Chromatographic purification method for 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 vaccine. Specifically, it relates to a method for purifying hepatitis A virus using a chromatography carrier containing cross-linked agarose having diethylaminopropyl as an anion exchange body functional group.

Background Art

[0002] Hepatitis A virus is an RNA virus of the Picornaviridae family with a diameter of 27 nm and an icosahedral shape. There are 7 genotypes, and there are differences in the prevalent types in different regions of the world. However, since there is only 1 serotype, there is no difference in immune antigenicity due to genotype.

[0003] Hepatitis A virus is proliferative in cultured cells, but the growth rate is slow, and it grows slowly over 2 to 3 weeks. Generally, it does not show a cytopathic effect and is retained in cells.

[0004] When hepatitis A virus infects humans, after a latent period of 2 to 6 weeks, serum transaminase increases following fever, malaise, etc. In addition to symptoms such as loss of appetite and vomiting, cases of jaundice, hepatomegaly, dark-colored urine, and grayish-white stools are also seen. Generally, it does not become chronic and recovers after 1 to 2 months, but rarely, fulminant hepatitis may occur. In recent years, due to the increase in overseas travelers, the risk of infection with hepatitis A virus has increased, and the demand for hepatitis A vaccine has been increasing.

[0005] The production method of hepatitis A vaccine as a conventional technology is as follows. (Patent Document 1) [1] Inoculate and infect hepatitis A virus for vaccine production into GL37 cells, which are established cells derived from African green monkey kidneys; [2] By performing roller bottle culture for 3 weeks, grow the infected hepatitis A virus in GL37 cells; [3] After culturing is complete, the cells are lysed with a surfactant 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 solution is inactivated with formalin to make the vaccine stock solution.

[0006] As an improvement to the manufacturing method of hepatitis A vaccine, a purification method is disclosed in which anion exchanger and gel filtration chromatography operations are performed in the presence of a surfactant (Patent Document 2). In the anion exchange chromatography described in Patent Document 2, conditions are set in which hepatitis A virus particles are temporarily held on a carrier and then eluted. However, the anion exchanger actually examined in the prior patent documents is only a specific DEAE-based carrier. Furthermore, a method for producing hepatitis A virus has been disclosed (Patent Document 3) that includes treating a hepatitis A virus preparation recovered after proliferation in a cell line with a protease to separate the hepatitis A virus from the protease-digested protein and then inactivating the virus. Here, it is also shown that an ion exchange step is performed as a purification step after protease digestion. Although prior art such as the above existed, further improvements were needed regarding the removal of impurities using anion exchangers. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 1-279843 [Patent Document 2] Japanese Patent Publication No. 6-279317 [Patent Document 3] Special Publication 2002-527105 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] Conventional hepatitis A vaccine manufacturing methods involve purifying the virus using harvested fluid (solubilized solution) after virus culture as the starting material, and many manual open-system operations such as centrifugation and extraction, which limits scale-up. While improved purification methods have been considered to increase production capacity and shorten processes, obtaining purified virus solution of the same quality as conventional methods remains a challenge, particularly in the efficient removal of impurities in the chromatography process.

[0009] Therefore, the present invention aims to provide a method for purifying hepatitis A virus that incorporates a chromatography step that can remove impurities more efficiently than the prior art. [Means for solving the problem]

[0010] As a result of repeated studies to achieve the above objective, the present inventors discovered that by using a carrier containing crosslinked agarose having diethylaminopropyl as a functional group as an anion exchange chromatography carrier in the chromatography process, impurities can be efficiently removed and hepatitis A virus can be purified, thus completing the present invention.

