Method for producing parvovirus having high infectivity titer and high purity
Provided are a parvovirus derived from an unconcentrated cell culture supernatant, having a infectivity titer of 10 9 TCID 50 /mL or more and an {infectivity titer (TCID 50 /mL)}:{impurity protein concentration (ng/mL)} ratio more than 5000:1; and a method of producing such a high-infectivity titer and high-purity parvovirus.
1. A method of producing a high-infectivity-titer and high-purity parvovirus, comprising:
(a) preliminarily calculating, every 24 hours, a time-dependent change of a cell density of a culture substrate when host cells are infected with a parvovirus for each cell density (A) when infecting virus;
(b) determining, based on the time-dependent change of a cell density calculated preliminarily in preliminarily calculating (a),
(b1) time (T max ) from infection to peak time of the time-dependent change of a cell density,
(b2) a cell density (B max ) at T max and A 1 which is A that satisfies the following equation (1),
(b3) the maximum (A max ) of the cell density A 1 when infecting virus, and
(b4) A 2 that satisfies the following equation (2);
B max /A 1 >1.2 Equation (1)
A max ≥A 2 ≥A max /10 Equation (2)
(c) inoculating a seed virus of the parvovirus into the culture substrate containing host cells having the cell density A 2 when infecting virus determined in (b4) of the determining (b) and a serum medium to give a multiplicity of infection (MOI) of from 0.001 to 0.1;
(d) culturing a cultured product containing the host cells and the parvovirus obtained in the inoculating (c) for a time period of T max or more to less than (T max +48) hours, wherein T max is determined in (b1) of the determining (b);
(e) replacing a culture supernatant obtained in the culturing (d) with a serum-free medium and culturing for 12 hours or more; and
(f) collecting the parvovirus-containing culture supernatant obtained by culturing in the replacing (e);
wherein determination of (b1)-(b4) via the preliminarily calculating (a) and determining (b) is followed by performance of (c)-(f), and
in the determining (b), when A satisfying the equation (1) is absent, the preliminarily calculating (a) and determining (b) are performed again by using another cell density A when infecting virus.
2. The method according to claim 1 , wherein the host cells are adhesion-dependent cells.
3. The method according to claim 1 , wherein the parvovirus is porcine parvovirus (PPV), canine parvovirus (CPV), minute virus of mice (MVM), rat virus (RV), H-1 virus (H-1), feline parvovirus (FPV), goose parvovirus (GPV), or bovine parvovirus (BPV).
4. The method according to claim 1 , wherein the replacing (e) includes replacing the culture supernatant with a serum-free medium and culturing for 24 hours or more.
5. The method according to claim 1 , wherein the determining (b) comprises calculating B max and A1′ which is A that satisfies the following equation (1′):
B max /A 1′ ≥2.0 Equation (1′).
6. The method according to claim 1 , wherein culturing in the culturing (d) and replacing (e) are performed at a temperature of 33° C. or more and 39° C. or less.
7. The method according to claim 1 , wherein in the culturing (d) and replacing (e), the host cells and the parvovirus grow concurrently.
8. The method according to claim 1 , wherein the collecting (f) comprises removing free host cells and host cell debris contained in the culture supernatant.
9. The method according to claim 8 , wherein the removing is performed using filtration through a membrane having a pore size of from 0.2 μm to 0.45 μm.