Rapid verification test of influenza virus production method and influenza virus antigen purification conditions using a disposable culture process system

Abstract

The present invention relates to an influenza virus production method using a disposable culture process system and a rapid confirmation test for influenza virus antigen purification conditions. According to the present invention, the conditions for obtaining (purifying) influenza surface antigens can be confirmed quickly and reliably using a unique method of the present invention without using radial immunodiffusion, which is currently used as a standard test method in influenza vaccine production. This significantly shortens the production period for influenza surface antigen subunit vaccines and enables rapid response to vaccine development / production even in situations where a new influenza virus is rapidly spreading. Furthermore, according to the influenza virus production method of the present invention, the use of a continuous low-speed centrifuge using disposable bags allows culture medium exchange to be carried out in a closed system, significantly reducing the possibility of contamination during the virus production process.

Description

[Technical Field]

[0001] This application claims priority to Korean Patent Application No. 10-2020-0052700 filed on April 29, 2020, and Korean Patent Application No. 10-2020-0052708 filed on April 29, 2020, the entire specifications of which are incorporated herein by reference.

[0002] The present invention relates to a method for producing influenza virus using a disposable culture process system and a rapid validation test for influenza virus antigen purification conditions. [Background technology]

[0003] While treatment is important for influenza-related diseases, prevention through vaccination is a realistic and effective method. If vaccines could be developed more quickly, it is predicted that the damage could be reduced in the event of a new influenza pandemic.

[0004] The recommended influenza virus strain for vaccine production changes every year, and given the characteristics of new strains of influenza, which spread worldwide at random times, the key to developing an influenza virus vaccine is to establish mass production conditions in a short period of time and produce the vaccine with high yield and purity.

[0005] The main antigen in influenza vaccines is hemagglutinin (HA) found in the outer coat of the influenza virus, and vaccine potency is measured based on the hemagglutinin concentration. Currently, the single radial immunodiffusion technique (SRID), recommended by the European Pharmacopoeia and the World Health Organization (WHO), is commonly used both domestically and internationally to measure the hemagglutinin concentration in influenza vaccines (Non-Patent Document 1). When measuring hemagglutinin content using radial immunodiffusion, standard antigens and standard antibodies provided by the National Institute for Biological Standards and Control (NIBSC) are used. The hemagglutinin concentration in influenza vaccines is measured by comparing and converting the size of the rings formed by agglutination of the influenza vaccine antigen and the standard antibody to the size of the rings formed by agglutination of the standard antigen and the standard antibody. However, this radial immunodiffusion method requires more than 24 hours to process samples, and most of the steps are performed manually, making it very labor-intensive compared to the throughput.

[0006] A pandemic refers to a state in which an infectious disease is widespread worldwide. In a pandemic situation, vaccine development must be rapid, so hemagglutinin content measurement must also be carried out quickly when developing a vaccine to deal with an influenza pandemic. In Korea, during the unexpected H1N1 pandemic in 2009, delays in the supply of standard antigens and standard antibodies from the World Health Organization (WHO) led to overall delays in vaccine production and subsequent clinical trials. When vaccine development must be rapid, as in a pandemic situation, a rapid method for measuring hemagglutinin content is required, but the development of standard antigens and standard antibodies used in radial immunodiffusion testing takes two to three months, which became an obstacle to rapid vaccine development.

[0007] Recently, research into new physicochemical testing methods has begun, but in order for these new physicochemical testing methods to replace the radial immunodiffusion test, data on any correlation between the results of the alternative test method and the radial immunodiffusion test is required, as well as the establishment and certification of the test method.

[0008] Currently, alternative testing methods such as reverse phase chromatography (RP-HPLC), isotope dilution liquid chromatography mass spectrometry (ID-LC / MS), and deglycosylated antigen SDS-PAGE analysis are known, but some methods still require standard antigens, and are known to have disadvantages such as the need for expensive equipment and specialized personnel, and low analytical sensitivity (Non-Patent Document 2).

[0009] There remains a need in the art for a method that can easily replace or eliminate the SRID method in vaccine production.

[0010] Meanwhile, influenza vaccines have been produced using fertilized eggs for the past 50 years. However, this method requires approximately one fertilized egg per dose, making production time and the manufacturing process complex. Furthermore, although quality-controlled specific pathogen-free (SPF) fertilized eggs are used for vaccine production, supply is limited, making it difficult to increase supply in the event of sudden demand unless planned in advance. Furthermore, fertilized egg-based vaccines contain trace amounts of egg-derived proteins, which may cause allergic reactions in individuals sensitive to these proteins. Furthermore, influenza viruses can undergo antigenic mutation when cultured in fertilized eggs. In some cases, fertilized egg culture is impossible or even fatal to chickens. These drawbacks could pose a major problem in supplying sufficient vaccines in the event of a global influenza pandemic. To overcome these drawbacks, development of techniques for culturing influenza viruses using animal cell cultures began about 15 years ago.

[0011] Meanwhile, cell culture for industrial-scale virus production is generally performed in stainless steel fermenters. However, this requires cleaning and sterilization of the fermenter before use, which increases the process preparation time and increases the risk of contamination with external substances. Furthermore, cross-contamination between lots or virus strains can occur during the cleaning process after use. In particular, influenza vaccines require the cultivation of three or four different viruses in trivalent or tetravalent vaccines, which increases the possibility of cross-contamination between strains. Therefore, a new virus production process that solves these problems is needed.

[0012] Korean Patent No. 10-1464783 discloses a method for culturing MDCK cells in a disposable bioreactor. However, the method provided in this document involves cell culture in the presence of microcarriers, and an additional step of removing the microcarriers, making the procedure cumbersome and increasing the risk of contamination. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] Korean Patent No. 10-1464783 [Patent Document 2] International Patent Publication WO05 / 108546 [Patent Document 3] International Patent Publication WO05 / 104706 [Patent Document 4] International Patent Publication WO05 / 10849 [Patent Document 5] Republic of Korea Patent Publication No. 10-2012-0024464 [Patent Document 6] Korean Patent Registration No. 10-1370512 [Patent Document 7] Republic of Korea Patent Application No. 10-2020-0052700 [Patent Document 8] Republic of Korea Patent Application No. 10-2020-0052708 Summary of the Invention [Problem to be solved by the invention]

[0014] In response to this, the present inventors have conducted research to enable the setting of process conditions during vaccine production without using radial immunodiffusion. They have found that the unique method of the present invention enables rapid and reliable confirmation of conditions for obtaining influenza surface antigens, thereby significantly shortening the production period for vaccines containing influenza antigens, and have thus completed the present invention.

[0015] Furthermore, as a result of extensive research into developing a cell culture system that reduces the preparation time for the process of cell culture for the propagation of influenza viruses and minimizes the risk of contamination, the inventors discovered that this objective can be achieved by culturing cells in a disposable cell incubator using disposable bags, and then replacing the culture medium before virus infection using a continuous low-speed centrifuge using disposable bags, leading to the completion of the present invention.

[0016] Therefore, an object of the present invention is to provide a method for purifying surface antigen proteins from influenza viruses using a detergent, characterized in that the amount of detergent to be applied to the sampled influenza viruses is determined by calculating the total protein quantification value (TPQV) per unit volume of the sampled influenza viruses and then substituting the value obtained into the following formula (1):

[0017] Surfactant treatment amount = [{(a*TPQV(μg / mL)) / (b μg / mL)} / c]* d (1)

[0018] (In the formula, a is 0.005 to 0.100 (v / v%), b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and the value closest to the TPQV is selected; c is the concentration (v / v%) of the surfactant stock being treated; d is the volume of the sampled influenza virus treated with the surfactant.

[0019] Another object of the present invention is to provide a method for rapidly confirming influenza antigen purification conditions, comprising the following first and second purification condition determination methods:

[0020] A first method for determining purification conditions, which is used in a step of purifying influenza virus from an influenza virus culture, and in which the conditions for purifying the influenza virus are determined based on a hemagglutination assay, SDS-PAGE, or a combination thereof, for influenza virus samples obtained by purification under different conditions; and A second method for determining purification conditions, which is used in the step of purifying a surface antigen protein from an influenza virus using a surfactant, and the amount of surfactant used during the purification of the surface antigen protein is determined based on a total protein quantification assay for an influenza virus sample.

[0021] Another object of the present invention is to provide a method for preparing a vaccine, which comprises purifying a surface antigen protein from an influenza virus, wherein the step is carried out under conditions in which a total protein quantification value (TPQV) per unit volume of a sampled influenza virus is determined and then substituted into the following equation (1), and a surfactant is treated according to the value obtained:

[0022] Surfactant treatment amount = [{(a*TPQV(μg / mL)) / (b μg / mL)} / c]* d (1)

[0023] (In the formula, a is 0.005 to 0.100 (v / v%), b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and the value closest to the TPQV is selected; c is the concentration (v / v%) of the surfactant stock being treated; d is the volume of the sampled influenza virus treated with the detergent.

[0024] Another object of the present invention is to provide a method for rapid purification of influenza surface antigens, comprising the steps of:

[0025] a) infecting cells with influenza virus and culturing them to obtain a virus culture; b) determining purification conditions based on hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus samples obtained by purification under different conditions in order to purify influenza virus from the culture of step a); c) purifying influenza virus from the culture of step a) according to the conditions determined in step b); d) determining the amount of surfactant to be used in purifying the surface antigen protein based on a total protein quantification assay for influenza virus in order to purify the surface antigen from the influenza virus purified in step c); e) Purifying the surface antigen protein from the influenza virus by treating with a detergent according to the conditions determined in step d).

[0026] Another object of the present invention is to provide a method for producing a vaccine containing an influenza surface antigen, characterized in that the production period of the vaccine is shortened, comprising the steps of:

[0027] a) infecting cells with influenza virus and culturing them to obtain a virus culture; b) determining purification conditions based on hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus samples obtained by purification under different conditions in order to purify influenza virus from the culture of step a); c) purifying influenza virus from the culture of step a) according to the conditions determined in step b); d) determining the amount of surfactant to be used in purifying the surface antigen protein based on a total protein quantification assay for influenza virus in order to purify the surface antigen from the influenza virus purified in step c); e) Purifying the surface antigen protein from the influenza virus by treating with a detergent according to the conditions determined in step d).

[0028] Another object of the present invention is to provide a method for producing a bioreactor comprising the steps of: (a) culturing cells in a single-use bioreactor (SUB); (b) replacing a portion of the cell culture medium in step (a) with fresh medium using a continuous low-speed centrifuge with a disposable bag; (c) infecting the expanded cells with influenza virus and culturing them under conditions permissive for influenza virus replication; and (d) A method for producing influenza virus, comprising the step of isolating influenza virus from the culture of step (c).

[0029] Another object of the present invention is to provide a method for producing a vaccine containing an influenza antigen, characterized in that the production period of the vaccine is shortened, comprising the steps of:

[0030] (a) culturing cells in a single-use bioreactor (SUB); (b) replacing a portion of the medium in the cell culture medium of step (a) with fresh medium using a continuous low-speed centrifuge; (c) infecting the grown cells with influenza virus and culturing them under conditions that allow influenza virus replication; and (d) isolating influenza virus from the culture of step (c). [Means for solving the problem]

[0031] In order to achieve the above-mentioned object, the present invention provides a method for purifying a surface antigen protein from an influenza virus using a detergent, characterized in that the amount of detergent to be treated with the sampled influenza virus is determined by calculating the total protein quantification value (TPQV) per unit volume of the sampled influenza virus and then substituting the value into the following formula (1):

[0032] Surfactant treatment amount = [{(a*TPQV(μg / mL)) / (b μg / mL)} / c]* d (1)

[0033] (In the formula, a is 0.005 to 0.100 (v / v%), b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and the value closest to the TPQV is selected; c is the concentration (v / v%) of the surfactant stock being treated; d is the volume of the sampled influenza virus treated with the detergent.

[0034] In order to achieve another object of the present invention, the present invention provides a method for rapidly confirming influenza antigen purification conditions, comprising the following first and second purification condition determination methods:

[0035] A first purification condition determination method used in the step of purifying influenza virus from an influenza virus culture, wherein the conditions for purifying the influenza virus are determined based on a hemagglutination assay, SDS-PAGE, or a combination thereof, for influenza virus samples obtained by purification under different conditions; and A second method for determining purification conditions, which is used in the step of purifying a surface antigen protein from an influenza virus using a surfactant, and the amount of surfactant used during the purification of the surface antigen protein is determined based on a total protein quantification assay for an influenza virus sample.

