WO2012128353A1 - タンパク性物質結合性低分子化合物 - Google Patents
タンパク性物質結合性低分子化合物 Download PDFInfo
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- WO2012128353A1 WO2012128353A1 PCT/JP2012/057521 JP2012057521W WO2012128353A1 WO 2012128353 A1 WO2012128353 A1 WO 2012128353A1 JP 2012057521 W JP2012057521 W JP 2012057521W WO 2012128353 A1 WO2012128353 A1 WO 2012128353A1
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- FTNJQNQLEGKTGD-UHFFFAOYSA-N C1Oc(cccc2)c2O1 Chemical compound C1Oc(cccc2)c2O1 FTNJQNQLEGKTGD-UHFFFAOYSA-N 0.000 description 1
- XBNNVJNFDLRRCF-UHFFFAOYSA-N Cc(cc1)ccc1OCc1nc(CSc2nc(N)cc(N)n2)c[s]1 Chemical compound Cc(cc1)ccc1OCc1nc(CSc2nc(N)cc(N)n2)c[s]1 XBNNVJNFDLRRCF-UHFFFAOYSA-N 0.000 description 1
- DMFYGFODGFMZTI-UHFFFAOYSA-N Nc(cc1)ccc1OC(C(C(Oc(cc1)ccc1N)=N1)F)C(F)=C1Oc(cc1)ccc1NI Chemical compound Nc(cc1)ccc1OC(C(C(Oc(cc1)ccc1N)=N1)F)C(F)=C1Oc(cc1)ccc1NI DMFYGFODGFMZTI-UHFFFAOYSA-N 0.000 description 1
- BYUPLADTBLGLEK-UHFFFAOYSA-N Oc(c(O)c1)ccc1-c1c[s]c(COc2ccccc2)n1 Chemical compound Oc(c(O)c1)ccc1-c1c[s]c(COc2ccccc2)n1 BYUPLADTBLGLEK-UHFFFAOYSA-N 0.000 description 1
- CLBCQYRZNKJNMZ-UHFFFAOYSA-N Oc(cc1)ccc1NS(c1cc(S(Nc(cc2)ccc2O)(=O)=O)c(cccc23)c2c1SC3=O)(=O)=O Chemical compound Oc(cc1)ccc1NS(c1cc(S(Nc(cc2)ccc2O)(=O)=O)c(cccc23)c2c1SC3=O)(=O)=O CLBCQYRZNKJNMZ-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
- B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
- B01J20/289—Phases chemically bonded to a substrate, e.g. to silica or to polymers bonded via a spacer
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
- B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
- B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3244—Non-macromolecular compounds
- B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
Definitions
- the present invention relates to a method for separating, purifying, characterizing, identifying or quantifying a proteinaceous substance using a low molecular compound that specifically binds to an antibody.
- Patent Document 1 For protein purification, chromatographic separation techniques such as ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography, gel filtration chromatography, and affinity chromatography are commonly used. Since many kinds of proteins are mixed, it is extremely difficult to obtain the target protein purely using one purification technique. For this reason, a purification process in which several techniques are combined is generally employed (Patent Document 1).
- antibody drugs are one of the medical proteins that have been actively developed.
- An antibody drug is a drug that uses the function of an antibody, and because it works specifically on the target molecule, it reduces the side effects of conventional drugs and is expected to have a high therapeutic effect. Yes.
- antibody drugs actually contribute to the improvement of various pathological conditions, they are said to have a large effect on the quality of purity when compared to other medical proteins because they are administered in large quantities in the living body. Yes.
- a technique such as affinity chromatography that uses a molecule that specifically binds to the antibody as a ligand is generally used.
- Protein A produced by Staphylococcus is well known as a ligand that is basically a monoclonal IgG antibody and has an affinity for IgG antibodies.
- an affinity chromatography column (hereinafter referred to as a protein A column) in which protein A is immobilized on a water-insoluble carrier as a ligand is generally used for the initial purification step (capture step) in the antibody drug manufacturing process.
- a protein A column in which protein A is immobilized on a water-insoluble carrier as a ligand is generally used for the initial purification step (capture step) in the antibody drug manufacturing process.
- Patent Document 2 Patent Document 3
- Patent Document 4 Patent Document 5
- Non-patent Document 6 sulfone derivatives
- Patent Document 7 triazine derivatives
- Patent Document 8 mercapto heterocyclic compounds
- Patent Document 3 thiazole derivatives
- Patent Document 9 thiazole derivatives
- Protein A alternative ligands that have been developed to date are related compounds derived from basic structures such as triazines and thiazoles and are not yet available as ligands for protein analysis, purification, characterization, identification or quantification. It was enough. Therefore, a broad search for a wide variety of ligands with high chemical stability and versatility was an issue.
- ArX represents a structure containing an optionally substituted aromatic 6-membered ring
- ArYHB represents a structure containing an optionally substituted aromatic 6-membered ring having a proton donor.
- Linker represents a group of 4 to 30 atoms that binds ArX and ArYHB), and found that the low molecular weight compound specifically binds to the Fc portion (IgG-Fc) of an IgG antibody, The present invention has been completed. That is, the present invention is as follows. [1] General formula (1):
- ArX represents a structure containing an optionally substituted aromatic 6-membered ring
- ArYHB represents a structure containing an optionally substituted aromatic 6-membered ring having a proton donor.
- Linker represents a 4 to 30 atomic group that binds ArX and ArYHB.
- ArYHB in the low molecular compound has a structure in which a hydroxyl group or an amino group is directly bonded to an aromatic 6-membered ring or a side chain thereof.
