Method of producing an immunogenic composition
The present invention relates to a method for purifying bacterial cytolysins such as pneumococcal pneumolysin. A single chromatography step produces excellent purification of the cytolysin by binding soluble aggregated cytolysin to a hydrophobic interaction chromatography material in the presence of detergent and high salt.
1. A process for the production of an immunogenic formulation comprising purified bacterial cytolysin, the process comprising the steps of:
a) providing a solution containing unpurified soluble aggregated bacterial cytolysin;
b) adding said solution to a hydrophobic interaction chromatography column, where the column material contains aromatic groups;
c) binding soluble aggregated bacterial cytolysin contained in the solution to the column material in the presence of an ionic detergent and a salt, where the salt concentration is from 0.6M to 2M;
d) eluting bound bacterial cytolysin using a solution containing the detergent and no more than 0.2M salt to provide purified bacterial cytolysin; and
e) mixing said purified bacterial cytolysin with a pharmaceutically acceptable excipient.
2. The process of claim 1 further comprising mixing an adjuvant with said purified bacterial cytolysin.
3. The process of claim 1 further comprising conjugating said purified bacterial cytolysin to a bacterial capsular polysaccharide.
4. The process of claim 1 further comprising the steps of:
(i) removing the detergent from the purified bacterial cytolysin;
(ii) solubilizing the purified bacterial cytolysin by addition of a denaturant; and
(iii) removing the denaturant from the purified bacterial cytolysin.
5. The process of claim 1 wherein the bacterial cytolysin is pneumococcal pneumolysin.
6. The process of claim 1 wherein the hydrophobic interaction chromatography material is phenyl-sepharose.
7. The process of claim 1 wherein the detergent is an aliphatic detergent.
8. The process of claim 1 wherein the detergent is sodium lauroyl sarcosinate.
9. The process of claim 1 wherein the salt used in step c) and d) is selected from the group consisting of sodium chloride, magnesium chloride, ammonium chloride, sodium sulphate, magnesium sulphate, ammonium sulphate, sodium phosphate, magnesium phosphate, and ammonium phosphate.
10. The process of claim 1 wherein step c) is carried out at a pH of between pH 6-8.
11. The process of claim 4 wherein said denaturant is guanidine hydrochloride.
12. The process of claim 1 comprising a further step of detoxifying the purified bacterial cytolysin by chemical treatment with a crosslinking agent.
13. The process of claim 12 wherein the crosslinking agent is reactive with amine and sulphydryl groups.
14. The process of claim 12 wherein the crosslinking agent is selected from the group consisting of: formaldehyde, glutaraldehyde, N-hydroxysuccinomido esters and GMBS.
15. The process of claim 1 wherein step (d) is carried out at a pH of between pH 6-8.
16. The process of claim 1 , where said salt concentration in step (c) is about 1M.
17. The process of claim 1 further comprising, after step (c) and prior to step (d), washing the column with a solution of salt of about 0.5M.
18. The process of claim 1 wherein step (d) is carried out with solutions of decreasing salt gradient, with no more than 0.2M salt.
19. The process of claim 1 , where the detergent is present at a concentration of 0.1% to 5% weight/volume.
20. The process of claim 1 , where the detergent is present at a concentration of 0.75% to 2% weight/volume.
21. The process of claim 1 , where the detergent is present at a concentration of about 1% weight/volume.
22. The process of claim 1 , where the detergent is a cationic detergent.
23. The process of claim 1 , where the detergent is an anionic detergent.
24. The process of claim 1 , where the detergent has no more than one aromatic ring.
25. The process of claim 1 , where the detergent has no aromatic rings.
26. The process of claim 1 where said solution containing unpurified soluble aggregated bacterial cytolysin is provided by a process comprising:
(a) disrupting cells expressing bacterial cytolysin to provide a solution containing bacterial cytolysin;
(b) adding a detergent to said solution; and
(c) centrifuging said solution to remove insoluble cellular debris.
