Chromatographic methods for purifying hepatitis a virus

Abstract

To provide methods for purifying hepatitis A virus comprising chromatography steps removing impurity with efficiency as compared with conventional techniques.SOLUTION: Disclosed is a method for purifying hepatitis A virus, comprising: a) culturing cells infected with hepatitis A virus to collect cultured cells; b) solubilizing collected cells to obtain a solubilized liquid; c) separating hepatitis A virus from the obtained solubilized liquid using an anion exchange chromatography comprising a chromatography carrier of crosslinked agarose having diethylamino propyl to obtain an eluate; d) subjecting the obtained eluate to gel filtration chromatography.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for purifying Hepatitis A virus by anion exchange chromatography in the production of Hepatitis A vaccines, and more particularly to a method for purifying Hepatitis A virus using a chromatographic carrier comprising cross-linked agarose having diethylaminopropyl as an anion exchange functional group. [Background technology]

[0002] Hepatitis A virus is an icosahedral RNA virus of the Picornaviridae family with a diameter of 27 nm. There are seven genotypes, and the prevalent types vary in different regions of the world, but there is only one serotype, so there is no difference in immunogenicity depending on the genotype.

[0003] Hepatitis A virus can grow in cultured cells, but the growth rate is slow, taking 2 to 3 weeks. It generally does not show cytopathic effects and remains intracellularly.

[0004] When hepatitis A virus infects humans, after an incubation period of 2-6 weeks, symptoms such as fever and fatigue are followed by an increase in serum transaminase. Other symptoms include loss of appetite and vomiting, as well as jaundice, hepatomegaly, dark urine, and gray-white stools. In general, the disease does not become chronic and people recover after 1-2 months, but in rare cases, fulminant hepatitis may develop. In recent years, the risk of infection with hepatitis A virus has increased due to an increase in the number of people traveling overseas, and the demand for hepatitis A vaccines is on the rise.

[0005] A conventional method for producing a Hepatitis A vaccine is as follows (Patent Document 1). [1] The hepatitis A virus for vaccine production is inoculated into GL37 cells, a cell line derived from the kidneys of African green monkeys, and infected; [2] Infected hepatitis A virus was propagated in GL37 cells by roller bottle culture for three weeks; [3] After the incubation, the cells are lysed with a detergent to recover the Hepatitis A virus and obtain a cell lysate; [4] The cell lysate is clarified by filtration, concentrated by salting out and centrifugation, and then treated with chloroform, enzymes, and organic solvents to obtain a purified virus solution; [5] The purified virus liquid is inactivated with formalin to create the original vaccine solution.

[0006] As an improvement to the method for producing a hepatitis A vaccine, a purification method has been disclosed in which chromatography operations using an anion exchanger and gel filtration are performed in the presence of a surfactant (Patent Document 2). In the anion exchange chromatography in Patent Document 2, conditions are set in which hepatitis A virus particles are temporarily retained on a carrier and then eluted. However, the anion exchanger actually examined in the prior patent document is only a specific DEAE-based carrier. Also disclosed is a method for producing hepatitis A virus, which comprises treating a hepatitis A virus preparation recovered after growth in a cell line with a protease, separating the hepatitis A virus from protease-digested proteins, and inactivating the virus (Patent Document 3), which also discloses an ion exchange step as a purification step after protease digestion. Although the above-mentioned prior art exists, further improvements are required for the removal of impurities by anion exchangers. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 1-279843 [Patent Document 2] Patent Publication No. 6-279317 [Patent Document 3] Special table 2002-527105 Summary of the Invention [Problem to be solved by the invention]

[0008] Conventional methods for purifying the virus in Hepatitis A vaccine production use the harvest liquid (solubilized liquid) after virus cultivation as the starting material, and involve many manual open-system operations such as centrifugation and extraction, which limits scale-up. Improved purification methods have been studied with the aim of improving production capacity and shortening the process, but there is a problem of efficiently removing impurities in the chromatography process in order to obtain purified virus liquid of the same quality as the conventional method.

