High-yielding Clone & Bioprocess for Production of Soluble, Tag-free Recombinant CRM197 Carrier Protein at Gram Scale

CRM197 is a genetically detoxified mutant of Diphtheria toxin (DTx, ~58 kDa). It features a single amino acid substitution at position 52, changing glycine to glutamic acid (G52E). This mutation eliminates its toxicity while preserving the immune-stimulatory and antigenic properties as the parent molecule, DTx. CRM197 is an integral component of conjugate vaccines used for the mass vaccination of millions of peoples, worldwide. Conjugated polysaccharide vaccines that include CRM197 as the carrier protein have been approved, including MENVEO®, a tetravalent conjugate vaccine targeting serogroups A, C, W-135, and Y of Neisseria meningitidis to prevent invasive meningococcal diseases; MENJUGATE®, targeting serotype C of Neisseria meningitidis; PREVNAR-13, PNEUBEVAX-14, a pneumococcal 13-valent and 14—valent conjugate vaccine, respectively for prevention of infants against Streptococcus pneumoniae infections; TYPHIBEV for prevention of Salmonella Typhi; Vaxem-Hib® and HibTITER®, which targets Haemophilus influenza type B, Hib. However, Indigenous technology to produce high-yield protein is not available in the country. We have developed a high yielding clone and scalable bioprocess for the production of soluble rCRM197 in E.coli.

CRM197 is a genetically detoxified mutant of Diphtheria toxin (DTx, ~58 kDa) possessing mild endonuclease enzymatic activities. Protein's enzymatic activity is an inherent property of this protein and is considered a hallmark of protein functional integrity. CRM197 is a very high value protein and is an integral part of conjugate vaccines. The worldwide CRM197 manufacturing capability falls behind the market demands. Therefore, many research groups have been developing new protocols for high-yield CRM197 expression in different host organisms during the last two decades. The demand for CRM197 is high and at a retail price of about $~500-5000 USD per milligram, depending on the category of the molecule (R&D or GMP grade). CRM197 is conventionally produced from its native host Corynebacterium diphtheriae C7 strain, but suffers from low yield (10-50 mg/L) and requires a sophisticated contained bioprocess BSL-2 facility for large-scale fermentation. Therefore, high-yield recombinant production processes of CRM197 are the unfilled gap in the domain. A bioprocess for large-scale, high-yield, and affordable production of protein is urgently required to meet the increasing global demand.

1. High yielding clones producing ~1- 1.5 g protein per litre @ 10L bioreactor)
2. Soluble expression of rCRM197.
3. Industry compatible clone and bioprocess
4. Tag-free clone design, suitable for Biopharma & vaccine application.
5. Easy, high-efficiency single-step purification process.
6. High-density fermentation process.