# US 9,040,296 B2 · Production of viral vaccines in suspension on avian embryonic derived stem cell lines

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- Number: US 9,040,296 B2
- Document ID: US9040296B2
- Status: Granted
- Kind: B2
- Application number: 13/423,195
- Filed: 2012-03-17
- Published: 2012-09-20
- Granted: 2015-05-26
- Earliest claimed: 2005-04-11
- Assignees: Valneva SE
- Applicants: Majid Mehtali; Patrick Champion-Arnaud; Arnaud Leon
- Inventors: Majid Mehtali; Patrick Champion-Arnaud; Arnaud Leon
- Web page: https://patentgrep.com/patent/US9040296B2

## 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 39/145: Orthomyxoviridae, e.g. influenza virus
- A61P 31/12: Antivirals
- A61P 31/16: for influenza or rhinoviruses
- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- A61K 2039/525: {Virus}
- A61K 2039/5252: {inactivated (killed)}
- C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components
- C12N 2500/99: Serum-free medium
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/125: Stem cell factor [SCF], c-kit ligand [KL]
- C12N 2501/13: Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
- C12N 2501/23: Interleukins [IL]
- C12N 2710/24121: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2710/24134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2710/24151: Methods of production or purification of viral material
- C12N 2760/16021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2760/16034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/16051: Methods of production or purification of viral material
- C12N 2760/16134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/16151: Methods of production or purification of viral material
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The present invention relates to the development and manufacturing of viral vaccines. In particular, the invention relates to the field of industrial production of viral vectors and vaccines, more in particular to the use of avian embryonic stem cells, preferably the EBx® cell line derived from chicken embryonic stem cells, for the production of viral vectors and viruses. The invention is particularly useful for the industrial production of viral vaccines to prevent viral infection of humans and animals.

## Claims

### Claim 1

A process of production of influenza virus which comprises replicating an influenza virus in cells of an avian cell line wherein said cell line has been obtained by a method comprising the following steps:
A) culturing avian embryonic stem cells in a cell culture medium containing the factors allowing their growth, wherein said factors comprise:
a. a trophic factor selected from the group consisting of stem cell factor (“SCF”), Insulin Growth Factor (“IGF-1”), and basic Fibroblast Growth Factor (“bFGF”), and
b. a cytokine whose action is through a receptor which is associated with the gp130 protein, selected from the group consisting of leukemia inhibitory factor (“LIF”), interleukin 11 (“IL-11”), interleukin 6 (“IL-6”), interleukin 6 receptor (“IL-6R”), ciliary neurotrophic factor (“CNTF”), oncostatin, and cardiotrophin
and supplemented with animal serum in the presence of a feeder layer;
B) passaging by modifying the culture medium so as to obtain the withdrawal of said exogenous growth factors and cytokines, of the serum, and of the feeder layer;
C) establishing adherent or non-adherent cell lines capable of proliferating in the absence of said exogenous growth factors, serum, and inactivated feeder layer from said cell culture medium;
said process of production of influenza virus comprising the steps of:
a) proliferating said cells in a cultivation vessel, in suspension, in a serum-free medium number 1;
b) infecting the cells with said influenza virus when the cell density is of at least 1.5 million cells/ml;
c) shortly before infection, simultaneously to infection, or shortly after infection adding to the cell culture serum-free medium number 2; and
d) further culturing said infected cells in order to allow virus replication; and
e) optionally, harvesting said virus,
wherein said process comprises an additional step of feeding the cells wherein said feeding step consists in a periodic addition of concentrated solutions of glutamine and D-glucose to the culture medium; and wherein glutamine concentration in the culture medium is maintained between 1 mM to 3 mM and D-glucose concentration in the culture medium is maintained between 1 g/l to 10 g/l.

### Claim 2 (depends on 1)

The process of claim 1, wherein the cell density in step b) is of at least 4 million cells/ml in a fed-batch process.

### Claim 3 (depends on 1)

The process of claim 1, wherein the cultivation vessel is a continuous stirred tank bioreactor.

### Claim 4 (depends on 1)

The process of claim 1, wherein the serum-free medium number 1 and the serum-free medium number 2 have a different composition.

### Claim 5 (depends on 1)

The process of claim 1, wherein the feeding occurs during steps b) to d).

### Claim 6 (depends on 1)

The process of claim 1, wherein the feeding occurs during steps a) to d).

### Claim 7 (depends on 1)

The process of claim 1, wherein the feeding occurs on a daily basis.

### Claim 8 (depends on 1)

The process of claim 1, wherein said process further comprises the step of adding a proteolytic enzyme in the culture medium in conditions allowing virus propagation.

### Claim 9 (depends on 1)

The process according to claim 1, wherein cell culture is performed at a temperature of 33° C.

### Claim 10 (depends on 1)

The process according to claim 1, wherein said influenza virus is a re-assorted virus.

### Claim 11 (depends on 1)

The process according to claim 1, wherein the glutamine concentration is maintained around 2 mM.

### Claim 12 (depends on 1)

The process according to claim 1, wherein the D-glucose concentration is maintained around 2 to 3 g/l.

### Claim 13 (depends on 1)

The process according to claim 1, wherein glutamine (2 mM) and D-glucose (2 g/l) concentrations are daily adjusted.