Separation material, method for manufacturing separation material, method for separating target molecules, mixture, and column for chromatography

Abstract

The problem addressed by the present invention is to provide: a separation material that has improved separation and refinement capabilities even for large molecules such as nucleic acids; and uses for the separation material. [Solution] The present invention is a separation material, formed by an anion exchange group bonding with a carrier, wherein the anion exchange group includes a structural unit represented by formula (A1) (n1 is at least 1 and no more than 10, Ra0 represents a hydrogen atom or an alkyl group with a carbon number of at least 1 and no more than 4, and the following may also be included as substituent groups: Ra1, representing O or NH; Ra2–Ra7, each of which independently represents a hydrogen atom or an alkyl group with a carbon number of at least 1 and no more than 4; and Ra2, representing a straight-chain or branched alkylene group with a carbon number of at least 1 and no more than 6.).

Description

Separation material, method for producing separation material, method for separating target molecules, mixture, and chromatography column

[0001] The present invention relates to a separation material, a method for manufacturing a separation material, a method for separating target molecules, a mixture, and a chromatography column. This application claims priority based on Japanese Patent Application No. 2024-103085 filed in Japan on June 26, 2024, Japanese Patent Application No. 2024-106985 filed in Japan on July 2, 2024, and Japanese Patent Application No. 2024-117565 filed in Japan on July 23, 2024, the contents of which are incorporated herein by reference.

[0002] In the research and development of biopolymers such as nucleic acids, chromatography is often used for their adsorption, separation, and purification. Known carriers used as separation materials for liquid chromatography include inorganic carriers such as silica gel and hydroxyapatite, natural polymer carriers such as agarose, dextran, cellulose, and chitosan, and synthetic polymer carriers such as polystyrene and poly(meth)acrylic acid esters. These carriers are used as is, or, if necessary, are provided with various functional groups to enable use in various separation modes. Furthermore, in recent years, further improvements in the separation and purification capabilities of nucleic acids have been desired.

[0003] For example, Patent Document 1 discloses a separation material in which anion exchange groups are fixed to a porous membrane via graft chains. According to this technology, a separation material with excellent nucleic acid separation performance can be obtained by graft polymerizing glycidyl methacrylate onto a porous membrane and then introducing anion exchange groups.

[0004] JP 2010-187615 A

[0005] However, in the method of purifying nucleic acids using a separation material disclosed in Patent Document 1, nucleic acids are first adsorbed into a module and then separated. This adsorption process depends on the ion exchange capacity of the separation material, but the separation material disclosed in Patent Document 1 has one anion exchange group for a monomer-derived structure, and the ion exchange capacity is insufficient to achieve the separation performance required in recent years.

[0006] Furthermore, the method of purifying nucleic acids using a separation material disclosed in Patent Document 1 is carried out by adding host cell debris, endotoxin, and BSA (albumin from bovine serum) as nucleic acid impurities. Meanwhile, chemical synthesis has become the mainstream method for synthesizing nucleic acids used in recent nucleic acid pharmaceutical applications, and since the impurities are monomeric differences of the target nucleic acid, further improvement in the separation performance of separation materials is required.

[0007] The present invention has been made in consideration of these problems, and relates to a separation material with improved separation and purification capabilities even for large molecules such as nucleic acids, a method for producing the separation material, a mixture, and application technologies thereof.

[0008] Regarding the first invention, the inventors conducted extensive research and discovered that the separation and purification ability of a separation material can be improved by immobilizing a (co)polymer having two or more ion exchange groups per structural unit derived from one monomer onto a carrier.

[0009] That is, the gist of the first invention is as follows: [1] A separation material comprising an anion exchange group bound to a carrier, the anion exchange group containing a structural unit represented by formula (A1). (In formula (A1), n1 is 1 or more and 10 or less. R ａ０ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ａ１ represents O or NH. ａ２ and R ａ３ R each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ａ４ R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ａ５ , R ａ６ and R ａ７ each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.) [2] The separation material according to [1], wherein the anion exchange group further contains a structural unit represented by formula (A2): (In formula (A2), m1 is 1 or more and 10 or less. R ａ８represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ａ９ represents O or NH. ａ１０ R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ａ１１ , R ａ１２ and R ａ１３ each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.) [3] The separation material of [1] or [2], which contains the structural unit represented by the above formula (A1) in a range of 10 mol% to 100 mol% based on all structural units contained in the anion exchange group. [4] The separation material of any of [1] to [3], which contains the structural unit represented by the above formula (A1) in a range of 20 mol% to 50 mol% based on all structural units contained in the anion exchange group. [5] The separation material of any of [1] to [3], which contains the structural unit represented by the above formula (A1) in a range of 40 mol% to 100 mol% based on all structural units contained in the anion exchange group. [6] The separation material of any of [1] to [5], which has an exchange capacity of 0.08 mmol / mL-R to 0.20 mmol / mL-R.

[0010] [7] A method for separating a target molecule, comprising the following steps (1-a) and (1-b): (1-a): contacting a solution containing the target molecule with the separation material of any one of [1] to [6] to adsorb the target molecule to the separation material, and (1-b): eluting the target molecule from the separation material treated in step (1-a). [8] The method for separating a target molecule of [7], wherein the target molecule is either a biopolymer, a partial structure of a biopolymer, or a chemically modified product of a biopolymer. [9] The method for separating a target molecule of [8], wherein the target molecule is either an oligonucleic acid, a partial structure of an oligonucleic acid, or a chemically modified product of an oligonucleic acid.

[10] A chromatography column comprising a separation material of any one of [1] to [6] as a packing material and having at least one container.

[0011] As a result of extensive research, the present inventors have found that the separation and purification ability of a separation material can be improved by immobilizing a (co)polymer having the following structure on a carrier.

[0012] That is, the gist of the second invention is as follows:

[11] A separation material comprising an anion exchange group bound to a carrier, wherein the anion exchange group contains a structural unit represented by formula (B1). (In formula (B1), n2 is 1 or more and 10 or less. R ｂ０ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｂ１ represents O or NH. ｂ２ R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ｂ３ , R ｂ４ and R ｂ５

[12] The separation material according to

[11] , wherein the carrier is composed of spherical particles.

[13] In the formula (B1), R ｂ１ is NH.

[14] The separation material of any of

[11] to

[13] , wherein in formula (B1), n2 is 2 or more and 3 or less.

[15] The separation material of any of

[11] to

[14] , wherein the anion exchange group further contains a structural unit represented by formula (B2). (In formula (B2), m2 is 1 or more and 10 or less. R ｂ６ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｂ７ represents O or NH. ｂ８ and R ｂ９ R each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｂ１０ R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ｂ１１ , R ｂ１２ and R ｂ１３each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.)

[16] The separation material of any of

[11] to

[15] , which contains the structural unit represented by the above formula (B1) in a range of 10 mol% to 100 mol% based on all structural units contained in the anion exchange group.

[17] The separation material of any of

[11] to

[15] , which contains the structural unit represented by the above formula (B1) in a range of 60 mol% to 100 mol% based on all structural units contained in the anion exchange group.

[18] The separation material of any of

[11] to

[15] , which contains the structural unit represented by the above formula (B1) in a range of 20 mol% to 60 mol% based on all structural units contained in the anion exchange group.

[19] The separation material of any of

[11] to

[18] , which has an exchange capacity of 0.08 mmol / mL-R to 0.20 mmol / mL-R.

[0013]

[20] A method for separating a target molecule, comprising the following steps (2-a) and (2-b): (2-a): contacting a solution containing the target molecule with the separation material of any one of [1] to

[19] to adsorb the target molecule to the separation material, and (2-b): eluting the target molecule from the separation material treated in step (2-a).

[21] The method for separating a target molecule of

[20] , wherein the target molecule is either a biopolymer, a partial structure of a biopolymer, or a chemically modified product of a biopolymer.

[22] The method for separating a target molecule of

[20] or

[21] , wherein the target molecule is either an oligonucleic acid, a partial structure of an oligonucleic acid, or a chemically modified product of an oligonucleic acid.

[23] A chromatography column comprising a separation material of any one of

[11] to

[19] as a packing material and having at least one container.

[0014] Regarding the third invention, the inventors conducted extensive research and discovered that the separation and purification ability of the separation material can be improved by binding a (co)polymer having two or more ion exchange groups per structural unit derived from one monomer to the carrier.

[0015] That is, the gist of the third invention is as follows:

[24] A method for producing a separation material having anion exchange groups bound to a carrier, the method comprising obtaining a polymer containing a structural unit represented by formula (C1) as the anion exchange group and having a number average molecular weight of 100,000 or less, and then performing a post-reaction to obtain a polymer containing a structural unit represented by formula (C2). (In formula (C1), n3 is 1 or more and 10 or less. R ｃ０ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｃ１ represents O or NH. ｃ２ and R ｃ３ each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. (In formula (C2), n3 is 1 or more and 10 or less. R ｃ０ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｃ１ represents O or NH. ｃ２ and R ｃ３ R each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｃ４ R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ｃ５ , R ｃ６ and R ｃ７ each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.)

[25] The method for producing a separation material according to

[24] , wherein a polymer further contains a structural unit represented by formula (C3) as the anion exchange group and has a number average molecular weight of 100,000 or less, and then a polymer containing structural units represented by formula (C2) and formula (C4) is obtained through a post-reaction. (In formula (C3), m3 is 1 or more and 10 or less. R ｃ８ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｃ９ represents O or NH.) (In formula (C4), m3 is 1 or more and 10 or less. R ｃ８ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｃ９ represents O or NH. ｃ１０R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ｃ１１ , R ｃ１２ and R ｃ１３ each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.)

[26] A method for producing a separation material according to

[24] or

[25] , in which the structural unit represented by the above formula (C2) is blended in an amount of 10 mol % to 100 mol % relative to the total structural units contained in the anion exchange group.

[27] A separation material produced by the method for producing a separation material according to any one of

[24] to

[26] .

[28] The separation material according to

[27] , having an exchange capacity of 0.08 mmol / mL-R to 0.20 mmol / mL-R.

[29] A mixture of the separation material according to

[27] and a polymer containing a structural unit represented by formula (C2) and having a number average molecular weight of 200,000 or less.

[0016]

[30] A method for separating a target molecule, comprising the following steps (3-a) and (3-b): (3-a): contacting a solution containing the target molecule with the separation material according to

[27] or the mixture according to

[29] to adsorb the target molecule to the separation material, and (3-b): eluting the target molecule from the separation material treated in step (3-a).

[31] The method for separating a target molecule according to

[30] , wherein the target molecule is any of a biopolymer, a partial structure of a biopolymer, and a chemically modified product of a biopolymer.

[32] The method for separating a target molecule according to

[31] , wherein the target molecule is any of an oligonucleic acid, a partial structure of an oligonucleic acid, and a chemically modified product of an oligonucleic acid.

[33] A chromatography column comprising the separation material according to

[27] or the mixture according to

[29] as a packing material and having at least one container.

[0017] The separation material and mixture of the present invention are suitable for the separation and purification of target molecules, particularly biopolymers and nucleic acids, and are also useful as chromatography packings. The method for producing a separation material of the present invention can produce a separation material that is suitable for the separation and purification of target molecules, particularly biopolymers and their partial structures or chemically modified products, and nucleic acids and their partial structures or chemically modified products, and is also useful as a chromatography packing. The method for separating target molecules of the present invention has good separation performance when separating target molecules from a solution of the target molecules containing impurities, and is useful for the separation and purification of target molecules, particularly biopolymers and their partial structures or chemically modified products, and nucleic acids and their partial structures or chemically modified products.

[0018] The present invention will be described in detail below, but the examples described below are examples (representative examples) of embodiments of the present invention, and the present invention is not limited to these contents as long as it does not deviate from the gist of the present invention.

[0019] In this specification, "(meth)acrylic" refers to either or both of "acrylic" and "methacrylic", and the same applies to "(meth)acrylate". Furthermore, "(co)polymerization" refers to either or both of "homopolymerization" and "copolymerization".

[0020] 1. First Invention 1.1. Separation Material One embodiment of the first invention relates to a separation material. The separation material of the first invention is a separation material in which anion exchange groups are bound to a carrier, and the anion exchange groups contain structural units represented by the following formula (A1). The anion exchange groups of the separation material of the first invention may further contain structural units represented by the following formula (A2). The anion exchange groups of the separation material of the first invention preferably contain structural units represented by the following formula (A1) in a range of 10 mol % to 100 mol % relative to the total structural units contained in the anion exchange groups.

