Surface-coated chip for viral vectors, and container

Abstract

The present invention addresses the problem of providing: a tool in which an adeno-associated virus vector is stabilized; an adeno-associated virus vector test kit; a method for decreasing the lower limit of detection in an adeno-associated virus vector test; a method for stabilizing an adeno-associated virus vector; and a method for retaining an infection ability of an adeno-associated virus vector and / or an ability of an adeno-associated virus vector to activate the expression of a gene in an infected cell.　The present invention provides a tool in which an adeno-associated virus vector is stabilized, the tool being provided with a coating film on at least a portion of the surface of the tool, in which the bubble contact angle in water on the surface of the coating film is 120° to 180°. It is preferred that the coating film contains a copolymer that contains a repeating unit containing a group represented by formula (a), a repeating unit containing a group represented by formula (b) and a repeating unit containing a group represented by formula (c) or a polymer or compound containing a hydroxyl group.

Description

Surface-coated chips and containers for viral vectors

[0001] The present invention relates to an instrument for stabilizing viruses, particularly adeno-associated virus vectors, an adeno-associated virus vector testing kit, a method for lowering the detection limit for adeno-associated virus vector testing, a method for stabilizing adeno-associated virus vectors, and a method for maintaining the infectivity of adeno-associated virus vectors and / or their gene expression activity in infected cells.

[0002] Viruses cannot replicate independently and therefore invade the cells of animals and plants, including humans, sometimes causing serious infectious diseases. For this reason, virus purification and storage are sometimes performed in the process of researching infectious disease prevention and treatment methods. Furthermore, to confirm or investigate viral infection, samples such as body fluids and sewage are collected, stored in containers, and then measured for viruses (see, for example, Non-Patent Document 1). However, viruses can adsorb to the surface of storage containers, resulting in loss of virus and reduced recoverability, posing a challenge. Similar to viruses, protein adsorption to container surfaces reduces recoverability and makes stable detection difficult. For example, ion complex materials capable of inhibiting the adhesion of biological substances and coating materials using such materials that inhibit the adhesion of biological substances have been reported (see, for example, Patent Document 1). While protein adsorption to container surfaces can sometimes be improved by adding additives such as surfactants, certain viruses are destroyed by surfactants, which can prevent adsorption but still cause problems with subsequent recovery and detection.

[0003] International Publication No. 2016 / 093293

[0004] Science of the Total Environment 739 (2020) 139076

[0005] The present invention aims to provide an instrument for stabilizing viruses, particularly adeno-associated viral vectors, an adeno-associated viral vector testing kit, a method for lowering the detection limit in adeno-associated viral vector testing, a method for stabilizing adeno-associated viral vectors, and a method for maintaining the infectivity of adeno-associated viral vectors and / or their gene expression activity in infected cells.

[0006] The present invention includes the following: [1] An instrument in which an adeno-associated virus vector is stabilized, the instrument having a coating film on at least a portion of its surface, wherein the coating film surface has a contact angle of an air bubble in water of 120° to 180°. [2] The instrument according to [1], wherein the stabilization is suppression of adhesion of the adeno-associated virus vector. [3] A copolymer in which the coating film comprises a repeating unit containing a group represented by the following formula (a), a repeating unit containing a group represented by the following formula (b), and a repeating unit containing a group represented by the following formula (c): [In the formula, U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ｃ represents a linear or branched alkyl group having 4 to 18 carbon atoms, a cyclic hydrocarbon group having 3 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, an aralkyl group having 7 to 14 carbon atoms, or an aryloxyalkyl group having 7 to 14 carbon atoms (wherein the aryl moiety may be substituted with a linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom); An －represents an anion selected from the group consisting of halide ions, inorganic acid ions, hydroxide ions, and isothiocyanate ions. [3] The instrument according to [1] or [2], wherein the coating film comprises a polymer or compound containing a hydroxy group. [5] The instrument according to any one of [1] to [4], which is a chip for adeno-associated virus vectors. [6] The instrument according to any one of [1] to [4], which is a container for storing adeno-associated virus vectors. [7] An adeno-associated virus vector testing kit comprising the instrument according to any one of [1] to [4]. [8] The instrument according to any one of [1] to [4], which is a material for producing adeno-associated virus vectors. [9] A method for reducing the lower limit of detection in adeno-associated virus vector testing, using the instrument according to any one of [1] to [4].

[10] A method for stabilizing an adeno-associated virus vector, comprising: (1) applying to at least a part of an instrument a coating film having a water-air bubble contact angle of 120° to 180° on the film surface; and (2) contacting the coating film with a composition containing an adeno-associated virus vector and a solvent.

[11] The coating film is a copolymer comprising a repeating unit containing a group represented by the following formula (a), a repeating unit containing a group represented by the following formula (b), and a repeating unit containing a group represented by the following formula (c): [In the formula, U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ｃ represents a linear or branched alkyl group having 4 to 18 carbon atoms, a cyclic hydrocarbon group having 3 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, an aralkyl group having 7 to 14 carbon atoms, or an aryloxyalkyl group having 7 to 14 carbon atoms (wherein the aryl moiety may be substituted with a linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom); An －represents an anion selected from the group consisting of halide ions, inorganic acid ions, hydroxide ions, and isothiocyanate ions.

[12] The method for stabilizing an adeno-associated virus vector according to

[10] , wherein the coating film comprises a polymer or compound containing a hydroxy group.

[13] The method for stabilizing an adeno-associated virus vector according to any one of

[10] to

[12] , wherein the composition does not contain a surfactant.

[14] A method for maintaining the infectivity of an adeno-associated virus vector and / or its gene expression activity in infected cells, using the instrument according to any one of [1] to [4].

[0007] The present invention provides an apparatus (e.g., a chip, a storage container, etc.) for stabilizing a virus, particularly an adeno-associated viral vector, an adeno-associated viral vector testing kit including the apparatus, a method for lowering the lower detection limit of an adeno-associated viral vector test using the apparatus, a method for stabilizing an adeno-associated viral vector, and a method for maintaining the infectivity of an adeno-associated viral vector and / or its gene expression activity in infected cells. Furthermore, the storage container of the present invention also has the effect of maintaining the viral infectivity (viral infectivity) of an adeno-associated viral vector even after storage for a certain period of time.

[0008] 1 is a graph showing the results of qPCR analysis of the nucleic acid content remaining in a surfactant-free rAAV1 sample when any of the devices of the present invention having the predetermined coating films obtained in Examples 1 to 3 was used, and when an uncoated device was used, in Test Example 1.