[0011] Therefore, the present invention includes the following inventions; [Section 1] A method for purifying hepatitis A virus, a) A process of culturing cells infected with hepatitis A virus and harvesting the cultured cells; b) A step of solubilizing the collected cells to obtain a solubilized solution; c) Separating the hepatitis A virus from the obtained solubilized solution using anion exchange chromatography with a chromatography support containing diethylaminopropyl crosslinked agarose, and then obtaining the eluate; and d) The obtained eluate is subjected to gel filtration chromatography. A method for purifying hepatitis A virus, including; [Section 2] The method according to item 1, wherein the isolated hepatitis A virus is eluted with a high-salt buffer in step c); [Section 3] 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 - 2.0 M NaCl and 0.002% Tween 80, the method according to item 2; [Item 4] The method according to any one of items 1 - 3, wherein the cell is a cell line derived from African green monkey kidney cells; [Item 5] The method according to item 4, wherein the cell line derived from African green monkey kidney cells is Vero cells or GL37 cells; [Item 6] The method according to any one of items 1 - 5, wherein the amount of antigen per protein (specific activity) in the eluate obtained in step c) is 0.5 mg antigen / mg or more; [Item 7] The method according to any one of items 1 - 6, including a step of inactivating hepatitis A virus after gel filtration in step d); [Item 8] A method for producing an immunogenic composition, characterized by purifying hepatitis A virus by the method according to any one of items 1 - 7 and using the obtained hepatitis A virus; [Item 9] A method for producing a hepatitis A vaccine, characterized by purifying hepatitis A virus by the method according to any one of items 1 - 7 and using the obtained hepatitis A virus; [Item 10] A method for producing hepatitis A virus, including a step of purifying hepatitis A virus by the method according to any one of items 1 - 7. [Advantages of the Invention]

[0012] In the present invention, a hepatitis A virus solution purified by using a chromatography carrier containing crosslinked agarose having diethylaminopropyl as a functional group is provided. The purified virus solution may be used for the production of hepatitis A vaccine. [Brief Description of the Drawings]

[0013] [Figure 1] This is a diagram showing a chromatogram (gradient elution) using DEAE - 650M in chromatographic carrier evaluation. The vertical axis is absorbance at a wavelength of 280 nm (mAu; milli - absorbance unit), and the horizontal axis is the chromatographic liquid volume (in mL units). [Figure 2] This is a diagram showing a chromatogram (gradient elution) using ANX Sepharose 4FF in chromatographic carrier evaluation. The vertical axis is absorbance at a wavelength of 280 nm (mAu; milli - absorbance unit), and the horizontal axis is the chromatographic liquid volume (in mL units). [Figure 3] This is a diagram showing a representative example of a chromatogram using ANX (gradient elution). The vertical axis is absorbance at a wavelength of 280 nm (mAu; milli - absorbance unit), and the horizontal axis is the chromatographic liquid volume (in mL units). [Figure 4] This is a diagram showing a representative example of a chromatogram using ANX (step - wise elution). The vertical axis is absorbance at a wavelength of 280 nm (mAu; milli - absorbance unit), and the horizontal axis is the chromatographic liquid volume (in mL units).

Embodiments for Carrying out the Invention

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

[0015] The cells used for the growth of hepatitis A virus are not particularly limited as long as they can grow hepatitis A virus, but preferably they are established cells derived from African green monkey kidney cells. Examples of established cells derived from African green monkey kidney cells include Vero cells and GL37 cells.

[0016] The culture medium can be any of the media commonly used for cell culture, such as MEM (Thermo Fisher Scientific, custom-made), Eagle MEM (Nissui Pharmaceutical), Dulbecco's Modified MEM (Nissui Pharmaceutical), or VP-SFM (Thermo Fisher Scientific). When culturing viruses, a medium supplemented with fetal bovine serum may also be used.

[0017] Cells in which hepatitis A virus has been grown are solubilized, for example, with a cell lysis solution containing a surfactant. This solubilized sample of purified hepatitis A virus may be subjected to processes such as ultrafiltration, enzymatic treatment for nucleolysis and proteolysis, anion exchange chromatography, and gel filtration chromatography. The same process may be performed two or more times; for example, the second enzymatic treatment may be performed simultaneously with the first enzymatic treatment, or immediately after another process following the first enzymatic treatment. When the same process is performed two or more times, it may be performed in the same manner or in different manner. For example, if enzymatic treatment is performed twice, the first step may involve treatment with a nuclease, and the second step may involve treatment with a protease.

[0018] Any of these steps may 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] Cells in which hepatitis A virus has been proliferated are solubilized, for example, with a cell lysis solution containing a surfactant. An example of such a surfactant is Nonidet P-40 (NP-40). Triton X-100 may also be used as a surfactant.

