Method for producing an inactivated SARS-CoV-2 vaccine, 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

Abstract

The present invention pertains to a method for manufacturing an inactivated SARS-CoV-2 vaccine, the method comprising a step for bringing a SARS-CoV-2-containing solution or an inactivated SARS-CoV-2-containing solution into contact with a cellulose sulfate ester gel at a pH of 8-10 inclusive to cause the SARS-CoV-2 or the inactivated SARS-CoV-2 to be adsorbed by the gel, then removing impurities, then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2.

Description

[Technical Field]

[0001] The present invention relates to a method for producing an inactivated SARS-CoV-2 vaccine, 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 technology]

[0002] The novel coronavirus disease (COVID-19), officially designated COVID-19 by the World Health Organization (WHO), originated in 2019 and is caused by a virus called SARS-CoV-2. COVID-19 is a respiratory tract infection typically presenting with symptoms such as headache, loss of smell and / or taste, nasal congestion and runny nose, cough, muscle pain, sore throat, fever, diarrhea, and difficulty breathing. While COVID-19 infections often present with mild, asymptomatic or cold-like symptoms that resolve spontaneously, severe cases can present with acute respiratory distress syndrome, sepsis, and / or multiple organ failure. SARS-CoV-2 is a single-stranded, positive-strand RNA virus with a diameter of 50–200 nm and belonging to the orthocoronavirus subfamily.

[0003] Vaccination is one method of preventing COVID-19. Various vaccines against SARS-CoV-2, including mRNA vaccines, viral vector vaccines, and recombinant protein vaccines, have been developed and approved, and some are already on the market (Non-Patent Document 1). In Japan, mRNA vaccines and viral vector vaccines are administered to many people aged 5 and older, with the hope of reducing the number of severe cases and deaths by preventing infection, aggravation, and onset of the disease. However, these vaccines have been reported to cause side effects after vaccination, such as pain at the injection site, headache, fatigue, and muscle pain. In rare cases, anaphylaxis has been reported after vaccination, and, depending on the type of vaccine, thrombosis, pericarditis, and myocarditis have also been reported.

[0004] Meanwhile, inactivated vaccines are also known as vaccines other than those mentioned above. Inactivated vaccines are used, for example, as seasonal influenza vaccines, Japanese encephalitis vaccines, and quadrivalent vaccines. Inactivated vaccines are produced using pathogens (such as viruses) that have lost their infectiousness, or components of those pathogens (such as parts of viruses), and are generally known to have few side effects and a high level of safety. Therefore, it is expected that inactivated SARS-CoV-2 vaccines will also be able to be administered to children under the age of five.

[0005] One method for producing an inactivated vaccine involves propagating the target virus using an established cell line as a virus culture substrate (host cell), then inactivating and purifying the propagated virus to obtain the purified inactivated virus as a viral antigen composition for vaccine production. This production method requires the establishment of a virus propagation method to achieve high productivity and a method for purifying the inactivated virus to achieve further safety. In inactivated SARS-CoV-2 vaccines, purification by ion exchange chromatography and / or size exclusion chromatography is typically used to purify the inactivated virus (Patent Document 1, Patent Document 2). Purification by affinity chromatography has also been reported (Patent Document 3, Non-Patent Document 2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2021 / 204825 [Patent Document 2] International Publication No. 2021 / 209060 [Patent Document 3] International Publication No. 2022 / 38642 [Non-patent literature]

[0007] [Non-Patent Document 1] Clinical Immunology 222(2021)108634 [Non-patent document 2] Purification of human coronavirus OC43 using Cellufine Sulfate, JNC Corporation Technical Data Sheet, https: / / www.jnc-corp.co.jp / fine / jp / cellufine / guide / pdf / affinity / TD_Sulfate_N5_V1_J.pdf (verified June 1, 2022) Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for producing a highly pure inactivated SARS-CoV-2 vaccine that has a low content (particularly, is substantially free of) impurities derived from host cells (e.g., animal cell lines) (e.g., host cell-derived proteins). Another object of the present invention is to provide a purification method for SARS-CoV-2 or inactivated SARS-CoV-2, which can yield a highly pure SARS-CoV-2 antigenic composition or inactivated SARS-CoV-2 antigenic composition that has a low content (particularly, is substantially free of) impurities derived from host cells (e.g., animal cell lines) (e.g., host cell-derived proteins). A further object of the present invention is to provide an inactivated SARS-CoV-2 vaccine, SARS-CoV-2 antigenic composition, or inactivated SARS-CoV-2 antigenic composition obtained by the production method or purification method. [Means for solving the problem]

[0009] As a result of extensive research to achieve the above-mentioned objective, the inventors discovered that by growing SARS-CoV-2 in an established animal cell line (Vero cells) as a virus culture substrate (host cells), and then purifying the inactivated SARS-CoV-2 by cellulose sulfate ester gel adsorption chromatography at a pH of 8 to 10, it is possible to obtain a more highly purified inactivated SARS-CoV-2 that is substantially free of impurities (proteins) derived from the virus culture substrate (host cells), thereby completing the present invention.

[0010] The present invention relates to a method for producing an inactivated SARS-CoV-2 vaccine, comprising the steps of contacting a SARS-CoV-2-containing solution or an inactivated SARS-CoV-2-containing solution with a cellulose sulfate ester gel at a pH of 8 to 10 to adsorb the SARS-CoV-2 or the inactivated SARS-CoV-2 onto the gel, removing impurities, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2.

