# US 10,767,166 B2 · Methods and compositions for production of vaccina virus

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- Number: US 10,767,166 B2
- Document ID: US10767166B2
- Status: Granted
- Kind: B2
- Application number: 16/270,585
- Filed: 2019-02-07
- Published: 2019-06-06
- Granted: 2020-09-08
- Earliest claimed: 2011-08-05
- Assignees: Sillajen Biotherapeutics, Inc.
- Applicants: Sillajen Biotherapeutics, Inc.
- Inventors: David Kirn; John Bell
- Web page: https://patentgrep.com/patent/US10767166B2

## 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 35/76: Viruses; Subviral particles; Bacteriophages
- A61P 35/00: Antineoplastic agents
- C12N 15/86: Viral vectors
- C12N 2710/24132: Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
- C12N 2710/24151: 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

Certain embodiments are directed to viral production processes for the large scale production of vaccinia virus.

## Claims

### Claim 1

A method for producing a vaccinia virus comprising:
(a) infecting HeLa cells adhered to a cell culture bioreactor surface with a Western Reserve strain vaccinia virus by contacting the HeLa cells with the vaccinia virus at a multiplicity of infection (m.o.i.) of between 0.005 and 1.0 plaque forming units (pfu)/cell;
(b) culturing the infected cells in an infection medium having a pH of 7.2 to 7.6 at temperature of 30° C. to 40° C.; and
(c) harvesting vaccinia virus from the culture, whereby at least 50 pfu of vaccinia virus per HeLa cell is produced, calculated as
total
⁢
⁢
virus
⁢
⁢
yield
⁢
⁢
from
⁢
⁢
step
⁢
⁢
(
c
)
⁢
⁢
in
⁢
⁢
pfu
total
⁢
⁢
number
⁢
⁢
of
⁢
⁢
viable
⁢
⁢
HeLa
⁢
⁢
cells
⁢
⁢
at
⁢
⁢
step
⁢
⁢
(
a
)
.

### Claim 2 (depends on 1)

The method of claim 1, wherein the cell culture bioreactor surface comprises a cumulative culture area of at least 168,000 cm2.

### Claim 3 (depends on 1)

The method of claim 1, wherein the pH of the infection medium is 7.2 to 7.4.

### Claim 4 (depends on 3)

The method of claim 3, wherein the pH of the infection medium is about 7.3.

### Claim 5 (depends on 1)

The method of claim 1, wherein the m.o.i. of step (a) is about 0.01 pfu/cell to about 0.05 pfu/cell.

### Claim 6 (depends on 5)

The method of claim 5, wherein the m.o.i. of step (a) is about 0.02 pfu/cell to about 0.03 pfu/cell.

### Claim 7 (depends on 1)

The method of claim 1, wherein steps (a) and (b) are performed at a temperature of 32° C. to 37.5° C.

### Claim 8 (depends on 7)

The method of claim 7, wherein steps (a) and (b) are performed at a temperature of 32° C., 33° C., 34° C., 35° C., 36° C. or 37° C.

### Claim 9 (depends on 1)

The method of claim 1, wherein the density of adhered HeLa cells is between 104 and 106 HeLa cells/cm2 during step (a).

### Claim 10 (depends on 1)

The method of claim 1, wherein the concentration of vaccinia virus during step (a) is between 103 and 105 pfu/ml.

### Claim 11 (depends on 1)

The method of claim 1, wherein step (b) is performed for a period of 40-80 hours.

### Claim 12 (depends on 11)

The method of claim 11, wherein step (b) is performed for a period of at least 44 hours.

### Claim 13 (depends on 1)

The method of claim 1, wherein at least 75 pfu vaccinia virus per HeLa cell is produced calculated as the total virus yield (pfu)/total number of viable HeLa cells at step (a).

### Claim 14 (depends on 1)

The method of any claim 1, wherein the medium comprises fetal bovine serum in an amount equal to or less than 10% by volume of the medium.

### Claim 15 (depends on 1)

The method of claim 1, wherein the medium does not comprise serum.

### Claim 16 (depends on 1)

The method of claim 1, wherein the harvested virus is subjected to one or more purification steps.

### Claim 17 (depends on 16)

The method of claim 16, wherein said purification comprises treatment of the harvested virus with a nuclease to remove HeLa cell nucleic acids, and/or a protease to remove HeLa cell proteins, and/or, a tangential flow filtration step, and/or a heparin affinity chromatography step and/or a membrane absorption chromatography step.

### Claim 18 (depends on 1)

The method of claim 1, wherein the vaccinia virus is a recombinant vaccinia virus, lacking a functional thymidine kinase gene and/or lacking a functional vaccinia growth factor gene and/or encoding a granulocyte macrophage colony-stimulating factor polypeptide.

### Claim 19 (depends on 18)

The method of claim 18, wherein the vaccinia virus lacks a functional thymidine kinase gene and lacks a functional vaccinia growth factor gene.