UMR 5247-CNRS-UM-ENSCM, Institut des Biomolécules Max Mousseron (IBMM), Université de Montpellier, Montpellier, France.
Département de Rhumatologie, Université de Montpellier, Hôpital Lapeyronie, Montpellier Cedex 5, 34295, France.
Electrophoresis. 2021 Jun;42(11):1229-1237. doi: 10.1002/elps.202000375. Epub 2021 Mar 10.
For their characterization and quality control, monoclonal antibodies are frequently analyzed at the bottom-up level to generate specific fingerprints that can be used to tackle post-translational modifications or ensure production consistency between lots. To circumvent time-consuming and labor-intensive off-line sample preparation steps, the implementation of integrated methodologies from sample preparation to separation and detection is highly valuable. In this perspective, capillary zone electrophoresis appears as a choice technique since the capillary can subsequently be used as a vessel for sample preparation and electrophoretic discrimination/detection of the reaction products. Here, a fast in-line methodology for the routine quality control of mAbs at the bottom-up level is reported. Simultaneous denaturation and reduction (pretreatment step) were conducted with RapiGest® surfactant and dithiothreitol before in-line tryptic digestion. Reactant mixing was realized by transverse diffusion of laminar flow profile under controlled temperature. In-line digestion was carried out with a resistant trypsin to autolysis. The main parameters affecting the digestion efficiency (trypsin concentration and incubation conditions) were optimized to generate mAb electrophoretic profiles free from trypsin interferences. An acidic MS-compatible BGE was used to obtain high resolution separation of released peptides and in-line surfactant cleavage. The whole methodology was performed in less than two hours with good repeatability of migration times (RSD = 0.91%, n = 5) and corrected peak areas (RSD = 9.6%, n = 5). CE-fingerprints were successfully established for different mAbs and an antibody-drug conjugate.
为了对单克隆抗体进行表征和质量控制，通常在自上而下的水平上进行分析，以生成特定的指纹图谱，用于解决翻译后修饰问题或确保批次间的生产一致性。为了避免耗时且劳动密集型的离线样品制备步骤，从样品制备到分离和检测的集成方法的实施具有很高的价值。从这个角度来看，毛细管区带电泳是一种不错的选择，因为毛细管随后可以用作样品制备和电泳区分/检测反应产物的容器。在此，报道了一种用于单克隆抗体自下而上常规质量控制的快速在线方法。在在线胰蛋白酶消化之前，用 Rapigest®表面活性剂和二硫苏糖醇同时进行变性和还原（预处理步骤）。反应物混合是通过层流剖面的横向扩散在受控温度下实现的。在线消化是用耐胰蛋白酶进行自溶的。优化了影响消化效率的主要参数（胰蛋白酶浓度和孵育条件），以生成无胰蛋白酶干扰的单克隆抗体电泳图谱。使用酸性 MS 兼容的 BGE 可实现释放肽的高分辨率分离和在线表面活性剂切割。整个方法在不到两个小时的时间内完成，迁移时间（RSD=0.91%，n=5）和校正峰面积（RSD=9.6%，n=5）的重复性良好。成功为不同的单克隆抗体和抗体药物偶联物建立了 CE 指纹图谱。
