JP5396933B2 - Liquid chromatography packing and biopolymer separation and purification method - Google Patents

Description

  The present invention relates to a filler suitable for separating and purifying or collecting and recovering ionic substances dissolved in an aqueous solution, in particular, biopolymers such as proteins and peptides, by adsorption / desorption and / or liquid chromatography. The present invention relates to a method for separating and purifying or collecting and recovering biopolymers.

  More specifically, under acidic aqueous solution conditions, the solute polymer is adsorbed using the interaction between the hydrophobic group of the filler and the surface hydrophobic group of a biopolymer such as protein or peptide, and the pH of the eluent is adjusted. Packing for liquid chromatography to change the packing material to hydrophilic by changing to neutral or weak alkalinity, and to recover by separating and purifying the adsorbed biopolymers such as proteins and peptides The present invention relates to an agent and a method for separating and purifying or collecting and collecting a biopolymer using the same.

  In many cases, the packing material for liquid chromatography that separates and purifies biopolymers such as proteins and peptides by adsorbing and desorbing them is based on a hydrophilic packing material that does not adsorb proteins and peptides in aqueous solution. It has a structure in which functional groups that interact with proteins are immobilized. As such a hydrophilic base material, for example, porous particles having pores having a size capable of penetrating a biopolymer and having a hydrophilic surface are used, and a functional group must be introduced. Each solute elutes in order of increasing molecular size.

  It is a nonionic polar group such as an alcoholic hydroxyl group or an amide group that imparts hydrophilicity to the substrate. In particular, the hydroxyl group is used as a reactive base point for immobilizing a specific functional group.

  A case where a hydrophobic group is introduced as a functional group is a filler for hydrophobic chromatography or a filler for reverse phase chromatography.

  In the case of eluting proteins or the like, the packing material for reverse phase chromatography requires an eluate containing an organic solvent and often denatures the protein, and is used for analysis but is not often used as a purification means.

  On the other hand, hydrophobic chromatography is a separation and purification method that allows proteins and the like to be eluted by adsorbing proteins and the like in a high-concentration salt solution and reducing the salt concentration without adding an organic solvent. Hydrophobic chromatography is widely used after ion exchange as a means for separating and purifying a target substance while retaining the complex physiological activity of biopolymers, and is often used in combination with ion exchange. The main reasons for this are, for example, that the protein can be separated with a stable solvent (composition, pH) and temperature, and that it is relatively stable with respect to regenerative washing, sterilization, endotoxin removal, etc. The life is long, and furthermore, biopolymers are those that adsorb and desorb based on hydrophobic interactions, and the separation mechanism is different from the widely used ion exchange method.

  Examples of the functional group used in the filler for hydrophobic chromatography include nonionic groups such as a butyl group, a hexyl group, an octyl group, and a phenyl group.

  In recent years, as a method for efficiently producing specific proteins (including peptides), a method for cultivating genetically modified cells using genetic recombination technology to produce proteins inside or outside the cells has been developed and used. Has been. The protein concentration in the culture supernatant and the homogenate is about several grams per liter even when it is large, and is usually lower. Accordingly, when producing a large amount of protein, it is required to rapidly process a culture solution of several hundred to several thousand times and collect a crude product containing the target protein. Therefore, regardless of the type of chromatography, increasing the load capacity of the target substance per unit volume helps to reduce costs by shortening the operation time and making the equipment compact, and includes proteins (including peptides). It is an important factor for purification technology.

  Here, in the hydrophobic chromatography, in order to adsorb proteins, it is necessary to contain ammonium sulfate, sodium sulfate, or the like at a high concentration (generally 1.5 mol / liter or more) in the adsorption buffer solution. Therefore, in order to process a large amount of culture supernatant and homogenate solution, a large amount of salt is required, and the disposal thereof becomes a problem, which increases the purification cost.

  On the other hand, the ion exchange method is suitable for adsorbing proteins from a solution having a low salt concentration, but the cell culture solution generally contains a salt of about physiological saline (about 0.15 mol / liter) or more. In many cases, since the salt concentration is too high to be collected by an ion exchanger, it is necessary to reduce the coexisting salt concentration by desalting or dilution. Therefore, it is necessary to preprocess with a dialysis or desalting column after one step, or to increase the volume of the culture solution by dilution, and in any case, it is suitable for quickly collecting the target protein from the cell culture solution. Absent.

  In recent years, V.M. Kasche et al. (See Non-Patent Document 1), S.K. C. Burton et al. (See Patent Literature 1, Patent Literature 2, and Non-Patent Literature 2), W.C. Adopted by Schwarz et al. (See Non-Patent Document 3) under neutral to weakly basic pH conditions using a filler in which a ligand having both a weak anion exchange group and a hydrophobic group is immobilized on the hydrophilic substrate. Adsorbs protein without being affected by the salt concentration of the solution, makes the eluate pH weakly acidic, ionizes the anion exchange group of the ligand, changes the filler to hydrophilic, and recovers the adsorbed protein by elution It was reported that it can be done. However, these fillers do not adsorb proteins having an isoelectric point of pH = 8.5 or more, or even when adsorbed, the amount of adsorption is limited to several milligrams / milliliter or less.

  A. According to Groenberg et al. (See Patent Document 3), using a filler in which a ligand having both a weak cation exchange group and a heteroaromatic ring composed of carbon, sulfur and oxygen is immobilized on a hydrophilic filler, a weak acidic pH condition Have been reported to selectively adsorb and elute under weakly basic pH conditions.

  However, in the case of a ligand-immobilized filler having both a weak anion exchange group and a hydrophobic group, the protein is adsorbed under neutral to weakly basic conditions. Since most anionic groups are ionized, the surface hydrophilicity is high, the hydrophobic adsorption force is weakened, and the adsorption capacity is small. On the other hand, in the case of a ligand-immobilized filler in which a ligand having both a weak cation exchange group and a heteroaromatic ring described in Patent Document 3 is immobilized on a hydrophilic filler, the specificity to a specific protein (antibody) is strongly adsorbed. However, it is difficult to apply to proteins in general and its application range is narrow.

  Therefore, the adsorption is not affected by the isoelectric point of the protein or the salt concentration of the adsorption solution, has a wide range of application to all proteins, and is eluted by controlling pH conditions during elution (for example, protein, Development of a filler capable of controlling peptides and the like and an adsorption / desorption method is desired.

  That is, with conventional fillers, the amount of adsorption changes depending on the physical properties of the protein (for example, isoelectric point) and the salt concentration of the solvent in which biopolymers such as proteins are dissolved, and from a large amount of diluted cell culture solution. It was difficult to concentrate and recover biopolymers such as targeted proteins widely.

  For example, in the case of a filler for ion exchange, although it is limited to a solution having a low ionic strength, a protein adsorption capacity of about 100 grams / liter (wet capacity) for a granular protein having a molecular weight of about 10,000 to 70,000. Is obtained. Moreover, in the ion exchange filler, the protein adsorption capacity can be further increased by immobilizing a hydrophilic graft polymer on the surface of the filler and introducing an ion exchange group onto the graft polymer (for example, patent document). 4).

  On the other hand, in hydrophobic chromatography and reverse-phase partition chromatography, a short-chain spacer (with a carbon-carbon bond and having about 3 to 10 carbon atoms in the carbon chain) is used without a spacer or in ordinary affinity chromatography. In the case of a protein having a molecular weight of 10,000 to several tens of thousands even when a base material having an appropriate pore size and porosity is used, the adsorption capacity is The maximum is 65 milligrams / milliliter or less.

  Also, in hydrophobic chromatography and reverse phase partition chromatography, hydrophobic groups can be introduced onto the graft polymer as in the above-described ion exchange filler, but proteins (including peptides) are retained. Under solvent conditions, the graft polymer introduced with a hydrophobic group aggregates and contracts, so that the protein adsorption capacity hardly increases but may decrease. Therefore, at present, the adsorption capacity is not increased by adsorption / desorption or chromatography based on hydrophobic bonds using this type of filler (see, for example, Patent Document 5).

US Pat. No. 5,652,348 US Pat. No. 5,945,520 International Publication No. 2005/082483 Pamphlet Japanese Patent Application Laid-Open No. 2008-232,764 Japanese Patent No. 3,059,443

Journal of Chromatography, 510 (1990) p. 149-154 Journal of Chromatography A, 814 (1998) p. 71-81 Journal of Chromatography A, 908, 1-2 (2001) p. 251-263

  The present invention has been made in view of the above background art, and its purpose is not affected by the isoelectric point of protein or the salt concentration of a solvent in which a biopolymer such as protein or peptide is dissolved, and the change in pH. To provide a novel liquid chromatography packing material that can be separated, purified, collected and recovered by adsorption and desorption of biopolymers such as proteins, and by using the packing material, a target can be obtained from a dilute and large amount of cell culture solution. It is to provide a method for concentrating and recovering biopolymers such as proteins.

  As a result of intensive studies in order to solve the above problems, the present inventors have obtained a specific ligand directly immobilized on the substrate and a specific ligand immobilized on the substrate via a specific spacer. The present inventors have found a liquid chromatography packing material and a method for separating and purifying and collecting and recovering proteins using the same, and have completed the present invention.

  That is, the present invention is a liquid chromatography packing material in which hydrophobic amino acids are immobilized as shown below, and a biopolymer separation / purification or collection / collection method using the same.

[1] A packing material for liquid chromatography having a ligand directly immobilized on a substrate and a ligand immobilized on the substrate via a spacer,
(1) The substrate is a hydrophilic substrate having an alcoholic hydroxyl group on the substrate surface,
(2) The spacer is a synthetic polymer or polysaccharide having an alcoholic hydroxyl group,
(3) The ligand is represented by the following formula (1)

（上記式中、Ｒは芳香族基又は炭素数５〜７個の非イオン性脂肪族基を表す。）
で示されるα−アミノ酸、及びアミノメチル安息香酸からなる群より選ばれる１種又は２種以上であり、
（４）基材に直接固定化されたリガンドが、上記式（１）で示されるα−アミノ酸又はアミノメチル安息香酸に含まれるアミノ基を介して、アミド結合又はウレタン結合で、上記基材に固定化されており、
（５）スペーサーを介して基材に固定化されたリガンドが、上記式（１）で示されるα−アミノ酸又はアミノメチル安息香酸に含まれるアミノ基を介して、アミド結合又はウレタン結合で、上記スペーサーに固定化されており、かつ
（６）基材に固定化されたリガンドの量が、液体クロマトグラフィー用充填剤１リットル（湿潤容量）当たり３０ミリモル以上である、液体クロマトグラフィー用充填剤。

(In the above formula, R represents an aromatic group or a nonionic aliphatic group having 5 to 7 carbon atoms.)
1 type or 2 types or more selected from the group consisting of an α-amino acid represented by and aminomethylbenzoic acid,
(4) The ligand directly immobilized on the base material is bonded to the base material by an amide bond or a urethane bond via the amino group contained in the α-amino acid or aminomethylbenzoic acid represented by the above formula (1). Is fixed,
(5) The ligand immobilized on the substrate via the spacer is an amide bond or urethane bond via the amino group contained in the α-amino acid or aminomethylbenzoic acid represented by the above formula (1), And (6) a packing material for liquid chromatography, wherein the amount of the ligand fixed to the substrate is 30 mmol or more per liter (wet volume) of the packing material for liquid chromatography.

