“Build Your Own” ADC Mimics: Identification of Nontoxic Linker/Payload Mimics for HIC-Based DAR Determination, High-Throughput Optimization, and Continuous Flow Conjugation

This manuscript reports the identification of hydrophobic interaction chromatography (HIC)-shifting, nontoxic linker-payload surrogates as tool molecules for the optimization of maleimide/cysteine conjugations relevant to antibody–drug conjugates (ADCs). These linker/payload (LP) mimics allow conjugation measurement via HIC with mAbs (monoclonal antibodies) bearing engineered or interchain cysteines as conjugation sites. Importantly, the tool molecules are employed to optimize maleimide/cysteine conjugations via modern methods of process development, including high-throughput experimentation and continuous flow. Overall, our studies provide confidence that commercially available, nontoxic LP mimics can be employed successfully to optimize ADC-type conjugations in batch and flow while minimizing materials needs and experimental work in specialized facilities required for potent compound handling.