KR102546626B1 - Rapid influenza virus antigen purification condition test - Google Patents

Abstract

The present invention relates to a rapid identification test of influenza virus antigen purification conditions. It is possible to check the conditions for obtaining (purifying) the surface antigen, and accordingly, the production period of the influenza surface antigen subunit vaccine is significantly shortened, so that even in the situation where the new influenza is rapidly spreading, response to vaccine development/manufacturing can be made quickly. .

Description

Influenza virus antigen purification condition rapid identification test {Rapid influenza virus antigen purification condition test}

The present invention relates to a rapid identification test for influenza virus antigen purification conditions.

For influenza-related diseases, treatment is important, but above all, prevention through vaccination is a realistic and effective method. predicted

Influenza virus vaccine production recommendations are changed every year, and according to the characteristics of new influenza outbreaks in which infections increase worldwide at an unspecified time, influenza virus vaccine development establishes mass production conditions in a short time to produce vaccines with high yield and high purity. It can be said that the key is to do it.

The main antigen of influenza vaccine is hemagglutinin (HA) in the envelope of influenza virus, and the vaccine titer is also based on hemagglutinin concentration. Currently, the single radial immunodiffusion technique (SRID) recommended by the European Pharmacopoeia and the World Health Organization (WHO) is commonly used domestically and internationally to measure the hemagglutinin concentration of influenza vaccines (B Patent Document 1). Standard antigens and standard antibodies distributed by the National Institute for Biological Standards and Control (NIBSC) are used when hemagglutinin content is measured using radioimmunoassay. The size of the ring in which influenza vaccine antigens and standard antibodies are aggregated The concentration of hemagglutinin in the influenza vaccine is measured by comparing and converting . However, this radioimmuno-diffusion method takes more than 24 hours to process samples, and most of the processes are manual, which is very labor-intensive compared to the throughput.

Pandemic refers to a state in which an infectious disease is prevalent worldwide. Since vaccine development must be prompt in a pandemic situation, the measurement of hemagglutinin content must also be performed promptly when developing a vaccine to cope with an influenza pandemic situation. will be. In Korea, in the context of an unexpected H1N1 pandemic in 2009, vaccine manufacturing and follow-up clinical trials were generally delayed due to delays in the supply of standard antigens and antibodies from the World Health Organization (WHO). When a vaccine needs to be developed quickly, such as in a pandemic situation, a rapid hemagglutinin content measurement method is required. it became

In addition, research on new physicochemical test methods has recently begun. In order for the newly attempted physicochemical test method to replace the radioimmunoassay test, data and test methods showing that there is some correlation between the alternative test method and the results of the radioimmunodiffusion method establishment, certification, etc.

Reverse-phase chromatography (RP-HPLC), isotope-diluted liquid chromatography-mass spectrometry (ID-LC/MS), deglycosylated antigen SDS-PAGE analysis, etc. are known as alternative test methods, but depending on the method, the standard antigen is still It is known that there are disadvantages such as requiring expensive equipment and professional manpower or low analytical sensitivity (Non-Patent Document 2).

Still, there is a need in the art for methods that can easily replace the SRID method or without the SRID method when preparing a vaccine.

Wood JM, Schild GC, Newman RW, Seagroatt V. An improved Single-Radial-Immunodiffusion technique for the assay of influenza haemagglutinin antigen application for potency determination of inactivated whole virus and subunit vaccines. J Biol Stand 1977;5:237-47. Wang Jin-ho, Pandemic Influenza Vaccine Antigen Content Rapid Test Method Commentary, Food and Drug Safety Evaluation Institute, 2015

Accordingly, while the present inventors were researching to enable setting of process conditions during vaccine production without using the radioimmunoassay, according to the method unique to the present invention, it is possible to quickly and reliably check the conditions for obtaining influenza surface antigens, and accordingly, influenza antigens The present invention was completed by confirming that the vaccine production period including a is remarkably shortened.

Therefore, an object of the present invention is a method for purifying a surface antigen protein from an influenza virus using a detergent, wherein the surfactant is a total protein quantity per unit dose of the influenza virus sampled. It is to provide a method characterized in that the amount of surfactant treated with the sampled influenza virus is determined by the value obtained by obtaining the quantification value (TPQV) and substituting it into Equation 1 below:

[Equation 1]

Surfactant Throughput = [{(a*TPQV(μg/mL))/(b μg/mL)}/c]* d

(In the above 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 (w/v%) of the treated surfactant stock;

d is the volume (volume) of the sampled influenza virus to be treated with surfactant.)

Another object of the present invention is to provide a test method for rapid confirmation of influenza antigen purification conditions, characterized by comprising the following first purification condition determination method and second purification condition determination method:

As the first purification condition determination method, it is used in the step of purifying influenza virus from influenza virus culture, and the conditions for purifying the influenza virus are the hemagglutination reaction method (Hemagglutination reaction) for influenza virus samples obtained by purification under different conditions. assay), SDS-PAGE, or a first purification condition determination method determined based on a combination thereof; and

As a second method for determining purification conditions, it is used in the step of purifying surface antigen protein from influenza virus using a surfactant. 2nd purification condition determination method determined based on assay)

Another object of the present invention is a vaccine manufacturing method comprising the step of purifying surface antigen protein from influenza virus, wherein the step is obtained after obtaining the total protein quantification value (TPQV) per unit dose of the sampled influenza virus It is to provide a method that is carried out under the condition of treating the surfactant with the value obtained by substituting it into the following formula 1. :

[Equation 1]

Surfactant Throughput = [{(a*TPQV(μg/mL))/(b μg/mL)}/c]* d

(In the above 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 (w/v%) of the treated surfactant stock;

d is the dose of the sampled influenza virus to treat the surfactant.)

Another object of the present invention is to provide a method for rapidly purifying influenza surface antigens, comprising the following steps:

a) infecting cells with an influenza virus and culturing to obtain a virus culture;

b) In order to purify the influenza virus from the culture of step a), purification conditions based on hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus samples obtained by purification under different conditions determining;

c) purifying the influenza virus from the culture of step a) according to the conditions determined in step b);

d) in order to purify the surface antigen from the influenza virus purified in step c), determining the amount of surfactant to be used when purifying the surface antigen protein based on a total protein quantification assay for the influenza virus;

e) purifying the surface antigen protein from the influenza virus by treating with a surfactant according to the conditions determined in step d).

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

a) infecting cells with an influenza virus and culturing to obtain a virus culture;

b) In order to purify the influenza virus from the culture of step a), purification conditions based on hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus samples obtained by purification under different conditions determining;

c) purifying the influenza virus from the culture of step a) according to the conditions determined in step b);

d) in order to purify the surface antigen from the influenza virus purified in step c), determining the amount of surfactant to be used when purifying the surface antigen protein based on a total protein quantification assay for the influenza virus;

e) purifying the surface antigen protein from the influenza virus by treating with a surfactant according to the conditions determined in step d).

In order to achieve the above object of the present invention, the present invention is a method for purifying a surface antigen protein from an influenza virus using a surfactant, wherein the surfactant is used as a whole per unit dose of the influenza virus sampled. Provided is a method characterized in that the amount of surfactant treated with the sampled influenza virus is determined by the value obtained by obtaining the total protein quantification value (TPQV) and then substituting it into Equation 1 below:

[Equation 1]

Surfactant Throughput = [{(a*TPQV(μg/mL))/(b μg/mL)}/c]* d

(In the above 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 (w/v%) of the treated surfactant stock;

d is the dose of the sampled influenza virus to treat the surfactant.)

In order to achieve another object of the present invention, the present invention provides a test method for rapid confirmation of influenza antigen purification conditions, characterized by comprising the following first purification condition determination method and second purification condition determination method:

As the first purification condition determination method, it is used in the step of purifying influenza virus from influenza virus culture, and the conditions for purifying the influenza virus are the hemagglutination reaction method (Hemagglutination reaction) for influenza virus samples obtained by purification under different conditions. assay), SDS-PAGE, or a first purification condition determination method determined based on a combination thereof; and

As a second method for determining purification conditions, it is used in the step of purifying surface antigen protein from influenza virus using a surfactant. 2nd purification condition determination method determined based on assay)

In order to achieve another object of the present invention, the present invention is a vaccine manufacturing method comprising the step of purifying a surface antigen protein from an influenza virus, wherein the step is a total protein quantification value per unit dose of the sampled influenza virus , TPQV) is obtained and then substituted into the following formula 1 to provide a method that is performed under conditions of treating a surfactant with a value obtained:

[Equation 1]

Surfactant Throughput = [{(a*TPQV(μg/mL))/(b μg/mL)}/c]* d

(In the above 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 (w/v%) of the treated surfactant stock;

d is the dose of the sampled influenza virus to treat the surfactant.)

