Antibody–Drug Conjugate Bioanalysis Using Lb-LC–MS/MS Hybrid Assays: Strategies, Methodology and Correlation to Ligand-Binding Assays

Abstract

Background: Antibody–drug conjugates (ADCs) are complex drug constructs with multiple species in the heterogeneous mixture that contribute to their efficacy and toxicity. The bioanalysis of ADCs involves multiple assays and analytical platforms. Methods: A series of ligand binding and LC–MS/MS (LB-LC–MS/MS) hybrid assays, through different combinations of anti-idiotype (anti-Id), anti-payload, or generic capture reagents, and cathepsin-B or trypsin enzyme digestion, were developed and evaluated for the analysis of conjugated-payload as well as for species traditionally measured by ligand-binding assays, total-antibody and conjugated-antibody. Results & conclusion: Hybrid assays are complementary or viable alternatives to ligand-binding assay for ADC bioanalysis and PK/PD modeling. The fit-for-purpose choice of analytes, assays and platforms and an integrated strategy from Discovery to Development for ADC PK and bioanalysis are recommended.

Keywords::

• antibody–drug conjugate
• antibody conjugated-payload
• DAR-bias
• DAR-sensitive ADC assay
• drug antibody ratio
• fit-for-purpose bioanalysis
• generic reagents
• immunocapture LC–MS/MS hybrid assay
• ligand binding-LC–MS/MS hybrid assay
• soluble target

Supplementary Data

Financial & competing interests disclosure

The authors of this article are current or past employees of Bristol-Myers Squibb Company (BMS). All financial support for the studies reported herein was provided by BMS. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Acknowledgements

The authors would like to thank the following for contributions to the assay strategy development, execution of the studies and experiments, and discussions: J Brailsford, J Zeng, R Iyer, X Gu, W Li, M Zhu, S Piccoli, D Passmore, D Desai, J Haulenbeek, F Zambito, S Gangwar, K Kelly, P Lemaire, A Davulcu, S, Brueggemeier, M Hay, C Yeung, A Tam, J Jackson.