Functionalized Membranes for Membrane Chromatography

大规模纯化单克隆抗体的ritchech技术在新疗法中变得越来越重要。亲和色谱法是一种有效的色谱方法，但成本高、通量低，使大规模操作变得困难。膜色谱法提供了更高的通量，如果容量、选择性和孔径分布可以得到改善，将是一个很好的选择。本提案的重点是使用功能化膜来应对这些挑战。功能化膜具有优异的通量和高容量，因为吸附位点位于孔隙中的聚合物接枝上，而不是孔表面。在本研究中，吸附位点基于苯基丙氨酸-132/酪氨酸-133基序的类似物，在固定在微滤膜孔中的聚合物刷中沿着每个功能化链多次结合。聚合物刷是由一系列取代苯乙烯的序贯阳离子聚合形成的。氯甲基苯乙烯重复单元的甘氨酸功能化将提供间隔物以限制位阻和抑制吸附抗体的变性。牛免疫球蛋白和牛血清白蛋白的个体和竞争性吸附将用作容量和选择性的测试系统。功能化的聚合物刷将被提取并通过1H NMR和x射线晶体学进行原位表征。此外，功能化膜将测试孔径分布，以表征孔径缩小。这项研究将导致改进膜吸附剂的蛋白质和抗体分离。此外，它将导致序列聚合的发现，以产生定制的结构，非肽聚合物的测序，以及设计合成的亲和配体。这项研究的结果将在经审定的期刊出版物和在区域和国家会议上的发言中广泛传播。

CBET-0756460RitchieTechnologies for large scale purification of monoclonal antibodies are becoming increasingly important for new therapies. Affinity chromatography is effective, but expense and low throughput complicate large scale operation. Membrane chromatography provides higher throughput, and would be an excellent option if the capacity, selectivity, and pore size distribution can be improved. The focus of this proposal is to meet each of these challenges using functionalized membranes. Functionalized membranes provide excellent throughput and high capacity because adsorption sites are located on polymer grafts in the pores, rather than on the pore surface. In the proposed research, the adsorption sites are based on an analog for the phenylalanine-132/tyrosine-133 motif, incorporated multiple times along each functionalized chain in a polymer brush immobilized in the pores of a microfiltration membrane. The polymer brush is formed by sequential cationic polymerization of a series of substituted styrenes. Glycine functionalization of chloromethyl styrene repeat units will provide spacers to limit steric hindrance and inhibit denaturing of the adsorbed antibody. Individual and competitive sorption of bovine immunoglobulin and bovine serum albumin will be used as a test system for capacity and selectivity. The functionalized polymer brushes will be extracted and fully characterized ex-situ by 1H NMR and X-ray crystallography. In addition, functionalized membranes will be tested for pore size distribution to characterize narrowing of the pore size. This research will result in improved membrane adsorbers for protein and antibody separation. In addition, it will lead to discoveries in sequential polymerization to generate customized structures, sequencing of non-peptide polymers, and design of synthetic affinity ligands. The results of this research will be broadly disseminated in refereed journal publications and in presentations at regional and national meetings.