Anionic exchange-hydrophobic mixed mode chromatography resins

Abstract

To provide anionic exchange-hydrophobic mixed mode chromatography resins suitable for purification of proteins from a biological sample.SOLUTION: A chromatography resin having an anionic exchange-hydrophobic mixed mode ligand is a chromatography matrix-(X)-N(R1)-(R2-L)n-Ar or an anionic salt thereof. X is a spacer. The chromatography matrix is a water-insoluble hydrophilic polymer, and polysaccharides such as agarose, dextran, and cellulose, as well as polymethacrylates, are suitable.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 896,196, filed September 5, 2019, the contents of which are incorporated by reference. [Background technology]

[0002] The extraction of immunoglobulins from source fluids, primarily mammalian body fluids or cell culture harvests, is important in obtaining immunoglobulins in a sufficiently concentrated or purified form for diagnostic and therapeutic applications, and for general laboratory research, as well as the purification of other types of proteins and other molecules from biological samples. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] U.S. Patent No. 5,645,717 [Patent Document 2] U.S. Patent No. 5,647,979 [Patent Document 3] U.S. Patent No. 5,935,429 [Patent Document 4] U.S. Patent No. 6,423,666 Summary of the Invention [Means for solving the problem]

[0004] A chromatography resin is provided that includes a chromatography matrix linked to an anion exchange-hydrophobic mixed mode ligand. In some embodiments, the chromatography resin is a chromatography matrix-(X)-N(R 1 )-(R 2 -L) n -Ar or an anion salt thereof. During the ceremony, X is a spacer, R1 is hydrogen or C1-C6 alkyl optionally substituted with -OH; 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 four unsubstituted alkyl groups; However, R 1 is hydrogen and R 2 is C2 alkyl and L is NR 4 or O, and when n is 1, then 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-CH2-CH(OH)-CH2-, -O-CH2-CH2-CH(OH)-CH2-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 a C2-C4 alkyl; L is O, n=1, Ar is a six-membered ring; When Ar is aryl, the aryl is optionally substituted with up to four C1-C2 unsubstituted alkyl, C3-C4 branched alkyl, or fluorine groups; or When Ar is heteroaryl, the heteroaryl is optionally substituted with up to three unsubstituted alkyl groups; However, R 1 is hydrogen and R 2 is C2 alkyl and n is 1, then Ar is not phenyl. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] A chromatography resin useful for purifying a target biomolecule using anion exchange and hydrophobic mixed-mode chromatography is provided. The chromatography resin allows for 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] definition Unless otherwise indicated, the terms used in this application, including the specification and claims, are defined as follows. As used in the specification and claims, the singular forms include plural referents unless clearly indicated otherwise. Definitions of standard chemical terms can be found in references including 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 employs, unless otherwise indicated, conventional methods of synthetic organic chemistry, mass spectrometry, chromatographic preparative and analytical methods, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology.

[0008] The term "alkyl" as used herein refers to a straight or branched chain saturated aliphatic radical having 1-10 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. Alkyl groups are typically monovalent, but may be divalent, such as when an alkyl group links two chemical groups together.

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

[0010] The term "aryl" as used herein refers to a monocyclic or fused bicyclic aromatic ring assembly. For example, aryl is phenyl or naphthyl. The aryl group may be optionally 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] The term "heteroaryl group" as used herein refers to an aromatic group containing one heteroatom as a member ring. Examples include, but are not limited to, pyrrole, furan, thiophene, and pyridine. Heteroaryl groups may be optionally substituted with one, two, three, or four alkyl groups.

[0013] An "anionic salt" is formed with a basic (e.g., amino or alkylamino) group in a ligand. Anionic 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] The term "spacer" as used herein refers to a molecule having 1-30 atoms selected from H, C, N, O and S. The spacer has a neutral charge and includes a cyclic group. The spacer connects the chromatography ligand to the chromatography matrix. The types of bonds used to connect the spacer to the chromatography matrix include, but are not limited to, amide, amine, ether, ester, carbamate, urea, thioether, thiocarbamate, thiocarbonate and thiourea. In some embodiments, the bond used to connect the spacer to the chromatography matrix is ​​an amine, ether or amide.