[0009] Therefore, an object of the present invention is to provide a method for purifying Hepatitis A virus that incorporates a chromatography step that can remove impurities more efficiently than conventional techniques. [Means for solving the problem]

[0010] As a result of extensive investigations to achieve the above-mentioned object, the inventors discovered that impurities can be efficiently removed and Hepatitis A virus can be purified by using a carrier containing cross-linked agarose having diethylaminopropyl as a functional group as an anion exchange chromatography carrier in the chromatography step, thereby completing the present invention.

[0011] Therefore, the present invention includes the following inventions: [Section 1] 1. A method for purifying Hepatitis A virus, comprising: a) culturing cells infected with Hepatitis A virus and recovering the cultured cells; b) solubilizing the collected cells to obtain a lysate; c) separating the Hepatitis A virus from the obtained lysate using anion exchange chromatography with a chromatographic support comprising cross-linked agarose with diethylaminopropyl, and then obtaining an eluate; and d) subjecting the obtained eluate to gel filtration chromatography; A method for purifying Hepatitis A virus, comprising: [Section 2] Item 2. The method according to Item 1, wherein in step c), the separated Hepatitis A virus is eluted with a high salt buffer; [Section 3] Item 3. The method according to Item 2, wherein in step c), the equilibration buffer is 10 mM phosphate buffer (pH 7.5) containing 0.2 M or less NaCl and 0.002% Tween 80, and the elution buffer is 10 mM phosphate buffer (pH 7.5) containing 1.0 to 2.0 M NaCl and 0.002% Tween 80; [Section 4] The method according to any one of items 1 to 3, wherein the cell is an established cell line derived from an African green monkey kidney cell; [Section 5] Item 5. The method according to Item 4, wherein the cell line derived from African green monkey kidney cells is a Vero cell or a GL37 cell; [Section 6] The method according to any one of Items 1 to 5, wherein the amount of antigen per protein (specific activity) of the eluate obtained in step c) is 0.5 mg antigen / mg or more; [Section 7] The method according to any one of Items 1 to 6, comprising a step of inactivating the Hepatitis A virus after the gel filtration step d); [Section 8] A method for producing an immunogenic composition, comprising purifying a hepatitis A virus by the method according to any one of items 1 to 7 and using the hepatitis A virus obtained; [Section 9] A method for producing a hepatitis A vaccine, comprising purifying a hepatitis A virus by the method according to any one of items 1 to 7 and using the hepatitis A virus obtained; [Section 10] A method for producing a hepatitis A virus, comprising a step of purifying the hepatitis A virus by the method according to any one of items 1 to 7. Effect of the Invention

[0012] The present invention provides a hepatitis A virus solution purified by using a chromatography carrier containing cross-linked agarose having diethylaminopropyl as a functional group. The purified virus solution may be used for producing a hepatitis A vaccine. [Brief description of the drawings]

[0013] [Figure 1] 1 shows a chromatogram (gradient elution) using DEAE-650M in a chromatography carrier evaluation, in which the vertical axis indicates absorbance at a wavelength of 280 nm (mAu; milliabsorbance unit), and the horizontal axis indicates the volume of the chromatography solution (mL unit). [Diagram 2] 1 shows a chromatogram (gradient elution) using ANX Sepharose 4FF in a chromatography carrier evaluation, where the vertical axis represents absorbance at a wavelength of 280 nm (mAu; milliabsorbance unit) and the horizontal axis represents the volume of the chromatography solution (mL unit). [Diagram 3] This is a representative example of a chromatogram (gradient elution) using ANX. The vertical axis indicates absorbance at a wavelength of 280 nm (mAu; milliabsorbance unit), and the horizontal axis indicates the volume of chromatographic solution (mL unit). [Figure 4] This is a representative example of a chromatogram (stepwise elution) using ANX. The vertical axis represents absorbance at a wavelength of 280 nm (mAu; milliabsorbance unit), and the horizontal axis represents the volume of chromatographic solution (mL unit). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention is used for purifying hepatitis A virus in the production of hepatitis A vaccine. Specifically, the present invention is characterized by a method for purifying hepatitis A virus, in which a chromatographic carrier containing cross-linked agarose having diethylaminopropyl as a functional group is incorporated as a chromatographic step.

