Method for culturing virus-infected cells

Abstract

The present disclosure relates to the field of methods for culturing virus-infected cells. More specifically, the present disclosure relates to methods for culturing cells with influenza A virus. The present disclosure further relates to the effects of certain culturing factors (such as trypsin concentration, pH level, incubation time, and combinations thereof) on the production of viral proteins, in particular hemagglutinin.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 562,853, filed March 8, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of methods for culturing virus-infected cells. More specifically, this disclosure relates to methods for culturing cells using influenza virus. Background Technology

[0004] Newer vaccine production methods may involve culturing the virus for vaccine production in cell cultures, such as Madin Darby canine kidney (MDCK) cells. However, such methods may have drawbacks, including low viral protein yields and high levels of contaminating proteins, such as host cell proteins. Therefore, there remains a need for cell culture methods for propagating viruses for vaccine production that can produce high yields of viral proteins while minimizing contaminating protein ingress. Summary of the Invention

[0005] This disclosure is based on the surprising finding that modifications to various cell culture conditions, such as pH, viral dose, and trypsin concentration, can be used to improve the yield and / or purity of influenza virus cultured on MDCK cells (as determined by hemagglutinin levels at the end of infection).

[0006] In a first aspect, this disclosure provides a method for determining the culture conditions of cells infected with influenza virus, the method comprising the following steps:

[0007] (a) Incubating influenza virus-infected cells in multiple test media, wherein one or more culture conditions of the cells are varied between each of the multiple test media, wherein the one or more culture conditions are selected from pH, trypsin concentration, and combinations thereof; and

[0008] (b) Select one or more culture conditions to optimize the yield and / or purity of the influenza virus.

[0009] In a specific example, changing one or more culture conditions between multiple test culture media includes: in the multiple test culture media,

[0010] (i) Increase trypsin concentration from baseline to its maximum level; and / or

[0011] (ii) Reduce the pH from the baseline level to its lowest level.

[0012] In some instances, the trypsin concentration was increased to a maximum level of 10,000 U / L and / or the pH was decreased to a minimum level of 6.0 in the multiple test media.

[0013] In other instances, the pH varies within a pH range of about 6.0 to about 8.0. More specifically, the pH may vary within a pH range of about 6.5 to about 7.5. Even more specifically, the pH may vary within a pH range of about 6.7 to about 7.3.

[0014] According to some examples, the trypsin concentration varies in the range of about 2000 U / L to about 10000 U / L. More specifically, the trypsin concentration can vary in the range of about 4000 U / L to about 8000 U / L.

[0015] Appropriately, the cells are used with about 10 -8 Incubate with a viral dose of approximately 10.

[0016] For various instances of the method disclosed herein, step (b) includes selecting one or more culture conditions to optimize the yield and / or purity of the protein produced by the influenza virus. In such instances, the protein is suitably a hemagglutinin protein.

[0017] In a second aspect, this disclosure provides a method for culturing cells infected with influenza virus, the method comprising the steps of incubating the influenza virus-infected cells at a pH of about 6.7 to about 7.3 and in a medium containing trypsin at a concentration of at least about 5000 U / L.

[0018] The method described in this aspect may further include the step of harvesting the influenza virus and / or influenza virus proteins from the culture medium.

[0019] In a third aspect, this disclosure provides a method for generating influenza virus and / or influenza virus proteins, the method comprising the following steps:

[0020] (a) Incubating influenza virus-infected cells at a pH of about 6.7 to about 7.3 and in a medium containing at least about 5000 U / L of trypsin; and

[0021] (b) Harvest the influenza virus and / or influenza virus protein thus generated from the culture medium.

[0022] Referring to the methods of the second and third aspects, the influenza virus-infected cells are appropriately cultured at a pH of about 6.8 to about 7.1.

[0023] For the methods of the second and third aspects, the culture medium appropriately contains trypsin at a concentration of about 5000 U / L to about 10000 U / L.

[0024] In some instances of the second and third aspects, the cells can be used with approximately 10 -8 Incubate or infect with a viral dose of approximately 10.

[0025] In other examples of the second and third aspects, the method may further include the step of purifying the influenza virus and / or the influenza virus protein harvested from the culture medium.

[0026] Suitablely, the influenza virus protein described in both of the above aspects is a hemagglutinin protein. In such examples, the hemagglutinin protein harvested from the culture medium may have a purity level of at least about 10% based on its total protein concentration. More specifically, the hemagglutinin protein may be present in the culture medium at a concentration of at least about 10 mg / L.

[0027] Appropriately, the influenza virus described above is an influenza A virus, such as an H1 or H3 subtype of influenza A virus, or more specifically, an H3N2 strain or an H1N1 strain.

[0028] Appropriately, the cells described above are or include MDCK cells.

[0029] Appropriately, the incubation steps described above are carried out for approximately 48 hours to approximately 96 hours.

[0030] In a fourth aspect, this disclosure provides influenza virus or influenza virus protein produced by the methods described in the second or third aspect.

[0031] In a fifth aspect, this disclosure provides a vaccine composition comprising the influenza virus protein described in the fourth aspect and a pharmaceutically acceptable carrier, diluent, or excipient.

[0032] In a sixth aspect, this disclosure provides a method for inducing an immune response in a subject, the method comprising the steps of administering to the subject a therapeutically effective amount of the influenza virus protein of the fourth aspect or the vaccine composition of the fifth aspect, thereby inducing an immune response in the subject.

[0033] In a seventh aspect, this disclosure provides a method for preventing and / or treating influenza-related diseases, symptoms, or conditions in a subject, the method comprising the steps of administering to the subject a therapeutically effective amount of the influenza virus protein of the fourth aspect or the vaccine composition of the fifth aspect, thereby preventing and / or treating the influenza-related diseases, symptoms, or conditions.

[0034] In an eighth aspect, this disclosure provides a method for culturing cells infected with influenza virus, the method comprising the steps of: culturing influenza virus-infected cells in a culture medium containing trypsin under one or more culture conditions selected in step (b) of the method of the first aspect. Attached Figure Description

[0035] The following figures form part of this specification and are included to further illustrate certain aspects of this disclosure. This disclosure can be better understood by referring to one or more of these figures in conjunction with the detailed description of the specific embodiments presented herein. Those skilled in the art will understand that many variations and / or modifications can be made to the above embodiments without departing from the broad general scope of this disclosure. Therefore, this disclosure should be considered illustrative rather than restrictive in all respects.

[0036] Figure 1 Model predictions from A / Florida / 50 / 2022.

[0037] Figure 2 Model predictions from A / Michigan / 41 / 2022.

[0038] Figure 3 Model predictions for CVR-88 (A / Darwin / 11 / 2021).

[0039] Figure 4 Model predictions from A / Canberra / 407 / 2019.

[0040] Figure 5 Model predictions from A / NorthCarolina / 01 / 2021.

[0041] Figure 6 Model predictions from A / Washington / 19 / 2020.

[0042] Figure 7 Model predictions from B / Philippines / 28 / 2019.

[0043] Figure 8 Model predictions from B / Connecticut / 01 / 2021.

[0044] Figure 9 Model predictions from A / Sydney / 5 / 2021.

[0045] Figure 10 Model predictions for A / Victoria / 10 / 2020.

[0046] Figure 11Model predictions for B / South Australia / 06 / 2019. Detailed Implementation

[0047] General techniques and definitions

[0048] Unless otherwise expressly defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in the fields of genomics, immunology, molecular biology, immunohistochemistry, biochemistry, oncology, and pharmacology).

[0049] Unless otherwise stated, this disclosure uses conventional techniques of molecular biology, microbiology, recombinant DNA technology, and immunology, without the need for excessive experimentation. These procedures are described in, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 4th edition (2012), Volumes I, II and III; DNA Cloning: A Practical Approach, Volumes I and II (DN Glover, 2nd edition, 1995), IRL Press, Oxford, full text; Oligonucleotide Synthesis: A Practical Approach (edited by MJ Gait, 1984), IRL Press, Oxford, full text, especially the following papers: Gait, pp. 1-22; Atkinson et al., pp. 35-81; Sproat et al., pp. 83-115; and Wu et al., pp. 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (edited by BD Hames & SJ Higgins, 1985), IRL Press, Oxford, full text; Immobilized Cells and Enzymes: A Practical Approach (1986), IRL Press, Oxford, full text; Perbal, B., Practical Guide to Molecular Cloning (1984) and Methods In Enzymology (edited by S. Colowick and N. Kaplan, Academic Press, Inc.), complete works.

[0050] Those skilled in the art will understand that this disclosure is readily adaptable to variations and modifications beyond the specific description. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes all steps, features, compositions, and compounds mentioned or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the stated steps or features.

[0051] The scope of this disclosure is not limited to the specific embodiments described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure, as described herein.

[0052] Each feature of any particular aspect, implementation, or embodiment of this disclosure may be applied to any other aspect, implementation, or embodiment of this disclosure with necessary modifications.

[0053] In this specification, unless otherwise specified or required by the context, references to a single step, a material composition, a set of steps, or a set of material compositions shall be construed as covering one or more (i.e., one or more) of such steps, material compositions, sets of steps, or sets of material compositions.

[0054] As used herein, the singular forms “a,” “an,” and “the” include the plural forms of these words unless the context explicitly specifies otherwise. For example, referring to “a bacterium” includes multiple such bacteria, and referring to “an allergen” refers to one or more allergens.

[0055] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y”, and should be regarded as providing explicit support for both meanings or either meaning.

[0056] In this specification, various aspects and components of this disclosure may be presented in a range format. The range format is included for convenience and should not be construed as a rigid limitation on the scope of this disclosure. Therefore, unless otherwise specifically stated, a description of a range should be considered as specifically disclosing all possible subranges and individual numerical values ​​within that range. For example, a description of a range such as 1 to 5 should be considered as specifically disclosing subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 5, 3 to 5, etc., and individual and partial numbers within said ranges, such as 1, 2, 3, 4, 5, 5.5, and 6, unless the context requires or implies integers. This applies regardless of the breadth of the disclosed range. Where specific values ​​are required, they will be indicated in the specification.

[0057] In this specification, the word “comprising” or variations such as “including” or “containing” will be understood to imply inclusion of the said element, integer or step, or group of elements, integers or steps, but does not exclude any other element, integer or step, or group of elements, integers or steps.

[0058] In the context of an amino acid sequence, “consistently made up of” means the amino acid sequence plus one, two, or three additional amino acids at the N- or C-terminus.

[0059] The term “substantially” does not exclude “completely” (e.g., a composition that is “substantially free” of Y can be completely free of Y).

[0060] The term "about" regarding the numerical value x is optional and means, for example, any number within 1%, 5%, or 10% of the referenced number. In some implementations, the term "about" covers the exact number stated.

[0061] All computer programs, algorithms, patents, and scientific literature cited in this article are incorporated herein by reference.

[0062] For the purposes of this disclosure, the database accession number or unique identifier of the gene, protein or viral strain provided herein, and one or more sequences of the associated gene and / or protein, are incorporated herein by reference.

[0063] Methods for determining the optimal culture conditions for virus-infected cells

[0064] The inventors have surprisingly demonstrated that, for cells infected with influenza virus, modification of specific culture conditions, such as one or more of pH, virus dose, and trypsin concentration, can be used to optimize (i.e., improve or enhance) the yield and / or purity of the virus at the end of the infection or incubation period.

[0065] Therefore, in one aspect, this disclosure provides a method for determining the culture conditions of cells infected with influenza virus, the method comprising the following steps:

[0066] (a) Incubating influenza virus-infected cells in multiple test media, wherein one or more culture conditions of the cells are varied between each of the multiple test media, wherein the one or more culture conditions are selected from pH, trypsin concentration, and combinations thereof; and

[0067] (b) Select one or more culture conditions to optimize the yield and / or purity of the influenza virus.

[0068] In a related aspect, this disclosure provides a method for culturing cells infected with influenza virus, the method comprising culturing the influenza virus-infected cells in a culture medium containing trypsin under one or more culture conditions selected in step (b) of the method described above. For example, such a method may include one or more additional culture steps provided herein, such as harvesting the influenza virus and / or its generated proteins from the culture medium.

[0069] For specific examples, the one or more culture conditions to be varied in the plurality of test media include the pH or pH level of the media. Thus, cell culture following influenza virus infection is appropriately performed at a specific pH, which is adjusted in each test media. According to some examples, the pH of the plurality of test media varies in the pH range of about 6.0 to about 8.0 (e.g., pH of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, or any range thereof), more particularly in the pH range of about 6.5 to about 7.5, even more particularly in the pH range of about 6.7 to about 7.3, or even more particularly in the pH range of about 6.8 to about 7.2. In this regard, at least one of the plurality of test media (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) suitably has a different pH than the other test media or is cultured at a different pH than the other test media. For example, the pH of the first test media may be about 6.5, the pH of the second test media may be about 7.0, and the pH of the third test media may be about 7.5.