[0011] The production method of the present invention involves purifying SARS-CoV-2 or inactivated SARS-CoV-2 using cellulose sulfate ester gel under specific pH conditions, thereby making it possible to obtain an inactivated SARS-CoV-2 vaccine that is substantially free of impurities (e.g., host cell-derived proteins) derived from host cells (e.g., animal cell lines).

[0012] The present invention also relates to an inactivated SARS-CoV-2 vaccine obtained by the above-mentioned production method and substantially free of host cell-derived proteins. Because the vaccine is obtained by the above-mentioned production method of the present invention, it is substantially free of impurities (e.g., host cell-derived proteins) derived from host cells (e.g., animal cell lines), making it safer.

[0013] The vaccine may further contain an adjuvant.

[0014] The present invention can also be considered as a method for purifying SARS-CoV-2 or inactivated SARS-CoV-2, comprising the steps of contacting a SARS-CoV-2-containing solution or an inactivated SARS-CoV-2-containing solution with cellulose sulfate gel at a pH of 8 to 10 to adsorb the SARS-CoV-2 or the inactivated SARS-CoV-2 onto the gel, removing impurities, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2.

[0015] The present invention further relates to a SARS-CoV-2 antigen composition or an inactivated SARS-CoV-2 antigen composition obtained by the above purification method, which is substantially free of host cell-derived proteins.

[0016] As a result of extensive research to achieve the above-mentioned objectives, the inventors have discovered that highly purified inactivated SARS-CoV-2 with a low content of impurities (proteins) derived from the virus culture substrate (host cells) can be obtained in high yields by growing SARS-CoV-2 in an established animal cell line (Vero cells) as a virus culture substrate (host cells), adsorbing the inactivated SARS-CoV-2 at a pH of 7 to 8, removing impurities at a pH of 8 to 10, and purifying the resultant product using cellulose sulfate ester gel adsorption chromatography, which then elutes and recovers the product. This discovery led to the completion of the present invention.

[0017] The present invention relates to a method for producing an inactivated SARS-CoV-2 vaccine, comprising the steps of contacting a SARS-CoV-2-containing solution or an inactivated SARS-CoV-2-containing solution with a cellulose sulfate ester gel at a pH of 7 to 8 to adsorb the SARS-CoV-2 or the inactivated SARS-CoV-2 onto the gel, removing impurities at a pH of 8 to 10, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2.

[0018] The production method of the present invention involves purifying SARS-CoV-2 or inactivated SARS-CoV-2 using cellulose sulfate gel under specific pH conditions, thereby enabling more efficient production of a highly pure inactivated SARS-CoV-2 vaccine with a low content of impurities (e.g., host cell-derived proteins) derived from host cells (e.g., animal cell lines).

[0019] The present invention also relates to an inactivated SARS-CoV-2 vaccine obtained by the above-mentioned production method. Because the vaccine is obtained by the above-mentioned production method of the present invention, the content of impurities (e.g., host cell-derived proteins) derived from host cells (e.g., animal cell lines) is low, thereby improving safety.

[0020] The vaccine may contain 30 ng or less of host cell-derived protein per μg of protein contained in the inactivated SARS-CoV-2 vaccine.

[0021] The vaccine may further contain an adjuvant.

[0022] The present invention can also be considered as a method for purifying SARS-CoV-2 or inactivated SARS-CoV-2, comprising the steps of contacting a SARS-CoV-2-containing solution or an inactivated SARS-CoV-2-containing solution with cellulose sulfate gel at a pH of 7 to 8 to adsorb the SARS-CoV-2 or the inactivated SARS-CoV-2 onto the gel, removing impurities at a pH of 8 to 10, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2.

[0023] The present invention further relates to a SARS-CoV-2 antigen composition or an inactivated SARS-CoV-2 antigen composition obtained by the above purification method.

[0024] The antigen composition may have a host cell-derived protein content of 30 ng or less per μg of protein contained in the SARS-CoV-2 antigen composition or the inactivated SARS-CoV-2 antigen composition. [Effects of the Invention]

[0025] The present invention provides a method for producing a highly pure inactivated SARS-CoV-2 vaccine that has a low content (particularly, is substantially free of) impurities derived from host cells (e.g., animal cell lines) (e.g., host cell-derived proteins).The present invention also provides a method for purifying SARS-CoV-2 or inactivated SARS-CoV-2, which can yield a highly pure SARS-CoV-2 antigen composition or inactivated SARS-CoV-2 antigen composition that has a low content (particularly, is substantially free of) impurities derived from host cells (e.g., animal cell lines) (e.g., host cell-derived proteins).The present invention further provides an inactivated SARS-CoV-2 vaccine, SARS-CoV-2 antigen composition, or inactivated SARS-CoV-2 antigen composition obtained by the production method or purification method.

[0026] The inactivated SARS-CoV-2 vaccine of the present invention is capable of inducing antibodies with neutralizing activity against SARS-CoV-2. [Brief explanation of the drawings]

[0027] [Figure 1] 1 is a graph showing the results of measuring neutralizing antibody titers against SARS-CoV-2 in serum in Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0028] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0029] [Manufacturing method for inactivated SARS-CoV-2 vaccine] The method for producing the inactivated SARS-CoV-2 vaccine according to this embodiment includes a step of contacting a SARS-CoV-2-containing solution or an inactivated SARS-CoV-2-containing solution with cellulose sulfate ester gel at a pH of 8 to 10 to adsorb SARS-CoV-2 or inactivated SARS-CoV-2 onto the gel, removing impurities, and then eluting and recovering the SARS-CoV-2 or inactivated SARS-CoV-2 (purification step A).

