Anion exchange-hydrophobic mixed mode chromatography resin
阅读说明：本技术 阴离子交换-疏水混合模式色谱树脂 (Anion exchange-hydrophobic mixed mode chromatography resin ) 是由 C·贝利斯勒 陈虹 徐越平 廖加利 何学梅 于 2019-03-08 设计创作，主要内容包括：具有阴离子交换-疏水混合模式配体的色谱树脂,其可用于使用阴离子交换(即,其中配体带正电)和疏水混合模式色谱来纯化靶分子。色谱树脂允许从样品高效纯化靶生物分子(例如,重组蛋白质、抗体、抗体药物偶联物、或抗体衍生物,所述抗体衍生物包括但不限于抗体片段和抗体融合体),并且已发现可用于从聚集体靶生物分子纯化单体靶生物分子。在一个实施方式中,色谱树脂可用于分离样品中的抗体与一种或多种组分(如污染物)。(Chromatography resins with anion exchange-hydrophobic mixed mode ligands that can be used to purify target molecules using anion exchange (i.e., where the ligands are positively charged) and hydrophobic mixed mode chromatography. Chromatography resins allow for efficient purification of target biomolecules (e.g., recombinant proteins, antibodies, antibody drug conjugates, or antibody derivatives including but not limited to antibody fragments and antibody fusions) from a sample and have been found useful for purification of monomeric target biomolecules from aggregate target biomolecules. In one embodiment, a chromatographic resin can be used to separate an antibody from one or more components (e.g., contaminants) in a sample.)
1. A chromatographic resin having the formula:
chromatography matrix- (X) -N (R)1)(R2)-(R3-L)n-Ar，
Wherein:
x is a spacer;
R1and R2Each independently is C optionally substituted by-OH1To C6An alkyl group;
R3is C2To C6Alkyl or C4-C6A cycloalkyl group;
l is NR4O, or S, wherein R4Is hydrogen, or C1To C4An alkyl group;
n is 1 or 2; and is
Ar is a 6-to 10-membered ring, and:
if Ar is aryl, the aryl group is substituted by up to 5C1To C3Unsubstituted alkyl, C3To C6A branched alkyl, unsubstituted aryl or fluoro group is optionally substituted; or
If Ar is heteroaryl, the heteroaryl is optionally substituted with up to 4 alkyl groups.
2. The chromatography matrix of claim 1, 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 (CH)3)2-CO-；
R1And R2Each independently is C1To C3An alkyl group;
R3is C2To C4An alkyl group;
l is O;
n is 1; and is
Ar is a 6-membered ring, and:
if Ar is aryl, the aryl group is substituted by up to 4C1To C2Unsubstituted alkyl, C3To C4Branched alkyl, or fluoro groups are optionally substituted; or
If Ar is heteroaryl, the heteroaryl is optionally substituted with up to 3 alkyl groups.
3. The chromatographic resin of claim 2, 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(CH2-OH)-(O-CH2-CH(OH)-CH2)2-；
R1And R2Each independently is C1Or C2An alkyl group;
R3is C2Or C3An alkyl group;
l is O;
n is 1; and is
Ar is a radical of at most 3C1To C2Unsubstituted alkyl optionally substituted phenyl, naphthyl or pyridyl.
4. The chromatographic resin of any of claims 1 to 3, wherein Ar is substituted with one or two C1To C2Unsubstituted alkyl optionally substituted phenyl.
5. A chromatography resin according to any one of claims 1 to 4, wherein- (X) -N (R)1)(R2)-(R3-L)nAr is any one of the ligands in Table 1.
6. A chromatographic resin having the formula:
chromatography matrix- (X) -N (R)1)-[(R3-L)n-Ar]2
Wherein:
x is a spacer;
R1is C optionally substituted by-OH1To C6An alkyl group;
R3is C2To C6Alkyl or C4-C6A cycloalkyl group;
l is NR4O, or S, wherein R4Is hydrogen, or C1To C4An alkyl group;
n is 1 or 2; and is
Ar is a 6-to 10-membered ring, and:
if Ar is aryl, the aryl group is substituted by up to 4C1To C3Unsubstituted alkyl, C3To C6A branched alkyl, unsubstituted aryl or fluoro group is optionally substituted; or
If Ar is heteroaryl, the heteroaryl is optionally substituted with up to 4 alkyl groups.