  [2] The packing material for liquid chromatography according to the above [1], wherein the α-amino acid is selected from the group consisting of phenylalanine, tryptophan, leucine, norleucine and α-aminooctanoic acid.

[3] The above [1] or [2], wherein the substrate is a chromatography carrier selected from the group consisting of a natural polymer carrier, a synthetic polymer carrier, and an inorganic carrier. [4] The packing material for liquid chromatography according to any one of [1] to [3], wherein the base material is a porous particle, and its exclusion limit molecular weight is 100,000 or more in terms of pullulan. The packing material for liquid chromatography as described in any one of.

  [5] The liquid chromatography according to any one of [1] to [4], wherein the polysaccharide is a polysaccharide having no anion exchange group and a weight average molecular weight of 10,000 or more, or a derivative thereof. filler.

  [6] After the alcoholic hydroxyl group in the substrate and the alcoholic hydroxyl group in the spacer are activated with 1,1-carbonylbis-1H-imidazole in an organic solvent, The liquid according to any one of [1] to [5], wherein the liquid is reacted with an amino group and a ligand is directly introduced into the base material through a urethane bond and is introduced into the base material through the spacer. A method for producing a chromatographic filler.

  [7] After introducing a carboxyl group into the substrate and the spacer, using carbodiimide as a catalyst, reacting it with an amino group in the ligand, and introducing the ligand directly into the substrate by an amide bond, The method for producing a packing material for liquid chromatography according to any one of the above [1] to [5], wherein the method is introduced into a base material via a substrate.

  [8] Using the liquid chromatography filler according to any one of [1] to [5] above, a biopolymer is adsorbed under an acidic aqueous solution condition of pH 5 or lower, and then neutral to a weak base of pH 9 or lower. A method for separating and purifying or collecting and recovering a biopolymer by liquid chromatography, which comprises desorbing a biopolymer adsorbed under a neutral condition.

  In the present invention, the ligand refers to a chemical substance to which a biopolymer such as a target protein or peptide specifically binds.

  In the present invention, the spacer refers to a chemical binding site used for adjusting the distance between the substrate and the ligand.

  The filler for liquid chromatography of the present invention has a ligand having a hydrophobic group and a carboxyl group immobilized on a hydrophilic base material, and adsorbs biopolymers such as proteins under acidic aqueous solution conditions. -Since the adsorbed biopolymers are desorbed under weakly basic conditions, they can be eluted and collected according to the hydrophobicity and ionicity of these biopolymers.

  Moreover, since the packing material for liquid chromatography of the present invention has the above ligand immobilized on a base material via a specific spacer (a synthetic polymer or polysaccharide having an alcoholic hydroxyl group), such a spacer is used. Compared with a filler in which the ligand is not immobilized on the substrate, the adsorption capacity of biopolymers such as proteins and peptides per unit volume of the filler can be increased quickly and efficiently. Biopolymers can be separated and purified or concentrated and recovered.

  Moreover, the packing material for liquid chromatography of the present invention is not affected by the salt concentration of a solvent in which biopolymers such as proteins are dissolved, and separates, purifies or collects them by adsorption / desorption of biopolymers due to pH change. It is a separation material that can be recovered.

  Furthermore, according to the separation and purification or collection and collection method of the biopolymer by the liquid chromatography of the present invention, for example, the cell culture supernatant containing physiological saline or a salt thereof can be used in a simple step such as pH adjustment. After the treatment, the biopolymer such as protein can be brought into contact with the filler of the present invention as it is without performing a desalting operation. Biopolymers such as unstable proteins can be separated and purified or concentrated and recovered quickly in large quantities with a more compact facility.

The packing material for liquid chromatography of the present invention is a packing material for liquid chromatography having a ligand directly immobilized on a substrate and a ligand immobilized on the substrate via a spacer,
(1) The substrate is a hydrophilic substrate having an alcoholic hydroxyl group on the substrate surface,
(2) The spacer is a synthetic polymer or polysaccharide having an alcoholic hydroxyl group,
(3) The ligand is represented by the following formula (1)

（上記式中、Ｒは芳香族基又は炭素数５〜７個の非イオン性脂肪族基を表す。）
で示されるα−アミノ酸、及びアミノメチル安息香酸からなる群より選ばれる１種又は２種以上であり、
（４）基材に直接固定化されたリガンドが、上記式（１）で示されるα−アミノ酸又はアミノメチル安息香酸に含まれるアミノ基を介して、アミド結合又はウレタン結合で、上記基材に固定化されており、
（５）スペーサーを介して基材に固定化されたリガンドが、上記式（１）で示されるα−アミノ酸又はアミノメチル安息香酸に含まれるアミノ基を介して、アミド結合又はウレタン結合で、上記スペーサーに固定化されており、かつ
（６）基材に固定化されたリガンドの量が、液体クロマトグラフィー用充填剤１リットル（湿潤容量）当たり３０ミリモル以上である、液体クロマトグラフィー用充填剤である。

(In the above formula, R represents an aromatic group or a nonionic aliphatic group having 5 to 7 carbon atoms.)
1 type or 2 types or more selected from the group consisting of an α-amino acid represented by and aminomethylbenzoic acid,
(4) The ligand directly immobilized on the base material is bonded to the base material by an amide bond or a urethane bond via the amino group contained in the α-amino acid or aminomethylbenzoic acid represented by the above formula (1). Is fixed,
(5) The ligand immobilized on the substrate via the spacer is an amide bond or urethane bond via the amino group contained in the α-amino acid or aminomethylbenzoic acid represented by the above formula (1), (6) a packing material for liquid chromatography in which the amount of the ligand immobilized on the substrate is 30 mmol or more per 1 liter (wetting capacity) of the packing material for liquid chromatography; is there.

  The substrate used in the liquid chromatography filler of the present invention is a hydrophilic substrate having an alcoholic hydroxyl group on its surface, and is not particularly limited. For example, it is generally used as a carrier for chromatography. Natural polymer carriers, synthetic polymer carriers, inorganic carriers and the like.

  In the present invention, examples of the natural polymer carrier include polysaccharides such as cellulose, agarose, and dextran. Examples of the synthetic polymer carrier include hydroxyl group-containing monomers such as 2-hydroxyethyl (meth) acrylate, 2-hydroxymethyl (meth) acrylate, hydroxypropyl (meth) acrylate, and ethylene glycol di (meth) acrylate. And those prepared by mixing with a crosslinkable monomer such as divinylbenzene and polymerizing in the presence of a polymerization initiator. Examples of the inorganic carrier include silica and zeolite.

  In the present invention, examples of the form of the substrate include, but are not particularly limited to, spherical particles, non-spherical particles, membranes, monoliths (continuous bodies), and the like.

  In the present invention, among these, a carrier for liquid chromatography that can be used as a filler for molecular size exclusion chromatography of water-soluble polymers (for example, proteins, peptides, etc.), wherein an alcoholic hydroxyl group is formed on the surface thereof. A certain carrier can be preferably used.

  Specifically, (meth) acrylic acid ester monomers, monomers typified by (meth) acrylamide monomers are copolymerized with crosslinkable monomers to form particles [for example, (meth) acrylic acid ester fillers , (Meth) acrylamide fillers, etc.], vinyl acetate and various cross-linking agents (monofunctional or higher monomers) are copolymerized into particles, and vinyl acetate monomer units are hydrolyzed, agarose, dextran, cellulose, etc. Those obtained by crosslinking polysaccharides represented by (polysaccharide fillers) can be suitably used.

  More specifically, as the (meth) acrylic ester filler, copolymer particles of 2-hydroxyethyl (meth) acrylate and ethylene glycol di (meth) acrylate, glycidyl methacrylate and ethylene glycol di (meth) acrylate Examples thereof include particles obtained by ring-opening addition of a glycidyl group with water or a polyhydric alcohol.

  Examples of the (meth) acrylamide filler include copolymer particles of 2-hydroxyethyl (meth) acrylamide and N, N′-methylenedi (meth) acrylamide.

  Furthermore, examples of the polysaccharide filler include a filler obtained by crosslinking polysaccharides such as agarose, dextran, and cellulose with epihalohydrin or polymethylene dihalogen having 2 to 8 carbon atoms between the hydroxyl groups of the polysaccharide.

  In the present invention, in order to ensure a sufficient adsorption capacity as a filler, the base material used is porous particles, and the pore size of the biopolymer (for example, protein, peptide, etc.) to be separated. It is preferable that the exclusion limit molecular weight is 100,000 or more in terms of pullulan.

  In addition, considering the liquid permeability when flowing the sample solution and the eluent at a practical flow rate, the filler is required to have physical strength. In the case of a porous filler, the degree of swelling with pure water is preferably 12.5 ml / gram or less.

  In the present invention, the spacer is preferably a synthetic polymer or polysaccharide having an alcoholic hydroxyl group and does not contain an anion exchange group. Specifically, pullulan, dextran, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose and the like are exemplified. If the molecular weight of these spacers is too small, the pores cannot be filled when immobilized on the pore inner wall of the substrate, and the effect of expanding the adsorption capacity of the biopolymer is limited. On the other hand, if it is too large, it cannot enter into the pores of the base material and can be immobilized only on the outer surface of the base material, and the effect of expanding the adsorption capacity is extremely small. Therefore, the weight average molecular weight of the spacer is preferably 10,000 or more. The upper limit of the weight average molecular weight depends on the exclusion limit molecular weight of the substrate pores. However, since the molecular weight distribution of these polymers is generally wide, the upper limit of the weight average molecular weight is not particularly limited.

  In the present invention, as a method for immobilizing a synthetic polymer or polysaccharide having an alcoholic hydroxyl group on a substrate, for example, first, epihalohydrin or polyglycidyl ether of polyalcohol and the substrate are added in a strong alkaline aqueous medium. And / or dehydrohalogenating and activating the substrate with epoxy, and then washing and removing the remaining epihalohydrin or polyglycidyl ether of polyalcohol, and then a synthetic polymer or polysaccharide having an alcoholic hydroxyl group dissolved in water Examples of the method include mixing, addition reaction under strong alkaline conditions, and immobilization.

  In the above method, as epihalohydrin, for example, epichlorohydrin, epibromohydrin, etc. can be used, and as polyglycidyl ether of polyalcohol, for example, ethylene glycol, butanediol, propylene glycol, glycerin, penta Polyglycidyl ethers such as erythritol, sorbitol and diglycerol can be used.

  In the present invention, the ligand has a hydrophobic group and a carboxyl group, and is specifically an α-amino acid having a hydrophobic group represented by the above formula (1) or aminomethylbenzoic acid. . As an α-amino acid having a hydrophobic group, an α-amino acid having an aromatic group or an α-amino acid having a nonionic aliphatic group having 5 to 7 carbon atoms exhibits the function of the present invention. It becomes. Specific examples of the α-amino acid having an aromatic group include phenylalanine and tryptophan. Specific examples of the α-amino acid having a nonionic aliphatic group include leucine, norleucine, α-aminooctanoic acid and the like. Although these α-amino acids have optical isomers, the functions of the present invention are exhibited regardless of the L-form, D-form and racemate.

