JP7315561B2 - chromatography carrier - Google Patents

Description

The present invention relates to chromatography carriers. More specifically, it relates to a chromatography carrier, a method for producing the carrier and intermediates for producing the carrier, a chromatography column, and a method for purifying a target substance.

In the field of biopharmaceuticals typified by antibody drugs and the like, techniques for expressing target substances such as proteins have made remarkable progress, and along with this, there is a demand for improved productivity in purification processes such as chromatography. In particular, when an antibody is used as a therapeutic or diagnostic agent, it is necessary to separate the antibody from cell by-products, culture fluid components, and the like with high purity.

Conventionally, as a method for highly purifying a target protein represented by an antibody, it was common to remove contaminants by affinity purification and then ion-exchange purification to further increase the purity. However, in ion-exchange purification, there was a problem that it was difficult to separate non-target proteins whose isoelectric points (pI) were close to the target protein from the target protein.
Therefore, in recent years, instead of ion exchange purification, mixed-mode purification using hydrophobic chromatography (HIC) and cation exchange chromatography has been performed for the purpose of separating both non-target proteins having an isoelectric point (pI) close to the target protein and non-target proteins having hydrophilicity and hydrophobicity close to the target protein from the target protein. Supports with mixed-mode ligands containing anionic functional groups and hydrophobic groups are used for this mixed-mode purification. As such mixed-mode carriers, for example, carriers having 2-(benzoylamino)-4-mercaptobutyric acid residues (Patent Documents 1 and 2), L-tryptophan residues (Patent Document 3) and 4-aminohippuric acid residues (Patent Document 4) as mixed-mode ligands have been proposed.

Japanese Patent Publication No. 2007-525501 Japanese Patent Publication No. 2008-505851 JP 2010-210497 A Japanese Patent Publication No. 2015-501310

However, in recent years when there is a demand for further purification of target substances such as target proteins, it is required to further improve the performance of separating contaminants such as non-target proteins from the target substances. In addition, the carriers described in Patent Documents 1 to 4 have room for improvement in terms of their dynamic binding capacities for target substances as well.
The problem to be solved by the present invention is to provide a chromatography carrier that has a large dynamic binding capacity for a target substance and excellent performance in separating the target substance and contaminants.

The problems of the present invention have been solved by the following means <1> to <15>.
<1> A chromatography carrier (hereinafter also referred to as "the chromatography carrier of the present invention") comprising a support and a ligand represented by the following formula (1) (hereinafter also referred to as "ligand (1)").

[In formula (1),
A represents an anionic functional group,
Q represents a divalent aromatic hydrocarbon group,
R ¹ represents a divalent organic group,
R ² represents a hydrogen atom or a hydrocarbon group,
X represents a thio group, a sulfinyl group or a sulfonyl group,
* indicates a bond. ]

<2> A is -C (= O) OH, -C (= O) O^-M.⁺, -C(=O)O^-, -S (=O)₂OH, -S (=O)₂O.^-M.⁺, -S (=O)₂O.^-, -P (=O) (OH)₂, -P (=O) (O^-M.⁺)₂, -P (=O) (O^-)₂, -P(=O)(OH)(O^-M.⁺), -P (= O) (OH) (O^-), -OP (= O) (OH)₂, -OP (=O) (O^-M.⁺)₂, -OP (=O) (O^-)₂, -OP (=O) (OH) (O^-M.⁺), or -OP (= O) (OH) (O^-) (above M⁺each represents a counter ion), the carrier according to <1>.

<3> The carrier according to <1> or <2>, wherein Q is a divalent group represented by the following formula (2) or (3).

[In formula (2),
R ³ represents a halogen atom, a hydroxy group, a carboxamide group or an organic group;
n represents an integer of 0 to 4,
* indicates a bond. ]

[In formula (3),
R ⁴ represents a halogen atom, a hydroxy group, a carboxamide group or an organic group;
m represents an integer from 0 to 6,
* indicates a bond. ]

<4> The carrier according to any one of <1> to <3>, wherein R ¹ is a divalent hydrocarbon group having 1 to 20 carbon atoms.

<5> The carrier according to any one of <1> to <4>, wherein X is a thio group.

<6> The carrier according to any one of <1> to <5>, wherein the ligand is represented by the following formula (α).

[In formula (α),
R ¹ represents an alkanediyl group having 1 to 10 carbon atoms,
* indicates a bond. ]

<7> The carrier according to any one of <1> to <6>, wherein the support is a synthetic polymer support.

<8> The carrier according to any one of <1> to <7>, wherein the ligand is bound to the support.

<9> The carrier according to any one of <1> to <8>, which is a protein purification chromatography carrier.

<10> The carrier according to any one of <1> to <8>, which is an antibody purification chromatography carrier.

<11> The carrier according to any one of <1> to <10>, which is a mixed mode chromatography carrier for hydrophobic chromatography and cation exchange chromatography.

<12> A method for producing the carrier according to any one of <1> to <11>, comprising a step of contacting a support having a functional group capable of reacting with a sulfanyl group in its molecule with a compound represented by the following formula (β) (hereinafter also referred to as “the production method of the present invention”).

[In formula (β),
A represents an anionic functional group,
Q represents a divalent aromatic hydrocarbon group,
R ¹ represents a divalent organic group,
^R2 represents a hydrogen atom or a hydrocarbon group. ]

<13> A chromatography column (hereinafter also referred to as "the chromatography column of the present invention"), in which the carrier according to any one of <1> to <11> is packed in a column vessel.

<14> A method for purifying a target substance using the carrier according to any one of <1> to <11> (hereinafter also referred to as “the method for purifying a target substance of the present invention”).

<15> A compound represented by the following formula (β) (hereinafter also referred to as “manufacturing intermediate of the present invention” or “compound (β)”).

[In formula (β),
A represents an anionic functional group,
Q represents a divalent aromatic hydrocarbon group,
R ¹ represents a divalent organic group,
^R2 represents a hydrogen atom or a hydrocarbon group. ]

The chromatographic carrier of the present invention has a large dynamic binding capacity for a target substance and is excellent in performance of separating the target substance and contaminants.
Therefore, according to the chromatography column and the method for purifying a target substance of the present invention, the target substance and contaminants can be easily and efficiently separated.
Moreover, according to the production method of the present invention, a chromatography carrier having a large dynamic binding capacity for a target substance and excellent performance in separating the target substance and contaminants can be produced easily and efficiently.
The production intermediate of the present invention is useful as a production intermediate of the chromatography carrier of the present invention.

<Chromatography carrier>
The chromatography carrier of the present invention is characterized by having a support and a ligand represented by the following formula (1).

[In formula (1),
A represents an anionic functional group,
Q represents a divalent aromatic hydrocarbon group,
R ¹ represents a divalent organic group,
R ² represents a hydrogen atom or a hydrocarbon group,
X represents a thio group (-S-), a sulfinyl group (-S(=O)-) or a sulfonyl group (-S(=O) ₂ -);
* indicates a bond. ]

(ligand)
In the present invention, the term "ligand" refers to a site capable of capturing a target substance represented by a protein, and the capture mechanism is not limited to chemical binding, but may be ion exchange, hydrophobic interaction, or the like. The chromatography carrier of the present invention has a ligand (1) having a specific chemical structure, thereby improving the dynamic binding capacity for the target substance and the ability to separate the target substance and contaminants.

Each symbol in formula (1) will be described below.
In Formula (1), A represents an anionic functional group. In the chromatography carrier of the present invention, the anionic functional group represented by A acts as a cation exchange group.
Specific examples of the anionic functional groups include -C(=O)OH, -C(=O)O ^- M ⁺ , -C(=O)O ^- , -S(=O) ₂ OH, -S(=O) ₂ O ^- M ⁺ , -S(=O) ₂ O ^- , -P(=O)(OH) ₂ , -P(=O)(O ^- M ⁺ ) ₂ , -P(=O)(O ^{- ) 2 , -P(=O)(OH)(O - M + ), -P(=O)(OH)(O - ), -OP(=O)(OH) 2 , -OP(=O)(O - M + ) 2 , -OP(=O)(O -} ₎ ^{2 , -OP (} ₌ ^{O )} _{( OH} )(O ^{- M +} ), -OP(=O)(OH) (O ⁻ ) and the like.
Each M ⁺ above represents a counter ion. Examples of counterions include alkali metal ions such as sodium ion and potassium ion; alkaline earth metal ions such as magnesium ion and calcium ion; ammonium ion; and organic ammonium ion.

Among these anionic functional groups, -C(=O)OH, -C(=O)O^-M.⁺, -C(=O)O^-, -S (=O)₂OH, -S (=O)₂O.^-M.⁺, -S (=O)₂O.^-, -P (=O) (OH)₂, -P (=O) (O^-M.⁺)₂, -P (=O) (O^-)₂, -P(=O)(OH)(O^-M.⁺), -P (= O) (OH) (O^-) is preferred, -C (= O) OH, -C (= O) O^-M.⁺, -C(=O)O^-, -S (=O)₂OH, -S (=O)₂O.^-M.⁺, -S (=O)₂O.^-is more preferred, -C (= O) OH, -C (= O) O^-M.⁺, -C(=O)O^-is particularly preferred.

Q represents a divalent aromatic hydrocarbon group. In the chromatography carrier of the present invention, the divalent aromatic hydrocarbon group represented by Q acts as a hydrophobic group that exhibits hydrophobic interaction.
The number of carbon atoms in the divalent aromatic hydrocarbon group is preferably 6-18, more preferably 6-12. As the divalent aromatic hydrocarbon group, an arylene group is preferable, and specific examples include a phenylene group, a naphthylene group, a phenanthrenylene group, and the like. Among these, a phenylene group and a naphthylene group are preferred, and a phenylene group is particularly preferred, in order to enhance the desired effects of the present invention.
The binding site of the divalent aromatic hydrocarbon group may be on any carbon on the ring.

