WO2011134660A1

WO2011134660A1 - Production of viral components

Info

Publication number: WO2011134660A1
Application number: PCT/EP2011/002135
Authority: WO
Inventors: Dick Smit; Marinus Adrianus Oerlemans
Original assignee: Abbott Biologicals B.V.
Priority date: 2010-04-28
Filing date: 2011-04-28
Publication date: 2011-11-03
Also published as: TW201202425A; AR080972A1

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.

Description

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 μΙ/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 μΙ 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 Mg/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 g/ml) in the culture medium is maintained. According to WO 96/15232, tests showed that a trypsin concentration of about 0.1 pg/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 pg/ml to 50 pg/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 pg/ml to 50 pg/ml, preferably in a concentration range of 1.5 pg/ml to 50 pg/ml, more preferably in a concentration range of 2.0 pg/ml to 50 pg/ml, and even more preferably in a concentration range of 2.5 pg/ml to 50 pg/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 1θ

(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.

(26) A process for testing whether the addition of a very low total number of infectious viral particles per cell necessary to infect a cell ("Multiplicity of Infection", 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 0.5x10⁶ cells/ml is reached,

b) adding to the cell culture composition with a total number of infectious viral particles of the pre-selected 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 10^~5. Preferably, the reference MOI is 10^~3.

In a further preferred embodiment, in step a), the cells are grown until a cell density of at least 3.0x10⁶ cells/ml, preferably of at least 5.0x10⁶ cells/ml, further preferred of at least 7.0x10⁶ cells/ml, even further preferred of at least about 9.0x10⁶ cells/ml, preferably of at least about 11.0x10⁶ cells/ml, or further preferred of at least 13.0x10⁶ cells/ml is reached.

(27) The testing process according to item (26), wherein the amount of viral particles 24 hours after virus addition is at least 6 when measured as 10log TCID₅₀/ml.

(28) The testing process according to item (26) or (27), wherein a harvesting and, optionally, further processing step of the produced viral particle is carried out prior to step c).

(29) The testing process according to any of items (26) to (28), wherein the amount of viral particles or processed viral particles obtained after adding to the cells the infectious viral particles of step b) is at least 1.2-fold, preferably at least 1.5-fold, more preferably at least 2- fold, and most preferably at least 3-fold the amount of viral particles or processed viral particles obtained after adding to the same type of cells the same type of infectious viral particles using a reference MOI in a range of at least 10^"5 or higher, wherein the amount of viral particles is preferably determined 24 hours after virus addition. It is particularly preferred to use a MOI of 10^"3 as the reference MOI.

(30) The testing process according to any of items (26) to (29), wherein after step a), preferably after step a) and prior to or during step b), the cell culture medium which is used for culturing the cells with a very low MOI and reference MOI is replaced with a virus propagation medium which has an osmolality of at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% compared to the osmolality of the cell culture medium which is previously used for the cultivation of the cells.

Regarding the preferred cell culture medium and medium used for virus propagation, reference is made to the respective culture media described herein. Whether the above- mentioned criteria with regard to the respective culture media are fulfilled, can be determined as described herein elsewhere.

(31) The testing process according to any of items (26) to (30), wherein the cells are cells as defined in item (20) or (21).

(32) The testing process according to any of items (26) to (31), wherein the infectious viral particle is an infectious influenza particle.

(33) The testing process according to any of items (26) to (32), wherein the processed viral particle is a processed viral particle as defined in item (23) or (24).

(34) The process according to any of items (1) to (25) or the testing process according to any of items (26) to (33), wherein the MOI is equal to or less than IxlO^"6, optionally equal to or less than 1x10^~7, further optionally equal to or less than 1x10^~8.

(35) The process according to any of items (1) to (25) or the testing process according to any of items (26) to (33), wherein the amount of the viral particles or processed viral particles is detected by Single Radial Immuno Diffusion (SRID) assay or by reversed phase high performance liquid chromatography (RT-HPLC) assay.

(36) A cell culture composition for the production of a viral particle and/or a processed viral particle, wherein

a) the cell culture composition has been prepared by using a starting amount of cells of at least 0.5x10⁶ cells/ml, i.e. the amount of cells in the cell culture at the time of virus addition was at least 0.5x10⁶ cells/ml, and

b) to the cell culture composition of step a) a total number of infectious viral particles using a very low MOI of less than 10^"5 has been added, and

c) the amount of living cells being present in the cell culture composition of step b) within a range of 1 day after virus addition corresponds to at least about 60%, preferably at least about 70%, more preferably at least about 75%, even more preferably at least about 80% and most preferably at least about 85% of the starting amount of cells being present in the cell culture composition, i.e. of the amount of living cells in the cell culture at the time of virus addition.

In a further preferred embodiment, the amount of living cells being present in the cell culture composition of step b) within a range of 24 hours after virus addition corresponds to at least about 100% or at least to about 110%of the starting amount of cells being present in the cell culture composition.

In a further preferred embodiment, the amount of living cells being present in the cell culture composition of step b) within a range of 48 hours after virus addition corresponds to at least about 5%, preferably at least about 10%, preferably at least about 15% of the amount of cells being present in the cell culture composition at the time of virus addition.

(37) The cell culture composition according to item (36), wherein to the cell culture composition of step a) infectious viral particles have been added by using a MOI tested according to the testing process of any of items (26) to (35).

(38) The cell culture composition according to item (36) or (37), wherein the haemagglutinin (HA)/ml ratio after 4 days after virus addition is higher than 15 Mg/ml, preferably higher than 20 Mg/ml.

(39) Use of a cell culture composition for the propagation of viral particles, wherein to a cell culture composition that contains an amount of cells of at least 0.5x10⁶ cells/ml at the time of virus addition infectious viral particles are added, wherein the used total number of infectious virus particles per cell necessary to infect a cell ("Multiplicity of Infection", MOI) is less than 10^"5 (very low MOI). In a further preferred embodiment, the MOI is equal to or less than 10^"6, equal to or less than 10^"7, or equal to or less than 10^"8. Reference is also made to the preferred values for the very low MOI as indicated above.

(40) Process for producing viral particles comprising immunogenic haemagglutinin (HA), or for producing immunogenic HA protein, comprising the steps of

a) propagation of viral particles comprising immunogenic HA, wherein to a cell culture composition that contains an amount of cells of at least 0.5x10⁶ cells/ml at the time of virus addition infectious viral particles are added, and wherein the used total number of infectious virus particles per cell necessary to infect a cell ("Multiplicity of Infection", MOI) is less than 10^"5; and b) obtaining the propagated viral particles, and, optionally, further processing the propagated viral particles to isolate immunogenic haemagglutinin (HA) protein and/or immunogenic neuraminidase (NA) protein.

(41) Process according to item (40), further defined as set forth in any one of items (1) to (25).

With regard to the preferred starting amount of cells, reference is made to the explanations above. Within the meaning of the present invention, the term "starting amount of cells" denotes the amount of cells in the cell culture at the time of virus addition. With the addition of virus to the cell culture the infection phase of the cells starts.

Detailed description of the invention

The present invention is now described in more detail by preferred embodiments and examples, which are however presented for illustrative purpose only and shall not be understood as limiting the scope of the present invention in any way.

Viruses such as influenza viruses are causative agents of emerging annual or seasonal outbreaks, epidemics or pandemics. These disease outbreaks are associated with considerable morbidity and mortality, especially in people at risk such as people suffering from heart or lung diseases, diabetics or a malfunction of the immune system. In order to protect the population against common pathogenic threats and in order to prevent endemic or pandemic spreads, vaccines are used. However, for instance in case of a pandemic, such as an influenza pandemic, a significant gap between vaccine manufacturing capacities and vaccine demands are expected on a global scale. Therefore, there is an urgent need to increase the number of available vaccine doses, which can for instance be achieved by improving, e.g. increasing, the virus yield or the yield of the viral components, respectively, in production cell lines.

The present invention provides a process for the propagation of influenza virus, leading to an increased yield of the propagated viral particles (i.e. the propagated influenza virus) and the obtained viral components. Within the meaning of the present invention, the term "viral components" denotes the processed viral particles. It has been surprisingly found that when a certain minimum cell density is started with, namely at least 0.5x10⁶ cells/ml, preferably at least 3.0x10⁶ cells/ml, 5.0x10⁶ cells/ml, 7.0x10⁶ cells/ml, 9.0x10⁶ cells/ml, 11.0x10⁶ cells/ml or 13.0x10⁶ cells/ml (starting amount of cells) a significantly increased yield in viral particles or in viral components can be provided by adding to such minimum amount of cells a total number of infectious viral particles per cell necessary to infect a cell (MOI) of less than 10^"5. Although a comparatively low starting amount of added virus per given amount of cells is used, it has unexpectedly been found that adding infectious virus particles to a minimum starting amount of cells with the aforementioned very low MOI is particularly advantageous and useful in a process for the propagation of viral particles, as an increased yield of propagated viral particles and an increased yield of viral components can be obtained. This is particularly advantageous for the production of influenza viral components, such as for the production of influenza viral components like the influenza virus antigens haemagglutinin (HA) or neuraminidase (NA). Without wishing to be bound by any theory, it appears that due to the addition of virus to the cell culture and thus the infection of cells with said very low MOI the percentage of cells that survive within a certain period of time after virus addition, e.g. a time period of up to about three days, is higher. This seems to lead to a more efficient virus propagation process especially in the critical initial period of ongoing cell infection, resulting in a more prolonged and productive virus production and consequently in an increased yield of propagated viral particles and in a higher yield of viral components. In particular, a high increase in viral haemagglutinin (HA) can be obtained when using a cell density (starting amount of cells, i.e. amount of cells in the cell culture at the time of virus addition) of between 1 and 4-5 x 10⁶ cells/ml, and wherein the MOI is in a range of from less than 10^"5 to about 10-⁷.