[0036] In order to achieve another object of the present invention, the present invention provides a method for producing a vaccine, which comprises a step of purifying a surface antigen protein from an influenza virus, wherein the step is carried out under conditions in which a total protein quantification value (TPQV) per unit volume of a sampled influenza virus is determined and then substituted into the following equation (1), to obtain a value, and the method treats the sample with a surfactant according to the value obtained:

[0037] Surfactant treatment amount = [{(a*TPQV(μg / mL)) / (b μg / mL)} / c]* d (1)

[0038] (In the formula, a is 0.005 to 0.100 (v / v%), b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and the value closest to the TPQV is selected; c is the concentration (v / v%) of the surfactant stock to be treated, and d is the volume of the sampled influenza virus to be treated with the surfactant.

[0039] In order to achieve another object of the present invention, the present invention provides a method for rapid purification of influenza surface antigens, comprising the steps of:

[0040] a) infecting cells with influenza virus and culturing them to obtain a virus culture; b) determining purification conditions based on hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus samples obtained by purification under different conditions in order to purify influenza virus from the culture of step a); c) purifying influenza virus from the culture of step a) according to the conditions determined in step b); d) determining the amount of surfactant to be used in purifying the surface antigen protein based on a total protein quantification assay for influenza virus in order to purify the surface antigen from the influenza virus purified in step c); e) Purifying the surface antigen protein from the influenza virus by treating with a detergent according to the conditions determined in step d).

[0041] In order to achieve another object of the present invention, the present invention provides a method for producing a vaccine containing an influenza surface antigen, which is characterized by shortening the production period of the vaccine, and includes the following steps:

[0042] a) infecting cells with influenza virus and culturing them to obtain a virus culture; b) determining purification conditions based on hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus samples obtained by purification under different conditions in order to purify influenza virus from the culture of step a); c) purifying influenza virus from the culture of step a) according to the conditions determined in step b); d) determining the amount of surfactant to be used in purifying the surface antigen protein based on a total protein quantification assay for influenza virus in order to purify the surface antigen from the influenza virus purified in step c); e) Purifying the surface antigen protein from the influenza virus by treating with a detergent according to the conditions determined in step d).

[0043] To achieve another object of the present invention, the present invention provides a method for producing influenza virus, comprising the steps of: (a) culturing cells in a single-use bioreactor (SUB); (b) replacing a portion of the medium in the cell culture solution of step (a) with fresh medium using a continuous low-speed centrifuge with a disposable bag; (c) infecting the grown cells with influenza virus and culturing them under conditions that allow influenza virus replication; and (d) isolating the influenza virus from the culture of step (c).

[0044] In order to achieve another object of the present invention, the present invention provides a method for producing a vaccine containing an influenza antigen, which is characterized by shortening the production period of the vaccine, and includes the following steps:

[0045] (a) culturing cells in a single-use bioreactor (SUB); (b) replacing a portion of the medium in the cell culture medium of step (a) with fresh medium using a continuous low-speed centrifuge; (c) infecting the grown cells with influenza virus and culturing them under conditions that allow influenza virus replication; and (d) isolating influenza virus from the culture of step (c).

[0046] The present invention will be described in detail below.

[0047] The present invention provides a method for purifying a surface antigen protein from an influenza virus using a detergent, characterized in that the amount of the detergent to be applied to the sampled influenza virus is determined by calculating the total protein quantification value (TPQV) per unit volume of the sampled influenza virus and then substituting the value into the following formula (1):

[0048] Surfactant treatment amount = [{(a*TPQV(μg / mL)) / (b μg / mL)} / c]* d (1)

[0049] (In the formula, a is 0.005 to 0.100 (v / v%), b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and the value closest to the TPQV is selected; c is the concentration (v / v%) of the surfactant stock being treated; d is the volume of the sampled influenza virus treated with the detergent.

[0050] The structure of the influenza virus virion is an envelope consisting of a double fatty layer on the outermost surface, from which two surface glycoproteins protrude: one is hemagglutinin (HA), a cylindrical hemagglutinin, and the other is neuraminidase (NA), a mushroom-shaped enzyme that breaks down neuramin.

[0051] The method provided by the present invention is a method for separating and purifying the influenza virus surface antigens, hemagglutinin and neuraminidase, by treating with a surfactant, and is characterized in that it enables the establishment of purification conditions for influenza virus surface antigens in a short period of time by quickly calculating the optimal treatment amount of the surfactant used to separate the influenza virus surface antigens from the virus core.

[0052] In the present invention, the term "surfactant" is used interchangeably with "detergent," and the surfactant is not limited to any particular type as long as it is one used in the art for separating and purifying viral surface antigens, and may include, for example, nonionic or ionic (e.g., cationic) surfactants. Non-limiting examples of surfactants may include alkyl glycosides, alkyl thioglycosides, acyl, sugars, sulfobetaine, betaine, polyoxyethylene alkyl ethers, N,N-dialkyl-glucamides, Hecameg, alkylphenoxy-polyethoxyethanols, quaternary ammonium compounds, sarkosyl, CTAB (cetyltrimethylammonium bromide), tri-N-butylphosphate, Cetavlon, myristyltrimethylammonium salts, lipofectin, lipofectamine, and DOTMA, octyl- or nonyl-phenoxypolyoxyethanols (e.g., Triton surfactants such as Triton X-100 or Triton N101), polyoxyethylene sorbitan esters (Tween surfactants), polyoxyethylene ethers, and polyoxyethylene esters.

[0053] In a preferred embodiment of the present invention, the surfactant may be a cationic surfactant.

[0054] In a further preferred embodiment of the present invention, the surfactant may be CTAB.

[0055] The influenza viruses of the present invention include types A, B, and C according to their molecular biological characteristics. Preferably, the influenza viruses may be type A or B. Among these, type A influenza viruses can be divided into various subtypes according to the types of hemagglutinin (HA) and neuraminidase (NA) glycoproteins that serve as surface antigens. More specifically, 18 types of hemagglutinin and 11 types of neuraminidase are known as surface antigens of influenza A viruses, and therefore, mathematically, a total of 198 types of influenza A virus subtypes can exist, and it can be understood that all of these are included in the influenza viruses of the present invention.

[0056] Meanwhile, the influenza viruses of the present invention may include, without limitation, influenza viruses of not only currently known subtypes but also new subtypes that may be identified in the future, including antigenic shifts in which the major antigens, HA and NA, change to new subtypes that are completely different from those previously known, or antigenic drift in which point mutations accumulate at the level of the HA and NA genes, resulting in changes to a small number of amino acids.

[0057] In a preferred embodiment of the present invention, the influenza virus may be influenza A, non-limiting examples of which may include H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, H6N1, etc.

[0058] In the present invention, the "sampled" influenza virus means that the virus has undergone one or more steps selected from the group consisting of culturing influenza viruses expressing surface antigen proteins in host cells to provide a culture, purifying and / or concentrating the culture, and inactivating the influenza viruses contained in the culture.

[0059] In one embodiment of the invention, the sampling means that the influenza virus has been treated by a method comprising the steps of:

[0060] (i) providing a cell-based viral growth medium or a fertilized egg-based viral growth medium in which influenza virus can grow; (ii) clarifying the culture medium to obtain a culture containing influenza virus; (iii) optionally purifying and optionally enriching said culture; and (iv) optionally inactivating influenza viruses contained in said culture.

[0061] In step (i), a culture medium containing influenza virus is provided. Such a culture medium can be derived from a cell-based virus growth medium or a fertilized egg-based virus growth medium. As known to those skilled in the art, viruses can be grown in cell-based culture systems or fertilized egg-based culture systems. Suitable cell lines used to grow influenza virus are typically of mammalian origin and therefore refer to mammalian cell lines, preferably animal cell lines. Suitable animal cell lines are known to those skilled in the art and may include cell lines of hamster, bovine, primate (including human and monkey), and canine origin. Non-limiting examples of such cell lines include Vero, PerC6, BHK, 293, COS, PCK, MRC-5, MDCK, MDBK, WI-38, and serum-free adapted versions thereof. Preferably, the cell line may be an MDCK cell.

[0062] The influenza virus can also be grown on cells by any suitable method known to those skilled in the art. For cell-based culture systems, any suitable medium can be used. In a preferred embodiment, cells can be cultured in serum-free and / or protein-free medium. For cell-based virus propagation, the virus or live virus preparation, respectively, is generally inoculated into the cell culture medium in which it is grown. Virus propagation can be performed in a batch, fed-batch, or perfusion mode.

[0063] Alternatively, the influenza virus can be propagated in a fertilized egg-based system. In the production of a fertilized egg-based system, a working seed virus is typically injected into embryonated eggs from laying hens. The virus then begins to grow in the allantoic fluid of the embryonated eggs, increasing the amount of virus. For example, after incubation for 2-3 days, the embryonated eggs are cooled to 2-8°C, and the allantoic fluid from each embryonated egg is harvested and pooled using a manual or automated vacuum harvesting system. The collected virus-containing allantoic fluid is a fertilized egg-based culture medium containing the virus, and is further processed in a subsequent purification process.

[0064] Depending on the culture system used to grow influenza viruses, the culture medium containing the grown virus particles may contain different contaminants. For example, potential contaminants present in fertilized egg-based culture medium are fertilized egg-based proteins, such as fertilized egg albumin, or antibiotics, while a typical contaminant present in cell-based culture medium is residual host cell DNA. Preferably, the method provided by the present invention is not limited by the system used to grow influenza viruses; it may be applied to both cell-based culture systems and culture medium containing viruses grown in fertilized egg-based culture systems.

[0065] In the present invention, the culture medium in step (i) may be produced using a disposable bioreactor. By using such a disposable bioreactor, for example, the obtained product itself may be improved in terms of quality and / or purity. This may be beneficial, particularly when the method is carried out on a large scale, for example, on an industrial scale.

[0066] The cell-based or egg-based culture medium containing the influenza virus may be clarified (step (ii)). The clarification step is performed on the culture medium containing virus particles, but not on sedimented or other pelleted material. In the present invention, the purpose of such a clarification step is to remove particulate matter, such as cell debris, protein impurities, and / or support materials used to grow the cells, such as microcarriers, from the culture medium. The protein impurities may be, for example, allantoin, collagen, and / or albumin, such as ovalbumin. For clarification, any suitable means or method known to those skilled in the art may be used. In particular, tangential flow filtration (TFF), depth filtration, or centrifugation may be applied. Thus, the respective settings of each applied clarification means (TFF, depth filtration, centrifugation) are selected in such a way that the above-mentioned objective is achieved. Generally, when such an objective is given, the respective settings for each means are known to those skilled in the art.

[0067] In the present invention, the clarified culture medium obtained by the above method is referred to as a "virus culture."

[0068] In one embodiment of the present invention, the virus culture may be the "sampled influenza virus" provided by the present invention.

[0069] In another embodiment of the present invention, the "sampled influenza virus" may be one that has been subjected to the following step (iii) and / or step (iv) after step (ii).

[0070] In one embodiment of the present invention, the (iii) stage culture may be purified by any method suitable for specifically purifying a viral antigen. Preferred purification methods include, for example, chromatography, such as ion exchange chromatography, hydrophobic interaction chromatography, pseudo-affinity chromatography, affinity chromatography, size exclusion chromatography, and liquid-liquid chromatography. In one embodiment, a preferred purification method is affinity chromatography, and a more preferred purification method is affinity chromatography using cellufine sulfate (CS) as a carrier.

[0071] In one embodiment of the present invention, when CS affinity chromatography is used to purify the culture in step (iii), the selection of column dimensions may be determined by a skilled technician. In the CS affinity chromatography, the CS column may be equilibrated with an equilibration buffer to load the influenza virus filtrate. In one embodiment of the present invention, the CS column is equilibrated with an equilibration buffer, which may contain 10 to 20 mM sodium phosphate and have a pH of 7.0 to 7.6.

[0072] In the purification process, the influenza virus attached to the CS column can be eluted with an elution buffer. In one embodiment of the present invention, the influenza virus attached to the CS column can be eluted when an elution buffer containing 0.01 to 1.0 M sodium chloride at a neutral pH is applied to the column. The pH of the elution buffer may be 6.5 to 7.4, and the buffer may contain 0.1 to 6.0 M sodium chloride.

[0073] In addition, to wash away any proteins bound to the CS column during the purification process, the column can be washed with an elution buffer containing 1.0 to 3.0 M sodium chloride. The used CS column can be optionally washed, stored in an appropriate agent, or reused.

[0074] In one embodiment of the present invention, the concentration in step (iii) is achieved by a suitable method that involves reducing the volume of the virus culture, with the goal of reducing the volume of the culture containing influenza virus to obtain a concentrated culture.

[0075] In a preferred embodiment, the volume can be reduced by applying TFF ultrafiltration or TFF ultrafiltration and diafiltration in a single step (TFF UF / DF), and ultracentrifugation or sedimentation, preferably TFF ultrafiltration, can be used to reduce the volume. Thus, the respective settings of the applied concentration means (TFF, TFF UF / DF, ultracentrifugation, or sedimentation) are selected in such a way that the above-mentioned objectives, i.e., volume reduction and influenza virus concentration, are achieved.