[0070] However, it is anticipated that two or more of the plurality of test media will suitably have or be cultured at the same or substantially the same pH as the other test media, while one or more of the other culture conditions described herein, such as viral dose and / or trypsin concentration, will vary or differ between these test media. For example, the first test medium may have a pH of about 6.5 and a trypsin concentration of about 5000 U / L, while the second test medium may have a pH of about 6.5 and a trypsin concentration of about 7500 U / L.

[0071] The pH or pH level of each test medium can be controlled by transferring CO2 gas from or to the test medium. In other instances, an acid or base, such as a buffer (e.g., sodium bicarbonate, HEPES), is added to the test medium to facilitate pH regulation and / or maintenance to the desired pH value. Cell culture yield and / or purity are generally maximized at an optimal pH, which is expected to be maintained during cell seeding and cell culture. Suitably, the pH of the test medium is maintained or substantially maintained (e.g., within ±1%, 2.5%, 5%, or 10% of the desired pH) throughout the incubation period or for at least about 70%, 75%, 80%, 85%, 90%, or 95% of the incubation period for the influenza virus-infected cells.

[0072] According to certain examples, the one or more culture conditions to be modified in the plurality of test media include the trypsin concentration of the culture medium. As used herein, the term "trypsin" generally refers to a proteolytic enzyme, more particularly a serine protease, classified as EC 3.4.21.4. Trypsin cleaves peptide chains primarily at the carboxyl side of the amino acid lysine or arginine, usually unless either of them is followed by proline. Trypsin can be derived from or isolated from natural and / or recombinant / synthetic sources. In a specific example, the trypsin described herein is a recombinant trypsin. Although trypsin is formed in vivo with a propeptide (i.e., trypsinogen), the term "trypsin" as used herein appropriately refers to a mature trypsin protein that does not contain any propeptide. The term "trypsin" also includes references to any type or isotype of trypsin known in the art, such as trypsin 1, trypsin 2, trypsin 3, trypsin 4, trypsin 5, and trypsin 6. Proteolytic cleavage using trypsin may also be referred to as "trypsin proteolysis" or "trypsin digestion," and the protein produced by trypsin cleavage is said to have been "trypsin digested."

[0073] Similar to pH, trypsin can be added to each test medium to achieve a trypsin concentration specific to or regulated within each test medium. According to some examples, the trypsin concentration for the multiple test media is from about 1000 U / L to about 12000 U / L. The concentration varies within a range of U / L (e.g., approximately 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000, 7250, 7500, 7750, 8000, 8250, 8500, 8750, 9000, 9250, 9500, 9750, 10000, 10250, 10500, 10750, 11000, 11250, 11500, 11750, 12000). (U / L or any range thereof), more particularly in the range of about 2000 U / L to about 10000 U / L, even more particularly in the range of about 3000 U / L to about 9000 U / L, even more particularly in the range of about 4000 U / L to about 8000 U / L, still even more particularly in the range of about 5000 U / L to about 10000 U / L, even more particularly in the range of about 4000 U / L to about 9000 U / L, or even more particularly in the range of about 4000 U / L to about 10000 U / L. As a result, for the plurality of test media, the trypsin concentration can vary in the range of about 10 mg / L to about 100 mg / L (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / L or any range thereof), more particularly in the range of about 20 mg / L to about 75 mg / L, even more particularly in the range of about 30 mg / L to about 70 mg / L, even more particularly in the range of about 35 mg / L to about 65 mg / L, even more particularly in the range of about 20 mg / L to about 80 mg / L, or even more particularly in the range of about 30 mg / L to about 80 mg / L. Therefore, at least one of the plurality of test media (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) appropriately has a different trypsin concentration than the other test media or is cultured at a different trypsin concentration than the other test media. For example, the trypsin concentration of the first test media may be about 3500 U / L, the trypsin concentration of the second test media may be about 5500 U / L, and the trypsin concentration of the third test media may be about 7500 U / L.

[0074] During incubation of infected cells according to the methods of this disclosure, trypsin reactivation can be performed by adding additional trypsin in a batch process or by continuously or intermittently adding trypsin solution in a perfusion system. Therefore, incubating cells using the methods provided herein may include replacing at least a portion of the cell culture medium with freshly prepared culture medium, culture medium concentrate, or defined components (e.g., amino acids, vitamins, lipid fractions, phosphates, trypsin, etc.) to optimize viral protein yield and / or purity.

[0075] Similarly, it is anticipated that two or more of the plurality of test media will suitably have the same or substantially the same trypsin concentration as the other test media, or be cultured at the same or substantially the same trypsin concentration as the other test media, while one or more of the other culture conditions described herein, such as virus dose and / or pH, vary or differ between these test media. For example, the first test medium may have a trypsin concentration of about 5000 U / L and a pH of about 6.5, while the second test medium may have a trypsin concentration of about 5000 U / L and a pH of about 7.0.

[0076] Trypsin functions by cleaving the precursor protein of hemagglutinin (HA), thereby facilitating the adsorption of influenza virus onto cells. Trypsin can be added shortly before, during, or shortly after infection with the influenza virus. If added concurrently with the infection step, trypsin can be added directly to the test medium or, for example, as a concentrate along with the influenza virus inoculum.

[0077] Those skilled in the art will understand that a single influenza virus strain may exhibit a preferred or optimal viral dose when infecting cells. Therefore, determining the optimal viral dose of an influenza virus strain can be included as a standard step prior to initiating commercial vaccine production in cells (e.g., MDCK cells) based on the influenza virus strain in question. Thus, this step of determining the optimal viral dose can be incorporated into the methods described herein. Thus, the one or more culture conditions in step (a) may further include a viral dose (e.g., MOI value, "multiple of infection," which corresponds to the number of viral units per cell at infection). Thus, the one or more culture conditions in step (a) can be selected from pH, trypsin concentration, viral dose, and any combination thereof. For this purpose, cells in each test medium can be suitably treated with approximately 10 -10 A viral dose of approximately 10 or an MOI infection (e.g., an MOI of approximately 10). -10 10 -9 10 -8 10 -7 10 -6 10 -5The viral doses are 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and any range thereof. Furthermore, at least one of the plurality of test media (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) suitably contains a different viral dose than the other test media. For example, the viral dose of the first test media may be approximately 10. -7 The viral dose in the second test culture medium can be approximately 10. -6 The viral dose in the third test culture medium can be approximately 10. -5 In alternative examples, the viral dose in each test culture medium was substantially the same or similar.

[0078] Similar to the culture conditions regarding pH and trypsin concentration, it is anticipated that two or more of the plurality of test media will suitably have the same or substantially the same viral dose as the other test media, or be cultured at the same or substantially the same viral dose as the other test media, while one or more of the other culture conditions described herein may vary or differ between these test media. For example, the first test medium may have approximately 10 -6 The viral dose and pH of approximately 6.5, while the second test medium can have approximately 10... -6 The viral dose and pH of approximately 7.0.

[0079] In some instances, the one or more culture conditions to be varied in the plurality of test media include the pH level of the medium and the trypsin concentration. This is intended to include any combination of those examples (including ranges) of pH levels and trypsin concentrations provided herein. For such examples, the pH is suitably varied in a pH range of about 6.0 to about 8.0, and the trypsin concentration is suitably varied in a concentration range of about 2000 U / L to about 10000 U / L. More specifically, the pH is suitably varied in a pH range of about 6.5 to about 7.5, and the trypsin concentration is suitably varied in a concentration range of about 3000 U / L to about 9000 U / L. Even more specifically, the pH is suitably varied in a pH range of about 6.7 to about 7.3, and the trypsin concentration is suitably varied in a concentration range of about 4000 U / L to about 8000 U / L.

[0080] In other instances, the one or more culture conditions to be varied in the plurality of test media include the pH level of the culture medium and the viral dose. This is intended to include any combination of those examples (including their ranges) of pH levels and viral doses provided herein. For such examples, the pH is suitably varied in a pH range of about 6.0 to about 8.0, and the viral dose is suitably varied in a range of about 10.-8 The concentration varies within a range of approximately 10. More specifically, the pH appropriately varies within a pH range of approximately 6.5 to approximately 7.5, and the viral dose appropriately varies within a range of approximately 10. -7 The concentration varies within a range of approximately 1. More specifically, the pH appropriately varies within a pH range of approximately 6.7 to approximately 7.3, and the viral dose appropriately varies within a range of approximately 10. -6 The concentration varies within the range of approximately 0.1.

[0081] In various instances, the one or more culture conditions to be varied in the plurality of test media include the trypsin concentration and viral dose of the culture medium. This is intended to include any combination of those examples (including ranges) of trypsin concentration and viral dose provided herein. For such examples, the trypsin concentration is suitably varied in the range of about 2000 U / L to about 10000 U / L, and the viral dose is suitably varied in the range of about 10 -8 The concentration varies within a range of approximately 10. More specifically, the trypsin concentration appropriately varies within a range of approximately 3000 U / L to approximately 9000 U / L, and the viral dose appropriately varies within a range of approximately 10. -7 The concentration varies within the range of approximately 1. More specifically, the trypsin concentration appropriately varies within the range of approximately 4000 U / L to approximately 8000 U / L, and the viral dose appropriately varies within the range of approximately 10. -6 The concentration varies within the range of approximately 0.1.

[0082] In specific examples, the one or more culture conditions to be varied in the plurality of test media include the pH level of the medium, the trypsin concentration, and the viral dose. This is intended to include any combination of those examples (including ranges) of pH levels, trypsin concentrations, and viral doses provided herein. For such examples, the pH is suitably varied in a pH range of about 6.0 to about 8.0, the trypsin concentration is suitably varied in a concentration range of about 2000 U / L to about 10000 U / L, and the viral dose is suitably varied in a concentration range of about 10 U / L. -8 The concentration varies within a range of approximately 10. More specifically, the pH appropriately varies within a pH range of approximately 6.5 to approximately 7.5, the trypsin concentration appropriately varies within a concentration range of approximately 3000 U / L to approximately 9000 U / L, and the viral dose appropriately varies within a range of approximately 10. -7 The concentration varies within a range of approximately 1. More specifically, the pH appropriately varies within a pH range of approximately 6.7 to approximately 7.3, the trypsin concentration appropriately varies within a concentration range of approximately 4000 U / L to approximately 8000 U / L, and the viral dose appropriately varies within a range of approximately 10.-6 The concentration varies within the range of approximately 0.1.

[0083] As demonstrated in the examples, the inventors have surprisingly shown that during incubation of virus-infected cells, lower pH levels and / or higher trypsin concentrations generally increase or maximize HA protein purity at the end of infection. Therefore, and according to certain examples, altering one or more culture conditions between each of the plurality of test media comprises: (i) increasing the trypsin concentration from a baseline level (e.g., a first level) to its maximum level (e.g., a second level) in the plurality of test media; and / or (ii) decreasing the pH from a baseline level (e.g., a first level) to its minimum level (e.g., a second level). Thus, the methods of this disclosure may include a first test media having a baseline trypsin concentration, a second test media having a maximum trypsin concentration, and optionally one or more additional test media having trypsin concentration levels between the baseline level and the maximum level. Similarly, the methods of this disclosure may include a first test media having a baseline pH level, a second test media having a minimum pH level, and optionally one or more additional test media having pH levels between the baseline level and the minimum level.

[0084] Appropriately, with respect to trypsin concentration and pH, the baseline level is the standard, average, typical, or acceptable level of these cell culture conditions used for incubating virus-infected cells. Similarly, in the context of trypsin concentration and pH, the maximum level is a level higher than the standard, average, typical, or acceptable level of these cell culture conditions used for incubating virus-infected cells. Conversely, the minimum level is a level lower than the standard, average, typical, or acceptable level of these cell culture conditions used for incubating virus-infected cells. For example, the baseline pH level can be from about 7.0 to about 7.5 (e.g., about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, 7.4, 7.45, 7.5 or any range thereof), and the minimum level can be from about 6.0 to about 7.0 (e.g., about 6.0, 6.05, 6.1, 6.15, 6.2, 6.25, 6.3, 6.35, 6.4, 6.45, 6.5, 6.55, 6.6, 6.65, 6.7, 6.75, 6.8, 6.85, 6.9, 6.95, 7.0 or any range thereof). Therefore, changing one or more culture conditions among each of the plurality of test media includes reducing the pH from a baseline level of about 7.0 to about 7.5, or more particularly about 7.0 to about 7.3, to a minimum level of about 6.0 to about 7.0, more particularly about 6.5 to about 7.0, or even more particularly about 6.5 to about 6.8. In addition, the baseline level of trypsin concentration can be from about 1,000 U / L to about 5,000 U / L (e.g., about 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000 U / L or any range thereof), and the maximum level can be from about 5,000 to about 12,000 U / L (e.g., about 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 10,500, 11,000, 11,500, 12,000 U / L or any range thereof). Therefore, altering one or more culture conditions among each of the plurality of test media includes increasing the trypsin concentration from a baseline level of about 1,000 U / L to about 5,000 U / L or more particularly about 2,000 U / L to about 4,000 U / L to about 5,000 U / L to about 12,000 U / L, more particularly about 5,000 U / L to about 10,000 U / L, or even more particularly about 5,000 U / L to about 8,000 U / L to a maximum level.

