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US 9,957,485 B2Granted
Systems and methods for virus propagation in cell cultures for vaccine manufacture
AssigneeInventprise, LLC
InventorSubhash V. Kapre
Life of this patent
Estimating the term…
Abstract
The present invention provides a closed system to propagate virus-infected cells without the effect of shear force, while providing quicker access to nutrients than is available conventionally. This system design allows for a high density of infected cell growth to increase the virus yields and to maintain homogeneity of the contents of the main container. The system further provides a nuclease to degrade the cellular DNA prior for purification of the virus or viral components. As the system is designed for maximum containment at low risk, the live virus can be a hazardous virus such as a Bio-safety Level 3 (BSL 3), BSL 4 or BSL5 virus.
Claims35
A method of producing bulk quantities of inactivated virus comprising:
- providing a closed-loop cell culture system that includes:
- a plurality of incubation vessels stacked one above another, each of which contains a permeable mesh affinity matrix material for supporting a population of adherent cells, each incubation vessel having at least one mesh surfaces secured to either end of the incubation vessel;
- a media tank containing cell culture media connected to the incubation vessels, wherein the cell culture media flows from the media tank to the incubation vessels and back to the media tank, wherein the flow rate of the cell culture media through the system is from about 100 mls/lar to about 950 mls/hr, wherein the media tank has a volume not more than three times the volume of the incubation vessels;
- a mixing system within the media tank;
- and
- a valve downstream of the incubation vessels that, when activated, closes a connection to the media tank and opens a connection to a column, wherein the column possesses an input end connected to the incubation vessels and an output end connected to a collection vessel;
- contacting the population of adherent cells to the permeable mesh affinity matrix material contained within the one or more incubation vessels;
- flowing the cell culture medium within the closed-loop cell culture system for a first period of time, wherein flow of cell culture media through the system is at a rate such that less than 1% of the adherent cells become un-adhered during the first period of time;
- injecting a live virus into the media tank to inoculate the population of adherent cells with live virus creating virus infected cells;
- culturing the virus-infected cells for a second period of time to generate replicated virus;
- adding an agent to the virus-infected cells after the second period of time to promote isolation of replicated virus from the virus-infected cells;
- activating the valve such that replicated virus flows to the column under conditions that promote the selective retention of replicated virus;
- removing the media tank from the closed-loop cell culture system and directed the flow to a desalting column;
- eluting the replicated virus in the desalting column and collecting virus in the collection vessel;
- inactivating the eluted virus in a first jacketed tank;
- repeating the inactivation in a second jacketed tank;
- collecting the inactivated bulk virus.
14 more elements
A method of producing bulk quantities of inactivated virus comprising:
- providing a closed-loop cell culture system that includes:
- a plurality of incubation vessels stacked one above another each of which contains a population of cells, each incubation vessel having at least one mesh surfaces secured to either end of the incubation chamber;
- one or more media tanks containing cell culture media connected to the incubation vessels, wherein the cell culture media flows from the one or more media tanks to the incubation vessels and back to the one or more media tanks, wherein the media tank has a volume not more than three times the volume of the incubation vessels;
- and
- a valve downstream of the incubation vessels that, when activated, closes a connection to the one or more media tanks and opens a connection to a column containing a permeable mesh affinity matrix material, wherein the column possesses an input end connected to the one or more incubation vessels and an output end connected to a collection vessel;
- adding the population of cells to the one or more incubation vessels and infecting the cells with virus or adding virus-infected cells to the one or more incubation vessels;
- flowing the cell culture medium within the closed-loop cell culture system, wherein the flow rate of the cell culture media through the system is from about 100 mls/hr to about 950 mls/hr such that less than 1% of the adherent cells become un-adhered;
- culturing the virus-infected cells for a period of time to generate replicated virus;
- activating the valve one or more times during the period of time such that replicated virus flows to the column under conditions that promote the selective binding of replicated virus to the permeable mesh affinity matrix material;
- eluting the replicated virus from the affinity material and collecting virus in the collection vessel;
- and
- inactivating the eluted virus to render the virus non-infectious and suitable for manufacture of a vaccine.
9 more elements
A closed system for producing bulk quantities of inactivated virus, comprising:
- a plurality of incubation chambers stacked one above another and containing at least one permeable mesh matrix secured to either end of each incubation chamber for supporting a population of cells;
- a media tank for maintaining cell culture medium in fluid communication with the at least one incubation chamber, wherein the flow rate of the cell culture media through the system is from about 100 mls/hr to about 950 mls/hr such that less than 1% of the adherent cells become un-adhered, wherein the media tank has a volume not more than three times the volume of the incubation vessels;
- a mixing system within the media tank;
- a virus capture loop in fluid communication with the media tank having an intake valve and an outflow valve;
- an intake valve control system coupled to the intake valve;
- an outflow valve control system coupled to the outflow valve;
- a virus inactivation vessel in fluid communication with the virus capture loop;
- and
- automated control systems and software for monitoring and adjusting one or more components of the cell culture medium and fluid flow.
6 more elements
Dependencies are inferred from the claim text.