- ArX in the low molecular weight compound has any one of a halogen group, an amino group, a hydroxyl group, and a methyl group as a substituent, or ArX represents a benzene ring, a naphthalene ring, or 1,2-
- the (Linker) is an optionally substituted tetrazole, an optionally substituted hydantoin, an optionally substituted pyrrole, an optionally substituted pyridine, or an optionally substituted 1,3 , 4-thiadiazole, optionally substituted triazole, optionally substituted aminopyrazolo [3,4-d] pyrimidine, optionally substituted thiazole, or optionally substituted pyrimidine
- CZ is a structure containing a benzene ring which may be substituted, or a structure containing a naphthalene ring which may be substituted.
- U and V are each a use or method according to any one of [1] to [3], which is composed of a non-hydrogen atom containing no ring structure.
- the low molecular weight compound is represented by the following structural formulas (2) to (17);
- the low molecular weight compound is immobilized on a carrier made of a water-insoluble substrate via a spacer that binds to the (Linker) of the low molecular weight compound according to any one of [1] to [9].
- An affinity separation matrix is provided.
- the low molecular weight compounds presented in the present invention can be used as useful and durable ligands for the separation, removal, isolation, purification, characterization, identification or quantification of proteinaceous substances, particularly immunoglobulins. it can.
- these low-molecular compounds that are versatile and versatile it is possible to inexpensively produce a selective desorbent for proteinaceous substances, particularly immunoglobulins, and it can be advantageously used for industrial purification. it can.
- 2 is a binding curve of low molecular compounds (2) to (5) (IgG-Fc binding low molecular compound) obtained by a Biacore measurement experiment in Example 2.
- 2 is a binding curve of low molecular compounds (6) to (9) (IgG-Fc binding low molecular compound) obtained by a Biacore measurement experiment in Example 2.
- 2 is a binding curve of low molecular compounds (10) to (13) (IgG-Fc binding low molecular compound) obtained by a Biacore measurement experiment in Example 2.
- 3 is a binding curve of low molecular compounds (14) to (17) (IgG-Fc binding low molecular compound) obtained by a Biacore measurement experiment in Example 2.
- the low molecular weight compound used in the present invention is represented by the following formula (1):
- ArX represents a structure containing an optionally substituted aromatic 6-membered ring
- ArYHB represents a structure containing an optionally substituted aromatic 6-membered ring having a proton donor.
- (Linker) is an optionally substituted tetrazole, an optionally substituted hydantoin, an optionally substituted pyrrole, an optionally substituted pyridine, an optionally substituted 1,3,4. -Thiadiazole, optionally substituted pyrimidine (such as 5-nitropyrimidine), optionally substituted triazole, optionally substituted aminopyrazolo [3,4-d] pyrimidine, or optionally substituted
- a structure containing thiazole or (Linker) is represented by (-U-CZ-V-)
- a structure containing a benzene ring which CZ may be substituted, or a naphthalene ring which may be substituted U and V are each composed of a non-hydrogen atom that does not contain a ring structure.
- the compound represented by the above formula (1) should be used as a useful and durable ligand for the separation, removal, isolation, purification, characterization, identification or quantification of proteinaceous substances, particularly immunoglobulins. Can do. In addition, by using these low-molecular compounds that are versatile and versatile, it is possible to produce a selective desorbent of proteinaceous substances, particularly immunoglobulins at low cost, which is advantageous for industrial purification. Can be used for
- ArX represents a structure containing an optionally substituted aromatic 6-membered ring, but the position, type, and number of the substituent are not particularly limited, and as long as the aromatic 6-membered ring part is included , A condensed aromatic structure may be used, and either a hydrocarbon ring or a heterocyclic ring may be used.
- the aromatic 6-membered ring includes various substituents such as halogen groups (fluorine group, chlorine group, bromine group, iodine group, etc.), aprotic polar groups (nitro group, nitrile group, carboxylic acid ester group, Sulfonic acid ester groups, alkoxy groups, etc.), proton donors (amino groups, hydroxyl groups, thiol groups, carboxylic acid groups, sulfonic acid groups, etc.), hydrocarbon groups (methyl groups, ethyl groups, etc.) may be bonded. .
- halogen groups fluorine group, chlorine group, bromine group, iodine group, etc.
- aprotic polar groups nitro group, nitrile group, carboxylic acid ester group, Sulfonic acid ester groups, alkoxy groups, etc.
- proton donors amino groups, hydroxyl groups, thiol groups, carboxylic acid groups, sulfonic acid groups, etc.
- ArX that exhibits good binding properties to immunoglobulins includes (i) halogen groups (preferably fluorine groups, chlorine groups, bromine groups, etc., especially fluorine groups, chlorine groups), proton donors (preferably amino groups, hydroxyl groups). ), Or a C 1-4 hydrocarbon group (preferably a methyl group) as a substituent, or (ii) a structure having a benzene ring unit, such as a benzene ring, a naphthalene ring, or Formula (18):
- ArX is a hydrocarbon ring such as a benzene ring or a naphthalene ring, and a proton donor (amino group, hydroxyl group, etc.) is not bonded to the hydrocarbon ring.
- ArYHB represents an optionally substituted aromatic 6-membered ring having a proton donor.
- the proton donor include those similar to ArX, and an amino group, a hydroxyl group and the like are preferable.
- the proton donor (group) may be directly bonded to the aromatic 6-membered ring or may be bonded to the side chain of the aromatic 6-membered ring, but is preferably directly bonded to the aromatic 6-membered ring.
