Institut des Biomolécules Max Mousseron (IBMM), UMR 5247-CNRS-UM-ENSCM, Université de Montpellier, Montpellier, France.
Département de Rhumatologie, Université de Montpellier, Hôpital Lapeyronie, 34295 Cedex 5, Montpellier, France.
J Chromatogr A. 2021 Jul 5;1648:462213. doi: 10.1016/j.chroma.2021.462213. Epub 2021 May 3.
Comprehensive characterization of physicochemical properties of monoclonal antibodies (mAbs) is a critical process to ensure their quality, efficacy, and safety. For this purpose, mAb analysis at different levels (bottom-up, middle-up) is a common approach that includes rather complex multistep sample preparation (reduction, digestion). To ensure high analysis performance, the development of fully integrated methodologies is highly valuable. Capillary zone electrophoresis is a particularly well-adapted technique for the multistep implementation of analytical strategies from sample preparation to detection. This feature was employed to develop novel integrated methodologies for the analysis of mAb at the middle-up level. Multiple in-line reactions (simultaneous reduction and digestion) were performed for the first time in the separation capillary. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was used as an effective reducing agent under a broad pH range and IdeS (Immunoglobulin degrading enzyme from Streptococcus) as a highly specific enzyme for mAb digestion. Transverse diffusion of laminar flow profile (TDLFP) was applied for reactants mixing. Both in-line sample preparation and separation parameters were optimized under non-denaturing and denaturing conditions. The developed in-line methodologies provided good reproducibility and higher peak efficiencies comparing with off-line assays. They were successfully applied to different mAbs.
全面表征单克隆抗体 (mAb) 的理化性质是确保其质量、疗效和安全性的关键过程。为此，在不同水平 (自下而上、中间向上) 对 mAb 进行分析是一种常见的方法，其中包括相当复杂的多步样品制备 (还原、消化)。为了确保高分析性能，完全集成的方法的开发非常有价值。毛细管区带电泳是一种特别适合于从样品制备到检测的分析策略的多步实施的技术。该特性被用于开发用于 mAb 中间向上水平分析的新型集成方法。在分离毛细管中首次进行了多次在线反应 (同时还原和消化)。三(2-羧乙基)膦盐酸盐 (TCEP) 在较宽的 pH 范围内用作有效还原剂，IdeS (来自链球菌的免疫球蛋白降解酶) 用作 mAb 消化的高度特异性酶。层流横向扩散 (TDLFP) 用于反应物混合。在非变性和变性条件下优化了在线样品制备和分离参数。与离线分析相比，开发的在线方法提供了更好的重现性和更高的峰效率。它们成功地应用于不同的 mAb。