[0021]

[0022] In formula (A1), n1 is 1 or more and 10 or less. That is, (CH ２ ) ｎ１ represents an alkylene group having 1 to 10 carbon atoms. ａ０represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ａ１ represents O or NH. ａ２ and R ａ３ R each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ａ４ R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ａ５ , R ａ６ and R ａ７ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

[0023]

[0024] In formula (A2), m1 is 1 or more and 10 or less. That is, (CH ２ ) ｍ１ represents an alkylene group having 1 to 10 carbon atoms. ａ８ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ａ９ represents O or NH. ａ１０ R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ａ１１ , R ａ１２ and R ａ１３ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

[0025] [Anion Exchange Group] The anion exchange group possessed by the separation material of the first invention contains a structural unit represented by the above formula (A1) and may further contain a structural unit represented by the above formula (A2). That is, the anion exchange group possessed by the separation material of the first invention is composed of a (co)polymer containing at least a structural unit represented by the above formula (A1) and may further contain a structural unit represented by the above formula (A2). Hereinafter, the structural unit represented by formula (A1) will also be referred to as "structural unit (A1)," and structural units represented by other formulas may also be referred to similarly. The anion exchange group possessed by the separation material of the first invention contains the structural unit (A1) because of its good polymerization reactivity with the monomer, ease of post-treatment and post-reaction, wide variety, and ease of industrial availability and synthesis.

[0026] In the above formula (A1), n1 is 1 or more and 10 or less, and from the viewpoint of the affinity of the separation material to water, it is preferably 1 or more and 4 or less. That is, (CH ２ ) ｎ１ is an alkylene group of 1 or more and 10 or less, and from the viewpoint of the affinity of the separation material to water, an alkylene group of 1 or more and 4 or less is preferred. ａ０ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. From the viewpoint of water solubility and high separability, R ａ０ is preferably a hydrogen atom or an alkyl group having 1 or 2 carbon atoms, more preferably a hydrogen atom or a methyl group. ａ１ represents O or NH. In order to improve the stability of the separation material, R ａ１ is preferably NH. ａ２ and R ａ３ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and from the viewpoint of water solubility and high separation performance, an alkyl group having 1 or 2 carbon atoms is preferred, and an alkyl group having 1 carbon atom, i.e., a methyl group, is more preferred.

[0027] R ａ４ is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. From the viewpoint of water solubility and high separation performance, R ａ４is preferably a linear alkylene group having 1 to 4 carbon atoms, more preferably a linear alkylene group having 1 to 4 carbon atoms and having a substituent, and even more preferably a linear alkylene group having 1 to 4 carbon atoms and having a hydroxyl group or an alkoxy group as a substituent. ａ４ The number of substituents that the alkylene group has may be one, two or more, for example, 1 to 4.

[0028] R ａ５ , R ａ６ and R ａ７ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and from the viewpoint of water solubility and high separation performance, an alkyl group having 1 or 2 carbon atoms is preferred.

[0029] As the structural unit (A1), the following structural units (A1-1) to (A1-3) are preferred, the structural unit (A1-2) and the structural unit (A1-3) are more preferred, and the structural unit (A1-3) is particularly preferred. Structural unit (A1-1): In the above formula (A1), n1 is 1 or more and 4 or less, R ａ０ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R ａ１ NH, R ａ２ and R ａ３ are each independently an alkyl group having 1 or 2 carbon atoms, R ａ４ is a linear alkylene group having 1 to 4 carbon atoms, R ａ５ ~R ａ７ are each independently an alkyl group having 1 or 2 carbon atoms. Structural unit (A1-2): a structural unit in which n1 in the above formula (A1) is 1 or more and 4 or less, R ａ０ is a hydrogen atom or a methyl group, R ａ１ NH, R ａ２ and R ａ３ are each independently an alkyl group having 1 or 2 carbon atoms, R ａ４ is a linear alkylene group having 1 to 4 carbon atoms and having a substituent, and R ａ５ ~R ａ７ are each independently an alkyl group having 1 or 2 carbon atoms. Structural unit (A1-3): a structural unit in which n1 in the above formula (A1) is 1 or more and 4 or less, R ａ０ is a hydrogen atom or a methyl group, R ａ１ NH, R ａ２ and Rａ３ is a methyl group, R ａ４ is a linear alkylene group having 1 to 4 carbon atoms and having a hydroxyl group or an alkoxy group as a substituent, and R ａ５ ~R ａ７ are each independently an alkyl group having 1 or 2 carbon atoms

[0030] In the above formula (A2), m1 is 1 or more and 10 or less, and from the viewpoint of the affinity of the separation material to water, it is preferably 1 or more and 4 or less. That is, (CH ２ ) ｍ１ is an alkylene group of 1 or more and 10 or less, and from the viewpoint of the affinity of the separation material to water, an alkylene group of 1 or more and 4 or less is preferred. ａ８ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. From the viewpoint of water solubility and high separability, R ａ８ is preferably a hydrogen atom or an alkyl group having 1 or 2 carbon atoms, more preferably a hydrogen atom or a methyl group. ａ９ represents O or NH. In order to improve the stability of the separation material, R ａ９ is preferably NH.

[0031] R ａ１０ is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. From the viewpoint of water solubility and high separation performance, R ａ１０ is preferably a linear alkylene group having 1 to 4 carbon atoms, more preferably a linear alkylene group having 1 to 4 carbon atoms and having a substituent, and even more preferably a linear alkylene group having 1 to 4 carbon atoms and having a hydroxyl group or an alkoxy group as a substituent. ａ１０ The number of substituents that the alkylene group has may be one, two or more, for example, 1 to 4.

[0032] R ａ１１ , R ａ１２ and R ａ１３ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and from the viewpoint of water solubility and high separation performance, an alkyl group having 1 or 2 carbon atoms is preferred.

[0033] As the structural unit (A2), the following structural units (A2-1) to (A2-3) are preferred, the structural unit (A2-2) and the structural unit (A2-3) are more preferred, and the structural unit (A2-3) is particularly preferred. Structural unit (A2-1): In the above formula (A2), m1 is 1 or more and 4 or less, R ａ８ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R ａ９ NH, R ａ１０ is a linear alkylene group having 1 to 4 carbon atoms, R ａ１１ ~R ａ１３ are each independently an alkyl group having 1 or 2 carbon atoms. Structural unit (A2-2): In the above formula (A2), m1 is 1 or more and 4 or less, R ａ８ is a hydrogen atom or a methyl group, R ａ９ NH, R ａ１０ is a linear alkylene group having 1 to 4 carbon atoms and having a substituent, and R ａ１１ ~R ａ１３ are each independently an alkyl group having 1 or 2 carbon atoms. Structural unit (A2-3): In the above formula (A2), m1 is 1 or more and 4 or less, R ａ８ is a hydrogen atom or a methyl group, R ａ９ NH, R ａ１０ is a linear alkylene group having 1 to 4 carbon atoms and having a hydroxyl group or an alkoxy group as a substituent, and R ａ１１ ~R ａ１３ are each independently an alkyl group having 1 or 2 carbon atoms

[0034] When the anion exchange group of the separation material of the first invention contains both the structural unit (A1) and the structural unit (A2), a combination of any of the structural units (A1-1) to (A1-3) with any of the structural units (A2-1) to (A2-3) is preferred, a combination of any of the structural units (A1-2) to (A1-3) with any of the structural units (A2-2) to (A2-3) is more preferred, and a combination of the structural unit (A1-3) with the structural unit (A2-3) is particularly preferred.

[0035] The anion exchange group of the separation material of the first invention preferably contains the structural unit (A1) in a range of 10 mol% to 100 mol%, more preferably 20 mol% to 100 mol%, and even more preferably 20 mol% to 50 mol%, of the total structural units contained in the anion exchange group. Furthermore, the content of the structural unit (A1) in the separation material of the first invention is also preferably 40 mol% to 100 mol%.

[0036] The anion exchange group of the separation material of the first invention may contain the structural unit (A2) in an amount of preferably 0 mol% to 90 mol%, more preferably 0 mol% to 80 mol%, and even more preferably 50 mol% to 80 mol%. The content of the structural unit (A2) in the separation material of the first invention is also preferably 0 mol% to 60 mol%.

[0037] The anion exchange group of the separation material of the first invention may contain another structural unit (A3) in addition to the structural unit (A1) and the structural unit (A2).

[0038] Examples of the other structural unit (A3) include a structural unit derived from an aromatic vinyl monomer such as styrene, a structural unit derived from (meth)acrylamide, and a structural unit derived from (meth)acrylic acid. The content of the other structural unit (A3) in the anion exchange group of the separation material of the first invention is preferably 5 mol% or less based on the total structural units contained in the anion exchange group.

[0039] The anion exchange group possessed by the separation material of the first invention is preferably an anion exchange group in which the content of the structural unit (A1) is 10 mol% to 100 mol%, the content of the structural unit (A2) is 0 mol% to 90 mol%, and the content of other structural units (A3) is 5 mol% or less, relative to the total structural units contained in the anion exchange group; more preferably an anion exchange group in which the content of the structural unit (A1) is 20 mol% to 100 mol%, the content of the structural unit (A2) is 0 mol% to 80 mol%, and the content of other structural units (A3) is 5 mol% or less; and even more preferably an anion exchange group in which the content of the structural unit (A1) is 20 mol% to 50 mol%, the content of the structural unit (A2) is 50 mol% to 80 mol%, and the content of other structural units (A3) is 5 mol% or less. The anion exchange group contained in the separation material of the first invention is also preferably an anion exchange group having a content of the structural unit (A1) of 40 mol% to 100 mol%, a content of the structural unit (A2) of 0 mol% to 60 mol%, and a content of the other structural unit (A3) of 5 mol% or less, provided that the total content of the structural units (A1) to (A3) does not exceed 100 mol%.

[0040] The number-average molecular weight of the polymer constituting the anion exchange group of the separation material of the first invention is preferably 100,000 or less, more preferably 52,000 or less. If the number-average molecular weight of the polymer is below the above upper limit, selectivity for nucleic acids and their impurities is increased. Furthermore, the number-average molecular weight of the polymer is preferably 20,000 or more, more preferably 30,000 or more. The lower and upper limits of the number-average molecular weight of the polymer can be arbitrarily combined, for example, from 20,000 to 100,000, or from 30,000 to 52,000. The number-average molecular weight of the polymer is measured by the method described in the Examples.

[0041] [Carrier] The carrier used in the first invention may be spherical particles, irregular particles, fibers, hollow fiber membranes, porous flat membranes, monolithic carriers, etc., with spherical particles being preferred because they are suitable for industrial scale-up and industrial operation, and spherical porous particles being particularly preferred because they have excellent oligonucleic acid loading characteristics. The material is not particularly limited, but from the viewpoint of physical strength, inorganic carriers such as silica gel and hydroxyapatite are preferred; natural polymer carriers such as agarose, dextran, cellulose, and chitosan; and synthetic polymers such as polystyrene and poly(meth)acrylic acid esters are more preferred.

[0042] [Separation Material] The separation material of the first invention is a separation material in which anion exchange groups are bound to a carrier. Examples of methods for binding the anion exchange groups to the carrier include a method in which a (co)polymer having anion exchange groups is prepared in advance and then bound to the carrier, a method in which a monomer having anion exchange groups is added to a reaction solution and the carrier is polymerized, and a method in which a carrier and a monomer having a reactive functional group are polymerized and then the functional group is modified. The separation material of the first invention can be obtained, for example, by a method similar to the manufacturing method of the separation material of the third invention described below.

[0043] The exchange capacity of the separation material of the first invention is preferably 0.08 mmol / mL-R or more and 0.20 mmol / mL-R or less, more preferably 0.10 mmol / mL-R or more and 0.20 mmol / mL-R or less, and even more preferably 0.12 mmol / mL-R or more and 0.17 mmol / mL-R or less. If the exchange capacity of the separation material is not less than the above-mentioned lower limit, the peak shape of nucleic acid is less likely to be distorted, and if it is not more than the above-mentioned upper limit, the selectivity between nucleic acid and its impurities is increased.

[0044] The separation material of the first invention has excellent separation performance between nucleic acids produced in the nucleic acid drug manufacturing process and their impurities, and can therefore be suitably used in the purification process of biopharmaceuticals, etc. Specifically, the separation material of the first invention is packed into a column, and a liquid containing a mixture of target molecules and impurities is passed through. In this liquid passing process, only the target molecules or the impurities are adsorbed and separated, or both the target molecules and impurities are adsorbed, and the salt concentration is increased during elution to separate the target molecules and impurities by utilizing the difference in adsorption properties to the separation material.

[0045] 1.2. Method for Separating Target Molecules Another embodiment of the first invention relates to a method for separating target molecules. The method for separating target molecules of the first invention is characterized by comprising the following steps (1-a) and (1-b): step (1-a): a step of contacting a solution containing target molecules with the separation material of the first invention to adsorb the target molecules to the separation material, and step (1-b): a step of eluting the target molecules from the separation material treated in step (1-a).

[0046] Examples of target molecules include inorganic ions, low molecular weight compounds such as amino acids and nucleic acids, and biopolymers, with biopolymers and their partial structures or chemically modified products being preferred in light of the separation targets that have been recently sought after. Examples of biopolymers and their partial structures or chemically modified products include proteins such as antibodies and peptides and their partial structures or chemically modified products, oligonucleic acids, and partial structures or chemically modified products of oligonucleic acids, with oligonucleic acids and partial structures or chemically modified products of oligonucleic acids being preferred in light of the significant progress that has been made in clinical development and practical application worldwide in recent years.