[0034] FIG. 1 is a graph showing the results of qPCR analysis of the nucleic acid content remaining in a surfactant-free rAAV1 sample when any of the devices of the present invention having the predetermined coating films obtained in Examples 1 to 3 was used, and when an uncoated device was used, in Test Example 2.

[0035] FIG. 1 is a graph showing the results of qPCR analysis of the nucleic acid content remaining in a surfactant-containing rAAV2 sample when any of the devices of the present invention having the predetermined coating films obtained in Examples 1 to 3 was used, and when an uncoated device was used, in Test Example 3.

[0036] FIG. 1 is a graph showing the results of qPCR analysis of the nucleic acid content remaining in a surfactant-containing rAAV2 sample when any of the devices of the present invention having the predetermined coating films obtained in Examples 1 to 3 was used, and when an uncoated device was used, in Test Example 4.

[0037] FIG. 1 is a graph showing the protein expression efficiency in cells when infected with AAV, as expressed as the mean fluorescence intensity of GFP in HeLaRC32 cells, when any of the devices of the present invention having the predetermined coating films obtained in Examples 1 and 2 was used, and when an uncoated device was used.

[0009] <Explanation of Terms> Unless otherwise specified, the terms used in the present invention have the following definitions.

[0010] In the present invention, unless otherwise specified, the "virus" refers to the "adeno-associated virus" described in detail below, and the term "virus" in this specification refers to the "adeno-associated virus."

[0011] In the present invention, the term "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

[0012] In the present invention, the term "alkyl group" refers to a monovalent group of a linear or branched saturated aliphatic hydrocarbon. Examples of "linear or branched alkyl groups having 1 to 5 carbon atoms" include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, and 1-ethylpropyl. Examples of "linear or branched alkyl groups having 1 to 18 carbon atoms" include, in addition to the examples of "linear or branched alkyl groups having 1 to 5 carbon atoms," hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl groups, and isomers thereof.

[0013] In the present invention, "a linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom" means the linear or branched alkyl group having 1 to 5 carbon atoms as described above, or the linear or branched alkyl group having 1 to 5 carbon atoms as described above which is substituted with one or more halogen atoms as described above. Examples of "a linear or branched alkyl group having 1 to 5 carbon atoms" are as described above. On the other hand, "a linear or branched alkyl group having 1 to 5 carbon atoms which is substituted with one or more halogen atoms" means the linear or branched alkyl group having 1 to 5 carbon atoms as described above in which one or more arbitrary hydrogen atoms have been replaced with a halogen atom, and examples thereof include a fluoromethyl group, a difluoromethyl group, a trifluoromethyl group, a chloromethyl group, a dichloromethyl group, a trichloromethyl group, a bromomethyl group, an iodomethyl group, a 2,2,2-trifluoroethyl group, a 2,2,2-trichloroethyl group, a perfluoroethyl group, a perfluorobutyl group, and a perfluoropentyl group.

[0014] In the present invention, an "ester bond" means -C(=O)-O- or -O-C(=O)-, an "amide bond" means -NHC(=O)- or -C(=O)NH-, and an ether bond means -O-.

[0015] In the present invention, the term "a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with one or more halogen atoms" refers to a linear or branched alkylene group having 1 to 10 carbon atoms, or a linear or branched alkylene group having 1 to 10 carbon atoms which is substituted with one or more halogen atoms. Here, the term "alkylene group" refers to a divalent organic group corresponding to the above alkyl group. Examples of the "linear or branched alkylene group having 1 to 10 carbon atoms" include a methylene group, an ethylene group, a propylene group, a trimethylene group, a tetramethylene group, a 1-methylpropylene group, a 2-methylpropylene group, a dimethylethylene group, an ethylethylene group, a pentamethylene group, a 1-methyl-tetramethylene group, a 2-methyl-tetramethylene group, a 1,1-dimethyl-trimethylene group, a 1,2-dimethyl-trimethylene group, a 2,2-dimethyl-trimethylene group, a 1-ethyl-trimethylene group, a hexamethylene group, an octamethylene group, and a decamethylene group. Of these, an ethylene group, a propylene group, an octamethylene group, and a decamethylene group are preferred, and a linear or branched alkylene group having 1 to 5 carbon atoms, such as an ethylene group, a propylene group, a trimethylene group, or a tetramethylene group, is more preferred, with an ethylene group or a propylene group being particularly preferred. The term "straight-chain or branched alkylene group having 1 to 10 carbon atoms substituted with one or more halogen atoms" refers to the above-mentioned alkylene group in which any one or more hydrogen atoms have been replaced with halogen atoms, and particularly preferred is an ethylene group or a propylene group in which some or all of the hydrogen atoms have been replaced with halogen atoms.

[0016] In the present invention, the term "cyclic hydrocarbon group having 3 to 10 carbon atoms" refers to a monocyclic or polycyclic, saturated or partially unsaturated, monovalent aliphatic hydrocarbon group having 3 to 10 carbon atoms. Among these, a monovalent, monocyclic or bicyclic, saturated aliphatic hydrocarbon group having 3 to 10 carbon atoms is preferred, and examples thereof include cycloalkyl groups having 3 to 10 carbon atoms, such as a cyclopropyl group, a cyclobutyl group, or a cyclohexyl group, and bicycloalkyl groups having 4 to 10 carbon atoms, such as a bicyclo[3.2.1]octyl group, a bornyl group, or an isobornyl group.

[0017] In the present invention, the term "aryl group having 6 to 10 carbon atoms" refers to a monovalent group of a monocyclic or polycyclic aromatic hydrocarbon having 6 to 10 carbon atoms, and examples thereof include a phenyl group, a naphthyl group, an anthryl group, etc. The "aryl group having 6 to 10 carbon atoms" may be substituted with one or more of the above-mentioned "linear or branched alkyl groups having 1 to 5 carbon atoms which may be substituted with a halogen atom".

[0018] In the present invention, the "aralkyl group having 7 to 14 carbon atoms" refers to a group -R-R' (wherein R represents the above-mentioned "linear or branched alkylene group having 1 to 5 carbon atoms" and R' represents the above-mentioned "aryl group having 6 to 10 carbon atoms"), and examples thereof include a benzyl group, a phenethyl group, and an α-methylbenzyl group. The aryl portion of the "aralkyl group having 7 to 14 carbon atoms" may be substituted with one or more of the above-mentioned "linear or branched alkyl groups having 1 to 5 carbon atoms which may be substituted with a halogen atom".

