0001]　The present invention relates to a carrier for mixed mode affinity chromatography.
BACKGROUND[0002]　Recently, genetic engineering, with the development of protein engineering and cell engineering, called antibody drugs, the development of drugs utilizing functions of the antibody has been actively conducted. Antibody drugs, compared with conventional drugs, in order to work more specifically against the target molecule, more and reduce the side effects, and are expected to be high therapeutic effect can be obtained, in practice various pathologies It has contributed to the improvement.
[0003]
　On the other hand, antibody drugs, since it is administered in large quantities to the living body, when compared with other recombinant proteins pharmaceuticals, is greatly affected its purity exerts quality. Antibody drugs because it is produced by purifying antibody expressed in the host cells by genetic recombination, contamination of impurities derived from the host cell and production process is a problem. For example, host cell derived proteins remaining as an impurity in the antibody drugs; it (HCP Host Cell Protein), since the association is suspected between anaphylaxis onset during antibody drugs administration, which improves the purification purity, reduce the impurity there is a demand.
[0004]　For example, Patent Document 1, Mixed Mode antibody affinity separation matrix having antibody affinity ligand and a cation-exchange groups in the same separation matrix is ​​described (claim 1). Furthermore, antibody affinity ligand, protein A, protein G, protein L, protein H, protein D, protein Arp, protein Fc [gamma] R, wherein it is at least one selected from the antibody binding synthetic ligands and their analogues is (claim 5).
[0005]　Further, for example, Patent Document 2, the synthetic, characterized in that on a polymeric support having a base and an affinity ligand comprising acid groups, mixed mode support is described (claim 11). Furthermore, as an antibody affinity ligand, protein A, protein G, protein L, protein H, protein D, protein Arp, protein Fc [gamma] R, antibody-binding synthetic peptide ligands and their related substances have been described (<0043>).
[0006]
　Further, for example, Patent Document 3, the surface-modified porous cross-linked particles, is described separating agent introduced an interactive functional group (<0011>), as interacting functional groups, ion exchange groups; affinity ligand ; alkyl group, a hydrophobic group such as a phenyl group and a polyalkyl ether group; more preferably, the separating agent obtained by introducing such interactive functional groups are in particular as an adsorbent polymeric materials such as protein, especially for chromatography that can be used effectively as a separating agent (<0026>) have been described. CITATIONPatent Document[0007]Patent Document 1: WO 2014/034457
Patent Document 2: JP 2016-069329 Patent Publication Patent Document 3: JP 2013-088398 JP Summary of the Invention
Problems that the Invention is to Solve
[0008]　Currently, the purification of therapeutic antibodies, it is generally performed by affinity chromatography using protein A as an affinity ligand. However, Protein A Staphylococcus aureus; a (Staphylococcus aureus Staphylococcus aureus) derived protein because of high immunogenicity against humans, when administered to the human body mixed in the purified antibody, the expected there is a case in which cause and not the immune response. Also, Protein A, since it is produced by genetic engineering methods using Escherichia coli (Escherichia coli), endotoxin from E. coli LPS (Lipopolysaccharide, lipopolysaccharide; Lipid A, Lipid A) is mixed in the purified antibody All it also. Therefore, it has a high degree of purification is required in the protein A, which is one of the factors to increase the antibody purification costs.
[0009]
　The present inventor has as an affinity ligand for use in mixed mode affinity chromatography matrix, as compared to protein A conceived the use of antibody binding peptides which can be produced at low cost, antibody adsorption capacity, impurity removal capacity and It was studied chemical resistance.
[0010]
　As a result, the present inventors, it a linear peptide in mixed mode affinity chromatography matrix used as an affinity ligand, there is room for any one or more improvements of the antibodies adsorption capacity, impurity removal capacity and chemical resistance was knowledge. For example, in Comparative Example 2 described later, is used a linear peptide as an affinity ligand, antibody adsorption capacity and chemical resistance is insufficient, it has been shown that there is a need for improvement.
[0011]
　The present invention aims antibody adsorption capacity, to provide either a mixed mode affinity chromatography matrix to which has excellent impurity removal capacity and chemical resistance.
Means for Solving the Problems
[0012]
　The present inventor has conducted extensive studies to achieve the above object, a substrate, a hydrophilic polymer, a cyclic peptide, and a cation-exchange groups, mixed mode affinity chromatography matrix having the antibody adsorbed capacity, become known that is excellent in both of the impurity removal capacity and chemical resistance, and completed the present invention.
[0013]
　That is, the present invention provides the following [1] to [21].
　[1] a substrate, a hydrophilic polymer, and an antibody-binding cyclic peptides, mixed mode affinity chromatography matrix having a cation exchange group, a.
　[2] the antibody binding cyclic peptides has an annular portion which is cyclized by intramolecular crosslinking between the side chains, mixed mode affinity chromatography matrix according to the above [1].
　[3] in the molecule crosslinking disulfide bonds, thioether bonds, triazole bond, and is selected from the group consisting of an amide bond include at least one, mixed mode affinity chromatography matrix according to the above [2].
　[4] in the molecule crosslinking disulfide bond, a thioether bond, and is selected from the group consisting of triazole coupling comprises at least one, [2] or [3] Mixed mode affinity chromatography carrier according to.
　[5] the disulfide bond, L- cysteine and D- first side other than the side chain thiol groups L- cysteine and D- cysteine amino acid residue in a side chain other than cysteine from an amino acid having a thiol group a disulfide bond formed between the side chain thiol group of a second amino acid residue derived from the amino acid having a thiol group at the chain, the thioether bond, the side chain other than L- cysteine and D- cysteine thioether formed between the side chain haloacetyl group of the second amino acid residue derived from the side chain thiol group and a side chain of the first amino acid residue derived from the amino acid having a thiol group in an amino acid having a haloacetyl group is a bond, mixed mode affinity chromatography matrix according to the above [4].
　[6] the disulfide bonds, cysteine, homocysteine and first side chain thiol group and the cysteine amino acid residues from an amino acid having a thiol group in the side chain selected from the group consisting of penicillamine, homocysteine and penicillamine a disulfide bond formed between the side chain thiol group of a second amino acid residue derived from the amino acid having a thiol group in the side chain selected from the group consisting of, the thioether bond, cysteine, homocysteine and the side chain thiol group and a side chain of the first amino acid residue derived from the amino acid having a thiol group in the side chain selected from the group consisting of penicillamine second amino acid residues from an amino acid having a haloacetyl group thioether bond formed between the side chain haloacetyl group, the [4 Mixed mode affinity chromatography carrier according to.
　[7] the disulfide bonds, from the group consisting of the first side chain thiol group and homocysteine and penicillamine amino acid residues from an amino acid having a thiol group in the side chain selected from the group consisting of homocysteine and penicillamine a disulfide bond formed between the side chain thiol group of a second amino acid residue derived from the amino acid having a thiol group in the side chain selected from the group the thioether bond, consists of homocysteine and penicillamine the side chain haloacetyl group of the second amino acid residue derived from the amino acid having a side chain thiol group and a side chain haloacetyl group of the first amino acid residue derived from the amino acid having a thiol group in the side chain selected thioether bonds formed between, mission according to any one of the above [4] to [6] Box mode affinity chromatography carrier.
　[8] the intramolecular crosslinking, the group consisting of the first side chain thiol group and homocysteine and penicillamine amino acid residues from an amino acid having a thiol group in the side chain selected from the group consisting of homocysteine and penicillamine side chain selected from the group consisting of formed disulfide bonds, or homocysteine and penicillamine with the side chain thiol group of a second amino acid residue derived from the amino acid having a thiol group in the side chain selected from formed between the side chain haloacetyl group of the second amino acid residue derived from the amino acid having a side chain thiol group and a side chain haloacetyl group of the first amino acid residue derived from the amino acid having a thiol group in a thioether bond, mixed mode affinity chromatography according to [2] or [3] Use carrier.
　[9] disulfide bond formed between the side chain thiol group of a second amino acid residue in which the disulfide bond is derived from the side chain thiol group and homocysteine first amino acid residue derived from the homocysteine There, formed between the side chain haloacetyl group of the second amino acid residue derived from the amino acid having a side chain thiol group and a side chain haloacetyl group of the first amino acid residue of the thioether linkage is derived from homocysteine It has been a thioether bond, mixed mode affinity chromatography matrix according to the above [8].
　[10] The number of amino acid residues of the annular portion is 8 to 14 residues, [2] to [9] Mixed mode affinity chromatography matrix of any one of.
　[11] the antibody binding cyclic peptide having straight portion, said [1] Mixed mode affinity chromatography matrix of any one of - [10].
　[12] the linear portion comprises an amino acid residue having at least one selected from the group consisting of hydroxy and carboxy groups in side chains, mixed mode affinity chromatography matrix according to the above [11].
　[13] is at least one said substrate is a polysaccharide, acrylate-based polymer is selected from the group consisting of methacrylate polymer and styrene polymer, any one of the above [1] to [12] mixed mode affinity chromatography carrier according to.
　[14] is at least one said substrate is a polysaccharide, selected from the group consisting of acrylate polymers and methacrylate polymer, mixed-mode according to any one of the above [1] to [13] affinity chromatography carrier.
　[15] is at least one said substrate is selected from the group consisting of agarose and cellulose, the above-mentioned [1] to [14] Mixed mode affinity chromatography matrix of any one of.
　[16] The hydrophilic polymer is a hydrophilic polysaccharide, the above-mentioned [1] to [15] Mixed mode affinity chromatography matrix of any one of.
　[17] The hydrophilic polysaccharide dextran, carboxymethyl dextran, pullulan, hydroxyethyl cellulose, and carboxymethyl is at least one member selected from the group consisting of methyl cellulose, mixed mode affinity chromatography matrix according to [16], .
　[18] The hydrophilic polysaccharide, dextran, carboxymethyl at least one selected from the group consisting of dextran and pullulan, mixed mode affinity chromatography matrix according to the above [16] or [17].
　[19] The molecular weight of the antibody-binding cyclic peptides is less than 5000, the above-mentioned [1] to [18] Mixed mode affinity chromatography matrix of any one of.
　[20] the cation exchange group is a carboxy group or a sulfoxy group, the above-mentioned [1] to [19] Mixed mode affinity chromatography matrix of any one of.
　[21] the cation exchange group is a carboxy group, the above-mentioned [1] to [20] Mixed mode affinity chromatography matrix of any one of.
Effect of the invention
[0014]
　According to the present invention, it is possible to provide antibodies adsorption capacity, either in mixed mode affinity chromatography matrix to which has excellent impurity removal capacity and chemical resistance.
DESCRIPTION OF THE INVENTION
[0015]
　Hereinafter, the mixed mode affinity chromatography matrix and a manufacturing method thereof of the present invention will be described in detail.
　In the present invention, when expressed by using "to" means a range, that range is intended to include both sides of the "~".
[0016]
[Mixed Mode affinity chromatography matrix]
　Mixed mode affinity chromatography matrix of the present invention includes a substrate, a hydrophilic polymer, and an antibody-binding cyclic peptide, and a cation-exchange group, a.
[0017]
　To take the mix mode affinity chromatography matrix such an arrangement of the present invention, when applied to affinity chromatography for antibody purification believed antibody adsorption capacity, but also excellent in any of the impurity removal capacity and chemical resistance . The reason is not necessarily limited, is presumed as roughly follows.
[0018]
　By having an antibody binding cyclic peptides and a cation exchange group on the same carrier, it acts both ligands in a concerted manner, by having the aggregate removing capability with specific adsorbability, antibody adsorption capacity, impurity removal It became excellent in any of the capacity and chemical resistance.
[0019]
　In the present invention, the impurity removal capacity, HCP amount before purification (ppm) and HCP purification factor [before purification HCP amount which is the ratio of HCP amount after purification (ppm) (ppm) / HCP amount after purification (ppm)] and it is a measure of the impurity removal performance of chromatography media.
[0020]

　The substrate used in the mixed mode affinity chromatography matrix of the present invention is not particularly limited, is preferably selected polysaccharides, acrylate-based polymer, from the group consisting of methacrylate polymer and styrene polymer is at least one that is at least one and more preferably is selected from the group consisting of polysaccharides, acrylate polymers and methacrylate polymer, at least 1 more preferably is selected from the group consisting of agarose and cellulose it is a species.
　The substrate may be used singly or may be used in combination of two or more kinds.
[0021]
"Polysaccharides"
　the polysaccharide is not particularly limited, for example, cellulose, agarose, dextran, chitosan, and natural, such as glucomannan polysaccharides, as well as crosslinked polysaccharides obtained by introducing a crosslinked structure into these natural polysaccharides and the like. Producing cross-linked polysaccharide, for example, with respect to hydroxyl groups of natural polysaccharides, epichlorohydrin, (poly) alkylene glycol diglycidyl ethers, and, by introducing a crosslinked structure with a crosslinking agent such as alkylene diisocyanates can do.
[0022]
　The polysaccharide is preferably cellulose, at least one selected from cross-linked cellulose, agarose and cross-linked agarose, at least one and more preferably is selected from agarose and crosslinked agarose.
[0023]
"Acrylate polymer"
　above acrylate-based polymer is not particularly limited, for example, methyl acrylate, ethyl acrylate, hydroxyethyl acrylate, hydroxypropyl acrylate, butyl acrylate, stearyl acrylate, 2 ethylhexyl, cyclohexyl acrylate, glycerin monoacrylate, glycidyl acrylate, 4,5-epoxy butyl acrylate, and 9,10-epoxy stearyl acrylate polymerization comprising polymer one acrylic acid esters such as 2 acrylic acid ester a copolymer obtained by copolymerizing the above species, as well, and copolymerized to become a copolymer and one or more 1 or more and a vinyl group-containing compound other than the acrylic acid ester of an acrylate ester. Examples of the vinyl group-containing compound other than acrylic acid esters, such as ethylene, and, monovinyl compound such as propylene, divinyl benzene, and aromatic polyvinyl compounds such as trivinylbenzene, as well as butadiene, methylene bisacrylamide, and include polyvinyl compounds such as triallyl isocyanurate. For these polymers or copolymers, epichlorohydrin, (poly) alkylene glycol diglycidyl ethers, and it may be introduced a crosslinked structure with a crosslinking agent such as alkylene diisocyanates.
[0024]
"Methacrylate polymer"
　said methacrylate polymer is not particularly limited, for example, methyl methacrylate, ethyl methacrylate, hydroxyethyl methacrylate, hydroxypropyl methacrylate, butyl methacrylate, stearyl methacrylate, 2-ethylhexyl methacrylate, cyclohexyl methacrylate, glycerol monomethacrylate, glycidyl methacrylate, 4,5-epoxy butyl methacrylate, and, 9,10 epoxy stearyl methacrylate polymer obtained by polymerizing one kind of methacrylic ester such as methacrylic a copolymer obtained by copolymerizing two or more kinds of esters, and I by copolymerizing a one or more 1 or more and a vinyl group-containing compound other than methacrylic acid esters of methacrylic acid ester Copolymers. Examples of the vinyl group-containing compound other than methacrylic acid esters, for example, compounds exemplified in the vinyl group-containing compound other than the acrylic acid ester described above. For these polymers or copolymers, it may be introduced a crosslinked structure with a crosslinking agent as described above which can be used in the acrylate polymer.
[0025]
"Styrene polymer"
　The styrene-based polymer is not particularly limited, for example, styrene, methyl styrene, ethyl styrene, hydroxystyrene, and, by polymerizing one of styrenic compounds such as chlorostyrene polymer comprising Te, a copolymer obtained by polymerizing two or more kinds of styrene compounds, and, by copolymerizing a one or more 1 or more and a styrene compound other than the vinyl group-containing compound of a styrene compound copolymers thereof made. Examples of the styrene compound other than the vinyl group-containing compounds, for example, compounds exemplified in the vinyl group-containing compound other than the acrylic acid ester described above. For these polymers or copolymers, it may be introduced a crosslinked structure with a crosslinking agent that may be used in the acrylate polymer.
[0026]
"Structure of the base material"
　substrate is preferably porous particles or porous membrane. By substrate is a porous particle or a porous membrane, the surface area is increased, it is possible to increase the processing capacity per unit time.
[0027]
(Pore volume)
　pore volume when the substrate is porous particles or porous film is not particularly limited, but the pore volume measured by a mercury porosimeter, preferably 0.2mL / g ~ 10mL / g , more preferably in the range of 0.2mL / g ~ 5.0mL / g. If the pore volume falls within this range, the antibody adsorption capacity increases. Further, the mechanical strength is not lowered.
[0028]
(Specific surface area)
　The specific surface area of when the substrate is porous particles or porous film is not particularly limited, BET (Brunauer, Emmett, Teller) measured specific surface area by method (BET specific surface area) is preferably 2m 2 / g ~ 1500 m 2 / g, more preferably 5 m 2 / g ~ 1000 m 2 in the range of / g. If the specific surface area is within this range, the antibody adsorption capacity increases.
[0029]
(Average particle diameter)
　The average particle diameter of the case where the substrate is porous particles is not particularly limited, preferably 0.5 [mu] m ~ 1000 .mu.m, more preferably 1 [mu] m ~ 250 [mu] m, more preferably in the range of 2 [mu] m ~ 150 [mu] m is there. When the average particle diameter is within this range, small pressure loss when passing liquid was packed in a column, can increase the liquid passage rate, together with the processing efficiency is improved, the antibody adsorption capacity increases. The average particle diameter of the porous particles can be measured in a known manner. For example, with an optical microscope, a particle diameter of 100 or more porous particles were measured, the average particle diameter is obtained by calculating the median size from the distribution.
[0030]
"Specific examples of substrates"
　Specific examples of the substrate, for example, a carrier of the agarose-based Sepharose series (GE Healthcare Inc.) ( "SEPHAROSE" are Registered Trade Mark), Cellufine a crosslinked carrier cellulosic Series (JNC Co., Ltd.) ( "CELLUFINE" is a registered trademark), allyl dextran and N, N'- methylene-bis-Sepharose (manufactured by GE Healthcare) acrylic series is a crosslinked polymer of acrylamide ( "SEPHACRYL" is a registered trademark), and, is a carrier of the acrylate Toyopearl HW series (manufactured by Tosoh Corporation) ( "Toyopearl" and "TOYOPEARL" is a registered trademark) include commercially available products such as, but not limited thereto.
[0031]
　It may also be used after introducing a hydroxyl group covalently bonded to functional group capable of an epoxy group into a commercially available base material as a base material. For example, the carrier of the agarose-based 2-chloromethyl-oxirane (aka: epichlorohydrin) by treatment with a hydroxy group of the agarose to glycidyl, it can be used a carrier to introduce epoxy groups on the surface.
[0032]

