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Enhanced Pharmacokinetic Bioanalysis of Antibody–drug Conjugates using Hybrid Immunoaffinity Capture and Microflow LC-MS/MS

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Abstract

The increasing complexity and diversity of antibody–drug conjugates (ADCs) have led to a need for comprehensive and informative bioanalytical methods to enhance pharmacokinetic (PK) understanding. This study aimed to evaluate the feasibility of a hybrid immunoaffinity (IA) capture microflow LC–MS/MS (μLC-MS/MS) method for ADC analysis, utilizing a minimal sample volume for PK assessments in a preclinical study. A robust workflow was established for the quantitative analysis of ADCs by the implementation of solid-phase extraction (SPE) and semi-automation in µLC-MS/MS. Utilizing the µLC-MS/MS approach in conjunction with 1 µL of ADC-dosed mouse plasma sample volume, standard curves of two representative surrogate peptides for total antibody (heavy chain, HC) and intact antibody (light chain, LC) ranged from 1.00 ng/mL (LLOQ) to 5000 ng/mL with correlation coefficients (r2) values of > 0.99. The linear range of the standard curve for payload as a surrogate for the concentration of total ADC was from 0.5 ng/mL (LLOQ) to 2000 ng/mL with high accuracy and precision (< 10% CV at all concentrations). Moreover, a high correlation of concentrations of total antibody between two assay approaches (µLC-MS and ELISA) was achieved with less than 20% difference at all time points, indicating that the two methods are comparable in quantitation of total antibody in plasma samples. The µLC-MS platform demonstrated a greater dynamic range, sensitivity, robustness, and good reproducibility. These findings demonstrated that the cost-effective µLC-MS method can reduce reagent consumption and minimize the use of mice plasma samples while providing more comprehensive information about ADCs being analyzed, including the total antibody, intact antibody, and total ADC.

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Acknowledgements

The authors thank bioscience teams for supporting ADCs for protein analysis by µLC-MS/MS. We also thank Xiaoru Chen for providing technical assistance.

Funding

This research was supported by AstraZeneca R&D.

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  1. Moo-jin Suh and Joshua B. Powers are authors contributed equally to this work

Authors and Affiliations

  1. Integrated Bioanalysis, Clinical Pharmacology & Safety Sciences, Biopharmaceuticals R&D, AstraZeneca, Gaithersburg, Maryland, 20878, USA

    Moo-jin Suh, Joshua B. Powers, Casey M. Daniels & Yuling Wu

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Suh, Mj., Powers, J.B., Daniels, C.M. et al. Enhanced Pharmacokinetic Bioanalysis of Antibody–drug Conjugates using Hybrid Immunoaffinity Capture and Microflow LC-MS/MS. AAPS J 25, 68 (2023). https://doi.org/10.1208/s12248-023-00835-0

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