In order to achieve another object of the present invention, the present invention provides a method for rapidly purifying influenza surface antigens, comprising the following steps:

a) infecting cells with an influenza virus and culturing to obtain a virus culture;

b) In order to purify the influenza virus from the culture of step a), purification conditions based on hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus samples obtained by purification under different conditions determining;

c) purifying the influenza virus from the culture of step a) according to the conditions determined in step b);

d) in order to purify the surface antigen from the influenza virus purified in step c), determining the amount of surfactant to be used when purifying the surface antigen protein based on a total protein quantification assay for the influenza virus;

e) purifying the surface antigen protein from the influenza virus by treating with a surfactant according to the conditions determined in step d).

In order to achieve another object of the present invention, the present invention provides a method for producing a vaccine containing influenza surface antigen, characterized in that the vaccine production period is shortened, including the following steps:

a) infecting cells with an influenza virus and culturing to obtain a virus culture;

b) In order to purify the influenza virus from the culture of step a), purification conditions based on hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus samples obtained by purification under different conditions determining;

c) purifying the influenza virus from the culture of step a) according to the conditions determined in step b);

d) in order to purify the surface antigen from the influenza virus purified in step c), determining the amount of surfactant to be used when purifying the surface antigen protein based on a total protein quantification assay for the influenza virus;

e) purifying the surface antigen protein from the influenza virus by treating with a surfactant according to the conditions determined in step d).

Hereinafter, the present invention will be described in detail.

The present invention is a method for purifying a surface antigen protein from an influenza virus using a surfactant, wherein the surfactant is a total protein quantification value (TPQV) per unit dose of the influenza virus sampled. ) is obtained and then the value obtained by substituting into Equation 1 below provides a method characterized in that the amount of surfactant treated with the sampled influenza virus is determined:

[Equation 1]

Surfactant Throughput = [{(a*TPQV(μg/mL))/(b μg/mL)}/c]* d

(In the above 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 (w/v%) of the treated surfactant stock;

d is the dose of the sampled influenza virus to treat the surfactant.)

The virion structure of the influenza virus has an outermost envelope composed of a double fat layer, and two surface glycoproteins protrude from it, one of which is hemagglutinin, a columnar hemagglutinin. One is hemagglutinin (HA), and the other is neuraminidase (NA), a mushroom-shaped neuraminase.

The method provided by the present invention is a method for separating and purifying the hemagglutinin and neuraminidase, which are surface antigens of influenza virus, by treatment with a surfactant, wherein the surface antigen of influenza virus is separated from the virus core. It is characterized in that it is possible to establish conditions for purifying influenza virus surface antigen in a short period of time by quickly calculating the optimal throughput of the surfactant used for separation.

In the present invention, the "surfactant" is used interchangeably with "detergent", and the type of the surfactant is not limited as long as it is used to isolate and purify the surface antigen of a virus in the art, for example Non-ionic or ionic (eg cationic) surfactants may be included. Non-limiting examples of the surfactant include alkylglycosides, alkylthioglycosides, acyls, sugars, sulfobetaines, betaines, polyoxyethylenealkylethers, N,N-dialkyl-glucamides, hecameg ( Hecameg), alkylphenoxy-polyethoxyethanol, quaternary ammonium compounds, sarcosyl, CTAB (cetyl trimethyl ammonium bromide), tri-N-butyl phosphate, cetavlon, myristyltrimethylammonium salt, lipofectin, lipofectamine, and DOTMA, octyl- or nonyl-phenoxy polyoxyethanol (e.g. Triton surfactants such as Triton X-1OO or Triton N101), polyoxyethylene sorbitan esters (Tween surfactants), polyoxy Ethylene ethers, polyoxyethylene esters may be included.

In one preferred embodiment of the present invention, the surfactant may be a cationic surfactant.

In a more preferred embodiment of the present invention, the surfactant may be CTAB.

The influenza virus of the present invention includes types A, B and C according to molecular biological characteristics. Preferably, the influenza virus may be type A or type B. Among them, the type A influenza virus can be classified into various subtypes according to the types of surface antigens, hemagglutinin (HA) and neuraminidase (NA) glycoproteins. More specifically, since 18 types of hemagglutinin and 11 types of neuraminidase are known as surface antigens of influenza A virus, a total of 198 types of influenza A virus subtypes can exist arithmetically, all of which are present in the present invention. It can be understood to be included in the influenza virus of the invention.

On the other hand, antigenic shift in which the HA and NA, which are the main antigens, are changed into new subtypes completely different from those previously known, or point mutations accumulate at the level of the HA and NA genes, resulting in a small number of Influenza viruses of currently known subtypes as well as new subtypes to be identified in the future, including antigenic drift in which amino acids are changed, can be included in the influenza virus of the present invention without limitation.

In a preferred aspect of the present invention, the influenza virus may be influenza A, and non-limiting examples thereof include H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, H6N1, and the like. can be included

In the present invention, the "sampling" influenza virus means culturing an influenza virus expressing surface antigen protein in a host cell to provide a culture, purifying and/or concentrating the culture, and culturing the culture It may mean passing through one or more steps selected from the group consisting of inactivating the influenza virus contained in the water.

In one aspect of the present invention, the sampling may mean that the influenza virus is treated according to a method comprising the following steps:

(a) providing a cell-based virus growth culture medium or fertilized egg-based virus growth culture medium in which influenza virus can proliferate;

(b) clarifying the culture medium to obtain a culture medium containing an influenza virus;

(c) optionally purifying and optionally concentrating the culture; and

(d) optionally, inactivating the influenza virus contained in the culture.

In the above (a), a culture medium containing an influenza virus is provided. Such cultures may be derived from cell-based virus growth cultures or fertilized egg-based virus growth cultures. As is known to those skilled in the art, viruses can be propagated in cell-based culture systems or in fertilized egg-based culture systems. Suitable cell lines used to grow influenza viruses are typically of mammalian origin and thus represent 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. As a non-limiting example of the cell line, cells of the mammalian cell line may include Vero, PerC6, BHK, 293, COS, PCK, MRC-5, MDCK, MDBK, WI-38, and serum-free adapted cells thereof. there is. Preferably, the cell line may be MDCK cells.

In addition, the influenza virus can be grown on cells according to any suitable method known to those skilled in the art. For cell-based culture systems, any suitable medium may be used. In a preferred embodiment, the cells may be cultured in serum-free and/or protein-free media. In the case of cell-based viral propagation, usually virus or live virus preparations are each inoculated into the cell culture in which they are grown. Virus propagation can be done in a batch mode, or fed-batch mode, or perfusion mode.

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

Depending on the culture system used to propagate the influenza virus, the culture containing the propagated viral particles may contain different contaminants. For example, potential contaminants present in fertilized egg-based cultures are fertilized egg-based proteins, such as fertilized egg albumin, or antibiotics, whereas typical contaminants present in cell-based cultures are residual host cell DNA. Preferably, the method provided by the present invention is not limited with respect to the system used for propagating influenza virus; Both in the cell-based culture system and similarly in the fertilized egg-based culture system, the treatment of the culture medium containing the propagated virus can be applied.

In the present invention, the culture solution of step (a) may be prepared by using a disposable bioreactor. By using such single-use bioreactors, for example, the resulting product itself can be improved, for example in terms of quality and/or purity. This may be advantageous in particular if the method is carried out on a large scale, eg on an industrial scale.

The cell-based or fertilized egg-based culture medium containing the influenza virus may be clarified (step (b)). The clarification step is performed on the culture containing the virus particles, but not on the sedimented or otherwise pelletized material. In the present invention, the purpose of this clarification step is to remove particulate matter, such as cell debris, protein impurities, and/or support materials used to grow cells, such as microcarriers, from the culture medium. The protein impurity may be, for example, allantoin, collagen and/or an albumin, such as ovalbumin. For clarification, any suitable means or method known to a person skilled in the art may be used. In particular, tangential flow filtration (TFF), depth filtration, or centrifugation can be applied. Hereby, each set of respectively applied clarification means (TFF, depth filtration, centrifugation) is selected in such a way that the stated objectives are reached. Generally, given this purpose, each of the above settings for each means is known to the person skilled in the art.

In the present invention, the clarified culture broth obtained according to the above method may be referred to as "virus culture".

In one aspect of the present invention, the virus culture may be the “sampled influenza virus” provided by the present invention.

In another aspect of the present invention, the "sampled influenza virus" may be obtained by further performing the following steps (c) and/or (d) after step (b).

In one aspect of the present invention, the purification of the culture in step (c) may be performed by any suitable method for specifically purifying viral antigens. Preferred purification methods include, for example, chromatographic methods such as ion exchange chromatography, hydrophobic interaction chromatography, pseudo-affinity chromatography, affinity chromatography, size exclusion chromatography, liquid-liquid chromatography, and the like. am. 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.

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

During the purification process, the influenza virus attached to the CS column may be eluted with an elution buffer. In one embodiment of the present invention, the influenza virus attached to the CS column could be eluted when an elution buffer containing 0.01 to 1.0 M sodium chloride at neutral pH was added to the column. The pH of the elution buffer may be 6.5 to 7.4, and may contain 0.1 to 6.0M sodium chloride.

In addition, washing may be performed using an elution buffer containing 1.0 to 3.0 M sodium chloride to remove any proteins bound to the CS column during the purification process. The spent CS column can optionally be washed, stored in a suitable agent, and optionally reused.

