METHOD FOR PRODUCING INACTIVATED SARS-CoV-2 VACCINE, INACTIVATED SARS-CoV-2 VACCINE, METHOD FOR PURIFYING SARS-CoV-2 OR INACTIVATED SARS-CoV-2, AND SARS-CoV-2 ANTIGEN COMPOSITION OR INACTIVATED SARS-CoV-2 ANTIGEN COMPOSITION

Abstract

The present invention relates to a method for producing an inactivated SARS-CoV-2 vaccine, the method comprising a step in which a solution containing SARS-CoV-2 or a solution containing inactivated SARS-CoV-2 is brought into contact with a cellulose sulfate gel at a pH of 8 to 10, whereby SARS-CoV-2 or inactivated SARS-CoV-2 is adsorbed to the gel, then impurities are removed, and then SARS-CoV-2 or inactivated SARS-CoV-2 is eluted and recovered.

Description

Technical Field

[0001] The present invention relates to a method for producing an inactivated SARS-CoV-2 vaccine and an inactivated SARS-CoV-2 vaccine, a method for purifying SARS-CoV-2 or inactivated SARS-CoV-2, and a SARS-CoV-2 antigen composition or an inactivated SARS-CoV-2 antigen composition. Background Art

[0002] The official international name of the novel coronavirus infection that occurred in 2019 is called COVID-19 by the World Health Organization (WHO), which is caused by the virus called SARS-CoV-2. COVID-19 is a respiratory infection disease that typically presents symptoms such as headache, loss of smell and / or taste, nasal congestion and rhinorrhea, cough, muscle pain, sore throat, fever, diarrhea, and dyspnea. In most cases, people infected with COVID-19 are asymptomatic or recover naturally with mild symptoms accompanied by cold-like symptoms, but in severe cases, they are accompanied by acute respiratory distress syndrome, scurvy and / or multiple organ failure. SARS-CoV-2 is a single-stranded positive-sense RNA virus with a diameter of 50 to 200 nm belonging to the subfamily of orthocoronavirus.

[0003] As one of the prevention methods for COVID-19, vaccination can be cited. As vaccines for SARS-CoV-2, various vaccines such as mRNA vaccines, viral vector vaccines, and recombinant protein vaccines have been developed and approved, and some have been listed (non-patent literature 1). In Japan, mRNA vaccines and viral vector vaccines have been vaccinated to many citizens over the age of 5, and it is expected that the serious cases and deaths will be reduced by infection prevention, prevention of severe illness and morbidity. On the other hand, as for these vaccines, as side effects after vaccination, pain at the vaccination site, headache, fatigue, muscle pain, etc. have been reported. In addition, allergic reactions after vaccination, and thrombosis, pericarditis, myocarditis, etc., which depend on the type of vaccine, are rarely reported.

[0004] On the other hand, as vaccines other than the above types, inactivated vaccines are also known. Inactivated vaccines are used, for example, as seasonal influenza vaccines, Japanese encephalitis vaccines, quadruple vaccines, etc. Known inactivated vaccines are vaccines made using pathogens (viruses, etc.) that have lost their infectivity, or components of the pathogens (parts of viruses, etc.), which usually have few side effects and high safety. Therefore, it is expected that the inactivated SARS-CoV-2 vaccine can also be inoculated in children less than 5 years old.

[0005] As a mode of the method for manufacturing inactivated vaccines, there is the following method: after using strain cells as virus culture substrate (host cell) to proliferate the target virus, the proliferated virus is inactivated and purified, and the purified inactivated virus is obtained as the virus antigen composition for manufacturing the vaccine. In this manufacturing method, it is necessary to establish a virus proliferation method for realizing high productivity and a purification method for the inactivated virus for further realizing safety. In the inactivated SARS-CoV-2 vaccine, as the purification method for inactivated virus, purification (patent document 1, patent document 2) based on ion exchange chromatography and / or size exclusion chromatography is generally adopted. In addition, there is also a report (patent document 3, non-patent document 2) utilizing affinity chromatography for purification.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: International Publication No. 2021 / 204825

[0009] Patent Document 2: International Publication No. 2021 / 209060

[0010] Patent Document 3: International Publication No. 2022 / 38642

[0011] Non-patent literature

[0012] Non-patent literature 1: Clinical Immunology 222(2021)108634

[0013] Non-patent document 2: Purification example of human coronavirus OC43 using Cellufine Sulfate, JNC Corporation technical parameter table, https: / / www.jnc-corp.co.jp / fine / jp / cellufine / guide / pdf / affinity / TD_Sulfa te_N5_V1_J.pdf (confirmed on June 1, 2022) Summary of the invention

[0014] Problems to be solved by the invention

[0015] The object of the present invention is to provide a method for producing a high-purity inactivated SARS-CoV-2 vaccine with a low content (especially substantially no) of impurities (such as proteins derived from host cells) derived from host cells (such as established animal cells). The object of the present invention is also to provide a method for purifying SARS-CoV-2 or inactivated SARS-CoV-2, which can obtain a high-purity SARS-CoV-2 antigen composition or inactivated SARS-CoV-2 antigen composition with a low content (especially substantially no) of impurities (such as proteins derived from host cells) derived from host cells (such as established animal cells). The object of the present invention is further to provide an inactivated SARS-CoV-2 vaccine, or a SARS-CoV-2 antigen composition or an inactivated SARS-CoV-2 antigen composition obtained by the manufacturing method or purification method.

[0016] Means for solving problems

[0017] The inventors of the present application have repeatedly conducted in-depth studies to achieve the above-mentioned purpose, and as a result, they have found that after SARS-CoV-2 is proliferated using strained animal cells (Vero cells) as a virus culture substrate (host cells), the inactivated SARS-CoV-2 is purified by cellulose sulfate gel adsorption chromatography under conditions of pH 8 to 10, thereby obtaining further highly purified inactivated SARS-CoV-2 that does not substantially contain impurities (proteins) derived from the virus culture substrate (host cells), thereby completing the present invention.

