Virus inactivation compound, virus inactivation agent, and virus inactivation method

Abstract

The virus inactivation compound according to the present invention is a compound represented by formula (1) or formula (2). (In formula (1), n is an integer of 3-19, and in formula (2), m is an integer of 3-19). The present invention makes it possible to provide a virus inactivation compound having an excellent virus inactivation effect.

Description

Virus Inactivation Compound, Virus Inactivator, and Virus Inactivation Method

[0001] The present invention relates to a virus inactivation compound, a virus inactivator, and a virus inactivation method.

[0002] Many biopharmaceuticals are manufactured using raw materials derived from living organisms, and there is a possibility of contamination with exogenous infectious substances such as viruses. Therefore, in general, a virus inactivation step is incorporated into the manufacturing process to reduce the risk of virus contamination in the product. Common virus inactivation methods include heat treatment, ultraviolet treatment, use of surfactants, virus removal filtration, low pH treatment, etc. Among these, Triton X-100 has been widely used for virus inactivation using surfactants. In this regard, Triton X-100 is considered to potentially affect human health and the environment, and its production, import, and use within the EU have been regulated by the REACH regulation. Therefore, the development of a surfactant having a virus inactivation effect to replace Triton X-100 is required.

[0003] For example, Patent Document 1 shows that the virus in the antibody solution can be sufficiently inactivated by incubating the antibody solution and the surfactant-containing solution for 5 to 120 minutes. Also, for example, Patent Document 2 discloses a method for inactivating enveloped viruses under low pH conditions using an N-methylglucamide solution as an alternative to Triton X-100.

[0004] Japanese Patent Application Laid-Open No. 2024-001035 International Publication No. WO2022 / 507369

[0005] However, neither the method of Patent Document 1 nor Patent Document 2 has a sufficient virus inactivation effect, and there is room for improvement from the perspective of replacing Triton X-100. In view of the above problems, an object of the present invention is to provide a virus inactivation compound having an excellent virus inactivation effect, a virus inactivator containing the virus inactivation compound, and a virus inactivation method using the virus inactivation compound.

[0006] In view of the above problems, the inventors of the present invention conducted diligent research and found that compounds represented by formula (1) or formula (2) have excellent virus inactivation effects, and thus completed the present invention. That is, the present invention provides the following [1] to [3].

[0007] [1] A virus inactivating compound represented by formula (1) or formula (2). [In equation (1), n ​​is an integer between 3 and 19.] [In formula (2), m is an integer from 3 to 19.] [2] A virus inactivator comprising the virus inactivating compound described in [1] above and at least one solvent selected from the group consisting of water and alcohol. [3] A method for inactivating a virus, comprising contacting the virus inactivating compound described in [1] above with a virus.

[0008] According to the present invention, it is possible to provide a virus inactivation compound with excellent virus inactivation effect, a virus inactivating agent containing the virus inactivation compound, and a virus inactivation method using the virus inactivation compound.

[0009] The present invention will be described in detail below.

[0010] [Virus-Inactivating Compound of the Present Invention] The virus-inactivating compound of the present invention is a compound represented by formula (1) or formula (2). The compound represented by formula (1) is as follows: [In equation (1), n ​​is an integer between 3 and 19.]

[0011] From the viewpoint of enhancing the virus inactivation effect, n in the compound of formula (1) is preferably 7 to 15, more preferably 11 to 15. The compound of formula (1) can be obtained, for example, by reacting 2-methacryloyloxyethyl-2-trimethylammonioethyl phosphate (MPC) with a 1-alkanethiol. For example, it can be synthesized by reacting 2-methacryloyloxyethyl-2-trimethylammonioethyl phosphate (MPC) and a 1-alkanethiol in a solvent using an amine catalyst such as diisopropylamine at room temperature (e.g., 25°C) for 10 to 50 hours. The solvent may be an alcohol or a solvent other than an alcohol, such as acetonitrile. The 1-alkanethiol is a 1-alkanethiol having 4 to 20 carbon atoms, with 1-alkanethiols having 8 to 16 carbon atoms being preferred, and 1-alkanethiols having 12 to 16 carbon atoms being more preferred.

