With newer and more complex molecules taking the spotlight these days, companies need better and faster ways to characterize these non-traditional antibodies. These new entities, coming in the different flavors of bispecifics, conjugates, multi-components, combinations, fusions etc., do not work well with the existing platforms. Coupled with the massive amount of data coming out from mass spectrometry and the regulatory demands for more accurate particle analysis and immunogenicity risk assessment, the industry is eager for novel approaches, proven methods and creative strategies that can help them analyze, interpret and translate information into innovation.
The Advances in Characterization Methods and Approaches conference arms scientists with the tools to develop the right strategies to speed up innovation, such as developing high throughput techniques for multi-parameter analysis; using in-process control and real-time release testing to improve CMC process; improved methods for biochemical and biophysical analysis, as well as advanced techniques for better understanding of the molecule’s structure-function dynamics, charge heterogeneity, post-translational modifications, and sequence variants.
TUESDAY, MARCH 15
12:30 pm Registration
1:30 Chairperson’s Opening Remarks
Baolin Zhang, Ph.D., Senior Investigator & Product Quality Reviewer, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. FDA (invited)
KEYNOTE PRESENTATION
1:40 Real-Time Release and Multi-Parameter/ Multi-Attribute Analysis
Hans-Martin Mueller, Ph.D., Director, BioProcess Development, MSD Merck
The modern biotech industry is constantly striving to modernize the analytical control of processes, drug substances and drug product batches. Today, the analytical characterization should be available earlier, at a higher quality and with more information content. The trend in our industry is to investigate practical approaches for on-line data generation, multi-attribute methods and batch releases in real-time. In biologics drug development the prerequisite for running these new techniques are “platform”-analytical methods, which can be applied to broad range of biologics.
2:10 Selected Poster Presentation:
Selected Poster Presentation:
Automatic Disulfide Bond Analysis for Structural Characterization of Biopharmaceuticals
ShengYu Huang, Ph.D., Director, Protein Science & Services Division, Mithra Biotechnology
Disulfide-linked peptides exhibit multiple enhanced a1 ion signals during CID/HCD fragmentation after dimethyl labeling. The distinct feature allows computational algorithm to specifically locate and identify disulfide bonds. Unlike the majority of the current approaches, this does not require prior knowledge of disulfide linkages or a high-end mass spectrometer and can avoid tedious labor work or deliberate interpretation. It has been proved useful for analyzing protein drugs, their scrambled forms and protein mixture.
2:25 Advancing Analytical Characterization for In-Process and Purified Protein Pharmaceuticals
Anastasiya Manuilov, MS, Senior Scientist, Analytical Research and Development, Pfizer
The development of analytical methods for the characterization of protein biopharmaceuticals faces complex challenges. 2D-LC method development will be presented for the effective separation of IgG1 and IgG2 isoforms, via pH-gradient cation exchange with an in-line protein-A column. This setup allows minimal sample preparation for direct analyses of conditioned cell culture media and for characterization of various product isoforms.
2:55 Automated de novo Identification and Profiling of Disulfide Bonds in Monoclonal Antibodies
Jason Wood, Ph.D., Market Manager, Biopharmaceuticals, Bruker
The location of disulfide bridges in biologics affects their spatial structures and can impact their safety and efficacy; therefore it is required during the characterization of biologics. It is often performed by a time-intensive, data-processing approach, involving the comparison of peptide maps of reduced and non-reduced samples. In order to avoid this differential approach, a workflow analyzing non-reduced protein digests by LC-MALDI-MS/MS was investigated where the disulfide bonded peptides are reduced in the mass spectrometer.
3:25 Refreshment Break in the Exhibit Hall with Poster Viewing
4:10 Monitoring Multiple mAb Attributes Using Peptide Mapping UPLC-MS
Nisana Andersen, Ph.D., Associate Scientist, Protein Analytical Chemistry, Genentech, Inc.
4:40 Q&A with Speakers
5:00 Close of Day Two
WEDNESDAY, MARCH 16
8:00 am Morning Coffee
8:30 Chairperson’s Remarks
Hans-Martin Mueller, Ph.D., Director, BioProcess Development, MSD Merck
KEYNOTE PRESENTATION
8:40 Fast Photochemical Oxidation of Proteins (FPOP) Complements HD Exchange for Protein Biophysics and Therapeutics
Michael Gross, Ph.D., Professor of Chemistry, Washington
University in St. Louis
We are developing methods for characterization of
protein therapeutics to meet the challenge that proteins,
unlike small-molecule drugs, can form various higher
order structures and can oligomerize. The overarching strategy
is mass-spectrometry-based protein footprinting, which has high
sensitivity and fast turnaround. We will review HD exchange,
specific amino-acid labeling, and fast photochemical oxidation of
proteins (FPOP) that meet the challenge to map epitopes, follow
oligomerization, and assure that structure of a protein has not
changed.
