Anion Exchange-Hydrophobic Mixed-Mode Chromatography Resin

Abstract

Chromatography resins with anion exchange-hydrophobic mixed-mode ligands and methods for using such resins are provided.

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Application No. 62 / 896,196, filed Sep. 5, 2019, the content of which is incorporated by reference herein.

Background Art

[0002] Primarily, the extraction of immunoglobulins from a feedstock fluid that is mainly a mammalian body fluid or cell culture harvest is important for obtaining immunoglobulins in a sufficiently concentrated or purified form for diagnostic, therapeutic uses, and general laboratory research. Similarly, the purification of other types of proteins and other molecules from biological samples is also important.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Summary of the Invention

Means for Solving the Problems

[0004] There is provided a chromatography resin comprising a chromatography matrix linked to an anion exchange - hydrophobic mixed - mode ligand. In some embodiments, the chromatography resin is chromatography matrix-(X)-N(R 1 )-(R 2 -L) n -Ar or its anion salt having . Wherein, X is a spacer, R 1 is hydrogen, or 、- C1-C6 alkyl substituted with OH, optionally wherein R R 2 is C2-C6 alkyl, or C4-C6 cycloalkyl, L is NR 4 , O, or S, n = 1 or 2, Ar is a 6- to 10-membered ring, when Ar is aryl, the aryl is optionally substituted with up to 5 C1-C3 unsubstituted alkyl, C3-C6 branched alkyl, unsubstituted aryl, or fluorine groups, or when Ar is heteroaryl, the heteroaryl is optionally substituted with up to 4 unsubstituted alkyl groups, provided that R 1 is hydrogen if and R 2 is C2 alkyl, L is NR 4 or O, n is 1 in the case of and Ar is not phenyl.

[0005] In some embodiments of the chromatography resin, X is selected from the group consisting of -O-CH2-, -O-CH2-CH2-, -O-CH2-CH2-CH2-, -O-CH2-CH2-CH2-CH2-, -O-CH2-CH(CH2-OH)-(O-CH2-CH(OH)-CH2)2-, -O-CH2-CH2-CH(CH2-OH)-(O-CH2-CH2-CH(OH)-CH2)2-, -O-CH2-CH(OH)-CH2-, -O-CH2-CH2-CH(OH)-CH2-CH2-, -O-CH2-CH(OH)-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH(OH)-CH2- and -CO-NH-C(CH3)2-CO-, R 1 is hydrogen or C1-C3 alkyl, R 2 is C2-C4 alkyl, L is O, n = 1, Ar is a 6-membered ring, When Ar is aryl, the aryl is optionally substituted with up to 4 C1-C2 unsubstituted alkyl, C3-C4 branched alkyl, or fluorine groups, or When Ar is heteroaryl, the heteroaryl is optionally substituted with up to 3 unsubstituted alkyl groups, provided that R 1 is hydrogen if and R 2 is C2 alkyl and n is 1 in the case of and Ar is not phenyl.

BEST MODE FOR CARRYING OUT THE INVENTION

[0006] There is provided a chromatography resin useful for purifying a target biomolecule using anion exchange and hydrophobic mixed-mode chromatography. This chromatography resin enables efficient purification of a target biomolecule (e.g., a recombinant protein) from a sample. In one embodiment, the chromatography resin is useful for separating a target protein from one or more components (e.g., contaminants) in a sample.

[0007] Definitions Unless otherwise specified, the terms used in this application, including the specification and claims, are defined as follows. As used in this specification and claims, the singular form includes plural referents unless the context clearly dictates otherwise. Definitions of standard chemical terms are found in references such as Carey and Sundberg (2007) "Advanced Organic Chemistry 5th Ed." Vols. A and B, Springer Science+Business Media LLC, New York. The practice of the present invention uses conventional methods of synthetic organic chemistry, mass spectrometry, chromatography preparation and analysis methods, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology unless otherwise noted.

[0008] As used herein, the term "alkyl" refers to a straight or branched chain saturated aliphatic radical having from 1 to 10 carbon atoms. For example, C1-C6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, and / or hexyl. Alkyl can contain any number of carbons such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6 and 5-6. An alkyl group is typically monovalent, but can be divalent, such as when an alkyl group links two chemical groups together.

[0009] As used herein, the term "cycloalkyl" refers to a monocyclic alkyl having the indicated number of carbon atoms. Examples of monocycles include, for example, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0010] As used herein, the term "aryl" refers to a monocyclic or fused bicyclic aromatic ring assembly. For example, aryl is phenyl or naphthyl. An aryl group can optionally be substituted with 1, 2, 3, 4, or 5 unsubstituted alkyl groups, unsubstituted aryl groups, or fluorine groups.

[0011] The term "heteroatom" refers to N, O and S.

[0012] As used herein, the term "heteroaryl group" refers to an aromatic group containing one heteroatom as a ring member. Examples include, but are not limited to, pyrrole, furan, thiophene, and pyridine. A heteroaryl group can optionally be substituted with 1, 2, 3, or 4 alkyl groups.

[0013] "Anion salt" is formed by a base (e.g., amino or alkylamino) group in the ligand. Examples of anion salts include, but are not limited to, halides, sulfonates, sulfates, carboxylates, phosphates, acetates, citrates, and nitrates. Examples of acid addition salts include, but are not limited to, hydrochlorides, hydrobromides, hydroiodides, sulfates, acetates, citrates, and nitrates.

[0014] As used herein, the term "spacer" refers to a molecule having 1 to 30 atoms selected from H, C, N, O, and S. The spacer has a neutral charge and contains a cyclic group. The spacer links the chromatography ligand to the chromatography matrix. The types of bonds used to link the spacer to the chromatography matrix include, but are not limited to, amides, amines, ethers, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas. In some embodiments, the bond used to link the spacer to the chromatography matrix is an amine, ether, or amide.

[0015] "Biological sample" refers to any composition containing a target molecule of biological origin (a "biomolecule") that is desired to be purified. In some embodiments, the target molecule to be purified is an antibody or a non-antibody protein.

[0016] "Antibody" refers to an immunoglobulin, complex (e.g., fusion), or fragment form thereof. The term includes polyclonal or monoclonal antibodies of the IgA, IgD, IgE, IgG, and IgM classes, including, but not limited to, naturally or genetically modified forms such as humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, grafted, and in vitro-generated antibodies, derived from human or other mammalian cell lines. "Antibody" also includes, but is not limited to, complex forms including fusion proteins containing an immunoglobulin portion. "Antibody" includes antibody fragments such as Fab, F(ab′)2, Fv, scFv, Fd, dAb, Fc, whether or not retaining antigen-binding functionality.

[0017] The term "protein" refers to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers (e.g., recombinant proteins).

