TW201202425A - Production of viral components - Google Patents

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 0.5x10<SP>6</SP> cells/ml and wherein the total number of infectious viral particles per cell added during the virus addition step (MOI) is less than 10<SP>-5</SP>, 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

201202425 VI. Description of the Invention: [Technical Field of the Invention] The present invention belongs to the field of the pharmaceutical industry and relates to a method for breeding influenza virus and the use of the method in the manufacture of influenza vaccine. Furthermore, the present invention relates to a method for testing whether the addition of a very low infectious virion number/cell (MO:[) preselected virus strain to a cell culture composition for propagating virions results in an increase in virion and/or processing virion production. Methods [Prior Art] Vaccines are used to protect groups from common pathogenic threats and have a significant impact on reducing disease burden and increasing life expectancy. The effective use of vaccines relies primarily on the ability to rapidly produce large amounts of vaccine material and increase the number of vaccine doses available, with different vaccine materials requiring different growth conditions to achieve acceptable yields. In this regard, patent application US 2006/0188977 Al (2006) "Non-tumorigenic MDCK cell line for propagating viruses", in particular, describes the production of vaccine materials (such as viruses) in cell culture. Herein, MDCK cells are propagated in a medium, infected with a virus, and cultured. The replicated virus is then separated. The cells are infected with the virus at an MOI of about 0.0001 to about 10.

Govorkova et al. ("African Green Monkey Kidney (Ve:ro)

"African green monkey kidney cell line (Vero) and Martinda The same dog kidney (Madin-Darby 154987.doc 201202425 canine kidney, MDCK) cells are also suitable for the production of a host cell system for replication of influenza virus, because Vero cell strain also provides a sufficient number of influenza A and B viruses to meet the pandemic. The vaccine needs faced by the disease. To infect two different cell lines with the influenza virus tested, 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, February 2004, pp. 1851-1857) describes the modified influenza virus mainly Viral strains with improved virus rescue and growth properties in Vero cell lines. It demonstrates that the improved properties are mediated by the PR8 NS gene replacing the Vero-adapted reassortant virus's PR8 NS gene (Eng53/v-a). Viral replication was assayed by infecting Vero cells at a multiplicity of infection (MOI) of 0.01 with Vero adapted reassortant virus.

Voeten et al. ("Functionization of high-growth reassortant influenza A viruses generated in MDCK cells cultured in serum-free medium", Vaccine 17, 1999, 1942-1950) describes the use of a continuous cell line (MDCK-SF1), which is capable of Growth without fetal bovine serum produces a high growth reassortant influenza A virus that can be used to produce viral antigens in such cells. The high growth phenotype of the reassortant strain can be demonstrated by comparing the reassortant virus produced by the infection with the hemagglutinating unit (HAU) of the corresponding wild strain. Cellular infection was carried out in 96-well plates (200 μl/well' cell density of about 2χ105 cells/mm 154987.doc 201202425 liters) with different MOIs ranging from 0.001 to 0.000001. The authors demonstrate that the high growth phenotype can be attributed to the matrix protein of the high growth laboratory strain PR 34 or HK 68, respectively. Voeten et al. measured viral propagation kinetics in hemagglutination units (HAU). However, this test, as well as the use of a low number of cells in each experiment (this test was performed in a medium of about 200 μΐ volume in a 96-well plate, which contained cells with a density of about 2.2×10 6 cells/ml) produced no significantness. Inaccurate results.

Audsley and Tannock ("The growth of attenuated influenza vaccine donor strains in continuous cell lines", Journal of Virological Methods 123 (2005), 187-193) compared three Russian live attenuated donor strains (Russian live attenuated) in their study. Donor strain) Growth characteristics in MDCK and Vero cell cultures at different MOIs. In this study, it showed that the best MOI for growth of all donor strains was 0.01. EP 2 022 849 A1 relates to a method for the large-scale production of influenza viruses which describes tissue cultures which 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 in which mammalian cells are infected and cultured while maintaining a trypsin concentration in the range of 0.05-1.0 pg/ml.

Brands et al. ("Influvac TC: A Safe Madin Darby Canine Kidney (MDCK) Cell Culture-Based Influenza Vaccine", Developments in Biological Standardization, Vol. 98, January 1, 1999) describes the overall manufacturing method of the influenza virus vaccine InfluvacTC. Among them, MDCK practical seed virus (WSV) was manufactured from 154987.doc 201202425 WHO-designated egg-adapted influenza virus in MDCK cell culture in a serum-free medium at a low infection rate. In addition, describe the subsequent processing performed during vaccine manufacturing (infecting cells with viruses, collecting virus-containing media, virus purification, processing viruses into vaccines), and the safety measures already in place (inactivation, virus removal, It is assumed that there is a test for host cell contaminants). Brands et al. did not mention a specific MOI at all. WO 2008/043805 relates to the use of macrolide polyene antibiotics or derivatives or analogues thereof for use as a culture supplement for virus propagation in order to increase the yield and quality of the virus propagated in a continuous cell line. . Figure 1 of WO 2008/043805 shows that for each MOI tested, the presence of amphotericin B in the virus growth medium has a positive effect on viral replication. WO 2008/043805 discloses that cells can be infected with an MOI of 0.001 or 0.0001 to 0.00001 or even lower by using a macrolide polyene antibiotic or a derivative or analog thereof. WO 2008/043805, for example, does not mention the amount of cells in the cell culture when the virus is added, and the viable cell density determined at a specific time point after the virus is added. WO 96/15232 relates to a method for ensuring replication of a human influenza virus in a mammalian cell line at a low infection rate, wherein the minimum membrane protease concentration (about 0.05 pg/ml) in the medium is maintained. According to WO 96/15232, the test shows that when the MOI is in the range of about ΙχΙΟ·5 and lxlCT6 TCID50 per cell, the trypsin concentration of about 0.1 gg/ml is optimal, and about 5×10·7 TCID5 per cell. Your Majesty has achieved satisfactory results. WO 96/1 5232 neither discloses the amount of cells in the cell culture at the time of virus addition, 154987.doc 201202425', nor does it reveal the density of viable cells at a particular time point after virus addition. Despite the above description of high growth disease strains and cell lines for propagation of virions in cell culture and vaccine manufacturing methods, there is still a need and therefore there are improved virus component manufacturing methods and improved cells for the production of viral components. The target of the culture. SUMMARY OF THE INVENTION The present invention provides the following aspects, subject matter, and preferred embodiments, each of which is used alone or in combination to help achieve the objectives of the present invention: (1) A breeding comprising immunogenic hemagglutinin (HA) a method of influenza virus, wherein the cells are cultured in a cell culture in a first step and wherein an infectious influenza particle (such as a type A, type 15 or C influenza virus) is subsequently added to the cell in a second step a culture wherein the medium for culturing the cells is replaced with a weight osmolality (〇Sm〇laHty) of the medium previously used for culturing the cells before or during the virus addition step and has at least a sail, preferably at least 85%, more preferably at least 9Q% and optimally at least the weight of the osmolality medium, and which does not have a significantly lower amount, preferably not less than 5%, compared to the medium previously used to culture the cells, More preferably, the amount of protein, growth factor and/or inorganic salt is not less than 65% and preferably not less than 75%, and the amount of cells in the cell culture of Dan 1f when the virus is added. For at least 〇5xl() 6 cells/ml, wherein within 12 to 36 hours after virus addition, the degree of acne fat of living cells is not less than 40% of the cell density at the time of infection, which is added during the virus addition step The total number of m virions / 154987.doc 201202425 The cell (infection magnification, MOI) is less than 10_5» In the meaning of the present invention, the expression "the medium for culturing cells", the cell culture medium for culturing cells, or the "cell culture medium" means The medium used before the cell culture is inoculated with the virus. The medium (displacement medium) in which the cell culture medium is replaced is represented by the term "culture medium J, "viral medium" or "virus propagation medium" for propagation of virus. It is the medium used in the breeding stage of the virus. In one embodiment, the cell culture medium for culturing cells and the medium for culturing the virus (ie, the replacement medium) differ only in the presence/absence of BSA (bovine serum albumin) (the amount of other components and components is basically The same, that is, the amount of all individual substances does not differ by more than 30%, preferably not more than 20%.) In another embodiment, a protein-rich medium is used as a medium for culturing cells and a protein lacking medium is used. A medium that acts on the virus. The terms "protein rich" and "protein deficiency" are defined. The total amount of protein is higher in "protein-rich medium" than in "protein-deficient medium". Or, alternatively, preferably, the total amount of free amino acid contained in the culture &amp; for propagation of the drug is not higher or substantially less than the total amount of amino acid in the cell culture medium used to culture the cells. . In addition, preferably, the medium for propagating the virus does not have a significantly lower amount than the culture medium for culturing the fine moon. (for example, not less than not less than 60%, or even more than 50%) Preferably, the individual is preferably no less than 65% and preferably no less than 750/ of individual substances or groups of substances. The substances are selected from the group consisting of proteins, bio-factors and/or inorganic salts. For example, in use In the medium of the reproductive disease 154987.doc 201202425, the amount of protein is preferably not less than 50%, more preferably not less than 6%, even more preferably not less than 65% and most preferably not less than 75%. Bsa is not considered when referring to the amount of individual substances in the protein group, but BSA is considered when comparing the total amount of protein. In the meaning of the present invention, the term "BSA" includes any kind of bSa, such as bSa Fraction V or Albumax I ( Bovine lipid-rich BSA. In the meaning of the present invention, a substance group, such as a protein, contains all individual substances belonging to the group, such as lactalbumin hydrolysate. Furthermore, in the meaning of the present invention, the term "protein" , growth factors and / or inorganic salts On the one hand, the total amount of individual substances belonging to the group of proteins, growth factors or inorganic salts (for example, the total amount of all individual substances belonging to the protein group, or the total amount of all individual substances belonging to the growth factor group)' In another aspect, the total amount of all individual substances belonging to the group of proteins, growth factors, and inorganic salts is indicated. In another preferred embodiment of the method, the medium for propagating the virus and the cell culture medium for culturing the cells are Comparing, three, or one selected from the group consisting of growth factors and/or inorganic salts, which are not significantly lower (less than 75% of the amount of cell culture medium). To determine whether the criterion is achieved, Methods in the art determine the amount of individual substances or groups of substances (such as protein populations, growth factor populations, and/or inorganic salt populations). Subsequent amounts of individual substances or groups of substances in the virus propagation medium are combined with cells. The amount of the medium is compared. In another embodiment, the cell culture medium is not supplemented with antibiotics. In another embodiment, the virus is propagated and cultured. No antibiotics were added. 154987.doc 201202425 In another embodiment, the cell culture medium and the virus propagation medium are not supplemented with antibiotics. In detail, the 'cell culture medium and/or virus propagation medium is not supplemented with macrocyclic vinegar polyene antibiotics. Or a derivative or the like. (2) The method of item (1), wherein the replacement medium does not contain BSA. In another preferred embodiment, the replacement medium is not supplemented with an antibiotic. (3) as an item (1) Or the method of (2), wherein after adding the infectious influenza particles, the protease is added to the medium at a concentration ranging from 1 pg/ml to 50 pg/ml. (4) The method according to item (3), wherein the protease More than 1〇 to 5〇