[0015] The cells used for propagating the hepatitis A virus are not particularly limited as long as they are capable of propagating the hepatitis A virus, but are preferably cell lines derived from African green monkey kidney cells, such as Vero cells and GL37 cells.

[0016] The medium may be any of those typically used for cell culture, such as MEM (Thermo Fisher Scientific, custom-made), Eagle MEM (Nissui Pharmaceutical), Dulbecco's modified MEM (Nissui Pharmaceutical), VP-SFM (Thermo Fisher Scientific), etc. For virus culture, a medium supplemented with fetal bovine serum may also be used.

[0017] The cells in which the hepatitis A virus has been propagated are solubilized, for example, with a cell lysis solution containing a surfactant. The solubilized sample of purified hepatitis A virus may be subjected to processes such as ultrafiltration, enzyme treatment for decomposing nucleic acids and proteins, anion exchange chromatography, and gel filtration chromatography. The same process may be performed two or more times, for example, the first enzyme treatment may be performed simultaneously, consecutively, or after another process following the first enzyme treatment. When the same process is performed two or more times, it may be performed in the same manner or in different manners. For example, when the enzyme treatment is performed twice, the first process is treated with a nucleic acid degrading enzyme, and the second process is treated with a protease.

[0018] Any of these steps can be performed alone or in combination before any other step, and may be performed before subjecting the sample to anion exchange chromatography to produce a purified Hepatitis A virus preparation.

[0019] The cells in which the hepatitis A virus has been propagated are solubilized with a cell lysis solution containing a surfactant, for example. The surfactant is, for example, Nonidet P-40 (NP-40). Triton X-100 or the like may also be used as the surfactant.

[0020] Enzymes for enzymatic treatment include, for example, proteases (eg, Proteinase K, trypsin), endonucleases (eg, DNase I, RNase A, Benzonase™), or other enzymes.

[0021] Examples of anion exchange chromatography supports available to those skilled in the art include strong anion exchangers and weak anion exchangers such as Q Sepharose™ Fast Flow, DEAE Sepharose™ Fast Flow, and ANX Sepharose™ 4 Fast Flow. Examples of anion exchangers available to those skilled in the art include strong anion exchangers and weak anion exchangers. Examples of strong anion exchangers include anion exchangers having quaternary ammonium groups as carriers, and examples of weak anion exchangers include anion exchangers having diethylaminoethyl (DEAE) or diethylaminopropyl as carriers. Examples of strong anion exchangers having quaternary ammonium groups as carriers include Q Sepharose™ Fast Flow, and examples of weak anion exchangers include DEAE Sepharose™ Fast Flow and ANX Sepharose™ 4 Fast Flow.

[0022] In the anion exchange chromatography step, an equilibration buffer may be used. Those skilled in the art can select an equilibration buffer capable of purifying the virus, taking into consideration the sample and carrier to be used. The equilibration buffer may contain any suitable salt. As the salt, sodium chloride (NaCl) is preferably used. The equilibration buffer typically contains a salt (e.g., NaCl) of about 0.05M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M or less, and more preferably contains NaCl of 0.2M or less. The equilibration buffer may be any suitable buffer, such as a phosphate buffer (e.g., 5, 10, 20, or 50 mM). As an example of an embodiment, the equilibration is preferably performed using a phosphate buffer having a pH of about 7.0, 7.2, or 7.5. The use of other equilibration buffers is also known in the art and may be used in carrying out the method of the present disclosure.

