This conference focuses on advances with analytical technology and their practical application. Pioneers will present their experiences with a range of technologies, particularly mass spec, epitope, peptide and computational mapping, microarray platforms, and fluorescent labeling. Presentations will include preparation of stable samples, measures to increase the speed of analytics and increase automation, advances with precision, the software used, and means of interpreting results. This leads onto applications of advanced technologies for a range of products, focusing on identification of forms of amidation and abnormal post translational modifications, protein and sequence variants, and glycosylation and glycan analysis. Practical examples will be presented, for example for clone and cell line selection, developability risk assessment, candidate selection, product optimization, affinity binding, the structure-function relationship, determining acceptance criteria and finally for product quality and manufacturing consistency and CQA assessment.
Monday, March 9
7:30 am Registration and Morning Coffee
8:30 Chairperson’s Opening Remarks
Taylor Zhang, Ph.D., Senior Scientist and Group Leader, Protein Analytical Chemistry, Genentech, Inc.
8:35 KEYNOTE PRESENTATION:
High-Throughput Methods to Measure Multiple Mechanisms of Action of Therapeutic Antibodies
T. Shantha Raju, Ph.D., Scientific Director, Biologics Research, Janssen Research & Development, LLC
9:05 Analytical Characterization for Biologics Candidate Selection and Optimization: Where Are We Now and Where Do We Go from Here?
Guodong Chen, Ph.D. Senior Principal Scientist, Bioanalytical and Discovery Analytical Sciences, Research and Development, Bristol-Myers Squibb Company
Biologics candidate selection and optimization is an important aspect of biologics discovery and development. This presentation highlights analytical strategies and methodologies in the selection and optimization of candidates, as illustrated in case studies.
9:35 Characterization of Higher Order Structure of Biotherapeutics
Maurizio Muroni, Ph.D., Scientist II, Development, Medimmune, UK
This presentation will cover a study of the oligomerization state of non-platform biotherapeutics by light scattering techniques and MALDI-TOF mass spectrometry, and the use of circular dichroism and mass spectrometry for investigation of secondary and tertiary structures.
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10:05 End-To-End Software Solution for Mass Spectrometry Based Biopharmaceutical Characterization
Joe Shambaugh, Head, Expressionist, Mass Spectrometry, US, Genedata Inc.
Biopharmaceutical R&D organizations employ mass spectrometry as the method of choice for the indepth characterization of therapeutic candidates. However, it remains challenging to optimize data processing, analysis, and reporting across multiple instrument platforms and laboratories within an organization. Here we discuss how the Genedata Expressionist for Mass Spectrometry enterprise software solution enables complete automation and standardization of the end-to-end characterization process from discovery and development to production and quality control.
10:35 Networking Coffee Break
11:00 Novel Methods to Speed Up PTM Profiling to Support Early Developability Risk Assessment of Biologics Candidates
Christian Graf, Ph.D., Principal Scientist, BPRD Integrated Biologics Profiling, Novartis Pharma
Characterization of critical post translational modifications is important for developability risk assessment of biotherapeutic drug candidates. However it usually involves labor-and time-consuming preparation of stressed samples, and also physicochemical analytics requiring large amounts of purified material. This talk presents new approaches and case studies using middle-down UPLC-MS and MS/MS methods combined with automated acquisition and data processing software, allowing rapid PTM profiling in much higher throughput with low concentrated antibody samples.
11:30 Tools to Examine the Driving Forces behind Chemical Degradation Hot Spots to Help with Candidate Selection
Hubert Kettenberger, Ph.D., Principal Scientist, Innovation Center, Penzberg, Large Molecule Research, Roche
Chemical degradation events such as asparagine deamidation or aspartate isomerization can be limiting factors for the stability of therapeutic proteins. Only a small subset of such residues actually degrades quickly, indicating that particular structural features define a degradation “hotspot”. Combining mass spectrometry, 3D structural modeling and machine learning, we developed an in-silico tool that reliably predicts hotspots and helps to select stable lead candidates.
12:00 pm Detection and Characterization of Reversible Self-Association of Therapeutic Monoclonal Antibodies
Sophia Levitskaya, Ph.D., Scientist, Analytical Biotechnology, MedImmune
This presentation discusses the nature of reversible self-association (RSA), and how we know if a protein self-associates. A case study of IgG2 (MAB A) will be presented. Factors affecting RSA, analytical methods used for RSA detection and characterization, and means of controlling self-association will all be discussed.
12:30 Luncheon Presentation (Sponsorship Opportunity Available, please contact Jon Stroup, jstroup@healthtech.com) or Lunch on Your Own
1:55 Chairperson’s Opening Remarks
Christian Graf, Ph.D., Principal Scientist, BPRD Integrated Biologics Profiling, Novartis Pharma
2:00 Case Study on Detection of Abnormal PTMs, Low Level Mutations, Sequence Variants, and Amino Acid Substitutions
Dingyi Wen, Ph.D., Principal Scientist, Analytical Biochemistry, Biologics Drug Discovery, Biogen Idec, Inc.