[0030] The production method according to this embodiment is characterized in that, in the process of producing an inactivated SARS-CoV-2 vaccine, the purification step, which includes adsorption and elution of SARS-CoV-2 or inactivated SARS-CoV-2 onto cellulose sulfate ester gel, is carried out under alkaline conditions (pH 8 to 10). This improves the efficiency of removing impurities (e.g., host cell-derived proteins) during the purification step, allowing for the production of a highly pure inactivated SARS-CoV-2 vaccine that is substantially free of impurities.

[0031] In another embodiment, a method for producing an inactivated SARS-CoV-2 vaccine includes a step of contacting a SARS-CoV-2-containing solution or an inactivated SARS-CoV-2-containing solution with cellulose sulfate ester gel at a pH of 7 to 8 to adsorb SARS-CoV-2 or inactivated SARS-CoV-2 onto the gel, followed by removing impurities at a pH of 8 to 10, and then eluting and recovering SARS-CoV-2 or inactivated SARS-CoV-2 (purification step B).

[0032] Another embodiment of the production method is a method for producing an inactivated SARS-CoV-2 vaccine, and is characterized in that, in the purification steps 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 adsorption is carried out under near-neutral conditions (pH 7 to 8), and the subsequent removal of impurities and elution are carried out under alkaline conditions (pH 8 to 10). This improves the efficiency of impurity removal in the purification step and also increases the yield of SARS-CoV-2 or inactivated SARS-CoV-2, enabling more efficient production of a highly pure inactivated SARS-CoV-2 vaccine with a low impurity content.

[0033] In addition to purification step A or B (hereinafter sometimes collectively referred to as the "purification step"), the manufacturing method of this embodiment may further comprise at least one step selected from the group consisting of a step of growing SARS-CoV-2 (growing step), a step of inactivating SARS-CoV-2 (inactivation step), and a step of mixing purified inactivated SARS-CoV-2 with other components to obtain an inactivated SARS-CoV-2 vaccine (formulation step).

[0034] (Proliferation process) The propagation step is a step of propagating SARS-CoV-2. Propagation of SARS-CoV-2 can be carried out by infecting host cells, which serve as a virus culture substrate, with SARS-CoV-2 and recovering the SARS-CoV-2 that has propagated in the infected host cells.

[0035] The host cells used for the propagation of SARS-CoV-2 are not particularly limited as long as they allow good growth of SARS-CoV-2 and produce highly immunogenic SARS-CoV-2 (antigen). For example, non-human animals (e.g., chickens) and animal-derived cell lines (animal cell lines) can be used. Specific examples of cell lines include 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. An example of a host cell suitable for use in the production method of this embodiment is Vero cells.

[0036] The SARS-CoV-2 virus strain used in the production method according to this embodiment is not particularly limited as long as it maintains protective antigens. Examples of the SARS-CoV-2 virus strain include the initial strain (Wuhan strain) and its mutant strains, such as the alpha strain, beta strain, gamma strain, delta strain, and omicron strain.

[0037] When an established cell line is used as the host cell, the medium used for expanding (growing) the host cell (hereinafter sometimes referred to as "growth medium") is selected from those commonly used in 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), EX-CELL293-S (Nichirei), TFBM-01 (Nichirei), and ASF104, and supplemented with amino acids, salts, antifungal / antibacterial agents, animal serum, etc. The preferred growth medium is D-MEM medium supplemented with amino acids, salts, antifungal / antibacterial agents, animal serum, etc.

[0038] After inoculation of SARS-CoV-2 into host cells, the medium used for SARS-CoV-2 growth (hereinafter sometimes referred to as "maintenance medium") is, for example, a serum-free or low-protein medium. While the above-mentioned growth medium without animal serum can be used as the maintenance medium, a medium containing VP-SFM supplemented with L-glutamic acid is preferably used. The use of serum-free medium facilitates purification because fewer impurities need to be removed, and also eliminates the possibility of contamination with unknown pathogens derived from serum.

[0039] Methods for growing host cells include, for example, static culture, roller bottle culture, and suspension culture. Among these, tank culture using microcarriers, which is one type of suspension culture, allows for high-density culture of host cells and is suitable for obtaining large quantities of SARS-CoV-2. Many microcarriers for this purpose are commercially available, and a preferred microcarrier for growing Vero cells is, for example, Cytodex® I (Cytiva (Global Life Science Technologies Japan, Inc.)). Cytodex® is preferably used at a concentration of 1 g / L or more and 5 g / L or less in the growth medium.

[0040] When infecting a host cell with SARS-CoV-2, the number of SARS-CoV-2 per host cell, i.e., the multiplicity of infection (MOI), may be, for example, in the range of 0.000001 to 0.01, or in the range of 0.00001 to 0.001.

[0041] The incubation temperature and incubation period for growing SARS-CoV-2 in infected host cells are adjusted depending on the type of host cell, the amount of SARS-CoV-2 inoculated, the incubation scale, and the incubation method. For example, the following method is used to grow SARS-CoV-2 in Vero cells using static incubation or roller bottle incubation.

