Part One: Analytical Methodology and PTMs


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.

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Monday, March 9

7:30 am Registration and Morning Coffee


ADVANCES WITH ANALYTICAL METHODS

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 RajuT. 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 ChenGuodong 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 MuroniMaurizio 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.

 

 Gene Data logo small10:05 Presentation to be Announced

 

10:35 Networking Coffee Break


POST-TRANSLATIONAL MODIFICATIONS

11:00 Novel Methods to Speed Up PTM Profiling to Support Early Developability Risk Assessment of Biologics Candidates

Christian GrafChristian 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 KettenbergerHubert 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 Sponsored Presentation (Opportunity available, please contact Jon Stroup, jstroup@healthtech.com)

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


POST-TRANSLATIONAL MODIFICATIONS Continued

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 ZhangTaylor 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 BeardsleyRichard 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.


PROTEIN AND SEQUENCE VARIANTS

5:00 Analysis of Protein Variants from Serum to Support CQA Assessment during Early Development

Fabian HigelFabian 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 DeGruttolaHeather DeGruttola, MS, Scientist, Mass Spectrometry and Biophysical Characterization, Pfizer, Inc.

Sequence variants (SV) are protein isoforms containing unintended amino acid substitutionstypically due to eitherDNA mutations ormisincorporationsfrom either transcription or translation errors. Our clone screening strategyinvolves a multi-tieredapproach using orthogonal mass spectrometric and genomic methods applied toSVdetection.Several case studies will be presented that demonstrate comprehensivedetection, identification and quantitation of sequence variants arising from both mutations and misincorporations.

6:00 Welcome Reception in the Exhibit Hall with Poster Viewing



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Tuesday, March 10

8:00 am Morning Coffee


PROTEIN AND SEQUENCE VARIANTS/CHARACTERIZATION OF GLYCOSYLATION AND GLYCAN PROFILING

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 AtoufFouad 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 LiHuijuan 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 ZhangLei 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.

Protein Metrics10:05 Making PTM and Sequence Variant Analysis Easy and Reliable 

 Chris BeckerChris 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 Sponsored Presentation(Opportunity available, please contact Jon Stroup, jstroup@healthtech.com) 

10:35 Coffee Break in the Exhibit Hall with Poster Viewing

11:05 Glycoprofiling and Structure-Activity-Relationship of a Highly Complex Glycoprotein

Kenneth MooreKenneth Moore, MSc, Associate Scientist II, Novel Molecules Group,
Analytical Biotechnology Division, MedImmune, Inc.

 

 

 

 

11:35 Case Study on Experiences with Glycosylation Analysis for Non-mAb Protein Therapeutics

Sherry CastleSherry 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 Close of Part One - Stay on for Part Two: Characterization & Compliance 




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