The concentration range of Mg/ml, preferably in the range of 丨5 μ§Ληι to 5〇^/mi, more preferably 2% 〇/〇11 to 50 μ§/ηιι, and even better 2 $ pg/ A concentration range of mi to 5 〇 Mg/mi was added to the medium. (5) The method according to item (3) or (4), wherein the protease is a prion protein (6), such as any fixation-dependent cells in the items (1) to (5) (anch〇rage depe_nt can) step = item (1) to (method of any of Ο ... add MO in the virus!); etc.; add the total number of pure virions / cells (infection magnification m 〇 i) 4 at or below 1 〇 -6. τ (8) If the step (m) of the item (1) to (6) is m, the day 4', the virus is added, and the infectious virus particle MOI is equal to or less than 10-7. +~number/cell (infection magnification (9) method according to any one of items (1) to (6) t 'in which the virus is added; 154987.doc 201202425 The added infection 枓', the total number of virions / cells (Infection rate, MOI) is specific to or less than 1〇-8. (10) For the items (1) to (9), the method of s &amp; ,, within 12 to 36 hours after the virus is added, The density of the / team /, · I is not lower than the cell density of the virus when added 60 ° /. : π) The method of any one of items (1) to (9), wherein within 36 hours of virus addition, living cells The density is not less than 80% of the cell density at the time of infection. - a method according to any one, wherein the virus density is not lower than the cell density at the time of infection (12), and within 12 to 36 hours after the items (1) to (9), the living cells are 100% 〇/13) The method of any one of the items (1) to (10), wherein the medium for culturing the cells is replaced with the prior use before or during the virus addition step. The medium in which the cells are cultured has a molar molar concentration of the medium (displacement medium) having a molar osmolality of at least 95%. The method of any one of the items (1) to (13), wherein the amount of the cells in the cell culture is at least 3 〇 6 cells when the virus is added. The method of any one of items (1) to (10), wherein the amount of cells in the cell culture at the time of virus addition is at least 5 〇χ1〇6 cells/ml. (10) The method according to any one of the items (1) to (13), wherein the amount of the cells in the culture 16 is at least 7.〇xl〇6 cells/ml when the virus is added. The method of any one of items (1) to (13), wherein the amount of cells in the cell culture is at least 9 liters when the virus is added. (10) The method of 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 η 6 cells. The method of 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 13.0 χ 106 cells/ml. The method of any one of the items (1) to (19), wherein the cells used are animal cells, preferably mammalian cells. In a preferred embodiment, the mammalian cell line is selected from the group consisting of %γ〇,

A group consisting of PerC6, BHK, 293, COS, PCK, MRC 5, mdck, MDBK and WI-38, and the cells are preferably MDCK cells. In another preferred embodiment, the cell line is cultured in the form of adherent cells. (21) The method of item (20), wherein the cells used are mdck cells. (22) The method of any one of items (1) to (21), wherein the method further comprises one or more steps of further processing the virion. (23) The method of item (22), wherein the processed virions comprise inactivated virions, and/or attenuated virions, and/or split virus antigens, and/or subunit viral antigens, and / or virosome 〇 (24) The method of item (23), wherein the processed virions comprise - or a plurality of influenza antigens. In a preferred embodiment, the 'process virion contains 154987.doc -12· 201202425 hemagglutinin (HA) and/or neuraminidase (να) » (25) The method according to any one of the preceding items It is used to make influenza vaccines. (26) A method for testing whether the total number of extremely low infectious virions/cells ("infection rate" 'ΜΟΙ) necessary for the addition of infected cells to the cell culture composition for breeding virions causes virions and And a method of increasing the yield of processed virions comprising the steps of: a) growing cells in a cell culture composition until a cell density of at least 0.5 X 106 cells/ml is reached, b) using a total number of very low MOIs Preselected virus strain infectious virions are added to the cell culture composition, wherein the very low MOI is less than the MOI of 丨〇_5, c) obtained by adding the infectious virions to the cells in step b) Production of virions and/or processed virions and virions and/or processing viruses obtained by adding the same type of infectious virions to the same type of cells using a reference MOI in the range of ΙΟ·5 or higher The amount of particles is compared. The reference MOI is preferably 1〇_3. In another preferred embodiment, in step a), the cells are grown until at least 3. 〇 X 〇 6 cells / ml, preferably at least 5 〇χΐ〇 6 cells / ml, further preferably at least 7.0Χ106 cells/ml, even more preferably at least about 9·〇χ1〇6 cells/ml, preferably at least about 1 〇χΐ〇6 cells/ml or even more preferably at least 13·〇χ1〇6 cells /ml of cell density. (27) The test method of the item (26), wherein the amount of the virion after the addition of the disease #2 is at least 6 when measured in the form of 101 og TCID 5 〇 / ml. 154987.doc •13· 201202425 (28) The test method of item (26) or (27), wherein prior to step c), the collection of the virions produced and other processing steps selected as appropriate are performed. (29) The test method according to any one of the items (26) to (28), wherein the amount of the virion or the processed virion obtained after the infectious virion of the step b) is added to the cells is used at least At least 1.2 times, preferably at least 1.5 times, more preferably at least 1.5 times, preferably the amount of virions or processed virions obtained after adding the same type of infectious virions to the same type of cells in the reference MOI in the range of 1〇-5 or 1〇-5 or more Preferably, it is at least 2 times and optimally at least 3 times, wherein the virions are preferably measured 24 hours after the virus is added. It is particularly preferable to use a 〇·3 ΜΟΙ as a reference ΜΟΙ. (3) The test method of any one of items (26) to (29), wherein after step a) preferably after step a) and before or during step b), with a very low MOI and reference The cell culture medium in which the MOI is used to culture the cells is replaced with a virus propagation medium having at least 80%, preferably at least 85%, more preferably at least 90% compared to the weight molar concentration of the cell culture medium previously used to culture the cells. An optimum osmotic concentration of at least 95% by weight. With respect to preferred cell culture media and media for propagation of viruses, reference may be made to the respective media described herein, "Whether the above criteria for individual media are implemented" can be determined as described elsewhere herein. (31) The test method according to any one of the items (26) to (30), wherein the cells are cells as defined in the item (20) or (21). (32) The test method according to any one of the items (26) to (31) wherein the infective virion is an infectious influenza particle. 154987.doc -14- 201202425 (33) A test method according to any one of items (26) to (32), wherein the processed virion is as in item (23) or (2). The test method of any one of the items (1) to (25), which is 荨 小于 1 〇 -7, 1 X 1 〇 8 as the case may be. The method of the item or the item (26) to M1 is equal to or less than 1 X 1 〇6, and the progress is equal to or less than (35) as in any one of the items (1) to (25) or as The amount of the virions or processed virions in the test method of any one of the items (26) to (33) is by one-way light-emitting immunodiffusion (Single Radial Im_〇)

