# US 6,194,210 B1 · Hepatitis A virus culture process

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- Number: US 6,194,210 B1
- Document ID: US6194210B1
- Status: Granted
- Kind: B1
- Application number: 08/634,011
- Filed: 1996-04-17
- Granted: 2001-02-27
- Earliest claimed: 1994-03-08
- Assignees: Merck & Co., Inc.
- Inventors: Frank S. Leu; Douglas B. Seifert
- Web page: https://patentgrep.com/patent/US6194210B1

## CPC

- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- A61K 39/12: Viral antigens
- A61K 39/165: Mumps or measles virus
- A61K 39/25: Varicella-zoster virus
- A61K 39/29: Hepatitis virus
- C12N 5/0075: {using microcarriers}
- A61K 2039/55555: {Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers}
- C12N 2531/00: Microcarriers
- C12N 2533/12: Glass
- C12N 2710/16734: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/18734: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2770/32434: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2770/32451: Methods of production or purification of viral material
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

A microcarrier based process to produce viral vaccines, of which one example is hepatitis A virus (HAV), is composed of an aggregated microcarrier system of glass coated polystyrene microcarriers and MRC-5 cells which creates a stable environment for the propagation of the virus over even extended infection periods. The microcarrier aggregates formed according to this process eliminate the sloughing of cells from the beads during long cultivations seen in other systems, allowing high virus productivity in microcarrier culture. The methodology is applicable where virus production can be enhanced by creating a stable culture during an extended infection period. Scalable stirred bioreactors are used instead of multiple parallel stationary surface bioreactors. This aggregated microcarrier process eliminates the capacity limitations of stationary surface bioreactors, it protects the cells upon which virus is cultured from shear, it provides enhanced cell to cell interactions, it provides a stable environment for virus growth, it provides void space for the convective transport of nutrients through the aggregate, and it provides a straight forward method for harvesting the viral lysate for downstream processing.

## Claims

### Claim 1

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, and
e) harvesting and recovering the HAV so produced.

### Claim 2 (depends on 1)

The method of claim 1 wherein cells are planted at a density of about 5-10 cells per microcarrier.

### Claim 3 (depends on 2)

The method of claim 2 wherein the microcarriers are glass coated polystyrene beads.

### Claim 4

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.

### Claim 5 (depends on 4)

The method of claim 4 wherein cells are planted at a density of about 5-10 cells per microcarrier.

### Claim 6 (depends on 5)

The method of claim 5 wherein the microcarriers are glass coated polystyrene beads.

### Claim 7

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.

### Claim 8 (depends on 7)

The method of claim 7 wherein cells are planted at a density of about 5-10 cells per microcarrier.

### Claim 9 (depends on 8)

The method of claim 8 wherein the microcarriers are glass coated polystyrene beads.

### Claim 10

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.

### Claim 11 (depends on 10)

The method of claim 10 wherein cells are planted at a density of about 5-10 cells per microcarrier.

### Claim 12 (depends on 11)

The method of claim 11 wherein the microcarriers are glass coated polystyrene beads.