Compositions and methods for producing viral vaccines with reduced particle size.

Abstract

Disclosed herein are methods and compositions for producing virus vaccines with reduced particle size, particularly for use in producing influenza virus vaccines. The methods include: a. purifying virus particles from a harvested cell culture; b. inactivating and dividing the virus particles to produce subvirions; and c. purifying the subvirions, wherein step b may include treating the virus particles with a reagent including a non-ionic detergent and an ionic detergent.

Description

[Technical Field]

[0001] Areas of disclosure A composition and method for producing a viral vaccine having reduced particle size are disclosed. [Background technology]

[0002] background Antigens in viral vaccines may contain purified components (e.g., recombinant viral proteins) or fragments of viruses modified to lose their toxicity (e.g., inactivated viruses). For example, some influenza vaccines contain influenza viruses that have been irreversibly inactivated by treatment with alkylating agents, thereby altering their viral nucleic acids or proteins. Regardless of the antigenic components, all vaccines must undergo extensive processing and purification after production and before they can be administered to patients. Purification often requires chromatography and filtration steps to remove unwanted by-products resulting from the production of the above-mentioned antigens (e.g., components of host cells or chemicals added during the process).

[0003] Some influenza vaccines are produced by inactivating live viruses. Viruses can be grown in pathogen-free chicken eggs or cultured cell lines before they are collected and purified from their host cells. The viruses can then be inactivated by heat or chemicals and subsequently subjected to a splitting process, during which the viral particles are broken down to form smaller subvilions. Depending on the conditions during splitting, the subvilions may not achieve monodispersity and / or the desired size. These can affect downstream purification processes. For example, high salt concentrations during splitting may provide inadequate shielding of the electrostatic charge, causing subvilion units to aggregate and form larger particles. These aggregates can then impair the purification column, clog the filter, and result in reduced throughput, low overall yield, and inability to produce the vaccine. Therefore, an improved process is needed, particularly during the intensification of seasonal influenza vaccine production or during pandemics, that can increase filter throughput and yield.

[0004] The inventors have surprisingly found that the size of fragmented subvilions can be reduced by treating the particles with a reagent comprising at least one component selected from the group consisting of nonionic surfactants, ionic surfactants, and salts, and that in this case, the at least one component is present in an effective amount to reduce the particle size of the subvilions. This disclosure provides methods and compositions that can be used to produce a viral vaccine having reduced particle size. This disclosure also provides methods and compositions for fragmenting viral particles to form subvilions, intended for use in the production of influenza virus vaccines.

[0005] As shown in the present disclosure, the use of subvirion particles correlates inversely with filter throughput. Therefore, strategies to increase filter throughput and yield during viral vaccine production may include reducing subvirion particle size via splitting, or maintaining ideal conditions throughout processing to prevent aggregation of subvirions. This can be achieved by the methods and compositions of the present invention, which includes varying the relative amounts of conditions and reagents (e.g., salts, surfactants) involved in the splitting step. Summary of the Invention Means for Solving the Problems

[0006] The present disclosure provides methods and compositions for producing viral vaccines having reduced particle size, and methods and compositions for splitting viral particles to form subvirions, and contemplates their use in the production of influenza virus vaccines.

[0007] The present disclosure provides a method of producing a viral vaccine formulated in a subvirion form, wherein the method comprises: a. purifying viral particles from harvested cell culture; b. inactivating and splitting the viral particles to produce subvirions; and c. purifying the subvirions, wherein the step of inactivating and splitting the viral particles in step b comprises treating the viral particles with a reagent comprising a nonionic surfactant and an ionic surfactant, wherein the nonionic surfactant and the ionic surfactant are present in an amount effective to reduce the particle size of the viral particles.

[0008] In some embodiments, the reagent further comprises a salt, wherein the salt is present in an amount effective to reduce the particle size of the viral particles.

[0009] In some embodiments, the average hydrodynamic radius of the subviral particles ranges from 150 nm to 350 nm.

[0010] In some embodiments, the nonionic surfactant is polysorbate 80. In some embodiments, the nonionic surfactant comprises at least about 1.0 g / L polysorbate 80, such as at least about 1.5 g / L, at least about 2.0 g / L, or at least about 2.5 g / L. In some embodiments, the ionic surfactant comprises cetrimonium bromide (CTAB). In some embodiments, the ionic surfactant comprises about 1.25 g / L to 3.0 g / L CTAB, such as about 1.5 g / L, about 2.0 g / L, about 2.5 g / L, or about 3.0 g / L. In some embodiments, the salt comprises sodium chloride (NaCl). In some embodiments, the salt comprises 0 to 200 mM NaCl, such as about 25 mM, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, or about 175 mM. In some embodiments, the nonionic surfactant comprises polysorbate 80, the ionic surfactant comprises CTAB, and the salt comprises sodium chloride NaCl.

[0011] In some embodiments of the method, the purifying step of step c comprises adsorption filtration. In some such embodiments, the adsorption filtration throughput is at least about 50 L / m 2 , such as at least about 100 L / m 2 , at least about 150 L / m 2 , at least about 200 L / m 2 , at least about 200 L / m 2 , at least about 300 L / m 2 , or at least about 350 L / m 2In some other embodiments, the particle size of the subvilion is less than about 500 nm, for example, less than about 400 nm, less than about 300 nm, less than about 200 nm, or less than about 150 nm. In some embodiments, the particle size of the subvilion is the hydrodynamic radius. In some embodiments, the size of the subvilion is measured by dynamic light scattering (DLS).

[0012] In some embodiments of the method, the virus particles are derived from influenza virus. In some embodiments, the virus particles are derived from influenza virus strain A.

[0013] The disclosure also provides a method for reducing the particle size of a virus, the method comprising the step of treating the virus particles with a reagent comprising a nonionic surfactant and an ionic surfactant, wherein the nonionic surfactant and the ionic surfactant are present in an amount effective for reducing the particle size of the virus particles. In some embodiments, the reagent further comprises a salt, wherein the salt is present in an amount effective for reducing the particle size of the virus particles.

[0014] In some embodiments, the nonionic surfactant comprises polysorbate 80. In some embodiments, the nonionic surfactant comprises at least about 1.0 g / L of polysorbate 80, for example, at least about 1.5 g / L, at least about 2.0 g / L, or at least about 2.5 g / L. In some embodiments, the ionic surfactant comprises cetrimonium bromide (CTAB). In some embodiments, the ionic surfactant comprises from about 1.25 g / L to 3.0 g / L of CTAB, for example, about 1.5 g / L, about 2.0 g / L, about 2.5 g / L, or about 3.0 g / L. In some embodiments, the salt comprises sodium chloride (NaCl). In some embodiments, the salt comprises 25 to 200 mM of NaCl, for example, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, or about 175 mM. In some embodiments, the nonionic surfactant comprises polysorbate 80, the ionic surfactant comprises CTAB, and the salt comprises sodium chloride NaCl.

[0015] In some embodiments, the method further comprises the step of filtering the treated virus particles through an adsorption filter. In some embodiments, the throughput of the adsorption filter is at least about 50 L / m 2 , for example, at least about 100 L / m 2 , at least about 150 L / m 2 , at least about 200 L / m 2 , at least about 250 L / m 2 , at least about 300 L / m 2 , or at least about 350 L / m 2 . In some embodiments, the virus particle size is less than about 500 nm, for example, less than about 400 nm, less than about 300 nm, less than about 200 nm, or less than about 150 nm. In some embodiments, the virus particle size is the hydrodynamic radius of the virus particle. In some embodiments, the virus particle size is measured by dynamic light scattering (DLS).

[0016] In some embodiments, the virus particles are derived from the influenza virus. In some embodiments, the virus particles are derived from influenza virus strain A.

[0017] This disclosure also relates to a method for reducing viral particle size in an influenza virus purification process, wherein the method is a. A process for purifying virus particles collected from cell cultures; b. A step of inactivating and dividing the above-mentioned virus particles to generate subvirions; and c. The process of purifying the above subvilions; The present invention provides a method comprising, where step b above, the step of inactivating and dividing the virus particles, comprising the step of treating the virus particles with a reagent comprising a nonionic surfactant and an ionic surfactant, wherein the nonionic surfactant and the ionic surfactant are present in amounts effective for reducing the particle size of the virus particles.