  In the present invention, depending on the type of ligand used, when the ligand density is too high, the hydrophobicity becomes too strong, and when the target water-soluble polymer (for example, protein, peptide, etc.) is adsorbed, it is denatured. The recovery rate may decrease. In such a case, although it does not function as a hydrophobic ligand, a neutral or acidic amino acid or a hydrophilic amine can be introduced together with the ligand of the present invention for hydrophobicity regulation.

  Examples of neutral to acidic amino acids include glycine, alanine, β-alanine, proline, serine, threonine, asparagine, glutamine, aspartic acid, glutamic acid, tyrosine and the like.

  Examples of the hydrophilic amine include ethanolamine, 2-amino- (2-hydroxymethyl), 1,3-propanediol and the like.

  In the present invention, as a method for introducing these ligands into the substrate, it is necessary that the ligand density be sufficiently high so that proteins and the like can be retained by hydrophobic bonds, and that the anion exchange groups are not substantially coexistent. . This is because when an anion exchange group coexists in the resulting filler, it is dissociated into ions at an acidic pH, increasing the hydrophilicity of the filler and hindering the hydrophobic bond. The ligand introduction method in the present invention is not particularly limited as long as it satisfies these conditions, and two methods are shown below as specific examples.

  In the first synthesis method, an alcoholic hydroxyl group in a substrate is activated with 1,1-carbonylbis-1H-imidazole (hereinafter abbreviated as CDI) in an organic solvent, and then an organic solvent or a hydrous organic solvent. In this method, the ligand is reacted with an amino group of the ligand, and the ligand is introduced into the substrate by urethane bonding.

  The second synthesis method is a method in which after introducing a carboxyl group into a substrate, the carbodiimide is used as a catalyst to react with the amino group of the ligand, and the ligand is introduced into the substrate through an amide bond.

  In the second synthesis method, a method for introducing a carboxyl group into the substrate is not particularly limited. For example, a method in which a halogenated carboxylic acid is reacted with an alcoholic hydroxyl group of the substrate under alkaline conditions. A method in which halohydrin is added under alkaline conditions, an epoxy group is introduced, and mercaptocarboxylic acid (for example, mercaptoacetic acid, mercaptopropionic acid, etc.) is reacted under neutral or weakly alkaline conditions, or allyl glycidyl ether is added, and allyl Examples include a method of introducing a group and reacting mercaptocarboxylic acid under acidic conditions.

  In the second synthesis method, as the carbodiimide, for example, diisopropylcarbodiimide, dicyclohexylcarbodiimide and the like can be used for an organic solvent system, and for an aqueous system or a mixture of water and an organic solvent, for example, 1 -Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-cyclohexyl-N '-(2-morpholinoethyl) carbodiimide, meso-p-toluenesulfonate, and the like can be used. In addition, after introducing a carboxyl group into a base material, when activating the carboxyl group with carbodiimides, N-hydroxysuccinimide (hereinafter sometimes abbreviated as NHS) or 1-hydroxybenzotriazole is allowed to coexist. Side reactions can be suppressed by reacting with a ligand having an amino group.

  B. H. J. et al. Hofste et al., Biochemical and Biophysical research communications, 63 (1975) p. 618-624, M.I. Kim et al., Journal of Chromatography, 585 (1991) p. As reported in 45-51, it is already known to introduce hydrophobic amino acids into a substrate by methods other than those described above. However, B. above. H. J. et al. As reported by Hofste et al., Sufficient hydrophobic amine cannot be introduced by the cyanogen bromide activation method, and the protein cannot be adsorbed unless the eluent is in a high-concentration salt solution.

  Moreover, after introducing an epoxy group or a formyl group into a substrate, an amino acid can be introduced by a secondary amine bond. However, in this method, the amino group is ionically dissociated under acidic conditions, the amino group and the carboxyl group are partially ionized under neutral conditions, and the carboxyl group is ionically dissociated under basic conditions. Therefore, the above M.I. As described in the report of Kim et al., Proteins can be adsorbed and retained by electrostatic interaction in an eluent with low ionic strength, but hydrophobic interaction does not work strongly, and by increasing the ionic strength of the eluent, Protein is released. Therefore, the above-described ligand introduction method in which an anion exchange group is generated by immobilization of the ligand is not adopted in the present invention.

  The packing material for liquid chromatography of the present invention obtained by the first and second synthesis methods has a ligand directly immobilized on a substrate and a ligand immobilized on the substrate via a spacer. It becomes a packing material for liquid chromatography.

  In the filler, the ligand directly immobilized on the base material is an amide bond or a urethane bond via the amino group contained in the α-amino acid or aminomethylbenzoic acid represented by the formula (1). The ligand fixed to the base material via the spacer and the base material is bonded to the amide via the amino group contained in the α-amino acid or aminomethylbenzoic acid represented by the formula (1). It is fixed to the spacer by a bond or a urethane bond.

  In the present invention, the amount of the ligand immobilized on the substrate is usually 30 mmol or more per 1 liter (wet volume) of the liquid chromatography filler of the present invention.

  When the liquid chromatography column of the present invention is packed in a liquid chromatography column and an eluent having a pH of 5 or less (preferably, a pH in the range of 3 to 5) is flowed, the pH in the column decreases and carboxyl The ion dissociation of the group is reduced, and the hydrophobicity of the above-described filler surface is increased. Here, when this filler is brought into contact with a sample solution in which a solute having a hydrophobic surface (for example, protein, peptide, etc.) is dissolved, the solute is adsorbed on the filler. In the present invention, it is preferable to add an acid or alkali to the sample solution to make the pH of the aqueous acid solution 5 or lower.

  Next, after washing the non-adsorbed component with an eluent having the same pH as the above eluent, when the pH of the eluent is gradually increased, ion dissociation (ionization ratio) of carboxyl groups in the filler increases, Conversely, the hydrophobicity of the filler surface decreases. Then, the adsorbed solute is desorbed and eluted from the filler according to the surface hydrophobicity under neutral to weakly basic conditions of pH 9 or less.

  Thus, in the present invention, by weakening the hydrophobic interaction between the solute and the filler, individual solutes can be separated and purified and recovered by elution based on the hydrophobicity and ionicity of the solute. Here, if the pH of the eluent is rapidly increased to neutral or weakly basic, the adsorbed solute can be eluted and recovered as a concentrated solution.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is limited to these and is not interpreted. The base materials used in the following examples and comparative examples are all carriers (hydrophilic base materials) having alcoholic hydroxyl groups on the surface thereof. The physical properties of the substrate in terms of porosity in water were evaluated by the exclusion limit molecular weight and the porosity. The measurement methods are as follows.

Measurement of exclusion limit molecular weight and porosity:
Using an aqueous gel slurry solution of a hydrophilic base, the base was packed into a stainless steel column having an inner diameter of 10.7 mm and a length of 150 mm so as to be closely packed. Next, the packed column was mounted on an HPLC system (manufactured by Tosoh Corporation) equipped with a RI-8020 detector (manufactured by Tosoh Corporation).

  Subsequently, dextran having a molecular weight of 40 million was used as a standard substance, pullulan and polyethylene glycol having various molecular weights described in Table 1, and 0.5 ml / min. Standard substances of various molecular weights were injected at a flow rate of 5 and the exclusion limit molecular weight was determined from the elution volume. The porosity was determined from the elution volume of dextran and ethylene glycol and the column volume.

  The hydrophilic base used for the measurement was a methacrylic ester porous filler [Toyopearl HW-65C, HW-55C, and HW-50C (above, manufactured by Tosoh Corporation)], a cross-linked agarose filler [Sepharose 6 -Fast flow (manufactured by GE Healthcare)] and five fillers of cross-linked dextran-based filler [Sephadex G-25 (manufactured by GE Healthcare)]. The obtained results are shown in Table 1.

製造例１．
その表面にアルコール性水酸基を有するメタクリル酸エステル系多孔性充填剤［トヨパール ＨＷ−６５Ｃ（東ソー社製）］を、グラスフィルター上で、純水で懸濁とろ過を繰返し洗浄し、当該基材スラリーの純水を吸引ろ過により除去して、サクションドライ・ゲルケーキを用意した。

Production Example 1
The base material slurry is obtained by repeatedly washing and filtering a methacrylic ester porous filler [Toyopearl HW-65C (manufactured by Tosoh Corp.)] having an alcoholic hydroxyl group on the surface with pure water on a glass filter. The suction water was removed by suction filtration to prepare a suction dry gel cake.

  Place 200 grams of this gel cake, 300 milliliters of pure water and 100 grams of chloromethyloxirane into a 1 liter separable flask, and drop 85 grams of 48% sodium hydroxide over 2 hours with stirring while maintaining the reaction temperature at 45 ° C. did. After the dropwise addition, the reaction was further continued for 1 hour, and the suspension and filtration were repeatedly washed with pure water on a glass filter to obtain a suction dry gel cake of an epoxy-activated substrate. The entire epoxy activated gel cake, 130 grams of dextran having a weight average molecular weight of 500,000 and 350 ml of pure water were added, and the dextran was dissolved while stirring at a temperature of 25 ° C. Then, 10 grams of 48% sodium hydroxide was added, reacted for another 16 hours after the addition, and suspension and filtration were repeatedly washed with pure water on a glass filter to obtain a dextran-immobilized substrate suction dry gel cake. It was. This is the intermediate substrate 1. The amount of dextran immobilized on the intermediate substrate 1 was measured by the following method.

Measurement of the amount of immobilized polysaccharide 1:
10 g of the intermediate substrate 1 (suction dry gel cake) was suspended in 15 ml of pure water, poured into a glass column with a glass filter and an inner diameter of 20 mm, and the solvent was removed by suction filtration. The volume of the substrate was measured from the height of the formed bed (the portion of the filler deposited on the column). Separately, 5 g of the intermediate substrate 1 (suction dry gel cake) was taken, dried under reduced pressure at 50 ° C., and the weight was measured. The dried gel and 20 ml of 2 mol / liter hydrochloric acid were placed in a 100 ml Erlenmeyer flask equipped with a reflux condenser, and dextran was hydrolyzed at 90 ° C. for 150 minutes. After the reaction, the base material was repeatedly washed and suspended with pure water on a glass filter, dried again under reduced pressure at 50 ° C., and the weight was measured. The amount of dextran immobilized was determined from the difference in dry weight of the substrate before and after hydrolysis. The measurement results are shown in Table 2.

次に、中間基材１（サクションドライ・ゲルケーキ）５０グラムをＮ，Ｎ−ジメチルホルムアミド（以下、ＤＭＦと略す。）溶媒で懸濁とろ過を繰返し、含有水分を除去し、当該充填剤スラリーの分散溶媒を吸引ろ過により除去して、サクションドライ・ゲルケーキを用意した。

Next, 50 g of the intermediate base material 1 (suction dry gel cake) was repeatedly suspended and filtered with an N, N-dimethylformamide (hereinafter abbreviated as DMF) solvent to remove the water content, and the filler slurry The dispersion solvent was removed by suction filtration to prepare a suction dry gel cake.

