A Case Study on the Impact of Thermal Hydrolysis and Lactone Ring Opening of Linker-Payload on Drug-to-Antibody Ratio Determination in a Topoisomerase I Inhibitor-Based ADC.

Accurate determination of the drug-antibody ratio (DAR) of an antibody-drug conjugate (ADC) requires multiple orthogonal analytical methods to ensure appropriate control of this critical quality attribute (CQA). This study presents the development considerations of two quality control (QC) methods: reversed-phase liquid chromatography (RP-HPLC) and hydrophobic interaction liquid chromatography (HIC-HPLC) to determine DAR of a topoisomerase I (TOP1) inhibitor-based ADC. We found that elevated column temperature used in the RP-HPLC method induced payload detachment from the linker, giving rise to characteristic shoulder peaks. This study systematically characterized the kinetics of this thermal hydrolysis process and elucidated its impact on DAR determination. To mitigate the underestimation of DAR by RP-HPLC, we developed an HIC-HPLC method and applied low-pH conditions for sample pretreatment, together with an optimized mobile phase, to suppress pH-dependent lactone ring opening of TOP1 inhibitors, thereby improving chromatographic resolution and DAR accuracy. Collectively, this study comprehensively evaluates the advantages and limitations of RP- and HIC-HPLC methods and provides guidance for future control strategies of ADC DAR.