An Overview of Liquid Chromatography-Mass Spectrometry (LC-MS) Methods for the Quantification of Antibody-Drug Conjugates

Antibody-drug conjugates (ADCs) are innovative drugs composed of cytotoxic molecules (payload) linked to antibodies, that selectively target and kill cancer cells upon internalization. In vivo, ADCs exist as intact molecules, naked antibodies, or released, unconjugated (linker-)payload. Accurate quantification of these entities is crucial for understanding ADCs pharmacokinetics. Ligand-binding assays are commonly used to measure ADC concentrations and total antibody concentrations, whereas LC-MS/MS is used to analyze the payload. Due to limitations in ligand-binding assays, this review focuses on quantitative LC-MS methods for the different ADC entities. Quantitative LC-MS assays were described for all ADC entities, available from full manuscripts and regulatory reviews of 12 ADCs evaluated by the European Medicine Agency, by January 2025. The review summarized sample pre-treatment, chromatography, mass spectrometry, validation, and stability data for each LC-MS method. Overall, critical details were often missing, particularly concerning sample pre-treatment, validation criteria, and sample stability. In conclusion, LC-MS quantification of ADC entities is feasible but current methods lack sufficient detail. Our review highlights the need for further research to develop reliable LC-MS assays for ADCs. This review may serve as a starting point and outlines key factors to consider in future LC-MS method development.