- the kind of substituent of ArYHB is not specifically limited, For example, the thing similar to ArX (except a proton donor) can be illustrated, The position and number are not specifically limited, either.
- a preferred substituent is a halogen group (preferably a fluorine group, a chlorine group, a bromine group, etc., particularly a bromine group).
- the aromatic 6-membered ring of ArYHB may have a condensed aromatic structure, and may be a heteroaromatic 6-membered ring such as pyrimidine in addition to a cyclic hydrocarbon ring (such as a benzene ring).
- a preferred aromatic 6-membered ring is a non-condensed hydrocarbon ring or a heterocyclic ring.
- tyrosine having a proton donor exists in a part of the structure of protein A that interacts with IgG-Fc, so that a hydroxyl group or amino group serving as a proton donor exists.
- a non-condensed aromatic 6-membered ring directly bonded to the ring structure is preferred.
- Linker means an atomic group of 4 to 30 that binds ArX and ArYHB in the formula (1), and may be in any form of linear, branched, or ring structure.
- the type of bond between ArX and ArYHB is not limited.
- the Linker and each of ArX or ArYHB may be combined at one place or at two places.
- the structure exhibiting good binding properties with immunoglobulins is preferably a tetrazole which may be substituted, a hydantoin which may be substituted, or a substituted, from the viewpoint that it is difficult to take a free structure with flatness.
- Pyrrole which may be substituted pyridine which may be substituted, 1,3,4-thiadiazole which may be substituted, pyrimidine which may be substituted (such as 5-nitropyrimidine), triazole which may be substituted , Having a cyclic structure such as an aminopyrazolo [3,4-d] pyrimidine which may be substituted, a structure containing thiazole which may be substituted, or a structure represented by (-U-CZ-V-) Is preferred.
- the CZ is a structure containing an optionally substituted benzene ring or an optionally substituted naphthalene ring, and U and V are both non-hydrogen atoms not containing a ring structure (eg, —O —, —S—, —SO—, —SO 2 —, —NH—, —NR—, —CH 2 —, —CHR—, —CR 2 —, etc., preferably —O—, —S—, —SO -, SO 2- , -NR-, -CR 2-, etc., a group having no hydrogen atom bonded thereto, wherein R is an organic group containing an alkyl group such as a methyl group) and the non-hydrogen One atom may be sufficient and two or more (especially two) may comprise U or V continuously.
- a ring structure eg, —O —, —S—, —SO—, —SO 2 —, —NH—, —
- the low molecular weight compound represented by the formula (1) can be further modified by binding to an immunoglobulin by chemical modification.
- Preferred low molecular compounds (1) include the following compounds (101), (201), (301), (401) and the like.
- Compound (101) is represented by the following formula.
- ArX1 is the same as ArX.
- Linker 1 represents a cyclic structure exemplified as the Linker or a divalent group in which —S— is bonded to the cyclic structure.
- R 11 represents a carbon atom or an oxygen atom, and in the case of a carbon atom, R 12 is bonded to this carbon atom.
- R 12 is a hydrogen atom or an organic group that forms a ring with R 11 .
- R 13 to R 15 are the same or different and are a hydrogen atom or a proton donor (particularly an amino group or a hydroxyl group), and at least one of R 13 to R 15 is a proton donor.
- Preferred ArX1 is an aromatic hydrocarbon ring such as a benzene ring or a naphthalene ring.
- Preferred Linker 1 is optionally substituted 1,3,4-thiadiazole, optionally substituted triazole, and optionally substituted aminopyrazolo [3,4-d] pyrimidine.
- Linker 1 and ArX1 may be directly bonded, and other bonds (for example, an ether bond, a thioether bond, a methylene chain, and combinations thereof.
- ArX1 and Linker may be bonded at two positions, directly bonded at one position, and may be bonded at another position through another bond.
- R 11 and R 12 form a ring
- the ring is preferably a 5- or 6-membered ring, and more preferably a tetradehydro- ⁇ -valerolactone ring.
- R 13 to R 15 are preferably at least two proton donors, and more preferably two adjacent proton donors.
- Compounds (2), (3), (4), and (5) described later are exemplified as specific examples of this compound (101).
- Compound (201) is represented by the following formula.
- Linker 2 represents a cyclic structure exemplified as the Linker or a divalent group in which the groups U and V are bonded to the cyclic structure.
- R 21 to R 26 are the same or different and are a hydrogen atom or a proton donor (particularly an amino group or a hydroxyl group), and at least one of R 24 to R 26 is a proton donor.
- Preferred Linker 2 is (-U-CZ-V-), an optionally substituted pyrimidine (especially 5-nitropyrimidine), an optionally substituted pyridine (for example, a halogen atom such as a fluorine atom, paraaminophenylene oxide, etc. Pyridine optionally substituted with an alkoxy group).
- a group U and a group V are bonded to the pyrimidine and pyridine, respectively.
- U, V are preferably, -O-, SO 2 -, - NH -, - NR -, - CR 2 - is a one or two, such as, more preferably -O-, SO 2 -, - One or two of NR-, -CR 2 -and the like.
- CZ of (-U-CZ-V-) is preferably a benzene ring, a naphthalene ring, a substituted naphthalene ring (particularly a substituted naphthalene ring in which the 1- and 8-positions are bridged to form a 5- or 6-membered ring, for example, 1-position and 8-position are naphthalene rings bridged by —S—C ( ⁇ O) —.
- At least one of R 21 to R 23 is preferably a proton donor.
- R 21 to R 23 and R 24 to R 26 preferably have the same proton donor.
