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Production of viral components
Field of the invention
The present invention belongs to the field of pharmaceutical industry and relates to a process for the propagation of influenza virus and to the use of said method in the production of influenza vaccine. Furthermore, it 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.
Description of the background art
Vaccines are used to protect the population against common pathogenic threats, providing a huge impact on lowering disease burden and increasing life expectancy. The effective use of vaccines is mainly dependent on being able to quickly produce large quantities of vaccine material and to increase the number of available vaccine doses, wherein different vaccine materials require different growth conditions in order to obtain acceptable yields.
In this respect, the patent application US 2006/0188977 A1 (August 24, 2006; “Non-tumorigenic MDCK cell line for propagating viruses”) describes inter alia the production of vaccine material such as virus, in cell culture. Herein, MDCK cells are proliferated in culture media, infected with virus and cultured. Afterwards, the replicated viruses are isolated. The infection of the cells with the virus is carried out at a MOI of about 0.0001 to about 10.
Govorkova et al. (“African Green Monkey Kidney (Vero) Cells Provide an Alternative Host Cell System for Influenza A and B Viruses”, Journal of Virology, Aug. 1996, p. 5519-5524) show that the African green monkey kidney cell line (Vero) is comparably suitable as Madin-Darby canine kidney (MDCK) cells as a host cell system for the productive replication of Influenza viruses, as the Vero cell lines also provided adequate quantities of influenza A and B viruses to meet the vaccine requirements imposed by an emerging pandemic. In order to infect the two different cell lines with the tested influenza viruses, the cell lines were infected with different multiplicity of infection (MOI), ranging from 0.01 to 0.001 PFU per cell.
Ozaki et al. (“Generation of High-Yielding Influenza A Viruses in African Green Monkey Kidney (Vero) Cells by Reverse Genetics”, Journal of Virology, Feb. 2004, p. 1851-1857) describe a modified influenza viral master strain that has improved viral rescue and growth properties in the Vero cell line. They could show that the improved properties were mediated by the substitution of the PR8 NS gene for that of a Vero-adapted reassortant virus (Eng53/v-a). The virus replication was assayed by infecting Vero cells with the Vero-adapted reassortant virus at a multiplicity of infection (MOI) of 0.01.
Voeten et al. (“Characterization of high-growth reassortant influenza A viruses generated in MDCK cells cultured in serum-free medium”, Vaccine 17,1999, 1942-1950) describe the use of continuous cell line (MDCK-SF1) that is able to grow without fetal bovine serum for the generation of high-growth reassortant influenza A viruses that can be used for viral antigen production in these cells. The high-growth phenotype of the reassortant strain could be demonstrated by a comparison from the hemagglutinating units (HAU) of reassortant viruses resulting from an infection with that of the corresponding field strain. The infection of the cells took place in 96-well plates (200 µl/well with a cell density of about 2x105 cells/ml) with different MOI, ranging from 0.001 to 0.000001. The authors demonstrated that the high-growth phenotype can be attributed to the matrix protein of the high-growth laboratory strains PR 34 or HK 68, respectively. Voeten et al. measured the kinetics of virus propagation in hemagglutinating units (HAU). However, this test as well as the low number of cells used in the respective experiments (the test was carried out in 96-wells plates in a volume of about 200 µl culture medium, containing cells with a density of about 0.2x106/ml) leads to inaccurate results exhibiting no significance.
Audsley and Tannock (“The growth of attenuated influenza vaccine donor strains in continuous cell lines”, Journal of Virological Methods 123 (2005), 187-193) compared in their study the growth characteristics of three Russian live attenuated donor strains in MDCK and Vero cell cultures at different MOI. In this study, they showed that the optimum MOI for the growth of all donor strains was 0.01.
EP 2 022 849 A1 relates to a method for producing influenza virus on a large scale, describing that tissue cultures can be infected with an MOI of 0.00001 to 0.01.
WO 97/38094 relates to the replication of high growth influenza virus strains wherein mammalian cells are infected with said strains and cultured while maintaining trypsin concentration in a range of 0.05-1.0 µg/ml.
