analytical methods. The use of platformed analytical programs, developed as a cost and time saver by the industry, is now diminishing as novel molecules and product
formats demand more individualized characterization steps. In addition, instrumentation suppliers are striving to support this new era with unique product features, software and feature combinations. The PEGS Characterization of Biotherapeutics meeting will explore these changes in the progression of analytical development, and offer a case study forum for those working in the field to share ideas, experiences and solutions that support the development of exciting new biotherapeutics.
MONDAY, MAY 4
7:00 am Registration and Morning Coffee
plenary keynote session
8:30 Chairperson’s Opening Plenary Remarks
8:40 Cancer Stem Cells and Mechanisms of Malignant Progression
Robert A. Weinberg, Ph.D., Founding Member, Whitehead Institute for Biomedical Research; Professor, Biology, Massachusetts Institute of Technology
The cell-biological program termed the epithelial-mesenchymal transition (EMT) plays a role in conferring aggressive traits on carcinoma cells. In addition, it generates cancer stem cells (CSCs) that have the ability, following dissemination,
to serve as founders of new metastatic colonies. The relationship between these CSCs and the SCs residing in normal tissues, and the participation of the CSCs in metastatic dissemination will be described.
9:25 Building an Antibody Discovery Company in a Crowded Field – the Adimab Story
Tillman Gerngross, Ph.D., CEO, Co-Founder, Adimab
The presentation will cover the evolution of Adimab from its founding in 2007 to becoming one of the few privately held profitable biotech companies in the last decade. Industry trends and specific strategic decisions along the way will be
discussed and used to illustrate the importance of integrating finance and scientific information to build successful capital efficient biotech companies.
10:10 Coffee Break
10:45 Chairperson’s Remarks
John F. Kellie, Ph.D., Investigator, Bioanalytical Sciences and Toxicokinetics, GlaxoSmithKline
10:50 KEYNOTE PRESENTATION
Native and Subunit Analysis of Antibody-Drug Conjugates
Alain Beck, Ph.D., Senior Director,
Antibody and ADC Physico-Chemistry, Center of Immunology, Pierre Fabre; Associate Editor, mAbs
Most of the current ADCs in clinical trials are controlled, but heterogeneous, mixtures of isomers and isoforms. Drug loading and distribution, amount of naked antibody and average drug to antibody ratio (DAR) are Critical Quality Attributes
for ADCs. At the top level, the advantages of cutting-edge mass spectrometry (MS) techniques such as native MS and Ion-Mobility MS will be compared to Hydrophobic Interaction Chromatography (HIC). In addition, optimization of middle up
and bottom up strategies will be presented allowing structural assessment of positional isomers.
11:20 Novel FRET Assay for the Evaluation of Intracellular Activation of ADC Linkers
Byoung-Chul Lee, Ph.D., Scientist, Protein Chemistry, Genentech, Inc.
Despite the recent success of ADCs, their mechanisms of action are not fully understood. In order to gain further understanding of the ADC intracellular uptake and payload release, we developed a novel fluorescence resonance energy transfer (FRET) ADC. This FRET assay will provide a facile and robust assessment of the intracellular processing and have significant implications for the future development and clinical use of ADCs.
Ten Lessons for the Formulation Development of Monoclonal Antibodies from Multimodal Thermal Unfolding Case Studies
Mark Brader, Ph.D., Principle Scientist, Protein
Pharmaceutical Development, Biogen Idec
The recent availability of technologies enabling simultaneous monitoring of spectroscopic and light scattering readouts creates new opportunities for high throughput analysis and expanded protein formulation parameter space. Importantly,
a better understanding of how formulation conditions affect domain stability and thermal unfolding becomes more readily accessible. This presentation will describe a series of case studies of multimodal thermal unfolding that
provide insights into various aspects of monoclonal antibody formulation design.
12:20 pm Luncheon Presentation I: Uncovering Receptor Targets and Off-Targets of Antibodies and Protein Ligands Using Human Cell Microarray Technology
Jim Freeth, Ph.D., Founder and Managing Director,
Human cell microarray screening provides a rapid and effective solution for discovering the cell surface targets of antibodies, proteins, viruses and small molecules. Case studies from pharmaceutical partners demonstrate how the platform
– which currently incorporates >65% of known plasma membrane proteins – is being used to: 1) Uncover novel targets from antibody phenotypic screening approaches; 2) Identify receptors for protein ligands in normal
and disease processes and 3) Uncover antibody secondary targets to guide lead selection.
