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.
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Antibody-drug conjugates (ADCs) have become promising therapy for the treatment of cancers. Among all the ADCs under development, 2/3 of them are interchain cysteine-linked ADCs. The ADCs are manufactured by partially reducing the 4 pairs of interchain disulfide bonds followed by conjugate cytotoxic payloads to the thiols, as a consequence, the antibodies are linked with 0, 2, 4, 6, 8 drugs. The drug-to-antibody ratio (DAR) and the drug linking position are important parameters that affect the therapeutic effects and need to be well characterized. In this study, we successfully characterize a method for the interchain cysteine linked ADC in a biological matrix.