[0023] In chromatography, elution is performed by contacting the hepatitis A virus bound to the carrier with an elution buffer. For example, a washing step may be used to remove contaminants so that the majority of the bound to the carrier is a component of the hepatitis A virus. In such a case, a single elution step may be used to elute the bound hepatitis A virus particles from the carrier. In elution, a salt solution may be used as an elution buffer. Any suitable salt may be used in the elution buffer. Sodium chloride (NaCl) may not be used as a salt. Sodium chloride (NaCl) is preferably used. The high salt buffer is typically about 300 mM, 500 mM, 1 M or 2 M salt (e.g., NaCl). For example, elution may be performed in a suitable buffer containing about 300 mM, 500 mM, 1 M or 2 M NaCl. As an example, any suitable buffer such as a phosphate (e.g., 5, 10, 20 or 50 mM) buffer may be used. As an example embodiment, elution is preferably carried out using a phosphate buffer with a high concentration of salt (e.g., 300 mM, 500 mM, 1 M or 2 M) at a pH of approximately 7.0, 7.2 or 7.5. The use of other elution buffers is well known in the art and may be used in the practice of the methods of the invention.

[0024] The buffer used may contain a surfactant. Examples of the surfactant include non-ionic surfactants such as Tween 80, and preferably Tween 80. The concentration of the surfactant is not particularly limited, but is about 0.0005, 0.001, 0.002, 0.003, 0.004, or 0.005% or less. Preferably, the concentration of the surfactant is about 0.002%.

[0025] The eluate after chromatography is preferably concentrated by ultrafiltration, the method of which can be appropriately determined by those skilled in the art depending on the purpose.

[0026] The eluate, preferably the concentrated eluate, is subjected to gel filtration. Suitable gel filtration carriers include Sephacryl S-400HR (Cytiva) and Sepharose CL-6B (Cytiva).

[0027] In the elution solution containing the purified hepatitis A virus, the amount of antigen per protein (specific activity) may be measured. The amount of antigen can be measured by quantifying HAV antigen by enzyme immunoassay (Biological Product Standard "Dried Tissue Culture Inactivated Hepatitis A Vaccine"). For example, the amount of antigen in the solution containing the purified hepatitis A virus is approximately 0.1 mg antigen / mg, 0.2 mg antigen / mg, 0.3 mg antigen / mg, 0.4 mg antigen / mg, 0.5 mg antigen / mg, 0.6 mg antigen / mg, 0.7 mg antigen / mg, 0.8 mg antigen / mg, 0.9 mg antigen / mg or more. The amount of antigen in the solution containing the purified hepatitis A virus is preferably approximately 0.5 mg antigen / mg or more.

[0028] In the present disclosure, the antigen yield of the chromatography may be calculated by the amount of antigen after treatment / the amount of antigen before treatment*100. In the present disclosure, the antigen yield of the chromatography is preferably 70%, 75%, 80%, 85%, 90%, 95%, 99% or more. In the present disclosure, the impurities include DNA and / or host cell-derived proteins (HCPs). The removal of chromatographic impurities may be examined by comparing the DNA content and / or HCP content before treatment with the DNA content and / or HCP content after treatment. The amount of impurities after the chromatography step of the present disclosure is 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 5, 2.5, 1% or less compared to the amount of impurities before the step. The amount of impurities after the chromatography step of the present disclosure may be below the detection limit. In the present disclosure, the degree of purification by chromatography may be comprehensively determined based on (i) whether adsorption of the antigen to the carrier is observed, (ii) whether an antigen peak is observed on a chromatogram upon elution, and (iii) whether the antigen peak is sufficiently separated from impurity peaks. In a preferred embodiment of the present disclosure, the antigen is determined to be purified when (i) adsorption of the antigen to the carrier is observed by chromatography, (ii) an antigen peak is observed on a chromatogram upon elution, and (iii) the antigen peak is sufficiently separated from impurity peaks.

[0029] Those skilled in the art can produce hepatitis A virus by using the purification method of hepatitis A virus included in the present disclosure. In addition, those skilled in the art can appropriately set a method for inactivating hepatitis A virus according to the purpose. The purified hepatitis A virus solution is inactivated, for example, in the presence of 0.025 vol% formalin, and then the hepatitis A vaccine is prepared through a formulation process.