[0042] First, Vero cells are cultured in a growth medium consisting of D-MEM medium supplemented with non-essential amino acids and bovine serum at a temperature of 32°C to 38°C, preferably 37°C, for a period of 2 to 7 days, preferably 5 to 7 days. Next, when the growth of Vero cells reaches a plateau, the medium is aspirated and the cells are washed several times with phosphate-buffered saline or the like. SARS-CoV-2 is then inoculated at an MOI of 0.000001 to 0.01, preferably 0.0001. VP-SFM is added as a maintenance medium, and the cells are cultured at a temperature of 32°C to 38°C for 3 to 8 days, preferably 37°C for 5 to 7 days. The resulting culture, i.e., the host cell lysate or culture supernatant, contains a large amount of SARS-CoV-2.

[0043] The SARS-CoV-2-containing solution obtained in the propagation step (e.g., host cell lysate or culture supernatant) is used to carry out the inactivation step or purification step.

[0044] (Inactivation process) The inactivation step is a step of inactivating SARS-CoV-2. The inactivation step can be carried out, for example, by contacting SARS-CoV-2 with an inactivating agent. SARS-CoV-2 can also be inactivated by adding the inactivating agent to a culture of host cells infected with SARS-CoV-2, or by adding the inactivating agent to the culture after impurities have been removed from the culture and / or to a concentrated version of the culture. Specifically, for example, insoluble matter can be removed from a culture of host cells infected with SARS-CoV-2 by coarse centrifugation or membrane filtration, and the resulting supernatant can then be further concentrated using an ultrafiltration membrane with a molecular weight exclusion limit of 100,000 to 750,000, preferably 300,000 to 750,000.

[0045] Examples of inactivating agents include formaldehyde, paraformaldehyde, glutaraldehyde, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, β-propiolactone, etc. β-propiolactone is preferred as the inactivating agent.

[0046] The amount of inactivating agent added when inactivating SARS-CoV-2 can be determined depending on the type of solution containing SARS-CoV-2, the type of inactivating agent, etc. For example, when insoluble matter is removed from a culture of host cells infected with SARS-CoV-2 by crude centrifugation or membrane filtration, and the resulting supernatant is further concentrated using an ultrafiltration membrane with an exclusion limit of 100,000 to 750,000, preferably 300,000 to 750,000, and β-propiolactone is added to the concentrate to inactivate SARS-CoV-2, the amount of inactivating agent added may be 0.01 w / v% to 0.1 w / v%, preferably 0.05 w / v%.

[0047] The temperature and reaction time (time of contact with the inactivating agent) when inactivating SARS-CoV-2 can be set depending on the type of solution containing SARS-CoV-2, the type and concentration of the inactivating agent, etc. For example, when insoluble matter is removed from a culture of host cells infected with SARS-CoV-2 by coarse centrifugation or membrane filtration, and the resulting supernatant is further concentrated using an ultrafiltration membrane with an exclusion limit of 100,000 to 750,000, preferably 300,000 to 750,000, and β-propiolactone is added to the concentrate to inactivate SARS-CoV-2, inactivation can be achieved by stirring at a reaction temperature of around 20°C for 16 hours or more.

[0048] The inactivation step may be performed before or after the purification step. When the inactivation step is performed before the purification step, the purification step may be performed using the inactivated SARS-CoV-2-containing solution obtained in the inactivation step (e.g., the reaction solution after the reaction with the inactivating agent).

[0049] (purification process) Purification step A is a step in which a solution containing SARS-CoV-2 or an inactivated SARS-CoV-2 is contacted with cellulose sulfate gel at a pH of 8 to 10 to adsorb SARS-CoV-2 or the inactivated SARS-CoV-2 onto the gel, impurities are removed, and then the SARS-CoV-2 or the inactivated SARS-CoV-2 is eluted and recovered.

[0050] Purification step B is a step in which a solution containing SARS-CoV-2 or an inactivated SARS-CoV-2 is contacted with cellulose sulfate gel at a pH of 7 to 8 to adsorb SARS-CoV-2 or an inactivated SARS-CoV-2 onto the gel, impurities are removed at a pH of 8 to 10, and then the SARS-CoV-2 or the inactivated SARS-CoV-2 is eluted and recovered.

[0051] The cellulose sulfate gel is a gel formed from a cellulose sulfate ester in which the hydroxy group of cellulose forms an ester with sulfuric acid. The cellulose sulfate is preferably an ester in which the hydroxy group at the 6-position of cellulose forms an ester with sulfuric acid. The particle size of the cellulose sulfate gel is, for example, 10 μm to 200 μm, preferably 20 μm to 170 μm, 30 μm to 150 μm, or 40 μm to 130 μm. Such a cellulose sulfate gel is available, for example, as Cellufine Sulfate (manufactured by JNC Corporation: the hydroxy group at the 6-position is sulfated, particle size 40-130 μm, spherical).

[0052] SARS-CoV-2 and inactivated SARS-CoV-2 can be adsorbed to cellulose sulfate gel at a pH range of 7 to 10. The production method according to this embodiment is characterized in that SARS-CoV-2 or inactivated SARS-CoV-2 is adsorbed to cellulose sulfate gel at a pH range of 8 to 10. Setting the pH within this range improves the efficiency of removing host cell-derived impurities (host cell-derived proteins), advantageously resulting in the production of more highly purified SARS-CoV-2 or inactivated SARS-CoV-2 that is substantially free of impurities. The pH during adsorption of SARS-CoV-2 or inactivated SARS-CoV-2 to cellulose sulfate gel is preferably 8.5 to 9.5, 8.6 to 9.4, 8.7 to 9.3, 8.8 to 9.2, 8.9 to 9.1, or 9.0.

