Method for the preservation of biologically-active material

• US 6,841,168 B1
• Filed: 06/21/2000
• Issued: 01/11/2005
• Est. Priority Date: 06/22/1999
• Status: Expired due to Fees

• Current Assignee: Anhydro Limited
• Original Assignee: Anhydro Limited
• Inventors: Worrall, Eric Edward
• Primary Examiner(s): Housel, James
• Assistant Examiner(s): Lucas, Zachariah
• Application Number:
  US10/018,834
• Time in Patent Office: 1,665 Days
• Field of Search: 435/374, 435/200, 435/2, 435/236, 435/238, 435/243, 436/18, 514/55, 424/78.08, 424/189.1, 424/185.1, 424/192.1, 424/206.1, 424/290.1, 424/93.1, 424/94.1, 424/536, 424/484, 424/486, 424/489, 424/490, 424/496, 424/497, 424/85.2, 424/198.1, 424/278.1, 536/123.13
• US Class Current: 424/484
• CPC Class Codes: A61K 2039/552 Veterinary vaccine
• CPC Class Codes: A61K 2039/70 Multivalent vaccine
• CPC Class Codes: A61K 39/0241 Mollicutes, e.g. Mycoplasma, Erysipelothrix
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: A61K 39/155 Paramyxoviridae, e.g. parainfluenza virus
• CPC Class Codes: A61K 9/1623 Sugars or sugar alcohols, e.g. lactose; Derivatives thereof; Homeopathic globules
• CPC Class Codes: A61K 9/1652 Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin homeopathic globules A61K9/1623
• CPC Class Codes: C12N 1/04 Preserving or maintaining viable microorganisms immobilised microorganisms C12N11/00
• CPC Class Codes: C12N 2760/18434 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Biologically-active material can be preserved by a method of desiccation, without lyophilisation, in a matrix of glassy trehalose. The method involves forming a coacervate of the biologically-active material and chitosan and then dehydrating mixture of coacervate and trehalose solution. In a cycle time much shorter than a typical freeze drying process biologically-active material, such as viruses, proteins and nucleic acids, can be preserved to provide a material that can be rehydrated. The invention is especially useful for the production of vaccines from preserved material.

Claims

1. A method of preserving biologically-active material comprising mixing an aqueous suspension of the biologically-active material with a sterile aqueous solution of chitosan or a non-toxic salt thereof to form a coacervate of the biologically-active material and chitosan or non-toxic salt thereof, adding to the coacervate a sterile aqueous solution of trehalose, subjecting the sterile mixture of coacervate and trehalose to drying at a pressure less than atmospheric and at a temperature which is initially less than or equal to 37°

C. and thereafter may fall provided that the temperature does not fall to, or below, 0°

C. to form a glassy porous matrix comprising metastable glassy trehalose containing, within the matrix, desiccated biologically-active material and chitosan or non-toxic salt thereof.

2. A method according to claim 1, wherein the biologically-active material is selected from viruses, bacteria, tertiary structured biologically-active protein and nucleic acid.

3. A method according to claim 2, wherein the biologically-active material is at least one virus selected from Rinderpest Virus, Peste de Petit Ruminants Virus, Measles, Mumps, Rubella, Yellow Fever, Polio, and Newcastle Disease Virus.

4. A method according to claim 2, wherein the biologically-active material is Mycoplasma mycoides.

5. A method according to any one of claims 1 to 4, wherein the sterile aqueous solution of chitosan or non-toxic salt thereof has a chitosan concentration of 0.01% w/v.

6. A method according to claim 5, wherein the sterile aqueous chitosan solution and the aqueous suspension of biologically-active material are mixed at a volume ratio of 1:

1 at pH 7.4.

7. A method according to claim 1, wherein the coacervate of biologically-active material and chitosan is subjected to vortex mixing.

8. A method according to claim 1, wherein the coacervate of biologically-active material and chitosan is mixed with a sterile aqueous trehalose solution having a trehalose concentration in the range of from 0.20 to 20% w/v.