The Diffusion ’ SRID assay is detected by reverse phase high performance liquid chromatography (RT-HPLC). (36) A cell culture composition for producing virions and/or processing virions, wherein a) the cell culture composition has been prepared by using an initial cell amount of at least 0.5 χ 1 (6 cells/ml). That is, the amount of cells in the cell culture at the time of virus addition is at least 0.5 χ 106 cells/ml, and b) the addition of a very low M0I of less than 1 〇.5 to the cell culture composition of step a) has been added. The total number of infectious virions, and c) the amount of viable cells present in the cell culture composition of step b) is equivalent to the amount of starting cells present in the cell culture composition within 1 day after virus addition ( That is, at least about 60%, preferably at least about 7%, more preferably at least about 75%, even more preferably at least about 80%, and most preferably at least about 85, of the amount of viable cells in the cell culture at the time of virus addition. 〇/〇. 154987.doc 15- 201202425 In another preferred embodiment, the amount of viable cells present in the cell culture composition of step b) is equivalent to that present in the cell culture composition within 24 hours of virus addition. At least about or at least about 110% of the starting cell amount. In another preferred embodiment, the amount of viable cells present in the cell culture composition of step b) is equivalent to 48 hours after virus addition. The amount of cells present in the cell culture composition at the time of virus addition is at least about 5%, preferably at least about 1%, preferably at least about 15%. (37) The cell culture composition according to item (36), wherein the cell culture of the step a) has been carried out by using the test method according to any one of the items (26) to (35) Infectious virions are added to the composition. (38) The cell culture composition according to item (36) or (37), wherein the hemagglutinin (HA)/mUb rate after 4 days after virus addition is higher than 15 gg/mi, preferably higher than 20 pg/ml . (39) A use of a cell culture composition for propagating virions, wherein an infectious virion is added to a cell culture composition containing a cell amount of at least 0.5 x 1 细胞6 cells/ml when the virus is added, wherein The total number of infectious virions/cells ("infection magnification", MOI) necessary for the infected cells used is less than ΙΟ·5 (very low M0I). In another preferred embodiment, the MOI is equal to or less than 1 -6, equal to or less than 10 -7, or equal to or less than the preferred value of the extremely low MOI as indicated above. (40) A method for producing a virion comprising immunogenic hemagglutinin (HA) or a method for producing an immunogenic HA protein, comprising the steps of: a) propagating a virion comprising an immunogenic HA, wherein the virus is present Adding infectious virus particles to a cell culture composition containing at least 0.5 χ 1 Ο 6 cells/ml of cells at 154987.doc -16-201202425, and the total number of infectious virions/cells necessary for the infected cells used therein ( "infection rate", Μ〇Ι) is less than 丨〇_5; and b) obtaining reproductive virions, and further processing such virions as appropriate to isolate immunogenic globulin (HA) protein and/or immunization The original neuroglycosylase (NA) protein. (41) The method of item (4), which is further defined as described in any one of items (丨) to (25). For the preferred starting cell amount, refer to the above description. In the meaning of the present invention, the term "starting cell amount" means the amount of cells in the cell culture at the time of virus addition. As the virus is added to the cell culture, the infection phase of the cells begins. The present invention is now described in more detail by the preferred embodiments and examples, which are set forth by way of illustration only and are not to be construed as limiting. A virus (such as an influenza virus) is a pathogen of an epidemic or pandemic that occurs annually or seasonally.疾病 疾病 爆 # # caused a considerable morbidity and mortality, especially in people at risk, such as those suffering from heart disease or lung disease, diabetes or immune system dysfunction. In order to protect groups from common disease threats and to prevent the spread of endemic diseases or pandemics, use vaccines such as in pandemics (such as influenza pandemics): 'The gap between vaccine production and vaccine demand on a global scale is expected Significant. Thus, there is an urgent need to increase the number of vaccine doses available, which can be achieved, for example, by modifying 154987.doc 201202425 in a cell line, for example to increase viral yield or viral component yield, respectively. The present invention provides a method of breeding an influenza virus which causes an increase in the production of viral particles (i.e., propagation of influenza virus) and the obtained viral components. In the meaning of the present invention, the term "viral component" means processing of viral particles. Surprisingly, it has been found that when the specific minimum cell density (i.e., at least 0.5 X 106 cells/ml, preferably at least 3 〇) &lt;1〇6 cells/ml, 5·〇χ106 cells/ml, 7.0, 6 cells, ml, 9〇χΐ〇6 cells/ml, η.οχπ) 6 cells/ml or 13〇xl 〇6 cells/ml (starting cell volume)), the total number of infectious virions/cells (Μ〇ι) required to add infected cells smaller than 〇_5 to the minimum amount of cells The production of virions or viral components is significantly increased. Although the use of a relatively low initial addition virus per used cell has been unexpectedly found to be particularly advantageous in the addition of infectious virions to the minimum starting amount of cells described above, and can be used to propagate virions, as Obtained increased production of virions and increased viral components in production. It is particularly advantageous for the manufacture of influenza virus components, such as the manufacture of influenza virus components such as the influenza virus antigen agglutinin (?alpha) or neuroglycosylsidase (?). Without wishing to be bound by any theory, it appears that since the virus is added to the cell culture and the cells are infected with this very low river 〇1, the percentage of viable cells within a certain period of time after the virus is added (for example, up to about three days) Higher. It appears to lead to a more efficient method of propagation of the virus, especially in the critical early stages of cell infection, resulting in a longer and more prolific virus production, thus increasing the production of reproductive virions and higher yields of viral components. When using a cell density between 1χ106 cells/ml and 4-5Χ106 fine 154987.doc -18· 201202425 cells/ml (starting cell volume, ie the amount of cells in cell culture at the time of virus addition) In particular, high increments of viral hemagglutinin (HA) are obtained, and wherein MOI is in the range of less than 1 〇-5 to about 1 〇7. In the method known to date using equal or large M1〇-52M〇I, when the virus propagation medium is used, compared with the cell culture medium for culturing cells, 2 is less as protein f, growth factor + etc. (4), and the maximum is obtained. Yield of reproductive virions. This means that the virus propagation medium is less enriched. However, it has been unexpectedly found that in the present invention, when the virus propagation medium is used in accordance with the present invention and the virus propagation medium is not significantly smaller than the cell culture medium, the virion yield can be further increased. Since the method of the present invention can propagate virions (e.g., influenza virions) and produce viral components (i.e., process viral particles) with increased yield, it accelerates the vaccine production process and reduces the vaccine delivery lead time. It is even possible to increase the production of viral components derived from + strains that have adapted to high growth (e.g., reassortant strains or strains that are better adapted to grow in a particular cell line) by the application of the invention. In general, the virus strain is added to a cell strain by using a virus strain having a specific Ι0Ι (infection rate) to propagate the virus strain. The virus strain that needs to be propagated is also referred to herein as a preselected virus strain. A particular Μ〇ι is typically in the range of equal to or higher than 1 〇-5 and is referred to herein as a "reference" MOI. The preferred reference MOI used is 1〇_3. However, as demonstrated in the course of the present invention, the "reference" MOI is not the most effective MOI for all strains of virus to be propagated, but has a very low virus (1) in the range of less than 1 〇 -5 to provide a more effective virus. Reproduction, thereby causing an increase in the production of virions and/or viral components. I54987.doc 19 201202425 The present invention therefore further provides a method for testing the addition of very low infectious virions, '·Number/cell preselected virus strains to cell cultures for propagation of virions, whether it is a chic virion or virus The yield of the components increased, and the winter one was β. "The application of this method can advantageously determine the maximum yield of virions and virus components in each of the pre-selected strains of the Qin strain, respectively, and especially in the extremely low MOI range that is not covered by the industrial vaccine manufacturing method. in. Furthermore, the limitation is that the minimum initial cell density described above is observed, and no incremental effect may be detected, which means that on a small scale or semi-industrial scale, for example, below about 3 1 or 31, or for example 3〇 The results obtained in a working volume below mi or 3 〇mi can be converted to a large/industrial scale, such as a bioreactor converted to a working volume having an ι 〇〇 1 or greater (eg, about 1200 丨). Thus, for example, multiple test cultures may be performed simultaneously on a small scale and/or on a semi-industrial scale to test whether the use of very low M〇I vaccination produces higher yield virions or viral components, respectively, and if High yield virions or viral components, this very low river 01 is the MOI that provides the highest possible yield for a given strain. This very low MOI can then be used to propagate preselected strains on a large/industrial scale. Furthermore, starting with a minimum cell density as described herein has, for example, the advantage of achieving a fit when compared to a period of time when the starting cell density is lower than the minimum cell density as described herein and is suitable for cell density for seeding. And the period of specific cell density used to inoculate virions is shorter. It also speeds up the vaccine production process, thereby reducing the vaccine delivery time. It is especially advantageous to use the following virus propagation medium, which has a weight molar osmotic concentration of at least 80%, preferably at least 85%, more preferably at least 154987, of the molar osmolality of the cell culture medium compared to the medium previously used to culture the cells. Doc -20- 201202425 最佳 and optimally at least 95%, and it does not have a significantly lower amount, preferably not less than 5 〇. /. More preferably, W6G%, or even better, the total amount of protein, growth factor and/or inorganic salt that is not less than 75/〇. It means that the weight of the molar is less than that of the medium used for culture, and the medium of the virus is used to culture the cells (before the virus is added). advantageous@. In another embodiment, the protein-rich medium is used as a medium for culturing cells and a medium lacking protein (4) for a medium for a thin virus. Alternatively or additionally, the total amount of free amine contained in the medium for propagating the virus is not higher than the total amount of amino acid in the cell culture medium for culturing the cells. This may additionally increase the production of viral components. Without being bound by any theory, the balance between the amount of preselected virus strain, the cells to which the infectious disease mother particles are added (cells/ml) and the μ〇ι used seems to make the virion particles in the cell culture composition and the obtained The production of viral components is increased. By applying the method of the present invention, an increase in the production of propagated viral particles and viral components can be achieved, thereby accelerating the vaccine production process and reducing the vaccine delivery time. In a particular aspect, the invention relates to a method of propagating a virion comprising an immunogenic lectin (HA), such as an influenza virus, wherein in a first step the cells are cultured in cell culture and wherein Infective influenza particles are added to the cell culture in a first step, wherein the medium used to culture the cells is replaced with the weight of the medium previously used to culture the cells before or during the virus addition step Infiltration of 154987.doc • 21 · 201202425 degrees compared to medium with at least 80% by weight of molar osmosis and which does not have a significantly lower amount than the medium previously used to culture the cells. The total amount of protein, growth factor and/or inorganic salt in 50%, wherein the amount of starting cells in the cell culture at the time of virus addition is y 0.5 1 cells/ml, wherein 12 to % after virus addition Within hours, the density of the field cells is not less than 4% of the cell density at the time of virus addition, and the total number of infectious virus particles/cells added during the virus addition step The dyeing ratio, MOI) is less than 1 () · 5. In another preferred embodiment, the density of living cells within 12 to 36 hours after virus addition is not less than 60% of the cell density at the time of virus addition. In a preferred embodiment, the density of viable cells within 36 hours after virus addition is not less than 8% of the cell density at the time of viral infection. In another preferred embodiment, viable cells are added after the virus is added to the publication hour. The density is not less than 1% by weight of the cell density at the time of virus addition. In another preferred embodiment, ΜΟΙ is equal to or less than 10-6, equal to or less than 1〇_7, or equal to or less than 1〇8. In the meaning of the present invention, the term "infection magnification" means the number/cell of infectious virions necessary for infecting cells. In other words, Μ0Ι is the ratio of infectious virions to cells. Thus, for example, 10_5 of 1 means that 1 virion is used per 100,000 cells. In the context of the present invention, it has been surprisingly found that inoculation of a specific starting amount of cells with less than 1 〇 5 of the total dish 可 1 is effective to increase the amount of reproductive virions, with a restriction of at least 〇 5xl 〇 6 cells /ml of a specific minimum cell density (initial, and thus the virus component to be manufactured. It can, for example, accelerate the vaccine preparation process and reduce the vaccine delivery time. If a viral infection occurs 154987.doc -22- 201202425 disease Seasonal 'especially epidemic or pandemic spread or outbreak, and especially when there is a spread of viral influenza. By applying the method of the invention, it is even possible to increase the source of adaptation to certain properties (such as high) The virions and viral a production of virus strains grown or grown in serum-free medium, which further reduces the time required to provide the vaccine composition. Furthermore, wild-type virions can even be propagated so that sufficient Yield of virions and/or viral components for industrial applications. According to the invention, the virions may suitably be selected from the desired virions. An infectious or non-infectious virion that can only be regenerated in a host cell. The term "infectious" means that a virus (or virion) is sufficient to produce a reproductive infection when introduced into a sputum, a cell. Ductive infecti〇n,|. Non-infectious virions or viruses cannot produce the infection, respectively; however, the particles will express their encoded genes. The virions are preferably infectious virions. In general, virions contain a nucleic acid surrounded by a shell (deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)), which may be formed by the same protein subunit capsid. The virions may also have a package that may be derived from a membrane of a host cell that has infected the virion. The nucleic acid can be a linear 'ringed or segmented single-stranded nucleic acid, a double-stranded nucleic acid, or a mixture thereof, and the strands can be positive-sense or negative-sense. In a preferred embodiment The virion contains RNA. The virions containing RNA and which have been found to efficiently multiply at very low initial M0I are, for example, influenza viruses. The influenza virus is composed of a segmented single-stranded RNA genome. The core of ribonucleoprotein and the outer lipoprotein envelope arranged by matrix proteins. Influenza virus 154987.doc •23- 201202425 Members are, for example, influenza A, B and C. Influenza A and B are each Containing eight single-stranded negative-sense RNA segments. The virions are preferably selected from the group consisting of: Orthomyxoviridae, such as influenza A, B or C; Paramyxoviridae , such as measles virus, mumps virus, parainfluenza virus, and respiratory syncytial virus; Togaviridae, such as Sindbis virus ( Sindbis virus) and rubella virus; Herpesviridae, such as Herpes Simplex virus, Epstein-Barr virus and varicella bandage Virus (Varicella Zoster virus); Rhabdoviridae, such as rabies virus; Retroviridae, 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; Acreaviridae ), such as lymphocytic choriomeningitis virus; and Poxviridae, such as variola virus; infectious virions are preferably selected from, for example, type A, type B or C A group of positive mucus virus families of influenza viruses. In another preferred embodiment, the virion is an influenza virion selected from the group consisting of 154987.doc -24 - 201202425 influenza A, influenza B or influenza C. Further preferably, the virion may also be a reassortant virion containing a combination of different parental gene segments, such as reassortant influenza virions. Reassortant viruses include those which include genetic and/or polypeptide components derived from more than one parental virus strain or source. For example, a reassortant virus is produced in the context of an approved major viral strain (also known as a primary donor virus (MDV)) to incorporate selected HA and NA antigens. For example, the reassortant virus includes seven viral genomic segments (or gene segments) derived from the first parental virus and one viral genomic region encoding the Η Α or Ν 来源于 derived from the second parental virus. segment. The 6:2 reassortant virus includes 6 genomic segments from the first parental virus (most typically 6 internal genes) and 2 genomic segments from the second parental virus (e.g., ΗΑ &amp; ΝΑ). The reassortant virus may include 6 genomic segments from the first parental virus (most commonly 6 internal genes), 1 genomic segment encoding the 亲 from the second parental virus, and ΝΑ a coding ΝΑ from the first Genomic segment of a triple parental virus. The six internal genes can also be internal genes of more than one parental virus. The reassortant virus can be produced by any method known to those skilled in the art, such as by classical reassortment techniques, such as by a synergistic infection method or by a particle rescue technique. The virions of the invention may also be inactivated or attenuated. Infectious virions can also be referred to as virions (vid〇n). In the method of the present invention, a cell 2 composition or a cell culture containing cells and a medium, respectively, is used. Cell culture is known to those skilled in the art: the components of the compound may vary depending on the cell used and the intended use. In a preferred embodiment, the cell culture composition is prepared for propagation of the virus I54987.doc -25 - 201202425 particles as described herein and/or for the manufacture of viral components. In addition, the virions can be further processed for the production of inactivated virions, and/or attenuated virions, and/or split virus antigens, and/or subunit viral antigens, and/or virions. For culture purposes, for example, cell cultures can comprise cells and suitable media for cell growth. In general, cells of any suitable cell line known to those skilled in the art can be used in the methods of the invention. The cells may be eukaryotic cells such as yeast cells, insect cells, amphibian cells, avian cells or mammalian cells (including human cells). In a preferred embodiment, the cells contained in the cell culture composition are animal cells, and the cells are preferably mammalian cells. The cells are preferably cultured in the form of 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 chicken kidney (PCK) cells, Martin Dabies kidney ( MDCK) cells, Martin Dabie cattle kidney (MDBK) cells, 293 cells (eg 293T cells or HEK-293 (human embryonic kidney cells) cells), WI-38 cells (derived from female fetuses obtained from approximately three months old) Cell line of cells; normal human fetal lung fibroblasts), MRC-5 cells (cell lines derived from normal lung tissues of 14-week-old male fetus, Nature 227: 168-170, 1970) and COS cells (eg COS 1) , C0S7 cells). Suitable cells also encompass combinations or mixtures of cells&apos; including, for example, mixed cultures of different cell types or cell lines (e.g., Vero and CEK cells). These cells are preferably MDCK cells. In another preferred embodiment, the cells of the present invention may be cells having a particular desired property, such as cells that are adaptable to growth in a particular medium used (e.g., in serum-free culture I54987.doc -26-201202425 base). 'Or cells that exhibit modified doubling time, modified tumorigenic profile, and/or modified viral manufacturing behavior. An example of a cell line adapted to serum-free growth is the MDCK-SF cell line. In a preferred embodiment, the cells used in the methods of the invention are cells which permit and/or support the replication and/or propagation of infectious virions inoculated with such cells. One indicator of the ability of a cell to support viral replication and/or reproduction is the production of viral virions obtained from inoculated cell cultures and infected cell cultures, respectively. In general, viral production, such as the amount of virions or the amount of viral components, can be determined by any suitable method known to those skilled in the art, respectively. The virus production is preferably quantified by measuring the concentration of virus or virions present in the sample, based on the measurement of the half of the tissue culture infectious dose (TCID5Q) of the infectious virus particles. TCID5Q, usually expressed as 1 〇 10 1 '(:1〇5()/1111, can be determined by any method known to those skilled in the art. Endpoint dilution techniques such as TCID5〇 assay are for measuring viral populations. Statistical methods. Several statistical methods for analyzing this data are available, such as Spearman-Karber, Reed & Muench, or Probabilistic Analysis. For example, serial dilutions of virus are added to cells (such as MDCK cells). After several days, record Cytopathic effect (CPE) and can be calculated as tissue culture half infection dose (TCIDsq). In addition, virions can also be expressed in the form of pfu (plaque forming unit) / ml. In a preferred embodiment, the cells support the virus. Replication and/or propagation 'including, but not limited to, viruses selected from the group consisting of the Orthomyxoviridae, such as influenza A, B or C viruses; the Paramyxoviridae, such as the measles virus, mumps virus , parainfluenza virus and respiratory tract fusion 154987.doc -27· 201202425 virus, togaviridae, such as Sindbis virus and rubella virus; herpesvirus family, such as simple Herpesvirus, Eberstein-Barr virus and varicella zoster virus, baculoviridae, such as rabies virus; retroviridae, such as human immunodeficiency virus (HIV); reoviridae, such as rotavirus And Corolla 蜱 heat transfer virus; Flaviviridae, such as yellow fever virus, adenoviridae, such as adenovirus; picornavirus family, such as poliovirus, genus, genus, such as lymphocytic choroid a meningitis virus; and a poxvirus family, such as variola virus; the cells preferably support replication of infectious virions selected from the group consisting of the positive mucinous virus family of influenza A, B, and/or c viruses and Furthermore, the cells used in the method of the invention also support replication and/or propagation of the reassortant virus. The cells used in the methods of the invention may further exhibit superior biological properties (eg, for virus production, tumorigenicity) Cells with sexual profiles and/or doubling times). Individual cells that exhibit these superior properties can be selected. These cells can be cloned at the same time, simultaneously or subsequently in individual cells. Specific culture conditions should be employed, such as for temperature, C〇2 concentration, p〇2 value, pH range, and conditions of the medium used. In a preferred embodiment of the method of the invention, separately present or infected cell culture is present The starting cell amount is at least about 〇5 χ丨〇 6 cells/ml, preferably at least about 3.0 M06 cells/ml, further preferably at least about 5.0 χ 106 cells/ml, even more preferably at least about 7. 〇χΐ〇6 cells/ml, even more preferably about 9 〇χ1〇6 cells/ml and optimally at least about 11·〇χ1〇6 cells/ml or at least about 13 〇χ1〇6 cells/ ML. It has been surprisingly found that by inoculating a cell culture composition having a minimum cell density of 154987.doc • 28-201202425 as described herein, the yield of the propagated virions can be significantly increased and thus Significantly increase the amount of viral components produced. If the cell culture composition is inoculated at a very low MOI, the yield of the propagated virions and the obtained virus components can be increased even more. The cells used in the method of the present invention may be cultured in suspension or adhered to the surface to which they adhere by adhering the cells. The cells are preferably cultured in the form of adherent cells. In another preferred embodiment, the cells are anchorage dependent! Adhesive surfaces on which cells can grow are well known in the art. Adhesive surfaces include, but are not limited to, surface modified polystyrene plastics, protein coated surfaces (eg, fibronectin and/or collagen coated glass/plastic), and are commercially available, for example, from Amersham Biosciences. Microcarriers (eg Cytodex 3 microcarriers). Microcarrier beads are small spheres that provide a large surface area for adherent cell growth per volume of cell culture. The choice of adhesion surface can be influenced by the method used to culture cells, such as MDCK cells, and can be determined by those skilled in the art. Suitable incubator blood or containers that can be used in the methods of the present invention, respectively, can be any vessel or trough known to those skilled in the art, such as spinners, boiled bottles, and fermenters. Or bioreactor, or tissue culture flask. The method of the invention may be carried out, for example, on a small scale in a vessel having a J volume (such as a tissue culture flask having a volume of, for example, about mi), for example, about 50 1 to a semi-industrial scale such as '.々100 1 The process of the invention is carried out on a scale of the invention in a process of the invention and in vessels having a large working volume, such as an acid fermentation tank or bioreactor having a working volume of, for example, 1000 1 or more. 154987.doc -29 201202425 In a preferred embodiment, the cells used in the methods of the invention can be cultured in a batch culture system, such as a batch feed culture system. In another preferred embodiment, the cells can also be cultured in a perfusion culture system. In the embodiment of the present invention, the cell line used in the method of the present invention is cultured under specific conditions. Those skilled in the art are aware of what cell types are appropriate for which cell type. The respective adaptation conditions are, for example, the concentration of c〇2, the value of p〇2, the pH, the temperature, and the medium used. The cell culture stage includes cell culture before inoculation and cell culture after inoculation (the individual conditions for virus propagation can remain the same; however, when the cell culture conditions are in the cell culture stage, that is, before (or during) the virus addition, respectively. It is also possible and ultimately more effective to change or change during cell culture after cell culture and inoculation/virus propagation, which cells can be changed at any time during the entire culture period prior to the inoculation/virus propagation phase, and such cultures The conditions may also be changed not only once but also several times during the cell culture process prior to the inoculation (ie virus addition)/virus propagation stage. Another effective operation is to make the conditional change (especially the medium change) coincide with the virus addition (ie The medium is replaced during virus addition. The cell culture conditions that can be altered are any cell culture conditions, such as temperature, c〇2 concentration, value, pH range, and conditions of the medium used. Another preferred embodiment of the method of the present invention Medium, virus propagation medium and cell culture medium (ie, first The medium for cell culture has a molar osmolality of at least 8%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% by weight. In addition, virus propagation The medium does not have a significant amount of 154987.doc 201202425, preferably not less than 50%, more preferably not less than 60%, even more preferably not less than 65% and optimally compared to the medium previously used to culture the cells. Not less than 75% of the total amount of protein, growth factor and/or inorganic salt, which means that the molar osmolality is not significantly lower than the weight of the medium used for cell culture and has a significantly lower amount of protein growth factor. And/or the viral propagation medium of the total amount of inorganic salts is advantageous for replacing the cell culture medium to be used for culturing the cells (before or during the addition of the infectious virions to the cells). In another embodiment the protein-rich medium A medium used as a cell culture medium for culturing cells and lacking a protein is used as a medium for propagating a virus. Or, or alternatively, a medium for propagating a virus The total amount of free amino acid contained in the cell is not greater than the total amount of amino acid in the cell culture medium used to culture the cells. It has been unexpectedly found (comparative pattern) to be used as described herein (with substances present in the cell culture medium) The amount of the cell culture medium does not have a significantly reduced amount and/or does not have a weight of 1 cell osmotic concentration of less than 80%, preferably less than 85%, more preferably less than 90% and most preferably less than 95% by weight of the cell culture medium. Molar osmolality of the virus propagation medium, and the use of a molar concentration of the cell culture medium having a weight molar concentration of less than 80%, less than 85%, less than 9% or less than 95% as described herein, and/or significant A reduced amount of material in the propagation medium is compared to 'providing an increased production of virions. The determination of the molar osmolality of the cell culture medium is described below. In another preferred embodiment of the invention, for the propagation of a virus Medium 2 has not been significantly lower in cell culture medium previously used to culture cells (preferably not less than 50%, more preferably not less than 55%, even more preferably not less than 60% and even optimally At 65%, and in another Protein preferred embodiment 154987.doc • 31- 201202425 preferred less than 70% or 75%) of the] long because? And / or the total amount of inorganic salts. Reference is also made to the embodiments described above. In another preferred embodiment, the medium for culturing the cells is replaced with a medium for virus propagation before or during the addition of the virus to the cells with the infectious 14 virions, wherein the medium for virus propagation is used. The medium has the same type as the cell culture medium used for cell culture, but does not contain BSA. In another preferred embodiment of the method of the present invention, the medium for propagating the virus does not have a lower amount of three selected from the group consisting of growth factors and/or inorganic salts, compared to the cell culture medium for culturing the cells. , two or one individual substance or group of substances. It is intended that, according to the present invention, the propagation step is preferably carried out by using a virus propagation medium which does not have a significantly reduced amount of individual substances as described above. In other words, 'as much as possible compared to the cell culture medium', the same or at least not significantly lower amounts of the individual substances or groups of substances are contained in the virus propagation medium, and the replacement medium is replaced, for example, the cell culture medium for cell culture is replaced by the virus propagation medium. It occurs in a certain period of time, which is generally determined by the size of the cell culture. If the cell culture is carried out on a small or semi-industrial scale (for example, below about 31 or 31, or for a working volume of, for example, 30 ml or less), the medium is replaced at a large/industrial scale (such as having 1 〇). Cell cultures under the above working volume of bioreactors require much faster replacement times. 154987.doc •32- 201202425 If the cell culture has been carried out on an industrial scale or on a large scale, the terms “before inoculation” or “before the virus addition step” mean that the medium replacement occurs within 12 hours or less, preferably 1〇. Hour, 9 hours, 8 hours, 7 hours or 6 hours or less, more preferably 5 hours or less, or even more preferably 4 hours or less. If the cell culture has been carried out on a small scale or semi-industrial scale, the medium replacement occurs 5 hours or less, preferably 4 hours, 3 hours, or 2 hours or less, more preferably 4 hours or less, more preferably i. Within hours or hours, more preferably 3 minutes or less, and even more preferably 15 minutes or less. Alternatively, the medium can be replaced during virus addition, in particular for both operations to be performed simultaneously. In the method of the present invention, any suitable cell culture medium known to those skilled in the art can be used. In general, a suitable cell culture medium contains a plurality of components (also referred to as additives and substances, respectively), such as inorganic salts, amino acids, nucleic acids, vitamins, lipids, sugar or carbon sources, proteins, growth factors, surfactants, and pH indicator. In addition, the medium used can be formulated such that the cultured cells retain desired characteristics, such as one or more of the following features including, but not limited to, non-tumorigenic, growth in the manner of adhering cells, support for infectivity during culture. Viral replication. In one embodiment, any suitable inorganic salt known to those skilled in the art may be present in the cell culture medium; the inorganic salt is preferably selected from the group consisting of CaCl2, CuS04, Fe(N03)3, FeS04, KC1. MgCl2

