Fermentation processes for cultivating Streptococci and purification processes for obtaining cps therefrom

Patent metadata

Field	Value
Publication number	US-9243271-B2
Application number	US-201313789501-A
Country	US
Kind code	B2
Filing date	Mar 7, 2013
Priority date	Oct 8, 2008
Publication date	Jan 26, 2016
Grant date	Jan 26, 2016

Abstract

This invention is in the field of bacterial cultures and specifically relates to the optimization of culture conditions to improve the production of bacterial capsular polysaccharides from Streptococcus strains in fed batch culture and to novel purification methods suitable for production scale purification of bacterial capsular polysaccharides from Streptococcus strains resulting in higher levels of purity than previously obtained for production scale.

First claim

The invention claimed is:

1. A fed batch method for cultivating Streptococcus for production of capsular polysaccharides (cps), wherein said fed batch method comprises (a) providing an inoculum of a strain of Streptococcus expressing the cps, and (b) cultivating the strain by fermentation, wherein said cultivating comprises a pH-independent linear addition of a carbon source to a cultivating medium.

2. The fed batch method of claim 1 further comprising step (c) recovering the capsular polysaccharide.

3. The fed batch method of claim 1 , wherein said strain of Streptococcus further comprises Streptococcus agalactiae.

4. The fed batch method of claim 3 , wherein said strain of Streptococcus agalactiae is O90, H36b, CBJ111, or M781.

5. The fed batch method of claim 1 , wherein an optical density (OD) of the inoculum is between about 0.6 and about 1.8.

6. The fed batch method of claim 1 , wherein a pH of the cultivating medium is between about 6.0 and about 7.5.

7. The fed batch method of claim 6 , wherein the pH is about 7.3.

8. The fed batch method of claim 1 , wherein a temperature of the cultivating medium is between about 34 and about 38° C.

9. The fed batch method of claim 8 , wherein the temperature is about 36° C.

10. The fed batch method of claim 1 , wherein said carbon source further comprises glucose.

11. The fed batch method of claim 1 , wherein said cultivating further comprises monitoring an OD of the cultivating medium such that when the OD reaches a designated level, said linear addition of a carbon source is initiated.

12. The fed batch method of claim 11 , wherein said designated level is between about 9.8 and about 10.2.

13. The fed batch method of claim 12 , wherein said designated level is about 10.

14. The fed batch method of claim 11 , wherein said cultivating further comprises monitoring an OD of the cultivating medium such that when the OD reaches a first and second instantaneous addition level, two instantaneous additions of yeast extract are initiated prior to said linear addition of a carbon source.

15. The fed batch method of claim 14 , wherein said first instantaneous addition level is between about 2.8 and about 3.2.

16. The fed batch method of claim 15 , wherein said first instantaneous addition level is about 3.0.

17. The fed batch method of claim 14 , wherein said second instantaneous addition level is between about 4.3 and about 4.7.

18. The fed batch method of claim 17 , wherein said second instantaneous addition level is about 4.5.

19. A fed batch method for cultivating Streptococcus for production of capsular polysaccharides (cps), wherein said fed batch method comprises:
(a) providing an inoculum of a strain of Streptococcus expressing the cps, and
(b) cultivating the strain by fermentation, wherein said cultivating comprises a pH-independent linear addition of a carbon source to a cultivating medium, wherein said cultivating medium is a defined medium comprising a phosphate source, a mineral source, a carbon source, a vitamin source, and an amino acid source to grow Streptococcus , wherein said vitamin source consists of six or fewer vitamins selected from the following list of seven vitamins: biotin, niacinamide, calcium pantothenate, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride and folic acid, wherein two of the vitamins are calcium pantothenate and niacinamide.

