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

项目摘要 这个STTR第一阶段项目的目标是论证开发第一个通用亲和力的可行性 用于快速、选择性、一步提纯难提纯蛋白质的膜。尽管迫在眉睫 扩大基因编辑系统的应用，加快疫苗和抗体检测的发展， CRISPR-Cas9和SARS-COV2刺突蛋白等关键蛋白的快速纯化 产率、高纯度和高活性仍然是一个艰巨的挑战。拟议中的创新将使公众受益 应对这一挑战，并对生物制药行业产生重大影响。这个 这一创新的产品将是市场上第一个基于万能亲和膜色谱柱的产品 TriAltus超高亲和力CL7/Im7纯化系统的研究研究将与克莱姆森合作进行 阿拉巴马大学伯明翰分校。大肠杆菌CRISPR-Cas9蛋白的纯化 裂解液将作为评价亲和膜性能的示范案例。 创新。这项第一阶段研究的具体目的是(1)合成和表征Im7激活的亲和力 (2)用于捕获纯化CRISPR-Cas9的原型亲和膜柱的测试。在……里面 具体目标1，我们将建立通过固定化四个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进行筛选实验中的容量、回收率和柱的可重用性。 在具体目标2中，我们将对CRISPR-Cas9捕获步骤纯化的性能进行量化和基准测试 来自E.Coli Lysate。我们将测量Cas9的结合能力、回收率、纯度和活性。在第二阶段，团队 计划综合评估合成条件和膜支撑结构所起的作用 结合能力；划定最大限度地提高生产率、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，以建立可扩展的流程来生产 用于外部验证的膜柱；与外部合作伙伴和潜在客户进行现场研究 顾客。商业化将把第一个通用亲和膜色谱柱带到 市场。进入市场的新栏目产品将由研发人员负责。栏目 将为快速、经济有效地高产率地纯化无标签蛋白质提供通用平台， 纯净和活力。通过简化提纯方案和减少提纯时间，从 这项膜创新将对开发新的蛋白质药物、基因疗法、 疫苗和抗体检测试剂盒。

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.