　By coating a substrate with a hydrophilic polymer, since the hydrophilic mixed mode affinity chromatography matrix surface of the present invention is increased, the adsorption of non-specific adsorbate is suppressed, purification purity there is an improvement to effect.
[0033]
　Hydrophilic polymers used in the mixed mode affinity chromatography matrix of the present invention is not particularly limited, at least one preferably selected from the group consisting of hydrophilic polysaccharides, more preferably dextran, carboxymethyl dextran, pullulan, at least one selected from the group consisting of hydroxyethyl cellulose and carboxymethyl cellulose, and more preferably at least one selected from the group consisting of dextran, carboxymethyl dextran, and pullulan.
[0034]
　The hydrophilic polymer may be used singly or may be used in combination of two or more kinds.
[0035]
　Further, the hydrophilic polymer to the substrate, preferably being covalently immobilized.
　If the substrate is porous particles, it is preferable to have a functional group capable of covalently binding with the hydroxyl groups on the surface. Examples of the functional group capable of covalently binding with the hydroxyl group, for example, an epoxy group and a glycidyl group; cyanogen bromide, N, N-disuccinimidyl hydroxy group is activated in such carbonate (DSC); aldehyde group (formyl group); and, N- hydroxysuccinimide esters, and activated carboxylic acid groups, such as carbonyl diimidazole activated ester; can be mentioned a reactive functional group such as and the like.
[0036]
"Hydrophilic polysaccharide"
　hydrophilic polysaccharide is not particularly limited, since a high effect of improving the purification purity is at least one preferably selected dextran, carboxymethyl dextran, and pullulan, and more preferably dextran.
[0037]
"The molecular weight of the hydrophilic polymer"
　molecular weight of the hydrophilic polymer is not particularly limited, in intrinsic viscosity, preferably 0.10 dL / g or more, more preferably 0.10dL / g ~ 0.90dL / g, more preferably 0.12dL / g ~ 0.40dL / g, more preferably 0.15dL / g ~ 0.30dL / g, even more preferably in the range of 0.15dL / g ~ 0.25dL / g.
　When the intrinsic viscosity is within this range, purification purity is further improved. Incidentally, the intrinsic viscosity, viscometry in the general measuring method is listed in the 16th revised Japanese Pharmacopoeia, in accordance with the first method, "capillary viscometer method", seeking viscosity of the polymer solution of several different concentrations Viscosity of the concentration dependence was measured, the concentration of the resulting straight line can be determined by extrapolating to zero.
[0038]
(Intrinsic viscosity)
　Incidentally, between the intrinsic viscosity η and the molecular weight M of the polymer is represented by the following marks - HOUWINK - since the equation of Sakurada (Mark-Houwink-Sakurada equation) is satisfied, a number a direct measurement determine the molecular weight of Kano sample, if decide K and α from the value of the molecular weight and each of the intrinsic viscosity, the same kind of polymer, the molecular weight M is by measuring the intrinsic viscosity eta, the molecular weight M intrinsic viscosity by measuring η, respectively, are determined.
　eta = KM alpha
　Here, K and alpha is a constant determined kind of polymer, the type and temperature of the solvent.
[0039]
　For example, the intrinsic viscosity (eta dextran Dextran ) and weight average molecular weight (Mw Dextran ) and are known to satisfy the following relationship.
　eta Dextran [dL / g] = 9 × 10 -4 × Mw Dextran 0.5 [dL / g]
[0040]
"Coverage of the hydrophilic polymer"
　coating amount of the hydrophilic polymer (hereinafter, simply referred to as "hydrophilic polymer coverage.") Is not particularly limited, preferably 3mg / g-dry gel ~ 450mg / g -dry gel, more preferably 3mg / g-dry gel ~ 250mg / g-dry gel, more preferably 3mg / g-dry gel ~ 230mg / g-dry gel, more preferably 10mg / g-dry gel ~ 230mg / g-dry gel, and even more preferably from 20mg / g-dry gel ~ 230mg / g-dry gel.
[0041]
　When the hydrophilic polymer coating amount is within this range, the adsorption is suppressed in mixed mode affinity chromatography for nonspecific adsorbates hydrophilicity increases moderately the surface of the carrier of the present invention, the antibody substrate since the antibody adsorption capacity increases are still many room for cementation, as well as improve the purification purity, the effect of reducing the purification cost. Incidentally, the hydrophilic polymer need not cover the entire surface of the substrate, it is sufficient to cover at least a portion of the substrate.
[0042]
　Incidentally, the hydrophilic polymer coating weight [unit: mg / g-dry gel] is the dry weight (W hydrophilic polymer covering P ) [unit: mg], and the dry weight of the coating before the substrate (W 0 ) [unit: g-dry gel] is a value obtained by dividing the. Further, the dry weight of the hydrophilic polymer (W P ), the dry weight (W carriers total amount 1 dry weight (W) 0 difference (W) 1 -W 0 ) [Unit: mg] in is there. 　Therefore, the hydrophilic polymer coating amount can be obtained by the following expression. 　Hydrophilic polymer coverage (mg / g-dry gel) = W P / W 0 = (W 1 -W 0 ) / W 0 　dry weight (W of the coating before the substrate 0 ) is calculated as follows can do. 　W 0 = W 0, Xg × W

0 / x
　where,
　W 0, xg : before coating dry weight of the wet gel xg substrates
　w 0 : wet gel mass of the hydrophilic polymer coating reaction before coating substrates using
　x: before coating used in the drying group wet gel mass of wood (xg)
is.
　The carrier the total amount of the dry weight (W 1 ) can also be calculated in the same manner.
[0043]
　By way of example of the measurement method of the hydrophilic polymer coverage.
　First, the wet gel 5g of the coated front substrate to measure the mass was dried under reduced pressure until no mass change at 50 ° C., than the product of the wet gel weight of the coating before the substrate used for the hydrophilic polymer coating reaction, coatings the dry weight of the pre-substrate.
　Then, the coating before the substrate moisture gel 5g of the support obtained by coating the hydrophilic polymer by measuring the mass was dried under reduced pressure until no mass change at 50 ° C., the support obtained after the hydrophilic polymer coating reaction than the product of the wet gel weight, the dry weight of the carrier.
　Further, the difference between the dry weight before coating and the dry weight of the carrier substrate, the dry weight of the hydrophilic polymer covering the coated front substrate.
　Finally, the hydrophilic polymer coating weight, calculated as dry weight of the hydrophilic polymer per dry weight of the coating before the substrate.
[0044]
(Hydrophilic)
　In the present invention, the hydrophilic for the polymer or polysaccharide means that comprises at least one hydrophilic group. The hydrophilic group is preferably a carboxy group, an alkali metal salt of a carboxyl group, a sulfonic acid group, an alkali metal salt of a sulfonic acid group, hydroxy group, an amido group, a carbamoyl group, a sulfonamido group, a sulfamoyl group, a phosphoric acid group, alkali metal salts of the phosphate group, oxyphosphorus acid group, and include functional groups such as alkali metal salts of oxyphosphorus acid group. These hydrophilic groups may be present at any position in the polymer, for example, the polymer main chain terminal and / or side chain, may be bonded directly or via a linking group. Further, the hydrophilic groups in the molecule, preferably a plurality are present.
[0045]

　antibody binding cyclic peptides used in the mixed mode affinity chromatography matrix of the present invention is not particularly limited, preferably has an annular portion which is cyclized by intramolecular crosslinking between the side chains.
[0046]
　In the molecule crosslinking group preferably disulfide bonds, thioether bonds, comprising at least one member selected from the group consisting of triazoles binding, and amide bond, more preferably, consisting of a disulfide bond, thioether bond, and triazole bonded It comprises at least one member selected from, more preferably comprises a disulfide bond or a thioether bond.
[0047]
　In this specification, simply "cyclic peptide" the antibody binding cyclic peptide, or, sometimes referred to as "affinity ligand".
[0048]
"Disulfide bond"
　disulfide bond is not particularly limited as long as "S-S" bond, for example, (also referred to as "mercapto group".) Two thiol groups include S-S bond formed between.
[0049]
(1) The above disulfide bonds, preferably, the second amino acid residue derived from the amino acid having a thiol group in the side chain thiol group and a side chain of the first amino acid residue derived from the amino acid having a thiol group in a side chain it is a disulfide bond formed between the side chain thiol group of groups.
[0050]
(2a) above disulfide bonds, more preferably, the side chain thiol group of the first amino acid residue derived from the amino acid having a thiol group in a side chain other than L- cysteine ​​and D- cysteine ​​L- cysteine ​​and D- is a disulfide bond formed between the side chain thiol group of a second amino acid residue derived from the amino acid having a thiol group in a side chain other than cysteine.
[0051]
(2b) The above disulfide bonds, more preferably, cysteine, homocysteine ​​and side chain thiol group and the cysteine ​​of the first amino acid residue derived from the amino acid having a thiol group in the side chain selected from the group consisting of penicillamine, it is a disulfide bond formed between the side chain thiol group of a second amino acid residue derived from the amino acid having a thiol group in the side chain selected from the group consisting of homocysteine ​​and penicillamine.
[0052]
　Such disulfide bonds include those represented by the following formula.
[0053]
[Formula 1]

[0054]
　However, the above formula:
　m and n are each independently 1 or
　2; when m = is 1, R 1 is a hydrogen atom or a methyl
　group; when a m = 2, R 1 is hydrogen ; be atoms
　when it is n = 1, R 2 is a hydrogen atom or a methyl group;
　when a n = 2, R 2 is a hydrogen atom;
　* the other amino acid residues or other substituents It represents the point of attachment.
[0055]
(3) The above disulfide bonds, more preferably, homocysteine ​​and first side chain thiol group and homocysteine ​​and penicillamine amino acid residues from an amino acid having a thiol group in the side chain selected from the group consisting of penicillamine side chain selected from the group consisting of a disulfide bond formed between the side chain thiol group of a second amino acid residue derived from the amino acid having a thiol group.
[0056]
　Such disulfide bonds include those represented by the following formula.
[0057]
[Formula 2]

[0058]
　However, the above formula:
　m and n are each independently 1 or
　2; when it is m = 1, R 1 is a methyl
　group; when a m = 2, R 1 is a hydrogen atom ;
　when it is n = 1, R 2 is a methyl group;
　when a n = 2, R 2 is a hydrogen atom;
　* denotes the point of attachment to the other amino acid residues or other substituents.
[0059]
(4) The above disulfide bonds, more preferably, formed between the side chain thiol group of a second amino acid residue derived from a first side chain thiol group and homocysteine ​​amino acid residues derived from the homocysteine It has been a disulfide bond.
[0060]
　Such disulfide bonds include those represented by the following formula.
[0061]
[Formula 3]

[0062]
　However, in the above formulas, * represents the point of attachment to other amino acid residues or other substituents.
[0063]
(A particularly preferred disulfide bond)
　disulfide bonds higher alkali resistance, mixed mode affinity chromatography carrier can be repeated washing with alkali is maintained antibody binding, it is possible to reduce the antibody purification costs. Such disulfide bonds, preferably disulfide bonds other than disulfide bond formed between two cysteine residues, which is formed between amino acid residues from an amino acid having a thiol group in a side chain other than cysteine disulfide bond is more preferable, disulfide bond formed between two homocysteine residue is particularly preferred.
[0064]
"Thioether"
　thioether bond is not particularly limited as long as the oxygen atom of the ether linkage "-O-" a "-S-" binding form substituted with a sulfur atom, for example, both a thiol group ( "mercapto group" say.) -S- bond formed between the haloacetyl groups and the like. Here, haloacetyl group is preferably a chloroacetyl group or a bromoacetyl group, more preferably a chloroacetyl group. Further, haloacetyl groups were introduced into the amino acid in the form of replacing a hydrogen atom of the side chain amino groups are preferred.
[0065]
　Amino acids with haloacetyl groups in side chains person represented by the following formula.
[0066]
[Chemical Formula 4]

[0067]
　However, in the above formulas, n is an integer of 1 or more, X is a halogen atom.
　Incidentally, n is preferably an integer that satisfies 1 ≦ n ≦ 4, X is preferably a chlorine atom or a bromine atom, more preferably chlorine atom.
[0068]
　Amino acid represented by the above
　formula, when it is n = 1, N 3 - haloacetyl -L-2,3-diaminopropane acid [(2S)-2-amino-3 - [(2-haloacetyl) amino] propane acid] or n 3 - haloacetyl -D-2,3-diamino propanoic acid [(2R)-2-amino-3 - a [(2-haloacetyl) amino] propanoic
　acid], when it is n = 2, n 4 - haloacetyl -L-2,4-diaminobutane acid [(2S)-2-amino-4 - [(2-haloacetyl) amino] butanoic acid] or N 4 - haloacetyl -D-2,4-diaminobutane acid [(2R)-2-amino-4 - [(2-haloacetyl) amino] butanoic acid]
　is, when n = a 3, n-.delta.-haloacetyl -L- ornithine [(2S)-2-amino - 5 - [(2-Haroa Cetyl) amino] pentanoic acid] or N-.delta.-haloacetyl -D- ornithine [(2R)-2-amino-5 - a [(2-haloacetyl) amino] pentanoic
　acid], when n = a 4, N -ε- haloacetyl -L- lysine [(2S)-2-amino-6 - [(2-haloacetyl) amino] hexanoic acid] or N-.epsilon.-haloacetyl -D- lysine [(2R)-2-amino-6 - is [(2-haloacetyl) amino] hexanoic acid].
[0069]
(1) The above thioether bond, preferably, the second amino acid residue derived from the amino acid having a side chain thiol group and a side chain haloacetyl group of the first amino acid residue derived from the amino acid having a thiol group in a side chain thioether bond formed between the side chain haloacetyl group group.
[0070]
(2a) above thioether bond, more preferably, the first side chain thiol group and a side chain haloacetyl groups of the amino acid residues from an amino acid having a thiol group in a side chain other than L- cysteine ​​and D- cysteine thioether bond formed between the side chain haloacetyl group of the second amino acid residue derived from the amino acid with.
[0071]
(2b) The above thioether bond, more preferably, cysteine ​​side chain thiol group and a side chain of the first amino acid residue derived from the amino acid having a thiol group in the side chain selected from the group consisting of homocysteine ​​and penicillamine a thioether bond formed between the side chain haloacetyl group of the second amino acid residue from an amino acid having a haloacetyl group.
[0072]
　Such thioether bond include those represented by the following formula.
[0073]
[Formula 5]

[0074]
　However, the above formula:
　m is 1 or 2;
　; n is an integer of 1 or more
　when it is m = 1, R is a hydrogen atom or a methyl group;
　when a m = 2, R is hydrogen It is atomic;
　* represents the point of attachment to other amino acid residues or end groups.
　Further, n is preferably an integer that satisfies 1 ≦ n ≦ 4.
[0075]
(3) The above thioether bond, more preferably, haloacetyl the side chain thiol group and a side chain of the first amino acid residue derived from the amino acid having a thiol group in the side chain selected from the group consisting of homocysteine ​​and penicillamine thioether bond formed between the side chain haloacetyl group of the second amino acid residue derived from the amino acid with a group.
[0076]
　Such thioether bond include those represented by the following formula.
[0077]
[Formula 6]

[0078]
　However, the above formula:
　m is 1 or 2;
　; n is an integer of 1 or more
　when it is m = 1, R is a methyl group;
　when a m = 2, R is a hydrogen atom ; *
　represents the point of attachment to other amino acid residues or other groups.
　Further, n is preferably an integer that satisfies 1 ≦ n ≦ 4.
[0079]
(4) The above thioether bond, more preferably, the side chain haloacetyl the second amino acid residue derived from the amino acid having a side chain thiol group and a side chain haloacetyl group of the first amino acid residue derived from the homocysteine thioether bond formed between the groups.
[0080]
　Such thioether bond include those represented by the following formula.
[0081]
[Chemical Formula 7]

[0082]
　However, in the above formulas, n is an integer of 1 or more, * represents the point of attachment to other amino acid residues or other substituents. Further, n is preferably an integer that satisfies 1 ≦ n ≦ 4.
[0083]
(Particularly preferred thioether bond)
　since thioether bond alkali resistance is high, mixed mode affinity chromatography carrier can be repeated washing with alkali is maintained antibody binding, it is possible to reduce the antibody purification costs. Such a thioether bond, particularly preferably a thioether bond formed between the homocysteine residue and N-.epsilon.-chloroacetyl lysine residues.
[0084]
"Triazole coupling"
　the triazole coupling is not particularly limited, preferably derived from the amino acid having an alkynyl group in the side chain azido group and the side chain of the first amino acid residue derived from the amino acid having an azido group in the side chain triazole linkage formed between the side chain alkynyl group of the second amino acid residue.
[0085]
　Between the side-chain alkynyl group of the second amino acid residue derived from the amino acid having a side chain azido group and the side chain alkynyl group of the first amino acid residue derived from the amino acid having an azido group in the side chain the formed triazole coupled, for example, those represented by the following formula.
[0086]
[Formula 8]