[0076] In other embodiments, when ultracentrifugation is used to reduce the volume, the ultracentrifugation conditions may be, for example, 30,000 g to 200,000 g for more than 10 minutes.

[0077] In another embodiment, when concentration is performed by applying precipitation, suitable chemicals are known to those skilled in the art and may be, for example, salt, methyl and ethyl alcohol, and polyethylene glycol, each at an appropriate concentration. Suitable chemicals at an appropriate concentration precipitate the product, e.g., viral antigen-containing particles, but have no or minimal negative effect on the viral antigen. Chemicals are selected so as not to react with the target antigen, e.g., influenza virus surface antigen protein, and not to alter the immunogenicity of the desired antigen.

[0078] In one embodiment, the volume of the virus culture can be reduced by 4 times or more, preferably 5 times or more, or 9 times or more, or preferably 12 times or more, or preferably 13 times or more, or 15 times or more, or preferably 20 times or more, or 25 times or more during the concentration step.

[0079] In one embodiment of the present invention, the virus culture purified and / or concentrated after step (iii) may be inactivated. Inactivation results in the removal of virus infectivity and may typically be carried out by treating the virus with an effective amount of one or more of the following chemical means: detergent, formaldehyde, β-propiolactone, methylene blue, psoralen, carboxyfullerene (C60), secondary ethylamine, acetylethyleneimine, or a combination thereof. Inactivation methods are also known to those skilled in the art and include, for example, treating the virus with physical means, such as gamma irradiation and / or UV light.

[0080] In one embodiment of the present invention, inactivation by application of the same detergent used to isolate the surface antigens of the influenza virus may not be performed.

[0081] In another embodiment of the present invention, the sampled influenza virus may be obtained by a method comprising the steps of:

[0082] (a) culturing cells in a single-use bioreactor (SUB); (b) replacing a portion of the culture medium from step (a) with fresh medium using a continuous low-speed centrifuge with disposable bags; (c) infecting the grown cells with influenza virus and culturing them under conditions that allow influenza virus replication; and (d) isolating influenza virus from the culture from step (c).

[0083] The steps (a) to (d) will be described in more detail as follows:

[0084] (a) culturing cells in a single-use bioreactor (SUB); In the present invention, step (a) is a step of culturing host cells for virus infection and proliferation, characterized in that the cells are cultured in a disposable bioreactor. In one embodiment, step (a) uses a bioreactor system including disposable components, such as flexible plastic bags for cell culture. Such bioreactor systems are well known in the art and are commercially available. See, for example, International Patent Publications WO 05 / 108546; WO 05 / 104706; and WO 05 / 10849 and Section 8.12 below. Bioreactor systems including disposable components (referred to herein as "single-use bioreactors" or "SUBs") may be pre-sterilized, eliminating the need for steam-in-place (SIP) or clean-in-place (CIP) for batch-to-batch or product-to-product conversion in a culture or production system. The SUB can be rapidly deployed with minimal or no preparation prior to use to facilitate the production of large quantities of vaccine material from cell cultures, ensuring zero contamination between batches and requiring less regulatory control. Therefore, it can be operated with significant cost and time advantages. In another aspect, the disposable bioreactor system can be a stirred tank reactor system, which can provide a hydrodynamic environment for mixing of the cell cultures, allowing for more efficient control of nutrients, O2, and pH.

[0085] In one aspect of the present invention, the cells may be used in the present invention without limitation, as long as they can be infected with a virus and cultured to achieve the purpose of viral propagation. In particular, suitable cells used to grow influenza viruses are typically of mammalian origin. Suitable animal cells are known to those skilled in the art and may include cell lines of hamster, bovine, primate (including human and monkey), and canine origin. Non-limiting examples of such cell lines include mammalian cells such as Vero, PerC6, BHK, 293, COS, PCK, MRC-5, MDCK, MDBK, WI-38, and serum-free adapted versions of these. Preferably, the cell line may be an MDCK cell.

[0086] The MDCK cells may be derived from, for example, the ATCC CCL-34 cell line, ATCC PTA-7909, or PTA-7910. In one exemplary embodiment, MDCK cell lines such as the ATCC CCL-34-derived MDCK Sky1023 (DSM ACC3112), MDCK Sky10234 (DSM ACC3114), or MDCK Sky3851 (DSM ACC3113) cell lines disclosed in Korean Patent Publication No. 10-2012-0024464, or the MDCKS-MG cell line (KCLRF-BP-00297) disclosed in Korean Patent Registration No. 10-1370512, may be used. Particularly preferred are MDCK Sky3851 cells, which do not require serum for cell growth, can be cultured in suspension without the need for a separate carrier for attachment, and have low tumorigenicity.

[0087] In a preferred embodiment, the medium for culturing the cells may be serum-free. Animal serum may contain other pathogenic substances, which may be potentially transmitted to humans or animals treated or vaccinated with the cell culture-produced vaccine, and may be fatal, especially to humans with weakened immune systems. The serum-free culture medium may be a well-known cell culture medium, such as MEM media (Eagle's minimal essential media) and Dulbecco's modified Eagle's medium (DMEM), or may be derived from or modified from such cell culture media. Commercially available culture media, such as VP-SFM manufactured by Gibco BRL / Life Technology, may also be used. Cell subculture may be induced using culture media derived from such culture media.

[0088] The medium may contain, for example, a plant hydrolysate obtained from one or more of corn, pea, soybean, malt, potato, and wheat; a lipid supplement such as cholesterol and saturated and / or unsaturated fatty acids; trace elements such as CuSO4·5H2O, ZnSO4·7H2O, iron citrate, etc.; one or more hormones such as growth factors; putrescine, amino acids, vitamins, fatty acids such as unsaturated fatty acids, nucleosides, sodium bicarbonate, a carbon source such as glucose, an iron-binding substance, etc. The amounts of these ingredients that can be added to the cell culture medium are well known in the art.

[0089] In one aspect of the invention, the cells may be cultured at a CO2 concentration of 1% or more, or 2% or more, or 3% or more, or 4% or more, or 5% or more, or 6% or more, or 7% or more, or 8% or more, or 9% or more, or 10% or more, or 20% or more.

[0090] In one aspect of the present invention, the dissolved oxygen (DO) concentration (pO2 value) in the disposable bioreactor is advantageously adjusted during the cultivation of the cells to a range of 5% to 95% (based on air saturation), or 10% to 60%. In certain embodiments, the dissolved oxygen (DO) concentration (pO2 value) may be 10% or more, 20% or more, 30% or more, or 50% or more.

[0091] In one aspect of the present invention, the pH of the culture medium used to culture the cells is adjusted during culture and may be in the range of pH 6.4 to pH 8.0, or in the range of pH 6.8 to pH 7.6. In certain embodiments, the pH of the culture medium may be 6.4 or higher, or 6.6 or higher, or 6.8 or higher, or 7.0 or higher, or 7.2 or higher.

[0092] In one aspect of the present invention, the cells may be cultured at a temperature of 25° C. to 39° C. In certain embodiments, the culture temperature may be 30° C. to 38° C., or 35° C. to 38° C., or 36° C. to 38° C.

[0093] In one embodiment of the present invention, the cells may be cultured in a stirred tank SUB while monitoring and / or controlling one or more parameters selected from the group consisting of temperature, agitation rate, pH, dissolved oxygen content (DO), O2, and CO2 flow rate. In one embodiment, the temperature is maintained at about 30°C to about 42°C, or about 33°C to about 39°C, or about 35°C to about 38°C. In a specific embodiment, the temperature is maintained at about 36°C to about 37°C. In one embodiment, the agitation rate is maintained at about 50 to 150 rpm. In a specific embodiment, the agitation rate is maintained at about 60 to about 120 rpm, or about 70 to about 100 rpm. The agitation rate is controlled by methods well known in the art. In yet another embodiment, the pH of the culture is maintained at about 6.0 to about 7.5. In a specific embodiment, the pH at the start of cultivation is about 6.0 to about 7.5, and the pH of the culture is maintained at about 7.0 to about 7.5 during cultivation. Those skilled in the art will recognize that the initial pH may be lower or higher than the preferred range, and that such pH can be increased or decreased to a desired level (e.g., 7.4) and maintained at that level. Such pH is maintained by any method known in the art. For example, the pH may be adjusted as needed by sparging with CO2 and / or adding an acid (e.g., HCl) or a base (e.g., NaOH). In yet another embodiment, an acceptable DO range is about 100% to about 35%. In certain embodiments, the DO is maintained at about 35% to about 70%, or about 50%. In yet other specific embodiments, the DO should not be lowered below about 35%. Those skilled in the art will recognize that the initial DO may be 100%, and that such DO can be decreased to a predetermined concentration (e.g., 50%) and maintained at this level. Such DO may be maintained by any method known in the art, for example, by sparging with O2.

[0094] (b) replacing a portion of the medium in the cell culture solution of step (a) with fresh medium using a continuous low-speed centrifuge with a disposable bag; The step (b) is a step of exchanging the medium before the cells cultured in the step (a) have grown sufficiently and become infected with a virus.

[0095] In one embodiment of the present invention, the medium exchange in step (b) is performed by continuously separating the cells and the medium using a continuous low-speed centrifuge with a disposable bag, without the need for a culture medium recovery and re-addition process, discarding a certain amount of the medium, and then introducing new medium into the disposable bag. Unlike the general method of recovering the entire amount of culture medium, dividing it into small portions into containers, and then centrifuging it, when a continuous low-speed centrifuge with a disposable bag is used, the medium exchange can be performed in a sealed system, significantly reducing the possibility of contamination.

[0096] In one embodiment of the present invention, the "part of the culture medium" may be 50% to 90% by volume of the total culture medium present in the disposable bag, preferably 60% to 80% by volume, and most preferably 70% to 80% by volume of the total culture medium present in the disposable bag.

[0097] In one embodiment, the medium is replaced with a medium of the same volume and composition. Specifically, when cells are sedimented using a continuous low-speed centrifuge, a portion of the upper medium is collected, and then a volume of medium equal to the collected medium is replenished to perform the medium exchange. In yet another embodiment, the medium is replaced with a reduced volume of medium, effectively concentrating the cells. The medium can be replaced with a medium having the same or a different composition. In one embodiment, the growth medium used for cell growth in step (a) may be replaced with the infection medium (i.e., the medium used during infection and viral replication) used in step (c) below. Optionally, the growth medium is supplemented with or contains additional components (e.g., glucose, trace minerals, amino acids, etc.) so that medium exchange is not necessary. In yet another specific embodiment, the infection medium contains a serine protease (e.g., trypsin, TrypLE, etc.). In other embodiments, where the medium is not changed, a serine protease (e.g., trypsin, TrypLE, etc.) is added immediately before, during, or immediately after infection. In certain embodiments, the protease is added prior to or simultaneously with infecting the cells with the virus.

[0098] (c) infecting the grown cells with influenza virus and culturing them under conditions that allow influenza virus replication; in one embodiment, the influenza virus may be administered to the culture medium at an MOI (multiplicity of infection) of approximately 0.00001 to 0.01, preferably 0.0001 to 0.002. This dosage is sufficient to infect the cells inoculated into the culture medium, and a desired amount of influenza virus proliferation can be expected.

[0099] In one embodiment, after infection, the cells are cultured at a temperature between 22°C and 40°C. In certain embodiments, after infection with the virus, the cells are cultured at a temperature of 39°C or below, or 38°C or below, or 37°C or below, or 36°C or below, or 35°C or below, or 34°C or below, or 33°C or below, or 32°C or below, or 30°C or below, or 28°C or below, or 26°C or below, or 24°C or below. In certain embodiments, after infection with the virus, the cells are cultured at a temperature of 33°C. In yet other embodiments, after infection with the virus, the cells are cultured at a temperature of 33°C or below. In yet other embodiments, after infection, the cells are cultured at a temperature of 31°C. In certain embodiments, the cell culture is carried out for 2 to 10 days. The culture may be carried out in a batch process.

[0100] In one embodiment of the present invention, the culturing in step (c) is carried out for a period sufficient to produce a virus at an appropriate yield, for example, 2 to 10 days after infection, or optionally, 3 to 7 days after infection. In another embodiment of the present invention, the culturing in step (c) is carried out for 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after infection.

[0101] (d) isolating influenza viruses from the culture obtained in step (c); step (d) is a step of purifying and isolating influenza viruses from the virus culture obtained in step (c).

[0102] The above explanation may be similarly applied to the method for purifying influenza virus in step (d).

[0103] In one embodiment of the present invention, step (d) may be performed by a method including: (d-1) determining purification conditions based on a hemagglutination assay for influenza virus samples obtained by purification under different conditions in order to purify influenza virus from the culture of step (c); and (d-2) purifying influenza virus from the culture of step (c) according to the conditions determined in step (d-1).