[0085] Assessing the yield and / or purity of influenza virus can be done by any method or means known in the art. Suitablely, step (b) of the method of this disclosure includes selecting one or more culture conditions that optimize (e.g., lead to the highest level) the yield and / or purity of the protein produced by the influenza virus. For some instances, step (b) of the method of this disclosure includes selecting one or more culture conditions that optimize the yield of the protein produced by the influenza virus. In these instances, the selected one or more culture conditions also suitably lead to a suitable or appropriate level of protein purity, such as those provided herein. In other instances, step (b) of the method of this disclosure includes selecting one or more culture conditions that optimize the purity of the protein produced by the influenza virus. For these instances, the selected one or more culture conditions also suitably lead to a suitable or appropriate level of protein yield, such as those provided herein. In a specific example, the influenza virus protein for which the yield and / or purity is to be assessed is a hemagglutinin protein.

[0086] Therefore, the method disclosed herein may further include the step of measuring or evaluating the yield of influenza virus proteins in each of the one or more culture conditions tested. The yield of influenza virus proteins can be measured by any protein quantification method known in the art, such as by gel electrophoresis (e.g., Western Blot); by ELISA; or by chromatographic methods, such as HPLC (high performance liquid chromatography), HPLC-UV, or mass spectrometry methods, such as LC-MS (liquid chromatography-mass spectrometry).

[0087] In addition to the above, the purity level of influenza virus and / or influenza virus protein in each of one or more culture conditions can be determined for the methods of this disclosure. For this purpose, extracts or isolates containing isolated influenza virus proteins typically contain impurities or contaminating proteins (e.g., non-viral proteins), such as host cell proteins derived from cells. The purity of the isolated influenza virus protein can be measured by any means known in the art. Exemplary methods may include calculating the ratio of the level, content, or concentration (e.g., determined by the methods described above) of a specific influenza virus protein in the isolated influenza virus protein to the total protein content of the isolated influenza virus protein. Alternatively, purity can be determined by measuring the ratio of the level, concentration, or content of one or more host cell proteins in the isolated influenza virus protein to its total protein content. The total protein content can be assessed by, for example, quinolinic acid (BCA) determination, gel electrophoresis (e.g., by Bradford assay, SDS-PAGE and Western Blot, or Coomassie staining), ELISA, chromatographic methods (such as HPLC), or mass spectrometry. In a specific example, the purity of the isolated influenza virus protein is determined by the ratio of its hemagglutinin protein level to the total protein content. For such cases, the level of hemagglutinin protein was appropriately measured by HPLC, and the total protein content was appropriately measured by BCA assay.

[0088] In various instances, the purity of the isolated influenza virus and / or isolated influenza virus proteins is also assessed after harvesting, isolating, purifying, or separating from cells and / or their respective test media, such as by methods described herein. For this purpose, cells or cell residues can be separated from their respective test media by methods known to those skilled in the art (e.g., centrifugation, separators, or filters). The influenza virus present in the test media can then be isolated, concentrated, or harvested by methods known to those skilled in the art (e.g., those described herein, such as gradient centrifugation, filtration, precipitation, etc.). In specific instances, the influenza virus and / or one or more proteins it produces are at least partially isolated or harvested from the test media by gradient ultracentrifugation. Therefore, the level of purity can be assessed relative to the isolated influenza virus proteins that have been at least partially purified or isolated from the test media (e.g., by gradient ultracentrifugation).

[0089] To determine or select one or more culture conditions that optimize the yield and / or purity of proteins produced by influenza virus, the methods disclosed herein may include comparing yield and / or purity levels obtained or measured under various culture conditions with one or more threshold levels. In various instances, if a yield and / or purity level exceeds its specific threshold level, it may be classified or selected as optimal. The nature and numerical value of the threshold levels for purity or yield typically vary depending on the influenza virus protein in question and / or the methods or means selected to determine the purity or yield level of that viral protein for its respective cell culture conditions.

[0090] For hemagglutinin, the appropriate yield threshold level for this viral protein may be at least about 10 mg / L (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 mg / L or any range thereof), at least about 15 mg / L, at least about 20 mg / L, or at least about 25 mg / L (in test medium at the end of infection or incubation period). Therefore, one or more culture conditions that produce hemagglutinin yields of at least about 10 mg / L (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 mg / L or any range thereof), at least about 15 mg / L, at least about 20 mg / L or at least about 25 mg / L in the test medium may be selected to optimize the yield in step (b) of the method of this disclosure. In some instances, one or more culture conditions that produce hemagglutinin yields of about 10 mg / L to about 50 mg / L, about 15 mg / L to about 45 mg / L, or about 20 mg / L to about 40 mg / L in the test medium can be selected to optimize the yield.

[0091] Similarly, for hemagglutinin, the appropriate purity threshold level for this viral protein may be at least about 10% (based on total protein concentration) (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50%), at least about 15% (based on total protein concentration), at least about 20% (based on total protein concentration), or at least about 25% (based on total protein concentration). Therefore, one or more culture conditions that produce a purity of at least about 10% (based on total protein concentration) (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50% or any range thereof), at least about 15% (based on total protein concentration), at least about 20% (based on total protein concentration), or at least about 25% (based on total protein concentration) of hemagglutinin may be selected in step (b) of the method of this disclosure to optimize purity. In some instances, one or more culture conditions that produce hemagglutinin purity of about 10% to about 50% of the total protein concentration, about 15% to about 45% of the total protein concentration, or about 20% to about 40% of the total protein concentration may be selected to optimize purity.

[0092] In some instances, one or more culture conditions that produce hemagglutinin yields of approximately 20 mg / L to approximately 50 mg / L and hemagglutinin purity of approximately 20% to approximately 50% of the total protein concentration in the test medium can be selected to optimize influenza virus yield and purity.

[0093] The method disclosed herein may also include one or more of the following steps:

[0094] (i) Cell proliferation prior to infection with influenza virus, for example in the culture medium described herein (e.g., suspension in serum-free medium);

[0095] (ii) Infecting cells with influenza virus;

[0096] (iii) Adding trypsin to cells shortly before, during, or shortly after infection with the influenza virus; and

[0097] (iv) Harvesting and isolating replicated influenza virus or virus-derived proteins after culturing or incubating infected cells; for example, by the methods described herein.

[0098] Methods for culturing virus-infected cells and producing viral proteins

[0099] Based on the above-described method for optimizing the culture conditions of virus-infected cells, the inventors have further demonstrated, surprisingly, that culturing influenza virus-infected cells under specific conditions of pH, trypsin concentration, and optional viral dose can optimize the yield and / or purity of influenza virus at the end of infection based on virus-derived proteins (such as hemagglutinin).

[0100] Therefore, in one aspect, this disclosure provides a method for culturing cells infected with influenza virus, the method comprising the steps of incubating the influenza virus-infected cells at a pH of about 6.7 to about 7.3 and in a medium containing trypsin at a concentration of at least about 5000 U / L.

[0101] Where appropriate, the method disclosed herein further includes the step of harvesting the influenza virus and / or influenza virus proteins from the culture medium.

[0102] In a related aspect, this disclosure provides a method for producing influenza virus and / or influenza virus protein, the method comprising the following steps:

[0103] (a) Incubating influenza virus-infected cells at a pH of about 6.7 to about 7.3 and in a medium containing at least about 5000 U / L of trypsin; and

[0104] (b) Harvest or isolate the influenza virus and / or the influenza virus protein from the culture medium.

[0105] In one particular aspect, this disclosure relates to a method for improving yield and / or purity in the manufacture of influenza vaccines, the method comprising the following steps:

[0106] (a) Incubating influenza virus-infected cells in multiple test media, wherein one or more culture conditions of the cells are changed between each of the multiple test media, wherein the one or more culture conditions are selected from pH, trypsin concentration and combinations thereof;

[0107] (b) Select one or more culture conditions to optimize the yield and / or purity of the influenza virus;

[0108] (c) Culturing suspension mammalian cells capable of being infected with influenza virus in serum-free medium; and

[0109] (d) Infecting the said mammalian cells with influenza virus; and

[0110] (e) Incubate the influenza virus-infected mammalian cells under one or more culture conditions selected in (b).

[0111] In another related aspect, this disclosure provides influenza virus or influenza virus protein produced according to the above method.

[0112] Therefore, the methods disclosed herein may include harvesting or isolating intact or whole viral particles from cell culture media. Alternatively or additionally, the methods disclosed herein may include harvesting or isolating lysed viral particles from culture media. In such instances, the harvesting or isolation step suitably includes contacting the influenza virus with a lysis agent (e.g., a detergent). The methods disclosed herein may alternatively or additionally include harvesting or isolating one or more specific influenza virus proteins, such as hemagglutinin, from culture media and / or from the harvested influenza virus, for example by affinity chromatography and / or gradient ultracentrifugation.

[0113] For the purposes of this disclosure, "separated" means material that has been removed from its natural state or has been artificially manipulated. Separated materials, such as influenza viruses and virus-derived proteins (e.g., hemagglutinin), may be substantially or essentially free of the components that normally accompany them in their natural state, or may be manipulated to be in an artificial state together with the components that normally accompany them in their natural state. Separated materials may be in natural, chemically synthesized, or recombinant forms.

[0114] In some instances, the methods described herein further include a step of proliferating or expanding cells prior to infection with an influenza virus, for example, in a culture medium as described herein (e.g., suspension in a serum-free medium). Pre-infection cell proliferation can be performed according to methods known to those skilled in the art. For example, cells can be cultured in a perfusion system or a fed-batch system prior to infection. In the context of this disclosure, the culture system is referred to as a fed-batch system, in which cells are initially cultured in a batch system and at least partially depleted of nutrients, and then grown in a medium replenished by controlled feeding to concentrate nutrients. The pH of the culture medium during pre-infection cell proliferation is suitably in the range of pH 6.6 to pH 7.8, and more particularly in the range of pH 7.2 to pH 7.3. Following infection with influenza virus, cells are appropriately cultured at a pH of about 6.7 to about 7.3 (e.g., about 6.7, 6.75, 6.8, 6.85, 6.9, 6.95, 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, and any range thereof), or more particularly at a pH of about 6.8 to about 7.1. Culture of cells to be infected with influenza virus typically occurs at a temperature of about 30°C to about 37°C (e.g., 30, 31, 32, 33, 34, 35, 36, 37°C, and any range thereof), more particularly at about 37°C. Following infection with influenza virus, cells can then be cultured at the temperatures described herein (e.g., about 30°C to about 36°C, or about 32°C to about 34°C, or about 33°C).

[0115] As described above, it is known in the art that a single viral strain may exhibit a preferred or optimal viral dose when infecting cells. Therefore, step (a) may further include exposing the cells to approximately 10 -10 A viral dose of approximately 10 or an MOI (e.g., an MOI of approximately 10). -10 10 -9 10 -8 10 -7 10 -6 10 -5 Cells can be contacted or incubated with 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and any range thereof. In a specific instance, the cells were incubated with approximately 10... -7 about 1 or more specifically about 10 -6 Incubate with a viral dose of approximately 0.1 or an MOI. Thus, the method disclosed herein may include the following steps: incubating cells with approximately 10 -8 Infected cells are exposed to a viral dose or MOI of approximately 10, and then incubated or cultured at a pH of approximately 6.7 to approximately 7.3 in a medium containing at least approximately 5000 U / L of trypsin. In other instances, the method disclosed herein includes the step of: exposing cells to approximately 10... -8 In contact with a viral dose or MOI of about 10, and then incubate or culture the infected cells at a pH of about 6.7 to about 7.3 and in a medium containing trypsin at a concentration of about 5000 U / L to about 12000 U / L. In various examples, the method of this disclosure includes the step of: contacting the cells with about 10 -8 Incubate or culture the infected cells at a viral dose or MOI of about 10, then at a pH of about 6.7 to about 7.3 and in a medium containing trypsin at a concentration of about 5000 U / L to about 10000 U / L (e.g., about 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000, 7250, 7500, 7750, 8000, 8250, 8500, 8750, 9000, 9250, 9500, 9750, or 10000 U / L). More specifically, the method disclosed herein may include the step of: exposing the cells to about 10 -7 Introduce or culture the infected cells with a viral dose or MOI of about 1, and then incubate or culture them at a pH of about 6.7 to about 7.2 in a medium containing trypsin at a concentration of about 5000 U / L to about 8000 U / L. More specifically, the method disclosed herein may include the step of: exposing the cells to about 10 -6Infected cells are exposed to a viral dose or MOI of about 0.1 and then incubated or cultured at a pH of about 6.8 to about 7.1 in a medium containing trypsin at a concentration of about 5000 U / L to about 7500 U / L.