Moreover, the divalent aromatic hydrocarbon group may be either a divalent aromatic hydrocarbon group having a substituent or an unsubstituted divalent aromatic hydrocarbon group. When the divalent aromatic hydrocarbon group has a substituent, the substitution position and number of substituents are arbitrary, and when it has two or more substituents, the substituents may be the same or different. Examples of substituents include halogen atoms such as a chlorine atom, a bromine atom and a fluorine atom; hydroxy groups; carboxamide groups; and organic groups. The organic group includes a hydrocarbon group and a group having one or more selected from an ether bond, an amide bond and an ester bond between carbon atoms of a hydrocarbon group having 2 or more carbon atoms. When the organic group is a hydrocarbon group, it preferably has 1 to 4 carbon atoms, more preferably 1 or 2 carbon atoms. The number of carbon atoms in the hydrocarbon group in the "group having one or more selected from an ether bond, an amide bond and an ester bond between carbon atoms of a hydrocarbon group having 2 or more carbon atoms" is preferably 2 to 4.
Moreover, as said hydrocarbon group, an alkyl group is preferable. The alkyl group may be linear or branched, and examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl and the like.

As the divalent aromatic hydrocarbon group represented by Q, in order to enhance the desired effect of the present invention, a divalent group represented by the following formula (2) or (3) is preferable, and a divalent group represented by formula (2) is particularly preferable.

[In formula (2),
R ³ represents a halogen atom, a hydroxy group, a carboxamide group or an organic group;
n represents an integer of 0 to 4,
* indicates a bond. ]

[In formula (3),
R ⁴ represents a halogen atom, a hydroxy group, a carboxamide group or an organic group;
m represents an integer from 0 to 6,
* indicates a bond. ]

Halogen atoms represented by R ³ and R ⁴ in formulas (2) and (3) include chlorine, bromine and fluorine atoms. Examples of organic groups represented by R ³ and R ⁴ include hydrocarbon groups and groups having one or more selected from an ether bond, an amide bond and an ester bond between carbon atoms of a hydrocarbon group having 2 or more carbon atoms. When the organic group is a hydrocarbon group, it preferably has 1 to 4 carbon atoms, more preferably 1 or 2 carbon atoms. The number of carbon atoms in the hydrocarbon group in the "group having one or more selected from an ether bond, an amide bond and an ester bond between carbon atoms of a hydrocarbon group having 2 or more carbon atoms" is preferably 2 to 4.
Moreover, as said hydrocarbon group, an alkyl group is preferable. The alkyl group may be linear or branched, and examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl and the like.

In formula (2), n represents an integer of 0 to 4, preferably 0 to 2, particularly preferably 0, in order to enhance the desired effects of the present invention. In formula (3), m represents an integer of 0 to 6, preferably 0 to 2, particularly preferably 0, in order to enhance the desired effects of the present invention.
When n is an integer of 2 to 4, n R ³ may be the same or different. Further, when m is an integer of 2 to 6, m R ⁴ may be the same or different.

R ¹ represents a divalent organic group. The divalent organic group represented by R ¹ includes a divalent hydrocarbon group and a group having one or more selected from an ether bond, an amide bond and an ester bond between the carbon-carbon atoms of a divalent hydrocarbon group having 2 or more carbon atoms, but a divalent hydrocarbon group is preferred in order to enhance the desired effects of the present invention.
When the divalent organic group represented by R ¹ is a divalent hydrocarbon group, its carbon number is preferably 1 to 20, more preferably 1 to 14, still more preferably 1 to 10, still more preferably 1 to 5, particularly preferably 1 or 2, in order to enhance the desired effect of the present invention. On the other hand, when the divalent organic group is "a group having one or more selected from an ether bond, an amide bond and an ester bond between carbon atoms of a divalent hydrocarbon group having 2 or more carbon atoms", the number of carbon atoms in the divalent hydrocarbon group in such a group is preferably 2 to 20, more preferably 2 to 14, still more preferably 2 to 10, still more preferably 2 to 5, particularly preferably 2, in order to enhance the desired effects of the present invention.
The divalent hydrocarbon group represented by R ¹ , the divalent hydrocarbon group in "a group having one or more selected from an ether bond, an amide bond and an ester bond between the carbon-carbon atoms of a divalent hydrocarbon group having 2 or more carbon atoms" may have a substituent. Examples of the substituent include halogen atoms such as a chlorine atom, a bromine atom and a fluorine atom, and a hydroxy group. The substitution position and number of substituents are arbitrary, and when two or more substituents are provided, the substituents may be the same or different.

The divalent hydrocarbon group represented by R ¹ , the divalent hydrocarbon group in "a group having one or more selected from an ether bond, an amide bond and an ester bond between the carbon-carbon atoms of a divalent hydrocarbon group having 2 or more carbon atoms" may be a divalent saturated hydrocarbon group or a divalent unsaturated hydrocarbon group. Examples of the divalent hydrocarbon group include a divalent aliphatic hydrocarbon group, a divalent alicyclic hydrocarbon group, and a divalent aromatic hydrocarbon group. In order to enhance the desired effect of the present invention, a divalent aliphatic hydrocarbon group and a divalent alicyclic hydrocarbon group are preferred. The divalent aliphatic hydrocarbon group may be linear or branched.

When the divalent organic group represented by R ¹ is a divalent aliphatic hydrocarbon group, its carbon number is preferably 1 to 20, more preferably 1 to 14, still more preferably 1 to 10, still more preferably 1 to 5, particularly preferably 1 or 2, in order to enhance the desired effects of the present invention. On the other hand, the divalent organic group is "a group having one or more selected from an ether bond, an amide bond and an ester bond between the carbon atoms of a divalent aliphatic hydrocarbon group having 2 or more carbon atoms".
Examples of the divalent aliphatic hydrocarbon group include an alkanediyl group and an alkenediyl group, and an alkanediyl group is preferred in order to enhance the desired effects of the present invention.
Specific examples of alkanediyl groups include methane-1,1-diyl, ethane-1,1-diyl, ethane-1,2-diyl, propane-1,1-diyl, propane-1,2-diyl, propane-1,3-diyl, propane-2,2-diyl, butane-1,1-diyl, butane-1,2-diyl, butane-1,3-diyl, and tan-1,4-diyl group, pentane-1,4-diyl group, pentane-1,5-diyl group, hexane-1,2-diyl group, hexane-1,5-diyl group, hexane-1,6-diyl group, heptane-1,7-diyl group, octane-1,8-diyl group, nonane-1,9-diyl group, decane-1,2-diyl group, decane-1,10-diyl group and the like. .

The divalent alicyclic hydrocarbon group preferably has 3 to 20 carbon atoms, more preferably 3 to 14 carbon atoms, still more preferably 3 to 10 carbon atoms, and particularly preferably 3 to 8 carbon atoms.
Examples of the divalent alicyclic hydrocarbon group include a cycloalkanediyl group and a divalent bridged cyclic hydrocarbon group such as an adamantylene group, with the cycloalkanediyl group being preferred.
Specific examples of the cycloalkanediyl group include a cyclohexanediyl group, a methylcyclohexanediyl group, a cycloheptanediyl group, and the like.

The number of carbon atoms in the divalent aromatic hydrocarbon group is preferably 6-18, more preferably 6-12. The divalent aromatic hydrocarbon group is preferably an arylene group, and specific examples thereof include a phenylene group and a naphthylene group.
The bonding site of the divalent alicyclic hydrocarbon group and the bonding site of the divalent aromatic hydrocarbon group may be on any carbon on the ring.

Among these divalent hydrocarbon groups, an alkanediyl group and a cycloalkanediyl group are preferred, and an alkanediyl group is particularly preferred, in order to enhance the desired effects of the present invention.

^R2 represents a hydrogen atom or a hydrocarbon group. The hydrocarbon group represented by R ² is a concept encompassing aliphatic hydrocarbon groups, alicyclic hydrocarbon groups and aromatic hydrocarbon groups, and may be saturated hydrocarbon groups or unsaturated hydrocarbon groups. The number of carbon atoms in the hydrocarbon group is preferably 1-4, more preferably 1 or 2. The hydrocarbon group is preferably an aliphatic hydrocarbon group, more preferably an alkyl group. The alkyl group may be linear or branched, and examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl and the like.
Also, among R ² , a hydrogen atom is preferred in order to enhance the desired effects of the present invention.

X represents a thio group, a sulfinyl group or a sulfonyl group, preferably a thio group.

As the ligand (1), one represented by the following formula (α) is preferable in order to enhance the desired effects of the present invention.

[In formula (α),
R ¹ has the same definition as R ¹ in formula (1) and represents a divalent organic group, preferably an alkanediyl group having 1 to 10 carbon atoms,
* indicates a bond. ]

In addition, when the ligand (1) has optical isomerism, any optical isomer may be used, and one optical isomer may be used alone, or a plurality of optical isomers may be used in combination.

