Functionalized Membranes for Membrane Chromatography

CBET-0756460Ritchie用于大规模纯化单抗的技术对于新的治疗方法正变得越来越重要。亲和层析是有效的，但费用和低通量使大规模操作变得复杂。膜层析提供了更高的吞吐量，如果能改善容量、选择性和孔径分布，将是一个很好的选择。这项提议的重点是使用功能化的膜来应对每一个挑战。功能化的膜提供了良好的通量和高容量，因为吸附位置位于孔内的聚合物接枝上，而不是孔表面。在拟议的研究中，吸附位置基于苯丙氨酸-132/酪氨酸-133基序的模拟，沿着每个官能化链多次并入固定在微滤膜孔中的聚合物刷子中。该聚合物刷是由一系列取代的苯乙烯顺序阳离子聚合而成的。氯甲基苯乙烯重复单元的甘氨酸官能化将提供间隔基，以限制空间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单独和竞争吸附将被用作容量和选择性的测试系统。官能化的聚合物刷将被提取，并通过1H核磁共振和X射线结晶学进行异位充分表征。此外，还将测试功能化膜的孔径分布，以表征孔径变窄的特征。这项研究将改进用于蛋白质和抗体分离的膜吸附器。此外，它还将导致顺序聚合的发现，以产生定制的结构，非肽聚合物的测序，以及合成亲和配体的设计。这项研究的结果将在被引用的期刊出版物以及区域和国家会议上的陈述中广泛传播。

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.