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The opening track of the Biotherapeutics Analytical Summit presents the identification, characterization and impact of Post-Translational Modifications. There will be a strong focus on new analytical technologies and their automation, on data analysis and on presentation to the regulatory authorities. We will focus on a range of PTMs, and present novel next-generation biotherapeutics as well as conventional ones as examples. We will investigate how these modifications impact on the properties of the product and on how they can be manipulated to advantage.


Monday, March 24


7:30 am Registration and Morning Coffee


PTM PROFILING AND STRUCTURE-FUNCTION RELATIONSHIP 

8:30 am Chairperson’s Opening Remarks

Hubert Kettenberger, Ph.D., Principal Scientist, Protein Analytics, Large Molecule Research, Pharma Research and Early Development (pRED), Roche Diagnostics


KEYNOTE

8:35 am Correlating the Role of PTMs with Structure-Functions for Therapeutic Antibodies

ShanthaRajuShantha T. Raju, Ph.D., Scientific Director, Biologics Research, Centocor, Inc.

Post-translational modifications (PTMs) of antibody therapeutics affect their functions and stability. Understanding the structure and functions of PTMs is necessary to successfully develop and market antibody therapeutics. Linking structure to function is critical to understand the mechanism of action (MOA), potential mechanism of toxicity (MOT), and critical quality attributes (CQAs). These correlations will be helpful for setting intelligent specifications. This presentation will illustrate the structural analyses of PTMs in relation to functional analyses, MOA and MOT.


9:05 am Rapid Profiling of N-Linked Glycans from Antibody Therapeutics Using Chip Nano LC-MS 

NiclasTanNiclas Tan, Ph.D., Scientist II, Analytical Development – Biologics, Takeda Pharmaceuticals International Co.

Rapid characterization of glycan post-translational modifications of monoclonal antibodies is of utmost importance due to their impact on efficacy, half-life, and mechanism of action. We present the results from the optimization of an integrated LC-MS chip for rapid online cleavage, purification, separation, identification and quantitation of label-free N-linked glycans based on accurate mass. The N-linked glycan characterization results from different production cell lines are presented to examine similarities and differences.

9:35 am Accurate Determination of PTMs and Micro-Variants by Stable Isotope labeling and LC-MS Analysis

Hongchen Liu, Ph.D., Scientist, Process Development, Alexion Pharmaceuticals

Heterogeneity is a common feature of recombinant monoclonal antibodies due to various chemical modifications. The nature and amounts of modifications are generally characterized by liquid chromatography and mass spectrometry analysis of peptides produced from protease digestion. However, the same modifications can also occur during sample preparation, which will result in overestimation of the levels of modifications. Several methods using the combination of stable isotope labeling and LC-MS to accurately determine the levels of modifications, including deamidation, oxidation and free sulfhydryl, will be discussed.

10:05 am Protein Phosphorylation: Regulation of Protein Binding and Pathway Cross-Talk

AnnaPanchenkoAnna Panchenko, Ph.D., Lead Scientist, NCBI, National Institutes of Health

We estimate the effect of phosphorylation on protein binding and function for different types of complexes from human proteome. First we find that phosphorylation sites tend to be located on protein-protein binding interfaces and may orthosterically modulate the strength of interactions and that binding hotspots have a high tendency to be phosphorylated. Our study of the effect of phosphorylation on protein binding in relation to intrinsic disorder shows that phosphorylation is correlated with disorder and disorder-to-order transitions upon binding. Finally we investigate how different phosphorylation patterns allow for dynamic regulation of cellular processes and provide the biological cross-talk between different biochemical pathways. 

10:35 am Coffee Break


CHARGE ISOFORMS, DEAMIDATION AND OXIDATION 

11:00 am Case Study on Analysis of Heterogeneity from Charge Isoforms

PatriciaCashPatricia W. Cash, Ph.D., Senior Director, Analytical Biochemistry, MedImmune, Inc.

Identification and characterization of charge isoform heterogeneity is important to biopharmaceutical product development. Charge heterogeneity generally arises from product-related variants such as C-terminal heterogeneity, glycation, or asparagine deamidation. Since these variants are often active, setting specifications based on a range of heterogeneity can be difficult. Deciding on acceptable levels of heterogeneity and the importance of prior knowledge will >be discussed.

11:30 am Case Studies on the Identification, Characterization and Data Analysis of Aspartic Acid Isomerization and Oxidation

DirkCheliusDirk Chelius, Ph.D., Fellow, Biologics Research & Development, Novartis Pharma AG

In this case study we show the development of a reversed-phase HPLC method for the quantification of isomerization in a monoclonal antibody. We used different techniques for the identification of the isomerization site, like enzymatic digestions and advance technology like ETD mass spectrometry. The amino acids most susceptible to oxidation were identified and the mass spectrometry data was used for the routine quantification of the oxidation levels during formulation development studies.

12:00 pm New Insights into Deamidation in Antibodies

YungHsiangKaoYung-Hsiang Kao, Ph.D., Principal Scientist and Senior Group Leader, Late Stage Pharmaceutical Development, Genentech, Inc.