The ion exchange capacity of the chromatography carrier of the present invention having ligand (1) is preferably 10-500 μeq, more preferably 50-200 μeq per 1 mL of the carrier.
The ion-exchange capacity can be calculated, for example, from the number of moles of HCl required for neutralization and the column capacity after equilibrating a chromatography column in which the chromatography carrier of the present invention is packed in a column container, neutralizing the column with an aqueous HCl solution.
More specifically, it can be measured by the following method. That is, a column container having a capacity of 4 mL (5 mmφ×200 mm length) is packed with the chromatography carrier of the present invention to a packing height of about 20 cm to prepare a chromatography column. After connecting the obtained column to AKTA Prime Plus manufactured by GE Healthcare, an aqueous 0.1 M NaOH/2.0 M NaCl solution is passed through the column at a linear flow rate of 600 cm/hr to wash the column by 5 times its capacity. Next, a 0.01 M NaOH aqueous solution is passed through the column at a linear flow rate of 600 cm/hr for 5 times the column volume for equilibration. Next, a 0.01 M HCl aqueous solution is passed through at a linear flow rate of 300 cm/hr until neutralization. It can be measured by a method such as calculation from the number of moles of HCl required for neutralization obtained here and the column capacity.

(support)
Examples of the support include organic supports such as synthetic polymer supports and natural polymer supports; inorganic supports; organic-organic composite supports and organic-inorganic composite supports combining these. Examples of synthetic polymer supports include those composed of polyvinyl alcohols, poly(meth)acrylates, poly(meth)acrylamides, polystyrenes, ethylene-maleic anhydride copolymers, and the like. Examples of natural polymer supports include those composed of polysaccharides such as cellulose (crystalline cellulose etc.), agarose and dextran. Examples of inorganic supports include those composed of glass beads, silica gel, metals, metal oxides, and the like. Among these, synthetic polymeric supports are preferred in order to enhance the desired effects of the present invention. Also, the support is preferably a water-insoluble support.

In addition, the chromatography carrier of the present invention is preferably one in which ligand (1) is bound to a support (that is, * in formula (1) is bound to the support) in order to enhance the desired effects of the present invention. Specifically, the support has a residue of a functional group capable of reacting with a sulfanyl group in the molecule, and this residue is chemically bonded to the ligand (1). The term "residue of a functional group capable of reacting with a sulfanyl group" means a residue remaining when a functional group capable of reacting with a sulfanyl group reacts with a sulfanyl group. Examples of the residue of a functional group capable of reacting with a sulfanyl group include those listed below as "a functional group capable of reacting with a sulfanyl group" which remain when reacted with a sulfanyl group. Preferred are divalent groups obtained by ring-opening a cyclic ether group, -C(=O)- and -NH-C(=O)-, and more preferred are divalent groups obtained by ring-opening a cyclic ether group.

Here, the "functional group capable of reacting with a sulfanyl group" includes one or more functional groups selected from the group consisting of a cyclic ether group, a carboxyl group, -C(=O)-OC(=O)-, a succinimideoxycarbonyl group, a formyl group, an isocyanate group, a maleimide group, a haloacetyl group (such as an iodoacetyl group and a bromoacetyl group), an α,β-unsaturated carbonyl group, an alkenyl group and an alkynyl group. Cyclic ether groups are preferred for enhancement.
As the “cyclic ether group” in the present specification, a cyclic ether group having 3 to 7 atoms constituting the ring is preferable. The cyclic ether group may have an alkyl group as a substituent. Specific examples of the cyclic ether group include cyclic ether groups represented by the following formulas (4) to (9), but in order to enhance the desired effect of the present invention, the cyclic ether group represented by formula (4) is preferred.

[In the formula, R ⁵ to R ⁸ each independently represent a hydrogen atom or an alkyl group, and * represents a bond. ]

The alkyl groups represented by R ⁵ to R ⁸ preferably have 1 to 4 carbon atoms, more preferably 1 or 2 carbon atoms. The alkyl group may be linear or branched, and examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl and the like. Moreover, hydrogen atoms are preferable as R ⁵ to R ⁸ . When the cyclic ether group is an epoxy group, the divalent group obtained by ring-opening the cyclic ether group is a ring-opened epoxy group (--CHOH--CH ₂ --).

Further, the support may have a group represented by the following formula (10) in its molecule.

[In formula (10),
R ⁹ represents a q+1 valent hydrocarbon group,
Y ¹ represents a thio group, a sulfinyl group or a sulfonyl group,
Y ² represents a hydrophilic group,
q represents an integer of 0 or more,
* indicates a bond. ]

In formula (10), R ⁹ represents a q+1 valent hydrocarbon group. That is, when q=0, ^R9 is a monovalent hydrocarbon group, when q=1, ^R9 is a divalent hydrocarbon group, and when q=2, ^R9 is a trivalent hydrocarbon group.
The monovalent hydrocarbon group represented by R ⁹ includes the same hydrocarbon groups as those represented by R ² . The divalent hydrocarbon group represented by R ⁹ includes the same divalent hydrocarbon groups as those represented by R ¹ . The number of carbon atoms in the trivalent hydrocarbon group represented by R ⁹ is preferably 1-12, more preferably 1-6. Preferred trivalent hydrocarbon groups are alkanetriyl groups such as methane-1,1,1-triyl, ethane-1,1,1-triyl, ethane-1,1,2-triyl, propane-1,2,3-triyl and propane-1,2,2-triyl groups.
Hydrophilic groups represented by Y ² include a hydroxy group, a sulfanyl group, a carboxy group and the like.
q represents an integer of 0 or more, preferably an integer of 0 to 4, more preferably an integer of 0 to 2. In addition, when q is an integer of 2 or more, q pieces of Y ² may be the same or different.

Moreover, when the support contains a polymer, the polymer preferably has a structural unit (A) having a residue of a functional group capable of reacting with a sulfanyl group. Moreover, it is preferable that some or all of the residues in the plurality of structural units (A) contained in the polymer are bound to the ligand (1). Moreover, some of the above residues in the plurality of structural units (A) contained in the polymer may be bonded to the group represented by the above formula (10).
In addition to the structural unit (A), the polymer may have a structural unit (B) having a functional group capable of reacting with a sulfanyl group.
The structural unit (A) is preferably a structural unit represented by the following formula (11), and the structural unit (B) is preferably a structural unit represented by the following formula (12).

[In formula (11),
R ¹⁰ represents a hydrogen atom or a methyl group,
R ¹¹ represents a single bond or a divalent linking group,
Z ¹ represents a residue of a functional group capable of reacting with a sulfanyl group,
** indicates a bond. ]

[In formula (12),
R ¹² represents a hydrogen atom or a methyl group,
R ¹³ represents a single bond or a divalent linking group,
Z ² represents a functional group capable of reacting with a sulfanyl group. ]

Examples of the residue of the functional group capable of reacting with the sulfanyl group represented by Z ¹ in formula (11) and the functional group capable of reacting with the sulfanyl group represented by Z ² of formula (12) are the same as those described above.

Examples of divalent linking groups represented by R ¹¹ in formula (11) and R ¹³ in formula (12) include an alkanediyl group, a group having an ether bond between carbon-carbon atoms of an alkanediyl group having 2 or more carbon atoms, an arylene group, -C(=O)OR ¹⁴ -*, -C(=O)NH-R ¹⁵ -*, -Ar-R ¹⁶ -*, or -Ar-OR ¹⁷ . -* etc. are mentioned. Here, Ar represents an arylene group, and * in R ¹¹ and R ¹³ represents a bond that binds to Z ¹ or Z ² above. The arylene group represented by R ¹¹ , R ¹³ and Ar includes phenylene group, naphthylene group, phenanthrenylene group and the like.
R ¹⁴ to R ¹⁷ each independently represent an alkanediyl group or a group having an ether bond between carbon atoms of an alkanediyl group having 2 or more carbon atoms.

The number of carbon atoms in the alkanediyl groups represented by R ¹¹ , R ¹³ , R ¹⁴ to R ¹⁷ is preferably 1-12, more preferably 1-6, and particularly preferably 1-3. The alkanediyl group may be linear or branched. Specific examples of alkanediyl groups include methane-1,1-diyl, ethane-1,1-diyl, ethane-1,2-diyl, propane-1,1-diyl, propane-1,2-diyl, propane-1,3-diyl, propane-2,2-diyl, butane-1,1-diyl, butane-1,2-diyl, butane-1,3-diyl and butane. -1,4-diyl group, pentane-1,1-diyl group, pentane-1,2-diyl group, pentane-1,3-diyl group, pentane-1,4-diyl group, pentane-1,5-diyl group, hexane-1,1-diyl group, hexane-1,2-diyl group, hexane-1,3-diyl group, hexane-1,4-diyl group, hexane-1,5-diyl group, hexane-1,6-diyl group , heptane-1,7-diyl group, octane-1,8-diyl group and the like.

The “group having an ether bond between the carbon-carbon atoms of an alkanediyl group having 2 or more carbon atoms” represented by R ¹¹ , R ¹³ , R ¹⁴ to R ¹⁷ is preferably a group represented by —R ^a (OR ^b ) _t OR ^c — (R ^a , R ^b and R ^c each independently represent an alkanediyl group having 1 to 4 carbon atoms, and t represents an integer of 0 to 30).
The alkanediyl groups represented by R ^a , R ^b and R ^c may be linear or branched. Specifically, methane-1,1-diyl group, ethane-1,1-diyl group, ethane-1,2-diyl group, propane-1,1-diyl group, propane-1,2-diyl group, propane-1,3-diyl group, propane-2,2-diyl group, butane-1,1-diyl group, butane-1,2-diyl group, butane-1,3-diyl group, butane-1,4-diyl group groups. t is preferably an integer of 0 to 20, more preferably an integer of 0 to 10, still more preferably an integer of 0 to 5, and particularly preferably 0. When t is an integer of 2 to 30, t R ^b may be the same or different. A preferred specific example of the group having an ether bond between carbon atoms of the alkanediyl group having 2 or more carbon atoms is a C _1-4 alkanediyloxy C _1-4 alkanediyl group.
The alkanediyl group represented by R ¹¹ , R ¹³ , R ¹⁴ to R ¹⁷ and the group having an ether bond between carbon atoms of the alkanediyl group having 2 or more carbon atoms may have a substituent. Examples of the substituent include a hydroxy group and the like.

