Identification of non-toxic linker/payload mimics for HIC-based DAR determination

Abstract

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 tool molecules are demonstrated to allow conjugation measurement via HIC with a mAb (monoclonal antibody) bearing engineered cysteines, and for conjugation to mAbinterchain cysteines. The linker/payload (LP) mimics were employed to optimize conjugations via high-throughput experimentation and employed to facilitate the development of continuous flow conjugations in a microfluidic reactor and on larger scale. Putative identification of the novel ADC mimics by HIC was confirmed by mass spectrometry. Overall, our studies provide confidence that commercially available, non-toxic 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.