Part Two: Characterization & Compliance

Characterization and Compliance focuses on application of advanced analytical technologies throughout development. It presents characterization of the complex heterogeneity of ADCs by means of sophisticated technologies such as new inductively-coupled plasma mass-spec ICP-MS, capillary electrophoresis and protein mass spectrometry. A range of technologies for aggregate characterization will also be presented together with an FDA study on pathways that lead to aggregation.  Because host cell proteins have become very hot, we have included a presentation (and short course) on the most advanced technologies for their characterization. In addition we will include practical case studies on control and monitoring of Critical Quality Attributes and on phase-specific characterization. Reference standards for product quality and manufacturing consistency will be presented by the FDA and backed up with examples from NIST and a case study from the industry. Regulatory issues are further addressed by regulatory and FDA on concerns such as reference standards and product comparability.

Tuesday, March 10

12:30 pm Registration


1:30 Chairperson’s Opening Remarks

Ned Mozier, Ph.D., Senior Director, Analytical R&D, Biotherapeutics Pharmaceutical Sciences, Pfizer, Inc.

New Approaches to Characterization of Biopharmaceutical Products

Igor A. KaltashovIgor A. Kaltashov, Ph.D., Professor, Chemistry, University of Massachusetts, Amherst

Quantitation of biotherapeutics in biological matrices remains a challenging problem in PK studies. One additional complication that is encountered frequently is the close similarity of the biopharmaceutical product and abundant endogenous proteins (e.g., transferrin- or albumin-based drug conjugates). We will discuss the application of traditional isotope labeling techniques (e.g., based on O18 labeling) and new approaches developed in our lab that utilize metal tagging protein therapeutics and their detection, quantitation and imaging based on inductively-coupled plasma mass spectrometry (ICP MS).

2:05 Overcoming Analytical Challenges in Characterization of Heterogeneities of Antibody-Drug Conjugates

April XuApril Xu, Senior Principle Scientist, BioTx – Analytical R&D, Pfizer, Inc.

An antibody drug conjugate exhibits additional heterogeneity compared to the corresponding monoclonal antibody. In this presentation, a case study will be presented on how heterogeneity in an antibody-drug conjugate is characterized. In particular, the application of improved analytical techniques using capillary electrophoresis and protein mass spectrometry are applied to characterize the heterogeneities due to drug load distribution.

2:35 Exploring the Structure and Flexibility of Dual Variable Domain Immunoglobulins Alone and with Bound Antigens

Czeslaw Radziejewski, Ph.D., Associate Director, Analytical Development, Process Sciences, AbbVie, Inc.

Dual-variable domain immunoglobulins (DVD-Ig) are engineered bispecific tetravalent molecules that are currently under development for multiple disease targets at AbbVie. We have applied electron microscopy to investigate DVD-Ig molecule and its complexes with antigens. This is the first time that the overall structure and flexibility of DVD-Ig molecules alone and accompanied by its antigens have been successfully interrogated by single particle electron microscopy.

3:05 Development of Mass Spectrometry-Based Approaches to Study the Dimerization of Therapeutic Monoclonal Antibodies

Shunhai WangShunhai Wang, Ph.D., Scientist, Analytical Chemistry, Regeneron Pharmaceuticals, Inc.

The formation of HMW species of therapeutic monoclonal antibodies raises great concerns on the drug safety and efficacy, while presenting a significant challenge for analytical characterization. Herein, we present two complementary mass spectrometry-based approaches to rapid and effective identification of dimerization interfaces in IgG HMW species. Particularly, the subdomains responsible for formation of HMW species can be confidently identified and their contributions to the drug activity can be speculated.

3:35 Epitope Mapping Enabled with Fast Photochemical Oxidation of Proteins Approach

Yingda Xu, Ph.D., Associate Director, Protein Analytics, Adimab, LLC

Characterization of the antibody/antigen interaction (epitope mapping) provides the fundamental understanding for rational design of therapeutic mAbs. Fast Photochemical Oxidation of Proteins (FPOP) has recently been developed in academic labs as an alternative method for HDX MS method. Here, we designed an adapted FPOP approach coupled with affinity precipitation and LC-MS/MS. As a result, we profiled the conformational epitope of target of interest #1 and #2 against commercialized mAbs and mAb leads from Adimab workflow.

4:05 Refreshment Break in the Exhibit Hall with Poster Viewing


4:45 Assessing Aggregation of Therapeutic Proteins in Human Plasma

Shen LuoShen Luo, Ph.D., Senior Staff Fellow, Division of Therapeutic Proteins, Office of Biotechnology Products, CDER/FDA

The formation of aggregates in therapeutic proteins could adversely impact product safety and efficacy. Therefore, the levels of aggregate must be adequately controlled throughout the formulation and clinical administration procedures. We are interested in understanding product aggregation under conditions mimicking clinical settings, which involve the active pharmaceutical ingredient, formulation excipients, diluent, and plasma components. This presentation describes our findings about the pathways leading to protein product aggregation in human plasma.