[0030] Those skilled in the art can prepare immunogenic compositions that are characterized by containing components derived from purified Hepatitis A virus fluids included in the present disclosure. The immunogenic compositions may contain, for example, stabilizers, surfactants, salts, pH adjusters, excipients, and the like.

[0031] Those skilled in the art can prepare a Hepatitis A vaccine that is characterized by containing components derived from the purified Hepatitis A virus solution contained in the present disclosure. The Hepatitis A vaccine may contain, for example, stabilizers, surfactants, salts, pH adjusters, excipients, etc.

[0032] As used herein, "approximately" means within ±10%, preferably ±5% of the indicated numerical value.

[0033] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0034] Example 1: Cultivation of Hepatitis A Virus Hereinafter, the present invention will be described in detail using a hepatitis A virus (derived from KRM003 strain) grown in GL37 cells, which are African green monkey kidney-derived cells, as an exemplary embodiment of the present invention, although the present invention is not limited to these methods.

[0035] GL37 cells were cultured in VP-SFM medium at an initial cell number of 1.2 × 10 9 The cells were suspended in 1600 mL of MEM medium supplemented with fetal bovine serum to obtain 1,600 cells, and inoculated with hepatitis A virus (moi 0.1). The MEM medium was poured into a high-density culture vessel (product name: BelloCell500A (registered trademark), CESCO) filled with a fibrous support (product name: BioNOCII (registered trademark), CESCO), and the medium was poured in and out at a volume equivalent to 10% of the volume of the culture vessel overnight to allow the cells to adhere to the support. After that, almost the entire volume of the medium was poured in and out to ensure adhesion. The cells were cultured for 3 weeks in an environment of 37°C and 5% CO2, and the old medium was discharged and new medium was added every day during the culture period to maintain the culture. The operation of the high-density culture apparatus was stopped, the medium was removed, and the cells were washed with PBS. The cells were solubilized in a cell lysis solution containing 1 V / V% NP-40, and the lysate was used as the starting material for purification of Hepatitis A virus.

[0036] Example 2: Purification of Hepatitis A virus (before anion exchange chromatography)

[0037] Ultrafiltration 1 The solubilized Hepatitis A virus solution was ultrafiltered at a transmembrane pressure difference of 0.04 to 0.06 MPa, and then replaced with a buffer solution (10 mM phosphate buffer) and concentrated.

[0038] Enzyme treatment (nucleic acid decomposition) Magnesium chloride was added to the Hepatitis A virus liquid after ultrafiltration 1 to a final concentration of 5 mM and mixed, and then a nuclease (product name: Benzonase (registered trademark), Merck) was added to a final concentration of 100 U / mL and mixed, followed by enzyme treatment at 37°C for 16 hours.

[0039] Enzyme treatment (protein hydrolysis) Furthermore, Proteinase K was added to a final concentration of 50 μg / mL, mixed, and enzyme treatment was carried out at 37° C. for 1.5 hours.

[0040] Ultrafiltration 2 The hepatitis A virus liquid after the protease treatment was ultrafiltered at a transmembrane pressure difference of 0.04 to 0.06 MPa, and then replaced with a buffer solution (10 mM phosphate buffer containing 0.002% Tween 80) and concentrated.