MgS04, NaC, NaHC03, Na2HP04, NaH2P〇4, Na2Se〇3, 154987.doc -33· 201202425 sodium acetate and ZnS04. In another preferred embodiment, any suitable amino acid known to those skilled in the art may be present in the cell culture medium. The amino acid is preferably selected from the group consisting of L-alanine, L-arginine-HC1, L-aspartame (free test), L-aspartate xH2, L-aspartate Acid, L-cysteine_HC1 (anhydrous), L-cysteine-HCl-H2, L-proline, L-proline X2HC1, L- faceamine, L- faceamine Amine, glycine, L-histidine-HC1xH20, isoleucine, L-lysine, L-lysine-HC1, L-oxime thioacid, L-phenylalanine, L-acantine , thio-L-amino acid (non-animal), serine, L-threonine, L-tryptophan, L-cool amine acid, L-cool amine acid x2Nax2H20 and L-proline . In another preferred embodiment, any suitable nucleic acid known to those skilled in the art can be present in the cell culture medium. Preferably, the nucleic acid is selected from the group consisting of adenine sulfate, adenosine-5-phosphate, adenosine-5-triphosphate, glutathione (reduction), and guanine Fib 〇naccj, jpg), guanine HC1, D-ribose, 2-deoxy-D-ribose, thymine, uridine. Ding, Astragalus and Hypoxanthine. In another preferred embodiment, any suitable vitamin known to those skilled in the art may be present in the cell culture medium. The vitamin is preferably selected from the group consisting of p-aminobenzoic acid, ascorbic acid, D. calcium pantothenate, D. biotin, gasified choline, cod liver oil, ergocalciferol, folic acid, inositol, and naphthoquinone. Nicotinamide, nicotinic acid, putrescine 2HC, pyridoxal _HC1, pyridoxine-HC1, riboflavin, thiamine Ηα, dithiooctanoic acid, thymidine, tocopherol phosphate disodium, DL-tocopherol acetate, vitamin a acetate and vitamin 154987.doc •34- 201202425 prime ~. In another preferred embodiment, any suitable lipid known to those skilled in the art may be present in the cell culture medium. The lipid is preferably selected from the group consisting of cholesterol, linoleic acid, linoleic acid, palm oleic acid, peanut tetrabasic acid, stearic acid 'myristic acid, palmitic acid, and oleic acid. In another preferred embodiment, any suitable sugar or carbon source known to those skilled in the art may be present in the cell culture medium. The sugar or carbon source is preferably selected from the group consisting of 1 glucose, D-fructose and sodium pyruvate. In another preferred embodiment, any suitable protein known to those skilled in the art can be present in the cell culture medium. Preferably, the protein is selected from the group consisting of BSA (bovine blood π albumin, part V) (such as Albumax I (bovine lipid-rich BSA)), lactalbumin hydrolysate, and Primatne RL. In another preferred embodiment, any growth factor known to those skilled in the art can be present in the cell culture medium. The growth factor is preferably selected from the group consisting of epidermal growth factor (murine 'recombinant murine or recombinant human epidermal growth factor), insulin (such as human tamsin (crystal biosynthesis human insulin)) and transferrin (metabolized whole bovine iron) a group of proteins). In another preferred embodiment, any suitable surfactant known to those skilled in the art can be used. The interfacial activity is preferably selected from the group consisting of P1_ic F68 and Tween 80. Ethanol can also be used. In another preferred embodiment, i11 -., J can be used, such as a pH indicator. Any suitable indicator known to those skilled in the art can be used. Preferably, the phenol red is used. In one embodiment, the cell culture medium is further preferably supplemented with antibiotics. 154987.doc •35- 201202425 In general, any suitable antibiotic known to those skilled in the art can be used in a concentration suitable for use alone or in combination with other antibiotics. In a preferred embodiment, no antibiotics are used. In another embodiment, the cell culture medium is not supplemented with antibiotics. In another embodiment, the viral propagation medium is not supplemented with antibiotics. In another embodiment, neither the cell culture medium nor the viral propagation medium is supplemented with antibiotics. The 'cell culture medium and/or virus propagation medium is not supplemented with a macrolide polyene antibiotic or a derivative or analog thereof. Having the cell culture medium and/or the viral propagation medium dispensed with the addition of antibiotics, particularly macrocyclic vinegar polyp antibiotics or derivatives or analogs thereof, provides an improved method of making viral components 'for example, regarding the robustness of the method or The operation has been improved. By applying the method of the present invention, despite the elimination of antibiotics as described above, an increase in viral yield can be achieved even when very low MOI is used as described herein. The medium for virus propagation (i.e., the replacement medium) is preferably a medium without Bsa or substantially no BSA. Substantially free of BSA means that the medium contains only traces of BSA, which does not have any effect on the cell culture and/or subsequent processing and/or propagation of the intended use of the virions and/or viral components, preferably without any Negative impact. Further, any suitable combination of the above components and/or additives may be used. An example of a suitable medium that can be used in the methods of the invention is, for example, Episerf, which is a commercially available medium (e.g., Invitrogen or Lonza). Episerf containing BSA and Episerf without BSA having a weight of about 360 mOsmol/kg 154987.doc • 36· 201202425 Molar osmotic concentration In the method of the present invention, when added to any virus, it contains at least 0.5 χΙΟ 6 cells/ml. The amount of cells or the preferred amount of cell plum, as it is, and the addition of infectious virus neutral to the sputum; two per particle. The time required to culture the cells until the corresponding ideal cell density has been reached depends primarily on the cell culture scale. Thus, if the cells are cultured on a large/industrial scale (see above, see above), the cells used in the method of the invention will proliferate for up to 4 G days, preferably up to 30 days, until a cell density of at least 0.5 cells/ml is achieved. Thus, if the cells are cultured on a semi-industrial/small scale, the cells used in the method of the invention will proliferate for up to 2 days, preferably up to 10 days or less, until the desired cell density is reached. However, the corresponding necessary time period required to reach the cell density is known to those skilled in the art because, for example, the cell density of the culture can be readily determined by conventional methods such as cell counting. In a preferred embodiment, the propagated cells are MDCK cells. In another preferred embodiment, the cells are cultured in tissue culture flasks or adhered to microcarriers, such as 3 microcarriers (e.g., available from Amersham Biosciences). The cell density per ml of cell culture can be determined by any method known to those skilled in the art. For example, NucleoCounter (Chemometec) can be used if cells are cultured in adherent cell cultures such as on microcarrier beads. Using this device, the total cell concentration can be determined in a sample that has not been enzymatically treated. The cell density of each cell culture vessel can be measured once a day, as many times as necessary. If necessary, the cell culture medium can be replaced as described herein during cell culture. 154987.doc • 37· 201202425 In another preferred embodiment of the method of the present invention, less than 1〇-s is added to at least about 0.5×10, the starting amount, and the cell (in terms of the ratio Mm per cell; See also elsewhere in this specification) and the total number of infectious virions added to the starting amount of cells. The term "number of infectious virions used" in the meaning of the present invention means that the total number of infectious virions externally administered to the cell culture composition is less than 1 〇 5. The term "inoculation" or "virus addition" means, in the meaning of the present invention, the external addition of infectious disease particles to the cell culture composition, respectively. In the meaning of the present invention, the total number of infectious virions can be added to the cell culture composition. However, the MOI of the present invention may also be added to the cell culture in two or more steps, for example, by using one of the total number of infectious virions for the first viral addition step, followed by another portion of the infectious virion. Two or more virus addition steps. The time lag between individual inoculation steps can vary. In a preferred embodiment, the virus is added as a whole step and/or within a period of 2 hours or less, preferably within 1 hour or less, or even more preferably 30 minutes or 30 minutes. The following time period is best completed within 15 minutes or 1 minute or less. In general, the addition of the virus to cells present in the cell culture can be carried out by any suitable method known to those skilled in the art. For example, LV can be seeded by simply adding the virus inoculum to the cell culture. In the meaning of the present invention, a cell culture medium containing the same components can be used throughout the culture stage (cell culture before inoculation and virus propagation), but the medium can also be replaced and used in a different group than the previously used medium. Sub-additive/additive/substance medium. In this regard, see 154987.doc -38- 201202425 for the above description. The cell culture medium for culturing the cells is preferably replaced with a virus propagation medium having a weight osmolality of at least 95% by weight of the osmotic concentration of the medium used to culture the cells (see above): as for example shown in FIG. And Figure 2 can be inferred that 'if the cell culture medium used to culture the cells is replaced with at least 95. /. The protein production and survival rate of the cell culture medium for culturing the cell culture medium can increase the protein production and survival rate. The right cell culture medium is replaced with a virus propagation nucleus that does not have a significantly lower total amount of protein, growth factor, and/or inorganic salt than the cell culture medium, or if the virus propagation medium has both properties, Similar beneficial effects can be found. In a preferred embodiment of the invention, the inoculation or virus addition is carried out as follows. At the end of the cell culture, before the addition of the infectious virions or by the door (5), it is suitable for washing with a person known to the skilled artisan. The liquid is subjected to a washing step to replace the cell culture medium with a virus medium. A virus inoculum prepared preferably in the presence of 刖J is added to the cell culture. After the virus was added and the medium was changed after this step, the cells were infected with the virus and the virus was propagated. In the meaning of the present invention, the term "after virus addition" or "after inoculation" means the period of time from the completion of the addition of the virus seed to the cell culture. Further, in the meaning of the present invention, the term "during the virus addition step" means that the period of the inoculation is performed or two: there is an overlap in time. The virus addition step begins with the addition of: di-gold: to the cell culture and ends with completing the entire virus inoculation." The cell density at the time of virus addition can be obtained by those skilled in the art 154987.doc •39· 201202425 Any suitable method for determining is described. As described above, the cells can be inoculated with a virus (e.g., with infectious particles, such as influenza particles) in a reduced medium volume compared to the final volume of the medium. For example, 'reduced The volume may correspond to about 7%, preferably about 50%, more preferably about 40%, and most preferably about 30% of the final volume of the medium. At the end of the inoculation phase, the reduced cell culture medium is supplemented to 100 with viral medium. The final concentration of %. As mentioned above, the viral medium used for this supplement may be the medium used during the inoculation phase, or it may be a different medium than that used during the inoculation phase. It is preferably the medium used for inoculation. After the cell inoculation has been completed (or the cells are separately cultured with the virus suspension), which is the case where the virus inoculum is just added to the cell culture ( See above), culture the cells for another specific period of time (virus propagation phase) until the desired CPE is observed (see below). In addition, the virus manufacturing steps can also be indicated by any indicator known to those skilled in the art. At the end, such as by empty microcarriers and/or by the addition of p〇2 into the bioreactor or by the reduction of 〇2 flow, generally έ, which is from about 5 hours to 8 days, preferably about 1 hour to A condition of about 6 days, about 10 hours to about 5 days, more preferably about 10 hours to about 4 days, and most preferably about 12 hours to about 3 days. CPE is a pathogenic effect, which in turn An adverse effect on cell growth or maintenance, especially in relation to microbial and/or viral infections. Pathogenic effects include, but are not limited to, cytopathic effects, cell rupture, growth inhibition, inhibition of protein synthesis or apoptosis. CPE is an observable change in cellular structure that can vary with cell type and cause of death, and can be determined according to the knowledge established in the art by 154987.doc • 40· 201202425. For example, some of the most common Virus infection The effect is morphological changes such as cell rounding and detachment from the substrate, cytolysis, syncytium formation, and inclusion body formation. CPE can be judged according to what is known to those skilled in the art. Microscopically observing and estimating the percentage of cells covering the reaction vessel and microcarriers. The method of the invention further comprises the step of further processing the propagation of virions in one or more preferred embodiments in a preferred embodiment. Purified reproductive virions (eg, influenza virions). The processed virions preferably comprise non-activated virions, and/or attenuated virions, and/or split virus antigens, and/or subunit viral antigens, and/or Or virion "mythomorphic virions" further preferably comprise one or more influenza virus antigens, such as HA and/or NA. Isolation and propagation of virions from cell culture media can be carried out by any suitable method known to those skilled in the art. For example, virions can be collected by separating cells or cell residues from the culture medium, for example, using a knife remover or filter. Known methods include, but are not limited to, filtration, ultrafiltration, adsorption to sulfur from "bee and dissolution" and centrifugation. For example, &amp; crude media from vaccination and infection of sigma can first be used by Centrifuge (continuously) at, for example, 2 Torr to 2 〇〇〇g for about 5 minutes to remove cell debris and other particulate matter for purification. Alternatively, filter the medium through a filter such as a 0.8 0 „! cellulose acetate filter to remove 7C whole cells and other particulate matter. The purified culture supernatant is then centrifuged, for example, at 15,000 g for about 3-5 hours, as appropriate to allow the virus (such as a virus) to form a pellet or to be concentrated by ultrafiltration using, for example, a 3 MWCO filter. Thereafter, the virion pellet can be resuspended in a suitable buffer such as STE buffer (0.01 M Tris-HCl; 0.15 Μ NaCl; 0.0001 Μ EDTA) or at pH 7.4. PBS (phosphate buffered saline) can then be concentrated and/or further purified by ultracentrifugation, for example, by density gradient of sucrose (60%-12%) or tartaric acid clock (5% to 10%). The gradient solution is centrifuged at a rate for a period of time sufficient to concentrate the virions into visible bands for recovery. The propagation virus particles can be further subjected to ion exchange chromatography or size exclusion chromatography. In addition, virions and/or nucleic acids contained therein can be propagated enzymatically. Furthermore, virions may not be propagated, killed or attenuated according to any suitable method known to those skilled in the art. Attenuation can be achieved, for example, by chemical biting by standard serial passage, wherein passage in sensitive cell cultures is used a sufficient number of times until the virions are not pathogenic without loss of immunogenicity. Further, the virions may not be activated, for example, by treatment with a detergent or formaldehyde. In another preferred embodiment of the invention, the obtained propagated virions can be further processed to obtain, for example, a viral antigen, such as 11 and/or NA ^ In a preferred embodiment, HA is obtained. The skilled person knows how to obtain viral antigens. The processed virions obtained can be used to make influenza vaccines. Other processing steps may be performed after the above collection and/or separation steps, and may include extracting viral components that can associate with cells or cell fragments, separating viral components, isolating viral components, and purifying them, such processing The steps are described in the example of WPaul A. Belter (author), EL Cussier (author), WeiShcm Hu (author) Biosep deputy i〇ns: D〇wnstream I54987.doc -42- 201202425