[0087]
　However, * is the point of attachment to the other amino acid residues or other substituents, m and n are each independently an integer of 1 or more, preferably, each independently a integer of 1 or more and at least one is an integer of 2 or more.
[0088]
　When a m = 1, amino acid residues from an amino acid having an azido group in the side chain is an amino acid residue derived from β- azide -L- alanine or β- azide -D- alanine, preferably β - an amino acid residue derived from azide -L- alanine.
　When a m-= 2, amino acid residues from an amino acid having an azido group in the side chain is an amino acid residue derived from γ- azide -L- homoalanine or γ- azide -D- homoalanine, preferably it is a γ- azide -L- homoalanine.
　When m = a 3 amino acid residues from an amino acid having an azido group in the side chain is an amino acid residue derived from δ- azide -L- ornithine or δ- azide -D- ornithine, preferably δ - is an azide -L- ornithine.
　When m = a 4 amino acid residues from an amino acid having an azido group in the side chain is an amino acid residue derived from ε- azide -L- lysine or ε- azide -D- lysine, preferably ε - it is an azide -L- lysine.
[0089]
　when n = 1, amino acid residues from an amino acid having an alkynyl group in the side chain, 2-propargyl -L- glycine [(2S)-2-amino-4-pentynoic acid] or D- propargylglycine [ (2R)-2-amino-4-pentyne is an amino acid residue derived from acid], amino acid residue preferably derived from 2-propargyl -L- glycine [(2S)-2-amino-4-pentyne acid] a group.
　when n = 2, amino acid residues from an amino acid having an alkynyl group in the side chain, 2-propargyl -L- alanine (L- homopropargyl glycine) [(2S)-2-amino-5-hexyne acid ] or 2-propargyl -D- alanine (D-homo propargyl glycine) [(2R) an amino acid residue derived from 2-amino-5-hexyne acid], preferably 2-propargyl -L- alanine (L - an amino acid residue derived from a homo propargyl glycine) [(2S)-2-amino-5-hexyne acid].
　When n = a 3 amino acid residues from an amino acid having an alkynyl group in the side chain, 2-propargyl -L- homoalanine (2-propargyl -L- bis homo glycine) [(2S)-2-amino - is 6-heptynoic acid] or 2-propargyl -D- homoalanine (2-propargyl -D- bis homo glycine) [(2R) amino acid residues derived from 2-amino-6-heptynoic acid], preferably 2 - an amino acid residue derived from propargyl -L- homoalanine (2-propargyl -L- bis homo glycine) [(2S)-2-amino-6-heptynoic acid].
[0090]
(A particularly preferred triazole coupling)
　since the triazole coupling alkali resistance is high, the triazole coupling intramolecular crosslinking, mixed mode affinity chromatography carrier is an antibody binding be repeated washing with alkali is maintained, it is possible to reduce the antibody purification costs. Such triazole linkages in particular, triazole bond formed between the γ- azidohomoalanine residue or ε- Ajidorishin residues homopropargyl glycine residue or bis homopropargyl glycine residue are preferred.
[0091]
"Amide bond"
　the amide bond is not particularly limited, preferably derived from the amino acid having a side chain amino group and the side chain carboxyl group of the first amino acid residue derived from the amino acid having an amino group in the side chain an amide bond formed between the side chain carboxyl group of a second amino acid residue.
[0092]
　Between the side chain carboxyl group of a second amino acid residue derived from the amino acid having a side chain amino group and the side chain carboxyl group of the first amino acid residue derived from the amino acid having an amino group in the side chain the formed amide bond, for example, those represented by the following formula.
[0093]
[Formula 9]

[0094]
　However, * is the point of attachment to the other amino acid residues or other substituents, m and n are each independently an integer of 1 or more, preferably m = 1, 2, 3 or 4, and, n = 1, 2 or 3, more preferably m = 2 or 3 and n = 1 or 2.
[0095]
　When a m = 1, amino acid residues from an amino acid having an amino group in the side chain, L-2,3-diaminopropane acid [(2S)-2,3-diamino propanoic acid] or D-2, an amino acid residue derived from 3-diaminopropane acid [(2R)-2,3-diaminopropane acid], preferably a L-2,3-diaminopropane acid [(2S)-2-amino-propanoic acid] it is derived from the amino acid residues.
　When a m = 2, amino acid residues from an amino acid having an amino group in the side chain, L-2,4-diaminobutane acid [(2S)-2,4-diaminobutane acid] or D-2, an amino acid residue derived from 4-diaminobutane acid [(2R)-2,4-diaminobutane acid], preferably L-2,4-diaminobutane acid [(2S)-2,4-diaminobutane acid an amino acid residue derived from a].
　When m = a 3 amino acid residues from an amino acid having an amino group in the side chain, L- ornithine [(2S)-2,5-diamino pentanoic acid] or D- ornithine [(2R) -2, 5 is a diamino acid residue derived from pentanoic acid], preferably amino acid residues derived from the L- ornithine [(2S)-2,5-diamino pentanoic acid].
　When m = a 4 amino acid residues from an amino acid having an amino group in the side chain, L- lysine [(2S)-2,6-diaminohexane acid] or D- lysine [(2R) -2, 6 is an amino acid residue derived from the di-amino hexanoic acid] is preferably an amino acid residue derived from a L- lysine [(2S)-2,6-diaminohexane acid].
[0096]
　when n = 1, amino acid residues from an amino acid having a carboxyl group in the side chain is an amino acid residue derived from the L- aspartic acid or D- aspartate, preferably derived from L- aspartic acid it is an amino acid residue.
　when n = 2, amino acid residues from an amino acid having a carboxyl group in the side chain is an amino acid residue derived from L- glutamic acid or D- glutamate, amino acid residue preferably derived from L- glutamic acid it is.
　When n = a 3 amino acid residues from an amino acid having a carboxyl group in the side chain, L- homoglutamine acid [(2S)-2-aminoadipic acid] or D- homoglutamic acid [(2R)-2- an amino acid residue derived from amino adipic acid], preferably amino acid residues derived from the L- homoglutamine acid [(2S)-2-aminoadipic acid].
[0097]
(A particularly preferred amide bond)
　amide bond higher alkali resistance, mixed mode affinity chromatography carrier can be repeated washing with alkali is maintained antibody binding, it is possible to reduce the antibody purification costs. Such amide bond, particularly preferably an amide bond formed between the lysine residues and glutamic acid residues.
[0098]
"Amino acid residues of the annular portion"
　amino acid residues of the annular portion is not particularly limited as long as less total number of amino acid residues in the cyclic peptide, preferably 8 to 14 residues, more preferably 9-13 is a residue, more preferably 10 to 12 residues, more preferably is 11 residues.
[0099]
"Straight section"
　the cyclic peptide may have a linear portion. The linear portion of the polypeptide chain of the cyclic peptide, refers to a moiety that is not included in the annular portion.
[0100]
　The linear portion may include amino acid residues having at least one selected from the group consisting of hydroxy and carboxy groups in the side chain. Will be described later amino acid residues having at least one selected from the group consisting of hydroxy and carboxy groups in the side chain.
[0101]
　The linear portion may include amino acid residues from an amino acid with immobilized functional groups in the side chain. It will be described later acids with immobilized functional groups and immobilized functional groups.
[0102]
"At least one and the binding of the substrate and the hydrophilic polymer of the cyclic peptide"
　the cyclic peptide is preferably linked via a covalent bond with at least one of the substrate and the hydrophilic polymer.
　Such covalent bond, (a) cyclic peptides linear portion amino acid residue side chains and substrate and covalent bond between at least one hydrophilic polymer, or (b) a cyclic peptide of the polypeptide chain covalent bond, etc. between at least one of the N-terminal or C-terminal and the substrate and the hydrophilic polymer.
　The covalent binding of said (a), capable of forming a covalent bond by reacting with at least one reactive functional group (immobilization functional group immobilized functional group and the base material and a hydrophilic polymer of amino acid residue side chains refers to functional groups.) bond and the like formed between the. For immobilization functional groups and reactive functional groups in this case will be described later.
　The covalent binding of said (b), via a linker between at least one of the polymer main chain or side chain of the N-terminal or C-terminal and the substrate and the hydrophilic polymer of the polypeptide chain, or going through without binding or the like and the like.
　Linkers of this case, PEG; include (Polyethylene Glycol Polyethylene glycol) linker and the like. Ethylene glycol unit number of PEG linker is not particularly limited as long as one or more, preferably 1 to 24, more preferably 1 to 12, more preferably from 4 to 8.
[0103]
"The molecular weight of the cyclic peptide"
　molecular weight of the cyclic peptide is not particularly limited, but is preferably 10000 or less from the viewpoint of synthesis cost, preferably 5000 or less from the viewpoint of antigenicity. The molecular weight of the cyclic peptides, more preferably about 4000 or less, more preferably about 3,000 or less, and most preferably about 2000 or less. Here, "about" is meant to include a range of ± 2%.
　Here, the molecular weight of the cyclic peptide is the molecular weight of the combination of the annular portion and the linear portion.
　If the molecular weight is less than 5000, antigenicity is reduced, it shows no substantial antigenicity.
　The amino acid residues of the cyclic peptide is not particularly limited, preferably not more than 100 residues, more preferably not more than 50 residues, more preferably not more than 40 residues, more preferably 30 and the residue less, and even more preferably 20 residues or less.
　Here, the amino acid residues of the cyclic peptide is amino acid residues obtained by combining the annular portion and the linear portion.
[0104]
"Antibody binding"
　in the present invention, the antibody binding, refers to the binding of the antibody and / or antibody derivatives.
　The higher the antibody binding, when used as an affinity ligand carrier for affinity chromatography, adsorption force antibodies is high, hardly desorbed even when washed.
　The antibody refers immunoglobulin or an analogue thereof, a fragment or fusion.
　Immunoglobulins, IgG (Immunoglobulin G; immunoglobulin G), IgM (Immunoglobulin M; immunoglobulin M), IgA (Immunoglobulin A; Immunoglobulin A), IgD (Immunoglobulin D; immunoglobulin D) and IgE (Immunoglobulin E; immune may be any of the five classes of immunoglobulin E) (isotypes), but preferably IgG or IgM, IgG is more preferable.
　Moreover, the analogs, immunoglobulins structure or feature is at least partially retained, natural or artificially created, refers to a protein or protein conjugate.
　Further, the fragment refers to a protein having been produced by enzymatic treatment or genetic engineering design, the immunoglobulin moiety.
　Further, the fusion, various cytokines, the functional part of a protein having the biological activity of the cytokine receptor like, all or part of an immunoglobulin and engineered to be fused refers to a protein produced by.
[0105]
　Further, the derivative of human immunoglobulin Fc region and a non-human mammalian immunoglobulins chimeric antibody that combines and Fab region of several human immunoglobulin Fc region and a number of non-human mammalian immunoglobulins chimeric antibodies that combines the Fv region of a non-human mammalian immunoglobulins CDR; non-human mammals that combines a CDR portion of the part and the human immunoglobulin remaining except the (complementarity determining region complementarity determining region) moiety animals antibodies, non-human mammal immunoglobulin Fc region and a human immunoglobulin Fab (fragment, antigen binding; antibody binding fragment) chimeric antibodies that combines the region, some of the Fc region of a non-human mammalian immunoglobulins with the human immune globulin Chimeric antibody that combines with some of the Fv region of the emission, the humanized antibody obtained by fusing the remainder of the non-human mammalian immunoglobulins CDR portion excluding the CDR portions of the human immunoglobulin, non-human mammal immunized globulin Fc region and the non-human mammal immunoglobulins chimeric antibodies and Fab regions were fused, and a number of the Fv region of some of the Fc region of a non-human mammalian immunoglobulins and non-human mammals immunoglobulin fusion chimeric antibodies were of non-human mammalian immunoglobulins CDR; non-human mammal antibody that combines the remainder of the non-human mammalian immunoglobulins CDR portion except for (complementarity determining region complementarity determining region) moiety and Fc a protein plus these chemical modifications It refers to a protein that holds the pass.
　Antibodies, fusion with a monoclonal antibody or immunoglobulin Fc region is preferred, a monoclonal antibody is more preferable.
[0106]
"More specific description of the cyclic peptide"
　describing the cyclic peptide more specifically.
　Here, described mixed mode affinity chromatography matrix of the present invention for cyclic peptides prior to introduction as an affinity ligand.
[0107]
　In the present invention, amino acids are, in principle, co-naming committee of the International Union of Pure and Applied Chemistry and the International Union of Biochemistry and Molecular Biology (INTERNATIONAL UNION OF PURE AND APPLIED CHEMISTRY and INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECULAR BIOLOGY IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN)) adopted by name, expressed using the abbreviation, and the like. The amino acid residues represented using amino acid abbreviations which the amino acid residue is derived. Note that the amino acid residues, including N-terminal amino acids (N-terminal residues) and the C-terminal amino acid (C-terminal residue).
　Unless otherwise indicated, (also referred to as "primary structure".) The amino acid sequence of a peptide or protein represented side by side amino acid residues so that the N-terminal to C-terminal from the left side to the right side in one dimension. If also identified, including positions of amino acid residues in the amino acid sequence of the peptide or protein will be given the number indicating what number of amino acid residues from the N-terminal side to the right side of abbreviations for amino acid residues there is a case to represent Te. For example, there is a case where the second L- lysine from the N-terminus representing the Lys2.
[0108]
　Further, in a case where the amino acid expressed by using the name of the isomers are in enantiomeric relationship, i.e., when the L-isomer and a D-isomer is present, explicitly stated another L-isomer and D-isomer If except denote the L-configuration in principle. For example, "isoleucine" is assumed to represent "L- isoleucine" enantiomer "isoleucine" represents a "D- isoleucine". The same applies to the amino acid residues.
[0109]
　Further, in a case where amino acids were expressed using the abbreviation (three letter abbreviation or single letter abbreviations) isomer in the enantiomer of relationship, i.e., when the L-isomer and a D-isomer is present, L-form and D except where explicitly stated another body, it is intended to refer to L-form as a rule. However, represents any amino acid "X" is not limited to this. For example, to represent a "Lys" and "L-Lys" Both "L- lysine", "D-Lys" represent the "D- lysine '. The same applies to the amino acid residues.
[0110]
　Further, in a case where the amino acid expressed by using the name of, if the isomers are in a relationship of diastereomers are present, and shall not be included in the amino acid specified by its name. Diastereomer is treated as different kinds of amino acids by using the prefix "allo". For example, "threonine" and "L- threonine" do not include "L- allo-threonine", "D- threonine" shall not include the "D- allo-threonine". The same applies to the amino acid residues.
[0111]
　1-letter abbreviations and 3-letter abbreviations were officially recognized amino acid names and abbreviations (1-letter abbreviations, three-letter abbreviations) is shown in Table 1.
[0112]
[Table 1]

[0113]
　Amino acids are not limited to those listed in Table 1, it is also possible to use the referred amino and unusual amino acids. Examples of unnatural amino acids are listed in Table 2 below, but is not limited thereto.
[0114]
[Table 2]

[0115]
"Formula (I)"
　The cyclic peptide is preferably a cyclic peptide represented by the following formula (I).
　R N -X g - [X i -X a -X m -X 1 -X 2 -X 3 -X n -X b -X j ] k -X h　-R C · · · (I)
[0116]
　In formula (I), for example, X n is intended to n-number of X are linked. In other words, - (X) N - and it can be said. Incidentally, X g , X i , X m , X j , and X h is also defined in the above X n is the same as.
[0117]
(Annular portion, a straight portion, bridge and antibody binding portion)
　in cyclic peptides used in the mixed mode affinity chromatography matrix of the present invention, among the polypeptide chain, the annulus portion of the closed ring by a bridge, the annular portion the called linear portion non part included in. Further, of the annular portion, it refers to a portion forming an intramolecular cross-linked structure of the cyclic peptide of the present invention and a crosslinking unit, contributing component strongly to the antibody binding cyclic peptides of the present invention that the antibody binding portion.
　Wherein the annular portion of the cyclic peptide represented by (I) is "X a -X m -X 1 -X 2 -X 3 -X n -X b is" part, straight part of the "X g ", " X h "," X i ", and" X j ", and the bridge is" X a "and" X b ", and the antibody binding portion" X 1 -X 2 -X 3 "it is.
　In the formula (1), [X i -X a -X m -X 1 -X 2 -X 3 -X n -X b -X j may be referred to repeatedly portion.
[0118]
(X 1 , X 2 and X 3 )
　In the above formula (1), X 1 is, L- leucine residue, L- isoleucine residue, L- methionine residue, L- lysine residue or L- arginine residue, a group, preferably L- leucine residue or L- isoleucine residue, more preferably L- leucine residue.
　In the above formula (1), X 2 is L- valine residue or L- isoleucine residue, preferably L- valine residue.
　In the above formula (1), X 3 is L- tryptophan residue or L- phenylalanine residue, preferably L- tryptophan residues.
[0119]
(X a and X b )
　formula (I), X a and X b are of the following (a) ~ (d), it is any one.
[0120]
(A) X a and X b are crosslinked by disulfide bonds.
　X a and X b are the preferably each independently, an amino acid residue derived from the amino acid having a thiol group in a side chain, more preferably, X a and X b At least one of L- cysteine and D- an amino acid residue derived from the amino acid having a thiol group in a side chain other than cysteine, and more preferably, X a and X b to the amino acid having a thiol group both in the side chain other than L- cysteine and D- cysteine it is derived from the amino acid residues.
[0121]
　That, X a and X b disulfide bond between is preferably a disulfide bond between the two amino acid residues from an amino acid having a thiol group in a side chain, more preferably, L- cysteine and D- cysteine a disulfide bond between amino acid residues from an amino acid having a thiol group at the amino acid residue and a side chain from an amino acid having a thiol group in a side chain other than, more preferably L- cysteine and D- cysteine in the side chain other than a disulfide bond between the two amino acid residue having a thiol group.
[0122]
　X a and X b chemical resistance among cyclic peptide comprising crosslinked by disulfide bonds typically, X a and X b in comparison with the case both of an amino acid residue derived from a L- cysteine or D- cysteine , X a and X b in the case one of is an amino acid residue derived from an amino acid having a thiol group in a side chain other than L- cysteine and D- cysteine, it made higher, X a and X b both There in the case of amino acid residues from an amino acid having a thiol group in a side chain other than L- cysteine and D- cysteine, becomes higher. That is, from the viewpoint of chemical resistance, disulfide bond, L- cysteine or D- more preferably disulfide bonds other than disulfide bond between the two amino acid residues derived from cysteine, L- cysteine and terminals other than D- cysteine disulfide bonds more preferably between two amino acid residues from an amino acid having a thiol group at the chain, and particularly preferably a disulfide bond between the two amino acid residues derived from the homocysteine.
[0123]
　The amino acid having a thiol group at the side chain, for example, L- cysteine, D- cysteine, L- homocysteine, D- homocysteine, L- penicillamine, and D- penicillamine and the like, cysteine, homocysteine and amino acid selected from the group consisting of penicillamine are preferred.
　As the amino acid having a thiol group in the side chain other than the L- cysteine and D- cysteine, for example, L- homocysteine, D- homocysteine, L- penicillamine, and D- penicillamine and the like, homocysteine and an amino acid selected from the group consisting of penicillamine are preferred.
[0124]
(B) X a and X b are cross-linked by thioether bonds.
　X a and X b are preferably, one is an amino acid residue derived from the amino acid having a thiol group in the side chain, amino acid residues other is derived from the amino acid having a haloacetyl group on the side chain.
[0125]
　Amino acid having a thiol group at the side chain is preferably an amino acid having a thiol group in a side chain other than L- cysteine and D- cysteine.
　The amino acid having a thiol group at the side chain is preferably, cysteine, an amino acid having a thiol group in the side chain selected from the group consisting of homocysteine and penicillamine.
　Furthermore, the amino acid having a thiol group at the side chain, more preferably an amino acid having a thiol group in the side chain selected from the group consisting of homocysteine and penicillamine, more preferably homocysteine.
[0126]
　Amino acids having a haloacetyl group on the side chain is preferably an amino acid represented by the following formula.
[0127]
[Formula 10]

