Conjugating amines
Inventors
Ryan, Edward T. • Kovac, Pavol • Qadri, Firdausi • Xu, Peng • Calderwood, Stephen B. • Vann, Willie Frank • Peterson, Dwight Christopher
Assignees
INTERNATIONAL CENTRE FOR DIARRHOEAL DISEASE RESEARCH BANGLADESH • General Hospital Corp • US Department of Health and Human Services
Publication Number
US-9616139-B2
Publication Date
2017-04-11
Expiration Date
2032-07-11
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Abstract
The disclosure provides directly conjugated polysaccharide vaccine molecules and methods related thereto.
Core Innovation
The invention provides chemical methods for directly conjugating amine-containing polysaccharides to amine-containing carriers, such as proteins, using squaric acid chemistry. The disclosed methods enable conjugation of free amine-containing species directly to amine-containing carriers without prior chemical modification of either species or the addition of external chemical linkers. Specifically, the disclosure exemplifies conjugation of bacterial polysaccharides, such as O-PS-core polysaccharides from lipopolysaccharide (LPS), to proteins to create conjugate molecules useful as vaccines.
The disclosed conjugate molecules comprise polysaccharides having one or more accessible amine groups, linked directly to amine group containing proteins via a squaramide bond. Polysaccharide sources include various bacterial O-PS-core polysaccharides from species such as Vibrio cholerae, Escherichia coli, Salmonella sp., and others, while carrier proteins include known immunogenic proteins such as tetanus toxoid, CRM197, bovine serum albumin, and fragments thereof. The methods also include detailed steps for manufacturing such conjugates, including treatment of the polysaccharide with alkyl squarate in buffered conditions followed by reaction with protein under separate buffered conditions, allowing control of conjugation ratios and purification of products.
The problem being solved addresses limitations in conventional conjugation techniques for polysaccharide vaccines, which are typically slow, costly, inefficient, and require chemical modifications of either polysaccharides or protein carriers through exogenous linkers. These limitations reduce reproducibility and complicate manufacturing. The disclosed squaric acid chemistry methods overcome these problems by enabling direct conjugation without prior modifications, thereby reducing cost, complexity, and increasing reproducibility, which is particularly significant for improving immunogenicity of polysaccharide antigens such as the O-PS of Vibrio cholerae and enhancing vaccine efficacy.
Claims Coverage
The patent includes multiple independent claims covering conjugate molecules and methods of their manufacture through direct conjugation of polysaccharides to proteins using squaric acid chemistry.
Direct conjugation of polysaccharides to proteins without intervening linkers
A conjugate molecule comprising a polysaccharide with at least one accessible amine group conjugated directly to an amine-containing protein carrier via a squaramide bond, explicitly excluding intervening linkers between polysaccharide, squarate, and protein.
Use of bacterial O-PS-core polysaccharides in conjugates
The polysaccharide component is a bacterial polysaccharide or antigenic fragment comprising O-PS and O-PS-Core from various bacteria including Escherichia coli, Vibrio cholerae (Inaba and Ogawa serotypes), Salmonella, Shigella, and others.
Manufacturing method employing alkyl squarate chemistry
A method of manufacturing the conjugate molecule by treating a polysaccharide possessing accessible amine groups from LPS with an alkyl squarate in a first buffer to form a polysaccharide squarate monoester, followed by reaction with protein in a second buffer to create the conjugate molecule without intervening linkers.
Buffer conditions for conjugation reactions
The first buffer comprises about pH 7.0 (phosphate buffer) and the second buffer comprises about pH 9.0 (borate buffer) to facilitate the conjugation reactions.
Selection of alkyl squarate reagents
The alkyl squarate used in conjugation can be selected from dimethyl squarate, diethyl squarate, dipropyl squarate, dibutyl squarate, or didecyl squarate, with dimethyl squarate specifically claimed.
Controlled molar ratios for conjugation
Molar ratio of polysaccharide to alkyl squarate and molar ratio of polysaccharide squarate monoester to protein are each controlled between about 1:1 to 50:1 to optimize conjugation.
Conjugate molecule comprising tetanus toxin fragments as carrier
Conjugate molecules specifically comprising tetanus toxin C-fragment as the carrier protein conjugated directly to Vibrio cholerae Inaba or Ogawa O-PS-Core polysaccharides.
The claims cover the novel direct conjugation of polysaccharides bearing inherent amine groups to amine-containing proteins via squaric acid chemistry, methods to produce such conjugates under specified conditions, and conjugate molecules incorporating various bacterial polysaccharides and carriers including tetanus toxin fragments.
Stated Advantages
Reduced cost and complexity of polysaccharide-protein conjugation by eliminating the need for chemical modification or addition of linkers.
Increased reproducibility of conjugate vaccine manufacturing through direct conjugation of unmodified polysaccharides and carriers.
Improved immunogenicity of polysaccharide antigens, producing conjugate compositions that serve as effective immunogens.
Documented Applications
Production and manufacture of monovalent and multivalent conjugate vaccines against bacterial pathogens including Vibrio cholerae and others.
Manufacture of conjugate vaccines utilizing bacterial polysaccharides such as O-PS-core antigens coupled to immunogenic carrier proteins.
Inducing immune responses in subjects to protect or treat bacterial infections such as cholera, by administration of conjugate vaccines comprising polysaccharide-protein conjugates made by the described methods.
Use of conjugate vaccines containing Vibrio cholerae O1 O-PS-Core conjugated to tetanus toxin heavy chain fragment for prevention of cholera in subjects at risk or exposed to Vibrio cholerae.
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