Methods for preparing squalene
An improved method for preparing squalene from a squalene-containing composition, said method comprising the steps of (a) a purification distillation carried out at a temperature T 1 (b) a denaturing distillation carried out at a temperature T 2 ; wherein steps (a) and (b) may be performed in either order; T 1 and T 2 are sufficient to cause squalene to boil; T 2 >T 1 ; and T 2 >200° C.
1. A method for the manufacture of an oil-in-water emulsion comprising:
(i) preparing purified squalene from a composition comprising squalene from a fish source by a process comprising steps of: (a) a purification distillation carried out at a temperature less than 140° C.; and (b) a denaturing distillation carried out at a temperature greater than or equal to 200° C.;
wherein the purification step and the denaturing distillation step may be performed in either order; the temperature of the purification step and the denaturing distillation step is sufficient to cause the squalene to boil; and the denaturing distillation denatures and/or removes potential contaminant proteins and viruses, thereby rendering the squalene safe for human use as compared with non-denatured squalene; and
(ii) preparing an oil-in-water emulsion using the squalene prepared in step (i).
2. The method of claim 1 , wherein the purified squalene prepared in step (i) is kept sterile following distillation treatment and prior to the preparation of the oil-in-water emulsion adjuvant in step (ii).
3. The method of claim 1 , wherein the composition used in step (i) comprises one or more proteins.
4. The method of claim 3 , wherein the composition comprises parvalbumin.
5. The method of claim 1 , wherein the denaturing distillation is carried out at a pressure of from 0.5 mm Hg to 5.0 mm Hg.
6. The method of claim 1 , wherein the purification distillation is carried out at a temperature of from 70 to 100° C.
7. The method of claim 1 , wherein the denaturing distillation is carried out at a temperature greater than or equal to 210° C.
8. The method of claim 1 , wherein the purification distillation is carried out at a pressure of from 0.5 μm Hg to 5 μm Hg.
9. The method of claim 1 , wherein the purification distillation is carried out prior to the denaturing distillation.
10. The method of claim 1 , wherein the composition used in step (i) is subjected to saponification.
11. The method of claim 10 wherein saponification comprises the addition of NaOH or KOH to the composition comprising squalene.
12. A method for the manufacture of an oil-in-water emulsion adjuvant comprising:
(i) preparing purified squalene from a composition comprising squalene from a fish source by a process comprising steps of: (a) a purification distillation carried out at a temperature less than 140° C.; and (b) a denaturing distillation carried out at a temperature greater than or equal to 200° C.;
wherein the purification step and the denaturing distillation step may be performed in either order; the temperature of the purification step and the denaturing distillation step is sufficient to cause the squalene to boil; and the denaturing distillation denatures and/or removes potential contaminant proteins and viruses, thereby rendering the squalene safe for human use as compared with non-denatured squalene; and
(ii) preparing an oil-in-water emulsion adjuvant using the squalene prepared in step (i).
13. The method of claim 12 , wherein the squalene prepared in step (i) is kept sterile following distillation treatment and prior to the preparation of the oil-in-water emulsion adjuvant in step (ii).
14. The method of claim 12 , wherein the composition used in step (i) comprises one or more proteins.
15. The method of claim 14 , wherein the composition comprises parvalbumin.
16. The method of claim 12 , wherein the denaturing distillation is carried out at a pressure of from 0.5 mm Hg to 5.0 mm Hg.
17. The method of claim 12 , wherein the purification distillation is carried out at a temperature of from 70 to 100° C.
18. The method of claim 12 , wherein the denaturing distillation is carried out at a temperature greater than or equal to 210° C.
19. The method of claim 12 , wherein the purification distillation is carried out at a pressure of from 0.5 μm Hg to 5 μm Hg.
20. The method of claim 12 , wherein the purification distillation is carried out prior to the denaturing distillation.
21. The method of claim 12 , wherein the composition used in step (i) is subjected to saponification.
22. The method of claim 21 , wherein saponification comprises the addition of NaOH or KOH to the composition comprising squalene.
23. The method of claim 12 , further comprising the step of combining the oil-in-water emulsion adjuvant with an antigen.
24. The method of claim 12 , further comprising the step of packaging the oil-in-water emulsion adjuvant into a kit as a kit component together with an antigen component.
25. The method of claim 24 , wherein the kit components are in separate vials.
26. The method of claim 25 , wherein the vials are made from borosilicate glass.
27. The method of claim 24 , wherein the antigen is an influenza virus antigen.
28. The method of claim 27 , wherein the combination of the oil-in-water emulsion adjuvant and the antigen forms a vaccine composition and wherein the vaccine composition includes about 15 μg, about 10 μg, about 7.5 μg, about 5 μg, about 3.8 μg, about 3.75 μg, about 1.9 μg, or about 1.5 μg of hemagglutinin per influenza virus strain.
29. The method of claim 28 , wherein the combination of the oil-in-water emulsion adjuvant and the antigen forms a vaccine composition and wherein the vaccine composition includes a thiomersal or 2-phenoxyethanol preservative.
30. The method of claim 12 , wherein the purified squalene comprises 99% squalene or more.
31. The method of claim 12 , wherein the oil-in-water emulsion adjuvant comprises between 2-10% squalene.
32. The method of claim 12 , wherein the oil-in-water emulsion adjuvant comprises about 5% squalene, about 0.5% polysorbate 80 and about 0.5% sorbitan trioleate by volume.
33. The method of claim 12 , wherein the fish oil is shark liver oil.