[0128]
　However, in the above formulas, n is an integer of 1 or more, X is a halogen atom.
　Incidentally, n is preferably an integer that satisfies 1 ≦ n ≦ 4, X is preferably a chlorine atom or a bromine atom, more preferably chlorine atom.
[0129]
　The above
　formula, when it is n = 1, N 3 - haloacetyl -L-2,3-diaminopropane acid [(2S)-2-amino-3 - [(2-haloacetyl) amino] propanoic acid] or N 3 - haloacetyl -D-2,3-diamino propanoic acid [(2R)-2-amino-3 - [(2-haloacetyl) amino] propanoic acid]
　represents, when it is n = 2, n 4 - haloacetyl -L 2,4 diaminobutane acid [(2S)-2-amino-4 - [(2-haloacetyl) amino] butanoic acid] or N 4 - haloacetyl -D-2,4-diaminobutane acid [(2R) - 2-amino-4 - [(2-haloacetyl) amino] represents butanoic
　acid], when n = a 3, n-.delta.-haloacetyl -L- ornithine [(2S)-2-amino-5 - [(2 - haloacetyl) amino] Bae Lanthanum acid] or N-.delta.-haloacetyl -D- ornithine [(2R)-2-amino-5 - [(2-haloacetyl) amino] represents pentanoic
　acid], when n = a 4, N-.epsilon.-haloacetyl -L- lysine [(2S)-2-amino-6 - [(2-haloacetyl) amino] hexanoic acid] or N-.epsilon.-haloacetyl -D- lysine [(2R)-2-amino-6 - [(2 - haloacetyl) represents an amino] hexanoic acid].
[0130]
　Amino acids having a haloacetyl group on the side chain, especially preferably N-.epsilon.-chloroacetyl -L- lysine, N-.epsilon. chloroacetyl -D- lysine, N-.delta. chloroacetyl -L- ornithine or, N- a .delta. chloroacetyl -D- ornithine, particularly preferably N-.epsilon.-chloroacetyl -L- lysine or N-.delta. chloroacetyl -L- ornithine, most preferably N-.delta. chloroacetyl -L- ornithine.
[0131]
(C) X a and X b are cross-linked by the triazole coupling.
　X a and X b are preferably, one is an amino acid residue derived from the amino acid having an azido group in the side chain, amino acid residues other is derived from the amino acid having an alkynyl group at a side chain.
[0132]
　Here, triazole bond is a bond that is an azide group and alkynyl group with Hyusugen reaction represented by the following formula was formed.
[0133]
[Of 11]

[0134]
　However, in the above formulas, R represents a moiety other than the azide group of amino acid residues having an azido group in the side chain, R 'represents a portion other than the ethynyl group of an amino acid residue having an alkynyl group at a side chain.
[0135]
　Amino acids with an azido group to the side chain, for example, those represented by the following formula. However, in the following formulas, m is an integer of 1 or more.
[0136]
[Chem. 12]

[0137]
　m is not particularly limited as long as it is 1 or more, from the viewpoint of economy, and preferably from 1 ≦ m ≦ 4, more preferably 2 ≦ m ≦ 4, more preferably 3 ≦ m ≦ 4, more and preferably m = 4.
　When a m = 1, amino acids having an azido group in the side chain represented by the above formula is a β- azido -L- alanine or β- azide -D- alanine, preferably β- azide -L- alanine it is.
　When a m = 2, amino acids having an azido group in the side chain represented by the above formula is γ- azido -L- homoalanine or γ- azide -D- homoalanine, preferably γ- azide -L- homoalanine it is.
　When m = a 3, amino acids having an azido group in the side chain represented by the above formula is δ- azido -L- ornithine or δ- azide -D- ornithine, preferably δ- azide -L- ornithine it is.
　When m = a 4, amino acids having an azido group in the side chain represented by the above formula is ε- azido -L- lysine or ε- azide -D- lysine, preferably ε- azide -L- lysine it is.
[0138]
　Amino acids having an azido group in the side chain, especially preferably ε- azide -L- lysine, .delta.-azido -L- ornithine or γ- azide -L- homoalanine, particularly preferably ε- azide -L- it is lysine or δ- azide -L- ornithine, most preferably ε- azide -L- lysine.
[0139]
　Amino acids with an alkynyl group on the side chain, for example, those represented by the following formula. However, in the following formulas, n represents an integer of 1 or more.
[0140]
[Formula 13]

[0141]
　n it is not particularly limited as long as it is 1 or more, from the viewpoint of economy, preferably 1 ≦ n ≦ 3, more preferably 2 ≦ n ≦ 3.
　when n = 1, amino acids having an alkynyl group at a side chain represented by the above formula is 2-propargyl -L- glycine or 2-propargyl -D- glycine, preferably 2-propargyl -L- glycine it is.
　when n = 2, amino acids having an alkynyl group at a side chain represented by the above formula is 2-propargyl -L- alanine (2-propargyl -L- Homogurishin) or 2-propargyl -D- alanine (2- propargyl -D- Homogurishin), preferably 2-propargyl -L- alanine (2-propargyl -L- Homogurishin).
　When n = a 3, amino acids having an alkynyl group at a side chain represented by the above formula is 2-propargyl -L- homoalanine (2-propargyl -L- bis homo glycine) or 2-propargyl -D- homoalanine ( is 2-propargyl -D- bis homo glycine), preferably 2-propargyl -L- homoalanine (2-propargyl -L- bis homo glycine).
[0142]
　Amino acids having an alkynyl group at the side chain, especially preferably 2-propargyl -L- alanine (2-propargyl -L- Homogurishin) or 2-propargyl -L- homoalanine (2-propargyl -L- bis homo glycine) There, particularly preferably 2-propargyl -L- homoalanine (2-propargyl -L- bis homo glycine).
[0143]
(D) X a and X b are cross-linked by an amide bond.
　X a and X b are preferably, one is an amino acid residue derived from the amino acid having an amino group in the side chain, amino acid residues other is derived from the amino acid having a carboxyl group in the side chain.
[0144]
　The amino acid having an amino group in the side chain, for example, those represented by the following formula. However, in the following formulas, m is an integer of 1 or more.
[0145]
[Formula 14]

[0146]
　m is not particularly limited as long as it is 1 or more, from the viewpoint of economy, and preferably from 1 ≦ m ≦ 4, more preferably 2 ≦ m ≦ 4, more preferably 3 ≦ m ≦ 4, more and preferably m = 4.
　When a m = 1, amino acid, L-2,3-diaminopropane acid [(2S)-2,3-diamino propanoic acid] or D-2 having an amino group in the side chain represented by the formula, a 3-diaminopropane acid [(2R)-2,3-diaminopropane acid], preferably L-2,3-diaminopropane acid [(2S)-2,3-diaminopropane acid].
　When a m = 2, amino acids, L-2,4-diaminobutane acid [(2S)-2,4-diaminobutane acid] or D-2 having an amino group in the side chain represented by the formula, 4- diaminobutane an acid [(2R)-2,4-diaminobutane acid], preferably L-2,4-diaminobutane acid [(2S)-2,4-diaminobutane acid].
　When m = a 3, amino acids having an amino group in the side chain represented by the above formula is L- ornithine or D- ornithine, preferably L- ornithine.
　When m = a 4, amino acids having an amino group in the side chain represented by the above formula is L- lysine or D- lysine, preferably L- lysine.
[0147]
　Amino acids with carboxyl groups on the side chains, for example, those represented by the following formula. However, in the following formulas, n represents an integer of 1 or more.
[0148]
[Formula 15]