[0018] "Binding elution mode" refers to an operational approach to chromatography in which buffer conditions are established such that, when a sample is applied to a ligand, the target molecule, and optionally undesirable contaminants, bind to the ligand. The fraction of interest is then achieved by changing the conditions such that the target elutes from the support. In some embodiments, the contaminants remain bound after the target elutes. In some embodiments, the contaminants are flowed through or bound and eluted prior to elution of the target.

[0019] "Flow-through mode" refers to an operational approach to chromatography in which buffer conditions are established such that removal of those from a sample is achieved by the purified target molecule flowing through a chromatography support containing a ligand while at least some sample contaminants are selectively retained.

[0020] Chromatography ligand In one embodiment, the mixed-mode chromatography support and ligand are has the following formula. Chromatography matrix-(X)-N(R 1 )-(R 2 -L) n -Ar or its anion salt Wherein X is a spacer, R 1 is hydrogen, or 、- is C1-C6 alkyl substituted with OH optionally and is R 2 is C2-C6 alkyl, or C4-C6 cycloalkyl, L is NR 4 , O, or S, n = 1 or 2, Ar is a 6- to 10-membered ring, When Ar is aryl, the aryl is optionally substituted with up to 5 C1-C3 unsubstituted alkyl, C3-C6 branched alkyl, unsubstituted aryl, or fluorine groups, or, When Ar is heteroaryl, the heteroaryl is optionally substituted with up to 4 unsubstituted alkyl groups, provided that R 1 is hydrogen if , R 2 is C2 alkyl, L is NR 4 or O, and n is 1 in the case of , Ar is not phenyl.

[0021] The charge of the nitrogen adjacent to the spacer is pH-dependent. Thus, these resins perform weak ion exchange.

[0022] In the first aspect of the first embodiment, R 1 is hydrogen or C1-C3 alkyl. Alternatively, R 1 is hydrogen or C1-C2 alkyl.

[0023] In the second aspect of the first embodiment, R 2 is C2-C4 alkyl. Alternatively, R 2 is C2 or C3 alkyl.

[0024] In the third aspect of the first embodiment, L is NR 4 or O, or NR 4 or S. Alternatively, L is O.

[0025] In the fourth aspect of the first embodiment, n is 1.

[0026] In the fifth aspect of the first embodiment, Ar is a 6-membered ring. When Ar is aryl, the aryl is optionally substituted with up to 4 C1-C2 unsubstituted alkyl, C3-C4 branched alkyl, or fluorine groups, or when Ar is heteroaryl, the heteroaryl is optionally substituted with up to 3 alkyl groups, provided that R 1 is hydrogen if , R 2 is C2 alkyl, L is NR 4 or O, n is 1 in the case of , Ar is not phenyl. Alternatively, Ar is optionally substituted with up to 3 C1-C2 unsubstituted alkyl or fluorine groups, provided that R 1 is hydrogen if , R 2 is C2 alkyl, L is NR 4 or O, n is 1 in the case of , Ar is not phenyl. Alternatively, Ar is optionally substituted with 1 or 2 C1-C2 unsubstituted alkyl, provided that R 1 is hydrogen if , R 2 is C2 alkyl, L is NR 4 or O, n is 1 in the case of , Ar is not phenyl. Alternatively, Ar is a chromatography matrix-(X)-N(R 1 )-(R 2 -L) n- is phenyl substituted at the para or meta position with one C1-C3 unsubstituted alkyl, C3-C6 branched alkyl, unsubstituted aryl, or fluorine group. Alternatively, Ar is heteroaryl, and the heteroatom in the heteroaryl is N. Alternatively, Ar is unsubstituted heteroaryl. In yet another option, Ar is pyridyl.

[0027] In a sixth aspect of the first embodiment, X is attached to the chromatography matrix via a bond selected from amide, amine, ether, ester, carbamate, urea, thioether, thiocarbamate, thiocarbonate and thiourea. Alternatively, the bond is amine, ether or amide.

[0028] In a seventh aspect of the first embodiment, X is -O-CH2-, -O-CH2-CH2-, -O-CH2-CH2-CH2-, -O-CH2-CH2-CH2-CH2-, -O-CH2-CH(CH2-OH)-(O-CH2-CH(OH)-CH2)2-, -O-CH2-CH2-CH(CH2-OH)-(O-CH2-CH2-CH(OH)-CH2)2-, -O-CH2-CH(OH)-CH2-, -O-CH2-CH2-CH(OH)-CH2-CH2-, -O-CH2-CH(OH)-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH(OH)-CH2- and -CO-NH-C(CH3)2-CO-. Alternatively, X is selected from the group consisting of -O-CH2-, -O-CH2-CH2-, -O-CH2-CH2-CH2-, -O-CH2-CH2-CH2-CH2- and -O-CH2-CH(OH)-CH2-.

[0029] In the second embodiment, the chromatography resin is has the following formula. chromatography matrix-(X)-N(R 1 )-(R 2 -L) n -Ar or its anion salt wherein X is selected from the group consisting of -O-CH2-, -O-CH2-CH2-, -O-CH2-CH2-CH2-, -O-CH2-CH2-CH2-CH2-, -O-CH2-CH(CH2-OH)-(O-CH2-CH(OH)-CH2)2-, -O-CH2-CH2-CH(CH2-OH)-(O-CH2-CH2-CH(OH)-CH2)2-, -O-CH2-CH(OH)-CH2-, -O-CH2-CH2-CH(OH)-CH2-CH2-, -O-CH2-CH(OH)-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH(OH)-CH2- and -CO-NH-C(CH3)2-CO-; R 1 is hydrogen or C1-C3 alkyl; R 2 is C2-C4 alkyl; L is O; n = 1, and Ar is a 6-membered ring, and when Ar is aryl, the aryl is optionally substituted with up to 4 C1-C2 unsubstituted alkyl, C3-C6 branched alkyl, or fluorine groups, or when Ar is heteroaryl, the heteroaryl is optionally substituted with up to 3 unsubstituted alkyl groups, provided that when R 1 is hydrogen, R if is C2 alkyl, n is 1, and 2 Ar is not unsubstituted phenyl. in the case of 。

[0030] In the first aspect of the second embodiment, R 1 is hydrogen or C1-C2 alkyl.

[0031] In the second aspect of the second embodiment, R 2 is C2 or C3 alkyl.

[0032] In the third aspect of the second embodiment, Ar is the most substituted with up to 3 C1-C2 unsubstituted alkyl or fluorine groups, optionally ​Replaced, phenyl, naphthyl, or pyridyl, provided that R 1 is hydrogen present 、R 2 is C2 alkyl, L is NR 4 or O, and n is 1 in the case of 、Ar is not phenyl. Alternatively, Ar is optionally phenyl substituted with one or two C1-C2 unsubstituted alkyls, provided that R 1 is hydrogen if 、R 2 is C2 alkyl, L is NR 4 or O, and n is 1 in the case of 、Ar is not phenyl. Alternatively, Ar is phenyl substituted with one C1-C2 unsubstituted alkyl at the para or meta position with respect to Chromatography matrix-(X)-N(R 1 )-(R 2 -L) n . Alternatively, Ar is heteroaryl, and the heteroatom in the heteroaryl is N. Alternatively, Ar is unsubstituted heteroaryl. In yet another option, Ar is pyridyl.

