Methods for preparing complex multivalent immunogenic conjugates
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-8557250-B2
Publication Date
2013-10-15
Expiration Date
2027-03-16
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Abstract
Methods for preparing complex multivalent immunogenic conjugates that include simultaneously reacting a plurality or immunogenic-distinct polysaccharides with at least one protein to make the complex multivalent immunogenic conjugates. The simultaneous reaction involves reaction of a hydrazide group on one reactant with an aldehyde or cyanate ester group on the other reactant.
Core Innovation
The invention provides methods for preparing complex multivalent immunogenic conjugates by simultaneously reacting a plurality of immunogenic-distinct polysaccharides with at least one protein to form complex multivalent conjugates. This conjugation involves reaction of a hydrazide group on one reactant with an aldehyde or cyanate ester group on the other reactant, followed by reduction of the formed C═N double bonds to C—N single bonds, resulting in stable conjugates. The invention enables the concurrent attachment of multiple distinct polysaccharides to protein carriers in a single batch reaction rather than multiple separate processes.
The problem solved pertains to limitations of conventional polysaccharide-protein conjugate vaccine production methods, which have low conjugation efficiency, require lengthy reaction times and complicated purification, and result in substantial loss of polysaccharide material. Many native polysaccharides cannot be directly linked to proteins without chemical activation. Prior methods commonly use reductive amination conjugation involving ε-amino groups of proteins with aldehyde groups on activated polysaccharide, which suffer poor reaction yields and solubility issues, necessitating tedious purification to remove unreacted components.
The inventive methods exploit hydrazide chemistry to efficiently activate proteins and polysaccharides, facilitating their high-yield conjugation under mild conditions. Novel protein activation is achieved by reacting proteins with hydrazine or dihydrazides in the presence of carbodiimides and amino acids or peptides, resulting in soluble hydrazide-activated proteins. Polysaccharides are activated with aldehyde or cyanate groups, which then react rapidly and simultaneously with hydrazide-activated proteins at controlled pH, producing complex multivalent conjugates in a simplified process with minimal purification requirements.
Claims Coverage
The patent includes one independent claim describing a method for making complex multivalent immunogenic conjugates using simultaneous conjugation of activated polysaccharides and hydrazide-activated proteins.
Method for making complex multivalent immunogenic conjugate using cyanylation and hydrazide chemistry
The method comprises reacting multiple immunogenic-distinct polysaccharides with a cyanylation agent to produce cyanate-activated polysaccharides, reacting at least one protein with hydrazine or similar hydrazide compounds to produce hydrazide-activated protein solution, and contacting the cyanate-activated polysaccharides with the hydrazide-activated protein at pH about 6 to 8. This results in simultaneous conjugation forming complex multivalent immunogenic conjugates via C—N bonds between each polysaccharide and the protein.
The claim covers an efficient method for simultaneous conjugation of multiple distinct polysaccharides activated by cyanylation agents to hydrazide-activated proteins, forming complex multivalent immunogenic conjugates with improved selectivity, solubility, and yield.
Stated Advantages
High conjugation efficiency leading to greater yield compared to conventional methods.
Rapid reaction rates allowing conjugation reactions to complete within 1 to 3 days.
Reduced need for purification steps due to low residual unreacted protein and polysaccharide content after conjugation.
Improved solubility and stability of activated proteins and conjugates.
Capability to prepare complex multivalent vaccines in a single batch reaction, reducing production costs and complexity.
Avoidance of toxic reagents such as sodium cyanoborohydride, preventing cyanide contamination.
Enhanced immunogenicity of the resulting conjugates, demonstrated by increased antibody titers compared to native polysaccharides.
Documented Applications
Preparation of multivalent polysaccharide-protein conjugate vaccines for prevention and treatment of bacterial infections including meningococcal disease caused by Neisseria meningitidis serogroups A, C, W135, and Y.
Vaccines incorporating multiple distinct polysaccharide antigens such as pneumococcal serotypes (e.g., 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 23F), Haemophilus influenzae type b polysaccharide, and Salmonella typhi Vi polysaccharide.
Production of multivalent vaccines using single protein carriers such as tetanus toxoid, diphtheria toxoid, CRM197, or mixtures of proteins like bovine thyroglobulin, ovalbumin, and bovine serum albumin.
Vaccines providing long-term immunity in infants, children, and adults against bacterial infections like meningitis, pneumonia, tetanus, and others.
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