[0020] Examples of enzymes used in enzymatic treatment include proteases (e.g., Proteinase K, Trypsin), endonucleases (e.g., 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. Anion exchangers available to those skilled in the art are, for example, strong anion exchangers or weak anion exchangers. Strong anion exchangers include anion exchangers having a quaternary ammonium group as a support, and weak anion exchangers include anion exchangers having diethylaminoethyl (DEAE) or diethylaminopropyl as a support. An example of a strong anion exchanger having a quaternary ammonium group as a support is Q Sepharose® Fast Flow, and examples of weak anion exchangers include DEAE Sepharose® Fast Flow and ANX Sepharose® 4 Fast Flow.

[0022] An equilibration buffer may be used in the anion exchange chromatography step. Those skilled in the art can select an equilibration buffer capable of purifying the virus, taking into account the sample and support used. The equilibration buffer may contain any suitable salt. Preferably, sodium chloride (NaCl) is used as the salt. The equilibration buffer typically contains a salt (e.g., NaCl) at a concentration of approximately 0.05 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, or 0.5 M or less, and more preferably contains 0.2 M or less of NaCl. Any suitable buffer may be used as the equilibration buffer, such as a phosphate buffer (e.g., 5, 10, 20, or 50 mM). As an example of embodiments, it is preferable to use a phosphate buffer with a pH of approximately 7.0, 7.2, or 7.5 as the equilibration buffer. The use of other equilibration buffers is also well known in the art and can be used in carrying out the method of this 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 conjugated material on the carrier is a component of hepatitis A virus. In such cases, 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 the elution buffer. Any suitable salt may be used as the elution buffer. Sodium chloride (NaCl) does not have to be used as the salt. Preferably, sodium chloride (NaCl) is used. High-salt concentration buffers are typically about 300 mM, 500 mM, 1 M, or 2 M of 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 of NaCl. As an example, any suitable buffer such as phosphate (e.g., 5, 10, 20, or 50 mM) buffer may be used. As an example of an embodiment, elution is preferably carried out using a phosphate buffer with a pH of approximately 7.0, 7.2, or 7.5 containing a high concentration of salt (e.g., 300 mM, 500 mM, 1 M, or 2 M). The use of other elution buffers is also well known in the art and may be used in carrying out the method of the present invention.

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

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

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

[0027] In an elution solution containing 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 using an enzyme immunoassay (Biological Products Standard "Dried Tissue Culture Inactivated Hepatitis A Vaccine"). For example, the amount of antigen in a solution containing 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, or 0.9 mg antigen / mg or more. Preferably, the amount of antigen in a solution containing purified hepatitis A virus is approximately 0.5 mg antigen / mg or more.

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

[0029] A person skilled in the art can produce hepatitis A virus by using the method for purifying hepatitis A virus contained in this disclosure. Furthermore, a person skilled in the art can appropriately set the method for inactivating hepatitis A virus according to the purpose. The purified hepatitis A virus solution is subjected to inactivation treatment, for example, in the presence of 0.025 vol% formalin, and then undergoes a formulation process to prepare a hepatitis A vaccine.

[0030] Those skilled in the art can prepare immunogenic compositions characterized by containing components derived from purified hepatitis A virus solution as contained in this disclosure. The immunogenic compositions may contain, for example, stabilizers, surfactants, salts, pH adjusters, and excipients.

[0031] Those skilled in the art can produce a hepatitis A vaccine characterized by containing components derived from purified hepatitis A virus solution as contained in this disclosure. The hepatitis A vaccine may contain, for example, stabilizers, surfactants, salts, pH adjusters, and excipients.

[0032] In this specification, "approximately" means a range of ±10%, preferably ±5%, of the indicated value.

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

[0034] Example 1: Culture of Hepatitis A virus The present invention will be described in detail below using hepatitis A virus (derived from strain KRM003) grown using GL37 cells, which are derived from the kidneys of African green monkeys, as an exemplary embodiment of the present invention. However, the present invention is not limited to these methods.