5:15 Reversible Self-Association and Stability in a Monoclonal Antibody

Julie Wei, Ph.D., Scientist II, Protein Pharmaceutical Development, Biogen Idec, Inc.

This work is a biophysical characterization study of a monoclonal antibody that displays reversible self-association and poor stability. A possible connection between the protein’s self-association with poor stability has been investigated by a suite of biophysical methods including AUC, FTIR, thermal and chemical denaturation, and HDX-MS, as well as molecular modeling. A structural basis for the reversible self-association and poor stability is proposed.

5:45 Close of Day / Short Course Registration

Wednesday, March 11

7:30 am Morning Coffee


8:00 Chairperson’s Remarks

Martin Vanderlaan, Ph.D., Director, Analytical Operations, Genentech, Inc.

Perspectives on FDA Expectations for Phase Specific Product Characterization

Emily ShacterEmily Shacter, Ph.D., Consultant ThinkFDA LLC

When do you have to do what in order to meet FDA expectations for the development and approval of protein products for clinical and commercial use? This talk is aimed at helping you understand regulatory expectations on many of the questions that arise during product development, including the extent of analytical characterization studies, method validation, bioactivity, CQA assessment, and process control required at each stage of development.

8:35 Integration of Product Comparability into Process Development Strategies to Ensure Regulatory Approval

Alain BernardAlain Bernard, Ph.D., Vice President, Technical Operations GPS, UCB Pharma

We will demonstrate how appropriately and safely implemented process changes have effectively improved product quality and how some changes can contribute significantly to major progress in process and product understanding with positive impacts for the patients. We will exemplify how an extensive array of analytical tools, including some low-throughput but highly indicative quality tests can be used to increase chances of success with the comparability exercise.

9:05 Case Study on Early and Late Stage Characterization of an HSA Fusion Protein to Align with Clinical Phases

Ping FengPing Feng, MSc, Director, Analytical Sciences, Biological CMC, Teva Pharmaceuticals, Inc.

The focus of early stage characterization is evaluation of product biological activity and overall purity profile, including assessment of primary degradation pathways, and PTMs to support initiation of early clinical studies (I&II) and setting-up a baseline for future comparability. Late stage characterization includes a full understanding of the purity/impurity profile, and compatibility and product stability to support drug licensure. A case study will be presented on variant characterization of an HSA fusion protein.

9:35 Breakout Discussions

Table 1: Practical Application of New Mass Spectrometry Technology

Moderator: Igor A. Kaltashov, Ph.D., Professor, Chemistry, University of Massachusetts, Amherst

  • Range of mass spec approaches for analytical characterization
  • Potential applications of mass spec: sequence errors, glycoforms,etc.
  • Application of mass spec for ADCs and bispecific antibody characterization
  • Applications for comparability and routine structural assessments

Table 2: Aggregation: Factors that Give Rise to Aggregation and Means of Characterizing Aggregates

Moderator: Shunhai Wang, Ph.D., Scientist, Analytical Chemistry, Regeneron Pharmaceuticals, Inc.

  • Aggregate heterogeneity and challenges for analytical characterization
  • Discussion on contributory factors to aggregation
  • Strategies for aggregate characterization
  • Additional challenges with aggregates

Table 3: Appropriate Use of Functional Assays to Support Product Characterization

Moderator: Tamer Eris, Principal Scientist, Attributes Sciences, Amgen, Inc.

  • Product dependent effector function characterization strategies
  • Understanding product quality attribute impact on effector function activity
  • Tools, technologies and assay formats, e.g. challenges with CDC, ADCC, binding assays, potency assays, surrogate assays

Table 4: Building an In-House Reference Standard System

Moderator: Baolin Zhang, Ph.D., Senior Investigator, Division of Therapeutic Proteins, Office of Biotechnology Products, CDER/FDA

  • Interpretations of the primary reference standard and the working reference standard
  • Discussion of when to do what (BLA stage, before process validation and/or after tech transfer)
  • Forward planning required to ensure there is enough reference material at every stage
  • Analytical testing that needs to be standardized
  • Comparing current reference standards with previous ones
  • Challenges with bioassays regarding reference standards

Table 5: Detection and Characterization of Process-Related Impurities

Maura Kibbey, Ph.D., Senior Scientist Liaison, Biologics and Biotechnology, USPharmacopeia

  • Importance of ensuring that most HCPs are detected and appropriate methods for doing this
  • Besides residual HCP and DNA, what are the other process-related impurities that are hot topics?
  • Comments on USP activities related to residual HCP and DNA detection

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


11:05 High Throughput Microfluidic Immunoassay Technology Supporting Bioprocess Development and its Application for Titer, Residual HCP, and Protein A Quantification

Jun Heo, Analytical Scientist, Bioprocess Technology and Expression (BTE), Merck Co., Inc.