  50 g of this gel cake and 100 ml of DMF were placed in a 300 ml separable flask and stirred. 60 mmol of CDI was dissolved in 30 grams of dioxane, and the CDI solution was added dropwise to the above separable flask at a constant temperature of 30 ° C. Stirring was continued for 1 hour after the dropping. Thereafter, the slurry was filtered through a glass filter, the gel was washed with a DMF solvent, unreacted CDI and by-products were removed, and a CDI-activated suction dry gel cake was synthesized.

  The total amount of the obtained gel cake was again put into a 300 ml separable flask, 100 ml of dimethylformamide (hereinafter abbreviated as DMF) was added and stirred. 24 mmol of L-phenylalanine and 6 mmol of glycine were dissolved in 25 ml of 1 mol / liter sodium hydroxide aqueous solution, and 50 ml of DMF was added and mixed. This amino acid solution was put into the separable flask at a time and stirred at room temperature for 16 hours to react.

  After completion of the reaction, the gel obtained in the order of DMF, 50% acetone, 0.1 mol / liter sodium hydroxide aqueous solution and pure water was washed again on the glass filter. The gel obtained by this reaction is designated as filler 1.

Measurement of ion exchange capacity:
10 g of washed filler 1 (suction dry gel cake) was suspended in 15 ml of pure water, poured into a glass column with a glass filter and an inner diameter of 20 mm, and the solvent was removed by suction filtration. Of the formed bed (filler portion deposited on the column), 10 ml or more of the filler portion is removed (that is, 10 ml of the filler in the column), and 0.5 mol / liter hydrochloric acid is added with 30 ml of hydrochloric acid. Then, the washing was repeated with 40 ml of pure water until the pH of the filtrate reached 5 or more. The washed filler is taken out, transferred to a 200 ml beaker, suspended in 100 ml of 0.5 mol / l saline, and 0% using an automatic titrator (COM-450, manufactured by Hiranuma Sangyo Co., Ltd.). Titration with 5 mol / liter aqueous sodium hydroxide. The end point was pH 8.5. When the ion exchange capacity was calculated from the amount of titrant until the end point, it was 125 meq / liter. The amount of ligand introduced in the total of phenylalanine and glycine in the filler 1 corresponds to the ion exchange capacity of the filler 1 and is 115 mmol / liter.

Production Example 2
In the same manner as in Production Example 1, Toyopearl HW-65C was used to synthesize an epoxy-activated base material suction dry gel cake under the same conditions. All of the epoxy activated gel cake, 150 grams of dextran having a weight average molecular weight of 200,000 and 350 ml of pure water were added, and the dextran was dissolved while stirring at a temperature of 25 ° C. Then, 10 grams of 48% sodium hydroxide was added, reacted for another 16 hours after the addition, and suspension and filtration were repeatedly washed with pure water on a glass filter to obtain a dextran-immobilized substrate suction dry gel cake. It was. This is the intermediate substrate 2. The amount of dextran immobilized on the intermediate substrate 2 was measured by the same method as in Production Example 1. The measurement results are also shown in Table 2.

  Next, a CDI-activated suction dry gel cake was synthesized using the intermediate substrate 2 in the same manner as in Production Example 1. Using the total amount of the CDI-activated gel cake obtained, L-phenylalanine and glycine were stirred at room temperature for 16 hours and reacted in the same manner as in Production Example 1.

  After completion of the reaction, the gel obtained in the order of DMF, 50% acetone, 0.1 mol / liter sodium hydroxide aqueous solution and pure water was washed again on the glass filter. The gel obtained by this reaction is designated as filler 2.

  When the ion exchange capacity was measured in the same manner as in Production Example 1, it was 110 meq / liter. The introduction amount of the ligand of phenylalanine and glycine in the filler 2 was 110 mmol / liter corresponding to the ion exchange capacity of the filler 2.

Production Example 3
In the same manner as in Production Example 1, Toyopearl HW-65C was used to synthesize an epoxy-activated substrate suction dry gel cake under the same conditions. The epoxy activated gel cake, 150 g of pullulan having a weight average molecular weight of 200,000 and 350 ml of pure water were added, and the pullulan was dissolved while stirring at a temperature of 25 ° C. After that, 10 grams of 48% sodium hydroxide was added, reacted for another 16 hours after the addition, and suspension and filtration were repeatedly washed on the glass filter with pure water to obtain a suction dry gel cake as a pullulan-immobilized substrate. It was. This is the intermediate substrate 3. The amount of pullulan immobilized on the intermediate substrate 3 was measured by the same method as in Production Example 1. The measurement results are also shown in Table 2.

  Next, a CDI-activated suction dry gel cake was synthesized using the intermediate substrate 2 in the same manner as in Production Example 1. Using the total amount of the CDI-activated gel cake obtained, L-phenylalanine and glycine were stirred at room temperature for 16 hours and reacted in the same manner as in Production Example 1.

After completion of the reaction, the gel obtained in the order of DMF, 50% acetone, 0.1 mol / liter sodium hydroxide aqueous solution and pure water was washed again on the glass filter. The gel obtained by this reaction is designated as filler 3.
When the ion exchange capacity was measured in the same manner as in Production Example 1, it was 135 meq / liter. The amount of the phenylalanine and glycine total ligand introduced into the filler 3 corresponds to the ion exchange capacity of the filler 2 and is 135 mmol / liter.

Production Example 4
A methacrylic ester porous filler [Toyopearl HW-55C (manufactured by Tosoh Corp.)] having an alcoholic hydroxyl group on its surface is repeatedly washed and suspended with pure water on a glass filter, and the base slurry The suction water was removed by suction filtration to prepare a suction dry gel cake.

  Under the same conditions as in Production Example 1, an epoxy-activated substrate suction dry gel cake was synthesized. The entire epoxy-activated gel cake, 150 grams of dextran having a weight average molecular weight of 70,000 and 350 ml of pure water were added, and the dextran was dissolved while stirring at a temperature of 25 ° C. Then, 10 grams of 48% sodium hydroxide was added, reacted for another 16 hours after the addition, and suspension and filtration were repeatedly washed with pure water on a glass filter to obtain a dextran-immobilized substrate suction dry gel cake. It was. This is the intermediate substrate 4. The amount of dextran immobilized on the intermediate substrate 4 was measured by the same method as in Production Example 1. The measurement results are also shown in Table 2.

  Next, a CDI activated suction dry gel cake was synthesized using the intermediate substrate 4 in the same manner as in Production Example 1. Using the total amount of the CDI-activated gel cake obtained, L-phenylalanine and glycine were stirred at room temperature for 16 hours and reacted in the same manner as in Production Example 1.

  After completion of the reaction, the gel obtained in the order of DMF, 50% acetone, 0.1 mol / liter sodium hydroxide aqueous solution and pure water was washed again on the glass filter. The gel obtained by this reaction is designated as filler 4.

  When the ion exchange capacity was measured in the same manner as in Production Example 1, it was 145 meq / liter. The introduction amount of the ligand of phenylalanine and glycine in the filler 4 was 145 mmol / liter corresponding to the ion exchange capacity of the filler 2.

Production Example 5
A methacrylic ester porous filler [Toyopearl HW-50C (manufactured by Tosoh Corp.)] having an alcoholic hydroxyl group on its surface is repeatedly washed with pure water on a glass filter and washed repeatedly. The suction water was removed by suction filtration to prepare a suction dry gel cake.

  Under the same conditions as in Production Example 1, an epoxy-activated substrate suction dry gel cake was synthesized. The entire epoxy activated gel cake, 150 grams of dextran having a weight average molecular weight of 10,000 and 350 ml of pure water were added, and the dextran was dissolved while stirring at a temperature of 25 ° C. Then, 10 grams of 48% sodium hydroxide was added, reacted for another 16 hours after the addition, and suspension and filtration were repeatedly washed with pure water on a glass filter to obtain a dextran-immobilized substrate suction dry gel cake. It was. This is the intermediate substrate 4. The amount of dextran immobilized on the intermediate substrate 4 was measured by the same method as in Production Example 1. The measurement results are also shown in Table 2.

  Next, a CDI-activated suction dry gel cake was synthesized using the intermediate substrate 2 in the same manner as in Production Example 1. Using the total amount of the CDI-activated gel cake obtained, L-phenylalanine and glycine were stirred at room temperature for 16 hours and reacted in the same manner as in Production Example 1.

  After completion of the reaction, the gel obtained in the order of DMF, 50% acetone, 0.1 mol / liter sodium hydroxide aqueous solution and pure water was washed again on the glass filter. The gel obtained by this reaction is designated as filler 5.

  When the ion exchange capacity was measured in the same manner as in Production Example 1, it was 188 meq / liter. The introduction amount of the ligand of phenylalanine and glycine in the filler 5 was 188 mmol / liter corresponding to the ion exchange capacity of the filler 2.

Production Example 6
The cross-linked agarose filler [Sepharose 6 Fast Flow (manufactured by GE Healthcare)] is repeatedly suspended and filtered with pure water on a glass filter, and the pure water of the base slurry is subjected to suction filtration. After removing, a suction dry gel cake was prepared.

  In the same manner as in Production Example 1, 120 grams of gel cake, 180 milliliters of pure water and 60 grams of chloromethyloxirane were placed in a 0.5 liter separable flask, and the reaction temperature was kept at 45 ° C. while stirring, 51 grams of 48. % Sodium hydroxide was added dropwise over 2 hours. After the dropwise addition, the reaction was further continued for 1 hour, and the suspension and filtration were repeatedly washed with pure water on a glass filter to obtain 126.9 g of an epoxy-activated base material as a suction dry gel cake. 5/6 of the total epoxy-activated gel cake, that is, 105.75 grams, 75 grams of dextran having a weight average molecular weight of 200,000 and 175 milliliters of pure water were added, and dextran was dissolved while stirring at a temperature of 25 ° C. Thereafter, 5 grams of 48% sodium hydroxide was added, and the reaction was continued for 16 hours after the addition. The suspension and filtration were repeatedly washed with pure water on a glass filter, and 114 suction-dried gel cake of the dextran-immobilized substrate was obtained. .7 grams were obtained. This is the intermediate substrate 6. The amount of dextran immobilized on the intermediate substrate 6 was measured by the following method 2 for measuring the amount of immobilized polysaccharide, because the substrate was also hydrolyzed with acid.

Measurement of immobilized amount of polysaccharide 2:
First, 10 grams of an epoxy-activated substrate suction dry gel cake was dried under reduced pressure at 50 ° C. and weighed, and the dry weight was measured from the product of the weight of the gel cake used in the dextran immobilization reaction. Next, 10 g of the intermediate substrate 6 (suction dry gel cake) was dried under reduced pressure at 50 ° C., and the weight was measured. The dry weight was measured from the product of the intermediate substrate 6 and the gel cake weight. The difference between the dry weight of the intermediate substrate 6 and the dry weight of the epoxy-activated substrate used for the reaction is the weight of the immobilized dextran. It was calculated as (dry weight of intermediate substrate 6) milligram (weight of immobilized dextran) per gram. The measurement results are also shown in Table 2.

