ADDITION OF NUCLEASES DIRECTLY TO CELL CULTURE TO FACILITATE DIGESTION AND CLEARANCE OF HOST CELL NUCLEIC ACIDS
The present invention provides an efficient process for culturing viruses in the presence of an endonuclease and for producing vaccines, typically from live attenuated viruses, under conditions to reduce the presence of host cell DNA and eliminate the need for a post-harvest DNA digestion step.
1 . A method for production of a virus, comprising the steps of:
a) infecting a culture of cells in a cell culture medium with a desired virus; and
b) incubating the cells in cell culture medium containing endonuclease for up to 8 days, wherein the endonuclease is added to the cell culture medium at any time from initial infection of the cells to prior to harvesting the cells and wherein the cells are adherent to a microcarrier or a static plastic surface.
2 . The method of claim 1 , wherein the endonuclease is added at the time of initial infection.
3 . The method of claim 1 , wherein the endonuclease is added from 0.5 to 8 days prior to harvesting the cells.
4 . The method of claim 1 , wherein the endonuclease may have DNA specificity, RNA specificity, or both.
5 . The method of claim 4 , wherein the endonuclease is a broad spectrum endonuclease derived from the bacterium Serratia marcescens.
6 . The method of claim 1 , wherein the cell culture medium comprises endonuclease at an initial concentration of 5 to 80 U per mL of medium.
7 . The method of claim 1 , wherein the culture of cells are mammalian cells.
8 . The method of claim 7 , wherein the mammalian cells are adherent to a microcarrier.
9 . The method of claim 8 , wherein the microcarrier is selected from microcarriers made of dextran, collagen, polystyrene, polyacrylamide, gelatine, glass, cellulose, polyethylene or plastic.
10 . The method of claim 1 , wherein the cell is selected from the group consisting of ARPE-19, Vero, BHK, CHO, RK, RK44, RK13, MRC-5, MDCK, CEF or diploid monolayer cells.
11 . The method of claim 1 , wherein the virus is selected from the group consisting of a primary viral isolate directly obtained from an infected individual, a genetically engineered attenuated virus, a genetically-engineered replication-deficient virus, a cell line passaged adapted virus, a cold-adapted virus, a temperature-sensitive mutant virus, and a genetically engineered re-assortant virus.
12 . The method of claim 1 , wherein the virus is a betaherpesvirus.
13 . The method of claim 12 , wherein the virus is a human cytomegalovirus.
14 . The method of claim 13 , wherein the virus is a genetically attenuated human cytomegalovirus
15 . the method of claim 1 further comprising:
a) harvesting infectious virus by collecting virus-containing supernatant obtained from centrifugation and/or filtration of the cell culture; and
b) preparing a vaccine by subjecting the virus-containing supernatant to at least one processing step selected from the group consisting of chromatography, filtration, centrifugation, precipitation, concentrating, freezing, freeze-drying, and stabilizing by addition of a stabilizing agent.
16 . A method for production of a human cytomegalovirus (HCMV), comprising the steps of:
a) infecting a culture of ARPE-19 cells in a cell culture medium with a HCMV; and
b) incubating the cells in cell culture medium containing endonuclease for 0.5 to 8 days, wherein the endonuclease is added to the cell culture medium at any time from initial infection of the cells to prior to harvesting the cells at an initial concentration of 5 to 80 U per mL of medium and wherein the cells are adherent to a microcarrier.
17 . The method of claim 16 , wherein the endonuclease is a broad spectrum endonuclease derived from the bacterium Serratia marcescens.
18 . The method of claim 16 , wherein the microcarrier is a dextran-based sphere with diethylaminoethyl functionality.
19 . The method of claim 16 , wherein the HCMV is a genetically attenuated HCMV.