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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

基本信息

批准号：
8581663
负责人：
FREDERICK R BLATTNER
金额：
$ 91.1万
依托单位：
SCARAB GENOMICS, LLC
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-27 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8581663
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

项目摘要

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)：生物疗法是制药市场中一个快速增长的领域，在每年1000亿美元的蛋白质疗法支出中，单克隆抗体几乎占到50%。细菌发酵约占所有蛋白质治疗药物生产的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。随着单链抗体等产品成为单抗疗法的廉价替代品，对高效和经济的细菌生物处理的需求变得更加重要，而单抗疗法是所有药物中最昂贵的。然而，常见的工业细菌菌株可能会有问题。未经改良的菌株会受到生产过剩诱导的压力反应，限制了稳定的生长，并可能导致细胞溶解。此外，生物制品，如单链抗体，往往是蛋白酶敏感的，很难在常见的工业菌株中生产。圣甲虫基因组学独特的生产平台Clean Genome(R)E.Coli是通过对K-12菌株MG1655进行基因组修改而设计的，消除了不需要或不必要的DNA，包括可移动插入元件和原噬菌体。其他缺失的目的是消除有害序列，这些序列可能会限制它们在生物加工中的生产力，并增强重组蛋白合成和质粒DNA生产。目前的应用建议在最先进的清洁基因组菌株的基础上，通过在这一起点上产生一种结合了低突变率和低蛋白酶活性的新菌株，目前包括69个缺失，使基因组减少20%。在第一阶段，我们建议去除蛋白酶基因和容易出错的聚合酶基因，以构建具有这些特性的单个菌株。该菌株将被测试其产生简单蛋白质和质粒DNA的能力。在第二阶段，将通过从基因组中删除剩余的23个应激诱导毒素/抗毒素基因和群体感应基因LuxS来进一步精炼遗传背景。这种独特的抗压菌株将被评估为最佳的生长和生产特性，包括它有效地产生单链抗体和其他对蛋白酶敏感的蛋白。此外，它还将被用于生产难以复制的二级结构的质粒。最后，为了达到商业化标准，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，这种单一的通用生产菌株将极大地简化单链抗体等生物制品的制造，并为制药业提供一种工具，通过它可以高效且廉价地制造最困难但高价值的生物制品。