[0033] In a third embodiment, the chromatography resin is has the following formula. Chromatography matrix-(X)-N(R 1 )-(R 2 -L) n -Ar or its anion salt wherein X is selected from the group consisting of -O-CH2-, -O-CH2-CH2-, -O-CH2-CH2-CH2-, -O-CH2-CH2-CH2-CH2- and -O-CH2-CH(OH)-CH2- R 1 is hydrogen or C1-C2 alkyl R 2 is C2-C3 alkyl L is O n = 1, and Ar is the most substituted with up to three C1-C2 unsubstituted alkyls optionallyis replaced by phenyl, naphthyl, or pyridyl, provided that R 1 is hydrogen if and R 2 is C2 alkyl and n is 1 in the case of and Ar is not unsubstituted phenyl.

[0034] In the first aspect of the third embodiment, Ar is optionally substituted with one or two C1-C2 unsubstituted alkyls, provided that when R 1 is hydrogen, R 2 is C2 alkyl and Ar is not phenyl. Alternatively, Ar is a phenyl substituted with a methyl group at the para or meta position with respect to chromatographic matrix-(X)-N(R 1 )-(R 2 -L) n -. Alternatively, Ar is unsubstituted phenyl, provided that R 1 is not hydrogen and R 2 is not C2 alkyl.

[0035] In the fourth embodiment, -(X)-N(R 1 )-(R 2 -L) n -Ar is any one of the ligands in Table 1.

[0036]

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

[0037] In the fifth embodiment, the chromatography resin is chromatography matrix-(X)-N-[(R 2 -L) n -Ar]2 or its anion salt. In the formula, X is a spacer, R 2 is C2-C6 alkyl, or C4-C6 cycloalkyl, L is NR 4 , O, or S, n = 1 or 2, and Ar is a 6- to 10-membered ring, and, when Ar is aryl, the aryl is optionally substituted with up to 5 C1-C3 unsubstituted alkyl, C3-C6 branched alkyl, unsubstituted aryl, or fluorine groups, or, when Ar is heteroaryl, the heteroaryl is optionally substituted with up to 4 unsubstituted alkyl groups.

[0038] In the first aspect of the fifth embodiment, R 2 is C2-C4 alkyl. Alternatively, R 2 is C2 or C3 alkyl.

[0039] In the second aspect of the fifth embodiment, L is NR 4 or O, or NR 4 or S. Alternatively, L is O.

[0040] In the third aspect of the fifth embodiment, n is 1.

[0041] In the fourth aspect of the fifth embodiment, Ar is a 6-membered ring. When Ar is aryl, the aryl is optionally substituted with up to 4 C1-C2 unsubstituted alkyl, C3-C4 branched alkyl, or fluorine groups, or when Ar is heteroaryl, the heteroaryl is optionally substituted with up to 3 alkyl groups. Alternatively, Ar is optionally phenyl, naphthyl, or pyridyl substituted with up to 3 C1-C2 unsubstituted alkyl or fluorine groups. Alternatively, Ar is phenyl substituted with 1 or 2 C1-C2 unsubstituted alkyls. Alternatively, Ar is -(R 2 -L) n - and is phenyl substituted with 1 C1-C3 unsubstituted alkyl, C3-C6 branched alkyl, unsubstituted aryl, or fluorine group at the para or meta position relative to it. Alternatively, Ar is heteroaryl and the heteroatom in the heteroaryl is N. Alternatively, Ar is unsubstituted heteroaryl. In yet another option, Ar is pyridyl.

[0042] In the fifth aspect of the fifth embodiment, X is attached to the chromatography matrix via a bond selected from amide, amine, ether, ester, carbamate, urea, thioether, thiocarbamate, thiocarbonate, and thiourea. Alternatively, the bond is an amine, ether, or amide.

[0043] In the sixth aspect of the fifth embodiment, X is selected from the group consisting of -O-CH2-, -O-CH2-CH2-, -O-CH2-CH2-CH2-, -O-CH2-CH2-CH2-CH2-, -O-CH2-CH(CH2-OH)-(O-CH2-CH(OH)-CH2)2-, -O-CH2-CH2-CH(CH2-OH)-(O-CH2-CH2-CH(OH)-CH2)2-, -O-CH2-CH(OH)-CH2-, -O-CH2-CH2-CH(OH)-CH2-CH2-, -O-CH2-CH(OH)-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH(OH)-CH2- and -CO-NH-C(CH3)2-CO-. Alternatively, X is selected from the group consisting of -O-CH2-, -O-CH2-CH2-, -O-CH2-CH2-CH2-, -O-CH2-CH2-CH2-CH2- and -O-CH2-CH(OH)-CH2-.

[0044] In the sixth embodiment, the chromatography resin is chromatography matrix-(X)-N-[(R 2 -L) n -Ar]]2 or its anion salt. In the formula,[[]] X is selected from the group consisting of -O-CH2-, -O-CH2-CH2-, -O-CH2-CH2-CH2-, -O-CH2-CH2-CH2-CH2-, -O-CH2-CH(CH2-OH)-(O-CH2-CH(OH)-CH2)2-, -O-CH2-CH2-CH(CH2-OH)-(O-CH2-CH2-CH(OH)-CH2)2-, -O-CH2-CH(OH)-CH2-, -O-CH2-CH2-CH(OH)-CH2-CH2-, -O-CH2-CH(OH)-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH(OH)-CH2- and -CO-NH-C(CH3)2-CO-, R 2 is C2-C4 alkyl, L is O, n = 1, and Ar is a 6-membered ring, and When Ar is aryl, the aryl is optionally substituted with up to 4 C1-C2 unsubstituted alkyl, C3 or C4 branched alkyl or fluorine groups, or When Ar is heteroaryl, the heteroaryl is optionally substituted with up to 3 unsubstituted alkyl groups.

[0045] In the first aspect of the sixth embodiment, R 2 is C2 or C3 alkyl.

[0046] In the second aspect of the sixth embodiment, Ar is optionally phenyl, naphthyl, or pyridyl substituted with up to 3 C1-C2 unsubstituted alkyl or fluorine groups. Alternatively, Ar is optionally phenyl substituted with 1 or 2 C1-C2 unsubstituted alkyls. Alternatively, Ar is phenyl substituted with 1 C1 or C2 unsubstituted alkyl at the para or meta position relative to -(R 2 -L) n -. Alternatively, Ar is heteroaryl, and the heteroatom in the heteroaryl is N. Alternatively, Ar is unsubstituted heteroaryl. In yet another option, Ar is pyridyl.

