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 诸如载体蛋白之类的成分。此外，使用使用蛋白质（例如）的新免疫疗法 单链抗体和类似病毒的颗粒正在越来越成功。满足生产需求 在这些扩展的领域中，圣甲虫基因组学提案延长其清洁基因组®e.coli的多功能性 生产系统，包括一种重组治疗蛋白传递到细胞的方法 可以将周期直接提取到培养基中，以方便纯化。结合圣甲虫的 扩展的发酵平台，可以使清洁基因组大肠杆菌的高密度生长和常数 产生靶蛋白，一种简化的重组蛋白提取方法将扩大圣甲虫的平台 按照FDA的要求，并促进持续生产。提取方法的开发将 减少生产时间和精力，大力简化了治疗蛋白（例如载体蛋白）的纯化 CRM197并大大降低了总生产成本。使用载体蛋白的初步实验 CRM197，嗜血杆菌蛋白D和假单胞菌量蛋白A表示微妙的变化对 生产宿主应变与专有提取缓冲液结合使用，可诱导多达90％的回收率 重组周质蛋白。此外，释放到培养基中的重组治疗蛋白是 稳定超过两个星期，进一步强调了系统的多功能性。在第1阶段，发酵和 测试载体蛋白CRM197的提取条件将得到改善，并需要进行优化的试剂 表达和提取将开发。第二阶段第2期提议的第一个目的是确定是否删除 在骨质稳定性中很重要的关键转肽酶的关键酶的酶 为了增强从周质中重组蛋白的释放，可以改善提取。同样， 将在生产质粒中表达的薄荷糖改良酶的影响将在 提取方法的上下文。第二个目标将检查四个目标的表达和提取 扩展发酵中的蛋白质以生产规模测试提取系统。除了载体 蛋白质嗜血杆菌蛋白D和假单胞菌异蛋白A，单链抗体和单链 将检查可变碎片。抗体片段也将进行纯化，以确认 将重组者提取到培养基中可以简化纯化，这是载体蛋白的证据。这 第三个目的建议是设计一种方法，以从提取溶液中去除细胞和细胞碎屑的方法 为连续色谱系统提供澄清材料的目标，以实现真正的连续过程。