[0019] In the present invention, the "aryloxyalkyl group having 7 to 14 carbon atoms" means a group -R-O-R' (wherein R represents the above-mentioned "linear or branched alkylene group having 1 to 5 carbon atoms" and R' represents the above-mentioned "aryl group having 6 to 10 carbon atoms"), and examples thereof include a phenoxymethyl group, a phenoxyethyl group, and a phenoxypropyl group. The aryl portion of the "aryloxyalkyl group having 7 to 14 carbon atoms" may be substituted with one or more of the above-mentioned "linear or branched alkyl groups having 1 to 5 carbon atoms which may be substituted with a halogen atom".

[0020] In the present invention, the term "halide ion" means a fluoride ion, a chloride ion, a bromide ion, or an iodide ion.

[0021] In the present invention, the term "inorganic acid ion" means a carbonate ion, a sulfate ion, a phosphate ion, a hydrogen phosphate ion, a dihydrogen phosphate ion, a nitrate ion, a perchlorate ion, or a borate ion.

[0022] The above An －Preferred are halide ions, sulfate ions, phosphate ions, hydroxide ions and isothiocyanate ions, and particularly preferred are halide ions.

[0023] In the present invention, the term "(meth)acrylate compound" refers to both acrylate compounds and methacrylate compounds. For example, "(meth)acrylic acid" refers to acrylic acid and methacrylic acid.

[0024] <Device for stabilizing adeno-associated virus vectors> The device for stabilizing adeno-associated virus vectors of the present application is characterized by comprising a coating film on at least a portion of its surface, and the contact angle of air bubbles in water on the coating film surface is 120° to 180°. In the present invention, "stabilization" typically refers to the suppression of adhesion of adeno-associated virus vectors to the device, and, when the device is a container, the suppression of aggregation of adeno-associated virus vectors in the solution present in the container. The suppression of adhesion refers to a nucleic acid content determined by qPCR analysis using primers for ITRs (inverted terminal repeats) after a dispensing test performed according to the method described in the Examples, which is 1.5-fold or more higher than that of a control without the coating of the present invention. The above-mentioned inhibition of aggregation in a solution refers to the fact that, after placing an adeno-associated virus vector solution (the adeno-associated virus vector has a concentration of, for example, 1.0 mg / mL) in a container and shaking it at 20 to 25°C for 24 hours, the particle concentration in the solution is measured using a flow imaging device (for example, FlowCam8100, manufactured by Fluid Imaging Technologies), and the particle concentration is found to be 1 / 100 or less compared to a control that does not contain the coating of the present invention.

[0025] <Coating Film> The coating film surface having a water-air bubble contact angle of 120° to 180° means, for example, that in static contact angle measurement using a contact angle meter (e.g., a fully automatic contact angle meter (Kyowa Interface Science Co., Ltd., DM-701)), the water-air bubble contact angle is 120° to 180°. The water-air bubble contact angle on the coating film surface is 130° to 180°, 140° to 180°, and preferably 150° to 180°.

[0026] The coating film may be provided on at least a portion of the surface of the device described below, but it is preferable that the coating film be formed over the entire surface that may come into contact with viruses, and it is even more preferable that the coating film be formed over the entire surface of the device.

[0027] In a preferred embodiment of the present invention, the coating film preferably contains a polymer or compound containing a hydroxy group. The polymer or compound containing a hydroxy group according to the present invention may be a polymer of an ethylenically unsaturated monomer having a hydroxy group, or a polysaccharide or a derivative thereof. Examples of the ethylenically unsaturated monomer include one or more ethylenically unsaturated monomers selected from the group consisting of (meth)acrylic acid and its esters; vinylpyrrolidone; and ethylene. Examples of polysaccharides or derivatives thereof include cellulose-based polymers such as hydroxyalkyl cellulose (e.g., hydroxyethyl cellulose or hydroxypropyl cellulose), starch, dextran, and curdlan.

[0028] The hydroxy group may be an alkylene glycol residue. The alkylene glycol residue refers to a hydroxyalkyl group (-Alk-OH) that remains after the hydroxy group at one terminal of alkylene glycol (HO-Alk-OH; where Alk is a linear or branched alkylene group having 1 to 10 carbon atoms) has reacted (condensed) with another compound, and may include a poly(alkyleneoxy) group in which alkyleneoxy units are repeated. Examples of polymers or compounds having such a structure include poly(2-hydroxyethyl(meth)acrylate), polyethylene glycol (meth)acrylate, and poloxamer, which is a block copolymer of polyoxyethylene chains and polyoxypropylene chains (for example, commercially available under the trade name Pluronic (registered trademark)).

[0029] In another preferred embodiment of the present invention, the coating film may be a coating film containing a copolymer including a repeating unit containing a group represented by the following formula (a), a repeating unit containing a group represented by the following formula (b), and a repeating unit containing a group represented by the following formula (c): [In the formula, U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ｃ represents a linear or branched alkyl group having 4 to 18 carbon atoms, a cyclic hydrocarbon group having 3 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, an aralkyl group having 7 to 14 carbon atoms, or an aryloxyalkyl group having 7 to 14 carbon atoms (wherein the aryl moiety may be substituted with a linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom); An － represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion.

[0030] <Formation of Coating Film> The coating film of the present invention can be formed by applying, by a known method, a known composition for forming a coating film, which is capable of forming a coating film on the surface of which the water bubble contact angle is 120° to 180°, to at least a part of the surface of the device described below, preferably to the surface of the device that may come into contact with viruses, and more preferably to the entire surface of the device. The coating film has been described above.

[0031] The device having the coating film of the present invention on at least a part of its surface is preferably a copolymer comprising a repeating unit containing a group represented by the following formula (a), a repeating unit containing a group represented by the following formula (b), and a repeating unit containing a group represented by the following formula (c): [In the formula, U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ｃ represents a linear or branched alkyl group having 4 to 18 carbon atoms, a cyclic hydrocarbon group having 3 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, an aralkyl group having 7 to 14 carbon atoms, or an aryloxyalkyl group having 7 to 14 carbon atoms (wherein the aryl moiety may be substituted with a linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom); An － represents an anion selected from the group consisting of halide ions, inorganic acid ions, hydroxide ions, and isothiocyanate ions] and a solvent, to at least a part of the surface of the appliance.

[0032] In a preferred embodiment of the present invention, the copolymer contained in the coating film-forming composition is a copolymer containing a repeating unit containing a group represented by formula (a), a repeating unit containing a group represented by formula (b), and a repeating unit containing a group represented by formula (c). In the present invention, the repeating unit containing a group represented by formula (c) is different from the repeating unit containing a group represented by formula (a) and the repeating unit containing a group represented by formula (b). The polymer is preferably obtained by radical polymerization of a monomer containing a group represented by formula (a), a monomer containing a group represented by formula (b), and a monomer containing a group represented by formula (c). However, a polymer obtained by polycondensation or polyaddition reaction can also be used. Examples of copolymers include vinyl polymers reacted with olefins, polyamides, polyesters, polycarbonates, polyurethanes, etc., among which vinyl polymers reacted with olefins or (meth)acrylic polymers polymerized with (meth)acrylate compounds are particularly preferred.