[0047] Oligonucleic acids are sequences of several to several hundred nucleic acids, and specific examples include nucleic acid drug modalities such as antisense oligos, siRNA, aptamers, and heteroduplex nucleic acids. The number of residues in an oligonucleic acid can be, for example, 3 to 200 or 4 to 150. Partial structures or chemically modified oligonucleic acids are oligonucleic acids whose inherent specific base pairing ability has been enhanced by chemical modification, and specific examples include oligonucleic acids that have been phosphorylated, aminated, biotinylated, thiolated, sulfurized (sulfurized), fluorescently modified, etc.

[0048] For example, steps (1-a) and (1-b) can be carried out by a chromatography method using a column packed with the separation material of the first invention, more specifically, by liquid chromatography such as HPLC. Apart from using a column packed with the separation material of the first invention, known chromatography methods can be used. Examples of known chromatography methods include anion exchange chromatography.

[0049] Examples of solutions containing target molecules include solutions containing a mixture of an oligonucleic acid, which is the target molecule, and impurities. Examples of impurities include those that are one or more residues shorter or longer than the oligonucleic acid and the partial structure or chemically modified product of the oligonucleic acid, or those that have not been subjected to phosphorylation, ammination, biotinylation, thiolation, sulfurization, fluorescent modification, or the like, or those in which a protecting group remains.

[0050] The process of adsorbing the target molecules onto the separation material and the process of eluting the target molecules from the separation material include, for example, a method of loading a solution containing the target molecules onto the separation material, passing a basic eluent with a predetermined salt concentration through the separation material, and changing the salt concentration from a low salt concentration to a high salt concentration, thereby separating the target molecules from impurities.

[0051] Examples of basic eluents include solutions containing at least one compound selected from the group consisting of inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and ammonium water, and organic bases such as morpholine, 4-methylmorpholine, piperazine, pyridine, ethylenediamine, diethylenetriamine, ethanolamine, diethanolamine, triethanolamine, trimethylamine, triethylamine, and tris(hydroxyethyl)aminomethane. In addition, inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, and citric acid may be added to adjust the pH of the eluent.

[0052] The pH of the basic eluent is preferably 8 or more and 14 or less. A pH of 8 or more improves the separation ability between the target molecule and the impurities. A pH of 14 or less improves the durability of the separation material.

[0053] Examples of salts to be added to the basic eluent include sodium chloride, potassium chloride, potassium bromide, sodium bromide, sodium iodide, sodium sulfate, and sodium perchlorate.

[0054] [Chromatography Column] Yet another embodiment of the first invention relates to a chromatography column. The chromatography column of the first invention is not particularly limited as long as it contains the separation material of the first invention as a packing material and has at least one container. That is, the chromatography column of the first invention can adopt any known form without particular limitation, except that the separation material of the first invention is used as a packing material.

[0055] 2. Second Invention 2.1. Separation Material One embodiment of the second invention relates to a separation material. The separation material of the second invention is a separation material in which anion exchange groups are bound to a carrier, and the anion exchange groups contain a structural unit represented by the following formula (B1). The anion exchange groups of the separation material of the second invention may further contain a structural unit represented by the following formula (B2). It is preferable that the structural unit represented by the following formula (B1) is contained in a range of 10 mol % to 100 mol % relative to the total structural units contained in the anion exchange groups of the separation material of the first invention.

[0056]

[0057] In formula (B1), n2 is 1 or more and 10 or less. That is, (CH ２ ) ｎ２ represents 1 to 10 alkylene groups. ｂ０ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｂ１ represents O or NH. ｂ２ R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ｂ３ , R ｂ４ and R ｂ５are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

[0058]

[0059] In formula (B2), m2 is 1 or more and 10 or less. That is, (CH ２ ) ｍ２ represents 1 to 10 alkylene groups. ｂ６ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｂ７ represents O or NH. ｂ８ and R ｂ９ R each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｂ１０ R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ｂ１１ , R ｂ１２ and R ｂ１３ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

[0060] [Anion Exchange Group] The anion exchange group possessed by the separation material of the second invention contains a structural unit represented by the above formula (B1) and may further contain a structural unit represented by the above formula (B2). That is, the anion exchange group possessed by the separation material of the second invention is composed of a (co)polymer containing at least a structural unit represented by the above formula (B1) and may further contain a structural unit represented by the above formula (B2). The anion exchange group possessed by the separation material of the second invention contains the structural unit (B1) because of its good polymerization reactivity with the monomer, ease of post-treatment and post-reaction, wide variety, and ease of industrial availability and synthesis.

[0061] In the above formula (B1), n2 is 1 or more and 10 or less, and from the viewpoint of the affinity of the separation material to water, it is preferably 1 or more and 4 or less. That is, (CH ２ ) ｎ２ is an alkylene group of 1 or more and 10 or less, and from the viewpoint of the affinity of the separation material to water, an alkylene group of 1 or more and 4 or less is preferred. ｂ０represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. From the viewpoint of water solubility and high separability, R ｂ０ is preferably a hydrogen atom or an alkyl group having 1 or 2 carbon atoms, more preferably a hydrogen atom or a methyl group. ｂ１ represents O or NH. In order to improve the stability of the separation material, R ｂ１ is preferably NH.

[0062] R ｂ２ is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. From the viewpoint of water solubility and high separation performance, R ｂ２ is preferably a linear alkylene group having 1 to 4 carbon atoms, more preferably a linear alkylene group having 1 to 4 carbon atoms and having a substituent, and even more preferably a linear alkylene group having 1 to 4 carbon atoms and having a hydroxyl group or an alkoxy group as a substituent. ｂ２ The number of substituents that the alkylene group has may be one, two or more, for example, 1 to 4.

[0063] R ｂ３ , R ｂ４ and R ｂ５ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and from the viewpoint of water solubility and high separation performance, an alkyl group having 1 or 2 carbon atoms is preferred.

[0064] As the structural unit (B1), the following structural units (B1-1) to (B1-3) are preferred, the structural unit (B1-2) and the structural unit (B1-3) are more preferred, and the structural unit (B1-3) is particularly preferred. Structural unit (B1-1): In the above formula (B1), n2 is 1 or more and 4 or less, R ｂ０ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R ｂ１ NH, R ｂ２ is a linear alkylene group having 1 to 4 carbon atoms, R ｂ３ ~R ｂ５ are each independently an alkyl group having 1 or 2 carbon atoms. Structural unit (B1-2): In the above formula (B1), n2 is 1 or more and 4 or less, Rｂ０ is a hydrogen atom or a methyl group, R ｂ１ NH, R ｂ２ is a linear alkylene group having 1 to 4 carbon atoms and having a substituent, and R ｂ３ ~R ｂ５ are each independently an alkyl group having 1 or 2 carbon atoms. Structural unit (B1-3): In the above formula (B1), n2 is 1 or more and 4 or less, R ｂ０ is a hydrogen atom or a methyl group, R ｂ１ NH, R ｂ２ is a linear alkylene group having 1 to 4 carbon atoms and having a hydroxyl group or an alkoxy group as a substituent, and R ｂ３ ~R ｂ５ are each independently an alkyl group having 1 or 2 carbon atoms

[0065] In the above formula (B2), m2 is 1 or more and 10 or less, and from the viewpoint of the affinity of the separation material to water, it is preferably 1 or more and 4 or less. That is, (CH ２ ) ｍ２ is an alkylene group of 1 or more and 10 or less, and from the viewpoint of the affinity of the separation material to water, an alkylene group of 1 or more and 4 or less is preferred. ｂ６ R represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. From the viewpoint of water solubility and high separability, a hydrogen atom or an alkyl group having 1 or 2 carbon atoms is preferred, and a hydrogen atom or a methyl group is more preferred. ｂ７ represents O or NH. In order to improve the stability of the separation material, R ｂ７ is preferably NH.

[0066] R ｂ８ and R ｂ９ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and from the viewpoint of water solubility and high separation performance, an alkyl group having 1 or 2 carbon atoms is preferred, and an alkyl group having 1 carbon atom, i.e., a methyl group, is more preferred.

[0067] R ｂ１０ is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. From the viewpoint of water solubility and high separation performance, R ｂ１０is preferably a linear alkylene group having 1 to 4 carbon atoms, more preferably a linear alkylene group having 1 to 4 carbon atoms and having a substituent, and even more preferably a linear alkylene group having 1 to 4 carbon atoms and having a hydroxyl group or an alkoxy group as a substituent. ｂ１０ The number of substituents that the alkylene group has may be one, two or more, for example, 1 to 4.

[0068] R ｂ１１ , R ｂ１２ and R ｂ１３ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and from the viewpoint of water solubility and high separation performance, an alkyl group having 1 or 2 carbon atoms is preferred.

[0069] As the structural unit (B2), the following structural units (B2-1) to (B2-3) are preferred, the structural unit (B2-2) and the structural unit (B2-3) are more preferred, and the structural unit (B2-3) is particularly preferred. Structural unit (B2-1): In the above formula (B2), m2 is 1 or more and 4 or less, R ｂ６ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R ｂ７ NH, R ｂ８ and R ｂ９ are each independently an alkyl group having 1 or 2 carbon atoms, R ｂ１０ is a linear alkylene group having 1 to 4 carbon atoms, R ｂ１１ ~R ｂ１３ are each independently an alkyl group having 1 or 2 carbon atoms. Structural unit (B2-2): a structural unit in which m2 in the above formula (B2) is 1 or more and 4 or less, R ｂ６ is a hydrogen atom or a methyl group, R ｂ７ NH, R ｂ８ and R ｂ９ are each independently an alkyl group having 1 or 2 carbon atoms, R ｂ１０ is a linear alkylene group having 1 to 4 carbon atoms and having a substituent, and R ｂ１１ ~R ｂ１３ are each independently an alkyl group having 1 or 2 carbon atoms. Structural unit (B2-3): a structural unit in which m2 in the above formula (B2) is 1 or more and 4 or less, R ｂ６ is a hydrogen atom or a methyl group, R ｂ７ NH, R ｂ８ and Rｂ９ is a methyl group, R ｂ１０ is a linear alkylene group having 1 to 4 carbon atoms and having a hydroxyl group or an alkoxy group as a substituent, and R ｂ１１ ~R ｂ１３ are each independently an alkyl group having 1 or 2 carbon atoms

[0070] When the anion exchange group of the separation material of the second invention contains both the structural unit (B1) and the structural unit (B2), a combination of any of the structural units (B1-1) to (B1-3) with any of the structural units (B2-1) to (B2-3) is preferred, a combination of any of the structural units (B1-2) to (B1-3) with any of the structural units (B2-2) to (B2-3) is more preferred, and a combination of the structural unit (B1-3) with the structural unit (B2-3) is particularly preferred.

[0071] The anion exchange group of the separation material of the second invention preferably contains the structural unit (B1) in a range of 10 mol% to 100 mol%, more preferably 30 mol% to 100 mol%, and even more preferably 60 mol% to 100 mol%, based on the total structural units contained in the anion exchange group. Furthermore, the content of the structural unit (B1) in the separation material of the second invention is also preferably 20 mol% to 60 mol%. When the content of the structural unit (B1) is above the above lower limit and below the above upper limit, the selectivity for nucleic acids and their impurities is improved.

[0072] The anion exchange group of the separation material of the second invention may contain the structural unit (B2) in an amount of preferably 0 mol% to 90 mol%, more preferably 0 mol% to 70 mol%, and even more preferably 0 mol% to 40 mol%. The content of the structural unit (B2) in the separation material of the second invention is also preferably 40 mol% to 80 mol%.

[0073] The anion exchange group of the separation material of the second invention may contain another structural unit (B3) in addition to the structural unit (B1) and the structural unit (B2).

[0074] Examples of the other structural unit (B3) include a structural unit derived from an aromatic vinyl monomer such as styrene, a structural unit derived from (meth)acrylamide, and a structural unit derived from (meth)acrylic acid. The content of the other structural unit (B3) in the anion exchange group of the separation material of the second invention is preferably 5 mol% or less based on the total structural units contained in the anion exchange group.

[0075] The anion exchange group possessed by the separation material of the second invention is preferably an anion exchange group in which the content of the structural unit (B1) is 10 mol% to 100 mol%, the content of the structural unit (B2) is 0 mol% to 90 mol%, and the content of other structural units (B3) is 5 mol% or less, relative to the total structural units contained in the anion exchange group; more preferably an anion exchange group in which the content of the structural unit (B1) is 30 mol% to 100 mol%, the content of the structural unit (B2) is 0 mol% to 70 mol%, and the content of other structural units (B3) is 5 mol% or less; and even more preferably an anion exchange group in which the content of the structural unit (B1) is 60 mol% to 100 mol%, the content of the structural unit (B2) is 0 mol% to 40 mol%, and the content of other structural units (B3) is 5 mol% or less. The anion exchange group contained in the separation material of the second invention is also preferably an anion exchange group having a content of the structural unit (B1) of 20 mol% to 60 mol%, a content of the structural unit (B2) of 40 mol% to 80 mol%, and a content of the other structural unit (B3) of 5 mol% or less, provided that the total content of the structural units (B1) to (B3) does not exceed 100 mol%.

