초록 ▼

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 t

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.

대표청구항 ▼

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,

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.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.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.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.