Of the above R ¹¹ and R ¹³ , -C(=O)OR ¹⁴ -* is particularly preferred.

As the monomer that provides the structural unit (A) or the structural unit (B), a monomer having a functional group capable of reacting with a sulfanyl group and a polymerizable unsaturated group is preferred. Such monomers include, for example, glycidyl (meth)acrylate, 3-oxiranylpropyl (meth)acrylate, 4-oxiranylbutyl (meth)acrylate, 5-oxiranylpentyl (meth)acrylate, 6-oxiranylhexyl (meth)acrylate, 7-oxiranylheptyl (meth)acrylate, 8-oxiranyloctyl (meth)acrylate, (3-methyloxiranyl)methyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate glycidyl ether, glycerin mono(meth)acrylate. ) Acrylate glycidyl ether, 3,4-epoxycyclohexylmethyl (meth)acrylate, 3,4-epoxycyclohexylethyl (meth)acrylate, 3,4-epoxycyclohexylpropyl (meth)acrylate, α-(meth)acrylic-ω-glycidyl polyethylene glycol, tetrahydrofurfuryl (meth)acrylate and other (meth)acrylate monomers having a cyclic ether group; (vinylbenzyl) glycidyl ether, (isopropenylbenzyl) glycidyl ether, (vinylphenethyl) glycidyl ether, ( Aromatic vinyl monomers having a cyclic ether group such as vinylphenylbutyl)glycidyl ether, (vinylbenzyloxyethyl)glycidyl ether, (vinylphenyl)glycidyl ether, (isopropenylphenyl)glycidyl ether, 1,2-epoxy-3-(4-vinylbenzyl)propane; allyl ether monomers having a cyclic ether group such as allyl glycidyl ether; (meth)acrylate monomers having an isocyanate group such as isocyanatoethyl (meth)acrylate; and unsaturated dicarboxylic anhydride monomers such as glutaconic anhydride, (meth)acrylic acid, 3,4-epoxy-1-butene, 3,4-epoxy-3-methyl-1-butene and the like. These monomers can be used individually by 1 type or in combination of 2 or more types.
Among these monomers, (meth)acrylate monomers having a cyclic ether group are preferred in order to enhance the desired effects of the present invention.

The total content of structural units (A) and (B) in the polymer contained in the support is preferably 50 to 100% by mass, more preferably 70 to 90% by mass, based on the total structural units in the polymer.

Moreover, the polymer contained in the support may have structural units other than the structural units (A) and (B). Monomers that provide such structural units (hereinafter also referred to as other monomers) include polymerizable unsaturated group-containing monomers that do not have a functional group capable of reacting with a sulfanyl group. Other monomers are broadly classified into non-crosslinking monomers and crosslinkable monomers, and either one of these may be used or both may be used.

Examples of the non-crosslinking monomers include (meth)acrylate-based non-crosslinking monomers, (meth)acrylamide-based non-crosslinking monomers, aromatic vinyl-based non-crosslinking monomers, vinyl ketone-based non-crosslinking monomers, (meth)acrylonitrile-based non-crosslinking monomers, N-vinylamide-based non-crosslinking monomers, and the like. These can be used individually by 1 type or in combination of 2 or more types. Among the non-crosslinking monomers, (meth)acrylate non-crosslinking monomers and aromatic vinyl non-crosslinking monomers are preferred.

Examples of the (meth)acrylate-based non-crosslinkable monomer include methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, 4-tert-butyl (meth)acrylate, isobutyl (meth)acrylate, n-octyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, cyclohexyl (meth)acrylate, methoxyethyl (meth)acrylate, hydroxyethyl (meth)acrylate, hydroxypropyl (meth)acrylate, glycerol mono (meth) acrylate, trimethylolethane mono (meth) ) acrylate, trimethylolpropane mono (meth) acrylate, butanetriol mono (meth) acrylate, polyethylene glycol mono (meth) acrylate, methoxypolyethylene glycol (meth) acrylate, pentaerythritol mono (meth) acrylate, dipentaerythritol mono (meth) acrylate, inositol mono (meth) acrylate and the like. These can be used individually by 1 type or in combination of 2 or more types.

Examples of the (meth)acrylamide-based non-crosslinkable monomers include (meth)acrylamide, dimethyl(meth)acrylamide, hydroxyethyl (meth)acrylamide, (meth)acryloylmorpholine, diacetone (meth)acrylamide, and the like. These can be used individually by 1 type or in combination of 2 or more types.

Examples of the aromatic vinyl-based non-crosslinkable monomer include styrene, α-methylstyrene, halogenated styrene, 4-methylstyrene, 2,4-dimethylstyrene, 2,4,6-trimethylstyrene, ethylvinylbenzene, 4-isopropylstyrene, 4-n-butylstyrene, 4-isobutylstyrene, 4-tert-butylstyrene and the like; vinylnaphthalenes such as 1-vinylnaphthalene and 2-vinylnaphthalene. These can be used individually by 1 type or in combination of 2 or more types.

Examples of the vinyl ketone-based non-crosslinking monomer include ethyl vinyl ketone, propyl vinyl ketone, isopropyl vinyl ketone, and the like. These can be used individually by 1 type or in combination of 2 or more types.
Examples of the (meth)acrylonitrile-based non-crosslinkable monomer include acrylonitrile and methacrylonitrile. These can be used individually by 1 type or in combination of 2 or more types.
Examples of the N-vinylamide-based non-crosslinkable monomers include N-vinylacetamide and N-vinylpropionamide. These can be used individually by 1 type or in combination of 2 or more types.

The content of structural units derived from non-crosslinkable monomers in the polymer contained in the support is preferably 0 to 50% by mass, more preferably 0 to 20% by mass, based on the total structural units in the polymer.

Examples of the crosslinkable monomers include (meth)acrylate crosslinkable monomers, aromatic vinyl crosslinkable monomers, and allyl crosslinkable monomers. These can be used individually by 1 type or in combination of 2 or more types. As the cross-linking monomer, a bi- to penta-functional cross-linking monomer is preferable, and a bi- or tri-functional cross-linking monomer is more preferable.
Among the crosslinkable monomers, (meth)acrylate crosslinkable monomers and aromatic vinyl crosslinkable monomers are preferred, and aromatic vinyl crosslinkable monomers are particularly preferred, in order to enhance the desired effects of the present invention.

Examples of the (meth)acrylate crosslinkable monomer include ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, tetraethylene glycol di(meth)acrylate, polyethylene glycol di(meth)acrylate, propylene glycol di(meth)acrylate, dipropylene glycol di(meth)acrylate, tripropylene glycol di(meth)acrylate, tetrapropylene glycol di(meth)acrylate, polypropylene glycol di(meth)acrylate, 1,4-butanediol di(meth)acrylate, 1,6-hexanediol di(meth)acrylate). (meth)acrylate, glycerol di(meth)acrylate, trimethylolethane di(meth)acrylate, trimethylolpropane di(meth)acrylate, trimethylolpropane tri(meth)acrylate, butanetriol di(meth)acrylate, pentaerythritol di(meth)acrylate, pentaerythritol tri(meth)acrylate, pentaerythritol tetra(meth)acrylate, glucose di(meth)acrylate, glucose tri(meth)acrylate, glucose tetra(meth)acrylate, dipentaerythritol di(meth)acrylate) ) acrylate, dipentaerythritol tri(meth)acrylate, dipentaerythritol tetra(meth)acrylate, dipentaerythritol penta(meth)acrylate, inositol di(meth)acrylate, inositol tri(meth)acrylate, inositol tetra(meth)acrylate, mannitol di(meth)acrylate, mannitol tri(meth)acrylate, mannitol tetra(meth)acrylate, mannitol penta(meth)acrylate and the like. These can be used individually by 1 type or in combination of 2 or more types.

Examples of the aromatic vinyl-based crosslinkable monomer include divinylbenzene, trivinylbenzene, divinyltoluene, divinylxylene, divinylethylbenzene, divinylnaphthalene, and the like. These can be used individually by 1 type or in combination of 2 or more types.

Examples of the allyl-based crosslinkable monomer include diallyl phthalate, diallyl isophthalate, diallyl terephthalate, diallyl maleate, diallyl fumarate, diallyl itaconate, diallyl trimellitate, triallyl trimellitate, triallyl cyanurate, diallyl isocyanurate, and triallyl isocyanurate. These can be used individually by 1 type or in combination of 2 or more types.
Further, examples of crosslinkable monomers include, in addition to those exemplified above, diaminopropanol, trishydroxymethylaminomethane, dehydration condensation reaction products of amino alcohols such as glucosamine and (meth)acrylic acid, and conjugated diolefins such as butadiene and isoprene.

The content of structural units derived from the crosslinkable monomer in the polymer contained in the support is preferably 5 to 50% by mass, more preferably 10 to 30% by mass, based on the total structural units in the polymer.

Also, the content of the support is preferably 50 to 99.9% by mass, more preferably 80 to 99% by mass, in the chromatography carrier of the present invention.

Moreover, the chromatography carrier of the present invention may be in any form as long as it can be used as a chromatography carrier. Such forms include, for example, particulate, monolithic, plate-like, film-like (including hollow fiber), fibrous, cassette-like, chip-like, etc., preferably particulate. In addition, porous carriers such as porous particles are preferable as the chromatography carrier of the present invention. As the porous particles, porous polymer particles are preferable.