[0018] The present invention relates to a method for producing an inactivated SARS-CoV-2 vaccine, which comprises the following steps: contacting a solution containing SARS-CoV-2 or a solution containing inactivated SARS-CoV-2 with a cellulose sulfate gel at a pH of 8 to 10, thereby adsorbing the SARS-CoV-2 or the inactivated SARS-CoV-2 to the gel, then removing impurities, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2.

[0019] The manufacturing method of the present invention includes purifying SARS-CoV-2 or inactivated SARS-CoV-2 using cellulose sulfate gel under specific pH conditions, so that an inactivated SARS-CoV-2 vaccine that substantially does not contain impurities (e.g., proteins derived from host cells) derived from host cells (e.g., strained animal cells) can be obtained.

[0020] The present invention also relates to an inactivated SARS-CoV-2 vaccine obtained by the above-mentioned manufacturing method and substantially free of host cell-derived proteins. The vaccine is obtained by the above-mentioned manufacturing method of the present invention, and therefore substantially does not contain impurities (e.g., host cell-derived proteins) derived from host cells (e.g., strained animal cells), and the safety is further improved.

[0021] The above-mentioned vaccine may further contain an adjuvant.

[0022] The present invention also covers a method for purifying SARS-CoV-2 or inactivated SARS-CoV-2, which comprises the following steps: contacting a solution containing SARS-CoV-2 or a solution containing inactivated SARS-CoV-2 with a cellulose sulfate gel at a pH of 8 to 10, thereby adsorbing the SARS-CoV-2 or the inactivated SARS-CoV-2 to the gel, removing impurities, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2.

[0023] The present invention also relates to a SARS-CoV-2 antigen composition or an inactivated SARS-CoV-2 antigen composition obtained by the above-mentioned purification method, which is substantially free of proteins derived from host cells.

[0024] The inventors of the present application have repeatedly conducted in-depth studies to achieve the above-mentioned purpose. As a result, they found that after SARS-CoV-2 is proliferated using strained animal cells (Vero cells) as a virus culture substrate (host cells), it is purified by the following cellulose sulfate gel adsorption chromatography, thereby obtaining a high yield of further highly purified inactivated SARS-CoV-2 with a low content of impurities (proteins) derived from the virus culture substrate (host cells), thereby completing the present invention. The cellulose sulfate gel adsorption chromatography adsorbs the inactivated SARS-CoV-2 under the condition of pH 7 to 8, removes impurities under the condition of pH 8 to 10, and elutes and recovers.

[0025] The present invention relates to a method for producing an inactivated SARS-CoV-2 vaccine, which comprises the following steps: contacting a solution containing SARS-CoV-2 or a solution containing inactivated SARS-CoV-2 with a cellulose sulfate gel at a pH of 7 to 8, thereby adsorbing the SARS-CoV-2 or the inactivated SARS-CoV-2 to the gel, then removing impurities at a pH of 8 to 10, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2.

[0026] The manufacturing method involved in the present invention includes purifying SARS-CoV-2 or inactivated SARS-CoV-2 using cellulose sulfate gel under specific pH conditions, thereby more efficiently manufacturing a high-purity inactivated SARS-CoV-2 vaccine with a low content of impurities (e.g., proteins derived from host cells) derived from host cells (e.g., strained animal cells).

[0027] The present invention also relates to an inactivated SARS-CoV-2 vaccine obtained by the above-mentioned manufacturing method. Since the vaccine is obtained by the above-mentioned manufacturing method of the present invention, the content of impurities (e.g., proteins derived from host cells) derived from host cells (e.g., strained animal cells) is low, and the safety is improved.

[0028] With regard to the above-mentioned vaccine, the content of protein derived from host cells may be less than 30 ng relative to 1 μg of protein contained in the above-mentioned inactivated SARS-CoV-2 vaccine.

[0029] The above-mentioned vaccine may further contain an adjuvant.

[0030] The present invention also covers a method for purifying SARS-CoV-2 or inactivated SARS-CoV-2, which comprises the following steps: contacting a solution containing SARS-CoV-2 or a solution containing inactivated SARS-CoV-2 with a cellulose sulfate gel at a pH of 7 to 8, thereby adsorbing the SARS-CoV-2 or the inactivated SARS-CoV-2 onto the gel, then removing impurities at a pH of 8 to 10, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2.

[0031] The present invention also relates to a SARS-CoV-2 antigen composition or an inactivated SARS-CoV-2 antigen composition obtained by the above purification method.

[0032] With respect to the above-mentioned antigen composition, the content of protein derived from host cells may be 30 ng or less relative to 1 μg of protein contained in the above-mentioned SARS-CoV-2 antigen composition or the above-mentioned inactivated SARS-CoV-2 antigen composition.

[0033] Effects of the Invention

[0034] According to the present invention, a method for producing a high-purity inactivated SARS-CoV-2 vaccine with a low content (especially substantially no) of impurities (such as proteins derived from host cells) derived from host cells (such as strained animal cells) can be provided. According to the present invention, a method for purifying SARS-CoV-2 or inactivated SARS-CoV-2 can also be provided, wherein the purification method can obtain a high-purity SARS-CoV-2 antigen composition or an inactivated SARS-CoV-2 antigen composition with a low content (especially substantially no) of impurities (such as proteins derived from host cells) derived from host cells (such as strained animal cells). According to the present invention, an inactivated SARS-CoV-2 vaccine, a SARS-CoV-2 antigen composition or an inactivated SARS-CoV-2 antigen composition obtained by the manufacturing method or purification method can also be provided.

