High-Throughput Experimentation Reveals Scope and\nLimitations of Selective Phosphine Reductants and Enables One-Pot\nmAb Reduction/Conjugation

This\nmanuscript describes the detailed evaluation of more than\n40 phosphine reductants via automated and nonautomated high-throughput\nexperimentation approaches with the goal of identifying selective\nreductants for cleaving the disulfide bonds of capped, engineered\ncysteines in a proprietary monoclonal antibody (mAb). As a point of\nreference, this study included phosphines that have previously been\ndocumented in the literature [4-diphenylphosphino benzoic acid (DPPBA),\ntris(3-sulfophenyl)phosphine (TSPP), and 3-(diphenylphosphino)benzenesulfonate\n(diPPBS)]; however, all known reductants showed the formation of undesired\nside products upon reduction (detectable by IEX), especially at higher\nphosphine loadings. The high-throughput study also revealed several\nphosphines with potential for selective reduction that had not been\npreviously studied for this type of transformation. These initial\nhits were further evaluated with regard to the phosphine/mAb ratio,\nsolubility in aqueous media, and air oxidation behavior. The best\nphosphine identified (1-(4-(diphenylphosphanyl)benzyl)-1-methylpyrrolidin-1-ium\nbromide (<b>P10</b>)) was then employed in a sequence of high-throughput\nstudies that established efficient one-pot reduction/conjugation reaction\nconditions. Overall, the work detailed herein demonstrates how high-throughput\nexperimental design enables rapid and resource-sparing insights into\nmAb reduction and conjugation reactivity with phosphine-based reductants.