Toxoid adjuvant CRM197 production in a stable reduced genome E. coli strain

在稳定的基因组减少的大肠杆菌菌株中产生类毒素佐剂 CRM197

• 批准号: 8252834
• 负责人: FREDERICK R BLATTNER
• 金额: $ 14.09万
• 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: 2012-09-05 至 2013-06-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8252834
• 关键词: ADP ribosylation; Active Sites; Adjuvant; Affect; Antibody Formation; Antigens; Bacteria; Bacterial Infections; Bacterial Meningitis; Biological Assay; Biological Products; Cell Death; Cells; Child; Childhood; Cleaved cell; Collection; Conjugate Vaccines; Consensus; Corynebacterium; Corynebacterium diphtheriae; Cytoplasm; Cytosol; DNA; Deletion Mutation; Diphtheria; Diphtheria Toxin; Diphtheria Toxoid; Disulfides; EWS/FLI 1 Type 2 gene; Elements; Elongation Factor; Endocytosis; Endosomes; Epidermal Growth Factor; Escherichia coli; Feasibility Studies; Fermentation; Gene Expression; Generations; Genes; Genome; Genomics; Gram-Positive Bacteria; Growth; Immune system; Improve Access; Inclusion Bodies; Infant; Link; Manufacturer Name; Marketing; Membrane; Meningitis; Methods; Mind; Molecular Chaperones; Mutation; Natural regeneration; Outcome; Peptide Hydrolases; Performance; Periplasmic Proteins; Pharmacologic Substance; Phase; Plasmids; Pneumonia; Polysaccharides; Population; Populations at Risk; Prevnar; Price; Process; Production; Protease Gene; Protein Synthesis Inhibition; Proteins; Proteolysis; Protocols documentation; Pseudomonas fluorescens; Reporting; Research; Series; Signal Transduction; Source; Streptococcus pneumoniae; Surface; System; Technology; Testing; Therapeutic; Toxin; Toxoids; Vaccine Adjuvant; Vaccines; Vulnerable Populations; Work; base; cost; cross reacting material 197; design and construction; immunogenicity; improved; infancy; mutant; overexpression; pathogenic bacteria; periplasm; polypeptide; promoter; receptor binding; research study; success; therapeutic protein; transposon/insertion element; vector

DESCRIPTION (provided by applicant): Scarab Genomics has developed valuable reduced-genome strains of Escherichia coli in which multiple deletions remove from the genome much unwanted and all potentially hazardous DNA. These Clean Genome(R) strains, along with Scarab's gene expression technology, provide a system for producing high yields of premium-quality proteins very efficiently. In this Phase I project the advantages of the system will be applied to production of a diphtheria toxoid known as CRM197. This protein is rendered non-toxic by a mutation in the active site, but remains highly antigenic. It forms the adjuvant portion in conjugate vaccines currently used to protect infants and children against bacterial meningitis. CRM197 is difficult to manufacture in quantity and therefore very expensive. These and other conjugate vaccines could become within the reach of wider populations if they could be manufactured much more efficiently. Using strains selected from Scarab's collection we will identify the optimal combination of host and vector to express CRM197. A panel of multiple combinations of useful mutations and deletions are available. These host strains will be used for expression tests in combination with a plasmid bearing a tightly controlled inducible promoter to drive CRM197 protein production. The expression plasmid can be used in any E. coli strain, so performance of different Clean Genome(R) hosts can be directly compared. Different modes of expression will also be evaluated. To produce soluble CRM197 signal-directed secretion into the periplasmic space between the inner and outer membranes will be used. Our recent work indicates new strategies to achieve high levels of periplasmic production. Alternatively, CRM197 produced at high levels in the cytoplasm, will be subjected to contemporary refolding methods to regenerate the soluble form. This is a feasible production strategy that has been used to produce a number of biopharmaceuticals. These experiments will indicate the best combination of host strain and expression mode. Finally, Scarab's optimization protocol will be applied to determine conditions for the highest yield of easily purifiable product. The Phase I project should establish the feasibility of an improved production process for this important protein. If successful, this system could enable Scarab to take advantage of a rapidly expanding market with a process aimed at improving access of vulnerable populations to needed vaccines. PUBLIC HEALTH RELEVANCE: The Clean Genome(R) expression system is expected to increase the efficiency and reduce the cost of manufacturing an important protein component of conjugate vaccines in current use. The protein will also be cleaner and safer than that produced in conventional bacteria with non-reduced genomes. These improvements could have a wide impact on production of pharmaceutical protein products and ultimately broaden access to vaccines for at-risk populations who need them.

描述(由申请人提供)：圣甲虫基因组学已经开发出有价值的减少基因组的大肠杆菌菌株，其中多个缺失从基因组中移除了许多不需要的和所有潜在危险的DNA。这些Clean Genome(R)菌株与Scarab的基因表达技术一起，提供了一种非常高效地生产高产量优质蛋白质的系统。在这一第一阶段项目中，该系统的优点将应用于生产一种名为CRM197的白喉类毒素。通过活性部位的突变，这种蛋白质变得无毒，但仍具有很强的抗原性。它形成了佐剂部分 目前用于保护婴儿和儿童免受细菌性脑膜炎影响的结合疫苗。CRM197很难批量生产，因此非常昂贵。如果这些疫苗和其他结合疫苗能够更高效地生产，它们可能会变得更广泛的人群能够承受。利用从Scarab收集的菌株，我们将确定表达CRM197的宿主和载体的最佳组合。有一组有用的突变和缺失的多种组合。这些宿主菌株将与含有严格控制的可诱导启动子的质粒一起用于表达测试，以驱动CRM197蛋白的生产。该表达载体可以在任何大肠杆菌菌株中使用，因此可以直接比较不同Clean Genome(R)宿主的性能。还将对不同的表达方式进行评估。为了产生可溶性CRM197，将使用信号定向分泌到内膜和外膜之间的周质空间。我们最近的工作表明了实现高水平周质生产的新策略。或者，在细胞质中高水平产生的CRM197将受到当代复性方法的影响，以再生可溶形式。这是一种可行的生产策略，已被用于生产许多生物制药。这些实验将指出宿主菌株和表达方式的最佳组合。最后，Scarab的优化方案将用于确定获得最高产量的易提纯产品的条件。第一阶段项目应该确定改进这种重要蛋白质的生产工艺的可行性。如果成功，这一系统可以使Scarab利用一个迅速扩大的市场，通过一个旨在改善弱势人口获得所需疫苗的过程。 与公共健康相关：Clean Genome(R)表达系统预计将提高效率并降低制造当前使用的结合疫苗的重要蛋白质成分的成本。这种蛋白质也将比在具有未还原基因组的传统细菌中产生的蛋白质更清洁、更安全。这些改进可能会对药用蛋白质产品的生产产生广泛影响，并最终扩大需要疫苗的高危人群获得疫苗的机会。