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

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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.