- Compounds (6), (7), (8), (11), and (12) described later are exemplified as specific examples of this compound (201).
- Compound (301) is represented by the following formula.
- Linker 3 represents an exemplary ring structure as the Linker.
- R 31 and R 32 are the same or different and each represents one of the non-hydrogen atoms or a ⁇ CH— group. Further, when a plurality of R 31 are present, they may be different from each other. The same applies when there are a plurality of R 32 .
- a and b are 0 or an integer of 1 or more.
- R 33 ⁇ R 38 are the same or different, a hydrogen atom, a proton donor (especially an amino group, a hydroxyl group), a halogen atom (chlorine group, bromine group), a hydrocarbon group (especially methyl), R 36 ⁇ At least one of R 38 is a proton donor.
- Preferred Linker 3 is an optionally substituted hydantoin, an optionally substituted thiazole, and the like.
- Preferred (R 31 ) a and (R 32 ) b are —CH 2 —, ⁇ CH—, —SO 2 —, —N (CH 3 ) —, —CH 2 —O—, —CH 2 —S— and the like. It is. Compounds (9), (10), (14), and (16) described later are exemplified as specific examples of this compound (301).
- Compound (401) is represented by the following formula.
- Linker 4 and Linker 5 show an exemplary annular structure as the Linker.
- R 41 represents one of the non-hydrogen atoms.
- c is 0 or 1
- one of d and e is 1 and the other is 0.
- R 42 ⁇ R 49 are the same or different, a hydrogen atom, a proton donor (especially an amino group, a hydroxyl group), a halogen atom (especially fluorine group), a hydrocarbon group (especially methyl), an alkoxy group, R 42 ⁇ Two of R 44 may be connected to form a ring, or two of R 47 to R 48 may be connected to form a ring. At least one of R 47 to R 49 is a proton donor.
- Preferred Linker 4 and Linker 5 are optionally substituted tetrazole and optionally substituted pyrrole (particularly dimethylpyrrole).
- Preferred R 41 is —CH 2 —.
- Compounds (13), (15), and (17) described later are exemplified as specific examples of this compound (401).
- the low molecular weight compound of the present invention has the following formulas (2) to (17):
- the compound (101), the compound (201) and the like are particularly preferable. It is preferable that the group U and the group V are bonded.
- the most preferred low molecular compounds are compounds (2), (3), (4), (5), (6), (7), and (8). These preferred low molecular weight compounds have particularly high binding ability to IgG-Fc.
- the present invention also relates to various methods of using the low molecular weight compound represented by the formula (1) as a low molecular weight proteinaceous substance using the high affinity for the Fc region of immunoglobulin.
- proteinaceous substance in the present invention refers to any molecule having a polypeptide structure, and is defined as a proteinaceous substance including a polypeptide chain that is fragmented or linked by peptide bonds.
- proteinaceous substance include immunoglobulins, immunoglobulin fragments, monoclonal antibodies, proteins containing immunoglobulin Fc region (IgG-Fc), Fc fusion proteins, and the like. These protein substances may be contained in the cell culture.
- the present invention is not limited to immunoglobulin molecules containing the Fc region without any deficiency, or derivatives thereof.
- a protein containing an Fc region of an immunoglobulin is a protein containing a site on the Fc region side, and if the low molecular weight compound can bind, the Fc region is completely retained. There is no need. Further, as long as it has an Fc region, an Fc region fusion compound, an Fc region fusion inorganic compound, or the like obtained by synthesis or the like may be used.
- the affinity for immunoglobulin is, for example, biosensors such as the Biacore system (GE Healthcare) using the surface plasmon resonance principle, and isothermal titration calorimeters (ITC) that detect exothermic and endothermic changes caused by binding, It can be evaluated by a biomolecule interaction detection / analysis device.
- biosensors such as the Biacore system (GE Healthcare) using the surface plasmon resonance principle, and isothermal titration calorimeters (ITC) that detect exothermic and endothermic changes caused by binding, It can be evaluated by a biomolecule interaction detection / analysis device.
- the present invention also includes an affinity ligand utilizing the characteristics of the low molecular weight compound. That is, an affinity separation matrix characterized in that the (Linker) portion of the ligand (low molecular compound) is immobilized on a carrier comprising a water-insoluble base material via a spacer is also included in the present invention.
- an “affinity ligand” is a substance (functional group) that selectively collects (binds) a target molecule from a set of molecules based on the affinity between specific molecules, represented by the binding between an antigen and an antibody. In the present invention, it particularly refers to a low molecular compound of the above formula (1) that specifically binds to immunoglobulin.
- the carrier comprising a water-insoluble substrate used in the present invention includes an inorganic carrier such as glass beads and silica gel, a synthetic polymer such as crosslinked polyvinyl alcohol, crosslinked polyacrylate, crosslinked polyacrylamide, crosslinked polystyrene, polymethacrylate and polyurethane, And organic carriers composed of polysaccharides such as crystalline cellulose, crosslinked cellulose, crosslinked agarose, crosslinked dextran, and organic-organic, organic-inorganic composite carriers obtained by combining these.
- an inorganic carrier such as glass beads and silica gel
- a synthetic polymer such as crosslinked polyvinyl alcohol, crosslinked polyacrylate, crosslinked polyacrylamide, crosslinked polystyrene, polymethacrylate and polyurethane
- organic carriers composed of polysaccharides such as crystalline cellulose, crosslinked cellulose, crosslinked agarose, crosslinked dextran, and organic-organic, organic-inorganic composite carriers obtained by combining these.