[0018] This disclosure also relates to a method for producing a viral vaccine formulated in sub-billion form, the method being: a. The process of purifying virus particles from the collected cell culture; b. A step of inactivating and dividing the above-mentioned virus particles to generate subvirions; and c. The process of purifying the above subvilions; The present invention provides a method comprising, where step b above, the step of inactivating and dividing the virus particles, comprising treating the virus particles with a reagent comprising a nonionic surfactant and an ionic surfactant, wherein the nonionic surfactant and the ionic surfactant are present in amounts effective for reducing the particle size of the virus particles.

[0019] This disclosure also relates to a method for producing an influenza virus vaccine formulated in subbilion form, the method being described as follows: a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the above-mentioned virus particles to generate subvirions; and c. The process of purifying the above subvilions; The present invention provides a method comprising, where step b above, the step of inactivating and dividing the virus particles, comprising treating the virus particles with a reagent comprising a nonionic surfactant and an ionic surfactant, wherein the nonionic surfactant and the ionic surfactant are present in amounts effective for reducing the particle size of the virus particles.

[0020] In some embodiments, the reagent further comprises a salt, which is present in an amount effective to reduce the particle size of the virus particles.

[0021] In some embodiments, the nonionic surfactant comprises polysorbate 80. In some embodiments, the nonionic surfactant comprises at least about 1.0 g / L of polysorbate 80, for example, at least about 1.5 g / L, at least about 2.0 g / L, or at least about 2.5 g / L. In some embodiments, the salt comprises sodium chloride (NaCl). In some embodiments, the salt comprises 25 to 200 mM NaCl, for example, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, or about 175 mM.

[0022] In certain embodiments, the concentration of polysorbate 80 in the nonionic surfactant is 0.3 g / L after inactivation and before the fission of the virus particles. In further embodiments, the concentration of polysorbate 80 in the nonionic surfactant increases in the range of 0 to 2.2 g / L during fission. Thus, possible concentrations of polysorbate 80 during fission may include 0.3 g / L, 1.4 g / L, and 2.5 g / L.

[0023] This disclosure also relates to a method for producing a formulated influenza virus vaccine comprising purified viral protein, wherein the method is a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the above-mentioned virus particles to generate subvirions; and c. A step of purifying the above subvirions to produce purified viral proteins; The present invention provides a method comprising, where step b above, the step of inactivating and dividing the virus particles, comprising treating the virus particles with a reagent comprising a nonionic surfactant and a salt, wherein the nonionic surfactant and the salt are present in an amount effective to reduce the particle size of the virus particles.

[0024] This disclosure also relates to a method for producing a formulated influenza virus vaccine comprising purified viral protein, wherein the method is a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the above-mentioned virus particles to generate subvirions; and c. A step of purifying the above subvirions to produce purified influenza virus protein; The present invention provides a method comprising, where step b above, the step of inactivating and dividing the virus particles, comprising treating the virus particles with a reagent comprising an ionic surfactant and a salt, wherein the ionic surfactant and the salt are present in an amount effective to reduce the particle size of the virus particles.

[0025] This disclosure also relates to a method for producing a formulated influenza virus vaccine comprising purified viral protein, wherein the method is a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating the purified virus particles by treating them with an alkylating agent; c. A step of treating the inactivated virus particles with an ionic surfactant to divide the inactivated virus particles and generate subvilions; and d) A step of purifying the above subvirions to produce purified influenza virus protein; The present invention provides a method comprising, wherein the step of inactivating and dividing the virus particles in step b and / or c above further comprises treating the virus particles with a reagent comprising a nonionic surfactant and an ionic surfactant, wherein the nonionic surfactant and the ionic surfactant are present in amounts effective for reducing the particle size of the virus particles.

[0026] This disclosure also relates to a method for producing an influenza virus vaccine formulated in subbilion form, the method being described as follows: a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the above-mentioned virus particles to generate subvirions; and c. The process of purifying the above subvilions; The present invention provides a method comprising, where step b above, the step of inactivating and dividing the virus particles, comprising treating the virus particles with a reagent comprising an effective amount of CTAB, NaCl, and polysorbate 80 to reduce the particle size of the virus particles.

[0027] This disclosure also relates to a method for producing an influenza virus vaccine formulated in subbilion form, the method being described as follows: a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the above-mentioned virus particles to generate subvirions; and c. The process of purifying the above subvilions; The present invention provides a method that includes, in the step of inactivating and dividing the virus particles in step b above, treating the virus particles with a reagent containing an amount effective to reduce the particle size of the virus particles, comprising 1.5-2.5 g / L CTAB, 0-150 mM NaCl, and 0-2.2 g / L polysorbate 80.

[0028] This disclosure also relates to a method for producing an influenza virus vaccine formulated in sub-billion form, the method being described as follows: a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the above-mentioned virus particles to generate subvirions; and c. The process of purifying the above subvilions; The present invention provides a method comprising, in which the step of inactivating and dividing the virus particles in step b above, treating the virus particles with a reagent comprising 1.5 to 2.5 g / L of an ionic surfactant, 0 to 150 mM of a salt, and 0 to 2.2 g / L of a nonionic surfactant in amounts effective to reduce the particle size of the virus particles.

[0029] In some embodiments, the ionic surfactant is CTAB. In some embodiments, the salt is NaCl. In some embodiments, the nonionic surfactant is polysorbate 80. [Brief explanation of the drawing]

[0030] [Figure 1] Figures 1A-C: Average particle size of influenza B / Victoria lineage subvilions generated under various splitting conditions, as measured by DLS. The relative amounts of CTAB, polysorbate 80, and NaCl were varied.

[0031] [Figure 2]Figures 2A-C: Average particle size of influenza A H3N2 subtype subvilions generated under various splitting conditions, as measured by DLS. The relative amounts of CTAB, polysorbate 80, and NaCl were varied.

[0032] [Figure 3] Figures 3A-C: Average particle size of influenza A H3N2 subtype subvilions generated under various splitting conditions, as measured by DLS. The relative amounts of CTAB, polysorbate 80, and NaCl were varied.

[0033] [Figure 4] Figure 4: Filter throughput of the adsorption filter during subvilion purification as a function of subvilion particle size. H1N1 strains designated IVR-180 and TT1384 ​​are shown. [Modes for carrying out the invention]

[0034] Detailed explanation The processes and compositions of this disclosure can be more readily understood by referring to the following detailed description, in conjunction with the accompanying drawings that form part of this disclosure.

[0035] Throughout this document, this description refers to processes and compositions for producing viral vaccines. Where this disclosure discloses or claims features or embodiments related to processes for producing viral vaccines, such features or embodiments are equally applicable to the viral vaccines. Similarly, where this disclosure discloses or claims features or embodiments related to viral vaccines, such features or embodiments are equally applicable to processes or compositions for producing the viral vaccines. Where a range of values ​​is expressed, it encompasses embodiments that use any particular value within that range. Furthermore, references to values ​​stated within a range include each and every value within that range. Where a value is expressed as an approximation by the use of the antecedent “about,” it is understood that the particular value forms another embodiment. The use of “or” means “and / or” unless otherwise specified by the particular context of its use. All references cited herein are incorporated herein by reference in their entirety. In the event of any conflict between the references and this document, this document shall prevail. It should be acknowledged that certain features of the processes and compositions disclosed herein are disclosed herein in the context of separate embodiments for clarity, but may also be provided in combination in a single embodiment. Conversely, various features of the processes and compositions disclosed herein are disclosed in the context of a single embodiment for conciseness, but may also be provided separately or in any partial combination.

[0036] Where used herein, the singular forms “a,” “an,” and “the” include the plural unless the context clearly indicates otherwise. Where used within the detailed description and / or any of the claims, the terms “including,” “includes,” “having,” “has,” “with,” or variations thereof are intended to be comprehensive in the same manner as the term “comprising.” Where used in the context of numbers and ranges, the terms “about” or “approximately” refer to a value or range that is close to or approximate the stated value or range, and as a result, the embodiments may be made to about ±10%, as intended, as will be apparent to those skilled in the art from the teachings contained herein. In some embodiments, about means ±10% of the quantity.

