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

说明(申请人提供)：CRM197是白喉毒素的一种非活性形式，当与疫苗抗原结合时，可增强免疫力，产生针对该抗原的长期疫苗接种。含有CRM197的结合疫苗很常见，目前市场上有七种含有CRM197的结合疫苗。最畅销的疫苗是含有CRM197的结合疫苗Prevnar(一种肺炎球菌疫苗)，2012年创收超过37亿美元。CRM197的需求很大，以每毫克约500美元的零售价计算，CRM197的市场估计高达10亿美元。由于每剂结合疫苗含有10到60微克的CRM197，每年使用超过1.5亿剂，因此很容易理解产生一种廉价的方法来生产纯化的CRM197的价值。此外，除了作为疫苗刺激剂的活性外，CRM197还可能在治疗和预防预后较差的癌症方面发挥重要作用。在第一阶段，Scarab基因组公司建议开发一种系统，将CRM197运送到我们的低突变率、基因清洁的基因组(R)大肠杆菌的周质隔间。周质定位能够产生一种系统，通过该系统可以很容易地从浓缩细菌中分离出大量可溶的CRM197。第一阶段取得的里程碑概述了产生CRM197的过程，该过程产生的产量是传统方法的10倍。摇瓶培养结果鉴定出四株使用我们专有表达系统产生高CMR197表达水平的Scarab菌株。在补料分批发酵试验中，可溶性CRM197的产量接近2g/L，与市场上最好的生产平台持平。第二阶段将由三个部分组成。首先，将根据我们发现在重组肽生产中重要的性质来优化发酵条件。这些因素包括pH、温度、诱导剂浓度和培养基数。Aim I的里程碑将是将市场上最好的CRM197生产平台的目前生产水平提高一倍，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CRM197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，CRM197将接受严格的下游产品分析，以在功能检测中验证纯度和活性。最后，将纯化的CRM197与商业上可用的产品进行比较，以评估最终的纯度，并证明它与目前可用的产品相当。为了开始商业化进程，提纯的CRM197将被冷冻干燥，并进行货架期和储存分析，为销售做准备。最后，斯卡拉布基因公司将准备并提交一份生物制品主文件，以提供文件，方便FDA审查客户对我们平台生产的CRM197的研究新药(IND)申请。

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.