METHOD FOR PURIFYING VIRUS
The present invention provides a method for purifying virus particles from host cells infected with virus, wherein the virus particles are treated with benzonase and polyethylene glycol and the use of said purified viruses for therapeutic compositions.
1 . A method for purifying virus particles from host cells infected with virus, comprising the following steps in the indicated order:
a) cultivating said host cells in a cell culture medium under conditions in which the virus particles are released into the cell culture medium,
b) separating supernatant comprising the virus particles from cell debris and cells by centrifugation,
c) incubating said supernatant with a nuclease,
d) precipitating the virus particles with polyethylene glycol (PEG),
e) centrifuging the virus particles, and
f) resuspending precipitated virus particles.
2 . The method according to claim 1 , wherein the nuclease is an endonuclease.
3 . The method according to claim 1 , wherein the virus is an RNA virus.
4 . The method according to claim 1 , wherein the host cells are provided without previous lysis treatment.
5 . The method according to claim 1 , wherein centrifugation is at least 2,000 g, at least 3,000 g, at least 4000 g, or at least 4100 g.
6 . The method according to claim 1 , wherein at least 7.5%, PEG is added.
7 . The method according to claim 1 , wherein the PEG has an average molecular weight of from about 5,000 (PEG5000) grams per mole to about 15,000 (PEG15000) grams per mole.
8 . The method according to claim 1 , wherein the virus is precipitated with PEG in the presence of sodium chloride.
9 . The method according to claim 1 , wherein the virus-infected host cells and the virus are not freeze thawed.
10 . The method according to claim 1 , wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% host cell protein is removed.
11 . The method according to claim 1 any one of claims 1 to 10 , wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the host cell DNA is removed.
12 . The method according to claim 1 , wherein the resuspended virus is sterile filtered.
13 . The method according to claim 1 , wherein the host cells are mammalian cells.
14 . The method according to claim 2 , wherein the nuclease is an endonuclease from Serratia marcescens.
15 . The method according to claim 3 , wherein the virus is an influenza virus.
16 . The method according to claim 15 , wherein the virus is an influenza A, B or C virus.
17 . The method according to claim 6 , wherein at least 8% PEG is added.
18 . The method according to claim 7 , wherein the PEG has an average molecular weight of about 6,000 (PEG 6000) grams per mole.
19 . The method according to claim 13 , wherein the host cells are Vero cells.