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

DESCRIPTION provided by applicant CRM 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 CRM are common and there are currently seven CRM containing conjugate vaccines on the market The top selling vaccine the CRM containing conjugate vaccine Prevnar a pneumococcal vaccine generated over $ billion USD in The demand for CRM is high and at a retail price of about $ USD per milligram the market for CRM is estimated to be as high as $ billion USD Since each dose of conjugate vaccine contains between and ug of CRM and over million doses are used each year it is easy to understand the value of generating an inexpensive method for producing purified CRM Further in addition to its activity as a vaccine stimulant CRM 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 CRM 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 CRM were readily available for isolation from concentrated bacteria Milestones achieved in Phase I outlined a process for generating CRM that delivered yields that were times that obtained by conventional methods Results in shake flask culture identified four Scarab strains that generated high CMR expression levels using our proprietary expression system A number of these were tested in fed batch fermentation where yields of soluble CRM were close to 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 CRM production platform on the market which is based on the bacteria Pseudomonas fluorescens Second CRM produced under optimized fermentation conditions will be purified using traditional protein purification techniques which has been performed successfully in preliminary studies Following purification CRM will undergo rigorous downstream product analysis to validate purity and activity in a functional assay Finally purified CRM 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 CRM 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 customerandapos s Investigational New Drug IND applications for CRM produced by our platform PUBLIC HEALTH RELEVANCE The Clean Genome R expression system is expected to increase the efficiency and reduce the cost of manufacturing an important protein component of conjugate vaccines in current use The protein called CRM will also be cleaner and safer than that produced in conventional bacteria with non reduced genomes These improvements could have a wide impact on production of pharmaceutical protein products and ultimately broaden access to vaccines for at risk populations who need them