In one aspect of the present invention, the concentration in step (c) may be achieved by a suitable method. The method leads to reducing the volume of the virus culture. The enrichment step aims to reduce the volume of the culture containing the influenza virus to obtain an enriched culture.

In a preferred embodiment, the volume can be reduced by applying TFF ultrafiltration or TFF ultrafiltration and diafiltration in a single step (TFF ultrafiltration/diafiltration), and ultracentrifugation or precipitation to reduce the volume, preferably TFF ultrafiltration can be used. Thus, each set of enrichment means applied (TFF, TFF ultrafiltration/diafiltration, ultracentrifugation or precipitation) is selected in such a way that the above indicated objectives, ie volume reduction and concentration of influenza virus are achieved.

In another embodiment, when ultracentrifugation is used to reduce the volume, conditions for ultracentrifugation can be, for example, 30,000g to 200,000g for more than 10 minutes.

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, salts, methyl and ethyl alcohol and polyethylene glycol, respectively, in appropriate concentrations. Suitable chemicals at appropriate concentrations precipitate the product, eg, particles containing viral antigens, but have no or minimal negative effects on the viral antigens. Chemicals are selected such that they do not react with the antigen of interest, eg influenza virus surface antigen protein, and do not alter the immunogenicity of the antigen of interest.

In one embodiment, the volume of the virus culture in the concentration step is at least 4-fold, preferably at least 5-fold, or at least 9-fold, also preferably at least 12-fold, also preferably at least 13-fold, or at least 15-fold, also preferably It can be reduced more than 20 times, or more than 25 times.

In one aspect of the present invention, the virus purified and/or concentrated virus culture may be inactivated after step (c). Inactivation results in infective clearance of the virus and can typically be accomplished by treating the virus with an effective amount of one or more of the following chemical means: detergents, formaldehyde, β-propiolactone, methylene blue, pso ralene, carboxyfullerene (C60), secondary ethylamine, acetyl ethylenimine, or combinations thereof. Methods of inactivation 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.

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

Thereafter, the total protein quantification value (TPQV) per unit dose of the influenza virus culture sampled by steps (a) to (d) was obtained and then substituted into Equation 1 below to treat the sampled influenza virus with the value obtained. It can be characterized in that the surfactant throughput is determined:

[Equation 1]

Surfactant Throughput = [{(a*TPQV(μg/mL))/(b μg/mL)}/c]* d

(In the above 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 (w/v%) of the treated surfactant stock;

d is the dose of the sampled influenza virus to treat the surfactant.)

Viral surface antigen vaccine is a high-quality vaccine with few side effects and high purity, and it is very important to set an appropriate amount of surfactant. When an excessive amount of surfactant is treated, the entire virus is disrupted, and the protein inside the virus core, which is an impurity, is exposed to the outside, resulting in a decrease in purity.

Therefore, in one aspect of the present invention, the surfactant throughput calculated according to the above formula is a concentration for selectively solubilizing only the surface antigen of influenza virus in a non-destructive state of the virus core or a state in which destruction of the virus core is minimized. characterized by

On the other hand, it is an important factor in the influenza vaccine production process to quickly identify an appropriate amount of surfactant for solubilization of viral surface antigens. Currently, the single radial immunodiffusion (SRID) method recommended by the European Pharmacopoeia and the World Health Organization (WHO) is commonly used at home and abroad to measure the surface antigen content of influenza vaccines, but the SRID method produces results There is a limitation that the time required to do so is too long.

Therefore, the present invention does not require a quantification process by SRID for the amount of hemagglutinin (HA) protein in the virus culture, and the amount of surfactant added based on the total protein quantification value per unit volume of the virus culture It provides a method characterized in that for determining.

In the method of the present invention, the total protein quantitative value (TPQV) per unit dose of virus culture can be determined according to any method used to calculate protein content in the art, and non-limiting examples thereof include BCA assay, lowry assay or Bradford assay may be included.

The a value in Equation 1 of the present invention means the surfactant stock treatment concentration (%) based on the protein content, and the optimal surfactant treatment concentration may vary depending on the virus strain, virus characteristics, etc. Therefore, by performing SDS-PAGE for any value within the range of 0.005 to 0.100 (v / v%), it is possible to confirm the appropriate surfactant stock processing concentration (%) according to the virus. Specifically, the virus solution is subdivided into small portions, treated with a surfactant for any value within the above numerical range, and ultra-high-speed centrifugation is performed to recover the surface antigen protein as a supernatant and separate the virus core protein as a precipitate. It can be determined by identifying the optimal surfactant treatment concentration.

In the present invention, the b value is 100, 200, 300, 400, 500, 600, 700, 800 or 900, and a value closest to the TPQV is selected. For example, if the TPQV of the sampled influenza virus is 220 μg / mL, 200, the closest value among the above values, becomes the b value, and if the TPQV is 370 μg / mL, 400, the closest value among the above values, is the b value It can be.

In the present invention, the TPQV may vary according to the degree of concentration of the sampled influenza virus described above. Therefore, in order to determine the optimal surfactant throughput according to Equation 1 of the present invention, it is preferable to use an influenza virus culture concentrated so that the TPQV of the sampled influenza virus can be 50 to 950 μg / mL.

The present invention also provides a test method for rapid confirmation of influenza antigen purification conditions, characterized in that it comprises the following first purification condition determination method and second purification condition determination method:

As the first purification condition determination method, it is used in the step of purifying influenza virus from influenza virus culture, and the conditions for purifying the influenza virus are the hemagglutination reaction method (Hemagglutination reaction) for influenza virus samples obtained by purification under different conditions. assay), SDS-PAGE, or a first purification condition determination method determined based on a combination thereof; and

As a second method for determining purification conditions, it is used in the step of purifying surface antigen protein from influenza virus using a surfactant. A second purification condition determination method determined based on assay).

In one aspect of the present invention, the first purification corresponds to the virus culture purification step of step (c) described above.

In another aspect of the present invention, the first purification is characterized in that a quantification process by single radial immunodiffusion (SRID) is not required for the amount of hemagglutinin (HA) protein in the virus culture.

In another aspect of the present invention, the step of purifying the influenza virus from the influenza virus culture may be characterized in that it is performed by chromatography.

In another aspect of the present invention, the chromatography, for example, ion exchange chromatography, hydrophobic interaction chromatography, pseudo-affinity chromatography, affinity chromatography, size exclusion chromatography, liquid-liquid chromatography, etc. includes 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.

In another aspect of the present invention, the first purification is performed after confirming conditions in which the influenza virus culture that has passed through the column can best bind to the resin, and then separating and purifying the virus bound with optimal yield and purity. It may be characterized by selecting even the elution conditions.

In another aspect of the present invention, the first purification condition determination method is to dilute the influenza virus culture by applying various ratios with equilibration buffer, apply to a chromatography column, and then pass through the column under different conditions. It can be characterized by selecting the condition with the lowest hematoglutinin (HA) titer by applying each hemagglutination assay (Hemagglutination assay). If, as a result of the hemagglutination reaction method, the HA titer of the liquid passing through the column is high, it is not preferable because the virus in the virus culture does not bind to the column and passes as it is.

In another aspect of the present invention, the first purification condition determination method is to dilute the influenza virus culture by applying various ratios with an equilibration buffer, apply it to a chromatography column under different conditions, and then apply the elution buffer to the column and The bound influenza virus was eluted, and the eluate according to each condition was applied to hemagglutination assay, SDS-PAGE, or a combination thereof, so that the HA titer was the highest and the hemagglutinin band was the thickest on SDS-PAGE. It may be characterized by selecting conditions.

In another aspect of the present invention, the first purification condition is a condition in which the virus best binds to the resin when the virus culture is passed through a column, and a condition in which the virus can best be eluted in the process of eluting the virus bound to the resin. It can be characterized as a combination of conditions. The conditions in which the virus binds best with the resin can be selected by applying the column flow through the column to hemagglutination assay, SDS-PAGE, or a combination thereof, and eluting the virus bound to the resin. Conditions that can best elute the virus in the process can be selected by applying the eluate that has passed through the column to hemagglutination assay, SDS-PAGE, or a combination thereof.

In another aspect of the present invention, the first purification condition determination method applies the virus culture (ie, column flow through) that has passed through the chromatography column under different conditions to hemagglutination reaction, SDS-PAGE, or a combination thereof It can be characterized by selecting conditions in which the influenza virus loses the least amount.

In another aspect of the present invention, the conditions may be at least one selected from the group consisting of the type of buffer, the concentration of the buffer, the pH and conductivity of the buffer. Preferably, the condition may be the pH or conductivity of the buffer.

In one embodiment of the present invention, the pH of the buffer used for the first purification was maintained at 7.0 to 7.4 to increase the stability of the protein, and the conditions for best binding of the virus to the resin were explored while lowering the conductivity. The conductivity of the influenza virus culture obtained in the influenza virus culture process is about 10.0 to 15.0 mS / cm. It was diluted with sodium phosphate buffer having low conductivity, and the binding ability was confirmed while lowering the conductivity of the sample to be loaded on the column. In order to quickly check the purification conditions, the time and variables required to check the purification conditions were reduced by adjusting only the conductivity except for the pH, which is set as one of the purification conditions.

