CLAIMS
1.
A method for the propagation of influenza virus comprising immunogenic
haemagglutinin (HA), wherein cells are cultivated in cell culture in a first step and wherein
subsequently infectious influenza particles are added to the cell culture in a second step,
wherein the culture medium used for the cultivation of the cells is prior to or during the virus
addition step replaced by culture medium with an osmolality of at least 80% compared to the
osmolality of the culture medium previously used for the cultivation of the cells and which
does not have a significantly lower amount, preferably not less than 50%, of the total amount
of proteins, growth factors and/or inorganic salts compared to the culture medium previously
used for the cultivation of the cells, wherein the amount of cells in the cell culture at the time
of virus addition is at least 0.5x106 cells/ml, wherein within 12 to 36 hour after virus addition
the density of living cells is not lower than 40% of the cell density at the time of virus addition,
wherein
the total number of infectious viral particles per cell added during the virus addition step
(Multiplicity of Infection, MOI) is less than 10-5.
2.
The method according to claim 1, wherein the replacement culture medium does not
contain BSA (bovine serum albumin).
3.
The method according to claim 1 or 2, wherein the replacement culture medium is not
supplemented with antibiotic/s.
4.
The method according to any of claims 1 to 3, wherein after the addition of infectious
influenza particles a protease is added to the culture medium in a concentration range of 1
µg/ml to 50 µg/ml.
5.
The method according to claim 4, wherein a protease is added to the culture medium
in a concentration range of more than 1.0 µg/ml to 50 µg/ml, preferably in a concentration
range of 1.5 µg/ml to 50 µg/ml, more preferably in a concentration range of 2.0 µg/ml to 50
µg/ml, and even more preferably in a concentration range of 2.5 µg/ml to 50 µg/ml.
6.
The method according to claim 4 or 5, wherein the protease is trypsin.
7.
The method according to any one of claims 1 to 6, wherein the cells are anchorage-
dependent cells.
2
8.
The method according to any one of claims 1 to 7, wherein the total number of
infectious viral particles per cell added during the virus addition step (Multiplicity of Infection,
MOI) is equal to or less than 10-6.
9.
The method according to any one of claims 1 to 7, wherein the total number of
infectious viral particles per cell added during the virus addition step (Multiplicity of Infection,
MOI) is equal to or less than 10-7.
10.
The method according to any one of claims 1 to 7, wherein the total number of
infectious viral particles per cell added during the virus addition step (Multiplicity of Infection,
MOI) is equal to or less than 10-8.
11.
The method according to any one of claims 1 to 10, wherein within 12 to 36 hours
after virus addition the density of living cells is not lower than 60% of the cell density at the
time of virus addition.
12.
The method according to any one of claims 1 to 10, wherein within 12 to 36 hours
after virus addition the density of living cells is not lower than 80% of the cell density at the
time of virus addition.
13.
The method according to any one of claims 1 to 12, wherein the culture medium used
for the cultivation of the cells is prior to or during the virus addition step replaced by culture
medium with an osmolality of at least 95% compared to the osmolality of the culture medium
previously used for the cultivation of the cells
14.
The method according to any one of claims 1 to 13, wherein the amount of cells in the
cell culture at the time of virus addition is at least 3.0x106 cells/ml.
15.
The method according to any one of claims 1 to 13, wherein the amount of cells in the
cell culture at the time of virus addition is at least 5.0x106 cells/ml.
16.
The method according to any one of claims 1 to 13, wherein the amount of cells in the
cell culture at the time of virus addition is at least 7.0x106 cells/ml.
17.
The method according to any one of claims 1 to 13, wherein the amount of cells in the
cell culture at the time of virus addition is at least 9.0x106 cells/ml.
3
18.
The method according to any one of claims 1 to 13, wherein the amount of cells in the
cell culture at the time of virus addition is at least 11.0x106 cells/ml.
19.
The method according to any one of claims 1 to 13, wherein the amount of cells in the
cell culture at the time of virus addition is at least 13.0x106 cells/ml.
20.
The method according to any one of claims 1 to 19, wherein the cells used are animal
cells, preferably mammalian cells.
21.
The method according to claim 20, wherein the cells used are MDCK cells.
22.
The method according to any one of claims 1 to 21, wherein the method further
comprises one or more steps of further processing the propagated viral particles.
23.
The method according to claim 22, wherein the processed viral particles comprise
inactivated viral particles, and/or attenuated viral particles, and/or split virus antigen, and/or a
subunit virus antigen, and/or virosomes.
24.
The method according to claim 23, wherein the processed viral particles comprise
one or more influenza antigens.
25.
The method according to any of the preceding claims for use in the production of
influenza vaccine.
26.
A process for testing whether the addition of a very low number of infectious virus
particles per cell necessary to infect a cell (MOI) of a pre-selected virus strain to a cell culture
composition for the propagation of viral particles leads to an increased yield of the viral
particles and/or processed viral particles, comprising the following steps:
a) growing of cells in a cell culture composition until a cell density of at least 0.5x10 6 cells/ml
is reached,
b) adding to the cell culture composition a total number of infectious viral particles of the preselected virus strain using a very low MOI, wherein the very low MOI is a MOI of less than
10-5,
c) comparing the yield of the viral particles and/or processed viral particles obtained after
adding the infectious viral particles of step b) to the cells with the amount of viral particles
and/or processed viral particles obtained when adding to the same type of cells the same
type of infectious viral particles using a reference MOI in a range of equal to or higher than
4
10-5.
5
Abstract
The present invention refers to a method for the propagation of influenza virus, wherein the
cell culture composition contains an amount of cells in the cell culture at the time of virus
addition of at least 0.5x106 cells/ml and wherein the total number of infectious viral particles
per cell added during the virus addition step (MOI) is less than 10-5, and to the use of said
method in the production of influenza vaccine. Furthermore, the present invention relates to a
process for testing whether the addition of a very low number of infectious virus particles per
cell (MOI) of a pre-selected virus strain to a cell culture composition for the propagation of
viral particles leads to an increased yield of the viral particles and/or processed viral particles.
6
Fig. 1
medium replacement CM; MOI 1x10^-3
medium replacement CM; MOI 1x10^-6
medium replacement Epi w/o BSA; MOI 1x10^-6
50
40
HA 30
protein
content
(µg/mL)
20
10
0
0
1
2
3
Time (days after virus addition)
4
5
7
Fig. 2
4,0E+06
medium replacement CM; MOI 1x10^-3
medium replacement CM; MOI 1x10^-6
medium replacement Epi w/o BSA; MOI 1x10^-6
3,0E+06
2,0E+06
Cell density
(cells/mL)
1,0E+06
0,0E+00
0
1
2
3
Time (days after virus addition)
4
5
8
Fig. 3
medium replacement CM; MOI 1x10^-3
medium replacement CM; MOI 1x10^-6
medium replacement Epi w/o BSA; MOI 1x10^-6
9
8
7
Log10
TCID50/ml
6
5
4
0
1
2
3
4
Time (days after virus addition)
5