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

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DESCRIPTION (provided by applicant): Scarab Genomics has developed valuable reduced-genome strains of Escherichia coli in which multiple deletions remove from the genome much unwanted and all potentially hazardous DNA. These Clean Genome(R) strains, along with Scarab's gene expression technology, provide a system for producing high yields of premium-quality proteins very efficiently. In this Phase I project the advantages of the system will be applied to production of a diphtheria toxoid known as CRM197. This protein is rendered non-toxic by a mutation in the active site, but remains highly antigenic. It forms the adjuvant portion in conjugate vaccines currently used to protect infants and children against bacterial meningitis. CRM197 is difficult to manufacture in quantity and therefore very expensive. These and other conjugate vaccines could become within the reach of wider populations if they could be manufactured much more efficiently. Using strains selected from Scarab's collection we will identify the optimal combination of host and vector to express CRM197. A panel of multiple combinations of useful mutations and deletions are available. These host strains will be used for expression tests in combination with a plasmid bearing a tightly controlled inducible promoter to drive CRM197 protein production. The expression plasmid can be used in any E. coli strain, so performance of different Clean Genome(R) hosts can be directly compared. Different modes of expression will also be evaluated. To produce soluble CRM197 signal-directed secretion into the periplasmic space between the inner and outer membranes will be used. Our recent work indicates new strategies to achieve high levels of periplasmic production. Alternatively, CRM197 produced at high levels in the cytoplasm, will be subjected to contemporary refolding methods to regenerate the soluble form. This is a feasible production strategy that has been used to produce a number of biopharmaceuticals. These experiments will indicate the best combination of host strain and expression mode. Finally, Scarab's optimization protocol will be applied to determine conditions for the highest yield of easily purifiable product. The Phase I project should establish the feasibility of an improved production process for this important protein. If successful, this system could enable Scarab to take advantage of a rapidly expanding market with a process aimed at improving access of vulnerable populations to needed vaccines. 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 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.