Kinetic Studies of the Partial Reduction and Conjugation Reactions in an Antibody-Drug Conjugate (ADC) Synthesis

Partially reducing native interchain disulfide bonds of antibodies and covalently conjugating the resulting cysteine thiol groups to potent small-molecule therapeutic agents via linkers result in a heterogeneous mixture of antibody-drug conjugates (ADCs). Since these conjugates have a varying number of drug-linker molecules attached at different positions of antibodies, this mixture has nonuniform pharmacological properties, stability, and therapeutic index (TI). To develop a better understanding of the ADC synthesis process that results in various drug-to-antibody ratios (DAR), kinetic studies of partial reduction and conjugation reactions were performed. It was found that the partial reduction reactions studied are slow, in comparison to the conjugation reactions, and are the rate-limiting steps in the ADC synthesis processes. Furthermore, it was observed that once one disulfide bond on the heavy–heavy chains is broken by the reducing agent, the remaining disulfide bonds on the heavy–heavy chains of an antibody are preferably reduced. This resulted in significant differences in the reduction rates of positional isomers, which have the same number of reduced cysteine thiols but at different positions. This gives rise to a wide product distribution during the partial reduction reactions. Kinetic models are developed to predict the impact of various process parameters on the distribution of products and are applied to identify the favorable design space for the ADC processes to maximize the yield of desired DAR species as well as offer a tool to evaluate other synthesis conditions and optimization objectives.