In addition, when the chromatography carrier of the present invention is a particulate carrier, the average particle size (volume average particle size) is preferably 20 to 150 μm, more preferably 40 to 100 μm in order to improve pressure resistance performance and breakage resistance performance. Also, the coefficient of variation of the average particle size is preferably 40% or less, more preferably 30% or less.
The specific surface area of the chromatography carrier of the present invention is preferably 1-500 m ² /g, more preferably 10-300 m ² /g.
Moreover, the volume average pore size of the chromatography carrier of the present invention is preferably 10 to 300 nm.
The average particle size, coefficient of variation, specific surface area and volume average pore size can be measured by laser diffraction/scattering particle size distribution measurement or the like.

<Method for producing carrier>
The chromatographic carrier of the present invention can be produced by appropriately combining conventional methods, but in order to produce the chromatographic carrier simply and efficiently, it is preferably produced by a method comprising a step of contacting a support having a functional group capable of reacting with a sulfanyl group in its molecule (hereinafter also referred to as a raw material support) with a compound represented by the following formula (β). Each symbol in formula (β) has the same meaning as in formula (1).

[In formula (β),
A represents an anionic functional group,
Q represents a divalent aromatic hydrocarbon group,
R ¹ represents a divalent organic group,
^R2 represents a hydrogen atom or a hydrocarbon group. ]

Examples of the production method of the present invention include a method of (Step P1-1) preparing a raw material support, (Step P1-2) preparing a compound (β), and (Step P2) bringing the raw material support and the compound (β) into contact.
Each of the above steps will be specifically described below.

(Step P1-1)
Step P1-1 is a step of preparing a raw material support.
The raw material supporters are specially available products, specialty, 2007-525501, specialty, 2008-505851, special opening, 2010-210497, specialties, specialty, 2015-501310, Open Open, 2015/119255, Special Open 2016-50897. The one prepared with reference to the known methods described in etc. may be used. Specific examples of the method for preparing the raw material support include a method of (co)polymerizing (preferably suspension polymerization) a monomer having a functional group capable of reacting with a sulfanyl group and a polymerizable unsaturated group.
Examples of the monomer having a functional group capable of reacting with a sulfanyl group and a polymerizable unsaturated group include those exemplified as the monomers giving the structural units (A) and (B). In addition, the monomer having a functional group capable of reacting with a sulfanyl group and a polymerizable unsaturated group may be copolymerized with the other monomers (non-crosslinkable monomer, crosslinkable monomer).
The total amount of the monomer having a functional group capable of reacting with a sulfanyl group and a polymerizable unsaturated group is preferably 50 to 100 parts by mass, more preferably 70 to 90 parts by mass, with respect to 100 parts by mass of the total monomer used in step P1-1.
The total amount of crosslinkable monomers used is preferably 5 to 50 parts by mass, more preferably 10 to 30 parts by mass, based on 100 parts by mass of the total amount of monomers used in step P1-1.
When non-crosslinkable monomers are used, the total amount used is the amount of the remainder other than the monomers having functional groups and polymerizable unsaturated groups capable of reacting with sulfanyl groups and the crosslinkable monomers.

Specific methods of step P1-1 include, for example, a method of dissolving a polymerization initiator in a mixed solution (monomer solution) containing a monomer and optionally a porosifying agent (monomer solution), suspending the polymerization initiator in an aqueous medium, and heating to a predetermined temperature to polymerize; A method of suspending it in a medium, heating it to a predetermined temperature, adding a polymerization initiator, and polymerizing it, and the like can be mentioned.

A radical polymerization initiator is preferred as the polymerization initiator. Examples of radical polymerization initiators include azo initiators, peroxide initiators, redox initiators, and specific examples include azobisisobutyronitrile, methyl azobisisobutyrate, azobis-2,4-dimethylvaleronitrile, benzoyl peroxide, di-tert-butyl peroxide, and benzoyl peroxide-dimethylaniline. The total amount of the polymerization initiator used is usually about 0.01 to 10 parts by mass per 100 parts by mass of the total amount of monomers.

The porosity agent is used to produce porous particles, exists together with the monomer in the polymerization in the oil droplets, and has a role of forming pores as a non-polymerized component. The porosity agent is not particularly limited as long as it can be easily removed from the porous surface. Examples thereof include linear polymers soluble in various organic solvents and mixed monomers, and these may be used in combination.

Examples of the porosifying agent include aliphatic hydrocarbons such as hexane, heptane, octane, nonane, decane and undecane; alicyclic hydrocarbons such as cyclopentane and cyclohexane; aromatic hydrocarbons such as benzene, toluene, xylene, naphthalene and ethylbenzene; halogenated hydrocarbons such as carbon tetrachloride, 1,2-dichloroethane, tetrachloroethane and chlorobenzene; Alicyclic alcohols such as 2-ethyl-1-hexanol; Alicyclic alcohols such as cyclohexanol; Aromatic alcohols such as 2-phenylethyl alcohol and benzyl alcohol; Ketones such as diethyl ketone, methyl isobutyl ketone, diisobutyl ketone, acetophenone, 2-octanone and cyclohexanone; and linear polymers such as homopolymers of non-crosslinkable vinyl monomers. A porosity agent can be used individually or in mixture of 2 or more types.
The total amount of the porosity agent used is usually about 40 to 600 parts by mass with respect to 100 parts by mass of the total amount of the monomers.

Examples of the aqueous medium include aqueous solutions of water-soluble polymers, and examples of water-soluble polymers include hydroxyethylcellulose, polyvinyl alcohol, carboxymethylcellulose, polyvinylpyrrolidone, starch, and gelatin.
The total amount of the aqueous medium used is usually about 200 to 7000 parts by mass per 100 parts by mass of the total amount of monomers.
Further, when water is used as the dispersion medium for the aqueous medium, for example, a dispersion stabilizer such as sodium carbonate, calcium carbonate, sodium sulfate, calcium phosphate, and sodium chloride may be used.

In step P1-1, various surfactants including anionic surfactants such as alkyl sulfates, alkylaryl sulfates, alkyl phosphates, and fatty acid salts may be used. In addition, polymerization inhibitors such as nitrites such as sodium nitrite, iodide salts such as potassium iodide, tert-butylpyrocatechol, benzoquinone, picric acid, hydroquinone, copper chloride and ferric chloride can also be used. Moreover, you may use polymerization regulators, such as dodecyl mercaptan.

The polymerization temperature in step P1-1 may be determined according to the polymerization initiator, but is usually about 2 to 100°C. When azobisisobutyronitrile is used as the polymerization initiator, the temperature is preferably 50 to 100°C, more preferably 60 to 90°C. The polymerization time is usually 5 minutes to 48 hours, preferably 10 minutes to 24 hours.

(Step P1-2)
Step P1-2 is a step of preparing compound (β).
Compound (β) can be obtained, for example, by contacting the following carboxylic acid (PR-A) with the following sulfanyl compound (PR-C). Further, among the compounds (β), the following compound (β') in which the anionic functional group A is -C(=O)OH can be obtained by contacting the following dicarboxylic acid anhydride (PR-B) with the following sulfanyl compound (PR-C). In this specification, the reaction between carboxylic acid (PR-A) and sulfanyl compound (PR-C) and the reaction between dicarboxylic anhydride (PR-B) and sulfanyl compound (PR-C) are collectively referred to as "compound (β) synthesis reaction".
When obtaining the compound (β'), it is preferably obtained by contacting the dicarboxylic anhydride (PR-B) with the sulfanyl compound (PR-C) in order to efficiently obtain the desired compound.

[Each symbol in the formula is as defined above. ]

Carboxylic acids (PR-A) include, for example, 3-sulfopropanoic acid, 4-sulfobutanoic acid, 5-sulfopentanoic acid, 3-phosphonopropionic acid, 4-phosphonobutyric acid and the like.
Dicarboxylic anhydrides (PR-B) include, for example, succinic anhydride, methylsuccinic anhydride, dimethylsuccinic anhydride, butylsuccinic anhydride, octylsuccinic anhydride, dichlorosuccinic anhydride, cyclopropane-1,2-dicarboxylic anhydride, hexahydrophthalic anhydride, methylhexahydrophthalic anhydride, glutaric anhydride, 2,4-diethylglutaric anhydride, sebacic anhydride, ginger Examples include oxalic anhydride, maleic anhydride, methylmaleic anhydride, and glutaconic anhydride.
Sulfanyl compounds (PR-C) include, for example, 2-aminobenzenethiol, 3-aminobenzenethiol, 4-aminobenzenethiol, 1-amino-2-naphthalenethiol and the like.
In the compound (β) synthesis reaction, the amount of the sulfanyl compound (PR-C) used is usually 1.0 to 3.0 molar equivalents, preferably 1.0 to 1.5 molar equivalents, per 1 mol of the carboxylic acid (PR-A) or dicarboxylic anhydride (PR-B).

The compound (β) synthesis reaction is preferably carried out in the presence of a solvent in order to efficiently obtain the desired compound. Examples of the solvent include ether solvents such as tetrahydrofuran, dibutyl ether and dimethoxyethane; aromatic hydrocarbon solvents such as benzene, toluene, xylene and cumene; aliphatic alkane solvents such as pentane, hexane, heptane and octane; cycloalkane solvents such as cyclohexane, cycloheptane and decalin; halogen solvents such as chloroform and methylene chloride; One of these may be used alone, or two or more may be used in combination.
The amount of the solvent used is usually 100 to 50,000 parts by mass, preferably 500 to 10,000 parts by mass, with respect to a total of 100 parts by mass of the carboxylic acid (PR-A) or dicarboxylic anhydride (PR-B) and the sulfanyl compound (PR-C).