[0037] The terms “comprise,” “comprising,” and “include,” “comprising,” “include,” “including” are unrestricted linking verbs. Any form or tense of one or more of these verbs (e.g., “comprises,” “comprising,” “has,” “having,” “includes,” and “including”) are likewise unrestricted. For example, any method “comprises,” “comprising,” or “comprising” one or more steps is not limited to having only those one or more steps, but may also cover other steps not listed. Similarly, any composition “comprises,” “comprising,” or “comprising” one or more features is not limited to having only those one or more features, but may also cover other features not listed. Any and all examples or illustrative language provided herein in reference to any particular embodiment (e.g., “such as”) is intended only to better illustrate the disclosure and not to impose any limitation on the scope of the claimed disclosure.

[0038] This disclosure relates to a method for producing a viral vaccine formulated in sub-billion form, the method being described as follows: a. The process of purifying virus particles from the collected cell culture; b. A step of inactivating and dividing the above-mentioned virus particles to generate subvirions; and c. The process of purifying the above subvilions; The present invention provides a method comprising, wherein the step of inactivating and dividing the virus particles in step b above comprises treating the virus particles with a reagent comprising at least one component selected from the group consisting of nonionic surfactants and ionic surfactants, wherein the nonionic surfactant and the ionic surfactant are present in an amount effective to reduce the particle size of the virus particles.

[0039] In some embodiments, the reagent further comprises a salt, which is present in an amount effective to reduce the particle size of the virus particles.

[0040] In some embodiments, the average hydrodynamic radius of the subbilion ranges from 150 nm to 350 nm.

[0041] In some embodiments, the nonionic surfactant is polysorbate 80. In some embodiments, the nonionic surfactant comprises at least about 1.0 g / L of polysorbate 80, for example, at least about 1.5 g / L, at least about 2.0 g / L, or at least about 2.5 g / L. In some embodiments, the ionic surfactant comprises cetrimonium bromide (CTAB). In some embodiments, the ionic surfactant comprises about 1.25 g / L to 3.0 g / L of CTAB, for example, about 1.5 g / L, about 2.0 g / L, about 2.5 g / L, or about 3.0 g / L. In some embodiments, the salt comprises sodium chloride (NaCl). In some embodiments, the salt comprises 0 to 200 mM NaCl, for example, about 25 mM, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, or about 175 mM. In some embodiments, the nonionic surfactant comprises polysorbate 80, the ionic surfactant comprises CTAB, and the salt comprises sodium chloride (NaCl).

[0042] In some embodiments of the method, the purification step c includes adsorption filtration. In some such embodiments, the adsorption filtration throughput is at least about 50 L / m³. 2 For example, at least about 100 L / m³ 2 , at least about 150 L / m³ 2 , at least about 200 L / m³ 2 , at least about 200 L / m³ 2 , at least about 300 L / m³ 2 , or at least about 350 L / m³ 2 In some other embodiments, the particle size of the subvilion is less than about 500 nm, for example, less than about 400 nm, less than about 300 nm, less than about 200 nm, or less than about 150 nm. In some embodiments, the particle size of the subvilion is the hydrodynamic radius. In some embodiments, the size of the subvilion is measured by dynamic light scattering (DLS).

[0043] In some embodiments of the method, the virus particles are derived from influenza virus. In some embodiments, the virus particles are derived from influenza virus strain A.

[0044] The disclosure also provides a method for reducing the size of a virus, the method comprising the step of treating the virus particles with a reagent comprising at least one component selected from the group consisting of a nonionic surfactant and an ionic surfactant, wherein the nonionic surfactant and the ionic surfactant are present in an amount effective for reducing the size of the virus particles.

[0045] In some embodiments, the nonionic surfactant comprises polysorbate 80. In some embodiments, the nonionic surfactant comprises at least about 1.0 g / L of polysorbate 80, for example, at least about 1.5 g / L, at least about 2.0 g / L, or at least about 2.5 g / L. In some embodiments, the ionic surfactant comprises cetrimonium bromide (CTAB). In some embodiments, the ionic surfactant comprises about 1.25 g / L to 3.0 g / L of CTAB, for example, about 1.5 g / L, about 2.0 g / L, about 2.5 g / L, or about 3.0 g / L. In some embodiments, the salt comprises sodium chloride (NaCl). In some embodiments, the salt comprises 25 to 200 mM NaCl, for example, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, or about 175 mM. In some embodiments, the nonionic surfactant comprises polysorbate 80, the ionic surfactant comprises CTAB, and the salt comprises sodium chloride (NaCl).

[0046] In some embodiments, the method further includes filtering the treated virus particles through an adsorption filter. In some embodiments, the throughput of the adsorption filter is at least about 50 L / m³. 2 For example, at least about 100 L / m³ 2, at least about 150 L / m³ 2 , at least about 200 L / m³ 2 , at least about 250 L / m³ 2 , at least about 300 L / m³ 2 , or at least about 350 L / m³ 2 In some embodiments, the virus particle size is less than about 500 nm, for example, less than about 400 nm, less than about 300 nm, less than about 200 nm, or less than about 150 nm. In some embodiments, the virus particle size is the hydrodynamic radius of the virus particle. In some embodiments, the virus particle size is measured by dynamic light scattering (DLS).

[0047] In some embodiments, the virus particles are derived from the influenza virus. In some embodiments, the virus particles are derived from influenza virus strain A.

[0048] This disclosure also relates to a method for reducing viral particle size in an influenza virus purification process, wherein the method is a. A process for purifying virus particles collected from cell cultures; b. A step of inactivating and dividing the above-mentioned virus particles to generate subvirions; and c. The process of purifying the above subvilions; The present invention provides a method comprising, where step b above, the step of inactivating and dividing the virus particles, comprising the step of treating the virus particles with a reagent comprising a nonionic surfactant and an ionic surfactant, wherein the nonionic surfactant and the ionic surfactant are present in amounts effective for reducing the particle size of the virus particles.

[0049] This disclosure also relates to a method for producing a viral vaccine formulated in sub-billion form, the method being: a. The process of purifying virus particles from the collected cell culture; b. A step of inactivating and dividing the above-mentioned virus particles to generate subvirions; and c. The process of purifying the above subvilions; The present invention provides a method comprising, where step b above, the step of inactivating and dividing the virus particles, comprising treating the virus particles with a reagent nonionic surfactant and an ionic surfactant, wherein the nonionic surfactant and the ionic surfactant are present in amounts effective for reducing the particle size of the virus particles.

[0050] This disclosure also relates to a method for producing an influenza virus vaccine formulated in subbilion form, the method being described as follows: a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the above-mentioned virus particles to generate subvirions; and c. The process of purifying the above subvilions; The present invention provides a method comprising, where step b above, the step of inactivating and dividing the virus particles, comprising treating the virus particles with a reagent comprising a nonionic surfactant and an ionic surfactant, wherein the nonionic surfactant and the ionic surfactant are present in amounts effective for reducing the particle size of the virus particles.

[0051] In some embodiments, the reagent further comprises a salt, which is present in an amount effective to reduce the particle size of the virus particles. In some embodiments, the nonionic surfactant comprises polysorbate 80. In some embodiments, the nonionic surfactant comprises at least about 1.0 g / L of polysorbate 80, for example, at least about 1.5 g / L, at least about 2.0 g / L, or at least about 2.5 g / L. In some embodiments, the salt comprises sodium chloride (NaCl). In some embodiments, the salt comprises 25-200 mM NaCl, for example, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, or about 175 mM.

[0052] This disclosure also relates to a method for producing a formulated influenza virus vaccine comprising purified viral protein, wherein the method is a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the above-mentioned virus particles to generate subvirions; and c. A step of purifying the above subvirions to produce purified viral proteins; The present invention provides a method comprising, where step b above, the step of inactivating and dividing the virus particles, comprising treating the virus particles with a reagent comprising a nonionic surfactant and a salt, wherein the nonionic surfactant and the salt are present in an amount effective to reduce the particle size of the virus particles.

