Abstract：
Antibody-drug conjugates (ADCs) represent the forefront of the next generation of biopharmaceuticals. An ADC typically comprises an antibody covalently linked to a cytotoxic drug via a linker, resulting in a highly heterogeneous product. This study focuses on the analysis of a custom-made cysteine-linked ADC. Initially, we developed a LC-MS-based characterization workflow using brentuximab vedotin (Adcetris®), encompassing native intact MS, analysis of reduced chains and subunits under denaturing condition, peptide mapping and online strong cation exchange chromatography coupled with UV and mass spectrometry detection (SCX-UV-MS) applied for brentuximab vedotin first time reported. Subsequently, we applied this in-depth characterization workflow to a custom-made cysteine-linked ADC. The measured drug-to-antibody ratio(DAR) of this ADC is 6.9, further analysis shown that there is a small amount of unexpected over-conjugation. Over-conjugation sites were successfully identified using multiple UHPLC-MS based characterization techniques. Also, one competitively cysteine-conjugated impurity was observed in native intact MS results, by combing native intact MS, reduced chains, subunit analysis and peptide mapping results, the impurity conjugation sites were also identified. Since this molecule is at early development stage, this provides important information for conjugation process improvement and link-drug material purification. SCX-UV-MS approach can separate the custom-made cysteine-linked ADC carrying different payloads and reduce the complexity of the spectra. The integrated approach underscores the significance of combining the SCX-UV-MS online coupling technique with other characterization methods to elucidate the heterogeneity of cysteine-linked ADCs.
摘要：
抗体-药物缀合物(ADC)代表了下一代生物药物的前沿。ADC通常包含通过接头与细胞毒性药物共价连接的抗体，导致高度异质的产品。这项研究的重点是分析定制的半胱氨酸连接的ADC。最初，我们使用本妥昔单抗vedotin(Adcetris®)开发了基于LC-MS的表征工作流程，包括原生完整的MS，变性条件下还原链和亚基的分析，首次报道了苯妥昔单抗vedotin的肽图谱和在线强阳离子交换色谱以及紫外和质谱检测（SCX-UV-MS）。随后，我们将这种深入的表征工作流程应用于定制的半胱氨酸连接ADC。测量的该ADC的药物与抗体比率(DAR)为6.9,进一步分析显示存在少量的意外过度缀合。使用多种基于UHPLC-MS的表征技术成功鉴定过缀合位点。此外，在天然完整MS结果中观察到一种竞争性半胱氨酸缀合的杂质，通过梳理原生完整的MS，减少的链条，亚基分析和肽图谱结果，还鉴定了杂质缀合位点。因为这个分子处于早期发展阶段,这为缀合过程改进和链接药物材料纯化提供了重要信息。SCX-UV-MS方法可以分离携带不同有效载荷的定制的半胱氨酸连接的ADC并降低光谱的复杂性。综合方法强调了将SCX-UV-MS在线偶联技术与其他表征方法相结合以阐明半胱氨酸连接的ADC的异质性的重要性。