[0035] GL37 cells cultured in VP-SFM medium with adherent cells, initial cell count 1.2 × 10⁶ 9 Fetal bovine serum was suspended in 1600 mL of MEM medium to form cells, and then hepatitis A virus (moi 0.1) was inoculated into it. The MEM medium was placed in a high-density culture vessel (product name: BelloCell500A®, CESCO) filled with a fibrous material support (product name: BioNOCII®, CESCO). Cells were adhered to the support by adding and removing 10% of the culture medium volume to the vessel overnight. Subsequently, adhesion was ensured by adding and removing almost the entire volume of culture medium. The cells were cultured for 3 weeks at 37°C and 5% CO2, and the culture was maintained by repeatedly draining the old culture medium and adding new culture medium daily during the culture period. The high-density culture apparatus was shut down, the culture medium was removed, and the cells were washed with PBS. The cells were solubilized with a cell lysis solution containing 1V / V%NP-40, and this solubilized solution was used as the starting material for hepatitis A virus purification.

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

[0037] Ultrafiltration 1 The solubilized hepatitis A virus solution was ultrafiltered under an intermembrane pressure differential of 0.04-0.06 MPa, and then replaced with a buffer solution (10 mM phosphate buffer) and concentrated.

[0038] Enzyme treatment (nucleic acid degradation) To the hepatitis A virus solution after ultrafiltration 1, magnesium chloride was added and mixed to a final concentration of 5 mM. Then, nuclease (trade name: Bensonase®, Merck) was added and mixed to a final concentration of 100 U / mL, and the mixture was treated with enzymes at 37°C for 16 hours.

[0039] Enzyme treatment (protein degradation) Furthermore, Proteinase K was added and mixed to a final concentration of 50 μg / mL, and the mixture was treated with enzymes at 37°C for 1.5 hours.

[0040] Ultrafiltration 2 The hepatitis A virus solution treated with proteolytic enzymes was ultrafiltered under an intermembrane pressure differential of 0.04-0.06 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 chromatography support) For the anion exchange chromatography step in the purification of hepatitis A virus, chromatographic supports were screened from the viewpoint of impurity removal and yield, and a suitable support was selected. As anion exchange resins, DEAE-650M (Tosoh), QAE-550C (Tosoh), NH2-750F (Tosoh), and ANX Sepharose 4FF (Cytiva) were evaluated. As hydrophobic carriers, Phenyl Sepharose 6FF (Cytiva), Butyl Sepharose 4FF (Cytiva), Octyl Sepharose 4FF (Cytiva), ToyoScreen® Hexyl-650C (Tosoh), ToyoScreen® Butyl-600M (Tosoh), ToyoScreen® Butyl-650M (Tosoh), ToyoScreen® Phenyl-650M (Tosoh), ToyoScreen® PPG-600M (Tosoh) and ToyoScreen® Ether-650M (Tosoh) were evaluated. For the screening of anion exchange resins, the solution was equilibrated with 10 mM phosphate buffer (pH 7.5) containing 0.002% Tween 80, followed by two ultrafiltration steps. This solution was used as the application solution, 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. For the screening of hydrophobic carriers, the solution was equilibrated 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 solution, 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, for DEAE-650M, QAE-550C, NH2-750F, and ANX Sepharose 4FF, the hepatitis A virus antigen yields were 77%, not measured, 61%, and 85% or higher, respectively, and the separation from impurities was △, △, △, and ◎. Here, △ means "adsorption of antigen to the carrier was observed, and an antigen peak was observed on the chromatogram at elution, but it could not be sufficiently separated from the impurity peak," and ◎ means "adsorption of antigen to the carrier was observed, a clear antigen peak was observed on the chromatogram at elution, and it could be sufficiently separated from the impurity peak." Furthermore, the specific activity (amount of antigen per unit of protein) was evaluated for DEAE-650M, NH2-750F, and ANX Sepharose 4FF, and it was confirmed that ANX Sepharose 4FF, which exhibited good separation, had relatively high specific activity. On the other hand, for Phenyl Sepharose 6FF, Butyl Sepharose 4FF, Octyl Sepharose 4FF, ToyoScreen® Hexyl-650C, ToyoScreen® Butyl-600M, ToyoScreen® Butyl-650M, ToyoScreen® Phenyl-650M, ToyoScreen® PPG-600M, and ToyoScreen® Ether-650M, all were either "not showing antigen adsorption to the carrier" or "showing antigen adsorption to the carrier, but unable to confirm an antigen peak during elution, or the antigen being present in the pass-through fraction and therefore unable to be separated and purified." Based on the above, it was found that ANX Sepharose 4FF is optimal for the purpose of purifying hepatitis A virus, removing impurities, and achieving a high antigen yield. The results described above are summarized in the table below, and the chromatograms for DEAE-650M and ANX Sepharose 4FF are shown in Figures 1 and 2, respectively. In DEAE-650M (Figure 1), there was significant overlap with the impurity peaks, resulting in insufficient separation of the target substance, hepatitis A virus, from the impurities.