[0033] The proportion of repeating units containing a group represented by formula (a) in the copolymer of the coating film of the present invention is 3 mol% to 80 mol%, preferably 3.5 mol% to 50 mol%, and more preferably 4 mol% to 30 mol%. The copolymer of the present invention may contain two or more repeating units containing a group represented by formula (a). The proportion of repeating units containing a group represented by formula (b) in the copolymer of the coating film of the present invention is 3 mol% to 80 mol%, preferably 5 mol% to 70 mol%, and more preferably 8 mol% to 65 mol%. The copolymer of the present invention may contain two or more repeating units containing a group represented by formula (b). The proportion of repeating units containing a group represented by formula (c) in the copolymer of the present invention may be the entire remainder after subtracting the repeating units represented by formulas (a) and (b) from the total copolymer, or the remainder after subtracting the combined proportions of the repeating units represented by formulas (a) and (b) and the fourth component described below. It is, for example, 1 mol% to 90 mol%, preferably 3 mol% to 88 mol%. The copolymer according to the present invention may contain two or more repeating units containing a group represented by formula (c).

[0034] The combination of proportions of repeating units containing groups represented by formula (a), formula (b) and formula (c) in the copolymer according to the present invention is preferably: formula (a): 3 mol% to 80 mol%, formula (b): 3 mol% to 80 mol%, formula (c): 1 mol% to 90 mol%, more preferably: formula (a): 3.5 mol% to 50 mol%, formula (b): 5 mol% to 70 mol%, formula (c): 3 mol% to 88 mol%, even more preferably: formula (a): 4 mol% to 30 mol%, formula (b): 8 mol% to 65 mol%, formula (c): 5 mol% to 87 mol%, and most preferably: formula (a): 4 mol% to 30 mol%, formula (b): 8 mol% to 65 mol%, formula (c): 50 mol% to 86 mol%.

[0035] As the copolymer contained in the composition for forming a coating film, copolymers containing repeating units of the following formulae (a1), (b1) and (c1) are particularly preferably used.

[0036] In the ceremony, T ａ , T ｂ , T ｃ , U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; Q ａ and Q ｂ each independently represents a single bond, an ester bond, or an amide bond; Q ｃ represents a single bond, an ether bond or an ester bond, R ａ and R ｂ each independently represents a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom; R ｃ represents a linear or branched alkyl group having 4 to 18 carbon atoms, a cyclic hydrocarbon group having 3 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, an aralkyl group having 7 to 14 carbon atoms, or an aryloxyalkyl group having 7 to 14 carbon atoms (wherein the aryl moiety may be substituted with a linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom), An － represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion; and m represents an integer of 0 to 6.

[0037] In formula (a1), m represents an integer of 0 to 6, preferably an integer of 1 to 6, more preferably an integer of 1 to 5, and particularly preferably 1.

[0038] The proportions of the copolymer containing repeating units of the above formulae (a1), (b1) and (c1) are the same as those described above as the proportions of repeating units containing groups represented by the above formulae (a), (b) and (c) in the copolymer according to the present invention, respectively.

[0039] The copolymer has the following formulas (A), (B) and (C): [In the formula, T ａ , T ｂ , T ｃ , Uａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; Q ａ and Q ｂ each independently represents a single bond, an ester bond, or an amide bond; Q ｃ represents a single bond, an ether bond or an ester bond; R ａ and R ｂ each independently represents a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom; R ｃ represents a linear or branched alkyl group having 4 to 18 carbon atoms, a cyclic hydrocarbon group having 3 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, an aralkyl group having 7 to 14 carbon atoms, or an aryloxyalkyl group having 7 to 14 carbon atoms (wherein the aryl moiety may be substituted with a linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom); An － represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion; and m represents an integer of 0 to 6], in a solvent, to react (polymerize).

[0040] T ａ , T ｂ and T ｃ is preferably a hydrogen atom, a methyl group or an ethyl group, more preferably a hydrogen atom or a methyl group. ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ is preferably a hydrogen atom, a methyl group, an ethyl group or a t-butyl group, and U in formula (a) ａ１ and U ａ２ represents a hydrogen atom, U in formula (b) ｂ１ , U ｂ２ and U ｂ３ is more preferably a hydrogen atom, a methyl group, an ethyl group or a t-butyl group.

[0041] In another embodiment of the present invention, the copolymer may further contain units derived from any fourth component. For example, the fourth component may contain a crosslinked structure derived from a (meth)acrylate compound having two or more functional groups. Examples of such fourth components include ethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, propylene glycol di(meth)acrylate, bis(methacryloyloxymethyl)phosphate, bis[(2-methacryloyloxy)ethyl]phosphate, bis[3-(methacryloyloxy)propyl]phosphate, and phosphinylidyne tris(oxy-2,1-ethanediyl)triacrylate.

[0042] For example, the proportion of crosslinked structures derived from the (meth)acrylate compound having two or more functional groups in the copolymer is 0 mol % to 50 mol %, preferably 5 mol % to 45 mol %, and most preferably 10 mol % to 40 mol %.

[0043] Examples of the solvent contained in the coating film-forming composition include water, phosphate buffered saline (PBS), and alcohol. Examples of the alcohol include alcohols having 2 to 6 carbon atoms, such as ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-heptanol, 2-heptanol, 2,2-dimethyl-1-propanol (neopentyl alcohol), 2-methyl-1-propanol, 2-methyl-1-butanol, 2-methyl-2-butanol (t-amyl alcohol), 3-methyl-1-butanol, 3-methyl-3-pentanol, cyclopentanol, 1-hexanol, 2-hexanol, 3-hexanol, 2,3-dimethyl-2-butanol, and 3,3-dimethyl Examples of the solvent include 1-butanol, 3,3-dimethyl-2-butanol, 2-ethyl-1-butanol, 2-methyl-1-pentanol, 2-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-1-pentanol, 3-methyl-2-pentanol, 3-methyl-3-pentanol, 4-methyl-1-pentanol, 4-methyl-2-pentanol, 4-methyl-3-pentanol, and cyclohexanol. These solvents may be used alone or in combination, but from the viewpoint of dissolving the copolymer, they are preferably selected from water, PBS, ethanol, propanol, and mixed solvents thereof, and more preferably selected from water, ethanol, and mixed solvents thereof.