[0104] The step (d-1) may be characterized by applying the influenza virus culture to a chromatography column, and then subjecting the column filtrates passed through the column under different conditions to a hemagglutination assay to select the conditions with the lowest hemagglutinin (HA) titer. If the HA titer of the virus culture passed through the column is high as a result of the hemagglutination assay, the virus in the virus culture cannot bind to the column and is not a suitable condition for passing through the column.

[0105] In another aspect of the present invention, step (d-1) may be characterized by applying the influenza virus culture to a chromatography column under different conditions, eluting the influenza virus bound to the column using a buffer solution, and applying the eluates obtained under each condition to a hemagglutination assay, SDS-PAGE, or a combination thereof, and selecting the conditions under which the HA titer is highest and HA is clearly visible on SDS-PAGE.

[0106] In another aspect of the present invention, step (d-1) may be characterized by a combination of conditions under which the virus best binds to the resin when the virus culture is passed through the column and conditions under which the virus best elutes during the process of eluting the virus bound to the resin. The conditions under which the virus best binds to the resin may be selected by subjecting the column effluent to a hemagglutination assay, SDS-PAGE, or a combination thereof, and the conditions under which the virus best elutes during the process of eluting the virus bound to the resin may be selected by subjecting the column effluent to a hemagglutination assay, SDS-PAGE, or a combination thereof.

[0107] In another aspect of the present invention, step (d-1) may be characterized by applying the virus cultures passed through the chromatography column under different conditions to a hemagglutination assay and SDS-PAGE to select the conditions that result in the least loss of influenza virus.

[0108] In one embodiment of the present invention, a step of concentrating the purified virus culture may be additionally performed after step (d). The above-mentioned explanations can be similarly applied to the method of concentrating the purified virus culture.

[0109] Thereafter, the total protein quantitative value (TPQV) per unit volume of the sampled influenza virus culture is determined, and the resulting value is substituted into the following formula (1), and the amount of surfactant to be treated on the sampled influenza virus is determined based on the obtained value:

[0110] Surfactant treatment amount = [{(a*TPQV(μg / mL)) / (b μg / mL)} / c]* d (1)

[0111] (In the formula, a is 0.005 to 0.100 (v / v%), b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and the value closest to the TPQV is selected; c is the concentration (v / v%) of the surfactant stock being treated; d is the volume of the sampled influenza virus treated with the detergent.

[0112] Viral surface antigen vaccines are high-quality vaccines with few side effects and high purity, so it is very important to set the appropriate amount of detergent to be used. If an excessive amount of detergent is used, the entire virus is disrupted, exposing impurities such as proteins inside the viral core to the outside, reducing purity. If the amount of detergent is insufficient, most of the surface antigens are not solubilized, reducing the yield of surface antigens.

[0113] Therefore, in one aspect of the present invention, the amount of surfactant used for treatment calculated by the above formula is characterized by being a concentration that selectively solubilizes only the surface antigens of influenza viruses while leaving the virus core intact or minimizing destruction of the virus core.

[0114] Meanwhile, determining the appropriate amount of surfactant to solubilize viral surface antigens within a short time is an important factor in influenza vaccine production. Currently, the single radial immunodiffusion (SRID) method, recommended by the European Pharmacopoeia and the World Health Organization (WHO), is commonly used both domestically and internationally to measure the surface antigen content of influenza vaccines. However, the SRID method has the limitation of requiring a long time to calculate results.

[0115] Therefore, the present invention provides a method that does not require a quantitative process using SRID to quantify the amount of hemagglutinin (HA) protein in the virus culture, and is characterized by determining the amount of surfactant to be added based on the quantitative value of total protein per unit volume of the virus culture.

[0116] In the method of the present invention, the total protein quantitative value (TPQV) per unit volume of the virus culture is determined by any method used in the art to calculate protein content, non-limiting examples of which may include the BCA assay, the Lowry assay, or the Bradford assay.

[0117] In the formula (1) of the present invention, the value a means the surfactant treatment concentration (%) based on the protein content, and since the optimal surfactant treatment concentration varies depending on the virus strain, virus characteristics, etc., SDS-PAGE can be performed at any value within the range of 0.005 to 0.100 (v / v%) to confirm the appropriate surfactant treatment concentration (%) for the virus. Specifically, the virus solution can be aliquoted and treated with a surfactant at any value within the above range, followed by ultra-high speed centrifugation to confirm the optimal surfactant treatment concentration at which the surface antigen protein is recovered as a supernatant and the viral core protein is separated as a precipitate.

[0118] In the present invention, the value is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and the value closest to the TPQV is selected. For example, if the TPQV of the sampled influenza virus is 220 μg / mL, the closest value among these values, 200, will be the b value, and if the TPQV is 370 μg / mL, the closest value among these values, 400, will be the b value.

[0119] In the present invention, the TPQV may vary depending on the concentration level of the sampled influenza virus. Therefore, in order to determine the optimal surfactant treatment amount according to the formula (1) of the present invention, it is preferable to use an influenza virus culture concentrated so that the TPQV of the sampled influenza virus is 50 to 950 μg / mL.

[0120] The present invention also provides a method for rapidly verifying influenza antigen purification conditions, comprising the following first and second purification condition determination methods:

[0121] A first method for determining purification conditions, which is used in a step of purifying influenza virus from an influenza virus culture, and in which the conditions for purifying the influenza virus are determined based on a hemagglutination assay, SDS-PAGE, or a combination thereof, for influenza virus samples obtained by purification under different conditions; and A second method for determining purification conditions, which is used in the step of purifying a surface antigen protein from an influenza virus using a surfactant, and the amount of surfactant used during the purification of the surface antigen protein is determined based on a total protein quantification assay for an influenza virus sample.

[0122] In one embodiment of the present invention, the first purification corresponds to the viral culture purification step (c) described above.

[0123] In another embodiment of the present invention, the first purification does not require a quantification process by single radial immunodiffusion (SRID) for the amount of hemagglutinin (HA) protein in the virus culture.

[0124] In another aspect of the present invention, the step of purifying the influenza virus from the influenza virus culture may be performed by chromatography.

[0125] In another aspect of the present invention, the chromatography includes, for example, ion exchange chromatography, hydrophobic interaction chromatography, pseudo-affinity chromatography, affinity chromatography, size exclusion chromatography, liquid-liquid chromatography, etc. In one embodiment, the preferred purification method is affinity chromatography, and a more preferred purification method is affinity chromatography using cellufine sulfate (CS) as a carrier.

[0126] In another aspect of the present invention, the first purification step may be characterized by determining the conditions under which the influenza virus culture passing through the column can best bind to the resin, and then selecting elution conditions under which the bound virus can be separated and purified with optimal yield and purity.

[0127] In another aspect of the present invention, the first purification condition determination method may be characterized by diluting the influenza virus culture with an equilibration buffer at various ratios, applying the diluted solution to a chromatography column, and then subjecting the column effluents passed through the column under different conditions to a hemagglutination assay to determine the conditions with the lowest hemagglutinin (HA) titer. If the HA titer of the column effluent is high as a result of the hemagglutination assay, this is undesirable because the virus in the virus culture cannot bind to the column and passes through intact.

[0128] In another aspect of the present invention, the first purification condition determination method may be characterized by diluting the influenza virus culture with an equilibration buffer at various ratios, applying the diluted solution to a chromatography column under different conditions, eluting the influenza virus bound to the column with an elution buffer, and applying the eluate obtained under each condition to a hemagglutination assay, SDS-PAGE, or a combination thereof, and selecting the conditions that result in the highest HA titer and the thickest hemagglutinin band on SDS-PAGE.

[0129] In another aspect of the present invention, the first purification conditions can be characterized by a combination of conditions under which the virus binds best to the resin when a virus culture is passed through a column and conditions under which the virus can be eluted best during the process of eluting the virus bound to the resin. The conditions under which the virus binds best to the resin can be selected by subjecting the column effluent to a hemagglutination assay, SDS-PAGE, or a combination thereof, and the conditions under which the virus can be eluted best during the process of eluting the virus bound to the resin can be selected by subjecting the column effluent to a hemagglutination assay, SDS-PAGE, or a combination thereof.

[0130] In another aspect of the present invention, the first purification condition determination method may be characterized by applying a virus culture (i.e., column flow-through) passed through a chromatography column under different conditions to a hemagglutination assay, SDS-PAGE, or a combination thereof to select conditions that result in the least loss of influenza virus.

[0131] In another aspect of the present invention, the condition may be one or more selected from the group consisting of the type of buffer, the concentration of the buffer, the pH of the buffer, and the conductivity of the buffer. Preferably, the condition may be the pH or the conductivity of the buffer.

[0132] In one embodiment of the present invention, the pH of the buffer solution used in the first purification step was maintained between 7.0 and 7.4 to enhance protein stability, while the conductivity was reduced to explore the conditions for optimal virus binding to the resin. The conductivity of the influenza virus culture obtained during the influenza virus cultivation process was approximately 10.0 to 15.0 mS / cm, and this was diluted with a low-conductivity sodium phosphate buffer to lower the conductivity of the sample loaded onto the column and confirm its binding ability. To rapidly confirm purification conditions, the time and variables required for confirmation were reduced by adjusting only the conductivity, excluding the pH, which is one of the purification conditions.

[0133] It is generally known that the lower the conductivity, the more effectively influenza viruses bind to the resin. However, lowering the conductivity increases the volume of sodium phosphate buffer solution added and the chromatography process time, which increases production time and production costs, making it important to determine the appropriate conductivity. In one embodiment of the present invention, the conductivity was reduced to 10.0 mS / cm or less by diluting the virus culture solution 0.5 to 4 times with sodium phosphate buffer solution. To determine whether the binding capacity was appropriate, a hemagglutination assay was performed on the virus culture passing through the column. When performing the hemagglutination assay, test results were obtained within one hour, and purification results under each condition were confirmed within one day.

[0134] In another embodiment of the present invention, the elution of the influenza virus bound to the resin may be carried out by searching for the optimal conductivity using a buffer solution of an appropriate concentration that can separate the binding of the influenza virus to the resin.

[0135] According to one embodiment of the present invention, a 2-5M sodium chloride buffer solution with increasing salt concentration was applied to a column using a linear gradient. As a result, a UV280 peak was observed as viruses and proteins eluted. The fractions were then collected and analyzed to determine the conductivity and salt concentration of the virus-containing fractions. The virus was eluted using a step gradient of 2-5M sodium chloride buffer solution and equilibration buffer to determine whether the majority of influenza viruses eluted at these elution concentrations, and the virus elution conditions were established. To determine whether the virus was eluted with high yield and purity, a hemagglutination assay and / or SDS-PAGE were performed.

[0136] In one aspect of the present invention, the second purification condition determination method may be characterized in that the total protein quantification value (TPQV) per unit volume of the influenza virus culture obtained through the first purification is determined, and then the amount of surfactant to be treated with the virus solution is determined based on the value obtained by substituting the value into the following formula (1):

[0137] Surfactant treatment amount = [{(a*TPQV(μg / mL)) / (b μg / mL)} / c]* d (1)

[0138] (In the formula, a is 0.005 to 0.100 (v / v%), b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and the value closest to the TPQV is selected; c is the concentration (v / v%) of the surfactant stock being treated; d is the volume of virus solution treated with detergent.)

[0139] The present invention also provides a method for producing a vaccine, which includes a step of purifying a surface antigen protein from an influenza virus, wherein the step is carried out under conditions in which a total protein quantification value (TPQV) per unit volume of a sampled influenza virus is determined and then substituted into the following equation (1), and a surfactant is treated according to the value obtained:

[0140] Surfactant treatment amount = [{(a*TPQV(μg / mL)) / (b μg / mL)} / c]* d (1)

[0141] (In the formula, a is 0.005 to 0.100 (v / v%), b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and the value closest to the TPQV is selected; c is the concentration (v / v%) of the surfactant stock being treated; d is the volume of virus solution treated with detergent.)

[0142] The present invention also provides a method for rapid purification of influenza surface antigens, comprising the steps of:

[0143] a) infecting cells with influenza virus and culturing them to obtain a virus culture; b) determining purification conditions based on hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus samples purified under different conditions in order to purify influenza virus from the culture of step a); c) purifying influenza virus from the culture of step a) according to the conditions determined in step b); d) determining the amount of surfactant to be used in purifying the surface antigen protein from the influenza virus solution purified in step c) based on a total protein quantification assay for the influenza virus solution; e) treating the virus solution with a surfactant according to the conditions determined in step d) to purify surface antigen proteins from the influenza virus.

[0144] In one aspect of the present invention, the method for rapid purification of influenza surface antigens may further comprise, before step b), a step of removing cells and cell debris from the virus culture.

[0145] In another aspect of the present invention, the removal of cells and cell debris from the virus culture may be performed by one or more methods selected from the group consisting of filtration, dialysis, and centrifugation.