Processing f〇r Biotechnology's general tutorial. As described above, the virions of the present invention (such as influenza virions) contain, for example, virions. The virion is a monolayer phospholipid bilayer vesicle having a suitable mean diameter, for example, in the range of about 70 nm to about 150 nm. The virion essentially represents a reconstituted empty viral envelope, and no nuclear material including the genetic material of the source virus. Capsid. The virion is not replicable, but is a pure fusion active vesicle that is inserted into a phospholipid bilayer membrane containing a functional viral envelope glycoprotein (such as influenza virus HA and NA). In addition, the processing virions comprise one or more influenza antigens such as HA and/or NA. The influenza virus antigens may be derived from strains that cause seasonal, pandemic and/or epidemic outbreaks of influenza. HA can be found, for example, on the surface of influenza viruses. It is an antigenic glycoprotein responsible for binding the virus to the infected cells. At least 16 different influenza HA antigens have been known to date. These subtypes are named H1 to H16. NA is an enzyme that cleaves the sugar bond of the neuroglycolic acid. At least 9 influenza neuraminidase enzyme subtypes have been known to date. Such subtypes can be found, for example, in a database known to those skilled in the art, such as the PubMed database (eg 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=l&itool=EntrezSystem2 .PEn trez .Sequence. Sequence - ResultsPanel.Sequence_RVDocSum ). Influenza viral proteins may also comprise any of these HA and/or NA coding sequences, either alone or in combination with each other. Influenza virus proteins may also contain only a portion of these sequences. Preferably, the influenza virus protein comprises a complete sequence encoding Η1, H2, H3, 154987.doc-43-201202425 H5, H6, H7, N1, N2, N3 or N7, or a portion of the sequences, alone or in combination, preferably containing The sequence encoding H5. In another preferred embodiment, the polynucleotide construct comprises a sequence encoding H1N1, H2n2, H3N2, H6N1, H7N3 or H7N7 or a portion of the sequences, preferably comprising a sequence encoding H5N1 or a portion of the sequences . Furthermore, the above method of the present invention can be used to manufacture a viral vaccine, preferably an influenza virus vaccine. To this end, the step of partially displacing the virions or one of the viral components or viral components into a vaccine composition can be carried out. A virion or a viral component or a portion of a viral component can be formulated by a suitable method known to those skilled in the art to provide a viral vaccine for administration to an individual. The deployment of virions and/or viral components suitable for use in a viral vaccine or portions thereof may include additional steps including, but not limited to, buffer replacement and sterilization steps. In general, viral vaccines can be administered prophylactically or therapeutically, separately with appropriate carriers or excipients. Preferably, the carrier is a pharmaceutically acceptable carrier such as sterile water, a buffered saline solution, a dextrose solution, a glycerol solution or a combination thereof. In addition, carriers which minimize allergic or other undue effects and which are suitable for the particular route of administration (e.g., subcutaneous, intramuscular, intranasal, etc.) are generally selected. In another preferred embodiment, the vaccine composition of the invention may contain other components and/or additives, such as adjuvants. An adjuvant is a substance that increases an immune response, such as a hydroxide of a plurality of metals, a bacterial cell wall component, an oil or a scutellarin. Furthermore, the present invention also relates to a method for testing a cell culture composition for the total number of very low infectious virions/cells (MOI) necessary for the addition of infected cells to a cell culture composition for use in the 154987.doc 201202425 virion to cause virions. And/or a method of processing increased virion yield comprising the steps of: a) growing cells in a cell culture composition until a cell density of at least x5×10 6 cells/ml is reached, b) using a very low MOI A total number of preselected strains of infectious virions are added to the cell culture composition, wherein the very low Μ〇ι is an MOI less than 丨〇·5, or wherein the 〇1 is preferably μ(1), c as defined herein, The yield of the glycoside particles and/or processed virions obtained by adding the infectious virions to the cells in step b) and the reference in the range of 10-5 or higher. 1 Compare the amount of virions and/or processed virions obtained when the same type of infectious virion is added to the same type of cells. In a preferred embodiment, the diseased granules and/or viral components are obtained after seeding the cells with the reference M0I in the same conditions as the diseased f particles and/or viral components obtained after seeding the cells at a very low ratio of 01. Obtained under. In the meaning of the present invention, the term "identical conditions" means the use of the same cell culture conditions, for example, with respect to temperature, CO2 concentration, P02 value, pH range, and medium. All of the preferred embodiments and parameters of the method for propagating virions as described herein are also suitable for the test method of the present invention. Surprisingly, it has been found that balancing the preselected virus strain used in (1), (ii) the cell density (cells/ml) when the infectious disease particles are added, and (iii) the MOI used to inoculate the cells, allowing the propagation of virions and/or viral components. The yield is increased by limiting the cell to a cell density of at least χ5χ1〇6 cells/ml at the time of inoculation, and the restriction is that the ΜΟΙ is extremely low 〇5〇. Other I54987.doc -45. 201202425 Suitable MOIs are described elsewhere in this document. In the prior art method, a "reference" MOI is added to the cell, which is an MOI equal to or higher than 10.5, and the reference MOI is preferably 1 〇 3 ^ However, it has been found in the present invention to reference the MOI Inoculation of cells does not necessarily result in the maximum production of reproductive virions or viral components, respectively. Instead, if the cells having a specific minimum cell density are seeded with the total number of preselected virus strain infectious virus particles, an improved optimized yield can be achieved, wherein the MOI is an extremely low MOI of less than 10.5. By applying the test method, it is determined that each of the preselected virus strains respectively provides the MOI of the improved production of the viremia virus particles and the virus components. In addition, since no incremental effect occurs, multiple small-scale test cultures can be simultaneously run in parallel by using the test method of the present invention, while observing the minimum cell density disclosed herein to test whether the use of very low MOI vaccination respectively produces High-yield virions or viral components, if so, the extremely low 〇1 is the MOI that provides the highest possible yield. This extremely low MOR can then be used for large/industrial-scale propagation of preselected strains' without re-measurement for large scale The method is extremely low Μοι. Surprisingly, the positive effects observed with small-scale cell cultures on the obtained viral particles and/or viral components are correspondingly converted into large-scale cell cultures. For the inoculated cells, the inoculation of the cell culture composition, the infectious virions used for inoculation and addition, the total number of infectious virions, the virions and virus components and the cell culture medium, please refer to the above description. In a preferred embodiment of the test method, in the first step a), the cells are grown in the medium until reaching at least χΐ〇5χΐ〇6 cells/ml 154987.doc •46-201202425 In the other preferred embodiment, the cells are grown until at least 3.0 x 10 cells/ml, preferably at least 5 〇χ 1 〇 6 cells/ml, more preferably at least 7.0 x 1 〇. 6 cells/ml, even more preferably at least about 9.0&gt;&lt; 10 cells/ml, preferably at least about 11 〇&gt;&lt;1〇6 cells/ml, or further preferably at least 13.〇 Cell density of χ1〇6 cells/ml e The cell density of the sample obtained from the culture vessel can be obtained by any method known to those skilled in the art. If the cells are cultured in adherent cell culture, the cell density can be determined by using Nucle® c〇u as described in Example i. In a preferred embodiment, the cells can be cultured and propagated as described above by adhering cells. In the preferred embodiment of the test method, the infectious virions of the virion strain are added to the cells using very low MCH, preferably infective (10) particles as described above. This extremely low is considered to be _ in the range of less than HT5 as described above. The cells can be seeded as described above, i.e., infectious virions are added. Furthermore, by carrying out the test method of the present invention and the method of (respectively) breeding influenza virus (or influenza virion), it is possible to add a protease 'the *ha pro-protein cleavage and thereby adsorb the virions or viruses on the cells, respectively. This, for example, additionally increases the yield of viral components. The protease may be added shortly before, simultaneously with, or shortly after the virus (e.g., influenza virus) is added to the cells. The protease may also be added after a period of 8 hours to 1 hour after the virus is added. If the protease is added simultaneously with the inoculation, the protease can be added directly to the cell culture or added to the cell culture, for example, as a concentrate with the infectious virions 154987.doc • 47-201202425 sub-inoculum. In a preferred embodiment, the protease is i to % after the addition of infectious influenza virions.