[0149]
　n it is not particularly limited as long as it is 1 or more, from the viewpoint of economy, preferably 1 ≦ n ≦ 3, more preferably n = 1 or 2, even more preferably n = 2.
　when n = 1, amino acids having a carboxyl group in the side chain represented by the above formula is L- aspartic acid or D- aspartate, preferably L- aspartic acid.
　when n = 2, amino acids having a carboxyl group in the side chain represented by the above formula is L- glutamic acid or D- glutamate, preferably L- glutamic acid.
　When n = a 3, amino acids having a carboxyl group in the side chain represented by the above formula is L- homoglutamic acid or D- homoglutamic acid, preferably L- homoglutamine acid.
[0150]
(X)
　in formula (I), X represents an amino acid residue, if X is plural, X may be the being the same or different.
　The above X may be any amino acid residue is not particularly limited, from an amino acid selected from the group consisting of amino acids shown in and Table 2 (. Except B, and Z and X) amino acids shown in Table 1 amino acid preferably residues are amino acids shown in Table 1 and more preferably amino acid residues from an amino acid selected from the group consisting of (B, except. Z and X). Further, if present, may be an amino acid residue derived from a enantiomers or diastereomers of these amino acids.
[0151]
(N-terminal group and C-terminal groups)
　formula (I), R N represents an N-terminal group.
　Examples of the N-terminal group, for example, include an amino group, the amino group, N- acetylation may be the N- formylated, or N- N-terminal modifications of the acyl and the like.
[0152]
　Wherein (I), R C represents a C-terminal group.
　As the C-terminal group, for example, it includes a carboxy group, the carboxy group may be the C-terminal modifications of the amide and the like.
[0153]
(G and h)
　wherein (I), g and h are each independently an integer of 0 or more.
　g preferably satisfies 0 ≦ g ≦ 20, more preferably satisfies the 0 ≦ g ≦ 10, more preferably satisfy 0 ≦ g ≦ 5.
　h is preferably satisfies 0 ≦ h ≦ 20, more preferably satisfies the 0 ≦ h ≦ 10, more preferably satisfy 0 ≦ h ≦ 5.
[0154]
(I and j)
　in formula (I), i and j are each independently an integer of 0 or more.
　i preferably satisfies 0 ≦ i ≦ 20, more preferably satisfies the 0 ≦ i ≦ 10, more preferably satisfy 0 ≦ i ≦ 5.
　j is preferably satisfies 0 ≦ j ≦ 20, more preferably satisfies the 0 ≦ j ≦ 10, more preferably satisfy 0 ≦ j ≦ 5.
[0155]
(M and n)
　in formula (I), m and n are each, 0 ≦ m ≦ 9, and is an integer satisfying 0 ≦ n ≦ 9.
　Further, m and n satisfies 3 ≦ m + n ≦ 9, preferably satisfies 4 ≦ m + n ≦ 8, more preferably satisfy 5 ≦ m + n ≦ 7.
[0156]
(Amino acid residues of the annular portion)
　in the above formula (I), the annular portion [X a -X m -X 1 -X 2 -X 3 -X n -X b ] amino acid residues of [(m + n + 5) residues groups] is a 8-14 acid radicals, preferably 9-13 residues, more preferably 10 to 12 residues.
　When amino acid residues of the annular portion is within this range, not too large distortion intramolecular cyclic peptides, since the conformation of such α-helix is stabilized, the antibody-binding cyclic peptides of the present invention excellent.
[0157]
(K) k
　is an integer satisfying k ≧ 1, preferably 1 ≦ k ≦ 3, more preferably 1 ≦ k ≦ 2, more preferably from k = 1.
　The number of repeating units is not particularly limited, it is possible as the number of repeating units often contains many annular portion, it may be able to improve antibody binding cyclic peptides, repeated because it can reduce the total number of amino acid residues as the number of units is small, it may be possible to suppress the antigenicity of cyclic peptides. From the viewpoint of synthesis cost of the cyclic peptide, preferably towards amino acid residues is small, it is preferable less number of repeating units.
[0158]
(K difference between repeating units in the case ≧ a 2)
　Moreover, if a k ≧ 2, i.e., the cyclic peptide repeating units [X represented by the formula (I) i -X a -X m -X 1 -X 2 -X 3 -X n -X b -X j when containing] a two or more, X in the repeating units 1 , X 2 , X 3 , X a , X b , X i , X j , X m , and X n , respectively, may be the same among the repeating units may be different.
[0159]
(Cyclic total number of amino acid residues of the peptide)
　In addition, in the above formula (I), the total number of amino acid residues in the cyclic peptide, preferably 8 to 50 residues, more preferably 9-40 residues, more preferably 10 to 30 residues, more preferably 10 to 20 residues.
　That formula (I), g, h, j, j, m, n, and k is preferably satisfies 8 ≦ g + h + (i + j + m + n + 5) × k ≦ 50, more preferably 9 ≦ g + h + (i + j + m + n + 5) × k ≦ met 40, more preferably satisfies 10 ≦ g + h + (i + j + m + n + 5) × k ≦ 30, more preferably satisfy 10 ≦ g + h + (i + j + m + n + 5) × k ≦ 20.
　Usually, since the number of amino acid residues number becomes higher synthesis cost becomes high from the economic point of view, the total number of amino acid residues is preferably small.
[0160]
"Formula (IA)"
　Further, the cyclic peptides, more preferably, a cyclic peptide represented by the following formula (IA).
　R N -X g - [X p2 -X 4 r -X p1 -X a -X m -X 1 -X 2 -X 3 -X n -X b -X q1 -X 5 s -X q2 ] k - X H -R C　　· · · (IA)
[0161]
　Wherein (IA), R N , R C , X 1 , X 2 , X 3 , X a , X b , X g , X h , X m , X n , X, g, h, m, n, and k are both the same meanings as those in the above formula (I).
　In the formula (IA), X n is, X in formula (I) n the same manner as, X is intended to have the n coupling. X m , X p1 , X p2 , X q1 , and X q2 is same for.
　In addition, in the formula (IA), X 4 RAnd X 5 s are each, X 4 that is the r connection, and, X 5 that are the s coupling, intended to.
[0162]
(Annular portion, a straight portion, bridge and antibody binding portion)
　linear portion of the cyclic peptide represented by formula (IA) is "X g ", "X h ", "X p2 -X 4 r -X p1 ", and" X Q1 -X 5 S -X Q2 is ". Annular portion, bridging portion, and the antibody binding portion is the same as the cyclic peptide represented by formula (I).
　Further, in compounds represented by formula (IA), the repeating unit [X p2 -X 4 r -X p1 -X a -X m -X 1 -X 2 -X 3 -X n -X b-X Q1 -X 5 S -X Q2 is].
[0163]
(X 4 and X 5 )
　In the formula (IA), X 4 and X 5 are each independently, from an amino acid having a hydroxy group on an amino acid residue or side chain from an amino acid having a carboxyl group in the side chain representing the amino acid residues.
[0164]
　Amino acids with carboxyl groups on the side chains, for example, L- aspartic acid, D- aspartic acid, L- glutamic acid, D- glutamate, L- homoglutamine acid, and D- homoglutamic acid and the like.
[0165]
　Amino acids with hydroxy groups in the side chains, for example, L- serine, D- serine, L- homoserine, D- homoserine, L- tyrosine, D- tyrosine, L- threonine, D- threonine, L- allothreonine and D- allothreonine and the like.
[0166]
　The X 4 and X 5 are preferably, each independently, L- serine residue, D- serine residue, L- homoserine residue, D- homoserine residue, L- aspartic acid, D- aspartate residue, L- glutamic acid residue, D- glutamic acid residue, L- homo-acid residues, D- homo-acid residues, L- tyrosine residue, D- tyrosine residue, L- homotyrosine residue, D- homotyrosine residues, L- threonine residue, a D- threonine residue, L- allo-threonine residue, and amino acid residue selected from the group consisting of D- allo-threonine residues, more preferably, each independently, L- aspartic acid residue, D- aspartic acid residue, an amino acid residue selected from the group consisting of L- threonine residues and D- threonine residues, more preferably X 4 is L- aspartic acid residue, and, X 5 is L- threonine residue.
[0167]
　The X 4 and X 5 are each independently, if it is an amino acid residue derived from the amino acid having a hydroxy group on an amino acid residue or side chain from an amino acid having a carboxyl group in the side chain, antibody binding ring portion the parts and antibodies and are hydrogen bond and / or electrostatic interactions can interact more strongly believed antibody binding is improved.
[0168]
(P1, p2, q1 and q2)
　in formula (IA), p1, p2, q1, and q2 are each independently an integer of 0 or more.
　p1 is preferably satisfies 0 ≦ p1 ≦ 20, more preferably satisfies 0 ≦ p1 ≦ 10, more preferably satisfies 0 ≦ p1 ≦ 5, more preferably satisfy 0 ≦ p1 ≦ 2.
　p2 preferably satisfies 0 ≦ p2 ≦ 20, more preferably satisfies 0 ≦ p2 ≦ 10, more preferably satisfies 0 ≦ p2 ≦ 5, more preferably satisfy 0 ≦ p2 ≦ 2.
　q1 preferably satisfies 0 ≦ q1 ≦ 20, more preferably satisfies 0 ≦ q1 ≦ 10, more preferably satisfies 0 ≦ q1 ≦ 5, more preferably satisfy 0 ≦ q1 ≦ 2.
　q2 preferably satisfies 0 ≦ q2 ≦ 20, more preferably satisfies 0 ≦ q2 ≦ 10, more preferably satisfies 0 ≦ q2 ≦ 5, more preferably satisfy 0 ≦ q2 ≦ 2.
[0169]
(R and s)
　in formula (IA), r and s are, respectively, 0 ≦ r ≦ 5,0 ≦ s ≦ 5, and, 1 ≦ Max (r, s) is an integer that satisfies ≦ 5, preferably , 0 ≦ r ≦ 4,0 ≦ s ≦ 4, and is an integer satisfying 1 ≦ Max (r, s) ≦ 4, more preferably, 0 ≦ r ≦ 3,0 ≦ s ≦ 3, and, 1 ≦ Max (r, s) is an integer satisfying ≦ 3.
　However, Max (r, s), when a r ≠ s, represents the larger of the two numbers r and s, when it is r = s, represent the r or s.
[0170]
(Amino acid residues of the annular portion)
　In the above formula (IA), the annular portion [X a -X m -X 1 -X 2 -X 3 -X n -X b ] amino acid residues of [(m + n + 5) residues group], like above-mentioned formula (I), a 8-14 acid radicals, preferably 9-13 residues, more preferably 10 to 12 residues.
　When amino acid residues of the annular portion is within this range, not too large distortion intramolecular cyclic peptides, since the conformation of such α-helix is stabilized, the antibody-binding cyclic peptides of the present invention excellent.
[0171]
(K difference between repeating units in the case ≧ a 2)
　Moreover, if a k ≧ 2, i.e., wherein the cyclic peptide repeating units represented by (IA) [X p2 -X 4 r -X p1 -X a -X m -X 1 -X 2 -X 3 -X n -X b -X q1 -X 5 s -X q2 If] a comprising two or more, X in the repeating units 1 , X 2 , X 3 , X A , X B , X 4 R , X 5 s , X m , X n , X p2 , X p1 , X q1 , and X q2 , respectively, may be the same among the repeating units may be different.
[0172]
(Cyclic total number of amino acid residues of the peptide)
　In addition, in the above formula (IA), the total number of amino acid residues in the cyclic peptide, preferably 8 to 50 residues, more preferably 9-40 residues, more preferably 10 to 30 residues, more preferably 10 to 20 residues.
　That is, in the formula (IA), g, h, m, n, p1, p2, q1, q2, r, s, and k is preferably satisfies 8 ≦ g + h + (m + n + p1 + p2 + q1 + q2 + r + s + 5) × k ≦ 50, more preferably It met 9 ≦ g + h + (m + n + p1 + p2 + q1 + q2 + r + s + 5) × k ≦ 40, more preferably satisfies 10 ≦ g + h + (m + n + p1 + p2 + q1 + q2 + r + s + 5) × k ≦ 30, more preferably satisfy 10 ≦ g + h + (m + n + p1 + p2 + q1 + q2 + r + s + 5) × k ≦ 20.
　Usually, since the number of amino acid residues number becomes higher synthesis cost becomes high from the economic point of view, the total number of amino acid residues is preferably small.
[0173]
"Formula (IB)"
　Further, the cyclic peptides, more preferably a cyclic peptide represented by the following formula (IB).
　R N -X v1 -X 6 t -X v2 - [X i -X a -X m -X 1 -X 2 -X 3 -X n -X b -X j ] k -X w2 -X 7 u - X W1 -R C　　· · · (IB)
[0174]
　Wherein (IB), R N , R C , X 1 , X 2 , X 3 , X a , X b , X i , X j , X m , X n , X, i, j, m, n, and k are both the same meanings as those in the above formula (I).
　In the formula (IB), X n is, X in formula (I) n the same manner as, X is intended to have the n coupling. X m , X v1 , X v2 , X w1 , and X w2 This also applies.
　In addition, in the formula (IB), X 6 TAnd X 7 u , respectively, X 6 that is the t connected, and, X 7 that is u-number connection is intended.
[0175]
(Annular portion, a straight portion, bridge and antibody binding portion)
　linear portion of the cyclic peptide represented by formula (IB) is "X i ", "X j ", "X v1 -X 6 t -X v2 ", and" X W2 -X 7 U -X W1 it is ". Annular portion, bridging portion, and the antibody binding portion is the same as the cyclic peptide represented by formula (I).
　In the formula (IB), repeating units are as for formula (I), [X i -X a -X m -X 1 -X 2 -X 3 -X n -X b -X j is] .
[0176]
(X 6 and X 7 )
　formula (IB), X 6 and X 7 each independently represent amino acid residues from an amino acid with immobilized functional groups in the side chain, X 6 or X 7 is more If it is, the plurality of X 6 or X 7 may be the being the same or different.
[0177]
　The above-mentioned "Immobilized functional group", the substrate and / or hydrophilic polymers on the functional group (sometimes referred to as "reactive functional group".) And the reaction was functional group capable of forming a covalent bond Say.
　Such immobilization functional groups, for example, an amino group, carboxy group, hydroxy group, thiol group, formyl group (aldehyde group), a carbamoyl group, an azido group, and, an alkynyl group.
　The combination of the immobilized functional group and the substrate and / or functional groups on the hydrophilic polymer in which the cyclic peptide has the amino and carboxy groups (amide bond formation reaction), an amino group and a formyl group (reductive amination ), amino group and an epoxy group, a carboxy group and a hydroxy group (ester bond formation reaction), a thiol group and a thiol group (disulfide bond), a thiol group and an epoxy group, and an azide group and alkynyl group (Hyusugen cycloaddition reaction) etc. the.
　By the functional groups on the immobilized functional group and the substrate and / or hydrophilic polymer in which the cyclic peptide has to form a covalent bond, the cyclic peptide is immobilized on a carrier. Incidentally, it is sufficient form a covalent bond at least a portion of the immobilized functional group the cyclic peptide has reacts with the functional groups on the substrate and / or a hydrophilic polymer, all of the immobilized functional group is a substrate and / or it may not react with the functional groups on the hydrophilic polymer.
[0178]
　In the amino acid with immobilized functional groups on the side chains, immobilized functional group is preferably at least one selected from the group consisting of amino group, thiol group and aldehyde group, more preferably an amino group and a thiol group at least one selected from the group consisting of.
[0179]
　Amino acids with immobilized functional groups on the side chains, at least preferably, L- lysine, D- lysine, L- cysteine, D- cysteine, is selected from the group consisting of L- homocysteine ​​and D- homocysteine ​​1 it is a kind of amino acid.
[0180]
　By using as the immobilized functional group an amino group, can be linked via an amide bond with a carboxyl group on the carrier, it can be immobilized cyclic peptides of the present invention as readily affinity ligand.
[0181]
　Further, by using thiol groups as the immobilized functional group, can be linked via a covalent bond with the epoxy group on the carrier, it can be immobilized cyclic peptides of the present invention as readily affinity ligand .
[0182]
　As an amino acid having an amino group in the side chain, there are L- lysine and D- lysine and the like, as an amino acid having a thiol group in the side chain, L- cysteine, there is a D- cysteine, that these are relatively inexpensive from, since it is possible to suppress the cost of synthesis of cyclic peptides of the present invention, preferred from an economical point of view.
[0183]
(T and u)
　formula (IB), t and u are each, 0 ≦ t ≦ 5,0 ≦ u ≦ 5, and, 1 ≦ Max (t, u) is an integer that satisfies ≦ 5, preferably 0 ≦ t ≦ 4,0 ≦ u ≦ 4,1 ≦ Max (t, u) is an integer satisfying ≦ 4, more preferably 0 ≦ t ≦ 3,0 ≦ u ≦ 3,1 ≦ Max (t, u ) is an integer satisfying ≦ 3.
　However, Max (t, u), when a t ≠ u, represents the larger of the two numbers t and u, when it is t = u, represents a t or u.
[0184]
(V1, v2, w1 and w2)
　in formula (IB), v1, v2, w1, and w2 are each independently an integer of 0 or more.
　v1 preferably satisfies 0 ≦ v1 ≦ 20, more preferably satisfies 0 ≦ v1 ≦ 10, more preferably satisfies 0 ≦ v1 ≦ 5, more preferably satisfy 0 ≦ v1 ≦ 2.
　v2 preferably satisfies 0 ≦ v2 ≦ 20, more preferably satisfies 0 ≦ v2 ≦ 10, more preferably satisfies 0 ≦ v2 ≦ 5, more preferably satisfy 0 ≦ v2 ≦ 2.
　w1 is preferably satisfies 0 ≦ w1 ≦ 20, more preferably satisfies 0 ≦ w1 ≦ 10, more preferably satisfies 0 ≦ w1 ≦ 5, more preferably satisfy 0 ≦ w1 ≦ 2.
　w2 is preferably satisfies 0 ≦ w2 ≦ 20, more preferably satisfies 0 ≦ w2 ≦ 10, more preferably satisfies 0 ≦ w2 ≦ 5, more preferably satisfy 0 ≦ w2 ≦ 2.
[0185]
(Amino acid residues of the annular portion)
　In the above formula (IB), the annular portion [X a -X m -X 1 -X 2 -X 3 -X n -X b ] amino acid residues of [(m + n + 5 ) residue], similar to above-mentioned formula (I), a 8-14 acid radicals, preferably 9-13 residues, more preferably 10 to 12 residues.
　When amino acid residues of the annular portion is within this range, not too large distortion intramolecular cyclic peptides, since the conformation of such α-helix is stabilized, the antibody-binding cyclic peptides of the present invention excellent.
[0186]
(K difference between repeating units in the case ≧ a 2)
　Moreover, if a k ≧ 2, i.e., the cyclic peptide repeating units [X represented by formula (IB) i -X a -X m -X 1 -X 2 -X 3 -X n -X b -X j when containing] a two or more, X in the repeating units 1 , X 2 , X 3 , X a , X b , X m , X n , X i , and X j , respectively, may be the same among the repeating units may be different.
[0187]
(Cyclic total number of amino acid residues of the peptide)
　In addition, in the above formula (IB), the total number of amino acid residues in the cyclic peptide, preferably 8 to 50 residues, more preferably 9-40 residues, more preferably 10 to 30 residues, more preferably 10 to 20 residues.
　That is, in the formula (IB), i, j, m, n, t, u, v1, v2, w1, w2, and k is preferably satisfies 8 ≦ (i + j + m + n + 5) × k + t + u + v1 + v2 + w1 + w2 ≦ 50, more preferably ( i + j + m + n + 5) meet × k + t + u + v1 + v2 + w1 + w2 ≦ 40, more preferably satisfies 10 ≦ (i + j + m + n + 5) × k + t + u + v1 + v2 + w1 + w2 ≦ 30, more preferably satisfies 10 ≦ (i + j + m + n + 5) × k + t + u + v1 + v2 + w1 + w2 ≦ 20.