In hitherto known processes which use an MOI of equal to or more than 10^"5, a maximum yield of the propagated viral particles is obtained when using a virus propagation medium which has less substances like protein, growths factors etc. compared to the cell culture medium which is used for cell cultivation. This means that the virus propagation medium is less enriched. However, it has unexpectedly been found within the context of the present invention that when using a very low MOI according to the invention, the viral particle yield can even be further increased when using a virus propagation medium which does not have significantly fewer amounts of substances than the cell culture medium.

As the process according to the present invention allows for the propagation of viral particles, e.g. influenza viral particles, and the production of viral components, i.e. the processed viral particles, with an increased yield, it accelerates the process of vaccine generation and reduces the vaccine release lead times. By applying the process according to the present invention, it is even possible to increase the yield of viral components derived from viral strains that are already adapted to high-growth, for instance reassortant virus strains or virus strains adapted to a better growth in specific cell lines.

In general, to a production cell line a virus strain is added by using a certain MOI (multiplicity of infection) of said virus strain in order to propagate said strain. The virus strain that has to be propagated is also referred to herein as a pre-selected virus strain. The certain MOI is usually in a range of equal to or higher than 10^"5 and is referred to herein as "reference" MOI. A preferred reference MOI used is10^"3. However, as has been demonstrated in the course of the present invention, this "reference" MOI is not the most effective MOI for all virus strains to be propagated, but that there are very low MOIs in the range of lower than 10^~5 that provide for a more effective virus propagation, thereby leading to enhanced yields of viral particles and/or viral components. Therefore, the present invention further provides a process for testing whether the addition of a very low total number of infectious viral particles per cell 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, or viral components, respectively. Advantageously, by applying said process, an MOI providing for a maximum yield of propagated viral particles and viral components, respectively, individual for each pre-selected virus strain, can be identified, and especially in very low MOI ranges which conventionally has not been contemplated for industrial vaccine production processes. Furthermore, provided that the aforementioned minimum starting cell density is observed, no up-scaling effect can be detected. This means that the results obtained at a small scale or in a semi- industrial scale, e.g. in a working volume of about 3 I or less, or e.g. 30 ml or less, may be translated to a large/industrial scale, such as to bioreactors having a working volume of above 100 I, e.g. of about 1200 I. Therefore, it is for instance possible to run multiple test cultures in parallel at small scale and/or at a semi-industrial scale in order to test whether an inoculation using a very low MOI leads to a higher yield of viral particles or viral components, respectively, and if so, which very low MOI is the one that provides for the highest possible yield for a given viral strain. This very low MOI can then be used for the propagation of the pre-selected virus strain at a large/industrial scale. Furthermore, starting with a minimum cell density as described herein has e.g. the advantage that the time period until a certain cell density suitable and used for being inoculated with viral particles is shorter compared to the time period until a cell density suitable and used for being inoculated is reached if the starting cell density is lower than the minimum cell density as described herein. This also accelerates the process of vaccine generation, thereby reducing the vaccine release lead time. It is in particular advantageous to use a virus propagation medium which has an osmolality of at least 80%, preferably at least 85%, more preferably at least 90% and most preferably of at least 95 % of the osmolality of the cell culture medium 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. This means that it is advantageous not to replace the cell culture medium to be used for cultivating cells (prior to the virus addition) by a virus propagation medium which has an osmolality that is significantly less than the osmolality of the culture medium which is used for the cultivation of the cells. In another embodiment, a protein rich medium is used as the cell culture medium that is used for the cultivation of the cells and a protein poor medium is used as the culture medium which is used for propagation of the virus. 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 higher than the total amount of amino acids in the cell culture medium that is used for the cultivation of the cells. Thereby, it may be possible to additionally enhance the yield of the viral component.

Without being bound to any theory, it appears that balancing the pre-selected virus strain used, the amount of cells (cells per ml) to which the infectious viral particles are added and the MOI used leads to an increase in the yield of propagated viral particles and obtained viral components in the cell culture composition.

All in all, by applying the method according to the present invention, an increased yield of propagated viral particles and viral components can be realized, thereby accelerating the process of vaccine generation and reducing the vaccine release lead times.

In one particular aspect, the present invention relates to a method for the propagation of viral particles, e.g. 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 starting 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 hours 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. In a further preferred embodiment, 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. In a further preferred embodiment, 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 infection. In a further preferred embodiment, 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 virus addition. In a further preferred embodiment, the MOI is equal to or less than 10^"6, equal to or less than 10^"7, or equal to or less than 10^~8.

Within the meaning of the present invention, the term "Multiplicity of Infection" denotes the number of infectious viral particles per cell necessary to infect a cell. In other words, the MOI is the ratio of infectious viral particles to cells. Therefore, for instance a MOI of 10^"5 means that one viral particle is used per 100000 cells.

In the context of the present invention, it has been unexpectedly found that the inoculation of a certain starting amount of cells with a total MOI of less than 10^"5 can effectively increase the amount of propagated viral particles, provided that a certain minimum cell density of at least 0.5x10⁶ cells/ml is started with, and, thus, the yield of a viral component to be produced. This allows for e.g. an accelerated process of vaccine preparation and a reduced vaccine release lead time. This is advantageous in case a seasonal, in particular for an epidemic or pandemic spread or outbreak of a viral infectious disease occurs, and especially when a spread of a viral influenza occurs. By applying the process according to the present invention, even the yield of viral particles and viral compounds derived from virus strains that have already been adapted to fulfill specific properties such as high growth, or growth in serum free culture medium, can be increased. This leads to a further reduction of the time needed to provide a vaccine composition. Furthermore, even wild type viral particles can be propagated such that a yield of viral particles and/ or viral components sufficient for industrial application can be obtained.

According to the present invention, the viral particles can be suitably selected from a desired viral particle.

A viral particle is an infectious or non-infectious viral particle that can only reproduce inside a host cell. The term "infectious" denotes a virus (or virus particle) capable of producing a productive infection when introduced into a host cell. A non-infectious viral particle or virus, respectively, is not able to produce such an infection; however this particle might be able to express the genes it encodes. Preferably, the viral particle is an infectious viral particle. In general, a viral particle contains nucleic acid, either desoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which can be surrounded by a capsid, which can be formed from identical protein subunits, the capsomers. Viral particles can also have an envelope that can be derived from the membrane of the host cell the virus particle has infected. The nucleic acid can be linear, circular or segmented, single-stranded, double-stranded, or a mixture thereof, and the strands can either be positive-sense, or negative-sense. In a preferred embodiment, the viral particles contain RNA. Viral particles that contain RNA and that have been found to be effectively propagated with very low starting MOI are for instance influenza viruses.

Influenza viruses are made up of an internal ribonucleoprotein core containing a segmented single-stranded RNA genome and an outer lipoprotein envelope lined by a matrix protein. Members of the influenza viruses are for instance Influenza virus A, B and C. Influenza A and influenza B viruses each contain eight segments of single stranded negative sense RNA.

Preferably, the viral particle is selected from the group consisting of the families of Orthomyxoviridae such as Influenzavirus A, B or C; Paramyxoviridae such as measles virus, mumps virus, parainfluenza virus and respiratory syncytial virus, Togaviridae such as Sindbis virus and rubella virus, Herpesviridae such as Herpes Simplex virus, Epstein-Barr virus and Varicella Zoster virus, Rhabdoviridae such as rabies virus, Retroviridae such as human immunodeficiency virus (HIV), Reoviridae such as rotavirus and Colorado tick fever virus, Flaviviridae such as yellow fever virus, Adenoviridae such as adenovirus, Picomaviridae such as poliovirus, Arenaviridae such as lymphocytic choriomeningitis virus, and Poxyviridae such as variola virus, preferably the infectious viral particle is selected from the group consisting of the families of Orthomyxoviridae such as Influenza virus A, B or C. In a further preferred embodiment, the viral particle is an influenza virus particle selected from the group consisting of influenza A, Influenza B, or influenza C. Further preferred, the viral particle can also be a reassortant viral particle, containing different combinations of parental gene segments, such as a reassortant influenza virus particle.

Reassortant viruses include viruses that include genetic and/or polypeptide components derived from more than one parental viral strain or source. For example, reassortant viruses are produced to incorporate selected HA and NA antigens in the context of an approved master strain also called a master donor virus (MDV). For example, a 7:1 reassortant includes 7 viral genomic segments (or gene segments) derived from a first parental virus, and 1 viral genomic segment, e.g., encoding HA or NA, from a second parental virus. A 6:2 reassortant includes 6 genomic segments, most commonly the 6 internal genes from a first parental virus, and two genomic segments, e.g., HA and NA, from a second parental virus. A 6: 1 : 1 reassortant may include 6 genomic segments, most commonly the 6 internal genes from a first parental virus, 1 genomic segment from a second parental virus encoding HA, and 1 genomic segment from a third parental virus encoding NA. The 6 internal genes may be those of more than one parental virus as well.

