Universal affinity membrane chromatography for rapid, one-step purification of proteins

用于快速、一步纯化蛋白质的通用亲和膜层析

• 批准号: 10250634
• 负责人: Scott M Husson
• 金额: $ 25.66万
• 依托单位: TRIALTUS BIOSCIENCE, LLC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2023-05-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10250634
• 关键词: Address; Affinity; Alabama; Benchmarking; Binding; Binding Proteins; Biological Products; Biological Sciences; COVID-19; CRISPR/Cas technology; Cells; Chimeric Proteins; Chromatography; Clustered Regularly Interspaced Short Palindromic Repeats; Column Chromatography; Complex; Cystic Fibrosis; Data; Development; Diagnostic Reagent Kits; Disease; Drops; Engineering; Enzymes; Escherichia coli; Genes; Goals; Guide RNA; Health; Heart Diseases; Human; Immobilization; Industry; Ligands; Malignant Neoplasms; Measures; Membrane; Mendelian disorder; Pathology; Performance; Pharmaceutical Preparations; Phase; Plant Resins; Play; Polymers; Process; Production; Productivity; Proteins; Protocols documentation; Public Health; Race; Recovery; Research; Ribonucleoproteins; Role; SARS-CoV-2 spike protein; Sales; Scientist; Sickle Cell Anemia; Small Business Technology Transfer Research; Speed; Structure; System; Technology; Testing; Time; Universities; Validation; Variant; Work; antibody test; base; commercial application; commercialization; cost effective; density; experimental study; gene therapy; genome editing; improved; innovation; interest; new technology; phase 1 study; prevent; prospective; protein E; protein purification; prototype; public health relevance; research and development; residence; screening; vaccine evaluation

Project Summary The goal of this STTR Phase I project is to demonstrate the feasibility of developing the first universal affinity membrane for the rapid, selective, one-step purification of difficult-to-purify proteins. Despite the urgency for expanding the application of gene editing systems and accelerating vaccine and antibody test development, the rapid purification of critical proteins such as CRISPR-Cas9 and the SARS-COV2 spike protein with high yield, high purity, and high activity remains a difficult challenge. The proposed innovation will benefit public health and have a significant impact on the biopharmaceutical industry by addressing this challenge. The products of this innovation will be first-in-market universal affinity membrane chromatography columns based on TriAltus’ ultrahigh affinity CL7/Im7 purification system. Research will be done in partnership with Clemson University and the University of Alabama at Birmingham. Purification of CRISPR-Cas9 protein from E. coli lysate will be used as a demonstration case for evaluating the performance of the affinity membrane innovation. The Specific Aims of this Phase I study are to (1) Synthesize and characterize Im7-activated affinity membranes and (2) Test prototype affinity membrane columns for capture purification of CRISPR-Cas9. In Specific Aim 1, we will establish the feasibility of synthesizing the affinity membranes by immobilizing four Im7 ligand variants at high density on polymer-grafted macroporous membranes. We will measure protein binding capacity, recovery, and column reusability in screening experiments using purified, CL7-tagged CRISPR-Cas9. In Specific Aim 2, we will quantify and benchmark performance for capture step purification of CRISPR-Cas9 from E. Coli lysate. We will measure Cas9 binding capacity, recovery, purity, and activity. In Phase II, the team plans to comprehensively evaluate the roles that synthesis conditions and membrane support structure play on binding capacity; delineate operating ranges that maximize productivity, recovery, purity, and activity; collaborate with a membrane technology company on research to establish a scalable process to produce membrane columns for external validation; and conduct field research with external partners and prospective customers. Commercialization will bring the first universal affinity membrane chromatography column to the market. Market entry for the new column products will be to research and development scientists. The columns will provide a universal platform for the rapid, cost-effective purification of tag-free proteins with high yield, purity, and activity. By simplifying purification protocols and reducing purification times, products derived from this membrane innovation will have a major impact in the race to develop new protein drugs, gene therapies, vaccines, and antibody test kits.

项目摘要 这个STTR第一阶段项目的目标是证明开发第一个通用亲和力的可行性。 膜用于快速，选择性，一步纯化难以纯化的蛋白质。尽管迫切需要 扩大基因编辑系统的应用，加速疫苗和抗体测试的发展， 快速纯化关键蛋白质，如CRISPR-Cas9和SARS-COV 2刺突蛋白， 产率、高纯度和高活性仍然是一个困难的挑战。这项创新将使公众受益。 通过应对这一挑战，对生物制药行业产生重大影响。的 这项创新的产品将是市场上第一个通用的亲和膜色谱柱， 在TriAltus的亲和CL 7/Im 7纯化系统上。研究将与克莱姆森合作进行 大学和亚拉巴马大学伯明翰分校。从大肠杆菌中纯化CRISPR-Cas9蛋白杆菌 裂解物将用作评价亲和膜性能的示范案例 创新该I期研究的具体目的是（1）合成和表征Im 7活化的亲和性。 （2）用于捕获纯化CRISPR-Cas9的测试原型亲和膜柱。在 具体目标1，我们将确定通过固定四个Im 7来合成亲和膜的可行性。 配体变体在聚合物接枝的大孔膜上的高密度。我们将测量蛋白质结合 在使用纯化的CL 7标记的CRISPR-Cas9的筛选实验中的容量、回收率和柱可重复使用性。 在具体目标2中，我们将量化和基准测试CRISPR-Cas9捕获步骤纯化的性能， 来自大肠大肠杆菌裂解物。我们将测量Cas9结合能力、回收率、纯度和活性。在第二阶段，团队 计划综合评价合成条件和膜支撑结构对 结合能力;描绘最大化生产率、回收率、纯度和活性的操作范围; 与一家膜技术公司合作进行研究，以建立一个可扩展的工艺来生产 用于外部验证的膜柱;并与外部合作伙伴和潜在合作伙伴进行实地研究 客户商业化将使第一个通用亲和膜色谱柱进入市场。 市场新色谱柱产品的市场准入将由研发科学家负责。的列 将提供一个通用平台，用于快速、经济高效地纯化无标签蛋白质， 纯度和活性。通过简化纯化方案和减少纯化时间， 这种膜的创新将在开发新的蛋白质药物，基因疗法， 疫苗和抗体检测试剂盒。