[0116] Infected cells are appropriately incubated under conditions that allow influenza virus replication. In this regard, the partial pressure of oxygen can also be adjusted during pre-infection culture, for example, to a partial pressure of oxygen of about 25% to about 95% (e.g., about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or any range thereof), and more particularly, to a partial pressure of oxygen of about 35% to about 60%. These partial pressure values ​​are appropriately based on air saturation.

[0117] Similar to the methods described above, trypsin can be added to the culture medium to achieve a trypsin concentration specific to or regulated within each test medium, as previously described herein. Therefore, in some instances, the methods disclosed herein include the step of adding trypsin to the culture medium shortly before, during, or shortly after infection with influenza virus. Trypsin is appropriately added to the culture medium to achieve a concentration of about 5000 U / L to about 12000 U / L (e.g., about 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000, 7250, 7500, 7750, 8000, 8250, 8500, 8750, 9000, 9250, 9500, 9750, 10000, 10250, 10500, 10750, 11000, 11250, 11500, 11750, 12000 U / L or any range thereof) or about 27.5 mg / L to about 100 mg / L (e.g., about 27.5, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / L or any range thereof). In some instances, trypsin is added to the culture medium to achieve a concentration of at least about 27.5 mg / L. In specific instances, trypsin has been added to or is present in the culture medium at concentrations of about 5000 U / L to about 10000 U / L, about 5000 U / L to about 9000 U / L, about 5000 U / L to about 8000 U / L, about 5000 U / L to about 7000 U / L, about 6000 U / L to about 10000 U / L, about 6000 U / L to about 9000 U / L, about 6000 U / L to about 8000 U / L, or about 6000 U / L to about 7000 U / L. In other instances, trypsin was added to or present in the culture medium at a concentration of approximately 5000 U / L to approximately 6000 U / L. In some instances, trypsin was added to or present in the culture medium at a concentration of approximately 7000 U / L to approximately 9000 U / L. In various instances, trypsin was added to or present in the culture medium at a concentration of approximately 5000 U / L. In some instances, trypsin was added to or present in the culture medium at a concentration of approximately 7500 U / L.

[0118] As can be clearly seen from the examples herein, the trypsin concentration required to achieve optimal or suitable levels of hemagglutinin purity and / or yield can vary depending on the viral strain and other cell culture conditions (e.g., pH, viral dose, incubation period). According to some examples, step (a) of the method disclosed herein includes at a pH of about 6.7 to about 7.3 (e.g., about 6.7, 6.75, 6.8, 6.85, 6.9, 6.95, 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3 and any range thereof) and in a solution containing a concentration of about 5000 U / L to about 12000 U / L. U / L (e.g., about 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000, 7250, 7500, 7750, 8000, 8250, 8500, 8750, 9000, 9250, 9500, 9750, 10000, 10250, 10500, 10750, 11000, 11250, 11500, 11750, 12000 U / L or any range thereof) or about 27.5 mg / L to about 100 mg / L (e.g., about 27.5, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 U / L). Cells infected with influenza virus are incubated in a culture medium containing trypsin at a concentration of about 5000 U / L to about 10000 U / L at a pH of about 6.7 to about 7.3. More specifically, step (a) of the method disclosed herein suitably includes incubating cells infected with influenza virus at a pH of about 6.7 to about 7.2 in a culture medium containing trypsin at a concentration of about 5000 U / L to about 8000 U / L. Even more specifically, step (a) of the method disclosed herein suitably includes incubating cells infected with influenza virus at a pH of about 6.8 to about 7.1 in a culture medium containing trypsin at a concentration of about 5000 U / L to about 7500 U / L.

[0119] The steps of harvesting or isolating replicated influenza virus and / or one or more proteins produced therefrom can be performed approximately 24 to approximately 240 hours after cell infection or incubation with influenza virus (e.g., approximately 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240 hours or any range thereof), more particularly approximately 36 to approximately 96 hours, even more particularly approximately 48 to approximately 72 hours, or even more particularly approximately 60 to approximately 72 hours. In a specific example, the step of harvesting or isolating replicated influenza virus is performed approximately 60 hours after cell infection or incubation with influenza virus. In other examples, the step of harvesting or isolating replicated influenza virus is performed approximately 72 hours after cell infection or incubation with influenza virus.

[0120] During the harvesting or isolation of influenza virus and / or influenza virus proteins in step (b), the cells can be separated from the culture medium by standard methods such as centrifugation, separation, filtration, or ultrafiltration. The influenza virus or viral proteins produced therefrom can then be concentrated and / or purified according to methods known to those skilled in the art, such as gradient centrifugation (e.g., gradient ultracentrifugation (GUC)), filtration, precipitation, chromatography, and any combination thereof. In a specific instance, the influenza virus and / or one or more proteins produced therefrom are isolated or harvested from the culture medium by gradient ultracentrifugation. Appropriately, the influenza virus is inactivated during or after purification. Virus inactivation can be achieved, for example, by contacting the influenza virus with an inactivating agent (e.g., the addition of β-propiolactone or formaldehyde) at any point during the purification process.

[0121] Appropriately, current methods achieve specific (e.g., threshold levels) yield and / or purity levels for harvested or isolated influenza viruses and / or influenza virus proteins. Therefore, the methods disclosed herein may further include the step of measuring or assessing the yield and / or purity of influenza viruses and / or influenza virus proteins harvested or isolated from the culture medium at the end of infection. For this purpose, the assessment of the yield and / or purity of isolated influenza viruses may be based at least in part on specific influenza virus proteins, such as hemagglutinin proteins, as described in more detail herein.

[0122] In a specific instance, the influenza virus harvested or isolated in step (b) has a purity level of at least about 10% (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50%, or any range thereof) based on its hemagglutinin protein, at least about 15%, at least about 20%, or at least about 25% (based on its total protein concentration). According to certain examples, the influenza virus harvested or isolated in step (b) has a hemagglutinin protein purity level of about 10% to about 50% of the total protein concentration, about 15% to about 45% of the total protein concentration, or about 20% to about 40% of the total protein concentration.

[0123] Regarding yield, after incubation step (a), hemagglutinin protein is appropriately present in the culture medium at the end of infection at a concentration of at least about 10 mg / L (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 mg / L or any range thereof), at least about 15 mg / L, at least about 20 mg / L or at least about 25 mg / L. Thus, incubation step (a) appropriately produces a hemagglutinin protein yield of at least about 10 mg / L, at least about 15 mg / L, at least about 20 mg / L, or at least about 25 mg / L in the culture medium at the end of infection. In various examples, incubation step (a) produces a hemagglutinin protein yield of about 10 mg / L to about 50 mg / L, about 15 mg / L to about 45 mg / L, or about 20 mg / L to about 40 mg / L in the culture medium at the end of infection.

[0124] Cultured cells

[0125] Regarding the cells described herein, the methods disclosed herein will generally use cell lines, although primary cells may be used as an alternative. The cells are typically mammalian cells. Suitable mammalian-derived cells include, but are not limited to, hamster, bovine, primate (including human and monkey) and canine cells. Various cell types may be used, such as kidney cells, fibroblasts, retinal cells, and lung cells, as known in the art. Suitable examples of hamster cells are cell lines named BHK21 or HKCC. Suitable monkey cells include African green monkey cells, such as kidney cells from the Vero cell line (Kistner et al. (1998) Vaccine 16:960-8; Kistner et al. (1999) Dev Biol Stand 98:101-110; Bruhl et al. (2000) Vaccine 19:1149-58). Suitable canine cell lines include canine kidney cells, such as the CLDK and MDCK cell lines (WO97 / 37000; Brands et al. (1999) Dev Biol Stand 98:93-100; Halperin et al. (2002) Vaccine 20:1240-7; Tree et al. (2001) Vaccine 19:3444-50). Therefore, suitable cell lines include, but are not limited to: MDCK; CHO; 293T; BHK; Vero; MRC-5; PER.C6 and WI-38 cell lines.

[0126] Based on specific examples, the cells or cell lines used for culturing influenza viruses described in this article include: MDCK cells derived from Madin Darby canine kidneys; cells derived from African green monkeys (…). Cercopithecus aethiops Vero cells from the kidney; or PER.C6 cells derived from human embryonic retinoblastoma cells (Pau et al. (2001) Vaccine 19:2716-21). These cell lines are widely available, for example from the American Type Culture Collection (ATCC), Coriell Cell Bank, and the European Collection of Cell Cultures (ECACC).

[0127] The influenza viruses described herein can be cultured on avian cell lines (see, for example, WO2003 / 076601; WO2005 / 042728; WO2003 / 043415), including cell lines derived from ducks (e.g., duck retinal cells) or chickens (e.g., chicken embryonic fibroblasts (CEF)). Examples include avian embryonic stem cells, including: EBx cell lines derived from chicken embryonic stem cells, EB45, EB14, EB14-074, and EB66.

[0128] Suitablely, the cells or cell lines used in the methods of this disclosure are MDCK cells derived from the kidneys of Madin Darby dogs. Original MDCK cells are available from ATCC under accession number CCL-34. Derivatives of MDCK cells may also be used. For example, MDCK cell lines can be adapted for suspension culture growth (e.g., 'MDCK 33016', accession number DSM ACC 2219). Similarly, WO2001 / 064846 discloses an MDCK-derived cell line grown in suspension in serum-free medium ('B-702', accession number FERM BP-7449). WO2006 / 071563 discloses non-tumorigenic MDCK cells, including 'MDCK-S' (ATCC PTA-6500), 'MDCK-SF101' (ATCC PTA-6501), 'MDCK-SF102' (ATCC PTA-6502), and 'MDCK-SF103' (PTA-6503). WO2005 / 113758 discloses MDCK cell lines with high susceptibility to infection, including 'MDCK.5F1' cells (ATCC CRL-12042). Any MDCK cell line, including those provided herein, can be used in the methods of this disclosure.

[0129] In order for the virus to grow or multiply on cell lines (such as MDCK cells), influenza virus can be cultured on suspension or adherent cells.

[0130] The cells described in this disclosure can be cultured in various serum-free or substantially serum-free media (e.g., containing less than 0.5, 0.1, 0.05, or 0.01 wt% serum) as known to those skilled in the art (e.g., Iscove's medium, ultra CHO medium (Bio Whittaker), EX-CELL (JRH Biosciences)). Alternatively, cells for replication can be cultured in serum-containing media (e.g., MEM or DMEM medium containing about 0.5% to about 10%, more particularly about 1.5% to about 5% fetal bovine serum) or protein-free media (e.g., PF-CHO (JRH Biosciences)). Suitable culture vessels that can be used in the process of the methods described in this disclosure are all containers known to those skilled in the art, such as roller bottles, flasks, or fermenters.

[0131] In specific instances, cells are appropriately cultured in serum-free and / or protein-free media, for example, for cell proliferation and / or to support influenza virus replication. In the context of this disclosure, a medium is referred to as serum-free if it contains no or substantially no (e.g., less than 0.5%, 0.25%, or 0.1% by weight) serum additives from human or animal sources. Protein-free means a medium in which cell proliferation occurs without proteins, growth factors, other protein additives, and non-serum proteins, but may optionally include proteins that may be essential for viral growth, such as trypsin or other proteases. Cells grown in such media naturally contain proteins.

[0132] Cell lines supporting influenza virus replication are appropriately cultured at temperatures below 37°C during viral replication (e.g., about 30°C to about 36°C, or about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or any range thereof). When the virus is grown on the cell lines, the culture medium and the viral inoculum used to initiate the culture are appropriately free of (e.g., tested and given a negative result for contamination) contaminating viruses such as herpes simplex virus, respiratory syncytial virus, parainfluenza virus type 3, SARS coronavirus, adenovirus, rhinovirus, reovirus, polyomavirus, dual RNA virus, circovirus, and / or parvovirus.