[0044] To form an appliance having a coating film of the present invention, the coating film-forming composition is applied to at least a portion of the surface of the appliance. The application method is not particularly limited, and conventional methods such as spin coating, dip coating, and solvent casting can be used.

[0045] The method for obtaining an appliance having a coating film of the present invention may include a step of drying the coating film following the above-mentioned application step. The drying step of the coating film is carried out in the atmosphere or under vacuum, preferably at a temperature in the range of −200° C. to 200° C. The drying step removes the solvent from the coating film-forming composition, and also forms ionic bonds between the copolymers of formula (a) and formula (b) according to the present invention, thereby completely fixing the composition to the container.

[0046] The coating film can be formed by drying at room temperature (10°C to 35°C, e.g., 25°C), but in order to form the coating film more quickly, drying at, for example, 40°C to 50°C may also be used. A drying process at extremely low to low temperatures (around -200°C to -30°C) using the freeze-drying method may also be used. Freeze-drying is also called vacuum freeze-drying, and is a method in which the material to be dried is cooled with a refrigerant and the solvent is removed by sublimation in a vacuum. Common refrigerants used in freeze-drying include a mixture of dry ice and methanol (-78°C) and liquid nitrogen (-196°C).

[0047] If the drying temperature is below -200°C, an uncommon refrigerant must be used, resulting in a lack of versatility and inefficient drying due to solvent sublimation. If the drying temperature is above 200°C, the ionic bonding reaction on the coating film surface will proceed too much, causing the surface to lose its hydrophilicity and failing to exhibit its ability to inhibit virus adhesion. A more preferred drying temperature is 10°C to 180°C, and a more preferred drying temperature is 25°C to 150°C.

[0048] After drying, the coating film may be washed with at least one solvent selected from water and an aqueous solution containing an electrolyte to remove impurities, unreacted monomers, and the like remaining on the coating film and to adjust the ion balance of the copolymer in the film. Washing with running water or ultrasonic cleaning is preferred. The aqueous solution containing water and an electrolyte may be heated, for example, to a temperature ranging from 40°C to 95°C. Preferred aqueous solutions containing electrolytes include PBS, saline (containing only sodium chloride), Dulbecco's phosphate-buffered saline, Tris-buffered saline, HEPES-buffered saline, and Veronal-buffered saline, with PBS being particularly preferred. After adhesion, the coating film remains firmly attached to the substrate without elution even when washed with water, PBS, alcohol, or the like. Even if biological materials adhere to the formed coating film, they can be easily removed by subsequent washing with water, etc.

[0049] If necessary, sterilization may be performed using radiation, electron beams, ethylene oxide, autoclave, or the like.

[0050] The thickness of the coating film of the present invention is preferably 10 to 1000 Å, more preferably 10 to 500 Å, and most preferably 20 to 400 Å.

[0051] The device of the present invention has a coating film formed from the above coating agent on at least a part of the surface of the device. Specifically, the coating film is present on the surface of the device that may come into contact with viruses, more preferably on the entire surface of the device.

[0052] Prior to the coating step, the surface of the tool may be subjected to a known plasma treatment. For example, UV irradiation or oxygen plasma treatment is known to hydrophilize oxide surfaces such as glass and ITO (indium tin oxide). Furthermore, a technique for hydrophilizing the surface of plastics or silicone rubber (polydimethylsiloxane) resins to promote adhesion with varnish has also been reported (Patent Nos. 5898703 and 4255911). Plasma can be generated using a device capable of creating a space containing a high density of active charged particles and active radicals, such as vacuum plasma generated from various oxygen-, nitrogen-, or fluorine-based gases or mixtures thereof, or plasma generated at atmospheric pressure or near atmospheric pressure.

[0053] The entire disclosure of WO 2016 / 093293 is incorporated herein by reference.

[0054] <Viruses> Generally, viruses (including vaccines and viral vectors) are broadly classified into enveloped and non-enveloped viruses. An envelope is a membranous structure composed of lipids and proteins derived from host cells, as well as viral glycoproteins. Viruses exist as enveloped and non-enveloped viruses. Furthermore, viruses are broadly classified into DNA viruses and RNA viruses based on the genetic structure they contain. In the present invention, "virus" refers to "adeno-associated virus (AAV)" unless otherwise specified, and their use as vaccines and vectors also falls within the scope of the "virus" defined in the present invention. Adeno-associated viruses are classified in the genus Dependovirus in the family Parvoviridae, and are non-enveloped, single-stranded DNA viruses. In recent years, the function of adeno-associated viruses as viral vectors has been enhanced and improved, and they are used in gene therapy and regenerative medicine. Various recombinant AAVs (rAAVs) are known and available from reagent suppliers, etc., and these are also included in the scope of the "virus" defined in the present invention. Furthermore, adeno-associated viruses have attracted considerable attention for their potential pharmaceutical applications because they exhibit natural tropism for specific cell and tissue types depending on their serotype.

[0055] <Instrument> The instrument of the present invention is not particularly limited as long as it is used for an adeno-associated virus, but it is preferable that it is one that comes into contact with the virus during use and is required to suppress viral adhesion. The shape of the instrument is also not particularly limited, and may be flat, curved, or uneven. Furthermore, the instrument of the present invention may be a material for producing an adeno-associated virus vector.

[0056] Specific examples include microwell plates, which usually have multiple wells (depressions), microplates, microtubes, screw-cap tubes, chips, culture flasks, biodevices, dispenser syringes, pre-filled syringes, filters, separation filtration membranes, sterilizing filters, nonwoven fabrics, and vials. In particular, materials for producing adeno-associated virus vectors include, in addition to those selected from the above specific examples, bioreactors, stirring blades, and production piping.

[0057] One embodiment of the device of the present invention may be a virus storage container. The storage container preferably has the above-mentioned coating film on the surface that comes into contact with the virus. The shape of the virus storage container is not particularly limited, and may be a bottle shape, a tube shape, or the like, as long as it is capable of storing a solution containing a virus (such as an aqueous solution containing a virus, which is normally liquid at room temperature). It is preferable that the container has a lid or the like on the top that can be sealed, allowing for hermetically sealed storage.

[0058] Another embodiment of the device of the present invention may be a tip. The tip is typically a pipette tip, and preferably has the above-mentioned coating film on the surface that comes into contact with the virus. The shape of the tip is not particularly limited, and tips of various capacities and shapes depending on the purpose are commercially available.