[0012] The compounds represented by formula (1) include, specifically, 2-[3-(octysulfanil)-2-methylpropionyloxy]ethyl-2-(trimethylammonio)ethyl phosphate (n=7 in formula (1)), 2-[3-(decylsulfanil)-2-methylpropionyloxy]ethyl-2-(trimethylammonio)ethyl phosphate (n=9 in formula (1)), and 2-[3-(dodecylsulfanil)-2-methylpropionyloxy Examples include 2-(3

[0013] The compound represented by formula (2) is as follows: [In equation (2), m is an integer between 3 and 19.]

[0014] From the viewpoint of enhancing the virus inactivation effect, the value of m in the compound of formula (2) is preferably 7 to 15, more preferably 11 to 15, and even more preferably 13 to 15. The compound of formula (2) can be obtained, for example, by reacting a compound obtained by reacting 2-chloro-2-oxo-1,3,2-dioxaphosphoran with 1-alkanol, and then further reacting it with trimethylamine (TMA). For example, it can be synthesized by reacting 2-chloro-2-oxo-1,3,2-dioxaphosphoran with 1-alkanol in a solvent such as acetonitrile using an amine catalyst such as diisopropylamine, then adding trimethylamine (TMA) to the resulting compound and reacting it at room temperature (e.g., 25°C) to 80°C for 10 to 50 hours. The 1-alkanol is a 1-alkanol having 4 to 20 carbon atoms, with a preference for 1-alkanols having 8 to 16 carbon atoms, a preference for 1-alkanols having 12 to 16 carbon atoms, and an even preference for 1-alkanols having 14 to 16 carbon atoms.

[0015] Examples of compounds represented by formula (2) include hexyl(2-[trimethylammonio]ethyl) phosphate (m=5 in formula (2)), octyl(2-[trimethylammonio]ethyl) phosphate (m=7 in formula (2)), decyl(2-[trimethylammonio]ethyl) phosphate (m=9 in formula (2)), dodecyl(2-[trimethylammonio]ethyl) phosphate (m=11 in formula (2)), tetradecyl(2-[trimethylammonio]ethyl) phosphate (m=13 in formula (2)), hexadecyl(2-[trimethylammonio]ethyl) phosphate (m=15 in formula (2)), and the like. Of these, the value of m in the compound of formula (2) is preferably 13 to 15.

[0016] [Virus Inactivating Agent of the Present Invention] The form of the virus inactivating agent of the present invention is not particularly limited as long as it can exert the effect of the agent. For example, the virus inactivating compound of the present invention can be dissolved in a solvent and used as a virus inactivating agent in solution form. As the solvent, water such as purified water, pure water, or deionized water, alcohols such as methanol, ethanol, or isopropanol, or a solution of these mixed in any proportion can be used. Furthermore, the virus inactivating compound of the present invention can be used dissolved in various buffer solutions containing water. Examples of various buffer solutions include phosphate buffer, Tris buffer, Good's buffer, glycine buffer, borate buffer, etc., and these may be mixed and used.

[0017] The amount of virus inactivating compound contained in the virus inactivating agent of the present invention is preferably 0.01% by mass or more. The amount of the virus inactivating compound is preferably 0.05% by mass or more, and more preferably 0.1% by mass or more. There is no particular upper limit to the amount of the virus inactivating compound as long as it is soluble in the solvent or buffer, but for example, it is 20% by mass or less, preferably 10% by mass or less. Within these ranges, the virus inactivating agent in solution form of the present invention exhibits an effective virus inactivation effect.

[0018] In addition to the compounds represented by formula (1) or formula (2), other compounds that may be contained in the virus inactivator include other reagents commonly used in this field for the purpose of further inactivating viruses, such as surfactants, proteins, sugars, and salts. Examples of surfactants include polyoxyethylene alkyl ethers and octylphenol ethoxylates. Examples of proteins include bovine serum albumin, gelatin, and casein. Examples of sugars include lactose, sucrose, and trehalose. Examples of salts include amino acids and amino acid salts such as glycine, alanine, serine, threonine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, and histidine, peptides such as glycylglycine, inorganic salts such as phosphates, borates, sulfates, and Tris salts, flavins, organic acids such as acetic acid, citric acid, malic acid, maleic acid, and gluconic acid, and salts of organic acids.

