SBIR-STTR Award

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.

Public Health Relevance Statement:
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.

NIH Spending Category:
Biotechnology; Genetics; Immunization; Infectious Diseases; Pediatric; Prevention; Vaccine Related

Project Terms:
Active Sites; Adjuvant; ADP ribosylation; Affect; Antibody Formation; Antigens; Bacteria; Bacterial Infections; Bacterial Meningitis; base; Biological Assay; Biological Products; Cell Death; Cells; Child; Childhood; Cleaved cell; Collection; Conjugate Vaccines; Consensus; Corynebacterium; Corynebacterium diphtheriae; cost; CRM197 (non-toxic variant of diphtheria toxin); Cytoplasm; Cytosol; Deletion Mutation; design and construction; Diphtheria; Diphtheria Toxin; Diphtheria Toxoid; Disulfides; DNA; Elements; Elongation Factor; Endocytosis; Endosomes; Epidermal Growth Factor; Escherichia coli; EWS/FLI 1 Type 2 gene; Feasibility Studies; Fermentation; Gene Expression; Generations; Genes; Genome; Genomics; Gram-Positive Bacteria; Growth; Immune system; immunogenicity; Improve Access; improved; Inclusion Bodies; infancy; Infant; Link; Manufacturer Name; Marketing; Membrane; Meningitis; Methods; Mind; Molecular Chaperones; mutant; Mutation; Natural regeneration; Outcome; overexpression; pathogenic bacteria; Peptide Hydrolases; Performance; periplasm; Periplasmic Proteins; Pharmacologic Substance; Phase; Plasmids; Pneumonia; polypeptide; Polysaccharides; Population; Populations at Risk; Prevnar; Price; Process; Production; Promotor (Genetics); Protease Gene; Protein Synthesis Inhibition; Proteins; Proteolysis; Protocols documentation; Pseudomonas fluorescens; receptor binding; Reporting; Research; research study; Series; Signal Transduction; Source; Streptococcus pneumoniae; success; Surface; System; Technology; Testing; Therapeutic; therapeutic protein; Toxin; Toxoids; transposon/insertion element; Vaccine Adjuvant; Vaccines; vector; Vulnerable Populations; Work

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.

Thesaurus Terms:
Acetylation;Adjuvant;Amino Acid Substitution;Antigens;Bacteria;Base;Biological Assay;Biological Testing;Bioreactors;Cancer Prevention;Carrier Proteins;Cellulose;Commercial Application;Commercialization;Conjugate Vaccines;Consult;Corynebacterium Diphtheriae;Cost;Crm197 (Non-Toxic Variant Of Diphtheria Toxin);Cultured Cells;Data;Deposition;Design;Diphtheria Toxin;Documentation;Dose;Endotoxins;Escherichia Coli;Feeding;Fermentation;Flasks;Freeze Drying;Genome;Genomics;Immunity;Immunogenicity;Investigational New Drug Application;Life;Manufacturer Name;Marketing;Methods;Milligram;Mutation;Norleucine;Norvaline;Outcome Forecast;Periplasm;Pharmacologic Substance;Phase;Phenols;Plant Resins;Play;Pneumococcal Vaccine;Populations At Risk;Positioning Attribute;Post-Translational Protein Processing;Pre-Clinical;Preparation;Prevnar;Price;Process;Production;Property;Protein Purification;Proteins;Protocols Documentation;Pseudomonas Fluorescens;Public Health Relevance;Recombinant Peptide;Research Study;Role;Running;Sales;Scale Up;Sepharose;Source;System;Techniques;Technological Innovation;Temperature;Testing;Time;Toxoids;Vaccination;Vaccine Antigen;Vaccine Development;Vaccine Production;Vaccines;Validation;Work;