[0047] In the seventh embodiment, the chromatography resin is chromatography matrix-(X)-N-[(R 2 -L) n -Ar]2 or its anion salt. In the formula, X is selected from the group consisting of O-CH2-, -O-CH2-CH2-, -O-CH2-CH2-CH2-, -O-CH2-CH2-CH2-CH2- and -O-CH2-CH(OH)-CH2-, R 1 is hydrogen, C1, or C2 alkyl, R 2 is C2 or C3 alkyl, L is O, n = 1, and Ar is optionally phenyl, naphthyl, or pyridyl, substituted with up to three C1-C2 unsubstituted alkyls.

[0048] In the first aspect of the seventh embodiment, Ar is optionally phenyl substituted with one or two C1-C2 unsubstituted alkyls. Alternatively, Ar is -(R 2 -L) n -phenyl substituted with a methyl group at the para or meta position with respect to -. Alternatively, Ar is unsubstituted phenyl.

[0049] In the eighth embodiment, -(X)-N-[(R 2 -L) n -Ar]2 is any one of the ligands in Table 2.

[0050]

Table 7

Table 8

Table 9

[0051] In some embodiments, the anion salt is a hydrochloride, phosphate, or sulfate.

[0052] The chromatography matrix is a polymer functionalized so as to be able to form a bond with spacer X. The polymer is preferably a hydrophilic polymer. The polymer is insoluble in water. Suitable polymers include, for example, polyhydroxy polymers based on polysaccharides such as agarose, dextran, cellulose, starch, pullulan; fully synthetic polymers such as polyacrylamide, polymethacrylamide, poly(hydroxyalkyl vinyl ether), poly(hydroxyalkyl acrylate) and polymethacrylate (e.g., polyglycidyl methacrylate), polyvinyl alcohol, polymers based on styrene and divinylbenzene; and copolymers containing two or more of the monomers corresponding to the above polymers. Suitable synthetic polymers include, but are not limited to, Fractogel and Eshmuno®-based beads from Sigma-Millipore-EMD-Merck, Toyoparl AF-Eposy-650M and Toyopearl AF-Tresyl-650M from Tosoh Bioscience, POROS media from ThermoFisher Scientific, Bio-Gel P and Macro Prep from Bio-Rad, HEMA and Separon from TESSEK, activated Sepharose 6B, activated Sepharose 4B, activated Sepharose 4 Fast Flow from GE Healthcare Life Sciences, and Hyper D and Trisacryl media from Pall. Polymers that are soluble in water are derivatized to be insoluble, for example, by crosslinking to an insoluble object via adsorption or covalent bonding, and by coupling. Hydrophilic groups are introduced by the polymerization of monomers that represent groups convertible to OH on a hydrophobic polymer (e.g., on a copolymer of monovinyl and divinylbenzene), or by the adsorption of a suitable compound such as a hydrophilic polymer, by hydrophilic modification of the final polymer.Examples of monomers polymerized to obtain a useful matrix are, in some cases, vinyl acetate having a functional group, vinylpropylamine, acrylic acid, methacrylate, butyl acrylate, acrylamide, methacrylamide, vinylpyrrolidone (vinylpyrrolidinone). Crosslinking agents are also useful in many embodiments and, when present, in some embodiments, constitute a molar ratio of about 0.1 to about 0.7 relative to all monomers. Examples of crosslinking agents include dihydroxyethylenebisacrylamide, diallyltartardiamide, triallylcitric triamide, ethylene diacrylate, bisacrylcystamine, N,N′-methylenebisacrylamide, and piperazine diacrylamide. In some embodiments, the matrix is a UNOsphere® (Bio-Rad, Hercules, Calif.) substrate, a polymer made from water-soluble hydrophilic monomers.

[0053] A chromatography matrix is in the form of particles, a chip, a membrane, or a monolith, i.e., a single block, pellet, or slab of material. Preferably, the chromatography matrix is porous. When used as a matrix, the particles are spheres or beads and can have either a smooth surface or a rough or textured surface. In some cases, some of the pores are through-holes that extend through the particles and serve as channels large enough to allow hydrodynamic flow or rapid diffusion through the pores. When in the form of spheres or beads, the median particle size (the term "particle size" refers to the longest external dimension of the particle) is from about 25 microns to about 150 microns. The disclosure of exemplary matrices and methods for producing them is in Patent Documents 1 to 4.

[0054] The ligand is linked to the chromatography matrix via spacer X. The linkage to the chromatography matrix depends on the particular chromatography matrix used and the chemical groups linked to the chromatography matrix. The ligand can be linked to the chromatography matrix by performing a reaction between the ligand and a functional group on the chromatography matrix. For a chromatography matrix without a suitable functional group, the chromatography matrix can be reacted with a suitable activating reagent to generate a suitable functional group to which the ligand can be added. Reductive amination, epoxidation, or azlactonization are examples of chemical actions that act on aldehyde, epoxide, or azlactone functional groups, respectively.

[0055] In some embodiments, the chromatography matrix includes a diol that is converted to an aldehyde, for example, by conversion with NaIO4. The primary or secondary amine of the ligand is coupled to the aldehyde on the chromatography matrix by a reductive amination reaction according to the following scheme. In this scheme, spacer X is -O-CH2-CH2-CH2-. In this synthetic scheme and other synthetic schemes in the present disclosure, the square represents the matrix, and all couplings are shown individually.

[0056]

Chemical formula

[0057] In some embodiments, the chromatography matrix includes an epoxide group, and the primary or secondary amine in the ligand is linked to the epoxide group via epoxidation according to the following scheme. In this scheme, spacer X is -O-CH2-CH(OH)-CH2-.

[0058]

Chemical formula

[0059] In some embodiments, the chromatography matrix comprises an azlactone ring, and the primary or secondary amine in the ligand is linked to the azlactone ring by the following scheme. In this scheme, the spacer X is -CO-NH-C(CH3)2-CO-.

[0060]

Chemical formula

[0061] In some embodiments, the chromatography matrix comprises a diol, and the primary or secondary amine is linked to the -OH group by activating the resin with two activating reagents, allyl glycidyl ether (AGE) and bromine, according to the following scheme. In this scheme, the spacer X is -O-CH2-CH(CH2-OH)-(O-CH2-CH(OH)-CH2)2-.

[0062]

Chemical formula

[0063] In certain embodiments, the chromatography matrix comprises an -OH group, and the primary or secondary amine is linked to the -OH group by activating the resin with epichlorohydrin according to the following scheme. In this scheme, the spacer X is -O-CH2-CH(OH)-CH2-.