Reassortant viruses may be generated by any method that is known to a skilled person, such as by classical reassortant techniques such as by co-infection methods or by plasmid rescue techniques. A viral particle according to the present invention can also be an inactivated or attenuated virus. The infectious viral particle can also be referred to as a virion.

In the process according to the present invention, a cell culture composition or a cell culture, respectively, containing cells and culture medium is used. It is known to a person skilled in the art that the components of a cell culture composition may vary, depending on the cells used and on the intended use. In a preferred embodiment, the cell culture composition is used for the propagation of viral particles as described herein and/or for the production of viral components. Moreover, the propagated viral particles can be further processed for preparing inactivated viral particles, and/or attenuated viral particles, and/or split virus antigen, and/or a subunit virus antigen, and/or virosomes. For cultivation purposes, for example a cell culture may comprise cells and a suitable medium for growth of the cells.

In general, cells from any suitable cell line that is known to a person skilled in the art can be used in the process according to the present invention. Such cells can be eukaryotic cells such as yeast, insect, amphibian, avian or mammalian cells, including human cells. In a preferred embodiment, the cells contained in the cell culture composition are animal cells, preferably the cells are mammalian cells. Preferably, the cells are cultivated as adherent cells.

Suitable cells include, but are not limited to, Vero (African green monkey kidney) cells, PerC6 cells (human embryonic retinal cells), BHK (baby hamster kidney) cells, primary chick kidney (PCK) cells, Madin-Darby Canine Kidney (MDCK) cells, Madin-Darby Bovine Kidney (MDBK) cells, 293 cells (e.g., 293T cells, or HEK-293 (human embryonic kidney cells) cells), WI-38 cells (cell line derived from cell taken from an about three months old female fetus; normal human fetal lung fibroblasts), MRC-5 cells (cell line derived from normal lung tissue of a 14- week old male fetus, Nature 227: 168-170, 1970), and COS cells (e.g., COSI, COS7 cells). Suitable cells also encompass combinations or mixtures of cells including, e.g., mixed cultures of different cell types or cell lines (e.g., Vero and CEK cells). Preferably, the cells are MDCK cells. In a further preferred embodiment, the cells according to the present invention can be cells that have specific desired properties, for instance the cells can be adapted to growth in a certain medium used, for instance in a serum-free medium, or the cells can exhibit modified doubling times, a modified tumorigenic profile and/or a modified viral production behaviour. One example of a cell line being adapted to serum-free growth is the MDCK-SF cell line. In a preferred embodiment, the cells used in the process according to the present invention are cells that allow for and/or support the replication and/ or propagation of infectious viral particles the cells are inoculated with. One indication of the ability of a cell to support viral replication and/or propagation is the yield of virus or viral particle, respectively, obtained from an inoculated and infected cell culture. In general, the viral yield, such as the amount of viral particles or the amount of viral components, respectively, can be determined by any suitable method that is known to a skilled person. Preferably, the viral yield is quantified by determining the concentration of virus or viral particles, respectively, present in a sample according to a median tissue culture infectious dose (TCID₅₀) assay that measures infectious virions. The TCID₅₀, which is often indicated as the Iog10 TCID₅₀/ml, can be determined by any method that is known to a skilled person. An endpoint dilution technique such as the TCID₅₀ assay is a statistical way of measuring virus populations. Several statistical methods for analysing such data are available e.g. Spearman-Karber, Reed & Muench or Probit analysis. For instance, to cells such as MDCK cells successive dilutions of virus are added. After several days, the cytopathogenic effect (CPE) is recorded and can be calculated into a tissue culture medium median infective dose (TCID₅₀). Furthermore, it is also possible to indicate the virus particles as pfu (plaque forming units)/ml.

In a preferred embodiment, the cells support the replication and/or propagation of viruses including, but not limited to, viruses selected from the group consisting of the families of Orthomyxoviridae such as Influenzavirus A, B or C, Paramyxoviridae such as measles virus, mumps virus, parainfluenza virus and respiratory syncytial virus, Togaviridae such as Sindbis virus and rubella virus, Herpesviridae such as Herpes Simplex virus, Epstein-Barr virus and Varicella Zoster virus, Rhabdoviridae such as rabies virus, Retroviridae such as human immunodeficiency virus (HIV), Reoviridae such as rotavirus and Colorado tick fever virus, Flaviviridae such as yellow fever virus, Adenoviridae such as adenovirus, Picornaviridae such as poliovirus, Arenaviridae such as lymphocytic choriomeningitis virus, and Poxyviridae such as variola virus, preferably the cells support the replication and/or propagation of an infectious virus particle selected from the group consisting of the families of Orthomyxoviridae such as influenza virus A, B and/or C. Furthermore, the cells used in the process according to the invention also support the replication and/or propagation of reassortant viruses. The cells used in the process according to the present invention can further be cells that exhibit superior biological properties, for instance with regard to viral production, tumorigenicity profile and/ or doubling times. Individual cells exhibiting such superior properties may be cloned. The adaption of the cells to certain culture conditions, for instance conditions relating to temperature, C0₂ concentration, p0₂ values, pH range, and the culture medium used, may take place prior to, concurrently with, or subsequently to the cloning of individual cells.

In a preferred embodiment of the process according to the present invention, the starting amount of cells being present in a cell culture that is inoculated or infected, respectively, is at least about 0.5x10⁶ cells/ml, preferably at least about 3.0x10⁶ cells/ml, further preferred at least about 5.0x10⁶ cells/ml, even further preferred at least about 7.0x10⁶ cells/ml, even more further preferred about 9.0x10⁶ cells/ml, and most preferred at least about 11.0x10⁶ cells/ml or at least about 13.0x10⁶ cells/ml. It has surprisingly been found out that by inoculating a cell culture composition having said minimum cell density with a very low MOI as described herein, the yield of propagated viral particles and, thus, the amount of viral components produced can be significantly enhanced. The yield of the propagated viral particles and the obtained viral components may be increased even more in case the cell culture composition is inoculated with a very low MOI.

The cells used in a process according to the present invention can be cultivated in suspension or as adherent cells on a surface to which they attach. Preferably, the cells are cultivated as adherent cells. In a further preferred embodiment, the cells are anchorage- dependent cells. Adherent surfaces on which cells can be grown are well known in the art. Adherent surfaces include, but are not limited to, surface modified polystyrene plastics, protein coated surfaces (e.g. fibronectin and/or collagen coated glass/plastic) as well as a large variety of commercially available microcarriers (e.g. Cytodex 3 microcarriers), available for instance from Amersham Biosciences. Microcarrier beads are small spheres that provide a large surface area for adherent cell growth per volume of cell culture. The choice of adherent surface can be influenced by methods utilized for the cultivation of the cells, such as MDCK cells, and can be determined by a person skilled in the art. Suitable culture vessels or containers, respectively, that can be employed in the process according to the present invention can be any vessels or containers that are known to skilled persons, such as spinner bottles, roller bottles, fermenters or bioreactors, or tissue culture flasks. It is possible to carry out the process according to the present invention in small scale, e.g. in vessels having a smaller volume such as tissue culture flasks with a volume of e.g. around 30 ml, a semi-industrial scale of e.g. about 50 I to e.g. about 100 I, and in industrial scale in vessels having a larger working volume such as fermenters or bioreactors with a working volume of e.g. 1000 I or more. In a preferred embodiment, the cells used in the process according to the present invention may be cultivated in a batch culture system, such as a fed batch culture system. In a further preferred embodiment, the cells can also be cultivated in a perfusion culture system.

In one embodiment of the present invention, the cells used in the process according to the present invention are cultivated at certain conditions. It is known to a person skilled in the art which respective conditions are suitable for which cell type. Respectively adapted conditions for instance refer to C0₂ concentration, p0₂ value, pH value, temperature and medium used. The cultivation period of the cells includes cell cultivation prior to the inoculation and cultivation of the cells after the inoculation (virus propagation).

Respective conditions can be maintained the same; however, it is also possible and eventually more effective when the cell culture conditions are changed or varied during the cultivation period of the cells, i.e. cell cultivation prior to (or during) virus addition and after inoculation/virus propagation, respectively. Said cell culture conditions can change at any time during the whole cultivation prior to inoculation/virus propagation period, and it is also possible that said culture conditions change not only once but also several times in the course of a cell cultivation prior to inoculation (i.e. virus addition)/virus propagation period. Another efficient operation is to let changing of conditions (in particular the medium change) and the virus addition proceed simultaneously (i.e. replacing medium during virus addition) Cell culture conditions that can change are any cell culture conditions, for instance conditions relating to temperature, C0₂ concentration, p0₂ values, pH range, and the culture medium used.

