DT is a 62 kDa single polypeptide chain containing two disulfide bonds, synthesized by Corynebacterium diphtheriae and released extracellularly. The intact DT molecule is non-enzymatically active. In the presence of certain thiols, the molecule is activated after mild treatment with trypsin or other proteases into fragment A of 24 kDa and fragment B of 38 kDa. Fragment A contains the catalytic C domain and fragment B consists of the transmembrane and receptor binding domains. Studies have shown that the role of fragment A and fragment B in immunology is quite different, and they do not cross-react in antitoxin immune diffusion. Anti-fragment A antibody can inhibit the enzymatic activity of isolated fragment A, but cannot neutralize endotoxin. Anti-fragment B antibodies neutralize the in vivo toxicity of toxins by preventing them from attaching to sensitive cell surfaces.
Fig.1 Different chemical approaches applied to obtain activated oligosaccharides or polysaccharides to be covalently coupled to the carrier protein. (Tontini, et al., 2016)
CD BioGlyco provides conjugation of DT to various polysaccharide antigens through chemistry-based polysaccharide conjugation and physical crosslinking-based polysaccharide conjugation technology.
Taking the development process of Men X polysaccharide-DT conjugate as an example, our development process is as follows.
The DT carrier protein is purified by affinity chromatography.
Polysaccharides are treated by reductive amination: Concentrations of polysaccharides are incubated in ammonium acetate and sodium cyanoborohydride for several days. Aminated polysaccharides are purified by diafiltration on regenerated cellulose membranes, and the number of introduced primary amino groups is determined colorimetrically.
Amino oligosaccharides are vacuum dried, dissolved in H2O and DMSO solution to final amino concentration, and reacted with excess adipate bis(N-hydroxysuccinimide). The reaction is maintained at room temperature with gentle stirring for 2 hours. Activated oligosaccharides are then obtained by precipitation with acetone and dried under vacuum. The content of introduced N-hydroxysuccinimide ester groups is determined.
Mice are subcutaneously immunized at solid time points to test the immune effect of the conjugate.
Fig.2 General steps in the development of polysaccharide-DT conjugates. (CD BioGlyco)
CD BioGlyco has an advanced Glyco™ Vaccine Development Platform and analysis tool that helps customers' Carbohydrate-based Vaccine Development projects systematically. If you are interested in our services, please contact us for more details without any hesitation.
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