[0015] A "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 immunoglobulin, conjugate (e.g., fusion), or fragment forms thereof. The term includes, but is not limited to, polyclonal or monoclonal antibodies of the IgA, IgD, IgE, IgG, and IgM classes derived from human or other mammalian cell lines, including natural or genetically modified forms such as humanized, human, single chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. "Antibody" also includes, but is not limited to, conjugate forms including fusion proteins containing an immunoglobulin portion. "Antibody" also includes antibody fragments, such as Fab, F(ab')2, Fv, scFv, Fd, dAb, Fc, whether or not they retain antigen-binding function.

[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 mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers (e.g., recombinant proteins).

[0018] "Bind-elute mode" refers to an operational approach to chromatography in which buffer conditions are established such that when a sample is applied to the ligand, the target molecule, and possibly undesirable contaminants, bind to the ligand. Fractionation of the targets is then achieved by changing conditions such that the targets are eluted from the support. In some embodiments, the contaminants remain bound after target elution. In some embodiments, the contaminants flow through or are bound and eluted prior to target elution.

[0019] "Flow-through mode" refers to an operational approach to chromatography in which the target molecule to be purified flows through a chromatographic support containing a ligand, while buffer conditions are established such that at least some sample contaminants are selectively retained, thereby achieving their removal from the sample.

[0020] Chromatography Ligands In one embodiment, the mixed-mode chromatography support and ligand have the formula: Chromatography matrix-(X)-N(R 1 )-(R 2 -L) n -Ar or its anion salt During the ceremony, X is a spacer, R 1 is hydrogen or C1-C6 alkyl optionally substituted with -OH; 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 four unsubstituted alkyl groups; However, R 1 is hydrogen and R 2 is C2 alkyl and L is NR 4 or O, and when n is 1, then Ar is not phenyl.

[0021] The charge on the nitrogen adjacent to the spacer is pH dependent, therefore these resins have weak ion exchange.

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

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

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

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

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

[0028] In a seventh aspect of the first 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-CH2-CH(OH)-CH2-, -O-CH2-CH2-CH(OH)-CH2-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 a second embodiment, the chromatography resin has the formula: Chromatography matrix-(X)-N(R 1 )-(R 2 -L) n -Ar or its anion salt During the ceremony, 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-CH2-CH(OH)-CH2-, -O-CH2-CH2-CH(OH)-CH2-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 six-membered ring, and When Ar is aryl, the aryl is optionally substituted with up to four C1-C2 unsubstituted alkyl, C3-C6 branched alkyl, or fluorine groups; or When Ar is heteroaryl, the heteroaryl is optionally substituted with up to three unsubstituted alkyl groups; However, R 1 is hydrogen and R 2 is C2 alkyl and n is 1, then Ar is not unsubstituted phenyl.

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

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

[0032] In a third aspect of the second embodiment, Ar is phenyl, naphthyl, or pyridyl, optionally substituted with up to three C1-C2 unsubstituted alkyl or fluorine groups, with the proviso that R 1 is hydrogen and R 2is C2 alkyl and L is NR 4 or O, and n is 1, then Ar is not phenyl; alternatively, Ar is phenyl optionally substituted with 1 or 2 C1-C2 unsubstituted alkyl, provided that R 1 is hydrogen and R 2 is C2 alkyl and L is NR 4 or O, and n is 1, then Ar is not phenyl. Alternatively, Ar is a chromatographic matrix -(X)-N(R 1 )-(R 2 -L) n - is phenyl substituted with one C1-C2 unsubstituted alkyl at the para or meta position to -. Alternatively, Ar is heteroaryl and the heteroatom in the heteroaryl is N. Alternatively, Ar is unsubstituted heteroaryl. In yet another alternative, Ar is pyridyl.