The concentration range of Hg/ml was added to the replacement medium. In another preferred embodiment, the protease is in a concentration range of more than 1.0 #§/1111 to 50 pg/ml2, preferably in a concentration range of 1.5 pg/ml to 50 Hg/m, more preferably 2 //(6) to a curse Kg. The concentration range of /ml and even more preferably the concentration range of 2·5 μ§ Λ1 to 5〇Rg/m丨 is added to the replacement medium. Suitable proteases to be added are known to those skilled in the art, for example, the protease is a serine protease, a cysteine protease or an aspartic protease. An example of a preferred suitable protease is trypsin. In another preferred embodiment, after step a) of the test method, preferably after step a) and before or during step b), the medium for culturing the cells with very low river (1) and reference MOI is used. Replace with virus propagation medium. With respect to the viral propagation medium, reference may be made to the above description, and the medium may be replaced according to any suitable method known to those skilled in the art, for example, as described above. Further, regarding the medium which can be used for the cell culture stage and the virus propagation stage, respectively, refer to the above description. The cell culture was inoculated and the cultured cells were cultured after the inoculation stage (virus propagation) as described above. After the end of the cultivation phase has been reached, the collection and/or separation steps are carried out as before step c) of the test method. This/these steps can be performed as described elsewhere herein. The obtained reproductive virions can be further processed. Processing virions 154987.doc • 48 · 201202425 (or viral components, respectively) containing non-activated virions, and/or attenuated virions, and/or split virus antigens, and/or subunit viral antigens, and/or viruses body. In addition, the amount of virions and/or viral components can be measured separately. In general, the amount of reproductive virions can be determined by any suitable method known to those skilled in the art. Preferably, the virus production can be quantified by measuring TCIDso as described above. The amount of viral component obtained can be determined by any suitable method known to those skilled in the art. An example of a suitable method is the one-way radiation immunodiffusion (SRID) test, which can be carried out according to the instructions of the European Pharmacopoeia (Monograph 2149 (04/2009: 2149) Influenza Vaccine (surface antigen, inactivated, prepared in cell cultures)). For the determination of HA. Immunochemical methods are based on the selective, reversible and non-covalent binding of antibodies to antigens. Use these methods to detect or quantify antigens or carcasses. The formation of an antigen-antibody complex can be detected and the amount of complex formed can be measured by a variety of techniques. Immunoprecipitation (SRID) is a simple quantitative immunodiffusion technique. When an equilibrium is established between the external reactants and the internal reactants, the annular precipitation zone originating from the external reactant sites is proportional to the amount of antigen used and inversely proportional to the concentration of antibody in the gel. Briefly, a gelatin gel containing a predetermined amount of antiserum was cast in a gel mold. The lyophilized hemagglutinin antigen reagent of each suitable virus strain having a predetermined content of HA per vial and the lyophilized anti-hemagglutinin sheep serum having a predetermined content of each suitable virus strain per ml of agarose are used. The reference antigen and the monovalent body were diluted to a predetermined concentration and treated with 1% amphoteric detergent (destroying virus particles) at room temperature for 30 knives. The treated antigen and body were then inoculated into wells pierced into a 154987.doc -49·201202425 coagulating gel and allowed to stand at room temperature for at least 18 hours overnight. Thereafter, the gel was dried on a gel-bonded film and dried with a filter paper (such as Whatman filter paper) and a paper towel. Once dried, the gel was stained with Coomassie bdlliant bhle G_25 to determine the presence of antibody/antigen agglutination. Use the appropriate soft body to measure the diameter of the Shen Dian area. The hemagglutinin content is then calculated by using a statistical analysis program. In step c) of the test method, the yield of virions and/or processed virions obtained after the addition of the infectious virions of step b) to the cells is used at or above 1〇-5. The reference within the range is compared to the amount of virions and/or processed virions obtained by adding the same type of infectious virions to the same type of cells. The reference Μ〇ι is preferably 10·3 〇. For the term "reference MOI", refer to the above description. To determine the M〇I method most suitable for the propagation of virions, at least two different MOIs were tested in culture vessels containing the same medium and using the same processing conditions. To this end, at least two culture vessels are used; the first culture vessel contains the cell culture composition inoculated with the reference MOI as described above and the second culture vessel contains the cell culture composition inoculated with the extremely low M〇I of the present invention. However, it is preferred to test one or more of the extremely low M〇I of the present invention in order to find the M〇I which is most suitable for the respective processing conditions. Therefore, it is preferred to refer to (1) for inoculation and/or to inoculate more than one time with a very low MOI. Further, a series of culture procedures can be carried out simultaneously using the extremely low 1^〇1 of the present invention. In addition, the test method can be carried out at any scale, which is carried out first on a small scale, and then on a semi-industrial or large-scale cell culture using very low bribes. Sensing the virions in the preferred step __ 'step b): the amount of virions or processed virions obtained afterwards is added to the reference MOI using rabbits in the range of at least 10 5 or more than 10 5 Preferably, at least U times 'the amount of virions or processed virions of the same type of infectious virions to the same type of cells is at least 15 times, more at least 3 times, wherein the amount of virions is wider and better. The virus was measured 24 hours after the addition. The amount of propagated virions and viral sites can be determined as described above. The invention also relates to a cell culture composition for the manufacture of virions and/or viral components, wherein a) has been activated by using at least x5 x 〇6 Λ, • 〇 cells/ml of starting cells The cell culture composition is prepared in an amount such that the amount of cells in the cell culture is at least 0.5 χ 106 cells/ml when the sputum is added, and b) has been added to the cell culture composition of step a) Using infectious virions that are less than the total number of extremely low MOIs, and C) the amount of viable cells present in the cell culture composition of step b) is equivalent to the cell culture composition within 1 day after virus addition. The amount of starting cells present (i.e., the amount of viable cells in the cell culture at the time of virus addition) is about 60%, preferably at least about 7%, more preferably at least about 5%, even more preferably at least About 80% and the best at least about. In another preferred embodiment, the amount of viable cells present in the cell culture composition of step b) is equivalent to at least the amount of cells present in the cell culture composition within the range of (1) days of virus addition. About 1% or at least about 154987.doc 51 201202425 110%. In another preferred embodiment, the amount of viable cells present in the cell culture composition of step b) is equivalent to the amount of starting cells present in the cell culture composition within a range of 48 hours after virus addition. At least about 5%, preferably at least about 10%, preferably at least about 5%. For example, cell culture compositions, virions and/or viral components, starting cell amounts, seed cell culture compositions (i.e., addition of virions to cell cultures), MOI, and assays for the amount of cells present in the cell culture Method (cell density, expressed in cells/ml), refer to the above description. The cells: the nutrient composition can advantageously improve the manufacture of the viral components, e.g., with respect to the yield obtained and/or the time required to manufacture the viral components. In a preferred embodiment, the cell culture composition of step a) has been inoculated with the M〇I tested according to the test methods as described herein. In another preferred embodiment, the cell culture composition exhibits greater than 8 _ml, preferably higher than 9 μβ/ηιΐ, more preferably Μ after 4 days after virus addition.