[0064]

Chemical formula

[0065] In some embodiments, the chromatography matrix contains -OH groups, and the primary or secondary amine is linked to the -OH group by activating the resin with 1,4-butanediol diglycidyl ether according to the following scheme. In this scheme, the spacer X is -O-CH2-CH(OH)-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH(OH)-CH2-.

[0066] [Chemical formula]

[0067] Other activating reagents include epibromohydrin (which reacts with -OH functional groups on the chromatography matrix to form epoxide groups), poly(ethylene glycol) diglycidyl ether (which reacts with -OH functional groups on the chromatography matrix to form epoxide groups), halogen-substituted aliphatic substances such as dichloropropanol (which reacts with -OH functional groups on the chromatography matrix to form epoxide groups), divinyl sulfone (which reacts with diol functional groups on the chromatography matrix to form vinyl groups), and sulfonyl chlorides such as tosyl chloride and tresyl chloride (which reacts with -OH functional groups on the chromatography matrix to form sulfonate esters), but are not limited thereto.

[0068] Other spacers include -O-CH2-, -O-CH2-CH2-, -O-CH2-CH2-CH2-CH2-, -O-CH2-CH2-CH(CH2-OH)-(O-CH2-CH2-CH(OH)-CH2)2- and -O-CH2-CH2-CH(OH)-CH2-CH2-, but are not limited thereto.

[0069] The chromatography matrix can be used in any conventional configuration including a packed column and a fluidized or expanded bed column, a monolith or a porous membrane, and by any conventional method including batch mode for loading, washing, and elution, as well as continuous or flow-through mode. In some embodiments, the column diameter ranges from 1 cm to 1 m and the height ranges from 1 cm to 30 cm or more.

[0070] Method A method for purifying a target biomolecule is also provided. In one embodiment, the method includes contacting a sample containing the biomolecule with a chromatography resin, thereby separating the biomolecule from contaminants. Subsequently, the obtained purified biomolecule is recovered. In some embodiments, the target biomolecule is a target protein and the method includes purifying the target protein from contaminants. In some embodiments, the target biomolecule is a monomeric antibody and the method includes purifying the monomeric antibody from aggregated antibodies in the sample.

[0071] The chromatography ligand is useful for purifying target molecules using anion exchange (i.e., when the ligand is positively charged) and hydrophobic mixed-mode chromatography. The conditions can be adjusted to perform the chromatography in bind-and-elute mode or flow-through mode.

[0072] The protein preparations to which the present method is applied include proteins derived from natural, synthetic, or recombinant sources. Unpurified protein preparations are derived from a variety of sources including, but not limited to, plasma, serum, ascitic fluid, milk, plant extracts, bacterial lysates, yeast lysates, or conditioned cell culture media. Partially purified protein preparations are derived from unpurified preparations that have been treated by at least one chromatography, precipitation, other fractionation process, or any combination of the foregoing. In some embodiments, the chromatography step uses any method including, but not limited to, size exclusion, affinity, anion exchange, cation exchange, protein A affinity, hydrophobic interaction, immobilized metal affinity chromatography, or hydroxyapatite chromatography. Precipitation steps include salt or polyethylene glycol (PEG) precipitation, precipitation with organic acids, organic bases, or other agents. Other fractionation processes include, but are not limited to, crystallization, liquid:liquid partitioning, or membrane filtration.

[0073] As is understood in the art, the loading, washing, and elution conditions used in mixed mode chromatography depend on the particular chromatography medium / ligand being used.

[0074] In some binding-elution mode embodiments, loading (i.e., binding the target protein to the matrix), and optionally, washing are performed at a pH above 6.5, e.g., 6.5 - 8, 7 - 9, etc. Some exemplary binding medium - elution conditions are as follows. Binding conditions: 0 - 1000 mM NaCl or 0 - 400 mM NaCl in a suitable buffer (e.g., Tris, Bis-Tris, sodium phosphate, sodium citrate, or sodium acetate), pH 6.5 - 8.5 or 7 - 9. Elution conditions: 10 - 1000 mM NaCl or 20 - 500 mM NaCl, pH 3 - 8.5 or 4 - 6 using a suitable buffer with sodium phosphate, sodium acetate, sodium citrate, arginine, or glycine.

[0075] Optionally, the matrix can be washed under conditions such that some components of the sample are removed from the chromatography matrix while the target biomolecule remains immobilized on the matrix. In some embodiments, the target biomolecule is subsequently eluted by reducing the salt concentration and / or by reducing the pH of the solution contacting the matrix.

[0076] Alternatively, some components of the sample are immobilized on the matrix, but the target biomolecule flows through the chromatography matrix (i.e., is flow-through) in a flow-through mode, and the sample is applied and collected. Some exemplary flow-through conditions are 0-150 mM NaCl, pH 4.0-8.5, and suitable buffers include, for example, 2-(N-morpholino)ethanesulfonic acid (MES), bis-tris, sodium acetate, or citrate-phosphate.

[0077] Examples The following examples are illustrative and not limiting. One of ordinary skill in the art should readily recognize various non-essential parameters that can be changed or modified to yield essentially the same or similar results. Example 1 - Generation of Chromatography Resin with the Ligands in Table 2

[0078] Primary amine ligands 5, 6, 11, and 16 in Table 3 For each of the primary amine ligands 5, 6, 11, and 16 in Table 3, UNOsphere diol (20 mL), a copolymer of 3-allyloxy-1,2-propanediol and vinylpyrrolidone cross-linked with N,N′-methylenebisacrylamide and having a diol density of 200 - 300 μmol / mL, was used in the form of spherical beads. The beads were suspended in either 20 mL of 0.1 M sodium acetate or water. Sodium periodate was added at a concentration within the range of 50 - 100 mM, and the resulting mixture was incubated at room temperature (about 70°F (21°C)) for 3 - 24 hours. The reaction resulted in the conversion of the diol groups to aldehyde groups at 150 - 250 μmol / mL. The resulting aldehyde-functionalized resin was transferred to a 20 mL column and washed there with 100 mL of water.

[0079] Next, for each primary amine ligand, 20 milliliters of UNOsphere aldehyde resin was suspended at pH 7.0 in 20 ml of 0.20 M sodium phosphate containing 0.6 g of the ligand. These mixtures were incubated at room temperature for 15 minutes (shaking, 200 rpm), after which 200 mg of NaBH3CN was added to each mixture and the reaction was continued for 3 - 20 hours. The ligand concentration in each reaction was in the range of 25 - 200 mM. At the end of the reaction, the resins were each transferred to 20 ml columns and washed with 3 column volumes (CV) of water, followed by 1 - 2 CV of 0.1 N HCl, and then 5 CV of water. The ligand density of each resin was in the range of 25 - 100 μmol / ml.

[0080] The structures and densities of the ligands 5, 6, 11, and 16 attached to the UNOsphere aldehyde resin are shown in Table 3.