[0033] In a third embodiment, the chromatography resin has the formula: Chromatography matrix-(X)-N(R 1 )-(R 2 -L) n -Ar or its anion salt During the ceremony, 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 phenyl, naphthyl, or pyridyl, optionally substituted with up to three C1-C2 unsubstituted alkyls; However, R 1 is hydrogen and R 2 is C2 alkyl and n is 1, then Ar is not unsubstituted phenyl.

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

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

[0036] [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6]

[0037] In a fifth embodiment, the chromatography resin comprises a chromatography matrix -(X)-N-[(R 2 -L) n --Ar]2 or an anionic salt thereof. During the ceremony, X is a spacer, R 2is 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 four unsubstituted alkyl groups.

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

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

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

[0041] In a fourth aspect of the fifth embodiment, Ar is a 6-membered ring, and if Ar is aryl, the aryl is optionally substituted with up to four C1-C2 unsubstituted alkyl, C3-C4 branched alkyl, or fluorine groups, or if Ar is heteroaryl, the heteroaryl is optionally substituted with up to three alkyl groups. Alternatively, Ar is phenyl, naphthyl, or pyridyl, optionally substituted with up to three C1-C2 unsubstituted alkyl or fluorine groups. Alternatively, Ar is phenyl substituted with one or two C1-C2 unsubstituted alkyl. Alternatively, Ar is -(R 2 -L) n- is phenyl substituted with one C1-C3 unsubstituted alkyl, C3-C6 branched alkyl, unsubstituted aryl, or fluorine group at the para or meta position to -. Alternatively, Ar is heteroaryl and the heteroatom in the heteroaryl is N. Alternatively, Ar is unsubstituted heteroaryl. In yet another alternative, Ar is pyridyl.

[0042] In a 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 a sixth aspect of the fifth embodiment, X is selected from the group consisting of -O-CH-, -O-CH-CH-, -O-CH-CH-, -O-CH-CH-CH-, -O-CH-CH-CH-, -O-CH-CH(CH-OH)-(O-CH-CH(OH)-CH)-, -O-CH-CH-CH(CH-OH)-(O-CH-CH-CH(OH)-CH)-, -O-CH-CH(OH)-CH-, -O-CH-CH-CH(OH)-CH-, -O-CH-CH(OH)-CH-, -O-CH-CH(OH)-CH-O-CH-CH-CH-CH-O-CH-CH(OH)-CH- and -CO-NH-C(CH)-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 a sixth embodiment, the chromatography resin comprises a chromatography matrix -(X)-N-[(R 2 -L) n —Ar]]2 or an anionic salt thereof. During the ceremony, 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-CH2-CH(OH)-CH2-, -O-CH2-CH2-CH(OH)-CH2-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 a C2-C4 alkyl; L is O, n=1, and Ar is a six-membered ring, and When Ar is aryl, the aryl is optionally substituted with up to four C3-C2 unsubstituted alkyl, C3 or C4 branched alkyl or fluorine groups; or When Ar is heteroaryl, the heteroaryl is optionally substituted with up to three unsubstituted alkyl groups.

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

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

[0047] In a seventh embodiment, the chromatography resin comprises a chromatography matrix -(X)-N-[(R 2 -L) n --Ar]2 or an anionic salt thereof. During the ceremony, X is selected from the group consisting of O-CH2-, -O-CH2-CH2-, -O-CH2-CH2-CH2-, -O-CH2-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 a C2 or C3 alkyl; L is O, n=1, and Ar is phenyl, naphthyl, or pyridyl optionally substituted with up to three C1-C2 unsubstituted alkyls.

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

[0049] In an 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 anionic salt is a hydrochloride, a phosphate, or a sulfate.

