The development of a process for the preparation of a conventional anti-Notch 3 antibody–drug conjugate (ADC) is described. The initial reaction conditions used for the conjugation of an auristatin payload to an anti-Notch 3 monoclonal antibody led to the formation of an ADC mixture with a significant level of aggregates. Further process optimization studies resulted in the identification of reaction conditions for formation of the conjugate with a low level of aggregates. The temperature of the antibody reduction step was found to have an impact on the formation of aggregates in the ADC mixture. Differences in the antibody reduction temperatures also caused changes in the distribution of conjugated payload on the ADC species. Stability studies of anti-Notch 3 ADCs prepared by two processes differing in the antibody reduction temperature showed subtle differences in their aggregation propensities. The aggregates produced in the crude ADC reaction mixture could be separated from the desired monomer on the hydroxyapatite column under mild conditions without significantly impacting the average drug loading of the purified ADC.

中文翻译：

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Notch 3抗体-药物偶联物的工艺开发和结构表征

描述了制备常规抗Notch 3抗体-药物偶联物（ADC）的方法的发展。用于将auristatin有效负载与抗Notch 3单克隆抗体缀合的初始反应条件导致形成具有大量聚集体的ADC混合物。进一步的工艺优化研究确定了形成聚集体含量低的偶联物的反应条件。发现抗体还原步骤的温度对ADC混合物中聚集体的形成有影响。抗体还原温度的差异还导致ADC物种上共轭有效负载分布的变化。通过两个在抗体还原温度不同的过程制备的抗Notch 3 ADC的稳定性研究表明，其聚集倾向存在细微差异。可以在温和条件下将粗ADC反应混合物中产生的聚集体与羟基磷灰石柱上的所需单体分离，而不会显着影响纯化ADC的平均载药量。