[0081]

Table 10

[0082] The secondary amine ligands 32, 34, 39, and 44 in Table 4 For each of the secondary amine ligands 32, 34, 39, and 44 in Table 4, UNOsphere diol (100 mL) was used in the form of spherical beads. The beads were suspended in 30 mL of water, 30 mL of 10 N NaOH, and 16 g of Na2SO4 in a shaker at 50 °C and 250 rpm for 10 minutes. 100 ml of allyl glycidyl ether (AGE) was added and held overnight at 50 °C in the same shaker. The resulting AGE-modified resin was washed with 3 × 2 column volumes (CV) of isopropyl alcohol (IPA) and 30 CV of water. The AGE-modified resin was mixed with 100 mL of water and 3.4 g of NaOAC. Bromine liquid was added dropwise to the slurry until an orange color remained (indicating the completion of the reaction of double bonds with bromine). Then, Na2SO3 was added until the orange color disappeared (reducing excess bromine to bromide). The resulting UNOsphere diol bromide resin was washed with 30 CV of water, and was ready for ligand coupling. For each secondary amine ligand, 100 mL of UNOsphere diol bromide resin was mixed with 50 mL of water and 50 mL of IPA. Next, 12.5 g of the ligand was added. Each mixture was incubated overnight at 50 °C in a shaker at 250 rpm. At the end of the reaction, each resin was washed with 2 CV IPA, 2 CV water, 2 CV 1 N HCl, 2 CV water, 2 CV 1 N NaOH, and then 30 CV water to obtain a tertiary amine resin.

[0083] Table 4 shows the number, structure, and density of ligands 32, 34, 39, and 44 attached to the UNOsphere diol bromide resin.

[0084]

Table 11

[0085] Example 2 - Evaluation of amine resins from Tables 3 and 4 using acidic proteins and binding - elution mode The static binding capacity, recovery rate, and purity of two model acidic proteins, CDP - D - glucose 4,6 - dehydratase and human serum albumin, were determined for the resins from Tables 3 and 4 using various binding and elution conditions.

[0086] Materials: 1. Resins from Tables 3 and 4 2. Disposable spin columns (Bio-Rad catalog number 732-6207) 3. Crude E. coli lysate containing CDP-D-glucose 4,6-dehydratase ("Eod") (pI is approximately 6 and is not stable below pH 6) 4. Human serum albumin ("HSA") (fraction V, Sigma catalog number A1653, pI is approximately 4.7) 5. Binding / washing buffer for Eod a. 25 mM sodium phosphate buffer at pH 6.5, 7.5, or approximately 8.5 b. 0, 25, or approximately 50 mM NaCl 6. Binding / washing buffer for HSA a. 25 mM sodium phosphate or sodium acetate buffer at pH 4, 6, or approximately 8 b. 0, 200, or approximately 400 mM NaCl 7. Elution buffer for Eod a. 25 mM sodium phosphate buffer at pH 6.5, 7.5, or approximately 8.5 b. 10, 500, or approximately 1000 mM NaCl 8. Elution buffer for HSA a. 25 mM sodium phosphate or sodium acetate buffer at pH 4, 6, or approximately 8 b. 20, 500, or approximately 1000 mM NaCl

[0087] Method Static Binding Capacity (SBC) Measurement: For each resin and target protein (i.e., Eod and / or HSA) tested, a spin column containing 0.1 ml of resin was pre-equilibrated with an appropriate binding buffer (depending on the target protein) and then incubated with an Eod-containing lysate or HSA solution for 5 minutes. For each resin and target protein, binding / washing buffers at three different pH values (e.g., pH 6.5, 7.5, 8.5 for Eod) were combined with three different NaCl concentrations (e.g., 0, 25, 50 mM NaCl for Eod) and tested with each of three combinations of elution buffer pH and NaCl concentration. Thus, each resin and target protein was tested with nine combinations of binding / washing buffers, and each combination of binding / washing buffers was tested with nine combinations of elution buffers. The column was centrifuged at 1000 xg for 1 minute to remove unbound protein / impurities, washed once with 5 column volumes of an appropriate binding / washing buffer, and then incubated with 5 CV of an appropriate elution buffer (depending on the target protein) to recover the bound target protein. The column was centrifuged again at 1000 xg for 1 minute, and the absorbance of the column effluent at 280 nm was determined along with the absorbance at 280 nm of the original protein solution loaded into the spin column. The static binding capacity was determined by SBC = (total protein loaded - protein in the flow-through fraction) / volume of resin. Target Protein Recovery: For each resin, the protein recovered in the eluate was quantified by the recovery rate (%) = (total protein in the eluate / total loaded protein) x 100 using the absorbance at 280 nm, an extinction coefficient of 1 for Eod, and an extinction coefficient of 0.534 for HSA (a 1 mg / mL solution of the target protein). Target Protein Purity: The purity of the target protein for each resin was evaluated by SDS-PAGE and Bio-Rad's ImageLab software. Optimal Binding and Elution Conditions: The JMP software (SAS Institute) was used to determine the optimal purification conditions for each target protein and resin.

[0088] Results: As a result of SBC, the resin recovery rates are shown in Table 5 (secondary amine resins 5, 6, 11, and 16) and Table 6 (tertiary amine resins 32, 34, 39, and 44). Tables 5 and 6 also list the optimal binding and elution conditions for each resin. In Table 5, "-" represents "about", and in both tables, "NaCl" represents the NaCl concentration.

[0089]

Table 12

[0090]

Table 13

[0091] The data from Tables 5 and 6 indicate the range of optimal binding and elution conditions according to the target protein and resin. For example, for Eod as the target protein, the optimal binding conditions, when tested with resin 5, were pH 6.5 / without NaCl, while for resin 16, the optimal binding conditions were pH 8.5 / 50 mM NaCl. Similarly, for HSA as the target protein, the optimal binding conditions with resin 6 were pH 8.0 / 400 mM NaCl, while for resin 34, the optimal binding conditions were pH 4.0 / without NaCl.

[0092] The optimal elution conditions for resins 5, 11, and 16 tested with Eod as the target protein were high pH (8 - 8.5) and high NaCl concentration (1000 mM). However, for resin 44 tested with HSA, the optimal elution conditions were low pH (4) and low NaCl concentration (20 mM).

[0093] According to the data from Tables 5 and 6, it can be seen that the resin can be used over a wide range of pH values and NaCl concentrations, and the selection of resin for a given application should depend on the target protein of interest.

