Methods for preparing complex multivalent immunogenic conjugates
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-9175033-B2
Publication Date
2015-11-03
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 conjugates. This simultaneous reaction involves the chemical reaction of a hydrazide group on one reactant with an aldehyde or cyanate ester group on the other reactant to create a complex multivalent conjugate product.
The problem being solved relates to the inefficiency and limitations of conventional methods for making polysaccharide-protein conjugate vaccines. Native bacterial polysaccharides are T-independent antigens that induce a short-lived immune response without memory, especially in infants. Converting these polysaccharides into T-dependent vaccines requires covalent attachment to carrier proteins, but prior conjugation reactions exhibit low efficiency (around 20%), long reaction times, and complications such as precipitation and the necessity for purification to remove unreacted components.
The disclosed methods propose hydrazide-based conjugation chemistry that achieves higher conjugation yields (up to about 60%), rapid reaction times, reduced by-products, and improved solubility of hydrazide-activated proteins. This allows for simultaneous conjugation of multiple immunogenic-distinct polysaccharides to one or more proteins in a single batch, simplifying production and lowering costs. The methods also include novel protein activation techniques that prevent aggregation by using amino acids during activation and maintaining the activated protein soluble at neutral or alkaline pH.
Claims Coverage
The patent includes several independent claims focusing on methods for making complex multivalent immunogenic conjugates, methods for preparing hydrazide-activated proteins, and the structures of the resulting conjugates.
Method for making complex multivalent conjugates via hydrazide chemistry and reductive amination
The method involves reacting a protein with 1-amino-2,3-propanediol (APDO) in the presence of a carbodiimide at pH about 5.5 to 7 to produce an APDO-modified protein, oxidizing this protein to create aldehyde-activated protein, and simultaneously reacting this with a mixture of hydrazide-activated immunogenic-distinct polysaccharides at pH about 5 to 8 to form a complex multivalent conjugate. The conjugate contains C═N double bonds that are subsequently reduced to C—N bonds.
Method for preparing hydrazide-activated proteins with improved solubility
The process includes reacting a protein with hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide, or mixtures thereof in the presence of (i) a carbodiimide catalyst and (ii) at least one amino acid selected from lysine, arginine, histidine, glycine, serine, threonine, glutamic acid, or cysteine, resulting in hydrazide-activated proteins soluble at neutral pH.
Complex multivalent immunogenic conjugate structures
The conjugates include at least one structure having multiple immunogenic-distinct polysaccharides conjugated to a single protein or protein construct with multiple proteins per construct, forming cross-linked lattice structures with multiple covalent bonds between proteins and polysaccharides.
Protein carrier selections for conjugation
The protein can include inactivated bacterial toxins such as diphtheria toxoid, CRM197, tetanus toxoid, pertussis toxoid, E. coli LT and ST, exotoxin A from Pseudomonas aeruginosa, or bacterial outer membrane proteins, as well as other proteins like ovalbumin, keyhole limpet hemocyanin, serum albumins, and purified protein derivatives of tuberculin.
The claims collectively cover improved methods for protein activation and conjugation to multiple polysaccharides simultaneously using hydrazide chemistry, resulting multivalent conjugates with enhanced immunogenic properties and improved manufacturing efficiency and product stability.
Stated Advantages
High efficiency conjugation with up to about 60% yield significantly surpassing conventional methods.
Rapid conjugation reaction times, reducing production from days to hours or 1-3 days.
Prevention of protein precipitation and aggregation by using amino acids during protein activation.
Elimination or reduction of purification steps due to lower levels of unreacted components.
Ability to simultaneously conjugate mixtures of multiple immunogenic-distinct polysaccharides in a single batch, simplifying manufacturing and lowering costs.
Conjugates produced induce strong and long-lasting immune responses including immune memory in infants, children, and adults.
Avoidance of toxic reagents such as sodium cyanoborohydride in the conjugation process, preventing cyanide contamination.
Documented Applications
Preparation of multivalent conjugate vaccines including combinations of pneumococcal, meningococcal, and Haemophilus influenzae type b polysaccharides conjugated to protein carriers such as tetanus toxoid.
Vaccines for preventing or treating bacterial infections such as bacterial meningitis, pneumonia, tetanus, and other bacterial diseases caused by various pathogens including Helicobacter pylori, Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, and Bacillus anthracis.
Cancer vaccines targeting various sarcomas, carcinomas, leukemias, lymphomas, and other tumors.
Formulation of vaccines in pharmaceutical compositions suitable for various administration routes including parenteral, intramuscular, intravenous, nasal, pulmonary, and topical delivery.
Combination vaccines containing multiple polysaccharide conjugates or mixtures of polysaccharides conjugated to single or multiple proteins.
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