A protease-deficient, low mutation rate E. coli for biotherapeutics production

用于生物治疗药物生产的蛋白酶缺陷型、低突变率大肠杆菌

• 批准号: 8727638
• 负责人: FREDERICK R BLATTNER
• 金额: $ 71.35万
• 依托单位: SCARAB GENOMICS, LLC
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-27 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8727638
• 关键词: Accounting; Antibodies; Antitoxins; Behavior; Biological Response Modifier Therapy; Cells; Characteristics; Cytolysis; Cytoplasm; Cytoplasmic Protein; DNA; DNA Insertion Elements; DNA Vaccines; DNA-Directed DNA Polymerase; Data; Drug Industry; Engineering; Escherichia coli; Fermentation; Genes; Genetic; Genome; Genome engineering; Genomic Segment; Genomics; Growth; Heat shock proteins; IS Elements; Individual; Industry; Liquid substance; Marketing; Methods; Modification; Monoclonal Antibodies; Mutation; Other Genetics; Peptide Hydrolases; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Plasmids; Polymerase Gene; Post-Translational Protein Processing; Process; Production; Productivity; Property; Prophages; Protease Gene; Protein Biosynthesis; Proteins; Recombinant Proteins; Replicon; Research; Resistance; Safety; Series; Stress; Structure; Testing; Therapeutic Monoclonal Antibodies; Time; Toxin; Traction; biological adaptation to stress; bioprocess; commercial application; commercialization; comparative; design; expectation; feeding; flasks; genetic element; genome sequencing; improved; meetings; novel; periplasm; plasmid DNA; quorum sensing; resistant strain; scale up; stress protein; technological innovation; therapeutic protein; tool; transposon/insertion element

DESCRIPTION (provided by applicant): Biotherapeutics is a fast-growth sector in the pharmaceutical market with monoclonal antibodies accounting for almost 50% of the 100 billion USD spent each year on protein therapies. Bacterial fermentation, which accounts for about 35% of all protein therapeutics manufacture, continues to be the most economical method of generating proteins that do not require eukaryotic post-translational modifications. The need for efficient and economical bacterial bioprocessing becomes even more important as products such as single chain antibodies gain traction as inexpensive alternatives to monoclonal antibody therapeutics, which are the most expensive of all drugs. The common industry bacterial strains, however, can be problematic. Unmodified strains suffer from overproduction-induced stress responses that limit stable growth and can cause cell lysis. Further, biologics, such as single-chain antibodies, are often protease-sensitive and difficult to produce in common industry strains. Scarab Genomics' unique production platform Clean Genome(R) E. coli was engineered by genomic modifications to the K-12 strain MG1655, eliminating unwanted or unnecessary DNA including mobile insertion elements and prophage. Other deletions were designed to eliminate noxious sequences that can limit their productivity in bioprocessing, and enhance recombinant protein synthesis and plasmid DNA production. The current application proposes to build on the most advanced Clean Genome strain now incorporating 69 deletions for a 20% genome reduction, by generating from this starting point, a new strain combining a low mutation rate with low protease activity. In Phase I we propose to remove protease genes and error-prone polymerase genes to construct a single strain with these properties. This strain will be tested for its capacity to produce simple proteins and plasmid DNA. In Phase II the genetic background will be further refined by deleting from the genome the 23 remaining stress-induced toxin/antitoxin genes and the quorum-sensing gene luxS. This unique stress-resistant strain will be evaluated for optimal growth and production characteristics including its ability to effectively produce single- chain antibodies and other protease-sensitive proteins. Further, it will be tested for production of plasmids with difficult-to-replicate secondary structure. Finally, to meet commercialization standards, the new strain will be compared with competing industry strains in fed-batch fermentations. When developed, the single all-purpose production strain will greatly simplify the manufacture of biologics such as single-chain antibodies and offer the pharmaceutical industry a tool by which the most difficult but high-value biologics can be made efficiently and cheaply.

描述（由申请人提供）：生物治疗是制药市场中快速增长的领域，单克隆抗体占每年用于蛋白质治疗的1000亿美元的近50%。细菌发酵占所有蛋白质治疗剂生产的约35%，仍然是产生不需要真核翻译后修饰的蛋白质的最经济的方法。对高效和经济的细菌生物处理的需求变得更加重要，因为诸如单链抗体的产品作为单克隆抗体治疗剂的廉价替代品而获得吸引力，单克隆抗体治疗剂是所有药物中最昂贵的。然而，常见的工业细菌菌株可能是有问题的。未修饰的菌株遭受过度生产诱导的应激反应，其限制稳定生长并可导致细胞裂解。此外，生物制剂，如单链抗体，通常是蛋白酶敏感的，难以在普通工业菌株中生产。Scarab Genomics的独特生产平台Clean Genome（R）E。大肠杆菌通过对K-12菌株MG 1655进行基因组修饰进行工程改造，消除不需要或不必要的DNA，包括移动的插入元件和原噬菌体。其他缺失被设计为消除可能限制其在生物加工中的生产力的有害序列，并增强重组蛋白合成和质粒DNA生产。本申请提出在最先进的Clean Genome菌株的基础上进行构建，该菌株现在并入了69个缺失以减少20%的基因组，通过从该起点产生结合了低突变率和低蛋白酶活性的新菌株。在第一阶段，我们建议删除蛋白酶基因和易错聚合酶基因，构建一个单一的菌株与这些属性。将测试该菌株产生简单蛋白质和质粒DNA的能力。在第二阶段的遗传背景将进一步完善删除基因组中的23个剩余的压力诱导的毒素/抗毒素基因和群体感应基因luxS。这种独特的抗应力应变将评估最佳的增长和生产特点，包括其能力， 产生单链抗体和其他蛋白酶敏感蛋白。此外，将检测其是否产生具有难以复制的二级结构的质粒。最后，为了满足商业化标准，新菌株将在补料分批发酵中与竞争的工业菌株进行比较。一旦开发出来，单一的通用生产菌株将大大简化单链抗体等生物制剂的生产，并为制药行业提供一种工具，通过这种工具，可以高效、廉价地生产最困难但价值最高的生物制剂。