7. The chromatographic resin as set forth in claim 6, 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 (CH)3)2-CO-；
R1Is C1To C3An alkyl group;
R3is C2To C4An alkyl group;
l is O;
n is 1; and is
Ar is a 6-membered ring, and:
if Ar is aryl, the aryl group is substituted by up to 4C1To C2Unsubstituted alkyl, C3To C4Branched alkyl, or fluoro groups are optionally substituted; or
If Ar is heteroaryl, the heteroaryl is optionally substituted with up to 3 alkyl groups.
8. The chromatographic resin as set forth in claim 7, 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(CH2-OH)-(O-CH2-CH(OH)-CH2)2-；
R1Is C1Or C2An alkyl group;
R3is C2Or C3An alkyl group; and is
L is O;
n is 1; and is
Ar is a radical of at most 3C1To C2Unsubstituted alkyl optionally substituted phenyl, naphthyl or pyridyl.
9. The chromatographic resin of any of claims 6 to 8, wherein Ar is substituted with one or two C1To C2Unsubstituted alkyl optionally substituted phenyl.
10. A chromatographic resin according to any of claims 6 to 9, wherein- (X) -N (R)1)-[(R3-L)n-Ar]Is any one of the ligands in table 2.
11. The chromatography resin of any one of claims 1 to 10, wherein Ar is unsubstituted.
12. The chromatography resin of claim 1 or 6, wherein Ar is a heteroaryl group and the heteroatom in the heteroaryl group is N.
13. The chromatography resin of any one of claims 1 to 12, wherein the anionic salt is a hydrochloride or sulfate salt.
14. The chromatography resin of any one of claims 1 to 13, wherein X is attached to the chromatography matrix by an amine, ether or amide bond.
15. A chromatography resin prepared by reacting any one of the ligands of table 1 with a chromatography matrix in any one of a reductive amination, an epoxide chemical reaction, or an azalide chemical reaction.
16. The chromatography resin of claim 15, wherein the chromatography matrix comprises aldehyde groups and any of the ligands of table 1 are reacted with the chromatography matrix by reductive amination.
17. The chromatography resin of claim 15, wherein the chromatography matrix comprises aldehyde groups and any of the ligands of table 1 are reacted with the chromatography matrix by epoxide chemistry.
18. A chromatography resin according to any one of claims 15 to 17, wherein prior to reaction of the chromatography matrix with the ligand, the chromatography matrix is reacted with: allyl glycidyl ether and bromine; 1, 4-butanediol diglycidyl group; or epichlorohydrin.
19. The chromatographic resin according to claim 18, wherein the chromatographic matrix contains-OH groups and is reacted with allyl glycidyl ether and bromine.
20. A chromatography resin prepared by reacting any one of the ligands of table 2 with a chromatography matrix in an epoxide chemical reaction.
21. A method of purifying a biomolecule, the method comprising:
contacting a sample containing biomolecules with a chromatography resin of any one of claims 1 to 20, thereby separating the biomolecules from the contaminants; and
collecting the purified biomolecule.
22. The method of claim 21, wherein the purified biomolecule is a protein.
23. The method of claim 22, wherein the protein is an antibody.
24. The method of any one of claims 21 to 23, wherein the sample comprises monomeric antibodies and antibody aggregates, the method comprising: the monomeric antibody is separated from the antibody aggregate, and the purified biomolecule comprises the monomeric antibody.
25. The method of any one of claims 24, wherein the purified biomolecule is a monomeric antibody.
26. The method of claim 25, wherein the contacting step comprises: immobilizing the monomeric antibody to a chromatography matrix, and the collecting step comprises: eluting the monomeric antibody from the chromatography matrix.
27. The method of claim 26, wherein the monomeric antibody is eluted by a process comprising: the pH of the solution contacted with the ligand is lowered from about 7-9 to about 4-6.