[0019] [Viruses to be inactivated by the virus inactivation compound of the present invention] The viruses to be inactivated by the virus inactivation compound of the present invention are not particularly limited. For example, enveloped viruses include the novel coronavirus, human influenza virus, herpesvirus, rubella virus, hepatitis B virus, hepatitis C virus, HIV, etc. Non-enveloped viruses include norovirus, rotavirus, poliovirus, adenovirus, etc. Of these, enveloped viruses are preferred. The virus to be inactivated may be a single virus or may include two or more types.

[0020] [Virus Inactivation Method of the Present Invention] The virus inactivation method of the present invention is a method comprising contacting a virus inactivation compound with a virus. Specifically, it is a method of inactivating a virus by contacting a treatment area or object in which a virus may be present with a virus inactivating agent containing a virus inactivation compound. That is, a virus can be inactivated by contacting the virus inactivating agent of the present invention with a treatment area or object. For example, the contact temperature between the virus and the virus inactivating agent in the virus inactivation method of the present invention is preferably 0 to 50°C, more preferably 10 to 40°C, and even more preferably 20 to 30°C. The contact time is preferably 0 to 60 minutes, more preferably 0 to 30 minutes, and even more preferably 1 to 10 minutes.

[0021] The present invention will be described in detail below with reference to examples and comparative examples, but the present invention is not limited thereto. In these examples, tests were conducted using viruses and bacteria that can be manipulated in a laboratory to confirm the effect of the virus inactivator containing the virus inactivating compound of the present invention.

[0022] [Synthesis Example 1-1] 53.50 g (0.3527 mol) of 2-methacryloyloxyethyl-2-trimethylammonioethyl phosphate (MPC) and 29.15 g (0.388 mol) of 1-octanthiol were dissolved in 123.17 g of acetonitrile (MeCN). 0.8067 g (0.01552 mol) of diisopropylamine was added as a catalyst, and the reaction was carried out at 30°C for 3 hours. After the reaction was complete, the reaction solution was diluted with 120.00 g of acetonitrile (MeCN), and reprecipitation in ethyl acetate was performed to obtain a white powder of the compound represented by formula (1), 2-[3-(octisulfanil)-2-methylpropionyloxy]ethyl-2-(trimethylammonio)ethyl phosphate (n=7 in formula (1)).

[0023] [Synthesis Example 1-2] Except for using 1-decanethiol instead of 1-octanthiol and changing the amount of charge so that the molar ratio was the same as in Synthesis Example 1-1, a white powder of the compound represented by formula (1), 2-[3-(decylsulfanyl)-2-methylpropionyloxy]ethyl-2-(trimethylammonio)ethyl phosphate (n=9 in formula (1)), was obtained in the same manner as in Synthesis Example 1-1.

[0024] [Synthesis Example 1-3] Except for using 1-dodecanethiol instead of 1-octanthiol and changing the amount of charge so that the molar ratio was the same as in Synthesis Example 1-1, a white powder of the compound represented by formula (1), 2-[3-(dodecylsulfanyl)-2-methylpropionyloxy]ethyl-2-(trimethylammonio)ethyl phosphate (n=11 in formula (1)), was obtained in the same manner as in Synthesis Example 1-1.

[0025] [Synthesis Example 1-4] Except for using 1-tetradecanethiol instead of 1-octanthiol and changing the amount of charge so that the molar ratio was the same as in Synthesis Example 1-1, a white powder of the compound represented by formula (1), 2-[3-(tetradecylsulfanyl)-2-methylpropionyloxy]ethyl-2-(trimethylammonio)ethyl phosphate (n=13 in formula (1)), was obtained in the same manner as in Synthesis Example 1-1.