[0076] The number-average molecular weight of the polymer constituting the anion exchange group of the separation material of the second invention is preferably 100,000 or less, more preferably 52,000 or less. If the number-average molecular weight of the polymer is below the above upper limit, the selectivity for nucleic acids and their impurities increases. Furthermore, the number-average molecular weight of the polymer is preferably 20,000 or more, more preferably 30,000 or more. The lower and upper limits of the number-average molecular weight of the polymer can be arbitrarily combined, for example, 20,000 or more and 100,000 or less, or 30,000 or more and 52,000 or less. The number-average molecular weight of the polymer is measured by the method described in the Examples.

[0077] [Carrier] The carrier used in the second invention may be spherical particles, irregular particles, fibers, hollow fiber membranes, porous flat membranes, monolithic carriers, etc., with spherical particles being preferred because they are suitable for industrial scale-up and industrial operation, and spherical porous particles being particularly preferred because they have excellent oligonucleic acid loading characteristics. The material is not particularly limited, but from the viewpoint of physical strength, inorganic carriers such as silica gel and hydroxyapatite are preferred; natural polymer carriers such as agarose, dextran, cellulose, and chitosan; and synthetic polymers such as polystyrene and poly(meth)acrylic acid esters are more preferred.

[0078] [Separation Material] The separation material of the second invention is a separation material in which anion exchange groups are bound to a carrier. Examples of methods for binding the anion exchange groups to the carrier include a method of preliminarily preparing a (co)polymer having anion exchange groups and then binding it to the carrier, a method of adding a monomer having anion exchange groups and the carrier to a reaction solution to polymerize, and a method of polymerizing a carrier and a monomer having a reactive functional group, followed by modifying the functional group. The separation material of the second invention can be obtained, for example, by a method similar to the manufacturing method of the separation material of the third invention described below. More specifically, the structural unit (B1) can be formed by a method similar to the structural unit (C4) described below, and the structural unit (B2) can be formed by a method similar to the structural unit (C2) described below.

[0079] The exchange capacity of the separation material of the second invention is preferably 0.08 mmol / mL-R or more and 0.20 mmol / mL-R or less, more preferably 0.10 mmol / mL-R or more and 0.15 mmol / mL-R or less, and even more preferably 0.10 mmol / mL-R or more and 0.13 mmol / mL-R or less. If the exchange capacity of the separation material is not less than the above-mentioned lower limit, the peak shape of nucleic acid is less likely to be distorted, and if it is not more than the above-mentioned upper limit, the selectivity between nucleic acid and its impurities is increased.

[0080] The separation material of the second invention has excellent separation performance between nucleic acids produced in the nucleic acid drug manufacturing process and their impurities, making it suitable for use in the purification process of biopharmaceuticals, etc. Specifically, the separation material of the second invention is packed into a column, and a liquid containing a mixture of target molecules and impurities is passed through. In this liquid passing process, only the target molecules or the impurities are adsorbed and separated, or both the target molecules and impurities are adsorbed, and the salt concentration is increased during elution to separate the target molecules and impurities by utilizing the difference in adsorption properties to the separation material.

[0081] 2.2. Method for Separating Target Molecules Another embodiment of the second invention relates to a method for separating target molecules. The method for separating target molecules of the second invention is characterized by comprising the following steps (2-a) and (2-b): step (2-a): a step of contacting a solution containing target molecules with the separation material of the second invention to adsorb the target molecules to the separation material, and step (2-b): a step of eluting the target molecules from the separation material treated in step (2-a).

[0082] Examples of target molecules include inorganic ions, low molecular weight compounds such as amino acids and nucleic acids, and biopolymers, with biopolymers and their partial structures or chemically modified products being preferred in light of the separation targets that have been recently sought after. Examples of biopolymers and their partial structures or chemically modified products include proteins such as antibodies and peptides and their partial structures or chemically modified products, oligonucleic acids, and partial structures or chemically modified products of oligonucleic acids, with oligonucleic acids and partial structures or chemically modified products of oligonucleic acids being preferred in light of the significant progress that has been made in clinical development and practical application worldwide in recent years.

[0083] Oligonucleic acids are sequences of several to several hundred nucleic acids, and specific examples include nucleic acid drug modalities such as antisense oligos, siRNA, aptamers, and heteroduplex nucleic acids. The number of residues in an oligonucleic acid can be, for example, 3 to 200 or 4 to 150. Partial structures or chemically modified oligonucleic acids are oligonucleic acids whose inherent specific base pairing ability has been enhanced by chemical modification, and specific examples include oligonucleic acids that have been phosphorylated, aminated, biotinylated, thiolated, sulfurized (sulfurized), fluorescently modified, etc.

[0084] For example, steps (2-a) and (2-b) can be carried out by a chromatography method using a column packed with the separation material of the second invention, more specifically, by liquid chromatography such as HPLC. Apart from using a column packed with the separation material of the second invention, any known chromatography method can be used.

[0085] Examples of solutions containing target molecules include solutions containing a mixture of an oligonucleic acid, which is the target molecule, and impurities. The impurities include those that are one or more residues shorter or longer than the oligonucleic acid and the partial structure or chemically modified product of the oligonucleic acid, or those that have not been subjected to phosphorylation, ammination, biotinylation, thiolation, sulfurization, fluorescent modification, or the like, or those in which a protecting group remains.

[0086] The process of adsorbing the target molecules onto the separation material and the process of eluting the target molecules from the separation material include, for example, a method of loading a solution containing the target molecules onto the separation material, passing a basic eluent with a predetermined salt concentration through the separation material, and changing the salt concentration from a low salt concentration to a high salt concentration, thereby separating the target molecules from impurities.

[0087] Examples of basic eluents include solutions containing at least one compound selected from the group consisting of inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and ammonium water, and organic bases such as morpholine, 4-methylmorpholine, piperazine, pyridine, ethylenediamine, diethylenetriamine, ethanolamine, diethanolamine, triethanolamine, trimethylamine, triethylamine, and tris(hydroxyethyl)aminomethane. In addition, inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, and citric acid may be added to adjust the pH of the eluent.

[0088] The pH of the basic eluent is preferably 8 or more and 14 or less. A pH of 8 or more improves the separation ability between the target molecule and the impurities. A pH of 14 or less improves the durability of the separation material.

[0089] Examples of salts to be added to the basic eluent include sodium chloride, potassium chloride, potassium bromide, sodium bromide, sodium iodide, sodium sulfate, and sodium perchlorate.

[0090] [Chromatography Column] Yet another embodiment of the second invention relates to a chromatography column. The chromatography column of the second invention is not particularly limited as long as it contains the separation material of the second invention as a packing material and has at least one container. That is, the chromatography column of the second invention can adopt any known form without particular limitation, except that the separation material of the second invention is used as a packing material.

[0091] 3. Third Invention 3.1. Method for Producing Separation Material One embodiment of the third invention relates to a method for producing a separation material. The method for producing a separation material of the third invention is a method for producing a separation material having anion exchange groups bound to a carrier, and includes obtaining a polymer containing a structural unit represented by the following formula (C1) as the anion exchange group and having a number average molecular weight of 100,000 or less, followed by a post-reaction to obtain a polymer containing a structural unit represented by the following formula (C2):

[0092] The carrier used in the method for producing a separation material of the third invention may be spherical particles, irregular particles, fibers, hollow fiber membranes, porous flat membranes, monolithic carriers, etc., with spherical particles being preferred because they are suitable for industrial scale-up and industrial operation, and spherical porous particles being particularly preferred because they have excellent oligonucleic acid loading characteristics. The material is not particularly limited, but from the viewpoint of physical strength, inorganic carriers such as silica gel and hydroxyapatite are preferred; natural polymer carriers such as agarose, dextran, cellulose, and chitosan; and synthetic polymers such as polystyrene and poly(meth)acrylic acid esters are more preferred.

[0093] In the method for producing a separation material of the third invention, a polymer further containing a structural unit represented by the following formula (C3) as the anion exchange group and having a number average molecular weight of 100,000 or less may be obtained, and then a polymer containing structural units represented by the following formulas (C2) and (C4) may be obtained through a post-reaction. In the method for producing a separation material of the third invention, it is preferable to blend the structural unit represented by the following formula (C2) in an amount of 10 mol % to 100 mol % relative to the total structural units contained in the anion exchange group. Hereinafter, a polymer containing a structural unit represented by formula (C1) and having a number average molecular weight of 100,000 or less will also be referred to as "polymer (X)," and a polymer containing structural unit (C2) obtained from polymer (X) through a post-reaction will also be referred to as "polymer (Y)."

[0094]

[0095] In formula (C1), n3 is 1 or more and 10 or less. That is, (CH ２ ) ｎ３ represents 1 to 10 alkylene groups. ｃ０ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｃ１ represents O or NH. ｃ２ and R ｃ３ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

[0096]

[0097] In formula (C2), n3 is 1 or more and 10 or less. That is, (CH２ ) ｎ３ represents 1 to 10 alkylene groups. ｃ０ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｃ１ represents O or NH. ｃ２ and R ｃ３ R each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｃ４ R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ｃ５ , R ｃ６ and R ｃ７ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

[0098]

[0099] In formula (C3), m3 is 1 or more and 10 or less. That is, (CH ２ ) ｍ３ represents 1 to 10 alkylene groups. ｃ８ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｃ９ represents O or NH.

[0100]

[0101] In formula (C4), m3 is 1 or more and 10 or less. That is, (CH ２ ) ｍ３ represents 1 to 10 alkylene groups. ｃ８ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｃ９ represents O or NH. ｃ１０ R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ｃ１１ , R ｃ１２ and R ｃ１３ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

[0102] [Anion Exchange Group] The anion exchange group possessed by the separation material produced by the third invention includes a structural unit (C2) formed from the structural unit (C1) via a post-reaction. The anion exchange group possessed by the separation material produced by the third invention may further include a structural unit (C4) formed from the structural unit (C3) via a post-reaction. That is, the anion exchange group possessed by the separation material produced by the third invention is composed of a (co)polymer containing at least a structural unit represented by the above formula (C2) and may further include a structural unit represented by the above formula (4). The anion exchange group contains the structural unit (C2) because of its good polymerization reactivity with the monomer, ease of post-treatment and post-reaction, wide variety, and ease of industrial availability and synthesis.

[0103] In the above formulas (C1) and (C2), n3 is 1 or more and 10 or less, and from the viewpoint of the affinity of the separation material to water, n3 is preferably 1 or more and 4 or less. That is, (CH ２ ) ｎ３ is an alkylene group of 1 or more and 10 or less, and from the viewpoint of the affinity of the separation material to water, an alkylene group of 1 or more and 4 or less is preferred. ｃ０ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. From the viewpoint of water solubility and high separability, R ｃ０ is preferably a hydrogen atom or an alkyl group having 1 or 2 carbon atoms, more preferably a hydrogen atom or a methyl group. ｃ１ represents O or NH. In order to improve the stability of the separation material, R ｃ１ is preferably NH. ｃ２ and R ｃ３ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and from the viewpoint of water solubility and high separation performance, an alkyl group having 1 or 2 carbon atoms is preferred, and an alkyl group having 1 carbon atom, i.e., a methyl group, is more preferred.

[0104] R ｃ４ is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. From the viewpoint of water solubility and high separation performance, R ｃ４is preferably a linear alkylene group having 1 to 4 carbon atoms, more preferably a linear alkylene group having 1 to 4 carbon atoms and having a substituent, and even more preferably a linear alkylene group having 1 to 4 carbon atoms and having a hydroxyl group or an alkoxy group as a substituent. ｃ４ The number of substituents that the alkylene group has may be one, two or more, for example, 1 to 4.

[0105] R ｃ５ , R ｃ６ and R ｃ７ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and from the viewpoint of water solubility and high separation performance, an alkyl group having 1 or 2 carbon atoms is preferred.

[0106] As the structural unit (C1), the following structural units (C1-1) to (C1-3) are preferred, the structural unit (C1-2) and the structural unit (C1-3) are more preferred, and the structural unit (C1-3) is particularly preferred. Structural unit (C1-1): n3 in the above formula (C1) is 1 or more and 4 or less, R ｃ０ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R ｃ１ NH, R ｃ２ and R ｃ３ are each independently an alkyl group having 1 or 2 carbon atoms. Structural unit (C1-2): a structural unit in which n3 in the above formula (C1) is 1 or more and 4 or less, R ｃ０ is a hydrogen atom or a methyl group, R ｃ１ NH, R ｃ２ and R ｃ３ are each independently an alkyl group having 1 or 2 carbon atoms. Structural unit (C1-3): a structural unit in which n3 in the above formula (C1) is 1 or more and 4 or less, R ｃ０ is a hydrogen atom or a methyl group, R ｃ１ NH, R ｃ２ and R ｃ３ is a structural unit in which the

[0107] As the structural unit (C2), the following structural units (C2-1) to (C2-3) are preferred, the structural unit (C2-2) and the structural unit (C2-3) are more preferred, and the structural unit (C2-3) is particularly preferred. Structural unit (C2-1): n3 in the above formula (C2) is 1 or more and 4 or less, Rｃ０ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R ｃ１ NH, R ｃ２ and R ｃ３ are each independently an alkyl group having 1 or 2 carbon atoms, R ｃ４ is a linear alkylene group having 1 to 4 carbon atoms, R ｃ５ ~R ｃ７ are each independently an alkyl group having 1 or 2 carbon atoms. Structural unit (C2-2): a structural unit in which n3 in the above formula (C2) is 1 or more and 4 or less, R ｃ０ is a hydrogen atom or a methyl group, R ｃ１ NH, R ｃ２ and R ｃ３ are each independently an alkyl group having 1 or 2 carbon atoms, R ｃ４ is a linear alkylene group having 1 to 4 carbon atoms and having a substituent, and R ｃ５ ~R ｃ７ are each independently an alkyl group having 1 or 2 carbon atoms. Structural unit (C2-3): a structural unit in which n3 in the above formula (C2) is 1 or more and 4 or less, R ｃ０ is a hydrogen atom or a methyl group, R ｃ１ NH, R ｃ２ and R ｃ３ is a methyl group, R ｃ４ is a linear alkylene group having 1 to 4 carbon atoms and having a hydroxyl group or an alkoxy group as a substituent, and R ｃ５ ~R ｃ７ are each independently an alkyl group having 1 or 2 carbon atoms

[0108] In the above formulas (C3) and (C4), m3 is 1 or more and 10 or less, and from the viewpoint of the affinity of the separation material to water, m3 is preferably 1 or more and 4 or less. That is, (CH ２ ) ｍ３ is an alkylene group of 1 or more and 10 or less, and from the viewpoint of the affinity of the separation material to water, an alkylene group of 1 or more and 4 or less is preferred. ｃ８ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. From the viewpoint of water solubility and high separability, R ｃ８ is preferably a hydrogen atom or an alkyl group having 1 or 2 carbon atoms, more preferably a hydrogen atom or a methyl group. ｃ９ represents O or NH. In order to improve the stability of the separation material, Rｃ９ is preferably NH.