- water-insoluble carrier such as GCL2000, which is a porous cellulose gel, Sephacryl® S-1000 in which allyldextran and methylenebisacrylamide are covalently crosslinked, Toyopearl, which is an acrylate carrier, and agarose.
- GCL2000 which is a porous cellulose gel
- Sephacryl® S-1000 in which allyldextran and methylenebisacrylamide are covalently crosslinked
- Toyopearl which is an acrylate carrier
- agarose agarose
- Sepharose CL4B which is a cross-linked carrier
- Cellufine which is a cellulosic cross-linked carrier.
- the water-insoluble carrier used in the present invention desirably has a large surface area and is preferably a porous material having a large number of pores of an appropriate size in view of the purpose and method of use of the affinity separation matrix.
- the form of the carrier can be any of beads, monoliths, fibers, membranes (including hollow fibers), and any form can be selected.
- a spacer molecule having an amino group, a carboxyl group, or a thiol group can be introduced into (Linker) of a low molecular compound, and can be bound to a carrier by a conventional coupling method.
- the “spacer” refers to a molecule that binds the low molecular compound and the matrix, and examples thereof include compounds having a reactive functional group such as an amino group, a carboxyl group, an ether group, and a thioether group. .
- the spacer molecule may be composed of a plurality of atoms.
- the carrier is reacted with cyanogen bromide, epichlorohydrin, diglycidyl ether, tosyl chloride, tresyl chloride, hydrazine, sodium periodate, etc.
- a low molecular weight compound that is immobilized as a ligand or in a system where a low molecular weight compound that is immobilized as a carrier and a ligand exists, such as carbodiimide And a condensing method such as glutaraldehyde, a reagent having a plurality of functional groups in the molecule, condensation, and crosslinking.
- the low molecular compound of the present invention used as a ligand may be immobilized by chemically modifying the carrier, or a spacer molecule useful for immobilization may be introduced.
- the essence of the present invention is that the function of the low molecular weight compound is similarly imparted to the matrix in which the low molecular weight compound is immobilized as a ligand, and how the low molecular weight compound is modified / modified for immobilization. And within the scope of the present invention.
- Antibody, antibody derivative, fragment antibody, and fragment containing immunoglobulin Fc region using the above affinity separation matrix in which the low molecular weight compound found by the present invention is immobilized on a water-insoluble carrier
- Antibody derivatives can be separated, purified and characterized by affinity column / chromatography purification methods.
- the “antibody derivative” refers to, for example, a chimeric antibody in which a part of the domain of human IgG is replaced with a domain of an IgG antibody of another species and fused, or the CDR portion of human IgG is a CDR of an antibody of another species.
- Examples include humanized antibodies that are fused by replacement with a portion, and ⁇ fragment antibodies '' include, for example, proteins consisting only of the Fc region of human IgG (IgG-Fc), and ⁇ fragment antibody derivatives ''
- An example is an artificial antibody in which the Fv region and Fc region of human IgG are fused.
- Purification methods for these antibodies, antibody derivatives, fragment antibodies, and fragment antibody derivatives can be achieved by procedures in accordance with affinity column chromatography purification methods using protein A columns that are already commercially available (Roque ACA Other, JournalJof Chromatography A, 2007, 1160, pp. 44-55).
- an antibody, an antibody derivative, a fragment antibody, and a buffer containing the fragment antibody derivative are adjusted to be neutral, and then the solution is passed through an affinity column packed with the affinity separation matrix of the present invention.
- the antibody derivative, the fragment antibody, and the fragment antibody derivative are adsorbed.
- an acidic buffer adjusted to an appropriate pH (which may contain a substance that promotes dissociation from the matrix) is passed through the column, and the desired antibody, antibody derivative, fragment antibody, and fragment antibody derivative are added. By elution, high purity purification is achieved.
- the antibody, antibody derivative, fragment antibody, and fragment antibody derivative adsorbed on the affinity separation matrix of the present invention can be characterized as having IgG-Fc.
- the affinity separation matrix of the present invention contains a pure buffer solution (suitable denaturing agent, or suitable strong acid or strong alkali) to such an extent that the ligand (low molecular weight compound) or the carrier substrate does not completely impair the function.
- a pure buffer solution suitable denaturing agent, or suitable strong acid or strong alkali
- it may be a solution containing an organic solvent), and it can be reused by washing it through.
- the chemical stability of the ligand is expected to be higher than when a protein such as protein A is used as the ligand.
- chemical stability means the property of retaining the function of a low molecular compound.
- “maintaining the function of a low molecular weight compound” refers to maintaining affinity for the Fc region (IgG-Fc) of an immunoglobulin, that is, “chemical stability of a ligand”. "Is higher, the degree of decrease in the affinity of the immunoglobulin for the Fc region (IgG-Fc) is smaller even when various chemical treatments are performed.
- Example 1 Preparation of Fc Fragment Derived from Human Plasma “Affinity to Fc region” in the present invention was examined using an Fc fragment that does not contain an immunoglobulin Fab region. Using an IgG preparation derived from human plasma as a raw material, this was fragmented into a Fab fragment and an Fc fragment with papain, and only the Fc fragment was separated and purified to prepare an Fc fragment.
- Gamma guard manufactured by Baxter
- an IgG preparation derived from human plasma is dissolved in papain digestion buffer (0.1 M AcOH-AcONa, 2 mM EDTA, 1 mM cysteine, pH 5.5), and Papain Agarose from papaya latex (papain fixation) Agarose (manufactured by SIGMA) was added, and the mixture was incubated at 37 ° C. for about 8 hours while mixing with a rotator.