Brands et al. ("InfluvacTC: A Safe Madin Darby Canine Kidney (MDCK) Cell Culture-Based Influenza Vaccine", Developments in Biological Standardization, vol. 98, 1 January 1999) describes the overall production process of the influenza virus vaccine InfluvacTC. Therein, MDCK working seed viruses (WSV) are produced from WHO-designated egg-adapted influenza virus in MDCK cell cultures at low multiplicity of infection in serum-free medium. Furthermore, the downstream processes that were carried out during vaccine production are described (infection of the cells with virus, harvesting of the virus-containing culture medium, virus purification, processing of the virus into vaccine), as well as safety measures that have been taken (inactivation of the virus, viral clearance, tests as to putative host cell contaminants being present). Brands et al. does not mention a specific MOI at all.
WO 2008/043805 refers to the use of macrolide polyene antibiotics or derivatives or analogues thereof as a culture supplement for the propagation of virus in order to increase the yield and quality of virus propagated in continuous cell lines. In Fig. 1 of WO 2008/043805 it is shown that for each MOI tested, the presence of amphotericin B in the virus growth medium has a positive effect on virus replication. WO 2008/043805 discloses that by using macrolide polyene antibiotics or derivatives or analogues thereof, MOI of 0.001 or of 0.0001 up to 0.00001 or even lower can be used for infection of cells. WO 2008/043805 is e.g. silent on the amount of cells in the cell culture at the time of virus addition, as well as on the defined density of living cells at a specific time point after virus addition.
WO 96/15232 refers to a process for ensuring replication of human influenza virus at a low multiplicity of infection in a mammalian cell line, wherein a consistent minimum concentration of trypsin (about 0.05 µg/ml) in the culture medium is maintained. According to WO 96/15232, tests showed that a trypsin concentration of about 0.1 µg/ml was optimal with regard to virus yields when the MOI ranged from about 1x10-5 and 1x10-6 TCID50 per cell, and that satisfactory results were obtained at about 5x10-7 TCID50 per cell. WO 96/15232 does neither disclose e.g. the amount of cells in the cell culture at the time point of virus addition, nor the density of living cells at a specific time point after virus addition.
Despite the above described high-growth viral strains and cell lines used for propagating viral particles in a cell culture, as well as the vaccine production process, there is still a need and thus an object for an improved process of production of viral components and improved cell cultures for the production of viral components.
Summary of the invention
The present invention provides the following aspects, subject-matters and preferred embodiments, which respectively taken alone or in combination, contribute to solving the object of the present invention:
(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 such as influenza virus A, B or C 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%, preferably of at least 85%, more preferably of at least 90% and most preferably of at least 95% 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%, more preferably not less than 60%, even more preferably not less than 65% and most preferably not less than 75%, 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 infection,
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.
Within the meaning of the present invention, the expression “culture medium used for the cultivation of the cells”, “cell culture medium that is used for the cultivation of the cells” or “cell culture medium” denotes the culture medium that is used prior to inoculation of the cell culture with virus. The culture medium that replaces said cell culture medium (replacement culture medium) is denoted by the term “culture medium which is used for propagation of the virus”, “virus culture medium” or “virus propagation medium”. This is the medium that is used in the virus propagation phase.
In one embodiment, the cell culture medium that is used for the cultivation of the cells and the culture medium which is used for propagation of the virus (i.e. the replacement medium) differ only in the presence/absence of BSA (bovine serum albumin) (other ingredients and amounts of ingredients are essentially the same, i.e. the amounts of all individual substances do not differ more than 30%, preferably not more than 20%).
In another embodiment, a protein rich medium is used as the cell culture medium for the cultivation of the cells and a protein poor medium is used as the culture medium for propagation of the virus. The terms “protein rich” and “protein poor” define that the total amount of protein is higher in the “protein rich medium” compared to the “protein poor medium”. Alternatively or additionally preferred, the total amount of free amino acids contained in the culture medium which is used for propagation of the virus is not or not essentially higher than the total amount of amino acids in the cell culture medium that is used for the cultivation of the cells.