　Usually, since the number of amino acid residues number becomes higher synthesis cost becomes high from the economic point of view, the total number of amino acid residues is preferably small.
[0188]
"Formula (IC)"
　In addition, the cyclic peptides, more preferably a cyclic peptide represented by the following formula (IC).
　R N -X v1 -X 6 t -X v2 - [X p2 -X 4 r -X p1 -X a -X m -X 1 -X 2 -X 3 -X n -X b -X q1 -X 5 S -X Q2 ] K -X W2 -X 7 ｕ－Ｘ ｗ１－Ｒ Ｃ　　・・・（ＩＣ）
[0189]
　Wherein (IC), R N , R C , X 1 , X 2 , X 3 , X a , X b , X m , X n , X, m, n, and k are both the formula (I ) have the same meanings as those in the, X 4 , X 5 , p1, p2, q1, q2, r, and s are all the same meaning as those in formula (IA), X 6 , X 7 , t, u, v1, v2, w1, and w2 are both the same meaning as in the formula (IB).
　In the formula (IC), X n is, X in formula (I) n the same manner as, X is intended to have the n coupling. X M , X P1 , X P2 , X q1 , X q2 , X v1 , X v2 , X w1 , and X w2 This also applies.
　Further, in the formula (IC), X 4 r , X 5 s , X 6 t , and X 7 u , respectively, X 4 that is the r connection, X 5 that are the s coupling, X 6 that is the t connected, and, X 7 that is u-number connection is intended.
[0190]
(Annular portion, a straight portion, bridge and antibody binding portion)
　linear portion of the cyclic peptide represented by formula (IC) is "X v1 -X 6 t -X v2 ", "X w2 -X 7 u - X W1 "," X P2 -X 4 R -X P1 ", and" X Q1 -X 5 S -X Q2 is ". Annular portion, bridging portion, and the antibody binding portion is the same as the cyclic peptide represented by formula (I).
　In the formula (IC), repeating units are as for formula (IA), [X p2 -X 4 r -X p1 -X a -X m-X 1 -X 2 -X 3 -X n -X b -X q1 -X 5 s -X q2 ] is.
[0191]
(Amino acid residues of the annular portion)
　In the above formula (IC), the annular portion [X a -X m -X 1 -X 2 -X 3 -X n -X b ] amino acid residues of [(m + n + 5 ) residue], similar to above-mentioned formula (I), a 8-14 acid radicals, preferably 9-13 residues, more preferably 10 to 12 residues.
　When amino acid residues of the annular portion is within this range, not too large distortion intramolecular cyclic peptides, since the conformation of such α-helix is stabilized, the antibody-binding cyclic peptides of the present invention excellent.
[0192]
(K ≧ 2 difference between repeating units where a)
　Further, k if ≧ a 2, i.e., the formula (IC) cyclic peptide repeating units [X represented by p2 -X 4 r -X p1 -X a -X m -X 1 -X 2 -X 3 -X n -X b -X q1 -X 5 s -X q2 If] a comprising two or more, X in the repeating units 1 , X 2 , X 3 , X A , X B , X 4 R , X 5 s , X m , X n , X p2 , X p1 , X q1 , and X q2 , respectively, may be the same among the repeating units may be different.
[0193]
(Cyclic total number of amino acid residues of the peptide)
　In addition, in the above formula (IC), total number of amino acid residues in the cyclic peptide, preferably 8 to 50 residues, more preferably 9-40 residues, more preferably 10 to 30 residues, more preferably 10 to 20 residues.
　That is, in the formula (IC), m, n, p1, p2, q1, q2, r, s, t, u, v1, v2, w1, w2, and k is preferably 8 ≦ (m + n + p1 + p2 + q1 + q2 + r + s + 5) × k + t + u + v1 + v2 + w1 + w2 ≦ met 50, more preferably satisfies the 9 ≦ (m + n + p1 + p2 + q1 + q2 + r + s + 5) × k + t + u + v1 + v2 + w1 + w2 ≦ 40, more preferably satisfies 10 ≦ (m + n + p1 + p2 + q1 + q2 + r + s + 5) × k + t + u + v1 + v2 + w1 + w2 ≦ 30, more preferably satisfies 10 ≦ (m + n + p1 + p2 + q1 + q2 + r + s + 5) × k + t + u + v1 + v2 + w1 + w2 ≦ 20.
　Usually, since the higher the cost of synthesis is the number of amino acid residues the greater the increases from the economic point of view, it is preferred that the total number of amino acid residues is small.
[0194]
"X m -X 1 -X 2 -X 3 -X n "
　Further, the above formula (I), (IA), (IB), or amino acid subsequence in (IC) X m -X 1 -X 2 -X 3 -X n is the amino acid sequence represented by the following formula (1) (SEQ ID NO: 1) or the following formula with the amino acid sequence represented by (2) (SEQ ID NO: 2), preferably 70% or more, more preferably at least 75%, more preferably at least 85%, more preferably has a sequence homology of 90% or more.
　-Y-A H-L 1 -G-E-L 2 -V-W · · ·
　(1) A-Y-H-R-G-E-L 2 -V-W · · · (2)
[0195]
　In the formula (1) and (2), A represents an L- alanine residue or D- alanine residue; Y represents L- tyrosine residue or D- tyrosine residue; H is L- histidine residues or D- histidine residues represents; L 1 represents a L- leucine residue or D- leucine residue; R represents an L- arginine residue or D- arginine residue; G is represents glycine residue; E represents an L- glutamic acid residue or D- glutamic acid residue; L 2 represents a L- leucine residue; V represents an L- valine residue; and, W represents a L- tryptophan residues.
[0196]
　Here, sequence homology of two amino acid sequences is determined as follows.
(I) performing an alignment of two amino acid sequences
　matched (matching) +1, mismatch (mismatch) -1, gaps given a score of -1, performing an alignment to alignment score is maximized.
(Ii) calculating sequence homology
　based on the alignment obtained, calculating sequence homology the following equation.
　Sequence homology [%] = (match position number / total Number of Positions) × 100 [%]
　total Number of Positions is the length of the alignment, the number of matched positions is the number of positions kinds of amino acids match.
　Here, determination of whether the type of amino acid residue matches shall be by whether the structure of the side chain of an amino acid underlying the amino acid residues (amino acid side chain) is identical. The structure of the side chain of an amino acid in the enantiomeric relationship is not the same.
(Iii) Calculation example of sequence homology
　example, consider the following amino acid sequence.
　Sequence A AYHRGELVW
　sequence B AWHGELVW
　this, when aligned under the conditions described above, as follows. Wherein the sequence A, the portion where the type of match amino acids (residues) between B, for clarity, homology string | wearing a "". In addition, "-" is the gap.
　Array A AYHRGELVW
　　　　　| | |||||
　sequence B AWHLGELVW
　score for this alignment, matching (+1) × 7 + mismatch (-1) × 1 + gap (-1) is a × 1 = 5.
　In this example, the total position count is 9, since the number of matched positions is seven, sequence homology was calculated according to the above formula is 7/9 × 100 = 77.8% .
[0197]
　In the present invention, in the above formula (I) ~ (IC), preferably k = 1.
[0198]
　When the repeating unit is one, it is possible to shorten the overall length of the cyclic peptide synthesis is facilitated. Further, it is possible to avoid the Hyusugen reaction in the cyclization to form a crosslinked in unintended locations.
[0199]
　As the cyclic peptides, particularly preferably, a cyclic peptide represented by the following formula (II).
　R N -X v0 -X 6 t0 -X e0 -X 4 r0 -X p0 -X a -A-Y-H-X 8 -G-E-L-V-W-X b -X q0 -X 5 s0 -X f0 -X 7 u0 -X w0 -R C　　· · · (II) 　formula (II), X a , X b , X, R N , and R
C have the same meanings as those in the above formula (I).
　In the formula (II), X e0 is, X in formula (I) n the same manner as, X is intended to be linked e0 pieces. X f0 , X p0 , X q0 , X v0 , and X w0 This also applies.
[0200]
　Annular portion of the cyclic peptide represented by formula (II) "X a -A-Y-H-X 8 -G-E-L-V-W-X b ", and the linear portion of the "X v0 -X 6 t0 -X e0 -X 4 r0 -X p0 - "and" X q0 -X 5 s0 -X f0 -X 7 u0 -X w0 is "bridge portions" X a "and" X b " , and the antibody binding portion is "L-V-W".
[0201]
　Wherein (II), X 4 and X 5 have the same meanings as those in the above formula (IA).
　Wherein (II), X 6 and X 7 have the same meanings as those in the above formula (IB).
[0202]
　Wherein (II), e0 and f0, respectively, 0 ≦ e0 ≦ 10, and an integer satisfying 0 ≦ f0 ≦ 10.
　e0 preferably satisfies 0 ≦ e0 ≦ 5, more preferably satisfies the 0 ≦ e0 ≦ 3, more preferably satisfy 0 ≦ e0 ≦ 2.
　f0 preferably satisfies 0 ≦ f0 ≦ 5, more preferably satisfies the 0 ≦ f0 ≦ 3, more preferably satisfy 0 ≦ f0 ≦ 2.
[0203]
　Wherein (II), p0 and q0, respectively, 0 ≦ p0 ≦ 5, and an integer satisfying 0 ≦ q0 ≦ 5.
　p0 preferably satisfies 0 ≦ p0 ≦ 3, more preferably satisfy 0 ≦ p0 ≦ 2.
　q0 preferably satisfies 0 ≦ q0 ≦ 3, more preferably satisfy 0 ≦ q0 ≦ 2.
[0204]
　In Formula (II), it is r0 and s0, respectively, 0 ≦ r0 ≦ 5, and an integer satisfying 0 ≦ s0 ≦ 5.
　r0 preferably satisfies 0 ≦ r0 ≦ 3, more preferably satisfy 0 ≦ r0 ≦ 2.
　s0 preferably satisfies 0 ≦ s0 ≦ 3, more preferably satisfy 0 ≦ s0 ≦ 2.
[0205]
　Wherein (II), t0 and u0, respectively, 0 ≦ t0 ≦ 5, and an integer satisfying 0 ≦ u0 ≦ 5.
　t0 preferably satisfies 0 ≦ t0 ≦ 3, more preferably satisfy 0 ≦ t0 ≦ 2.
　s0 preferably satisfies 0 ≦ s0 ≦ 3, more preferably satisfy 0 ≦ s0 ≦ 2.
[0206]
　Wherein (II), v0 and w0, respectively, 0 ≦ v0 ≦ 5, and is an integer satisfying 3 ≦ w0 ≦ 5.
　v0 preferably satisfies 0 ≦ v0 ≦ 3, more preferably satisfy 0 ≦ v0 ≦ 2.
　w0 preferably satisfies 0 ≦ w0 ≦ 3, more preferably satisfy 0 ≦ w0 ≦ 2.
[0207]
　Further, in the above formula (II), A represents an L- alanine residue or D- alanine residue; Y represents L- tyrosine residue or D- tyrosine residue, H is L- histidine residue or D - represents a histidine residue, L 1 represents a L- leucine residue or D- leucine residue, G represents a glycine residue, E is represents L- glutamic acid residue or D- glutamic acid residue, L 2 represents an L- leucine residue, V is represents L- valine residue, W represents a L- tryptophan residues.
[0208]
　Further, in the above-mentioned formula (II), the total number of amino acid residues in the cyclic peptide is 11 to 50 residues, preferably 11-40 residues, more preferably from 11 to 30 residues, more preferably is 11 to 20 residues.
　That is, in the formula (II), e0, f0, p0, q0, r0, s0, t0, u0, v0, and w0 satisfies 0 ≦ e0 + f0 + p0 + q0 + r0 + s0 + t0 + u0 + v0 + w0 ≦ 39, preferably satisfies 0 ≦ e0 + f0 + p0 + q0 + r0 + s0 + t0 + u0 + v0 + w0 ≦ 29, more preferably satisfies 0 ≦ e0 + f0 + p0 + q0 + r0 + s0 + t0 + u0 + v0 + w0 ≦ 19, more preferably satisfy 0 ≦ e0 + f0 + p0 + q0 + r0 + s0 + t0 + u0 + v0 + w0 ≦ 9.
[0209]
"Specific examples of the cyclic part"
　shows an example of the cyclic peptide ring section below, but the invention is not limited thereto.
[0210]
(Those crosslinked by thioether bond)
-Xaa1-Ala-Tyr-His-Leu-Gly-Glu-Leu-Val-Trp-Xaa11- · · · (i)
　formula (i), Xaa1 is L- homocysteine an amino acid residue derived from, Xaa @ 11 is an amino acid residue from the N-.epsilon.-chloroacetyl -L- lysine, and Xaa1 and Xaa @ 11, and thiol group in the side chain of L- homocysteine N-epsilon - cross-linked to form a thioether bond between the chloroacetyl group in the side chain of chloroacetyl -L- lysine (SEQ ID NO: 3).
[0211]
-Xaa1-Ala-Tyr-His- Leu-Gly-Glu-Leu-Val-Trp-Xaa11- ··· (ii)
　In the formula (ii), Xaa1 is derived from N-.epsilon.-chloroacetyl -L- lysine is an amino acid residue, Xaa @ 11 is an amino acid residue derived from L- homocysteine, and Xaa1 and Xaa @ 11, N-.epsilon. of chloroacetyl -L- lysine side chain of chloroacetyl groups and L- homocysteine forming a thioether bond between the thiol groups of the side chains are crosslinked (SEQ ID NO: 4).
[0212]
-Xaa1-Ala-Tyr-His- Leu-Gly-Glu-Leu-Val-Trp-Xaa11- ··· (iii)
　in formula (iii), Xaa1 is a amino acid residues derived from L- cysteine, Xaa @ 11 is an amino acid residue from the N-.epsilon.-chloroacetyl -L- lysine, and Xaa1 and Xaa @ 11, L-cysteine side chain of the thiol group and N-.epsilon.-chloroacetyl -L- lysine side chain of the forming a thioether bond between the chloroacetyl group are crosslinked (SEQ ID NO: 5).
[0213]
-Xaa1-Ala-Tyr-His- Leu-Gly-Glu-Leu-Val-Trp-Xaa11- ··· (iv)
　in formula (iv), Xaa1 is derived from N-.epsilon.-chloroacetyl -L- lysine is an amino acid residue, Xaa @ 11 is an amino acid residue derived from L- penicillamine, and Xaa1 and Xaa @ 11, N-.epsilon. chloroacetyl -L- chloroacetyl groups and L- cysteine side chain of the side chain of lysine are crosslinked by forming a thioether bond between the thiol group (SEQ ID NO: 6).
[0214]
(Disulfide bond by those cross-linked)
-Xaa1-Ala-Tyr-His-Leu-Gly-Glu-Leu-Val-Trp-Xaa11- · · · (v)
　Formula (v), Xaa1 and Xaa11 are both also, an amino acid residue derived from L- homocysteine, to form a disulfide bond between the thiol group of the side chain of L- homocysteine are crosslinked (SEQ ID NO: 7).
[0215]
-Xaa1-Ala-Tyr-His- Leu-Gly-Glu-Leu-Val-Trp-Xaa11- ··· (vi)
　In the formula (vi), the Xaa1 and Xaa11, either, amino acid derived from the L- penicillamine a residue, are crosslinked to form a disulfide bond between the thiol group of the side chain of L- cysteine (SEQ ID NO: 8).
[0216]
(Those crosslinked with triazole coupling)
-Xaa1-Ala-Tyr-His-Leu-Gly-Glu-Leu-Val-Trp-Xaa11- · · · (vii)
　formula (vii), Xaa1 is 2- propargyl - an amino acid residue derived from L- Homogurishin, Xaa @ 11 is an amino acid residue derived from β- azide -L- alanine, and Xaa1 and Xaa @ 11 is a propargyl group in the side chain of 2-propargyl -L- Homogurishin are crosslinked to form the triazole coupling between the azide group of the side chain of β-azido -L- alanine (SEQ ID NO: 9).
[0217]
-Xaa1-Ala-Tyr-His- Arg-Gly-Glu-Leu-Val-Trp-Xaa11- ··· (viii)
　formula (viii), the amino acid residues Xaa1 is derived from β- azide -L- alanine in it, Xaa @ 11 is an amino acid residue derived from 2-propargyl -L- Homogurishin, Xaa1 and is Xaa @ 11, beta azido group and 2-propargyl -L- side chain of Homogurishin side chain azide -L- alanine are crosslinked to form the triazole coupling between propargyl group (SEQ ID NO: 10).
[0218]
(Those crosslinked by amide bond)
-Xaa1-Ala-Tyr-His-Leu-Gly-Glu-Leu-Val-Trp-Xaa11- · · · (ix)
　formula (ix), Xaa1 the L- lysine a derived amino acid residues, Xaa @ 11 is L- glutamic acid at an amino acid residue derived form an amide bond between the carboxy group of the side chain of the amino groups and L- glutamic acid side chain of L- lysine cross-linked Te (SEQ ID NO: 11).
[0219]
-Xaa1-Ala-Tyr-His- Leu-Gly-Glu-Leu-Val-Trp-Xaa11- ··· (x)
　in formula (ix), Xaa1 is a amino acid residues derived from L- glutamic acid, Xaa @ 11 the L- lysine is an amino acid residue derived from, are crosslinked to form an amide bond between the amino group of the side chain with the carboxyl group of L- lysine side chain of L- glutamic acid (SEQ ID NO: 12 ).
[0220]
"Affinity ligand-introduced amount"
　the amount of introduction of the affinity ligand of mixed mode affinity chromatography matrix of the present invention (hereinafter, simply referred to as "affinity ligand-introduced amount".) Is not particularly limited, preferably 0.01mmol / L-gel ~ 100mmol / L -gel, more preferably 0.05mmol / L-gel ~ 50mmol / L-gel, more preferably 0.10mmol / L-gel ~ 10mmol / L-gel, more preferably 0. 10mmol / L-gel ~ 5.0mmol / L-gel, and even more preferably 0.10mmol / L-gel ~ 2.5mmol / L-gel.
[0221]
"Method for synthesizing a cyclic peptide"
　method for synthesizing the cyclic peptide is not particularly limited, for example, it can be synthesized by organic synthetic chemical peptide synthesis methods or genetic engineering methods for peptide synthesis.
　The organic synthetic chemical peptide synthesis methods, may be any one of liquid-phase synthesis, solid phase synthesis. Method for synthesizing polypeptides of the present invention, solid phase synthesis method using an automated peptide synthesizer is simple preferred.
　Genetic engineering methods for peptide synthesis are then transfected into cells, a method for synthesizing peptides. The cells, bacteria, nematode cell, an insect cell, a mammalian cell, and an animal cell or the like is used.
　For example, using a four base codon method, it can be synthesized by introducing non-natural amino acids. Also, to synthesize the linear peptide was cyclized by reacting crosslinkable functional groups of the side chains of amino acid residues introduced into the annulus can be synthesized.
　When forming a disulfide bond, for example, a side chain thiol group of long amino acid residue of the methylene chain than homocysteine residue side chain thiol group of another or side-chain thiol groups and homocysteine homocysteine residue, it is reacted under oxidizing conditions capable of forming a disulfide bond.
　When forming a thioether bond, for example, be formed with lysine residues or ornithine residues chloroacetyl the amino group of the side chain, a thioether bond by reacting a side chain thiol group of homocysteine residues it can.
　Incidentally, thioether bond formation, chloroacetyl the side chain amino group of a lysine residue (-NH-C (= O) -CH 2-Cl; number 1), or 3-chloropropionic oxidation of methylene units (-NH-C (= O) - (CH 2 ) 2 -Cl; After the chlorinated by number 2), and methylene units, thiol group for those to be reacted with the compound used in the chlorination is not limited to those exemplified here, the number of methylene units is small, i.e., increases the higher the cyclization efficiency as methylene chain is shorter, preferably.
[0222]
　When forming a triazole bond, for example, as crosslinkable functional groups, using azide and alkynyl groups. To synthesize the polypeptide chains comprising the amino acid residues introduced azide group or alkynyl group, the method incorporates the amino acids was introduced azide group, or an alkynyl group in the polypeptide chain during peptide synthesis, or, a polypeptide chain after synthesized, there is a method of introducing an azide group or alkynyl group in the side chain of the desired amino acid residues. It may be by any of the methods.
[0223]
　After synthesizing a polypeptide chain comprising an amino acid residue has been introduced an azide or alkynyl group, the Hyusugen (Huisgen) reaction, to cause the addition reaction between the alkynyl group and the azido group, bridging between amino acid residues. Hyusugen reaction azide (-N = N + = N - compounds having an atomic group) and alkynes - forming a 1,2,3-triazole from (a carbon-carbon triple bond compound) 1,3-dipolar cycloaddition reaction it is. Azide and alkynyl groups inert to many functional groups or biomolecules, reaction for producing triazole ring from both an exothermic thermodynamically favorable reactions. Since Hyusugen reaction reaction in the presence of a copper catalyst accelerates dramatically, it is preferred to use a copper catalyst.
[0224]