9. A method according to claim 8, wherein the sterile aqueous solution of trehalose has a trehalose concentration in the range of from 2.6 to 8% w/v.

10. A method according to claim 9, wherein the sterile aqueous solution of trehalose has a trehalose concentration of about 5% w/v.

11. A method according to claim 1, wherein the drying stage is carried out at a pressure of not greater than 800 mbar.

12. A method according to claim 1, wherein the resulting trehalose matrix containing desiccated biologically-active material and chitosan or non-toxic salt thereof. Is subjected to a secondary drying procedure for 10 to 30 hours at a pressure not greater than 0.1 mbar and at a temperature which is in the range of from 40 to 45°

C. to form a trehalose matrix having a residual moisture content of not greater than 2% containing, within the matrix, desiccated biologically-active material and chitosan or non-toxic salt thereof.

13. A method according to claim 12, wherein secondary drying is carried out for 20 to 30 hours.

14. A method according to claim 12, wherein secondary drying is carried out for 16 to 17 hours at a temperature of about 37°

C. and the temperature is, thereafter, raised gradually over the remaining secondary drying time to a final temperature in the range of from 40 to 45°

C.

15. A method according to claim 12, wherein the residual moisture content at the end of the secondary drying step is 1.0% or lower.

16. A method of making a vaccine comprising preserving a biologically-active material according to the method of claim 1 and rehydrating the glassy product obtained thereby in an appropriate aqueous medium.

17. A method according to claim 16, wherein the vaccine is for oral or intranasal use.

18. A method according to claim 16, wherein the vaccine is a Measles, Mumps, Rubella (MMR) vaccine.

19. A rehydratable composition comprising trehalose in the form of a metastable glass matrix containing, within the matrix, a coacervate of a desiccated biologically-active material and chitosan or a non-toxic salt thereof.

20. A rehydratable composition according to claim 19 which has a residual moisture content of not greater than 2%.

21. A rehydratable composition according to claim 20 which has a residual moisture content of not greater than 1%.

22. A rehydratable composition according to claim 19, useful on rehydration for making a vaccine.

23. A rehydratable composition according to claim 19, wherein the biologically-active material is selected from viruses, bacteria, tertiary structured biologically-active proteins and nucleic acids.

24. A rehydratable composition according to claim 23, wherein the biologically-active material is at least one virus selected from Rinderpest Virus, Peste de Petit Ruminants Virus, Measles, Mumps, Rubella, Yellow Fever, Polio, and Newcastle Disease Virus.

25. A rehydratable composition according to claim 23, wherein the biologically-active material is Mycoplasma mycoides.

26. A method of preserving biologically-active material comprising mixing an aqueous suspension of the biologically-active material with a sterile aqueous solution of chitosan or a non-toxic salt thereof to form a coacervate of the biologically-active material and chitosan or non-toxic salt thereof, adding to the coacervate a sterile aqueous solution of trehalose, subjecting the sterile mixture of coacervate and trehalose to drying for a period of 30 to 60 minutes at a pressure less than atmospheric and at a temperature, which is initially less than or equal to 37°

C. and thereafter may fall provided that the temperature does not fall to, or below 0°

C. and wherein the final temperature is less than or equal to 40°

C. to form a glassy porous matrix comprising metastable glassy trehalose having a residual moisture content of less than or equal to 10% containing, within the matrix, desiccated biologically-active material and chitosan or non-toxic salt thereof.

27. A method according to claim 26, wherein the biologically-active material is selected from viruses, bacteria, tertiary structured biologically-active protein, and nucleic acid.

28. A method according to claim 27, wherein the biologically-active material is at least one virus selected from Rinderpest Virus, Peste de Petit Ruminants Virus, Measles, Mumps, Rubella, Yellow Fever, Polo, and Newcastle Disease Virus.