20. The fed batch method of claim 19 further comprising step (c) recovering the capsular polysaccharide.

21. The fed batch method of claim 19 , wherein said strain of Streptococcus further comprises Streptococcus agalactiae.

22. The fed batch method of claim 21 , wherein said strain of Streptococcus agalactiae is O90, H36b, CJB111, or M781.

23. The fed batch method of claim 19 , wherein an optical density (OD) of the inoculum is between about 0.6 and about 1.8.

24. The fed batch method of claim 19 , wherein a pH of the cultivating medium is between about 6.0 and about 7.5.

25. The fed batch method of claim 24 , wherein the pH is about 7.3.

26. The fed batch method of claim 19 , wherein a temperature of the cultivating medium is between about 34 and about 38° C.

27. The fed batch method of claim 26 , wherein the temperature is about 36° C.

28. The fed batch method of claim 19 , wherein said carbon source further comprises glucose.

29. The fed batch method of claim 19 , wherein said cultivating further comprises monitoring an OD of the cultivating medium such that when the OD reaches a designated level, said linear addition of a carbon source is initiated.

30. The fed batch method of claim 29 , wherein said cultivating further comprises monitoring an OD of the cultivating medium such that when the OD reaches a first and second instantaneous addition level, two instantaneous additions of yeast extract are initiated prior to said linear addition of a carbon source.

31. The fed batch method of claim 30 , wherein said first instantaneous addition level is between about 2.8 and about 3.2.

32. The fed batch method of claim 31 , wherein said first instantaneous addition level is about 3.0.

33. The fed batch method of claim 30 , wherein said second instantaneous addition level is between about 4.3 and about 4.7.

34. The fed batch method of claim 33 , wherein said second instantaneous addition level is about 4.5.

35. The fed batch method of claim 19 , wherein said designated level is between about 9.8 and about 10.2.

36. The fed batch method of claim 35 , wherein said designated level is about 10.

37. The fed batch method of claim 19 further comprising step (c) recovering the capsular polysaccharide.

38. A fed batch method for cultivating Streptococcus for production of capsular polysaccharides (cps), wherein said fed batch method comprises:
(a) providing an inoculum of a strain of Streptococcus expressing the cps, and
(b) cultivating the strain by fermentation, wherein said cultivating comprises a pH-independent linear addition of a carbon source to a cultivating medium, wherein said cultivating medium is a complex medium comprising a yeast extract, a phosphate source, a carbon source, a vitamin source, and optionally an amino acid source to grow Streptococcus , wherein said vitamin source consists of four or fewer vitamins selected from the following list of five vitamins: biotin, niacinamide, riboflavin, thiamine hydrochloride and pyridoxine hydrochloride, wherein one of the vitamins is biotin.

39. The fed batch method of claim 38 , wherein said strain of Streptococcus further comprises Streptococcus agalactiae.

40. The fed batch method of claim 39 , wherein said strain of Streptococcus agalactiae is O90, H36b, CJB111, or M781.

41. The fed batch method of claim 38 , wherein an optical density (OD) of the inoculum is between about 0.6 and about 1.8.

42. The fed batch method of claim 38 , wherein a pH of the cultivating medium is between about 6.0 and about 7.5.

43. The fed batch method of claim 42 , wherein the pH is about 7.3.

44. The fed batch method of claim 38 , wherein a temperature of the cult

Assignees

• Costantino Paolo
• Norelli Francesco
• Berti Francesco
• Olivieri Roberto
• Bazzocchi Giulia
• Cicala Concetta
• Fontani Silvia
• Novartis Ag

Inventors

• Costantino Paolo
• Norelli Francesco
• Berti Francesco
• Olivieri Roberto
• Bazzocchi Giulia
• Cicala Concetta
• Fontani Silvia

Classifications

• A61P31/04

  Antibacterial agents · CPC title

• C12N2500/32

  Amino acids · CPC title

• C12N2500/74

  from fungi, e.g. yeasts · CPC title

• C12N2500/34

  Sugars · CPC title

• C12N1/20 Primary

  Bacteria; Culture media therefor · CPC title