[0109] R ｃ１０ is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. From the viewpoint of water solubility and high separation performance, R ｃ１０ is preferably a linear alkylene group having 1 to 4 carbon atoms, more preferably a linear alkylene group having 1 to 4 carbon atoms and having a substituent, and even more preferably a linear alkylene group having 1 to 4 carbon atoms and having a hydroxyl group or an alkoxy group as a substituent. ｃ１０ The number of substituents that the alkylene group has may be one, two or more, for example, 1 to 4.

[0110] R ｃ１１ , R ｃ１２ and R ｃ１３ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and from the viewpoint of water solubility and high separation performance, an alkyl group having 1 or 2 carbon atoms is preferred.

[0111] As the structural unit (C3), the following structural units (C3-1) to (C3-3) are preferred, the structural unit (C3-2) and the structural unit (C3-3) are more preferred, and the structural unit (C3-3) is particularly preferred. Structural unit (C3-1): In the above formula (C3), m3 is 1 or more and 4 or less, R ｃ８ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R ｃ９ Structural unit (C3-2): In the above formula (C3), m3 is 1 or more and 4 or less, R ｃ８ is a hydrogen atom or a methyl group, R ｃ９ Structural unit (C3-3): In the above formula (C3), m3 is 1 or more and 2 or less, R ｃ８ is a hydrogen atom or a methyl group, R ｃ９ is a structural unit in which

[0112] As the structural unit (C4), the following structural units (C4-1) to (C4-3) are preferred, the structural unit (C4-2) and the structural unit (C4-3) are more preferred, and the structural unit (C4-3) is particularly preferred. Structural unit (C4-1): In the above formula (C4), m3 is 1 or more and 4 or less, R ｃ８ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R ｃ９ NH, R ｃ１０ is a linear alkylene group having 1 to 4 carbon atoms, R ｃ１１ ~R ｃ１３ are each independently an alkyl group having 1 or 2 carbon atoms. Structural unit (C4-2): In the above formula (C4), m3 is 1 or more and 4 or less, R ｃ８ is a hydrogen atom or a methyl group, R ｃ９ NH, R ｃ１０ is a linear alkylene group having 1 to 4 carbon atoms and having a substituent, and R ｃ１１ ~R ｃ１３ are each independently an alkyl group having 1 or 2 carbon atoms. Structural unit (C4-3): In the above formula (C4), m3 is 1 or more and 2 or less, R ｃ８ is a hydrogen atom or a methyl group, R ｃ９ NH, R ｃ１０ is a linear alkylene group having 1 to 4 carbon atoms and having a hydroxyl group or an alkoxy group as a substituent, and R ｃ１１ ~R ｃ１３ are each independently an alkyl group having 1 or 2 carbon atoms

[0113] When polymer (X) contains both the structural unit (C1) and the structural unit (C3), a combination of any of the structural units (C1-1) to (C1-3) with any of the structural units (C3-1) to (C3-3) is preferred, a combination of any of the structural units (C1-2) to (C1-3) with any of the structural units (C3-2) to (C3-3) is more preferred, and a combination of the structural unit (C1-3) with the structural unit (C3-3) is particularly preferred. When polymer (Y) contains both the structural unit (C2) and the structural unit (C4), preferred combinations are the same as when polymer (X) contains both the structural unit (C1) and the structural unit (C3).

[0114] In the anion exchange group of the separation material produced by the third invention, the structural unit (C2) is preferably blended in a range of 10 mol% to 100 mol% relative to the total structural units contained in the anion exchange group, more preferably in a range of 20 mol% to 100 mol%, and even more preferably in a range of 30 mol% to 80 mol%.

[0115] The anion exchange group possessed by the separation material produced by the third invention may contain the structural unit (C4) relative to the total structural units contained in the anion exchange group, and the structural unit (C4) is preferably blended in the range of 0 mol% to 90 mol%, more preferably in the range of 0 mol% to 80 mol%, and even more preferably in the range of 20 mol% to 70 mol%.

[0116] The anion exchange group possessed by the separation material produced by the third invention may contain another structural unit (C5) in addition to the structural unit (C2) and the structural unit (C4).

[0117] Examples of the other structural unit (C5) include structural units derived from aromatic vinyl monomers such as styrene, structural units derived from (meth)acrylamide, and structural units derived from (meth)acrylic acid. The structural units (C1) and (C3) that remain unreacted after the post-reaction are included in the other structural units (C5). The content of the other structural units (C5) in the anion exchange groups of the separation material produced by the third invention is 5 mol% or less based on the total structural units containing the anion exchange groups.

[0118] The anion exchange group possessed by the separation material produced by the third invention is preferably an anion exchange group in which the content of the structural unit (C2) is 10 mol% to 100 mol%, the content of the structural unit (C4) is 0 mol% to 90 mol%, and the content of other structural units (C5) is 5 mol% or less, relative to the total structural units contained in the anion exchange group; more preferably an anion exchange group in which the content of the structural unit (C2) is 20 mol% to 100 mol%, the content of the structural unit (C4) is 0 mol% to 80 mol%, and the content of other structural units (C5) is 5 mol% or less; and even more preferably an anion exchange group in which the content of the structural unit (C2) is 30 mol% to 80 mol%, the content of the structural unit (C4) is 20 mol% to 70 mol%, and the content of other structural units (C5) is 5 mol% or less. However, the total content of the structural units (C2), (C4) and (C5) does not exceed 100 mol %.

[0119] The number-average molecular weight of polymer (X) is 100,000 or less, and preferably 52,000 or less. If it is below the upper limit, selectivity for nucleic acids and their impurities increases. Furthermore, the number-average molecular weight of polymer (X) is preferably 20,000 or more, and more preferably 30,000 or more. The lower and upper limits of the number-average molecular weight of polymer (X) can be arbitrarily combined, and can be, for example, 20,000 or more and 100,000 or less, or 30,000 or more and 52,000 or less.

[0120] The number average molecular weight of polymer (Y) is preferably 100,000 or less, more preferably 52,000 or less. If the number average molecular weight of polymer (Y) is equal to or less than the above upper limit, selectivity for nucleic acids and their impurities is increased. Furthermore, the number average molecular weight of polymer (Y) is preferably 20,000 or more, more preferably 30,000 or more. The lower and upper limits of the number average molecular weight of polymer (Y) can be arbitrarily combined, and can be, for example, 20,000 or more and 100,000 or less, or 30,000 or more and 52,000 or less. The number average molecular weight of the polymer is measured by the method described in the Examples.

[0121] [Production of Polymer (X)] Polymer (X) can be produced by polymerizing, using a known polymerization initiator, a monomer component containing at least a monomer represented by the following formula (c-1), preferably further containing a monomer represented by the following formula (c-2), and optionally containing an aromatic vinyl monomer such as styrene and other monomers such as (meth)acrylamide, by a known method. Hereinafter, the monomer represented by formula (c-1) will also be referred to as "monomer (c-1)," and the same will be used for monomers represented by other formulas.

[0122]

[0123] n3, m3, and R in the above formulas (c-1) and (c-2) ｃ０ , R ｃ１ ~R ｃ３ , R ｃ８ , R ｃ９ has the same meaning as the above formulas (C1) and (C3), and the preferred embodiments and combinations are also the same.

[0124] With respect to the proportion of each monomer relative to the total monomer components used in the production of polymer (X), the proportion of monomer (c-1) is preferably 10 mol% to 100 mol% and the proportion of monomer (c-2) is preferably 0 mol% to 90 mol%, more preferably 20 mol% to 100 mol% and the proportion of monomer (c-2) is 0 mol% to 80 mol%, and even more preferably 30 mol% to 80 mol% and the proportion of monomer (c-2) is 20 mol% to 70 mol%, provided that the sum of the proportions of monomer (c-1) and monomer (c-2) does not exceed 100 mol%.

[0125] As the polymerization method, for example, known polymerization methods such as solution polymerization, suspension polymerization, bulk polymerization, and emulsion polymerization can be used.

[0126] The polymerization initiator is preferably a radical polymerization initiator, for example, azo-based radical polymerization initiators such as 2,2'-azobisisobutyronitrile, 2,2'-azobis(2-methylbutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2,4-dimethyl-4-methoxyvaleronitrile), 2,2'-azobis(2-methylpropionamidine) dihydrochloride, and 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine]; 1,1,3,3-tetramethylbutylperoxy 2-ethylhexanoate, t-hexylperoxypivalate, 2,4-dihydroxybenzoylperoxybenzoate, 1,1,3,3-tetramethylbutyl ... Examples of the radical polymerization initiator include peroxide radical polymerization initiators such as chlorobenzoyl peroxide, t-butyl peroxypivalate, o-methylbenzoyl peroxide, bis-3,5,5-trimethylhexanoyl peroxide, octanoyl peroxide, t-butylperoxy-2-ethylhexanoate, cyclohexanone peroxide, benzoyl peroxide, methyl ethyl ketone peroxide, dicumyl peroxide, lauroyl peroxide, diisopropylbenzene hydroperoxide, t-butyl hydroperoxide, and di-t-butyl peroxide. These radical polymerization initiators can be used alone or in combination of two or more.

[0127] In the radical polymerization, a chain transfer agent can be used. Examples of the chain transfer agent include butanethiol, octanethiol, decanethiol, dodecanethiol, hexadecanethiol, octadecanethiol, cyclohexyl mercaptan, thiophenol, octyl thioglycolate, octyl 2-mercaptopropionate, octyl 3-mercaptopropionate, 2-ethylhexyl mercaptopropionate, 2-ethylhexyl thioglycolate, butyl-3-mercaptopropionate, mercaptopropyltrimethoxysilane, methyl-3-mercaptopropionate, 2,2-(ethylenediaminetetraacetic acid)-2,2-dimethyl-2,2-propanol, ... Examples of suitable chain transfer agents include thiol compounds such as mercaptohydroxydiethanethiol, ethanethiol, 4-methylbenzenethiol, octanoic acid 2-mercaptoethyl ester, 1,8-dimercapto-3,6-dioxaoctane, decane trithiol, dodecyl mercaptan, diphenyl sulfoxide, dibenzyl sulfide, 2,3-dimethylmercapto-1-propanol, mercaptoethanol, thiosalicylic acid, α-thioglycerol, thioglycolic acid, 3-mercaptopropionic acid, thiomalic acid, mercaptoacetic acid, mercaptosuccinic acid, and 2-mercaptoethanesulfonic acid. These chain transfer agents can be used alone or in combination of two or more.

[0128] In the polymerization, an acid such as sulfuric acid, hydrochloric acid, or nitric acid may be used for the purpose of carrying out the reaction under acidic conditions. These acids may be used alone or in combination of two or more. Among them, sulfuric acid is preferred as a strong divalent acid.

[0129] [Production of Polymer (Y)] Polymer (Y) can be obtained by reacting polymer (X) with a glycidyl group-containing quaternary ammonium compound such as glycidyltrimethylammonium chloride. This reaction can be carried out, for example, in water. The reaction temperature for the post-reaction to obtain polymer (Y) from polymer (X) is preferably 5 to 70°C, more preferably 25 to 50°C.

[0130] [Carrier] The carrier used in the third invention is not particularly limited, but from the viewpoint of physical strength, inorganic carriers such as silica gel and hydroxyapatite; natural polymer carriers such as agarose, dextran, cellulose and chitosan; and synthetic polymers such as polystyrene and poly(meth)acrylic acid esters are preferred. Polystyrene and poly(meth)acrylic acid esters are more preferred.

