Hepatitis A virus culture process

A method for producing large quantities of hepatitis A virus (HAV) in an aggregated microcarrier-cell culture which comprises retaining an HAV-infected MRC-5 cell population in an aggregated and attached state, with no observable floating MRC-5 cells due to cell sloughing from the microcarriers, comprising:

• a) planting MRC-5 cells in a suitable culture medium at a planting concentration sufficient to achieve uniform attachment of the cells to glass coated microcarriers, such that essentially every microcarrier has at least one cell attached,
• b) growing the cells so attached to the microcarriers in a culturing vessel using a rate of agitation equal to the critical off bottom suspension rate,
• c) infecting the microcarrier aggregates in late log phase or early stationary phase with HAV, wherein said MRC-5 cells remain in an attached state, with no observable floating cells due to cell sloughing from the microcarriers,
• d) culturing the infected microcarrier-cell aggregates from about 14-28 days after infection, and
• e) harvesting and recovering the HAV so produced by forcing the aggregated microcarrier cell culture through a series of orifices of decreasing diameter such that the smallest orifice is approximately only twice the size of a single microcarrier.

A method for producing large quantities of hepatitis A virus (HAV) in an aggregated microcarrier-cell culture which comprises retaining an HAV-infected MRC-5 cell population in an aggregated and attached state, without sloughing of MRC-5 cells from the microcarriers, comprising:

• a) planting MRC-5 cells in a suitable culture medium at a planting concentration sufficient to achieve uniform attachment of the cells to glass coated microcarriers, such that essentially every microcarrier has at least one cell attached,
• b) growing the cells so attached to the microcarriers in a culturing vessel using a rate of agitation equal to the critical off bottom suspension rate,
• c) infecting the microcarrier aggregates in late log phase or early stationary phase with HAV, wherein said MRC-5 cells remain in an attached state, without sloughing from the microcarriers,
• d) culturing the infected microcarrier-cell aggregates, and
• e) harvesting and recovering the HAV so produced.

A method for producing large quantities of hepatitis A virus (HAV) in an aggregated microcarrier-cell culture which comprises retaining an HAV-infected MRC-5 cell population in an aggregated and attached state, without sloughing of MRC-5 cells from the microcarriers, comprising:

• a) planting MRC-5 cells in a suitable culture medium at a planting concentration sufficient to achieve uniform attachment of the cells to glass coated microcarriers, such that essentially every microcarrier has at least one cell attached,
• b) growing the cells so attached to the microcarriers in a culturing vessel using a rate of agitation equal to the critical off bottom suspension rate,
• c) infecting the microcarrier aggregates in late log phase or early stationary phase with HAV, wherein said MRC-5 cells remain in an attached state, without sloughing from the microcarriers,
• d) culturing the infected microcarrier-cell aggregates from about 14-28 days after infection, and
• e) harvesting and recovering the HAV so produced by forcing the aggregated microcarrier cell culture through a series of orifices of decreasing diameter such that the smallest orifice is approximately only twice the size of a single microcarrier.