[0053] This disclosure also relates to a method for producing a formulated influenza virus vaccine comprising purified viral protein, wherein the method is a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the above-mentioned virus particles to generate subvirions; and c. A step of purifying the above subvirions to produce purified influenza virus protein; The present invention provides a method comprising, where step b above, the step of inactivating and dividing the virus particles, comprising treating the virus particles with a reagent comprising an ionic surfactant and a salt, wherein the ionic surfactant and the salt are present in an amount effective to reduce the particle size of the virus particles.

[0054] This disclosure also relates to a method for producing a formulated influenza virus vaccine comprising purified viral protein, wherein the method is a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating the purified virus particles by treating them with an alkylating agent; c. A step of treating the inactivated virus particles with an ionic surfactant to divide the inactivated virus particles and generate subvilions; and d) A step of purifying the above subvirions to produce purified influenza virus protein; The present invention provides a method comprising, wherein the step of inactivating and dividing the virus particles in step b and / or c above further comprises treating the virus particles with a reagent comprising a nonionic surfactant and an ionic surfactant, wherein the nonionic surfactant and the ionic surfactant are present in amounts effective for reducing the particle size of the virus particles.

[0055] This disclosure also relates to a method for producing an influenza virus vaccine formulated in subbilion form, the method being described as follows: a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the above-mentioned virus particles to generate subvirions; and c. The process of purifying the above subvilions; The present invention provides a method comprising, where step b above, the step of inactivating and dividing the virus particles, comprising treating the virus particles with a reagent comprising an effective amount of CTAB, NaCl, and polysorbate 80 to reduce the particle size of the virus particles.

[0056] This disclosure also relates to a method for producing an influenza virus vaccine formulated in subbilion form, the method being described as follows: a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the above-mentioned virus particles to generate subvirions; and c. The process of purifying the above subvilions; The present invention provides a method that includes, in the step of inactivating and dividing the virus particles in step b above, treating the virus particles with a reagent containing an amount effective to reduce the particle size of the virus particles, comprising 1.5-2.5 g / L CTAB, 0-150 mM NaCl, and 0-2.2 g / L polysorbate 80.

[0057] This disclosure also relates to a method for producing an influenza virus vaccine formulated in sub-billion form, the method being described as follows: a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the above-mentioned virus particles to generate subvirions; and c. The process of purifying the above subvilions; The present invention provides a method comprising, in which the step of inactivating and dividing the virus particles in step b above, treating the virus particles with a reagent comprising 1.5 to 2.5 g / L of an ionic surfactant, 0 to 150 mM of a salt, and 0 to 2.2 g / L of a nonionic surfactant in amounts effective to reduce the particle size of the virus particles.

[0058] In some embodiments, the ionic surfactant is CTAB. In some embodiments, the salt is NaCl. In some embodiments, the nonionic surfactant is polysorbate 80.

[0059] Generation method

[0060] A process for producing a viral vaccine formulated in subvirion form is disclosed herein. As used herein, the term “viral vaccine” means a biological preparation made from a virus that, when administered to a subject, can provide immunity against a particular disease. The vaccine composition may contain one or more antigens derived from one or more viruses or virus strains, and shall contain a sufficient amount of such antigens to produce an immunological response in a subject. As used herein, the term “subvirion form” means that the vaccine composition of this disclosure is generally formulated in the form of a split virus, if its viral lipid envelope is lysed or destroyed, or (b) in the form of a subunit virus containing one or more purified viral proteins. The process of forming a subvirion, i.e., “splitting,” is described herein.

[0061] Growth in culture

[0062] The viral vaccines disclosed herein may be produced from viruses grown in chicken eggs or in cell cultures. In some embodiments, the viruses used herein are grown in specific pathogen-free (SPF) embryogenerating chicken eggs and purified from the contents of the eggs (aloulanal fluid). Although not bound by theory, viral vaccines produced from viruses grown in animal cell cultures may cause some allergic reactions. Exemplary animal cell lines that may be used to produce the viruses used herein include, but are not limited to, hamster, cattle, primate (including human and monkey) and canine cells. In some embodiments, the viruses used herein are grown in animal cell cultures. In some embodiments, the viruses used herein are grown in mammalian cell cultures. Exemplary mammalian cell types that may be used to produce the viruses used herein include, but are not limited to, kidney cells, fibroblasts, retinal cells, and lung cells. Either the original MDCK cell line (available from the American Type Culture Collection (ATCC) as CCL 34) or its derivative cell lines may be used. In some embodiments, the viruses used herein are grown in MDCK cells. With respect to growth in cell lines (e.g., MDCK cells), the viruses may be grown in cells in suspension cultures or adherent cultures. One suitable MDCK cell line for suspension culture is MDCK 33016 (received as DSM ACC 2219). Further examples of suitable cell lines, as well as cell culture methods, can be found in WO 2007 / 052055 (which is incorporated herein by reference in its entirety). Methods for purifying viruses (e.g., influenza viruses) from cell cultures are well known in the art.

[0063] The methods and compositions disclosed herein may be used to produce viral vaccines from any virus known in the art. Exemplary viruses include, but are not limited to, adenovirus, influenza virus, herpesvirus, hepatitis virus, human papillomavirus, measles virus, mumps virus, poliomyelitis virus, Japanese encephalitis virus, rabies virus, rubella virus, smallpox virus, varicella-zoster virus, and yellow fever virus. In some embodiments, the viral particles are derived from influenza virus. In some embodiments, the viral particles are derived from influenza virus strain A. In some embodiments, the viral particles are derived from influenza virus strain B.

[0064] Virus inactivation and division

[0065] As used herein, the term “inactivating” refers to a process that renders a virus inactive, unable to replicate, and / or unable to infect by chemical or surface alterations of its components. Methods for inactivating viruses (e.g., influenza viruses) are well known in the art (see, e.g., Budowsky et al., Vaccine, (1999) 9(6):398-402; WO2011 / 138229; and WO2011 / 138682). Exemplary methods for inactivating viruses include, but are not limited to, pasteurization, dry heat, steam heat, solvents / detergents, alkylating agents, and / or treatment at low pH. Although not bound by theory, alkylating agents may modify viral proteins, thereby inhibiting viral entry into cells or release of the viral genome. Alkylating agents may also make viral protein capsids permeable and chemically inactivate viral nucleic acids. Exemplary alkylating agents that may be used to inactivate viruses for use herein include, but are not limited to, formalin, β-propiolactone (β-PL), and N-acetylaziridine. In some embodiments, inactivation of viral particles is carried out by treating the viral particles with an alkylating agent. In some embodiments, inactivation of viral particles is carried out by treating the viral particles with β-propiolactone.

[0066] As used herein, the term “splitting” means the destruction or fragmentation of a complete virus with a disruptive concentration of a splitting agent, whether infectious (wild-type or attenuated) or non-infectious (e.g., inactivated). While not theoretically bound, splitting agents generally include, typically, agents capable of degrading and lysing lipid membranes, having a hydrophobic tail bound to a hydrophilic head. The destruction may result in the complete or partial solubilization of viral proteins, thereby altering the integrity of the virus.