[0041] Example 3: Purification of Hepatitis A Virus (Selection of Anion Exchange Chromatographic Support) For the anion exchange chromatography step in the purification of hepatitis A virus, we screened chromatographic supports from the viewpoints of impurity removal and yield, and selected the most suitable support. The anion exchange resins evaluated were DEAE-650M (Tosoh), QAE-550C (Tosoh), NH2-750F (Tosoh), and ANX Sepharose 4FF (Cytiva), and the hydrophobic carriers evaluated were Phenyl Sepharose 6FF (Cytiva), Butyl Sepharose 4FF (Cytiva), Octyl Sepharose 4FF (Cytiva), ToyoScreen (registered trademark) Hexyl-650C (Tosoh), ToyoScreen (registered trademark) Butyl-600M (Tosoh), ToyoScreen (registered trademark) Butyl-650M (Tosoh), ToyoScreen (registered trademark) Phenyl-650M (Tosoh), ToyoScreen (registered trademark) PPG-600M (Tosoh), and ToyoScreen (registered trademark) Ether-650M (Tosoh). In the screening of anion exchange resin, after equilibration with 10 mM phosphate buffer (pH 7.5) containing 0.002% Tween 80, the solution after two ultrafiltration steps was used as the application liquid, and anion exchange chromatography was performed by gradient elution with 10 mM phosphate buffer (pH 7.5) containing 500 mM to 5 M NaCl and 0.002% Tween 80. In addition, in the screening of hydrophobic carrier, after equilibration with 10 mM phosphate buffer (pH 7.5) containing 2 M NaCl and 0.002% Tween 80, the purified hepatitis A virus solution was used as the application liquid, and hydrophobic chromatography was performed by linear gradient elution with 10 mM phosphate buffer (pH 7.5) containing 0.002% Tween 80. As a result, the antigen yields of Hepatitis A virus for DEAE-650M, QAE-550C, NH2-750F and ANX Sepharose 4FF were 77%, not determined, 61% and 85% or more, respectively, and the separation from impurities was rated as △, △, △ and ◎. Here, △ means that "adsorption of the antigen to the carrier was observed, and an antigen peak was observed on the chromatogram at the time of elution, but it was not sufficiently separated from the impurity peak," and ◎ means that "adsorption of the antigen to the carrier was observed, and a clear antigen peak was observed on the chromatogram at the time of elution, and it was sufficiently separated from the impurity peak." Furthermore, the specific activity (amount of antigen per protein) was evaluated for DEAE-650M, NH2-750F and ANX Sepharose 4FF, and it was confirmed that ANX Sepharose 4FF, which provided good separation, had a relatively high specific activity. On the other hand, for Phenyl Sepharose 6FF, Butyl Sepharose 4FF, Octyl Sepharose 4FF, ToyoScreen (registered trademark) Hexyl-650C, ToyoScreen (registered trademark) Butyl-600M, ToyoScreen (registered trademark) Butyl-650M, ToyoScreen (registered trademark) Phenyl-650M, ToyoScreen (registered trademark) PPG-600M and ToyoScreen (registered trademark) Ether-650M, "no antigen adsorption to the carrier was observed" or "adsorption of the antigen to the carrier was observed but no antigen peak was confirmed during elution or the antigen was contained in the flow-through fraction and separation and purification was not possible." From the above, it was found that ANX Sepharose 4FF is optimal for purifying Hepatitis A virus, removing impurities, and achieving a high antigen yield. The above results are summarized in the table below, and the chromatograms for DEAE-650M and ANX Sepharose 4FF are shown in Figures 1 and 2, respectively. With DEAE-650M (Figure 1), there was a large overlap with the impurity peaks, and separation of the target hepatitis A virus from the impurities was insufficient.

[0042] [Table 1]

[0043] Example 4: Purification of Hepatitis A virus (optimization of anion exchange chromatography conditions) For the selected ANX Sepharose 4FF, optimization of chromatographic conditions (gradient elution) such as buffer composition and pH was performed. The optimal conditions were determined as follows, based on the antigen yield, the amount of antigen per protein, and good separation of the Hepatitis A virus antigen peak from impurities (FIG. 3).

[0044] [Table 2]

[0045] For the selected ANX Sepharose 4FF, optimization of chromatographic conditions (stepwise elution) such as buffer composition and pH was performed. The optimal conditions were determined as follows, based on the antigen yield, the amount of antigen per protein, and good separation of the Hepatitis A virus antigen peak from impurities (FIG. 4).