27. The process of claim 26 where said centrifugation is at 30,000 rpm for 20 minutes.
28. The process of claim 1 where said solution containing unpurified soluble aggregated bacterial cytolysin is provided by a process comprising:
(a) disrupting cells expressing bacterial cytolysin to provide a solution containing bacterial cytolysin;
(b) adding a detergent to said solution, where said detergent is the same detergent as in steps (c) and (d) of claim 1 ; and
(c) centrifuging said solution to remove insoluble cellular debris.
29. The process of claim 26 where said detergent is 1% sodium lauroyl sarcosinate.
30. The process of claim 26 further comprising, prior to the addition of detergent in step (b), adjusting the pH of said solution containing unpurified soluble aggregated bacterial cytolysin to a pH of less than 7.5 for a period of at least eight hours to form aggregates of bacterial cytolysin.
31. The process of claim 1 wherein the hydrophobic interaction chromatography material contains phenyl groups.
32. A process for the production of an immunogenic formulation comprising purified Streptococcus pneumoniae pneumolysin, the process comprising the steps of:
a) providing a solution containing unpurified soluble aggregated S. pneumoniae pneumolysin;
b) adding said solution to a hydrophobic interaction chromatography column, where the column material contains aromatic groups;
c) binding soluble aggregated Streptococcus pneumoniae pneumolysin contained in the solution to the column material in the presence of sodium lauroyl sarcosinate at a concentration of about 1% weight/volume and a salt at a concentration of from 0.6M to 2M;
d) eluting bound Streptococcus pneumoniae pneumolysin using a solution containing sodium lauroyl sarcosinate at a concentration of about 1% weight/volume and no more than 0.2M salt to provide purified S. pneumoniae pneumolysin; and
e) mixing said purified S. pneumoniae pneumolysin with a pharmaceutically acceptable excipient.
33. The process of claim 32 further comprising mixing an adjuvant with said purified S. pneumoniae pneumolysin.
34. The process of claim 32 further comprising conjugating said purified S. pneumoniae pneumolysin to a bacterial capsular polysaccharide.
35. The process of claim 32 further comprising the steps of:
(i) removing the detergent from the purified S. pneumoniae pneumolysin;
(ii) solubilizing the purified S. pneumoniae pneumolysin by addition of a denaturant; and
(iii) removing the denaturant from the purified S. pneumoniae pneumolysin.
36. The process of claim 35 where said denaturant is guanidine hydrochloride.
37. The process of claim 32 wherein the hydrophobic interaction chromatography material is phenyl-sepharose.
38. The process of claim 32 wherein the salt used in step (c) and (d) is selected from the group consisting of sodium chloride, magnesium chloride, ammonium chloride, sodium sulphate, magnesium sulphate, ammonium sulphate, sodium phosphate, magnesium phosphate, and ammonium phosphate.
39. The process of claim 32 wherein steps (c) and (d) are carried out at a pH of between pH 6-8.
40. The process of claim 32 , where said salt concentration in step (c) is about 1M.
41. The process of claim 32 further comprising, after step (c) and prior to step (d), washing the column with a solution of salt of about 0.5M.
42. The process of claim 32 wherein step (d) is carried out with solutions of decreasing salt gradient, with no more than 0.2M salt.
43. The process of claim 32 wherein said Streptococcus pneumoniae pneumolysin is a point mutated S. pneumoniae pneumolysin.
44. A process for the production of an immunogenic formulation comprising purified Streptococcus pneumoniae pneumolysin, the process comprising the steps of:
a) providing a solution containing unpurified soluble aggregated S. pneumoniae pneumolysin;
b) adding said solution to a phenyl-sepharose hydrophobic interaction chromatography column;
c) binding soluble aggregated S. pneumoniae pneumolysin contained in the extract to the column material in the presence of sodium lauroyl sarcosinate at a concentration of about 1% weight/volume and a salt at a concentration from 0.6M to 2M;
d) eluting bound S. pneumoniae pneumolysin using a solution containing sodium lauroyl sarcosinate at a concentration of about 1% weight/volume and no more than 0.2 M salt to provide purified S. pneumoniae pneumolysin; and
e) mixing said purified S. pneumoniae pneumolysin with a pharmaceutically acceptable excipient.