[0052] The chromatography matrix is ​​a polymer functionalized to allow the formation of bonds with the spacer X. The polymer is preferably a hydrophilic polymer. The polymer is insoluble in water. Suitable polymers are, for example, polyhydroxypolymers based on polysaccharides such as agarose, dextran, cellulose, starch, pullulan, etc.; fully synthetic polymers such as polyacrylamides, polymethacrylamides, poly(hydroxyalkyl vinyl ethers), poly(hydroxyalkyl acrylates) and polymethacrylates (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® base beads from Sigma-Millipore-EMD-Merck, Toyopearl AF-Eposy-650M and Toyopearl AF-Tresyl-650M from Tosoh Bioscience, POROS medium 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 medium from Pall. Water-soluble polymers are derivatized to become insoluble, for example, by crosslinking and by coupling to insoluble bodies via adsorption or covalent bonds. Hydrophilic groups are introduced onto the hydrophobic polymer (e.g. on copolymers of monovinyl and divinylbenzene) by polymerization of monomers exhibiting groups which are converted to OH, or by hydrophilization of the final polymer, e.g. by adsorption of suitable compounds such as hydrophilic polymers.Examples of monomers that may be polymerized to obtain a useful matrix are vinyl acetate, vinylpropylamine, acrylic acid, methacrylate, butyl acrylate, acrylamide, methacrylamide, vinylpyrrolidone (vinylpyrrolidinone), optionally with functional groups. Crosslinkers are also useful in many embodiments, and when present, in some embodiments, comprise a molar ratio of about 0.1 to about 0.7 relative to the total monomers. Examples of crosslinkers include dihydroxyethylenebisacrylamide, diallyltardiamide, triallylcitric triamide, ethylenediacrylate, bisacrylcystamine, N,N'-methylenebisacrylamide, and piperazinediacrylamide. In some embodiments, the matrix is ​​a UNOsphere® (Bio-Rad, Hercules, Calif.) substrate, which is a polymer made from water-soluble hydrophilic monomers.

[0053] A chromatography matrix is ​​a particle, chip, membrane, or monolith, i.e., a single block, pellet, or slab of material in the form of a particle. Preferably, the chromatography matrix is ​​porous. The particles, when used as a matrix, are spheres or beads, and either have a smooth surface or a rough or textured surface. In some cases, some of the pores are through-holes, extending through the particle and acting 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 about 25 microns to about 150 microns. Disclosures of exemplary matrices and methods for producing them can be found in US Pat. Nos. 5,993,523, 5,993, 6,103,142, 6,103,152, 6,103,162, 6,103,17 ...

[0054] The ligand is linked to the chromatography matrix via a spacer X. Linking to the chromatography matrix depends on the particular chromatography matrix used and the chemical group to be linked to the chromatography matrix. The ligand can be linked to the chromatography matrix by carrying out a reaction between the ligand and a functional group on the chromatography matrix. For chromatography matrices that do not have suitable functional groups, the chromatography matrix is ​​reacted with a suitable activating reagent to generate a suitable functional group to which the ligand can be attached. Reductive amination, epoxidation, or azlactonization are examples of chemistries that act on aldehyde, epoxide, or azalactone functional groups, respectively.

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

[0056] [ka]

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

[0058] [ka]

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

[0060] [ka]

[0061] In some embodiments, the chromatography matrix comprises a diol and a 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, where the spacer X is -O-CH2-CH(CH2-OH)-(O-CH2-CH(OH)-CH2)2-.

[0062] [ka]

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

[0064] [ka]

[0065] In some embodiments, the chromatography matrix comprises an -OH group and a 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, where the spacer X is -O-CH2-CH(OH)-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH(OH)-CH2-.

[0066] [ka]

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

[0068] Other spacers include, but are not limited to, -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-.

[0069] The chromatography matrices can be utilized in any conventional configuration, including packed columns and fluidized or expanded bed columns, monoliths or porous membranes, and by any conventional method, including batch mode, as well as continuous or flow-through modes for loading, washing, and elution. In some embodiments, the diameter of the columns ranges from 1 cm to 1 m, and the height ranges from 1 cm to 30 cm or more.

