Antibody-drug conjugates (ADCs) present unique challenges for ligand-binding assays primarily due to the dynamic changes of the drug-to-antibody ratio (DAR) distribution in vivo and in vitro. Here, an automated on-tip affinity capture platform with subsequent mass spectrometry analysis was developed to accurately characterize the DAR distribution of ADCs from biological matrices. A variety of elution buffers were tested to offer optimal recovery, with trastuzumab serving as a surrogate to the ADCs. High assay repeatability (CV 3%) was achieved for trastuzumab antibody when captured below the maximal binding capacity of 7.5 μg. Efficient on-tip deglycosylation was also demonstrated in 1 h followed by affinity capture. Moreover, this tip-based platform affords higher throughput for DAR characterization when compared with a well-characterized bead-based method.

中文翻译：

---

自动化的亲和力捕获与质谱联用，可表征血液中完整的抗体-药物结合物

抗体-药物偶联物（ADC）对配体结合测定提出了独特的挑战，这主要是由于体内和体外药物与抗体之比（DAR）分布的动态变化所致。在此，开发了具有后续质谱分析功能的自动尖端亲和力捕获平台，以准确表征生物基质中ADC的DAR分布。测试了多种洗脱缓冲液以提供最佳的回收率，曲妥珠单抗可作为ADC的替代品。当曲妥珠单抗抗体的最大结合量低于7.5μg时，可实现较高的测定重复性（CV 3％）。还证明了在1小时内有效的尖端去糖基化，然后进行了亲和力捕获。而且，