[0133] When influenza viruses are grown on mammalian cell lines, vaccine compositions or analogues derived from them will advantageously be free of oval proteins (e.g., ovalbumin and ovomucoid) and chicken DNA, thereby reducing their allergenicity. Avoiding allergens is useful for minimizing Th2 responses. When cells are used as culture hosts in the methods of this disclosure, the cell culture conditions described herein (e.g., temperature, cell density, pH, trypsin concentration, virus dose / MOI, etc.) can be varied over a wide range depending on the cell line and influenza virus strain used, and can be adapted to the requirements of the methods of this disclosure, as described in more detail herein.

[0134] Influenza virus and influenza virus protein

[0135] Influenza viruses are enveloped RNA viruses belonging to the Orthomyxoviridae family (Palese and Shaw (2007) Orthomyxoviridae: The Viruses and Their Replication, 5th ed. Fields' Virology, edited by BN Fields, DM Knipe, and PM Howley, Wolters Kluwer Health / Lippincott Williams & Wilkins, Philadelphia, USA, pp. 1647-1689). Influenza A and B viruses are the primary human pathogens, causing a respiratory illness ranging in severity from subclinical infection to fatal primary viral pneumonia. The clinical outcome of infection varies depending on the virulence of the influenza strain and the host's exposure, medical history, age, and immune status. The natural host of influenza viruses is primarily birds, but influenza viruses, especially influenza A viruses (including avian-origin ones), can also infect humans and other animal hosts (bats, canines, pigs, horses, marine mammals, and mustelids) and cause disease.

[0136] The influenza virus disclosed herein can be either influenza A or influenza B. In some instances, the influenza virus is influenza A. In alternative instances, the influenza virus is influenza B. The influenza A or B virus can be any viral strain. For example, the influenza A virus provided herein may include HA subtypes selected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16. In various instances, the influenza A virus is the H1 or H3 subtype. Furthermore, such viruses may contain NA subtypes of influenza A virus, N1, N2, N3, N4, N5, N6, N7, N8, or N9. Specifically, influenza A viruses can be strains selected from the group consisting of H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H7N1, H7N2, H7N3, H7N7, H9N2, and H10N7. In some instances, the influenza A virus is the H3N2 strain. In alternative instances, the influenza A virus is the H1N1 strain.

[0137] Given the foregoing, the influenza virus referred to herein is appropriately a seasonal influenza virus strain. As used herein, the term "seasonal influenza virus strain" refers to an influenza virus strain to which a subject population has been exposed on a seasonal basis. Thus, in some instances, the term "seasonal influenza virus strain" refers to an influenza A virus strain. In other instances, the term "seasonal influenza virus strain" refers to an influenza A virus strain belonging to the H1 or H3 subtype (i.e., the two subtypes currently persisting or circulating in the population). In some instances, the term "seasonal influenza virus strain" refers to an influenza B virus strain.

[0138] The influenza virus referred to in this article encompasses any type, subtype, or strain of virus, including but not limited to naturally occurring strains, variants or mutants, mutagenic viruses, reassortant viruses, and / or genetically modified viruses.

[0139] Appropriately, influenza viruses are reassortant viruses. The term "reassortant virus" refers to a virus that contains genetic material produced by a combination of genetic material from at least two donor viruses. When reassortant viruses are used to prepare vaccine compositions, their genetic material typically contains at least the HA and NA genes from seasonal or pandemic influenza viruses, while other genes (i.e., backbone genes) are from one or more other donor viruses that have been selected to be readily grown on production substrates used to prepare influenza vaccines (e.g., allantoic cavities of chicken embryos or permitted cell lines) and / or to be less pathogenic or non-pathogenic to humans. Examples of donor or seed viruses that serve as backbone gene donors include A / Puerto Rico / 8 / 1934 (PR8), A / Texas / 1 / 1977, A / New York / 55 / 2004, A / Ann Arbor / 6 / 60, A / Leningrad / 134 / 17 / 57, B / AnnArbor / 1 / 66, B / Florida / 4 / 2006, B / Panama / 45 / 1990, and B / Lee / 1940. Additional exemplary donor or seed virus strains are described in International Patent Application PCT / IB2024 / 055963, which is incorporated herein by reference. Reassortant viruses can be generated by any method known in the art, including reverse genetics and classical reassortment.

[0140] The influenza virus proteins referred to herein may include any such proteins known in the art. Typically, influenza virus proteins include: for influenza A virus: PB1, PB2, PA, HA, NP, NA, M1, M2, NS1, and NS2 / NEP proteins; or for influenza B virus: PB1, PB2, PA, HA, NP, NA, NB, M1, BM2, NS1, and NS2 / NEP proteins.

[0141] In some instances of this disclosure, the influenza virus protein is a hemagglutinin protein. The terms "hemagglutinin" and "HA" refer to any hemagglutinin protein known to those skilled in the art (e.g., influenza A HA subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16). In some instances, the hemagglutinin protein is an influenza hemagglutinin protein, such as influenza A hemagglutinin protein or influenza B hemagglutinin protein. Typical hemagglutinin proteins comprise a signal peptide (optionally herein), a stem domain, a globular head domain, a lumen domain, a transmembrane domain, and a cytoplasmic domain. In some instances, the hemagglutinin protein comprises a single polypeptide chain, such as HA0. In other instances, the hemagglutinin protein comprises more than one polypeptide chain with quaternary association (e.g., HA1 and HA2). In some instances, the hemagglutinin protein lacks a signal peptide (i.e., the hemagglutinin protein is a mature hemagglutinin). The hemagglutinin protein provided in this article can also be modified through post-translational processing, such as signal peptide cleavage, disulfide bond formation, glycosylation (e.g., N-linked glycosylation), protease cleavage, and lipid modification (e.g., S-palmitoylation).

[0142] vaccine

[0143] This disclosure contemplates that influenza viruses produced according to the methods described herein and / or influenza virus proteins derived from said influenza viruses can be used in vaccine compositions.

[0144] Influenza vaccines are typically based on attenuated live or inactivated viruses. Inactivated vaccines can be based on intact virus particles, “lysed” virus particles, or purified surface antigens. Antigens can also be presented in virion form. The methods disclosed herein can be used to prepare any of these types of vaccines. When using inactivated influenza virus, the vaccine may contain intact virus particles, lysed virus particles, or purified surface antigens (e.g., hemagglutinin and optionally neuraminidase). Chemical methods for inactivating viruses include treatment with an effective amount of one or more of the following inactivating agents: detergents, formaldehyde, β-propiolactone, methylene blue, psoralen, carboxyfullerene (C60), diethylamine, acetylenine, or combinations thereof. Non-chemical methods for virus inactivation are also known in the art, such as ultraviolet light or gamma ray irradiation.

[0145] Viral particles can be harvested from virus-containing liquids (e.g., cell culture supernatants) using various methods. For example, purification methods may involve regional centrifugation or affinity chromatography using a linear sucrose gradient solution (optionally containing a detergent to disrupt the viral particles). Antigens can be purified by percolation after optional dilution.

[0146] Vaccine compositions may contain pharmaceutically acceptable carriers, diluents, or excipients. "Pharmaceutically acceptable carriers, diluents, or excipients" refers to solid or liquid fillers, diluents, or encapsulating substances that can be safely used for systemic administration. Depending on the specific route of administration, a variety of carriers, diluents, and excipients well known in the art may be used. These may be selected from the group consisting of sugars, starch, cellulose and their derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer solutions, emulsifiers, isotonic saline and salts [such as inorganic acid salts (including hydrochlorides, bromides, and sulfates), organic acid salts (such as acetates, propionates, and malonates)], water, and pyrogen-free water.

[0147] A useful reference describing acceptable carriers, diluents, and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. NJ USA, 1991), which is incorporated herein by reference.

[0148] Appropriately, for the purpose of inducing an immune response, certain immunological or immunogenic agents may be used in combination with the immunogenic proteins described herein. The term "immunogenic agent" includes, within its scope, carriers, delivery agents, immunostimulants, and / or adjuvants well known in the art. As will be understood in the art, immunostimulants and adjuvants refer to or include one or more substances that enhance the immunogenicity and / or efficacy of a composition. Non-limiting examples of suitable immunostimulants and adjuvants include squalane and squalene (or other plant or animal-derived oils), including squalene oil-in-water emulsions (e.g., MF59, AS03, and AF03); block copolymers; TLR agonists, such as pathogen-derived compounds, including lipopeptides, glycolipids, nucleotides, small molecule inhibitors, and bacterial-derived components, such as flagellins; detergents, such as Tween®-80; Quil® A; mineral oils, such as Drakeol or Marcol; vegetable oils, such as peanut oil; and Corynebacterium-derived adjuvants, such as Corynebacterium breve (…). Corynebacterium parvum Propionibacterium-derived adjuvants, such as Propionibacterium acnes (… Propionibacterium acne Mycobacterium bovis (); Mycobacterium bovis (BCG or BCG vaccine); Bordetella pertussis ( Bordetella pertussisAntigens; tetanus toxoid; diphtheria toxoid; surfactants such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysophosphatidylcholine, dimethyl dioctadecyl ammonium bromide, N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl-propanediamine), methoxyhexadecylglycerol, and Pranic polyol; polyamines such as pyran, dextran sulfate, and polyinosinic acid (IC) carbomer; peptides such as muramyl dipeptide and its derivatives, dimethylglycine, and tuftsin; oil emulsions; and mineral gels such as aluminum phosphate, aluminum hydroxide, or alum; interleukins such as interleukin-2 and interleukin-12; mononuclear factors such as interleukin-1; tumor necrosis factor; interferons such as gamma interferon; and immunostimulatory DNA such as CpG. DNA; combinations such as saponin-aluminum hydroxide or Quil-A aluminum hydroxide; saponins such as Matrix-M; liposomes; ISCOM® and ISCOMATRIX® adjuvants; mycobacterial cell wall extracts; synthetic glycopeptides such as muramyl dipeptide or other derivatives; avridin; lipid A derivatives; dextran sulfate; DEAE-dextran alone or in combination with aluminum phosphate; carboxylated polymethylene, such as Carbopol'EMA; acrylic copolymer emulsions such as Neocryl A640 (e.g., US Patent No. 5,047,238); water-in-oil emulsifiers such as Montanide ISA 720; poliovirus, vaccinia virus, or animal poxvirus proteins; or mixtures thereof.

[0149] Immunogenic agents may include: carriers, such as thyroglobulin; albumins, such as human serum albumin; toxins, toxoids, or any mutant cross-reactive substances (CRMs) from tetanus, diphtheria, pertussis, Pseudomonas, Escherichia coli, Staphylococcus, and Streptococcus; polyamino acids, such as poly(lysine:glutamate); influenza; rotavirus VP6; parvovirus VP1 and VP2; hepatitis B virus core protein; recombinant hepatitis B virus vaccines, etc. Alternatively, fragments or epitopes of carrier proteins or other immunogenic proteins may be used. For example, T-cell epitopes of bacterial toxins, toxoids, or CRMs may be used. For this purpose, reference may be made to U.S. Patent No. 5,785,973, which is incorporated herein by reference.

[0150] Oil-in-water emulsions have been found to be particularly suitable for use as adjuvants in influenza virus vaccines. Several such emulsions are known, and they typically comprise at least one oil and at least one surfactant, wherein both the oil and surfactant are biodegradable (metabolizable) and biocompatible. The oil droplets in the emulsions are typically less than 5 μm in diameter, and can even be submicron in diameter; these small sizes are achieved using microfluidics to provide a stable emulsion. Droplets with an average size of less than 220 nm are preferred because they can be sterilized by filtration.

[0151] In various instances, oil-in-water emulsions are homogeneous. A homogeneous emulsion is characterized by the fact that the majority of droplets (particles) dispersed therein are within a specified size range (e.g., diameter). Suitable specified size ranges may be, for example, 50–220 nm (e.g., 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220 nm or any range therewith), 50–180 nm, 80–180 nm, 100–175 nm, 120–185 nm, 130–190 nm, 135–175 nm, 150–175 nm. In some instances, homogeneous emulsions contain less than 10% of droplets (particles) exceeding the specified diameter range. In some instances, the average droplet size in the oil-in-water emulsion formulation is 135–175 nm, for example 155 nm ± 20 nm as measured by dynamic light scattering, and the formulation contains no more than 1 x 10⁻⁶ ppm. 7 Large particles / mL formulation (measured by optical particle sensing). As used herein, “large particles” refers to those with a diameter ≥ 1.2 μm, typically in the range of 1.2–400 μm. In specific examples, homogeneous emulsions contain less than 10%, less than 5%, or less than 3% of droplets outside the preferred size range. In some instances, the average droplet size of particles in oil-in-water emulsion formulations is 125–185 nm, for example, about 130 nm, about 140 nm, about 150 nm, about 155 nm, about 160 nm, about 170 nm, or about 180 nm, and the oil-in-water emulsion is homogeneous because less than 5% of the droplets in the formulation exceed the 125–185 nm range.

