WO2020045541A1 - Support de chromatographie - Google Patents

Support de chromatographie Download PDF

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Publication number
WO2020045541A1
WO2020045541A1 PCT/JP2019/033820 JP2019033820W WO2020045541A1 WO 2020045541 A1 WO2020045541 A1 WO 2020045541A1 JP 2019033820 W JP2019033820 W JP 2019033820W WO 2020045541 A1 WO2020045541 A1 WO 2020045541A1
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Prior art keywords
group
carrier
divalent
chromatography
hydrocarbon group
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PCT/JP2019/033820
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English (en)
Japanese (ja)
Inventor
高典 岸田
智哉 則信
Original Assignee
Jsr株式会社
ジェイエスアール マイクロ インコーポレイテッド
ジェイエスアール マイクロ エヌ．ブイ．
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Application filed by Jsr株式会社, ジェイエスアール マイクロ インコーポレイテッド, ジェイエスアール マイクロ エヌ．ブイ． filed Critical Jsr株式会社
Priority to JP2020539567A priority Critical patent/JP7315561B2/ja
Publication of WO2020045541A1 publication Critical patent/WO2020045541A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/285—Porous sorbents based on polymers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes

Definitions

  • the present invention relates to a chromatography carrier. More specifically, the present invention relates to a chromatography carrier, a method for producing the carrier and a production intermediate thereof, a chromatography column, and a method for purifying a target substance.
  • a carrier having a mixed mode ligand containing an anionic functional group and a hydrophobic group is used.
  • mixed mode carriers include 2- (benzoylamino) -4-mercaptobutyric acid residue (Patent Documents 1 and 2), L-tryptophan residue (Patent Document 3) and 4-amino horse.
  • a carrier having a uric acid residue (Patent Document 4) as a mixed mode ligand has been proposed.
  • Patent Documents 1 to 4 have room for improvement in dynamic binding capacity to a target substance in the first place.
  • the problem to be solved by the present invention is to provide a chromatography carrier having a large dynamic binding capacity to a target substance and excellent in the performance of separating a target substance from contaminants.
  • a chromatography carrier (hereinafter, also referred to as “the chromatography carrier of the present invention”) having a support and a ligand represented by the following formula (1) (hereinafter, also referred to as “ligand (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.
  • 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.
  • R 4 represents a halogen atom, a hydroxy group, a carboxamide group or an organic group, m represents an integer of 0 to 6, * Indicates a bond.
  • ⁇ 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 ( ⁇ ).
  • 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.
  • the carrier according to any one of ⁇ 1> to ⁇ 8> which is a protein purification chromatography carrier.
  • the carrier according to any one of ⁇ 1> to ⁇ 10> which is a mixed mode chromatography carrier of hydrophobic chromatography and cation exchange chromatography.
  • ⁇ 12> A method for producing the carrier according to any one of ⁇ 1> to ⁇ 11>, wherein the support has a functional group capable of reacting with a sulfanyl group in a molecule, and is represented by the following formula ( ⁇ ): (Hereinafter, also referred to as “the production method of the present invention”).
  • 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.
  • ⁇ 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 target substance purification method of the present invention”).
  • 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.
  • the chromatography carrier of the present invention has a large dynamic binding capacity for a target substance and has excellent performance for separating a target substance from contaminants. Therefore, according to the chromatography column and the method for purifying a target substance of the present invention, the target substance and impurities can be easily and efficiently separated. Further, according to the production method of the present invention, a chromatography carrier having a large dynamic binding capacity to a target substance and excellent in the performance of separating a target substance from contaminants can be simply and efficiently produced.
  • the production intermediate of the present invention is useful as a production intermediate of the chromatography carrier of the present invention.
  • the chromatography carrier of the present invention is characterized by having a support and a ligand represented by the following 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
  • ligand refers to a site capable of capturing a target substance represented by a protein, and the mechanism of the capture is not limited to chemical bonding, but may be based on ion exchange, hydrophobic interaction, or the like.
  • the chromatographic support of the present invention has improved dynamic binding capacity to a target substance and performance of separating a target substance from contaminants by having a ligand (1) having a specific chemical structure.
  • A represents an anionic functional group.
  • the anionic functional group represented by A acts as a cation exchange group.
  • M + represents a counter ion.
  • the counter ion include an alkali metal ion such as a sodium ion and a potassium ion; an alkaline earth metal ion such as a magnesium ion and a calcium ion; an ammonium ion; and an organic ammonium ion.
  • the divalent aromatic hydrocarbon group represented by Q acts as a hydrophobic group having a hydrophobic interaction.
  • the carbon number of the divalent aromatic hydrocarbon group is preferably from 6 to 18, and more preferably from 6 to 12.
  • an arylene group is preferable, and specific examples include a phenylene group, a naphthylene group, and a phenanthrylene group. Among them, a phenylene group and a naphthylene group are preferable, and a phenylene group is particularly preferable, in order to enhance a desired effect of the present invention.
  • the binding site of the divalent aromatic hydrocarbon group may be on any carbon on the ring.
  • the divalent aromatic hydrocarbon group may be any of a divalent aromatic hydrocarbon group having a substituent and an unsubstituted divalent aromatic hydrocarbon group.
  • the substitution position and the number of the substituent are arbitrary.
  • the divalent aromatic hydrocarbon group has two or more substituents, the substituents may be the same or different.
  • the substituent include a halogen atom such as a chlorine atom, a bromine atom and a fluorine atom; a hydroxy group; a carboxamide group; and an organic group.