[0035] The inactivated SARS-CoV-2 vaccine of the present invention can induce antibodies with neutralizing activity against SARS-CoV-2. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] [ Figure 1 ] is a graph showing the results of measuring the neutralizing antibody titer against SARS-CoV-2 in serum in Example 5. DETAILED DESCRIPTION

[0037] Hereinafter, the mode for implementing the present invention will be described in detail. However, the present invention is not limited to the following embodiment.

[0038] [Method for producing inactivated SARS-CoV-2 vaccine]

[0039] The method for producing an inactivated SARS-CoV-2 vaccine involved in this embodiment comprises the following step (purification step A): by contacting a solution containing SARS-CoV-2 or a solution containing inactivated SARS-CoV-2 with a cellulose sulfate gel at a pH of 8 to 10, thereby adsorbing SARS-CoV-2 or inactivated SARS-CoV-2 to the gel, then removing impurities, and then eluting and recovering the above-mentioned SARS-CoV-2 or the above-mentioned inactivated SARS-CoV-2.

[0040] The manufacturing method involved in this embodiment has the following characteristics: in the manufacturing process of the inactivated SARS-CoV-2 vaccine, the purification process including the adsorption and elution of SARS-CoV-2 or inactivated SARS-CoV-2 to the cellulose sulfate gel is carried out under alkaline conditions (pH 8 or more and pH 10 or less). As a result, the removal efficiency of impurities (for example, proteins derived from host cells) in the purification process is improved, and a high-purity inactivated SARS-CoV-2 vaccine that is substantially free of impurities can be obtained.

[0041] The method for producing an inactivated SARS-CoV-2 vaccine involved in other embodiments comprises the following step (purification step B): by contacting a solution containing SARS-CoV-2 or a solution containing inactivated SARS-CoV-2 with a cellulose sulfate gel at a pH of 7 to 8, thereby adsorbing SARS-CoV-2 or inactivated SARS-CoV-2 to the gel, then removing impurities at a pH of 8 to 10, and then eluting and recovering SARS-CoV-2 or inactivated SARS-CoV-2.

[0042] The manufacturing method involved in other embodiments has the following characteristics: in the manufacturing method of the inactivated SARS-CoV-2 vaccine, in the purification process including adsorption of SARS-CoV-2 or inactivated SARS-CoV-2 to cellulose sulfate gel, removal of impurities, and elution of SARS-CoV-2 or inactivated SARS-CoV-2, the above-mentioned adsorption is carried out under conditions near neutrality (pH 7 or more and pH 8 or less), and the subsequent removal of impurities and the above-mentioned elution are carried out under alkaline conditions (pH 8 or more and pH 10 or less). As a result, the efficiency of removing impurities in the purification process is improved, and on this basis, the yield of SARS-CoV-2 or inactivated SARS-CoV-2 is further improved, and a high-purity inactivated SARS-CoV-2 vaccine with a low impurity content can be manufactured more efficiently.

[0043] The manufacturing method involved in this embodiment may include, in addition to the purification step A or B (hereinafter sometimes also collectively referred to as the "purification step"), at least one step selected from the group consisting of a step of proliferating SARS-CoV-2 (proliferation step), a step of inactivating SARS-CoV-2 (inactivation step), and a step of mixing the purified inactivated SARS-CoV-2 with other components to obtain an inactivated SARS-CoV-2 vaccine (formulation step).

[0044] (Growth process)

[0045] The proliferation step is a step of propagating SARS-CoV-2. The propagation of SARS-CoV-2 can be carried out by causing SARS-CoV-2 to infect host cells serving as a virus culture substrate and recovering SARS-CoV-2 that has propagated in the infected host cells.

[0046] As the host cell used in the proliferation of SARS-CoV-2, as long as the proliferation of SARS-CoV-2 is good and the cell produces SARS-CoV-2 (antigen) with high immunogenicity, there is no particular limitation, for example, animals other than humans (e.g., chickens), strained cells derived from animals (strained animal cells) can be used. As a specific example of strained cells, for example, Calu-3 cells and A549 cells derived from human lung epithelial adenocarcinoma cells, and Vero cells, VeroE6 cells and VeroE6 / TMPRSS2 cells derived from African green monkey kidneys can be cited. As the host cell preferably used in the manufacturing method involved in the present embodiment, Vero cells can be cited.

[0047] The SARS-CoV-2 virus strain used in the manufacturing method of the present embodiment is not particularly limited as long as it is a virus strain that maintains a protective antigen. As the SARS-CoV-2 virus strain, for example, the original strain and its variants such as the alpha strain, beta strain, gamma strain, and Omicron strain can be cited.

[0048] When using strained cells as host cells, as a medium used for expansion (cell proliferation) of host cells (hereinafter, sometimes referred to as "proliferation medium"), a medium commonly used for tissue culture such as M199-earle base, minimum essential medium (MEM), Dulbecco's minimum essential medium (D-MEM), SC-UCM102 (Nissui), VP-SFM (GIBCO BRL), EX-CELL302 (Nichirei Corporation), EX-CELL293-S (Nichirei Corporation), TFBM-01 (Nichirei Corporation), ASF104, etc. can be appropriately selected, and a medium to which amino acids, salts, antimycotics and antibacterial agents, animal serum, etc. are added can be used. As a proliferation medium, D-MEM medium and a medium to which amino acids, salts, antimycotics and antibacterial agents, animal serum, etc. are added are preferably used.

[0049] As a medium used for the proliferation of SARS-CoV-2 after inoculating SARS-CoV-2 into host cells (hereinafter, sometimes referred to as a "maintenance medium"), for example, a serum-free or low-protein concentration medium can be used. As a maintenance medium, the above-mentioned proliferation medium without animal serum can be used, and preferably a medium in which L-glutamic acid is added to VP-SFM. By using a serum-free medium, the impurities to be removed are reduced, so purification becomes easy, and in addition, the possibility of mixing with unknown pathogens derived from serum can be eliminated.

