Patent EA-004131-B1 (EA)
Title: Method for preservation of viruses and micoplasma
Inventor: WORRALL ERIC EDWARD
IPC: C12N1/04
Filed: 2000-05-03
Abstract: 1. A method of preserving a biologically-active material comprising a live virus or mycoplasma which method comprises the steps: (i) mixing an aqueous suspension of the biologically-active material with a sterile aqueous solution of trehalose to give a trehalose concentration in the mixture in the range of from 0.2 to 10% w/v; (ii) subjecting the mixture to primary drying, for 30 to 60 minutes, at a pressure of less than atmospheric and at a temperature initially no greater than 37 degree C, and which is controlled not to fall to 0 degree C or below and which finally is no greater than 40 degree C to form a glassy porous matrix comprising glassy trehalose having a residual moisture content of not greater than 10% and containing, within the matrix, desiccated biologically-active material; and (iii) subjecting the glassy porous matrix of step (ii) to secondary drying for 10 to 30 hours at a pressure not greater than 0.1 mbar and at a temperature which finally is in the range of from 40 to 45 degree C to form a trehalose matrix having a residual moisture content of not greater than 2% containing, within the matrix, desiccated biologically-active material. 2. A method according to claim 1, wherein secondary drying in step (iii) is carried out for 20 to 30 hours. 3. A method according to claim 2, wherein secondary drying is carried out for 15 to 17 hours at a temperature of about 37 degree 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 degree C. 4. A method according to any one of claims 1 to 3, wherein the primary drying in step (ii) is carried out at a pressure of not greater than 800 mbar. 5. A method according to any one of claims 1 to 4, wherein the residual moisture content of the glassy trehalose matrix at the end of the primary drying in step (ii) is about 10%. 6. A method according to any one of claims 1 to 5, wherein the residual moisture content at the end of the secondary drying in step (iii) is 1.0% or lower. 7. A method according to any one of claims 1 to 6, wherein the live virus is selected from Rinderpest virus, Peste des Petits Ruminants virus, Measles virus, Mumps virus, Rubella virus, Yellow Fever virus, Polio virus and Newcastle Disease virus. 8. A method according to claim 7, wherein the live virus is Rinderpest virus or Peste des Petits Ruminants virus. 9. A method according to any one of claims 1 to 8, wherein the mycoplasma is Contagious Bovine Pleuropneumonia mycoplasma. 10. A rehydratable composition for making a vaccine comprising trehalose in the form of a metastable glass matrix having a residual moisture content of not greater than 1% containing, within the matrix, desiccated biologically-active material selected from desiccated live virus and desiccated mycoplasma. 11. A composition according to claim 10, wherein the biologically-active material is a live virus selected from Rinderpest virus, Pest de Petits Ruminants virus, Measles virus, Mumps virus, Rubella virus, Yellow Fever virus, Polio virus and Newcastle Disease virus. 12. A composition according to claim 11, wherein the biologically-active material is a live virus selected from Rinderpest virus and Peste des Petits Ruminants virus. 13. A composition according to claim 12, wherein the biologically-active material is Contagious Bovine Pleuropneumonia mycoplasma. 14. A method of making a vaccine comprising rehydrating the composition claimed in any one of claims 11 to 13 in an appropriate aqueous medium.