Luncheon Presentation II: Advanced Characterization of Antibody Drug Conjugates (ADC’s) by Liquid Chromatography and Mass Spectrometry (LC/MS)
John C. Gebler, Ph.D., Director, Biopharmaceuticals, Waters Corporation
Antibody drug conjugates (ADC’s) are a complex the amalgamation of monoclonal antibodies (mAb) and low molecular weight toxins. The combination is often a mAb targeted to cancer to deliver a potent cyotoxin to a specific site
or cell. The resulting conjugate is complex and typically heterogeneous making physical chemical characterization challenging. Liquid chromatography coupled to mass spectrometry (LC/MS) is a powerful tool for the characterization
of proteins including mAb. At Waters we have placed a focus on the LC/MS for ADC’s based on cysteine, lysine, and engineered conjugates. An ADC specific work flow will be shown for intact, sub-unit, peptide level, and glycan
characterization by LC/MS.
1:20 Session Break
1:50 Chairperson’s Remarks
John F. Kellie, Ph.D., Investigator, Bioanalytical Sciences and Toxicokinetics, GlaxoSmithKline
1:55 FTiH Support of an ADC: Stability, Assay Development, and Clinical Experiences
John F. Kellie,
Ph.D., Investigator, Bioanalytical Sciences and Toxicokinetics, GlaxoSmithKline
Characterization of circulating ADC species (conjugated antibody, total antibody, and payload) is critical to understanding the safety and efficacy of ADC therapeutics. Current methodology requires development and validation of
immunoassays and liquid chromatography-mass spectrometry assays. This presentation will share experiences from assay validation through first time in human bioanalytical study support along with an update and outlook for state-of-the-art
technologies set to drive ADC method development in the future.
2:25 Comparative Clinical Pharmacokinetics of Antibody-Drug Conjugates in First-in-Human Phase 1 Studies
Amara, Senior Pharmacokineticist, Sanofi
Comparison of the clinical pharmacokinetics (PK) for ADCs now in development is challenging because of the large number of targets, ADC constructs, dosing regimens, and patient populations. This presentation presents an evaluation
of ADC clinical PK properties, dosing regimens, determination of doses ranges and associated maximum tolerated doses. The effect of structural characteristics and target types (hematological vs. solid tumors) on PK will also
2:55 Bioanalytical Strategy for Development and Validation of Ligand Binding Assays for ADCs
Kumar, Ph.D., Principal Scientist, Pfizer, Inc.
The dynamic and heterogeneous mixture of ADCs containing various drug-to-antibody ratios (DAR) species and different conjugation sites may have different binding affinities for the capture and detection reagents typically used
in ligand binding assays (LBA). These differences in binding affinity may translate into a variation in the ability of the LBA to accurately detect various DAR species. The case studies presented will evaluate various bioanalytical
strategies employed for the development and validation of DAR independent LBAs for ADC bioanalysis.
3:25 Characterization of Fusion Proteins
Wilma Lau, Ph.D., Senior Scientist, Large Molecule Research, Roche Pharma Research & Early Development, Roche Innovation
The engineering of multi-domain proteins such as Fab-Cytokine or Fab-Toxin fusion proteins promises the advancement of superior biotherapeutics. Their development requires design and characterization strategies tailored to the
properties and the critical quality attributes of the combined domains. Here, we present examples for early design testing using Hapten and Sortase coupling technologies, for adaption of developability concepts for fusion proteins
and for equitable evaluation of fusion protein characteristics.
3:55 Refreshment Break in the Exhibit Hall with Poster Viewing
4:35 Problem-Solving Breakout Discussions
New and Emerging Biophysical Tools for Characterization of Higher Order Structure
Moderator: George Bou-Assaf, Ph.D., Scientist, Biogen Idec
- What new technologies are available?
- How are they different from current ones? Advantages and disadvantages?
- What are the practical considerations to conduct experiments with these new technologies? (Sample requirement? Sample preparation? Time required to carry the experiment? Sensitivity? Reproducibility? Etc..)
- How do we introduce these new technologies into our regulatory filings without causing panic from the reviewer’s side?
Progress and Applications of Intact Protein Mass Spectrometry
Moderator: John Kellie, Ph.D., Investigator, Bioanalytical Sciences and Toxicokinetics, GlaxoSmithKline
- Intact versus digestion-based methods of protein analysis and characterization
- Applications of high-resolution mass spectrometry for intact protein analysis
- Intact antibody/biosimilar characterization: Practicality, limitations, and current MS instrumentation.