In general, it is known that the lower the conductivity, the better the influenza virus binds to the resin. However, when the conductivity is lowered, the amount of sodium phosphate buffer used and the chromatography process time increase, which increases the production period and production cost. It is important. Therefore, in one embodiment of the present invention, the virus culture solution is diluted 0.5 to 4 times using sodium phosphate buffer to lower the conductivity to 10.0 mS/cm or less, while passing through the column to determine whether the binding capacity is appropriate. Hemagglutination assay was performed on water. When the hemagglutination reaction method was performed, the test results could be confirmed within 1 hour, so the purification results according to each condition were confirmed within 1 day.

In another aspect of the present invention, the elution of the influenza virus bound to the resin may be performed by using a buffer solution having an appropriate concentration capable of separating the resin and the influenza virus, and searching for optimal conductivity.

According to one embodiment of the present invention, as a result of injecting the buffer into the column while increasing the salt concentration under 2-5 M sodium chloride buffer / linear gradient conditions, UV280 peaks appeared as viruses and proteins were eluted, and the fractions were recovered The analysis confirmed the conductivity and salt concentration of the fraction containing the virus. Viruses were eluted under step gradient conditions using the 2-5 M sodium chloride buffer and equilibration buffer, and after confirming that most influenza viruses were eluted at the elution concentration, virus elution conditions were established. Hemagglutination assay and/or SDS-PAGE were performed to confirm that the virus was eluted with high yield and high purity.

In one aspect of the present invention, the second purification condition determination method calculates the total protein quantification value (TPQV) per unit volume of the influenza virus culture obtained through the first purification and then substitutes it into the following formula 1 It can be characterized that the amount of surfactant to be treated with the virus solution is determined by the value obtained:

[Equation 1]

Surfactant Throughput = [{(a*TPQV(μg/mL))/(b μg/mL)}/c]* d

(In the above 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 (w/v%) of the treated surfactant stock;

d is the volume of the virus solution to treat the surfactant.)

The present invention is also a vaccine manufacturing method comprising the step of purifying surface antigen protein from influenza virus, wherein the step is obtained by the following formula after obtaining the total protein quantification value (TPQV) per unit dose of the sampled influenza virus The value obtained by substituting 1 into 1 provides a method which is carried out under conditions of treatment with a surfactant:

[Equation 1]

Surfactant Throughput = [{(a*TPQV(μg/mL))/(b μg/mL)}/c]* d

(In the above 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 (w/v%) of the treated surfactant stock;

d is the volume of the virus solution to treat the surfactant.)

The present invention also provides a method for rapidly purifying influenza surface antigens, comprising the following steps:

a) infecting cells with an influenza virus and culturing to obtain a virus culture;

b) In order to purify the influenza virus from the culture of step a), purification conditions based on hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus samples obtained by purification under different conditions determining;

c) purifying the influenza virus from the culture of step a) according to the conditions determined in step b);

d) In order to purify the surface antigen from the influenza virus solution purified in step c), determining the amount of surfactant to be purified during surface antigen protein purification based on the total protein quantification assay for the influenza virus solution ;

e) purifying the surface antigen protein from the influenza virus by treating the virus solution with a surfactant according to the conditions determined in step d).

In one aspect of the present invention, the method for rapidly purifying influenza surface antigens may further include, prior to step b), removing cells and cell debris from the virus culture. can

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. there is.

In another aspect of the present invention, step b) may be characterized in that purification conditions are determined based on hemagglutination assay and SDS-PAGE.

In another aspect of the present invention, the rapid purification method of the influenza surface antigen may be characterized in that it comprises an additional purification process by at least one method selected from the group consisting of ultrafiltration and diafiltration after step c). there is.

In another aspect of the present invention, the step e) may be characterized in that the surface antigen protein is separated by a method of recovering the supernatant by centrifugation (preferably, ultracentrifugation) after treatment with a surfactant. The surface antigen protein separated from the virus core by the surfactant treatment can be efficiently separated by centrifugation. This separation can be performed by pelleting the viral core, eg by ultracentrifugation. Viral surface proteins such as hemagglutinin and/or neuraminidase may be present in the supernatant and the viral core may be present in the pellet. The centrifugation conditions selected for pelleting the viral cores are known to those skilled in the art. For example, ultracentrifugation may be either a batch method or a continuous method, and the ultracentrifugation conditions used are each at a suitable temperature, for example, 2 to 25 ° C., more preferably 2 to 8 ° C. It may be performed at 30,000g for more than 10 minutes (min.) at 200,000g for more than 10 minutes.

In another aspect of the present invention, the rapid purification method of influenza surface antigen may be characterized in that it further comprises a surfactant removal process after step e). Removal of these surfactants may be effected by any method suitable for this purpose, for example by Amberlite or TFF treatment. When using amberlite treatment, after step e) has been performed, e.g., after pelleting the viral core, amberlite is added to the collected supernatant at a mass ratio of amberlite to mass of cationic surfactant of from about 1:1 to about 1:1. Add 100:1 and incubate at a suitable temperature, eg a temperature within the range of 2 to 8° C. for a suitable time, eg 8 to 24 hours, suitably 16 hours. Amberlite may then be removed by any suitable method known to those skilled in the art, for example by using conventional flow filtration/dead end filtration, sieves, or TFF. If a filter is used, it is preferably selected in such a way as to retain particles larger than 5 μm or larger than 7 μm. Amberlite can also be removed by using a sieve. The opening of a suitable sieve may be in the range of 0.1 to 200 μm, preferably less than 150 μm, more preferably less than 100 μm.

According to one embodiment of the present invention, CTAB used as a surfactant was removed by adding Amberlite XAD-4 (macroreticular cross-linked aromatic polymer). Since CTAB has a large molecular weight and is highly ionic, a method of removing it by direct adsorption was carried out. CTAB was adsorbed by treating Amberlite XAD-4 to the supernatant of ultra-high-speed centrifugation, reacting with stirring for more than 3 hours under refrigeration conditions at 2-8 ° C, and then filtering through a 0.22 μm filter to remove Amberlite XAD-4.

In another aspect of the present invention, the rapid purification method of the influenza surface antigen may include an additional chromatographic process 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, and the like, preferably may be anion exchange chromatography, most preferably TMAE (trimethylaminoethyl) anion exchange chromatography. Since DNA has an anion, it can be easily separated and removed by using the property of binding to anion exchange resin.

In another aspect of the present invention, the TMAE column may be equilibrated using a sodium phosphate buffer for equilibration, and the equilibration buffer may contain 1 to 20 mM sodium phosphate and have a pH of 6.8 to 7.6.

In one embodiment of the present invention, a sodium phosphate buffer containing pH 6.5 to 7.4 and 1 to 6M sodium chloride is used in the Amberlite XAD-4 adsorption filtration process to bind DNA to the resin and recover influenza surface antigens as a column flow through the column. Conductivity was adjusted by adding it to the finished influenza surface antigen process solution, and an appropriate level of surface antigen recovery was confirmed at a conductivity of 30 to 120 mS/cm.

In another aspect of the present invention, the rapid purification method of the influenza surface antigen further comprises an additional purification process by at least one method selected from the group consisting of ultrafiltration and diafiltration after chromatography for removing impurities It can be characterized by doing.

The present invention also provides a method for producing a vaccine comprising influenza surface antigen, characterized in that the vaccine production period is shortened, including the following steps:

a) infecting cells with an influenza virus and culturing to obtain a virus culture;

b) In order to purify the influenza virus from the culture of step a), purification conditions based on hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus samples obtained by purification under different conditions determining;

c) purifying the influenza virus from the culture of step a) according to the conditions determined in step b);

d) In order to purify the surface antigen from the influenza virus solution purified in step c), determining the surfactant throughput during surface antigen protein purification based on the total protein quantification assay for the influenza virus solution ;

e) purifying the surface antigen protein from the influenza virus by treating the virus solution with a surfactant according to the conditions determined in step d).

In one aspect of the present invention, the cells to be used for viral infection in step a) or the cells infected with the virus in step a) may be cultured in a single-use bioreactor (SUB). In the case of a disposable bioreactor, there is an advantage in that process preparation time is reduced and contamination concerns are very low because a process of washing and sterilization before use is unnecessary. In addition, since the disposable bioreactor can be discarded without a washing process after use and a new disposable bioreactor can be used, cross-contamination between lots or virus strains can be prevented. In particular, the influenza vaccine is a trivalent or tetravalent vaccine, which requires culturing three or four different viruses, so the use of a disposable bioreactor simplifies the process and can prevent cross-contamination between strains, which is preferable. .