[0094] All patents, patent applications, and other published references cited in this specification are hereby incorporated by reference in their entirety. Additional disclosure and claimed subject matter

[0095] Item 1 A chromatography matrix covalently bonded to a ligand, having the following formula Chromatography matrix-(X)-N(R 1 )-(R 2 -L) n -Ar or its anion salt (wherein X is a spacer, R 1 is hydrogen, or 、- C1-C6 alkyl substituted with OH optionally is, R 2 is C2-C6 alkyl, or C4-C6 cycloalkyl, L is NR 4 , O, or S, n = 1 or 2, and Ar is a 6- to 10-membered ring, and when Ar is aryl, the aryl is optionally substituted with up to 5 C1-C3 unsubstituted alkyl, C3-C6 branched alkyl, unsubstituted aryl, or fluorine groups, or when Ar is heteroaryl, the heteroaryl is optionally substituted with up to 4 unsubstituted alkyl groups, provided that R 1 is hydrogen if , R 2 is C2 alkyl, L is NR 4 or O, n is 1 in the case of, has , and Ar is not phenyl) having , the chromatography matrix.

[0096] Item 2 X is selected from the group consisting of -O-CH2-, -O-CH2-CH2-, -O-CH2-CH2-CH2-, -O-CH2-CH2-CH2-CH2-, -O-CH2-CH(CH2-OH)-(O-CH2-CH(OH)-CH2)2-, -O-CH2-CH2-CH(CH2-OH)-(O-CH2-CH2-CH(OH)-CH2)2-, -O-CH2-CH(OH)-CH2-, -O-CH2-CH2-CH(OH)-CH2-CH2-, -O-CH2-CH(OH)-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH(OH)-CH2- and -CO-NH-C(CH3)2-CO-; R 1 is hydrogen or C1-C3 alkyl; R 2 is C2-C4 alkyl; L is O; n = 1; Ar is a 6-membered ring; and when Ar is aryl, the aryl is optionally substituted with up to 4 C1-C2 unsubstituted alkyl, C3-C4 branched alkyl, or fluorine groups, or when Ar is heteroaryl, the heteroaryl is optionally substituted with up to 3 unsubstituted alkyl groups; provided that when R 1 is hydrogen if and R 2 is C2 alkyl and n is 1 in the case of then Ar is not phenyl; The chromatography matrix according to item 1.

[0097] Item 3 X is selected from the group consisting of -O-CH2-, -O-CH2-CH2-, -O-CH2-CH2-CH2-, -O-CH2-CH2-CH2-CH2- and -O-CH2-CH(OH)-CH2-; R 1 is hydrogen or C1-C2 alkyl; R 2 is C2-C3 alkyl; L is O; n = 1; and Ar is the most phenyl, naphthyl, or pyridyl, optionally substituted with up to three C1-C2 unsubstituted alkyls, provided that R 1 is hydrogen if and R 2 is C2 alkyl and n is 1 in the case of and Ar is not phenyl, the chromatography matrix according to item 2.

[0098] Item 4 Ar is phenyl substituted with one or two C1-C2 unsubstituted alkyls at the para or meta position with respect to chromatography matrix-(X)-N(R 1 )-(R 2 -L) n the chromatography resin according to any one of items 1 to 3.

[0099] Item 5 -(X)-N(R 1 )-(R 2 -L) n -Ar is any one of the ligands in Table 1, the chromatography resin according to any one of items 1 to 4.

[0100] Item 6 a chromatography resin, chromatography matrix-(X)-N-[(R 2 -L) n -Ar]2 or its anion salt (wherein, X is a spacer, R 2 is C2-C6 alkyl or C4-C6 cycloalkyl, L is NR 4 , O, or S, n = 1 or 2, and Ar is a 6- to 10-membered ring, ​When Ar is aryl, the aryl is optionally substituted with up to 5 C1-C3 unsubstituted alkyl, C3-C6 branched alkyl, unsubstituted aryl, or fluorine groups, or When Ar is heteroaryl, the heteroaryl is optionally substituted with up to 4 unsubstituted alkyl groups), A chromatographic resin which is

[0101] Item 7 X is selected from the group consisting of -O-CH2-, -O-CH2-CH2-, -O-CH2-CH2-CH2-, -O-CH2-CH2-CH2-CH2-, -O-CH2-CH(CH2-OH)-(O-CH2-CH(OH)-CH2)2-, -O-CH2-CH2-CH(CH2-OH)-(O-CH2-CH2-CH(OH)-CH2)2-, -O-CH2-CH(OH)-CH2-, -O-CH2-CH2-CH(OH)-CH2-CH2-, -O-CH2-CH(OH)-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH(OH)-CH2- and -CO-NH-C(CH3)2-CO-; R 2 is C2-C4 alkyl, L is O, n = 1, and Ar is a 6-membered ring, When Ar is aryl, the aryl is optionally substituted with up to 4 C1-C2 unsubstituted alkyl, C3 or C4 branched alkyl or fluorine groups, or When Ar is heteroaryl, the heteroaryl is optionally substituted with up to 3 unsubstituted alkyl groups, the chromatographic matrix according to item 6.

[0102] Item 8 X is selected from the group consisting of -O-CH2-, -O-CH2-CH2-, -O-CH2-CH2-CH2-, -O-CH2-CH2-CH2-CH2- and -O-CH2-CH(OH)-CH2-; R 1 is hydrogen or C1-C2 alkyl, R 2is C2 or C3 alkyl, L is O, n = 1, and Ar is optionally phenyl, naphthyl, or pyridyl, substituted with up to 3 C1-C2 unsubstituted alkyls. The chromatography matrix according to item 7.

[0103] Item 9 Ar is -(R 2 -L) n - at the para or meta position to -, and is substituted with 1 or 2 C1-C2 unsubstituted alkyls, the chromatography resin according to any one of items 6-9.

[0104] Item 10 -(X)-N-[(R 2 -L) n -Ar]2 is any one of the ligands in Table 2, the chromatography resin according to any one of items 6-9.

[0105] Item 11 Ar is heteroaryl, and the heteroatom in the heteroaryl is N, the chromatography resin according to item 1 or 6.

[0106] Item 12 The anion salt is hydrochloride, phosphate, or sulfate, the chromatography resin according to any one of items 1-11.

[0107] Item 13 X is added to the chromatography matrix via an amine, ether, or amide bond, the chromatography resin according to any one of items 1-12.

[0108] Item 14 The chromatography resin prepared by reacting any one of the ligands in Table 1 with the chromatography matrix by any one of reductive amination, epoxidation, or azalactonization.

[0109] Item 15 The chromatography matrix contains an aldehyde group, and any one of the ligands in Table 1 reacts with the chromatography matrix by reductive amination. The chromatography resin described in Item 14

[0110] Item 16 The chromatography matrix contains an epoxide group, and any one of the ligands in Table 1 reacts with the chromatography matrix by epoxidation. The chromatography resin described in Item 14

[0111] Item 17 Before reacting the chromatography matrix with the ligand, the chromatography matrix is reacted with allyl glycidyl ether and bromine, 1,4-butanedial diglycidyl or epichlorohydrin. The chromatography resin described in any one of Items 14 to 16

[0112] Item 18 A chromatography resin prepared by reacting any one of the ligands in Table 2 with a chromatography matrix by epoxidation

[0113] Item 19 A method for purifying a biomolecule, comprising Contacting a sample containing the biomolecule with any one of the chromatography resins of Items 1 to 18, thereby separating the biomolecule from contaminants Collecting the purified biomolecule. A method

[0114] Item 20 The method according to Item 19, wherein the purified biomolecule is a protein

[0115] Item 21 The method according to item 20, wherein the contacting step includes immobilizing the protein on a chromatography matrix, and the collecting step includes eluting the protein from the chromatography matrix.

