高容量蛋白A色谱介质理性设计的分子基础及方法学研究

In this proposal, the rational design of high-capacity Protein A adsorbents is proposed to solve the bottleneck of low binding capacity in commercial Protein A adsorbents during the production of monoclonal antibody. Based on the in-depth investigation of the Protein A – antibody interaction at interfaces and the multi-scale relationship of the architecture of chromatographic materials and chromatographic performance, the underlying fundamental of architecture – performance relationship of protein A chromatography is profoundly demonstrated at a multiscale way and the molecular basis of protein A– antibody interaction at interfaces is elaborated on nanostructure afforded by scaffolded DNA origami. The result provides scientific insight and theoretical guidance for the rational design and synthesis of Protein A adsorbents. In this research, Protein A ligands are coupled onto the matrices to investigate the evolution of the orientation and conformation of Protein A ligands at interfaces while physicochemical regulation of Protein A immobilization is studied to optimize the conditions for the immobilization and stabilize the ligand. Hence, a guideline for the rational design of Protein A adsorbent is proposed. After a composite particle is prepared as a matrix for Protein A adsorbents, novel Protein A adsorbents are synthesized with the help of the guideline. The performance of the adsorbents is evaluated to optimize the synthesis of high-capacity Protein A adsorbents. Finally, high-performance Protein A chromatography is achieved based on the synthesis of high-capacity Protein A adsorbents and the technical integration. The research will provide an insight of Protein A adsorption, and material fundamental for the innovation of Protein A chromatography.

针对当前单抗药物制备工艺中蛋白A色谱介质吸附容量低的“瓶颈”问题，本项目提出了基于理性设计开发高容量蛋白A色谱介质的研究思路。通过纳米（分子）尺度上对固液界面蛋白A与抗体相互作用以及色谱材料结构与蛋白A色谱介质性能关系两个科学问题的深入剖析，实现多尺度视角下深刻揭示色谱材料结构与蛋白A色谱介质性能的关系，进而科学地阐述固液界面蛋白A与抗体相互作用的分子机制，由此为蛋白A色谱介质的理性设计提供科学依据和理论指导。在对科学问题深入阐述的基础上，系统地研究蛋白A色谱配基固定化过程中蛋白A分子构象演化及稳定性，建立蛋白A色谱介质理性设计策略，指导固定化蛋白A色谱配基性能和稳定性的调控；合成多尺度复合色谱材料，制备高容量蛋白A色谱介质，最终通过技术集成实现蛋白A色谱过程的高效化。本项目研究工作将深化蛋白A色谱机制的科学认知并为蛋白A色谱的技术创新奠定材料学基础。

针对单抗药物制备中蛋白A色谱介质吸附容量低的“瓶颈”问题，本项目提出了基于理性设计开发高容量蛋白A色谱介质的研究目标，开展了（1）固液界面蛋白A与抗体相互作用机制，（2）色谱材料结构与色谱介质性能关系，（3）蛋白A色谱配基固定化与调控，（4）复合色谱材料的合成以及（5）高容量蛋白A色谱介质理性设计与合成等系统的研究工作。本项目将DNA折纸技术与原子力显微镜相结合，实现了固液界面结合抗体分子取向的单分子表征。该方法为蛋白质吸附行为的研究平台完善了分子表征的新模块。单分子成像的结果显示，那些与C末端耦联于DNA折纸表面的蛋白A配基结合的抗体分子呈现出“end-on”和“side-on”取向，反映出与蛋白A配基结合抗体分子的柔性特征。这一结果实现了从分子水平深入理解固液界面上蛋白A配基与抗体的结合行为及其分子构象的演化。本项目进一步解析了聚合物分子层内抗体等蛋白质分子的结合行为，揭示了不同聚合物分子层结构对于蛋白质吸附层及其分子构象的演化规律，从而为聚合物接枝的高容量蛋白A色谱介质的设计与合成提供了科学的指导。在此基础上，本项目探索了高容量蛋白A色谱介质的合成方法，合成了抗体吸附容量接近100 mg/g gel的聚合物接枝高容量蛋白A色谱介质，开发了仿生肽亲和色谱纯化mAb的新工艺，实现了不同物系中抗体的高效纯化，实验室规模制备中产品的HCP和DNA残留均达到了临床应用要求。本项目的研究工作为创制高容量蛋白A色谱介质、实现抗体药物分离纯化的高效化奠定了科学的基础和技术的支撑。