Activation of polysaccharides via the cyanylating agent, 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT), in the preparation of polysaccharide/protein conjugate vaccines
This invention provides novel reagents for cyanating polysaccharides in aqueous or part aqueous solutions so that they may be covalently linked to proteins either directly or through a spacer. These reagents include 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT), 1-cyano-imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), or 2-cyanopyridazine-3(2H)one (2-CPO), or a functional derivative or modification thereof. The examples illustrate the use of these reagents with a variety of polysaccharides and proteins showing that the methods are generally applicable.
1. A process of conjugation of a carbohydrate comprising:
mixing 1-cyano-4-pyrrolidinopyridinium tetrafluorborate (CPPT) with the carbohydrate to create an activated chemical compound; and
mixing the activated compound with an antigenic second compound to create a conjugate, wherein the antigenic second compound is a peptide, a polypeptide or a protein.
2. The process of claim 1 , wherein the carbohydrate is a natural or synthetic carbohydrate, polysaccharide, oligosaccharide, or combination thereof.
3. The process of claim 1 , wherein conjugation is direct or indirect with the addition of functional groups that facilitate conjugation.
4. The process of claim 1 , further comprising removing components with a lower molecular weight than the conjugate by dialysis, filtration, chromatography, or a combination thereof.
5. The process of claim 1 , wherein the conjugate is a vaccine or a diagnostic agent.
6. The process of claim 1 where the steps are performed together.
7. The process of claim 1 , further comprising mixing the activated compound with hexanediamine, ethylenediamine, hydrazine, adipic dihydrazide, or 1,6-diaminooxyhexane to form a linking group between the activated compound and the antigenic second compound.
8. A vaccine or diagnostic agent produced by the process of claim 1 .