In addition, the reaction time of the compound (β) synthesis reaction is not particularly limited, but it is usually about 15 minutes to 96 hours, preferably 1 to 24 hours. Moreover, the reaction temperature may be appropriately selected at the boiling point of the solvent or lower, but it is usually about 20 to 100°C.

The compound (β) obtained in step P1-2 is a novel compound and useful as an intermediate for producing the chromatography carrier of the present invention.

(Process P2)
Step P2 is a step of contacting the raw material support with the compound (β).
The amount of compound (β) to be used is preferably 0.1 to 20 molar equivalents, more preferably 0.5 to 5 molar equivalents, relative to 1 mol of functional groups capable of reacting with sulfanyl groups contained in the raw material support.

Step P2 is preferably carried out in the presence of a solvent in order to efficiently obtain the desired carrier. Examples of the solvent include water; alcohols such as methanol, ethanol, propanol, isopropyl alcohol and n-butyl alcohol; sulfoxides such as dimethylsulfoxide. One of these may be used alone, or two or more may be used in combination.
The amount of the solvent to be used is generally 300 to 3000 parts by mass, preferably 500 to 2000 parts by mass, per 100 parts by mass of the raw material support.

The pH of step P2 is preferably 8-16, more preferably 10-14, in order to efficiently obtain the desired carrier.
In addition, the reaction time in step P2 is not particularly limited, but it is usually about 0.5 to 72 hours, preferably 1 to 48 hours. Moreover, the reaction temperature may be appropriately selected at the boiling point of the solvent or lower, but it is usually about 10 to 100°C.

When producing a carrier in which X in formula (1) is a sulfinyl group or a sulfonyl group, the carrier in which X in formula (1) is a thio group obtained in step P2 above may be oxidized.
The oxidation may be performed with reference to known methods described in International Publication No. 2015/119255, Japanese Patent Application Laid-Open No. 2016-50897, etc. Specific examples include a method using an oxidizing agent. The oxidizing agent includes organic oxidizing agents such as peracetic acid, perbenzoic acid, meta-chloroperbenzoic acid and tert-butyl hydroperoxide; and inorganic oxidizing agents such as hydrogen peroxide, chromic acid and permanganate. In addition, these oxidizing agents can be used individually by 1 type or in combination of 2 or more types.

Alternatively, the carrier obtained as described above may be brought into contact with a sulfanyl compound such as methanethiol or thioglycerol to open unreacted functional groups. This processing may be performed with reference to the description in International Publication No. 2015/119255 pamphlet and the like.
In addition, the reaction product obtained in each of the above steps may be purified by separation means such as distillation, extraction, and washing.

The chromatography carrier of the present invention that can be produced as described above has a large dynamic binding capacity for a target substance and is excellent in the performance of separating the target substance and contaminants. In particular, as described in the Examples below, each protein can be easily and efficiently separated from a mixture containing multiple types of proteins, and thus the method is useful for separating target proteins and non-target proteins.
The reason why such an effect is exhibited is not necessarily clear, but it is not only the fact that it has a ligand containing a divalent aromatic hydrocarbon group and an anionic functional group, but also that the divalent aromatic hydrocarbon group represented by Q in formula (1) is adjacent to the thio group, sulfinyl group or sulfonyl group represented by X, and the divalent aromatic hydrocarbon group represented by Q and the anionic functional group represented by A are -N(-R ² )-C(=O)-R ¹ . The present inventors presume that this is because they are linked via a specific linking group -.
The chromatographic carrier of the present invention is therefore suitable for protein purification and also suitable for antibody purification. In addition, the chromatography carrier of the present invention is extremely useful for mixed mode chromatography of hydrophobic chromatography and cation exchange chromatography.

<Chromatography column>
The chromatography column of the present invention is a column container filled with the chromatography carrier of the present invention.
The chromatographic column of the invention is suitable for protein purification and also suitable for antibody purification. Moreover, it is extremely useful for mixed mode chromatography of hydrophobic chromatography and cation exchange chromatography.

<Purification method>
The method for purifying a target substance of the present invention is characterized by using the chromatography carrier of the present invention.
The method for purifying the target substance of the present invention can be carried out in the same manner as known methods except for using the chromatographic carrier of the present invention. For example, it may be carried out with reference to known methods described in JP-A-2007-525501, JP-A-2008-505851, JP-A-2010-210497, JP-A-2015-501310, and the like. Specifically, a method including the step of contacting the chromatography carrier of the present invention with a sample containing a target substance can be mentioned. Moreover, it is preferable to further include an elution step of eluting the target substance captured by the carrier in this step. A dissociation solution that dissociates the ligand and the target substance is usually used in the elution step.
Purification may also be performed using the chromatography column of the present invention. Examples of such a method include a method comprising passing a sample containing a target substance through the chromatography column of the present invention, and preferably further comprising an elution step as described above.

Examples of target substances include bio-related substances such as proteins, peptides, amino acids, sugars, polysaccharides, lipids, vitamins, DNA, and RNA. Proteins and peptides are preferred, and proteins are more preferred. Target proteins include antigens, antibodies, virus particles and the like, with antibodies being preferred. Antibodies include polyclonal antibodies, monoclonal antibodies, bispecific antibodies, synthetic antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, Fab fragments, Fab′ fragments, F(ab′)2 fragments, Fv fragments, Fv′ fragments, fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies, epitope-binding fragments of any of the above. Antibody classes include IgG, IgE, IgM, IgD, and IgA. Note that the subclass is not particularly limited.
Examples of the sample containing the target substance include blood components such as whole blood, serum, plasma, blood components, various blood cells, clots, and platelets, body fluids such as urine, semen, breast milk, sweat, interstitial fluid, interstitial lymph, bone marrow fluid, tissue fluid, saliva, gastric juice, joint fluid, pleural fluid, bile, ascitic fluid, amniotic fluid, bacterial fluid, cell culture medium, cell culture supernatant, and various liquids such as tissue cell lysate. As the sample, it is possible to use a sample that has undergone other purification treatment such as affinity purification one or more times, or to use a sample that has not been subjected to such other purification treatment.
Contaminants include, for example, off-target proteins. Specifically, when an antibody is the target substance, it is a protein other than the antibody. DNA and host cell-derived proteins are used when virus particles are the target substance.

More specifically, the target substance purification method of the present invention includes a method including the following steps S1 and S2. Then, the target substance eluted from the carrier in step S2 can be recovered.
(Step S1) A step of contacting the chromatography carrier of the present invention with a sample containing a target substance (Step S2) A step of contacting a dissociation solution for dissociating the ligand possessed by the carrier and the target substance with the carrier that has captured the target substance in Step S1.

(Step S1)
Step S1 is a step of bringing the chromatography carrier of the present invention into contact with a sample containing a target substance. A target substance is captured by the chromatography carrier of the present invention by step S1.
Step S1 is usually performed in the presence of a buffer. The pH (25° C.) of the buffer used in step S1 is preferably in the range of 2 to 9, more preferably in the range of 3 to 8, in order to efficiently separate the target substance from contaminants. Also, the salt concentration of the buffer is preferably 0-0.5M.

(Step S2)
Step S2 is a step of bringing a dissociation solution for dissociating the target substance and the ligand possessed by the chromatography carrier of the present invention into contact with the carrier that captured the target substance in step S1. By step S2, the target substance captured by the carrier is eluted, and the target substance can be separated from the contaminants by utilizing the fact that the affinity between the chromatography carrier of the present invention and the target substance is different from the affinity between the chromatography carrier of the present invention and contaminants.
Step S2 is usually performed in the presence of a buffer. The pH (25° C.) of the buffer used in step S2 is preferably in the range of 2 to 9, more preferably in the range of 3 to 8, in order to efficiently separate the target substance from contaminants.
In step S2, the target substance can be eluted from the carrier by a stepwise method, a gradient method, or a method in which both are combined. In addition, the salt concentration of the buffer may be increased to generally 1.0-3.0M.
The stepwise method refers to a method in which a plurality of buffers with different salt concentrations are used and the salt concentration is changed stepwise to elute the target substance from the carrier.
The gradient method refers to a method in which the target substance is eluted from the carrier by continuously changing the salt concentration. When elution is performed by a gradient method, it is preferable to increase the concentration by preferably 0.001 to 0.2 M/min, more preferably 0.01 to 0.1 M/min.

EXAMPLES The present invention will be described in detail below with reference to Examples, but the present invention is not limited to these Examples.

[Synthesis Example 1]
Compound (E-A1) was obtained according to the following synthetic route.

That is, 11.27 g of 4-aminobenzenethiol and 9.01 g of succinic anhydride were added to a 3-necked flask equipped with a Dimroth, and deaeration and nitrogen substitution were performed. After that, 400 mL of dehydrated toluene and 450 mL of dehydrated THF were added, and the mixture was stirred at 50° C. for 4 hours. After allowing to cool, the solvent was distilled off to obtain 20.22 g of compound (E-A1).

¹ H NMR (600 MHz, DMSO-d ₆ ): δ2.53(m, 4H), 7.21(d, J = 8.4 Hz, 2H), 7.47(d, J = 8.4 Hz, 2H), 9.94(s, 1H), 12.10(brs, 1H)

[Synthesis Example 2]
Except for changing 9.01 g of succinic anhydride to 10.27 g of glutaric anhydride, the same procedure as in Synthesis Example 1 was performed to obtain 21.40 g of the following compound (E-A2).