[0067] Methods for splitting viruses (e.g., influenza viruses) are well known in the art (see, for example, WO02 / 28422, WO02 / 067983, WO02 / 074336, WO01 / 21151, etc.). Splitting virus particles may involve treating the virus particles with one or more nonionic surfactants and / or ionic (e.g., cationic) surfactants. As used herein, “surfactant” means an agent that can reduce the surface tension (or interfacial tension) between two phases. Surfactants can act as detergents, wetting agents, emulsifiers, foaming agents, and dispersants. Although not bound by theory, surfactants are generally amphiphilic, having a hydrophobic “head” and one or two hydrophilic “tails.” As used herein, the term "non-ionic surfactant" refers to a surfactant that does not have a charged group in its head, while the term "ionic surfactant" refers to a surfactant that has a net positive charge (cationic) or a net negative charge (anionic) in its head. Examples of nonionic surfactants include, but are not limited to, alkyl glycosides, alkyl thioglycosides, acyl sugars, polyoxyethylene sorbitan esters (e.g., polysorbate 20 or Tween® 20, polysorbate 40, polysorbate 60 or polysorbate 80), octyl- or nonylphenoxy polyoxyethanol (e.g., Triton® surfactants (e.g., Triton® X-100 or Triton® N101)), polyoxyethylene ethers, polyoxyethylene esters, polyoxyethylene alkyl ethers, Hecameg, N,N-dialkyl-glucamides, and alkylphenoxy polyethoxyethanol.Examples of ionic surfactants include, but are not limited to, sulfobetaines, betaines, sarcosyls, and quaternary ammonium compounds, such as CTAB (cetyltrimethylammonium bromide), Cetrimide (myristyltrimethylammonium bromide), lipofectin, lipofectamine, and DOT-MA.

[0068] In some embodiments, the step of splitting the virus particles includes treating the virus particles with a reagent containing a nonionic surfactant. In some embodiments, the nonionic surfactant includes polysorbate 80. In some embodiments, the nonionic surfactant includes about 0.2 g / L, about 0.4 g / L, about 0.6 g / L, about 0.8 g / L, about 1.0 g / L, about 1.2 g / L, about 1.4 g / L, about 1.6 g / L, about 1.8 g / L, about 2.0 g / L, about 2.2 g / L, about 2.4 g / L, about 2.6 g / L, about 2.8 g / L, or about 3.0 g / L of polysorbate 80. In some embodiments, the nonionic surfactant includes at least about 1.0 g / L of polysorbate 80, for example, at least about 1.5 g / L, at least about 2.0 g / L, or at least about 2.5 g / L. In some embodiments, the nonionic surfactant comprises about 0.0 g / L to 2.2 g / L of polysorbate 80. In some embodiments, the step of dividing the virus particles comprises treating the virus particles with a reagent containing an ionic surfactant. In some embodiments, the ionic surfactant comprises cetyltrimethylammonium bromide or cetrimonium bromide (CTAB). In some embodiments, the ionic surfactant comprises about 1.25 g / L to 3.0 g / L of CTAB, for example, about 1.5 to 2.5 g / L, about 2.0 g / L, about 2.5 g / L, or about 3.0 g / L of CTAB.

[0069] The splitting of virus particles may also involve treating the virus particles with one or more salts. Although not constrained by theory, salts can stabilize virus particles and subvirions. Exemplary salts that may be used include, but are not limited to, sodium chloride, potassium chloride, magnesium chloride, potassium phosphate, and calcium phosphate. In some embodiments, the step of splitting the virus particles involves treating the virus particles with a reagent containing a salt. In some embodiments, the salt is NaCl. In some embodiments, the salt contains 0 to 200 mM NaCl, for example, about 25 mM, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, or about 175 mM NaCl. In some embodiments, the nonionic surfactant, the ionic surfactant, and / or the salt are each present in an amount effective to reduce the particle size of the virus particles.

[0070] The steps for inactivating and splitting the viral particles may also advantageously include adding enzymes that degrade matrix proteins and reduce subbillion particle size. Exemplary enzymes include, but are not limited to, proteases (e.g., proteinase K, trypsin, pepsin, elastase, thrombin, chymotrypsin, papain) and nucleases (e.g., benzonase, RNAse A, RNAse H). In some embodiments, the steps for inactivating and splitting the viral particles further include adding a protease. In some embodiments, the protease is trypsin. In some embodiments, the protease is proteinase K. In some embodiments, the steps for inactivating and splitting the viral particles further include adding a nuclease. In some embodiments, the protease is benzonase.

[0071] Refinement of divided vilions

[0072] Methods for purifying subvirions, individual proteins, or antigens from viruses are well known to those skilled in the art and include, for example, filtration, chromatography, centrifugation, ultrafiltration, and diafiltration. In some embodiments, the subvirions are purified by size exclusion chromatography (SEC). In some embodiments, the subvirions are purified by ultracentrifugation. In some embodiments, the subvirions are purified by adsorption filtration. In some embodiments, the subvirions are purified by adsorption filtration with a polymer resin. In some embodiments, the subvirions are purified by ultrafiltration or diafiltration. In some embodiments, the subvirions are purified by one or more of ultracentrifugation, adsorption filtration, ultrafiltration, and diafiltration.

[0073] For example, methods and compositions disclosed herein, involving nonionic surfactants, ionic surfactants, and salts, can advantageously produce viral vaccines formulated in subbillion forms with reduced particle size compared to those produced without surfactants or salts. Viral particles with reduced particle size can result in improved filtration throughput and higher yields. In some embodiments, the adsorption filtration throughput is at least about 5 L / m³. 2 For example, at least about 10 L / m³ 2 at least approximately 15 L / m³ 2 , or at least about 20 L / m³ 2 In an alternative large-scale generation embodiment of the present disclosure, the adsorption throughput is at least about 50 L / m³. 2 ~350L / m 2 For example, at least about 50 L / m³ 2 , at least about 100 L / m³ 2 , at least about 200 L / m³ 2 , at least about 300 L / m³ 2 , or at least about 350 L / m³ 2 That is the case.

[0074] Methods for measuring viral particle size are well known to those skilled in the art and include, but are not limited to, small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), resonance mass measurement, size exclusion chromatography (SEC), and laser diffraction. Other particle sizing techniques (e.g., liquid chromatography) may be unsuitable due to intrusive sample preparation, undesirable surface chemical phenomena of the column resin interacting with the test sample, or high column pressures that may potentially alter the higher-order structure. DLS is a rapid, non-contact, and non-intrusive particle sizing technique that allows analytes to be tested in their natural state without compromising the integrity of their higher-order structure. In some embodiments, viral particle size is measured by dynamic light scattering (DLS). In some embodiments, viral and subbillion particle size is measured by SAXS. In some embodiments, viral and subbillion particle size is measured by resonance mass measurement. In some embodiments, viral and subbillion particle size is measured by size exclusion chromatography (SEC). In some embodiments, the particle size is the hydrodynamic radius (R) of the virus particles. H ) is defined by the radius of gyration (R) of the virus particles. In some embodiments, particle size is defined by the radius of gyration (R) of the virus particles. G Defined by ). In some embodiments, the particle size of the subbillions is less than about 500 nm, for example, less than about 400 nm, less than about 300 nm, less than about 200 nm, or less than about 150 nm.

[0075] This disclosure is further illustrated by the following embodiments, which should not be construed as limiting. All references, patents, and published patent applications and drawings referenced throughout this application are incorporated herein by reference in whole for all purposes. [Examples]

[0076] The following embodiments are illustrative and should be construed as not being limitations on the scope of the present disclosure disclosed above.

[0077] Example 1:

[0078] Method: Purification of virus particles from collected cell cultures.

[0079] A clarified sample from an MCDK cell culture containing influenza virus was concentrated and diafiltration was performed using a hollow fiber membrane. The clarified sample was then filled into a retentate vessel (UF0) of an ultrafiltration tank, concentrated to the target volume, and subsequently diafiltration with salt-containing Tris buffer. After diafiltration, the retentate was concentrated again and then flushed with the same buffer to recover the product. The product was then processed in a chromatographic step to remove host cell proteins (HCPs) and purify the virus particles. The concentrated and diafiltrationd cell sample was loaded onto a column through a pre-equilibrium filter. The product was collected in a flow-through and added to a retentate vessel (UF1) of an ultrafiltration tank with a hollow fiber membrane. The product was concentrated to the target volume and then diafiltration was performed at a constant volume with a buffer containing MgCl2. Benzonase solution was added, and the product was then recycled through an ultrafilter to enable DNA digestion. After digestion, a second diafiltration was performed with sodium phosphate at a fixed volume, and the product was exchanged for a suitable buffer for inactivation.