[0026] [Synthesis Example 1-5] Except for using 1-hexadecanethiol instead of 1-octanthiol and changing the amount of charge so that the molar ratio was the same as in Synthesis Example 1-1, a white powder of the compound represented by formula (1), 2-[3-(hexadecylsulfanyl)-2-methylpropionyloxy]ethyl-2-(trimethylammonio)ethyl phosphate (n=15 in formula (1)), was obtained in the same manner as in Synthesis Example 1-1.

[0027] [Synthesis Example 2-1] 10.27 g (0.07 mol) of 2-chloro-2-oxo-1,3,2-dioxaphosphoran and 7.36 g (0.07 mol) of 1-hexanol were dissolved in 100 g of acetonitrile (MeCN). 14.58 g (0.14 mol) of diisopropylamine was added as a base, and the mixture was reacted at 0°C for 2 hours. The reaction solution was then filtered and purified. Next, 51.57 g (0.13 mol) of trimethylamine (TMA, approximately 13% acetonitrile solution) was added to the obtained filtrate, and the mixture was reacted at 75°C for 14 hours. After desolvation and purification by recrystallization, a white powder of hexyl(2-[trimethylammonio]ethyl) phosphate (m=5 in formula (2)), which is the compound represented by formula (2), was obtained.

[0028] [Synthesis Example 2-2] Except for using 1-octanol instead of 1-hexanol and changing the amount of ingredients charged so that the molar ratio was the same as in Synthesis Example 2-1, a white powder of octyl(2-[trimethylammonio]ethyl) phosphate (m=7 in formula (2)), which is the compound represented by formula (2), was obtained in the same manner as in Synthesis Example 2-1.

[0029] [Synthesis Example 2-3] Except for using 1-decanol instead of 1-hexanol and changing the amount of ingredients charged so that the molar ratio was the same as in Synthesis Example 2-1, a white powder of decyl(2-[trimethylammonio]ethyl) phosphate (m=9 in formula (2)), which is the compound represented by formula (2), was obtained in the same manner as in Synthesis Example 2-1.

[0030] [Synthesis Example 2-4] Except for using lauryl alcohol instead of 1-hexanol and changing the amount of charge so that the molar ratio was the same as in Synthesis Example 2-1, a white powder of dodecyl(2-[trimethylammonio]ethyl) phosphate (m=11 in formula (2)), which is the compound represented by formula (2), was obtained in the same manner as in Synthesis Example 2-1.

[0031] [Synthesis Example 2-5] Except for using 1-tetradecanol instead of 1-hexanol and changing the amount of charge so that the molar ratio was the same as in Synthesis Example 2-1, a white powder of tetradecyl(2-[trimethylammonio]ethyl) phosphate (m=13 in formula (2)), which is the compound represented by formula (2), was obtained in the same manner as in Synthesis Example 2-1.

[0032] [Synthesis Example 2-6] Except for using 1-hexadecanol instead of 1-hexanol and changing the amount of charge so that the molar ratio was the same as in Synthesis Example 2-1, a white powder of hexadecyl (2-[trimethylammonio]ethyl) phosphate (m=15 in formula (2)), which is the compound represented by formula (2), was obtained in the same manner as in Synthesis Example 2-1.

[0033] [Example 1-1: Examples 1-1-1 to 1-1-3] <Preparation of virus inactivating agent> The virus inactivating compounds shown in Synthesis Example 1-1 were dissolved in purified water to the concentrations listed in Table 1 to prepare the virus inactivating agents.

[0034] <Confirmation of the inactivation effect of phage Φ6> Preparation of culture medium, Pseudomonas syringae (hereinafter referred to as P bacteria) solution, and phage Φ6 test solution Phage diluent: 1.8 g of ordinary broth medium (manufactured by Eiken Chemical Co., Ltd.) was added to a 200 mL Erlenmeyer flask, dissolved in 100 mL of purified water, and autoclaved. 1.0 g of autoclaved ordinary broth medium was added to 500 mL of sterile purified water and diluted. This was used as the phage diluent.

[0035] SCDLP medium: 12.0 g of SCDLP liquid medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a 500 mL medium bottle, dissolved in 250 mL of purified water, and autoclaved. This was used as the SCDLP medium.