[0152] The vaccine compositions described herein may use oils, such as those derived from animals (e.g., fish) or plants. Sources of plant oils include nuts, seeds, and grains. Peanut oil, soybean oil, coconut oil, and olive oil are the most common, for example, nut oils. Jojoba oil obtained from jojoba beans may also be used. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame oil, etc. Among grains, corn oil is the most readily available, but oils from other grains (such as wheat, oats, rye, rice, Ethiopian thrush, black wheat, etc.) may also be used. While 6-10 C fatty acid esters of glycerol and 1,2-propanediol are not naturally present in seed oils, they can be prepared by hydrolyzing, separating, and esterifying suitable materials starting from nut and seed oils. Fats and oils from mammalian milk are metabolizable and therefore can be used in the vaccine compositions described herein. Methods for the separation, purification, saponification, and other processes necessary to obtain pure oils from animal sources are well known in the art. Most fish contain easily recyclable metabolizable oils. For example, cod liver oil, shark liver oil, and whale oil (such as whale wax) exemplify several fish oils that can be used in this article.

[0153] Many branched oils are biochemically synthesized in 5-carbon isoprene units and are commonly referred to as terpenes. Shark liver oil contains a branched unsaturated terpene called squalene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-docosahexaene. Squalane, a saturated analog of squalene, may also be used in this vaccine composition. Fish oils, including squalene and squalane, are readily available from commercial sources or obtained by methods known in the art. Other suitable oils are tocopherols. Mixtures of oils are also contemplated.

[0154] Surfactants can be classified according to their 'HLB' (hydrophilic / lipophilic balance). Suitablely, the surfactants described herein have an HLB of at least 10, more particularly at least 15, and even more particularly at least 16. The vaccine composition may include one or more surfactants, including but not limited to: polyoxyethylene sorbitan ester surfactants (commonly known as Tween), particularly polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butyl oxide (BO), sold under the trade name DOWFAX™, such as linear EO / PO block copolymers; octylphenol polyoxyethylene ethers, which may vary in the number of repeating ethoxy (oxy-1,2-ethylenedimethyl) groups, such as octylphenol polyoxyethylene ether-9 (Triton X-100, or tert-octylphenoxypolyethoxyethanol); (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids, such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl, hexadecyl, stearyl, and oleyl alcohol (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij). 30); and sorbitan esters (commonly known as SPAN), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Nonionic surfactants are preferred. Exemplary surfactants used in the emulsion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin, and Triton X-100.

[0155] Mixtures of surfactants (e.g., a Tween 80 / Span 85 mixture) can also be used. Combinations of polyoxyethylene sorbitan esters (such as polyoxyethylene sorbitan monooleate (Tween 80)) and octylphenol polyoxyethylene ethers (such as tert-octylphenoxypolyethoxyethanol (Triton X-100)) are also suitable. Another anticipated combination includes lauryl ether-9 plus polyoxyethylene sorbitan esters and / or octylphenol polyoxyethylene ethers.

[0156] Exemplary amounts (by weight%) of surfactants are: 0.01% to 1% polyoxyethylene sorbitan ester (such as Tween 80), particularly about 0.1%; 0.001% to 0.1% octyl or nonylphenoxy polyoxyethylene alcohol (such as Triton X-100, or other detergents in the Triton series), particularly 0.005% to 0.02%; and 0.1% to 20% polyoxyethylene ether (such as lauryl ether-9), more particularly 0.1% to 10%, even more particularly 0.1% to 1% or about 0.5%.

[0157] In specific examples, the oil-in-water emulsion is a squalene oil-in-water emulsion, and more particularly a submicron squalene oil-in-water emulsion.

[0158] Any suitable procedure is intended for the production of the vaccine composition. Exemplary procedures include, for example, those described in NewGeneration Vaccines (1997, Levine et al., Marcel Dekker, Inc. New York, Basel, Hong Kong), which are incorporated herein by reference.

[0159] It can be administered via any safe route, including, but not limited to, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intramuscular, intradermal, subcutaneous, inhalation, intranasal, intraocular, intraperitoneal, intraventricular, local, mucosal, and transdermal administration.

[0160] Dosage forms include tablets, dispersants, suspensions, injections, solutions, syrups, lozenges, capsules, nasal sprays, suppositories, aerosols, transdermal patches, etc. These dosage forms may also include injection or implantable controlled-release devices specifically designed for this purpose, or other forms of implants modified to additionally function in this manner. Controlled release can be achieved by coating with hydrophobic polymers, including acrylic resins, waxes, higher fatty alcohols, polylactic acid and polyglycolic acid, and certain cellulose derivatives such as hydroxypropyl methylcellulose. Furthermore, controlled release can be achieved using other polymer matrices, liposomes, and / or microspheres.

[0161] The compositions can be presented in discrete units, such as capsules, sachets, functional foods / feeds, or tablets, each containing a predetermined amount of one or more therapeutic agents of this disclosure, as powders or granules, or as solutions or suspensions in aqueous liquids, non-aqueous liquids, oil-in-water emulsions, or water-in-oil liquid emulsions. Such compositions can be prepared by any pharmaceutical method, but all methods include the step of combining one or more of the aforementioned agents with a carrier constituting one or more essential components. Typically, the compositions are prepared by uniformly and tightly mixing the agents of this disclosure with a liquid carrier or a finely chopped solid carrier, or both, and then, if desired, shaping the product into the desired presentation.

[0162] The above-described composition can be administered in a manner compatible with dosage forms and in an effective amount. The dose administered to the subject, in the context of this disclosure, should be sufficient to produce a beneficial response (e.g., a protective immune response) in the subject over an appropriate period of time. The amount administered may depend on the subject to be treated, including their age, sex, weight, and general health condition, which will depend on the practitioner's judgment.

[0163] This document also discloses containers for containing the immunogenic or vaccine compositions disclosed herein. Any suitable container known in the art can be used. For example, containers may be selected from the group consisting of vials, syringes, ampoules, flasks, fermenters, bioreactors, bags, cans, ampoules, cartridges, and disposable pens. In one example, the container is a vial, ampoule, or syringe.

[0164] The container may be made of glass, metal (e.g., steel, stainless steel, aluminum, etc.) and / or polymer (e.g., thermoplastic, elastomer, thermoplastic elastomer). The container may be at least partially siliconized.

[0165] The vaccine compositions disclosed herein may further comprise a buffer solution. The buffer solution may be any suitable buffer solution known in the art. For example, the buffer solution may be a TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycine, histidine, glycine, succinate, or triethanolamine buffer solution. In one example, the buffer solution is a phosphate buffer. In another example, the buffer solution is a succinate buffer. In yet another example, the buffer solution is a histidine buffer. In yet another example, the buffer solution is a citrate buffer.

[0166] The buffer solution can be selected from USP-compatible parenteral buffer solutions, especially when the pharmaceutical preparation is intended for parenteral use. For example, the buffer solution can be selected from the group consisting of: monobasic acids, such as acetic acid, benzoic acid, gluconic acid, glyceric acid, and lactic acid; dibasic acids, such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, and tartaric acid; polybasic acids, such as citric acid and phosphoric acid; and bases, such as ammonia, diethanolamine, glycine, triethanolamine, and TRIS.

[0167] Methods used to trigger immune responses and for treatment

[0168] The influenza virus protein and vaccine compositions described herein are suitable for administration to human or non-human animal subjects, thereby providing methods for inducing an immune response and / or preventing and / or treating influenza-related illnesses, symptoms, or conditions in subjects. This disclosure also provides for the use of the compositions described herein as pharmaceuticals, and for the use of such compositions in the preparation of pharmaceuticals for inducing an immune response in subjects and / or preventing and / or treating influenza-related illnesses, symptoms, or conditions.

[0169] Therefore, in one aspect, this disclosure provides a method for inducing an immune response in a subject, the method comprising the step of administering a therapeutically effective amount of the influenza virus, influenza virus protein or vaccine composition provided herein to the subject, thereby inducing an immune response in the subject.

[0170] In a related respect, this disclosure provides a method for preventing and / or treating influenza-related illnesses, symptoms, or conditions in a subject, the method comprising the steps of administering to the subject a therapeutically effective amount of the influenza virus, influenza virus protein, or vaccine composition described herein, thereby preventing and / or treating the influenza-related illnesses, symptoms, or conditions.

[0171] For the purposes of this document, the terms “subject,” “patient,” and “individual” include, but are not limited to, mammals, including humans, performing animals (such as horses, camels, and greyhounds), livestock (such as cattle, sheep, and horses), and companion animals (such as cats and dogs). Appropriately, a subject is a human.

[0172] "Initiating an immune response" means the generation or activity of one or more components of the immune system, including the cellular immune system, the humoral immune system (i.e., antibodies), and / or the innate immune system. Appropriately, the immune response described herein includes one or more components of the immune system, such as T lymphocytes, B lymphocytes, antibodies, neutrophils, dendritic cells (including plasmacytoid dendritic cells), cytokines, and / or chemokines. Non-limiting examples of cytokines include pro-inflammatory cytokines such as TNF-α, IL-2, IL-6, IL-8, IL-17A, and IL-1 (e.g., IL-1β). A non-limiting example of a chemokine is the neutrophil chemical attractant IL-8. In some instances, the immune response induced by the vaccine compositions described herein is protective.

[0173] As commonly used, the terms “immunization,” “vaccination,” and “vaccine” refer to methods and / or compositions that elicit a protective immune response against an influenza virus, thereby providing at least partial prevention or minimization of subsequent infection with the influenza virus or related serotypes, strains, or variants.

[0174] As used herein, “treatment,” “management,” or “therapy” means a therapeutic intervention that at least partially improves, eliminates, or alleviates the symptoms or pathological signs of an influenza-related illness, symptom, or condition (such as influenza infection) after the onset of those symptoms or pathological signs. Treatment need not be absolute to be beneficial to the subject. Beneficial effects can be determined using any method or standard known to a person skilled in the art.

[0175] As used herein, “prevention,” “avoidance,” or “prophylactic” refers to a process of action initiated before infection or exposure to the influenza virus or its molecular components and / or before the onset of symptoms or pathological signs of a disease, condition, or illness, thereby preventing infection and / or alleviating symptoms or pathological signs. It should be understood that such prevention need not be absolute to be beneficial to the subject. “Prophylactic” treatment is administered to subjects who do not exhibit signs of a disease, condition, or illness, or who exhibit only early signs, with the aim of reducing the risk of developing symptoms or pathological signs of a disease, condition, or illness.

[0176] The term "therapeuticly effective amount" describes the amount of a specified agent (such as the influenza virus, influenza virus protein, or vaccine composition described herein) sufficient to achieve the desired effect in a subject treated with that agent or composition. For example, this could be the amount of the agent and optionally one or more other therapeutic agents (e.g., one or more other antiviral agents) required to reduce, alleviate, and / or prevent influenza-related illness, symptoms, or conditions (e.g., influenza infection). Appropriately, a "therapeuticly effective amount" is sufficient to reduce or eliminate the symptoms of influenza-related illness, symptoms, or conditions. More specifically, a "therapeuticly effective amount" can be an amount sufficient to achieve the desired biological effect, such as an amount that effectively reduces or prevents disease progression.

[0177] Ideally, the therapeutically effective amount of the reagent is sufficient to induce the desired outcome without causing substantial cytotoxic effects in the subject. The therapeutically effective amount of the reagent used to reduce, alleviate, and / or prevent the diseases, conditions, and ailments described herein will depend on the subject being treated, the type and severity (e.g., disease progression) of any associated diseases, conditions, and / or ailments, and the manner of administration of the therapeutic composition. Appropriately, the therapeutically effective amount of the reagent is administered parenterally to the subject.

[0178] The vaccines described in this article can be used to treat children and adults. Influenza vaccines are currently recommended for immunization of children and adults, starting at 6 months of age. Therefore, human subjects can be under 1 year old, 1–5 years old, 5–15 years old, 15–55 years old, or at least 55 years old. Preferred subjects for vaccination are the elderly (e.g., ≥50 years, ≥60 years, preferably ≥65 years), young children (e.g., ≤5 years), hospitalized subjects, healthcare workers, armed forces and military personnel, pregnant women, patients with chronic diseases, immunocompromised subjects, subjects who have taken antiviral compounds within 7 days prior to vaccination, those with egg allergies, and those who have traveled abroad. However, vaccines are not only suitable for these groups but can also be used more generally in the population. For pandemic strains, priority is given to all age groups.