[0131] 3.2. Separation Material Another embodiment of the third invention relates to a separation material. The separation material of the third invention is a separation material manufactured by the above-described method for manufacturing a separation material of the third invention, and is a separation material in which anion exchange groups are bound to a carrier. Methods for binding the anion exchange groups to the carrier include, for example, a method in which a polymer (X) is prepared in advance and then post-reacted to form a polymer (Y), and then the polymer (X) is prepared in advance and then post-reacted after binding to the carrier to form a polymer (Y), and a method in which a monomer having a reactive functional group is polymerized with the carrier and then the functional group is modified.

[0132] The exchange capacity of the separation material produced by the third invention is preferably 0.08 mmol / mL-R or more and 0.20 mmol / mL-R or less, more preferably 0.10 mmol / mL-R or more and 0.16 mmol / mL-R or less, and even more preferably 0.10 mmol / mL-R or more and 0.14 mmol / mL-R or less. If the exchange capacity of the separation material is not less than the above-mentioned lower limit, the peak shape of nucleic acid is less likely to be distorted, and if it is not more than the above-mentioned upper limit, the selectivity between nucleic acid and impurities is increased.

[0133] The separation material of the third invention may also be a mixture of a polymer (hereinafter also referred to as "polymer (Z)") containing the structural unit (C2) and having a number average molecular weight of 200,000 or less. That is, a mixture containing the separation material of the third invention and a polymer (Z) that is not bonded to a carrier may also be used. The polymer (Z) may further contain the structural unit (C4) and another structural unit (C5).

[0134] The content of the structural unit (C2) in the polymer (Z) is preferably 10 mol% to 100 mol%, more preferably 20 mol% to 100 mol%, and even more preferably 30 mol% to 80 mol%, based on the total structural units contained in the polymer (Z). The content of the structural unit (C4) in the polymer (Z) is preferably 0 mol% to 90 mol%, more preferably 0 mol% to 80 mol%, and even more preferably 20 mol% to 70 mol%, based on the total structural units contained in the polymer (Z). The content of the other structural unit (C5) in the polymer (Z) is 5 mol% or less based on the total structural units containing anion exchange groups based on the total structural units contained in the polymer (Z).

[0135] As the polymer (Z), a polymer in which the content of the structural unit (C2) is 10 mol% to 100 mol%, the content of the structural unit (C4) is 0 mol% to 90 mol%, and the content of other structural units (C5) is 5 mol% or less, based on the total structural units, is preferred, a polymer in which the content of the structural unit (C2) is 20 mol% to 100 mol%, the content of the structural unit (C4) is 0 mol% to 80 mol%, and the content of other structural units (C5) is 5 mol% or less is more preferred, and a polymer in which the content of the structural unit (C2) is 30 mol% to 80 mol%, the content of the structural unit (C4) is 20 mol% to 70 mol%, and the content of other structural units (C5) is 5 mol% or less is even more preferred, provided that the total content of the structural units (C2), (C4), and (C5) does not exceed 100 mol%.

[0136] The number average molecular weight of polymer (Z) is preferably 20,000 or more and 200,000 or less, more preferably 40,000 or more and 120,000 or less. If the number average molecular weight of polymer (Z) is equal to or more than the above lower limit, the peak shape of nucleic acid is less likely to be distorted, and if the number average molecular weight of polymer (Z) is equal to or less than the above upper limit, the selectivity between nucleic acid and its impurities is increased. The number average molecular weight of the polymer is measured by the method described in the Examples. Polymer (Z) can be produced by the same method as polymer (Y) except that it is not bound to a carrier.

[0137] The proportion of polymer (Z) in the mixture is preferably 5 to 50 parts by mass, more preferably 5 to 20 parts by mass, relative to 100 parts by mass of the separation material of the third invention. If the proportion of polymer (Z) is equal to or greater than the above-mentioned lower limit, the peak shape of the nucleic acid is less likely to be distorted, and if the proportion of polymer (Z) is equal to or less than the above-mentioned upper limit, the selectivity between the nucleic acid and its impurities is increased.

[0138] 3.3. Method for Separating Target Molecules Yet another embodiment of the third invention relates to a method for separating target molecules. The method for separating target molecules of the third invention is characterized by comprising the following steps (3-a) and (3-b): step (3-a): a step of contacting a solution containing target molecules with the separation material or mixture of the third invention to adsorb the target molecules to the separation material, and step (3-b): a step of eluting the target molecules from the separation material treated in step (3-a).

[0139] Examples of target molecules include inorganic ions, low molecular weight compounds such as amino acids and nucleic acids, and biopolymers, with biopolymers and their partial structures or chemically modified products being preferred in light of the separation targets currently in demand. Examples of biopolymers and their partial structures or chemically modified products include proteins such as antibodies and peptides and their partial structures or chemically modified products, oligonucleic acids, and partial structures or chemically modified products of oligonucleic acids, with oligonucleic acids and partial structures or chemically modified products of oligonucleic acids being preferred in light of the significant progress in clinical development and practical application worldwide in recent years.

[0140] Oligonucleic acids are sequences of several to several hundred nucleic acids, and specific examples include nucleic acid drug modalities such as antisense oligos, siRNA, aptamers, and heteroduplex nucleic acids. The number of residues in an oligonucleic acid can be, for example, 3 to 200 or 4 to 150. Partial structures or chemically modified oligonucleic acids are oligonucleic acids whose inherent specific base pairing ability has been enhanced by chemical modification, and specific examples include oligonucleic acids that have been phosphorylated, aminated, biotinylated, thiolated, sulfurized (sulfurized), fluorescently modified, etc.

[0141] For example, steps (3-a) and (3-b) can be carried out by a chromatography method using a column packed with the separation material of the third invention, more specifically, by liquid chromatography such as HPLC. Apart from using a column packed with the separation material of the third invention, any known chromatography method can be used.

[0142] Examples of solutions containing target molecules include solutions containing a mixture of an oligonucleic acid, which is the target molecule, and impurities. The impurities include those that are one or more residues shorter or longer than the oligonucleic acid and the partial structure or chemically modified product of the oligonucleic acid, or those that have not been subjected to phosphorylation, ammination, biotinylation, thiolation, sulfurization, fluorescent modification, or the like, or those in which a protecting group remains.

[0143] The process of adsorbing the target molecules onto the separation material and the process of eluting the target molecules from the separation material include, for example, a method of loading a solution containing the target molecules onto the separation material, passing a basic eluent with a predetermined salt concentration through the separation material, and changing the salt concentration from a low salt concentration to a high salt concentration, thereby separating the target molecules from impurities.

[0144] Examples of basic eluents include solutions containing at least one compound selected from the group consisting of inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and ammonium water, and organic bases such as morpholine, 4-methylmorpholine, piperazine, pyridine, ethylenediamine, diethylenetriamine, ethanolamine, diethanolamine, triethanolamine, trimethylamine, triethylamine, and tris(hydroxyethyl)aminomethane. In addition, inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, and citric acid may be added to adjust the pH of the eluent.

[0145] The pH of the basic eluent is preferably 8 or more and 14 or less. A pH of 8 or more improves the separation ability between the target molecule and the impurities. A pH of 14 or less improves the durability of the separation material.

[0146] Examples of salts to be added to the basic eluent include sodium chloride, potassium chloride, potassium bromide, sodium bromide, sodium iodide, sodium sulfate, and sodium perchlorate.

[0147] 3.4. Chromatography Column Yet another embodiment of the third invention relates to a chromatography column. The chromatography column of the third invention is not particularly limited as long as it contains the separation material of the third invention as a packing material and has at least one container. That is, the chromatography column of the third invention can adopt any known form without particular limitation, except that it uses the separation material of the third invention as a packing material.

[0148] 4. Experimental Results The present invention will be described in more detail below using examples, but the present invention is not limited to the following examples as long as it does not deviate from the gist of the invention. In the following description, "parts" and "%" mean "parts by mass" and "% by mass", respectively, unless otherwise specified.

[0149] 4.1. Regarding the First Invention [Evaluation and Evaluation Method] <Measurement of Exchange Capacity> A slurry aqueous solution containing a separation material was placed in a 10 mL measuring cylinder, and the separation material was allowed to settle while tapping, and 5.0 mL of separation material was measured out. This separation material was filtered, placed in a column, and regenerated by passing 100 mL of 2 M sodium hydroxide aqueous solution through it. It was then thoroughly washed with water, and it was confirmed that the pH of the effluent water was neutral. The resulting separation material was filtered and mixed with 60 mL of 5% sodium chloride aqueous solution. The liberated acid was titrated, and the titration value was divided by the volume of the separation material to obtain the exchange capacity per mL of separation material.

[0150] <Separation of S-modified nucleic acid containing impurities> (1) Equipment and reagents used LC system: Shimadzu CBM-20A Buffer A: 10 mM aqueous sodium hydroxide solution Buffer B: 2 M NaCl + 10 mM aqueous sodium hydroxide solution Column: diameter 4.6 mm, length 50 mm Sample: 21-mer S-modified nucleic acid (containing 26% 20-mer S-modified nucleic acid as an impurity)

[0151] (2) Measurement method A column packed with the separation material was connected to an LC system and equilibrated with Buffer A. Next, Buffer B was increased to 25% by volume and equilibrated. A 21-mer S-modified nucleic acid solution containing 26% 20-mer S-modified nucleic acid as an impurity was applied to the column to a concentration of 10 mg / mL-R. After application, Buffer B was increased from 25% by volume to 100% by volume, and the adsorbed 21-mer S-modified nucleic acid was eluted. Aliquots were collected at 0.4 mL intervals, and the recovery rate of 21-mer S-modified nucleic acid at a purity of 95% was calculated. The flow rate was 1.0 mL / min in all cases.

[0152] <Method for calculating the recovery rate of sulfur-modified nucleic acids containing impurities> (1) Equipment and reagents used LC system: Shimadzu CBM-20A Buffer A: 10 mM aqueous sodium hydroxide solution Buffer B: 1 M NaClO ４ + 10 mM sodium hydroxide aqueous solution Column: CQA35S (Mitsubishi Chemical Corporation)

[0153] (2) Analysis of S-modified nucleic acid solution The column was connected to an LC system and equilibrated with buffer A. Next, buffer B was increased to 38% by volume and equilibrated. 10 μL of the collected 21-mer S-modified nucleic acid solution was applied, and analysis was performed at a flow rate of 0.8 mL / min with buffer B increased to 58% by volume. This was repeated for each sample collected.

[0154] (3) Method for calculating the recovery rate of 21-mer S-modified nucleic acid The analysis in (2) gave a peak of 21-mer S-modified nucleic acid and a peak of impurities. The area value of the absorbance of each peak at 295 nm was plotted against the fractionation time to draw a chromatogram. The sum of the area values ​​of 21-mer S-modified nucleic acid in a certain range of fractionated samples was calculated as A Ｓ The sum of the area values ​​of the 21-mer sulfur-modified nucleic acid and impurities in all the fractionated samples is A ｔｏｔａｌ The purity P was calculated from the formula (I) as follows. Next, the sum of the area values ​​of the 21-mer S-modified nucleic acid at a purity of 95% was calculated as A ９５ , the sum of the area values ​​of the 21-mer S-modified nucleic acid is A Ｓ２１ The recovery rate R of the 21-mer sulfur-modified nucleic acid at a purity of 95% was calculated from formula (II) as follows: P = A Ｓ / A ｔｏｔａｌ × 100 Formula (I) R = A９５ / A Ｓ２１ ×100 Formula (II)

[0155] <Calculation of the Resolution of 19- and 20-mer Oligonucleic Acids> (1) Equipment and Reagents Used LC System: Shimadzu CBM-20A Buffer A: 10 mM aqueous sodium hydroxide solution Buffer B: 2 M NaCl + 10 mM aqueous sodium hydroxide solution Column: diameter 4.6 mm, length 50 mm Sample: mixture of 19-mer oligonucleic acid and 20-mer oligonucleic acid (50:50 wt%) 19-mer oligonucleic acid: oligonucleic acid having a sequence represented by the following formula (SEQ ID NO: 1)

[0156]

[0157] 20-mer oligonucleic acid: oligonucleic acid having a sequence represented by the following formula (SEQ ID NO: 2)

[0158]

[0159] The formulas of the above 19-mer oligonucleic acid and 20-mer oligonucleic acid are both shown in two columns for convenience, but the bonds between riboses, including the portions shown by dotted lines, are also normal phosphodiester bonds.

[0160] (2) Measurement method A column packed with the separation material was connected to an LC system and equilibrated with Buffer A. Next, Buffer B was increased to 15% by volume and equilibrated. A 19-mer and 20-mer oligonucleic acid mixture was applied at 1.0 mg / mL-R. After application, Buffer B was increased from 15% by volume to 50% by volume, and the 19-mer and 20-mer oligonucleic acid mixture was eluted.