In another aspect of the present invention, before infecting cells with the virus of step a), a part of the medium in the cell culture medium to be used for virus infection is exchanged with a fresh medium through a continuous low-speed centrifugation. . Through this, the medium can be exchanged in such a way that the medium is continuously separated from the cells and the medium is discarded and a new medium is introduced into the bioreactor without the process of recovering and reintroducing the culture medium. Unlike the general method of centrifuging by subdividing the medium into containers after recovering the entire amount, when using a continuous low-speed centrifugal separator, the medium can be exchanged in a closed system, which has the advantage of greatly reducing the possibility of contamination.

In another aspect of the present invention, some of the medium is 50 to 90%, preferably 60 to 80%, most preferably 70 to 80% of the existing medium is discarded, and the same amount of fresh medium is injected. can

In another aspect of the present invention, the rapid purification method of the influenza surface antigen may be characterized in that it further comprises a DNA cutting enzyme treatment process after step c). Since DNA derived from cells used for culturing influenza virus may be incorporated into the virus culture, a process of cutting and removing DNA to a size of 100 bp or less by treating with a DNA cutting enzyme may be added. In the present invention, the DNA cutting enzyme is not particularly limited in its type, but may be Benzonase, Exonuclease, Ribozyme, or a mixture thereof.

In another aspect of the present invention, the rapid purification method of influenza surface antigen may be characterized in that it further comprises a virus inactivation process after step c). Inactivation of the virus can be performed by treatment with surfactant, formaldehyde, β-propiolactone, methylene blue, psoralen, carboxyfullerene (C60), secondary ethylamine, acetyl ethylenimine or a combination thereof. can In addition, the inactivation method may be performed by physical means known to those skilled in the art, for example, by treatment with gamma irradiation and/or UV light.

It is possible to quickly and reliably check the conditions for obtaining (purifying) influenza surface antigens according to the unique method of the present invention without using the radioimmunodiffusion method, which has been previously used as a standard test method for influenza vaccine production, and thus the influenza surface antigen subunits As the vaccine production period is remarkably shortened, it has the expected effect of being able to quickly respond to vaccine development/manufacturing even in a situation where new influenza is quickly circulating.

Figure 1 is a supernatant or inactivated virus obtained by determining the CTAB throughput for a virus (B/Maryland/15/2016) based on the protein content according to the method of the present invention, treating it with an inactivated virus culture medium, and then ultra-high-speed centrifugation. This is the result of analyzing the protein contained in the liquid by SDS-PAGE (HA: Hemagglutinin, NP: Nucleoprotein, M: Matrix protein).
Figure 2 is a result of confirming the conductance conditions that minimize the loss of hemagglutinin in the column flow through SDS-PAGE and HA assay after performing the linear gradient purification process to determine the optimal conditions for virus binding to the CS column resin. (IVR-190 virus, linear gradient purification - chromatogram).
Figure 3 is a result of confirming the conductance conditions that minimize the loss of hemagglutinin in the column flow through SDS-PAGE and HA assay after performing the linear gradient purification process to determine the optimal conditions for virus binding to the CS column resin. (IVR-190 virus, linear gradient - chromatogram (Eluate fraction enlarged)).
Figure 4 is a result of confirming the conductance conditions that minimize the loss of hemagglutinin in the column flow through SDS-PAGE and HA assay after performing the linear gradient purification process to determine the optimal conditions for virus binding to the CS column resin. (IVR-190 virus, linear gradient purification - SDS-PAGE result).
Figure 5 is the result of analyzing the virus eluate under each condition by performing the HA assay and SDS-PAGE used in the method of the present invention.

Hereinafter, the present invention will be described in detail.

However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited to the following examples.

Example 1: New establishment of rapid search strategy for purification conditions in the process of purifying surface antigen from influenza virus

In the process of purifying surface antigens from influenza viruses, surfactants (ionic or nonionic) can be added to influenza viruses to separate and purify surface antigen proteins through fractionation means such as centrifugation. In this case, for example, cetyl trimethyl ammonium bromide (CTAB ) It is common to use an ionic surfactant such as

It is preferable to determine the amount of surfactant treated with influenza virus according to the content of hemagglutinin, a surface antigen of the virus. Since it takes about 3 days, it is not suitable as an in-process confirmation test, and in particular, if the supply of SRID standard products from NIBSC is not done or is delayed, the test cannot be performed.

On the other hand, in the surface antigen purification process from influenza virus, the amount of surfactant capable of selectively separating only the surface antigens hemagglutinin and neuraminidase without crushing the virus core is different for each virus. Surface antigen vaccine is a high-quality vaccine with few side effects and high purity, and it is very important to set an appropriate amount of surfactant. Excessive surfactant treatment disrupts the entire virus and exposes the protein inside the virus core, which is an impurity, to the outside, so the purity of surface antigens in the purified product decreases. As a result, the yield of surface antigen is reduced. In addition, it is an important factor in the influenza vaccine production process to quickly confirm the appropriate surfactant throughput.

The present inventors first invented the method of the present invention, which will be described later, after various comparative experiments using various analytical techniques, enabling prompt setting of surfactant treatment conditions suitable for surface antigen separation without relying on the SRID method, as well as substantially existing It was also confirmed that there was a significant relationship with the method based on the standard test method SRID, and reliability and reproducibility were supported. The following examples support that the effect according to the method unique to the present invention is a special effect that is difficult to predict from conventional conventional processes. The following examples typically show an example of using CTAB as a surfactant, which is abbreviated as CTCCT (CTAB treatment concentration confirmation test).

1-1. New establishment of SRID-independent surface antigen purification process condition setting method

The present inventors used a total protein quantification value (TPQV) per unit dose of influenza virus sampled from an inactivated influenza virus concentrate to obtain an optimized surfactant (here as an example) for surface antigen purification. CTAB) by implementing an algorithm for estimating the throughput, a calculation relation such as Equation 1 below was derived. The measurement of the total protein quantitative value (total protein content value) could be confirmed within 1 hour by performing a BCA assay (or lowry assay, Bradford assay, etc.). The total protein quantification value as a standard may vary depending on the strain of the virus, but as a result of analyzing the protein content of the inactivated virus concentrate used in the experiment in this study, it was confirmed to be 200 ~ 600 μg/mL, and the protein content of the inactivated virus solution After measuring 100, 200, 300, 400, 500, 600, 700, 800 or 900 μg / mL, the value corresponding to the approximate value of the TPQV measurement was set as the standard value of protein content for CTAB treatment. The CTAB treatment amount was calculated according to Equation 1 below, and the CTAB stock treatment concentration (w/v%) relative to the protein content of the inactivated virus solution was determined within 0.005% to 0.100% (v/v).

[Equation 1]

Surfactant Throughput = [{(a*TPQV(μg/mL))/(b μg/mL)}/c]* d

(In the above 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 (w/v%) of the treated surfactant stock;

d is the dose of the sampled influenza virus to treat the surfactant.)

Since the CTAB stock processing concentration (w/v%) based on protein content differs depending on the strain and virus characteristics, SDS-PAGE is performed for an arbitrary range to obtain an appropriate CTAB stock processing concentration (w/v%). v%) was determined by checking. That is, after subdividing the inactivated virus solution in small portions and treating it with CTAB for a certain range, ultra-high-speed centrifugation is performed under the condition of 30,000 rpm or more according to the SKYCellflu manufacturing process, and the surface antigen protein is recovered in the supernatant, and the virus core The optimal CTAB treatment concentration at which proteins were separated as precipitates was confirmed.

The optimal CTAB treatment concentration identified was applied to the manufacturing process. As a result of performing SDS-PAGE using the sample (B/Maryland/15/2016 virus) obtained by performing the above-described process, the results shown in FIG. 1 were obtained. When CTAB is treated at a concentration of 0.01% to 0.10%, impurities other than hemagglutinin found in the inactivated viral fluid such as nucleoprotein and matrix protein are removed when ultra-high-speed centrifugation is performed after CTAB treatment, and only hemagglutinin It was confirmed that the supernatant was recovered by ultra-high-speed centrifugation.

1-2. Confirmation of reliability and reproducibility of surface antigen purification process condition setting method according to the present invention _ Correlation with existing SRID method-based method

Table 1 summarizes the 2,000L scale commercial production data, and according to Equation 1, the surfactant treatment amount was obtained based on the protein content, and the CTAB treatment process was performed.

In addition, the HA content of the inactivated virus solution and the HA content of the ultra-high-speed centrifugation supernatant obtained by performing the ultra-high-speed centrifugation process after CTAB treatment of the inactivated virus solution were obtained and compared. Sufficient commerciality was confirmed at a level of 70% to 80% compared to the HA content of the liquid. When CTAB is excessively treated to increase the HA recovery rate, it is very important to obtain an appropriate amount of CTAB treatment because it may be produced in the form of a split vaccine rather than a subunit vaccine to be produced.

Therefore, it was confirmed that the surfactant throughput calculation method according to Equation 1 established in the present invention is a method capable of quickly and accurately confirming the CTAB throughput required in the process of purifying surface antigens of influenza viruses.