[0059] The material of the device of the present invention is not particularly limited. Examples include glass, metal-containing compounds or metalloid-containing compounds, and resins. Glass or resin moldings are preferred for their versatility. Examples of metal-containing or metalloid-containing compounds include ceramics, which are sintered bodies whose basic component is a metal oxide and are sintered by high-temperature heat treatment; inorganic solid materials such as molded bodies of inorganic compounds such as semiconductors (e.g., silicon), metal oxides or metalloid oxides (e.g., silicon oxide, alumina), metal carbides or metalloid carbides, metal nitrides or metalloid nitrides (e.g., silicon nitride), and metal borides or metalloid borides; aluminum, nickel titanium, and stainless steel (e.g., SUS304, SUS316, SUS316L). The resin may be either a natural resin or a derivative thereof, or a synthetic resin. Examples of natural resins or derivatives thereof include cellulose, cellulose triacetate (CTA), nitrocellulose (NC), and cellulose immobilized with dextran sulfate. Examples of synthetic resins include polyacrylonitrile (PAN), polyimide (PI), polyester polymer alloy (PEPA), polystyrene (PS), polysulfone (PSF), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), polyurethane (PU), and ethylene vinyl alcohol. Preferably used are EVAL, polyethylene (PE), polyester, polypropylene (PP), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polycarbonate (PC), cycloolefin polymer (COP) (for example, ZEONOR (registered trademark), ZEONEX (registered trademark) (manufactured by Zeon Corporation)), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), ultra-high molecular weight polyethylene (UHPE), polydimethylsiloxane (PDMS), acrylonitrile-butadiene-styrene resin (ABS), or Teflon (registered trademark).

[0060] <Virus Stabilization Method> The virus stabilization method of the present invention comprises the steps of applying a coating film, the surface of which has a water-air bubble contact angle of 120° to 180°, to at least a portion of an appliance, and contacting the coating film applied to the appliance with a composition containing a virus and a solvent, thereby stabilizing the virus by reducing the amount of virus adhering to the appliance through the coating film. For example, if the appliance is a container, this refers to placing a virus-containing solution in the container and storing it for a certain period of time, and thereafter the amount of virus in the solution changes little from the initial amount (e.g., a change of 30% or less from the initial amount). The certain period of time is, for example, one hour to one year. The temperature may be frozen (e.g., −100°C to −20°C or lower), refrigerated (e.g., below −20°C to 10°C or lower), or room temperature (e.g., below 10°C to 35°C). This means that after storage, the virus in the solution is maintained at, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, and preferably 100% of the initial virus amount. The composition may or may not contain a surfactant, but the stabilization method of the present invention makes it possible to suppress adhesion of adeno-associated viruses to the surface of a container even if the composition does not contain a surfactant.

[0061] The meanings of the terms and preferred embodiments of the stabilization method are as described above.

[0062] Therefore, the present invention also relates to use of a coating film having a water-air bubble contact angle of 120° to 180° on the film surface for reducing virus adhesion to an instrument. The meanings and preferred embodiments of each term are as described above.

[0063] <Virus detection methods> Generally, virus detection methods can be broadly divided into four types: the first is a method that detects nucleic acids contained in viruses, the second is a method that detects proteins contained in viruses, the third is a method that utilizes the properties of viruses, and the fourth is a method that detects the virus particles themselves.

[0064] Methods for detecting nucleic acids contained in viruses can identify and quantify viruses by detecting nucleic acid sequences specific to the virus, and include PCR, LAMP, TMA, NASBA, liquid-phase nucleic acid hybridization, Southern blot hybridization, Northern blot hybridization, in situ hybridization, and microarray.

[0065] Methods for detecting viral proteins allow for virus identification and quantification by detecting proteins specific to the virus, and include enzyme immunoassay, fluorescent antibody assay, immunochromatography, Western blotting, chemiluminescence immunoassay, and radioimmunoassay.

[0066] Methods that utilize the properties of viruses can quantify viruses by detecting phenotypes that are characteristic of the virus (such as cytopathic effect or hemagglutination), and examples of such methods include TCID50, PFU, HA, and LD50.

[0067] Methods for detecting virus particles themselves allow for identification and quantification of viruses by detecting one or more virus particles on the order of tens to hundreds of nanometers. These methods include electron microscopy (TEM, AFM, cryo-EM, etc.), nanoparticle tracking analysis (NTA (NanoSight, Zeta View, etc.)), nanopore current measurement (qNano, etc.), analytical ultracentrifugation (AUC), and mass photometry (Refyen One).

[0068] <Virus detection device> The virus detection method may use a specific detection device (e.g., a virus test kit), and by applying the coating of the present invention to parts or instruments of such devices where viruses are expected to adhere, the lower detection limit for virus detection can be further lowered and virus detection performance can be improved. There are no particular restrictions on the detection device, and commercially available products may be used. The coating of the present invention may be applied to the sample adhesion part of the extraction container used in the detection device.

[0069] <Specific Proteins Used for Virus Detection> In the case of adeno-associated viruses, AAV capsid proteins are preferably used as antigens for detection using antibodies.

[0070] <Virus Sample Collection Kit> The virus sample collection kit of the present invention is one embodiment of the device, and comprises a combination of a tip to be attached to a pipette or the like for sample collection, a virus extraction container, and a virus storage container.

[0071] <Method for reducing the lower detection limit in virus testing> When a virus test is performed using the device of the present invention, specifically a virus testing kit, the amount of virus adhering to the device is suppressed, making it possible to detect trace amounts of virus present in a sample and reducing the lower detection limit.

[0072] Therefore, the present invention also relates to the use of a hydrophilic coating film for reducing the lower limit of virus detection. The meanings of the respective terms are as described above.

[0073] <Method for Preserving Viral Infectivity and / or Gene Expression Activity in Infected Cells> By using the device of the present invention, the amount of virus adhering to the device is suppressed, and the infectivity and / or gene expression activity in infected cells of the virus whose attachment has been suppressed is maintained. Therefore, the device of the present invention is also a device for preserving the infectivity and / or gene expression activity in infected cells of the virus. By using the device of the present invention, the infectivity and / or gene expression activity in infected cells of the virus is maintained, thereby enabling appropriate storage, research, and evaluation of the virus. In the present invention, "maintaining infectivity" means that the viral infectivity is maintained, and the infectivity is measured using a method known to those skilled in the art, for example, as the TCID indicating a 50% tissue culture cell infection rate. 50 The "ability to activate gene expression in infected cells" can be evaluated using the Tissue Culture Infectious Dose 50 as an index. Furthermore, the "ability to activate gene expression in infected cells" can be evaluated by methods known to those skilled in the art, for example, the method described in Test Example 4 below.

[0074] Therefore, the present invention also relates to the use of a hydrophilic coating film for preserving the infectivity of a virus. The meanings of the respective terms are as described above.