[0050] As a method for propagating host cells, for example, static culture, roller bottle culture and suspension culture can be enumerated. Among these, the tank culture using a microcarrier as one of the suspension culture methods can achieve high-density culture of host cells, which is suitable as a method for obtaining SARS-CoV-2 in large quantities. The microcarrier used for this purpose has been sold, and as a preferred microcarrier for propagating Vero cells, for example, Cytodex (registered trademark) I (Cytiva (Global Life Sciences Technologies Japan KK)) can be enumerated. Cytodex (registered trademark) is preferably used with a concentration of 1g / L or more and 5g / L or less relative to the proliferation medium.

[0051] The number of SARS-CoV-2 relative to one host cell when SARS-CoV-2 infects a host cell, i.e., the multiplicity of infection (MOI), can be, for example, in the range of 0.000001 to 0.01, or in the range of 0.00001 to 0.001.

[0052] The culture temperature and culture time when SARS-CoV-2 is propagated in host cells infected with SARS-CoV-2 are adjusted according to the combination of the type of host cells, the amount of SARS-CoV-2 inoculation, the culture scale, the culture method, etc. For example, when SARS-CoV-2 is propagated in Vero cells by static culture or roller bottle culture, the following method is adopted.

[0053] First, Vero cells are cultured in a proliferation medium composed of a D-MEM medium supplemented with non-essential amino acids and bovine serum at a culture temperature of 32°C to 38°C (preferably 37°C) and a culture time of 2 days to 7 days (preferably 5 days to 7 days). Next, when the proliferation of Vero cells reaches a stable phase, the culture medium is removed by aspiration, washed several times with phosphate buffered saline or the like, and then SARS-CoV-2 is inoculated under an MOI of 0.000001 to 0.01 (preferably MOI of 0.0001), and VP-SFM is added as a maintenance medium, and then cultured at a culture temperature of 32°C to 38°C for 3 days to 8 days, preferably 5 days to 7 days. A large amount of SARS-CoV-2 is present in the culture obtained in this way, that is, the host cell disrupted liquid or culture supernatant.

[0054] Using the solution containing SARS-CoV-2 obtained in the proliferation step (for example, a host cell disrupted solution or a culture supernatant), an inactivation step or a purification step is performed.

[0055] (Inactivation process)

[0056] The inactivation process is a process for inactivating SARS-CoV-2. The inactivation process can be implemented, for example, by contacting SARS-CoV-2 with an inactivating agent. In addition, the inactivation of SARS-CoV-2 can be implemented, for example, by adding an inactivating agent to a culture of host cells infected with SARS-CoV-2, and can also be implemented by adding an inactivating agent to a substance after impurities have been removed from the culture and / or a substance after the culture has been concentrated. Specifically, for example, the following concentrated solution can be used for implementation, wherein the concentrated solution is obtained by removing insoluble matter from a culture of host cells infected with SARS-CoV-2 by coarse centrifugation or membrane filtration, and then further concentrating the obtained supernatant using an ultrafiltration membrane with an exclusion limit molecular weight of more than 100,000 and less than 750,000 (preferably more than 300,000 and less than 750,000).

[0057] Examples of the inactivator include formaldehyde, paraformaldehyde, glutaraldehyde, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and β-propiolactone. The inactivator is preferably β-propiolactone.

[0058] The amount of the inactivating agent added when inactivating SARS-CoV-2 can be set according to the type of solution containing SARS-CoV-2, the type of inactivating agent, etc. For example, when β-propiolactone is added to the following concentrated solution for inactivation, the amount of the inactivating agent added can be 0.01 w / v% to 0.1 w / v%, preferably 0.05 w / v%, wherein the concentrated solution is obtained by removing insoluble matter from a culture of host cells infected with SARS-CoV-2 by coarse centrifugation or membrane filtration, and then concentrating the obtained supernatant using an ultrafiltration membrane with an exclusion limit molecular weight of 100,000 to 750,000 (preferably 300,000 to 750,000).

[0059] The temperature and reaction time (contact time with the inactivating agent) when inactivating SARS-CoV-2 can be set according to the type of solution containing SARS-CoV-2, the type and concentration of the inactivating agent, etc. For example, in the case of adding β-propiolactone to the following concentrated solution for inactivation, the inactivation can be carried out by stirring at a reaction temperature of about 20°C for more than 16 hours, wherein the concentrated solution is obtained by removing insoluble matter from a culture of host cells infected with SARS-CoV-2 by coarse centrifugation or membrane filtration, and then concentrating the obtained supernatant using an ultrafiltration membrane with an exclusion limit molecular weight of 100,000 to 750,000 (preferably 300,000 to 750,000).

[0060] The inactivation step can be performed before the purification step or after the purification step. When the inactivation step is performed before the purification step, the purification step can be performed using the solution containing inactivated SARS-CoV-2 obtained in the inactivation step (for example, the reaction solution after reacting with the inactivating agent).

[0061] (Purification process)

[0062] Purification step A is a step in which a solution containing SARS-CoV-2 or a solution containing inactivated SARS-CoV-2 is brought into contact with a cellulose sulfate gel at a pH of 8 to 10, thereby adsorbing SARS-CoV-2 or inactivated SARS-CoV-2 onto the gel, removing impurities, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2.

[0063] Purification step B is a step in which a solution containing SARS-CoV-2 or a solution containing inactivated SARS-CoV-2 is brought into contact with a cellulose sulfate gel at a pH of 7 to 8, thereby adsorbing SARS-CoV-2 or inactivated SARS-CoV-2 to the gel, and then removing impurities at a pH of 8 to 10, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2.