[0053] In another embodiment, the method is characterized in that SARS-CoV-2 or inactivated SARS-CoV-2 is adsorbed onto 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. Setting the pH within this range improves the efficiency of removing host cell-derived impurities and also improves the yield of SARS-CoV-2 or inactivated SARS-CoV-2, resulting in the efficient production of highly purified SARS-CoV-2 or inactivated SARS-CoV-2. The pH during adsorption of SARS-CoV-2 or inactivated SARS-CoV-2 onto cellulose sulfate gel is preferably 7.1 to 7.9, 7.1 to 7.8, 7.2 to 7.7, 7.2 to 7.6, 7.3 to 7.5, or 7.4. In addition, the pH during removal of impurities from the cellulose sulfate ester gel and elution of SARS-CoV-2 or inactivated SARS-CoV-2 is preferably between pH 8.5 and 9.5, between pH 8.6 and 9.4, between pH 8.7 and 9.3, between pH 8.8 and 9.2, between pH 8.9 and 9.1, or between pH 9.

[0054] Examples of buffers suitable for maintaining the pH within the above range include carbonate-bicarbonate buffer (sometimes referred to as carbonate buffer), Tris-hydrochloric acid buffer, glycine-sodium hydroxide buffer, etc. The concentration of the above buffers is typically the concentration used for ion exchange chromatography, etc., for example, 10 mM to 100 mM, preferably 10 mM to 50 mM.

[0055] First, a solution containing SARS-CoV-2 or an inactivated SARS-CoV-2-containing solution (e.g., a concentrated solution) is dialyzed against an appropriate buffer (solvent replacement), and then the solution is brought into contact with cellulose sulfate gel that has been pre-equilibrated with the same buffer to adsorb SARS-CoV-2 or an inactivated SARS-CoV-2. Contact of the SARS-CoV-2-containing solution or an inactivated SARS-CoV-2-containing solution with the cellulose sulfate gel can be carried out, for example, by passing the SARS-CoV-2-containing solution or an inactivated SARS-CoV-2-containing solution through a column packed with cellulose sulfate gel.

[0056] The cellulose sulfate gel onto which SARS-CoV-2 or inactivated SARS-CoV-2 has been adsorbed is washed with the same buffer solution used for adsorption or a similar buffer solution at the same pH.

[0057] SARS-CoV-2 or inactivated SARS-CoV-2 is eluted from the cellulose sulfate gel by increasing the salt concentration of the buffer solution, typically using salts used in ion exchange and adsorption chromatography, such as sodium chloride (0.2 M to 1 M).

[0058] The purification step may be performed only once or multiple times. When the purification step is performed multiple times, the first and subsequent purification steps may be performed under the same conditions as the first purification step or under different conditions. When the purification steps are performed under different conditions, for example, the pH conditions during adsorption may be changed between the first and subsequent purification steps.

[0059] (Formulation process) The formulation process involves mixing purified inactivated SARS-CoV-2 with other ingredients to obtain an inactivated SARS-CoV-2 vaccine.

[0060] Other components include, for example, adjuvants such as aluminum hydroxide, aluminum phosphate, potassium phosphate, mineral oil or non-mineral oil, and stabilizers such as amino acids and sugars.

[0061] The purified inactivated SARS-CoV-2 may be replaced with an appropriate buffer solution by ultrafiltration or other methods as necessary, and then sterile filtered through a membrane filter before being mixed with other components, or it may be mixed with other components and then sterile filtered through a membrane filter.

[0062] The production method of this embodiment produces an inactivated SARS-CoV-2 vaccine that is substantially free of host cell-derived proteins. Here, "substantially free of host cell-derived proteins" means that the amount of host cell-derived proteins contained in the inactivated SARS-CoV-2 vaccine is 10 ng or less, preferably 5 ng or less, 4 ng or less, 3 ng or less, or 2 ng or less, per μg of protein contained in the inactivated SARS-CoV-2 vaccine. The amount of protein contained in the inactivated SARS-CoV-2 vaccine can be quantified by subjecting the vaccine to a protein quantification method (e.g., a standard method such as the TCA-Lowry method). The amount of host cell-derived proteins contained in the inactivated SARS-CoV-2 vaccine can be quantified by subjecting the vaccine to a quantification method that can specifically detect host cell-derived proteins (e.g., when the host cells are Vero cells, the method described in the Examples below).

[0063] Furthermore, a production method according to another embodiment enables the efficient production of a highly pure inactivated SARS-CoV-2 vaccine with a low content of host cell-derived proteins. Here, "a low content of host cell-derived proteins" means that the amount of host cell-derived proteins per 1 μg of protein contained in the inactivated SARS-CoV-2 vaccine is 30 ng or less, preferably 20 ng or less, 18 ng or less, 16 ng or less, 14 ng or less, or 12 ng or less, more preferably 10 ng or less, and even more preferably 5 ng or less, 4 ng or less, 3 ng or less, or 2 ng or less.