  • Examples of the organic group include a hydrocarbon group and a group having at least one selected from an ether bond, an amide bond, and an ester bond between carbon-carbon atoms of a hydrocarbon group having 2 or more carbon atoms.
  • the organic group is a hydrocarbon group, it preferably has 1 to 4 carbon atoms, more preferably 1 or 2.
  • the hydrocarbon group in the “group having at least one selected from ether bond, amide bond and ester bond between carbon-carbon atoms of a hydrocarbon group having 2 or more carbon atoms” preferably has 2 to 4 carbon atoms.
  • the hydrocarbon group is preferably an alkyl group.
  • the alkyl group may be linear or branched, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group and a tert-butyl group.
  • divalent aromatic hydrocarbon group represented by Q a divalent group represented by the following formula (2) or (3) is preferable in order to enhance the desired effect of the present invention.
  • the divalent group represented by is particularly preferable.
  • 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.
  • R 4 represents a halogen atom, a hydroxy group, a carboxamide group or an organic group, m represents an integer of 0 to 6, * Indicates a bond.
  • the halogen atom represented by R 3 and R 4 includes a chlorine atom, a bromine atom and a fluorine atom.
  • the organic group represented by R 3 or R 4 has at least one selected from an ether bond, an amide bond and an ester bond between carbon-carbon atoms of a hydrocarbon group or a hydrocarbon group having 2 or more carbon atoms. Groups. When the organic group is a hydrocarbon group, it preferably has 1 to 4 carbon atoms, more preferably 1 or 2.
  • the hydrocarbon group in the “group having at least one selected from ether bond, amide bond and ester bond between carbon-carbon atoms of a hydrocarbon group having 2 or more carbon atoms” preferably has 2 to 4 carbon atoms.
  • the hydrocarbon group is preferably an alkyl group.
  • the alkyl group may be linear or branched, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group and a tert-butyl group.
  • n represents an integer of 0 to 4, but is preferably 0 to 2 and particularly preferably 0 in order to enhance the desired effect of the present invention.
  • m represents an integer of 0 to 6, and is preferably 0 to 2 and particularly preferably 0 in order to enhance a desired effect of the present invention.
  • n R 3 s may be the same or different.
  • m R 4 s may be the same or different.
  • R 1 represents a divalent organic group.
  • the divalent organic group represented by R 1 is selected from a divalent hydrocarbon group, an ether bond, an amide bond and an ester bond between carbon-carbon atoms of a divalent hydrocarbon group having 2 or more carbon atoms. Although a group having more than one kind is mentioned, a divalent hydrocarbon group is preferable in order to enhance a desired effect of the present invention.
  • the divalent organic group represented by R 1 is a divalent hydrocarbon group
  • the number of carbon atoms is preferably 1 to 20, more preferably 1 to 14 in order to enhance the desired effect of the present invention. , More preferably 1 to 10, more preferably 1 to 5, particularly preferably 1 or 2.
  • the divalent organic group is “a group having at least one selected from an ether bond, an amide bond, and an ester bond between carbon-carbon atoms of a divalent hydrocarbon group having 2 or more carbon atoms”
  • the carbon number of the divalent hydrocarbon group in such a group is preferably from 2 to 20, more preferably from 2 to 14, still more preferably from 2 to 10, and further preferably from 2 to increase the desired effect of the present invention. To 5, particularly preferably 2.
  • a divalent hydrocarbon group represented by R 1 “a group having at least one selected from an ether bond, an amide bond, and an ester bond between carbon-carbon atoms of a divalent hydrocarbon group having 2 or more carbon atoms”
  • the divalent hydrocarbon group in "" may have a substituent.
  • the substituent include a chlorine atom, a bromine atom, a halogen atom such as a fluorine atom, a hydroxy group, and the like.
  • the substitution position and the number of the substituents are arbitrary. When two or more substituents are present, the substituents may be the same or different.
  • the divalent hydrocarbon group represented by R 1 “a group having at least one selected from an ether bond, an amide bond, and an ester bond between carbon-carbon atoms of a divalent hydrocarbon group having 2 or more carbon atoms”
  • the divalent hydrocarbon group 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 increase the value, a divalent aliphatic hydrocarbon group and a divalent alicyclic hydrocarbon group are preferable. Note that the divalent aliphatic hydrocarbon group may be linear or branched.
  • 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 20 in order to enhance the desired effect of the present invention.
  • more preferably 1 to 10 more preferably 1 to 5, particularly preferably 1 or 2.
  • the divalent organic group is “a group having at least one selected from ether bond, amide bond and ester bond between carbon-carbon atoms of a divalent aliphatic hydrocarbon group having 2 or more carbon atoms”.
  • the carbon number of the divalent aliphatic hydrocarbon group in such a group is preferably 2 to 20, more preferably 2 to 14, still more preferably 2 to 10, in order to enhance the desired effect of the present invention.
  • the divalent aliphatic hydrocarbon group examples include an alkanediyl group and an alkenediyl group, and an alkanediyl group is preferable in order to enhance a desired effect of the present invention.
  • alkanediyl group examples include methane-1,1-diyl, ethane-1,1-diyl, ethane-1,2-diyl, propane-1,1-diyl, and propane-1.
  • the divalent alicyclic hydrocarbon group preferably has 3 to 20, more preferably 3 to 14, more preferably 3 to 10, and particularly preferably 3 to 8, carbon atoms.
  • Examples of the divalent alicyclic hydrocarbon group include a cycloalkanediyl group and a divalent bridged ring hydrocarbon group such as an adamantylene group, and a cycloalkanediyl group is preferable.