[0080] Method: Inactivation and fragmentation of virus particles

[0081] Polysorbate 80 (PS80) in sodium phosphate was added to the product from UF1, and the mixture was incubated at room temperature. The influenza virus was then inactivated using β-propiolactone (BPL). Briefly, the virus and PS80 mixture was cooled to 5°C, and then BPL was added to inactivate the influenza virus. After inactivation, the mixture was heated to 37°C to hydrolyze the BPL. The mixture was then brought to room temperature before the addition of PS80, as discussed below.

[0082] Next, additional PS80 was added to the mixture at the concentrations shown in Table 1. Then, sodium chloride was added at the concentrations shown in Table 1. Finally, CTAB was added at the concentrations shown in Table 1, followed by incubation at room temperature and then transfer to an ultracentrifuge.

[0083] Method: Subbillion purification

[0084] The solubilized surface antigen was separated from the viral core by continuous ultracentrifugation. The ultracentrifugation was flushed with sodium phosphate to enhance product recovery. The flow-through containing the viral antigen was collected in an adsorption vessel. PS80 was added to bring the final concentration to 2.5 g / L. Next, a polymer resin in sodium phosphate was added and incubated with the mixture for a sufficient time for CTAB adsorption to occur. After incubation, the product was removed from the resin and filtered through a 0.5 / 0.2 μm filter before ultrafiltration. The overall product recovery yield from the adsorption filter was measured by throughput as shown in Figure 4. [Table 1]

[0085] result

[0086] The tests were performed on three different influenza virus samples, as detailed in the Methods section above: (1) influenza B / Victoria lineage (shown in Figures 1A-C), (2) influenza A H3N2 from Maine (shown in Figures 2A-C), and (3) influenza A H3N2 from South Carolina (shown in Figures 3A-C). The average particle size measured by DLS under different conditions for the three samples is shown in Figures 1A-C, 2A-C, and 3A-C, respectively. Error bars indicate the range in repeated measurements. As shown in Figures 1A-C, 2A-C, and 3A-C, generally, lower levels of CTAB and higher levels of polysorbate 80 produce subbilions with smaller particle sizes.

[0087] Example 2: Particle size determination by dynamic light scattering method

[0088] The particle size of virus particles in subbillion morphology was measured using dynamic light scattering (DLS) with a Wyatt Technologies DynaPro Plate Reader II instrument. Before and during measurement, dust was removed by centrifugation of the sample in a conical tube using a 0.1 μm anotop syringe filter or by centrifugation at 3,000 rpm for 10 minutes in a fixed-angle benchtop centrifuge. A reference standard of 14 kDa lysozyme at 0.25 mg / mL in aqueous solution was used as a benchmark. The above sample and reference standard were placed on a 384-well glass-bottom microplate (Greiner P / N 781892), and the microplate was centrifuged at 2,000 × g for 2 minutes in a swing-bucket centrifuge to remove air bubbles. The microplate was loaded into the DLS instrument, and 14 data acquisitions were performed at 25°C with acquisition times of 25 seconds each.

[0089] Data analysis was performed using the DYNAMICS software package to generate and adjust the sum-of-squares difference from the Gaussian cumulant results. Monomodal analyses were performed for results with ≤57% multidispersion using one of the Gaussian distributions of the correlation rates during curve fitting. Results showing a low sum-of-squares value (≤20) showed a reasonable mathematical agreement between the measured correlation curve and the cumulant fitted curve, suggesting that the above samples were likely monomodal with low multidispersion, particularly a tight size distribution. Results showing SOS > 20 and / or % multidispersion > 57% were reported as multimodal, and the results from their adjusted graphs are reported.

[0090] The results of Experiment 2 are shown in Figure 4. Here, filter throughput versus DLS average particle size was measured and compared. As shown, as the particle size decreases, the overall throughput through the filter increases, which results in increased yield.

[0091] Example 3: Adsorption filtration experiment with A / turkey / Turkey strain

[0092] Adsorption filtration experiments were conducted, and four split conditions were tested using A / turkey / Turkey / 1 / 2005 NIBRG-23(H5N1). More specifically, the tested split conditions concerned the adsorption filtration capacity at various concentrations of CTAB, NaCl, and PS80, as shown in Table 2 below. Follow-up adsorption filtration experiments for A / turkey / Turkey / 1 / 2005 NIBRG-23(H5N1) were performed as shown in Table 3 below. [Table 2]

[0093] The results in Table 2 suggest that higher concentrations of CTAB and PS80, and lower concentrations of NaCl, result in optimal filtration performance for the A / turkey / Turkey(H5N1) strain (see, for example, filtration conditions 4a and 4b). Although the splitting conditions were the same, throughput was 36 to 72 times better than existing conditions. [Table 3]

[0094] The results in Table 3 suggest that at constant concentrations of 2.5 g / L CTAB and 2.2 g / L PS80, increased NaCl correlates with increased supernatant turbidity and decreased filter throughput. Therefore, reduced NaCl appears to provide a significant increase in the filtration capacity of the A / turkey / Turkey (H5N1) strain. At constant concentrations of CTAB (2.5 g / L) and PS80 (2.2 g / L), filtration capacity (throughput) improved by approximately seven times as NaCl decreased from 75 mM to zero.

[0095] [Table 4]

[0096] Example 4: Method: Purification of viral particles from collected cell cultures under large-scale production conditions.

