Cysteine‐based conjugation is one of the main antibody conjugation strategies for generating both heterogeneous and homogeneous Antibody Drug Conjugates (ADCs), being the reduction of the antibody a crucial step in these processes. In this work we have analysed the reduction conditions for the site‐directed conjugation of an anti‐HER2 (Human Epidermal Receptor 2) Trastuzumab_cys114 antibody to the cytotoxic drug vcMMAE (valine‐citrulline monomethyl auristatin E), with an aimed average DAR (Drug‐Antibody Ratio) of 2. Initial reduction was found to have a direct impact on the availability of free thiols for the conjugation: increasing of reducing agent concentration (until a molar excess of 50x) in the reduction step resulted in a lower proportion of naked antibody in the final ADC product and allowed us to obtain an ADC close to the DAR of interest. This work shows that reduction conditions must be adjusted in order to obtain the desired homogeneous ADC product.

中文翻译：

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还原步骤对抗HER2半胱氨酸工程抗体的定点缀合影响的见解

基于半胱氨​​酸的结合是产生异质抗体和均相抗体药物结合物（ADC）的主要抗体结合策略之一，是减少抗体在这些过程中的关键步骤。在这项工作中，我们分析了针对细胞毒性药物vcMMAE（缬氨酸-瓜氨酸单甲基auristatin E）的抗-HER2（人类表皮受体2）Trastuzumab_cys114抗体的定点缀合的还原条件，以及目标平均DAR（Drug-抗体比率）为2。发现初始还原对游离硫醇的结合有直接影响：还原步骤中还原剂浓度的增加（直到摩尔过量50x）导致裸抗体的比例降低在最终的ADC产品中使用，并允许我们获得接近目标DAR的ADC。