  • Specific examples of the cycloalkanediyl group include a cyclohexanediyl group, a methylcyclohexanediyl group, and a cycloheptanediyl group.
  • the bivalent aromatic hydrocarbon group preferably has 6 to 18 carbon atoms, more preferably 6 to 12 carbon atoms.
  • an arylene group is preferable, and specific examples 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.
  • an alkanediyl group and a cycloalkanediyl group are preferable, and an alkanediyl group is particularly preferable, in order to enhance a desired effect of the present invention.
  • R 2 represents a hydrogen atom or a hydrocarbon group.
  • the hydrocarbon group represented by R 2 is a concept including an aliphatic hydrocarbon group, an alicyclic hydrocarbon group, and an aromatic hydrocarbon group, and may be a saturated hydrocarbon group or an unsaturated hydrocarbon group.
  • the carbon number of the hydrocarbon group is preferably 1 to 4, more preferably 1 or 2.
  • the hydrocarbon group is preferably an aliphatic hydrocarbon group, and more preferably an alkyl group.
  • the alkyl group may be linear or branched, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group and a tert-butyl group.
  • R 2 a hydrogen atom is preferable in order to enhance a desired effect of the present invention.
  • X represents a thio group, a sulfinyl group or a sulfonyl group, and a thio group is preferable.
  • the ligand (1) is preferably a ligand represented by the following formula ( ⁇ ) in order to enhance the desired effect of the present invention.
  • R 1 has the same meaning 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. ]
  • any of the optical isomers may be used, or one kind of optical isomer may be used alone, or a plurality of kinds of optical isomers may be used in combination. Is also good.
  • the ion exchange capacity of the chromatography carrier of the present invention having the ligand (1) is preferably from 10 to 500 ⁇ eq, more preferably from 50 to 200 ⁇ eq per mL of the carrier.
  • the ion exchange capacity is determined, for example, by equilibrating a chromatography column in which the chromatography carrier of the present invention is packed in a column container, neutralizing by passing an aqueous HCl solution, and converting the molar amount of HCl required for the neutralization. It can be calculated from the number and column capacity. More specifically, it can be measured by the following method.
  • a chromatography container of the present invention is packed into a column container having a capacity of 4 mL (5 mm ⁇ ⁇ 200 mm length) at a packing height of about 20 cm to prepare a chromatography column.
  • AKTA Prime Plus manufactured by GE Healthcare
  • it is washed by passing a 0.1 M NaOH / 2.0 M NaCl aqueous solution at a linear flow rate of 600 cm / hr 5 times the column volume.
  • equilibration is performed by passing a 0.01 M NaOH aqueous solution at a linear flow rate of 600 cm / hr 5 times the column volume.
  • a 0.01 M HCl aqueous solution is passed at a linear flow rate of 300 cm / hr until neutralization. It can be measured by a method such as calculating from the number of moles of HCl required for neutralization obtained here and the column capacity.
  • Examples of the support include an organic support such as a synthetic polymer support and a natural polymer support; an inorganic support; an organic-organic composite support or an organic-inorganic composite support obtained by combining these.
  • Examples of the synthetic polymer support include those composed of polyvinyl alcohols, poly (meth) acrylates, poly (meth) acrylamides, polystyrenes, ethylene-maleic anhydride copolymers, and the like.
  • Examples of the natural polymer support include those composed of polysaccharides such as cellulose (eg, crystalline cellulose), agarose, and dextran.
  • Examples of the inorganic support include those composed of glass beads, silica gel, metal, metal oxide, and the like. Among these, a synthetic polymer support is preferable in order to enhance the desired effect of the present invention. Further, the support is preferably a water-insoluble support.
  • the chromatography carrier of the present invention has a ligand (1) bound to a support in order to enhance the desired effect of the present invention (that is, * in the formula (1) is bound to the support).
  • the support may have a functional group residue capable of reacting with a sulfanyl group in the molecule, and this residue may be chemically bonded to the ligand (1).
  • the “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 the functional group capable of reacting with the sulfanyl group include residues remaining when those listed below as the ⁇ functional group capable of reacting with the sulfanyl group '' react with the sulfanyl group, and a cyclic ether group is exemplified.
  • cyclic ether group As the “cyclic ether group” in the present specification, a cyclic ether group having 3 to 7 atoms constituting a 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). In order to enhance the desired effect of the present invention, the cyclic ether group is represented by the following formula (4). Cyclic ether groups are preferred.
  • R 5 to R 8 each independently represent a hydrogen atom or an alkyl group, and * represents a bond.
  • the alkyl group represented by R 5 to R 8 preferably has 1 to 4 carbon atoms, more preferably 1 or 2.
  • the alkyl group may be linear or branched, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group and a tert-butyl group.
  • a hydrogen atom is preferable.
  • the cyclic ether group is an epoxy group
  • the divalent group formed by ring opening of the cyclic ether group is a ring-opened epoxy group (—CHOH—CH 2 —).
  • the support may have a group represented by the following formula (10) in the molecule.
  • 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
  • the trivalent hydrocarbon group represented by R 9 preferably has 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms.
  • Examples of the trivalent hydrocarbon group include methane-1,1,1-triyl, ethane-1,1,1-triyl, ethane-1,1,2-triyl, propane-1,2,3- Alkanetriyl groups such as a triyl group and a propane-1,2,2-triyl group are preferred.
  • Examples of the hydrophilic group represented by Y 2 include a hydroxy group, a sulfanyl group, and a carboxy group.
  • q represents an integer of 0 or more, preferably an integer of 0 to 4, more preferably an integer of 0 to 2. Incidentally, when q is an integer of 2 or more, q number of Y 2 may or may not be the same.