[0161] (3) Calculation method of resolution (Rs) The peaks of the 19-mer oligonucleic acid and the 20-mer oligonucleic acid were obtained by the analysis in (2). The time when the peak of the 19-mer oligonucleic acid reached its peak top was defined as t １ (min), the half-width of the peak is W ０．５ｈ１ t ２ (min), the half-width of the peak is W ０．５ｈ２ Rs was calculated using the following formula: Rs = 1.18 × (t ２ -t １ ) / (W０．５ｈ１ +W ０．５ｈ２ )

[0162] [Example A1] A smeared ... ２ 100 parts of a (meth)acrylic polymer carrier having a pH of 1.0 / g, a pore volume of 0.90-1.20 mL / g, and a pore diameter of 500-1250 Å were mixed with 883 parts of water, 184 parts of N-hydroxyethylacrylamide (Tokyo Chemical Industry Co., Ltd.), 62 parts of N-[3-(dimethylamino)propyl]acrylamide (Tokyo Chemical Industry Co., Ltd.), 83 parts of 47% sulfuric acid (Fujifilm Wako Pure Chemical Industries, Ltd.), 1 part of α-thioglycerol (Tokyo Chemical Industry Co., Ltd.), and 2 parts of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.), and dissolved. This reaction solution was reacted at 50°C, and the particles were filtered and washed after the reaction. 600 parts of glycidyl trimethylammonium chloride, 260 parts of water, and 260 parts of 2M sodium hydroxide were added to this carrier and reacted at 30°C. After the reaction, the particles were filtered and washed. Here, the molar ratio of N-[3-(dimethylamino)propyl]acrylamide to N-hydroxyethylacrylamide was 20:80. The structural unit derived from N-[3-(dimethylamino)propyl]acrylamide becomes a structural unit represented by formula (A1) upon reaction with glycidyltrimethylammonium chloride. The structural unit derived from N-hydroxyethylacrylamide becomes a structural unit represented by formula (A2) upon reaction with glycidyltrimethylammonium chloride. The separation performance of the separation material of Example A1 was evaluated. The recovery rate of 21-mer sulfur-modified nucleic acid at a purity of 95% was 92.0%. The results are shown in Table 1.

[0163] [Examples A2 to A7] Separation materials were obtained in the same manner as in Example A1, except that the amounts of N-hydroxyethylacrylamide, N-[3-(dimethylamino)propyl]acrylamide, and 47% sulfuric acid were changed as shown in Table 2, and the separation performance was evaluated. The results are shown in Table 1.

[0164] [Comparative Example A1] A smeared ... ２ 100 parts of a (meth)acrylic polymer carrier having a density of 0.90-1.20 mL / g, a pore volume of 0.90-1.20 mL / g, and a pore diameter of 500-1250 Å were mixed with 883 parts of water, 69 parts of N-hydroxyethylacrylamide (Tokyo Chemical Industry Co., Ltd.), 386 parts of a 75% aqueous solution of (3-acrylamidopropyl)trimethylammonium chloride (Tokyo Chemical Industry Co., Ltd.), 1 part of α-thioglycerol (Tokyo Chemical Industry Co., Ltd.), and 2 parts of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.) and dissolved. This reaction solution was reacted at 50°C, and the particles were filtered and washed after the reaction. Here, the ratio (molar ratio) of (3-acrylamidopropyl)trimethylammonium chloride to N-hydroxyethylacrylamide was 70:30. The separation performance of the separation material of Comparative Example A1 was evaluated in the same manner as in Example A1. The results are shown in Table 1.

[0165]

[0166]

[0167] As shown in Table 1, Comparative Example A1 is inferior to Examples A1 to A7 in terms of exchange capacity, recovery rate, and separation rate Rs. Furthermore, Examples A1 to A4 are significantly superior in recovery rate, and Examples A3 to A6 are superior in separation rate Rs.

[0168] 4.2. Regarding the Second Invention [Evaluation and Evaluation Method] <Measurement of Exchange Capacity> A slurry aqueous solution containing a separation material was placed in a 10 mL measuring cylinder, and the separation material was allowed to settle while tapping, and 5.0 mL of separation material was measured out. This separation material was filtered, placed in a column, and regenerated by passing 100 mL of 2 M sodium hydroxide aqueous solution through it. It was then thoroughly washed with water, and it was confirmed that the pH of the effluent water was neutral. The obtained separation material was filtered, mixed with 60 mL of 5% sodium chloride aqueous solution, and the liberated acid was titrated. The titration value was divided by the volume of the separation material to obtain the exchange capacity per mL of separation material.

[0169] <Calculation of the resolution of 20-mer and 21-mer S-modified nucleic acid> (1) Equipment and reagents used LC system: Shimadzu CBM-20A Buffer A: 10 mM aqueous sodium hydroxide solution Buffer B: 2 M NaCl + 10 mM aqueous sodium hydroxide solution Column: diameter 4.6 mm, length 50 mm Sample: 20-mer S-modified nucleic acid and 21-mer S-modified nucleic acid

[0170] (2) Measurement method A column packed with the separation material was connected to an LC system and equilibrated with Buffer A. Next, Buffer B was increased to 25% by volume and equilibrated. 20-mer S-modified nucleic acid was applied at 0.24 mg / mL-R. After application, Buffer B was increased from 25% by volume to 100% by volume, and the 20-mer S-modified nucleic acid was eluted. The same procedure was performed for 21-mer S-modified nucleic acid.

[0171] (3) Method for calculating resolution (Rs) The analysis in (2) gave peaks for the 20-mer S-modified nucleic acid and the 21-mer S-modified nucleic acid. The time at which the peak of the 20-mer S-modified nucleic acid reached its peak top was determined as t １ (min), the half-width of the peak is W ０．５ｈ１ The time when the peak of the 21-mer S-modified nucleic acid reaches its peak is t ２ (min), the half-width of the peak is W ０．５ｈ２ Rs was calculated using the following formula: Rs = 1.18 × (t ２ -t １ ) / (W ０．５ｈ１ +W ０．５ｈ２ )

[0172] <Calculation of the resolution of 19- and 20-mer oligonucleic acids> (1) Equipment and reagents used LC system: Shimadzu CBM-20A Buffer A: 10 mM aqueous sodium hydroxide solution Buffer B: 2 M NaCl + 10 mM aqueous sodium hydroxide solution Column: diameter 4.6 mm, length 50 mm Sample: mixture of 19-mer oligonucleic acid and 20-mer oligonucleic acid (50:50 wt%)

[0173] (2) Measurement method A column packed with the separation material was connected to an LC system and equilibrated with Buffer A. Next, Buffer B was increased to 15% by volume and equilibrated. A 19-mer and 20-mer oligonucleic acid mixture was applied at 1.0 mg / mL-R. After application, Buffer B was increased from 15% by volume to 50% by volume, and the 19-mer and 20-mer oligonucleic acid mixture was eluted.

[0174] (3) Calculation method of resolution (Rs) The peaks of the 19-mer oligonucleic acid and the 20-mer oligonucleic acid were obtained by the analysis in (2). The time when the peak of the 19-mer oligonucleic acid reached its peak top was defined as t １ (min), the half-width of the peak is W ０．５ｈ１ t ２ (min), the half-width of the peak is W ０．５ｈ２ Rs was calculated using the following formula: Rs = 1.18 × (t ２ -t １ ) / (W ０．５ｈ１ +W ０．５ｈ２ )

[0175] [Example B1] A granular ... ２100 parts of a (meth)acrylic polymer carrier having a pH of 1.0 / g, a pore volume of 0.90-1.20 mL / g, and a pore diameter of 500-1250 Å were mixed with 883 parts of water, 46 parts of N-hydroxyethylacrylamide (Tokyo Chemical Industry Co., Ltd.), 250 parts of N-[3-(dimethylamino)propyl]acrylamide (Tokyo Chemical Industry Co., Ltd.), 334 parts of 47% sulfuric acid (Fujifilm Wako Pure Chemical Industries, Ltd.), 1 part of α-thioglycerol (Tokyo Chemical Industry Co., Ltd.), and 2 parts of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.), and dissolved. This reaction solution was reacted at 50°C, and the particles were filtered and washed after the reaction. 600 parts of glycidyl trimethylammonium chloride, 260 parts of water, and 260 parts of 2M sodium hydroxide were added to this carrier and reacted at 30°C. After the reaction, the particles were filtered and washed. Here, the ratio (molar ratio) of N-hydroxyethylacrylamide to N-[3-(dimethylamino)propyl]acrylamide was 20:80. The structural unit derived from N-hydroxyethylacrylamide becomes a structural unit represented by formula (B1) upon reaction with glycidyltrimethylammonium chloride. The structural unit derived from N-[3-(dimethylamino)propyl]acrylamide becomes a structural unit represented by formula (B2) upon reaction with glycidyltrimethylammonium chloride. The separation performance of the separation material of Example B1 was evaluated.

[0176] [Examples B2 to B7] Separation materials were obtained in the same manner as in Example B1, except that the amounts of N-hydroxyethylacrylamide, N-[3-(dimethylamino)propyl]acrylamide, and 47% sulfuric acid were changed as shown in Table 4, and the separation performance was evaluated. The results are shown in Table 3.

[0177] [Comparative Example B1] A smear consisting of 70 parts of glycidyl methacrylate and 30 parts of ethylene glycol dimethacrylate, with an average particle size of 30 μm and a specific surface area of ​​30 to 56 m ２100 parts of a (meth)acrylic polymer carrier having a pore volume of 0.90-1.20 mL / g, a pore diameter of 500-1250 Å, 883 parts of water, 69 parts of N-hydroxyethylacrylamide (Tokyo Chemical Industry Co., Ltd.), 386 parts of a 75% aqueous solution of (3-acrylamidopropyl)trimethylammonium chloride (Tokyo Chemical Industry Co., Ltd.), 1 part of α-thioglycerol (Tokyo Chemical Industry Co., Ltd.), and 2 parts of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed and dissolved. This reaction solution was reacted at 50°C, and the particles were filtered and washed after the reaction. Here, the ratio (molar ratio) of (3-acrylamidopropyl)trimethylammonium chloride to N-hydroxyethylacrylamide was 70:30. The separation performance of the separation material of Comparative Example B1 was evaluated in the same manner as in Example B1. The results are shown in Table 3.

[0178]

[0179]

[0180] As shown in Table 3, Comparative Example B1 is inferior to Examples B1 to B7 in terms of exchange capacity, recovery rate, and separation rate Rs. Furthermore, Examples B3 to B7 are significantly superior in recovery rate, and Examples B1 to B4 are superior in separation rate Rs.

[0181] 4.3. Regarding the third invention [Evaluation and evaluation method] <Number average molecular weight of copolymer> (1) Equipment and reagents used Polyethylene oxide: GL Science LC system: Shimadzu CBM-20A Mobile phase: 0.1 M sodium nitrate aqueous solution Column: TSKgel G5000PW ＸＬ -CP

[0182] (2) Method for measuring number average molecular weight: A column was connected to the system and equilibrated with the mobile phase. After filtering the polymerization reaction solution of the example to remove particles, the reaction solution was diluted 10 times to prepare a sample. This sample was applied to the column and subjected to gel filtration chromatography. The number average molecular weight was then calculated using a calibration curve prepared from polyethylene oxides with different molecular weights.

[0183] <Measurement of exchange capacity> The aqueous slurry containing the separation material was placed in a 10 mL measuring cylinder, the separation material was allowed to settle while tapping, and 5.0 mL of separation material was measured out. This separation material was filtered, placed in a column, and regenerated by passing 100 mL of 2 M sodium hydroxide aqueous solution through it. It was then thoroughly washed with water, and it was confirmed that the pH of the effluent water was neutral. The obtained separation material was filtered, mixed with 60 mL of 5% sodium chloride aqueous solution, and the liberated acid was titrated. The titration value was divided by the volume of the separation material to obtain the exchange capacity per mL of separation material.

[0184] <Separation of S-modified nucleic acid containing impurities> (1) Equipment and reagents used LC system: Shimadzu CBM-20A Buffer A: 10 mM aqueous sodium hydroxide solution Buffer B: 2 M NaCl + 10 mM aqueous sodium hydroxide solution Column: diameter 4.6 mm, length 50 mm Sample: 21-mer S-modified nucleic acid (containing 26% 20-mer S-modified nucleic acid as an impurity)

[0185] (2) Measurement method A column packed with the separation material was connected to an LC system and equilibrated with Buffer A. Next, Buffer B was increased to 25% by volume and equilibrated. A 21-mer S-modified nucleic acid solution containing 26% 20-mer S-modified nucleic acid as an impurity was applied to the column to a concentration of 10 mg / mL-R. After application, Buffer B was increased from 25% by volume to 100% by volume, and the adsorbed 21-mer S-modified nucleic acid was eluted. Aliquots were collected at 0.4 mL intervals, and the recovery rate of 21-mer S-modified nucleic acid at a purity of 95% was calculated. The flow rate was 1.0 mL / min in all cases.

[0186] <Calculation method for oligonucleotide recovery rate> (1) Equipment and reagents used LC system: Shimadzu CBM-20A Buffer A: 10 mM sodium hydroxide aqueous solution Buffer B: 1 M NaClO ４ + 10 mM sodium hydroxide aqueous solution Column: CQA35S (Mitsubishi Chemical Corporation)

[0187] (2) Analysis of Oligonucleotide Solution The column was connected to an LC system and equilibrated with Buffer A. Next, Buffer B was increased to 38% by volume and equilibrated. 10 μL of the collected 21-mer S-modified nucleic acid solution was applied, and Buffer B was increased to 58% by volume, and analysis was performed at a flow rate of 0.8 mL / min. This was repeated for each sample collected.

