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.

30%的重组治疗蛋白是在大肠杆菌中生产的。包括重要的疫苗 载体蛋白等成分。此外，新兴的使用蛋白质的新免疫疗法， 单链抗体和病毒样颗粒正获得越来越多的成功。满足生产需要 在这些不断扩展的领域中，Scarab Genomics公司提议扩展其Clean Genome®大肠杆菌的多功能性， 生产系统，包括将重组治疗性蛋白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纯化。结合圣甲虫的 扩展的发酵平台，可实现Clean Genome E的高密度生长。大肠杆菌和常数 生产的目的蛋白，一个简化的重组蛋白提取方法将扩大圣甲虫的平台 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，大大简化治疗性蛋白质如载体蛋白质的纯化， CRM 197和显着降低总生产成本.使用载体蛋白的初步实验 CRM 197、嗜血杆菌蛋白D和假单胞菌胞外蛋白A表明， 生产宿主菌株与专有提取缓冲液组合可诱导高达90%的 重组周质蛋白此外，释放到培养基中的重组治疗蛋白被 稳定超过两周，进一步强调了系统的多功能性。在第1阶段，发酵和 待测载体蛋白CRM 197的提取条件有待改进，试剂有待优化 将开发表达和提取。第二阶段的第一个目标是确定是否删除 已被证明对周质稳定性至关重要的关键转肽酶 以增强重组蛋白从周质的释放，可以改善提取。类似地， 从生产质粒表达的肽聚糖修饰酶的影响将在 提取方法的上下文。第二个目标是研究四个目标的表达和提取 在延长发酵中的蛋白质，以测试在生产规模的提取系统。除了承运人 蛋白质嗜血杆菌蛋白质D和假单胞菌胞外蛋白质A，单链抗体和单链 将检查可变片段。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，这对于载体蛋白是显而易见的。的 第三个目的提出设计一种从提取溶液中去除细胞和细胞碎片的方法， 目的是为真正的连续过程提供具有澄清材料的连续色谱系统。