  • the polymer When the support contains a polymer, the polymer preferably has a structural unit (A) having a functional group residue capable of reacting with a sulfanyl group. Further, it is preferable that some or all of the residues in the plurality of structural units (A) contained in the polymer are bonded to the ligand (1). Further, some of the residues in the plurality of structural units (A) contained in the polymer may be bonded to the group represented by the formula (10).
  • the polymer may have a structural unit (B) having a functional group capable of reacting with a sulfanyl group, in addition to the structural unit (A).
  • a structural unit represented by the following formula (11) is preferable
  • the structural unit (B) a structural unit represented by the following formula (12) is preferable.
  • 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.
  • 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.
  • Ar represents an arylene group
  • * in R 11 and R 13 represents a bond bonded to Z 1 or Z 2 .
  • Examples of the arylene group represented by R 11 , R 13 , and Ar include a phenylene group, a naphthylene group, and a phenanthrylene group.
  • R 14 to R 17 each independently represent an alkanediyl group or a group having an ether bond between carbon-carbon atoms of an alkanediyl group having 2 or more carbon atoms.
  • the carbon number of the alkanediyl group represented by R 11 , R 13 , and R 14 to R 17 is preferably 1 to 12, more preferably 1 to 6, and particularly preferably 1 to 3.
  • the alkanediyl group may be linear or branched.
  • alkanediyl group examples include a methane-1,1-diyl group, an ethane-1,1-diyl group, an ethane-1,2-diyl group, a propane-1,1-diyl group, a 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, 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,
  • Examples of the “group having an ether bond between carbon and carbon atoms of an alkanediyl group having 2 or more carbon atoms” represented by R 11 , R 13 and R 14 to R 17 include —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.
  • t is preferably an integer of 0 to 20, more preferably an integer of 0 to 10, further preferably an integer of 0 to 5, and particularly preferably 0.
  • t R b 's may be the same or different.
  • Specific preferable examples of the group having an ether bond between carbon atoms include C 1-4 alkanediyl-oxy C 1-4 alkanediyl group.
  • the alkanediyl group represented by R 11 , R 13 , and R 14 to R 17 or the group having an ether bond between the carbon atoms of the alkanediyl group having 2 or more carbon atoms may have a substituent. Good. Examples of the substituent include a hydroxy group.
  • a monomer having a functional group capable of reacting with a sulfanyl group and a polymerizable unsaturated group is preferable.
  • 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 glycidyl ether, 3,4-epoxycyclohexyl
  • the total content of the structural units (A) and (B) in the polymer contained in the support is preferably from 50 to 100% by mass, more preferably from 70 to 100% by mass, based on all the structural units in the polymer. 90% by mass.
  • the polymer contained in the support may have a structural unit in addition to the structural units (A) and (B).
  • Examples of a monomer that provides such a structural unit include a polymerizable unsaturated group-containing monomer having no functional group capable of reacting with a sulfanyl group.
  • Other monomers are roughly classified into non-crosslinkable monomers and crosslinkable monomers, and one of these may be used or used in combination.
  • non-crosslinkable monomer examples include (meth) acrylate non-crosslinkable monomers, (meth) acrylamide non-crosslinkable monomers, aromatic vinyl non-crosslinkable monomers, vinyl ketone non-crosslinkable monomers, and (meth) acrylonitrile.
  • Non-crosslinkable monomers and N-vinylamide non-crosslinkable monomers can be used alone or in combination of two or more.
  • (meth) acrylate-based non-crosslinkable monomers and aromatic vinyl-based non-crosslinkable monomers are preferable.
  • 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, and 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 , Methoxy polyethylene
  • Examples of the (meth) acrylamide non-crosslinkable monomer include (meth) acrylamide, dimethyl (meth) acrylamide, hydroxyethyl (meth) acrylamide, (meth) acryloylmorpholine, and diacetone (meth) acrylamide. Can be These can be used alone or in combination of two or more.
  • aromatic vinyl non-crosslinkable monomer examples include styrene, ⁇ -methylstyrene, halogenated styrene, 4-methylstyrene, 2,4-dimethylstyrene, 2,4,6-trimethylstyrene, and ethylvinyl.
  • Styrenes such as benzene, 4-isopropylstyrene, 4-n-butylstyrene, 4-isobutylstyrene, and 4-tert-butylstyrene; and vinylnaphthalenes such as 1-vinylnaphthalene and 2-vinylnaphthalene. These can be used alone or in combination of two or more.
  • Examples of the vinyl ketone-based non-crosslinkable monomer include ethyl vinyl ketone, propyl vinyl ketone, and isopropyl vinyl ketone. These can be used alone or in combination of two or more.
  • Examples of the (meth) acrylonitrile-based non-crosslinkable monomer include acrylonitrile and methacrylonitrile. These can be used alone or in combination of two or more.
  • Examples of the N-vinylamide non-crosslinkable monomer include N-vinylacetamide and N-vinylpropionamide. These can be used alone or in combination of two or more.
  • the content ratio of the structural unit derived from the non-crosslinkable monomer in the polymer contained in the support is preferably 0 to 50% by mass, more preferably 0 to 20% by mass, based on all the structural units in the polymer. %.
  • crosslinkable monomer examples include (meth) acrylate crosslinkable monomers, aromatic vinyl crosslinkable monomers, and allyl crosslinkable monomers. These can be used alone or in combination of two or more.
  • a bifunctional to pentafunctional crosslinkable monomer is preferable, and a bifunctional or trifunctional crosslinkable monomer is more preferable.