[0070] method Also provided is a method for purifying a target biomolecule. In one embodiment, the method comprises contacting a sample containing the biomolecule with a chromatography resin, thereby separating the biomolecule from contaminants. The resulting purified biomolecule is then recovered. In some embodiments, the target biomolecule is a target protein, and the method comprises purifying the target protein from contaminants. In some embodiments, the target biomolecule is a monomeric antibody, and the method comprises purifying the monomeric antibody from aggregated antibodies in the sample.

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

[0072] Protein preparations to which the method is applied include proteins from natural, synthetic, or recombinant sources. Non-purified protein preparations are derived from a variety of sources, including, but not limited to, blood plasma, plasma, ascites 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 step, or any combination of the above. 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 steps include, but are not limited to, crystallization, liquid:liquid partitioning, or membrane filtration.

[0073] As is understood in the art, the load, wash and elution conditions used in mixed-mode chromatography will depend on the particular chromatographic medium / ligand used.

[0074] In some bind-elute mode embodiments, loading (i.e., binding of 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 an appropriate 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 an appropriate 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 chromatographic matrix, but the target biomolecules remain immobilized on the matrix, hi some embodiments, the target biomolecules are subsequently eluted by decreasing the salt concentration and / or decreasing the pH of the solution in contact with the matrix.

[0076] Alternatively, samples are applied and collected in a flow-through mode, where some components of the sample are immobilized on the matrix, but the target biomolecules flow (i.e., are flow-passed) through the chromatographic matrix. 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] Working Example The following examples are illustrative and not limiting. Those of ordinary skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially the same or similar results. Example 1 - Preparation of chromatographic resins with 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 vinylpyrrolidinone crosslinked with N,N′-methylenebisacrylamide and with a diol density of 200-300 μmol / mL, was used in the form of spherical beads. The beads were suspended in 20 mL of either 0.1 M sodium acetate or water. Sodium periodate was added at a concentration ranging from 50 to 100 mM, and the resulting mixture was incubated at room temperature (approximately 70° F. (21° C.)) for 3 to 24 hours. The reaction resulted in the conversion of 150-250 μmol / mL of diol groups to aldehyde groups. The resulting aldehyde-functionalized resin was transferred to a 20 mL column where it was washed with 100 mL of water.

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

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

[0081] [Table 10]

[0082] 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 at 50°C in a shaker at 250 rpm for 10 minutes. 100 ml of allyl glycidyl ether (AGE) was added and kept overnight at 50°C in the same shaker. The resulting AGE modified resin was washed with 3 x 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 completion of the reaction of the double bond with bromine). Then, additional Na2SO3 was added (reducing the excess bromine to bromide) until the orange color disappeared. 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. Then, 12.5 g of 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 1N HCl, 2 CV water, 2 CV 1N NaOH, and then 30 CV water to obtain the tertiary amine resin.

[0083] The number, structure, and density of ligands 32, 34, 39, and 44 attached to UNOsphere diol bromide resin are shown in Table 4.

[0084] [Table 11]

[0085] Example 2 - Evaluation of amine resins from Tables 3 and 4 using acidic proteins and bind-elute mode The static binding capacity, recovery 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 a variety of binding and elution conditions.

[0086] material: 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 about 4.7) 5.Eod Binding / Wash Buffer a. 25 mM sodium phosphate buffer at pH 6.5, 7.5, or about 8.5 b. 0, 25 or about 50 mM NaCl 6. Binding / Wash Buffer for HSA a. 25 mM sodium phosphate or sodium acetate buffer at pH 4, 6, or about 8 b. 0, 200 or about 400 mM NaCl 7.Elution buffer for Eod a. 25 mM sodium phosphate buffer at pH 6.5, 7.5, or about 8.5 b. 10, 500 or about 1000 mM NaCl 8.Elution buffer for HSA a. 25 mM sodium phosphate or sodium acetate buffer at pH 4, 6, or about 8 b. 20, 500, or about 1000 mM NaCl

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

[0088] Results: The SBC results and recovery rates of the resins 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, "-" stands for "about" and in both tables, "NaCl" stands for NaCl concentration.

