High Resolution Protein Separations via Charged Block Copolymer Membranes

 描述(由申请人提供)：从活生物体或生物制品中提取的治疗性生物分子的最新进展导致它们在疾病治疗中得到广泛应用。从单抗到重组蛋白，数以百计的生物制品现在正在上市。随着这种以蛋白质为基础的处理方法的使用获得了吸引力，从复杂混合物中提纯活性生物制品的要求变得更加迫切。将实验室规模的蛋白质组学研究转化为商业规模的药物制造的一个主要挑战是纯化过程。只分离感兴趣的蛋白质的能力以及完成这一任务的速度是提高疗效和降低生物制造成本的重要因素。该项目致力于使用基于膜的分离技术从复杂混合物中有效地分离蛋白质。基于膜的分级可以在短时间内过滤大量材料。该项目优化了膜的物理和化学特性，以实现高度选择性和高通量的蛋白质纯化。利用膜技术实现快速、高质量蛋白质纯化的一个关键是适当地调整膜结构和化学成分。Terapore的超滤膜是由一种独特的、自组装的有机分子制成的，这种分子可以产生高密度的非常均匀的毛孔。高的孔密度导致了高的膜渗透率，使得能够以相对较小的膜面积处理大量的分子，而统一的孔径使得基于尺寸排斥原理能够有效地分离相似大小的分子。在这个项目中，将解决通过化学调节膜表面和孔壁化学来提高膜选择性的关键问题，而不是结构上的考虑。特别是，这个项目探索了膜结构的表面定制将如何导致基于目标蛋白质的化学来区分相似大小的蛋白质的能力。该项目的具体目标包括使用终端和交联剂对膜表面进行定量功能化，这将使用核磁共振光谱和傅里叶变换红外光谱进行监测。将评估功能化膜的渗透性，并使用死端搅拌细胞过滤装置将其与未经处理的膜的吞吐量进行比较。膜还将受到单蛋白和多蛋白缓冲饲料溶液的挑战，以评估在不同溶液处理条件下的拒绝特性。饲料和渗透蛋白浓度将使用UV-Vis光谱和凝胶渗透色谱进行监测，并通过高分辨率高效液相分析加以证实。最后，用发酵液对膜进行挑战，以确定针对工业饲料流的性能。

 DESCRIPTION (provided by applicant): Recent advances in the development of therapeutic biomolecules derived from living organisms, or biologics, have lead to their widespread applications in the treatment of disease. From monoclonal antibodies to recombinant proteins, hundreds of biologics are now in the market pipeline. As the use of such protein-based treatments gains traction, the requirements for purifying active biologics from complex mixtures becomes more pressing. A major challenge in translating bench-scale proteomics research to commercial-scale drug manufacturing is the purification process. The ability to isolate only the protein of interest as well as the speed by which this can be accomplished are important factors in increasing therapeutic efficacy and reducing biologic manufacturing costs. This project seeks to enable the efficient fractionation of proteins from complex mixtures using membrane- based separations technology. Membrane-based fractionation enables large volumes of material to be filtered over short periods of time. This project optimizes both physical and chemical characteristics of membranes to achieve very selective and high throughput protein purification. One key to achieving rapid, high quality protein purifications using membrane technology is the appropriate tailoring of membrane structure and chemistry. Terapore's ultrafiltration membranes are made from a unique, self-assembling organic molecule that creates a high density of very uniform pores. The high pore density results in high membrane permeability, enabling large volumes to be processed with relatively small membrane areas, while the uniform pore sizes enable similarly size molecules to be efficiently isolated based on size- exclusion principles. In this project, the critical issue for enhancing the membrane selectivity beyond structural considerations through chemically tuning the membrane surface and pore wall chemistry will be addressed. In particular, this project explores how surface customization of the membrane structure will result in the ability to distinguish between similarly sized proteins based on the chemistry of the target protein. Specific goals in the project include the quantitative functionalization of membrane surfaces using both terminal and cross-linking agents, which will be monitored using nuclear magnetic resonance spectroscopy and Fourier-transform infrared spectroscopy. The functionalized membrane will be evaluated for permeability and compared with throughput for untreated membranes using a dead-end stirred cell filtration setup. The membranes will also be challenged both single-protein and multi-protein buffered feed solutions to evaluate rejection characteristics under various solution processing conditions. Feed and permeate protein concentrations will be monitored using UV-vis spectroscopy and gel permeation chromatography, and corroborated using high-resolution HPLC analyses. Finally, the membranes will be challenged with fermentation broth feed solutions to determine performance against industrial feed streams.