基于高通量优化的重组人血白蛋白纯化过程研究

Aiming at the requirement of high purity of recombinant human serum albumin (rHSA) for the pharmaceutical applications, new strategy based on the high-throughput process development (HTPD) and optimization would be introduced and used to improve the efficiency of process development. A HTPD-based platform would be established for the development and optimization of chromatographic separation of rHSA. The experimental factors and the screening space would be enhanced significantly. The optimized separation conditions for single-step chromatography and the combination of several chromatographic steps would be studied. The complicated impurities in the yeast culture broth would be removed efficiently. On the other hand, new concept of displacement separation would be proposed for the purification of the binding impurities on rHSA. With the computer-aided molecular simulation methods, the structure of displacement molecules would be designed and analyzed. The hydrophobic interactions would be forcued to improve the binding of displacement molecule onto rHSA and the electrostatic repulsion would be used for the departure of displacement molecule from rHSA. With the displacement molecules designed, the binding impurities on rHSA would be removed significantly. Based on the separation of the fermentation impurities and the binding impurities on rHSA, the downstream process would be designed for the production of rHSA with high purity from the yeast culture broth. The process efficiency would be improved significantly. In this project new methods based on HTPD and molecular simulation-aided design would be established, which would certainly improve the optimization of downstream process and develop new process for bioseparation.

针对临床药用重组人血白蛋白（rHSA）分离纯化的高要求，引入高通量的过程开发和优化方法，提高过程开发效率，实现分离过程的最优化。一方面，构建适用于层析分离优化的高通量过程开发方法，显著提高实验通量和优化空间，探讨酵母发酵液中复杂杂质的有效去除，实现单级层析优化和多级优化组合；另一方面，针对难分离的rHSA结合杂质，提出置换分离新概念，借助计算机分子模拟的高通量筛选，优化设计置换分子，组合疏水和静电作用，协调疏水结合和静电排斥解离，实现rHSA结合杂质的置换分离；在此基础上，优化rHSA分离流程，提高过程整体效率，实现高纯rHSA制备。本项目将建立基于"高通量过程开发"和"分子模拟辅助设计"的研究新方法，为生物分离过程优化和新方法构建提供指导，可推广到其它药用蛋白和生物活性物质的分离纯化过程。

针对重组人血白蛋白（rHSA）的高效分离，集中于高通量过程开发和分离新方法，一方面从高通量方法入手，构建高通量实验筛选平台，筛选分离介质，优化分离条件；另一方面从分子模拟入手，探讨配基-HSA相互作用，实现计算机辅助配基筛选和结构设计；在此基础上，针对毕赤酵母发酵液中分离rHSA，构建混合模式层析分离新方法，优化分离过程，提高分离效率。主要成果包括三个方面：第一，高通量过程开发和HSA分离条件优化：针对蛋白分离纯化，基于微孔过滤板，建立了高通量过程开发实验方法，系统分析了典型离子交换介质和混合模式介质的HSA吸附性能，优化了酵母发酵液中分离rHSA的工艺参数，发现混合模式介质MX-Trp-650M的rHSA吸附容量高，洗脱纯度达到97%，宿主细胞蛋白去除率约98%。第二，HSA-配基相互作用的分子模拟和配基设计：采用计算机分子模拟方法，探讨了HSA-有机小分子的结合机制，实现了结合过程的动态模拟，针对HSA位点II进行配基虚拟筛选，开展了HSA-色胺配基的相互作用研究，并基于HSA位点II进行了配基理性设计，实现多种相互作用的优化组合。第三，酵母发酵液rHSA的分离新方法和过程优化：基于分子模拟辅助配基设计，制备了系列混合模式介质，发现色胺介质性能最优，实现了介质制备优化和过程放大；完成了混合模式层析从毕赤酵母发酵液中分离rHSA，进一步提出了混合模式扩张床吸附新方法，直接从含酵母细胞的发酵液中分离rHSA，rHSA纯度和收率分别为99%和92%，显示出良好的应用潜力；此外，针对rHSA微量杂质，实现混合模式层析分离，有效去除宿主细胞蛋白，为高纯rHSA制备提供了备选方案。本项目研究对高通量过程开发、分子模拟辅助配基设计、新型混合模式介质研发、重组蛋白药物分离纯化等具有很好的指导和推广作用。

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