[0097] A clarified sample from an MCDK cell culture containing influenza virus is concentrated and diafiltration is performed using a hollow fiber membrane in salt-containing Tris buffer. The collected product is then further concentrated, and the system is flushed with the same buffer to extract the product. This material is passed through a 1.2 μm column guard filter and applied to a chromatography column to remove HCP, resulting in purified complete virus. The purified complete virus is concentrated and diafiltration is performed using a hollow fiber membrane in magnesium-containing buffer (UF / DF1). Benzonase is added to remove DNA. A second diafiltration is performed to exchange the purified complete virus for phosphate buffer, and the system is flushed with the same buffer to extract the product. The purified complete virus is inactivated at 2-8°C in the presence of polysorbate 80 and BPL. After inactivation, the BPL is hydrolyzed at 37°C. Viral splitting occurs with strain-specific addition of polysorbate 80, sodium chloride, and CTAB to enable surface antigens in the ranges of 1.0–2.5 g / L, 0–200 mM, and 1.25–3.0 g / L, respectively, or alternatively, as shown in Table 1. The viral core is removed by continuous flow ultracentrifugation, and the soluble fraction is contacted with a polymer resin to remove the CTAB. The resin is removed by passing the product slurry through a 60 μm mesh bag, and the soluble viral surface antigens are then processed through a 0.5 / 0.2 μm filter. The filtered antigens are concentrated and diafiltration into the final formulation buffer using a cassette membrane before processing through a 0.2 μm filter (UF / DF2). In certain embodiments, for example, the following are provided: (Item 1) A method for producing a viral vaccine formulated in subviron form, wherein the method is: a. The process of purifying virus particles from the collected cell culture; b. A step of inactivating and dividing the virus particles to generate subvirions; and c. A step of purifying the subvilion, A method comprising, wherein the step of inactivating and dividing the virus particles in step b comprises treating the virus particles with a reagent comprising a nonionic surfactant and an ionic surfactant, wherein the nonionic surfactant and the ionic surfactant are present in amounts effective for reducing the particle size of the virus particles. (Item 2) The method according to item 1, wherein the reagent further comprises a salt, the salt present in an amount effective to reduce the particle size of the virus particles. (Item 3) The average hydrodynamic radius of the subbillion is in the range of 150 nm to 350 nm, as described in any one of items 1 to 2. (Item 4) The method according to any one of items 1 to 3, wherein the nonionic surfactant is polysorbate 80. (Item 5) The nonionic surfactant comprises at least about 1.0 g / L of polysorbate 80, for example, at least about 1.5 g / L, at least about 2.0 g / L, or at least about 2.5 g / L, according to any one of items 1 to 4. (Item 6) The ionic surfactant is the method described in any one of items 1 to 5, comprising cetrimonium bromide (CTAB). (Item 7) The ionic surfactant is the method according to any one of items 1 to 6, comprising approximately 1.25 g / L to 3.0 g / L CTAB, for example, approximately 1.5 g / L, approximately 2.0 g / L, approximately 2.5 g / L, or approximately 3.0 g / L. (Item 8) The salt is a salt comprising sodium chloride (NaCl) as described in any one of items 1 to 7. (Item 9) The salt comprises 0 to 200 mM NaCl, for example, about 25 mM, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, or about 175 mM, as described in any one of items 1 to 8. (Item 10) The method according to any one of items 1 to 9, wherein the nonionic surfactant comprises polysorbate 80, the ionic surfactant comprises CTAB, and the salt comprises sodium chloride (NaCl). (Item 11) The purification step in step c is the method described in any one of items 1 to 10, including adsorption filtration. (Item 12) The adsorption filtration throughput is at least approximately 50 L / m³. 2 For example, at least about 100 L / m³ 2 , at least about 150 L / m³ 2 , at least about 200 L / m³ 2 , at least about 200 L / m³ 2 , at least about 300 L / m³ 2 , or at least about 350 L / m³ 2 The method described in item 11. (Item 13) The method according to any one of items 1 to 12, wherein the particle size of the subbilion is less than about 500 nm, for example, less than about 400 nm, less than about 300 nm, less than about 200 nm, or less than about 150 nm. (Item 14) The particle size of the subbilion is the hydrodynamic radius, as described in items 1 to 13. (Item 15) The size of the subbilion is measured by dynamic light scattering (DLS) as described in any one of items 1 to 14. (Item 16) The virus particles are derived from the influenza virus, according to the method described in any one of items 1 to 15. (Item 17) The virus particles are derived from influenza virus strain A, according to the method described in any one of items 1 to 16. (Item 18) The method according to any one of items 4 to 17, wherein the concentration of the polysorbate 80 is 0.3 g / L before division. (Item 19) The method according to any one of items 4 to 18, wherein the concentration of the polysorbate 80 increases in the range of 0 to 2.2 g / L during the division. (Item 20) The method according to any one of items 4 to 19, wherein the concentration of the polysorbate 80 is 0.3 g / L, 1.4 g / L, or 2.5 g / L during the division. (Item 21) NaCl concentrations ranging from 0 to 25 mM are associated with increased filtration throughput, as described in any one of items 8 to 20. (Item 22) A method for reducing the particle size of a virus, the method comprising the step of treating the virus particles with a reagent comprising a nonionic surfactant and an ionic surfactant, wherein the nonionic surfactant and the ionic surfactant are present in an amount effective for reducing the particle size of the virus particles. (Item 23) The method according to item 22, wherein the reagent further comprises a salt, the salt present in an amount effective to reduce the particle size of the virus particles. (Item 24) The nonionic surfactant is the method according to any one of items 22 to 23, comprising polysorbate 80. (Item 25) The nonionic surfactant comprises at least about 1.0 g / L of polysorbate 80, for example, at least about 1.5 g / L, at least about 2.0 g / L, or at least about 2.5 g / L, according to any one of items 22 to 24. (Item 26) The ionic surfactant is the method described in any one of items 22 to 25, comprising cetrimonium bromide (CTAB). (Item 27) The ionic surfactant is the method according to any one of items 22 to 26, comprising about 1.25 g / L to 3.0 g / L CTAB, for example, about 1.5 g / L, about 2.0 g / L, about 2.5 g / L, or about 3.0 g / L. (Item 28) The salt is the method described in any one of items 22 to 27, comprising sodium chloride (NaCl). (Item 29) The salt comprises 25–200 mM NaCl, for example, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, or about 175 mM, as described in any one of items 22–28. (Item 30) The method according to any one of items 22 to 29, wherein the nonionic surfactant comprises polysorbate 80, the ionic surfactant comprises CTAB, and the salt comprises sodium chloride (NaCl). (Item 31) The method according to any one of items 22 to 30, further comprising the step of filtering the treated virus particles through an adsorption filter. (Item 32) The throughput of the adsorption filter is at least about 50 L / m³. 2 For example, at least about 100 L / m³ 2 , at least about 150 L / m³ 2 , at least about 200 L / m³ 2 , at least about 250 L / m³ 2 , at least about 300 L / m³ 2 , or at least about 350 L / m³ 2 The method described in item 31. (Item 33) The method according to any one of items 22 to 32, wherein the virus particle size is less than approximately 500 nm, for example, less than approximately 400 nm, less than approximately 300 nm, less than approximately 200 nm, or less than approximately 150 nm. (Item 34) The method according to items 22 to 33, wherein the virus particle size is the hydrodynamic radius of the virus particle. (Item 35) The aforementioned virus particle size is measured by dynamic light scattering (DLS) according to the method described in any one of items 22 to 34. (Item 36) The virus particles are derived from the influenza virus, as described in any one of items 22 to 35. (Item 37) The virus particles are derived from influenza virus strain A, according to the method described in any one of items 22 to 36. (Item 38) The method according to any one of items 24 to 37, wherein the concentration of the polysorbate 80 is 0.3 g / L before division. (Item 39) The method according to any one of items 24 to 38, wherein the polysorbate 80 increases in the range of 0 to 2.2 g / L during the division. (Item 40) The method according to any one of items 24 to 39, wherein the concentration of the polysorbate 80 is 0.3 g / L, 1.4 g / L, or 2.5 g / L during the division. (Item 41) NaCl concentrations ranging from 0 to 25 mM are associated with increased filtration throughput, as described in any one of items 28 to 40. (Item 42) A method for reducing the size of a virus in an influenza virus purification process, wherein the method is a. A process for purifying virus particles collected from cell cultures; b. A step of inactivating and dividing the virus particles to generate subvirions; and c. A step of purifying the subvilion; A method comprising, wherein the step of inactivating and dividing the virus particles in step b comprises treating the virus particles with a reagent comprising a nonionic surfactant and an ionic surfactant, wherein the nonionic surfactant and the ionic surfactant are present in an amount effective to reduce the particle size of the virus particles. (Item 43) A method for producing a viral vaccine formulated in subviron form, wherein the method is: a. The process of purifying virus particles from the collected cell culture; b. A step of inactivating and dividing the virus particles to generate subvirions; and c. A step of purifying the subvilion; A method comprising, wherein the step of inactivating and dividing the virus particles in step b comprises treating the virus particles with a reagent comprising a nonionic surfactant and an ionic surfactant, wherein the nonionic surfactant and the ionic surfactant are present in amounts effective for reducing the particle size of the virus particles. (Item 44) A method for producing an influenza virus vaccine formulated in subbillion form, wherein the method is: a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the virus particles to generate subvirions; and c. A step of purifying the subvilion; A method comprising, wherein the step of inactivating and dividing the virus particles in step b comprises treating the virus particles with a reagent comprising a nonionic surfactant and an ionic surfactant, wherein the nonionic surfactant and the ionic surfactant are present in amounts effective for reducing the particle size of the virus particles. (Item 45) The method according to any one of items 42 to 44, wherein the reagent further comprises a salt, the salt present in an amount effective to reduce the particle size of the virus particles. (Item 46) The nonionic surfactant is the method according to any one of items 42 to 45, comprising polysorbate 80. (Item 47) The nonionic surfactant comprises at least about 1.0 g / L of polysorbate 80, for example, at least about 1.5 g / L, at least about 2.0 g / L, or at least about 2.5 g / L, according to any one of items 42 to 46. (Item 48) The salt is a salt comprising sodium chloride (NaCl) as described in any one of items 42 to 47. (Item 49) The salt comprises 25–200 mM NaCl, for example, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, or about 175 mM, as described in any one of items 42–48. (Item 50) The method according to any one of items 46 to 49, wherein the concentration of the polysorbate 80 is 0.3 g / L before division. (Item 51) The method according to any one of items 46 to 50, wherein the concentration of the polysorbate 80 increases in the range of 0 to 2.2 g / L during the division. (Item 52) The method according to any one of items 46 to 51, wherein the concentration of the polysorbate 80 is 0.3 g / L, 1.4 g / L, or 2.5 g / L during the division. (Item 53) NaCl concentrations ranging from 0 to 25 mM are associated with increased filtration throughput, as described in any one of items 48 to 52. (Item 54) A method for producing a formulated influenza virus vaccine containing purified viral protein, wherein the method is: a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the virus particles to generate subvirions; and c. A step of purifying the subvirions to produce purified viral proteins; A method comprising, wherein the step of inactivating and dividing the virus particles in step b comprises treating the virus particles with a reagent comprising a nonionic surfactant and a salt, wherein the nonionic surfactant and the salt are present in an amount effective to reduce the particle size of the virus particles. (Item 55) A method for producing a formulated influenza virus vaccine containing purified viral protein, wherein the method is: a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the virus particles to generate subvirions; and c. A step of purifying the subvirion to produce purified influenza virus protein; A method comprising, wherein the step of inactivating and dividing the virus particles in step b comprises treating the virus particles with a reagent comprising an ionic surfactant and a salt, wherein the ionic surfactant and the salt are present in an amount effective to reduce the particle size of the virus particles. (Item 56) A method for producing a formulated influenza virus vaccine containing purified viral protein, wherein the method is: a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating the purified virus particles by treating them with an alkylating agent; c. A step of treating the inactivated virus particles with an ionic surfactant to divide the inactivated virus particles and generate subvilions; and d. A step of purifying the subvirion to produce purified influenza virus protein; A method comprising, wherein the step of inactivating and fragmenting the virus particles in step b and / or c further comprises treating the virus particles with a reagent comprising a nonionic surfactant and an ionic surfactant, wherein the nonionic surfactant and the ionic surfactant are present in amounts effective for reducing the particle size of the virus particles. (Item 57) A method for producing an influenza virus vaccine formulated in subbillion form, wherein the method is: a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the virus particles to generate subvirions; and c. A step of purifying the subvilion; A method comprising, wherein the step of inactivating and fragmenting the virus particles in step b comprises treating the virus particles with a reagent comprising an amount of CTAB, NaCl, and polysorbate 80 effective in reducing the particle size of the virus particles. (Item 58) The method according to item 57, wherein the concentration of the polysorbate 80 is 0.3 g / L before division. (Item 59) The method according to any one of items 57 to 58, wherein the concentration of the polysorbate 80 increases in the range of 0 to 2.2 g / L during the division. (Item 60) The method according to any one of items 57 to 59, wherein the concentration of the polysorbate 80 is 0.3 g / L, 1.4 g / L, or 2.5 g / L during the division. (Item 61) NaCl concentrations ranging from 0 to 25 mM are associated with increased filtration throughput, as described in any one of items 57 to 60. (Item 62) A method for producing an influenza virus vaccine formulated in subbillion form, wherein the method is: a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the virus particles to generate subvirions; and c. A step of purifying the subvilion; This includes, where the step of step b, which inactivates and divides the virus particles, is performed in an amount of 1.5 to 2.5 g / L that is effective in reducing the particle size of the virus particles. A method comprising treatment with a reagent containing CTAB, 0-150 mM NaCl, and 0-2.2 g / L polysorbate 80. (Item 63) A method for producing an influenza virus vaccine formulated in subbilion form, wherein the method is: a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the virus particles to generate subvirions; and c. A step of purifying the subvilion; A method comprising, wherein the step of inactivating and fragmenting the virus particles in step b comprises treating the virus particles with a reagent comprising 1.5 to 2.5 g / L of an ionic surfactant, 0 to 150 mM of a salt, and 0 to 2.2 g / L of a nonionic surfactant in an amount effective to reduce the particle size of the virus particles. (Item 64) The method according to item 63, wherein the ionic surfactant is CTAB. (Item 65) The method according to any one of items 63 to 64, wherein the salt is NaCl. (Item 66) The method according to any one of items 63 to 65, wherein the nonionic surfactant is polysorbate 80. (Item 67) The method according to item 66, wherein the concentration of the polysorbate 80 is 0.3 g / L before division. (Item 68) The method according to any one of items 66 to 67, wherein the concentration of the polysorbate 80 increases in the range of 0 to 2.2 g / L during the division. (Item 69) The method according to any one of items 66-68, wherein the concentration of the polysorbate 80 is 0.3 g / L, 1.4 g / L, or 2.5 g / L during the division. (Item 70) NaCl concentrations ranging from 0 to 25 mM are associated with increased filtration throughput, as described in any one of items 65 to 69. (Item 71) A method for producing an influenza virus vaccine formulated in subbilion form, wherein the method is: a. A process for purifying virus particles from a cell culture obtained from MDCK cells; b. A step of inactivating and dividing the virus particles to generate subvirions; and c. A step of purifying the subvilion; A method comprising, wherein the step of inactivating and dividing the virus particles in step b comprises treating the virus particles with a reagent comprising an amount selected from the group consisting of conditions 1 to 27 in Table 1, comprising CTAB, NaCl, and polysorbate 80.