[0064] [Method for purifying SARS-CoV-2 or inactivated SARS-CoV-2] The method for purifying SARS-CoV-2 or inactivated SARS-CoV-2 according to this embodiment includes the steps of contacting a solution containing SARS-CoV-2 or inactivated SARS-CoV-2 with cellulose sulfate gel at a pH of 8 to 10 to adsorb SARS-CoV-2 or inactivated SARS-CoV-2 to the gel, removing impurities, and then eluting and recovering SARS-CoV-2 or inactivated SARS-CoV-2.

[0065] Another embodiment of a method for purifying SARS-CoV-2 or inactivated SARS-CoV-2 comprises the steps of contacting a solution containing SARS-CoV-2 or inactivated SARS-CoV-2 with cellulose sulfate gel at a pH of 7 to 8 to adsorb SARS-CoV-2 or inactivated SARS-CoV-2 to the gel, removing impurities at a pH of 8 to 10, and then eluting and recovering SARS-CoV-2 or inactivated SARS-CoV-2.

[0066] As a specific embodiment of the purification method according to this embodiment, the embodiments described in the purification step of the production method according to this embodiment can be applied without any particular limitations.

[0067] The purification method of this embodiment allows for the production of a SARS-CoV-2 antigen composition or an inactivated SARS-CoV-2 antigen composition that is substantially free of host cell-derived proteins. Here, "substantially free of host cell-derived proteins" means that the amount of host cell-derived proteins contained in the SARS-CoV-2 antigen composition or inactivated SARS-CoV-2 antigen composition is 10 ng or less, preferably 5 ng or less, 4 ng or less, 3 ng or less, or 2 ng or less, per μg of protein contained in the SARS-CoV-2 antigen composition or inactivated SARS-CoV-2 antigen composition. The amount of protein contained in the SARS-CoV-2 antigen composition or inactivated SARS-CoV-2 antigen composition can be quantified by subjecting the composition to a protein quantification method (e.g., a standard method such as the TCA-Lowry method). The amount of host cell-derived proteins contained in the SARS-CoV-2 antigen composition or inactivated SARS-CoV-2 antigen composition can be quantified by subjecting the composition to a quantification method capable of specifically detecting host cell-derived proteins (e.g., when the host cells are Vero cells, the method described in the Examples below).

[0068] Another embodiment of the purification method also allows for the production of a highly purified and high-content SARS-CoV-2 antigen composition or inactivated SARS-CoV-2 antigen composition, which has a low content of host cell-derived proteins and a high content of SARS-CoV-2 or inactivated SARS-CoV-2. Here, "a low content of host cell-derived proteins" means that the amount of host cell-derived proteins per μg of protein contained in the SARS-CoV-2 antigen composition or inactivated SARS-CoV-2 antigen composition is 30 ng or less, preferably 20 ng or less, 18 ng or less, 16 ng or less, 14 ng or less, or 12 ng or less, more preferably 10 ng or less, and even more preferably 5 ng or less, 4 ng or less, 3 ng or less, or 2 ng or less. [Example]

[0069] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples in any way.

[0070] Example 1: Propagation of SARS-CoV-2 Vero cells (CCL-81®) were obtained from the American Type Culture Collection (ATCC) as a virus propagation substrate (host cells). Vero cells were grown using appropriate media and culture methods to create a cell bank. The cells from this cell bank were expanded (cell cultured) in D-MEM medium containing bovine serum. Initially, the cells were cultured in flasks, then expanded into culture bags. Finally, Vero cell medium (VP-SFM, Thermo Fisher Scientific) containing cell culture microcarriers (Cytodex®, Cytiva, Global Life Science Technologies Japan, Inc.) at a concentration of 1 g / L to 5 g / L was added to the culture tank, and Vero cells were seeded and grown. The grown Vero cells were inoculated with the virus (SARS-CoV-2) at an MOI of 0.0001, and the virus-infected Vero cells were further cultured. The virus suspension obtained during culture was harvested.

[0071] Example 2: Inactivation of SARS-CoV-2 The harvested virus solution was concentrated using an ultrafiltration membrane (hollow fiber cartridge, Cytiva (Global Life Science Technologies Japan, Inc.)). β-propiolactone was added to the concentrated solution to a concentration of 0.05 w / v%, and the solution was then inactivated by leaving it at 20°C for at least 16 hours to obtain a solution containing inactivated SARS-CoV-2.

[0072] Example 3: Purification by cellulose sulfate gel chromatography (1) Inactivated SARS-CoV-2 was purified by column chromatography using a gel-packed column (height 117 mm) packed with cellulose sulfate ester gel (Cellufine Sulfate, manufactured by JNC Corporation: sulfated at the 6-hydroxyl group, particle size 40-130 μm, spherical) as a carrier in an empty column (inner diameter 50 mm, Cytiva (Global Life Science Technologies Japan Co., Ltd.)). Column chromatography was performed under the conditions of pH 7.4 and pH 9.0 described in Non-Patent Document 2.

[0073] For the pH 7.4 condition, the column was equilibrated with 10 mM phosphate buffer (pH 7.4) containing 140 mM sodium chloride. The solvent in the inactivated SARS-CoV-2-containing solution was then replaced with this phosphate buffer (pH 7.4) to form the application solution. This application solution (466 mL) was then applied to the column, allowing the inactivated SARS-CoV-2 to adsorb onto the carrier. The phosphate buffer (pH 7.4) was then applied to the column, and the carrier was washed after adsorption. The inactivated SARS-CoV-2 was then eluted with 10 mM phosphate buffer (pH 7.4) containing an even higher concentration of sodium chloride (NaCl). Elution was performed stepwise using eluents with increasing NaCl concentrations (0.34 M → 0.6 M → 1 M).