[0146] In another embodiment of the present invention, step b) may be characterized by determining purification conditions based on a hemagglutination assay and SDS-PAGE.

[0147] In another aspect of the present invention, the method for rapid purification of influenza surface antigens may be characterized by comprising, after step c), an additional purification step using one or more methods selected from the group consisting of ultrafiltration and diafiltration.

[0148] In another aspect of the present invention, step e) may be characterized by isolating the surface antigen protein by a method of centrifuging (preferably, ultracentrifugation) after detergent treatment and recovering the supernatant. The surface antigen protein separated from the viral core by the detergent treatment can be efficiently separated by centrifugation. Such separation may be performed by pelleting the viral core, for example, by ultracentrifugation. The viral surface protein, e.g., hemagglutinin and / or neuraminidase, may be present in the supernatant, while the viral core may be present in the pellet. The centrifugation conditions selected to pellet the viral core are well known to those skilled in the art. For example, ultracentrifugation may be performed in either a batch or continuous mode, and the ultracentrifugation conditions used may be at a suitable temperature, for example, 2 to 25°C, more preferably 2 to 8°C, at 30,000 g for more than 10 minutes (min.) to 200,000 g for more than 10 minutes.

[0149] In another aspect of the present invention, the rapid purification method for influenza surface antigens may further include a detergent removal step after step e). The detergent removal may be performed by any method suitable for this purpose, such as Amberlite or TFF treatment. When Amberlite treatment is used, Amberlite is added to the supernatant collected after pelleting the virus cores after step e), at a mass ratio of Amberlite to cationic surfactant of about 1:1 to 100:1, and the mixture is incubated at a suitable temperature, for example, within the range of 2 to 8°C, for a suitable time, for example, 8 to 24 hours, preferably 16 hours. Subsequently, the Amberlite may be removed by any suitable method known to those skilled in the art, such as conventional flow filtration / dead-end filtration, sieving, or TFF. When a filter is used, it is preferably selected to retain particles larger than 5 μm or larger than 7 μm. Alternatively, the Amberlite may be removed by sieving. Suitable sieve openings are in the range of 0.1 to 200 μm, preferably less than 150 μm, and more preferably less than 100 μm.

[0150] In one embodiment of the present invention, Amberlite XAD-4 (a macroreticular crosslinked aromatic polymer) was added to remove CTAB, which was used as a surfactant. CTAB has a large molecular weight and is ionic, so a direct adsorption method was used to remove it. The supernatant from ultra-high speed centrifugation was treated with Amberlite XAD-4 to adsorb CTAB, and the mixture was stirred and reacted for at least 3 hours under refrigerated conditions at 2-8°C. The mixture was then filtered through a 0.22 μm filter to remove the Amberlite XAD-4.

[0151] In another aspect of the present invention, the method for rapid purification of influenza surface antigens may include an additional chromatography step for removing impurities such as DNA after step e). The chromatography may include, for example, ion exchange chromatography, hydrophobic interaction chromatography, pseudo-affinity chromatography, affinity chromatography, size exclusion chromatography, liquid-liquid chromatography, etc., and preferably anion exchange chromatography, most preferably TMAE (trimethylaminoethyl) anion exchange chromatography. DNA carries an anion, and therefore can be easily separated and removed by utilizing its ability to bind to anion exchange resins.

[0152] In another embodiment of the present invention, the TMAE column can be equilibrated with a sodium phosphate buffer for equilibration, and the equilibration buffer can contain 1 to 20 mM sodium phosphate and have a pH of 6.8 to 7.6.

[0153] In one embodiment of the present invention, DNA was bound to a resin, and influenza surface antigens were recovered as a column flow-through solution. To do this, a sodium phosphate buffer solution containing 1-6 M sodium chloride and a pH of 6.5-7.4 was added to the influenza surface antigen process solution after the Amberlite XAD-4 adsorptive filtration process to adjust the conductivity. An appropriate level of surface antigen recovery was confirmed at a conductivity of 30-120 mS / cm.

[0154] In another aspect of the present invention, the method for rapid purification of influenza surface antigens may further comprise, after the chromatography for removing the impurities, an additional purification step using one or more methods selected from the group consisting of ultrafiltration and diafiltration.

[0155] The present invention also provides a method for producing a vaccine containing an influenza surface antigen, characterized in that the production period of the vaccine is shortened, comprising the steps of:

[0156] a) infecting cells with influenza virus and culturing them to obtain a virus culture; b) determining purification conditions based on hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus samples obtained by purification under different conditions in order to purify influenza virus from the culture of step a); c) purifying influenza virus from the culture of step a) according to the conditions determined in step b); d) determining the amount of surfactant to be used in purifying the surface antigen protein from the influenza virus solution purified in step c) based on a total protein quantification assay for the influenza virus solution; e) treating the virus solution with a surfactant according to the conditions determined in step d) to purify surface antigen proteins from the influenza virus.

[0157] In one embodiment of the present invention, the cells used for viral infection in step a) or the cells infected with a virus in step a) may be cultured in a single-use bioreactor (SUB). Disposable bioreactors eliminate the need for cleaning and sterilization before use, thereby reducing process preparation time and significantly reducing the risk of contamination. Furthermore, after use, a disposable bioreactor can be discarded without cleaning and replaced with a new one, preventing cross-contamination between lots or virus strains. In particular, influenza vaccines are trivalent or tetravalent vaccines, which require the cultivation of three or four different viruses. Therefore, using a disposable bioreactor is preferred because it greatly simplifies the process and prevents cross-contamination between strains.

[0158] In another aspect of the present invention, before infecting cells with a virus in step a), a portion of the cell culture medium used for virus infection may be replaced with fresh medium through a continuous low-speed centrifuge. This allows the medium to be replaced by continuously separating the cells from the medium, discarding the medium, and then introducing fresh medium into the bioreactor, without the need for a culture medium recovery and reintroduction process. Unlike the general method of recovering the entire medium, dividing it into small portions into containers, and then centrifuging it, the use of a continuous low-speed centrifuge has the advantage that the medium replacement can be performed in a closed system, significantly reducing the possibility of contamination.

[0159] In another aspect of the present invention, a portion of the medium may be characterized by discarding 50 to 90%, preferably 60 to 80%, and most preferably 70 to 80% of the existing medium, and injecting the same amount of fresh medium.

[0160] In another aspect of the present invention, the method for rapid purification of influenza surface antigens may further comprise a DNA cleaving enzyme treatment step after step c). Because DNA from cells used in influenza virus culture may be contaminated in the virus culture, a step of cleaving the DNA to lengths of 100 bp or less by treating with a DNA cleaving enzyme and removing it may be added. In the present invention, the DNA cleaving enzyme is not particularly limited, and may be benzonase, exonuclease, ribozyme, or a mixture thereof.

[0161] In another aspect of the present invention, the method for rapid purification of influenza surface antigens may further include a virus inactivation step after step c). The virus may be inactivated by treatment with a detergent, formaldehyde, β-propiolactone, methylene blue, psoralen, carboxyfullerene (C60), secondary ethylamine, acetylethyleneimine, or a combination thereof. Alternatively, the virus may be inactivated by physical means known to those skilled in the art, such as treatment with gamma irradiation and / or UV light.

[0162] The present invention also provides a method for producing influenza viruses, comprising the steps of:

[0163] (a) culturing cells in a single-use bioreactor (SUB); (b) replacing a portion of the cell culture medium in step (a) with fresh medium using a continuous low-speed centrifuge with a disposable bag; (c) infecting the expanded cells with influenza virus and culturing them under conditions permissive for influenza virus replication; and (d) isolating influenza virus from the culture of step (c).

[0164] In one aspect of the present invention, the cells may be characterized as being MDCK (Madin-Darby Canine Kidney) cells.

[0165] In another aspect of the present invention, step (b) may be characterized by replacing the medium by continuously separating the cells and the medium using a continuous low-speed centrifuge, discarding a portion of the medium, and introducing new medium into the cell incubator, without a process of recovering and re-introducing the entire amount of culture medium.

[0166] In another embodiment of the present invention, the portion of the medium may be 50% to 80% of the total culture solution.

[0167] The present invention also provides a method for producing a vaccine containing an influenza antigen, which is characterized by shortening the production period of the vaccine, comprising the steps of:

[0168] (a) culturing cells in a single-use bioreactor (SUB); (b) replacing a portion of the medium in the cell culture solution of step (a) with fresh medium using a continuous low-speed centrifuge; (c) infecting the expanded cells with influenza virus and culturing them under conditions permissive for influenza virus replication; and (d) isolating influenza virus from the culture of step (c).

[0169] In one aspect of the present invention, the step (d) may further include the following steps:

[0170] (d-1) determining purification conditions based on a hemagglutination assay for influenza virus samples obtained by purification under different conditions in order to purify influenza virus from the culture of step (c); and (d-2) Purifying influenza virus from the culture of step (c) according to the conditions determined in step (d-1).

[0171] In another aspect of the present invention, the method may further comprise, before step (d-1), a step of removing cells and cell debris from the virus culture.

[0172] In another aspect of the present invention, the removal of cells and cell debris from the virus culture may be carried out by one or more methods selected from the group consisting of filtration, dialysis, and centrifugation.

[0173] In another aspect of the present invention, (d-1) may be characterized by determining the concentration of influenza virus in culture samples purified under different conditions by hemagglutination assay, SDS-PAGE, or a combination thereof.

[0174] In another embodiment of the present invention, the method may include, after step (d-2), an additional purification step using one or more methods selected from the group consisting of ultrafiltration and diafiltration.

[0175] In another embodiment of the present invention, the method may further comprise a treatment step with Benzonase, Exonuclease, Ribozyme, or a mixture thereof after step (d).

[0176] In another embodiment of the present invention, the method may further comprise a virus inactivation step after step (d).

[0177] In another aspect of the present invention, the virus inactivation may be carried out by treatment with a detergent, formaldehyde, β-propiolactone, methylene blue, psoralen, carboxyfullerene (C60), secondary ethylamine, acetylethyleneimine, or a combination thereof.

[0178] In another aspect of the present invention, the method may further comprise the steps of:

[0179] (e) isolating surface antigen proteins from influenza viruses.

[0180] In another embodiment of the present invention, step (e) may involve separating the surface antigen protein by a method of treating with a surfactant, then centrifuging the mixture and recovering the supernatant.

[0181] In another embodiment of the present invention, step (e) may be carried out by including the following steps:

[0182] (e-1) determining the amount of surfactant to be used in purifying the surface antigen protein from the influenza virus purified in step (d) based on a total protein quantification assay for the influenza virus sample; (e-2) Purifying surface antigen proteins from the influenza virus of step (d) by treating with a detergent according to the conditions determined in step (e-1).

[0183] In another embodiment of the present invention, the method may further comprise a surfactant removal step after step (e).

[0184] In another aspect of the present invention, the step (e-1) may be characterized in that the total protein quantification value (TPQV) per unit volume of the influenza virus isolated in the step (d) is calculated and then substituted into the following formula (1) to determine the amount of surfactant to be treated: surfactant treatment amount=[{(a*TPQV(μg / mL)) / (b μg / mL)} / c]*d (1)

[0185] (In the formula, a is 0.005 to 0.100 (v / v%), b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and the value closest to the TPQV is selected; c is the concentration (v / v%) of the surfactant stock being treated; d is the volume of the sampled influenza virus treated with the detergent.

[0186] In another embodiment of the present invention, the method may include an additional chromatography step for removing impurities after step (e).

[0187] In another aspect of the present invention, the chromatography may be characterized as being TMAE (trimethylaminoethyl) anion exchange chromatography.

[0188] In another aspect of the invention, the method may include an additional purification step after chromatography by one or more methods selected from the group consisting of ultrafiltration and diafiltration. [Effects of the Invention]

[0189] The unique method of the present invention makes it possible to quickly and reliably confirm the conditions for obtaining (purifying) influenza surface antigens without using radial immunodiffusion, which has been used as a standard test method in the production of previous influenza vaccines. This is expected to significantly shorten the production period of influenza surface antigen subunit vaccines, and enable rapid vaccine development / production in the event of a sudden pandemic of a new strain of influenza.