[0064] Cellulose sulfate gel is a gel formed of cellulose sulfate, which is obtained by forming an ester between the hydroxyl group of cellulose and sulfuric acid. Cellulose sulfate is preferably obtained by forming an ester between the hydroxyl group at the 6th position of cellulose and sulfuric acid. The particle size of the cellulose sulfate gel is, for example, not less than 10 μm and not more than 200 μm, preferably not less than 20 μm and not more than 170 μm, not less than 30 μm and not more than 150 μm, and not less than 40 μm and not more than 130 μm. Such a cellulose sulfate gel can be obtained, for example, as CellufineSulfate (manufactured by JNC Co., Ltd.: a substance in which the hydroxyl group at the 6th position is esterified with sulfuric acid, with a particle size of 40-130 μm, and spherical).

[0065] SARS-CoV-2 and inactivated SARS-CoV-2 can be adsorbed on cellulose sulfate gel in the range of pH 7 to 10. On the other hand, the manufacturing method involved in the present embodiment is characterized in that SARS-CoV-2 or inactivated SARS-CoV-2 is adsorbed on cellulose sulfate gel at a pH of 8 to 10. By setting the pH to this range, the following advantageous effects are obtained: the removal efficiency of impurities (proteins derived from host cells) derived from host cells is improved, and further highly purified SARS-CoV-2 or inactivated SARS-CoV-2 substantially free of impurities is obtained. The pH when SARS-CoV-2 or inactivated SARS-CoV-2 is adsorbed on cellulose sulfate gel is preferably pH 8.5 to 9.5, pH 8.6 to 9.4, pH 8.7 to 9.3, pH 8.8 to 9.2, pH 8.9 to 9.1, or pH 9.

[0066] The production method according to another embodiment is characterized in that SARS-CoV-2 or inactivated SARS-CoV-2 is adsorbed to a cellulose sulfate gel at a pH of 7 to 8, and impurities are removed from the cellulose sulfate gel and SARS-CoV-2 or inactivated SARS-CoV-2 is eluted at a pH of 8 to 10. By setting the pH to this range, the following advantageous effects are obtained: the removal efficiency of impurities derived from host cells is improved, the yield of SARS-CoV-2 or inactivated SARS-CoV-2 is further increased, and highly purified SARS-CoV-2 or inactivated SARS-CoV-2 can be efficiently produced. The pH when SARS-CoV-2 or inactivated SARS-CoV-2 is adsorbed to a cellulose sulfate gel is preferably pH 7.1 to 7.9, pH 7.1 to 7.8, pH 7.2 to 7.7, pH 7.2 to 7.6, pH 7.3 to 7.5, or pH 7.4. In addition, the pH when removing impurities from the cellulose sulfate gel and eluting SARS-CoV-2 or inactivated SARS-CoV-2 is preferably pH 8.5 to 9.5, pH 8.6 to 9.4, pH 8.7 to 9.3, pH 8.8 to 9.2, pH 8.9 to 9.1, or pH 9.

[0067] As a buffer suitable for maintaining the pH in the above range, for example, carbonic acid-bicarbonate buffer (carbonic acid-bicarbonate buffer) (sometimes also referred to as carbonate buffer), Tris-hydrochloric acid buffer, glycine-sodium hydroxide buffer, etc. can be cited. In addition, the concentration of the above buffer is usually the concentration used in ion exchange chromatography, etc., for example, 10 mM or more and 100 mM or less, preferably 10 mM or more and 50 mM or less.

[0068] First, after dialyzing a solution containing SARS-CoV-2 or a solution containing inactivated SARS-CoV-2 (e.g., a concentrated solution) with an appropriate buffer (solvent replacement), it is contacted with a cellulose sulfate gel that has been equilibrated with the same buffer in advance, thereby adsorbing SARS-CoV-2 or inactivating SARS-CoV-2. The contact of the solution containing SARS-CoV-2 or the solution containing inactivated SARS-CoV-2 with the cellulose sulfate gel can be implemented, for example, by passing the solution containing SARS-CoV-2 or the solution containing inactivated SARS-CoV-2 through a column filled with cellulose sulfate gel.

[0069] In washing the cellulose sulfate gel to which SARS-CoV-2 or inactivated SARS-CoV-2 has been adsorbed, the same buffer as used for the adsorption or a similar buffer with the pH maintained is used.

[0070] SARS-CoV-2 or inactivated SARS-CoV-2 can be eluted from the cellulose sulfate gel by increasing the salt concentration of the same buffer. To increase the salt concentration, common salts used in ion exchange, adsorption chromatography, etc., such as sodium chloride at 0.2 M or more and 1 M or less, can be used.

[0071] The purification process may be performed only once or multiple times. When performed multiple times, the purification processes after the first time may be performed under the same conditions as the first purification process or under different conditions. When performed under different conditions, for example, the pH conditions during adsorption may be changed between the first purification process and the subsequent purification processes.

[0072] (Formulation process)

[0073] The formulation process is the process of mixing the purified inactivated SARS-CoV-2 with other ingredients to obtain an inactivated SARS-CoV-2 vaccine.

[0074] Examples of other components include aluminum hydroxide, aluminum phosphate, potassium phosphate, adjuvants such as mineral oil or non-mineral oil, and stabilizers such as amino acids and sugars.

[0075] The purified inactivated SARS-CoV-2 can be replaced with an appropriate buffer by ultrafiltration or the like as needed, and then sterile filtered through a membrane filter and mixed with other components, or mixed with other components and then sterile filtered through a membrane filter.

[0076] Utilizing the manufacturing method involved in the present embodiment, an inactivated SARS-CoV-2 vaccine that is substantially free of proteins derived from host cells is obtained. Here, the so-called “substantially free of proteins derived from host cells” means that the protein derived from host cells is less than 10 ng, preferably less than 5 ng, less than 4 ng, less than 3 ng, or less than 2 ng, relative to 1 μg of protein contained in the inactivated SARS-CoV-2 vaccine. The content of protein contained in the inactivated SARS-CoV-2 vaccine can be quantified by subjecting the vaccine to a protein quantification method (e.g., conventional methods such as the TCA-Lowry method). The content of protein derived from host cells contained in the inactivated SARS-CoV-2 vaccine can be quantified by subjecting the vaccine to a quantification method that can specifically detect proteins derived from host cells (e.g., when the host cells are Vero cells, the method described in the embodiments described later).

