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US 7,223,585 B2Granted
Viral purification methods
AssigneeOncolytics Biotech Inc.
InventorMatthew C. Coffey
Life of this patent
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Abstract
The present invention is directed to an improved method of purifying virus, particularly reovirus. Infectious virus can be extracted from a cell culture with a detergent to produce high titers of virus, and the virus can then be purified by simple steps such as filtration and column chromatography. Viruses and compositions comprising the viruses prepared according to the present invention are also provided.
Claims14
A method of producing virus from a culture of cells, comprising the steps of:
- (a) providing a culture of mammalian cells in suspension which has been infected by the virus in culture;
- (b) extracting the virus from the cells by adding a detergent to the culture of mammalian cells in suspension and incubating for a period of time to result in a cell lysate;
- (c) separating cell debris from the virus in the cell lysate by step-wise filtration comprising:
- (i) filtering through a prefilter having a pore size of 5 μM or 8 μM, and
- (ii) filtering after step (i) through a combination filter having pore sizes of 3 μM and 0.8 μM;
- (d) purifying the virus by a combination of ion exchange and size exclusion chromatography;
- and
- (e) collecting the virus,
- provided that after infection by the virus and before extraction of the virus, the mammalian cells are not pelleted or resuspended.
6 more elements
A method of producing infectious reovirus, comprising:
- (a) providing a culture of HEK 293 cells in suspension which has been infected by reovirus in culture;
- (b) extracting the reovirus from the HEK 293 cells by adding octoxynol-9 to 10 to the culture of HEK 293 cells in suspension and incubating at about 25° C. to about 37° C.;
- (c) treating the mixture from step (b) with a DNA-cleaving enzyme;
- (d) separating cell debris from the reovirus in the mixture from step (c) by step-wise filtration comprising:
- (i) filtering through a prefilter having a pore size of 5 μM or 8 μM, and
- (ii) filtering after step (i) through a combination filter having pore sizes of 3 μM and 0.8 μM;
- (e) concentrating the filtrate by ultrafiltration or diafiltration;
- (f) purifying the reovirus by a combination of ion exchange and size exclusion chromatography;
- and
- (g) collecting the reovirus,
- provided that after infection by the reovirus and before extraction of the reovirus, the HEK 293 cells are not pelleted or resuspended.
8 more elements
Dependencies are inferred from the claim text.