29. A method according to claim 27, wherein the biologically-active material is Mycoplasma mycoides.

30. A method according to claim 26, wherein the sterile aqueous solution of chitosan or non-toxic salt thereof has a chitosan concentration of 0.01% w/v.

31. A method according to claim 30, wherein the sterile aqueous chitosan solution and the aqueous suspension of biologically-active material are mixed at a volume ratio of 1:

1 at pH 7.4.

32. A method according to claim 26, wherein the coacervate of biologically-active material and chitosan is subjected to vortex mixing.

33. A method according to claim 26, wherein the coacervate of biologically-active material and chitosan is mixed with a sterile aqueous trehalose solution having a trehalose concentration in the range of from 0.20 to 20% w/v.

34. A method according to claim 33, wherein the sterile aqueous solution of trehalose has a trehalose concentration in the range of from 2.5 to 8% w/v.

35. A method according to claim 26, wherein the drying stage is carried out at a pressure of not greater than 800 mbar.

45. A method of making a vaccine comprising preserving a biologically-active material according to the method of claim 26 and rehydrating the glassy product obtained thereby in an appropriate aqueous medium.

36. A method of preserving biologically-active material comprising mixing an aqueous suspension of the biologically-active material with a sterile aqueous solution of chitosan or a non-toxic salt thereof to form a coacervate of the biologically-active material and chitosan or non-toxic salt thereof, adding to the coacervate a sterile aqueous solution of trehalose, subjecting the sterile mixture of coacervate and trehalose to drying for a period of from 30 to 60 minutes at a pressure not greater than 800 mbar and at a temperature which is initially less than or equal to 37°

C. and thereafter may fall provided that the temperature does not fall to, or below, 0°

C. to form a glassy porous matrix comprising metastable glassy trehalose having a residual moisture content of less than or equal to 10% containing, within the matrix, desiccated biologically-active material and chitosan or non-toxic salt thereof, and then subjecting the resulting trehalose matrix containing desiccated biologically-active material and chitosan or non-toxic salt thereof to a secondary drying procedure for 10 to 30 hours at a pressure not greater than 0.1 mbar and at a temperature which is in the range of from 40 to 45°

C. to form a trehalose matrix having a residual moisture content of not greater than 2% containing, within the matrix, desiccated biologically-active material and chitosan or non-toxic salt thereof.

37. A method according to claim 36, wherein the biologically-active material is selected from viruses, bacteria, tertiary structured biologically-active protein, and nucleic acid.

38. A method according to claim 37, wherein the biologically-active material is at least one virus selected from Rinderpest Virus, Peste de Petit Ruminants Virus, Measles, Mumps, Rubella, Yellow Fever, Polio, and Newcastle Disease Virus.

39. A method according to claim 37, wherein the biologically-active material is Mycoplasma mycoides.

40. A method according to claim 36, wherein the coacervate of biologically-active material and chitosan is mixed with a sterile aqueous trehalose solution having a trehalose concentration in the range of from 0.20 to 20% w/v.

41. A method according to claim 40, wherein the sterile aqueous solution of trehalose has a trehalose concentration in the range of from 2.5 to 8% w/v.

42. A method according to claim 36, wherein secondary drying 19 carried out for 20 to 30 hours.

43. A method according to claim 36, wherein secondary drying is carried out for 15 to 17 hours at a temperature of about 37°

C. and the temperature is, thereafter, raised gradually over the remaining secondary drying time to a final temperature in the range of from 40 to 45°

C.

44. A method according to claim 36, wherein the residual moisture content at the end of the secondary drying step is 1.0% or lower.

46. A method of making a vaccine comprising preserving a biologically-active material according to the method of claim 36 and rehydrating the glassy product obtained thereby in an appropriate aqueous medium.

47. A method according to claim 46, wherein the vaccine is for oral or intranasal use.

48. A method according to claim 46, wherein the vaccine is a Measles, Mumps, Rubella (MMR) vaccine.