[0116] Item 22 The method according to item 21, wherein the protein is eluted by a step including reducing the pH of the solution contacting the ligand from about 7-9 to about 4-6.

[0117] Item 23 The method according to item 20, wherein the contacting step includes flowing the protein through the chromatography matrix, and the collecting step includes collecting the flowing protein.

Claims

1. A chromatography resin comprising a chromatography matrix covalently bonded to a ligand via a spacer, wherein the chromatography resin has the following formula Chromatography matrix -(X)-N-[(R 2 -L) n -Ar] 2 or its anion salt (wherein, X is a spacer, R ２ is C 2 to C 6 alkylene, or C 4 to C 6 cycloalkylene, and L is O, n = 1 or 2, and Ar is a 6- to 10-membered ring, and When Ar is aryl, the aryl may have up to 5 C １ -C ３ unsubstituted alkyl, C 3 -C 6 branched alkyl, unsubstituted aryl, or may be substituted with a fluorine group, or when Ar is heteroaryl, the heteroaryl may be substituted with up to 4 unsubstituted alkyl groups) represented by the chromatography resin.

2. X is -O-CH ２ -, -O-CH ２ -CH ２ -, -O-CH ２ -CH ２ -CH ２ -, -O-CH ２ -CH ２ -CH ２ -CH ２ -, -O-CH ２ -CH(CH ２ -OH)-(O-CH ２ -CH(OH)-CH ２ ) ２ -, -O-CH ２ -CH ２ -CH(CH ２ -OH)-(O-CH ２ -CH ２ -CH(OH)-CH ２ ) ２ -, -O-CH ２ -CH(OH)-CH ２ -, -O-CH ２ -CH ２ -CH(OH)-CH ２ -CH ２ -, -O-CH ２ -CH(OH)-CH ２ -O-CH2-CH ２ -CH ２ -CH ２ -O-CH ２ -CH(OH)-CH ２ - and -CO-NH-C(CH ３ ) ２ selected from the group consisting of -CO- R 2 is C 2 to C 4 alkylene, and L is O, n = 1, and Ar is a 6-membered ring, and When Ar is aryl, the aryl may have up to 4 C1-C ２ unsubstituted alkyl, C 3 or C 4 branched alkyl, or may be substituted with a fluorine group, or when Ar is heteroaryl, the heteroaryl may be substituted with up to 3 unsubstituted alkyl groups, The chromatography resin according to claim 1.

3. X is -O-CH 2 -, -O-CH 2 -CH 2 -, -O-CH 2 -CH 2 -CH 2 -, -O-CH 2 -CH 2 -CH 2 -CH 2 - and -O-CH 2 -CH(OH)-CH 2 selected from the group consisting of, R 2 is C 2 or C ３ alkylene, L is O, n = 1, and Ar is phenyl, naphthyl, or pyridyl, each of which may be substituted with up to three C 1 -C 2 unsubstituted alkyl, The chromatography resin according to claim 2.

4. Ar is phenyl substituted with one or two C 1 -C 2 unsubstituted alkyl, and is substituted at the para or meta position relative to the bonding point of the phenyl to L The chromatography resin according to claim 1.

5. The chromatography resin includes a structure selected from the groups consisting of 【Table 1】 wherein the circle is the chromatography matrix and the spacer, The chromatography resin according to any one of claims 1 to 4.

6. Ar is heteroaryl, and the heteroatom in the heteroaryl is N, The chromatography resin according to claim 1.

7. The anion salt is hydrochloride, phosphate, or sulfate, The chromatography resin according to any one of claims 1 to 6.

8. X is added to the chromatography matrix via an amine, ether, or amide bond, The chromatography resin according to any one of claims 1 to 7.

9. A method for preparing a chromatography resin in which a chromatography matrix is linked to a ligand via a spacer containing a functional group, By reacting the functional group bonded to the chromatography matrix with the ligand containing an amine group, the ligand is bonded to the functional group via the amine group, and the chromatography matrix is linked to the ligand via the spacer containing the functional group, and the functional group is an epoxide group, The method for preparing a chromatography resin, wherein the ligand is selected from the group consisting of bis(2-phenoxyethyl)amine, bis[2-(2-phenoxyethoxy)ethyl]amine, bis[2-(3,5-xilyloxy)ethyl]amine, bis[2-(4-biphenylyloxy)ethyl]amine, bis[2-(p-tolyloxy)ethyl]amine, bis[2-(o-tolyloxy)ethyl]amine, bis[2-(m-tolyloxy)ethyl]amine, bis[2-(p-ethylphenoxy)ethyl]amine, bis[2-(p-cumenyloxy)ethyl]amine, bis{2-[p-(tert-butyl)phenoxy]ethyl}amine, bis[2-(1-naphthyloxy)ethyl]amine, bis[2-(p-fluorophenoxy)ethyl]amine, bis[2-(2,5-difluorophenoxy)ethyl]amine, bis[2-(m-fluorophenoxy)ethyl]amine, bis[2-(p-cumenyloxy)ethyl]amine, bis[2-(3,4-difluorophenoxy)ethyl]amine, bis[2-(3,4,5-trifluorophenoxy)ethyl]amine, bis[2-(2,3,4,5,6-pentafluorophenoxy)ethyl]amine, bis[2-(2,6-dimethyl-4-pyridyloxy)ethyl]amine, bis[2-(4-pyridyloxy)ethyl]amine, bis[2-(3-pyridyloxy)ethyl]amine, bis(3-phenoxycyclobutyl)amine, bis(3-phenoxycyclopentyl)amine, and bis(4-phenoxycyclohexyl)amine.

10. A method for purifying a biomolecule, comprising: contacting a sample containing the biomolecule with the chromatography resin according to any one of Claims 1 to 8, thereby separating the biomolecule from contaminants; and collecting the purified biomolecule.

11. The method according to Claim 10, wherein the purified biomolecule is a protein.

12. The method according to Claim 11, wherein the contacting step includes immobilizing the protein on the chromatography resin, and the collecting step includes eluting the protein from the chromatography resin.

13. The method according to Claim 12, wherein the protein is eluted by a step including lowering the pH of the solution in contact with the chromatography resin from 7-9 to 4-6.

14. The method according to claim 11, wherein the contacting step comprises flowing the protein over the chromatography resin, and the collecting step comprises collecting the flowing protein.

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