[0190] Furthermore, according to the method for producing influenza virus of the present invention, cells are cultured using disposable bags, and in the virus infection step, the medium is replaced using a continuous low-speed centrifuge using disposable bags before the virus is infected, thereby minimizing the possibility of contamination and providing a culture process that can shorten the production period and maximize production cost savings. Unlike the general method in which the entire culture medium is collected and then divided into small containers for centrifugation, when a continuous low-speed centrifuge using disposable bags is used, the medium can be replaced in a closed system, significantly reducing the possibility of contamination. [Brief explanation of the drawings]

[0191] [Figure 1] The amount of CTAB to be applied to the virus (B / Maryland / 15 / 2016) was determined based on the protein content using the method of the present invention, and this was applied to an inactivated virus culture medium. After ultra-high speed centrifugation, the supernatant or the inactivated virus medium was analyzed by SDS-PAGE (HA: Hemagglutinin, NP: Nucleoprotein, M: Matrix protein). [Figure 2] To determine the optimal conditions for virus binding to the CS column resin, a linear gradient purification process was performed, followed by SDS-PAGE and an HA assay to confirm the conductivity conditions that minimize the loss of hemagglutinin in the column flow-through (IVR-190 virus, linear gradient purification - chromatogram). [Figure 3] To determine the optimal conditions for virus binding to the CS column resin, a linear gradient purification process was performed, followed by SDS-PAGE and an HA assay to confirm the conductivity conditions that minimize the loss of hemagglutinin in the column flow-through (IVR-190 virus, linear gradient chromatogram (enlarged eluate fraction)). [Figure 4] To determine the optimal conditions for virus binding to the CS column resin, a linear gradient purification process was performed, followed by SDS-PAGE and an HA assay to confirm the conductivity conditions that minimize the loss of hemagglutinin in the column flow-through (IVR-190 virus, linear gradient purification-SDS-PAGE results). [Figure 5] 1 shows the results of analyzing virus eluates under various conditions by performing the HA assay and SDS-PAGE used in the method of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0192] The present invention will be described in detail below.

[0193] However, the following examples are merely for the purpose of illustrating the present invention, and the content of the present invention is not limited to the following examples.

[0194] Example 1: Establishment of a new strategy for influenza virus culture using the disposable bag and continuous low-speed centrifuge unique to this invention 1)MDCK cell culture The culture method of the present invention provides a method for efficiently culturing MDCKSky-3851 cells using a disposable cell incubator that uses disposable bags.

[0195] One aspect of the present invention is a culture method comprising culturing MDCK cells using a spinner flask in an incubator at 37°C and 5% CO2. The agitation speed should be set at 40-150 rpm for a 125 mL spinner flask, and 80-150 rpm for a 500 mL or 1 L spinner flask. The culture medium used is a chemically defined serum-free medium supplemented with L-glutamine at a concentration of 3-6 mM as a nutrient necessary for cell growth. Culture using a spinner flask is carried out in a volume of 1 L or less.

[0196] In another embodiment of the present invention, the cell culture is performed in a cell incubator using a disposable bag after flask culture. The culture volume can be increased using cell incubators with a capacity of 10 L, 50 L, 200 L, or 2000 L. To facilitate cell culture and enable large-scale culture, culture conditions such as dissolved oxygen and pH are controlled and maintained. Dissolved oxygen and pH are controlled using gases (air, oxygen, carbon dioxide, nitrogen) and base (sodium bicarbonate) required for culture. In a specific aspect, the temperature, dissolved oxygen concentration, and pH during the culture stage are maintained at 37±3°C, DO 50±10%, and pH 7.2±0.2, respectively. The medium used for this culture contains 3-6 mM L-glutamine.

[0197] 2) Viral infection The present invention is intended to obtain a virus culture medium by infecting the cell culture medium obtained in the main culture step with influenza viruses. Once a sufficient number of cells have been obtained through the main culture, the culture medium is replaced before virus infection.

[0198] Before virus infection, the medium is replaced using a continuous low-speed centrifuge with disposable bags to continuously separate the cells and medium without the need for a culture medium recovery and re-addition process. A certain amount of medium (70%-80%) is then discarded and new medium is added to the cell incubator. Unlike the general method of recovering the entire culture medium, then dividing it into small containers for centrifugation, the use of a continuous low-speed centrifuge with disposable bags allows medium replacement to be carried out in a closed system, significantly reducing the possibility of contamination. Contamination that occurs during this process can waste the medium used in the culture stage and extend the process period, resulting in time and economic losses.

[0199] In the present invention, a culture process is established in which cells are cultured using a single-use bag and infected with a virus after medium replacement using a continuous low-speed centrifuge in the virus infection step, thereby minimizing the possibility of contamination and maximizing production time and production cost savings. This type of cell culture and virus infection process is referred to as the "virus cultivation process using a single-use bioreactor and low speed continuous centrifuge (SBCC process)."

[0200] The medium change and virus infection steps involve removing the medium that has become depleted of nutrients during the main culture and introducing new medium and virus from the outside, making them the steps with the highest risk of contamination. If contamination occurs during the medium change step, it will result in a loss of production time. However, if virus infection is carried out in a 10L cell incubator from the cell thawing step, where frozen MDCK-Sky3851 cells for vaccine production are thawed, it will take about 13 days to change the medium. In the case of commercial production of 2000L, it will take about 24 days to change the medium for virus infection. Additionally, production costs will increase due to the additional purchase of medium and disposable bags.

[0201] After medium exchange using the SBCC process, the virus stock for production is prepared. The virus in an amount calculated based on the determined multiplicity of infection (MOI) and 2.5 to 20 μg / mL of trypsin are aseptically introduced into a cell incubator to infect the cells. After culturing for about 2 to 4 days, when the virus titer, cell number, and viability reach appropriate levels, the virus culture is terminated and the culture medium is collected. In one embodiment of the present invention, 3 x 10 6 MDCK cells with a cell count of ≥ 1000 cells / ml were treated with virus at an MOI of 0.0001-0.01 and trypsin at concentrations of 1-20 μg / mL, and cultured for approximately 3 days.

[0202] Example 2: Establishment of a new strategy for rapid search of purification conditions during the purification process of surface antigens from influenza viruses During the process of purifying surface antigens from influenza viruses, a surfactant (ionic or nonionic) can be added to the influenza viruses and the surface antigen proteins can be separated and purified by fractionation such as centrifugation. For example, an ionic surfactant such as cetyltrimethylammonium bromide (CTAB) is commonly used.

[0203] The amount of surfactant used to treat influenza viruses should preferably be determined based on the content of hemagglutinin, the viral surface antigen. However, the currently commonly used standard HA content test, single radial immunodiffusion (SRID), takes approximately 2-3 days, making it unsuitable as a confirmation test during processing. In particular, if the supply of SRID standard samples from NIBSC or other institutions is unavailable or delayed, testing cannot be performed.

[0204] Meanwhile, during the surface antigen purification process from influenza viruses, the amount of surfactant required to selectively separate only the surface antigens hemagglutinin and neuraminidase without disrupting the viral core varies for each virus. Surface antigen vaccines are high-quality vaccines with minimal side effects and high purity, so it is extremely important to determine the appropriate amount of surfactant. Treatment with an excessive amount of surfactant disrupts the entire virus, exposing impurities such as proteins inside the viral core, resulting in a decrease in the purity of the surface antigen in the purified product. Insufficient surfactant treatment results in most of the surface antigen remaining in the pellet, resulting in a decrease in surface antigen yield. Furthermore, determining the appropriate amount of surfactant treatment within a short period of time is an important factor in the influenza vaccine production process.

[0205] The inventors first invented the method of the present invention, described below, as a result of various comparative experiments using various analytical techniques. They confirmed that the method not only allows for rapid establishment of surfactant treatment conditions suitable for surface antigen isolation without relying on the SRID method, but also has a significant correlation with existing standard SRID-based methods, demonstrating reliability and reproducibility. The following examples demonstrate that the unique effects of the method of the present invention are special effects that are difficult to predict from existing conventional processes. The following examples demonstrate an example using CTAB as a representative surfactant, which is abbreviated as CTCCT (CTAB treatment concentration confirmation test).

[0206] 2-1. Establishment of a new method for setting the purification process conditions for SRID-independent surface antigens The present inventors implemented an algorithm to calculate the amount of surfactant (e.g., CTAB) optimized for surface antigen purification using the total protein quantification value (TPQV) per unit volume of influenza virus sampled from an inactivated influenza virus concentrate, and derived the following equation (1): The total protein quantification value (total protein content) could be confirmed within one hour by BCA assay (or Lowry assay, Bradford assay, etc.). Although the standard total protein quantification value may vary depending on the virus strain, the protein content of the inactivated virus concentrate used in this study was confirmed to be 200-600 μg / mL. After measuring the protein content of the inactivated virus solution, the value closest to the TPQV measurement value was set as the standard protein content for CTAB treatment among 100, 200, 300, 400, 500, 600, 700, 800, or 900 μg / mL. The CTAB treatment amount was calculated using the following formula (1), and the CTAB treatment concentration (%) relative to the protein content of the inactivated virus solution was determined to be within the range of 0.005%-0.100% (v / v).

[0207] Surfactant treatment amount = [{(a*TPQV(μg / mL)) / (b μg / mL)} / c]* d (1)

[0208] (In the formula, a is 0.005 to 0.100 (v / v%), b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and the value closest to the TPQV is selected; c is the concentration (v / v%) of the surfactant stock being treated; d is the volume of the sampled influenza virus treated with the detergent.

[0209] The CTAB treatment concentration (%) based on protein content varies depending on the strain and virus characteristics, so the appropriate CTAB treatment concentration (%) was confirmed and determined by performing SDS-PAGE over a desired range. Specifically, the inactivated virus solution was aliquoted and treated with CTAB over a desired range, and then ultra-high speed centrifugation was performed at 30,000 rpm or higher according to the SkyCellFull manufacturing process to confirm the optimal CTAB treatment concentration at which the surface antigen protein was recovered as the supernatant and the viral core protein was separated as a precipitate.

[0210] The confirmed optimal CTAB treatment concentration was applied to the manufacturing process. SDS-PAGE was performed on the sample (B / Maryland / 15 / 2016 virus) obtained through the above process, and the results shown in Figure 1 were obtained. When CTAB was treated at concentrations of 0.01% to 0.10%, impurities other than hemagglutinin found in the inactivated virus solution, such as nucleoprotein and matrix protein, were removed during the ultra-high speed centrifugation process after CTAB treatment, and only hemagglutinin was recovered in the supernatant from the ultra-high speed centrifugation.

[0211] 2-2. Confirmation of reliability and reproducibility of the method for setting surface antigen purification process conditions according to the present invention - correlation with existing SRID-based methods

[0212] [Table 1]

[0213] Table 1 above summarizes the data of 2,000 L scale commercial production, and the amount of surfactant to be treated was calculated based on the protein content using the formula (1) above, and the CTAB treatment step was carried out.

[0214] In addition, the HA content of the inactivated virus solution was compared with that of the supernatant obtained by ultra-high-speed centrifugation after CTAB treatment of the inactivated virus solution. The results showed that the HA content of the supernatant obtained by ultra-high-speed centrifugation was sufficient for commercial use at a level of 70-80% of the HA content of the inactivated virus solution. If excessive CTAB is used to increase the HA recovery rate, a split vaccine may be produced instead of the intended subunit vaccine, so it is very important to determine the appropriate amount of CTAB treatment.

[0215] Therefore, it was confirmed that the method for calculating the amount of surfactant to be treated according to the formula (1) established in the present invention is a method that can quickly and accurately determine the amount of CTAB to be treated during the purification process of influenza virus surface antigens.

[0216] 2-3. Comparison of the execution time between the refinement condition setting based on the existing SRID method (Comparative Example 1) and the refinement condition setting according to the present invention As shown in Example 2-2, the process time required for setting surface antigen purification conditions using the conventional radial immunodiffusion (SRID) method was compared with that required for setting the purification conditions of the present invention (see Examples 2-1 and 2-2). As a result, as shown in Table 2 below, it was confirmed that the process time required for the method of the present invention is much shorter than that required for the conventional radial immunodiffusion method, making it more efficient for determining the purification conditions for influenza surface antigens.

[0217] [Table 2]

[0218] Example 3: Establishment of a Rapid Influenza Virus Antigen Purification Condition Test Set (RIVPCT) for Virus Cultivation and After The purification process for the production of subunit vaccines containing influenza surface antigens requires two major steps: the purification of influenza virus from influenza virus-infected cell cultures and the isolation of surface antigen proteins from influenza virus. Existing vaccine production processes can optimize process conditions using the standard test method, single radial immunodiffusion (SRID), at these two steps. However, as mentioned above, SRID has various drawbacks in vaccine production. In response to this, the present inventors sought a novel method for determining suitable process conditions without SRID and developed a rapid confirmation test set for influenza virus antigen purification conditions, which includes the following first and second purification condition determination methods (based on the new strategy established in Example 2). In this invention, the set of the first and second purification condition determination methods is referred to as RIVPCT.

[0219] 3-1. First purification condition determination method: Isolation / purification / concentration of influenza virus from influenza virus culture The present invention provides a determination / discrimination method for quickly determining optimal conditions for each virus without SRID measurement when setting purification conditions used in the step of purifying influenza virus from influenza virus culture. Specifically, the present invention provides a test / judgment method for determining the conditions for influenza virus purification based on the hemagglutination assay, SDS-PAGE, and discrimination criteria specific to the present invention, which will be described later, for influenza virus samples obtained by purification under different conditions.