  • a (meth) acrylate-based crosslinkable monomer and an aromatic vinyl-based crosslinkable monomer are preferable, and an aromatic vinyl-based crosslinkable monomer is particularly preferable, in order to enhance a desired effect of the present invention.
  • Examples of the (meth) acrylate-based crosslinkable monomer include, for example, ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, tetraethylene glycol di (meth) acrylate, and polyethylene glycol di.
  • aromatic vinyl-based crosslinkable monomer examples include divinylbenzene, trivinylbenzene, divinyltoluene, divinylxylene, divinylethylbenzene, divinylnaphthalene, and the like. These can be used alone or in combination of two or more.
  • allyl crosslinkable monomer examples include, for example, diallyl phthalate, diallyl isophthalate, diallyl terephthalate, diallyl maleate, diallyl fumarate, diallyl itaconate, diallyl trimellitate, triallyl trimellitate, triallyl cyanurate , Diallyl isocyanurate, triallyl isocyanurate and the like. These can be used alone or in combination of two or more.
  • crosslinkable monomer examples include, in addition to those exemplified above, a dehydration-condensation reaction product of an amino alcohol such as diaminopropanol, trishydroxymethylaminomethane, and glucosamine with (meth) acrylic acid, and a conjugated diene such as butadiene and isoprene. Olefin and the like can be mentioned.
  • the content ratio of the structural unit derived from the crosslinkable monomer in the polymer contained in the support is preferably from 5 to 50% by mass, more preferably from 10 to 30% by mass, based on all the structural units in the polymer. It is.
  • 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.
  • the chromatography carrier of the present invention may be in any form as long as it can be used as a chromatography carrier.
  • a form include a particle form, a monolith form, a plate form, a film form (including a hollow fiber), a fibrous form, a cassette form, a chip form and the like, and preferably a particulate form.
  • a porous support such as a porous particle is preferable.
  • porous particles porous polymer particles are preferable.
  • the average particle size is preferably from 20 to 150 ⁇ m, more preferably from 20 to 150 ⁇ m, in order to enhance pressure resistance and breakage resistance. It is 40 to 100 ⁇ m.
  • 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 support of the present invention is preferably 1 to 500 m 2 / g, and more preferably 10 to 300 m 2 / g.
  • the volume average pore diameter of the chromatography support of the present invention is preferably 10 to 300 nm.
  • the average particle diameter, coefficient of variation, specific surface area and volume average pore diameter can be measured by laser diffraction, scattering particle diameter distribution measurement, and the like.
  • the chromatography carrier of the present invention can be produced by appropriately combining conventional methods, but in order to easily and efficiently produce the chromatography carrier, a support having a functional group capable of reacting with a sulfanyl group in the molecule. (Hereinafter, also referred to as a raw material support) and a compound comprising a compound represented by the following formula ( ⁇ ): Each symbol in the formula ( ⁇ ) has the same meaning as in the 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.
  • Step P1-1 a raw material support is prepared, (Step P1-2) a compound ( ⁇ ) is prepared, and (Step P2) a raw material support and a compound ( ⁇ ) are prepared. And a method of contacting Hereinafter, each of the above steps will be specifically described.
  • Step P1-1 is a step of preparing a raw material support. Even if a commercially available product is used as the raw material support, JP-T-2007-525501, JP-T-2008-505851, JP-A-2010-210497, JP-T-2015-501310, International Publication No. Those prepared with reference to known methods described in, for example, JP-A-119255 and JP-A-2016-50897 may be used.
  • 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 is exemplified.
  • the monomer having a functional group capable of reacting with a sulfanyl group and a polymerizable unsaturated group include those exemplified as the monomer providing the structural units (A) and (B).
  • the other monomer may be copolymerized with a monomer having a functional group capable of reacting with a sulfanyl group and a polymerizable unsaturated group.
  • the total amount of the monomer having a functional group capable of reacting with the sulfanyl group and the monomer having a polymerizable unsaturated group is preferably 50 to 100 parts by mass, and more preferably 90 parts by mass is more preferred.
  • the total amount of the crosslinkable monomer 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 the monomers used in step P1-1.
  • the total amount thereof is the remaining amount other than a monomer having a functional group capable of reacting with a sulfanyl group and a polymerizable unsaturated group or a crosslinkable monomer.
  • a polymerization initiator is dissolved in a mixed solution (monomer solution) containing a monomer and, if necessary, a porogen, and suspended in an aqueous medium.
  • a polymerization method by heating to a predetermined temperature, or dissolving a polymerization initiator in a mixed solution (monomer solution) containing a monomer and, if necessary, a porogen, and adding it to an aqueous medium heated to a predetermined temperature.
  • a radical polymerization initiator is preferable as the polymerization initiator.
  • the radical polymerization initiator include an azo-based initiator, a peroxide-based initiator, a redox-based initiator, and the like. Specifically, azobisisobutyronitrile, methyl azobisisobutyrate, azobis-2, 4-dimethylvaleronitrile, benzoyl peroxide, di-tert-butyl peroxide, benzoyl peroxide-dimethylaniline and the like.
  • the total amount of the polymerization initiator is usually about 0.01 to 10 parts by mass based on 100 parts by mass of the total amount of the monomers.
  • the above-mentioned porogen is used for producing porous particles, and is present together with the monomer in the polymerization in oil droplets, and has a role of forming pores as a non-polymerized component.
  • the porosifying agent is not particularly limited as long as it can be easily removed from the porous surface, and examples thereof include linear polymers soluble in various organic solvents and mixed monomers. You may use together.
