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TUESDAY, APRIL 13
7:30am Registration & Morning Coffee
8:30 Chairperson’s Remarks
Daryl Fernandes, Ph.D., Chief Executive Officer, Ludger Ltd.
8:35 High-Resolution Glycan Analysis of Monoclonal Antibodies
Hansjoerg Toll, Ph.D., Head, Characterization, Biopharmaceutical Operations, Sandoz GmbH
Glycosylation is an important parameter when assessing biopharmaceutical product quality. It can impact drug safety and efficacy and, in addition, is an independent indicator for manufacturing consistency. In glycan characterization, results from a toolbox of methods need to be combined to obtain a comprehensive picture of the glycosylation pattern. The talk will compare different methods for the determination and quantification of glycans deriving from monoclonal antibodies, focusing on chromatographic methods in combination with mass spectrometry as methodologies for the identification and structural characterization of glycan species.
9:05 Characterization and Control of Protein Glycosylation in Cell Bioprocesses
Michael Butler, Ph.D., Professor, Microbiology, University of Manitoba
The glycosylation profile of proteins secreted from mammalian cells in culture is dependent upon critical parameters associated with the host cell line, the culture media, the mode of culture and the specific protein synthesized. It is important to control these parameters in an industrial bioprocess to ensure consistency of the final product and maximum bioactivity. The critical culture parameters will be discussed in the context of bioprocesses in which fine control is necessary to synthesize glycoproteins for the production of highly efficacious biopharmaceuticals with consistent structural profiles.
9:35 Determination of the A-Fucosylation Distribution within an Antibody Preparation
Christiane Jaeger, Ph.D., Head, Process Biochemistry, F. Hoffmann-La Roche
Glycosylation of an IgG has an important effect on Fc-mediated effector functions. The lack of a fucose residue which is usually attached to the first GlcNAc residue in the glycan has an affinity improving effect on the interaction between FcγRIIIa and IgG. IgG’s can have either two, one or no fucose attached. For a proper characterization it is mandatory to analyze this distribution of the a-fucosylation rate biochemically, but current state-of-the-art methods provide only the information of an overall a-fucosylation degree. By applying a newly developed method, described in this talk, it is now possible to determine the a-fucosylation degree per single Fc within an antibody pool.
10:05 Speed Networking
10:25 Networking Coffee Break, Poster and Exhibit Viewing
Featured Presentation
11:10 USP Standards for the Characterization of Post-Translational Modifications
Tina S. Morris, Ph.D., Vice President, Biologics and Biotechnology, United States Pharmacopeial Convention
Reliable and well-characterized standards play a key role in method development, qualification and validation. In the area of protein and glycoprotein characterization the USP has been working on developing both documentary and physical standards that aid in these areas. The presentation will give an overview of USP glycoprotein and glycan standards in development, specifically USP Chapter <1084> Glycoprotein and Glycan Ananlysis – General Considerations published for comment in Pharmacopeial Forum 36(2), as well as physical reference standards currently in development.
11:40 Development of Analytical Methods to Monitor Glycation
Eduard Orvisky, Ph.D., Senior Scientist I, Analytical Sciences, Human Genome Sciences, Inc.
Glycation is the non-enzymatic post-translational covalent binding of a sugar molecule, such as glucose, to protein amine groups. Glycation is generally an undesirable product attribute that may impair the function of the protein. Glycation of the product may occur either inside the body (endogenous glycation) or outside the body (exogenous glycation). This presentation will focus on development and qualification of analytical methods specific for measuring protein glycation. Two methods to monitor glycation and two case studies will be presented.
12:10Luncheon Presentation or Lunch on Your Own
(Opportunity available, please contact Jon Stroup, jstroup@healthtech.com)
2:00 Chairperson’s Remarks
Tina S. Morris, Ph.D., Vice President, Biologics and Biotechnology, United States Pharmacopeial Convention
Keynote
2:05 Assessment of Deamidation Sites in Antibody Products
Alistair Kippen, Ph.D., Associate Director, Analytical Biochemistry, MedImmune Ltd.
Daniel Higazi, Ph.D., Scientist, Analytical Biochemistry, MedImmune Ltd.
Discussion of an analytical strategy for assessment of deamidation, as a potential critical quality attribute in relation to post-translation modifications to support antibody product development. Description of the methods used to identify and measure deamidation propensity at specific sites within the CDR of novel antibody products using peptide mapping LC-MS, and determination of the effect on product potency and structure. Review of the hydrophobic and steric nature of residues surrounding deamidation motifs, and presenting a case study on the effect of adjacent site-specific amino acid substitutions on the rate of deamidation, alongside assessment of any changes to structure and potency.
2:35 Distribution of Free Sulfhydryl in Recombinant Antibodies
Hongcheng Liu, Ph.D., Associate Research Fellow, Process Sciences, Abbott Bioresearch Center
A low percentage of free sulfhydryl is a common feature of recombinant monoclonal antibodies although, in theory, all cysteine residues should be bound in disulfide bonds. A differential alkylation method, using 12C iodoacetic acid, 13C iodoacetic acid and mass spectrometry analysis, was developed to determine the percentage of free sulfhydryl at each cysteine residue of several recombinant monoclonal antibodies. Different levels of free sulfhydryl were observed in different antibodies. However, free sulfhydryl was distributed similarly in the domain structures of different antibodies.
