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）：生物治疗是制药市场中一个快速增长的领域，单克隆抗体占每年蛋白质疗法支出 1000 亿美元的近 50%。细菌发酵约占所有蛋白质治疗药物制造的 35%，仍然是生成不需要真核翻译后修饰的蛋白质的最经济方法。随着单链抗体等产品作为单克隆抗体疗法（所有药物中最昂贵的）的廉价替代品而受到关注，对高效且经济的细菌生物加工的需求变得更加重要。然而，常见的工业菌株可能存在问题。未修饰的菌株会遭受过度生产引起的应激反应，限制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，通常对蛋白酶敏感并且难以在常见工业菌株中生产。 Scarab Genomics 独特的生产平台 Clean Genome(R) 大肠杆菌是通过对 K-12 菌株 MG1655 进行基因组修饰而设计的，消除了不需要的或不必要的 DNA，包括移动插入元件和原噬菌体。其他删除旨在消除可能限制其生物加工生产力的有害序列，并增强重组蛋白合成和质粒 DNA 生产。当前的申请建议以最先进的清洁基因组菌株为基础，该菌株目前包含 69 个缺失，基因组减少了 20%，从这个起点生成一种结合了低突变率和低蛋白酶活性的新菌株。在第一阶段，我们建议去除蛋白酶基因和易错聚合酶基因，以构建具有这些特性的单一菌株。将测试该菌株生产简单蛋白质和质粒 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，进一步完善遗传背景。将评估这种独特的抗逆菌株的最佳生长和生产特性，包括其有效地产生单链抗体和其他蛋白酶敏感蛋白。此外，还将测试具有难以复制二级结构的质粒的生产。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，单一通用生产菌株将大大简化单链抗体等生物制剂的制造，并为制药行业提供一种工具，可以高效、廉价地制造最困难但高价值的生物制剂。