45. The process of claim 44 further comprising mixing an adjuvant with said purified S. pneumoniae pneumolysin.
46. The process of claim 44 further comprising conjugating said purified S. pneumoniae pneumolysin to a bacterial capsular polysaccharide.
47. The process of claim 44 wherein the salt used in step (c) and (d) is selected from the group consisting of sodium chloride, magnesium chloride, ammonium chloride, sodium sulphate, magnesium sulphate, ammonium sulphate, sodium phosphate, magnesium phosphate, and ammonium phosphate.
48. The process of claim 44 wherein steps (c) and (d) are carried out at a pH of between pH 6-8.
49. The process of claim 44 , where said salt concentration in step (c) is about 1M.
50. The process of claim 44 further comprising, after step (c) and prior to step (d), washing the column with a solution of salt of about 0.5M.
51. The process of claim 44 wherein step (d) is carried out with solutions of decreasing salt gradient, with no more than 0.2M salt.
52. The process of claim 44 comprising a further step of detoxifying the purified S. pneumoniae pneumolysin by chemical treatment with formaldehyde.
53. The process of claim 44 wherein said Streptococcus pneumoniae pneumolysin is a point mutated S. pneumoniae pneumolysin.
54. The process of claim 44 further comprising the steps of:
(i) removing the detergent from the purified S. pneumoniae pneumolysin;
(ii) solubilizing the purified S. pneumoniae pneumolysin by addition of a denaturant; and
(iii) removing the denaturant from the purified S. pneumoniae pneumolysin.
55. The process of claim 54 where said denaturant is guanidine hydrochloride.
56. A process for the production of an immunogenic formulation comprising purified Streptococcus pneumoniae pneumolysin, the process comprising the steps of:
a) providing a solution containing unpurified soluble aggregated S. pneumoniae pneumolysin;
b) adding said solution to a phenyl-sepharose hydrophobic interaction chromatography column;
c) binding soluble aggregated S. pneumoniae pneumolysin contained in the extract to the column material in the presence of sodium lauroyl sarcosinate at a concentration of about 1% weight/volume and a salt at a concentration of about 1M, at a pH of between pH 6-8;
d) eluting bound S. pneumoniae pneumolysin using a solution containing sodium lauroyl sarcosinate at a concentration of about 1% weight/volume and no more than 0.2 M salt, at a pH of between pH 6-8, to provide purified S. pneumoniae pneumolysin; and
(e) mixing said S. pneumoniae purified pneumolysin with a pharmaceutically acceptable excipient.
57. The process of claim 56 further comprising mixing an adjuvant with said purified S. pneumoniae pneumolysin.
58. The process of claim 56 further comprising conjugating said purified S. pneumoniae pneumolysin to a bacterial capsular polysaccharide.
59. The process of claim 56 wherein the salt used in step (c) and (d) is selected from the group consisting of sodium chloride, magnesium chloride, ammonium chloride, sodium sulphate, magnesium sulphate, ammonium sulphate, sodium phosphate, magnesium phosphate, and ammonium phosphate.
60. The process of claim 56 further comprising, after step (c) and prior to step (d), washing the column with a solution of salt of about 0.5M.
61. The process of claim 56 wherein step (d) is carried out with solutions of decreasing salt gradient, with no more than 0.2M salt.
62. The process of claim 56 comprising a further step of detoxifying the purified S. pneumoniae pneumolysin by chemical treatment with formaldehyde.
63. The process of claim 56 wherein said Streptococcus pneumoniae pneumolysin is a point mutated S. pneumoniae pneumolysin.
64. The process of claim 56 further comprising the steps of:
(i) removing the detergent from the purified S. pneumoniae pneumolysin;
(ii) solubilizing the purified S. pneumoniae pneumolysin by addition of a denaturant; and
(iii) removing the denaturant from the purified S. pneumoniae pneumolysin.
65. The process of claim 64 where said denaturant is guanidine hydrochloride.