In a further preferred embodiment of the process according to the present invention the virus propagation medium has an osmolality of at least 80%, preferably at least 85%, more preferably at least 90% and most preferably of at least 95 % compared to the osmolality of the cell culture medium, i.e. the culture medium previously used for the cultivation of the cells. Moreover, the virus propagation medium 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. This means that it is advantageous not to replace the cell culture medium to be used for cultivating cells (prior to or during the addition of infectious viral particles to the cells) by a virus propagation medium which has an osmolality that is significantly less than the osmolality of the culture medium which is used for the cultivation of the cells and that has a significant lower amount of the total amount of proteins, growth factors and/or inorganic salts. In another embodiment, a protein rich medium is used as the cell culture medium that is used for the cultivation of the cells and a protein poor medium is used as the culture medium which is used for propagation of the virus. 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 higher than the total amount of amino acids in the cell culture medium that is used for the cultivation of the cells.

It has been unexpectedly found (compare figures) that using a medium for virus propagation that does not have significantly decreased amounts of substances as described herein (compared to the amounts of substances being present in the cell culture medium) and/or that does not have an osmolality of less than 80 %, preferably less than 85 %, more preferably less than 90 % and most preferably less than 95 % of the osmolality of the cell culture medium provides for an increased yield of viral particles compared to the use of a culture medium for propagation which has an osmolality of less than 80 %, of less than 85 %, of less than 90 % or less than 95 % of the osmolality of the cell culture medium and/or which has significantly decreased amounts of substances as described herein. The determination of the osmolality of the cell culture medium is described below.

In a further preferred embodiment according to the present invention, the culture medium which is used for propagation of the virus does not have a significantly lower amount (preferably not less than 50%, more preferably not less than 55%, even more preferably not less than 60% and even most preferably not less than 65%, and in a further preferred embodiment preferably not less than 70%, or 75%, of the total amount of proteins, growth factors and/or inorganic salts compared to the cell culture medium previously used for the cultivation of the cells.

Reference is also made to the embodiments as described above.

In a further preferred embodiment, the culture medium which is used for cultivation of the cells is replaced prior to or during the addition of virus to the cells with an infectious viral particle by a culture medium which is used for virus propagation, wherein the culture medium to be used for virus propagation is of the same type as the cell culture medium which is used for cell cultivation but does not contain BSA.

In a further preferred embodiment of the process according to the present invention, the culture medium which is used for propagation of the virus does not have a lower amount of three, two, or one, individual substance/s or group of substances 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.

This means that according to the present invention it is preferred to carry out the propagation step by using a virus propagation medium which does not have a significant decreased amount of individual substances as described above. In other words, it is advantageous that as many individual substances or groups of substances, respectively, of the virus propagation medium as possible are contained in the same or at least not a significantly lower amount compared to the cell culture medium.

The replacement of the medium, e.g. the replacement of the cell culture medium used for cell culture by virus propagation medium, takes place within a time period that, in general, also depends on the scale of the cell culture. In case the cell culture takes place at a small scale or in a semi-industrial scale, e.g. in a working volume of about 3 I or less, or e.g. 30 ml or less, the medium replacement can be carried out much faster compared to the replacement time needed for cell cultures in a large/industrial scale (such as bioreactors having a working volume of above 100 I).

The term "prior to inoculation" or "prior to the virus addition step", respectively, denotes that the replacement of the medium takes place within a time period of 12 hours (h) or less, preferably of 10 h, 9 h, 8 h, 7 h or 6 h or less, more preferably of 5 h or less, even more preferably of 4 h or less if the cell culture has been carried out at a industrial or large scale. In case the cell culture has been carried out at small scale or semi-industrial scale, the replacement of the medium takes place within a time period of 5 h or less, preferably of 4 h, 3 h, or 2 h or less, more preferably 1 h or less, more preferably 30 min or less, and even more preferred 15 min or less before the inoculation of the cells starts. Alternatively, it is possible to replace the medium during virus addition, in particular to let both operations proceed simultaneously.

In the method according to the present invention, any suitable cell culture medium that is known to a skilled person can be used. In general, a suitable cell culture medium comprises various components (also denoted as additives and substances, respectively) such as inorganic salts, amino acids, nucleic acids, vitamins, lipids, sugars or carbon sources, proteins, growth factors, surfactants and pH indicators.

Furthermore, it is possible that the culture medium used is formulated such that the cultivated cells retain desired characteristics, such as one or more characteristics including, but not limited to, being non-tumorgenic, growing as adherent cells, supporting the replication of the infectious viral particle when cultured.

In one embodiment, any suitable inorganic salt that is known to a person skilled in the art can be present in the cell culture medium; preferably, the inorganic salts are chosen from the group consisting of CaCI₂, CuS0₄, Fe(N0₃)₃, FeS0₄, KCL, MgCI₂, MgS0₄, NaCI, NaHC0_3, Na₂HP0₄, NaH₂P0₄, Na₂Se0₃, Na Acetate, and ZnS0₄.

In a further preferred embodiment, any suitable amino acid that is known to a person skilled in the art can be present in the cell culture medium. Preferably, the amino acid is chosen from the group consisting of L-alanine, L-arginine-HCI, L-asparagine (free base), L- asparagine x H₂0, L-aspartic acid, L-cysteine-HCI (anhydrous), L-cysteine-HCI-H₂0, L- cystine, L-cystine x 2HCI, L-glutamic acid, L-glutamine, glycine, L-histidine-HCI x H₂0, L- isoleucine, L-lysine, L-lysine-HCI, L-methionine, L-phenylalanine, L-proline, hydroxyl-L- proline (non-animal), L-serine, L-threonine, L-tryptophan, L-tyrosine, L-tyrosine x 2Na x 2H₂0, and L-valine.

In a further preferred embodiment, any suitable nucleic acid that is known to a person skilled in the art can be present in the cell culture medium. Preferably, the nucleic acid is chosen from the group consisting of adenine sulphate, adenosine-5-phosphate, adenosine-5- triphosphate, glutathione (reduced), guanine Fibonacci (FB), guanine HCI, D-ribose, 2- deoxy-D-ribose, thymine, uracil, xanthine, and hypoxanthine.

In a further preferred embodiment, any suitable vitamin that is known to a person skilled in the art can be present in the cell culture medium. Preferably, the vitamin is chosen from the group consisting of P-amino benzoic acid, ascorbic acid, D-Ca pantothenate, D-biotin, choline chloride, cod liver oil, ergocalciferol, folic acid, l-inositol, menadione, nicotinamide, nicotinic acid, putrescine 2HCI, pyridoxal-HCI, pyridoxine-HCI, riboflavin, thiamine-HCI, dithiooctanoic acid, thymidine, Na₂ tocopherol ph, DL-tocopherol acetate, vitamin A acetate, and vitamin B ₂.

In a further preferred embodiment, any suitable lipid that is known to a person skilled in the art can be present in the cell culture medium. Preferably, the lipid is chosen from the group consisting of cholesterol, linolenic acid, linoleic acid, palmitoleic acid, arachidonic acid, stearic acid, myristic acid, palmitic acid, and oleic acid. In a further preferred embodiment, any suitable sugar or carbon source that is known to a person skilled in the art can be present in the cell culture medium. Preferably, the sugar or carbon source is chosen from the group consisting of D-glucose, D-fructose and Na pyruvate.

In a further preferred embodiment, any suitable protein that is known to a person skilled in the art can be present in the cell culture medium. Preferably, the protein is chosen from the group consisting of BSA (bovine serum albumin; fraction V) such as Albumax I (lipid-rich BSA, bovine), lactalbumine hydrolysate, and Primatone RL.

In a further preferred embodiment, any growth factor that is known to a person skilled in the art can be present in the cell culture medium. Preferably, the growth factor is chosen from the group consisting of epidermal growth factor (murine, recombinant murine or recombinant human), insulin such as human insulin (crystalline biosynthetic), and transferrin (holo bovine).

In a further preferred embodiment, any suitable surfactant that is known to a person skilled in the art can be used. Preferably, the surfactant is chosen from the group consisting of Pluronic F68 and Tween 80. Furthermore, it is also possible to use ethanol.

In a further preferred embodiment, it is possible to use indicators such as pH indicators. Any suitable indicator that is known to a person skilled in the art can be used. Preferably, the indicator used is phenol red.

In one embodiment it is further preferred that the cell culture medium is supplemented with antibiotics. In general, any suitable antibiotic that is known to a person skilled in the art can be used in a suitable concentration, either alone or in combination with other antibiotics. In a preferred embodiment, no antibiotic/s is/are used.

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.

Omitting supplementing the cell culture medium and/or the virus propagation medium with antibiotics, in particular with macrolide polyene antibiotic/s or derivatives or analogues thereof, provides for an improved production process of viral components, e.g. with regard to robustness or handling of the process. By applying the method according to the present invention, despite the above described omittance of antibiotic/s, it is possible to arrive at an enhanced viral yield, even when using a very low MOI as described herein.

Preferably, the medium used for virus propagation, i.e. the replacement culture medium, is BSA-free or essentially BSA-free. Essentially BSA-free means that only trace amounts of BSA are contained in the medium that do not have any influence, preferably not any negative influence, on the cell culture and/or on the following processes carried out and/or on the intended use of the propagated viral particles and/or viral components.

