Process for preparing polysaccharide-protein conjugate vaccines
Inventors
Jessouroun, Ellen • Da Silveira, Ivna Alana Freitas Brasileiro • Bastos, Renata Chagas • Frasch, Carl E. • Lee, Che-Hung Robert
Assignees
DEPARTMENT OF HEALTH AND HUMAN SERVICES GOVERNMENT OF United States, AS REPRESENTED BY SECRETARY • FIOCRUZ • US Department of Health and Human Services
Publication Number
US-9173931-B2
Publication Date
2015-11-03
Expiration Date
2024-08-06
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Abstract
Methods for the manufacture of polysaccharide-protein conjugate vaccines at high yield are provided. The methods involve reaction of a hydrazide group on one reactant with an aldehyde group on the other reactant. The reaction proceeds rapidly with a high conjugation efficiency. Simplified purification processes can be employed to separate the conjugate product from the unconjugated protein and polysaccharide and other small molecule by-products.
Core Innovation
The invention provides methods for manufacturing polysaccharide-protein conjugate vaccines at high yield by utilizing a reaction between hydrazide groups on one reactant and aldehyde groups on another. This hydrazide-based conjugation chemistry enables a rapid reaction with high conjugation efficiency, and facilitates simplified purification processes to separate the conjugate from unreacted protein, polysaccharide, and small molecule by-products.
The problem addressed by the invention arises from conventional conjugation methods that rely on the ε-amino groups of lysine residues on the protein to react with functional groups on activated polysaccharides. These methods typically have low efficiency, about 20%, and require long reaction times of two to three days. The limited availability of free amino groups on carrier proteins such as toxoids, combined with reduced solubility and precipitation issues of activated proteins, contribute to low yields and complicated purification steps.
The invention overcomes these limitations by introducing hydrazide groups onto the protein through carbodiimide reaction with hydrazine or dihydrazides, maintaining the activated protein in solution at alkaline pH, and reacting it with aldehyde-activated polysaccharides under controlled pH and buffer conditions. This approach achieves a conjugation efficiency up to about 60%, reduces reaction times to one or two days, and results in higher quality conjugates with fewer by-products. The enhanced yield and faster reaction facilitate large-scale commercial production of conjugate vaccines capable of stimulating effective immune responses.
Claims Coverage
The patent contains two independent claims directed to methods for preparing meningococcal-tetanus toxoid conjugate vaccines at commercial scale using hydrazide-aldehyde conjugation chemistry with defined process parameters.
Method for preparing meningococcal-tetanus toxoid conjugate vaccine in commercial volumes
A process comprising: reacting meningococcal polysaccharide with an oxidizing agent to generate aldehyde-activated polysaccharide; reacting tetanus toxoid protein with hydrazine dichloride at acidic pH to generate hydrazine-activated protein; purifying the hydrazine-activated protein by diafiltration at volumes ≥5 liters; reacting the aldehyde-activated polysaccharide with hydrazine-activated protein at pH 5-7 in the presence of sodium cyanoborohydride; neutralizing unreacted aldehyde groups with adipic acid dihydrazide; purifying the resulting conjugate by diafiltration at volumes ≥2 liters; concentrating purified conjugate by tangential flow ultrafiltration to yield a conjugate vaccine capable of stimulating immune response.
Specific buffer and pH control steps to optimize conjugation
Buffer exchanging aldehyde-activated polysaccharide with HEPES buffer at pH 7-8; buffer exchanging hydrazine-activated tetanus toxoid protein with Na2CO3 buffer and adjusting pH from 7 to 11 before diafiltration; reacting polysaccharide-to-protein ratios from 1:1.6 to 1:5; optionally adding saccharose as a stabilizer; and freeze drying the concentrated conjugate to obtain a dried vaccine product.
The claims comprehensively cover a scalable method for producing meningococcal-tetanus toxoid conjugate vaccines featuring hydrazine-based protein activation and aldehyde-activated polysaccharides, purification by diafiltration with controlled buffer and pH conditions, and steps ensuring high yield, product stability, and immunogenicity suitable for commercial vaccine manufacture.
Stated Advantages
Higher conjugation efficiency (up to about 60%) compared to conventional methods (around 20%) leads to improved yields.
Faster conjugation reaction completed within one or two days instead of several days.
Simplified purification processes due to reduced amounts of unconjugated protein and polysaccharide and fewer by-products.
Improved solubility and stability of activated proteins maintained at alkaline pH, reducing precipitation.
Capability for scaled-up commercial production yielding large volumes of conjugate vaccine.
Reduced product contamination risk by avoidance of sodium cyanoborohydride in some embodiments.
Production of conjugates that are immunogenic and suitable for inducing long-lasting protective immune responses.
Documented Applications
Preparation of polysaccharide-protein conjugate vaccines against Neisseria meningitidis serogroups A and C.
Vaccine formulations capable of conferring long-term protection against bacterial infections such as meningitis, influenza, tetanus, Haemophilus influenzae type b, Streptococcus pneumoniae, Salmonella typhi, and group B Streptococcus.
Conjugate vaccines for immunization protocols in infants, children, and adults to stimulate effective immune responses and immunological memory.
Potential use in vaccines directed against a variety of bacterial infections including Helicobacter pylori, Borelia burgdorferi, Mycobacteria, Staphylococcus aureus, Neisseria gonorrhoeae, Streptococcus species, Bacillus anthracis, and others.
Use in preparing vaccines for treatment or prevention of cancers such as sarcomas, carcinomas, leukemias, lymphomas, and other malignancies.
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