[0179] Treatment can be administered in single-dose or multi-dose regimens. Multi-dose regimens can be used for primary immunization and / or booster immunization. In multi-dose regimens, different doses can be administered via the same or different routes (e.g., parenteral primary immunization and mucosal booster, mucosal primary immunization and parenteral booster). Administering more than one dose (usually two doses) is particularly useful in patients who are immunologically unexposed to the antigen (e.g., for subjects who have never been vaccinated against influenza before) or specifically for vaccination against a new HA subtype (e.g., during a pandemic outbreak). Multi-dose regimens are typically administered at intervals of at least one week (e.g., approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 6 weeks, approximately 8 weeks, approximately 10 weeks, approximately 12 weeks, approximately 16 weeks, etc.).

[0180] In order to fully understand and put into practice the preferred embodiments of this disclosure, reference is made to the following non-limiting embodiments.

[0181] Example

[0182] Example 1.

[0183] This embodiment aims to optimize purity at the end of infection by studying how pH, viral dose, and trypsin concentration affect purity at the end of infection, and to investigate how the optimal conditions for maximizing GUC purity compare to the optimal conditions for maximizing HA production across a range of influenza virus strains.

[0184] Materials and Methods

[0185] All cell expansions were performed according to the following procedures:

[0186] Suspension MDCK cells were grown in DM134 medium at 37°C for 4-6 passages in increasing volume. After the growth phase, MDCK cells were planted at a rate of 2.5 x 10⁻⁶ cells / year. 6Cells / mL were seeded in suitable benchtop bioreactor vessels and infected at different pH, viral dilutions, and trypsin levels as determined by the experimental design (DOE; Table 1). A central composite design was created using three consecutive factors: pH (6.8–7.2), trypsin (4000–8000 U / L), and viral dilution (-4 to -9). Influenza-infected cultures were grown for 60–72 hours and harvested by centrifugation. After cell removal, ultracentrifugation (UC) was performed to precipitate the virus, and HA yield was quantified by HA-HPLC. In addition to the virus, the UC precipitate would also contain host cell contaminants. BCA analysis was performed to determine the total protein for each sample, and relative purity was calculated using the HA / BCA ratio. This ratio has been shown to correlate well with purity after downstream purification.

[0187] Experimental Design

[0188] The experimental design for each strain studied used the response surface methodology.

[0189] A three-factor experiment with three levels (pH, viral dilution, and trypsin) was used in the experimental design (Table 1). I-optimal or central composite designs were created to optimize the effects of each factor on HA yield and percentage of purity (% purity). Continuous factors evaluated were pH (6.8 to 7.2), trypsin (4000 to 8000), and viral dilution (10...). -4 Up to 10 -9 The response is HA yield and purity percentage (% purity).

[0190] Table 1: Coding levels of experimental factors and their independent variables in experimental design.

[0191]

[0192] result

[0193] A / Florida / 50 / 2022 (H3N2)

[0194] Figure 1 The model predictions leading to the highest HA yield and % purity of this viral strain are shown. This indicates that at pH 6.8 and a trypsin concentration of 8000 U / L, the purity at the end of infection should be approximately 37.9%, and the HA titer approximately 30.1 mg / kg. The pH trends for HA yield and purity are similar, with an optimum at 6.8. Trypsin data show similar curves for HA yield and purity; increasing trypsin concentration leads to an increase in both readings, with the maximum values ​​for both HA yield and purity reached at a trypsin concentration of 8000 U / L. Suitable viral dilutions were observed for this strain within the tested dilution range (i.e., dilutions -6 to -9).

[0195] A / Michigan / 41 / 2022 (H3N2)

[0196] Figure 2 The model predictions for maximizing HA yield and % purity for this strain are shown. Based on the predicted model, maximum HA yield and % purity were obtained at pH 6.9 and a trypsin concentration of 8000 U / L. The model predicts that approximately 34 mg / kg of HA should be produced with a purity of approximately 34% under optimized infection parameters. Suitable viral dilutions were observed for this strain within the tested dilution range (i.e., dilutions from -7 to -9).

[0197] A / Darwin / 11 / 2021 CVR-88 (H3N2)

[0198] Figure 3 The model predictions for maximizing HA yield for this strain are shown. The figure reveals an inverse relationship between HA yield and purity with respect to pH, with the optimal range for HA yield being pH 7.1–7.2 and for purity being pH 6.8–6.9. Trypsin data show similar curves for HA yield and purity; increasing trypsin concentration leads to increases in both readings, with the maximum values ​​for both HA yield and purity reached at a trypsin concentration of 7500 U / L. Suitable viral dilutions were observed for this strain across the entire tested dilution range (i.e., dilutions -6 to -8).

[0199] A / Canberra / 407 / 2019 (H3N2)

[0200] Figure 4 The model predictions for maximizing HA yield for this strain are shown. The figure reveals an inverse relationship between HA yield and purity with respect to pH, with the optimal range for HA yield being pH 7.0–7.2 and for purity being pH 6.8–6.9. Trypsin data show similar curves for HA yield and purity; increasing trypsin concentration leads to increases in both readings, with the maximum values ​​for both HA yield and purity reached at a trypsin concentration of 8000 U / L. Suitable viral dilutions for this strain were observed between -7 and -9.

[0201] A / NorthCarolina / 01 / 2021 (H1N1)

[0202] Figure 5The model predictions for maximizing HA yield for this strain are shown. The figure reveals an inverse relationship between HA yield and purity with respect to pH; the optimal range for HA yield is pH 7.1–7.4, and for purity, it is pH 6.8–7.0. Trypsin data show a similar curve for HA yield and purity; increasing trypsin concentration leads to increases in both readings up to a trypsin concentration of 6000 U / L, above which HA purity and yield decrease slightly. Suitable viral dilutions for this strain were observed between -6.5 and -8.

[0203] A / Washington / 19 / 2020 (H1N1)

[0204] Figure 6 The model predictions for optimizing HA yield and purity for this strain are shown. The figure indicates that pH has little effect on purity, but HA yield increases with increasing pH. The optimal range for HA yield is pH 7.1–7.2, and the optimal range for purity is pH 6.9–7.2. Trypsin data show similar curves for HA yield and purity; increasing trypsin concentration leads to increases in both readings until the trypsin concentration reaches 7000–8000 U / L. Above this concentration, HA purity and yield plateau or slightly decrease. Suitable viral dilutions were observed for this strain between -7 and -9.

[0205] B / Philippines / 28 / 2019 (BVIC)

[0206] Figure 7 The model predictions for optimizing HA yield and purity for this strain are shown. As can be seen from the figure, both HA yield and purity increase with increasing pH to approximately pH 7.2, decreasing slightly above this concentration. The optimal range for HA yield and purity is pH 7.0–7.2. Trypsin data show similar curves for HA yield and purity; increasing trypsin concentration leads to increases in both readings, with the maximum values ​​for both HA yield and purity reached at a trypsin concentration of 7000 U / L. Suitable viral dilutions were observed for this strain from -4 to -8.

[0207] B / Connecticut / 01 / 2021 (BVIC)

[0208] Figure 8The model predictions for optimizing HA yield and purity for this strain are shown. The figure shows that HA yield increases with increasing pH to approximately pH 7.2, while purity increases with increasing pH to approximately pH 7.1. Above these values, the parameter slightly decreases. The optimal range for HA yield is pH 7.1–7.2, and the optimal range for purity is pH 7.0–7.1. Trypsin data show that HA yield decreases with increasing trypsin concentration, while purity increases with increasing trypsin concentration. Optimal yield is achieved at a trypsin concentration of 5000 U / L, and optimal purity is achieved at a trypsin concentration of 8000 U / L. Suitable viral dilutions were observed for this strain from -7 to -9.

[0209] A / Sydney / 5 / 2021 CVR-112 (H1N1)

[0210] Figure 9 The model predictions for optimizing HA yield and purity for this strain are shown. The figure reveals an inverse relationship between HA yield and purity with respect to pH, similar to what was observed with A / NorthCarolina / 01 / 2021. The optimal range for HA yield is pH 7.0–7.2, and for purity, it is pH 6.8–7.0. Trypsin data show a similar curve for HA yield and purity; increasing trypsin concentration leads to increases in both readings until a trypsin concentration of 7000 U / L is reached, above which HA purity and yield plateau.

[0211] A / Victoria / 10 / 2020 (H3N2)

[0212] Figure 10 The model predictions for optimizing HA yield and purity for this strain are shown. The figure reveals an inverse relationship between HA yield and purity with respect to pH, similar to what was observed with A / Canberra / 407 / 2019 (H3N2). The optimal range for HA yield is pH 7.0–7.1, and for purity, it is pH 6.8–6.9. Trypsin data show a similar curve for HA yield and purity; increasing trypsin concentration leads to increases in both readings. The maximum values ​​for both HA yield and purity are reached at a trypsin concentration of 7500 U / L; above this concentration, both HA yield and purity plateau.

[0213] B / South Australia / 06 / 2019 (B / Yam)

[0214] Figure 11The model predictions for optimizing HA yield and purity for this strain are shown. As can be seen from the figure, both HA yield and purity increase with increasing pH to approximately 7.0 or 7.1, and decrease slightly above these values. The optimal range for HA yield is pH 7.0–7.2, and the optimal range for purity is pH 6.8–7.1. Trypsin data show similar curves for HA yield and purity; increasing trypsin concentration leads to increases in both readings. The maximum values ​​for both HA yield and purity are reached at trypsin concentrations of 6000–7000 U / L; above this concentration, HA purity and yield plateau or decrease slightly.

[0215] in conclusion

[0216] This example demonstrates that altering infection conditions (pH and trypsin concentration) can affect purity and yield at the end of infection. Generally, for influenza A strains, decreasing pH and increasing trypsin activity tend to lead to increased HA purity at the end of infection. Conversely, increasing pH (and also increasing trypsin) tends to lead to increased HA yield in influenza A strains at the end of infection. Increased trypsin concentration also tends to increase the purity level of influenza B strains at the end of infection.

[0217] Itemized list of the implementation plan

[0218] 1. A method for determining the culture conditions of cells infected with influenza virus, the method comprising the following steps:

[0219] (a) Incubating influenza virus-infected cells in multiple test media, wherein one or more culture conditions of the cells are varied between each of the multiple test media, wherein the one or more culture conditions are selected from pH, trypsin concentration, and combinations thereof; and

[0220] (b) Select one or more culture conditions to optimize the yield and / or purity of the influenza virus.

[0221] 2. The method of embodiment 1, wherein changing one or more culture conditions between each of the plurality of test culture media comprises: in the plurality of test culture media,

[0222] (i) Increase trypsin concentration from baseline to its maximum level; and / or

[0223] (ii) Reduce the pH from the baseline level to its lowest level.

[0224] 3. The method of embodiment 2, wherein in the plurality of test culture media, the trypsin concentration is increased to a maximum level of 10,000 U / L and / or the pH is decreased to a minimum level of 6.0.

[0225] 4. The method of any one of the foregoing embodiments, wherein the pH varies in a pH range of about 6.0 to about 8.0.

[0226] 5. The method of embodiment 4, wherein the pH varies in a pH range of about 6.5 to about 7.5.

[0227] 6. The method of embodiment 4 or embodiment 5, wherein the pH varies in a pH range of about 6.7 to about 7.3.

[0228] 7. The method of any one of the foregoing embodiments, wherein the trypsin concentration varies in the range of about 2000 U / L to about 10000 U / L.

[0229] 8. The method of embodiment 7, wherein the trypsin concentration varies in the range of about 4000 U / L to about 8000 U / L.

[0230] 9. The method of any one of the foregoing embodiments, wherein the cells are used with about 10 -8 Incubate with a viral dose of approximately 10.

[0231] 10. The method of any one of the foregoing embodiments, wherein step (b) includes selecting one or more culture conditions to optimize the yield and / or purity of the protein produced by the influenza virus.

[0232] 11. The method of embodiment 10, wherein the protein is a hemagglutinin protein.

[0233] 12. A method for culturing cells infected with influenza virus, the method comprising the steps of incubating the influenza virus-infected cells at a pH of about 6.7 to about 7.3 and in a medium containing trypsin at a concentration of at least about 5000 U / L.

[0234] 13. The method of embodiment 10, the method further comprising the step of harvesting the influenza virus and / or influenza virus protein from the culture medium.

[0235] 14. A method for producing influenza virus and / or influenza virus proteins, the method comprising the steps of:

[0236] (a) Incubating influenza virus-infected cells at a pH of about 6.7 to about 7.3 and in a medium containing at least about 5000 U / L of trypsin; and

[0237] (b) Harvest the influenza virus and / or influenza virus protein thus generated from the culture medium.

[0238] 15. The method of any one of embodiments 12 to 14, wherein the influenza virus-infected cells are cultured at a pH of about 6.8 to about 7.1.