28. The method of claim 25, wherein the contacting step comprises: flowing the monomeric antibody through a chromatography matrix, and the collecting step comprises: the transfused monomeric antibody was collected.
Background
The extraction of immunoglobulins from source liquids (mainly mammalian body fluids or cell cultures) is valuable for obtaining immunoglobulins in a substantially concentrated or purified form, typically for diagnostic and therapeutic use and laboratory studies. Similarly, purification of other types of proteins and other molecules from biological samples can be valuable.
Disclosure of Invention
Chromatography resins are provided that comprise a chromatography matrix linked to an anion exchange-hydrophobic mixed mode ligand. In some embodiments, the chromatographic resin has the formula or an anionic salt thereof:
chromatography matrix- (X) -N (R)1)(R2)-(R3-L)n-Ar，
Wherein:
x is a spacer;
R1and R2Each independently is C optionally substituted by-OH1To C6An alkyl group;
R3is C2To C6Alkyl or C4-C6A cycloalkyl group;
l is NR4O, or S, wherein R4Is hydrogen, or C1To C4An alkyl group;
n is 1 or 2; and is
Ar is a 6-to 10-membered ring, and:
if Ar is aryl, the aryl group is substituted by up to 5C1To C3Unsubstituted alkyl, C3To C6A branched alkyl, unsubstituted aryl or fluoro group is optionally substituted; or
If Ar is heteroaryl, the heteroaryl is optionally substituted with up to 4 alkyl groups.
In some embodiments of the chromatographic 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 (CH)3)2-CO-；
R1And R2Each independently is C1To C3An alkyl group;
R3is C2To C4An alkyl group;
l is O;
n is 1; and is
Ar is a 6-membered ring, and:
if Ar is aryl, the aryl group is substituted by up to 4C1To C2Unsubstituted alkyl, C3To C4Branched alkyl, or fluoro groups are optionally substituted; or
If Ar is heteroaryl, the heteroaryl is optionally substituted with up to 3 alkyl groups.
Detailed Description
Chromatography resins are provided that can be used to purify target biomolecules using anion exchange (i.e., where the ligands are positively charged) and hydrophobic mixed mode chromatography. Chromatography resins allow for efficient purification of target biomolecules (e.g., recombinant proteins, antibodies, antibody drug conjugates, or antibody derivatives including but not limited to antibody fragments and antibody fusions) from a sample and have been found useful for purification of monomeric target biomolecules from aggregate target biomolecules. In one embodiment, a chromatographic resin can be used to separate an antibody from one or more components (e.g., contaminants) in a sample.
Definition of
As used herein (including the specification and claims), the following terms have the meanings given below, unless otherwise indicated. As used in this specification and the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Definitions of standardized chemical terms can be found in reference works including Carey and Sundberg (2007) volumes A and B of "fifth edition of Advanced organic chemistry" (Advanced organic chemistry 5th Ed.), Springer science, N.Y., Business Media, LLC. The practice of the present invention will employ, unless otherwise indicated, conventional methods of synthetic organic chemistry, mass spectrometry, chromatographic methods of preparation and analysis, protein chemistry, biochemistry, recombinant DNA technology and pharmacology.
"antibody" refers to an immunoglobulin, complex (e.g., fusion) or fragment form 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 naturally occurring or genetically modified forms such as humanized, human, single chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted and in vitro generated antibodies. "antibody" also includes complex forms, including but not limited to fusion proteins containing an immunoglobulin moiety. "antibodies" include antibody fragments, such as Fab, F (ab')2、Fv、scFv、Fd、dAb、Fc。
As used herein, the term "alkyl" refers to a straight or branched, saturated aliphatic group having 1 to 10 carbon atoms. E.g. C1-C6Alkyl groups include, but are not limited to: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, and/or hexyl. Alkyl groups may include 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 may be divalent, for example, when an alkyl group connects two chemical groups together.
As used herein, the term "cycloalkyl" refers to a monocycloalkyl group having the specified number of carbon atoms. A single ring includes: for example, cyclobutyl, cyclopentyl and cyclohexyl.