  Next, a CDI activated suction dry gel cake was synthesized using the intermediate substrate 6 in the same manner as in Production Example 1. Using the total amount of the CDI-activated gel cake obtained, L-phenylalanine and glycine were stirred at room temperature for 16 hours and reacted in the same manner as in Production Example 1.

  After completion of the reaction, the gel obtained in the order of DMF, 50% acetone, 0.1 mol / liter sodium hydroxide aqueous solution and pure water was washed again on the glass filter. The gel obtained by this reaction is designated as filler 6.

  When the ion exchange capacity was measured in the same manner as in Production Example 1, it was 85 meq / liter. The introduced amount of the ligand of phenylalanine and glycine in the filler 6 was 85 mmol / liter corresponding to the ion exchange capacity of the filler 2.

Production Example 7
The crosslinked dextran filler [Sephadex G-25 (manufactured by GE Healthcare)] is repeatedly suspended and filtered with pure water on a glass filter, and the pure water of the base slurry is removed by suction filtration. Suction dry gel cake was prepared.

  In the same manner as in Production Example 1, 120 grams of this gel cake, 180 milliliters of pure water and 60 grams of chloromethyloxirane were placed in a 0.5 liter separable flask, and the reaction temperature was kept at 45 ° C. while stirring, 48% sodium hydroxide was added dropwise over 2 hours. After the dropwise addition, the reaction was further continued for 1 hour, and suspension and filtration were repeatedly washed with pure water on a glass filter to obtain 123.6 g of an epoxy-activated substrate suction dry gel cake. 5/6 of the total amount of the epoxy activated gel cake, that is, 103.0 grams, 75 grams of dextran having a weight average molecular weight of 200,000 and 175 milliliters of pure water were added, and dextran was dissolved while stirring at a temperature of 25 ° C. Thereafter, 5 grams of 48% sodium hydroxide was added, reacted for another 16 hours after the addition, and suspension and filtration were repeatedly washed with pure water on a glass filter, and 103 suction-dried gel cakes of a dextran-immobilized substrate were obtained. .5 grams were obtained. This is the intermediate substrate 7. The amount of dextran immobilized on the intermediate substrate 7 was measured by the above-described method 2 for measuring the amount of immobilized polysaccharide, because the substrate is also hydrolyzed with acid. The measurement results are also shown in Table 2, and the weight increase was within the measurement error range.

  Next, a CDI activated suction dry gel cake was synthesized using the intermediate substrate 6 in the same manner as in Production Example 1. Using the total amount of the CDI-activated gel cake obtained, L-phenylalanine and glycine were stirred at room temperature for 16 hours and reacted in the same manner as in Production Example 1.

  After completion of the reaction, the gel obtained in the order of DMF, 50% acetone, 0.1 mol / liter sodium hydroxide aqueous solution and pure water was washed again on the glass filter. The gel obtained by this reaction is designated as filler 6.

  When the ion exchange capacity was measured in the same manner as in Production Example 1, it was 163 meq / liter. The introduced amount of the ligand of phenylalanine and glycine in the filler 6 corresponds to the ion exchange capacity of the filler 2, and was 163 mmol / liter.

  The polysaccharide immobilization amount of the intermediate substrate prepared in Production Example 1 to Production Example 7 is shown in Table 2 together.

Production Example 8
Toyopearl HW-65C was repeatedly suspended and filtered with a dioxane solvent on a glass filter, the water content was removed, and the dispersion solvent of the filler slurry was removed by suction filtration to prepare a suction dry gel cake.

  50 g of this gel cake and 100 ml of dioxane were placed in a 300 ml separable flask and stirred. 60 mmol of CDI was dissolved in 30 grams of dioxane, and the CDI solution was added dropwise to the above separable flask at a constant temperature of 30 ° C. Stirring was continued for 1 hour after the dropping. Thereafter, the slurry was filtered through a glass filter, the gel was washed with a dioxane solvent, unreacted CDI and by-products were removed, and a CDI-activated suction dry gel cake was synthesized.

  The total amount of the obtained gel cake was again put into a 300 ml separable flask, and 100 ml of DMF was added and stirred. 24 mmol of L-phenylalanine and 6 mmol of glycine were dissolved in 25 ml of 1 mol / liter sodium hydroxide aqueous solution, and 50 ml of DMF was added and mixed. This amino acid solution was put into the separable flask at a time and stirred at room temperature for 16 hours to react.

  After completion of the reaction, the gel obtained in the order of DMF, 50% acetone, 0.1 mol / liter sodium hydroxide aqueous solution and pure water was washed again on the glass filter. The gel obtained by this reaction is designated as filler 8. When the ion exchange capacity was measured in the same manner as in Production Example 1, it was 80 meq / liter. The ligand introduction amount of the total of phenylalanine and glycine in the filler 8 corresponds to the ion exchange capacity of the filler 8, and was 80 mmol / liter.

Production Example 9
Cross-linked agarose-based filler [Sepharose 6 Fast Flow (GE Healthcare)] is suspended and filtered with a dioxane solvent on a glass filter, the water content is removed, and a dispersion solvent of the filler slurry is removed. It was removed by suction filtration to prepare a suction dry gel cake.

  A gel obtained by reacting and treating in the same manner as in Production Example 8 using 50 grams of this gel cake is used as a filler 9. When the ion exchange capacity was measured in the same manner as in Production Example 1, it was 100 meq / liter. The ligand introduction amount of the phenylalanine and glycine in the filler 9 was 100 mmol / liter corresponding to the ion exchange capacity of the filler 5.

Production Example 10
Using 50 g of the intermediate substrate 1 (suction dry gel cake) synthesized in Production Example 1, a CDI-activated suction dry gel cake was synthesized in the same manner as in Production Example 1.

  Half of the gel cake obtained was placed in a 100 ml separable flask, and 50 ml of DMF was added and stirred. 12 mmol of 4-aminomethylbenzoic acid and 3 mmol of glycine were dissolved in 12.5 ml of a 1 mol / liter sodium hydroxide aqueous solution, and 25 ml of DMF was added and mixed. This amino acid solution was put into the separable flask at a time and stirred at room temperature for 16 hours to react.

  After completion of the reaction, the gel obtained in the order of DMF, 50% acetone, 0.1 mol / liter sodium hydroxide aqueous solution and pure water was washed again on the glass filter. The gel obtained by this reaction is designated as filler 10.

  When the ion exchange capacity was measured in the same manner as in Production Example 1, it was 115 meq / liter. The total amount of ligands introduced into the filler 10 as 4-aminomethylbenzoic acid and glycine was 115 mmol / liter corresponding to the ion exchange capacity of the filler 10.

Production Example 11
The remaining half of the CDI-activated suction dry gel cake synthesized in Production Example 10 was placed in a 100 ml separable flask, and 50 ml of DMF was added and stirred. 15 mmol of α-aminooctanoic acid was dissolved in 12.5 mL of a 1 mol / L sodium hydroxide aqueous solution, and 25 mL of DMF was added and mixed. This amino acid solution was put into the separable flask at a time and stirred at room temperature for 16 hours to react.

  After completion of the reaction, the gel obtained in the order of DMF, 50% acetone, 0.1 mol / liter sodium hydroxide aqueous solution and pure water was washed again on the glass filter. The gel obtained by this reaction is referred to as filler 11.

  When the ion exchange capacity was measured in the same manner as in Production Example 1, it was 105 meq / liter. The amount of α-aminooctanoic acid ligand introduced into the filler 11 corresponds to the ion exchange capacity of the filler 11 and was 105 mmol / liter.

  Table 3 shows the base material, activator, ligand, and ion exchange capacity of each filler prepared in Production Example 1 to Production Example 11.

製造例１２．
製造例２で合成した中間基材２のゲルケーキ１２０グラムとクロロ酢酸ナトリウム０．８モル、純水２４０ミリリットルを５００ミリリットルのセパラブルフラスコに入れ、攪拌しながら、反応温度５０℃で４８％水酸化ナトリウム水溶液を１．１モルの水酸化ナトリウム相当を１時間かけて上記セパラブルフラスコに滴下した。滴下終了後３時間反応を継続し、得られたゲルを純水で洗浄した。この反応で得られた、イオン交換基としてカルボキシメチル基を有するゲルをＣＭ化中間基材２とする（ここで、ＣＭはカルボキシメチルの略である。以下同じ。）。製造例１と同様にして、そのイオン交換容量を測定すると、１６０ミリ当量／リットルであった。

Production Example 12.
120 g of the intermediate base 2 gel cake synthesized in Production Example 2, 0.8 mol of sodium chloroacetate and 240 ml of pure water were placed in a 500 ml separable flask and stirred at a reaction temperature of 50 ° C. with 48% hydroxylation. An aqueous sodium solution equivalent to 1.1 mol of sodium hydroxide was added dropwise to the above separable flask over 1 hour. Reaction was continued for 3 hours after completion | finish of dripping, and the obtained gel was wash | cleaned with the pure water. The gel obtained by this reaction and having a carboxymethyl group as an ion exchange group is used as the CM-converted intermediate substrate 2 (where CM is an abbreviation for carboxymethyl; the same applies hereinafter). When the ion exchange capacity was measured in the same manner as in Production Example 1, it was 160 meq / liter.

  60 grams of the resulting CMized intermediate substrate 2 gel cake was washed on a glass filter with 0.5 mol / liter hydrochloric acid and then with pure water until the filtrate was neutral. Further, suspension and filtration were repeated with a DMF solvent to remove the contained water, and the dispersion solvent of the filler slurry was removed by suction filtration to prepare a suction dry gel cake.

  60 g of this gel cake and 150 ml of DMF are placed in a 300 ml separable flask, and 35 mmol of N-hydroxysuccinimide (hereinafter abbreviated as NHS) and 30 mmol of diisopropylcarbodiimide (hereinafter abbreviated as DIC) are added and stirred. did. Stirring is continued at 30 ° C. for 2 hours, the slurry is filtered through a glass filter, the gel is washed with a dioxane solvent, unreacted products and by-products are removed, and 63.5 g of dioxane / suction dry gel cake is obtained. It was. The gel cake obtained by this reaction is referred to as NHS activated filler 1.

  Take 20 grams of this NHS activated filler 1 and place in a 100 milliliter separable flask. 10 milliliters of dioxane, 40 milliliters of 0.1 mol / liter phosphate buffer (pH 6.9) and 6 millimoles of L-tryptophan. Was added and stirred. After reacting at 25 ° C. for 16 hours, the reaction solution is removed by filtration, and the resulting gel is washed in the order of 50% acetone, 0.1 mol / liter hydrochloric acid, pure water, and 0.1 mol / liter sodium hydroxide. Then, unreacted products and by-products were removed. The filler obtained by this reaction is designated as filler 12. When the ion exchange capacity of the filler 12 was measured in the same manner as in Production Example 1, it was 152 meq / liter. Further, the swelling degree of the filler 12 was measured and found to be 4.0 ml / gram.