- Immunopurification and front-end separation techniques applied to intact protein mass spectrometry
Emerging Computational Technologies to Streamline the Development of Protein Therapeutics
Moderator: Dennis Livesay, Ph.D., Professor, Bioinformatics and Genomics, University of North Carolina
- Computational design of proteins and protein-protein interfaces
- Computational approaches to screen for increased protein stability
- Computational approaches to characterize protein-solvent and protein-cosolute interactions
- Computational approaches to simultaneously optimize binding affinity and formulation
- Computational identification of allosteric sites
Liquid-Liquid Phase Separation in Pharmaceutical Protein Solutions
Moderator: Ying Wang, Ph.D., Research Associate, Physics and Biological Physics, Massachusetts Institute of Technology
- Challenges in the development of concentrated formulation of pharmaceutical proteins
- Liquid-liquid phase separation in concentrated antibody formulations
- The experimental approaches to observe liquid-liquid phase separation in protein solutions
- The relationship between liquid-liquid phase separation and other phenomena in concentrated protein solutions, such as crystallization, aggregation and solution viscosity
- Use liquid-liquid phase separation to our advantage in industrial applications: e.g. evaluation of colloidal stability; purification of proteins
5:35 Welcome Reception in the Exhibit Hall with Poster Viewing
6:50 End of Day
TUESDAY, MAY 5
8:00 am Morning Coffee
8:25 Chairperson’s Remarks
Yelena Lyubarskaya, Ph.D., Senior Principal Scientist, Biogen Idec
8:30 Application of in vitro Models to Understand and Optimize Pharmacokinetic Properties of Biologic-Based Molecules
Carlson, Scientific Director, Pharmacokinetics & Drug Metabolism, Amgen, Inc.
Proteins and peptides are attractive drug candidates due to their biological activity, but they often have suboptimal pharmacokinetic properties. These molecules are prone to high clearance and low bioavailability, limiting their
duration of action and systemic exposure. We are developing and applying in vitro approaches to predict the in vivo stability of peptides and proteins. The goal is to characterize pharmacokinetic liabilities and ultimately
to design molecules with more optimal properties.
9:00 In vitro Biological Characterization of IFN-β-1a Major Glycoforms
Horst Bierau, Ph.D.,
Scientific Advisor and Relation Manager, Merck Serono S.p.A.
Interferon β-1a glycoforms were subjected to physico-chemical and biological characterization by means of mass spectrometry, sialic acid content, thermal denaturation and various in vitro bioassays. The in vitro bioassay responses
revealed a correlation mainly with the glycan antennarity. It is therefore suggested that all glycoforms are having biological activity and play a role in modulating the overall IFN-β biological activity with higher-antennarity
glycoforms being able to better sustain IFN-β-1a bioactivity over time.
9:30 Characterization and Control of Product Variants Applying QbD Principles
Kepert, Ph.D., Senior Manager, Pharma Technical Development Europe, Roche Diagnostics GmbH
The application of quality by design principles on characterizing and controlling product variants for a recently approved therapeutic monoclonal antibody is illustrated. During development several product variants and process
related impurities were characterized and categorized into critical and non-critical quality attributes (CQAs) applying a risk ranking and filtering tool. The identified CQAs were further monitored in process development studies
and an attribute testing strategy was developed encountering process capability and CQA impact.
10:00 Coffee Break in the Exhibit Hall with Poster Viewing
10:50 Protein Characterization for APC: Monitoring Product Quality Attributes for Process Development and Manufacturing
Lyubarskaya, Ph.D., Senior Principal Scientist, Biogen Idec
Advanced process control can provide flexibility and efficiency in biopharmaceutical manufacturing. Understanding of the effect of manufacturing process variables on product quality attributes is required in support of APC development.
The use of mass spectrometry for monitoring glycan distribution of a glycoprotein during cell culture process will be demonstrated. The measurements are performed to obtain early readout of product quality to enhance process
understanding and explore potential process analytical tools.
11:20 Novel Method for Assessing Host Cell Protein Assays
Susan Flor, Senior Research Associate, Analytical Operations, Genentech
Immunoassays are typically used to monitor residual host cell protein (HCP) clearance from biopharmaceuticals. Antibodies to a complex mixture of total HCPs are generated for this purpose. Health authorities often request a quantitative
assessment of HCP coverage with the antibody used in the immunoassay. 2D gels and Westerns have inherent limitations that prevent accurate quantification of coverage. We explore a new method for HCP reagent coverage characterization.