As used herein, the term "aryl" refers to a monocyclic or fused bicyclic aromatic ring composition. For example, aryl may be phenyl, naphthyl, or pyridyl. The aryl group can be optionally substituted with one, two, three, four, or five unsubstituted alkyl groups, unsubstituted aryl groups, or fluoro groups.
The term "heteroatom" refers to N, O and S.
As used herein, the term "heteroaryl" refers to an aromatic group that contains one heteroatom as a ring member. Examples include, but are not limited to: pyrrole, furan, thiophene and pyridine. The heteroaryl group may be optionally substituted with one, two, three or four alkyl groups.
An "anionic salt" is formed on a basic (e.g., alkylamino) group of the 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, hydrochloride, hydrobromide, hydroiodide, sulfate, acetate, citrate, and nitrate salts.
As used herein, the term "spacer" refers to a molecule having 1 to 30 atoms selected from the group consisting of: H. c, N, O and S. The spacer has a neutral charge and may include a cyclic group. The spacer couples the chromatographic ligand to the chromatographic matrix. Types of bonds for attaching 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 attach the spacer to the chromatography matrix is an amine, ether, or amide.
By "biological sample" is meant any composition containing a target molecule of biological origin ("biomolecule") which it is desired to purify. In some embodiments, the target molecule to be purified is an antibody protein or a non-antibody protein (e.g., a hormone or an enzyme).
"Bind-elute mode" refers to a method of operation of chromatography in which buffer conditions are established such that the target molecule and optionally undesirable contaminants will Bind to the ligand when the sample is applied to the ligand. Fractionation of the target can then be achieved by changing the conditions such that the target elutes from the support. In some embodiments, the contaminants remain bound after the target is eluted. In some embodiments, the contaminants flow through (flow-through) or bind and elute prior to elution of the target.
"Flow-through mode" refers to a method of operation of chromatography in which buffer conditions are established to allow target molecules to be purified to Flow through a chromatographic support containing a ligand while selectively retaining at least some sample contaminants, thereby effecting removal thereof from the sample.
Chromatography resin
In a first embodiment, the chromatographic resin has the formula or an anionic salt thereof:
chromatography matrix- (X) -N (R)1)(R2)-(R3-L)n-Ar，
Wherein:
x is a spacer;
R1and R2Each independently is C optionally substituted by-OH1To C6An alkyl group;
R3is C2To C6Alkyl or C4-C6A cycloalkyl group;
l is NR4O, or S, wherein R4Is hydrogen, or C1To C4An alkyl group;
n is 1 or 2; and is
Ar is a 6-to 10-membered ring, and:
if Ar is aryl, the aryl group is substituted by up to 5C1To C3Unsubstituted alkyl, C3To C6A branched alkyl, unsubstituted aryl or fluoro group is optionally substituted; or
If Ar is heteroaryl, the heteroaryl is optionally substituted with up to 4 alkyl groups.
Due to its structure, the nitrogen adjacent to the spacer is positively charged, and therefore the charge is independent of pH. Thus, these resins provide strong ion exchange.
In a first aspect of the first embodiment, R1And R2Each independently is C1To C3An alkyl group. Or, R1And R2Each independently is C1Or C2An alkyl group.
In a second aspect of the first embodiment, R3Is C2To C4An alkyl group. Or, R3Is C2Or C3An alkyl group.
In a third aspect of the first embodiment, L is NR4Or O, or NR4Or S. Alternatively, L is O.
In a fourth aspect of the first embodiment, n is 1.
In a fifth aspect of the first embodiment, Ar is a six membered ring, and if Ar is aryl, the aryl is substituted with up to 4C1To C2Unsubstituted alkyl, C3To C4Branched alkyl, or fluoro groups are optionally substituted; or if Ar is heteroaryl, the heteroaryl is optionally substituted with up to 3 alkyl groups. Or Ar is a radical of at most 3C1To C2Phenyl, naphthyl or pyridyl optionally substituted with unsubstituted alkyl or fluoro groups. Or Ar is substituted by one or two C1To C2Unsubstituted alkyl optionally substituted phenyl. Alternatively, Ar is unsubstituted phenyl. Alternatively, Ar is heteroaryl and the heteroatom in heteroaryl is N. Alternatively, Ar is unsubstituted heteroaryl. In another alternative, Ar is pyridyl.