¹ H NMR (600 MHz, DMSO-d ₆ ): δ1.79(m,2H), 2.26(dd, J = 7.5 Hz, 2H), 2.32(dd, J = 7.5 Hz, 2H), 7.21(d, J = 8.6 Hz, 2H), 7.48(d, J = 8.6 Hz, 2H), 9.88(s, 1H), 12.05 (brs, 1H)

[Comparative Synthesis Example 1]
Compound (E-B1) was obtained according to the following synthetic route.

(1) That is, 4.75 g of 4-aminohippuric acid and 25.5 g of 1M sodium hydroxide aqueous solution were added to a flask and stirred in an ice bath. 4.25 g of bromoacetic chloride and 6.50 g of a 5M sodium hydroxide aqueous solution were alternately added dropwise thereto. After the addition was completed, the solution was removed from the ice bath and stirred at room temperature for 30 minutes. Thereafter, a 2M hydrochloric acid aqueous solution was added until a cloudy precipitate was generated from the solution, and ethyl acetate was further added to carry out a liquid separation/extraction operation. After distilling off under reduced pressure, vacuum drying was performed to obtain 3.80 g of intermediate compound (E-PB1).
(2) Subsequently, 3.10 g of the intermediate compound (E-PB1) was added to another flask and dissolved in 150 mL of acetone. 1.13 g of potassium thioacetate was added thereto and stirred at 40° C. for 4 hours. After acetone was distilled off under reduced pressure, ethyl acetate was added to dissolve the reactant. A 5% by mass citric acid aqueous solution was added thereto to perform a washing operation. The resulting organic phase was distilled off under reduced pressure and then dried in vacuo to obtain 3.01 g of intermediate compound (E-PB2).
(3) Subsequently, 2.80 g of the intermediate compound (E-PB2) and 20 g of methanol were added to a new flask and stirred. Furthermore, 20.0 g of 1M sodium hydroxide aqueous solution was added, and the mixture was stirred at room temperature for 1 hour. After removing methanol by distillation under reduced pressure, a precipitate was generated by adding a 2M hydrochloric acid aqueous solution. After that, ethyl acetate was added and an extraction operation was performed. After washing the obtained organic phase with saturated saline, water was removed with magnesium sulfate. Further, the obtained organic phase was distilled off under reduced pressure and dried in a vacuum to obtain 2.30 g of compound (E-B1).

[Comparative Synthesis Example 2]
Compound (E-B2) was obtained according to the following synthetic route.

(1) That is, 5.00 g of DL-homocysteine thiolactone hydrochloride and 150 mL of dehydrated DMF were added to a degassed, nitrogen-substituted flask, and the mixture was stirred in an ice bath. Then, after adding 5.49 g of benzoyl chloride and 5.05 g of N,N-diisopropylethylamine, the solution was removed from the ice bath and stirred at room temperature for 2 hours. DMF was removed by distillation under reduced pressure, and ethyl acetate was added thereto. After washing with saturated saline and distilled water, the organic phase was distilled under reduced pressure and dried in vacuum to obtain 6.12 g of intermediate compound (E-PB3).
(2) Subsequently, 5.00 g of the intermediate compound (E-PB3) and 100 mL of THF were added to another flask and stirred. Furthermore, 25.0 g of 1M sodium hydroxide aqueous solution was added, and the mixture was stirred at room temperature for 1 hour. After removing THF by distillation under reduced pressure, a 2M hydrochloric acid aqueous solution was added to cause precipitation. After that, ethyl acetate was added and an extraction operation was performed. After washing the obtained organic phase with saturated saline, water was removed with magnesium sulfate. Further, the obtained organic phase was distilled off under reduced pressure and vacuum dried to obtain 5.33 g of compound (E-B2).

[Comparative Synthesis Example 3]
Compound (E-B3) was obtained according to the following synthetic route.

(1) That is, 5.00 g of L-tryptophan and 25.5 g of 1M sodium hydroxide aqueous solution were added to a flask and stirred in an ice bath. 4.25 g of bromoacetic chloride and 6.50 g of a 5M sodium hydroxide aqueous solution were alternately added dropwise thereto. After the addition was completed, the solution was removed from the ice bath and stirred at room temperature for 30 minutes. Thereafter, a 2M hydrochloric acid aqueous solution was added until a cloudy precipitate was generated from the solution, and ethyl acetate was further added to carry out a liquid separation/extraction operation. After distilling off under reduced pressure, vacuum drying was performed to obtain 3.48 g of intermediate compound (E-PB4).
(2) Subsequently, 3.20 g of the intermediate compound (E-PB4) was added to another flask and dissolved in 150 mL of acetone. 1.13 g of potassium thioacetate was added thereto and stirred at 40° C. for 4 hours. After acetone was distilled off under reduced pressure, ethyl acetate was added to dissolve the reactant. A 5% by mass citric acid aqueous solution was added thereto to perform a washing operation. The resulting organic phase was distilled off under reduced pressure and dried in vacuo to obtain 3.05 g of intermediate compound (E-PB5).
(3) Subsequently, 2.90 g of the intermediate compound (E-PB5) and 20 g of methanol were added to a new flask and stirred. Furthermore, 20.0 g of 1M sodium hydroxide aqueous solution was added, and the mixture was stirred at room temperature for 1 hour. After removing methanol by distillation under reduced pressure, a 2M hydrochloric acid aqueous solution was added to cause precipitation. After that, ethyl acetate was added and an extraction operation was performed. After washing the obtained organic phase with saturated saline, water was removed with magnesium sulfate. Further, the obtained organic phase was distilled off under reduced pressure and dried in a vacuum to obtain 2.31 g of compound (E-B3).

[Example 1]
(1) Add 2.69 g of polyvinyl alcohol (PVA-217 manufactured by Kuraray Co., Ltd.) to 448 g of pure water, heat and stir to dissolve the polyvinyl alcohol, cool, then sodium dodecyl sulfate (manufactured by Wako Pure Chemical Industries) 0.045 g was added and stirred to prepare an aqueous solution S.
On the other hand, a monomer composition consisting of 3.63 g of divinylbenzene (manufactured by Wako Pure Chemical Industries), 0.36 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo) and 14.15 g of glycidyl methacrylate (manufactured by Mitsubishi Gas Chemical Co., Ltd.) was dissolved in 29.38 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) to prepare a monomer solution.
Next, the entire aqueous solution S was put into a separable flask, equipped with a thermometer, a stirring blade and a cooling tube, set in a warm water bath, and stirred under a nitrogen atmosphere. The entire amount of the monomer solution was put into a separable flask, and when the inner temperature reached 85°C by heating with a warm water bath, 1.34 g of 2,2'-azoisobutyronitrile (manufactured by Wako Pure Chemical Industries, Ltd.) was added to raise the inner temperature to 86°C.

(2) After that, the mixture was stirred for 3 hours while maintaining the temperature at 86°C. Then, after cooling the reaction liquid, the reaction liquid was filtered and washed with pure water and ethanol. The washed particles were dispersed in pure water and decanted three times to remove small particles. Next, the particles were dispersed in pure water so that the concentration of the particles was 10% by mass to obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous carrier 1".
(3) After that, 20 g of the porous particle dispersion was taken out, and after removing water, 9.5 g of 1M aqueous sodium hydroxide solution and 0.976 g of compound (E-A1) were added and stirred at 50° C. for 15 hours. The reaction solution was then filtered and washed with pure water.
(4) Next, 7.30 g of α-thioglycerol (manufactured by Tokyo Kasei Kogyo Co., Ltd.) and 2.16 g of sodium hydroxide were added and stirred at 70° C. for 3 hours. Then, the reaction solution was filtered and washed with pure water, and then the particles were dispersed in pure water so that the particle concentration was 10% by mass. The porous particles contained in this dispersion are referred to as "chromatographic carrier V1".

[Example 2]
The same operations as steps (1) to (4) of Example 1 were performed except that 0.976 g of compound (E-A1) in step (3) of Example 1 was changed to 1.036 g of compound (E-A2). The porous particles contained in the resulting dispersion are referred to as "chromatographic carrier V2".

[Comparative Example 1]
The same operations as in steps (1) to (4) of Example 1 were performed, except that 0.976 g of compound (E-A1) in step (3) of Example 1 was changed to 1.162 g of compound (E-B1). The porous particles contained in the resulting dispersion are referred to as "chromatographic carrier X1".

[Comparative Example 2]
The same operations as steps (1) to (4) of Example 1 were performed except that 0.976 g of compound (E-A1) in step (3) of Example 1 was changed to 1.036 g of compound (E-B2). The porous particles contained in the resulting dispersion are referred to as "chromatographic carrier X2".

[Comparative Example 3]
The same operations as steps (1) to (4) of Example 1 were performed except that 0.976 g of compound (E-A1) in step (3) of Example 1 was changed to 1.205 g of compound (E-B3). The porous particles contained in the resulting dispersion are referred to as "chromatographic carrier X3".

[Example 3]
The same operations as steps (1) to (4) of Example 1 were performed, except that 3.63 g of divinylbenzene (manufactured by Wako Pure Chemical Industries) and 0.36 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo) in step (1) of Example 1 were changed to 5.56 g of ethylene glycol dimethacrylate. The porous particles contained in the resulting dispersion are referred to as "chromatographic carrier V3". A porous carrier prepared instead of porous carrier 1 by using ethylene glycol dimethacrylate is referred to as "porous carrier 2".

