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 该STTR I阶段项目的目标是证明开发第一个普遍亲和力的可行性 膜，用于难以纯化蛋白质的快速，选择性的一步纯化。尽管紧迫 扩展基因编辑系统的应用，并加速疫苗和抗体测试开发， 临界蛋白（例如CRISPR-CAS9）和SARS-COV2峰值蛋白的快速纯化 产量，高纯度和高活动性仍然是一个困难的挑战。拟议的创新将使公众受益 健康，通过应对这一挑战对生物制药行业产生重大影响。这 这项创新的产品将是基于市场的第一范围内膜膜色谱柱 在试验的超高亲和力CL7/IM7纯化系统上。研究将与克莱姆森合作进行 大学和阿拉巴马大学伯明翰。从大肠杆菌中纯化CRISPR-CAS9蛋白 裂解物将用作评估亲和力膜性能的演示案例 创新。该阶段研究的具体目的是（1）合成和表征IM7激活的亲和力 机理和（2）测试原型亲和力膜柱用于捕获CRISPR-CAS9的纯化。在 具体目标1，我们将通过固定四个IM7来确定合成亲和力膜的可行性 在聚合物接枝的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标签CRISPR-CAS9在筛选实验中的容量，恢复和柱可重复使用。 在特定目标2中，我们将量化和基准性能以捕获CRISPR-CAS9的步骤净化 来自大肠杆菌裂解物。我们将测量CAS9的结合能力，恢复，纯度和活动。在第二阶段，团队 计划全面评估合成条件和膜支撑结构发挥作用的作用 结合能力；描述运行范围，可最大程度地提高产品生产率，恢复，纯度和活动； 与一家膜技术公司合作进行研究，以建立可扩展的过程来生产 外部验证的膜列；并与外部合作伙伴和潜在的现场研究 顾客。商业化将使第一个通用亲和力膜色谱柱带入 市场。新专栏产品的市场进入将是研发科学家。列 将提供一个通用平台，用于对具有高收率的无标签蛋白的快速，具有成本效率的纯化， 纯度和活​​动。通过简化纯化协议并减少纯化时间，从 这种膜创新将对开发新蛋白质药物，基因疗法的种族产生重大影响， 疫苗和抗体测试试剂盒。