Product characterization, Alexion Pharmaceuticals, 100 College Street, New Haven, CT 06510, United states.
Product characterization, Alexion Pharmaceuticals, 100 College Street, New Haven, CT 06510, United states..
J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Aug 15;1092:15-18. doi: 10.1016/j.jchromb.2018.05.010. Epub 2018 May 19.
Recombinant monoclonal antibodies have been routinely characterized at intact, subunit and peptide levels by LC-MS. Papain and pepsin have been the enzymes commonly used to cleave IgG into various fragments to facilitate in-depth characterization. However, non- specific cleavage for both papain and pepsin and the need for a reducing reagent for papain has limited their usage. In contrast, IdeS has gained popularity due to its specificity and independence of reducing reagent. Results presented in the current study demonstrated that the readily available endoprotease Lys-C can cleave IgG2 at a specific peptide bond in CH2 domain to generate a homogeneous F(ab')2 fragment, and the Fc regions was digested to peptides under limited digestion condition. The generated F(ab)2 fragment is suitable for further analysis because of its homogeneity. The posttranslational modifications located in the Fc region including glycosylation, deamidation and C-terminal heterogeneity can be rapidly analyzed by LC-MS.
通过 LC-MS 对完整的、亚基和肽水平的重组单克隆抗体进行了常规表征。木瓜蛋白酶和胃蛋白酶通常被用来将 IgG 切割成不同的片段，以促进深入的表征。然而，木瓜蛋白酶和胃蛋白酶的非特异性切割以及对木瓜蛋白酶还原剂的需求限制了它们的使用。相比之下，IdeS 因其特异性和还原剂的独立性而受到欢迎。本研究结果表明，易得的内切蛋白酶 Lys-C 可以在 CH2 结构域中特异性切割 IgG2 的特定肽键，生成均一的 F(ab')2 片段，并且在有限的消化条件下，Fc 区域被消化成肽。由于其均一性，生成的 F(ab)2 片段适合进一步分析。位于 Fc 区域的翻译后修饰，包括糖基化、脱酰胺和 C 末端异质性，可以通过 LC-MS 快速分析。
