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: 8782091
• 负责人: FREDERICK R BLATTNER
• 金额: $ 73.3万
• 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 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8782091
• 关键词: Acetylation; Adjuvant; Amino Acid Substitution; Antigens; Bacteria; Biological Assay; Biological Testing; Bioreactors; Carrier Proteins; Cellulose; Conjugate Vaccines; Consult; Corynebacterium diphtheriae; Cultured Cells; Data; Deposition; Diphtheria Toxin; Documentation; Dose; Endotoxins; Escherichia coli; Fermentation; Freeze Drying; Genome; Genomics; Immunity; Investigational New Drug Application; Life; Manufacturer Name; Marketing; Methods; Mutation; Norleucine; Pharmacologic Substance; Phase; Phenols; Plant Resins; Play; Pneumococcal vaccine; Populations at Risk; Positioning Attribute; Post-Translational Protein Processing; Preparation; Prevnar; Price; Process; Production; Property; Proteins; Protocols documentation; Pseudomonas fluorescens; Role; Running; Sales; Sepharose; Source; System; Techniques; Temperature; Testing; Time; Toxoids; Vaccination; Vaccine Antigen; Vaccine Production; Vaccines; Validation; Work; base; cancer prevention; commercial application; commercialization; cost; cross reacting material 197; design; feeding; flasks; immunogenicity; milligram; norvaline; outcome forecast; periplasm; pre-clinical; protein purification; public health relevance; recombinant peptide; research study; scale up; technological innovation; vaccine development

DESCRIPTION (provided by applicant): CRM197 is an inactive form of diphtheria toxin that, when conjugated to a vaccine antigen, enhances immunity to generate long-lasting vaccination against the antigen. Conjugate vaccines containing CRM197 are common and there are currently seven CRM197-containing conjugate vaccines on the market. The top-selling vaccine, the CRM197-containing conjugate vaccine Prevnar (a pneumococcal vaccine) generated over $3.7 billion USD in 2012. The demand for CRM197 is high and at a retail price of about $500 USD per milligram, the market for CRM197 is estimated to be as high as $1 billion USD. Since each dose of conjugate vaccine contains between 10 and 60 ug of CRM197 and over 150 million doses are used each year, it is easy to understand the value of generating an inexpensive method for producing purified CRM197. Further, in addition to its activity as a vaccine stimulant, CRM197 may also play an important role in the treatment and prevention of cancers that have poor prognoses. In Phase I, Scarab Genomics proposed to develop a system for the delivery of CRM197 to the periplasmic compartment of our low mutation rate, genetically Clean Genome(R) E. coli. Periplasmic targeting was able to produce a system by which high amounts of soluble CRM197 were readily available for isolation from concentrated bacteria. Milestones achieved in Phase I outlined a process for generating CRM197 that delivered yields that were 10 times that obtained by conventional methods. Results in shake-flask culture identified four Scarab strains that generated high CMR197 expression levels using our proprietary expression system. A number of these were tested in fed-batch fermentation where yields of soluble CRM197 were close to 2 g/L, on par with the best production platform on the market. Phase II will be composed of three parts. First, fermentation conditions will be optimized based on properties that we have found to be important in recombinant peptide production. These include pH, temperature, inducer concentration and medium. The milestone within aim I will be to double the current production levels of the best CRM197 production platform on the market, which is based on the bacteria Pseudomonas fluorescens. Second, CRM197 produced under optimized fermentation conditions will be purified using traditional protein purification techniques, which has been performed successfully in preliminary studies. Following purification, CRM197 will undergo rigorous downstream product analysis to validate purity and activity in a functional assay. Finally, purified CRM197 will be compared to commercially available product to assess final purity and demonstrate that it is comparable to currently available products. To begin the process of commercialization, purified CRM197 will be lyophilized and subjected to shelf-life and storage analysis in preparation for sale. Finally, Scarab Genomics will prepare and submit a Biologics Master File to provide documentation to facilitate the FDA review of customer's Investigational New Drug (IND) applications for CRM197 produced by our platform.

描述（由申请人提供）：CRM 197是一种无活性形式的白喉毒素，当与疫苗抗原结合时，可增强免疫力以产生针对抗原的持久疫苗接种。含有CRM 197的结合疫苗很常见，目前市场上有7种含有CRM 197的结合疫苗。最畅销的疫苗是含有CRM 197的结合疫苗Prevnar（一种肺炎球菌疫苗），2012年的收入超过37亿美元。对CRM 197的需求很高，零售价约为每毫克500美元，CRM 197的市场估计高达10亿美元。由于每剂缀合物疫苗含有10至60 μ g的CRM 197，并且每年使用超过1.5亿剂，因此容易理解产生用于生产纯化的CRM 197的廉价方法的价值。此外，除了作为疫苗刺激剂的活性外，CRM 197还可以在治疗和预防具有不良免疫力的癌症中发挥重要作用。在第一阶段，圣甲虫基因组公司提出开发一种系统，用于将CRM 197递送到我们的低突变率的周质区室，遗传上称为Clean Genome（R）E。杆菌周质靶向能够产生一种系统，通过该系统可以容易地从浓缩的细菌中分离出大量的可溶性CRM 197。在第一阶段中实现的里程碑概述了用于产生CRM 197的方法，其提供的产率是通过常规方法获得的产率的10倍。摇瓶培养的结果鉴定了四种金龟子菌株，其使用我们的专有表达系统产生高CMR 197表达水平。其中许多在补料分批发酵中进行了测试，其中可溶性CRM 197的产量接近2 g/L，与市场上最好的生产平台相当。第二阶段将由三个部分组成。首先，发酵条件将基于我们已经发现的在重组肽生产中重要的性质进行优化。这些因素包括pH值、温度、诱导剂浓度和培养基。目标I中的里程碑将是将目前市场上最好的CRM 197生产平台的生产水平提高一倍，该平台基于细菌荧光假单胞菌。其次，在优化的发酵条件下生产的CRM 197将使用传统的蛋白质纯化技术进行纯化，该技术已在初步研究中成功进行。纯化后，CRM 197将进行严格的下游产品分析，以验证功能测定中的纯度和活性。最后，将纯化的CRM 197与市售产品进行比较，以评估最终纯度，并证明其与目前可用的产品相当。为了开始商业化过程，将冻干纯化的CRM 197并进行有效期和储存分析，以准备销售。最后，Scarab Genomics将准备并提交一份生物制剂主文件，以提供文件，促进FDA对客户在我们平台上生产的CRM 197的研究性新药（IND）申请进行审查。