Methods for purifying bacterial polysaccharides

1 . A method for purifying a bacterial polysaccharide from a solution comprising said polysaccharide together with contaminants, wherein said method comprises a flocculation step comprising the addition of a flocculating agent.

2 . The method of claim 1 wherein the flocculating agent comprises a multivalent cation selected from aluminium, iron, calcium and magnesium.

3 . The method of claim 1 wherein the flocculating agent comprises an agent selected from alum (e.g. potassium alum, sodium alum or ammonium alum), aluminium chlorohydrate, aluminium sulphate, calcium oxide, calcium hydroxide, iron(II) sulphate (ferrous sulphate), iron(III) chloride (ferric chloride), polyacrylamide, modified polyacrylamides, polyDADMAC, polyethylenimine (PEI), sodium aluminate and sodium silicate.

4 . The method of claim 1 wherein the flocculating agent is selected from alum (e.g. potassium alum, sodium alum or ammonium alum), aluminium chlorohydrate, aluminium sulphate, calcium oxide, calcium hydroxide, iron(II) sulphate (ferrous sulphate), iron(III) chloride (ferric chloride), polyacrylamide, modified polyacrylamides, polyDADMAC, sodium aluminate and sodium silicate.

5 . The method of claim 1 wherein the concentration of flocculating agent is between about 0.1 and about 20% (w/v).

6 . The method of claim 1 wherein the solution is hold for some time to allow settling of the flocs prior to downstream processing.

7 . The method of claim 6 wherein said flocculation step is performed at an acidic pH.

8 . The method of claim 6 wherein the settling step, if present, is performed at a temperature between about 4° C. and about 30° C.

9 . The method of claim 6 wherein the settling step, if present, is performed at a temperature of between about 30° C. to about 95° C.

10 . The method of claim 1 wherein, following flocculation the suspension is clarified by decantation, sedimentation, filtration or centrifugation.

11 . The method of claim 10 wherein, the polysaccharide containing solution is filtrated.

12 . The method of claim 11 wherein, said filtration is depth filtration.

13 . The method of claim 11 wherein the filtrate is subjected to microfiltration.

14 . The method of claim 11 wherein the filtrate is further treated by Ultrafiltration and Diafiltration.

15 . The method of claim 14 wherein said ultrafiltration step is performed at temperature between about 20° C. to about 90° C.

16 . The method of claim 14 wherein the diafiltration comprises a replacement solution comprising a chelating agent.

17 . The method of claim 14 wherein said diafiltration step is performed at temperature of between about 20° C. to about 90° C.

18 . The method of claim 10 wherein the solution containing the polysaccharide is treated by an activated carbon filtration step.

19 . The method of claim 18 , wherein the filtrate is subjected to microfiltration.

20 . The method of claim 18 , wherein the filtrate is further clarified by ultrafiltration and diafiltration.

21 . The method of claim 20 wherein the diafiltration comprises a replacement solution comprising a chelating agent.

22 . The method of claim 20 wherein said diafiltration step is performed at temperature of between about 20° C. to about 90° C.

23 . The method of claim 1 wherein said purified solution of polysaccharide is homogenized by sizing.

24 . The method of claim 1 wherein said purified solution of polysaccharide is sterilely filtered.

25 . The method of claim 1 wherein said bacterial polysaccharide is a capsular polysaccharide.

26 . A glycoconjugate of a purified bacterial polysaccharide obtained by the method of claim 1 .

27 . An immunogenic composition comprising a glycoconjugate of claim 26 .