Kg/ml, even better than 15 and optimally higher than the HA/ml ratio of 丨. For the term "after virus addition", refer to the above description. Further, 'the invention (d) is for use in a cell culture composition for propagating virions, wherein the infectious virions are inoculated with a fine composition containing at least MW cells/ml of starting cells or other preferred cell amounts as defined herein. The infection virion infection rate necessary for the infected cells used is less than.... Other preferred embodiments for the virion rabbit colonization method or test method as described above are also suitable for use. 54987.doc • 52-201202425 Preparation of the cell culture composition β of the present invention Regarding cell culture composition, virion, and starting cell Amount, seed cell culture, infectious virions and sputum, again reference to the above description. In addition, virions may be collected and/or isolated for propagation as described above, and other processing steps as described above may be performed as appropriate. The invention also relates to a method for producing an immunogenic viral antigen, preferably an influenza virus antigen, and more preferably an immunogenic hemagglutinin protein, comprising the steps of: a) propagating a virion comprising an immunogenic sputum, Wherein infectious virus particles are added to a cell culture composition containing a cell amount of at least 0.5 X 106 cells/ml when the virus is added, and the total number of infectious virus particles/cells (infection magnification) necessary for the infected cells used therein is added. , M〇I) is less than 丨〇_5; and b) obtaining reproductive virions, and further processing the virions to separate immunogenic hemagglutinin (HA) protein and/or immunogenic ceramide Acid zymase (NA) protein. In a preferred embodiment, the additional processing steps comprise a separation step for propagating viral particles and optionally a purification step. This step/steps are followed by a separation step of the immunogenic viral antigen, preferably the influenza virus antigen and more preferably the immunogenic globulin (ΗΑ) protein. The other processing steps described above are known to those skilled in the art and are conventional methods. It can be any processing step suitable for obtaining the isolated immunogenic viral antigen as described above. In a preferred embodiment, the separation step of the following propagation virions and the separation step of the immunogenic HA protein are performed by dissolving, for example,

Michael W. Wolff and Udo Reichl's "D〇Wnstream Processi 1 s · 154987.doc •53· 201202425

