Insight into Temperature Dependency and Design of Experiments towards Process Development for Cysteine‐Based Antibody‐Drug Conjugates

Abstract In the synthetic organic community, antibody‐drug conjugates (ADCs) have become a popular topic of interest. The field is expanding beyond cytotoxic drug synthesis, and into other bioconjugation applications to have a greater impact on drug discovery and development. Bioconjugation chemistry is a complex domain for synthetic chemists, and there are limited published reports that can serve as guidance to overcome technical gaps between small molecule synthesis and large molecule bioconjugation to make ADCs. Here, we report a thorough evaluation of the temperature dependency for the tris‐(2‐carboxyethyl)‐phosphine (TCEP) reduction of antibody interchain disulfide bonds, which is a crucial step for creating cysteine‐conjugated ADCs. This investigation was conducted using three different antibodies with different isoelectric points, and under equivalent reaction conditions. Additionally, an initial stability assessment of the resulting ADCs was performed and revealed appropriate material storage conditions. The experimental data described herein can be a useful guide for synthetic organic chemists designing future ADC preparation processes.