　cation exchange groups, antibody-binding cyclic peptides and concerted to act, by having the aggregate removing capability and specific adsorption capacity, the mixed mode affinity chromatography matrix of the present invention It believed to improve the impurity removal capability.
[0225]
　Cation exchange group used in mixed mode affinity chromatography matrix of the present invention is not particularly limited, preferably a carboxy group or a sulfoxy group, more preferably a carboxy group. While any of the carboxy group and sulfoxy group excellent in aggregate removal capability, the carboxy group is weak ionic strength than a sulfonic acid group, it is possible to suppress low nonspecific adsorption, is particularly advantageous.
[0226]
"Cation exchange group introduction amount"
　introduction amount of the cation exchange groups of the mixed mode affinity chromatography matrix of the present invention (hereinafter, simply referred to as "cation exchange group introduced amount".) Is not particularly limited , the ion exchange capacity, preferably 15mmol / L-gel ~ 60mmol / L-gel, more preferably from 15mmol / L-gel ~ 55mmol / L-gel, more preferably 15mmol / L-gel ~ 40mmol / it is an L-gel.
[0227]
　When cation exchange groups introduced amount is within this range, the affinity ligand introduction amount can be within an appropriate range, it is possible to suppress the adsorption of non-specific adsorbates.
[0228]
[Method for mixed mode affinity chromatography matrix]
　Mixed mode affinity chromatography matrix of the present invention is to coat the substrate with a hydrophilic polymer, by introducing a cation exchange group, further substrate affinity ligand and hydrophilic it can be produced by introducing at least one of sex polymer.
[0229]
　Substrate, the hydrophilic polymer and the affinity ligand are as described already "mixed mode affinity chromatography matrix."
[0230]
(1) coating a substrate with a hydrophilic polymer
　a method of coating a substrate with a hydrophilic polymer is not particularly limited as long as it can be covalently attached to a hydrophilic polymer to the substrate but, for example, the base material 2-chloromethyl-oxirane (aka: epichlorohydrin) method by reacting introducing an epoxy group, is reacted with it to the hydrophilic polymer, in a solvent in the presence of an alkali, the substrate the 2-chloromethyl-oxirane (aka: epichlorohydrin) is reacted with a crosslinking agent such as a method of reacting the reaction product obtained with a hydrophilic polymer, a halogen group such as a chloro group (chlorine atom) (halogen the atoms) introduced into the substrate using a halogenating agent, and a method for fixing the hydrophilic polymer with ether synthesis of Williamson and the like.
[0231]
　When coating a substrate with a hydrophilic polymer, the hydrophilic polymer coating amount is preferably 3mg / g-dry gel ~ 450mg / g-dry gel, more preferably 3mg / g-dry gel ~ 250mg / g- dry gel, more preferably 3mg / g-dry gel ~ 230mg / g-dry gel more preferably 10mg / g-dry gel ~ 230mg / g-dry gel, and even more preferably 20mg / g-dry gel ~ 230mg / g made to be -dry gel.
[0232]
　In this specification, the base material just prior to coating with a hydrophilic polymer referred to as "covering front substrate", a material obtained by coating a hydrophilic polymer on the coated front substrate may be referred to as "coated carrier".
[0233]
(2) introducing a cation exchange group into coated carrier
　method for introducing cation exchange groups in the coating carrier, particularly as long as it can be covalently attached to a compound having a cation exchange group coated carrier but are not limited to, for example, by reacting chloroacetic acid in coated carrier under alkaline conditions, a method of introducing a part carboxymethyl group of the hydroxy group of the coated carrier surface, introducing a formyl group into coated carrier the method for introducing a cation exchange group and cation exchange group into a formyl group by reductive amination via the amino group of the compound having an amino group such as an amino acid, and the like.
[0234]
　When introducing cation-exchange groups in the coating carrier, 15 mmol amount introduced cation exchange group in the ion exchange amount / L-gel ~ 60mmol / L-gel, preferably 15mmol / L-gel ~ 55mmol / L-gel , more preferably made to be 15mmol / L-gel ~ 40mmol / L-gel.
[0235]
　In this specification, a material obtained by introducing a cation exchange group coated carrier may be referred to as "cation exchange group introducing carrier". In particular, "carboxy group-introduced carrier" a material obtained by introducing a carboxyl group into the coating carrier, those obtained by introducing a sulfoxy group to coated carrier may be referred to as "sulfoxy group introducing carrier".
[0236]
(3) introducing the affinity ligands cation exchange group introducing carrier (immobilized) to
　a method of introducing the affinity ligands cation exchange group introducing carrier (immobilization) is a covalent bond affinity ligand cation exchange group introducing carrier is not particularly limited as long as it can be bound by, e.g., a portion of the cation exchange groups EDC (N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide; N- (3- dimethylaminopropyl) - N'- ethylcarbodiimide) / NHS (N-hydroxysuccinimide; N- hydro kiss succinimide) turned into, is reacted with an affinity ligand, a method of reproducing a cation exchange group is EDC / NHS of unreacted after the reaction, the amino group Afinite with When the ligand is to protect the cation exchange groups, by introducing a formyl group, and a method of introducing the affinity ligands formyl group by reductive amination via an amino group.
[0237]
　When introducing an affinity ligand to a cation exchange group introducing carrier, the introduction of the affinity ligand, preferably 0.01mmol / L-gel ~ 100mmol / L-gel, more preferably 0.05mmol / L-gel ~ 50mmol / L-gel, more preferably 0.10mmol / L-gel ~ 10mmol / L-gel, more preferably 0.10mmol / L-gel ~ 5.0mmol / L-gel, and even more preferably 0.10 mmol / made to be L-gel ~ 2.5mmol / L-gel.
[0238]
　Incidentally, the cation exchange group introduction amount of mixed mode affinity chromatography matrix when combined with affinity ligands to a portion of the cation exchange groups of the cation exchange group introducing carrier, "cation exchange group introducing carrier cations exchange groups introduced amount - represents an affinity ligand-introduced amount "of mixed mode affinity chromatography matrix.
[0239]
Purification Method of the biological material purified biological material by the purification process]
　biological material is purified by mixed mode affinity chromatography matrix of the present invention is not particularly limited, preferably an antibody or antibody derivative , more preferably is an immunoglobulin G or a derivative thereof.
　These are used as a raw material for antibody drugs.
[0240]
　Hereinafter, a detailed description of the purification method using a mixed mode affinity chromatography matrix of the present invention, biological material is illustrated for the case of the immunoglobulin G, the present invention is not limited thereto.
[0241]
　Mixed mode affinity chromatography matrix biological substances using (especially antibodies) purification of the large, the adsorption step, washing step, the ionic strength adjusting step, and, in addition consists of four steps of the elution step, subsequent regeneration process and / or CIP (cleaning-in-place; cleaning in place) process, process may include for reuse, such as re-equilibration step.
[0242]
　In the adsorption step, typical affinity chromatographic purification methods can be used. That is, in one example, after the pH of the protein solution containing the immunoglobulin G was adjusted to around neutral, the solution mixed mode affinity chromatography matrix of the present invention is passed through a column packed with affinity ligand specifically adsorbed on mixed mode affinity chromatography matrix immunoglobulin G through. If the antibody binding cyclic peptides and affinity ligands, their load pH, i.e. during addition the pH of the biological material is preferably 5.0 to 9.0, more preferably 5.3 to 9.0 still more preferably from 5.5 to 9.0, more preferably 6.0 to 8.5. In the purification of immunoglobulin G produced by cultured mammalian cells, in particular addition that does not require preparation of ionic strength, it can be further suppressed nonspecific adsorption in advance increased ionic strength.
[0243]
　In the washing step, a buffer condition range to which the affinity ligand will function to an appropriate amount passed, to wash the column interior. That is, the preferred range of pH of the buffer may be the same range as when the load, for example, preferably pH 5.0 ~ 9.0. Immunoglobulin G at this time is adsorbed on the mixed mode affinity chromatography matrix of the present invention. At this time, by optimizing the ionic strength and / or composition at near neutral pH, there is a case where impurities can be effectively removed. During cleaning, the condition is preferably cation exchange groups does not work, i.e., preferably the use of the cleaning liquid above a certain ionic strength as well as to the near neutral pH, the carrier for mixed mode affinity chromatography in the process and / or it can be cleaned nonspecifically impurity remaining on the column via immunoglobulin G. Ionic strength, for example, preferably at 0.2M or more, and more preferably 0.5M or more.
[0244]
　The ionic strength adjustment step, the ionic strength in the vicinity of neutral replace column lower buffer comprises the expression of ionic strength-dependent elution functions by cation exchange group at elution.
[0245]
　The elution step, an acidic pH, the combination of ionic strength, during elution from the affinity ligand is functional cation exchange separation mode and concerted action of both ligands, low ionic strength elution high fractions of monomer content it can be recovered in fractions. The pH of the eluate can be applied elution pH of the immunoglobulin G from the affinity ligand. The pH, since it is determined mainly separation conditions determined by the type of mixed mode affinity chromatography matrix and immunoglobulin G, does not require special conditions set.
[0246]
　Elution time pH is preferably set to 2.0-5.0. However, the purpose of avoiding the acid-modified biological materials, more preferably pH2.8 or higher, more preferably pH3.0 or higher, more preferably at pH3.2 or higher. pH is preferably 5.0 or less, more preferably 4.8 or less.
[0247]
　The alkali resistance of the cyclic peptide to be used as an affinity ligand, typically eluted at pH preferably is set 3.2 to 4.0, but is not limited thereto. Also, elution ionic strength, in addition to depending on the introduction ratio of the affinity ligand and the cation exchange group, but it depends on the load amount of immunoglobulin G per unit volume, optimized point by Gurajien Bok experiment or stepwise elution experiments It can be easily set me.
[0248]
　Antibodies eluted from mixed mode affinity chromatography matrix prepared by the present invention is also applicable in also stepwise eluted at a salt concentration Gurajien Bok elution step with ionic strength when the purpose of reducing the volume of eluate wise elution is preferred. Furthermore, for simplicity of operation, the conditions set achievable recovery and highly purified purity of antibody by one step elution is preferred.
[0249]
　Incidentally, if the aggregate a combination of ionic strength and acidic pH of the washing step is not mixed in the eluted fraction remaining in the column, it is possible to omit the ionic strength adjusting step.
[0250]
　Biological material, especially an antibody or antibody derivative that is purified by the purification method of the invention, the structure itself before and after purification, properties do not change, purified purity is high. However, if it is how much higher, because it depends on the purification before the solution or the like, it is not possible to say categorically.
[0251]
　Immunoglobulin G purified using mixed mode affinity chromatography matrix prepared according to the present invention exhibit high monomer selectivity than affinity chromatography matrix based on a single isolation mode, the eluate high monomer content.
[0252]
　Even when using the affinity chromatography matrix based on a single separation mode, by optimizing such elution pH and ionic strength, but the monomer content is possible to increase somewhat, the effect is low, the effect expression It is accompanied by a decrease in greater recovery. By using the mixed mode affinity chromatography matrix of the present invention, a highly specific affinity purification, mainly the improvement of the monomer content which can be achieved by cation exchange chromatography, a single while maintaining high recovery rate because it is effectively achievable chroma Bok operation, it is possible to reduce the load on the subsequent process, it can contribute to the improvement of the yield improvement and the monomer content of the entire process. That is, the use of the novel mixed-mode affinity chromatography matrix of the present invention, can contribute to improve productivity and purity of the manufacturing process of the antibody drugs.
Example
[0253]
　Hereinafter, more detailed description of the present invention to examples, the present invention is in no way limited thereto.
[0254]
[Measurement methods and evaluation
methods] A. Method of measuring the cation exchange group introduction amount
　were suspended carboxy pellets 1g of pure water 3 mL, poured into a disposable column having an inner diameter 15 mm, and the solvent was removed by suction filtration. It washed 4 times with hydrochloric acid 3mL of 0.2 mol / L, was repeated then washed with pure water 4 mL. After washing, by measuring the height bed (carrier moiety deposited column) and calculate the volume of carboxylated carrier. Removed carboxylated carrier, transferred to a 100mL beaker, and suspended in saline 40mL of 0.1 mol / L, using an automatic titrator "COM-1600" (Hiranuma Sangyo Co., Ltd.), water 0.1 mol / L It was titrated with aqueous sodium hydroxide. It was calculated ion-exchange capacity per carboxylated carrier 1L (meq / L-gel) from titrant volume to the end point. In addition, by converting the units to "mmol / L-gel" (1meq / L-gel = 1mmol / L-gel). Cation exchange group introduction amount was expressed by the ion exchange capacity.
[0255]
B. Method of measuring the affinity ligand-introduced amount (peptide-immobilized amount)
　peptide solution after peptide immobilization reaction, the reaction liquid and washing liquid, respectively, subjected to gel filtration chromatography (below), the peak area value of the absorbance at 280 nm, each solution It was calculated amount of peptide contained in the.
　Peptide immobilization reaction before peptide solution, than the difference between the reaction solution and the amount of peptide contained in the washing solution were calculated peptides immobilized amount.
(Gel filtration chromatography conditions)
　chromatographic system: actor A Wand 25 (AKTA avant 25) (GE Healthcare
Inc.) ( "AKTAAVANT" is a registered trademark)
　Column: peptide purification gel filtration chromatography column "Superdex Peptide 10/300 GL (GE Healthcare Inc.) "
　buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.4
　flow rate: 0.5 mL / min
[0256]
C. Evaluation of antibody adsorption capacity
(a) antibody adsorption capacity measurements
　Examples and Comparative Examples in the manufactured mixed mode affinity chromatography matrix 1mL glass column "Torikon 10/20 column (Tricorn 10/20 column)" (GE Health manufactured Care Corporation) ( "Tricorn" fills the registered trademark), chromatographic systems "actor a Wand 25 (AKTA avant 25)" (GE Healthcare Inc.) ( "AKTAAVANT" is connected to a registered trademark), an antibody adsorption capacity It was of measurement.
　The column equilibration solution (20 mM phosphate buffer, 150 mM NaCl, pH 7.4) after equilibrating with human IgG antibody (Immunoglobulin G: Immunoglobulin G) standard buffer (20 mM phosphate buffer, 150 mM NaCl, pH 7.4 the solution 15mL adjusted to 5 mg / mL was added at a flow rate of 0.42 mL / min at). Thereafter, the load after the washing liquid (20 mM phosphate buffer, 150 mM NaCl, pH 7.4) After washing by flowing 5mL at the same flow rate, elution preclean solution (20 mM phosphate buffer, 1M NaCl, pH 7.4) 5mL of the same flow rate shed. Thereafter, the eluate (100 mM citrate buffer, 500 mM NaCl, pH 3.2) was flowed 5mL at the same flow rate. Further continuously, cleaning in place (CIP; cleaning in place) fluid flow 5mL of (0.1M aqueous sodium hydroxide) at the same flow rate, then re-equilibrated solution (20 mM phosphate buffer, 150 mM NaCl, pH 7.4) It was flushed 5mL at the same flow rate. At this time, from the IgG elution peak obtained by monitoring absorbance at 280nm was calculated antibody elution amount of eluate in each fraction. The amount of antibody eluted until CIP solution from elution preclean solution was calculated as the antibody adsorption capacity.
[0257]
(B) Evaluation of antibody adsorption capacity
　evaluation of antibody adsorption capacity was carried out according to the following criteria.
　Antibody adsorption capacity is 30g / L super ... rated "A"
　antibody adsorption capacity is 3.5g / L greater than, 30g / L or less ... evaluation "B"
　antibody adsorption capacity is 3.5g / L or less ... evaluation "E"
　rated a and B represent the antibody adsorption capacity is sufficient, evaluation E represents the antibody adsorption capacity is not sufficient.
　The mixed mode affinity chromatography matrix of the present invention with a sufficient antibody adsorption capacity when used to purify antibodies can enhance the purification efficiency of the antibody, it is possible to further reduce the antibody purification costs.
[0258]
D. Evaluation method for impurity removal capability
(a) HCP calculated purification factor
　mixed mode affinity chromatography matrix 1mL a glass column prepared in Example or Comparative Example "Torikon 10/20 column (Tricorn 10/20 column)" (GE Health made Care, Inc.) ( "TRICORN" is filled in a registered trademark), chromatographic system "actor a Wand 25 (AKTA avant 25)" (GE Healthcare Co., Ltd.) ( "AKTAAVANT" is connected to a registered trademark), each fraction in the eluted HCP (host cell protein; host cell derived protein) mass and IgG; it was measured (immunoglobulin G immunoglobulin G) amount.
[0259]
(IgG amount and HCP amount of measurement)
　equilibrated liquid column (20 mM phosphate buffer, 150 mM NaCl, pH 7.4) after equilibrating in, IgG, CHO-S (Chinese Hamster Ovary S) cell-derived HCP, respectively , 1.6mg / mL, 0.32mg / mL to become as standard buffer (20 mM phosphate buffer, 150 mM NaCl, pH 7.4) and IgG / HCP mixture prepared with, obtained by the above-described C the solution volume, such as 80% of the amount of antibody antibody adsorption capacity is loaded was added at a flow rate of 0.21 mL / min. Thereafter, the load after the washing liquid (20 mM phosphate buffer, 150 mM NaCl, pH 7.4) After washing by flowing 5mL at the same flow rate, elution preclean solution (20 mM phosphate buffer, 1M NaCl, pH 7.4) with a flow rate of 0.42mL / was passed 5mL in min. Then, ionic strength adjustment liquid (20 mM phosphate buffer, pH 7.4) was flowed 5mL at the same flow rate. Thereafter, the eluate 1 (100 mM citrate buffer, pH 3.2) flowed 5mL at the same flow rate, further continuously eluate 2 (100 mM citrate buffer, 50 mM NaCl, pH 3.2), eluent 3 (100 mM citric acid buffer, 75 mM NaCl, pH 3.2), eluent 4 (100 mM citrate buffer, shed 500 mM) at the same flow rate by 5 mL. Further successively, flowed 5mL CIP solution (0.1M sodium hydroxide solution) at the same flow rate, then re-equilibrated solution (20 mM phosphate buffer, 150 mM NaCl, pH 7.4) was flowed 5mL at the same flow rate.
　Than IgG elution peak obtained by monitoring absorbance at 280nm was calculated IgG elution amount of eluate in each fraction.
　The eluate of each fraction was collected, 1M Tris-HCl solution was a pH of the eluate 5-6 using host cell-derived protein detection kit "CHO ​​HCP 3rd Generation ELISA kit" (Cygnus Technologies Inc. ) by ELISA method was used to calculate the HCP amount of eluate in each fraction.
[0260]
(HCP calculated purification factor)
　using IgG amount and HCP the calculated amount of eluate, as HCP amount per amount of IgG in the eluate HCP contamination amount in the eluate, was calculated from the following equation.
　HCP mixed amount of eluate 1-3 (ppm) = eluate 1-3 total HCP amount / total amount of IgG eluate 1-3
　HCP contamination of the eluate (ppm) and the load was IgG / HCP mixture HCP purification factor from HCP mixture amount (ppm) of was calculated by the following equation.
　HCP mixed amount of HCP purification factor = load the IgG / HCP mixture (ppm) / HCP mixed amount of eluate 1 ~ 3 (ppm)
[0261]
(B) Evaluation of the impurity removal capacity
　assessment of the impurity removing ability was carried out according to the following criteria.
　HCP purification factor of 5000 super ... rated "A"
　HCP purification factor of 1500, more than 5000 or less ... evaluation "B"
　HCP purification factor of 1500 less than ... rated "E"
　rated A and B impurities removal capability indicates that it is sufficient, evaluation E represents the impurity removal ability is not sufficient.
　With mixed mode affinity chromatography matrix of the present invention with sufficient impurity removing ability to purify antibodies, it is possible to increase the purification purity of the antibody, it is possible to further improve the purification purity of the antibody.
[0262]
E. Evaluation method of chemical resistance
calculation of (a) bonds change rate
　Filling the mixed mode affinity chromatography matrix 1mL prepared in Examples and Comparative Examples glass column "Torikon 10/20 column (Tricorn 10/20 column)" (GE Healthcare Inc.) ( "Tricorn" is a registered trademark) to and, chromatographic system "actor a Wand 25 (AKTA avant 25)" (GE Healthcare Co., Ltd.) ( "AKTAAVANT" is a registered trademark) is connected to, measurements were made of the antibody binding capacity. The column equilibration solution (20 mM phosphate buffer, 150 mM NaCl, pH 7.4) after equilibrating at was adjusted human IgG antibody standard buffer (20 mM phosphate buffer, 150 mM NaCl, pH 7.4) at 5 mg / mL the solution 15mL was added at a flow rate of 0.21 mL / min. Thereafter, the load after the washing liquid (20 mM phosphate buffer, 150 mM NaCl, pH 7.4) After washing by flowing 5mL at the same flow rate, elution preclean solution (20 mM phosphate buffer, 1M NaCl, pH 7.4) 5mL of the same flow rate shed. Thereafter, the eluate (100 mM citrate buffer, pH 3.2) was flowed 5mL at the same flow rate. Further continuously, CIP (cleaning in place; cleaning in place) fluid flowing 5mL of (0.1M sodium hydroxide) at the same flow rate, then re-equilibrated solution (20 mM phosphate buffer, 150 mM NaCl, pH 7.4) to It was flowed 5mL at the same flow rate. At this time, it was IgG obtained by monitoring absorbance at 280nm; than (Immunoglobulin G Immunoglobulin G) elution peak, the amount of antibodies to the carrier is bound to 10% of the antibody stock solution leaks from the carrier as an antibody binding capacity It was measured. Then, after standing meets 6 hours at 25 ° C. The mixed mode affinity chromatography matrix to 0.2 M NaOH aqueous solution, the same measurement of antibody binding capacity of the carrier, the amount bound from the amount of bound antibody before and after the alkali treatment to calculate the rate of change.
[0263]
(B) Evaluation of chemical resistance
　evaluation of chemical resistance was carried out according to the following criteria.
　Binding amount change rate is 75% ultra ··············· AAA
　bond content change rate is greater than 65%, 75% or less ········· AA
　binding rate of change There than 40%, 65% or less ········· A
　bonding amount change rate is more than 15%, 40% or less ········· B
　binding rate of change is 15% or less ... ············ E
　evaluation AAA, AA, a and B represents that chemical resistance is sufficient, evaluation E represents the chemical resistance is not sufficient.
　With mixed mode affinity chromatography matrix of the present invention with sufficient chemical resistance to purify antibodies, after washing also can repeatedly bind specifically to an antibody, a long period of time, it is possible to purify antibodies , it is possible to further reduce the refining costs antibody.
[0264]
F. Evaluation of contamination antigenic ligand
　Evaluation contaminating antigenic ligand was carried out according to the following criteria.
　Introduced affinity ligand is low antigenicity (molecular weight less than 4,000) ... rated "A"
　introduced affinity ligand is low antigenicity (molecular weight less than 5,000) ... rated "B"
　introduced affinity ligand is an antigen sex (molecular weight of 5,000 or higher) ... rated "E"
　rated a and B indicates that the antigenic ligand purified antibody does not substantially contaminated evaluation E antigenic ligand is mixed in purified antibody fear indicating that there is.
　When administering an antibody that antigenic ligand is mixed in a living body, it may induce an unintended immune responses, there is a risk of causing undesirable side effects.
[0265]
[Example
1] 1. Manufacture of mixed mode affinity chromatography carrier
(1) wet preparation of gel
　cross-linked agarose-based substrate "Sepharose 4 Fast Flow (Sepharose 4 Fast Flow)" (GE Healthcare Co., Ltd.) ( "SEPHAROSE" is a registered trademark) and on a glass filter, washed by repeated suspension and filtration with pure water to obtain a substrate slurry. From this base slurry, pure water was removed by suction filtration to obtain a wet gel.
[0266]
(2) Preparation of coating before the substrate - the introduction of an epoxy group
　resulting wet gel 50 g, of pure water 76mL and 2-chloromethyl-oxirane 25 g, placed in a three-necked flask 300mL volume, in the 45 ° C. bath, the flask contents and stirring was started of things. Temperature in the flask was stirred until 45 ° C.. In the 45 ° C. bath, while maintaining the temperature in the flask at about 45 ° C., it was added dropwise to the flask over a period 50% (w / w) 2 hours aqueous sodium 20.4g hydroxide. It was added dropwise the whole amount of the aqueous sodium hydroxide solution, and further reacted for 1 hour, on a glass filter, washed by repeated suspension and filtration with pure water to obtain wet gel before coating substrates.
[0267]
(3) Preparation of the carrier - coating with a hydrophilic polymer
　Dextran 40 (weight average molecular weight of about 40,000) 31 g and pure water 72 mL, placed in a three-necked flask 300mL volume, at room temperature, and stirring was started the flask contents. Dextran 40 was stirred until complete dissolution. After dissolution, supplying dampening gel 42g of coating before the substrate in the three-necked flask, at room temperature and further stirred. From getting mixed solution in the three-necked flask is uniform, was added 50% (w / w) aqueous sodium hydroxide 2.0 g. After addition of aqueous sodium hydroxide solution, and reacted for 16 hours at room temperature, on a glass filter, washed by repeated suspension and filtration with pure water to obtain wet carrier gel.
[0268]
(4) Preparation of carboxylated carriers - introduction of cation exchange groups
　obtained carrier wet gel 15 g, sodium chloroacetate 5.1g and pure water 31 mL, placed in a three-necked flask 100mL volume, in the 50 ° C. bath , and stirring was started of the contents of the flask. Sodium chloroacetate was stirred until complete dissolution. After dissolution, in the 50 ° C. bath, while maintaining the flask temperature at about 50 ° C., it was added dropwise over 50% (w / w) 1 hour aqueous sodium 11g hydroxide. It was added dropwise the whole amount of the aqueous sodium hydroxide solution, and further 3 hours, on a glass filter, washed by repeated suspension and filtration with pure water to obtain wet gel carboxy pellets.
[0269]
(5) Preparation of the cyclic peptide fixed carboxylated carrier - the introduction of cyclic peptide
　the resulting carboxy pellets, 2.5 mL min as a wet volume, placed in a disposable column, by repeating the suspension and filtered through a pure water washing to obtain a carrier slurry. From this support slurry, pure water was removed by suction filtration to obtain a wet gel.
　The resulting wet gel 2.0g taken in a reaction vessel, EDC (N- (3-dimethylaminopropyl ) -N'-ethylcarbodiimide; N- (3- dimethylaminopropyl) -N '- ethylcarbodiimide) solution 2mL added, at room temperature in 10 minutes, and fall stirring. Thereafter, NHS in the reaction vessel; the (N-hydroxysuccinimide N- hydro kiss succinimide) solution 2mL added, at room temperature, 30 minutes, and fall stirring. Here, EDC solution, DMSO; are those prepared by dissolving EDC 0.2 g to (dimethyl Sulfoxide dimethylsulfoxide) 2 mL, NHS solution, which was prepared by dissolving NHS 0.2 g in DMSO 2 mL is there.
　The reaction gel solution was transferred to a disposable column, and washed with 1mM hydrochloric acid 6mL of ice-cold to give the EDC / NHS activated carboxy pellets.
[0270]
　The resulting EDC / NHS activated carboxy pellets 1.5g placed in a reaction vessel, followed by a 5 mg / mL cyclic peptide A solution 1.5mL was added to the reaction vessel, at 25 ° C., 2 h, and shaken.
　Here, the 5 mg / mL cyclic peptide A volume liquid is one in which the cyclic peptide A (SEQ ID NO: 13) represented by the following formula (A) was prepared by dissolving DMSO.
　Lys-Lys-Lys-Asp- Xaa5-Ala-Tyr-His-Leu-Gly-Glu-Leu-Val-Trp-Xaa15-Thr ··· (A)
　However, in the formula (A), Xaa5 is N-ε - an amino acid residue derived from chloroacetyl -L- lysine, Xaa15 is an amino acid residue derived from L- homocysteine, and Xaa5 and Xaa15, N-ε- chloroacetyl -L- side chain of a lysine forming a thioether bond are crosslinked with the thiol group of the side chain of the chloroacetyl groups and L- homocysteine.
[0271]
　The solvent was replaced with 1M aqueous sodium chloride solution and 0.5M ethanolamine aqueous solution was washed, to obtain a cyclic peptide fixed carboxy pellets.
[0272]
2. Measurement of carboxyl group introduced amount
　of carboxyl groups introduced amount of the resulting carboxy pellets was determined according to the "method of measuring the cation exchange group introduction amount" described above. As a result, a carboxyl group introduced amount was 30mmol / L-gel.
[0273]
3. Measurement of cyclic peptides immobilized amount
　cyclic peptide immobilization amount of the resulting cyclic peptide fixed carboxylated carrier (affinity ligand introduced amount) was measured according to the "method of measuring the affinity ligand-introduced amount (peptide immobilization amount)" described above . As a result, the cyclic peptide immobilized amount was 1.7mmol / L-gel.
[0274]
4. Evaluation of antibody adsorption capacity
　antibody adsorption capacity of the resulting cyclic peptide fixed carboxy pellets were evaluated according to the "method of evaluating an antibody adsorption capacity" described above. The evaluation results are shown in "antibody adsorption capacity" column of Table 3.
[0275]
5. Evaluation of the impurity removal capacity
　of the impurities removal capability of the resulting cyclic peptide fixed carboxy pellets were evaluated according to the "method of evaluating the impurity removal performance" described above. The evaluation results are shown in "impurity removal ability" column of Table 3.
[0276]
6. Evaluation of chemical resistance
　The resulting cyclic peptide chemical resistance of the fixed carboxy pellets were evaluated according to "Evaluation method of chemical resistance" described above. The evaluation results are shown in "chemical resistance" column in Table 3.
[0277]
7. Evaluation of contamination antigenic ligand
　antigenic ligand contamination resulting cyclic peptide fixed carboxy pellets were evaluated according to the "Evaluation Method of contaminating antigenic ligand" described above. As a result, the evaluation of contamination antigenic ligand was "A".
[0278]
Example
2 1. Manufacture of mixed mode affinity chromatography matrix
　- in the "preparation of the cyclic peptide fixed carboxy pellets cyclic introduction of peptides", the following formula in place of the cyclic peptide A cyclic peptide B represented by (B) (SEQ ID NO: 14) the except that was used to prepare a cyclic peptide fixed carboxy pellets in the same manner as in example 1.
　Lys-Lys-Lys-Asp- Xaa5-Ala-Tyr-His-Leu-Gly-Glu-Leu-Val-Trp-Xaa15-Thr ··· (B)
　However, in the formula (B), is Xaa5 and Xaa15, both, L- homo a cysteine from the amino acid residues, and Xaa5 and Xaa15, are crosslinked to form a disulfide bond between the thiol group of the side chain of L- homocysteine.
[0279]
2. Measurement of cyclic peptides immobilized amount
　was measured according to the above-described cyclic peptide immobilization amount of the resulting cyclic peptide fixed carboxylated carrier (affinity ligand introduced amount) "method of measuring the affinity ligand-introduced amount (peptide immobilization amount)". As a result, the cyclic peptide introduction amount was 1.6mmol / L-gel.
[0280]
3. Antibody adsorption capacity, evaluation of the impurity removal capacity and chemical resistance
　resulting cyclic peptide antibody adsorption capacity of the fixed carboxylated carriers, the impurity removal capacity and chemical resistance were evaluated in the same manner as in Example 1. The evaluation results are shown in "antibody adsorption capacity" column, "impurity removal performance" column and "chemical resistance" column in Table 3.
[0281]
4. Evaluation of contamination antigenic ligand
　antigenic ligand contamination resulting cyclic peptide fixed carboxy pellets were evaluated according to the "Evaluation Method of contaminating antigenic ligand" described above. As a result, the evaluation of contamination antigenic ligand was "A".
[0282]
[Example
3] 1. Manufacture of mixed mode affinity chromatography matrix
　- in the "carboxy pellets prepared introduction of the cation exchange group", except for changing sodium chloroacetate amount 6.1 g, in the same manner as in Example 1, carboxylation carrier was prepared.
[0283]
　Further, - in the "preparation of the cyclic peptide fixed carboxy pellets introducing cyclic peptide", that using a carboxylated carrier prepared as described above, and is represented by the following formula (C) in place of the cyclic peptide A except using the cyclic peptide C (SEQ ID NO: 15), was prepared cyclic peptide fixed carboxy pellets in the same manner as in example 1.
　Lys-Lys-Lys-Asp- Xaa5-Ala-Tyr-His-Leu-Gly-Glu-Leu-Val-Trp-Xaa15-Thr ··· (C)
　However, in the formula (C), Xaa5 is N-ε - an amino acid residue derived from chloroacetyl -L- lysine, Xaa15 is an amino acid residue derived from L- penicillamine, and Xaa5 and Xaa15, the side chains of N-.epsilon.-chloroacetyl -L- lysine forming a thioether bond are crosslinked with the chloroacetyl group and L- penicillamine thiol group of the side chain of.
[0284]
2. Measurement of carboxyl group introduced amount
　of carboxyl groups introduced amount of the resulting carboxy pellets was determined according to the "method of measuring the cation exchange group introduction amount" described above. As a result, a carboxyl group introduced amount was 36mmol / L-gel.
[0285]
3. Measurement of cyclic peptides immobilized amount
　cyclic peptide immobilization amount of the resulting cyclic peptide fixed carboxylated carrier (affinity ligand introduced amount) was measured according to the "method of measuring the affinity ligand-introduced amount (peptide immobilization amount)" described above . As a result, the cyclic peptide immobilized amount was 2.0mmol / L-gel.
[0286]
4. Antibody adsorption capacity, evaluation of the impurity removal capacity and chemical resistance
　resulting cyclic peptide antibody adsorption capacity of the fixed carboxylated carriers, the impurity removal capacity and chemical resistance were evaluated in the same manner as in Example 1. The evaluation results are shown in "antibody adsorption capacity" column, "impurity removal performance" column and "chemical resistance" column in Table 3.
[0287]
5. Evaluation of contamination antigenic ligand
　antigenic ligand contamination resulting cyclic peptide fixed carboxy pellets were evaluated according to the "Evaluation Method of contaminating antigenic ligand" described above. As a result, the evaluation of contamination antigenic ligand was "A".
[0288]
[Example
4] 1. Manufacture of mixed mode affinity chromatography matrix
　- in the "preparation of the cyclic peptide fixed carboxy pellets cyclic introduction of peptides", the point of using the carboxy pellets prepared as described above, and the following formula in place of the cyclic peptide A except using the cyclic peptide D (SEQ ID NO: 16) represented by (D), were prepared cyclic peptide fixed carboxy pellets in the same manner as in example 3.
　Lys-Lys-Lys-Asp- Xaa5-Ala-Tyr-His-Leu-Gly-Glu-Leu-Val-Trp-Xaa15-Thr ··· (D)
　However, in the formula (D), Xaa5 2-propargyl an amino acid residue derived from -L- Homogurishin, Xaa15 is, beta is an amino acid residue derived from azide -L- alanine, and Xaa5 and Xaa15, 2-propargyl -L- propargyl group in the side chain of Homogurishin to form a triazole bond cross-linked with the azide group of the side chain of β-azido -L- alanine and.
[0289]
2. Measurement of cyclic peptides immobilized amount
　cyclic peptide immobilization amount of the resulting cyclic peptide fixed carboxylated carrier (affinity ligand introduced amount) was measured according to the "method of measuring the affinity ligand-introduced amount (peptide immobilization amount)" described above . As a result, the cyclic peptide immobilized amount was 1.7mmol / L-gel.
[0290]
3. Antibody adsorption capacity, evaluation of the impurity removal capacity and chemical resistance
　resulting cyclic peptide antibody adsorption capacity of the fixed carboxylated carriers, the impurity removal capacity and chemical resistance were evaluated in the same manner as in Example 1. The evaluation results are shown in "antibody adsorption capacity" column, "impurity removal performance" column and "chemical resistance" column in Table 3.
[0291]
4. Evaluation of contamination antigenic ligand
　antigenic ligand contamination resulting cyclic peptide fixed carboxy pellets were evaluated according to the "Evaluation Method of contaminating antigenic ligand" described above. As a result, the evaluation of contamination antigenic ligand was "A".
[0292]
[Example
5] 1. Manufacture of mixed mode affinity chromatography matrix
　- in the "preparation of the cyclic peptide fixed carboxy pellets cyclic introduction of peptides", the point of using the carboxy pellets prepared as described above, and the following formula in place of the cyclic peptide A except using the cyclic peptide E (SEQ ID NO: 17) represented by (E), were prepared cyclic peptide fixed carboxy pellets in the same manner as in example 3.
　Lys-Lys-Lys-Asp- Xaa5-Ala-Tyr-His-Leu-Gly-Glu-Leu-Val-Trp-Xaa15-Thr ··· (E)
　However, in the formula (E), Xaa5 is N-ε - an amino acid residue derived from chloroacetyl -L- lysine, Xaa15 is an amino acid residue derived from cysteine, and Xaa5 and Xaa15, chloro side chain of N-.epsilon.-chloroacetyl -L- lysine forming a thioether bond are crosslinked with the thiol group of a side