[0089] [Table 12]

[0090] [Table 13]

[0091] The data from Tables 5 and 6 show the range of optimal binding and elution conditions depending on the target protein and resin. For example, for Eod as the target protein, the optimal binding conditions were pH 6.5 / no NaCl when tested with resin 5, 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 were pH 8.0 / 400 mM NaCl with resin 6, while for resin 34, the optimal binding conditions were pH 4.0 / no 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] The data from Tables 5 and 6 show that the resins can be used over a wide range of pH values ​​and NaCl concentrations, and that the choice of resin for a given application depends on the target protein of interest.

[0094] All patents, patent applications, and other published references cited herein are hereby incorporated by reference in their entirety. Additional Disclosure and Claimed Subject Matter

[0095] Item 1 A chromatography matrix covalently bound to a ligand having the formula: Chromatography matrix-(X)-N(R 1 )-(R 2 -L) n -Ar or its anion salt (In the formula, X is a spacer, R 1 is hydrogen or C1-C6 alkyl optionally substituted with -OH; 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 four unsubstituted alkyl groups; However, R 1 is hydrogen and R 2 is C2 alkyl and L is NR 4 or O, and when n is 1, Ar is not phenyl. 4. A chromatography matrix comprising:

[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-CH2-CH(OH)-CH2-, -O-CH2-CH2-CH(OH)-CH2-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 a C2-C4 alkyl; L is O, n=1, Ar is a six-membered ring, and When Ar is aryl, the aryl is optionally substituted with up to four C1-C2 unsubstituted alkyl, C3-C4 branched alkyl, or fluorine groups; or When Ar is heteroaryl, the heteroaryl is optionally substituted with up to three unsubstituted alkyl groups; However, R 1 is hydrogen and R 2 is C2 alkyl, and when n is 1, Ar is not phenyl; Item 1. A 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 a C2-C3 alkyl group; L is O, n=1, and Ar is phenyl, naphthyl, or pyridyl, optionally substituted with up to three C1-C2 unsubstituted alkyls; However, R 1 is hydrogen and R 2 is C2 alkyl, and when n is 1, Ar is not phenyl; Item 2. A chromatography matrix according to item 2.

[0098] Item 4 Ar is the chromatography matrix -(X)-N(R 1 )-(R 2 -L) n - is a phenyl substituted with 1 or 2 C1-C2 unsubstituted alkyl at the para or meta position to -; 4. 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 one of the ligands in Table 1; 5. The chromatography resin according to any one of items 1 to 4.

[0100] Item 6 1. A chromatography resin comprising: 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; 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 four unsubstituted alkyl groups; Chromatography resin.

[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-CH2-CH(OH)-CH2-, -O-CH2-CH2-CH(OH)-CH2-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 a C2-C4 alkyl; L is O, n=1, and Ar is a six-membered ring; When Ar is aryl, the aryl is optionally substituted with up to four C3-C2 unsubstituted alkyl, C3 or C4 branched alkyl or fluorine groups; or 7. The chromatography matrix according to item 6, wherein when Ar is heteroaryl, the heteroaryl is optionally substituted with up to three unsubstituted alkyl groups.

[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 2 is C2 or C3 alkyl, L is O, n=1, and Ar is phenyl, naphthyl, or pyridyl, optionally substituted with up to three C1-C2 unsubstituted alkyls; 8. A chromatography matrix according to item 7.

[0103] Item 9 Ar is -(R 2 -L) n - is substituted at the para or meta position to - with one or two C1-C2 unsubstituted alkyl.

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

[0105] Item 11 7. The chromatography resin according to item 1 or 6, wherein Ar is heteroaryl and the heteroatom in the heteroaryl is N.

