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US 6,528,305 B2Granted
Method of producing infectious reovirus
AssigneeOncolytics Biotech Inc.
InventorsBradley G. Thompson, Matthew C. Coffey
Life of this patent
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Abstract
A simple and efficient method of producing mammalian reovirus is developed using HEK 293 cells. The method provides for fast production of reovirus in high yield. Furthermore, this method provides for a simpler purification procedure of the produced reovirus.
Claims21
A method of producing mammalian reovirus, comprising the steps of:
- (a) contacting human embryo kidney 293 (HEK 293) cells with a mammalian reovirus under conditions which result in reoviral infection of said HEK 293 cells;
- (b) incubating the culture of said infected cells for a period of time sufficient to allow for viral replication;
- and
- (c) harvesting the virus produced.
1 more element
A method of producing infectious reovirus comprising the steps of:
- (a) culturing human embryo kidney 293 (HEK 293) cells in a culture medium containing 293 Serum Free Medium supplemented with 4 mM L-glutamine at 36° C.±2° C., 6%±2% CO2 and 80%±5% relative humidity in spinner flasks at an impeller speed of 35-40 rpm until the cells reach a cell density of about 106 cells/ml;
- (b) infecting the cells with the Dearing strain reovirus at a multiplicity of infection of 0.5;
- (c) incubating the culture of said infected cells in the same conditions as in step (a) until the percentage of viable cells drops to 50-80%;
- (d) harvesting the virus produced by centrifugation of the culture, freezing-thawing to release the virus from the cells, and purifying the virus by a CsCl gradient;
- and
- (e) storing the virus at −60° C. or below.
3 more elements
Dependencies are inferred from the claim text.