Wu Gang, Du Jialiang, Li Meng, Xu Gangling, Fu Zhihao, Liu Yu, Zhang Xiaoxi, Wu Guanghao, Yu Chuanfei, Wang Junzhi
School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang, Liaoning 110016, China.
Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, Daxing District, Beijing 102629, China.
J Pharm Biomed Anal. 2024 Sep 15;248:116331. doi: 10.1016/j.jpba.2024.116331. Epub 2024 Jul 4.
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.
抗体药物偶联物（ADCs）代表了下一代生物制药的前沿领域。一个ADC通常由一个通过连接子与细胞毒性药物共价连接的抗体组成，从而产生一种高度异质性的产物。本研究聚焦于对一种定制的半胱氨酸连接的ADC进行分析。最初，我们使用brentuximab vedotin（Adcetris®）开发了一种基于液相色谱-质谱的表征流程，包括天然完整质谱、变性条件下还原链和亚基的分析、肽图分析以及首次报道应用于brentuximab vedotin的在线强阳离子交换色谱与紫外和质谱检测联用（SCX-UV-MS）。随后，我们将这种深入的表征流程应用于一种定制的半胱氨酸连接的ADC。该ADC测得的药物与抗体比率（DAR）为6.9，进一步分析表明存在少量意外的过度偶联。使用多种基于超高效液相色谱-质谱的表征技术成功鉴定了过度偶联位点。此外，在天然完整质谱结果中观察到一种竞争性的半胱氨酸偶联杂质，通过结合天然完整质谱、还原链、亚基分析和肽图分析结果，也鉴定了该杂质的偶联位点。由于该分子处于早期研发阶段，这为偶联过程改进和连接子-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payload的定制半胱氨酸连接的ADC，并降低光谱的复杂性。这种综合方法强调了将SCX-UV-MS在线联用技术与其他表征方法相结合以阐明半胱氨酸连接的ADCs异质性的重要性。
