Trisulfide Modification Impacts the Reduction Step in Antibody-Drug Conjugation Process
Trisulfide Modification Impacts the Reduction Step in Antibody-Drug Conjugation Process
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DOI：
10.1021/bc4000299
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发表时间：
2013-07-01
影响因子：
4.7
通讯作者：
Jacobson, Fredric
中科院分区：
文献类型：
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作者：
Cumnock, Katherine;Tully, Timothy;Jacobson, Fredric
利用半胱氨酸导向的接头化学的抗体-药物缀合物（ADC）具有与天然重链和重链轻链链间二硫键共价结合的细胞毒性药物。这些ADC的生产涉及还原步骤，然后是缀合步骤。当使用三（2-羧乙基）膦（TCEP）作为还原剂时，反应化学计量预测，对于每个加入的TCEP分子，应还原一个链间二硫化物，生成两个用于药物连接的游离巯基。在实践中，达到所需药物-抗体比所需的TCEP量通常超过预测值，并且对于不同批次的单克隆抗体起始材料是可变的。我们已经确定这种变异性的原因是单克隆抗体中链间三硫键的水平不一致。我们提出TCEP与每个三硫键反应形成硫代膦和二硫键，不产生用于缀合的净抗体游离硫醇。具有更高水平的三硫键的抗体需要更大的TCEP：抗体摩尔比以实现靶向的药物与抗体比。
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.