Furthermore, any suitable combination of the aforementioned components and/or additives can be used.

An example for a suitable medium that can be used for the process of the present invention is for instance Episerf, which is commercially available (e.g. Invitrogen or Lonza). Episerf containing BSA and Episerf without BSA have an osmolality of about 360 mOsmol/kg.

In the method according to the present invention, to a cell culture composition containing an amount of cells of at least 0.5 x 10⁶ cells/ml at the time of virus addition, or of the preferred amounts as defined herein, infectious viral particles are added. The time period necessary for culturing the cells until the respective desired cell density has been reached mainly depends on the scale of the cell culture. Therefore, in case the cells are cultured in large/industrial scale (see above), the cells used for the process of the present invention are proliferated for up to 40 days, preferably for up to 30 days until a cell density of at least 0.5 x 10⁶ cells pro ml is reached. Accordingly, in case the cells are cultured in semi-industrial/small scale, the cells used for the process according to the present invention are proliferated for up to 20 days, preferably for up to 10 days or less until the desired cell density is reached. However, the respective necessary time period needed for reaching said cell density it is known to a person skilled in the art, as e.g. the cell density of the culture can easily be determined by routine methods such as cell counting. In a preferred embodiment, the cells that are propagated are MDCK cells. In a further preferred embodiment, the cells are cultivated as adherent cells, in tissue culture flasks or being attached to micro carriers such as Cytodex 3 micro carriers (e.g. obtainable from Amersham Biosciences). The cell density per ml cell culture can be determined by any method that is known to a person skilled in the art. For example, if the cells are cultivated in an adherent cell culture such as on microcarrier beads, a NucleoCounter (Chemometec) can be used. With this apparatus, it is possible to determine the total concentration of cells in a sample without enzymatic treatment. The cell density of the respective cell culture vessel can be determined once a day, optionally more times a day.

If necessary, in the course of the cultivation of the cells, an exchange of the cell culture medium can be carried out, as described herein.

In a further preferred embodiment in the process according to the present invention, to the starting amount of cells, which is at least about 0.5x10⁶, a total number of infectious viral particles, which is used and added to such starting amount of cells, of less than 10^"5 (indicated in a ratio per cell, MOI; also see elsewhere in the specification) is added. The term "used total number of infectious viral particles" within the meaning of the present invention denotes that the total number of infectious viral particles that is externally given to the cell culture composition is less than 10^"5. The term "inoculation" or "virus addition", respectively, within the meaning of the present invention denotes that infectious viral particles are added externally to the cell culture composition. Within the meaning of the present invention, it is possible to add to the cell culture composition the total number of infectious viral particles in one step. However, it is also possible to add to the cell culture the inventive MOI in two or more steps, e.g. a first virus addition step is carried out with one part of the total number of infectious viral particles, followed by a second or multiple virus addition step/s with the other part/s of infectious viral particles. The time lag between the respective inoculation steps can vary. In a preferred embodiment, the virus addition as a whole is carried out in one step and/or is completed within a time period of 2 h or less, preferably within a time period of 1 h or less, even more preferably within a time period of 30 min. h or less and most preferably within a time period of 15 min. or 10 min. or less.

In general, the addition of virus to the cells being present in the cell culture can be carried out by any suitable method that is known to a person skilled in the art. For instance, the cells can be inoculated by simply adding the virus inoculum to the cell culture. Within the meaning of the present invention, it is possible to use the cell culture medium containing the same components during the whole cultivation period (cell cultivation prior to inoculation and virus propagation), but it is also possible to replace the medium and to use a medium containing different components/additives/substances compared to the medium used before. With this regard, reference is made to the description above.

Preferably, the cell culture medium used for the cultivation of the cells is replaced with virus propagation medium, which has an osmolality of at least 95% of the osmolality of the culture medium which is used for the cultivation of the cells (see above): As can be deduced e.g. from figures 1 and 2, the protein yield and the viability of the cells can be increased in case the cell culture medium used for the cultivation of the cells is replaced with a virus propagation medium having at least 95% of the osmolality of the cell culture medium that is used for the cultivation of the cells. A similar favourable effect can be seen in case the cell culture medium is replaced with a virus propagation medium not having a significantly lower total amount of proteins, growth factors and/or inorganic salts compared to the cell culture medium, or in case the virus propagation medium has both properties at the same time.

In a preferred embodiment according to the present invention, the inoculation or virus addition, respectively, is carried out as follows: At the end of cell cultivation, prior to or during adding infectious virus particles, the cell culture medium is replaced by virus culture medium by means of carrying out washing steps with suitable washing solutions that are known to a skilled person. The virus inoculum, which is prepared preferably less than one hour before adding, is added to the cell culture. After having added the virus inoculum, optionally with exchanging medium at this step simultaneously, the infection of the cells with virus and virus propagation starts. Within the meaning of the present invention, the term "after virus addition" or "after inoculation" denotes the time period starting with the finished adding of the virus inoculum to the cell culture. Furthermore, within the meaning of the present invention, the term "during the virus addition step" denotes the time period during which the inoculation is carried out, or at least that there is an overlap in time. The virus addition step starts with the addition of infectious virus particles to the cell culture and ends when the addition of the complete virus inoculum is finished. The cell density at the time of virus addition can be determined by any suitable method that is known to a person skilled in the art.

As described above, the inoculation of the cells with virus (e.g. with the infectious particle such as an influenza particle) can be carried out in a reduced culture medium volume compared to the final volume of the culture medium. For instance, the reduced volume can correspond to about 70 %, preferably to about 50 %, more preferably to about 40 % and most preferably to about 30 % of the final volume of the culture medium. At the end of the inoculation period, the reduced cell culture medium is filled up with virus culture medium to 100 % of the final concentration. As described above, the virus culture medium used for this filling up can be the medium used during the inoculation period, or it can be a culture medium different from the medium used during the inoculation period. Preferably, it is the medium used for inoculation.

After the inoculation of the cells (or the incubation of the cells with the viral suspension, respectively) has been finished, which is the case as soon as the addition of the virus inoculum to the cell culture has been finished (see above), the cells are cultivated for a further certain period of time (virus propagation phase) until a desired CPE can be observed (see below). Furthermore, the end of the virus production step can also be indicated by any indicator that is known to a skilled person, such as by empty microcarriers and/or by an increase of pO_z or decreased 0₂-flow into the bioreactor. In general, this is the case after a further cultivation period of about 5 h to 8 days, preferably of about 10 h to about 6 days, about 10 h to about 5 days, more preferably of about 10 h to about 4 days and most preferably of about 12 h to about 3 days. A CPE is a pathogenic effect which, in turn, is an adverse effect on the growth or maintenance of a cell, particularly the effects associated with microbial and/ or viral infections. Pathogenic effects include, but are not limited to, cytopathic effects, cell rupture, inhibition of growth, inhibition of protein synthesis, or apoptosis. A CPE is an observable change in cell structure which may vary with cell types and cause of death, and can be determined according to established knowledge in the art. For example, some of the most common effects of viral infection are morphological changes such as cell rounding and detachment from the substrate, cell lysis, syncytium formation and inclusion body formation. The CPE can be determined according to any suitable method that is known to a person skilled in the art, e.g. by microscopic observation and estimating the percentage of cell coverage of the reaction vessels and microcarriers.

In a preferred embodiment, the process according to the present invention further comprises one or more steps of further processing the propagated viral particles, preferably for preparing isolated or purified propagated viral particles, e.g. the influenza viral particles. Preferably, 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. Further preferred, the processed viral particle comprise one or more influenza virus antigen/s, such as HA and/or NA.

The isolation of the propagated viral particles from the cell culture medium can be carried out by any suitable method that is known to a skilled person. For instance, the viral particles can be harvested by e.g. separating the cells or cell residues from the culture medium by seperators or filters. Known methods include, but are not limited to, filtration, ultrafiltration, adsorption on barium sulphate and elution, and centrifugation. For example, crude medium from inoculated and infected cultures can first be clarified by (continuous) centrifugation at e.g. 200 to 2000 g for about 5 minutes (min) to remove cell debris and other particulate matter. Alternatively, the medium is filtered through an e.g. 0.8 m cellulose acetate filter to remove intact cells and other particulate matter. Optionally, the clarified medium supernatant is then centrifuged to pellet the viruses, such as the influenza viruses at, e.g., 15.000 g for about 3-5 hours (hrs) or concentrated by ultra filtration using e.g. 300 KD MWCO filter membranes. Afterwards, the viral particles pellet may be resuspended in an appropriate buffer such as a STE buffer (0.01 M Tris-HCI; 0.15 M NaCI; 0.0001 M EDTA) or PBS (phosphate buffered saline) at pH 7.4, then the isolated viral particles may be concentrated and/or further purified for instance by density gradient ultra centrifugation on sucrose (60% - 12%) or potassium tartrate (50% - 10%). The gradients are centrifuged at a speed and for a time sufficient for the viral particles to concentrate into a visible band for recovery. Further, the propagated viral particles can be subjected to an ion exchange chromatography or size exclusion chromatography. Furthermore, the propagated viral particle and/or the nucleic acid contained can be subjected to enzymatic treatment.

