Intensified purification and refolding of inclusion body protein through multi-column simulated moving bed chromatography

蛋白质复性是赋予蛋白质生物活性的关键，影响治疗效果。蛋白质复性面临的挑战包括产量低、产品稀释度高、工艺量大，这意味着高昂的产品成本。目前常用的蛋白质纯化方法是单层析柱，但存在介质利用率低、溶剂消耗量大、生产效率低等缺点。基于模拟移动床(SMB)技术的多柱连续层析(MCC)技术是一种很有发展前途的手性分离技术，但由于其加工难度较大，尚未在生物处理中得到广泛应用。这项研究旨在通过MCC加强对少数商业相关蛋白质的复性和纯化，以提高加工效率和生产率。这项基础性研究产生的新知识将解决导致SMB在很大程度上没有被探索用于大规模生物加工的技术挑战。特别是，这将使MCC工艺的创新合理设计和放大方法成为可能，旨在提高连续蛋白质生物加工的生产率。这项工作的成功将对高通量进入临床试验的新生物制品具有广泛的应用价值，降低药物制造成本，加快新蛋白药物上市的速度。预计它将立即适用于基于蛋白质的新药和“生物仿制药”，这两种药物在成本基础上与即将失去专利的药物竞争。PI拥有一台独特的MCC实验台，能够承担拟议的研究。Waters UPLC H-Class Bio系统所需的分析基础设施将有助于在LC-MS工作流程中对极少量的目标分子进行可靠的高分辨率快速分析，包括完整的蛋白质、蛋白质复合体和质谱图上游的多肽。这项计划是独一无二的，因为加拿大任何地方都没有正在进行的类似的MCC开发，因此，这项投资将在该国创建一种独特的、最先进的大规模蛋白质折叠和纯化研究能力。

Protein refolding is essential to impart biological activity on proteins which affects therapeutic efficacy. Challenges in protein refolding include low yield, high product dilution, and large process volumes, which translates to exorbitant product costs. Protein purification using single chromatographic columns are commonly used, but disadvantaged by low media utilization, high solvent consumption, and low productivity. Multi-column Continuous Chromatography (MCC) processing based on Simulated Moving Bed (SMB) technology is a promising technology that has had significant impact on pharmaceutical chiral separations, but has not found widespread use in bio-processing due to challenges in processing. This research aims to intensify refolding and purification of few commercially-relevant proteins, for improved process efficiency and productivity via MCC. The new knowledge generated from this fundamental study will address technical challenges that have caused SMB to be largely unexplored for large-scale bio-processing. Particularly, this will enable innovative rational design and scale-up methodology of MCC process aimed to enhance productivity of continuous protein bio-processing. The successful outcome of this work will have broad application for high-throughput of new biologics entering clinical trials reducing drug manufacturing costs and the speed with which new protein drugs can be brought to market. It is expected to have immediate applicability for both new protein-based drugs and "bio-generics", which are competing with drugs coming off-patent on a cost basis. The PI has a unique MCC experimental rig capable be undertaking the proposed research. The requested analytical infrastructure of Waters UPLC H-Class Bio systems will help perform reliable high-resolution rapid analysis of extremely small amounts of target molecules as diverse as intact proteins, protein complexes, and peptides upstream of mass spectrometry in LC-MS workflows. This initiative is unique as there is no similar MCC development on-going anywhere in Canada, and therefore, this investment will be creating a distinctive state-of-the-art research capability on large-scale protein refolding and purification within this country.