GB2082202A - Method of increasing virus yield - Google Patents

Abstract

Viral yields are increased by removing the virus-infected cells from the growth surface and replanting them in the same vessel with fresh growth medium.

Description

SPECIFICATION Method of increasing virus yield Background of the Invention Conventional procedure in growing viruses is to incubate the cell sheet for a period of time necessary to produce a visible cytopathic effect.

Because the time required to produce a visible cytopathic effect varies from lot to lot, harvest time is unpredictable.

Objects of the Invention It is an object of the present invention to provide a method which results in predictable production cycle. Another object is to provide a method which results in a shorter production cycle. A further object is to provide higher yields.

Yet another object is to provide reduced requirements of labor, materials and seed. These and other objects of the present invention will be apparent from the following description.

Summary of the Invention Higher yields of virus are obtained in shorter time by treating an infected cell sheet to remove the cells from their growth surface and then replanting the cells in the same vessel with fresh growth medium.

Detailed Description The present invention relates to a method of growing viruses in vitro and, more particularly, to a method of increasing the yield of viruses grown in vitro.

According to the present invention the cells which are to be infected and the virus are added to a culture vessel such as, for example, roller bottles, a mass cell culture vessel such as disclosed for instance in U.S. patent 3,407,120, or a rotating disc propagator such as disclosed for instance in U.S. patents 3,839,1 55 and 3,905,865. The cells and virus are incubated in the presence of a suitable nutrient medium under appropriate temperature and aeration conditions and allowed to form a cell sheet. Alternatively, the virus may be added after the cells have formed a cell sheet. Typically the incubation temperature is about 370C and the oxygenating atmosphere contains about 5% CO2, although certain viruses may grow better somewhat above or below 370C.

After a period of at least about 1 day following incubation, typically for from about 1 to about 10 days, the nutrient medium is decanted and the cells removed from their growth surface. Removal of the cells may be effected by treating the cells with an enzyme such as, for example, trypsin, pronase or coilagenase or with a chelating agent such as, for example, ethylene diamine tetraacetic acid (EDTA) or by vigorous agitation, or by mechanical displacement. The enzyme is used in an aqueous solution which may contain from about 0.01 to about 2% (wt/vol), and may be added at about room temperature or at elevated temperature.Preferably the enzyme solution has a concentration of about 0.25% (wt/vol) and is added at a temperature of about 37"C. The cells are agitated for a period of from about several seconds to several minutes, typically for from about 5 seconds to about 2 minutes after the enzyme is added to loosen the cells. The enzyme solution is then discarded and fresh nutrient medium is added. The culture vessel then is shaken vigorously to displace the cells from the growth surfaces. The incubation is then resumed for a period of from about 8 to about 48 hours, typically for a period of from about 1 6 to about 32 hours, after which the cells are harvested, preferably by trypsinization.

In lieu of enzyme treatment the cells may be displaced by treatment with a chelating agent such as, for example, EDTA which is used in aqueous solution at a concentration of from about 100 to about 300 mg/liter. Like the enzyme, the chelating agent may be added at about room temperature or at elevated temperature, preferably at a temperature of about 370C. The EDTA, however, is allowed to contact the cells for longer periods than the enzyme, generally for a period of from about 5 minutes to about 30 minutes.

The cells may also be displaced without the addition of a chemical agent such as an enzyme or a chelating agent simply by vigorously shaking the culture vessel. In this case the shaking is continued until the cells are dislodged.

Alternatively, the cells may be removed mechanically, for example, by means of cell scraping devices disclosed in U.S. patents 4,004,981 and 4,065,359.

The following examples illustrate the present invention without, however, limiting the same thereto.

EXAMPLE 1 Twenty-four roller bottles are each planted Wil,-, 160 x 106 whole chick embryo cells and 0.2 m aj sonicated turkey herpes virus seed in medit;m 1 containing 5% fetal calf serum. The bottles are incubated at 370C in a 5% CO2 atmosphere on roller racks at 0.25 rpm. After 3 days the supernatant liquid from 12 of the bottles is decanted and 10 ml of a prewarmed (370C) 0.25% (wt/vol) trypsin preparation is added to each bottle. These bottles are rotated for 10 seconds and the trypsin then discarded and replaced with 100 ml of fresh medium 199 containing 5% fetal calf serum. The cells are shaken vigorously off the inner walls of the bottles. These bottles are then replaced on the roller racks and incubated at 37 C for an additional 24 hours. The remaining 12 bottles serve as controls.

On day 4 post inoculation the cells are harvested by trypsinization. The cell slurry is centrifuged at 1,800 x G for 10 minutes in a refrigerated centrifuge and resuspended at a concentration of 107 cells/ml in medium 1 99 containing 10% DMSO and 10% fetal calf serum.

Each yroup of 12 bottles is treated separately an.

cells are tested for titer by plaque formation on chick embryo monolayers. The results are as follows: PFU/Dose Run 1 Run 2 Control bottles 1,050 850 Treated bottles 3,250 2,750

Claims (10)

1. A method for increasing the yield of a virus grown in vitro which comprises removing nutrient medium from an infected cell sheet on a growth surface, treating the cell sheet to loosen the cells in the cell sheet from the growth surface, replacing the removed nutrient medium with fresh nutrient medium, shaking the cells from the growth surface, incubating for an additional period of time, and harvesting the virus.

2. A method according to claim 1 wherein the treating to loosen the cells is effected with an enzyme or a chelating agent for a period of from about 5 seconds to about 2 minutes.

3. A method according to claim 2 wherein the enzyme is trypsin, pronase, or collagenase.

4. A method according to claim 3 wherein the enzyme is employed in the form of an aqueous solution which contains from about 0.01 to about 2% (wt/vol) of enzyme.

5. A method according to claim 2 wherein the chelating agent is ethylene diamine tetraacetic acid.

6. A method according to claim 2 wherein the chelating agent is employed in the form of an aqueous solution which contains from about 100 to about 300 mg/liter of chelating agent.

7. A method according to claim 1 wherein the enzyme or chelating agent is prewarmed to a temperature of about 370C before contacting the cells.

8. A method according to claim 1 wherein the incubation for an period of time is from about 8 to about 48 hours.

9. A method according to claim 1 wherein the treating to loosen the cells is effected solely by agitation.

10. A method according to claim 2 wherein the treating to loosen the cells is effected solely by mechanical displacement.