[0075] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0076] Synthesis Example 1 260 g of acid phosphooxyethyl methacrylate (product name: Phosmer M, manufactured by Unichemical Co., Ltd.; non-volatile content after hardening to dryness at 100°C for 1 hour: 91.8%; mixture of acid phosphooxyethyl methacrylate (44.2% by mass), bis[2-(methacryloyloxy)ethyl]phosphate (28.6% by mass), and other substances (27.2% by mass)) was added to 390 g of ethanol, and while cooling to below 35°C, 310 g of choline (48-50% aqueous solution: manufactured by Tokyo Chemical Industry Co., Ltd.) was added and stirred until homogenous. To this mixture were added 220 g of 80% aqueous solution of methcroylcholine chloride (manufactured by Tokyo Chemical Industry Co., Ltd.) and 300 g of butyl methacrylate (manufactured by Tokyo Chemical Industry Co., Ltd.), and an additional 260 g of ethanol was added and stirred. Furthermore, an aqueous solution prepared by dissolving 22 g of 2,2'-azobis(N-(2-carboxyethyl)-2-methylpropionamidine) n-hydrate (product name: VA-057, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) in 230 g of pure water was added to the above solution while maintaining the temperature at 35°C or below. After thorough stirring, the resulting homogeneous mixture was introduced into a three-necked flask via a dropping pump. Separately, 650 g of pure water and 980 g of ethanol were added to a three-necked flask equipped with a condenser, and nitrogen was passed through the flask, followed by stirring while heating to the reflux temperature. While maintaining this state, the above mixture was added dropwise to a boiling solution of pure water and ethanol over 1.5 hours using a dropping pump connected to a Teflon tube. After the dropwise addition, the mixture was heated and stirred for 2 hours while maintaining the above environment. After cooling for 2 hours, 3610 g of a copolymer-containing varnish with a solids content of approximately 24.20% by mass was obtained. The resulting liquid had a weight average molecular weight of about 23,225 by GFC.

[0077] Preparation Example 1: 157 g of 1 mol / L hydrochloric acid (1N) (Kanto Chemical Co., Inc.), 1697 g of pure water, and 4456 g of ethanol were added to 568 g of the copolymer-containing varnish obtained in Synthesis Example 1, and the mixture was thoroughly stirred to prepare a composition for forming a coating film. The pH was 2.6.

[0078] Preparation Example 2 1 g of poly(2-hydroxyethyl methacrylate) (manufactured by Aldrich, product number: P3932-25G) was added to 99 g of an aqueous ethanol solution (ethanol 91.7 wt %) and dissolved with stirring in a water bath at 50° C. to prepare a composition for forming a coating film.

[0079] Preparation Example 3 4 g of Pluronic (registered trademark) F-127 (manufactured by Aldrich, product number: P2443-250G) was added to 96 g of pure water, and the mixture was stirred and dissolved at room temperature to prepare a composition for forming a coating film.

[0080] Example 1 1.0 mL of the coating film-forming composition obtained in Preparation Example 1 was placed in 1.0 mL polypropylene (PP) CryoTube Vials (manufactured by Thermo Fisher Scientific, 377224) and 1.5 mL in a PP microtube, and the mixture was allowed to stand at 25°C for 0.5 hours. The coating film-forming composition was removed from the tube / vial, and then dried at 25°C for 3 hours. The tube / vial was then thoroughly washed with pure water to obtain a PP tube and vial with a coating film formed on its inner surface.

[0081] Example 2 The tips of PP pipetting tips epT.IPS Reloads 0.5-20 μL, 2-200 μL, and 50-1000 μL (manufactured by Eppendorf, product numbers: 0030073401, 0030073428, and 0030073460) were capped with parafilm, a hole was made with a 27G syringe needle, and 2, 200, or 1000 μL of the coating film-forming composition obtained in Preparation Example 1 was added, followed by standing at 25°C for 1 second. After removing the coating film-forming composition, the pipetting tips were dried at 25°C for 3 hours. The pipetting tips were then thoroughly washed with pure water to obtain PP pipetting tips with a coating film formed on their inner surfaces.

[0082] Example 3 The coating film-forming composition obtained in Preparation Example 1 was placed in a PP qPCR plate (manufactured by Applied Biosystems, REF4346906) at 200 μL / well and allowed to stand at 25° C. for 0.5 hours. The coating film-forming composition was removed from the wells and then dried at 25° C. for 3 hours. The wells were then thoroughly washed with pure water to obtain a PP qPCR plate on which a coating film had been formed.

[0083] Example 4 2 mL of the coating film-forming composition obtained in Preparation Example 1 was placed in a polystyrene (PS) Aznol Petri dish φ90 × 20 mm (manufactured by AS ONE Corporation, product number: 1-8549-04), and allowed to stand at 25°C for 0.5 hours. After removing the coating film-forming composition, the dish was dried at 25°C for 3 hours. Thereafter, the dish was thoroughly washed with pure water to obtain a PS substrate on which a coating film had been formed.

[0084] Example 5 The composition for forming a coating film obtained in Preparation Example 2 was coated under the same conditions as in Example 4 to obtain a PS substrate on which a coating film was formed.

[0085] Example 6 The composition for forming a coating film obtained in Preparation Example 3 was coated under the same conditions as in Example 4 to obtain a PS substrate on which a coating film was formed.

[0086] Test Example 1: An adeno-associated virus vector rAAV1 sample (surfactant-free) was dispensed using the pipetting tips of Example 2 and uncoated pipetting tips, and qPCR analysis was performed using primers targeting the ITR (inverted terminal repeat). The results obtained using the coated tips were compared with those obtained using uncoated tips. Similarly, the qPCR results for the cryotube vials (Example 1) used during dispensing and the qPCR plate (Example 3) used during measurement were compared with those obtained using the uncoated cryotube vials and plates of Examples 1 and 3, respectively. As a result, the nucleic acid content obtained using coated pipetting tips was 12.9-fold higher than that obtained using uncoated ones. The nucleic acid content obtained using coated pipetting tips was 6.3-fold higher than that obtained using uncoated cryotube vials and 20.7-fold higher than that obtained using uncoated qPCR plates. The results are shown in Figure 1.

[0087] Test Example 2: Adeno-associated virus vector rAAV1 samples (surfactant-free) were dispensed into the coated and uncoated cryotube vials of Example 1, pipetted using the coated and uncoated pipetting tips of Example 2, and qPCR analysis was performed in triplicate in the coated and uncoated qPCR plates of Example 3. The same primers were used as in Test Example 1. The results of a preparation using all coated samples were compared with the results of a preparation using all uncoated samples. As a result, the nucleic acid content was 1.7-3.8 times higher when using coated cryotube vials, pipetting tips, and qPCR plates than when using uncoated samples. The results are shown in Figure 2.