[0077] In addition, by using the manufacturing method involved in other embodiments, it is possible to more efficiently manufacture an inactivated SARS-CoV-2 vaccine with a low content of protein derived from host cells and a high purity. Here, the so-called "low content of protein derived from host cells" means that the protein derived from host cells is 30 ng or less, preferably 20 ng or less, 18 ng or less, 16 ng or less, 14 ng or less, 12 ng or less, more preferably 10 ng or less, and further preferably 5 ng or less, 4 ng or less, 3 ng or less, or 2 ng or less, relative to 1 μg of protein contained in the inactivated SARS-CoV-2 vaccine.

[0078] [SARS-CoV-2 or purification method for inactivated SARS-CoV-2]

[0079] The method for purifying SARS-CoV-2 or inactivated SARS-CoV-2 involved in this embodiment comprises the following steps: contacting a solution containing SARS-CoV-2 or a solution containing inactivated SARS-CoV-2 with a cellulose sulfate gel at pH 8 to 10, thereby adsorbing SARS-CoV-2 or inactivated SARS-CoV-2 to the gel, then removing impurities, and then eluting and recovering SARS-CoV-2 or inactivated SARS-CoV-2.

[0080] Other embodiments involve a method for purifying SARS-CoV-2 or inactivated SARS-CoV-2, which comprises the following steps: contacting a solution containing SARS-CoV-2 or a solution containing inactivated SARS-CoV-2 with a cellulose sulfate gel at a pH of 7 to 8, thereby adsorbing SARS-CoV-2 or inactivated SARS-CoV-2 to the gel, then removing impurities at a pH of 8 to 10, and then eluting and recovering SARS-CoV-2 or inactivated SARS-CoV-2.

[0081] As a specific aspect of the purification method according to the present embodiment, the aspects described in the purification step in the production method according to the present embodiment can be applied without particular limitation.

[0082] Utilizing the purification method involved in the present embodiment, it is possible to obtain a SARS-CoV-2 antigen composition or an inactivated SARS-CoV-2 antigen composition that does not substantially contain a protein derived from a host cell. Here, the so-called "substantially does not contain a protein derived from a host cell" refers to that the protein derived from the host cell is less than 10ng, preferably less than 5ng, less than 4ng, less than 3ng, or less than 2ng relative to the protein 1μg contained in the SARS-CoV-2 antigen composition or the inactivated SARS-CoV-2 antigen composition. The content of the protein contained in the SARS-CoV-2 antigen composition or the inactivated SARS-CoV-2 antigen composition can be quantified by implementing a protein quantification method (for example, conventional methods such as the TCA-Lowry method) to the composition. The content of the protein derived from the host cell contained in the SARS-CoV-2 antigen composition or the inactivated SARS-CoV-2 antigen composition can be quantified by implementing a quantification method (for example, when the host cell is a Vero cell, the method described in the embodiment described later) that can specifically detect the protein derived from the host cell to the composition.

[0083] Through the purification methods involved in other embodiments, it is also possible to obtain a SARS-CoV-2 antigen composition or an inactivated SARS-CoV-2 antigen composition with a low content of host cell-derived protein and a high content of SARS-CoV-2 or inactivated SARS-CoV-2. Here, the so-called "low content of host cell-derived protein" means that the host cell-derived protein is 30 ng or less, preferably 20 ng or less, 18 ng or less, 16 ng or less, 14 ng or less, 12 ng or less, more preferably 10 ng or less, and further preferably 5 ng or less, 4 ng or less, 3 ng or less, or 2 ng or less, relative to 1 μg of protein contained in the SARS-CoV-2 antigen composition or the inactivated SARS-CoV-2 antigen composition.

[0084] Example

[0085] Hereinafter, the present invention will be described in detail based on Examples, but the present invention is not limited to these Examples at all.

[0086] [Example 1: Proliferation of SARS-CoV-2]

[0087] Vero cells (CCL-81 (registered trademark)) as virus proliferation substrate (host cell) are obtained from ATCC (American Type Culture Collection). Vero cells are propagated using appropriate culture medium and culture method to make a cell bank. The cells of the cell bank are expanded (cell culture) using D-MEM culture medium containing bovine serum. First, culture with a culture bottle (flask), and then expand to a culture bag. Finally, a Vero cell culture medium (VP-SFM, ThermoFisher Scientific system) containing a cell culture microcarrier (Cytodex (registered trademark), Cytiva (GlobalLife Sciences Technologies Japan KK)) with a concentration of more than 1g / L and less than 5g / L is put into a culture tank, and then Vero cells are inoculated to proliferate. Viruses (SARS-CoV-2) are inoculated into proliferated Vero cells with an MOI of 0.0001, and the Vero cells infected by the virus are further cultured. Virus liquid is harvested from the virus suspension obtained by culture.

[0088] [Example 2: Inactivation of SARS-CoV-2]

[0089] The harvested virus solution was concentrated using an ultrafiltration membrane (hollow fiber cartridge, Cytiva (Global Life Sciences Technologies Japan KK)). β-propiolactone was added to the concentrate to a concentration of 0.05 w / v%, and then inactivated at 20°C for more than 16 hours to obtain a solution containing inactivated SARS-CoV-2.

[0090] [Example 3: Purification by cellulose sulfate gel chromatography (1)]

[0091] Purification of inactivated SARS-CoV-2 was performed by column chromatography using the following gel-filled column (height 117 mm), wherein a cellulose sulfate gel (Cellufine Sulfate, manufactured by JNC Co., Ltd.: a substance in which the hydroxyl group at the 6th position is sulfated, particle size 40-130 μm, spherical) was filled in an empty column (inner diameter 50 mm, Cytiva (Global Life Sciences Technologies Japan K.K.)) as a carrier. Column chromatography was performed under the conditions of pH 7.4 described in Non-Patent Document 2 and pH 9.0.

