High Resolution Protein Separations via Charged Block Copolymer Membranes

通过带电嵌段共聚物膜进行高分辨率蛋白质分离

• 批准号: 8898442
• 负责人: Rachel Dorin
• 金额: $ 22.49万
• 依托单位: TERAPORE TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2016-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8898442
• 关键词: Address; Area; Bovine Serum Albumin; Buffers; Cell Membrane Permeability; Cells; Characteristics; Charge; Chemicals; Chemistry; Complex Mixtures; Confidential Information; Data; Disease; Drug Formulations; Electrostatics; Equation; Exclusion; Fermentation; Filtration; Fourier Transform; Fractionation; Gel Chromatography; Goals; Hemoglobin; High Pressure Liquid Chromatography; Isoelectric Point; Lead; Life; Marketing; Measures; Membrane; Methodology; Monitor; Monoclonal Antibodies; Myoglobin; NMR Spectroscopy; Organism; Performance; Permeability; Pharmaceutical Preparations; Procedures; Process; Proteins; Proteomics; Publishing; Reaction Time; Recombinant Proteins; Reporting; Research; Resolution; Route; Sampling; Solutions; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Speed; Stream; Structure; Surface; Technology; Temperature; Testing; Text; Therapeutic; Time; Traction; Translating; Treatment Efficacy; Ultrafiltration; Work; base; bioprocess; commercialization; copolymer; cost; crosslink; density; feeding; improved; infrared spectroscopy; interest; novel; protein complex; protein purification; public health relevance; research study; solute; therapeutic development; therapeutic protein

 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.

 描述（由申请人提供）：在开发源自活生物体或生物制剂的治疗性生物分子方面的最新进展已导致其在疾病治疗中的广泛应用。从单克隆抗体到重组蛋白质，数百种生物制剂现在正在市场上销售。随着这种基于蛋白质的治疗方法的使用获得牵引力，从复杂混合物中纯化活性生物制剂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主要挑战是纯化过程。仅分离目的蛋白质的能力以及可以实现这一点的速度是增加治疗功效和降低生物制品制造成本的重要因素。 该项目旨在利用膜分离技术从复杂的混合物中有效分离蛋白质。基于膜的分级分离能够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，以实现高选择性和高通量的蛋白质纯化。 使用膜技术实现快速、高质量蛋白质纯化的一个关键是膜结构和化学的适当定制。Terapore的超滤膜由独特的自组装有机分子制成，可形成高密度的非常均匀的孔。高孔密度导致高膜渗透性，使得能够用相对小的膜面积处理大体积，而均匀的孔径使得能够基于尺寸排阻原理有效地分离类似尺寸的分子。在这个项目中，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将得到解决。特别是，该项目探索了膜结构的表面定制如何能够根据目标蛋白的化学性质区分类似大小的蛋白质。 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立叶变换红外光谱进行监测。将评价官能化膜的渗透性，并使用死端搅拌细胞过滤装置与未处理膜的通量进行比较。还将对膜进行单蛋白和多蛋白缓冲进料溶液挑战，以评价各种溶液处理条件下的截留特性。将使用紫外-可见光谱和凝胶渗透色谱法监测进料和渗透物蛋白浓度，并使用高分辨率HPLC分析进行确证。最后，将用发酵液进料溶液对膜进行挑战，以确定对工业进料流的性能。