Department of Protein Analytical Chemistry, Genentech, Inc., 1 DNA way, South San Francisco, CA 94080-4990, USA.
Bioconjug Chem. 2013 Jul 17;24(7):1154-60. doi: 10.1021/bc4000299. Epub 2013 Jun 11.
Antibody-drug conjugates (ADCs) utilizing cysteine-directed linker chemistry have cytotoxic drugs covalently bound to native heavy-heavy and heavy-light interchain disulfide bonds. The manufacture of these ADCs involves a reduction step followed by a conjugation step. When tris(2-carboxyethyl)phosphine (TCEP) is used as the reductant, the reaction stoichiometry predicts that for each molecule of TCEP added, one interchain disulfide should be reduced, generating two free thiols for drug linkage. In practice, the amount of TCEP required to achieve the desired drug-to-antibody ratio often exceeds the predicted, and is variable for different lots of monoclonal antibody starting material. We have identified the cause of this variability to be inconsistent levels of interchain trisulfide bonds in the monoclonal antibody. We propose that TCEP reacts with each trisulfide bond to form a thiophosphine and a disulfide bond, yielding no net antibody free thiols for conjugation. Antibodies with higher levels of trisulfide bonds require a greater TCEP:antibody molar ratio to achieve the targeted drug-to-antibody ratio.
抗体偶联药物（ADCs）利用半胱氨酸定向连接化学将细胞毒性药物共价连接到天然的重链-重链和重链-轻链间二硫键上。这些 ADC 的制造涉及还原步骤和连接步骤。当使用三(2-羧乙基)膦（TCEP）作为还原剂时，反应化学计量学预测，每添加一个 TCEP 分子，就应该还原一个链间二硫键，生成两个用于药物连接的游离硫醇。实际上，为了达到所需的药物与抗体的比例，所需的 TCEP 量常常超过预测值，并且对于不同批次的单克隆抗体起始材料是可变的。我们已经确定这种可变性的原因是单克隆抗体中链间三硫键水平不一致。我们提出 TCEP 与每个三硫键反应形成硫代膦和二硫键，对于连接没有净抗体游离硫醇。具有更高三硫键水平的抗体需要更大的 TCEP:抗体摩尔比才能达到目标的药物与抗体的比例。