It is additionally preferred that the culture medium which is used for propagation of the virus does not have a significant lower amount (e.g. not less than 50%, more preferably not less than 60%, even more preferably not less than 65% and most preferably not less than 75%) of an individual substance or group of substances, respectively, selected from the group consisting of proteins, growth factors and/or inorganic salts compared to the cell culture medium which is used for cultivation of the cells. For example it is preferred that the amount of proteins is not less than 50%, more preferably not less than 60%, even more preferably not less than 65% and most preferably not less than 75% in the culture medium used for propagation of the virus. BSA is not taken into account when comparing the amounts of individual substances belonging to the group of proteins, however it is taken into account when comparing the total amount of proteins. Within the meaning of the present invention, the term “BSA” includes any kind of BSA, such as BSA Fraction V or Albumax I (lipid-rich BSA, bovine). Within the meaning of the present invention, a “group of substances”, e.g. proteins, comprises all individual substances, e.g. lactalbumin hydrolysate, that belong to said group.
Furthermore, within the meaning of the present invention, the term “total amount of proteins, growth factors and/or inorganic salts” denotes on the one hand the total amount of the individual substances respectively belonging to the group of proteins, growth factors or inorganic salts (e.g. the total amount of all individual substances belonging to the group of proteins, or the total amount of all individual substances belonging to the group of growth factors), and on the other hand it denotes the total amount of all individual substances belonging to the group of proteins, growth factors and inorganic salts.
In a further preferred embodiment of the process, the culture medium which is used for propagation of the virus does not have a significantly lower amount (less than 75% of the amount of the cell culture medium) of three, two, or one substance/s selected from the group consisting of growth factors and/or inorganic salts compared to the cell culture medium which is used for cultivation of the cells.
In order to determine whether such criteria are fulfilled, either the amount/s of the respective individual substances or the group of substances such as the group of proteins, growth factors and/or inorganic salts, is/are determined by methods which are commonly used in this field. Then, the respective amounts either of the individual substances or the group of substances of the virus propagation medium are compared with the amounts of the cell culture medium.
In a further embodiment, the cell culture medium is not supplemented with antibiotic/s.
In a further embodiment, the virus propagation medium is not supplemented with antibiotic/s.
In a further embodiment, neither the cell culture medium nor the virus propagation medium is supplemented with antibiotic/s.
In particular, the cell culture medium and/or the virus propagation medium is/are not supplemented with macrolide polyene antibiotic/s or derivatives or analogues thereof.
(2)The method according to item (1), wherein the replacement culture medium does not contain BSA.
In a further preferred embodiment, the replacement culture medium is not supplemented with antibiotic/s.
(3)The method according to item (1) or (2), 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.
(4)The method according to item (3), 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.
(5)The method according to item (3) or (4), wherein the protease is trypsin.
(6)The method according to any one of items (1) to (5), wherein the cells are anchorage-dependent cells.
(7)The method according to any one of items (1) to (6), 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.
(8)The method according to any one of items (1) to (6), 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.
(9)The method according to any one of items (1) to (6), 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.
(10)The method according to any one of items (1) to (9), 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.
(11)The method according to any one of items (1) to (9), 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 infection.
(12)The method according to any one of items (1) to (9), wherein within 12 to 36 hours after virus addition the density of living cells is not lower than 100% of the cell density at the time of infection.
(13)The method according to any one of items (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 (replacement 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 items (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 items (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 items (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 items (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.
(18)The method according to any one of items (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 items (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 items (1) to (19), wherein the cells used are animal cells, preferably mammalian cells.
In a preferred embodiment, the mammalian cells are selected from the group consisting of Vero, PerC6, BHK, 293, COS, PCK, MRC-5, MDCK, MDBK and WI-38, preferably the cells are MDCK cells.
In a further preferred embodiment, the cells are cultivated as adherent cells.
(21)The method according to item (20), wherein the cells used are MDCK cells.
(22)The method according to any one of items (1) to (21), wherein the method further comprises one or more steps of further processing the propagated viral particles.
(23)The method according to item (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 item (23), wherein the processed viral particles comprise one or more influenza antigens. In a preferred embodiment, the processed viral particles comprise haemagglutinin (HA) and/or neuraminidase (NA).
(25)The method according to any of the preceding items for use in the production of influenza vaccine.