(184l) High-Throughput Experimentation Reveals Scope and Limitations of Selective Phosphine Reductants and Enables One-Pot Mab Reduction/Conjugation

This work describes the detailed evaluation of more than 40 phosphine reductants via automated and non-automated high-throughput experimentation approaches with the goal to identify selective reductants for cleaving the monoclonal antibody (mAb) disulfide bonds of capped, engineered cysteines. As a point of reference, this study included phosphines that have previously been documented in the literature [4-diphenylphosphino benzoic acid (DPPBA), tris(3-sulfophenyl)phosphine (TSPP), and 3-(diphenylphosphino)benzenesulfonate (diPPBS)] but showed relatively poor selectivity. The high-throughput experiments performed in this study revealed several phosphines with potential for selective reduction, which have not been previously studied for this type of transformation. These initial hits are further evaluated with regard to phosphine/mAb ratio, solubility in aqueous media, and air oxidation behavior. The best phosphine identified through these studies (1-(4-(diphenylphosphanyl)benzyl)-1-methylpyrrolidin-1-ium bromide) was then employed in a high-throughput study that established efficient one-pot reduction/conjugation reaction conditions. Overall, the studies detailed herein demonstrate how high-throughput experimental design enables rapid and resource-sparing insights into mAb reduction and conjugation reactivity with phosphine-based reductants.