The main purpose of the present work is to provide a fully integrated miniaturized electrophoretic methodology in order to facilitate the quality control of monoclonal antibodies (mAbs). This methodology called D-PES, which stands for Diffusion-mediated Proteolysis combined with an Electrophoretic Separation, permits to perform subsequently mAb tryptic digestion and electrophoresis separation of proteolysis products in an automated manner. Tryptic digestion conditions were optimized regarding the influence of enzyme concentration and incubation time in order to achieve similar enzymatic digestion efficiency to that obtained with the classical methodology (off-line). Then, the optimization of electrophoretic separation conditions concerning the nature of background electrolyte (BGE), ionic strength and pH was realized. Successful and repeatable electrophoretic profiles of three mAbs digests (Trastuzumab, Infliximab and Tocilizumab), comparable to the off-line digestion profiles, were obtained demonstrating the feasibility and robustness of the proposed methodology. In summary, the use of the proposed and optimized in-line approach opens a new, fast and easy way for the quality control of mAbs.

本工作的主要目的是提供一种完全集成的微型电泳方法，以促进单克隆抗体（mAb）的质量控制。这种称为D-PES的方法学代表扩散介导的蛋白水解结合电泳分离，允许随后以自动化方式进行mAb胰蛋白酶消化和蛋白水解产物的电泳分离。针对酶浓度和孵育时间的影响，对胰蛋白酶消化条件进行了优化，以实现与经典方法（离线）相似的酶消化效率。然后，实现了关于背景电解质（BGE）的性质，离子强度和pH的电泳分离条件的优化。获得了三种mAb消化物（曲妥珠单抗，英夫利昔单抗和托珠单抗）的成功且可重复的电泳图谱，与离线消化图谱相当，证明了所提出方法的可行性和稳健性。总之，使用建议的和优化的在线方法为mAb的质量控制开辟了一种新的，快速而简便的方法。