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（由应用提供）：理论生物分子的发展最新进展，从活物组织或生物制剂中得出，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，现在有数百种生物制剂。随着这种基于蛋白质的治疗的使用获得了吸引，因此从复杂混合物中净化活性生物制剂的要求变得更加紧迫。将基准规模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。仅分离感兴趣的蛋白质以及可以实现的速度的能力是提高治疗效率和降低生物制造成本的重要因素。该项目旨在使用基于膜的分离技术使蛋白质从复杂混合物中有效分馏。基于膜的分级使大量材料在短时间内被过滤。该项目优化了膜的物理和化学特征，以实现非常有选择性和高吞吐量蛋白质纯化。使用膜技术实现快速，高质量蛋白质净化的关键是膜结构和化学的适当调整。 Terapor的超滤机制是由独特的，自组装的有机分子制成的，该分子产生了高密度非常均匀的孔。高孔密度可导致高膜渗透性，从而使大量的膜可以使用相对较小的膜区域处理，而均匀的孔径可以根据大小排除原理有效地分离出相似大小的分子。在该项目中，将解决通过化学调整膜表面和孔隙壁化学的关键问题，以增强膜选择性。特别是，该项目探讨了膜结构的表面自定义如何根据靶蛋白的化学性能区分相似大小的蛋白质的能力。该项目中的特定目标包括使用末端和交联剂对膜表面进行定量功能化，将使用核磁共振光谱和傅立叶转换红外光谱法对其进行监测。将评估功能化的膜的渗透性，并与未经处理的膜使用死端搅拌的细胞过滤设置进行比较。膜还将受到挑战单蛋白和多蛋白缓冲饲料溶液，以评估各种溶液处理条件下的排斥特性。将使用UV-VIS光谱和凝胶渗透色谱法监测饲料和渗透蛋白浓度，并使用高分辨率HPLC分析进行佐证。最后，膜将受到发酵肉汤饲料解决方案的挑战，以确定针对工业饲料流的性能。