WE CLAIMS

[Requested item 1]　A substrate, a hydrophilic polymer, and an antibody-binding cyclic peptides, mixed mode affinity chromatography matrix having a cation exchange group, a.
[Requested item 2]　Said antibody binding cyclic peptides has an annular portion which is cyclized by intramolecular crosslinking between the side chains, mixed mode affinity chromatography matrix of claim 1.
[Requested item 3]　The intramolecular bridge is a disulfide bond, a thioether bond, triazole bond, and is selected from the group consisting of an amide bond include at least one, mixed mode affinity chromatography matrix of claim 2.
[Requested item 4]
　The intramolecular bridge is a disulfide bond, thioether bond, and is selected from the group consisting of triazole coupling comprises at least one, mixed mode affinity chromatography matrix of claim 2 or 3.
[Requested item 5]　The disulfide bond, and a side chain thiol group of the first amino acid residue derived from the amino acid having a thiol group in a side chain other than L- cysteine and D- cysteine, in a side chain other than L- cysteine and D- cysteine the second is a disulfide bond formed between the side chain thiol group of the amino acid residues, derived from the amino acid having a thiol group
　said thioether bond, a thiol group on the side chain other than L- cysteine and D- cysteine thioether bond formed between the side chain thiol group of the first amino acid residue derived from the amino acid, the side chain haloacetyl group of the second amino acid residue from an amino acid having a haloacetyl group on the side chain having a in it, mixed mode affinity chromatography matrix of claim 4.
[Requested item 6]
　The disulfide bond is cysteine, and a side chain thiol group of the first amino acid residue derived from the amino acid having a thiol group in the side chain selected from the group consisting of homocysteine and penicillamine, consisting cysteine, homocysteine and penicillamine a disulfide bond formed between the side chain thiol group of a second amino acid residue derived from the amino acid having a thiol group in the side chain selected from the group,
　the thioether bond, cysteine, homocysteine and penicillamine and a side chain thiol group of the first amino acid residue derived from the amino acid having a thiol group in the side chain selected from the group consisting of the side of the second amino acid residues from an amino acid having a haloacetyl group on the side chain thioether bond formed between the chain haloacetyl groups, in claim 4 Mixed mode affinity chromatography carrier of the mounting.
[Requested item 7]
　The disulfide bond is selected and a side chain thiol group of the first amino acid residue derived from the amino acid having a thiol group in the side chain selected from the group consisting of homocysteine and penicillamine, from the group consisting of homocysteine and penicillamine that the side chain is a disulfide bond formed between the side chain thiol group of a second amino acid residue derived from the amino acid having a thiol group,
　the thioether bond is selected from the group consisting of homocysteine and penicillamine between the side-chain thiol group of the first amino acid residue derived from the amino acid having a thiol group in a side chain that includes a side-chain haloacetyl group of the second amino acid residue from an amino acid having a haloacetyl group on the side chain is in the formed thioether bond, mix mode according to any one of claims 4-6 Affinity chromatography carrier.
[Requested item 8]
　The intramolecular bridge is selected and a side chain thiol group of the first amino acid residue derived from the amino acid having a thiol group in the side chain selected from the group consisting of homocysteine ​​and penicillamine, from the group consisting of homocysteine ​​and penicillamine disulfide bond formed between the side chain thiol group of a second amino acid residue derived from the amino acid having a thiol group in the side chains, or, in the side chain selected from the group consisting of homocysteine ​​and penicillamine and a side chain thiol group of the first amino acid residue derived from the amino acid having a thiol group, which is formed between the side chain haloacetyl group of the second amino acid residue from an amino acid having a haloacetyl group on the side chain a thioether bond, mixed-mode affinity chromatography according to any one of claims 2-4 Over for the carrier.
[Requested item 9]
　The disulfide bond, be a disulfide bond formed between a side-chain thiol group of the first amino acid residue derived from the homocysteine side chain thiol group of a second amino acid residue derived from the homocysteine ,
　is formed between the side chain haloacetyl group of the second amino acid residue derived from the amino acid having a side chain thiol group and a side chain haloacetyl group of the first amino acid residue of the thioether linkage is derived from homocysteine and a thioether bond, mixed mode affinity chromatography matrix of claim 8.
[Requested item 10]
　It said amino acid residues of the annular portion is 8 to 14 residues, mixed mode affinity chromatography carrier according to any one of claims 2-9.
[Requested item 11]
　It said antibody binding cyclic peptide having straight portions, mixed mode affinity chromatography matrix of any one of claims 1 to 10.
[Requested item 12]
　The linear portion comprises an amino acid residue having at least one selected from the group consisting of hydroxy and carboxy groups in side chains, mixed mode affinity chromatography matrix of claim 11.
[Requested item 13]
　Said substrate, polysaccharides, acrylate-based polymer is at least one selected from the group consisting of methacrylate polymer and styrene polymer, mixed-mode according to any one of claims 1 to 12 affinity chromatography carrier.
[Requested item 14]
　Said substrate, polysaccharides, is at least one selected from the group consisting of acrylate polymers and methacrylate polymer, mixed mode affinity chromatography matrix of any one of claims 1 to 13 .
[Requested item 15]
　Wherein the substrate is at least one selected from the group consisting of agarose and cellulose, mixed mode affinity chromatography carrier according to any one of claims 1 to 14.
[Requested item 16]
　Wherein the hydrophilic polymer is a hydrophilic polysaccharide, mixed mode affinity chromatography carrier according to any one of claims 1 to 15.
[Requested item 17]
　Wherein the hydrophilic polysaccharide is dextran, of at least one selected carboxymethyl dextran, pullulan, hydroxyethyl cellulose, and from the group consisting of carboxymethyl cellulose, mixed mode affinity chromatography matrix of claim 16.
[Requested item 18]
　Wherein the hydrophilic polysaccharide is dextran, of at least one selected from the group consisting of carboxymethyl dextran and pullulan, mixed mode affinity chromatography matrix of claim 16 or 17.
[Requested item 19]
　The molecular weight of the antibody-binding cyclic peptides is less than 5000, mixed mode affinity chromatography carrier according to any one of claims 1 to 18.
[Requested item 20]
　The cation exchange group, a carboxy group or a sulfoxy group, mixed mode affinity chromatography carrier according to any one of claims 1 to 19.
[Requested item 21]　The cation exchange group is a carboxy group, mixed mode affinity chromatography carrier according to any one of claims 1 to 20.