- IgG-Fc Fc fragment
- a reaction solution (mixed of Fab and Fc fragments) diluted with phosphate buffer (20 mM NaH 2 PO 4 -Na 2 HPO 4 , 150 mM NaCl, pH 7.4) to be close to pH 7.4
- phosphate buffer 20 mM NaH 2 PO 4 -Na 2 HPO 4 , 150 mM NaCl, pH 7.4
- elution buffer B 100 mM citrate, pH 3.0
- Example 2 Analysis of affinity between low molecular weight compounds and IgG-Fc
- the affinity analysis between low molecular weight compounds and IgG-Fc was performed using Biacore 3000 (manufactured by GE Healthcare) using surface plasmon resonance as follows. Carried out. The sensor chips and reagents used below are all manufactured by GE Healthcare. In addition, low molecular weight compounds (2) to (17) for which the binding ability to IgG-Fc was evaluated were obtained from Namiki Shoji.
- IgG-Fc obtained in Example 1 is diluted 50 times with an immobilization buffer (10 mM CH 3 COOH-CH 3 COONa, pH 4.5), and IgG-Fc is immobilized on sensor chip CM5 according to the protocol attached to Biacore 3000. did. Immobilization of IgG-Fc to sensor chip CM5 is performed by an amine coupling method using N-hydroxysuccinimide (NHS) and N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochroride (EDC). Ethanolamine was used for blocking. In addition, a reference cell serving as a negative control was prepared by performing treatment for immobilizing Ethanolamine after activation with EDC / NHS for another flow cell on the chip.
- an immobilization buffer 10 mM CH 3 COOH-CH 3 COONa, pH 4.5
- Immobilization of IgG-Fc to sensor chip CM5 is performed by an amine coupling method using
- a ligand consisting of any one of various low molecular compounds (2) to (17) was allowed to flow on the sensor chip to detect the interaction.
- a running buffer (20 mM NaH 2 PO 4 —Na 2 HPO 4 , 150 mM NaCl, 0.005% P-20, 5% DMSO) , pH 7.4), 3 or 4 diluted solutions having different concentrations were prepared in the range of 25 ⁇ M to 100 ⁇ M.
- Each prepared low molecular weight compound solution was added to the sensor chip for 120 seconds at a flow rate of 30 ⁇ L / min.
- a binding reaction curve at the time of addition (binding phase, 120 seconds) and after completion of the addition (dissociation phase, 120 seconds) was observed in order. After each observation, 40 mM NaOH was added for 15 seconds to regenerate the sensor chip.
- the binding curve of the sample containing DMSO was corrected for the obtained binding curve using a Biacore correction tool manufactured by GE Healthcare (see FIGS. 1 to 4). Represents the passage of detection (measurement) time (sec), and the vertical axis RU represents the detection intensity).
- the binding constant K A for IgG-Fc of various low molecular weight compounds (2) to (17) is 10 3 - 10 4 M about -1, strong binding to IgG-Fc The results shown are obtained.
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Abstract
Description
[1]一般式(1):
[1]上記式(1)で表される低分子化合物とタンパク性物質とを接触させて、タンパク性物質を低分子化合物に結合させる方法。
[2]前記低分子化合物におけるArYHBが、芳香族6員環又はその側鎖に水酸基もしくはアミノ基が直接結合した構造を有することを特徴とする、[1]に記載の使用又は方法。
