This manuscript describes the detailed evaluation of more than 40 phosphine reductants via automated and nonautomated high-throughput experimentation approaches with the goal of identifying selective reductants for cleaving the disulfide bonds of capped, engineered cysteines in a proprietary monoclonal antibody (mAb). 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)]; however, all known reductants showed the formation of undesired side products upon reduction (detectable by IEX), especially at higher phosphine loadings. The high-throughput study also revealed several phosphines with potential for selective reduction that had not been previously studied for this type of transformation. These initial hits were further evaluated with regard to the phosphine/mAb ratio, solubility in aqueous media, and air oxidation behavior. The best phosphine identified (1-(4-(diphenylphosphanyl)benzyl)-1-methylpyrrolidin-1-ium bromide (P10)) was then employed in a sequence of high-throughput studies that established efficient one-pot reduction/conjugation reaction conditions. Overall, the work detailed herein demonstrates how high-throughput experimental design enables rapid and resource-sparing insights into mAb reduction and conjugation reactivity with phosphine-based reductants.

中文翻译：

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高通量实验揭示了选择性膦还原剂的范围和局限性，并实现了一锅法 mAb 还原/偶联


本手稿描述了通过自动和非自动高通量实验方法对 40 多种膦还原剂进行的详细评估，目的是确定用于在专有单克隆抗体 （mAb） 中裂解加帽工程半胱氨酸的二硫键的选择性还原剂。作为参考，本研究包括先前在文献中记录的膦 [4-二苯基膦基苯甲酸 （DPPBA）、三（3-磺基苯基）膦 （TSPP） 和 3-（二苯基膦基）苯磺酸盐 （diPPBS）];然而，所有已知的还原剂在还原时都显示出不需要的副产物的形成（可通过 IEX 检测），尤其是在较高的膦含量下。这项高通量研究还揭示了几种具有选择性还原潜力的膦，这些膦以前没有针对这种类型的转化进行过研究。进一步评估了这些初始命中结果的膦/mAb 比值、在水性介质中的溶解度和空气氧化行为。然后将鉴定出的最佳膦化氢（1-（4-（二苯基膦基）苄基）-1-甲基吡咯烷-1-溴化铵 （P10）） 用于一系列高通量研究，以建立高效的一锅法还原/偶联反应条件。总体而言，本文详述的工作展示了高通量实验设计如何能够快速且节省资源地深入了解 mAb 还原和与膦基还原剂的偶联反应性。