Highly pure polysialic acid and process for preparation thereof

1. A process for the production of polysialic acid having purity of greater than 99.0% and an average molecular weight of 70 KDa to 100 KDa, comprising the steps of:

(a) preparing a nutrient medium containing sorbitol in an amount of 8 g/L to 12 g/L, yeast extract in an amount of 0.4 g/L to 0.6 g/L, casamino acids in an amount of 8 g/L, potassium hydrogen phosphate in an amount of 8 g/L to 12 g/L, copper sulphate in an amount of 0.001 g/L to 0.003 g/L, and magnesium sulphate in an amount of 0.4 g/L to 0.6 g/L;

(b) inoculating a fermentor comprising the nutrient medium with a polysialic acid producing bacterium;

(c) incubating the inoculated medium at a temperature of 30 to 40° C., a pH of 6.5 to 7.5, a dissolved oxygen percentage of 35 to 45% and an agitation of 100 to about 150 rpm;

(d) harvesting the polysialic acid produced in step (c); and

(e) concentrating or purifying the polysialic acid obtained in step (d).

2. The process for the production of polysialic acid according to claim 1 , wherein said copper sulphate is in an amount of 0.002 g/L.

3. The process for the production of polysialic acid according to claim 1 , wherein said polysialic acid producing bacterium belongs to the Escherichia coli ( E. coli ) sero group 07:K1 and does not show O-acetylation of polysialic acid.

4. The process for the production of polysialic acid according to claim 1 , wherein said polysialic acid producing bacterium is E. coli LP 1674.

5. The process for the production of polysialic acid according to claim 1 , wherein said inoculation is by an inoculum having an optical density of 2 to 3.

6. The process for the production of polysialic acid according to claim 1 , wherein said incubation is performed at a temperature of 37° C., a pH of 7, a dissolved oxygen percentage of 40%, and an agitation of about 150 rpm for 4 to 6 hours.

7. The process for the production of polysialic acid according to claim 1 , wherein said harvesting is performed by cross flow microfiltration of the fermentation broth with 0.45 micron cassettes in a closed system or centrifugation of the fermentation broth at 4000 rpm.

8. The process for the production of polysialic acid according to claim 1 , wherein said concentration of polysialic acid is performed by ultrafiltration using 10 KDa centrifugal filters and subjecting the retentate to buffer exchange at pH 6.5 to 7.5 with optional ultrafiltration using a 0.2 micron filter.

9. The process for the production of polysialic acid according to claim 1 , wherein said purification of polysialic acid is performed by separating the impurities by anion exchange chromatography followed by washing the separated polysialic acid with 6M Urea at pH 4.5, and optional ultrafiltration using a 0.2 micron filter.

10. The process for the production of polysialic acid according to claim 1 , wherein said polysialic acid contains less than 1% total impurities, the impurities comprising proteins, nucleic acids and endotoxins.

11. The process for the production of polysialic acid according to claim 1 , wherein the endotoxin content of the polysialic acid is less than 10 EU/mg.

12. The process according to claim 1 , wherein the process is fed-batch.