In a sixth aspect of the first embodiment, X is attached to the chromatography matrix by a bond selected from the group consisting of: amides, amines, ethers, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas. Alternatively, the bond is an amine, ether or amide.
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-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 (CH)3)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(CH2-OH)-(O-CH2-CH(OH)-CH2)2-。
In a second embodiment, the chromatographic resin has the formula or an anionic salt thereof:
chromatography matrix- (X) -N (R)1)(R2)-(R3-L)n-Ar，
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 (CH)3)2-CO-；
R1And R2Each independently is C1To C3An alkyl group;
R3is C2To C4An alkyl group;
l is O;
n is 1; and is
Ar is a 6-membered ring, and:
if Ar is aryl, the aryl group is substituted by up to 4C1To C2Unsubstituted alkyl, C3To C4Branched alkyl, or fluoro groups are optionally substituted; or
If Ar is heteroaryl, the heteroaryl is optionally substituted with up to 3 alkyl groups.
In a first aspect of the second embodiment, R1And R2Each independently is C1Or C2An alkyl group.
In a second aspect of the second embodiment, R3Is C2Or C3An alkyl group.
In a third aspect of the second embodiment, Ar is C, with up to 31To C2Unsubstituted alkyl-substituted phenyl, naphthyl or pyridyl. Or Ar is substituted by one or two C1To C2Unsubstituted alkyl optionally substituted phenyl. Alternatively, Ar is unsubstituted phenyl. Alternatively, Ar is heteroaryl and the heteroatom in heteroaryl is N. Alternatively, Ar is unsubstituted heteroaryl.
In a third embodiment, the chromatographic resin has the formula or an anionic salt thereof:
chromatography matrix- (X) -N (R)1)(R2)-(R3-L)n-Ar，
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(CH2-OH)-(O-CH2-CH(OH)-CH2)2-；
R1And R2Each independently is C1Or C2An alkyl group;
R3is C2Or C3An alkyl group;
l is O;
n is 1; and is
Ar is a radical of at most 3C1To C2Unsubstituted alkyl optionally substituted phenyl, naphthyl or pyridyl.
In a first aspect of the third embodiment, Ar is substituted with one or two C1To C2Unsubstituted alkyl-substituted phenyl. Alternatively, Ar is unsubstituted phenyl.
In a fourth embodiment, - (X) -N (R)1)(R2)-(R3-L)nAr is any one of the ligands in Table 1.
TABLE 1
In a fifth embodiment, the chromatographic resin has the formula or an anionic salt thereof:
chromatography matrix- (X) -N (R)1)-[(R3-L)n-Ar]2，
Wherein:
x is a spacer;
R1is C optionally substituted by-OH1To C6An alkyl group;
R3is C2To C6Alkyl or C4-C6A cycloalkyl group;
l is NR4O, or S, wherein R4Is hydrogen, or C1To C4An alkyl group;
n is 1 or 2; and is
Ar is a 6-to 10-membered ring, and:
if Ar is aryl, the aryl group is substituted by up to 5C1To C3Unsubstituted alkyl, C3To C6A branched alkyl, unsubstituted aryl or fluoro group is optionally substituted; or
If Ar is heteroaryl, the heteroaryl is optionally substituted with up to 4 alkyl groups.
In the first aspect of the fifth embodiment, R1Is C1To C3An alkyl group. Or, R1Is C1Or C2An alkyl group.
In a second aspect of the fifth embodiment, R3Is C2To C4An alkyl group. Or, R3Is C2Or C3An alkyl group.
In a third aspect of the fifth embodiment, L is NR4Or O, or NR4Or S. Alternatively, L is O.
In a fourth aspect of the fifth embodiment, n is 1.