  • the porosifying agent examples include aliphatic hydrocarbons such as hexane, heptane, octane, nonane, decane and undecane; alicyclic hydrocarbons such as cyclopentane and cyclohexane; benzene, toluene, xylene, naphthalene, and ethylbenzene.
  • Aromatic hydrocarbons such as carbon tetrachloride, 1,2-dichloroethane, tetrachloroethane, chlorobenzene, etc .; butanol, pentanol, hexanol, heptanol, 4-methyl-2-pentanol, 2- Aliphatic alcohols such as ethyl-1-hexanol; alicyclic alcohols such as cyclohexanol; aromatic alcohols such as 2-phenylethyl alcohol and benzyl alcohol; diethyl ketone, methyl isobutyl ketone, diisobutyl ketone, acetophenone, -E Ketones such as tanone and cyclohexanone; ethers such as dibutyl ether, diisobutyl ether, anisole and ethoxybenzene; esters such as isopentyl acetate, butyl acetate, -3-methoxy
  • the aqueous medium examples include an aqueous solution of a water-soluble polymer, and examples of the water-soluble polymer include hydroxyethyl cellulose, polyvinyl alcohol, carboxymethyl cellulose, polyvinyl pyrrolidone, starch, and gelatin.
  • the total amount of the aqueous medium used is usually about 200 to 7000 parts by mass based on 100 parts by mass of the total amount of the monomers.
  • a dispersion stabilizer such as sodium carbonate, calcium carbonate, sodium sulfate, calcium phosphate, and sodium chloride may be used.
  • various surfactants such as anionic surfactants such as alkyl sulfates, alkyl aryl sulfates, alkyl phosphates, and fatty acid salts may be used.
  • nitrites such as sodium nitrite
  • iodide salts such as potassium iodide
  • polymerization inhibitors such as tert-butyl pyrocatechol, benzoquinone, picric acid, hydroquinone, copper chloride, and ferric chloride can also be used.
  • a polymerization modifier such as dodecyl mercaptan may be used.
  • the polymerization temperature in step P1-1 may be determined according to the polymerization initiator, but is usually about 2 to 100 ° C., and when azobisisobutyronitrile is used as the polymerization initiator, the polymerization temperature is 50 to 100 ° C. , And more preferably 60 to 90 ° C.
  • the polymerization time is generally 5 minutes to 48 hours, preferably 10 minutes to 24 hours.
  • Step P1-2 is a step of preparing compound ( ⁇ ).
  • Compound ( ⁇ ) can be obtained, for example, by bringing the following carboxylic acid (PR-A) into contact with the following sulfanyl compound (PR-C).
  • PR-A carboxylic acid
  • PR-C sulfanyl compound
  • the reaction between a carboxylic acid (PR-A) and a sulfanyl compound (PR-C) and the reaction between a dicarboxylic anhydride (PR-B) and a sulfanyl compound (PR-C) are described as " Compound ( ⁇ ) synthesis reaction ”.
  • the compound ( ⁇ ′) is obtained, it is preferably obtained by contacting a dicarboxylic anhydride (PR-B) with a sulfanyl compound (PR-C) in order to obtain a desired compound efficiently.
  • Examples of the carboxylic acid (PR-A) include 3-sulfopropanoic acid, 4-sulfobutanoic acid, 5-sulfopentanoic acid, 3-phosphonopropionic acid, and 4-phosphonobutyric acid.
  • Examples of the dicarboxylic anhydride (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, succinic anhydride , Maleic anhydride, methyl maleic anhydride, glutaconic anhydride and the like.
  • the sulfanyl compound (PR-C) for example, 2-aminobenzenethiol, 3-aminobenzenethiol, 4-aminobenzenethiol, 1-amino-2-naphthalenitol and the like can be mentioned.
  • the amount of the sulfanyl compound (PR-C) to be used is generally 1.0 to 3.0 mol per 1 mol of the carboxylic acid (PR-A) or dicarboxylic anhydride (PR-B). It is 0 molar equivalent, preferably 1.0 to 1.5 molar equivalent.
  • the compound ( ⁇ ) synthesis reaction is preferably performed in the presence of a solvent in order to efficiently obtain a desired compound.
  • a 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; cyclohexane; Cycloalkane solvents such as cycloheptane and decalin; halogen solvents such as chloroform and methylene chloride; ketone solvents such as acetone and methyl ethyl ketone; amide solvents such as dimethylformamide.
  • the amount of the solvent to be used is generally 100 to 50,000 parts by mass based on 100 parts by mass of the total of the carboxylic acid (PR-A) or the dicarboxylic anhydride (PR-B) and the sulfanyl compound (PR-C). Preferably it is 500 to 10,000 parts by mass.
  • the reaction time of the compound ( ⁇ ) synthesis reaction is not particularly limited, but is usually about 15 minutes to 96 hours, preferably 1 to 24 hours.
  • the reaction temperature may be appropriately selected below the boiling point of the solvent, but is usually about 20 to 100 ° C.
  • the compound ( ⁇ ) obtained in the step P1-2 is a novel compound and is useful as an intermediate for producing the chromatography carrier of the present invention.
  • Step P2 is a step of bringing the raw material support into contact with compound ( ⁇ ).
  • the amount of compound ( ⁇ ) to be used is preferably 0.1 to 20 molar equivalents, more preferably 0.5 to 5 molar equivalents, per 1 mol of the functional group capable of reacting with the sulfanyl group contained in the raw material support. is there.
  • Step P2 is preferably performed in the presence of a solvent in order to efficiently obtain a desired carrier.