Biologicals, a class of drugs of growing importance, require careful characterization because of their large size and complexity. This talk will describe the mass spectrometry-based characterization strategy currently used in our laboratory to detect and quantify low levels of sequence variants, PTMs and other modifications in biological drug candidates. Specific cases, such as those involving amino acid substitution and O-Xylosylation, will be discussed, as well as cell culture conditions that affect PTMs and sequence variation.
2:30 Antibody Fc Deamidation: The Well-Known and Less Well-Known
Taylor Zhang, Ph.D., Senior Scientist and Group Leader, Protein Analytical Chemistry, Genentech, Inc.
Deamidation is one of the most common degradation pathways and frequently occurs at “hot spots” with NG, NS or NT sequences. Here we will present the use of a chymotryptic peptide mapping method to identify and characterize a not-“hot spot” deamidated form of an IgG1 which was observed as an acidic peak. The formation of this unique deamidation and other well-known deamidation sites will be discussed.
3:00 Breakout Discussions
Table 1: Practical Application of Glycosylation Characterization
Moderators: T. Shantha Raju, Ph.D., Scientific Director, Biologics Research, Janssen Research & Development, LLC, and Kenneth Moore, MSc, Associate Scientist II, Novel Molecules Group, Analytical Biotechnology Division, MedImmune, Inc.
- How much glycosylation characterization is required?
- When do we evaluate the impact of glycosylation on structure and function?
- How do we control glycan microheterogeneity?
- How close are we to ‘dialling-in’ a glycan profile?
Table 2: Analysis of Heterogeneity of Charge Isoforms
Moderator: Taylor Zhang, Ph.D.,Senior Scientist and Group Leader, Protein Analytical Chemistry, Genentech, Inc.
- Technologies for characterizing and quantifying charged isoforms
- Measures to ensure you identify all the species
- How you decide on acceptable levels of heterogeneity
- Setting specifications based on a range of heterogeneity
- Importance of prior knowledge of the product
Table 3: Analytical Characterization for Candidate Selection and Optimization
Moderator: Christian Graf, Ph.D., Principal Scientist, BPRD Integrated Biologics Profiling, Novartis Pharma
- How to best quantify fragmentation, deamidation, isomerization, glycation, glycosylation, conjugates?
- Ranking criteria for critical PTMs and the selection of different candidates
- Prediction, optimization and re-engineering of molecule liabilities
- New technologies for characterization of structural integrity and modifications
Table 4: Challenges of Structure and Sequence Analysis
Moderator: Richard Beardsley, Ph.D., Scientist, Protein Analytical Chemistry, Genentech, Inc.
- Advances being made in analytical technology that allow the discovery of new variants
- Variants observed
- Identifying and avoiding pitfalls arising from sequence variants over the course of product development
- Differences that can be tolerated / what matters and what doesn’t
- Comparing sequences with products from different expression systems
- Consequences of sequence variants from a regulatory perspective
- Means of examining the impact of new variants on safety and efficacy
- Risk assessment of results in terms of immunogenicity, potency and stability
4:00 Networking Refreshment Break
4:30 Discovery and Characterization of a Novel Free Drug Species in Antibody-Drug Conjugates
Richard Beardsley, Ph.D., Technical Development Scientist, Protein Analytical Chemistry, Genentech, Inc.
This presentation will focus on the factors leading to the formation of a novel free drug species that can be found in antibody-drug conjugate materials. The impurity is a dimerized form of the drug species used to conjugate the antibody, and can appear at low levels depending, in-part, on the presence of inter-chain trisulfides in the antibody prior to the conjugation reaction.
5:00 Analysis of Protein Variants from Serum to Support CQA Assessment during Early Development
Fabian Higel, Ph.D., Scientist, PK Profiling, Analytical Characterization, Sandoz Biopharmaceuticals
MAbs and Fc fusion proteins are highly complex protein mixtures containing multiple different modifications that might impact the pharmacokinetics, efficacy or safety. The identification and evaluation of these critical quality attributes is important and should be implemented during early development. Case studies investigating the effect of e.g. N-glycosylation or disulfide variants on the pharmacokinetics of mAbs and Fc fusion proteins are shown, demonstrating their feasibility and usefulness in early development.
5:30 Sequence Variant Analysis of Antibody Biotherapeutics with Ultra High-Resolution Mass Spectrometry
Heather DeGruttola, MS, Scientist, Mass Spectrometry and Biophysical Characterization, Pfizer, Inc.