[Example 4]
Step (1) of Example 1 except that 3.63 g of divinylbenzene (manufactured by Wako Pure Chemical Industries) and 0.36 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo) in step (1) of Example 1 were changed to 5.56 g of ethylene glycol dimethacrylate, and further that 0.976 g of compound (E-A1) in step (3) of Example 1 was changed to 1.036 g of compound (E-A2). The same operation as in (4) was performed. The porous particles contained in the resulting dispersion are referred to as "chromatographic carrier V4".

[Comparative Example 4]
Step (1) of Example 1 except that 3.63 g of divinylbenzene (manufactured by Wako Pure Chemical Industries) and 0.36 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo) in step (1) of Example 1 were changed to 5.56 g of ethylene glycol dimethacrylate, and further that 0.976 g of compound (E-A1) in step (3) of Example 1 was changed to 1.162 g of compound (E-B1). The same operation as in (4) was performed. The porous particles contained in the resulting dispersion are referred to as "chromatographic carrier X4".

[Comparative Example 5]
Step (1) of Example 1 except that 3.63 g of divinylbenzene (manufactured by Wako Pure Chemical Industries) and 0.36 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo) in step (1) of Example 1 were changed to 5.56 g of ethylene glycol dimethacrylate, and further that 0.976 g of compound (E-A1) in step (3) of Example 1 was changed to 1.036 g of compound (E-B2). The same operation as in (4) was performed. The porous particles contained in the resulting dispersion are referred to as "chromatographic carrier X5".

[Comparative Example 6]
Step (1) of Example 1 except that 3.63 g of divinylbenzene (manufactured by Wako Pure Chemical Industries) and 0.36 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo) in step (1) of Example 1 were changed to 5.56 g of ethylene glycol dimethacrylate, and further that 0.976 g of compound (E-A1) in step (3) of Example 1 was changed to 1.205 g of compound (E-B3). The same operation as in (4) was performed. The porous particles contained in the resulting dispersion are referred to as "chromatographic carrier X6".

[Test Example 1 (Measurement of DBC)]
Using AKTA Prime Plus manufactured by GE Healthcare, the DBC of carriers V1 to V4 and X1 to X6 of each example and comparative example against protein (human IgG antibody, HGG-1000 manufactured by Equitech Bio) at a linear flow rate of 300 cm/hr was measured. A column vessel with a capacity of 4 mL (5 mm diameter × 200 mm length) was used, and protein was dissolved in 50 mM sodium acetate/100 mM sodium chloride aqueous solution (pH 4.7) at 5 mg/mL, respectively. Table 1 shows the results.

[Test Example 2 (Protein Separation)]
The protein separation ability was evaluated by separating each protein from a liquid sample containing three types of proteins (trypsinogen (pI = 10, Mw = 23 kDa), cytochrome C (pI = 10, Mw = 12 kDa), lysozyme (pI = 11, Mw = 14 kDa)) using the carrier of each example and comparative example. Specific procedures are shown below.
A column was prepared by filling a column container having a capacity of 4 mL (5 mmφ×200 mm length) with each carrier to a packing height of about 5 cm. The resulting column was connected to AKTA Prime Plus manufactured by GE Healthcare. A 20 mM sodium phosphate buffer (pH 7.0) was passed through the column four times its volume for equilibration.
Next, trypsinogen, cytochrome C and lysozyme were dissolved in 20 mM sodium phosphate buffer (pH 7.0) to prepare a protein mixture (trypsinogen: 0.2 mg/mL, cytochrome C: 0.1 mg/mL, lysozyme: 0.2 mg/mL).
Next, 100 μL of the protein mixture was passed through the column at a linear flow rate of 300 cm/hr.
Then, linear gradient elution from 100% 20 mM sodium phosphate buffer (pH 7.0) to 100% 20 mM sodium phosphate/1.5 M sodium chloride buffer (pH 7.0) was performed over 40 minutes, followed by elution with 100% 20 mM sodium phosphate/1.5 M sodium chloride buffer (pH 7.0) for 10 minutes. The elution volume V and peak width W of each protein peak at the time of elution were obtained using a spectrophotometer attached to AKTA Prime Plus manufactured by GE Healthcare (UV detection wavelength: 280 nm).
Elution volume V is the volume of the eluate determined from the point of intersection with the baseline when a perpendicular line is drawn from the point of highest absorbance value of the peak to the baseline.
The peak width W is a volume value obtained from the width between two points of contact with the baseline when two tangent lines passing through the points of inflection on the left and right of the peak are drawn. However, when the elution peaks overlapped, a perpendicular line was drawn from the point of the lowest absorbance value between these overlapping peaks to the baseline, and it was determined from the point of intersection with the baseline. It can be said that the smaller the value of the peak width W, the higher the protein concentration recovered, and the better the protein separation ability.
Also, using the values of the elution volume V and the peak width W, the resolution Rs1 and Rs2 were calculated from the following formulas. It can be said that the larger the values of the separation ability Rs1 and Rs2, the better the protein separation ability. Table 1 shows the results of Test Example 2.

Rs1 = (2( _V2 - _V1 ))/( _W2 + _W1 )
Rs2 = (2( _V3 - _V2 ))/( _W3 + _W2 )

V ₁ : elution volume of trypsinogen, V ₂ : elution volume of cytochrome C, V ₃ : elution volume of lysozyme, W ₁ : peak width of trypsinogen, W ₂ : peak width of cytochrome C, W ₃ : peak width of lysozyme

As shown in Table 1, when using a carrier to which compounds (E-A1) and (E-A2) were bound as ligands (Examples 1 and 2, Examples 3 and 4), the separation ability Rs2 was greater than when a carrier with a 4-aminohippuric acid residue as a ligand was used (Comparative Examples 1 and 4). In addition, the measured value of DBC was also large.
In addition, when a carrier to which compounds (E-A1) and (E-A2) were bound as ligands was used (Examples 1 and 2, Examples 3 and 4), the separation ability Rs1 was greater than when a carrier having a 2-(benzoylamino)-4-mercaptobutyric acid residue or an L-tryptophan residue as a ligand was used (Comparative Examples 2 and 3 and Comparative Examples 5 and 6). In addition, the measured value of DBC was also large.
In addition, as shown in the results of Comparative Examples 2 and 3, with the carrier having a 2-(benzoylamino)-4-mercaptobutyric acid residue or an L-tryptophan residue as a ligand, lysozyme did not even elute depending on the type of support (base particle), and the separation ability Rs2 could not be calculated.

Claims (13)

A synthetic polymer support containing a polymer and a ligand represented by the following formula (1), wherein the polymer has a structural unit (A) having a residue of a functional group capable of reacting with a sulfanyl group and a structural unit (B) having a functional group capable of reacting with the sulfanyl group, the total content of the structural units (A) and (B) in the polymer being 50 to 100% by mass with respect to all structural units in the polymer, and the structural unit (A) being a (meth)acrylate system having a cyclic ether group. Chromatographic carriers (excluding those with steroid residues) derived from monomers .

[In formula (1),
A represents an anionic functional group,
Q represents a divalent aromatic hydrocarbon group,
R ¹ represents a divalent organic group,
R ² represents a hydrogen atom or a hydrocarbon group,
X represents a thio group, a sulfinyl group or a sulfonyl group,
* indicates a bond. ] A is -C(=O)OH, -C(=O)O^-M.⁺, -C(=O)O^-, -S (=O)₂OH, -S (=O)₂O.^-M.⁺, -S (=O)₂O.^-, -P (=O) (OH)₂, -P (=O) (O^-M.⁺)₂, -P (=O) (O^-)₂, -P(=O)(OH)(O^-M.⁺), -P (= O) (OH) (O^-), -OP (= O) (OH)₂, -OP (=O) (O^-M.⁺)₂, -OP (=O) (O^-)₂, -OP (=O) (OH) (O^-M.⁺), or -OP (= O) (OH) (O^-) (above M⁺each represent a counterion). 3. The carrier according to claim 1, wherein Q is a divalent group represented by the following formula (2) or (3).

[In formula (2),
R ³ represents a halogen atom, a hydroxy group, a carboxamide group or an organic group;
n represents an integer of 0 to 4,
* indicates a bond. ]

[In formula (3),
R ⁴ represents a halogen atom, a hydroxy group, a carboxamide group or an organic group;
m represents an integer from 0 to 6,
* indicates a bond. ] The carrier according to any one of claims 1 to 3, wherein R ¹ is a divalent hydrocarbon group having 1 to 20 carbon atoms. A carrier according to any one of claims 1 to 4, wherein X is a thio group. The carrier according to any one of claims 1 to 5, wherein the ligand is represented by the following formula (α).

[In formula (α),
R ¹ represents an alkanediyl group having 1 to 10 carbon atoms,
* indicates a bond. ] A carrier according to any one of claims 1 to 6 , wherein said ligand is bound to said support. The carrier according to any one of claims 1 to 7 , which is a protein purification chromatography carrier. The carrier according to any one of claims 1 to 7 , which is an antibody purification chromatography carrier. The carrier according to any one of claims 1 to 9 , which is a mixed mode chromatography carrier for hydrophobic chromatography and cation exchange chromatography. A method for producing the carrier according to any one of claims 1 to 10 , wherein a support having in the molecule a structural unit derived from a monomer having a functional group capable of reacting with a sulfanyl group and a polymerizable unsaturated group, and the following formula (β) .

[In formula (β),
A represents an anionic functional group,
Q represents a divalent aromatic hydrocarbon group,
R ¹ represents a divalent organic group,
^R2 represents a hydrogen atom or a hydrocarbon group. ] A chromatography column, wherein the carrier according to any one of claims 1 to 10 is packed in a column container. A method for purifying a target substance using the carrier according to any one of claims 1 to 10 .