CRM197 protein is a modified variant of diphtheria toxin that lacks toxicity. It is developed as an alternative carrier protein to address the limitations of diphtheria toxin. Diphtheria toxoids consist of a complex mixture of substances that require detoxification before they can be used as carrier proteins. However, the detoxification process with formaldehyde reduces the purity of diphtheria toxoid from over 90% to only 60%-70%. Moreover, after dilution and removal of excess formaldehyde, diphtheria toxoid regains toxicity. To prevent the reoccurrence of toxicity, lysine is added during the detoxification process. However, the chemical modifications of diphtheria toxin can result in alterations to its native structure and potential loss of certain T-cell and B-cell epitopes.
CRM197 protein, a non-toxic mutant protein of diphtheria toxin, has advantages of high purity, no formaldehyde treatment steps, and strong immunogenicity, and has become a star carrier of protein-polysaccharide vaccines.
Fig.1 CRM197 as a carrier protein for carbohydrate conjugate vaccines targeted at bacterial and fungal pathogens. (Khatuntseva & Nifantiev, 2022)
The unique advantages of CRM197 protein make it a preferred carrier for polysaccharide vaccines. CD BioGlyco provides CRM197-based vaccine development for a variety of vaccines, including Haemophilus influenzae type B vaccine, Streptococcus pneumoniae vaccine, and Meningococcal serogroup C conjugate vaccine.
Taking the development of S. pneumoniae polysaccharide vaccines as an example, our conjugation and characterization steps are shown below.
Each polysaccharide and CRM197 protein is sonicated and molecular weight assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then sterile-filter (0.22 μm pore size) and refrigerated until initiation of conjugation experiments. For conjugation, the polysaccharide is activated using sodium periodate, then binds to the CRM197 carrier protein, saccharifies under vacuum drying at high temperature, and caps with sodium borohydride to generate the polysaccharide-CRM conjugate. The polysaccharide-CRM conjugate is suspended in water for injection and prepared for in vitro characterization and mixing using dialysis and sterile filtration.
The polysaccharide-CRM197 protein conjugate is characterized by size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS), SDS-PAGE, Bradford protein assay, and anthrone sugar assay. Then refrigerate until used to formulate the investigational vaccine.
Multiple conjugates are combined in QS to obtain a final multivalent vaccine at a certain concentration. Sodium chloride solution is used as a diluent, and aluminum phosphate is used as an experimental adjuvant. Fill the vaccine into sterile vials through an aseptic technique. All vials, seals, and vial contents are directly visually inspected before sterility testing, refrigeration, shipment, and immediate subsequent use in animal studies.
Fig.2 Conjugation procedure of S. pneumoniae polysaccharide-CRM197 carrier protein. (CD BioGlyco)
CD BioGlyco has a professional Glyco™ Vaccine Development Platform with multiple world-leading technologies to help customers comprehensively promote the Development of Carbohydrate-based vaccines. If you are interested in our services, please contact us for more details without any hesitation.
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