  • a solvent include water; alcohols such as methanol, ethanol, propanol, isopropyl alcohol, and n-butyl alcohol; and sulfoxides such as dimethyl sulfoxide. 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 in step P2 is preferably 8 to 16, more preferably 10 to 14, in order to efficiently obtain a desired carrier.
  • the reaction time in step P2 is not particularly limited, but is usually about 0.5 to 72 hours, and preferably 1 to 48 hours.
  • the reaction temperature may be appropriately selected below the boiling point of the solvent, but is usually about 10 to 100 ° C.
  • the carrier wherein X in the formula (1) is a sulfinyl group or a sulfonyl group
  • the carrier wherein X in the formula (1) obtained in the above step P2 is a thio group may be oxidized.
  • the above oxidation may be performed with reference to known methods described in International Publication No. WO 2015/119255, JP-A-2016-50897, and the like, and specific examples thereof include a method using an oxidizing agent.
  • the oxidizing agent examples include organic oxidizing agents such as peracetic acid, perbenzoic acid, metachloroperbenzoic acid, and tert-butyl hydroperoxide; and inorganic oxidizing agents such as hydrogen peroxide, chromic acid, and permanganate.
  • organic oxidizing agents such as peracetic acid, perbenzoic acid, metachloroperbenzoic acid, and tert-butyl hydroperoxide
  • inorganic oxidizing agents such as hydrogen peroxide, chromic acid, and permanganate.
  • these oxidizing agents can be used alone or in combination of two or more.
  • a sulfanyl compound such as methanethiol or thioglycerol may be brought into contact with the carrier obtained as described above to open an unreacted functional group.
  • This process may be performed with reference to the description in International Publication No. WO 2015/119255.
  • 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 which can be produced as described above, has a large dynamic binding capacity for a target substance and has excellent performance for separating a target substance and contaminants.
  • each protein can be easily and efficiently separated from a mixed solution containing a plurality of types of proteins, which is useful for separating a target protein from a non-target protein.
  • the reason why such an effect is exerted is not necessarily clear, but is not limited to having a ligand containing a divalent aromatic hydrocarbon group and an anionic functional group, but also represented by Q in the formula (1).
  • a divalent aromatic hydrocarbon group is adjacent to a thio group, a sulfinyl group or a sulfonyl group represented by X, and a divalent aromatic hydrocarbon group represented by Q and an anionic functional group represented by A
  • the chromatography column of the present invention is one in which the chromatography carrier of the present invention is packed in a column container.
  • the chromatography column of the present invention is suitable for protein purification and also for antibody purification. It is also very useful for mixed mode chromatography of hydrophobic chromatography and cation exchange chromatography.
  • 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 performed in the same manner as a known method except that the chromatography carrier of the present invention is used.
  • the method may be performed with reference to known methods described in JP-T-2007-525501, JP-T-2008-505851, JP-A-2010-210497, JP-T-2015-501310, and the like.
  • a method including a step of contacting the chromatography carrier of the present invention with a sample containing a target substance may be mentioned.
  • the method further includes an elution step of eluting the target substance captured by the carrier in this step.
  • a dissociation solution for dissociating the ligand and the target substance is usually used.
  • purification may be performed using the chromatography column of the present invention. Examples of such a method include a method including a step of passing a sample containing a target substance through the chromatography column of the present invention, and preferably further include an elution step as described above.
  • Examples of the target substance include biologically relevant substances such as proteins, peptides, amino acids, sugars, polysaccharides, lipids, vitamins, DNA, and RNA. Proteins and peptides are preferable, and proteins are more preferable.
  • Examples of the target protein include an antigen, an antibody, and a virus particle, and an antibody is preferable. 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.
  • the class of the antibody includes IgG, IgE, IgM, IgD, and IgA.
  • the subclass is not particularly limited.
  • sample containing the target substance examples include, for example, whole blood, serum, plasma, blood components, various blood cells, blood clots, blood components such as platelets, urine, semen, breast milk, sweat, interstitial fluid, interstitial lymph, Examples include various fluids such as bone marrow fluid, tissue fluid, saliva, gastric fluid, synovial fluid, pleural effusion, bile, ascites fluid, amniotic fluid, and other body fluids, bacterial fluid, cell culture medium, cell culture supernatant, and crushed tissue cell fluid.
  • Contaminants include, for example, non-target proteins. Specifically, when an antibody is used as the target substance, it is a protein other than the antibody. When a virus particle is used as a target substance, DNA or a host cell-derived protein is used.
  • the method for purifying the target substance 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 collected.
  • Step S1 a step of contacting the chromatography carrier of the present invention with a sample containing a target substance
  • Step S2 a dissociation solution for dissociating the ligand of the carrier and the target substance, and capturing the target substance in Step S1 Contacting the carrier
  • Step S1 is a step of bringing the chromatography carrier of the present invention into contact with a sample containing a target substance.
  • the target substance is captured on the chromatography carrier of the present invention.
  • 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 3 to 8, in order to efficiently separate the target substance from contaminants.
  • the salt concentration of the buffer is preferably 0 to 0.5M.
  • Step S2 is a step of contacting a dissociation solution for dissociating the ligand of the chromatography carrier of the present invention and the target substance with the carrier having captured the target substance in step S1.
  • the target substance captured by the carrier is eluted by step S2
  • 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 the contaminant.
  • the target substance can be separated from impurities.
  • 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 3 to 8, in order to efficiently separate the target substance from contaminants.
  • the target substance can be eluted from the carrier by a stepwise method, a gradient method, or a combination of both.
  • the salt concentration of the buffer may be usually increased to 1.0 to 3.0M.
