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.

项目概要 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 尽管迫切需要，但仍可用于快速、选择性、一步纯化难以纯化的蛋白质。 扩大基因编辑系统的应用并加速疫苗和抗体测试的开发， 快速纯化关键蛋白，如 CRISPR-Cas9 和 SARS-COV2 刺突蛋白，具有高 产量、高纯度和高活性仍然是一个艰巨的挑战，所提出的创新将使公众受益。 通过应对这一挑战，可以改善健康，并对生物制药行业产生重大影响。 这项创新产品将是市场上第一个基于通用亲和膜色谱柱的产品 TriAltus 超高亲和力 CL7/Im7 纯化系统的研究将与克莱姆森大学合作进行。 伯明翰大学和阿拉巴马大学从大肠杆菌中纯化 CRISPR-Cas9 蛋白。 裂解液将作为评估亲和膜性能的示范案例 该 I 期研究的具体目标是 (1) 合成和表征 Im7 激活的亲和力。 (2) 测试用于 CRISPR-Cas9 捕获纯化的原型亲和膜柱。 具体目标1，我们将建立通过固定四个Im7来合成亲和膜的可行性 聚合物接枝大孔膜上高密度的配体变体我们将测量蛋白质结合。 使用纯化的 CL7 标记的 CRISPR-Cas9 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 CRISPR-Cas9 捕获步骤纯化的性能并对其进行基准测试 在第二阶段，我们将测量 Cas9 的结合能力、回收率、纯度和活性。 计划综合评估合成条件和膜支撑结构所发挥的作用 结合能力；划定可最大限度提高生产率、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，建立可扩展的生产工艺 用于外部验证的膜柱；并与外部合作伙伴和潜在客户进行实地研究 商业化将为客户带来第一款通用亲和膜色谱柱。 新色谱柱产品的市场进入将由色谱柱研发人员负责。 将为快速、经济高效地高产量纯化无标签蛋白质提供一个通用平台， 通过简化纯化方案并减少纯化时间，衍生出产品。 这种膜创新将对开发新蛋白质药物、基因疗法、 疫苗和抗体检测试剂盒。