Viral extraction from cell culture
AssigneeNational Research Council of Canada
InventorsHue Tran, Matthew Coffey, Allison Hagerman, Robert Voyer, Krishna Raj Tiwari, Jyoti Latawa, Mehul Patel, Allan Matte
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Abstract
Provided herein are methods of producing virus from a culture of host cells. The methods include providing a culture of host cells which has been infected by the virus; contacting the host cells with a first buffer comprising a detergent and incubating the host cells in the presence of the first buffer for a first period of time thereby producing a cell lysate; contacting the cell lysate with a second buffer comprising an endonuclease for a second period of time to degrade the host cell nucleic acids; and collecting the virus.
Claims28
A method of producing reovirus, comprising:
- (a) providing a culture of HEK 293 cells which has been infected by reovirus;
- (b) contacting the cells with a first buffer comprising polysorbate 20 and at least 20 mM phosphate and incubating the HEK 293 cells in the presence of the first buffer for a first period of time thereby producing a cell lysate, wherein the incubating occurs at a temperature of about 30° C. to about 40° C. and a pH of about 7.5 to about 8.0, with the proviso that polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether is absent from the first buffer;
- (c) contacting the cell lysate with a second buffer comprising an endonuclease for a second period of time to degrade nucleic acids of the HEK 293 cells;
- (d) removing cell debris by filtration;
- (e) concentrating the filtrate by ultrafiltration and diafiltration;
- (f) purifying the reovirus by a combination of ion exchange and size exclusion chromatography;
- and
- (g) collecting the reovirus.
Dependencies are inferred from the claim text.