Determination of drug-to-antibody ratio of antibody–drug conjugate in biological samples using microflow-liquid chromatography/high-resolution mass spectrometry: Supplementary materials

Background: Antibody–drug conjugates (ADCs) are a promising modality for cancer treatment; however, considering their complicated nature, analytical complexity in understanding their pharmacokinetics and pharmacodynamics in the body presents a significant challenge. Results: Vorsetuzumabmaleimidocaproyl valine-citrulline p-aminobenzyloxycarbonyl monomethyl auristatin E was used to develop pretreatment and analytical workflows suitable for ADCs. Monomethyl auristatin E release and drug-to-antibody ratio retention were consistent in mouse plasma but inconsistent in monkey and human plasma. Further, metabolites were species-specific. Microflow-liquid chromatography/high-resolution mass spectrometry (LC–HRMS) resulted in a 4–7-fold improvement in detection sensitivity compared with conventional flow LC–HRMS. Conclusion: Microflow-LC–HRMS can be a useful tool in understanding the complex properties of ADCs in the body from a drug metabolism and pharmacokinetics point of view.

背景：抗体偶联药物（Antibody–drug conjugates, ADCs）是极具前景的癌症治疗手段，但其结构特性复杂，使得解析其体内药代动力学与药效动力学的分析工作面临重大挑战。结果：本研究采用维妥珠单抗-马来酰亚胺己酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基-单甲基澳瑞他汀E，开发了适用于ADCs的前处理与分析工作流程。单甲基澳瑞他汀E（Monomethyl auristatin E）的释放量与药物抗体比保留情况在小鼠血浆中表现稳定，但在猴血浆与人类血浆中则表现不稳定。此外，其代谢产物具有物种特异性。微流液相色谱/高分辨质谱（Microflow-liquid chromatography/high-resolution mass spectrometry, LC–HRMS）的检测灵敏度相较于常规流路LC–HRMS提升了4~7倍。结论：微流LC–HRMS可作为从药物代谢与药代动力学视角解析ADCs体内复杂特性的有效工具。