[0074] At 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 10 mM phosphate buffer (pH 7.4) containing 140 mM sodium chloride, and elution was carried out stepwise using eluents with gradually increasing NaCl concentrations (0.2 M → 0.4 M → 0.6 M → 0.8 M → 1 M).

[0075] The inactivated SARS-CoV-2 and protein contents in the applied solution and eluate were quantified by the TCA-Lowry method. The host cell protein contents in the applied solution and eluate were quantified by the Immunoenzymetric Assay for Measurement of Vero Cell Host Cell Proteins (Catalog #F500, Cygnus Technologies).

[0076] The yield of inactivated SARS-CoV-2, protein yield, and host cell-derived protein yield in the eluate are summarized in Table 1. In Table 1, "yield" is the ratio (%) of the amount contained in the total eluate to the amount contained in the application solution.

[0077] Under pH 7.4 conditions, the yield of inactivated SARS-CoV-2 was 64%, the protein yield was 53%, and the host cell-derived protein yield was 58%. On the other hand, under pH 9.0 conditions, the yield of inactivated SARS-CoV-2 was 76%, the protein yield was 64%, and the host cell-derived protein yield was 28%. These results show that the yield of host cell-derived protein relative to the yield of inactivated SARS-CoV-2 and protein was significantly lower when using purification conditions at pH 9.0. In other words, purification conditions at pH 9.0 resulted in an eluate in which more host cell-derived proteins in the application solution were removed, confirming that the eluate had fewer impurities and was of higher purity.

[0078] [Table 1]

[0079] Example 4: Purification by cellulose sulfate gel chromatography (2) We compared purification by ion exchange chromatography, which is commonly used in the production of inactivated SARS-CoV-2 vaccines, with purification by chromatography using cellulose sulfate ester gel as a carrier.

[0080] (Purification by cellulose sulfate gel chromatography) A column (inner diameter 140 mm, length 150 mm, Cytiva (Global Life Science Technologies Japan, Inc.)) packed with cellulose sulfate ester gel (Cellufine Sulfate, manufactured by JNC Corporation: sulfated at the 6-hydroxyl group, particle size 40-130 μm, spherical) was equilibrated by repeated washing with 10 mM carbonate buffer adjusted to pH 9.0. The inactivated SARS-CoV-2 solution, replaced with the same buffer, was then applied to the column, and the inactivated SARS-CoV-2 was adsorbed onto the carrier. The inactivated SARS-CoV-2 was eluted by gradually adding 1 M sodium chloride solution. The collected eluate was replaced with 10 mM phosphate buffer (pH 7.2) containing 140 mM sodium chloride by ultrafiltration or other methods, and used as the drug substance for vaccine production (inactivated SARS-CoV-2 antigen composition).

[0081] (Purification by ion exchange chromatography) An anion chromatography membrane (Zaltobind Q, manufactured by Sartorius) was used as the carrier for ion exchange chromatography. A solution containing inactivated SARS-CoV-2, adjusted to a sodium chloride concentration of 340 mM, was applied to the anion chromatography membrane equilibrated with phosphate buffer containing 340 mM sodium chloride, and the flow-through liquid was collected. The collected flow-through liquid was replaced with 10 mM phosphate buffer (pH 7.2) containing 140 mM sodium chloride by ultrafiltration or other methods to obtain the drug substance (inactivated SARS-CoV-2 antigen composition) for vaccine production.

[0082] The protein content and host cell-derived protein content in each drug substance were quantified by ELISA using anti-Vero protein antibodies prepared by immunizing rabbits and guinea pigs with proteins obtained from Vero cell culture supernatant. The results are shown in Table 2. In Table 2, the host cell-derived protein content per dose indicates the host cell-derived protein content when 1 dose has a protein content of 10 μg.

[0083] [Table 2]

[0084] The host cell-derived protein content in the drug substance obtained when an anion chromatography membrane was used as the carrier was 337.3 ng per dose, whereas the host cell-derived protein content in the drug substance obtained when cellulose sulfate ester gel was used was 14.7 ng per dose. In other words, the drug substance (inactivated SARS-CoV-2 antigen composition) obtained by purification by chromatography using cellulose sulfate ester gel as a carrier was shown to have few impurities and high purity.

[0085] Example 5: Production and evaluation of inactivated SARS-CoV-2 vaccines The drug substance (inactivated SARS-CoV-2 antigen composition) obtained by purification by chromatography using cellulose sulfate gel as a carrier in Example 4 was used directly as an inactivated SARS-CoV-2 vaccine (non-adjuvanted vaccine). Furthermore, aluminum hydroxide was added to the drug substance as an adjuvant to obtain an inactivated SARS-CoV-2 vaccine (adjuvanted vaccine). Balb / c mice (N=6) and C57BL / 6N mice (N=6) were intramuscularly administered 0.0625 μg / dose, 0.25 μg / dose, or 1.00 μg / dose of the adjuvanted or unadjuvanted vaccine twice, two weeks apart, and serum neutralizing antibody titers against SARS-CoV-2 were measured 14 days after the second administration. The measurement results are shown in Figure 1. As shown in Figure 1, immunization of mice with the vaccine induced high titers of neutralizing antibodies against SARS-CoV-2 in the serum of the mice.