Sequence variants (SV) are protein isoforms containing unintended amino acid substitutions typically due to either DNA mutations or misincorporations from either transcription or translation errors. Our clone screening strategy involves a multi-tiered approach using orthogonal mass spectrometric and genomic methods applied to SV detection. Several case studies will be presented that demonstrate comprehensive detection, identification and quantitation of sequence variants arising from both mutations and misincorporations.
6:00 Welcome Reception in the Exhibit Hall with Poster Viewing
Tuesday, March 10
8:00 am Morning Coffee
8:30 Chairperson’s Opening Remarks
T. Shantha Raju, Ph.D., Scientific Director, Biologics Research,
Janssen Research & Development, LLC
8:35 KEYNOTE PRESENTATION:
USP Perspective on Analysis of Variants
Fouad Atouf, Ph.D., Director, Biologics and Biotechnology, U.S. Pharmacopeia
Test specifications for recombinant proteins variants are an important component of public standards and are very often included in pharmacopeial monographs. This presentation will discuss approaches used to include these requirements into monographs and how these requirements evolve throughout the lifecycle of a public standard, as well as the type of reference standards used to support this type of tests.
9:05 High-Throughput Methodology in Characterization of Glycoproteins
Huijuan Li, Ph.D., Director, Sterile Product and Analytical Development, Bioprocess Development, Merck & Co., Inc.
The production of recombinant therapeutic glycoproteins has been an active area of research and drug development over the last 20 years. Multiple high-throughput methods for protein characterization with rapid cycle times will be discussed.
9:35 Application of Lectin-Based Microarray in Direct Glycan Profiling of Therapeutic Proteins
Lei Zhang, Ph.D., ORISE Fellow, Office of Biotechnology Products, CDER/FDA
Glycan moieties of therapeutic glycoproteins are a critical product attribute that directly affect protein folding, bioactivity, pharmacokinetics, and immunogenicity. Therefore, glycan variants must be adequately analyzed and controlled to ensure product quality and manufacturing consistency. This presentation describes our FDA research aimed at assessing the suitability of lectin-based microarray platforms for the quantitation of glycan variants in therapeutic proteins and monoclonal antibodies.
10:05 Making PTM and Sequence Variant Analysis Easy and Reliable
Chris Becker, Ph.D., President & CEO, Protein Metrics Inc.
Mass spectrometry is
relied on within biopharma throughout the development pipeline. Learn about novel, risk-reducing, scalable
software tools from Protein Metrics for rapid peptide
mapping, similarity studies, and sensitive analysis and reporting of variants
and post-translational modifications.
10:20 Utility of OneStep SPR Injection Technology in the Detection of Protein Aggregates
Eric Reese, Ph.D., Vice President, Sales and Marketing, SensiQ Technologies Inc
10:35 Coffee Break in the Exhibit Hall with Poster Viewing
11:05 Glycoprofiling of a Highly Complex Glycoprotein
Kenneth Moore, M.Sc., Associate Scientist II, Novel Molecules Group
Analytical Biotechnology Division, MedImmune, Inc.
Glycosylation is a major source of heterogeneity in recombinant proteins and requires thorough characterization. The N-linked glycan structures of a highly complex glycoprotein were characterized using Hydrophilic Interaction Liquid Chromatography (HILIC) and Matrix-assisted laser desorption/ionization Time-of-flight (MALDI-TOF) spectrometry. The glycan sequence of the protein was determined using these techniques in combination with monosaccharide-specific exoglycosidases. This approach revealed the presence of a complex, heterogeneous glycoprofile with several distinct oligosaccharide populations, including some atypical glycan structures.
11:35 Case Study on Experiences with Glycosylation Analysis for Non-mAb Protein Therapeutics
Sherry Castle, Senior Analytical Development Specialist, R&D, Shire, Inc.
Existing N-linked glycan analysis platforms are not sufficient for several classes of complex glycoprotein therapeutics.Potential quality attributes may be overlooked when performing established glycan assays that were developed using mAbs.Unique technical challenges include the presence of highly charged glycan moieties, such as sialic acid, phosphate and sulfate; multi-antennarity; and complex microheterogenity.Adoption of emerging technologies provides continuous improvement to the analytical lifecycle, but thesetechnologies must function within the framework of past reportable values. Here we share a glycan analysismodification of an established glycan methodology to test for unique properties of complex glycosylation.
12:05 pm Analysis of O-Glycosylation - Case Studies
Berangere Tissot, Ph.D., Technical Client Manager, Biopharmaceuticals, SGS Life Sciences
Amongst the tools to characterize protein glycosylation, the tollkit for O-glycosylation is not as diverse and not as extensive as the one for N-glycans. Case studies of O-glycan characterization will be presented.
12:20 Close of Part One - Stay on for Part Two: Characterization & Compliance