Challenges in the Characterization of Antibody Drug Conjugates

By Glenn Petrie, Ph.D.
Senior Scientific Advisor
ABC Laboratories
www.abclabs.com

Antibody Drug Conjugates (ADC) provide a unique new treatment for a variety of cancers. ADCs consist of a monoclonal antibody (mAb) targeted to the receptor of interest and linked to a highly cytotoxic payload. The mAb binds to the cell and enters the cytoplasm. Once inside the cell, the linker is cleaved and the toxin released. This provides the ability to use highly cytotoxic compounds without the serious side effects of systematic chemotherapy. It is estimated that there are 100-150 ADCs currently in preclinical development. ADCs present unique analytical challenges.

In addition to the complicated mAb, there is the added complexity of combining a cleavable linker and a cytotoxic drug. This introduces the necessity for determining drug loading, linkage sites and Drug Antibody Ratio (DAR). The critical technique for analysis on ADCs is ultra-high resolution QToF mass spectroscopy. This allows for determination of relative loading, DAR and linkage sites, as well as PTMs, disulfide linkages and related substances/degradants (deamidation/oxidation, truncations, and amino acid substitutions). Due to their high resolution, UHR-QTof instruments have the ability to sequence proteins up to 25-30 kDa. The specific protease IdeS (which cleaves just below the hinge region of IgG) under reducing conditions results in three polypeptide chains of ~ 25kDa: Fd region, Fc/2 region and the LC region. Analysis of these digests by UHR QTof MS yields complete sequencing of each polypeptide, DAR, payload and glycan distribution. If necessary, other proteases may be utilized for more detailed analysis. Additional characterization includes the following:

• Intact mass
• Deglycosylated mass
• IEX
• Imaging CIEF
• CE-SDS
• SEC
• N-linked glycan analysis
• HIC (secondary DAR analysis)
• Binding assay
• Bioassay
• Higher Order Analysis (CD, AUC, DSC, etc.)

The FDA considers the mAb a drug substance so both the mAb and ADC must be fully characterized. In addition, the agency has been requesting characterization of charge variants of the mAb and ADC. This necessitates preparative IEX followed by characterization of the acidic and basic fractions. Based on all these considerations, characterization of ADCs require careful planning and attention to detail.

Upcoming ABC Webinar: "The Use of High Resolution Accurate Mass Spectrometry in Environmental Fate and Metabolism Studies"

The use of liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) can be a powerful tool in the characterization and identification of metabolites and degradation products of environmental pollutants in environmental fate and metabolism studies, whether conducted in the laboratory or the field. Advantages of HRMS include:

• Improved accuracy – important in distinguishing analytes from isobaric interferences in complicated matrices

• Improved post-acquisition data processing – important in reducing the number of analyses and experiments required to get results

• Scientific and regulatory necessity – important owing to the required non-targeted analysis of novel compounds dosed at ever lower levels

This webinar will review the merits of employing LC-HRMS by addressing these basic concepts:

• Scientific and regulatory importance of metabolite identification

• Instrument Platforms

• Mass resolution and accurate mass measurements

• Molecular Formula Information (Most Probable Elemental Composition)

• Data-Processing Techniques (mass defect filtering, isotope pattern, etc.)

• The Importance of Separations Chemistry

• Future Developments of HRMS in Environmental Fate and Metabolism

The presentation will also review case studies in the scientific literature and examples from our laboratory to illustrate how HRMS can be employed in environmental fate and metabolism studies to answer both scientific and regulatory questions.