[3]前記低分子化合物におけるArXが、ハロゲン基、アミノ基、水酸基、もしくは、メチル基のいずれかを置換基として有するか、または、ArXが、ベンゼン環、ナフタレン環、もしくは、1,2-メチレンジオキシベンゼンのいずれかを含む構造を有するか、或いはそれらの両方であることを特徴とする、[1]または[2]に記載の使用又は方法。
[4]前記(Linker)が、置換されていてもよいテトラゾール、置換されていてもよいヒダントイン、置換されていてもよいピロール、置換されていてもよいピリジン、置換されていてもよい1,3,4-チアジアゾール、置換されていてもよいトリアゾール、置換されていてもよいアミノピラゾロ[3,4-d]ピリミジン、置換されていてもよいチアゾール、もしくは置換されていてもよいピリミジンを含む構造であるか、或いは(Linker)を(-U-CZ-V-)で表したとき、CZが置換されていてもよいベンゼン環を含む構造、もしくは、置換されていてもよいナフタレン環を含む構造であり、UおよびVは、いずれも環構造を含まない非水素原子で構成される[1]~[3]のいずれかに記載の使用又は方法。
[5]前記低分子化合物が、以下の構造式で示される化合物(2)~(17);
[6]タンパク性物質が、免疫グロブリン、免疫グロブリン断片であることを特徴とする、[1]~[5]のいずれかに記載の使用又は方法。
[7]タンパク性物質が、モノクローナル抗体であることを特徴とする、[1]~[6]のいずれかに記載の使用又は方法。
[8]タンパク性物質が、IgG-Fc、または、Fc融合タンパク質であることを特徴とする、[1]~[7]のいずれかに記載の使用又は方法。
[9]タンパク性物質が細胞培養物中に含まれることを特徴とする、[1]~[8]のいずれかに記載の使用又は方法。
[10][1]~[9]のいずれかに記載の低分子化合物の(Linker)に結合するスペーサーを介して、水不溶性の基材からなる担体に前記低分子化合物が固定化されたことを特徴とする、アフィニティー分離マトリックス。
化合物(101)は、下記式で表される。
好ましいLinker 1は、置換されていてもよい1,3,4-チアジアゾール、置換されていてもよいトリアゾール、置換されていてもよいアミノピラゾロ[3,4-d]ピリミジンである。なおLinker 1とArX1とは、直接結合していてもよく、他の結合(例えば、エーテル結合、チオエーテル結合、メチレン鎖、及びこれらの組合せ。好ましくはチオエーテル結合、或いはチオエーテル結合とメチレン鎖からなる結合)を介して結合してもよく、さらにはArX1とLinkerとが2箇所で結合しており、その1箇所では直接結合し、残りの1箇所では他の結合を介して結合してもよい。
R13~R15は、少なくとも2つがプロトンドナーであるのが好ましく、さらには隣接する2つがプロトンドナーであるのが好ましい。
なお後述する化合物(2)、(3)、(4)、(5)がこの化合物(101)の具体例として例示される。
なお後述する化合物(6)、(7)、(8)、(11)、(12)がこの化合物(201)の具体例として例示される。
なお後述する化合物(9)、(10)、(14)、(16)がこの化合物(301)の具体例として例示される。
なお後述する化合物(13)、(15)、(17)がこの化合物(401)の具体例として例示される。
(2)ArX=ベンゼン環、ArYHB=水酸基が結合した、置換されている芳香族6員環、(Linker)=アミノピラゾロ[3,4-d]ピリミジン、
(3)ArX=ベンゼン環、ArYHB=水酸基が結合した、置換されている芳香族6員環、(Linker)=アミノピラゾロ[3,4-d]ピリミジン、
(4)ArX=ベンゼン環、ArYHB=水酸基が結合した、置換されている芳香族6員環、(Linker)=トリアゾール、
(5)ArX=ナフタレン環、ArYHB=水酸基が結合した、置換されている芳香族6員環、(Linker)=1,3,4-チアジアゾール、
(6)ArX=水酸基を置換基として有する芳香族6員環、ArYHB=水酸基が結合した芳香族6員環、(Linker)=ベンゼン環、
(7)ArX=水酸基を置換基として有する芳香族6員環、ArYHB=水酸基が結合した芳香族6員環、(Linker)=ニトロピリミジン、
(8)ArX=アミノ基を置換基として有している芳香族6員環、ArYHB=アミノ基が結合した芳香族6員環、(Linker)=ベンゼン環、
(9)ArX=ハロゲン基を置換基として有する芳香族6員環、ArYHB=水酸基が結合した、置換されている芳香族6員環、(Linker)=ヒダントイン、
(10)ArX=ベンゼン環、ArYHB=水酸基が結合した、置換されている芳香族6員環、(Linker)=チアゾール、
(11)ArX=水酸基を置換基として有する芳香族6員環、ArYHB=水酸基が結合した芳香族6員環、(Linker)=置換されたナフタレン環、
(12)ArX=アミノ基を置換基として有する芳香族6員環、ArYHB=アミノ基が結合した芳香族6員環、(Linker)=ピリジン、
(13)ArX=ハロゲン基を置換基として有する芳香族6員環、ArYHB=水酸基が結合した芳香族6員環、(Linker)=テトラゾール、
(14)ArX=ベンゼン環、ArYHB=水酸基が結合した芳香族6員環、(Linker)=チアゾール、
(15)ArX=メチル基を置換基として有する芳香族6員環、ArYHB=水酸基が結合した芳香族6員環、(Linker)=テトラゾール、
(16)ArX=メチル基を置換基として有する芳香族6員環、ArYHB=アミノ基が結合した、置換されている芳香族6員環、(Linker)=チアゾール、
(17)ArX=(18)式を含む構造、ArYHB=アミノ基が結合したピリミジン(複素芳香族6員環)、(Linker)=ピロール。
本発明における低分子化合物としては、化合物(101)、化合物(201)などが特に好ましく、この化合物(201)は、Linker 2が置換されていてもよいベンゼン環又は置換されていてよいピリミジン環に基U、基Vが結合したものであるのが好ましい。最も好ましい低分子化合物は、化合物(2)、(3)、(4)、(5)、(6)、(7)、(8)である。これら好ましい低分子化合物は、IgG-Fcとの結合能が特に高い。
本発明に用いる、水不溶性の基材からなる担体としては、ガラスビーズ、シリカゲルなどの無機担体、架橋ポリビニルアルコール、架橋ポリアクリレート、架橋ポリアクリルアミド、架橋ポリスチレン、ポリメタクリレート、ポリウレタンなどの合成高分子や、結晶性セルロース、架橋セルロース、架橋アガロース、架橋デキストランなどの多糖類からなる有機担体、さらにはこれらの組み合わせによって得られる有機-有機、有機-無機などの複合担体などが挙げられる。
リガンドの固定化方法としては、例えば、低分子化合物の(Linker)にアミノ基、カルボキシル基、または、チオール基を有するスペーサー分子を導入し、従来のカップリング法で担体に結合することができる。
カップリング法としては、例えば、担体を臭化シアン、エピクロロヒドリン、ジグリシジルエーテル、トシルクロライド、トレシルクロライド、ヒドラジン、および、過ヨウ素酸ナトリウムなどと反応させて担体を活性化し(あるいは担体表面に反応性官能基を導入し)、リガンドとして固定化する低分子化合物とカップリング反応を行い固定化する方法、または、担体とリガンドとして固定化する低分子化合物が存在する系にカルボジイミドのような縮合試薬、もしくは、グルタルアルデヒドのように分子中に複数の官能基を持つ試薬を加えて縮合、架橋することによる固定化方法などが挙げられる。