From Egg to Cell Culture-Derived Influenza Virus

Particles, Chem. Eng. Technol. 2008, 31, No. 6, 846_857. Preferably, the virions are first isolated and then the immunogenic hemagglutinin (HA) protein is separately separated or purified from the virions, and then the immunogenic hemagglutinin is isolated. As described in more detail above, 'other processing steps are preferably performed after the virion collection and/or separation step, and may include extracting viral components that can associate with cells or cell fragments, separating viral components, and isolating viral components. And purify it. Immunization prepared by the method of the present invention Further, the present invention relates to a primary hemagglutinin (HA). The present invention is illustrated in more detail in the following figures and examples, however, it is intended to be presented for illustrative purposes only and is to be construed as limiting in any way the method of the invention: the volumetric molar concentration (-y) of the nutrient base and By using osmolality, by using any known formula, such as by making the glutamine osmolality/weight of the osmotic osmotic leaf, the same method is used to determine the fineness of the cultured cells. = The conditions are: the volume of the culture medium, the molar osmotic concentration / weight of the molar (four), the virus, the measurement of the concentration of the D point, the determination of the weight of the molar and the determination of the protein content of the medium. The protein content of the medium can be determined by using any known method. The 154987.doc -54 - 201202425 conditions are used to determine the protein content of the cell culture medium for cultured cells and the medium for virus propagation using the same method. Several methods are available, such as the L〇wry or Bradford assay. The total amount of the substance or the amount of the individual substance can be determined by any known method for the determination of the amino acid, the growth factor and the inorganic salt. The limitation is that the cell culture medium for culturing the cell is determined by the same method and used for virus propagation. Individual content in the cell culture medium. The amount of the amino acid can be determined, for example, by using HPLC (high pressure liquid chromatography) and the amount of the growth factor can be determined, for example, by mass spectrometry. The inorganic salt content can be determined by measuring the metal ion concentration of the inorganic salt in the medium with an ion selective electrode. EXAMPLES Example 1: Culture of MDCK cells MDCK cells were cultured on a Cytodex 3 microcarrier by adhering cells. Preparation of Cytodex 3 microcarriers: Cytodex 3 microcarriers for culturing MDCK cells were prepared and sterilized according to the following protocol: The required amount of dried microcarriers was weighed and added to a phosphate buffered saline (PBS) solution to swell the microcarriers. After swelling, the microcarriers were washed with PBS and then sterilized at 121 °C. After sterilization, the PBS was replaced with cell culture medium and used. The MDCK cells were cultured in a bioreactor having a working volume of 31 as follows: After preparing (correcting the sensor and sterilizing) the bioreactor for cell culture, the cell culture medium and the microcarriers were added to the bioreactor. The cells were seeded to the bioreactor at a cell density of about 0.4 χ 1 Ο 6 cells/ml at 154987.doc • 55· 201202425. The temperature was maintained at 37 ° C, pH 7.1 and dissolved oxygen (DO) concentration greater than 40% air saturation. The cells will grow into monolayers of cells in 3 to 4 days. 0 Replacement Cell Culture Medium The cell culture medium is replaced with a concentrated cell culture medium before inoculation of MDCK cells with infectious virions. The medium was replaced according to the following protocol: Stirring was stopped and the microcarriers were allowed to settle at the bottom of the bioreactor. After the microcarriers settle, they are passed through 80 °/ by overpressure. The dipping tube removes about 80% of the medium and then refills the bioreactor with the concentrated medium. Determination of cell density (cells/ml) The cell density of cells present in individual cell cultures was determined by using 1^11 (:160 (1:011110111616::). A representative sample was taken from the reactor and after the microcarriers were settled in a test tube, the medium was removed. The microcarriers were then treated with a lysis buffer and a neutralizing buffer to enable counting of the nuclei. Example 2: Virus culture: at 35 ° C , pH 7.1 and DO &gt; 40% culture strain A/Wisc〇nsin/ 67/2005 (H3N2) X-161B. Analysis of virus propagation in cell community reached about 3 · 0 χ 106 cells / ml with about 4 The cell culture was inoculated with virus strain A/Wiscansin/67/2005 (H3N2) X-161B after 〇χ1 〇6 cells/ml. The cell culture composition was inoculated according to the following protocol: 154987.doc -56- 201202425 After replacement of the cell culture medium, add the calculated amount of virus and add a small amount of membrane protease (0.5 mL 2.5% TGase solution) separately. Continue to propagate the virus until it reaches about 100% cytopathic effect (CPE), which usually It takes about 3 days. The CPE was visually inspected by microscopy to estimate the cell coverage of the microcarriers. The virus was then collected. Analysis of the virus sample TCID5〇 analysis Infected MDCK cells with serial dilutions of the virus; several days later, the cytopathic effect (CPE) was recorded and calculated Half of the infectious dose (TCID5G) is cultured for tissue. Endpoint dilution techniques such as the TCID5G assay are statistical methods for measuring viral populations. Several statistical methods for analyzing such data are available, such as Spearman-Karber, Reed & Muench, or Probabilistic Analysis. Analysis (HA concentration): The influenza virus is treated with an amphoteric detergent to split the HA antigen into diffusible particles. After diffusion through the agarose layer containing the specific antiserum, the antigen forms a precipitation ring with the antiserum. This precipitated ring was visualized by staining with Coomassie brilliant blue. The diameter of the precipitated ring was compared to a standard solution treated in the same manner. The content was expressed in HA/mL. (Monograph 2149 (04/2009: 2149) Influenza Vaccine (surface antigen, Inactivated, prepared in cell cultures)). Immunochemical methods are based on antibodies to antigens Optional, reversible and non-covalent binding using such methods to detect or quantify antibody or antigen-antigen can be detected. - antibody complexes form it into, and may be by the amount of complex formed by measuring a variety of techniques. Immunoprecipitation (SRID) is a simple quantitative immunodiffusion technique. When an equilibrium is established between the external reactants 154987.doc -57-201202425 and the internal reactants, the annular chamber region originating from the external reactant sites is proportional to the amount of antigen used and inversely proportional to the antibody concentration in the gel. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows prion kinetics (obtained by srid assay) produced by infecting cells with virus strain A/Wisc〇nsin χ·161Β. The upper line is the condition "medium replacement EPI w/o BSA, ΙχΙΟ ΙχΙΟ · 6". It means that the extremely low MOi of the present invention and the virus propagation medium having a significantly lower amount of protein, growth factor and/or inorganic salt and/or at least 95% by weight of the osmolality of the medium for cell culture have been used. (Ερι w/o BSA) (The virus propagation medium contains only BSA). This preferred method provides the best results. The intermediate curve is the condition "medium replacement CM; MOI 1 X1 〇·6", which provides an improvement using the extremely low M0] of the present invention. Under these conditions, the cell culture medium has been replaced with a virus propagation medium having a significantly lower amount of protein, growth factor and/or inorganic salt than the cell culture medium, and/or the weight per osmotic concentration of the medium is less than The medium used for cell culture weighed 95% of the molar osmolality. This virus propagation medium is called "CM" medium. The lower curve is the condition "medium replacement Cm; ΜΟΙ 1χΐ〇·3". Under this condition, the cell culture medium has been replaced with CM medium and has been inoculated with an MOI of 1χ1〇3. This prior art method provides a minimum of 11 protein content. The condition "medium replacement Epi w/o BSA, MOI lxio·6" after infection was 154987.doc •58·201202425 on day 4, the average hemagglutinin protein production was 37〇. The condition "medium replacement CM; MOI lxio·6" produced a yield of 19 Mg/ml on the fourth day after virus addition, and the condition "medium replacement CM; M〇I lxl〇.3j had 8.7 on the fourth day after virus addition. Bg/ml is reduced by 4 times the average HA yield. This figure shows that compared to inoculation of cells with reference MOI (eg MOI 1 X1 〇·3, lower curve), 'very low MOI (eg ΜΟΙ 1χΐ〇-6, middle curve) And the upper curve) the sputum yield increased significantly after inoculation of the cell culture. In addition: It is shown that this effect is attributed to the reduction of ΜΟΙ (the increase in ΗΑ yield, comparing the lower curve with the middle curve), and if the medium has been used for cell culture, Substituting CM medium, but replacing it with a medium that does not have a significant reduction in the amount of protein, growth factor and/or inorganic salt and/or at least 95% by weight of the osmotic concentration of the medium used for cell culture, this effect can be further Figure 2 shows the cell kinetics of adherent cells during propagation of virus strain a/Wisconsin X-161B. The continuous curve is the condition "medium replacement CM; MOI lxlO·3". This condition shows that the cell density measured on the first day after infection is faster and larger. ✓J, 〇 The imaginary curve indicates the condition "medium replacement CM; MOI 1 X 1 〇-6". Here, the measured cell density is slightly reduced. The dotted line is the condition "medium replacement EPI w/o BSA, MOI 1χΐ〇-6". Here, it can be seen that the cell density measured on the first day after infection increased slightly. This figure shows that, as indicated by the individual cell density (cells/ml), compared to cells seeded with reference MOI (eg MOI 1 X 10·3), at very low MOI (eg 154987.doc -59·201202425 eg MOI 1 χ 1 0·6) After inoculation of cells, more cells survived. Furthermore, this effect is shown to be due to a decrease in sputum, and this effect can be further increased according to the present invention by replacing the medium prior to infection. Figure 3 shows the tcid50 during the breeding period of virus strain A/Wisconsin Χ-161.曲: 曰 培养基 培养基 培养基 CM ; ; M ; ; ; ; ; ; ; M ; M ; ; M M M M ; ; ; ; M ; ; ; M M ; ; M M ; ; M M M ; M ; M ; ; ; ; ; ; ; ; ; ; ; ; Line. Car exchange f (10), ΜΟΪ lxl0·6") is a high protein yield with a similar value above the two 踝 higher TCI 〇 5 。. 154987.doc 6〇,

Claims (1)

201202425 VII. Patent application scope: ι_ A method for breeding an influenza virus comprising immunogenic hemagglutinin (HA), wherein cells are cultured in cell culture in a first step and then infectivity is added in a second step Influenza particles to the cell culture, wherein the medium in which the cells are cultured is replaced with the osmolality of the medium previously used to culture the cells during or during the virus addition step, At least 8% by weight of the molar osmolality of the base, J^ does not have a significantly lower amount, preferably not less than 50% of the protein, growth factor and/or compared to the medium previously used to culture the cells. Or the total amount of inorganic salts, wherein the amount of cells in the cell culture at the time of virus addition is at least 0.5×10 6 cells/ml, wherein the viable cell density is not lower than the virus addition within 12 to 36 hours after virus addition. 40% of the cell density, wherein the total number of infectious virions added per cell during the virus addition step/cell (Multiplicity 〇f infecti〇n), MOI) is less than 1 〇-ί. 2. The method of claim 1, wherein the replacement medium does not contain bsa (bovine serum albumin). 3. The method of claim 1 or 2, wherein the replacement medium does not supplement the antibiotic. The method of claim 1 or 2, wherein after the addition of the infectious influenza particles, the protease is added to the medium at a concentration ranging from 1 pg/ml to 5 〇 μδ/ιη1. 5. The method of claim 4, wherein the protease has a concentration range of more than 1 ton / per unit to ^ pg / ml, preferably 15 ton / claw 1 to 5 〇 / / concentration range 154987.doc 201202425 circumference, more A concentration range of from 2,0 pg/ml to 5 〇pg/ml, even more preferably from 2 $ pg/ml to 5 〇pg/mi is added to the medium. 6. The method of claim 4 wherein the protease is trypsin. 7. The method of claim 2, wherein the cells are anchorage-dependent cells. 8. The method of claim 1 or 2, wherein the total number of infectious virions/cell (infection magnification, Μ〇ι) added during the virus addition step is equal to or less than 10.6. The method of claim 1 or 2, wherein the total number of infected virions/cell (infection magnification, m〇i) added during the viral addition step is equal to or less than 10.7. 10. The method of claim 1 or 2 wherein the total number of infectious virions/cell (infection magnification, Μ(7)) added during the virus addition step is equal to or less than 10.8. ' 11. The method of claim 2 or 2 wherein the activity of the cell in the period of 12 to 36 hours after the virus is added is not lower than the cell density at the time of virus addition. 12. The method of claim 1 or 2, wherein the viable cell density is not less than the virus density when the virus is added for up to 36 hours after the virus is added. 13. If the clearing item 1 or 2 is square, Gans The medium in which the cells are cultured is replaced by the amount of the molar concentration of the re-introduction of the medium used to culture the cells during the virus addition step. A medium having a molar osmolality of at least 95% by weight. 14. The method of claim 1 or 2, wherein the amount of cells in the cell culture of Danja when the virus is added is at least 3.〇xl〇6 cells/ml. The method of claim 1 or 2, wherein the amount of cells in the cell culture is at least 5.0×10 6 cells/ml when the virus is added. 16. The method of claim 1 or 2, wherein the amount of cells in the cell culture is at least 7.Ox 1〇6 cells/ml when the disease is added. 17. The method of claim 1 or 2, wherein the amount of cells in the cell culture is at least 9. 6 cells/ml when the diseased silkworm is added. 18. The method of claim 1 or 2, wherein the amount of cells in the cell culture is at least 11.0 Χ 106 cells/ml per virus addition. 19. The method of claim 1 or 2, wherein the amount of cells in the cell culture is at least 13. 6 cells/ml when the disease is added to the silkworm. 20. The method according to claim 1 or 2, wherein the fine cells used are &gt;, the field cells are animal cells, preferably mammalian cells. • The method of claim 20, wherein the cells used are mdck cells. 22. The method of claim </RTI> wherein the method further comprises one or more steps of further processing the propagated virions. 23. The method of claim 22, wherein the processed virions comprise inactivated virions, and/or attenuated virions and/or split virus antigens, and/or subunit viral antigens, and/or viruses Body (virosomes). 24. The method of claim 23, wherein the processed virions comprise one or more influenza antigens. 25. The method of claim 2 or 2 is for the manufacture of influenza vaccine. 26. The method of claim 1, wherein the replacement medium does not contain bsa (bovine serum albumin), wherein the replacement medium is not supplemented with antibiotics, and wherein the protease is added after adding 154987.doc 201202425 plus infectious influenza particles! A concentration ranging from pg/m丨 to 5〇 μ§/ηι1 was added to the medium. 27. A method of testing whether a very low number of infectious virions/cells (ΜΟΙ) pre-selected virus strains required to infect a cell are added to a cell culture composition for propagating virions, such that the virions and/or processing virions are caused A method of increasing yield comprising the steps of: a) growing cells in a cell culture composition until a cell density of at least X5 X1 〇 6 cells/ml is reached, b) using the very low Ο Ο I for the preselected virus The total number of infectious virions of the strain is added to the cell culture composition, wherein the extremely low enthalpy is less than 1 〇 -5, c) the virus obtained by comparing the infectious virions of step b) to the cells The amount of virions and/or processed virions obtained by adding the same type of infectious virions to the same type of cells when the yield of particles and/or processed virions is equal to the reference MOI in the range of 1 〇 5 . ’ 154987.doc