3:05 Comparability of Biopharmaceutical Glycosylation during Scale-Up
Daryl Fernandes, D. Phil., Chief Executive Officer, Ludger Ltd.
Glycosylation can significantly influence the safety and efficacy profiles of biopharmaceuticals. Consequently, there is an essential need to maintain a consistent glycosylation pattern during product scale-up and to demonstrate that consistency through comparability studies. This can be very challenging due to the complexity and variability of drug glycosylation and its sensitivity to fermentation conditions and downstream processing. This talk overviews a practical QbD (Quality by Design) based approach to comparability of drug glycosylation during scale-up that deals with these issues and that allows development scientists to make meaningful assessments of the variations in glycosylation that occur as production scale is changed.
3:35 High Throughput N-glycan Analysis: Profiling Through Characterization 
Bradley Prater, Sr. Associate Scientist, Analytical Sciences Lead Aranesp, AMGEN
We present a multi-methodological, high-throughput approach for glycan analysis at various stages in the product life cycle, incorporating lab automation and PhyTipTM technology. From the use of stable isotopes and MS-based profiling at the clone selection stage, through complete characterization via permethylated MSn analysis during product characterization at licensure, to UPLC-based separarations in support of lot release and routine analysis, it is critical to apply the appropriate tool at the appropriate time.
3:55 Networking Refreshment Break, Poster and Exhibit Viewing
Featured Presentation
4:30 High Throughput Glycan Profiling for Monoclonal Antibody Development
Heidi Zhang, Ph.D., Fellow, Head of Mass Spectrometry PSP-ARD, Novartis Biologics
Because glycan composition can be affected by the type of cell line, clonal selection, and specific production conditions, it is important to closely monitor and control glycosylation during antibody development and production. Glycan screening during process development requires fast analysis of large sample sets and presents a challenge for conventional methods. We present a MALDI-MS based method in which glycans are enzymatically released and directly subjected to MALDI-MS analysis. Advantages include quick turnaround time and compatibility with high throughput automated sample preparation. The limit of detection and reproducibility will be compared with the traditional approach using 2AB labeling and NP-LC-FLD-MS. In addition, key considerations for instrument set up to ensure successful MALDI measurement will be discussed.
5:00 Characterization of Protein Oxidation and Deamidation to Support Process Development, Product Stability and Comparability
Zhuchun Wu, Ph.D., Senior Scientist, Analytical Sciences, Human Genome Sciences, Inc.
5:30 Break Out Sessions
Break out sessions are interactive moderated discussions on topics of interest to investigators in the field of Post-Translational Modifications. Problems are discussed and solutions are shared.
Table 1: Relationship Between PTMs and Scale-Up
Moderator: Daryl Fernandes, PhD., CEO, Ludger Ltd.
- Difficulties experienced by the industry
- Challenges with interpreting the results: what is relevant and how to simplify
- Means of overcoming this in terms of ensuring safety and efficacy
- What is and is not considered comparable regarding the regulatory authorities?
Table 2: Characterization Protocol for PTMs throughout Development
Moderator: Boyan Zhang Ph.D., Scientist & Group Leader, Protein Analytical Chemistry, Genentech, Inc.
- Summary table of PTMs and their characterization methodologies
- Pros and cons of different approaches
- Approaches most acceptable to the regulatory authorities
- Means of making PTMs less variable with changes in manufacturing
- Prioritizing: what must be done early and what can be left until later
Table 3: How Di-sulphide Bridges Affect the Folding of the Protein and Impact on the Product Safety and Efficacy
Moderator: Hongcheng Liu, Ph.D., Associate Research Fellow, Process Sciences, Abbott Bioresearch Center
- Technologies for analysis
- Impact of folding on the product
Table 4: Experiences with New Technologies and Technology Advances
Moderator: Heidi Zhang, Ph.D., Fellow, Head of Mass Spectrometry, PSP-ARD Novartis Biologics
Advances in mass spec instrumentation and methodology New sample preparation methods to ease PTM detection and characterization Novel non-MS methods for detecting and quantitating PTMs Challenges with data analysis Advances in automation Experiences with method validation Measures to increase speed
Table 5: Linking PTMs with Safety and Efficacy
Moderator: Alistair Kippen, Ph.D., Associate Director, Analytical Biochemistry, MedImmune Ltd
- How PTMs may affect product structure and interfere with receptor binding function
- Changes within the CDR of antibodies that can affect product potency
- Can a knowledge base be built and used to identify liability sites
- Can accelerated (stressed) studies reliably predict long term changes to stability and efficacy
- Sharing of experiences regarding binding assays, functional bioassays, in-vivo studies
- Significance of charged species: deamidation, glycation and oxidation
- Consideration of bridging studies when necessary
- Effect of PTMs on stability
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6:30 Networking Reception in the Exhibit Hall
7:30 End of Day One