Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, China.
CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, China, 201203.
Sci Rep. 2017 Aug 10;7(1):7763. doi: 10.1038/s41598-017-08151-2.
Drug-antibody ratio (DAR) of antibody-drug conjugates (ADCs) is important for their therapeutic efficacy and pharmacokinetics, therefore control on DAR in synthesis process is a key for ADC quality control. Although various analytical methods were reported, the real-time monitoring on DAR is still a challenge because time-consuming sample preparation is usually needed during the analysis. Antibody deglycosylation of ADC simplifies DAR measurement, however long-time PNGaseF digestion for deglycosylation hampers the real-time detection. Here, we report a rapid DAR analysis within 15 min by robust deglycosylation treatment and LC-MS detection that enables real-time DAR monitoring for optimization on ADC synthetic process. With this approach, we were able to screen suitable conjugation conditions efficiently and afford the ADCs with expected DARs. To the best of our knowledge, this is the first report on real-time DAR analysis of ADCs for conjugation optimization and quality control, compatible with random lysine-linked ADCs, glycosite-specific ADCs, and the complicated dual-payload ADCs.
抗体药物偶联物（ADC）的药物抗体比（DAR）对于其治疗效果和药代动力学非常重要，因此在合成过程中控制 DAR 是 ADC 质量控制的关键。尽管已经报道了各种分析方法，但由于分析过程中通常需要耗时的样品制备，因此实时监测 DAR 仍然是一个挑战。ADC 的抗体去糖基化简化了 DAR 的测量，但长时间的 PNGaseF 消化用于去糖基化会阻碍实时检测。在这里，我们通过稳健的去糖基化处理和 LC-MS 检测报告了在 15 分钟内进行快速 DAR 分析的方法，从而能够实时监测 ADC 合成过程中的 DAR，以进行优化。通过这种方法，我们能够有效地筛选合适的偶联条件，并提供具有预期 DAR 的 ADC。据我们所知，这是第一个用于偶联优化和质量控制的实时 ADC DAR 分析的报告，与随机赖氨酸连接的 ADC、糖基化位点特异性 ADC 和复杂的双重有效载荷 ADC 兼容。