[0046] [Table 3]

[0047] Example 5: Purification of Hepatitis A Virus (after anion exchange chromatography)

[0048] Ultrafiltration 3 The Hepatitis A virus liquid after anion exchange chromatography obtained using ANX Sepharose 4FF under the optimal conditions of Example 4 was concentrated by ultrafiltration at a transmembrane pressure difference of 0.04 to 0.06 MPa.

[0049] Gel filtration The hepatitis A virus solution after ultrafiltration 3 was developed on a dextran gel filtration column, and the peak portion of the hepatitis A virus was collected. The column unit was packed with Sephacryl S-400HR (manufactured by Cytiva), and chromatography was carried out at room temperature using a phosphate buffer solution containing 0.002% polysorbate 80 at a linear velocity of 9 cm / hour. The obtained peak fraction of hepatitis A virus was used as purified hepatitis A virus solution. An outline of an embodiment relating to purification of Hepatitis A virus is given below:

[0050] [Table 4]

[0051] Example 6: Evaluation of Hepatitis A virus purification flow using ANX Sepharose 4FF We evaluated each step in the purification flow of Hepatitis A virus using ANX Sepharose 4FF as the carrier in the anion exchange chromatography step, and the quality of the purified virus solution obtained. In the purified virus fluid, impurities remaining after ultrafiltration 2 were efficiently removed by ANX Sepharose 4FF, no impurity bands were observed on SDS-PAGE, and the DNA content and HCP (host cell-derived protein) content were below the lower limit of detection, confirming that a highly pure purified Hepatitis A virus fluid was obtained. The table below shows the change in the amount of Hepatitis A virus antigen per protein (specific activity) over time during the purification process as an index of the degree of purification of Hepatitis A virus.

[0052] [Table 5]

[0053] Example 7: Inactivation of Hepatitis A Virus and Vaccine Preparation The purified hepatitis A virus solution obtained in Example 5 was inactivated in the presence of 0.025 vol% formalin at 37°C for 12 days, and the hepatitis A vaccine was prepared through final bulk preparation, filling and freeze-drying. [Industrial Applicability]

[0054] According to the present invention, there is provided a method for purifying hepatitis A virus by anion exchange chromatography in the production of a hepatitis A vaccine.

Claims

1. 1. A method for purifying Hepatitis A virus, comprising: a) culturing cells infected with Hepatitis A virus and recovering the cultured cells; b) solubilizing the collected cells to obtain a lysate; c) separating the Hepatitis A virus from the obtained lysate using anion exchange chromatography with a chromatographic support comprising cross-linked agarose with diethylaminopropyl, and then obtaining an eluate; and d) subjecting the obtained eluate to gel filtration chromatography; A method for purifying Hepatitis A virus, comprising:

2. The method according to claim 1, wherein in step c) the separated Hepatitis A virus is eluted with a high salt buffer.

3. 3. The method according to claim 2, wherein in step c), the equilibration buffer is 10 mM phosphate buffer (pH 7.5) containing 0.2 M NaCl or less and 0.002% Tween 80, and the elution buffer is 10 mM phosphate buffer (pH 7.5) containing 1.0 to 2.0 M NaCl and 0.002% Tween 80.

4. The method according to any one of claims 1 to 3, wherein the cells are an established cell line derived from African green monkey kidney cells.

5. The method according to claim 4, wherein the cell line derived from African green monkey kidney cells is a Vero cell or a GL37 cell.

6. The method according to claim 1, wherein the amount of antigen per protein (specific activity) of the eluate obtained in step c) is 0.5 mg antigen / mg or more.

7. The method according to claim 1, further comprising a step of inactivating the Hepatitis A virus after the gel filtration in step d).

8. A method for producing an immunogenic composition, comprising purifying a hepatitis A virus by the method according to claim 1 and using the hepatitis A virus obtained.

9. A method for producing a hepatitis A vaccine, comprising purifying a hepatitis A virus by the method according to claim 1 and using the hepatitis A virus obtained.

10. A method for producing hepatitis A virus, comprising the step of purifying hepatitis A virus by the method according to claim 1.

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