Furthermore, the propagated viral particles can be inactivated, killed or attenuated according to any suitable method that is known to a skilled person. Attenuation can for instance be accomplished chemically or by standard serial passages wherein a sufficient number of passages in a susceptible cell culture is employed until the virus particle is rendered nonpathogenic without the loss of immunogenicity. Furthermore, the viral particles may be inactivated for instance by detergent or formaldehyde treatment.

In a further preferred embodiment of the present invention, the obtained propagated viral particle can be further processed in order to obtain e.g. a viral antigen such as HA and/or NA. In a preferred embodiment, HA is obtained. It is known to a skilled person how viral antigens can be obtained. The obtained processed viral particles can be used in the production of influenza vaccine.

The further processing step can optionally be carried out after the aforementioned harvesting and/or isolation steps and may include the extraction of a viral component, which can be associated with the cells or cell fragments, separating the viral component, isolating the viral component, and purifying it, described in general textbooks e.g. Bioseparations: Downstream Processing for Biotechnology by Paul A. Belter (Author), E. L. Cussler (Author), WeiShou Hu (Author). As mentioned above, the viral particles such as the influenza viral particles according to the present invention comprise for instance virosomes. A virosome is a unilamellar phospholipid bilayer vesicle with a suitable mean diameter, for example in the range of from about 70 nm to about 150 nm. Essentially, virosomes represent reconstituted empty virus envelopes, devoid of the nucleocapsid including the genetic material of the source virus. Virosomes are not able to replicate but are pure fusion-active vesicles that contain functional viral envelope glycoproteins such as influenza virus HA and NA intercalated in the phospholipid bilayer membrane.

Furthermore, the processed viral particle comprises one or more influenza antigens, such as HA and/or NA. This influenza virus antigen can be derived from a virus strain leading to a seasonal, pandemic and/or epidemic outbreak of influenza.

HA can for instance be found on the surface of the influenza viruses. It is an antigenic glycoprotein that is responsible for binding the virus to the cell that is being infected. To date, at least 16 different influenza HA antigens are known. These subtypes are named H1 through H16. NA is an enzyme which cleaves the glycosidic linkages of neuraminic acid. To date, at least nine subtypes of influenza neuraminidase are known. These subtypes can be found, for instance, in databases that are known to persons skilled in the art, such as the PubMed database (e.g. http://www.ncbi.nlm.nih.gov/, http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html, http://www.ncbi.nlm.nih.gov/nuccore and

http://www.ncbi. nlm.nih.gov/nuccore/145284465?ordinalpos=1&itool=EntrezSystem2.PEntre z.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum). It is also possible that the influenza vir_tal protein comprises any of these HA and/or NA coding sequences, either alone or in combination with each other. It is also possible that the influenza viral protein contains only parts of these sequences. Preferably, the influenza viral protein contains the complete sequence or a part of the sequence coding for H1 , H2, H3, H5, H6, H7, N1 , N2, N3 or N7, either alone or in combination, preferably for H5. In a further preferred embodiment, the polynucleotide construct comprises sequences or part of the sequences coding for H1 N1 , H2N2, H3N2, H6N1 , H7N3 or H7N7, preferably the sequences or part of the sequences coding for H5N1.

Furthermore, the above described method according to the present invention can be used in the production of a viral vaccine, preferably of an influenza viral vaccine. For this purpose, a step of formulating said viral particles or said viral components or parts of viral components in a vaccine composition can be earned out. The viral particles or viral components or parts of viral components can be formulated by suitable methods that are known to a skilled person to provide a viral vaccine for administration to a subject. The formulation of viral particles and/or components or parts thereof suitable for a viral vaccine may comprise additional steps including, but not limited to, buffer exchange and sterilization steps. In general, viral vaccines can be administered prophylactically or therapeutically with an appropriate carrier or excipient, respectively. Preferably, such a carrier is a pharmaceutically acceptable carrier such as sterile water, buffered saline solution, dextrose solution, glycerol solution, or a combination thereof. Furthermore, in general a carrier is selected to minimize allergic reactions or other unwanted effects, and to suit the particular route of administration, e.g. subcutaneous, intramuscular, intranasal, and so on.

In a further preferred embodiment, the vaccine composition according to the present invention can contain further components and/or additives, such as adjuvants. Adjuvants are substances which increase the immune response, e.g. hydroxides of various metals, constituents of bacterial cell walls, oils or saponins.

Furthermore, the present invention is also related to 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:

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, or wherein the MOI is a preferred MOI as defined herein,

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 10^"5

In a preferred embodiment, the viral particles and/or viral components obtained after inoculating the cells with said reference MOI are obtained under the same conditions as the viral particles and/or viral components obtained after inoculating the cells with a very low MOI. The term "same conditions" within the meaning of the present invention denotes that the same cell culture conditions are used, e.g. with regard to temperature, C0₂ concentration, p0₂ values, pH range, and the culture medium. All preferred embodiments and parameters as described herein with respect to the process for propagation of the viral particles are also suitable with respect to the testing method according to the invention.

It has surprisingly been found out that balancing (i) the pre-selected virus strain used, (ii) the density of cells (cells per ml) to which with infectious viral particles are added and (iii) the MOI which is used for the inoculation of the cells leads to an increase in the propagated viral particles and/or in the yield of viral components, provided that the cells have a cell density of at least 0.5x10^"6 cells/ml when inoculated, and provided that the MOI is a very low MOI of less than 10 ^s. Further suitable MOIs are described elsewhere herein. In prior art processes, to cells a "reference" MOI, which is a MOI being equal to or higher than 10^"5, preferably the reference MOI is 10³ is added. However, it has been found out in the present invention that the inoculation of the cells with this reference MOI does not necessarily lead to a maximum yield of propagated virus particles or viral components, respectively. Instead, an improved, optimized yield can be reached if cells having a certain minimum cell density are inoculated with a total number of infectious viral particles of a pre-selected virus strain, wherein the MOI is a very low MOI of less than 10^"5. By applying said testing process, an MOI providing for such improved yield of propagated viral particles and viral components, respectively, individual for each pre-selected virus strain, can be identified. Furthermore, due to the fact that no up-scaling effect does appear, by using the testing process according to the present invention multiple small scale test cultures can be run in parallel at the same time while observing the minimum cell density disclosed herein, in order to test whether an inoculation using a very low MOI leads to a higher yield of virus particles or viral components, respectively, and if so, which very low MOI is the one that provides for the highest possible yield. This very low MOI can then be used for the propagation of the pre-selected virus strain at a large/industrial scale; it is not necessary to determine anew a suitable very low MOI for a large scale process. Surprisingly, the positive effect with regard to obtained viral particles and/or viral components that can be observed in small scale cell cultures, correspondingly transforms into large scale cell cultures.

With regard to the cells being inoculated, the inoculation of a cell culture composition, the infectious viral particles used for inoculation and addition, respectively, the total number of infectious viral particles, the propagated viral particles and viral components, and the cell culture medium, reference is made to the specification above.

In a preferred embodiment of the testing process, in a first step a), cells are grown in a culture medium until a cell density of at least 0.5x10⁶ cells/ml is reached. In a further preferred embodiment, the cells are grown until a cell density of at least 3.0x10⁶ cells/ml, preferably of at least 5.0x10⁶ cells/ml, further preferred of at least 7.0x10⁶ cells/ml, even further preferred of at least about 9.0x10⁶ cells/ml, preferably of at least about 11.0x10⁶ cells/ml, or further preferred of at least 13.0x10⁶ cells/ml is reached.

The cell density in a sample obtained from the culture vessel can be determined by any method that is known to a person skilled in the art. If the cells are cultivated in an adherent cell culture, the cell density can be determined by using a NucleoCounter as described in Example 1.

In a preferred embodiment, the cells can be cultured and proliferated as adherent cells as described above.

In a preferred embodiment of the testing process, in step b) to the cells infectious viral particles of the pre-selected virus strain are added, preferably an infectious viral particle as described above, using a very low MOI. The very low MOI is a MOI in the range of less than 10^"5, as described above. The inoculation of the cells, i.e. the addition of the infectious viral particles, can be carried out as described above.

Furthermore, by carrying out the testing process and the method for the propagation of influenza virus (or influenza viral particles, respectively) according to the present invention, it is possible that a protease is added which brings about the cleavage of the precursor protein of HA and thus the adsorption of the viral particle or virus, respectively, on the cells, thereby e.g. additionally increasing the yield of the viral components. The protease can be added shortly before, simultaneously or shortly after the addition of the virus, e.g. the influenza virus, to the cells. The protease can also be added after a time period of 8 h to 10 h after the addition of the virus. In case the addition of the protease is carried out simultaneously to the inoculation, the protease can either be added directly to the cell culture, or e.g. as a concentrate together with the infectious viral particle inoculum. In a preferred embodiment, a protease is added to teh replacement culture medium after the addition of the infectious influenza viral particles in a concentration range of i g/ml to 50 g/ml. In a further preferred embodiment, the protease is added to the replacement culture medium in a concentration range of more than 1.0 pg/rnl to 50 g/ml, preferably in a concentration range of 1.5 pg/rnl to 50 pg/ml, more preferably in a concentration range of 2.0 pg/ml to 50 pg/ml, and even more preferably in a concentration range of 2.5 pg/ml to 50 pg/ml. Suitable proteases that can be added are known to a skilled person, for instance the protease is a serine protease, a cysteine protease or an asparagines protease. An example for a preferred, suitable protease is trypsin.

