Capsular polysaccharide solubilisation and combination vaccines
Precipitated bacterial capsular polysaccharides can be efficiently re-solubilised using alcohols as solvents. The invention provides a process for purifying a bacterial capsular polysaccharide, comprising the steps of (a) precipitation of said polysaccharide, followed by (b) solubilisation of the precipitated polysaccharide using ethanol. CTAB can be used for step (a). The material obtained, preferably following hydrolysis and sizing, can be conjugated to a carrier protein and formulated as a vaccine. Also, in vaccines comprising saccharides from both serogroups A and C, the invention provides that the ratio (w/w) of MenA saccharide:MenC saccharide is >1.
1 . A process for conjugating a bacterial capsular polysaccharide to a carrier protein, comprising:
purifying the polysaccharide, comprising the steps of (a) precipitation of the polysaccharide using one or more cationic detergents, followed by (b) solubilisation of the precipitated polysaccharide using an alcohol,
conjugation of the polysaccharide to a carrier protein, wherein the carrier protein is a bacterial toxin or toxoid, and
mixing with saccharide antigens from Neisseria meningitidis strains A, C, W135 and/or Y which are conjugated to a bacterial toxin or toxoid,
wherein the bacterial capsular polysaccharide is from Neisseria meningitidis serogroup A, W135 or Y, or from Haemophilus influenzae , or from Streptococcus pneumoniae.
2 . The process claim 1 , wherein the cationic detergent(s) comprise a cetyltrimethylammonium salt, a tetrabutylammonium salt, a myristyltrimethylammonium salt and/or hexadimethrine bromide.
3 . The process of claim 1 or claim 2 , wherein the alcohol used in step (b) comprises ethanol, and wherein the ethanol has a final concentration of between 50% and 95%.
4 . The process of claim 1 , wherein purification further comprises the step of (c) treating the polysaccharide obtained in step (b) to remove contaminants.
5 . The process of claim 4 , wherein step (c) comprises one or more steps of filtration.
6 . The process of claim 5 , wherein step (c) comprises depth filtration, filtration through activated carbon, size filtration and/or ultrafiltration.
7 . The process of claim 1 or claim 4 , wherein the polysaccharide obtained in step (b) or step (c) is then precipitated.
8 . The process of claim 7 , wherein precipitation is by addition of calcium or sodium salts.
9 . The process of claim 1 or claim 4 , wherein the saccharide is activated prior to conjugation.
10 . The process of claim 7 , wherein the saccharide is activated prior to conjugation.
11 . The process of claim 9 , wherein activation involves a cyanylating reagent.
12 . The process of claim 10 , wherein activation involves a cyanylating reagent.
13 . The process of claim 1 or claim 4 , wherein conjugation is with a linker.
14 . The process of claim 7 , wherein conjugation is with a linker.
15 . The process of claim 9 , wherein conjugation is with a linker.
16 . The process of claim 1 or claim 4 , wherein the conjugated saccharide has a saccharide:protein ratio (w/w) between 0.5:1 and 5:1.
17 . The process of claim 7 , wherein the conjugated saccharide has a saccharide:protein ratio (w/w) between 0.5:1 and 5:1.
18 . The process of claim 9 , wherein the conjugated saccharide has a saccharide:protein ratio (w/w) between 0.5:1 and 5:1.
19 . The process of claim 13 , wherein the conjugated saccharide has a saccharide:protein ratio (w/w) between 0.5:1 and 5:1.
20 . The process of claim 1 or claim 4 , wherein the carrier protein is diphtheria toxoid or tetanus toxoid.
21 . The process of claim 7 , wherein the carrier protein is diphtheria toxoid or tetanus toxoid.
22 . The process of claim 9 , wherein the carrier protein is diphtheria toxoid or tetanus toxoid.
23 . The process of claim 13 , wherein the carrier protein is diphtheria toxoid or tetanus toxoid.
24 . The process of claim 1 or claim 4 , wherein, after conjugation, free and conjugated saccharides are separated.
25 . The process of claim 7 , wherein, after conjugation, free and conjugated saccharides are separated.
26 . The process of claim 9 , wherein, after conjugation, free and conjugated saccharides are separated.
27 . The process of claim 13 , wherein, after conjugation, free and conjugated saccharides are separated.
28 . The process of claim 24 , wherein separation uses hydrophobic chromatography, tangential ultrafiltration, or diafiltration.
29 . The process of claim 1 or claim 4 , wherein mixing gives a composition comprising a mixture of saccharides from more than one serogroup of N. meningitidis , and wherein the composition comprises saccharides from serogroups A+C, A+W135, A+Y, C+W135, C+Y, W135+Y, A+C+W135, A+C+Y, C+W135+Y or A+C+W135+Y.
30 . The process of claim 16 , wherein the composition comprises capsular saccharides from both serogroups A and C and the ratio (w/w) of MenA saccharide:MenC saccharide is 2:1.
31 . The process of claim 16 , wherein the composition comprises capsular saccharides from serogroup Y and one or both of serogroups C and W135, and wherein the ratio (w/w) of MenY saccharide:MenW135 saccharide is greater than 1 and/or that the ratio (w/w) of MenY saccharide:MenC saccharide is less than 1.
32 . The process of claim 16 , wherein the composition comprises capsular saccharides from serogroups A, C, W135 and Y, and wherein serogroups A:C:W135:Y have ratios (w/w) of 1:1:1:1; 1:1:1:2; 2:1:1:1; 4:2:1:1; 8:4:2:1; 4:2:1:2; 8:4:1:2; 4:2:2:1; 2:2:1:1; 4:4:2:1; 2:2:1:2; 4:4:1:2; or 2:2:2:1.
33 . The process of claim 1 or claim 4 , further comprising step(s) of vaccine formulation comprising mixing the saccharide antigen(s) with an adjuvant which is an aluminium phosphate and/or an aluminium hydroxide.