1-3. Comparison of purification condition setting based on the existing SRID method (Comparative Example 1) and purification condition setting execution time according to the present invention

As performed in Example 1-2, surface antigen purification conditions were set by the existing SRID (radioimmuno-diffusion method), and purification conditions were set according to the present invention (see Examples 1-1 and 1-2). As a result of comparing the process execution time, as shown in Table 2 below, it was confirmed that the execution time according to the method of the present invention is much shorter than that of the conventional radioimmunodiffusion method and is more efficient in confirming the conditions for purifying influenza surface antigens.

Example 2: Establishment of rapid influenza virus antigen purification condition test set (abbreviated as RIVPCT) after virus culture

In the process of producing subunit vaccines containing influenza surface antigens, the purification process consists of two major steps: purification of influenza virus from cell culture infected with influenza virus and separation of surface antigen protein from influenza virus. it is needed In the existing vaccine production process, process conditions can be optimized using the standard test method single radioimmunoassay (SRID) in the above two process steps, but as described above, SRID has several disadvantages in vaccine production. Accordingly, the present inventors searched for a novel method for setting suitable process conditions without using a single radioimmunoassay diffusion method (SRID), and developed the following method for determining the first purification condition and the method for determining the second purification condition (based on the new strategy established in Example 1). A set of rapid confirmation test sets containing influenza virus antigen purification conditions was developed. A set of methods for determining first purification conditions and methods for determining second purification conditions in the present invention is referred to herein as RIVPCT.

2-1. Method for determining the first purification conditions _ Separation/purification/concentration of influenza virus from influenza virus culture

In setting purification conditions used in the step of purifying influenza virus from influenza virus culture, a decision/discrimination method for quickly setting optimal conditions for each virus without measuring SRID is provided. Specifically, the conditions for the purification of the influenza virus were determined based on the Hemagglutination assay, SDS-PAGE, and criterion specific to the present invention for influenza virus samples obtained by purification under different conditions. Provides a test method/criterion for determining

In the following examples, an example of using chromatography as a representative virus purification means is shown. Before the virus culture medium is introduced into the chromatography column, pretreatment in which cells and cell debris are primarily removed from the virus culture medium may be performed. can be performed

In order to proceed with the chromatography process, it is necessary to set different conditions for each influenza virus. Due to the nature of influenza viruses, which are subject to change in vaccine production recommendations every year, it is necessary to quickly check the purification conditions for each virus strain. In order to set chromatographic purification conditions, after confirming the conditions in which the influenza virus can best bind to the resin, a test for elution conditions that can separate and purify the bound virus with optimal yield and purity is performed. Hereinafter, as a representative example, a method for determining influenza virus purification conditions (binding conditions and elution conditions for CS chromatography column resin) when using Cellufine Sulfate (CS) chromatography is shown, and this is referred to as CS Chromatography Purification Condition Test (CSPCT). abbreviation

The principle of virus binding to the resin of the chromatography column is an ionic bond by affinity between the surface protein of influenza virus and the sulfate group of the resin, and the binding ability is generally determined by conductivity and pH. This example exemplarily shows that the neutral pH of the buffer used in the process is maintained at 7.0 to 7.4 to increase the stability of the protein and to find conditions in which the virus can bind to the maximum while lowering the conductivity. The conductivity of the generally obtained influenza virus culture (liquid) is about 10.0 to 15.0 mS/cm, and the conductivity of the sample to be loaded on the column is lowered by diluting it with sodium phosphate buffer having low conductivity to confirm the binding ability. In this example, in order to quickly check process conditions, it is shown as an example that the time and variables required for checking the purification conditions are reduced by a simple method of adjusting only the conductivity except for the pH, which is generally set as one of the purification conditions.

In general, it is known that the lower the conductivity, the better the influenza virus binds to the CS resin. It's important to check. Therefore, hemagglutinin confirmation test (HA) for the column flow through column to check whether the binding capacity is appropriate while lowering the conductivity to 10.0 mS/cm or less by diluting the virus culture medium 0.5 to 4 times using sodium phosphate buffer. assay) was performed.

Specifically, the HA assay was performed according to the protocol described in the ‘Manual for the laboratory diagnosis and virological surveillance of influenza’ prepared and distributed by the WHO. According to the above method, the appropriate conductivity of the loading sample was confirmed based on the decreasing trend of the HA titer of the column flow through, and SDS-PAGE was also performed at the same time to confirm whether or not influenza virus was lost to the column flow through for each condition.

After determining the optimal conditions for virus binding to the CS column resin as described above, conditions suitable for virus elution are sought. Elution of the influenza virus bound to the resin was performed by using a buffer solution containing sodium chloride at an appropriate concentration capable of separating the resin from binding, and searching for optimal conductivity. When it is added to the column while increasing the salt concentration under linear gradient conditions with 2-5M sodium chloride buffer, viruses and proteins are eluted and UV peaks appear. These fractions are recovered and the conductivity and salt concentration of the fraction containing the virus are analyzed by HA assay and SDS- It was confirmed by performing PAGE. Based on the virus elution conditions identified here and the conductivity conditions for which the virus binding capacity was determined to be appropriate, the virus was eluted under step gradient conditions using 2-5 M sodium chloride buffer and sodium phosphate buffer, and most of the virus was eluted at the elution concentration. Virus elution conditions were established after confirming that the influenza virus was eluted. In order to confirm that the virus was eluted with high yield and high purity, HA assay was performed and confirmed according to the same method and criteria as described above. The confirmation was performed with SDS-PAGE.

The CS chromatography process condition test (CSPCT) for purifying the virus from the recovered virus culture medium, which is performed in a series of processes as described above, can be completed within about 6 to 12 hours, and the strain for vaccine production is changed every year. Due to the nature of the influenza vaccine being produced, this reduction in time is also associated with a reduction in the overall production period.

When setting chromatographic conditions, the standard test method, Single Radial Immunodiffusion technique (SRID), is performed to check the hemagglutinin content in the column flow through. Cons there is When HA assay and SDS-PAGE are performed according to the present invention, the test results can be checked within 3 hours, so the purification condition test for each virus is performed within 1 day, so it is very fast and there are no issues such as supply of standard products, so the test is performed It can be seen that there are few restrictions on The method of confirming the binding ability of the influenza virus by appropriately lowering the conductivity and quickly confirming the conditions for eluting the virus with high yield and high purity within 12 hours can be said to be the characteristics of the above-mentioned CSPCT, and the principle and procedure are as described above. .

2-2. Method for determining the second purification conditions_ Separation and purification of surface antigens from influenza virus

For separation and purification of surface antigens from influenza virus, this example presents an example using CTAB as a surfactant. The CTAB throughput was quickly confirmed by performing CTCCT among RIVPCTs in the same manner as in Example 1 above.

2-3. Purification effect of surface antigen protein according to the method for determining the first purification condition and the method for determining the second purification condition

If the conditions necessary for the purification processes necessary for vaccine production are obtained based on the HA content confirmed by performing SRID, more accurate purification conditions can be set. It has the advantage of being able to identify them more quickly. When performing the method using SRID, it takes more than 2 days even excluding the availability of standard products and the warehousing period. To shorten this time, the RIVPCT method using SDS-PAGE, HA assay, etc. was invented.

Example 3: Influenza subunit vaccine production process with reduced production period

3-1. Infection and culture of influenza virus in cells

1) MDCK (Madin-Darby Canine Kidney cells) cell culture

1 vial of the MDCK-Sky3851 cell line for vaccine production in frozen storage was thawed in a constant temperature water bath at 37° C. and diluted with a culture medium. Next, the diluted cells were centrifuged to separate the cells from the medium, and then the cell pellet was released into fresh medium again, and samples were taken to measure cell number and viability. The cells were inoculated in a 125 mL spinner flask at an inoculation concentration of 1x10 ⁵ to 5x10 ⁵ cells/ml, and cultured for 3 to 4 days under agitation at 80 rpm at 37°C and 5% CO ₂ .

The cell number and viability of MDCK cells cultured in the 125mL spinner flask were measured, and the cell culture medium was inoculated enough to start the culture in a 250mL scale in a 500mL spinner flask, and stirred at 100rpm at 37°C and 5% CO ₂ conditions. The primary seed culture was performed for ~4 days. The cells were cultured in a batch process, and a chemically defined medium was used as the cell culture medium and L-glutamine was added at a concentration of 3 to 6 mM/L. After the primary seed culture, after measuring the cell number and viability, cells were inoculated into two 1L spinner flasks to start the culture at a scale of 500 mL, and cultured for 3 to 4 days under the same conditions as the primary seed culture.

After taking a sample from the 1L spinner flask in the 500mL culture and measuring the cell number and viability, aseptically inoculate the culture medium of the 1L spinner flask to the cell culture medium enough to start culture at a 5L scale in a 10L disposable cell culture medium, Incubated for 3-4 days. Pre-sterilize and prepare the instruments and pH and DO probes necessary for inoculating the cell culture medium in the spinner flask into the cell culture medium. After installing the pH and DO probes and injecting the culture medium, set the temperature to 37±1℃ and set the DO value. was calibrated to prepare the cells for inoculation.

