Lysis-free extraction of biopharmaceuticals from the periplasm of Clean Genome E. coli

从清洁基因组大肠杆菌周质中免裂解提取生物药物

• 批准号: 9926039
• 负责人: FREDERICK R BLATTNER
• 金额: $ 91.12万
• 依托单位: SCARAB GENOMICS, LLC
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9926039
• 关键词: Antibodies; Area; Bacteria; Biological Products; Bioreactors; Buffers; Carrier Proteins; Cell Extracts; Cells; Centrifugation; Coupled; Cytolysis; Development; Enzymes; Escherichia coli; Excision; Fermentation; Genome; Genomics; Goals; Growth; Health; Hemophilus; Human; Immunoglobulin Fragments; Immunotherapeutic agent; Immunotherapy; Methods; Modification; Peptidoglycan; Peptidyltransferase; Periplasmic Proteins; Pharmaceutical Preparations; Phase; Plasmids; Post-Translational Protein Processing; Process; Production; Property; Proteins; Pseudomonas; Reagent; Recombinant Proteins; Recombinants; Recovery; Research; Scheme; System; Technology; Temperature; Testing; Therapeutic; Time; Vaccines; Virus-like particle; cost; cross reacting material 197; delta protein; density; experimental study; feeding; flasks; improved; novel; periplasm; polyclonal antibody; protein purification; success; therapeutic protein

Thirty percent of recombinant therapeutic proteins are made in E. coli and include important vaccine components such as carrier proteins. Further, burgeoning new immunotherapies that use proteins such as single chain antibodies and virus-like particles are enjoying increasing success. To meet the production needs of these expanding areas, Scarab Genomics proposes to extend the versatility of its Clean Genome® E.coli production system to include a method by which recombinant therapeutic proteins delivered into the cell periplasm can be extracted directly into the culture medium for easy purification. Combined with Scarab’s extended fermentation platform, which enables high-density growth of Clean Genome E. coli and the constant production of target protein, a simplified recombinant protein extraction method will expand Scarab’s platform and facilitate continuous production, as mandated by the FDA. The development of an extraction method will reduce production time and effort, greatly simplify purification of therapeutic proteins such as the carrier protein CRM197 and dramatically reduce total production costs. Preliminary experiments using the carrier proteins CRM197, Haemophilus protein D and Pseudomonas exoprotein A indicate that subtle changes to the production host strain combined with a proprietary extraction buffer can induce up to 90% recovery of recombinant periplasmic protein. Further, recombinant therapeutic proteins released into the medium were stable for more than two weeks further emphasizing the versatility of the system. In Phase 1, fermentation and extraction conditions for the test carrier protein CRM197 will be improved and reagents needed to optimize expression and extraction will be developed. The first aim of Phase 2 proposes to determine whether deletions of key transpeptidase enzymes that are important in the stability of the periplasm, and which have been shown to enhance the release of recombinant proteins from the periplasm, can improve extraction. Similarly, the influence of peptidoglycan-modifying enzymes expressed from the production plasmid will be examined in the context of the extraction method. The second aim will examine the expression and extraction of four target proteins in extended fermentation to test the extraction system at production scale. In addition to the carrier proteins Haemophilus protein D and Pseudomonas exoprotein A, a single chain antibody and a single-chain variable fragment will be examined. The antibody fragments will also be subjected to purification to confirm that extraction of recombinants into culture medium simplifies purification, as was evident for carrier proteins. The third aim proposes to devise a method for removing cells and cell debris from the extracted solution with the goal of providing continuous chromatographic systems with clarified material for a truly continuous process.

百分之三十的重组治疗蛋白是在大肠杆菌中产生的，包括重要的疫苗 此外，使用诸如载体蛋白之类的蛋白质的新免疫疗法正在兴起。 单链抗体和病毒样颗粒在满足生产需求方面取得了越来越大的成功。 在这些扩展领域中，Scarab Genomics 提议扩展其 Clean Genome® 大肠杆菌的多功能性 生产系统包括将重组蛋白治疗剂递送到细胞中的方法 周质可以直接提取到培养基中，以便于与 Scarab 结合纯化。 扩展的发酵平台，可实现清洁基因组大肠杆菌的高密度生长和恒定 生产目标蛋白，简化的重组蛋白提取方法将扩展 Scarab 的平台 并按照 FDA 的要求促进连续生产。提取方法的开发将 减少生产时间和精力，大大简化载体蛋白等治疗蛋白的纯化 CRM197 并显着降低了使用载体蛋白的初步实验的总生产成本。 CRM197、嗜血杆菌蛋白 D 和假单胞菌外蛋白 A 表明， 生产宿主菌株与专有的提取缓冲液相结合可诱导高达 90% 的回收率 此外，释放到培养基中的重组治疗蛋白。 稳定超过两周，进一步强调了系统在第一阶段、发酵和过程中的多功能性。 测试载体蛋白CRM197的提取条件将得到改进，所需试剂也将优化 第二阶段的第一个目标是确定是否存在删除。 关键转肽酶对周质的稳定性很重要，并且已被证明 增强重组蛋白从周质的释放，可以改善提取。 从生产质粒表达的肽聚糖修饰酶的影响将在 第二个目标将检查四个目标的表达和提取。 延长发酵中的蛋白质，以测试生产规模的提取系统。 蛋白质 嗜血杆菌蛋白 D 和假单胞菌外蛋白 A、单链抗体和单链 还将对抗体片段进行纯化以确认。 将重组体提取到培养基中简化了纯化，这对于载体蛋白来说是显而易见的。 第三个目标是设计一种从提取溶液中去除细胞和细胞碎片的方法 目标是为连续色谱系统提供澄清的材料，以实现真正的连续过程。