[0036] 702 liquid medium: 1.3 g of condensate solution 702 "Daigo" powder (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a 250 mL medium bottle, dissolved in 100 mL of purified water, and autoclaved. This was prepared as the 702 liquid medium.

[0037] 802 Agar Medium Plate: 8.4 g of the powder of the recovery culture medium 802 "Daigo" (manufactured by Fujifilm Wako Pure Chemical Corporation) was added to a 500 mL Erlenmeyer flask, dissolved in 300 mL of purified water, and subjected to autoclave treatment. The medium after autoclave treatment was used as the 802 agar medium. After cooling to about 60°C, 10 mL portions were added to 10 cmΦ plates before the agar solidified, and left at room temperature. After drying, it was stored in a constant temperature bath at 30°C until use. This was designated as the 802 agar medium plate.

[0038] 702 Soft Agar Medium: 1.3 g of the powder of the rehydration solution 702 "Daigo" (manufactured by Fujifilm Wako Pure Chemical Corporation) and 0.7 g of BD Bacto Agar (manufactured by Becton Dickinson) were added to a 200 mL Erlenmeyer flask, dissolved in 100 mL of purified water, and subjected to autoclave treatment. After cooling to about 60°C, 3 mL portions were dispensed into 15 mL conical tubes before the agar solidified, and stored in a constant temperature bath at 45°C. This was designated as the 702 soft agar medium.

[0039] P Bacterium Solution: One platinum loopful was collected from the P bacterium solution (NBRC14084 strain) using asepsis and plated on the entire 802 agar medium plate. It was left at 30°C for 5 days, and the resulting plate was designated as the P bacterium storage plate. 10 mL of the 702 liquid medium was dispensed into a 50 mL conical tube, and a single colony was collected with a platinum loop from the P bacterium storage plate and inoculated. It was cultured with shaking overnight at 30°C in a constant temperature bath, and after the shaking culture was completed, it was stored at room temperature. This was designated as the P bacterium solution.

[0040] Phage Φ6 Test Solution: The phage Φ6 solution (NBRC105899 strain) was diluted with a phage diluent so that it became 1.0×10 7 (PFU / mL). This was designated as the phage Φ6 test solution.

[0041] - Phage Φ6 Inactivation Effect Confirmation Test 4.5 mL of the prepared virus inactivator solution was collected into a 50 mL conical tube. 0.5 mL of the phage Φ6 test solution was added thereto, mixed, and allowed to contact for 1 minute. After 1 minute, 0.5 mL of the test sample that had been contacted with the phage Φ6 test solution was collected and added to a 50 mL conical tube to which 4.5 mL of SCDLP medium had been previously added, and then mixed. This solution was 10 １Using a 1.5 mL Eppendorf tube as the dilution solution, 0.9 mL of SCDLP medium was mixed. This step was repeated to prepare a 10 １ to 10 ７ times dilution solution. For 3 mL of 702 soft agar medium, 0.1 mL of P bacterial solution and 0.1 mL of each dilution solution were mixed and added to an 802 agar medium plate pre-warmed to 30 °C. After incubating overnight at 25 °C, the number of plaque-forming units was counted and the phage infection titer was calculated. The results are shown in Table 1. Phage infection titer (PFU / mL): Number of plaque-forming units (PFU) × Dilution factor / 0.1 (mL) Phage infection titer reduction value [Δlog(PFU / mL)]: Phage infection titer [log(PFU / mL)] in the sample - Phage infection titer [log(PFU / mL)] in Comparative Example 1-1 (SCDLP medium, negative control) Phage inactivation rate: (1 - 10^(-[Phage infection titer reduction value])) × 100

[0042] [Example 1-2: Examples 1-2-1 to 1-2-3] The phage Φ6 inactivation effect was confirmed in the same manner as in Example 1-1, except that the virus-inactivating compound obtained in Synthesis Example 1-2 was used instead of the virus-inactivating compound shown in Synthesis Example 1-1. The results are shown in Table 1.

[0043] [Example 1-3: Examples 1-3-1 to 1-3-3] The phage Φ6 inactivation effect was confirmed in the same manner as in Example 1-1, except that the virus-inactivating compound obtained in Synthesis Example 1-3 was used instead of the virus-inactivating compound shown in Synthesis Example 1-1. The results are shown in Table 1.