  • the stepwise method refers to a method in which a target substance is eluted from a carrier by changing the salt concentration stepwise using a plurality of types of buffers having different salt concentrations.
  • the gradient method is a method in which the target substance is eluted from the carrier by continuously changing the salt concentration.
  • the concentration is preferably increased to 0.001 to 0.2 M / min, more preferably 0.01 to 0.1 M / min.
  • Example 1 (1) 2.69 g of polyvinyl alcohol (PVA-217 manufactured by Kuraray Co., Ltd.) was added to 448 g of pure water, and the polyvinyl alcohol was dissolved by heating and stirring. After cooling, sodium dodecyl sulfate (manufactured by Wako Pure Chemical Industries) 0 0.045 g was added and stirred to prepare an aqueous solution S.
  • PVA-217 manufactured by Kuraray Co., Ltd.
  • a monomer composition composed 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 Company).
  • the product was dissolved in 29.38 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) to prepare a monomer solution.
  • the whole amount of the aqueous solution S was put into a separable flask, and a thermometer, a stirring blade and a cooling tube were attached, and the flask was set in a hot water bath, and stirring was started under a nitrogen atmosphere.
  • the whole amount of the monomer solution was put into a separable flask, and heated in a hot water bath.
  • 2,2′-azoisobutyronitrile manufactured by Wako Pure Chemical Industries
  • porous carrier 1 The porous particles contained in this dispersion are referred to as “porous carrier 1”.
  • Example 2 The same as steps (1) to (4) of Example 1 except that 0.976 g of the compound (EA1) in Step (3) of Example 1 was changed to 1.036 g of the compound (EA2) The operation was performed.
  • the porous particles contained in the obtained dispersion are referred to as “chromatography carrier V2”.
  • Example 3 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. Except for the above, the same operation as in steps (1) to (4) of Example 1 was performed.
  • the porous particles contained in the obtained dispersion are referred to as “chromatography carrier V3”.
  • the porous carrier prepared in place of the porous carrier 1 by using ethylene glycol dimethacrylate is referred to as “porous carrier 2”.
  • Example 4 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 the steps (1) to (4) of Example 1 were repeated except that 0.976 g of the compound (EA1) in Step (3) of Example 1 was changed to 1.036 g of the compound (EA2). The same operation was performed.
  • the porous particles contained in the obtained dispersion are referred to as “chromatography carrier V4”.
  • a 20 mM sodium phosphate buffer (pH 7.0) was passed four times the column volume to equilibrate.
  • trypsinogen, cytochrome C and lysozyme were dissolved in a 20 mM sodium phosphate buffer (pH 7.0), and a protein mixture (trypsinogen: 0.2 mg / mL, cytochrome C: 0.1 mg / mL, lysozyme: 0. 2 mg / mL).
  • 100 ⁇ L of the above protein mixture was passed through the column at a linear flow rate of 300 cm / hr.
  • the elution volume V is the volume value of the eluate obtained from the contact point that intersects the baseline when a perpendicular line is drawn from the point of the peak maximum absorbance value to the baseline.
  • the peak width W is a volume value obtained from the width between two contact points that intersect the base line when two tangents passing through the inflection points on the left and right of the peak are drawn.
  • the peak width W was determined from the contact point that intersected the baseline. It can be said that the smaller the value of the peak width W, the higher the concentration of the protein recovered, and the better the protein separation ability.
  • the resolving powers Rs1 and Rs2 were calculated from the following formulas. It can be said that the larger the value of the resolving power Rs1 and Rs2, the better the protein resolving power. Table 1 shows the results of Test Example 2.
  • 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.
  • the resolving power Rs1 was larger than in the case where a carrier having a butyric acid residue or L-tryptophan residue as a ligand was used (Comparative Examples 2-3 and Comparative Examples 5-6).
  • the measured value of DBC was also large.
  • the carrier having 2- (benzoylamino) -4-mercaptobutyric acid residue or L-tryptophan residue as a ligand depends on the type of the support (base particle). In the first place, lysozyme was not eluted, and the resolution Rs2 could not be calculated.

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Abstract

L'invention concerne un support de chromatographie qui comprend une capacité de liaison cinétique élevée à une substance cible et présente une excellente performance pour séparer une substance cible vis-à-vis de contaminants. Ce support de chromatographie comprend un corps de support et un ligand représenté par la formule (1). [Dans la formule (1), A représente un groupe fonctionnel anionique, Q représente un groupe hydrocarboné aromatique divalent, R1 représente un groupe organique divalent , R2 représente un atome d'hydrogène ou un groupe hydrocarboné , X représente un groupe thio, un groupe sulfinyle ou un groupe sulfonyle, et représente une liaison.]
PCT/JP2019/033820 2018-08-30 2019-08-29 Support de chromatographie WO2020045541A1 (fr)

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Citations (4)

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US20020156288A1 (en) * 2001-01-03 2002-10-24 Giuseppe Caputo Symmetric, monofunctionalised polymethine dyes labelling reagents
JP2013033040A (ja) * 2011-07-27 2013-02-14 Pall Corp 混合モード配位子
JP2015501310A (ja) * 2011-10-19 2015-01-15 バイオ−ラッド ラボラトリーズ インコーポレーティッド タンパク質の混合モードクロマトグラフィー精製用の固相
JP2017138149A (ja) * 2016-02-02 2017-08-10 日立化成株式会社 分離材及びカラム

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020156288A1 (en) * 2001-01-03 2002-10-24 Giuseppe Caputo Symmetric, monofunctionalised polymethine dyes labelling reagents
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