[0092] Under the condition of pH 7.4, after the column is balanced with 140mM sodium chloride + 10mM phosphate buffer (pH 7.4), the solvent of the solution containing the inactivated SARS-CoV-2 is replaced with the phosphate buffer (pH 7.4) as the injection liquid, and the injection liquid (466mL) is injected into the column to adsorb the inactivated SARS-CoV-2 on the carrier. The phosphate buffer (pH 7.4) is injected into the column, and the adsorbed carrier is washed. Then, the inactivated SARS-CoV-2 is further eluted with a 10mM phosphate buffer (pH 7.4) containing a high concentration of sodium chloride (NaCl). For elution, it is implemented in stages using an eluent with a step-by-step increase in the NaCl concentration (0.34M→0.6M→1M).

[0093] Under the condition of pH 9.0, purification was carried out in the same manner as at pH 7.4, except that 10 mM carbonate buffer (pH 9.0) was used instead of 140 mM sodium chloride + 10 mM phosphate buffer (pH 7.4), and elution was carried out stepwise using an eluent with a stepwise increase in NaCl concentration (0.2 M → 0.4 M → 0.6 M → 0.8 M → 1 M).

[0094] The inactivated SARS-CoV-2 content and protein content in the injection solution and the eluate were quantified by the TCA-Lowry method. The host cell-derived protein content in the injection solution and the eluate was quantified by the Immunoenzymetric Assay for Measurement of Vero Cell Host Cell Proteins (Catalog # F500, manufactured by CYGNUS Technologies).

[0095] The yield of inactivated SARS-CoV-2 in the eluate, the yield of protein, and the yield of protein derived from host cells are summarized in Table 1. In Table 1, "yield" is the ratio (%) of the amount contained in the total amount of the eluate to the amount contained in the injection solution.

[0096] Under the condition of pH 7.4, the yield of inactivated SARS-CoV-2 was 64%, the protein yield was 53%, and the protein yield from host cells was 58%. On the other hand, under the condition of pH 9.0, the yield of inactivated SARS-CoV-2 was 76%, the protein yield was 64%, and the protein yield from host cells was 28%. According to the results, when the purification condition of pH 9.0 is used, the protein yield from host cells is greatly reduced relative to the inactivated SARS-CoV-2 yield and protein yield. That is, it is confirmed that under the purification condition of pH 9.0, an eluate in which the protein from host cells in the feed solution is further removed is obtained, with few impurities and high purity.

[0097] [Table 1]

[0098] Yield of inactivated SARS-CoV-2 Protein yield Protein yield from host cells pH7.4 64％ 53％ 58％ pH 9.0 76％ 64％ 28％

[0099] [Example 4: Purification by cellulose sulfate gel chromatography (2)]

[0100] Purification based on ion exchange chromatography, which is commonly used in the production of inactivated SARS-CoV-2 vaccines, was compared with purification based on chromatography using cellulose sulfate gel as a support.

[0101] (Purification based on cellulose sulfate gel chromatography)

[0102] A column (140 mm in inner diameter, 150 mm in length, Cytiva (Global Life Sciences Technologies Japan K.K.)) filled with cellulose sulfate gel (Cellufine Sulfate, manufactured by JNC Co., Ltd.: a substance in which the hydroxyl group at the 6th position is sulfated, with a particle size of 40-130 μm and a spherical shape) as a carrier was repeatedly washed and balanced with a 10 mM carbonate buffer adjusted to pH 9.0. Next, a solution containing inactivated SARS-CoV-2 replaced with the same buffer was injected into the column to adsorb the inactivated SARS-CoV-2 to the carrier. The elution of the inactivated SARS-CoV-2 was carried out by slowly adding a 1 M sodium chloride solution. The recovered eluate was replaced with 140 mM sodium chloride + 10 mM phosphate buffer (pH 7.2) by ultrafiltration or the like as a raw drug for vaccine manufacturing (inactivated SARS-CoV-2 antigen composition).

[0103] (Purification based on ion exchange chromatography)

[0104] As a carrier for ion exchange chromatography, an anion chromatographic membrane (Sartobind Q, manufactured by Sartorius) was used. A solution containing inactivated SARS-CoV-2 adjusted to a sodium chloride concentration of 340 mM was injected into an anion chromatographic membrane equilibrated with 340 mM sodium chloride + phosphate buffer, and the flow-through liquid was recovered. The recovered flow-through liquid was replaced with 140 mM sodium chloride + 10 mM phosphate buffer (pH 7.2) by ultrafiltration or the like, as a raw drug for vaccine manufacturing (inactivated SARS-CoV-2 antigen composition).

[0105] The protein content in each original drug and the protein content derived from the host cell were quantified by an ELISA system using an anti-Vero protein antibody, which was prepared by immunizing rabbits and guinea pigs with proteins obtained from the culture supernatant of Vero cells. The results are shown in Table 2. In Table 2, the protein content derived from the host cell relative to 1 dose indicates the protein content derived from the host cell when 1 dose is set to 10 μg of protein content.

[0106] [Table 2]

[0107]

[0108] The content of host cell-derived protein contained in the original drug obtained when an anion chromatography membrane was used as a carrier was 337.3 ng per dose, while the content of host cell-derived protein contained in the original drug obtained when cellulose sulfate gel was used was 14.7 ng per dose. That is, it is shown that the original drug (inactivated SARS-CoV-2 antigen composition) obtained by purification by chromatography using cellulose sulfate gel as a carrier has few impurities and is of high purity.