Measurement of swelling degree:
The CM ion exchange filler 1 was washed twice with 30 ml of 0.5 mol / liter sodium hydroxide, and then washed with 40 ml of pure water until the pH of the filtrate was 8.5 or less. 10 g of the washed filler (suction dry gel cake) was suspended in 15 ml of pure water, poured into a glass column with a glass filter and an inner diameter of 20 mm, and suction filtered to remove the solvent. In the formed bed, 10 ml or more of the filler was removed, and the remaining 10 ml of the filler was transferred to a glass filter and washed twice with 30 ml of 0.5 mol / liter hydrochloric acid. Thereafter, the washing of the filler was repeated with 40 ml of pure water until the pH of the filtrate was 5 or more. After washing twice with 40 ml of acetone, the washed filler was taken out, dried under reduced pressure at 40 ° C., the weight of 10 ml of the filler was measured, and the degree of swelling was calculated [degree of swelling (ml / g) = Volume (ml) / weight (g)]. The swelling degree of this filler was 5.2 ml / gram.

  Further, using this dry filler as a sample for elemental analysis, a nitrogen weight percentage was measured using a CHN fully automatic analyzer (manufactured by PerkinElmer, Model 2400II). In addition, the elemental analysis of the dry filler is similarly carried out in Production Example 12 and thereafter.

Production Example 13
Using 120 g of the gel cake of the intermediate substrate 3 synthesized in Production Example 3, a gel CM-converted intermediate substrate 3 having a carboxymethyl group as an ion exchange group was synthesized in the same manner as in Production Example 12. When the ion exchange capacity was measured in the same manner as in Production Example 1, it was 180 meq / liter.

  60 grams of the resulting CMized intermediate substrate 3 gel cake was washed on a glass filter with 0.5 mol / liter hydrochloric acid and then with pure water until the filtrate was neutral. Further, suspension and filtration were repeated with a DMF solvent to remove the contained water, and the dispersion solvent of the filler slurry was removed by suction filtration to prepare a suction dry gel cake.

  NHS was reacted in the same manner as in Production Example 12 using 60 grams of this gel cake to synthesize an NHS activated filler.

  The slurry after the reaction was filtered through a glass filter, the gel was washed with a dioxane solvent, unreacted products and by-products were removed, and 63.3 g of dioxane / suction dry / gel cake was obtained.

  20 grams of the gel cake NHS activated filler obtained by this reaction was taken, and L-tryptophan was added again in the same manner as in Production Example 12. The obtained gel was washed in the same manner to remove unreacted products and by-products. The filler obtained by this reaction is designated as filler 13. When the ion exchange capacity of the filler 13 was measured in the same manner as in Production Example 1, it was 172 meq / liter. The swelling degree of the filler 13 was measured and found to be 4.0 ml / gram.

Production Example 14
Using 120 g of the gel cake of the intermediate substrate 4 synthesized in Production Example 4, a gel CM-converted intermediate substrate 4 having a carboxymethyl group as an ion exchange group was synthesized in the same manner as in Production Example 12. When the ion exchange capacity was measured in the same manner as in Production Example 1, it was 185 meq / liter.

  60 g of the obtained gel cake of the CMized intermediate substrate 4 was washed on a glass filter with 0.5 mol / liter hydrochloric acid and then with pure water until the filtrate became neutral. Further, suspension and filtration were repeated with a DMF solvent to remove the contained water, and the dispersion solvent of the filler slurry was removed by suction filtration to prepare a suction dry gel cake. The gel cake NHS activated filler obtained by this reaction is referred to as NHS activated filler 2.

  20 grams of the NHS activated filler 2 gel cake was taken, and L-tryptophan was added again in the same manner as in Production Example 12. The obtained gel was washed in the same manner to remove unreacted products and by-products. The filler obtained by this reaction is designated as filler 14. When the ion exchange capacity of the filler 14 was measured in the same manner as in Production Example 1, it was 178 meq / liter. Further, the swelling degree of the filler 14 was measured and found to be 4.2 ml / gram.

Production Example 15.
Using 120 g of the gel cake of the intermediate substrate 6 synthesized in Production Example 6, a gel CM-converted intermediate substrate 4 having a carboxymethyl group as an ion exchange group was synthesized in the same manner as in Production Example 12. When the ion exchange capacity was measured in the same manner as in Production Example 1, it was 115 meq / liter.

  60 g of the obtained gel cake of the CMized intermediate substrate 4 was washed on a glass filter with 0.1 mol / liter hydrochloric acid and then with pure water until the filtrate became neutral. Further, suspension and filtration were repeated with a DMF solvent to remove the contained water, and the dispersion solvent of the filler slurry was removed by suction filtration to prepare a suction dry gel cake. The gel cake NHS activated filler obtained by this reaction is referred to as NHS activated filler 3.

  20 grams of the NHS activated filler 3 gel cake was taken, and L-tryptophan was added again in the same manner as in Production Example 12. The obtained gel was washed in the same manner to remove unreacted products and by-products. The filler obtained by this reaction is designated as filler 15. When the ion exchange capacity of the filler 15 was measured in the same manner as in Production Example 1, it was 103 meq / liter. Further, when the degree of swelling of the filler 15 was measured, it was 4.5 ml / gram.

Production Example 16.
30 grams (equivalent to 35 milliliters) of the gel cake of the CM intermediate substrate 2 synthesized in Production Example 12 and 35 milliliters of pure water are placed in a 300 milliliter separable flask, and 0.5 mol / liter hydrochloric acid is gradually added. Then, the pH was adjusted to 5.2. Next, 30 ml of dioxane, 10.9 mmol of NHS and 6 mmol of 4-aminomethylbenzoic acid were added and dissolved by stirring. 10.9 mmol of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (hereinafter sometimes abbreviated as EDC) was dissolved in 3.5 ml of pure water, and the separable flask was charged with 25 ° C. After the reaction at 25 ° C. for 16 hours, the reaction solution was removed by filtration, and the gel obtained was washed with 50% acetone, 0.1 mol / liter sodium hydroxide and pure water in this order. Unreacted products and by-products were removed. The gel obtained by this reaction is used as filler 16. When the ion exchange capacity of the filler 16 was measured in the same manner as in Production Example 1, it was 151 meq / liter. Moreover, when the swelling degree of the filler 16 was measured similarly to manufacture example 12, it was 4.0 milliliters / gram.

Production Example 17
30 grams (equivalent to 35 milliliters) of the gel cake of the CM intermediate substrate 2 synthesized in Production Example 12 and 35 milliliters of pure water are placed in a 100 milliliter separable flask, and 0.5 mol / liter hydrochloric acid is gradually added. Then, the pH was adjusted to 5.2. Next, 30 ml of dioxane, 10.9 mmol of NHS and 6 mmol of DL-phenylalanine were added and dissolved by stirring. 10.9 mmol of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was dissolved in 3.5 ml of pure water, added to the above separable flask at 25 ° C., and reacted at 25 ° C. for 16 hours. The reaction solution was removed by filtration, and the gel obtained was washed in the order of 50% acetone, 0.1 mol / liter sodium hydroxide and pure water to remove unreacted products and by-products. The gel obtained by this reaction is used as filler 17. When the ion exchange capacity of the filler 17 was measured in the same manner as in Production Example 1, it was 153 meq / liter. Moreover, when the swelling degree of the filler 16 was measured similarly to manufacture example 12, it was 4.0 milliliters / gram.

Production Example 18.
30 grams (equivalent to 35 milliliters) of the gel cake of the CM intermediate substrate 4 synthesized in Production Example 14 and 35 milliliters of pure water are placed in a 100 milliliter separable flask, and 0.5 mol / liter hydrochloric acid is gradually added. Then, the pH was adjusted to 5.2. Next, 30 ml of dioxane, 10.9 mmol of NHS, 6 mmol of DL-phenylalanine and 2-ethanolamine were added and dissolved by stirring. 10.9 mmol of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was dissolved in 3.5 ml of pure water, added to the above separable flask at 25 ° C., and reacted at 25 ° C. for 16 hours. The reaction solution was removed by filtration, and the gel obtained was washed in the order of 50% acetone, 0.1 mol / liter sodium hydroxide and pure water to remove unreacted products and by-products. The gel obtained by this reaction is used as filler 17. When the ion exchange capacity of the filler 17 was measured in the same manner as in Production Example 1, it was 130 meq / liter. Moreover, when the swelling degree of the filler 16 was measured similarly to manufacture example 12, it was 4.2 milliliters / gram.

Production Example 19.
30 grams (corresponding to 35 milliliters) of the gel cake of CMized intermediate base material 3 synthesized in Production Example 13 and 35 milliliters of pure water were placed in a 100 milliliter separable flask, and 0.5 mol / liter hydrochloric acid was gradually added. Then, the pH was adjusted to 5.2. Next, 30 ml of dioxane, 10.9 mmol of NHS and 6 mmol of L-tryptophan were added and dissolved by stirring. 10.9 mmol of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was dissolved in 3.5 ml of pure water, added to the above separable flask at 25 ° C., and reacted at 25 ° C. for 16 hours. The reaction solution was removed by filtration, and the gel obtained was washed in the order of 50% acetone, 0.1 mol / liter sodium hydroxide and pure water to remove unreacted products and by-products. The gel obtained by this reaction is used as filler 19. When the ion exchange capacity of the filler 19 was measured in the same manner as in Production Example 1, it was 170 meq / liter. Moreover, when the swelling degree of the filler 19 was measured similarly to manufacture example 12, it was 4.0 milliliters / gram.

Production Example 20.
30 grams (corresponding to 35 milliliters) of the gel cake of CMized intermediate base material 6 synthesized in Production Example 15 and 35 milliliters of pure water are placed in a 100 milliliter separable flask, and 0.5 mol / liter hydrochloric acid is gradually added. Then, the pH was adjusted to 5.2. Next, 30 ml of dioxane, 10.9 mmol of NHS and 6 mmol of L-tryptophan were added and dissolved by stirring. 10.9 mmol of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was dissolved in 3.5 ml of pure water, added to the above separable flask at 25 ° C., and reacted at 25 ° C. for 16 hours. The reaction solution was removed by filtration, and the gel obtained was washed in the order of 50% acetone, 0.1 mol / liter sodium hydroxide and pure water to remove unreacted products and by-products. The gel obtained by this reaction is used as filler 20. When the ion exchange capacity of the filler 20 was measured in the same manner as in Production Example 1, it was 105 meq / liter. Moreover, when the swelling degree of the filler 20 was measured like manufacture example 12, it was 4.2 milliliters / gram.

Production Example 21.
CM-Toyopearl 650M (manufactured by Tosoh Corporation) was a CM ion exchange filler based on HW-65C and had an ion exchange capacity of 110 meq / liter. This was repeatedly suspended and filtered with pure water on a glass filter, purged with pure water, and suction filtered to prepare a suction dry gel cake.