9:10 How Precise Is that Hydrogen-Deuterium Exchange Mass Spectrometry Measurement?
Jeffrey W. Hudgens, Ph.D., Research Chemist, Biomolecular Measurement Division, Institute for Bioscience and Biotechnology Research, National Institute of Standards and Technology
Adoption of hydrogen-deuterium exchange mass spectrometry (HDX-MS) for certifying properties of biotherapeutics cannot occur without a thorough knowledge of its measurement uncertainties. For an international study NIST enlisted experts from the biopharma, non-profit, and academic sectors who conducted HDX-MS measurements on the Fab and Fc domains of the antibody reference material, NIST mAb. Analyses of these data have produced the first determinations of HDX-MS reproducibility for measurements involving mAb materials.
9:40 Potency Assays for Biopharmaceuticals: A Regulatory Perspective
Baolin Zhang, Ph.D., Senior Investigator & Product Quality Reviewer, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. FDA (invited)
Because of the complex nature of biopharmaceuticals, it can be scientifically challenging to develop appropriate potency assays for each product. A regulatory evaluation of adequacy of potency assays is made on a case-by-case basis, taking into account multiple factors including, but not limited to, product type, MoA, associated risk, and phases of development. This presentation provides an overview of regulatory expectations regarding potency assays and discusses several case studies that highlight some of the relevant issues commonly seen in the regulatory submissions.
10:10 Bioassay and Immunoassay Assessment of an ADC
Jeffrey Glenn, Ph.D., Associate Director, Analytical Development, Bristol-Myers Squibb
Potency of ADCs can be measured using ELISA and cell-based assays. Both methodologies are sensitive to changes in antibody-drug ratios and modifications to the CDR-regions of the molecule. By employing both ELISA and cell-based bioassay a complete picture of the ADC can be obtained. This talk will present data from both types of assays, and describe how they can be used to evaluate manufacturing and lot-to-lot changes.
10:40 Coffee Break in the Exhibit Hall with Poster Viewing
11:10 New MS- and LC/MS- Based Approaches to Characterization of Highly Heterogeneous Biopharmaceuticals
Igor A. Kaltashov, Ph.D., Professor and Graduate Program Director, Department of Chemistry, University of Massachusetts-Amherst
Characterization of biopharmaceuticals focuses on their structure at a variety of levels, and the heterogeneity inherent to many protein-based therapeutics makes it a truly challenging task. This presentation will feature several analytical methods recently developed in our laboratory that allow this task to be accomplished for a variety of highly heterogeneous targets including stressed biopharmaceuticals, PEGylated proteins and other protein conjugates. The main focus will be on size exclusion and ion exchange chromatography with on-line detection by native MS and top-down MS/MS.
11:40 Applications of LC-MS Towards the Understanding and Characterization of High Molecular Weight Variants in the Development of Biotherapeutics
Richard Ludwig, Ph.D., Principal Scientist, Molecular Analytics and Development, Bristol-Myers Squibb
LC MS has traditionally played a significant role in the characterization of biotherapeutics. This role has until recently been limited to assessment of the primary structure of proteins. Recent advancements in LC-MS techniques have enabled the use of LC-MS as a means of investigating higher order interactions. The application of hydrogen deuterium exchange as a means of characterizing a high molecular weight species within a developmental monoclonal antibody biotherapeutic is described.
12:10 pm Deconvolution of Intact Mass Spectra: Novel Methods for Deep Characterization of Biotherapeutics
Dominik Mertens, Ph.D., Scientific Account Manager, Genedata
Intact protein analysis is a key analytical assay for the identification and relative quantification of large biomolecules. Sophisticated data processing and analysis methods for the accurate identification and quantification of biomolecules are presented. It is demonstrated that prior to the intact mass deconvolution, refined noise removal and smoothing algorithms are critically important for accurate and reliable quantification, which is exemplified by the determination of drug-to-antibody ratios (DAR) of antibody-drug conjugates (ADCs).
12:40 Luncheon Presentation: Application of Analytical Techniques for Product Quality Driven Process Development and Routine cGMP Manufacturing
Greg Adams, Ph.D., Director, Analytical Development, FUJIFILM Diosynth Biotechnologies
This presentation will discuss using analytical methods to optimize expression of proteins with specific qualities or properties, and the utilization of rapid methods during clone selection, cell culture, recovery and purification development. These methods are then directly adapted to routine manufacturing testing thereby ensuring consistent analytical data throughout the clinical development lifecycle.