In the same way, cell culture was sequentially performed up to a 50L scale, a 100L scale, a 500L scale, and a 2000L scale.

2) Viral infection

The cell number and viability were measured by taking the culture medium of the cell culture medium being cultured on the 2000L scale, and when a cell number of 1x10 ⁶ to 3x10 ⁶ cells/ml or more was obtained, the existing culture medium was replaced with a fresh medium. The culture medium was replaced by removing 70 to 80% of the medium based on the cell culture medium using a continuous low-speed centrifuge in a disposable bag for cell culture, and then reintroducing fresh medium.

During the medium exchange and virus infection process, the inside of the cell culture medium is maintained in an airtight environment, and the material used for medium exchange is a sterile disposable bag, so the possibility of contamination is very low. The process time required for the process was 3 to 4 hours for a 10L scale and 7 to 12 hours for a 2000L scale commercial production.

Virus infection and propagation were carried out under DO 50±10%, pH 7.2±0.2 conditions, and the influenza virus strain to be prepared was melted, and the amount of virus calculated at MOI of 0.0001 to 0.002 and trypsin at a concentration of 2.5 to 20 μg/mL were used. was aseptically put into a cell incubator to infect cells, and cultured for about 3 to 4 days. When the virus titer, cell number, and viability reached an appropriate level, the culture was terminated and the culture medium was recovered.

In the cell culture and virus infection methods, microcarriers are not used, and in particular, cells are cultured and viruses are propagated in a batch cell culture system rather than a perfusion system. Since the influenza virus proliferates inside the cells after being infected with the MDCK-Sky3851, bursts the cells, and ejects to the outside, when the cells are cultured in a perfusion system, the number of cells gradually decreases, which is not suitable for the system of the present invention. did not

3-2. Conditioning and Virus Purification for Influenza Virus Purification from Viral Cultures

1) Centrifugation and filtration

When the virus culture in the cell incubator is completed, the virus culture medium is centrifuged at a speed of 8,000 to 20,000 rpm using a continuous low-speed centrifuge to remove cells and cell debris by recovering the supernatant. The collected supernatant was filtered through a 1.0/0.5 μm prefilter and then filtered again through a 0.45 μm size filter to remove particles larger than a certain size.

2) Setting purification conditions and purification of influenza virus using cellufine sulfate (CS) affinity resin

The filtered virus culture medium was subjected to the first chromatography process using a resin (cellufine sulfate) having affinity for influenza virus. After equilibrating the column using sodium phosphate buffer for equilibration, the virus filtrate was added. Purification conditions were rapidly set by the CSPCT method as described in Example 2-1 above. A purification condition setting test was performed using IVR-190 (A/Brisbane/02/2018 (H1N1) pdm09-like virus), and the results between the CSPCT method and the SRID method were compared and analyzed.

First, in order 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 HA assay to confirm conductivity conditions that minimized loss of hemagglutinin in the column flow through ( 2 to 4, Table 4).

IVR-190 virus culture medium and sodium phosphate buffer for equilibration were diluted 1:1 to lower the conductivity from 13.03 mS/cm to 7.54 mS/cm, and a linear gradient purification condition test was performed. On SDS-PAGE, no HA was lost as the column flow through, and it was confirmed that impurities except for HA were eluted. HA assay results using 0.5% chicken RBC also confirmed that 0 HAU/50μL was confirmed in all column flow-through fractions, confirming that the conductivity of the loading sample used in the test was suitable for the first chromatographic purification.

Regarding the confirmation of the virus elution concentration, sodium phosphate buffer for elution containing sodium chloride and sodium phosphate buffer were diluted in an appropriate ratio, and the UV280 peak was confirmed and the eluted virus fraction was collected. As a result, the concentration of 0.1 M to 0.2 M NaCl conditions, the highest peak (UV280 value) and HA titer were confirmed on the chromatogram, and at the same time, it was confirmed that the peak and HA titer gradually decreased until the 0.3 M NaCl concentration condition. Confirmed. Summarizing the test results, it was determined that the conductivity of the sample to be loaded on the CS column was 7.54 mS/cm, and 0.4 M NaCl was determined to be appropriate for the virus elution concentration.

After setting the conductivity conditions of the loading sample according to the CSPCT method described above, in order to compare with the SRID-based method, the virus elution concentration was set to 0.3 M, 0.35 M, and 0.4 M NaCl to perform a step gradient purification process, and each The HA content of the virus eluate of the batch was compared. In addition, SDS-PAGE and HA assay were performed to confirm the correlation between the results obtained by the CSPCT method of the present invention and the method using SRID.

As a result of SRID, it was confirmed that the best yield and purity were obtained when the virus was eluted at a concentration of 0.4 M NaCl (Table 5).

HA assay used in the CSPCT method and SDS-PAGE were performed using the sample used for SRID to analyze the virus eluate under each condition.

Referring to the SDS-PAGE results of FIG. 5, it was confirmed that the HA band became thicker as the virus elution concentration increased, and through this, it was found that the HA contained in the virus eluate increased. In the case of the HA titer of the virus eluate, it was the same as 1,024 HAU/50μL under the 0.3 M and 0.35 M NaCl concentration conditions, but increased to 2,048 HAU/50μL under the 0.4 M condition. As such, the virus elution conditions confirmed by the CSPCT method also confirmed that the virus elution concentration was 0.4 M NaCl, the same as the SRID method, confirming the correlation between the two methods (Table 6).

On the other hand, the pH of the equilibration buffer used in the purification process is neutral at 7.0 to 7.4, and the virus filtrate is diluted with the equilibration buffer to lower the conductivity to which the virus can bind as much as possible. At the end of the virus culture, the conductivity of the virus culture medium is about 10.0 ~ 15.0 mS / cm. As a result of diluting this with an equilibration buffer with low conductivity and lowering the conductivity, the binding ability with the resin was confirmed. An appropriate level of binding capacity was shown under the conditions of 4.0 to 10.0 mS/cm.

After adding the virus filtrate diluted with equilibration buffer to the CS column, the column passing liquid that flowed out without binding to the column was discarded after sampling, and 2 to 3 CV of equilibration buffer was introduced to thoroughly wash the column, including sodium chloride. Sodium phosphate buffer for elution and sodium phosphate buffer diluted in an appropriate ratio were added, and the eluted virus fraction was collected while confirming the UV280 peak. Elution of the influenza virus was performed by performing CSPCT (see Example 2-1) among RIVPCT to establish conditions by confirming an elution buffer containing an appropriate concentration of sodium chloride capable of separating the binding between the virus and the resin, and most viruses It was eluted at a sodium chloride concentration of 1 M or less.

3-3. Virus enrichment and desalting

A first ultrafiltration is performed to concentrate the virus eluate and exchange it into a PBS buffer. Concentrate to 10% or less based on the volume of the cell culture medium using an ultrafiltration filter of 300 kDa to 800 kDa size, and dilute with a PBS buffer that is 8 times or more of the concentrated volume until the conductivity is equal to that of the PBS buffer. Filtration was performed.

3-4. MDCK cell-derived DNA removal in virus concentrate

In order to remove cell line-derived DNA from the recovered virus concentrate, DNA cleavage enzyme benzonase (Merck Co.) was treated with 0.5 to 50 U/mL and reacted at room temperature for 30 minutes to 24 hours with slow stirring. As a cofactor for benzonase, 0.5 to 5 mM magnesium chloride hexahydrate was added to increase the activity of benzonase.

3-5. virus inactivation

Influenza virus concentrate after completion of benzonase reaction was inactivated to remove infectivity. Any chemical means for inactivating the virus can be used as long as it is known, and examples include detergent, formaldehyde, betapropiolactone, methylene blue, psoralen, carboxyfullerene (C60), and the like. In this embodiment, formaldehyde The virus was inactivated by treating with an aldehyde solution at a level of 0.01 to 0.1%.

3-6. Separation of surface antigen using detergent

Surface antigen proteins can be separated and purified by treating the influenza virus with a surfactant (ionic or nonionic) or a solvent. As a representative example, it is common to use an ionic surfactant such as cetyl trimethyl ammonium bromide (CTAB).

In this Example, CTAB was treated based on the protein content of the inactivated virus concentrate, and the amount of CTAB treated was confirmed by performing CTCCT among RIVPCTs as in Example 2-2. After reacting at refrigeration or room temperature for 1 hour or more, centrifugation using a continuous ultra-high-speed centrifuge at 30,000 rpm or more, and then the supernatant is recovered to separate only the surface antigen protein.

In order to remove CTAB in the ultra-high-speed centrifugation supernatant, adsorption resin (Amberlite XAD-4) was added to the supernatant to remove CTAB. Then, the resin for adsorption was removed by filtration through a filter.

3-7. Residual DNA Removal Using TMAE Anion Exchange Resin

In order to further remove DNA derived from the host agent, a second chromatography process was performed using an anion exchange chromatography resin. After equilibrating the column using sodium phosphate buffer for equilibration, the virus surface antigen recovery filtrate is introduced. At this time, it is diluted with sodium phosphate buffer containing 1 to 6 M sodium chloride with high conductivity, and a chromatography process is performed with the conductivity that the surface antigen does not bind to the resin and the DNA binds to the resin. It is recovered as a surface antigen fraction.