[0220] In the following examples, chromatography is used as a representative virus purification method. Before the virus culture solution is loaded into the chromatography column, it is pretreated to remove cells and cell debris from the virus culture solution. Such pretreatment may be carried out by, for example, centrifugation, dialysis, filtration, or the like.

[0221] Different conditions must be set for each influenza virus during the chromatography process. However, due to the nature of influenza viruses, where the recommended strain for vaccine production changes annually, it is necessary to quickly confirm the purification conditions for each virus strain. To set the chromatography purification conditions, the conditions under which influenza virus can best bind to the resin are identified, and then elution conditions that can separate and purify the bound virus with optimal yield and purity are tested. Below, we will show a representative example of how to determine influenza virus purification conditions (binding and elution conditions for the target substance on the CS chromatography column resin) when using cellulosic sulfate (CS) chromatography, and this is abbreviated as CSPCT (CS chromatography purification condition test).

[0222] Viruses bind to the resin of a chromatography column through ionic bonds due to the affinity between influenza virus surface proteins and sulfate groups on the resin. The binding capacity is generally determined by conductivity and pH. This example demonstrates how to find conditions that maximize virus binding by maintaining a neutral pH of 7.0-7.4 for the buffer solution used in the process, enhancing protein stability, and lowering conductivity. The conductivity of a typical influenza virus culture (liquid) is approximately 10.0-15.0 mS / cm. The sample is diluted with a low-conductivity sodium phosphate buffer to lower the conductivity of the sample loaded onto the column, thereby confirming binding capacity. This example demonstrates how a simple method for quickly confirming process conditions reduces the time and variables required to confirm purification conditions by adjusting only the conductivity, excluding pH, which is typically set as one of the purification conditions.

[0223] It is generally known that the lower the conductivity, the better the influenza virus binds to CS resin, but since the volume of sodium phosphate buffer added to lower the conductivity and the CS chromatography process time increase, production time and production costs also increase, so it is important to confirm the appropriate conductivity.To this end, we lowered the conductivity to below 10.0 mS / cm by diluting the virus culture solution 0.5 to 4 times with sodium phosphate buffer, and then performed a hemagglutinin confirmation test (HA assay) on the column flow-through to confirm whether the binding capacity was appropriate.

[0224] Specifically, the HA assay was performed according to the protocol described in the WHO Manual for the Laboratory Diagnosis and Virological Surveillance of Influenza. Based on the decreasing trend of HA titer in the column flow-through, the appropriate conductivity of the loading sample was confirmed. SDS-PAGE was also performed simultaneously to confirm whether influenza virus was lost in the column flow-through under each condition.

[0225] After determining the optimal conditions for virus binding to the CS column resin, we then identified the optimal conditions for virus elution. Elution of resin-bound influenza virus was performed by searching for the optimal conductivity using a buffer solution containing an appropriate concentration of sodium chloride to dissociate the virus from the resin. A linear gradient of 2-5 M sodium chloride buffer solution with increasing salt concentration was applied to the column, resulting in the elution of virus and proteins, as indicated by UV peaks. These fractions were collected, and the conductivity and salt concentration of the virus-containing fractions were confirmed by HA assay and SDS-PAGE. Based on the virus elution conditions identified here and the conductivity conditions previously determined to be appropriate for virus binding, the virus was eluted using a step gradient of 2-5 M sodium chloride buffer and sodium phosphate buffer. After confirming whether the majority of influenza virus eluted at these elution concentrations, the virus elution conditions were established. To confirm whether the virus was eluted with high yield and purity, an HA assay was performed using the same method and criteria as described above. This confirmation was performed using SDS-PAGE.

[0226] The CS chromatography process condition test (CSPCT), which is carried out through the above series of steps to purify the virus from the collected virus culture medium, can be completed within approximately 6 to 12 hours. Given the nature of influenza vaccines, where the vaccine production strain changes every year, such a time reduction also contributes to a reduction in the overall production period.

[0227] The most accurate method is to determine the hemagglutinin content of the column flow-through using the single radial immunodiffusion technique (SRID), a standard test method used when setting chromatography conditions. However, it takes approximately 2–3 days to confirm one purification condition, significantly increasing the condition testing period. Furthermore, if SRID standards are not available or are delayed at NIBSC (National Institute for Biological Standards and Control) or TGA (Therapeutic Goods Administration), the test cannot be performed. When performing HA assay and SDS-PAGE according to the present invention, test results can be obtained within 3 hours. Purification condition testing for each virus can be completed within one day, making it extremely rapid and eliminating issues such as the need for standards, and there are few limitations on test execution. The key feature of the CSPCT described above is the rapid confirmation within 12 hours of the influenza virus elution conditions, achieved by appropriately lowering the conductivity and confirming the binding capacity of the influenza virus. The principles and procedures are as described above.

[0228] 3-2. Second purification condition determination method: Separation and purification of surface antigens from influenza viruses This example presents an example in which CTAB was used as a representative surfactant for the isolation and purification of surface antigens from influenza viruses. The amount of CTAB used was rapidly confirmed by performing CTCCT in RIVPCT using the same method as in Example 2.

[0229] 3-3. Purification effect of surface antigen proteins by the first and second purification condition determination methods While determining the necessary conditions for the purification process required for vaccine production based on the HA content confirmed by SRID allows for more accurate purification conditions to be set, the RIVPCT of the present invention has the advantage of allowing for more rapid determination of the necessary conditions for the purification process required for vaccine production.Since performing a method using SRID requires more than two days, excluding the availability of standards and the storage period, we have invented a RIVPCT method using SDS-PAGE, HA assay, etc. to shorten this required time.

[0230] [Table 3]

[0231] Example 4: Influenza subunit vaccine production process with reduced production time 4-1. Infection and cultivation of cells with influenza virus 1) MDCK (Madin-Darby Canine Kidney cells) cell culture One vial of frozen MDCK-Sky3851 cell line for vaccine production was thawed in a 37°C water bath and diluted with culture medium. The diluted cells were then centrifuged to separate the cells from the medium, and the cell pellet was dissolved in fresh medium again. A sample was taken and the cell count and viability were measured. 1 x 10 5 ~5×10 5 The cells were inoculated into a 125 mL spinner flask at an inoculation concentration of 100 cells / mL and cultured at 37°C, 5% CO with agitation at 80 rpm for 3 to 4 days.

[0232] The cell count and viability of MDCK cells cultured in the 125 mL spinner flask were measured, and a cell culture medium sufficient to initiate a 250 mL culture was inoculated into a 500 mL spinner flask. Primary seed culture was carried out for 3–4 days at 37°C and 5% CO2 with agitation at 100 rpm. The cells were cultured in a batch process using a chemically defined cell culture medium supplemented with L-glutamine at a concentration of 3–6 mM / L. After primary seed culture, the cell count and viability were measured. Then, two 1 L spinner flasks were inoculated with enough cells to initiate a 500 mL culture and cultured for 3–4 days under the same conditions as the primary seed culture.

[0233] A sample was taken from the 1L spinner flask during the 500mL culture to measure the cell count and viability, and then the culture medium from the 1L spinner flask, enough to start a 5L culture in a 10L disposable cell incubator, was aseptically inoculated into the cell incubator and cultured for 3-4 days. The equipment and pH and DO probes required for inoculating the spinner flask cell culture medium into the cell incubator were sterilized and prepared in advance. After attaching the pH and DO probes and adding the culture medium, the temperature was set to 37±1°C and the DO value was corrected to prepare for cell inoculation.

[0234] In the same manner, cell culture was carried out in succession on a scale of 50 L, 100 L, 500 L, and 2000 L.

[0235] 2) Viral infection The culture medium in the cell incubator during the 2000 L-scale culture was sampled to measure the cell number and viability, and 1 × 10 6 ~3×10 6 Once the cell count reached 300 cells / ml, the existing culture medium was replaced with fresh medium. The culture medium was replaced by using a continuous low-speed centrifuge in a disposable cell culture bag to remove 70-80% of the medium (based on the cell culture solution) and then adding fresh medium.

[0236] During the medium exchange and virus infection process, the cell incubator maintains a sealed environment, and the materials used for medium exchange are sterilized disposable bags, greatly reducing the possibility of contamination. The process takes 3-4 hours for a 10L scale, and 7-12 hours for a 2000L scale commercial production.

[0237] Viral infection and growth were carried out under conditions of DO 50±10% and pH 7.2±0.2. The influenza virus strain to be produced was dissolved, and the virus calculated at an MOI of 0.0001-0.002 and trypsin at a concentration of 2.5-20 μg / mL were aseptically added to a cell incubator to infect the cells. The cells were cultured for approximately 3-4 days. When the virus titer, cell count, and viability reached appropriate levels, the culture was terminated and the culture medium was collected.

[0238] The cell culture and virus infection methods did not use microcarriers, and in particular, the cells were cultured in a batch cell culture system rather than a perfusion system, and the virus was propagated. After infection with MDCK-Sky3851, influenza viruses propagate within the cells, causing the cells to burst and be expelled to the outside. Therefore, when cells are cultured in a perfusion system, the number of cells gradually decreases, making it unsuitable for the system of the present invention.

[0239] 4-2. Condition setting and virus purification for influenza virus from virus culture 1) Centrifugation and filtration After the virus culture in the cell incubator was completed, the virus culture was centrifuged at 8,000-20,000 rpm using a continuous low-speed centrifuge to collect the supernatant, which was then filtered through a 1.0 / 0.5 μm prefilter and then filtered again through a 0.45 μm filter to remove particles larger than a certain size.

[0240] 2) Purification conditions and influenza virus purification using cellufine sulfate (CS) affinity resin The filtered virus culture medium was subjected to a primary chromatography step using a resin (cellufine sulfate) that has affinity for influenza viruses. After equilibrating the column with sodium phosphate buffer, the virus filtrate was loaded. Purification conditions were quickly established using the CSPCT method described in Example 3-1. Purification condition establishment tests were conducted using IVR-190 (A / Brisbane / 02 / 2018(H1N1) pdm09-like virus), and the results were compared between the CSPCT method and the SRID method.

[0241] First, to determine the optimal conditions for virus binding to the CS column resin, linear gradient purification was performed, followed by SDS-PAGE and HA assay to confirm the conductivity conditions that minimized the loss of hamaglutinin in the column flow-through (Figures 2 to 4, Table 4).

[0242] [Table 4]

[0243] A linear gradient purification test was performed by diluting the JVR-190 virus culture medium with sodium phosphate buffer (equilibration buffer) 1:1 to lower the conductivity from 13.03 mS / cm to 7.54 mS / cm. SDS-PAGE confirmed that no HA was lost in the column flow-through, and impurities other than HA were eluted. HA assay results using 0.5% chicken RBCs also confirmed 0 HAU / 50 μL in all column flow-through fractions, confirming that the conductivity of the loading sample used in the test was suitable for primary chromatographic purification.

[0244] To confirm the virus elution concentration, sodium phosphate buffer containing sodium chloride and sodium phosphate buffer were diluted at appropriate ratios and added, and the UV280 peak was confirmed and the eluted virus fraction was collected.The highest peak (UV280 value) and HA titer were observed on the chromatogram at NaCl concentrations of 0.1M to 0.2M, and the peak and HA titer gradually decreased up to 0.3M NaCl, confirming that 0.4M NaCl is the appropriate virus elution concentration for increasing yield.Taking the test results together, it was determined that a conductivity of 7.54mS / cm for the sample loaded onto the CS column is appropriate, and 0.4M NaCl is therefore determined to be the appropriate virus elution concentration.

[0245] After setting the conductivity conditions for the loading sample according to the CSPCT method, we performed a step gradient purification process using virus elution concentrations of 0.3M, 0.35M, and 0.4M NaCl to compare with the SRID-based method, and then compared the HA content of the virus eluate from each batch. Additionally, SDS-PAGE and HA assays were performed to confirm the correlation between the results obtained by the CSPCT method of the present invention and the SRID-based method.

[0246] The SRID results confirmed that the best yield and purity were obtained when the virus was eluted at a NaCl concentration of 0.4M (Table 5).

[0247] [Table 5]

[0248] The samples used in SRID were used to analyze the virus eluates under each condition by HA assay and SDS-PAGE, which are used in the CSPCT method.

[0249] Referring to the SDS-PAGE results in Figure 5, it was confirmed that the HA band thickened as the virus elution concentration increased, indicating an increase in the amount of HA contained in the virus eluate. The HA titer of the virus eluate was 1,024 HAU / 50μL under the 0.3M and 0.35M NaCl concentration conditions, but increased to 2,048 HAU / 50μL under the 0.4M condition. As such, the virus elution conditions confirmed by the CSPCT method also resulted in a virus elution concentration of 0.4M NaCl, similar to the SRID method, confirming a correlation between the two methods (Table 6).