  30 grams (equivalent to 35 milliliters) of this gel cake and 35 milliliters of pure water were placed in a 100 milliliter separable flask, and 0.5 mol / liter hydrochloric acid was gradually added to adjust the pH to 5.2. Next, 30 ml of dioxane, 10.9 mmol of NHS and 6 mmol of L-tryptophan were added and dissolved by stirring. 10.9 mmol of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was dissolved in 3.5 ml of pure water, added to the above separable flask at 25 ° C., and reacted at 25 ° C. for 16 hours. The reaction solution was removed by filtration, and the gel obtained was washed in the order of 50% acetone, 0.1 mol / liter sodium hydroxide and pure water to remove unreacted products and by-products. The gel obtained by this reaction is used as filler 21. When the ion exchange capacity of the filler 21 was measured in the same manner as in Production Example 1, it was 102 meq / liter. Moreover, when the swelling degree of the filler 21 was measured like manufacture example 12, it was 4.0 milliliters / gram.

Production Example 22.
The ion exchange capacity of the cross-linked agarose weak cation exchange gel [CM-Sepharose Fast Flow (GE Healthcare)] was measured and found to be 105 meq / liter.

  This cross-linked agarose weak cation exchange gel was suspended and filtered with pure water repeatedly on a glass filter, purged with pure water, and then suction filtered to prepare a suction dry gel cake.

  17 grams (equivalent to 20 milliliters) of this gel cake and 36 milliliters of pure water were placed in a 100 milliliter separable flask, and 0.5 mol / liter hydrochloric acid was gradually added to adjust the pH to 5.0. Next, 20 ml of dioxane, 4.2 mmol of NHS, and 2.1 mmol of DL-phenylalanine were added to the above separable flask and mixed by stirring to dissolve. 4.2 mmol of EDC was dissolved in 2 ml of pure water, added to the above separable flask at 25 ° C., and stirred for 16 hours to carry out the reaction. The reaction solution is removed by filtration through a glass filter, and the gel obtained is washed in order of 50% acetone, 0.1 mol / liter sodium hydroxide, and pure water to remove unreacted products and by-products. did. The gel obtained by this reaction is used as the filler 22. When the ion exchange capacity was measured in the same manner as in Production Example 1, it was 98 meq / liter. Moreover, when the swelling degree was measured like manufacture example 12, it was 10.6 milliliters / gram.

Production Example 23.
In the same manner as in Production Example 1, using Toyopearl HW-65C, an epoxy-activated substrate suction dry gel cake was synthesized under the same conditions. A solution prepared by dissolving 100 g of hydroxyethyl cellulose having a weight average molecular weight of 400,000 in 100 ml of pure water was added and mixed. Then, while stirring at a temperature of 25 ° C., 10 grams of 48% sodium hydroxide was added. After the addition, the reaction was continued for another 16 hours, and the suspension and filtration were washed repeatedly with pure water on a glass filter. An immobilization base material suction dry gel cake was obtained. This is referred to as an intermediate base material 23. The amount of hydroxyethyl cellulose immobilized on the intermediate substrate 23 was measured by the same method as in Production Example 1. The measurement results are also shown in Table 2.

  Using 120 g of the gel cake of the synthesized intermediate substrate 23, a gel CM intermediate substrate 23 having a carboxymethyl group as an ion exchange group was synthesized in the same manner as in Production Example 12. When the ion exchange capacity was measured in the same manner as in Production Example 1, it was 86 meq / liter.

  30 grams (corresponding to 35 milliliters) of the gel cake of CM intermediate substrate 23 and 35 milliliters of pure water were placed in a 100 milliliter separable flask, 0.5 mol / liter hydrochloric acid was gradually added, and the pH was adjusted to 5 Adjusted to .2. Next, 30 ml of dioxane, 10.9 mmol of NHS and 6 mmol of L-tryptophan were added and dissolved by stirring. 10.9 mmol of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was dissolved in 3.5 ml of pure water, added to the above separable flask at 25 ° C., and reacted at 25 ° C. for 16 hours. The reaction solution was removed by filtration, and the gel obtained was washed in the order of 50% acetone, 0.1 mol / liter sodium hydroxide and pure water to remove unreacted products and by-products. The gel obtained by this reaction is used as filler 23. When the ion exchange capacity of the filler 23 was measured in the same manner as in Production Example 1, it was 86 meq / liter. Moreover, when the swelling degree of the filler 23 was measured like manufacture example 12, it was 4.2 milliliters / gram.

  Table 4 shows the base material, activator, ligand, ion exchange capacity, and elemental analysis results of each filler prepared in Production Example 12 to Production Example 23.

実施例１．
製造例１〜製造例７で得られた充填剤１〜充填剤７について、各充填剤ごとに各タンパク質サンプルの主ピークの溶出時間、ウシ血清アルブミン（以下ＢＳＡと略す。）吸着量、及びヒトγ−グロブリン（以下ＩｇＧと略す。）吸着量を測定した。それらの結果を表３にあわせて示す。

Example 1.
For the fillers 1 to 7 obtained in Production Examples 1 to 7, the elution time of the main peak of each protein sample, the bovine serum albumin (hereinafter abbreviated as BSA) adsorption amount, and human for each filler. The amount of γ-globulin (hereinafter abbreviated as IgG) adsorbed was measured. The results are also shown in Table 3.

In addition, protein adsorption and elution by the pH gradient elution method, measurement of BSA and IgG adsorption capacities, and measurement of the recovery rate were performed as follows.
(1) Protein adsorption and elution by pH gradient elution method:
Each of the packing materials shown in Table 2 was packed in a stainless steel column having an inner diameter of 7.5 mm and a length of 75 mm. These are filled in a liquid chromatograph system (manufactured by Tosoh Corporation) consisting of a liquid feed pump (CCPM-II), an autosampler (AS-8020), an ultraviolet / visible absorbance meter (UV-8020), and a system controller (SC-8020). A column was attached. By operating under the following chromatographic conditions, the elution time of the main peak of each sample was measured.

Chromatographic conditions 1:
Eluent 1: 50 mmol / L acetate buffer (containing 0.15 mol / L sodium chloride, pH 4.5),
Eluent 2: 50 mmol / liter phosphate buffer (containing 0.15 mol / liter sodium chloride, pH 7.2),
Elution method: linear gradient elution from eluent 1: 100% to eluent 2: 100% for 60 minutes, followed by eluent 2: 100% for 5 minutes and finally regenerated for 15 minutes with eluent 1: 100% Equilibration,
Eluent flow rate: 1.0 ml / min,
Sample: soybean trypsin inhibitor (hereinafter abbreviated as STI), bovine serum albumin (hereinafter abbreviated as BSA), human γ-globulin (hereinafter abbreviated as IgG), bovine α-chymotrypsinogen A (hereinafter abbreviated as CHY) Abbreviated),
Sample concentration: 2.0 g / liter each (dissolved in eluent 1),
Sample injection volume: 0.2 ml,
Temperature: 25 ° C
Detection: UV absorption, wavelength: 280 nanometers.
(2) Measurement of BSA adsorption capacity and recovery rate:
A 200 ml Erlenmeyer flask was charged with 30 ml of an adsorption buffer and 1.0 ml of the filler shown in Tables 3 and 4. 10 ml of a solution of BSA dissolved in a buffer solution for adsorption at a concentration of 15 g / l is added to the Erlenmeyer flask and shaken at 25 ° C. for 3.0 hours to adsorb BSA, and then adsorb the supernatant. Absorbance was measured by diluting 2.5 times with the buffer solution. A blank without a filler was diluted in the same manner as described above, and the absorbance was measured. The amount of BSA adsorption was determined from the difference between the two.

Absorbance difference: ΔI = Ib−W × Is
Ib: Absorbance of 2.5-fold diluted blank,
Is: absorbance of 2.5-fold diluted supernatant,
W: Coefficient of moisture brought into the filler (W = 1.015 for all fillers).

BSA adsorption amount: A = 80 × F (ΔI)
F (ΔI): function of absorbance and BSA concentration relationship.

  In determining the amount of BSA adsorption, BSA solutions having concentrations of 0.75 / liter and 1.5 gram / liter were prepared, and their absorbance was measured in advance at a wavelength of 280 nm, and the BSA concentration and ultraviolet 280 nanometers were measured. The relational expression of the absorbance was prepared.

  Next, the BSA-adsorbed packing material was washed away with 30 ml of an adsorption buffer solution, transferred to a column with a filter (inner diameter 10 mm), and unadsorbed BSA was further poured out with 10 ml of the adsorption buffer solution. Next, the elution buffer is applied to the column, and 45 mL or more of the eluate is collected and collected in a 50 mL volumetric flask, diluted with the elution buffer, and the absorbance is measured. The amount of BSA recovered was calculated from a function of the relationship between absorbance and BSA concentration. The recovery rate was calculated from the calculated adsorption amount and recovery amount.

Buffer for adsorption: 50 mmol / liter acetate buffer (containing 0.15 mol / liter sodium chloride, pH 4.0),
Elution buffer: 0.1 mol / liter Tris-HCl buffer (containing 0.3 mol / liter sodium chloride, pH 8.5).
(3) Measurement of IgG adsorption capacity and recovery rate:
A 200 ml Erlenmeyer flask was charged with 30 ml of an adsorption buffer and 1.0 ml of the filler shown in Tables 3 and 4. Human serum γ-globulin (manufactured by Chemical and Serological Therapy Research Laboratories) About 150 milligrams / milliliter of 5 milliliters dissolved in an adsorption buffer, 10 milliliters of a solution up to 50 milliliters was added to the Erlenmeyer flask, After shaking for 3.0 hours at a temperature of 25 ° C. to adsorb IgG, the supernatant was diluted 5-fold with an adsorption buffer, and the absorbance was measured. A blank without a filler was diluted in the same manner as described above, and the absorbance was measured. The IgG adsorption amount was determined from the difference between the two.

Absorbance difference: ΔI = Ib−W × Is
Ib: Absorbance of 5-fold diluted blank,
Is: absorbance of 5-fold diluted supernatant,
W: Coefficient related to moisture carried by the filler (W = 1.015 for all fillers).

IgG adsorption amount: A = 200 × ΔI / 1.4
(The absorbance per 1.0 milligram of IgG is 1.4).

  Next, the IgG adsorbed packing material was washed away with 30 ml of an adsorption buffer solution, transferred to a column with a filter (inner diameter 10 mm), and unadsorbed BSA was further poured out with 10 ml of the adsorption buffer solution. Next, the elution buffer was passed through the column, and 50 ml or more of the eluate was collected and collected. The elution buffer was diluted with a buffer for elution in a 200 ml measuring flask, and the absorbance was measured. The amount of recovered IgG was calculated from the absorbance. The recovery rate was calculated from the calculated adsorption amount and recovery amount.

IgG recovery amount: R = 200 × Ir / 1.4
Ir: Absorbance of recovered IgG solution The same buffer solution for adsorption and elution was used for measuring the BSA adsorption capacity.

  The fillers 1 to 7 shown in Table 3 use an intermediate base material in which a nonionic polysaccharide is immobilized on a base material having an exclusion limit molecular weight of 10,000 to 2.1 million by pullulan or an exclusion limit molecular weight of 3,000 by polyethylene glycol. Thus, after CDI activation, L-phenylalanine and glycine are introduced. As is clear from Table 3, it was confirmed that these can adsorb and retain various proteins under weakly acidic conditions and can be eluted by increasing the pH.