[0088] Test Example 3: Adeno-associated virus vector rAAV2 samples (containing surfactant) were dispensed into the coated and uncoated cryotube vials of Example 1, pipetted using the coated and uncoated pipetting tips of Example 2, and subjected to qPCR analysis in the coated and uncoated qPCR plates of Example 3. The same primers were used as in Test Example 1. The results of a preparation using all coated primers were compared with the results of a preparation using all uncoated primers. As a result, the nucleic acid content was 1.6-fold higher when using coated cryotube vials, pipetting tips, and qPCR plates than when using uncoated ones. The results are shown in Figure 3.

[0089] Test Example 4: The effect of coating was measured by measuring the protein expression efficiency in cells infected with an adeno-associated virus vector. AAV1 carrying a gene encoding GFP was added to a medium containing HeLaRC32 cells using the coated cryotube vial of Example 1 and the pipetting tip of Example 2, or using the untreated, uncoated tube and pipetting tip of Examples 1 and 2, and the cells were cultured. As a result, the average fluorescence intensity of GFP from the cells using the coated cryotube vial and pipetting tip was 1.8-fold higher than that using the uncoated ones. The results are shown in Figure 4.

[0090] Test Example 5 The hydrophilicity of the inner surfaces of the untreated microtube and coated microtube of Example 1 and each of the tubes in Reference Examples 1 to 5 below, and the untreated polystyrene substrates and each of the coated polystyrene substrates of Examples 4 to 6 was evaluated using a fully automatic contact angle meter (DM-701, manufactured by Kyowa Interface Science Co., Ltd.). Reference Example 1: Proteosave SS 1.5 mL microtube (manufactured by Sumitomo Bakelite Co., Ltd., product number: MS-4215M), Reference Example 2: 1.5 mL protein adsorption control sampling tube (manufactured by Sarstedt Co., Ltd., product number: 72.41152.006), Reference Example 3: Protein LoBind Tube 1.5 mL (manufactured by Eppendorf Co., Ltd., product number: 0030108116), Reference Example 4: 1.5 mL siliconized microtube round bottom (manufactured by Fukae Kasei Co., Ltd., product number: 131-615CH), Reference Example 5: TORAST (registered trademark) PP Vial (manufactured by Shimadzu GLC Co., Ltd., product number: 370-04051-01). Note that the contact angle was evaluated not only by measuring the contact angle of a water droplet in the air but also by measuring the contact angle in water by placing various substrates upside down in water and measuring the contact angle of an air bubble. The measurement results are shown in Table 1.

[0091]

[0092] The measurement results in air showed no significant difference between the coated and uncoated cases, whereas the measurement results in water confirmed that the tubes in Example 1, Examples 4 to 6, and Reference Example 1 (commercially available tubes in the low protein adsorption container series) were significantly more hydrophilic than the uncoated cases.

[0093] The present invention provides an instrument in which virus adhesion is suppressed and a method for reducing virus adhesion using the instrument. Specifically, it provides a virus storage container with low virus loss and a virus test kit with improved virus detection sensitivity. It also provides an instrument (e.g., a chip or storage container) in which viruses, particularly adeno-associated virus vectors, can be stabilized, an adeno-associated virus vector test kit including the instrument, a method for lowering the detection limit in adeno-associated virus vector tests using the instrument, a method for stabilizing adeno-associated virus vectors, and a method for maintaining the infectivity of adeno-associated virus vectors and / or gene expression activity in infected cells. Furthermore, the storage container of the present invention also has the effect of maintaining the viral infectivity (viral infectivity) of an adeno-associated virus vector even after storage for a certain period of time.

Claims

1. An instrument for stabilizing an adeno-associated virus vector, comprising a coating film on at least a portion of its surface, wherein the contact angle of an air bubble in water on the surface of the coating film is 120° to 180°.

2. The device of claim 1 , wherein the stabilization inhibits adhesion of an adeno-associated virus vector.

3. The coating film is a copolymer including a repeating unit including a group represented by the following formula (a), a repeating unit including a group represented by the following formula (b), and a repeating unit including a group represented by the following formula (c): 【Chemistry 1】 [In the formula, U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ｃ represents a linear or branched alkyl group having 4 to 18 carbon atoms, a cyclic hydrocarbon group having 3 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, an aralkyl group having 7 to 14 carbon atoms, or an aryloxyalkyl group having 7 to 14 carbon atoms (wherein the aryl portion may be substituted with a linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom); A － The device according to claim 1, which is a coating film containing an anion selected from the group consisting of halide ions, inorganic acid ions, hydroxide ions and isothiocyanate ions.

4. The device of claim 1 , wherein the coating comprises a polymer or compound containing a hydroxy group.

5. The device according to any one of claims 1 to 4, which is a chip for an adeno-associated virus vector.

6. The device according to any one of claims 1 to 4, which is an adeno-associated virus vector storage container.

7. An adeno-associated virus vector test kit comprising the device according to any one of claims 1 to 4.

8. The device according to any one of claims 1 to 4, which is a material for producing an adeno-associated virus vector.

9. A method for reducing the lower limit of detection in an adeno-associated virus vector test, comprising using the device according to any one of claims 1 to 4.

10. A method for stabilizing an adeno-associated virus vector, comprising: (1) applying a coating film having a water-air bubble contact angle of 120° to 180° on a surface of at least a part of the device; (2) A method for stabilizing an adeno-associated virus vector, comprising a step of contacting the coating film with a composition containing the adeno-associated virus vector and a solvent.

11. The coating film is a copolymer including a repeating unit including a group represented by the following formula (a), a repeating unit including a group represented by the following formula (b), and a repeating unit including a group represented by the following formula (c): 【Chemistry 2】 [In the formula, U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R ｃ represents a linear or branched alkyl group having 4 to 18 carbon atoms, a cyclic hydrocarbon group having 3 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, an aralkyl group having 7 to 14 carbon atoms, or an aryloxyalkyl group having 7 to 14 carbon atoms (wherein the aryl portion may be substituted with a linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom); A － The method for stabilizing an adeno-associated virus vector according to claim 10, comprising:

12. The method for stabilizing an adeno-associated virus vector according to claim 10 , wherein the coating film comprises a polymer or compound containing a hydroxyl group.

13. The method for stabilizing an adeno-associated virus vector according to any one of claims 10 to 12, wherein the composition does not contain a surfactant.

14. A method for retaining the infectivity of an adeno-associated virus vector and / or gene expression activity in an infected cell, comprising using the device according to any one of claims 1 to 4.

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