Claims

1. A method for producing a viral vaccine, wherein the method is a. The process of purifying virus particles from the collected cell culture; b. A step of inactivating and dividing the virus particles to generate subvirions; and c. A step of purifying the subvilion, A method comprising, wherein the step of inactivating and dividing the virus particles in step b comprises treating the virus particles with a reagent comprising polysorbate 80 at a concentration of 1.1 g / L to 2.2 g / L and cetrimonium bromide (CTAB) at a concentration of 1.5 g / L to 2.5 g / L, wherein the reagent is either free of sodium chloride (NaCl) or contains NaCl at a concentration of up to 25 mM.

2. The method according to claim 1, wherein the average hydrodynamic radius of the subbillion is in the range of 150 nm to 350 nm.

3. The method according to claim 1, wherein the particle size of the subbillions is less than approximately 500 nm.

4. The method according to claim 1, wherein the particle size of the subbilion is the hydrodynamic radius.

5. The method according to claim 1, wherein the virus particles are derived from the influenza virus.

6. A method for reducing the size of a virus in a virus manufacturing process, wherein the method is a. A process for purifying virus particles collected from cell cultures; b. A step of inactivating and dividing the virus particles to generate subvirions; and c. A step of purifying the subvilion; A method comprising, wherein the step of inactivating and dividing the virus particles in step b comprises treating the virus particles with a reagent comprising polysorbate 80 at a concentration of 1.1 g / L to 2.2 g / L and cetrimonium bromide (CTAB) at a concentration of 1.5 g / L to 2.5 g / L, wherein the reagent is either free of sodium chloride (NaCl) or contains NaCl at a concentration of up to 25 mM.

7. A method for producing an influenza virus vaccine preparation containing purified viral protein, wherein the method is: a. A process for purifying virus particles from cell cultures obtained from MDCK cells; b. A step of inactivating and dividing the virus particles to generate subvirions; and c. A step of purifying the subvirions to produce purified viral proteins; A method comprising, wherein the step of inactivating and dividing the virus particles in step b comprises treating the virus particles with a reagent comprising polysorbate 80 at a concentration of 1.1 g / L to 2.2 g / L and cetrimonium bromide (CTAB) at a concentration of 1.5 g / L to 2.5 g / L, wherein the reagent is either free of sodium chloride (NaCl) or contains NaCl at a concentration of up to 25 mM.

8. A method for producing an influenza virus vaccine preparation containing purified viral protein, wherein the method is: a. A process for purifying virus particles from cell cultures obtained from MDCK cells; b. A step of inactivating the purified virus particles by treating them with at least one alkylating agent; c. A step of treating the inactivated virus particles with at least one ionic surfactant to divide the inactivated virus particles and generate subvilions; and d. A step of purifying the subvirion to produce purified influenza virus protein; A method comprising, wherein the step of inactivating and dividing the virus particles in step b and / or c, further comprises treating the virus particles with a reagent comprising polysorbate 80 at a concentration of 1.1 g / L to 2.2 g / L and cetrimonium bromide (CTAB) at a concentration of 1.5 g / L to 2.5 g / L, wherein the reagent is either free of sodium chloride (NaCl) or contains NaCl at a concentration up to 25 mM.

9. The method according to claim 1, wherein the viral vaccine is formulated in a subvilion form.

10. The method according to claim 1, wherein the particle size of the subbilions ranges from greater than about 150 nm to less than about 500 nm.

Images