  In addition, the BSA and IgG adsorption capacities of the fillers 1 to 4 and the filler 6 derived from a substrate having an exclusion limit molecular weight of 300,000 or more by pullulan are 85 mg / milliliter or more, which is a very high adsorption capacity. It was confirmed that there was.

  Further, the fillers 1 to 3 derived from the base material having a large exclusion limit molecular weight have a large effect of increasing the adsorption capacity of IgG having a large molecular weight (molecular weight: 155,000), and the exclusion limit molecular weight is slightly smaller than those. The fillers 4 and 6 derived from the base material had a large effect of increasing the BSA (molecular weight: 66,000) adsorption capacity, but had a relatively small effect of increasing the IgG adsorption capacity.

  On the other hand, the effect of increasing the BSA and IgG adsorption capacities of the filler 5 and the filler 7 having an exclusion limit molecular weight of 10,000 or less was not significant. However, in the case of the filler 5, it is estimated that the adsorption capacity of a protein having a molecular weight smaller than that of BSA (a protein having a molecular weight of 50,000 or less) or a peptide may be increased. In addition, since only the outer surface of the particle is used for the filler 7, the increase in the absolute value of the adsorption capacity was not large.

  Furthermore, the protein recovery rate was as high as 94% or more for all fillers.

Comparative Example 1
The filler 8 and the filler 9 obtained in Production Example 8 and Production Example 9 were introduced with L-phenylalanine and glycine after CDI activation using a base material having an exclusion limit molecular weight of 400,000 or 2.1 million by pullulan. It is a filler. In the same manner as in Example 1, the elution time of the main peak and the BSA and IgG adsorption capacity of each protein sample were measured for each of these fillers. The results are also shown in Table 3.

  As is apparent from Table 3, it was confirmed that these fillers can adsorb and retain various proteins under weakly acidic conditions and can be eluted by increasing pH. However, even with the filler 9 whose pore properties are suitable for these protein adsorption capacities, the BSA and IgG adsorption capacities did not reach 65 mg / ml. In the case of the filler 8 having a large exclusion limit molecular weight and a smaller effective surface area, the adsorption capacity is further smaller, and the adsorption capacities of the fillers 1 to 4 and the filler 6 synthesized using the intermediate substrate on which the polysaccharide is immobilized. It was about one third of the above, and the performance did not reach at all.

  The protein recovery rate was as high as 94% or more for all fillers.

Example 2
The filler 10 and the filler 11 obtained in Production Example 10 and Production Example 11 were obtained by using 4-aminobenzoic acid or α-aminooctane after activation of CDI using a substrate having an exclusion limit molecular weight of 2.1 million by pullulan. It is a filler into which an acid is introduced. In the same manner as in Example 1, the elution time and BSA adsorption amount of the main peak of each protein sample were measured for each of these fillers. The results are also shown in Table 3.

  As is apparent from Table 3, it was confirmed that these fillers can adsorb and retain various proteins under weakly acidic conditions and can be eluted by increasing pH. Moreover, it was confirmed that both of the fillers have very high adsorption capacities of BSA and IgG of 95 mg / ml or more. Furthermore, the recovery rate was 92% or more, and the recovery rate was high.

Example 3
Filler 12 to Filler 15 obtained in Production Example 12 to Production Example 15 synthesize an intermediate base material on which a nonionic polysaccharide is immobilized using a base material having an exclusion limit molecular weight of 300,000 or 2.1 million based on pullulan. Then, after introducing a carboxyl group, carbodiimide is used in an organic solvent system, NHS activation is performed, and then L-tryptophan is introduced. In the same manner as in Example 1, the elution time, the BSA adsorption amount, and the IgG adsorption amount of the main peak of each protein sample were measured for each of these fillers. The results are also shown in Table 4.

  As is apparent from Table 4, it was confirmed that these fillers can adsorb and retain various proteins under weakly acidic conditions and can be eluted by increasing pH. In addition, it was confirmed that each of the fillers had a very high adsorption capacity of BSA and IgG adsorption capacity of 85 mg / ml or more. In the filler 12 and the filler 13 derived from the base material having a large exclusion limit molecular weight, the effect of increasing the IgG adsorption capacity having a large molecular weight is large. With the filler 15, the effect of increasing the BSA adsorption capacity was large, but the effect of increasing the IgG adsorption capacity was relatively small. Furthermore, the protein recovery rate was as high as 94% or more for all fillers.

Example 4
The fillers 16 to 18 obtained in Production Example 16 to Production Example 18 synthesize an intermediate base material on which a nonionic polysaccharide is immobilized using a base material having an exclusion limit molecular weight of 300,000 or 2.1 million by pullulan. Then, after introduction of the carboxyl group, NHS activation and a ligand are simultaneously introduced using water-soluble carbodiimide in a mixed system of an organic solvent and water. In the same manner as in Example 1, the elution time, the BSA adsorption amount, and the IgG adsorption amount of the main peak of each protein sample were measured for each of these fillers. The results are also shown in Table 4.

  As is apparent from Table 4, it was confirmed that these fillers can adsorb and retain various proteins under weakly acidic conditions and can be eluted by increasing pH. In addition, it was confirmed that any of the fillers had a very high adsorption capacity of BSA and IgG adsorption capacity of 100 mg / ml or more. In the filler 16 and the filler 17 derived from the substrate having a large exclusion limit molecular weight, the effect of increasing the IgG adsorption capacity having a large molecular weight is large, and in the filler 18 derived from the substrate having a slightly smaller exclusion limit molecular weight. Although the effect of increasing the BSA adsorption capacity was large, the effect of increasing the IgG adsorption capacity was relatively small. Furthermore, the protein recovery rate was as high as 93% or more for all fillers.

Example 5 FIG.
The filler 19, the filler 20 and the filler 23 obtained in Production Example 19, Production Example 20 and Production Example 23 were prepared by using a nonionic polysaccharide or Filler in which NHS activation and ligand (L-tryptophan) are simultaneously introduced using water-soluble carbodiimide in a mixed system of an organic solvent and water after synthesizing an intermediate base material on which a polysaccharide derivative is immobilized and introducing a carboxyl group It is. In the same manner as in Example 1, the elution time, the BSA adsorption amount, and the IgG adsorption amount of the main peak of each protein sample were measured for each filler. The results are also shown in Table 4.

  As is apparent from Table 4, it was confirmed that these fillers can adsorb and retain various proteins under weakly acidic conditions and can be eluted by increasing pH. In addition, it was confirmed that each of the fillers had a very high adsorption capacity of BSA and IgG adsorption capacity of 92 mg / ml or more. In the filler 19 and the filler 23 derived from the substrate having a large exclusion limit molecular weight, the effect of increasing the IgG adsorption capacity having a large molecular weight is large, and in the filler 20 derived from the substrate having a slightly smaller exclusion limit molecular weight. Although the effect of increasing the BSA adsorption capacity was large, the effect of increasing the IgG adsorption capacity was relatively small. Furthermore, the protein recovery rate was as high as 94% or more for all fillers.

Comparative Example 2
The filler 21 and filler 22 obtained in Production Example 21 and Production Example 22 correspond to the exclusion molecular weights of the base material corresponding to the filler 19 and the filler 20, respectively, but the polysaccharide spacer is not immobilized. . That is, an ion-exchange filler in which a carboxyl group is directly introduced into a base material, and a filler in which NHS activation and a ligand (L-tryptophan) are simultaneously introduced using water-soluble carbodiimide in a mixed system of an organic solvent and water. It is. In the same manner as in Example 1, the elution time, the BSA adsorption amount, and the IgG adsorption amount of the main peak of each protein sample were measured for each of these fillers. The results are also shown in Table 4.

  As is apparent from Table 4, it was confirmed that these fillers can adsorb and retain various proteins under weakly acidic conditions and can be eluted by increasing pH. However, even with the filler 22 whose pore properties are suitable for these protein adsorption capacities, the BSA and IgG adsorption capacities did not reach 65 milligrams / milliliter. In the case of the filler 21 having a large exclusion limit molecular weight and a smaller effective surface area, the adsorption capacity is further small, and about 3 minutes of the adsorption capacity of the filler 19 and the filler 20 synthesized using the intermediate substrate on which the polysaccharide is immobilized. The performance was not at all. The protein recovery rate was as high as 93% for any filler.

Claims (8)

A filler for liquid chromatography having a ligand directly immobilized on a substrate and a ligand immobilized on a substrate via a spacer, wherein (1) the substrate has an alcoholic hydroxyl group on the surface of the substrate (2) the spacer is a synthetic polymer or polysaccharide having an alcoholic hydroxyl group,
(3) The ligand is represented by the following formula (1)

(In the above formula, R represents an aromatic group or a nonionic aliphatic group having 4 to 7 carbon atoms) 1 or 2 selected from the group consisting of an α-amino acid and aminomethylbenzoic acid More than seeds,
(4) The ligand directly immobilized on the base material is bonded to the base material by an amide bond or a urethane bond via the amino group contained in the α-amino acid or aminomethylbenzoic acid represented by the above formula (1). Is fixed,
(5) The ligand immobilized on the substrate via the spacer is an amide bond or urethane bond via the amino group contained in the α-amino acid or aminomethylbenzoic acid represented by the above formula (1), And (6) a packing material for liquid chromatography, wherein the amount of the ligand fixed to the substrate is 30 mmol or more per liter (wet volume) of the packing material for liquid chromatography. The packing material for liquid chromatography according to claim 1, wherein the α-amino acid is selected from the group consisting of phenylalanine, tryptophan, leucine, norleucine and α-aminooctanoic acid. The liquid chromatograph according to claim 1 or 2, wherein the substrate is a chromatographic carrier selected from the group consisting of a natural polymer carrier, a synthetic polymer carrier, and an inorganic carrier. Graphic filler The packing material for liquid chromatography according to any one of claims 1 to 3, wherein the base material is a porous particle, and its exclusion limit molecular weight is 100,000 or more in terms of pullulan. The packing material for liquid chromatography according to any one of claims 1 to 4, wherein the polysaccharide is a polysaccharide having a weight average molecular weight of 10,000 or more or a derivative thereof having no anion exchange group. The alcoholic hydroxyl group in the substrate and the alcoholic hydroxyl group in the spacer are activated with 1,1-carbonylbis-1H-imidazole in an organic solvent, and then reacted with an amino group in the ligand in an organic solvent or a hydrous organic solvent. The ligand for liquid chromatography according to any one of claims 1 to 5, wherein the ligand is directly introduced into the base material through a urethane bond, and is introduced into the base material through the spacer. Manufacturing method. After introducing a carboxyl group into the base material and the spacer, carbodiimide is used as a catalyst to react with an amino group in the ligand, and the ligand is directly introduced into the base material through an amide bond, and the group is introduced via the spacer. The method for producing a filler for liquid chromatography according to any one of claims 1 to 5, wherein the filler is introduced into the material. Using the liquid chromatography filler according to any one of claims 1 to 5, a biopolymer is adsorbed under an acidic aqueous solution condition of pH 5 or less, and thereafter adsorbed under a weak basic condition of neutral to pH 9 or less. It was characterized by desorbing the biopolymer separated and purified to collect a method of recovering a biopolymer by liquid chromatography.