[0188] (3) Method for calculating the recovery rate of 21-mer S-modified nucleic acid The analysis in (2) gave a peak of 21-mer S-modified nucleic acid and a peak of impurities. The area value of the absorbance of each peak at 295 nm was plotted against the fractionation time to draw a chromatogram. The sum of the area values ​​of 21-mer S-modified nucleic acid in a certain range of fractionated samples was calculated as A Ｓ The sum of the area values ​​of the 21-mer sulfur-modified nucleic acid and impurities in all the fractionated samples is A ｔｏｔａｌ The purity P was calculated from the formula (I) as follows. Next, the sum of the area values ​​of the 21-mer S-modified nucleic acid at a purity of 95% was calculated as A ９５ , the sum of the area values ​​of the 21-mer S-modified nucleic acid is A Ｓ２１ The recovery rate R of the 21-mer sulfur-modified nucleic acid at a purity of 95% was calculated from formula (II) as follows: P = A Ｓ / A ｔｏｔａｌ × 100 Formula (I) R = A ９５ / A Ｓ２１ ×100 Formula (II)

[0189] <Calculation of the resolution of 19- and 20-mer oligonucleic acids> (1) Equipment and reagents used LC system: Shimadzu CBM-20A Buffer A: 10 mM aqueous sodium hydroxide solution Buffer B: 2 M NaCl + 10 mM aqueous sodium hydroxide solution Column: diameter 4.6 mm, length 50 mm Sample: mixture of 19-mer oligonucleic acid and 20-mer oligonucleic acid (50:50 wt%)

[0190] (2) Measurement method A column packed with the separation material was connected to an LC system and equilibrated with Buffer A. Next, Buffer B was increased to 15% by volume and equilibrated. A 19-mer and 20-mer oligonucleic acid mixture was applied at 1.0 mg / mL-R. After application, Buffer B was increased from 15% by volume to 50% by volume, and the 19-mer and 20-mer oligonucleic acid mixture was eluted.

[0191] (3) Calculation method of resolution (Rs) The peaks of the 19-mer oligonucleic acid and the 20-mer oligonucleic acid were obtained by the analysis in (2). The time when the peak of the 19-mer oligonucleic acid reached its peak top was defined as t １ (min), the half-width of the peak is W ０．５ｈ１ t ２ (min), the half-width of the peak is W ０．５ｈ２ Rs was calculated using the following formula: Rs = 1.18 × (t ２ -t １ ) / (W ０．５ｈ１ +W ０．５ｈ２ )

[0192] [Example C1] A cellulose ester containing 70 parts of glycidyl methacrylate and 30 parts of ethylene glycol dimethacrylate, having an average particle size of 30 μm and a specific surface area of ​​30 to 56 m ２ 100 parts of a (meth)acrylic polymer carrier having a pH of 1.0 / g, a pore volume of 0.90 to 1.20 mL / g, and a pore diameter of 500 to 1250 Å were mixed with 883 parts of water, 312 parts of N-[3-(dimethylamino)propyl]acrylamide (Tokyo Chemical Industry Co., Ltd.), 417 parts of 47% sulfuric acid (Fujifilm Wako Pure Chemical Industries, Ltd.), 4 parts of α-thioglycerol (Tokyo Chemical Industry Co., Ltd.), and 2 parts of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.), and dissolved. This reaction solution was polymerized at 50°C to obtain a polymerization reaction solution. The particles were then filtered, the polymerization reaction solution was recovered, and the particles were washed. 600 parts of glycidyl trimethylammonium chloride, 260 parts of water, and 260 parts of 2M sodium hydroxide were added to this carrier and reacted at 30°C. After the reaction, the particles were filtered and washed. The structural unit derived from N-[3-(dimethylamino)propyl]acrylamide represented by formula (C1) reacts with glycidyltrimethylammonium chloride to form a structural unit represented by formula (C2). The separation performance of the separation material of Example C1 was evaluated. The recovery rate of 21-mer sulfur-modified nucleic acid at a purity of 95% was 93.0%. The results are shown in Table 5.

[0193] Examples C2 to C7 Separation materials were obtained in the same manner as in Example C1, except that the amounts of N-[3-(dimethylamino)propyl]acrylamide, N-hydroxyethylacrylamide, 47% sulfuric acid, and α-thioglycerol were changed as shown in Table 6, and their separation performance was evaluated. The structural unit derived from N-hydroxyethylacrylamide represented by formula (C3) becomes a structural unit represented by formula (C4) by reaction with glycidyltrimethylammonium chloride. The results are shown in Table 5.

[0194] [Comparative Example C1] A smear consisting of 70 parts of glycidyl methacrylate and 30 parts of ethylene glycol dimethacrylate, with an average particle size of 30 μm and a specific surface area of ​​30 to 56 m ２ 100 parts of a (meth)acrylic polymer carrier having a pore volume of 0.90 to 1.20 mL / g, a pore diameter of 500 to 1250 Å, 883 parts of water, 551 parts of a 75% aqueous solution of (3-acrylamidopropyl)trimethylammonium chloride (manufactured by Tokyo Chemical Industry Co., Ltd.), 1 part of α-thioglycerol (manufactured by Tokyo Chemical Industry Co., Ltd.), and 2 parts of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed and dissolved. This reaction solution was polymerized at 50°C to obtain a polymerization reaction solution. The particles were then filtered and washed. The separation performance of the separation material of Comparative Example C1 was evaluated in the same manner as in Example C1. The results are shown in Table 5.

[0195]

[0196]

[0197] As shown in Table 5, Comparative Example C1 is inferior to Examples C1 to C7 in both recovery rate and resolution Rs. Furthermore, Examples C1, C2, and C5 to C7 are significantly superior in recovery rate, and Examples C1 to C3 and C7 are superior in resolution Rs.

[0198] The separation material of the present invention and the separation material produced by the production method of the present invention exhibit separation and adsorption performance with high selectivity, particularly for oligonucleic acids, and are of great practical value in the fields of medicine and diagnosis.

Claims

1. A separation material comprising an anion exchange group bound to a carrier, the anion exchange group comprising a structural unit represented by formula (A1). (In formula (A1), n1 is 1 or more and 10 or less. R ａ０ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ａ１ represents O or NH. ａ２ and R ａ３ R each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ａ４ R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ａ５ , R ａ６ and R ａ７ each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

2. The separation material according to claim 1, wherein the anion exchange group further contains a structural unit represented by formula (A2). (In formula (A2), m1 is 1 or more and 10 or less. R ａ８ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ａ９ represents O or NH. ａ１０ R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ａ１１ , R ａ１２ and R ａ１３ each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

3. The separation material according to claim 1 or 2, wherein the structural unit represented by the above formula (A1) is contained in an amount of 10 mol % to 100 mol % based on the total structural units contained in the anion exchange group.

4. The separation material according to claim 3, wherein the structural unit represented by the above formula (A1) is contained in an amount of 20 mol % to 50 mol % based on the total structural units contained in the anion exchange group.

5. The separation material according to claim 3, wherein the structural unit represented by the above formula (A1) is contained in an amount of 40 mol % or more and 100 mol % or less based on the total structural units contained in the anion exchange group.

6. The separation material according to claim 1 or 2, which has an exchange capacity of 0.08 mmol / mL-R or more and 0.20 mmol / mL-R or less.

7. A method for separating a target molecule, comprising the following steps (1-a) and (1-b): (1-a): contacting a solution containing the target molecule with the separation material according to claim 1 or 2 to adsorb the target molecule to the separation material; and (1-b): eluting the target molecule from the separation material treated in step (1-a).

8. The method for isolating a target molecule according to claim 7, wherein the target molecule is either a biopolymer, a partial structure of a biopolymer, or a chemically modified product of a biopolymer.

9. The method for isolating a target molecule according to claim 8, wherein the target molecule is any one of an oligonucleic acid, a partial structure of an oligonucleic acid, and a chemically modified oligonucleic acid.

10. A chromatography column comprising the separation material according to claim 1 or 2 as a packing material and having at least one container.

11. A separation material comprising an anion exchange group bound to a carrier, the anion exchange group comprising a structural unit represented by formula (B1). (In formula (B1), n2 is 1 or more and 10 or less. R ｂ０ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｂ１ represents O or NH. ｂ２ R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ｂ３ , R ｂ４ and R ｂ５ each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

12. The separation material according to claim 11, wherein the carrier comprises spherical particles.

13. In the formula (B1), R ｂ１ The separation material according to claim 11, wherein is NH.

14. The separation material according to claim 11, wherein in formula (B1), n2 is 2 or more and 3 or less.

15. The separation material according to claim 11, wherein the anion exchange group further contains a structural unit represented by formula (B2). (In formula (B2), m2 is 1 or more and 10 or less. R ｂ６ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｂ７ represents O or NH. ｂ８ and R ｂ９ R each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｂ１０ R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ｂ１１ , R ｂ１２ and R ｂ１３ each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

16. The separation material according to claim 11 or 15, wherein the structural unit represented by the above formula (B1) is contained in an amount of 10 mol % to 100 mol % relative to the total structural units contained in the anion exchange group.

17. The separation material according to claim 16, wherein the structural unit represented by the above formula (B1) is contained in an amount of 60 mol % to 100 mol % relative to the total structural units contained in the anion exchange group.

18. The separation material according to claim 16, wherein the structural unit represented by the above formula (B1) is contained in an amount of 20 mol % to 60 mol % relative to the total structural units contained in the anion exchange group.

19. The separation material according to claim 11 or 15, having an exchange capacity of 0.08 mmol / mL-R or more and 0.20 mmol / mL-R or less.

20. A method for separating a target molecule, comprising the following steps (2-a) and (2-b): (2-a): contacting a solution containing the target molecule with the separation material according to claim 11 or 15 to adsorb the target molecule to the separation material; and (2-b): eluting the target molecule from the separation material treated in step (2-a).

21. The method for isolating a target molecule according to claim 20, wherein the target molecule is either a biopolymer, a partial structure of a biopolymer, or a chemically modified product of a biopolymer.

22. The method for isolating a target molecule according to claim 21, wherein the target molecule is any one of an oligonucleic acid, a partial structure of an oligonucleic acid, and a chemically modified oligonucleic acid.

23. A chromatography column comprising the separation material according to claim 11 or 15 as a packing material and having at least one container.

24. A method for producing a separation material in which anion exchange groups are bound to a carrier, comprising obtaining a polymer containing a structural unit represented by formula (C1) as the anion exchange group and having a number average molecular weight of 100,000 or less, and then performing a post-reaction to obtain a polymer containing a structural unit represented by formula (C2). (In formula (C1), n3 is 1 or more and 10 or less. R ｃ０ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｃ１ represents O or NH. ｃ２ and R ｃ３ each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. (In formula (C2), n3 is 1 or more and 10 or less. R ｃ０ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｃ１ represents O or NH. ｃ２ and R ｃ３ R each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｃ４ R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ｃ５ , R ｃ６ and R ｃ７ each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

25. A method for producing a separation material according to claim 24, wherein a polymer further containing a structural unit represented by formula (C3) as the anion exchange group and having a number average molecular weight of 100,000 or less is obtained, and then a polymer containing structural units represented by formula (C2) and formula (C4) is obtained through a post-reaction. (In formula (C3), m3 is 1 or more and 10 or less. R ｃ８ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｃ９ represents O or NH.) (In formula (C4), m3 is 1 or more and 10 or less. R ｃ８ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. ｃ９ represents O or NH. ｃ１０ R is a linear or branched alkylene group having from 1 to 6 carbon atoms, which may have one or more substituents selected from a hydroxy group, an alkoxy group, an amino group, a carboxy group, a sulfo group, and a halogen. ｃ１１ , R ｃ１２ and R ｃ１３ each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

26. A method for producing a separation material according to claim 24 or 25, wherein the structural unit represented by the above formula (C2) is blended in an amount of 10 mol % or more and 100 mol % or less relative to the total structural units contained in the anion exchange group.

27. A separation material manufactured by the method for manufacturing a separation material according to claim 24 or 25.

28. The separation material according to claim 27, having an exchange capacity of 0.08 mmol / mL-R or more and 0.20 mmol / mL-R or less.

29. A mixture of the separation material according to claim 27 and a polymer containing a structural unit represented by formula (C2) and having a number average molecular weight of 200,000 or less.

30. A method for separating a target molecule, comprising the following steps (3-a) and (3-b): (3-a): contacting a solution containing the target molecule with the separation material according to claim 27 or the mixture according to claim 29, thereby adsorbing the target molecule onto the separation material; and (3-b): eluting the target molecule from the separation material treated in step (3-a).

31. The method for isolating a target molecule according to claim 30, wherein the target molecule is either a biopolymer, a substructure of a biopolymer, or a chemically modified biopolymer.

32. The method for isolating a target molecule according to claim 31, wherein the biopolymer is any one of an oligonucleic acid, a partial structure of an oligonucleic acid, and a chemically modified oligonucleic acid.

33. A chromatography column comprising the separation material of claim 27 or the mixture of claim 29 as a packing material and having at least one container.

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