[0239] 16. The method according to any one of embodiments 12 to 15, wherein the culture medium contains trypsin at a concentration of about 5000 U / L to about 10000 U / L.

[0240] 17. The method according to any one of embodiments 12 to 16, wherein the cells are used with about 10 -8 Incubate with a viral dose of approximately 10.

[0241] 18. The method according to any one of embodiments 13 to 17, the method further comprising the step of purifying the influenza virus and / or the influenza virus protein harvested from the culture medium.

[0242] 19. The method according to any one of embodiments 13 to 18, wherein the influenza virus protein is a hemagglutinin protein.

[0243] 20. The method of embodiment 19, wherein the hemagglutinin protein harvested from the culture medium has a purity level of at least about 10% based on its total protein concentration.

[0244] 21. The method of embodiment 19 or embodiment 20, wherein the hemagglutinin protein is present in the culture medium at a concentration of at least about 10 mg / L.

[0245] 22. The method of any one of the foregoing embodiments, wherein the influenza virus is an influenza A virus.

[0246] 23. The method of implementation scheme 22, wherein the influenza A virus is an H3N2 strain or an H1N1 strain.

[0247] 24. The method of any one of the foregoing embodiments, wherein the cell is or comprises MDCK cells.

[0248] 25. The method of any one of the foregoing embodiments, wherein the incubation step is performed for about 48 hours to about 96 hours.

[0249] 26. Influenza virus or influenza virus protein produced by any one of embodiments 13 to 25.

[0250] 27. A vaccine composition comprising the influenza virus protein as described in embodiment 26 and a pharmaceutically acceptable carrier, diluent, or excipient.

[0251] 28. A method for inducing an immune response in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of the influenza virus protein of embodiment 26 or the vaccine composition of embodiment 27, thereby inducing an immune response in the subject.

[0252] 29. A method for preventing and / or treating influenza-related illness, symptoms, or conditions in a subject, the method comprising the steps of administering to the subject a therapeutically effective amount of the influenza virus protein of embodiment 26 or the vaccine composition of embodiment 27, thereby preventing and / or treating the influenza-related illness, symptoms, or conditions.

[0253] 30. A method for culturing cells infected with influenza virus, the method comprising the step of culturing the influenza virus-infected cells in a culture medium containing trypsin under one or more culture conditions selected in step (b) of any one of embodiments 1 to 11.

[0254] 31. A method for improving yield and / or purity in influenza vaccine manufacturing, the method comprising the steps of:

[0255] (a) Incubating influenza virus-infected cells in multiple test media, wherein one or more culture conditions of the cells are changed between each of the multiple test media, wherein the one or more culture conditions are selected from pH, trypsin concentration and combinations thereof;

[0256] (b) Select one or more culture conditions to optimize the yield and / or purity of the influenza virus;

[0257] (c) Culturing suspension mammalian cells capable of being infected with influenza virus in serum-free medium; and

[0258] (d) Infecting the said mammalian cells with influenza virus; and

[0259] (e) Incubate the influenza virus-infected mammalian cells under one or more culture conditions selected in (b).

[0260] 32. The method of embodiment 31, wherein changing one or more culture conditions between each of the plurality of test culture media comprises: in the plurality of test culture media,

[0261] (i) Increase trypsin concentration from baseline to its maximum level; and / or

[0262] (ii) Reduce the pH from the baseline level to its lowest level.

[0263] 33. The method of embodiment 32, wherein in the plurality of test culture media, the trypsin concentration is increased to a maximum level of 10,000 U / L and / or the pH is decreased to a minimum level of 6.0.

[0264] 34. The method of any one of the foregoing embodiments, wherein the pH varies in a pH range of about 6.0 to about 8.0.

[0265] 35. The method of embodiment 4, wherein the pH varies in a pH range of about 6.5 to about 7.5.

[0266] 36. The method of embodiment 4 or embodiment 5, wherein the pH varies in a pH range of about 6.7 to about 7.3.

[0267] 37. The method of any one of the foregoing embodiments, wherein the trypsin concentration varies in the range of about 2000 U / L to about 10000 U / L.

[0268] 38. The method of embodiment 7, wherein the trypsin concentration varies in the range of about 4000 U / L to about 8000 U / L.

[0269] 39. The method of any one of the foregoing embodiments, wherein the cells are used with about 10 -8 Incubate with a viral dose of approximately 10.

[0270] 40. The method of any one of the foregoing embodiments, wherein step (b) includes selecting one or more culture conditions to optimize the yield and / or purity of the protein produced by the influenza virus.

[0271] 41. The method of embodiment 40, wherein the protein is a hemagglutinin protein.

Claims

1. A method for determining the culture conditions of cells infected with influenza virus, the method comprising the following steps: (a) Incubating influenza virus-infected cells in multiple test media, wherein one or more culture conditions of the cells are changed between each of the multiple test media, wherein the one or more culture conditions are selected from pH, trypsin concentration and combinations thereof; as well as (b) Select one or more culture conditions to optimize the yield and / or purity of the influenza virus.

2. The method of claim 1, wherein changing one or more culture conditions between each of the plurality of test culture media comprises: In the plurality of test culture media, (i) Increase the trypsin concentration from baseline to its maximum level; and / or (ii) Reduce the pH from the baseline level to its lowest level.

3. The method of claim 2, wherein in the plurality of test culture media, the trypsin concentration is increased to a maximum level of 10,000 U / L and / or the pH is decreased to a minimum level of 6.

0.

4. The method of claim 1, wherein the pH varies in a pH range of about 6.0 to about 8.

0.

5. The method of claim 4, wherein the pH varies in a pH range of about 6.5 to about 7.

5.

6. The method of claim 4, wherein the pH varies in a pH range of about 6.7 to about 7.

3.

7. The method of claim 1, wherein the trypsin concentration varies in the range of about 2000 U / L to about 10000 U / L.

8. The method of claim 7, wherein the trypsin concentration varies in the range of about 4000 U / L to about 8000 U / L.

9. The method of claim 1, wherein the cells are used with about 10 -8 Incubate with a viral dose of approximately 10.

10. The method of claim 1, wherein step (b) comprises selecting one or more culture conditions to optimize the yield and / or purity of proteins produced by the influenza virus.

11. The method of claim 10, wherein the protein is a hemagglutinin protein.

12. The method of claim 1, wherein the influenza virus is an influenza A virus.

13. The method of claim 12, wherein the influenza A virus is an H1 subtype or an H3 subtype.

14. The method of claim 12, wherein the influenza A virus is an H3N2 strain or an H1N1 strain.

15. The method of claim 1, wherein the cell is or comprises MDCK cells.

16. The method of claim 1, wherein the incubation step is performed for about 48 hours to about 96 hours.

17. A method for culturing cells infected with influenza virus, the method comprising the steps of incubating the influenza virus-infected cells at a pH of about 6.7 to about 7.3 and in a medium containing trypsin at a concentration of at least about 5000 U / L.

18. The method of claim 17, the method further comprising the step of harvesting the influenza virus and / or influenza virus protein from the culture medium.

19. The method of claim 17, wherein the influenza virus-infected cells are cultured at a pH of about 6.8 to about 7.

1.

20. The method of claim 17, wherein the culture medium contains trypsin at a concentration of about 5000 U / L to about 10000 U / L.

21. The method of claim 17, wherein the cells are used with about 10 -8 Incubate with a viral dose of approximately 10.

22. The method of claim 18, the method further comprising the step of purifying the influenza virus and / or the influenza virus protein harvested from the culture medium.

23. The method of claim 18, wherein the influenza virus protein is a hemagglutinin protein.

24. The method of claim 23, wherein the hemagglutinin protein harvested from the culture medium has a purity level of at least about 10% based on its total protein concentration.

25. The method of claim 23, wherein the hemagglutinin protein is present in the culture medium at a concentration of at least about 10 mg / L.

26. The method of claim 17, wherein the influenza virus is an influenza A virus.

27. The method of claim 26, wherein the influenza A virus is an H1 subtype or an H3 subtype.

28. The method of claim 26, wherein the influenza A virus is an H3N2 strain or an H1N1 strain.

29. The method of claim 17, wherein the cell is or comprises MDCK cells.

30. The method of claim 17, wherein the incubation step is performed for about 48 hours to about 96 hours.

31. Influenza virus or influenza virus protein produced by the method of claim 18.

32. A method for producing influenza virus and / or influenza virus protein, the method comprising the following steps: (a) Incubate influenza virus-infected cells at a pH of about 6.7 to about 7.3 in a medium containing at least about 5000 U / L of trypsin; as well as (b) Harvest the influenza virus and / or influenza virus protein thus generated from the culture medium.

33. The method of claim 32, wherein the influenza virus-infected cells are cultured at a pH of about 6.8 to about 7.

1.

34. The method of claim 32, wherein the culture medium contains trypsin at a concentration of about 5,000 U / L to about 10,000 U / L.

35. The method of claim 32, wherein the cells are used with about 10 -8 Incubate with a viral dose of approximately 10.

36. The method of claim 32, the method further comprising the step of purifying the influenza virus and / or the influenza virus protein harvested from the culture medium.

37. The method of claim 32, wherein the influenza virus protein is a hemagglutinin protein.

38. The method of claim 37, wherein the hemagglutinin protein harvested from the culture medium has a purity level of at least about 10% based on its total protein concentration.

39. The method of claim 37, wherein the hemagglutinin protein is present in the culture medium at a concentration of at least about 10 mg / L.

40. The method of claim 32, wherein the influenza virus is an influenza A virus.

41. The method of claim 40, wherein the influenza A virus is an H1 subtype or an H3 subtype.

42. The method of claim 40, wherein the influenza A virus is an H3N2 strain or an H1N1 strain.

43. The method of claim 32, wherein the cell is or comprises MDCK cells.

44. The method of claim 32, wherein the incubation step is performed for about 48 hours to about 96 hours.

45. Influenza virus or influenza virus protein produced by the method of claim 32.

46. ​​A vaccine composition comprising the influenza virus protein of claim 31 and a pharmaceutically acceptable carrier, diluent, or excipient.

47. A vaccine composition comprising the influenza virus protein of claim 45 and a pharmaceutically acceptable carrier, diluent, or excipient.

48. A method for inducing an immune response in a subject, the method comprising the steps of: The subject is given a therapeutically effective amount of the influenza virus protein of claim 31, thereby inducing an immune response in the subject.

49. A method for preventing and / or treating influenza-related illnesses, symptoms, or conditions in subjects, the method comprising the following steps: The subject is given a therapeutically effective amount of the influenza virus protein of claim 31 to prevent and / or treat the influenza-related disease, symptoms or condition.

50. A method for culturing cells infected with influenza virus, the method comprising the following steps: In the step (b) of the method of claim 1, influenza virus-infected cells are cultured in a culture medium containing trypsin under one or more culture conditions selected.

51. A method for improving yield and / or purity in the manufacture of influenza vaccines, the method comprising the following steps: (a) Incubating influenza virus-infected cells in multiple test media, wherein one or more culture conditions of the cells are changed between each of the multiple test media, wherein the one or more culture conditions are selected from pH, trypsin concentration and combinations thereof; (b) Select one or more culture conditions to optimize the yield and / or purity of the influenza virus; (c) Culturing suspension mammalian cells capable of being infected with influenza virus in serum-free medium; and (d) Infecting the said mammalian cells with influenza virus; and (e) Incubate the influenza virus-infected mammalian cells under one or more culture conditions selected in (b).

52. The method of claim 51, wherein changing one or more culture conditions between each of the plurality of test culture media comprises: In the plurality of test culture media, (i) Increase the trypsin concentration from baseline to its maximum level; and / or (ii) Reduce the pH from the baseline level to its lowest level.

53. The method of claim 52, wherein in the plurality of test culture media, the trypsin concentration is increased to a maximum level of 10,000 U / L and / or the pH is decreased to a minimum level of 6.

0.

54. The method of claim 51, wherein the pH varies in a pH range of about 6.0 to about 8.

0.

55. The method of claim 51, wherein the pH varies in a pH range of about 6.5 to about 7.

5.

56. The method of claim 51, wherein the pH varies in a pH range of about 6.7 to about 7.

3.

57. The method of claim 51, wherein the trypsin concentration varies in the range of about 2000 U / L to about 10000 U / L.

58. The method of claim 51, wherein the trypsin concentration varies in the range of about 4000 U / L to about 8000 U / L.

59. The method of claim 51, wherein the cells are used with about 10 -8 Incubate with a viral dose of approximately 10.

60. The method of claim 51, wherein step (b) comprises selecting one or more culture conditions to optimize the yield and / or purity of proteins produced by the influenza virus.

61. The method of claim 60, wherein the protein is a hemagglutinin protein.

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