ヒト血漿由来Fcフラグメントの調製
本発明における「Fc領域への親和性」については、免疫グロブリンのFab領域を含まないFcフラグメントを用いて調べた。ヒト血漿由来IgG製剤を原料として、これをパパインによってFabフラグメントとFcフラグメントに断片化し、Fcフラグメントのみを分離精製することでFcフラグメントを調製した。ヒト血漿由来IgG製剤のガンマガード(バクスター社製)を、パパイン消化用緩衝液(0.1 M AcOH-AcONa、2 mM EDTA、1 mM システイン、pH 5.5)に溶解し、Papain Agarose from papaya latex(パパイン固定化アガロース、SIGMA社製)を添加し、ローテーターで混和させながら、37℃で約8時間インキュベートした。パパイン固定化アガロースから分離した反応溶液(FabフラグメントとFcフラグメントが混在)から、Protein Aカラム(MabSelect, GEヘルスケア社製)を利用したアフィニティークロマトグラフィーにより、Fcフラグメント(以後、IgG-Fcと表記する)を分離精製した。具体的には、リン酸緩衝液(20 mM NaH2PO4-Na2HPO4, 150mM NaCl, pH 7.4)で、pH 7.4に近くなるよう希釈した反応溶液(FabフラグメントとFcフラグメントが混在)を、リン酸緩衝液にて平衡化したProtein Aカラムに添加し、リン酸緩衝液で洗浄後、溶出緩衝液B(100 mM クエン酸, pH 3.0)を利用し溶出されるIgG-Fcを分取し、IgG-Fc溶液を得た。なおクロマトグラフィーによるタンパク質精製は、AKTAprime plusシステム(GEヘルスケア社製)を利用して実施した。
低分子化合物のIgG-Fcとの親和性の解析
低分子化合物とIgG-Fcとの親和性の解析は表面プラズモン共鳴を利用したBiacore 3000(GEヘルスケア社製)を用いて、以下の様にして実施した。なお以下で用いたセンサーチップや試薬は全てGEヘルスケア社製である。またIgG-Fcとの結合力の評価を行った低分子化合物(2)~(17)はナミキ商事から入手した。
実施例1で得たIgG-Fcを固定化用緩衝液(10mM CH3COOH-CH3COONa, pH 4.5)で50倍に希釈し、Biacore3000付属のプロトコルに従い、IgG-FcをセンサーチップCM5に固定した。IgG-FcのセンサーチップCM5への固定化は、N-hydroxysuccinimide (NHS)、および、N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochroride(EDC)を用いたアミンカップリング法にて行い、ブロッキングにはEthanolamineを用いた。また、チップ上の別のフローセルに対して、EDC/NHSにより活性化した後にEthanolamineを固定化する処理を行うことで、ネガティブ・コントロールとなるリファレンスセルも用意した。
Claims (10)
- 前記低分子化合物におけるArYHBが、芳香族6員環又はその側鎖に水酸基またはアミノ基が直接結合した構造を有することを特徴とする、請求項1に記載の使用。
- 前記低分子化合物におけるArXが、ハロゲン基、アミノ基、水酸基、もしくは、メチル基のいずれかを置換基として有するか、または、ArXが、ベンゼン環、ナフタレン環、もしくは、1,2-メチレンジオキシベンゼンのいずれかを含む構造を有するか、或いはそれらの両方であることを特徴とする、請求項1または2に記載の使用。
- 前記(Linker)が、置換されていてもよいテトラゾール、置換されていてもよいヒダントイン、置換されていてもよいピロール、置換されていてもよいピリジン、置換されていてもよい1,3,4-チアジアゾール、置換されていてもよいトリアゾール、置換されていてもよいアミノピラゾロ[3,4-d]ピリミジン、置換されていてもよいチアゾール、もしくは置換されていてもよいピリミジンを含む構造であるか、或いは(Linker)を(-U-CZ-V-)で表したとき、CZが置換されていてもよいベンゼン環を含む構造、もしくは、置換されていてもよいナフタレン環を含む構造であり、UおよびVは、いずれも環構造を含まない非水素原子で構成される請求項1~3のいずれかに記載の使用。
- タンパク性物質が、免疫グロブリン、または、免疫グロブリンの断片であることを特徴とする、請求項1~5のいずれかに記載の使用。
- タンパク性物質が、モノクローナル抗体であることを特徴とする、請求項1~6のいずれかに記載の使用。
- タンパク性物質が、IgG-Fc、または、Fc融合タンパク質であることを特徴とする、請求項1~7のいずれかに記載の使用。
- タンパク性物質が細胞培養物中に含まれることを特徴とする、請求項1~8のいずれかに記載の使用。
- 請求項1~9のいずれかに記載の低分子化合物の(Linker)に結合するスペーサーを介して、水不溶性の基材からなる担体に前記低分子化合物が固定化されたことを特徴とする、アフィニティー分離マトリックス。
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US8674073B2 (en) | 2006-02-21 | 2014-03-18 | Protenova Co., Ltd. | Immunoglobulin affinity ligand |
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WO2009141384A2 (en) * | 2008-05-21 | 2009-11-26 | Novo Nordisk A/S | Process for the purification of factor vii polypeptides using affinity resins comprising specific ligands |
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Also Published As
Publication number | Publication date |
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JP6038774B2 (ja) | 2016-12-07 |
US9273151B2 (en) | 2016-03-01 |
EP2690106A4 (en) | 2015-04-22 |
US20140018524A1 (en) | 2014-01-16 |
EP2690106A1 (en) | 2014-01-29 |
JPWO2012128353A1 (ja) | 2014-07-24 |
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