Centre d'Immunologie Pierre Fabre, 5 Avenue Napoleon III BP 60497, 74164 Saint-Julien-en-Genevois, France.
Centre d'Immunologie Pierre Fabre, 5 Avenue Napoleon III BP 60497, 74164 Saint-Julien-en-Genevois, France.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Feb 15;981-982:9-13. doi: 10.1016/j.jchromb.2014.12.017. Epub 2014 Dec 24.
Antibody-drug conjugates (ADCs) are becoming a major class of oncology therapeutics. Because ADCs combine the monoclonal antibody specificity with the high toxicity of a drug, they can selectively kill tumor cells while minimizing toxicity to normal cells. Most of the current ADCs in clinical trials are controlled, but heterogeneous mixtures of isomers and isoforms. Very few protocols on ADC characterization at the peptide level have been published to date. Here, we report on the improvement of an ADC peptide mapping protocol to characterize the drug-loaded peptides by LC-MS analysis. These methods were developed on brentuximab vedotin (Adcetris), a commercial ADC with an average of four drugs linked to interchain cysteine residues of its antibody component. Because of the drug hydrophobicity, all the steps of this protocol including enzymatic digestion were improved to maintain the hydrophobic drug-loaded peptides in solution, allowing their unambiguous identification by LC-MS. For the first time, the payloads positional isomers observed by RP-HPLC after IdeS-digestion and reduction of the ADC were also characterized.
抗体药物偶联物（ADCs）正在成为一类主要的肿瘤治疗药物。由于 ADC 结合了单克隆抗体的特异性和药物的高毒性，它们可以选择性地杀死肿瘤细胞，同时最大限度地减少对正常细胞的毒性。目前大多数临床试验中的 ADC 都是受控制的，但存在异构混合物的异构体和同型物。迄今为止，只有少数关于 ADC 在肽水平上的表征的方案已经发表。在这里，我们报告了一种 ADC 肽图谱分析方法的改进，该方法通过 LC-MS 分析来表征载药肽。这些方法是在 brentuximab vedotin（Adcetris）上开发的，这是一种商业 ADC，其抗体成分的链间半胱氨酸残基上连接有平均四个药物。由于药物的疏水性，该方案的所有步骤，包括酶解，都得到了改进，以保持疏水性载药肽在溶液中，允许通过 LC-MS 进行明确的鉴定。这是第一次在 IdeS 消化和 ADC 还原后，通过 RP-HPLC 观察到负载物的位置异构体。