In a further preferred embodiment, after step a), preferably after step a) and prior to or during step b), of the testing process, the culture medium which is used for culturing the cells with a very low MOI and reference MOI is replaced with a virus propagation medium. With regard to said virus propagation medium, reference is made to the description above. The medium replacement can be carried out according to any suitable method that is known to a skilled person, e.g. it can be carried out as described above.

Furthermore, with regard to the culture media that can be used in the cell culture phase and in the virus propagation phase, respectively, reference is made to the description above.

The inoculation of the cell culture as well as the cultivation of the cell culture after the inoculation period (virus propagation) is carried out as described above.

After the end of the cultivation period has been reached, optionally, prior to step c) of the testing process, a harvesting and/or isolation step is carried out. This/these step/s can be carried out as described elsewhere herein.

The obtained propagated viral particles can be further processed. The processed viral particles (or viral components, respectively) comprise inactivated viral particles, and/or attenuated viral particles, and/or split virus antigen, and/or a subunit virus antigen, and/or virosomes. Furthermore, the amount or yield, respectively, of viral particles and/or components can be determined.

In general, the amount of propagated viral particles can be determined by any suitable method that is known to a person skilled in the art. Preferably, the viral yield can be quantified by determining the TCID₅₀ as described above. The amount of the obtained viral components can be determined by any suitable method that is known to a person skilled in the art. An example of a suitable method is the Single Radial Immuno Diffusion (SRID) test which can be used for the determination of HA, carried out according to the instructions of the European Pharmacopoeia (Monograph 2149 (04/2009:2149) "Influenza Vaccine (surface antigen, inactivated, prepared in cell cultures). Immunochemical methods are based on the selective, reversible and non-covalent binding of antigens by antibodies. These methods are employed to detect or quantify either antigens or antibodies. The formation of an antigen- antibody complex may be detected, and the amount of complex formed may be measured by a variety of techniques. The immunoprecipitation method, SRID, is a simple quantitative immunodiffusion technique. When the equilibrium between the external and the internal reactant has been established, the circular precipitation area, originating from the site of the external reactant, is directly proportional to the amount of the antigen applied and inversely proportional to the concentration of the antibody in the gel. In brief, an agarose gel containing a predetermined amount of anti-serum is cast in a gel mould. Lyophilized haemagglutinin antigen reagents from the respective suitable virus strain with a predetermined content of HA/vial as well as lyophilized anti-haemagglutinin sheep serum of the respective suitable virus strain with a predetermined content per ml of agarose is used. Reference antigen and monovalent bulks are diluted to predetermined concentrations and treated with 1 %

Zwittergent (to disrupt virions) for 30 min. at room temperature. Treated antigen and bulks are then inoculated into holes punched into the solidified gel and left to diffuse at least 18 hrs, overnight at room temperature. Afterwards, the gel is dried onto a gel bond film and dried with filter paper, such as Whatman filter paper, and paper towels. Once dry, the gel is stained with Coomassie brilliant blue G-250 in order to ascertain the presence of antibody/ antigen aggregates. The diameters of the resulting precipitation zone are measured using suitable software. Haemagglutinin content is then calculated by using a statistical analysis program.

In step c) of the testing process, the yield of the viral particles and/or processed viral particles obtained after adding the infectious viral particles of step b) to the cells is compared 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 10^"5. Preferably, the reference MOI is 10^"3.

With regard to the term "reference MOI", reference is made to the description above.

In order to determine the most suitable MOI for the process of propagation viral particles, at least two different MOIs are tested in culture vessels containing the same culture medium and using the same process conditions. For this purpose, at least two culture vessels are used; the first culture vessel contains the cell culture composition that is inoculated with the reference MOI as described above and the second culture vessel contains the cell culture composition that is inoculated using a very low MOI according to the invention. However, it is preferred to test more than one very low MOI according to the invention in order to find the most suitable MOI for the respective process conditions. Consequently, it is preferred that more than one inoculation with a reference MOI and/or more than one inoculation with a very low MOI is carried out. Furthermore, it is possible to carry out a series of cultivation procedures using different very low MOIs according to the invention at the same time. It is further possible to carry out the testing process at any scale, preferably the testing process is firstly carried out at small scale, followed by using the very low MOI at semi-industrial or large scale cell culture.

In a further preferred embodiment, the amount of viral particles or processed viral particles obtained after adding to the cells the infectious viral particles of step b) is at least 1.2-fold, preferably at least 1.5-fold, more preferably at least 2-fold, and most preferably at least 3-fold the amount of viral particles or processed viral particles obtained after adding to the same type of cells the same type of infectious viral particles using a reference MOI in a range of at least 10^"5 or higher, wherein the amount of viral particles is preferably determined 24 hours after virus addition. The amount of propagated viral particles and viral components can be determined as described above.

The present invention is also directed to a cell culture composition for the production of a viral particle and/or a viral component, wherein

b) to the cell culture composition of step a) a total number of infectious viral particles using a very low MOI of less than 10^~5 has been added, and

In a further preferred embodiment, the amount of living cells being present in the cell culture composition of step b) within a range of 1 day after virus addition corresponds to at least about 100% or at least to about 110% of the starting amount of cells being present in the cell culture composition.

In a further preferred embodiment, the amount of living cells being present in the cell culture composition of step b) within a range of 48 hours after virus addition corresponds to at least about 5%, preferably at least about 10%, preferably at least about 15% of the starting amount of cells being present in the cell culture composition.

With regard to e.g. the cell culture composition, the viral particle and/or viral component, the starting amount of cells, the inoculation of a cell culture composition (i.e. the addition of viral particles to the cell culture), the MOI, and the method of determining the amount of cells being present in a cell culture (cell density, expressed in cells/ml), reference is made to the description above. Advantageously, such a cell culture composition allows for an improved production of viral components, for instance with regard to the yield obtained and/or with regard to the time needed for the production of said viral components.

In a preferred embodiment, the cell culture composition of step a) has been inoculated with a MOI tested according to the testing process as described herein.

In a further preferred embodiment, the cell culture composition exhibits a HA/ml ratio after 4 days after virus addition that is higher than 8 pg/rnl, preferably higher than 9 pg/rnl, more preferably higher than 10 pg/ml, even more preferably higher than 15 pg/ml, and most preferably higher than 20 pg/ml.

With regard to the term "after virus addition", reference is made to the description above.

Furthermore, the present invention is also related to the use of a cell culture composition for the propagation of viral particles, wherein a cell culture composition containing a starting amount of cells of at least 0.5x10⁶ cells/ml, or other preferred amounts of cells as defined herein, is inoculated with infectious viral particles, wherein the used total number of infectious virus particles per cell necessary to infect a cell ("Multiplicity of Infection", MOI) is less than 10^"5. Other preferred embodiments as described above with respect to the process for propagation of viral particles or the testing method are also suitable for preparing the cell culture compositions according to the invention.

Again, with regard to the cell culture composition, viral particles, starting amount of cells, inoculation of the cell culture, infectious viral particles and MOI reference is made to the description above. Furthermore, the propagated viral particle can be harvested and/or isolated as described above, and, optionally, further processing steps as described above can be carried out.

The present invention also relates to a process for producing immunogenic viral antigen, preferably influenza viral antigen, and more preferably immunogenic haemagglutinin (HA) protein, comprising the steps of

In a preferred embodiment, the further processing step comprises an isolation step, and, optionally, a purification step of the propagated viral particles. This step/these steps is/are followed by an isolation step of the immunogenic viral antigen, preferably of the influenza viral antigen, and more preferably of the immunogenic haemagglutinin (HA) protein. The above-mentioned further processing steps are known to a skilled person and are routine methods. They can be any processing step(s) that is (are) suitable for arriving at an isolated immunogenic viral antigen as described above. In a preferred embodiment, the following isolation step of the propagated viral particles and isolation step of Immunogenic HA protein is carried out by solubilisation e.g. reviewed in "Downstream Processing: From Egg to Cell Culture-Derived Influenza Virus Particles", Chem. Eng. Technol. 2008, 31 , No. 6, 846-857 by Michael W. Wolff and Udo Reichl.

Preferably, the viral particles are firstly isolated and then immunogenic haemagglutinin (HA) protein is separated or purified, respectively, from the viral particles and, subsequently, immunogenic haemagglutinin is isolated. As described in more detail above, the further processing step is preferably carried out after the harvesting and/or isolation steps of the viral particles and may include the extraction of a viral component, which can be associated with the cells or cell fragments, separating the viral component, isolating the viral component, and purifying it.

In addition, the present invention relates to an immunogenic haemagglutinin (HA) prepared by a process according to the invention.

The following drawings and examples illustrate the present invention in more detail, which are however presented for illustrative purpose only and shall not be understood as limiting the scope of the present invention in any way.

Brief description of the drawings

Fig. 1 shows the HA protein kinetics (obtained by the SRID assay) produced by the cells infected with the virus strain A/Wisconsin X-161 B.