In a fifth aspect of the fifth embodiment, Ar is a six membered ring, and if Ar is aryl, the aryl is substituted with up to 4C1To C2Unsubstituted alkyl, C3To C4Branched alkyl, or fluoro groups are optionally substituted; or if Ar is heteroaryl, the heteroaryl is optionally substituted with up to 3 alkyl groups. Or Ar is a radical of at most 3C1To C2Phenyl, naphthyl or pyridyl optionally substituted with unsubstituted alkyl or fluoro groups. Or Ar is substituted by one or two C1To C2Unsubstituted alkyl optionally substituted phenyl. Alternatively, Ar is unsubstituted phenyl. Alternatively, Ar is heteroaryl and the heteroatom in heteroaryl is N. Alternatively, Ar is unsubstituted heteroaryl.
In a sixth embodiment, the chromatographic resin has the formula or an anionic salt thereof:
chromatography matrix- (X) -N (R)1)-[(R3-L)n-Ar]2，
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-；
R1Is C1To C3An alkyl group;
R3is C2To C4An alkyl group;
l is O;
n is 1; and is
Ar is a 6-membered ring, and:
if Ar is aryl, the aryl group is substituted by up to 4C1To C2Unsubstituted alkyl, C3To C4Branched alkyl, or fluoro groups are optionally substituted; or
If Ar is heteroaryl, the heteroaryl is optionally substituted with up to 3 alkyl groups.
In the first aspect of the sixth embodiment, R1Is C1Or C2An alkyl group.
In a second aspect of the sixth embodiment, R3Is C2Or C3An alkyl group.
In a third aspect of the sixth embodiment, Ar is at most 3C1To C2Unsubstituted alkyl-substituted phenyl, naphthyl or pyridyl. Or Ar is substituted by one or two C1To C2Unsubstituted alkyl optionally substituted phenyl. Alternatively, Ar is unsubstituted. Alternatively, Ar is heteroaryl and the heteroatom in heteroaryl is N. Alternatively, Ar is unsubstituted heteroaryl.
In a seventh embodiment, the chromatographic resin has the formula or an anionic salt thereof:
chromatography matrix- (X) -N (R)1)-[(R3-L)n-Ar]2，
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(CH2-OH)-(O-CH2-CH(OH)-CH2)2-；
R1Is C1Or C2An alkyl group;
R3is C2Or C3An alkyl group; and is
L is O;
n is 1; and is
Ar is a radical of at most 3C1To C2Unsubstituted alkyl optionally substituted phenyl, naphthyl or pyridyl.
In a first aspect of the seventh embodiment, Ar is substituted with one or two C1To C2Unsubstituted alkyl-substituted phenyl. Alternatively, Ar is unsubstituted.
In the eighth embodiment, - (X) -N (R)1)-[(R3-L)n-Ar]2Is any one of the ligands in table 2.
TABLE 2
In some embodiments, the anionic salt is a hydrochloride or sulfate salt.
The chromatography matrix is a polymer that is functionalized so that a bond can be formed with the spacer X. Preferably, the polymer is a hydrophilic polymer. The polymer is insoluble in water. Suitable polymers are polyhydroxy polymers, for example, based on: polysaccharides, such as agarose, dextran, cellulose, starch, amylopectin (pullulan), and fully synthetic polymers, such as polyacrylamide, polymethacrylamide, polyhydroxyalkylvinylether, polyhydroxyalkylacrylate and polymethacrylate (e.g. polyglycidyl methacrylate), polyvinyl alcohol, and polymers based on styrene and divinylbenzene, and combinations comprising two or more thereofCopolymers of monomers corresponding to the above polymers. Suitable synthetic polymers include, but are not limited to, the following: fractogel from Toso-Haas, POROS media from Sammer Feishel scientific, Bio-Gel P and Macro Prep from Boyle (Bio-Rad), HEMA and Separon from TESSEK, and Hyper D and triacryl media (Trisacryl media) from Polle (Pall). Water-soluble polymers can be derivatized to be insoluble, for example, by crosslinking and coupling to insoluble bodies by adsorption or covalent bonding. Hydrophilic groups can be introduced onto hydrophobic polymers (e.g., copolymers of monovinyl and divinylbenzene) by: monomers which exhibit groups which can be converted into hydroxyl groups are polymerized, or by hydrophilizing the final polymer, for example by adsorption of suitable compounds (e.g.hydrophilic polymers). Examples of monomers that can be polymerized to obtain a useful matrix are: vinyl acetate, vinyl propylamine, acrylic acid, methacrylate, butyl acrylate, acrylamide, methacrylamide, vinyl pyrrolidone (vinyl pyrrolidone), in some cases with a functional group. Crosslinking agents may also be used in many embodiments, and in some embodiments when present, may constitute a molar ratio of about 0.1 to about 0.7 relative to the total monomers. Examples of crosslinking agents are: dihydroxyethylene bisacrylamide, diallyl tartaric acid diamide, triallyl citric acid triamide, ethylene glycol diacrylate (ethylene diacrylate), cysteamine bisacryloyl, N' -methylenebisacrylamide, and piperazine bisacrylamide. In some embodiments, the substrate is UNOsphereTMA carrier, a polymer produced from water-soluble hydrophilic monomers (burle corporation, herkles, ca).
