Covalently-modified bacterial polysaccharides, stable covalent conjugates of such polysaccharides and immunogenic proteins with bigeneric spacers and methods of preparing such polysaccharides and conjugataes and of confirming covalency
AssigneeMerck & Co., Inc.
InventorsStephen Marburg, Peter J. Kniskern, Richard L. Tolman
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Abstract
Covalently modified bacterial polysaccharides and proteins; covalent conjugates of such polysaccharides linked by a bigeneric spacer, which permits proof of covalency and facilitates purification of conjugated materials, with immunogenic bacterial membrane or other proteins, which conjugates are useful components of bacterial vaccines; and methods of preparing such polysaccharides, proteins and conjugates and of confirming the covalency of the linkage between polysaccharides and proteins.
Claims17
A process for solubilizing polyanionic polysaccharides having acid groups comprising
- (a) replacing the acid hydrogens of the polysaccharide with large hydrophobic cations selected from the group consisting of tri or tetra (C.sub.1 -C.sub.5) alkylammonium, 1-azabicyclo [2.2.2.]octane and 11,8-diazabicyclo undec-7-ene, thereby creating the salt form of the polysaccharide, then
- (b) dissolving the salt form of the polysaccharide in a non-aqueous, polar, aprotic solvent.
A process for covalently-modifying a polyanionic polysaccharide to form a modified polysaccharide capable of covalent bonding to a functionalized protein comprising
- (a) solubilizing the polysaccharide in a non-aqueous, polar, aprotic solvent;
- (b) activating the polysaccharide with a bifunctional reagent;
- then
- (c) reacting this activated polysaccharide with a bis-nucleophile.
A process for preparing polysacchar protein conjugates comprising bacterial capsular polysaccharides having acid groups and immunogenic proteins coupled through bigeneric spacers containing thioether bonds, comprising
- (a) solubilizing the bacterial capsular polysaccharide having acid groups by
- (i) replacing the acid hydrogens of the polysaccharide, then, with large hydrophobic cations selected from the group consisting of tri or tetra (C.sub.1 -C.sub.5) alkylammonium, 1-azabicyclo [2.2.2]octane and 1,8-diazabicyclo [5.4.0]undec-7-ene, thus creating the salt form of the polysacchardide, then
- (ii) dissolving the salt form of the polysaccharide in in a non-aqueous, polar, aprotic solvent;
- (b) activating the polysaccharide with a bifunctional reagent;
- (c) reacting this activated polysaccharide with a bis nucleophile;
- (d) reacting this activated polysaccharide which has been reacted with a bis-nucleophile, with a reagent generating electrophilic sites, thus forming a polysaccharide with pendant electrophilic sites;
- (e) independently reacting the immunogenic protein with a reagent generating thiol groups, to form a protein with pendant thiol groups;
- then
- (f) removing low molecular weight thiol groups from the protein which has pendant thiol groups by ultracentrifugation;
- then
- (g) reacting the polysaccharide with pendant electrophilic sites with the protein with pendant thiol groups to form a polysaccharide-protein conjugate which is coupled through a covalent thioether bond;
- then
- (h) centrifuging the resulting mixture to remove non-covalently-bonded polysaccharides and proteins.
Dependencies are inferred from the claim text.