In this talk, high throughput technologies in bioprocess development are introduced with focus on analytical tools that support cell line and purification development. The automated microfluidic immunoassay technology enabled five times faster titer assay for screening of IgG products with no hook effect. Its application for more complex assays such as residual host cell protein and residual protein A makes it excellent tool for rapid process development.

11:35 Case Study: Development of Specific Host Cell Protein  ELISAs to Monitor Process Development

Chris FongChris Fong, Senior Supervisor, Analytical Operations, Genentech, Inc.

Host cell proteins (HCPs) are routinely monitored during manufacturing. When routine methods are not able to quantify HCPs accurately, then specific HCP assays must be developed to demonstrate clearance. These HCP specific assays can be used to help collegaues with process improvements. Strategies for integrating Process Validation clearance data with GMP lot release testing will be discussed.

12:05pm USP Chapter <1132> Residual Host Cell Protein Measurement in Biopharmaceuticals: Chapter Development, Anti-HCP Characterization, and Method Validation

Maura KibbeyMaura Kibbey, Ph.D., Senior Scientist Liaison, Biologics and Biotechnology, USPharmacopeia




Ned MozierNed Mozier, Ph.D., Senior Director, Analytical R&D, Biotherapeutics Pharmaceutical Sciences, Pfizer, Inc.


The USP draft monograph for HCP measurements was drafted, then published last fall in the Pharmacopeial Forum 40 (4) issue for public comment. The presentation will cover chapter development, finalization, and public comment resolution, key points from the chapter, and recommendations for reagent development, characterization, and method validation. 

12:35 Luncheon Presentation: Comprehensive Identification and Quantification of Host Cell Proteins by Mass Spectrometry

Daniel ChelDaniel Chelsky, Ph.D., CSO, Caprion Proteomics

New advances in mass spectrometry allow for the direct identification and quantification of low levels of host cell proteins that copurify with biologics.  Proteins in the low ppm range can be detected without antibodies or gel separation after manufacture in CHO, E. coli or yeast.  Quantitative assays with labeled reference standards for each protein can then be rapidly constructed and used to monitor process improvements and consistency.


2:00 Chairperson’s Remarks

Baolin Zhang, Ph.D., Senior Investigator, Division of Therapeutic Proteins, Office of Biotechnology Products, CDER/FDA

2:05 Regulative Perspectives on the Use of Reference Standards in the Development of Therapeutic Proteins

Baolin ZhangBaolin Zhang, Ph.D., Senior Investigator, Division of Therapeutic Proteins, Office of Biotechnology Products, CDER/FDA

Many therapeutic proteins are new molecular entities lacking relevant international or national reference standards, which require the development of in-house reference standards from representative(s) of the actual product lots. When adequately qualified, reference standards ensure the assay performance and validity of testing results regarding product quality and manufacturing consistency. This presentation will focus on regulatory expectations on the use and qualification of in-house reference standards for novel therapeutic protein products.

2:35 The NIST mAb: A Reference Material to Supplement Biopharmaceutical Characterization and Compliance

John E. SchielJohn E. Schiel, Ph.D., Research Chemist, Biomolecular Measurement Division, National Institute for Science and Technology

This presentation will discuss the establishment of an IgG1k Reference Material expected to more firmly underpin regulatory decisions and facilitate the development of originator and follow-on biologics. The RM is intended for a variety of uses including, but not necessarily limited to: system suitability tests, establishing method or instrument performance and variability, comparing changing analytical methods, assisting in method qualification, etc.

3:05 Strategy for Building an In-House Reference Standard System for Early Development through to Commercialization

Michael Huang, Ph.D., Senior Scientist, Analytical Biotechnology, MedImmune

3:35 Refreshment Break in the Exhibit Hall with Poster Viewing


4:05 Hamster Phospholipase B-Like 2 (PLBL2), a host cell protein impurity in CHO-derived therapeutic monoclonal antibodies

Martin VanderlaanMartin Vanderlaan, Ph.D., Director, Analytical Operations, Genentech, Inc.

Discovery and development of analytical methods for PLBL2 will be discussed. This HCP impurity has been identified in multiple therapeutic Mabs from multiple sources and is a potential platform impurity.


4:35 Case Study on Control and Monitoring of Critical Quality Attributes during Process and Product Development

Tamer ErisTamer Eris, Principal Scientist, Attributes Sciences, Amgen, Inc.

Development of biotherapeutic molecules requires in-depth knowledge of the product quality attributes critical for the safety and efficacy of the drug candidate. Consequently, appropriate understanding and assessment of these critical attributes is necessary to develop robust manufacturing processes that deliver consistent product quality and yield. Strategies for control and monitoring of biologically relevant attributes for a monoclonal antibody process will be presented and discussed.

5:05 Networking Reception in the Exhibit Hall with Poster Viewing

6:05 Close of Part Two - Stay on for Part Three: Comparability for Biologics