The upper line is the condition "medium replacement EPI w/o BSA, MOI 1x10^"6". This means that a very low MOI according to the invention and a virus propagation medium (EPI w/o BSA) which has not a significant lower amount of proteins, growth factors and/or inorganic salts and/or which has at least an osmolality of 95% of the medium used for cell culture has been used (only BSA is not contained in the virus propagation medium). This preferred process provides the best results.

The graph in the middle is the condition "medium replacement CM; MOI 1x10^"6"", which provides the improvement of using very low MOI according to the invention. In this condition, the cell culture medium has been replaced by a virus propagation medium that has a significant lower amount of proteins, growth factors and/or inorganic salts compared to the cell culture medium, and/or this medium has an osmolality of less than 95% of the osmolality of the medium used for cell culture. This virus propagation medium is referred to as "CM"- medium.

The lower graph is the condition "medium replacement CM; MOI 1x10³ " In this condition, the cell culture medium has been replaced by CM medium and has been inoculated with a MOI of 1 x10^"3 . This process according to the prior art provides the lowest HA protein content.

The average haemagglutinin protein yield for the condition "medium replacement Epi w/o BSA, MOI IxlO^"6" was 37.0 pg/ml at day 4 post infection. The condition "medium replacement CM, MOI 1x10^"6"" resulted in a HA yield of 19 g/ml at day 4 after virus addition, and the condition "medium replacement CM; MOI 1x10^"3"" had an average HA yield, 4 times lower with 8.7 pg/ml at day 4 after virus addition.

This figure shows that there is a significant increase in HA yield after the inoculation of the cell culture with very low MOI (e.g. MOI IxlO^"6, graph in the middle and upper graph) compared to the inoculation of the cells with reference MOI (e.g. MOI 1x10^"3, lower graph). Furthermore, it is shown that this effect is due to the lowering of the MOI (enhanced yield of HA, compare lower graph and middle graph), and that this effect can further be enhanced in case the medium which has been used for cell cultivation is not replaced with CM-medium, but with culture medium which has not a significant lower amount of proteins, growth factors and/or inorganic salts and/or which has at least an osmolality of 95% of the medium used for cell culture.

Fig. 2 shows the cell kinetics of attached cells during the propagation of the virus strain A/Wisconsin X-161 B

The continuous graph is the condition "medium replacement CM; MOI 1x10^"3'. This condition shows a fast and big decrease of cell density measured at day 1 post infection.

The dashed graph represents the condition "medium replacement CM; MOI IxlO^"6". Here, a slight decrease of the cell density measured can be seen. The spotted line is the condition "medium replacement EPI w/o BSA, MOI IxlO^"6". Here, a slight increase of the cell density measured at day 1 post infection can be seen.

This figure shows that after inoculation of cells with very low MOI (e.g. MOI IxlO^"6) more cells survive compared to the inoculation of cells with a reference MOI (e.g. MOI 1x10^~3), as indicated by the respective cell density (cells/ml). Furthermore, it shows that this effect is due to the lowering of the MOI, and that this effect can further be enhanced by a replacement of the medium prior to infection according to the invention.

Fig. 3 shows the TCID₅₀ during the propagation of the virus strain A/Wisconsin X-161 B.

The condition "medium replacement CM; MOI 1x10^{3 n} is the lowest, continuous graph, and the other conditions ("medium replacement Epi w/o BSA, MOI IxlO^'and "medium replacement, CM MOI 1x10^"6"") are the upper two lines with similar values. A higher TCID₅₀ indicates a higher infectious viral yield which results in a higher protein yield.

Methods

Determination of the osmolality and of the osmolality of a culture medium

The osmolarity/osmolality can be determined by using any known method (e.g. by using an osmometer) provided that the same method is used for determining the osmolarity/osmolality of the cell culture medium which is used for cultivation of the cells and of the culture medium which is used for virus propagation.

Osmolality is determined by measurement of the depression of freezing point. Determination of the protein content of a culture medium

The protein content of a medium can be determined by using any known method provided that the same method is used for determining the protein content of the cell culture medium which is used for cultivation of the cells and of the culture medium which is used for virus propagation. Several methods are available e.g. Lowry or Bradford assay.

Determination of the total amount of a substance or of the amount of an individual substance

The amount of amino acids, growth factors and inorganic salts can be determined by any known method, provided that the same method is used for determining the respective content in the cell culture medium which is used for cultivation of the cells and of the cell culture medium which is used for virus propagation.

The amount of amino acids can for instance be determined by using HPLC (High Pressure Liquid Chromatography) and the amount of growth factors can for instance be determined by Mass Spectroscopy. The content of inorganic salts can be determined by measuring the metal ion concentration of its salt(s) in the medium with an ion-selective electrode.

Examples

Example 1 : Cultivation of MDCK cells

The MDCK cells were cultured as adherent cells on Cytodex 3 microcarrier. Preparation of the Cytodex 3 microcarrier:

The Cytodex 3 microcarrier on which the MDCK cells were cultivated were prepared and sterilized according to the following protocol: The necessary amount of dry microcarriers is weighed and added to a phosphate buffered saline (PBS) solution for swelling of the microcarriers. After swelling, the microcarriers are washed with PBS and subsequently sterilized at 121 °C. After sterilization PBS is replaced by cell culture medium and ready to use.

The MDCK cells were cultivated in a bioreactor having a working volume of 3 I as follows: After preparation (calibration sensors and sterilization) of the bioreactor for cell culture, cell culture medium and microcarriers are added to the bioreactor. Cells are inoculated to the bioreactor with a cell density of about 0.4^*10^Λ6 cells/mL. Temperature is kept at 37°C, pH at 7.1 and dissolved oxygen (DO) concentration >40% air saturation. Cells will grow to a confluent monolayer of cells within 3 to 4 days.

Replacement of cell culture medium

Before the inoculation of the MDCK cells with the infectious viral particles took place, the cell culture medium was replaced by an enriched cell culture medium. The replacement of the culture medium was carried out according to the following protocol: Stirring is stopped and microcarriers are settled on the bottom of the bioreactor. After settling the microcarriers about 80% of the medium is removed by overpressure via a 80% dip pipe and subsequently the bioreactor is refilled with the enriched culture medium.

Determining the cell density (cells/ml) The cell density of the cells being present in the respective cell culture has been determined by using a NucleoCounter (Chemometec). A representative sample is taken from the bioreactor and after settling microcarriers in a test tube, the culture medium is removed. Subsequently the microcarriers are treated with a lysis buffer and a neutralizing buffer enabling to count the nuclei of the cells.

Example 2: Virus cultivation:

The cultivation of the virus strain A/Wisconsin/67/2005 (H3N2) X-161 B takes place at 35°C, pH 7.1 and DO >40%.

Analysis of the virus propagation

After a cell density was reached that was in a range of about 3.0x10⁶ cells/ml and about 4.0x10⁶ cells/ml, the cell culture was inoculated with the virus strain A/Wisconsin/67/2005 (H3N2) X-161 B. The inoculation of the cell culture composition was carried out according to the following protocol:

After replacement of cell culture medium the calculated amount of virus and separately a small amount of trypsin (0.5 ml_ 2.5% trypsin solution) is added. The virus propagation continues until about 100% cyto pathic effect (CPE) is reached, which usually takes about 3 days. CPE can be visually checked by microscope to estimate the cell coverage of microcarriers. Then the virus can be harvested.

Analysis of the virus sample

TCIDsn analysis

MDCK cells are infected with successive dilutions of virus; after several days the cytopathogenic effect (CPE) is recorded and can be calculated into a tissue culture medium median infective dose (TCID₅₀). The endpoint dilution technique such as the TCID₅₀ assay is a statistical way of measuring virus populations. Several statistical methods for analysing such data are available e.g. Spearman-Karber, Reed & Muench or Probit analysis.

SRID analysis (HA concentration):

Influenza virus is treated with Zwittergent to split the HA antigen into diffusible particles. After diffusion through an agarose layer which contains specific antiserum, the antigen forms a precipitating ring with the antiserum. By staining with Coomassie brilliant blue, this precipitating ring is made clearly visible. The diameter of the precipitating ring is compared with a standard solution treated in the same way. The contents is expressed as HA per ml_. (Monograph 2149 (04/2009:2149) "Influenza Vaccine (surface antigen, inactivated, prepared in cell cultures). Immunochemical methods are based on the selective, reversible and non- covalent binding of antigens by antibodies. These methods are employed to detect or quantify either antigens or antibodies. The formation of an antigen-antibody complex may be detected, and the amount of complex formed may be measured by a variety of techniques. The immunoprecipitation method, SRID, is a simple quantitative immunodiffusion technique. When the equilibrium between the external and the internal reactant has been established, the circular precipitation area, originating from the site of the external reactant, is directly proportional to the amount of the antigen applied and inversely proportional to the concentration of the antibody in the gel.

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 g/ml to 50 pg/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 pg/ml to 50 g/ml, preferably in a concentration range of 1.5 pg/ml to 50 pg/ml, more preferably in a concentration range of 2.0 pg/ml to 50 pg/ml, and even more preferably in a concentration range of 2.5 pg/ml to 50 pg/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.

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. 8. 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 o

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:

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 10