Interchain cysteine linked antibody–drug conjugates (ADCs) are emerging therapeutic products that antagonize cancers. The toxic payloads are selectively linked to the interchain cysteines and generate heterogeneous mixtures of positional isomers. These positional isomers might contribute differently to the therapeutic efficacy because of the variation in conjugation stability, and thus they need to be well characterized. However, the characterization of the positional isomers of interchain cysteine linked ADCs is very challenging, mainly because of the high similarity between those isomers. In this research, we developed a novel mass spectrometry method for the characterization of positional isomers of interchain cysteine linked ADCs. The subunit analysis and the bottom-up analysis provided abundant information about the drug numbers and drug linking positions on each chain. Because the method can provide accurate data on drug linking numbers and positions on each chain, it will be very useful for researchers in cancer drug development and cancer treatment.

In this eBook, we explore the various ways LC-MS is utilized for the bioanalysis of ADCs and the strategies used for ADCs in biotherapeutics and clinical development.

Accurate determination of the drug-to-antibody ratio (DAR) of interchain cysteine-linked antibody–drug conjugates (ADCs) is challenging. High-resolution mass spectrometry (HRMS) analysis of the ADCs at the intact or subunit level provides a feasible way to measure the DAR. However, the measured DAR is usually lower than the true DAR because of the variation in ionization efficiency between different DAR species. In this work, we developed a novel standard-free HRMS method involving isotope-labeled payload conjugation, protease digestion, and liquid chromatography–HRMS (LC-HRMS) analysis for accurate determination of the DAR of the interchain cysteine-linked ADCs with cleavable or non-cleavable linkers. Isotope-labeled payload conjugations eliminated the structural and chemical differences between different DAR species and ensured that the drugs or payload-containing peptides could be separated from each other in the mass spectrometer. A papain digestion strategy for ADCs with cleavable linkers showed a DAR of 3.79, with a relative standard deviation (RSD) of 0.48 (n = 3). Similarly, the trypsin and chymotrypsin digestion strategy that is applicable to ADCs with non-cleavable linkers showed a DAR of 3.77 and an RSD of 0.86 (n = 3). The DAR determined by this method was consistent with the DAR of the ADCs that was measured by the UV/Vis method. This method will be very useful to researchers working in the field of ADC discovery and development.