Methods for preparing squalene

1. A method for the manufacture of an oil-in-water emulsion comprising:

i. obtaining squalene prepared from a fish oil source by a process comprising subjecting fish oil comprising squalene to: (a) a purification distillation carried out at near vacuum; and (b) a denaturing distillation carried out at a temperature greater than or equal to 210° C. at a pressure of 0.8 to 5.0 mmHg,

wherein the temperature in the denaturing distillation exceeds the temperature in the purification distillation; and

wherein the conditions for both the purification and the denaturing distillation are sufficient to cause squalene to boil; and

wherein 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 from step (i).

2. The method of claim 1 , wherein the purification distillation occurs at a temperature of less than 140° C.

3. The method of claim 1 , wherein the squalene prepared in step (i) is kept sterile following distillation treatment and prior to the preparation of the oil-in-water emulsion.

4. The method of claim 1 , wherein the fish oil comprises one or more contaminant proteins.

5. The method of claim 4 , wherein the contaminant protein comprises parvalbumin.

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 purification distillation is carried out at a pressure of from 0.5 μm Hg to 5 μm Hg.

8. The method of claim 1 , wherein the purification distillation is carried out prior to the denaturing distillation.

9. The method of claim 1 , which further comprises a step of subjecting the fish oil to saponification prior to conducting the purification distillation and denaturing distillation steps.

10. The method of claim 1 , which further comprises a step of subjecting the fish oil to saponification before conducting the denaturing distillation step.

11. The method of claim 9 , wherein saponification comprises the addition of NaOH or KOH to the fish oil.

12. The method of claim 10 , wherein saponification comprises the addition of NaOH or KOH to the fish oil.

13. The method of claim 1 , wherein the purification distillation results in a composition comprising 99% squalene or more.

14. The method of claim 1 , wherein the fish oil is shark liver oil.

15. The method of claim 1 , further comprising the step of combining the oil-in-water emulsion with an antigen.

16. The method of claim 1 , further comprising the step of packaging the oil-in-water emulsion into a kit as a kit component together with an antigen component.

17. The method of claim 16 , wherein the kit components are in separate vials.

18. The method of claim 17 , wherein the vials are made from borosilicate glass.

19. The method of claim 15 , wherein the antigen is an influenza virus antigen.

20. The method of claim 19 , wherein the combination of the oil-in-water emulsion 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.

21. The method of claim 19 , wherein the combination of the oil-in-water emulsion and the antigen forms a vaccine composition and wherein the vaccine composition includes a thiomersal or 2-phenoxyethanol preservative.

22. The method of claim 1 , wherein the oil-in-water emulsion comprises between 2-10% squalene.

23. The method of claim 1 , wherein the oil-in-water emulsion comprises squalene, polysorbate 80 and sorbitan trioleate.

24. A method for processing squalene from a fish source, said method comprising the steps of:

i providing a composition comprising distilled squalene which has been obtained by a process of distillation, and wherein the squalene is from a fish source; and

ii re-distilling the composition from (i) at a temperature sufficient to cause the distilled squalene to boil, so as to provide a composition comprising re-distilled squalene;

wherein the process of distillation is performed at a temperature less than 140° C. and at a near vacuum and the process of re-distillation is performed at a temperature greater than or equal to 210° C. and a pressure of 0.8 to 5.0 mmHg; and

wherein the conditions for both the distillation and the re-distillation are sufficient to cause the squalene to boil; and

wherein the denaturing distillation denatures and/or removes potential contaminant proteins and viruses, thereby rendering the squalene safer for human use as compared with non-denatured squalene.

25. A method for the manufacture of an oil-in-water emulsion comprising:

i. obtaining squalene prepared from a fish oil source by a process comprising subjecting fish oil comprising squalene to: (a) a purification distillation carried out at near vacuum at a temperature of less than 140° C.; and (b) a denaturing distillation carried out at a temperature greater than or equal to 210° C.,

wherein the conditions for both the purification and the denaturing distillation are sufficient to cause squalene to boil; and

wherein 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 from step (i).

26. The method of claim 25 , wherein the squalene prepared in step (i) is kept sterile following distillation treatment and prior to the preparation of the oil-in-water emulsion.

27. The method of claim 25 , wherein the fish oil comprises one or more contaminant proteins.

28. The method of claim 27 , wherein the contaminant protein comprises parvalbumin.

29. The method of claim 25 , wherein the purification distillation is carried out at a temperature of from 70 to 100° C.

30. The method of claim 25 , wherein the purification distillation is carried out at a pressure of from 0.5 μm Hg to 5 μm Hg.

31. The method of claim 25 , wherein the purification distillation is carried out prior to the denaturing distillation.

32. The method of claim 25 , which further comprises a step of subjecting the fish oil to saponification prior to conducting the purification distillation and denaturing distillation steps.

33. The method of claim 25 , which further comprises a step of subjecting the fish oil to saponification before conducting the denaturing distillation step.

34. The method of claim 32 , wherein saponification comprises the addition of NaOH or KOH to the fish oil.

35. The method of claim 33 , wherein saponification comprises the addition of NaOH or KOH to the fish oil.

36. The method of claim 25 , wherein the purification distillation results in a composition comprising 99% squalene or more.

37. The method of claim 25 , wherein the fish oil is shark liver oil.

38. The method of claim 25 , further comprising the step of combining the oil-in-water emulsion with an antigen.

39. The method of claim 25 , further comprising the step of packaging the oil-in-water emulsion into a kit as a kit component together with an antigen component.

40. The method of claim 39 , wherein the kit components are in separate vials.

41. The method of claim 40 , wherein the vials are made from borosilicate glass.

42. The method of claim 39 , wherein the antigen is an influenza virus antigen.

43. The method of claim 42 , wherein the combination of the oil-in-water emulsion 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.

44. The method of claim 42 , wherein the combination of the oil-in-water emulsion and the antigen forms a vaccine composition and wherein the vaccine composition includes a thimersal or 2-phenoxyethanol preservative.

45. The method of claim 25 , wherein the oil-in-water emulsion comprises between 2-10% squalene.

46. The method of claim 25 , wherein the oil-in-water emulsion comprises squalene, polysorbate 80 and sorbitan trioleate.

47. A method for the manufacture of an oil-in-water emulsion comprising:

i. obtaining squalene prepared from a fish oil source by a process comprising subjecting fish oil comprising squalene to: (a) a purification distillation carried out at near vacuum; and (b) a denaturing distillation carried out at a temperature greater than or equal to 210° C. at a pressure of 0.8 to 5.0 mmHg,

wherein the temperature in the denaturing distillation exceeds the temperature in the purification distillation; and

wherein the conditions for both the purification and the denaturing distillation are sufficient to cause squalene to boil; and

wherein 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 from step (i) by: (a) preparing a first emulsion having a first average droplet size; (b) microfluidizing the first emulsion to form a second emulsion having a second average droplet size which is less than the first average oil droplet size; and (c) filtering the second emulsion;

wherein the oil droplets in the oil-in-water emulsion have a diameter less than 5 μm.