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

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

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

[0109] Item 15 15. The chromatography resin according to item 14, wherein the chromatography matrix comprises an aldehyde group and any one of the ligands of Table 1 is reacted with the chromatography matrix by reductive amination.

[0110] Item 16 Item 15. The chromatography resin according to item 14, wherein the chromatography matrix comprises an epoxide group and any one of the ligands of Table 1 reacts with the chromatography matrix by epoxidation.

[0111] Item 17 17. The chromatography resin according to any one of items 14 to 16, wherein the chromatography matrix is ​​reacted with allyl glycidyl ether and bromine, 1,4-butanediaryl diglycidyl or epichlorohydrin prior to reacting the chromatography matrix with the ligand.

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

[0113] Item 19 1. A method for purifying a biomolecule, comprising: contacting a sample containing said biomolecule with a chromatography resin according to any one of items 1 to 18, thereby separating said biomolecule from contaminants; and collecting the purified biomolecule.

[0114] Item 20 20. The method of claim 19, wherein the purified biomolecule is a protein.

[0115] Item 21 21. The method of claim 20, wherein the contacting step comprises immobilizing the protein on a chromatography matrix and the collecting step comprises eluting the protein from the chromatography matrix.

[0116] Item 22 22. The method of claim 21, wherein the protein is eluted by a step comprising lowering the pH of a solution in contact with the ligand to about 7-9 to about 4-6.

[0117] Item 23 21. The method of claim 20, wherein the contacting step comprises flowing the protein through the chromatography matrix and the collecting step comprises collecting the protein flowing through.

Claims

1. A matrix resin comprising a chromatography matrix covalently attached to a ligand via a spacer, The chromatography resin has the formula: Chromatography matrix -(X)-N(R １ ) - (R ２ -L) ｎ -Ar or its anion salt (In the formula, X is a spacer, -N(R １ ) - (R ２ -L) ｎ -Ar is a ligand, R 1 is hydrogen or C １ ~C 6 alkyl, １ ~C 6 The alkyl is optionally substituted with -OH; R 2 is C 2 ~C 6 Alkylene, or C 4 ~C 6 is cycloalkylene, L is O; n=1 or 2, and Ar is a 6- to 10-membered ring; When Ar is aryl, the aryl may be selected from up to five C １ ~C ３ Unsubstituted alkyl, C 3 ~C 6 may be substituted with branched alkyl, unsubstituted aryl, or fluorine groups; or When Ar is heteroaryl, said heteroaryl may be optionally substituted with up to four unsubstituted alkyl groups; However, R １ is hydrogen, R 2 is C 2 When Ar is alkyl, L is O, and n is 1, then Ar is not unsubstituted phenyl. A chromatography resin represented by:

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-CH ２ -CH ２ -CH ２ -CH ２ -O-CH ２ -CH(OH)-CH ２ -and -CO-NH-C(CH ３ ) ２ -CO-, and is selected from the group consisting of, R １ is hydrogen or C １ ~C ３ is alkyl, R ２ is C ２ ~C ４ is alkylene, L is O, n=1, and Ar is a six-membered ring, and When Ar is aryl, the aryl may be selected from up to four methyl or ethyl groups, C 3 Or C ４ It may be substituted with a branched alkyl group or a fluorine group, or When Ar is heteroaryl, said heteroaryl is optionally substituted with up to three unsubstituted alkyl groups; However, R １ is hydrogen, R 2 is C 2 When Ar is alkylene, L is O, and n is 1, then Ar is not unsubstituted phenyl; The chromatography resin of 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 １ is hydrogen or C １ ~C ２ is alkyl, R ２ is C ２ ~C ３ is alkylene, L is O, n=1, and Ar is phenyl, naphthyl, or pyridyl, each of which is optionally substituted with up to three methyl or ethyl groups; However, R 1 is hydrogen, R 2 is C 2 When Ar is alkylene, L is O, and n is 1, then Ar is not unsubstituted phenyl; The chromatography resin of claim 1.