3-8. Concentration and desalting of surface antigen fractions

The surface antigen fraction was concentrated using an ultrafiltration filter having a size of 30 to 50 kDa. After the concentration process was completed, buffer exchange was performed using a PBS buffer solution until the conductivity was the same as that of the PBS buffer solution, and the concentrated solution was recovered. The recovered concentrate was bactericidally filtered with a 0.5/0.2um filter to produce a stock solution and stored at 2-8°C.

It is possible to quickly and reliably check the conditions for obtaining (purifying) influenza surface antigens according to the unique method of the present invention without using the radioimmunodiffusion method, which has been previously used as a standard test method for influenza vaccine production, and thus the influenza surface antigen subunits The vaccine production period is remarkably shortened, so that even in a situation where a new type of influenza is rapidly spreading, response to vaccine development/manufacturing can be made quickly, so industrial applicability is very high.

Claims (31)

In the method for purifying surface antigen proteins from influenza viruses using CTAB (cetyl trimethyl ammonium bromide), the CTAB is a total protein quantification value (TPQV) per unit dose of the sampled influenza virus After the calculation, the CTAB stock throughput to be treated with the sampled influenza virus is determined by the value obtained by substituting it into Equation 1 below, and the TPQV of the sampled influenza virus is 50 to 950 μg / mL. :
[Equation 1]
CTAB stock throughput = [{(a*TPQV(μg/mL))/(b μg/mL)}/c]* d
(In the above formula,
a is 0.04 to 0.08,
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 (w/v%) of the treated CTAB stock;
d is the dose of the sampled influenza virus to treat the CTAB stock.)
The method of claim 1, wherein the CTAB stock throughput is,
A method characterized in that for selectively isolating only the virus core in a non-destructive state or in a state in which destruction of the virus core is minimized.
The method of claim 1, wherein the CTAB stock throughput determination is
A method characterized in that a quantification process by SRID (Single Radial Immunodiffusion) is not required for the amount of hemagglutinin (HA) protein in the sample.
delete delete The method according to claim 1, wherein the total protein quantification value (TPQV) is obtained by BCA assay, Lowry assay or Bradford assay.
delete A test method for rapid confirmation of influenza antigen purification conditions, comprising the following first purification condition determination method and second purification condition determination method:
As the first purification condition determination method, it is used in the step of purifying influenza virus from influenza virus culture, and the conditions for purifying the influenza virus are the hemagglutination reaction method (Hemagglutination reaction) for influenza virus samples obtained by purification under different conditions. assay), SDS-PAGE, or a first purification condition determination method determined based on a combination thereof; and
As a method for determining the second purification conditions, it is used in the step of purifying the surface antigen protein from the influenza virus using CTAB, and the CTAB stock throughput during the purification of the surface antigen protein is the influenza virus obtained through the first purification. The second purification condition determination method, characterized in that the throughput of the CTAB stock is determined by the value obtained by obtaining the total protein quantification value (TPQV) per unit volume of culture and then substituting it into Equation 1 below:
[Equation 1]
CTAB stock throughput = [{(a*TPQV(μg/mL))/(b μg/mL)}/c]* d
(In the above formula,
a is 0.04 to 0.08,
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 (w/v%) of the treated CTAB stock;
d is the volume of the influenza virus culture to treat the CTAB stock;
The TPQV of the influenza virus culture is 50 to 950 μg/mL.)
The method of claim 8, wherein the influenza antigen purification condition rapid confirmation test method does not require a quantification process by SRID (Single Radial Immunodiffusion) for the amount of hemagglutinin (HA) protein in the influenza virus sample. Characterized in that method.
The method of claim 8, wherein the step of purifying the influenza virus from the influenza virus culture is performed by chromatography.
11. The method of claim 10, wherein the chromatography is performed by chromatography filled with Cellufine Sulfate (CS).
The method of claim 8, wherein the first purification condition determination method is determined by performing a hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus culture samples purified under different conditions, respectively. How to.
13. The method of claim 12, wherein the conditions include buffer type, buffer concentration, pH, and conductivity.
13. The method of claim 12, wherein said condition is conductivity.
delete A vaccine manufacturing method comprising the step of purifying surface antigen protein from influenza virus, wherein the step is to calculate the total protein quantification value (TPQV) per unit dose of the sampled influenza virus and substitute it into the following formula 1 to obtain The obtained value is carried out under the conditions of processing the CTAB stock, and the sampled influenza virus has a TPQV of 50 to 950 μg / mL:
[Equation 1]
CTAB stock throughput = [{(a*TPQV(μg/mL))/(b μg/mL)}/c]* d
(In the above formula,
a is 0.04 to 0.08,
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 (w/v%) of the treated CTAB stock;
d is the dose of the sampled influenza virus to treat the CTAB stock.)
A rapid purification method of influenza surface antigen, comprising the following steps:
a) infecting cells with an influenza virus and culturing to obtain a virus culture;
b) In order to purify the influenza virus from the culture of step a), purification conditions based on hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus samples obtained by purification under different conditions determining;
c) purifying the influenza virus from the culture of step a) according to the conditions determined in step b);
d) In order to purify the surface antigen from the influenza virus purified in step c), the total protein quantification value (total protein quantification value, TPQV) and then substituting it into Equation 1 to determine the throughput of the CTAB stock;
[Equation 1]
CTAB stock throughput = [{(a*TPQV(μg/mL))/(b μg/mL)}/c]* d
(In the above formula,
a is 0.04 to 0.08,
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 (w/v%) of the treated CTAB stock;
d is the volume of the influenza virus culture to treat the CTAB stock;
The purified influenza virus has a TPQV of 50 to 950 μg/mL.)
e) purifying the surface antigen protein from the influenza virus by treating the CTAB stock according to the conditions determined in step d).
18. The method according to claim 17, wherein the rapid purification method of influenza surface antigen further comprises removing cells and cell debris from the virus culture before step b).
The method of claim 18, wherein the removal of cells and cell debris from the virus culture is performed by one or more methods selected from the group consisting of filtration, dialysis, and centrifugation.
The method of claim 17, wherein the rapid purification method of the influenza surface antigen comprises an additional purification process by at least one method selected from the group consisting of ultrafiltration and diafiltration after step c).
The method of claim 17, wherein step e) is to separate the surface antigen protein by centrifuging the supernatant after treatment with CTAB stock.
The method of claim 17, wherein the rapid purification method of influenza surface antigen further comprises a CTAB removal process after step e).
18. The method of claim 17, wherein the rapid purification method of the influenza surface antigen comprises an additional chromatography to remove impurities after step e).
24. The method of claim 23, wherein the chromatography is TMAE (trimethylaminoethyl) anion exchange chromatography.
25. The method of claim 24, wherein the rapid purification method of the influenza surface antigen comprises an additional purification process by at least one method selected from the group consisting of ultrafiltration and diafiltration after chromatography.
A method for producing a vaccine containing influenza surface antigen, characterized in that the vaccine production period is shortened, comprising the following steps:
a) infecting cells with an influenza virus and culturing to obtain a virus culture;
b) In order to purify the influenza virus from the culture of step a), purification conditions based on hemagglutination assay, SDS-PAGE, or a combination thereof for influenza virus samples obtained by purification under different conditions determining;
c) purifying the influenza virus from the culture of step a) according to the conditions determined in step b);
d) In order to purify the surface antigen from the influenza virus purified in step c), the total protein quantification value (total protein quantification value, TPQV) and then substituting it into Equation 1 to determine the throughput of the CTAB stock;
[Equation 1]
CTAB stock throughput = [{(a*TPQV(μg/mL))/(b μg/mL)}/c]* d
(In the above formula,
a is 0.04 to 0.08,
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 (w/v%) of the treated CTAB stock;
d is the volume of the influenza virus culture to treat the CTAB stock;
The purified influenza virus has a TPQV of 50 to 950 μg/mL.)
e) purifying the surface antigen protein from the influenza virus by treating the CTAB stock according to the conditions determined in step d).
27. The method of claim 26, wherein the cells to be used for virus infection in step a) or the cells infected with virus in step a) are cultured in a single-use bioreactor (SUB).
27. The method according to claim 26, wherein, before infecting the cells with the virus of step a), a part of the medium in the cell culture to be used for virus infection is exchanged with a fresh medium through a continuous low-speed centrifugal separator.
The method of claim 26, wherein the rapid purification method of influenza surface antigen further comprises a process of treating Benzonase, Exonuclease, Ribozyme, or a mixture thereof after step c). method.
27. The method of claim 26, wherein the rapid purification method of influenza surface antigen further comprises a virus inactivation process after step c).
The method of claim 30, wherein the virus inactivation agent (detergent), formaldehyde, β-propiolactone, methylene blue, psoralen, carboxyfullerene (C60), secondary ethylamine, acetyl ethyleneimine or a combination thereof A method characterized in that it is performed by processing.

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