[0042] [Table 1]

[0043] Example 4: Purification of Hepatitis A virus (optimization of anion exchange chromatography conditions) For the selected ANX Sepharose 4FF, we investigated the optimization of chromatographic conditions (gradient elution), including buffer solution (composition, pH). The following optimal conditions were determined based on antigen yield, antigen amount per protein, and good separation of the hepatitis A virus antigen peak from impurities (Figure 3).

[0044] [Table 2]

[0045] For the selected ANX Sepharose 4FF, we investigated the optimization of chromatographic conditions (stepwise elution), including buffer solution (composition, pH). The following optimal conditions were determined based on antigen yield, antigen amount per protein, and good separation of the hepatitis A virus antigen peak from impurities (Figure 4).

[0046] [Table 3]

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

[0048] Ultrafiltration 3 The hepatitis A virus solution obtained after anion exchange chromatography using ANX Sepharose 4FF under the optimal conditions of Example 4 was ultrafiltered at an intermembrane pressure of 0.04-0.06 MPa and concentrated.

[0049] Gel filtration The hepatitis A virus solution, after ultrafiltration 3, was spread onto a dextran-based gel filtration column, and the peak portion of the hepatitis A virus was collected. A Sephacryl S-400HR (manufactured by Cytiva) column was packed into the column unit, and chromatography was performed at room temperature by flowing phosphate buffer 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. The following is an overview of an embodiment related to the purification of hepatitis A virus:

[0050] [Table 4]

[0051] Example 6: Evaluation of the hepatitis A virus purification flow using ANX Sepharose 4FF. The quality of each step and the resulting purified virus solution were evaluated using a hepatitis A virus purification flow using ANX Sepharose 4FF as a carrier in the anion exchange chromatography process. In the purified virus solution, impurities remaining after ultrafiltration 2 were efficiently removed by ANX Sepharose 4FF, and no impurity bands were observed on SDS-PAGE. The DNA content and HCP (host cell-derived protein) content were below the detection limit, confirming that a highly pure purified hepatitis A virus solution was obtained. The table below shows the changes in the amount of hepatitis A virus antigen per unit of protein (specific activity) during the purification process, which serves as an indicator 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 then the hepatitis A vaccine was prepared by final bulk preparation, filling, and freeze-drying. [Industrial applicability]

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

Claims

1. A method for purifying hepatitis A virus, a) A process of culturing cells infected with hepatitis A virus and harvesting the cultured cells; b) A step of solubilizing the collected cells to obtain a solubilized solution; c) Separating hepatitis A virus from the obtained solubilized solution using anion exchange chromatography with a chromatography support containing crosslinked agarose having diethylaminopropyl, and then obtaining an eluate with an elution buffer containing 500 mM to 2.0 M of salt; and d) The obtained eluate is subjected to gel filtration chromatography. A method for purifying hepatitis A virus, including [specific details omitted].

2. The method according to claim 1, wherein the elution buffer contains a 1.0 to 2.0 M salt.

3. The method according to claim 2, wherein in step c), the equilibration buffer is a 10 mM phosphate buffer (pH 7.5) containing 0.2 M NaCl or less and 0.002% Tween 80, and the elution buffer is a 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 a 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 Vero cells or GL37 cells.

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 the step of inactivating hepatitis A virus after gel filtration in step d).

8. A method for producing an immunogenic composition, characterized by purifying hepatitis A virus by the method described in claim 1 and using the obtained hepatitis A virus.

9. A method for producing a hepatitis A vaccine, characterized by purifying the hepatitis A virus by the method described in claim 1 and using the obtained hepatitis A virus.

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

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