[0250] [Table 6]

[0251] Meanwhile, the equilibration buffer used in the purification process is maintained at a neutral pH of 7.0-7.4, and the virus filtrate is diluted with the equilibration buffer to lower the conductivity to maximize virus binding. At the end of virus cultivation, the conductivity of the virus culture solution was approximately 10.0-15.0 mS / cm. The conductivity was then lowered by diluting it with an equilibration buffer with a lower conductivity to check the resin binding ability. While this varied depending on the type of influenza virus, an appropriate level of binding ability was observed at approximately 4.0-10.0 mmS / cm.

[0252] The virus filtrate diluted with equilibration buffer was applied to the CS column, and the flow-through liquid that did not bind to the column was sampled and discarded. The column was thoroughly washed with 2-3 CV of equilibration buffer, and then an appropriate ratio of sodium phosphate buffer containing sodium chloride for elution was diluted and applied. The eluted virus fraction was collected while checking the UV280 peak. The influenza virus was eluted by performing CSPCT (see Example 3-1) during RIVPCT to confirm the conditions for an elution buffer containing an appropriate concentration of sodium chloride that could separate the binding of the virus to the resin, and most of the virus was eluted at a sodium chloride concentration of 1 M or less.

[0253] 4-3. Virus concentration and desalting The virus eluate was concentrated and exchanged with PBS buffer by primary ultrafiltration. The eluate was concentrated to less than 10% of the volume of the cell culture liquid using a 300-800 kDa ultrafiltration filter, and then diafiltered with PBS buffer equivalent to a volume of at least 8 times the concentrated volume until the conductivity was the same as that of the PBS buffer.

[0254] 4-4. Removal of MDCK cell-derived DNA from virus concentrate To remove cell line-derived DNA from the collected virus concentrate, the solution was treated with 0.5-50 U / mL of benzonase (Merck), a DNA-cleaving enzyme, and incubated at room temperature for 30 minutes to 24 hours with gentle stirring. Benzonase activity was enhanced by adding 0.5-5 mM magnesium chloride hexahydrate as a cofactor.

[0255] 4-5.Virus inactivation The influenza virus concentrate after the benzonase reaction was inactivated to remove infectivity. Any known chemical means for inactivating the virus can be used, such as detergent, formaldehyde, β-propiolactone, methylene blue, psoralen, or carboxyfullerene (C60). In this example, the virus was inactivated by treating the concentrate with a formaldehyde solution at a concentration of 0.01 to 0.1%.

[0256] 4-6. Isolation of surface antigens using detergent Surface antigen proteins can be separated and purified by treating influenza viruses with detergents (ionic or nonionic) or solvents, and a typical example is the use of ionic detergents such as cetyltrimethylammonium bromide (CTAB).

[0257] In this example, CTAB was treated based on the protein content of the inactivated virus concentrate, and the amount of CTAB treatment was confirmed by performing CTCCT in RIVPCT as in Example 3-2. After incubation at room temperature or in a refrigerator for at least one hour, the mixture was centrifuged at 30,000 rpm or higher using a continuous ultra-high speed centrifuge, and the supernatant was collected to separate only the surface antigen protein.

[0258] To remove CTAB from the supernatant after ultra-high speed centrifugation, an adsorption resin (Amberlite XAD-4) was added to the supernatant, and the adsorption resin was then removed by filtration.

[0259] 4-7.Removal of residual DNA using TMAE anion exchange resin To further remove host cell-derived DNA, a second chromatography step was performed using an anion exchange chromatography resin. After equilibrating the column with sodium phosphate buffer, the viral surface antigen recovery filtrate was loaded. The column was then diluted with sodium phosphate buffer containing 1-6M sodium chloride, which has a high conductivity, so that the surface antigens do not bind to the resin, but the DNA does. The chromatography step was then performed at a conductivity that allows the resin to bind, and the unbound column flow-through was collected as the surface antigen fraction.

[0260] 4-8. Concentration and desalting of surface antigen fraction The surface antigen fraction was concentrated using a 30-50 kDa ultrafiltration filter. Once the concentration process was complete, buffer exchange was performed using PBS buffer until the conductivity was the same as that of PBS buffer, and the concentrate was collected. The collected concentrate was then sterilized and filtered through a 0.5 / 0.2 μm filter to produce a stock solution, which was then stored at 2-8°C. [Industrial Applicability]

[0261] The unique method of the present invention makes it possible to quickly and reliably confirm the conditions for obtaining (purifying) influenza surface antigens without using radial immunodiffusion, which has previously been used as a standard test method in influenza vaccine production. This significantly shortens the time required for influenza surface antigen subunit vaccine production, allowing for rapid vaccine development / production even in situations where a new influenza pandemic is rapidly occurring, and has extremely high industrial applicability.

Claims

1. A method for purifying a surface antigen protein from an influenza virus using a detergent, characterized in that the amount of the detergent to be treated with the sampled influenza virus is determined by calculating the total protein quantification value (TPQV) per unit volume of the sampled influenza virus and then substituting the value into the following formula (1): Surfactant treatment amount = [{(a * TPQV (μg / mL)) / (b μg / mL)} / c] * d ... (1) (In the formula, a is 0.005 to 0.100 (v / v%), b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and the value closest to the TPQV is selected; c is the concentration (v / v%) of the surfactant stock being treated; d is the volume of the sampled influenza virus treated with detergent.

2. The method according to claim 1, wherein the amount of the surfactant used is a concentration that selectively separates only surface antigens without destroying the viral core or with minimal destruction of the viral core.

3. 2. The method of claim 1, wherein the determination of the amount of surfactant to be treated does not require a quantification process using single radial immunodiffusion (SRID) for the amount of hemagglutinin (HA) protein in the sample.

4. 2. The method of claim 1, wherein the surfactant is a cationic surfactant.

5. 5. The method of claim 4, wherein the cationic surfactant is CTAB (cetyltrimethylammonium bromide).

6. The method of claim 1, wherein the total protein quantification value (TPQV) is obtained by BCA assay, lowry assay, or Bradford assay.

7. The method according to claim 1, characterized in that the sampled influenza virus is obtained by a method comprising the steps of: (a) culturing cells in a single-use bioreactor (SUB); (b) replacing a portion of the medium in the cell culture solution of step (a) with fresh medium using a continuous low-speed centrifuge using a disposable bag; (c) infecting the expanded cells with influenza virus and culturing them under conditions permissive for influenza virus replication; and (d) isolating influenza viruses from the culture of step (c).

8. The method according to claim 7, wherein the cells are MDCK (Madin-Darby Canine Kidney) cells.

9. 8. The method of claim 7, wherein step (b) involves continuously separating the cells and the medium using a continuous low-speed centrifuge, discarding a portion of the medium, and then introducing new medium into the cell incubator, without a process of recovering and re-introducing the entire amount of the culture medium.

10. 10. The method of claim 9, wherein the portion of the culture medium is 50% to 80% of the total culture liquid.

11. The method of claim 7, wherein step (d) comprises the steps of: (d-1) determining purification conditions based on a hemagglutination assay for influenza virus samples obtained by purification under different conditions in order to purify influenza virus from the culture of step (c); and (d-2) purifying influenza viruses from the culture of step (c) according to the conditions determined in step (d-1).

12. The method according to claim 1, wherein the quantitative value of the total protein per unit volume of the sampled influenza virus is 50 to 950 μg / mL.

13. A method for rapidly confirming influenza antigen purification conditions, comprising the following first and second purification condition determination methods: A first purification condition determination method is used in a step of purifying influenza virus from an influenza virus culture, and the conditions for purifying the influenza virus are determined based on a hemagglutination assay, SDS-PAGE, or a combination thereof, for influenza virus samples obtained by purification under different conditions; and The second purification condition determination method is used in a step of purifying a surface antigen protein from an influenza virus using a surfactant, and the amount of surfactant used in the purification of the surface antigen protein is determined based on a total protein quantification assay for an influenza virus sample, The second purification condition determination method is characterized in that the total protein quantification value (TPQV) per unit volume of the influenza virus culture obtained through the first purification is determined, and then the amount of surfactant treatment is determined based on the value obtained by substituting TPQV into the following formula (1): Surfactant treatment amount = [{(a * TPQV (μg / mL)) / (b μg / mL)} / c] * d ... (1) (In the formula, a is 0.005 to 0.100 (v / v%), b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and the value closest to the TPQV is selected; c is the concentration (v / v%) of the surfactant stock being treated, and d is the volume of the influenza virus culture treated with detergent.

14. The method according to claim 13, wherein the method for rapidly confirming influenza antigen purification conditions does not require a quantification process using single radial immunodiffusion (SRID) for the amount of hemagglutinin (HA) protein in an influenza virus sample.

15. 14. The method of claim 13, wherein the step of purifying influenza virus from the influenza virus culture is carried out by chromatography.

16. The method according to claim 15, wherein the chromatography is carried out using a column packed with Cellufine sulfate (CS).

17. The method of claim 13 , wherein the conditions include buffer type, buffer concentration, pH, and conductivity.

18. The method of claim 13, wherein the condition is conductivity.

19. 1. A method for producing a vaccine, comprising the step of purifying a surface antigen protein from an influenza virus, wherein the step is carried out under conditions in which a total protein quantification value (TPQV) per unit volume of a sampled influenza virus is determined and then substituted into the following formula (1) to obtain the value, and a surfactant is treated according to the value: Surfactant treatment amount = [{(a * TPQV (μg / mL)) / (b μg / mL)} / c] * d ... (1) (In the formula, a is 0.005 to 0.100 (v / v%), b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and the value closest to the TPQV is selected; c is the concentration (v / v%) of the surfactant stock being treated; d is the volume of the sampled influenza virus treated with detergent.

20. A method for rapid purification of influenza surface antigens, comprising the steps of: a) infecting cells with influenza virus and culturing them to obtain a virus culture; b) determining purification conditions based on hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus samples obtained by purification under different conditions in order to purify influenza virus from the culture of step a); c) purifying influenza virus from the culture of step a) according to the conditions determined in step b); d) determining the amount of surfactant to be used in purifying the surface antigen protein from the influenza virus purified in step c) based on a total protein quantification assay for influenza virus; e) treating the influenza virus with a surfactant according to the conditions determined in step d) to purify the surface antigen protein from the influenza virus, wherein the total protein quantification value (TPQV) per unit volume of the influenza virus separated in step d) is determined and then substituted into the following formula (1) to determine the amount of surfactant to be treated: Surfactant treatment amount = [{(a * TPQV (μg / mL)) / (b μg / mL)} / c] * d ... (1) (In the formula, a is 0.005 to 0.100 (v / v%), b is 100, 200, 300, 400, 500, 600, 700, 800, or 900, and the value closest to the TPQV is selected; c is the concentration (v / v%) of the surfactant stock being treated, and d is the volume of the sampled influenza virus treated with detergent.

21. 21. The method of claim 20, wherein the method for rapid purification of influenza surface antigens further comprises, prior to step b), removing cells and cell debris from the virus culture.

22. 22. The method of claim 21, wherein the removal of cells and cell debris from the virus culture is carried out by one or more methods selected from the group consisting of filtration, dialysis, and centrifugation.

23. 21. The method of claim 20, wherein the rapid purification method for influenza surface antigens comprises, after step c), an additional purification step by one or more methods selected from the group consisting of ultrafiltration and diafiltration.

24. The method according to claim 20, wherein step e) separates the surface antigen protein by treating with a surfactant, followed by centrifugation to collect the supernatant.

25. The method of claim 20, wherein the method for rapid purification of influenza surface antigens further comprises a detergent removal step after step e).

26. 21. The method of claim 20, wherein the rapid purification method for influenza surface antigens comprises an additional chromatography step for removing impurities after step e).

27. 27. The method of claim 26, wherein the chromatography is TMAE (trimethylaminoethyl) anion exchange chromatography.

28. 28. The method of claim 27, wherein the rapid purification method for influenza surface antigens comprises, after chromatography, an additional purification step by one or more methods selected from the group consisting of ultrafiltration and diafiltration.

29. The method according to claim 20, wherein the cells used for viral infection in step a) or the cells infected with the virus in step a) are cultured in a single-use bioreactor (SUB).

30. The method according to claim 20, characterized in that, before infecting the cells with the virus in step a), a portion of the medium in the cell culture used for viral infection is replaced with fresh medium through a continuous low-speed centrifuge.

31. 21. The method of claim 20, wherein the method for rapid purification of influenza surface antigens further comprises a treatment step with benzonase, exonuclease, ribozyme, or a mixture thereof after step c).

32. The method of claim 20, wherein the method for rapid purification of influenza surface antigens further comprises a virus inactivation process after step c).

33. The method according to claim 32, wherein the virus inactivation is carried out by treatment with a detergent, formaldehyde, β-propiolactone, methylene blue, psoralen, carboxyfullerene (C60), secondary ethylamine, acetylethyleneimine, or a combination thereof.

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