[0109] [Example 5: Preparation and evaluation of inactivated SARS-CoV-2 vaccine]

[0110] The original drug (inactivated SARS-CoV-2 antigen composition) obtained by purification by chromatography using cellulose sulfate gel as a carrier in Example 4 was directly used as an inactivated SARS-CoV-2 vaccine (vaccine without adjuvant). In addition, aluminum hydroxide was added to the original drug as an adjuvant to obtain an inactivated SARS-CoV-2 vaccine (vaccine with adjuvant). The adjuvanted vaccine or the unadjuvanted vaccine was administered intramuscularly twice to Balb / c mice (N=6) and C57BL / 6N mice (N=6) at intervals of 2 weeks at 0.0625μg / dose, 0.25μg / dose or 1.00μg / dose, respectively. 14 days after the second administration, the neutralizing antibody titer against SARS-CoV-2 in the serum was measured. The measurement results are shown in Figure 1 .like Figure 1 As shown, it was found that when mice were immunized with the vaccine, high-titer neutralizing antibodies against SARS-CoV-2 were induced in the serum of the mice.

[0111] [Example 6: Purification by cellulose sulfate gel chromatography (3)]

[0112] The column filled with cellulose sulfate gel used in Example 4 was repeatedly washed and balanced using a 10mM carbonate buffer adjusted to pH 9.0. Next, a solution containing inactivated SARS-CoV-2 replaced with 100mM sodium chloride + 50mM phosphate buffer (pH 7.4) was injected into the column to adsorb the inactivated SARS-CoV-2 to the carrier. Then, it was washed and balanced with a capacity of 5 columns using a 10mM carbonate buffer adjusted to pH 9.0. The elution of the inactivated SARS-CoV-2 was carried out by slowly adding a 1M sodium chloride solution (pH 9.0). The recovered eluate was replaced with 140mM sodium chloride + 10mM phosphate buffer (pH 7.2) by ultrafiltration or the like as a raw drug for vaccine manufacture (inactivated SARS-CoV-2 antigen composition).

[0113] The protein content in the original drug and the protein content derived from the host cells were quantified by an ELISA system using an anti-Vero protein antibody prepared by immunizing rabbits and guinea pigs with proteins obtained from the culture supernatant of Vero cells. The results were compared with those of Example 4 and are shown in Table 3. In Table 3, the protein content derived from the host cells relative to one dose indicates the protein content derived from the host cells when one dose is set to a protein content of 10 μg.

[0114] By setting the pH of the solution containing inactivated SARS-CoV-2 at 7.4 during adsorption, the protein content derived from the host cells is 152.4 ng relative to one dose. It is shown that although it contains more impurities than the result (14.7 ng) of Example 4 in which the pH of the solution containing inactivated SARS-CoV-2 is set to 9.0 during adsorption, the impurities are reduced compared to the result (337.3 ng) of the case where an anion chromatography membrane is used as a carrier. On the other hand, when focusing on the protein content, by setting the pH of the solution containing inactivated SARS-CoV-2 at 7.4 during adsorption, the protein content is 408.1 μg / mL, which is more than the result (129.2 μg / mL) of Example 4, and it can be seen that the method of this embodiment is effective for the purpose of increasing protein yield.

[0115] [Table 3]

[0116]

Claims

1. A method for producing an inactivated SARS-CoV-2 vaccine, comprising the steps of: contacting a solution containing SARS-CoV-2 or a solution containing inactivated SARS-CoV-2 with a cellulose sulfate gel at a pH of 8 to 10, thereby adsorbing the SARS-CoV-2 or the inactivated SARS-CoV-2 to the gel, removing impurities, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2.

2. An inactivated SARS-CoV-2 vaccine obtained by the production method described in claim 1 and substantially free of host cell-derived proteins.

3. The vaccine according to claim 2, further comprising an adjuvant.

4. A method for purifying SARS-CoV-2 or inactivated SARS-CoV-2, comprising the following steps: contacting a solution containing SARS-CoV-2 or a solution containing inactivated SARS-CoV-2 with a cellulose sulfate gel at a pH of 8 to 10, thereby adsorbing the SARS-CoV-2 or the inactivated SARS-CoV-2 to the gel, then removing impurities, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2.

5. A SARS-CoV-2 antigen composition or an inactivated SARS-CoV-2 antigen composition, which is obtained by the purification method according to claim 4 and substantially does not contain proteins derived from host cells.

6. A method for producing an inactivated SARS-CoV-2 vaccine, comprising the following steps: contacting a solution containing SARS-CoV-2 or a solution containing inactivated SARS-CoV-2 with a cellulose sulfate gel at a pH of 7 to 8, thereby adsorbing the SARS-CoV-2 or the inactivated SARS-CoV-2 to the gel, then removing impurities at a pH of 8 to 10, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2.

7. An inactivated SARS-CoV-2 vaccine obtained by the manufacturing method according to claim 6.

8. The vaccine according to claim 7, wherein The content of host cell-derived protein is less than 30 ng relative to 1 μg of protein contained in the inactivated SARS-CoV-2 vaccine.

9. The vaccine according to claim 7 or 8, further comprising an adjuvant.

10. A method for purifying SARS-CoV-2 or inactivated SARS-CoV-2, comprising the following steps: contacting a solution containing SARS-CoV-2 or a solution containing inactivated SARS-CoV-2 with a cellulose sulfate gel at a pH of 7 to 8, thereby adsorbing the SARS-CoV-2 or the inactivated SARS-CoV-2 to the gel, then removing impurities at a pH of 8 to 10, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2.

11. A SARS-CoV-2 antigen composition or an inactivated SARS-CoV-2 antigen composition obtained by the purification method according to claim 10.

12. The antigen composition according to claim 11, wherein The content of host cell-derived protein is less than 30 ng relative to 1 μg of protein contained in the SARS-CoV-2 antigen composition or the inactivated SARS-CoV-2 antigen composition.

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