[0086] Example 6: Purification by cellulose sulfate gel chromatography (3) A column packed with the cellulose sulfate gel used in Example 4 was equilibrated by repeated washing with 10 mM carbonate buffer adjusted to pH 9.0. Next, a solution containing inactivated SARS-CoV-2, which had been replaced with 50 mM phosphate buffer (pH 7.4) containing 100 mM sodium chloride, was applied to the column, and inactivated SARS-CoV-2 was adsorbed onto the carrier. The column was then equilibrated by washing with five column volumes of 10 mM carbonate buffer adjusted to pH 9.0. Elution of inactivated SARS-CoV-2 was performed by gradually adding 1 M sodium chloride solution (pH 9.0). The collected eluate was replaced with 10 mM phosphate buffer (pH 7.2) containing 140 mM sodium chloride by ultrafiltration or other methods, and used as a drug substance for vaccine production (inactivated SARS-CoV-2 antigen composition).

[0087] The protein content and host cell-derived protein content in the drug substance were quantified by an ELISA system using anti-Vero protein antibodies prepared by immunizing rabbits and guinea pigs with proteins obtained from Vero cell culture supernatant. The results are shown in Table 3 in comparison with the results of Example 4. In Table 3, the host cell-derived protein content per dose indicates the host cell-derived protein content when 1 dose has a protein content of 10 μg.

[0088] By adjusting the pH of the inactivated SARS-CoV-2-containing solution to 7.4 during adsorption, the host cell-derived protein content per dose was 152.4 ng. Although the impurity content was higher than in Example 4 (14.7 ng) when the inactivated SARS-CoV-2-containing solution was adjusted to pH 9.0 during adsorption, the impurity content was reduced compared to the result (337.3 ng) when an anion chromatography membrane was used as the carrier. Meanwhile, focusing on the protein content, adjusting the pH of the inactivated SARS-CoV-2-containing solution to 7.4 during adsorption resulted in a content of 408.1 μg / mL, which was higher than the result (129.2 μg / mL) in Example 4, demonstrating that the method of this example is effective for increasing protein yield.

[0089] [Table 3]

Claims

1. 1. A method for producing an inactivated SARS-CoV-2 vaccine, comprising: the method comprises the steps of contacting a SARS-CoV-2-containing solution or an inactivated SARS-CoV-2-containing solution with a cellulose sulfate gel at a pH of 8 to 10 to adsorb the SARS-CoV-2 or the inactivated SARS-CoV-2 onto the gel, removing impurities, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2; The SARS-CoV-2-containing solution or the inactivated SARS-CoV-2-containing solution contains a carbonate-bicarbonate buffer solution of 10 mM or more and 50 mM or less.

2. An inactivated SARS-CoV-2 vaccine obtained by the production method of claim 1, which is substantially free of host cell-derived proteins.

3. 3. The vaccine of claim 2, further comprising an adjuvant.

4. 1. A method for purifying SARS-CoV-2 or inactivated SARS-CoV-2, comprising: the method comprises the steps of contacting a SARS-CoV-2-containing solution or an inactivated SARS-CoV-2-containing solution with a cellulose sulfate gel at a pH of 8 to 10 to adsorb the SARS-CoV-2 or the inactivated SARS-CoV-2 onto the gel, removing impurities, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2; The SARS-CoV-2-containing solution or the inactivated SARS-CoV-2-containing solution contains a carbonate-bicarbonate buffer solution of 10 mM or more and 50 mM or less.

5. A SARS-CoV-2 antigen composition or an inactivated SARS-CoV-2 antigen composition obtained by the purification method according to claim 4, which is substantially free of host cell-derived proteins.

6. 1. A method for producing an inactivated SARS-CoV-2 vaccine, comprising: the method comprises the steps of contacting a SARS-CoV-2-containing solution or an inactivated SARS-CoV-2-containing solution with a cellulose sulfate gel at a pH of 7 to 7.9 to adsorb the SARS-CoV-2 or the inactivated SARS-CoV-2 onto the gel, removing impurities at a pH of 8 to 10, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2; The SARS-CoV-2-containing solution or the inactivated SARS-CoV-2-containing solution contains 10 mM or more and 50 mM or less of phosphate buffer.

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

8. The vaccine of claim 7, wherein the content of host cell-derived proteins is 30 ng or less per μg of protein contained in the inactivated SARS-CoV-2 vaccine.

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

10. 1. A method for purifying SARS-CoV-2 or inactivated SARS-CoV-2, comprising: the method comprises the steps of contacting a SARS-CoV-2-containing solution or an inactivated SARS-CoV-2-containing solution with a cellulose sulfate gel at a pH of 7 to 7.9 to adsorb the SARS-CoV-2 or the inactivated SARS-CoV-2 onto the gel, removing impurities at a pH of 8 to 10, and then eluting and recovering the SARS-CoV-2 or the inactivated SARS-CoV-2; The SARS-CoV-2-containing solution or the inactivated SARS-CoV-2-containing solution contains 10 mM or more and 50 mM or less of phosphate buffer.

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 30 ng or less per 1 μg of protein contained in the SARS-CoV-2 antigen composition or the inactivated SARS-CoV-2 antigen composition.

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