The chromatography matrix may be in the form of particles, chips, membranes or monoliths (monoliths), i.e. chunks, monoliths, or pellets of material. Preferably, the chromatography matrix is porous. When used as a matrix, the particles may be in the form of spheres or beads, and are smooth-surfaced or have a rough or textured surface. In some cases, some of the pores are through-holes that extend through the particle to serve as channels large enough to allow hydrodynamic flow or rapid diffusion through the pores. When in spherical or beaded form, the median particle diameter is from about 25 microns to about 150 microns, wherein the term "diameter" refers to the longest outer dimension of the particle. Exemplary matrices and methods for their preparation are disclosed in U.S. patent No. 6498236 to Lihme et al, U.S. patent No. 5,645,717 to Hjerten et al, U.S. patent No. 5,647,979 to Liao et al, U.S. patent No. 5,935,429 to Liao et al, and U.S. patent No. 6,423,666 to Liao et al.
The ligand is attached to the chromatography matrix by a spacer X. Attachment to the chromatography matrix will depend on the particular chromatography matrix used and the chemical groups to be attached to the chromatography matrix. The ligand may be attached to the chromatography matrix by a reaction between a functional group on the chromatography matrix and the ligand. For chromatographic matrices that do not have suitable functional groups, the chromatographic matrix is reacted with a suitable activating reagent to produce the appropriate functional group to which the ligand can be attached. Reductive amination, epoxide chemistry, or azalide chemistry are examples of chemistries that act on the aldehyde, epoxide, or azalide functional groups, respectively.
In some embodiments, the chromatography matrix comprises an epoxide group, and the tertiary amine in the ligand is attached to the epoxide group by epoxide chemistry through the following scheme. In this scheme, the spacer X is-O-CH2-CH(OH)-CH2-. In this and other synthetic schemes of the present disclosure, the squares represent the matrix, while all coupling chemistry moieties are shown separately.
In some embodiments, the chromatography matrix comprises an azlactone ring, and the primary amine in the ligand is attached to the azlactone ring by the following scheme. In this scheme, the spacer X is-CO-NH-C (CH)3)2-CO-。
In some embodiments, the chromatography matrix comprises a diol and the tertiary amine in the ligand is attached 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-。
In certain embodiments, the chromatography matrix comprises-OH groups, and the tertiary amine in the ligand is attached to the-OH groups by activating the resin with epichlorohydrin, as described below. In this scheme, the spacer X is-O-CH2-CH(OH)-CH2-。
In some embodiments, the chromatography matrix comprises-OH groups, and the tertiary amine in the ligand is attached to the-OH groups 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-。
Other activating agents include, but are not limited to: epibromohydrin (which reacts with-OH functional groups on the chromatography matrix to produce epoxy groups), polyethylene glycol diglycidyl ether (which reacts with-OH functional groups on the chromatography matrix to produce epoxy groups), and sulfonyl chlorides (such as tosyl chloride and tresyl chlorides) (which react with-OH functional groups on the chromatography matrix to produce sulfonic acid esters).
