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：
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
项目类别：
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

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）：生物治疗学是制药市场中一个快速增长的领域，单克隆抗体占每年1亿美元用于蛋白质疗法的1000亿美元的50％。细菌发酵约占所有蛋白质疗法生产的35％，它仍然是产生不需要真核后翻译后修饰的蛋白质的最经济的方法。对有效和经济的细菌生物处理的需求变得更加重要，因为诸如单链抗体之类的产品成为单克隆抗体疗法的廉价替代品，这是所有药物中最昂贵的。但是，常见的行业细菌菌株可能会出现问题。未修饰的菌株受到生产过量诱导的应激反应的限制，这些反应限制了稳定生长并可能导致细胞裂解。此外，生物制剂（例如单链抗体）通常对蛋白酶敏感，难以在常见的行业菌株中产生。 Scarab Genomics独特的生产平台清洁基因组（R）大肠杆菌是通过对K-12菌株MG1655的基因组修饰而设计的，它消除了不需要或不必要的DNA，包括移动插入元件和预言。其他缺失旨在消除可以限制其在生物处理中生产力的有害序列，并增强重组蛋白合成和质粒DNA的产生。当前的应用提议建立在最先进的清洁基因组菌株上，现在结合了69个缺失，以减少20％的基因组，这是从该起点产生的，这是一种新的菌株，将低突变率与低蛋白酶活性结合在一起。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。该菌株将测试其产生简单蛋白质和质粒DNA的能力。在II期中，将通过从基因组中删除23个剩余应力诱导的毒素/抗毒素基因和群体感应基因luxs来进一步完善遗传背景。将评估这种独特的抗压力菌株的最佳生长和生产特征，包括其有效的能力产生单链抗体和其他蛋白酶敏感蛋白。此外，将测试它的生产具有难以复制的二级结构的质粒。最后，为了达到商业化标准，将与美联储发酵中的竞争行业菌株进行比较。开发时，单一通用生产应变将极大地简化生物制剂的生产，例如单链抗体，并为制药行业提供一种工具，可以通过该工具有效，便宜地制造出最困难但高价值的生物制剂。