Deamidation of asparagine is a major degradation pathway for proteins. We have used different methods, including a domain ion exchange chromatography method and several different biophysical techniques, to evaluate the effects of deamidation in antibodies and the dependence of demaidation rate on buffer type, pH and temperature. We also used a mutagenesis approach to model the deamidation and to evaluate the impact of deamidation on antibody functions. A summary of our findings will be presented.

12:30 pm Luncheon Presentation (Opportunity available, please contact Jon Stroup, jstroup@healthtech.com) or Lunch on Your Own


PTMS AND DEVELOPABILITY ASSESSMENT 

2:00 pm Chairperson’s Opening Remarks

JeffreyHudgensJeffrey W. Hudgens, Ph.D., Research Chemist, Institute for Bioscience and Biotechnology Research, Biomolecular Measurement Division, National Institute of Standards and Technology




2:05 pm Early Stage Characterization for Developability Assessment of Biologics

HonglingHanHongling Han, Ph.D., Senior Scientist, Integrated Biologics Profiling, Novartis, Inc.

Developability of biologics candidates should be assessed in the early stage of development to minimize the risk of technical hurdles. The concept of developability assessment presented here includes molecular profiling for biochemical (e.g., sensitivity to clipping, deamidation, aspartate isomerization and oxidation) & biophysical (e.g., high molecular weight species, viscosity and melting temperature) properties and pre-formulation assessment for evaluation of short-term stability profiles.

2:35 pm Identification of Antibodies Free from Chemical Degradation Sites: Prediction versus Reality

HubertKettenbergerHubert Kettenberger, Principal Scientist, Pharma Research and Early Development (pRED), Roche Diagnostics

Besides function, chemical stability is an important selection criterion in the discovery of therapeutic proteins. Degradation “hotspots”, i.e. amino acids which degrade particularly quickly during manufacturing, storage or >in vivo, should be avoided whenever possible. By combining mass spectrometry, structural biology and machine learning, we developed a reliable in silico approach to predict Aspartate and Asparagine degradation hotspots in the variable regions of monoclonal antibodies.

3:05 pm Refreshment Break in the Exhibit Hall with Poster Viewing


3:35 pm Structural Characterization, Quality Attribute Risk Assessment and Control of Atypical Modifications in Therapeutic Monoclonal Antibody Product Development Following the Quality by Design Principle

Jerry Lian ZhiruiZhirui (Jerry) Lian, Ph.D., Senior Research Scientist and Group Leader, Bioproducts Research & Development, Eli Lilly and Company

Two atypical post-translational modifications in a therapeutic mAb in development were identified and thoroughly characterized using mass spectrometry and biophysical techniques. Their impacts on biological activities of the molecule including antigen binding and potency were assessed. The critical quality attribute (CQA) risks were assessed based on the data. Some aspects of the control strategy will also be discussed.

4:05 pm Featured Presentation: Strategies to Control Reproducibility of Glycosylation in the Manufacturing Process

MichaelButlerMichael Butler, Ph.D., Professor, Microbiology, University of Manitoba

The glycan profile of the final product of a bioprocess can depend upon the producer cell line, the growth media, the culture conditions as well as the protein structure. The presentation will review bioprocess parameters that can be controlled in order to minimize batch to batch variation of Mab glycosylation. Strategies will also be discussed to produce Mabs with pre-defined glycan structures.

4:35- 5:30 pm Breakout Discussions


Table 1: Analysis of Heterogeneity of Charge Isoforms

Moderator: Hongcheng Liu, Ph.D, Development Scientist III, Protein Characterization, Alexion Pharmaceuticals, Inc.


Table 2: Glycosylation as a Strategy to Improve Antibody-Based Therapeutics

Moderator: David Spencer, BSc, Scientist II, Analytical Biochemistry, MedImmune, Inc.


Table 3: Practical Application of Glycosylation Characterization

Moderator: Dietmar Reusch, Director, Pharma Development Analytics, Pharma Biotech Production and Development Characterization, Roche Diagnostics GmbH


Table 4: Challenges of Controlling Reproducibility of Glycosylation in the Manufacturing Process

Moderator: Michael Butler, Ph.D., Professor, Microbiology, University of Manitoba


Table 5: Application of New Technologies for Analysis of PTMs

Moderator: Bing Gong, Ph.D., Associate Principal Scientist, GlycoFi, Biologics Discovery


Table 6: Characterization of Native and Scrambled Disulfide Bonds

Moderator: Yung-Hsiang Kao, Ph.D., Principal Scientist and Senior Group Leader, Late Stage Pharmaceutical Development, Genentech, Inc.


Table 7: Evaluation of Impact of PTMs on Product Properties and Development of Control Strategies

Moderator: Zhirui (Jerry) Lian, Ph.D., Senior Research Scientist and Group Leader, Bioproducts Research and Development, Eli Lilly and Company

 

5:30 pm Welcome Reception in the Exhibit Hall with Poster Viewing


6:30 pm End of Day One of Post Translational Modifications


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