1:10 Luncheon Presentation (Sponsorship Opportunity Available) or Lunch on Your Own
1:55 Chairperson’s Remarks
Jeffrey W. Hudgens, Ph.D., Research Chemist, Biomolecular Measurement Division, Institute for Bioscience and Biotechnology Research, National Institute of Standards and Technology
2:00 Comparability and Monitoring N-Linked Oligosaccharides Using HPLC and Mass Spectrometry
Xiaoyang Zheng, Ph.D., Senior Scientist, Analytical Development, Sanofi US
One of the most important structural features of recombinant therapeutics is the N-linked oligosaccharide profile. These profiles impact recombinant therapeutics in a wide range of ways by affecting their pharmacokinetics, potency, safety, and stability. Oligosaccharide profiling using HPLC, sequential digestion with exoglycosidases, and mass spectrometric analysis will be presented. And the potential impact of oligosaccharides on function and stability of the glycoprotein therapeutics will be discussed in this talk.
2:30 Fc Effector Functions, FcRn and Their Impact on PK/PD of Antibodies, and Potential Implications for Comparability and Biosimilarity
Shanmuuga Sundaram, Ph.D., Research Advisor, Bioanalytical Sciences, Bioproduct R&D, Eli Lilly and Company
Monoclonal antibodies have demonstrated safety and efficacy with favorable clinical response, resulting in many commercial products on the market. Several factors determine the efficacy, including target specificity through Fab, effector functions and half-life through Fc. The regulatory expectations and requirements for detailed characterization of Fc’s part are increasing. This presentation will share our strategy.
3:00 Strategies for the Control of Glycosylation of a Chimeric Human-Llama Antibody
Michael Butler, Ph.D., Scientific Director, MabNet, Distinguished Professor, Department of Microbiology, University of Manitoba
The glycoform profile of a chimeric monoclonal antibody can be controlled to pre-defined levels- galactosylation, fucosylation and sialylation. These common variants of the conserved Fc glycan have a significant effect on the functional properties of the molecule that may affect therapeutic efficacy. The presentation will review the bioprocess parameters that can be controlled in order to minimize batch to batch variation of glycosylation and obtain a desired glycan profile.
3:30 An Insight into the LabChip GXII and Alpha Technology Platforms for Biologics Method Development
Roger Bosse, Strategic Product Development/PerkinElmer Human Health, Human Health, PerkinElmer
Sensitive and reproducible characterization of large molecule drugs within the biopharmaceutical development space presents a complex challenge that requires various technologies that can bridge the gap from Method Development to GMP Lot Release Testing Laboratories. During this presentation, we will introduce and highlight the usage of the Microfluidics-based LabChip GXII for titer, purity and N-linked Glycan analysis as well as the homogenous no-wash AlphaLISA Technology for ligand binding and functional bioassays.
4:00 Refreshment Break in the Exhibit Hall with Poster Viewing
4:30 Sequence Variants and Sequence Variant Analysis in Biotherapeutic Proteins
Oleg V. Borisov, Ph.D., Director, Analytical Development, Novavax
A “sequence variant” is a surrogate term covering any unintentional amino acid substitutions, omissions, or insertions during protein biosynthesis. The occurrence of sequence variants contributes to heterogeneity of recombinant protein therapeutics. Establishing a sequence variant profile of a biotherapeutic product is essential in providing proof of its structure, its manufacturing consistency, and the stability of the producing cell line. This talk will discuss analytical methodologies for detecting sequence variants in biotherapeutic proteins, and will discuss the results of an intra-laboratory study for sequence variants analysis in the NIST monoclonal antibody (mAb) by peptide mapping.
5:00 Assessment of Critical Quality Attributes in Biopharmaceuticals and Their Impact on Different Stages of Product Development
Robert Mayer, Ph.D., Scientist, Characterization Group, Sandoz GmbH
The adequate control of CQAs at different stages of the manufacturing process is essential, as they can directly be linked to clinical safety and efficacy. Other elements like process risk assessments or drug substance and drug product control strategies are being established based on the CQA assessments. This talk will focus on the identification and classifications of quality attributes according to their criticalities based on the available knowledge and (un)certainty thereof. The impact of CQAs on different stages of the product development will be discussed.
5:30 Networking Reception in the Exhibit Hall with Poster Viewing
6:30 Close of Part Two: Advances in Characterization Methods and Approaches - Stay on for Part Three: Comparability and Biosimilarity