4. Ar is 1 or 2 C 1 ~C ２ phenyl substituted with unsubstituted alkyl, substituted in the para or meta position relative to the point of attachment of said phenyl to L; The chromatography resin according to any one of claims 1 to 3.

5. The chromatography resin is 【Table 1】 wherein the circle is the chromatography matrix and the spacer X; The chromatography resin according to any one of claims 1 to 4.

6. 2. The chromatography resin of claim 1, wherein Ar is heteroaryl and the heteroatom in the heteroaryl is N.

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

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

9. 1. A method for preparing a chromatography resin comprising reacting a chromatography matrix comprising a functional group bound to said chromatography matrix with a ligand comprising an amine group, such that said ligand is bound to said functional group via an amine, comprising the steps of: the functional group comprises an aldehyde group with which the amine reacts; or the functional group comprises an epoxy group with which the amine reacts; or the functional group comprises an azalactone ring with which the amine reacts; or the functional group comprises a bromine with which the amine reacts; The ligand may be 2-(p-tolyloxy)ethylamine, 2-(o-tolyloxy)ethylamine, 2-(m-tolyloxy)ethylamine, 3,5-dimethylphenoxyethanamine, 4-ethylphenoxyethanamine, 4-isopropylphenoxyethanamine, 4-t-butylphenoxyethanamine, 4-phenylphenoxyethanamine, 1-naphthaleneoxyethanamine, 3-fluorophenoxyethanamine, 4-fluorophenoxyethanamine, 3,5-difluorophenoxyethanamine, 2 ... Phenoxyethanamine, 3,4-difluorophenoxyethanamine, 3,4,5-trifluorophenoxyethanamine, perfluorophenoxyethanamine, 4-pyridinylethanamine, 3-pyridinylethanamine, 4-(2,6-dimethylpyridinyl)ethanamine, phenoxypropylamine, 2-phenoxycyclobutylamine, 2-phenoxycyclopentylamine, 4-phenoxycyclohexylamine, 2-(2-phenoxyethoxy)ethanamine, N-methyl-2-phenoxyethan-1-amine, N-methyl-3 -phenoxyethan-1-amine, N-methyl(2-phenoxycyclobutyl)amine, N-methyl(2-phenoxycyclopentyl)amine, N-methyl(4-phenoxycyclohexyl)amine, N-methyl-2-(p-tolyloxy)ethan-1-amine, 2-(3,5-dimethylphenoxy)-N-methylethan-1-amine, 2-(4-ethylphenoxy)-N-methylethan-1-amine, 2-(4-isopropylphenoxy)-N-methylethan-1-amine, 2-(4-(tert-butyl)phenoxy)-N-methylethane -1-amine, 2-([1,1'-biphenyl]-4-yloxy)-N-methylethan-1-amine, N-methyl-2-(naphthalen-1-yloxy)ethan-1-amine, 2-((2-phenoxyethyl)amino)ethan-1-ol, 2-(3-fluorophenoxy)-N-methylethan-1-amine, 2-(4-fluorophenoxy)-N-methylethan-1-amine, 2-(2,5-difluorophenoxy)-N-methylethan-1-amine, 2-(3,4-difluorophenoxy)-N-methylethan-1-amine, 2-(3,2-((2,6-dimethylpyridin-4-yl)oxy)-N-methylethan-1-amine, and N-methyl-2-(2-phenoxyethoxy)ethan-1-amine, Methods for preparing chromatography resins.

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

11. The method of claim 10, wherein the purified biomolecule is a protein.

12. 12. The method of claim 11, wherein the contacting step comprises immobilizing the protein on a chromatography resin and the collecting step comprises eluting the protein from the chromatography resin.

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

14. 12. The method of claim 11, wherein the contacting step comprises flowing the protein onto the chromatography resin and the collecting step comprises collecting the protein in the flow.

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