[0044] [Example 1-4: Examples 1-4-1 to 1-4-3] The phage Φ6 inactivation effect was confirmed in the same manner as in Example 1-1, except that the virus-inactivating compound obtained in Synthesis Example 1-4 was used instead of the virus-inactivating compound shown in Synthesis Example 1-1. The results are shown in Table 1.

[0045] [Example 1-5: Examples 1-5-1 to 1-5-3] The phage Φ6 inactivation effect was confirmed in the same manner as in Example 1-1, except that the virus-inactivating compound obtained in Synthesis Example 1-5 was used instead of the virus-inactivating compound shown in Synthesis Example 1-1. The results are shown in Table 1.

[0046] [Example 2-1: Examples 2-1-1 to 2-1-3] The phage Φ6 inactivation effect was confirmed in the same manner as in Example 1-1, except that the virus inactivation compound obtained in Synthesis Example 2-1 was used instead of the virus inactivation compound shown in Synthesis Example 1-1. The results are shown in Table 2.

[0047] [Example 2-2: Examples 2-2-1 to 2-2-3] The phage Φ6 inactivation effect was confirmed in the same manner as in Example 1-1, except that the virus inactivation compound obtained in Synthesis Example 2-2 was used instead of the virus inactivation compound shown in Synthesis Example 1-1. The results are shown in Table 2.

[0048] [Examples 2-3: Examples 2-3-1 to 2-3-3] The phage Φ6 inactivation effect was confirmed in the same manner as in Example 1-1, except that the virus inactivation compound obtained in Synthesis Example 2-3 was used instead of the virus inactivation compound shown in Synthesis Example 1-1. The results are shown in Table 2.

[0049] [Example 2-4: Examples 2-4-1 to 2-4-3] The phage Φ6 inactivation effect was confirmed in the same manner as in Example 1-1, except that the virus inactivation compound obtained in Synthesis Example 2-4 was used instead of the virus inactivation compound shown in Synthesis Example 1-1. The results are shown in Table 2.

[0050] [Example 2-5: Examples 2-5-1 to 2-5-3] The phage Φ6 inactivation effect was confirmed in the same manner as in Example 1-1, except that the virus inactivation compound obtained in Synthesis Example 2-5 was used instead of the virus inactivation compound shown in Synthesis Example 1-1. The results are shown in Table 2.

[0051] [Example 2-6: Examples 2-6-1 to 2-6-3] The phage Φ6 inactivation effect was confirmed in the same manner as in Example 1-1, except that the virus inactivation compound obtained in Synthesis Example 2-6 was used instead of the virus inactivation compound shown in Synthesis Example 1-1. The results are shown in Table 2.

[0052] [Comparative Example 1-1] The phage Φ6 inactivation effect was confirmed in the same manner as in Example 1-1, except that no virus inactivator was used and instead SCDLP medium was used. The results are shown in Table 2.

[0053] [Comparative Example 1-2: Comparative Examples 1-2-1 to 1-2-3] The phage Φ6 inactivation effect was confirmed in the same manner as in Example 1-1, except that Triton X-100 was used instead of the virus inactivation compound shown in Synthesis Example 1-1. The results are shown in Table 2.

[0054]

[0055]

[0056] As is clear from Tables 1 and 2, the virus inactivators using the compounds according to the embodiments of the present invention in Examples 1-1 to 2-6 have the same inactivating effect as Comparative Example 1-2. Therefore, the virus inactivator of the present invention has an excellent virus inactivating effect and can serve as a substitute for Triton X-100.

Claims

1. A virus inactivating compound represented by formula (1) or formula (2). [In equation (1), n ​​is an integer between 3 and 19.] [In equation (2), m is an integer between 3 and 19.] 2. A virus inactivator comprising the virus inactivating compound described in claim 1 and at least one solvent selected from the group consisting of water and alcohol.

3. A method for inactivating a virus, comprising contacting a virus inactivating compound according to claim 1 with a virus.

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