快流速高载量抗体纯化介质设计与应用基础研究

The upstream process of antibody preparation has been well developed. But its downstream process has not reached an efficient state, which has become the bottleneck of large scale preparation of antibody. The dynamic binding capacity (DBC) and throughput of Protein A affinity medium for antibody purification is relatively low, which can't meet the large scale preparation of antibody. In order to eliminate the disadvantage of antibody purification medium, this research devotes to design a novel and efficient antibody purification medium. Dextran molecules are grafted on the surface of rigid, macroporous poly (glycidyl methacrylate) (PGMA) microspheres. Structure controlled tentacle is introduced onto the macroporous microsphere when the hydrophobic microspheres are modified with hydrophilic molecules of dextrans. The tentacle structure provides a three-dimensional structure for ligand coupling and antibody absorption. A controllable and site-specific ligand coupling method is developed to improve the utilization ratio of protein A ligand. The coupling site is characterized by a method coupling of high performance liquid chromatography-mass spectrometry (HPLC-MS) and enzymatic digestion. Finally, a high-speed antibody purification medium with high capacity is developed, and antibody is purified quickly and efficiently with such a novel protein A medium. This research provides insight into the understanding of the structure-performance relationship of chromatographic medium, establishes a controllable and site-specific ligand coupling technique, which are beneficial for the development of high-performance protein A medium and the efficient preparation of antibody.

抗体制备的上游技术已日趋成熟，但其纯化过程仍未实现高效化，已成为抗体规模化制备的技术瓶颈。本项目针对抗体纯化介质动态载量低、通量小的不足，设计新型、高效的抗体纯化介质，在刚性、超大孔聚甲基丙烯酸缩水甘油酯微球上接枝葡聚糖分子，在对超大孔聚合物微球进行亲水改性的同时引入结构可控的葡聚糖"触须"(tentacle)结构单元，为配基偶联和抗体吸附提供丰富的三维结构空间；建立定点、可控的Protein A配基偶联方法和偶联位点的表征方法，提高配基的利用率；最终实现快流速、高载量抗体纯化介质的制备以及抗体的快速、高效纯化。本研究对揭示介质的"结构-性能"关系、建立配基的可控、定点偶联技术，实现抗体的高效制备具有重要的科学意义，为推动抗体药物的研发提供高性能的分离介质和奠定坚实的技术基础。

抗体制备的上游技术已日趋成熟，但其纯化过程仍未实现高效化，已成为抗体规模化制备的技术瓶颈。本项目针对抗体纯化介质动态载量低、通量小的不足，设计和开发了新型、高效的抗体纯化介质，在刚性、超大孔聚甲基丙烯酸缩水甘油酯微球上可控接枝葡聚糖分子，在对超大孔聚合物微球进行亲水改性的同时引入结构可控的葡聚糖“触须”(tentacle)结构单元，为配基偶联和抗体吸附提供丰富的三维结构空间；设计了高耐碱性的Protein A配基，建立了定点、可控的Protein A配基偶联方法和偶联位点的表征方法，提高了配基的利用率和结合抗体的能力；最终实现了快流速、高载量抗体纯化介质的制备以及抗体的快速、高效纯化。Protein A亲和介质在多种抗体体系中得到了初步应用，结果表明该介质具有耐碱性好（可耐受0.5 M氢氧化钠）、载量高（大于60 mg/ml介质）、纯化效果好（单步纯化抗体纯度达到95%以上，甚至电泳纯和色谱纯的高纯度）等特点和优势，具有重要的应用前景。本研究对揭示介质的“结构-性能”关系、建立配基的可控、定点偶联技术，实现抗体的高效制备具有重要的科学意义，为推动抗体药物的研发提供高性能的分离介质和奠定坚实的技术基础。