Other spacers include, but are not limited to: -O-CH2-,–O-CH2-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-。
The chromatography matrix may be used in any conventional configuration, including packed and fluidized or expanded bed columns, monoliths or porous membranes, and may be used by any conventional method, including batch mode for loading, washing and elution, and continuous or flow-through mode. In some embodiments, the column ranges from 1cm to about 1m in diameter and 1cm to about 30cm or more in height.
Method of producing a composite material
Methods for purifying a target biomolecule are also provided. In one embodiment, the method comprises: contacting a sample containing biomolecules with a chromatography resin, thereby separating the biomolecules from the contaminants. The resulting purified biomolecules are then collected. In some embodiments, the target biomolecule is a monomeric antibody, and the method comprises purifying the monomeric antibody from aggregated antibodies in the sample.
Chromatography resins can be used to purify target biomolecules using anion exchange (i.e., where the ligands are positively charged) and hydrophobic mixed mode chromatography. Conditions can be adjusted so that the chromatography is run in bind-elute mode or flow-through mode.
Protein preparations to which the method may be applied may include unpurified or partially purified antibodies (e.g., IgG) from natural, synthetic, or recombinant sources. For example, an unpurified antibody preparation can be from a variety of sources, including but not limited to: plasma, serum, ascites fluid, milk, plant extracts, bacterial lysates (bacterial lysates), yeast lysates, or conditioned cell culture medium. The partially purified protein preparation may be derived from an unpurified preparation that has been treated by at least one chromatography, precipitation, other fractionation step, or any combination of the foregoing. In some embodiments, one or more chromatography steps employ any method, including, but not limited to: size exclusion chromatography, affinity chromatography, anion exchange chromatography, cation exchange chromatography, protein a affinity chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography, or hydroxyapatite chromatography. One or more precipitation steps may include salt or polyethylene glycol (PEG) precipitation, or precipitation with an organic acid, organic base, or other reagent. Other fractionation steps may include, but are not limited to: crystallization, liquid-liquid partitioning (liquid: liquid partitioning), or membrane filtration.
As will be understood in the art, the loading, washing and elution conditions for mixed mode chromatography will depend on the particular chromatography media/ligand used.
In some embodiments of the bind-elute mode, the loading (i.e., binding of the antibody to the chromatography resin) and optionally washing is performed at a pH above 7 (e.g., 7-8, 7-9, etc.). Some exemplary bind-elute conditions are:
the combination condition is as follows: in a suitable buffer (e.g., Tris, Bis-Tris or phosphate), pH 6.5-8.5, 0-1000mM NaCl or 100-;
elution conditions: an appropriate buffer with sodium acetate, citrate, arginine, or glycine, pH 3-8.5 or 4.0-6.0, 1-1000mM NaCl or 0-150mM NaCl is used.
Optionally, the chromatography resin may be washed under conditions such that some components of the sample are removed from the chromatography resin but the target biomolecule remains immobilized on the chromatography resin. In some embodiments, the target biomolecule is subsequently eluted by changing (e.g., decreasing or increasing) the salt concentration and/or decreasing the pH of the solution in contact with the substrate.
Alternatively, the sample may be applied in a flow-through mode, wherein some components of the sample are immobilized on the chromatography resin, but the target biomolecule flows through (i.e., flows through) the chromatography resin and is collected. Some exemplary flow-through conditions are: 0-150mM NaCl, pH 4.0-8.0; suitable buffers may include: for example, 2- (N-morpholino) ethanesulfonic acid (MES), Bis-Tris, sodium acetate, citrate-phosphate.
Drawings
FIG. 1 is a graph of the Dynamic Binding Capacity (DBC) of various mixed mode chromatography resins to monoclonal antibody S (mAb S).
Figure 2 is the chromatographic elution profile of mAb S on prototype 13.
FIG. 3 shows a flow-through mode (flow-through mode) purification chromatogram for monoclonal antibody T (mAb T) on prototype 13.
Fig. 4 is a binding-elution mode (bind-elute mode) purification chromatogram of mAb S on prototype 12.
Fig. 5 is a binding-elution pattern purification chromatogram for mAb S on prototype 13.
Examples
The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that may be altered or modified to produce substantially the same or similar results.
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