11:50 High-Throughput Analytics in Support of Process Development and Identification of Critical Quality Attributes
Hui Cai, Ph.D., Research Investigator,
Process Development Analytics, Bristol Myers Squibb
The Quality by Design (QbD) initiative aims to thoroughly understand the product and the manufacturing process for better product quality and faster product development. One of the first steps in the QbD approach consists in identification
of the critical quality attributes (CQA). At BMS, we use automation for both process development and process analytics. Here we present data that show the use of automation in the facilitation of CQA establishment.
pm Luncheon Presentation I: Assessing IgG Fc Variant Cross-Reactivity between Human and Rhesus Macaque Fc-gamma Receptors using Array-Based SPR
Austin Boesch, Ph.D., Candidate,
A number of antibody therapies rely on Fc receptor (FcR)-mediated effector functions for optimal activity, prompting the need to understand how IgG scaffolds engineered to differentially bind to the human receptors translate in
non-human primate (NHP) models. Use of a high-throughput array-based surface plasmon resonance (SPR) platform enabled efficient characterization of the affinity between an IgG Fc variant panel (including subclass, Fc mutants
and glycosylation) and major human and rhesus FcR allotypic variants.
12:50 Luncheon Presentation II (Sponsorship Opportunity Available)
1:20 Ice Cream Break in the Exhibit Hall with Poster Viewing
2:00 Chairperson’s Remarks
Timothy Fenn, Ph.D., Principal Scientist, Boehringer Ingelheim
2:05 Analytical Development for Bispecific Antibodies: Optimization of a Murine Surrogate Platform
Matthew Bunce, Ph.D., Principal Research Scientist, Janssen
Murine disease models provide an important early step to assess the safety and efficacy of new therapeutic candidates. The DuoBody platform offers an efficient and convenient method to generate bispecific antibodies; unfortunately,
initial work with murine antibodies failed to recapitulate the efficient heterodimerization seen with human IgGs. Here we summarize efforts to optimize a murine surrogate DuoBody platform by “humanizing” the murine
Fc domain, thereby facilitating heterodimerization.
2:35 Pharmacology of Blood–Brain Barrier-Permeable Bispecific Antibodies
Sue Twine, Ph.D., Team Leader, Human Health Therapeutics Portfolio, National Research
This talk will focus on the development of targeted SRM-ILIS and 2D-LC-SRM methods with improved sensitivity for quantification of BBB-crossing bi-specific antibodies in small samples of cerebrospinal fluid (less than 2 μl)
and in complex protein matrices of brain tissue. These analytical techniques can be multiplexed to allow quantification of several antibodies in the same sample and is well suited for serum-CSF-brain PK analyses of co-injected
antibodies as well as for simultaneous measurements of target-engagement biomarkers.
Biologics Developability Assessment and Formulations Optimization Using Isothermal Chemical Denaturation (ICD)
Richard K. Brown, Ph.D., President,
Stability optimization and aggregation minimization are two of the most important hurdles in the development of biologics. ICD provides the most accurate way of measuring protein stability under different formulation conditions.
Additionally, ICD experiments performed at different protein concentrations provide a quantitative assessment of protein aggregation in the native and denatured states. ICD is ideally suited to optimize the formulation of highly
concentrated formulations, bispecific antibodies and antibody drug conjugates. In this presentation, the fundamentals of ICD and its application to the evaluation of protein stability and optimization of formulation conditions
will be discussed.
3:35 Refreshment Break in the Exhibit Hall with Poster Viewing
4:25 Development and Characterization of Novel Antibody Formats
Geddie, Ph.D., Principal Scientist, Merrimack Pharmaceuticals
Multispecific antibodies and antibody-like molecules broaden the therapeutic application of IgGs, but can be challenging to engineer and manufacture. Using a network biology approach to identify key design parameters, we have engineered
novel formats for specific biological targeting. We then use rapid design cycles followed by high-throughput characterization of these formats to select for potential therapeutic candidates with robust pharmaceutical properties.
4:55 FDA/Sponsor Interaction on the Development of a Control Strategy for the Albumin Domain of an Albumin-fusion Protein
Weijie Wang, Ph.D., Senior Scientist, Teva Biopharmaceutical USA, Inc.
Fully recombinant Albumin fusion proteins are an established platform for extending the serum half-life of therapeutic proteins. While control strategies for the therapeutically active domain are common, including a cell-based
potency assay, the control of the albumin portion of the product needed to be developed. This talk will present a case study where a comprehensive control strategy was designed with FDA input based on a fundamental structure/function
assessment of the criticality of Quality Attributes.
5:25 End of Conference
5:30 Registration for Dinner Short Courses