# US 2016/0257940 A1 · Duck embryonic derived stem cell lines for the production of viral vaccines

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2016/0257940 A1
- Document ID: US20160257940A1
- Status: Published application
- Kind: A1
- Application number: 15/013,079
- Filed: 2016-02-02
- Published: 2016-09-08
- Earliest claimed: 2007-04-24
- Assignees: Valneva SE
- Inventors: Fabienne Guehenneux; Karine Moreau; Magali Esnault; Majid Mehtali
- Web page: https://patentgrep.com/patent/US20160257940A1

## 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 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- A61K 39/12: Viral antigens
- A61K 39/145: Orthomyxoviridae, e.g. influenza virus
- A61K 39/17: Newcastle disease virus
- A61K 39/285: Vaccinia virus or variola virus
- A61P 31/16: for influenza or rhinoviruses
- C12N 5/00: Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; (plant reproduction by tissue culture techniques A01H4/00)
- C12N 5/0603: {Embryonic cells (production of embryos, nuclear transfer A01K67/027); Embryoid bodies}
- 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}
- C12N 2501/105: Insulin-like growth factors [IGF]
- 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 2501/2306: Interleukin-6 (IL-6)
- 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 2760/16034: 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 2760/18134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/18151: Methods of production or purification of viral material
- C12N 2760/18434: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/18451: 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 duck 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 at position 1

-23. (canceled)

### Claim 24

A process of production of a virus which comprises replicating a virus in cells of an avian cell line wherein said cell line has been obtained by a method comprising the following steps:
a) isolating a duck embryo around oviposition;
b) dissociating the embryo of step a) into cells;
c) seeding the dissociated cells of step b) in basal medium comprising insulin like growth factor 1 (IGF-1), ciliary neurotrophic factor (CNTF), animal serum, and a layer of feeder cells;
d) culturing the cells of step c) for at least one passage;
e) withdrawing the IGF-1 and CNTF from the culture of step d);
f) culturing the cells of step e) for at least one passage;
g) progressively withdrawing the feeder cells from the culture of step f);
h) culturing the cells of step g) for at least one passage;
i) progressively withdrawing the animal serum from the culture medium of step h) over several passages; and
j) adapting the cells of step i) to suspension culture conditions such that a duck cell line capable of proliferating in basal medium in the absence of exogenous growth factors, a feeder layer, and animal serum is obtained, wherein the duck cell line does not produce replication-competent endogenous retrovirus particles;
said process of production of virus comprising the steps of:
1) proliferating said cells in suspension, in a serum-free medium;
2) infecting the cells with said virus;
3) culturing said infected cells in order to allow virus replication; and
4) harvesting said virus.

### Claim 25 (depends on 24)

The process of claim 24, wherein said process further comprises an additional step of feeding the cells, wherein said feeding step consists of a periodic addition of concentrated solutions of glutamine and D-glucose to the culture medium.

### Claim 26 (depends on 24)

The process of claim 24, wherein the cell density in step 2) is of at least 1.5 million cells/ml in a fed-batch process.

### Claim 27 (depends on 24)

The process of claim 24, wherein cells are cultured in a continuous stirred tank bioreactor.

### Claim 28 (depends on 25)

The process of claim 25, wherein the feeding occurs during steps 2) and 3) of the process.

### Claim 29 (depends on 25)

The process of claim 25, wherein the feeding occurs during steps 1) to 3) of the process.

### Claim 30 (depends on 25)

The process of claim 25, wherein the feeding occurs on a daily basis.

### Claim 31 (depends on 24)

The process of claim 24, wherein said process further comprises the step of adding a proteolytic enzyme in the culture medium in conditions allowing virus propagation.

### Claim 32 (depends on 24)

The process according to claim 24, wherein cell culture is performed at a temperature of 33° C.

### Claim 33 (depends on 25)

The process according to claim 25, wherein the glutamine concentration is maintained between 1 mM to 3 mM, preferably around 2 mM.

### Claim 34 (depends on 25)

The process according to claim 25, wherein the D-glucose concentration is maintained between 1 g/l to 10 g/l, preferably around 2 to 3 g/l.

### Claim 35 (depends on 24)

The process according to claim 24, wherein the withdrawal of the growth factors IGF-1 and CNTF from the culture medium in step e) is performed simultaneously.

### Claim 36 (depends on 24)

The process according to claim 24, wherein said duck is a Pekin duck.

### Claim 37 (depends on 24)

The process according to claim 24, wherein said duck is a Muscovy duck.

### Claim 38 (depends on 24)

The process according to claim 24, wherein said duck cell line is diploid.

### Claim 39 (depends on 24)

The process according to claim 24, wherein said duck cell line does not produce replication competent endogenous avian leucosis (ALV-E) and/or endogenous avian virus (EAV) retroviral particles.

### Claim 40 (depends on 24)

The process according to claim 24, wherein the genome of said duck cell line does not contain a proviral sequence of an avian leucosis (ALV-E) and/or an endogenous avian virus (EAV) susceptible to produce replication competent endogenous retroviral particles.

### Claim 41 (depends on 24)

The process according to claim 24, wherein said virus is selected from the group comprising poxviruses, recombinant poxvirus, orthomyxoviruses, recombinant orthomyxovirus, reassorted orthomyxovirus, paramyxoviruses, recombinant paramyxovirus, herpes viruses, hepadnaviruses, adenoviruses, parvoviruses, recombinant parvoviruses, reoviruses, circoviruses, coronaviruses, flaviviruses, togaviruses, birnaviruses and retroviruses.

### Claim 42 (depends on 41)

The process according to claim 41, wherein
the poxvirus or the recombinant poxvirus is selected from the group consisting of Modified Vaccinia Ankara (MVA) virus, Lister-Elstree vaccinia virus, LC16m8 vaccinia virus, CVI78 vaccinia virus, Fowl pox virus, canary pox virus, ALVAC, NYVAC, juncopox virus, mynah pox virus, pigeonpox virus, psittacine pox virus, quail pox virus, sparrow poxvirus, starling pox virus, and Turkey pox virus;
the paramyxovirus or the recombinant paramyxovirus is selected from the group consisting of measles virus, mumps virus, rubella virus, Sendai virus, Respiratory Syncythial virus (RSV), human para-influenza types I and III, Rinderpest virus, canine distemper virus, Newcastle disease virus, and duck para-influenza virus;
the orthomyxovirus, the recombinant orthomyxovirus, or the reassorted orthomyxovirus is selected from the group consisting of human influenza virus, avian influenza virus, swine influenza virus, equine influenza virus, and feline influenza virus;
the togavirus is selected from the group consisting of Sinbis virus, Semliki forest virus, O'nyong'nyong virus, Chikungunya virus, Mayaro virus, Ross river virus, Eastern equine encephalitis virus, Western Equine encephalitis virus, Venezuelan Equine encephalitis virus, and a recombinant togavirus thereof;
the retrovirus is selected from the group consisting of reticulo-endotheliosis virus, duck infectious anemia virus, duck spleen necrosis virus, and a recombinant retrovirus thereof;
the parvovirus or recombinant parvovirus is a duck parvovirus;
the adenovirus is selected from the group consisting of fowl adenovirus, goose adenovirus, duck adenovirus and pigeon adenovirus and a recombinant adenovirus thereof;
the birnavirus is Infectious Bursal Disease virus; or
the flavivirus is selected from the group consisting of Dengue virus, Japanese encephalitis virus and West Nile virus.