JP6834128B2 - Separator and column - Google Patents

Description

The present invention relates to a separating material and a column.

Conventionally, when biopolymers typified by proteins are separated and purified, generally, an ion exchanger based on a porous synthetic polymer and an ion exchanger based on a crosslinked gel of a hydrophilic natural polymer are used. Etc. are used. In the case of an ion exchanger based on a porous synthetic polymer, the volume change due to salt concentration is small, so when it is packed in a column and used for chromatography, it tends to have excellent pressure resistance during liquid passage. However, when this ion exchanger is used for separating proteins and the like, non-specific adsorption such as irreversible adsorption based on hydrophobic interaction occurs, so peak asymmetry occurs, or ion exchange occurs due to the hydrophobic interaction. There is a problem that the protein adsorbed on the body cannot be recovered while being adsorbed.

On the other hand, in the case of an ion exchanger whose parent is a crosslinked gel of a hydrophilic natural polymer typified by a polysaccharide such as dextran or agarose, there is an advantage that there is almost no non-specific adsorption of proteins. However, this ion exchanger has the disadvantages that it swells remarkably in an aqueous solution, the volume changes due to the ionic strength of the solution, and the volume change between the free acid type and the loaded type is large, and the mechanical strength is not sufficient. .. In particular, when the crosslinked gel is used for chromatography, there is a drawback that the pressure loss during liquid passage is large and the gel is consolidated by liquid passage.

In order to overcome the drawbacks of crosslinked gels of hydrophilic natural polymers, complexes in which gels such as natural polymer gels are held in the pores of porous polymers are known in the field of peptide synthesis (for example, See Patent Document 1). Further, a separating material in which an inorganic porous body such as Celite holds a xerogel such as a polysaccharide such as dextran or cellulose is known (see, for example, Patent Documents 2 and 3).

Further, there is known an ion exchanger of a hybrid copolyma in which the pores of a copolima having a macro network structure are filled with a crosslinked copolymer gel synthesized from a monoma (see, for example, Patent Document 4). A composite filler in which a crosslinked gel of a hydrophilic natural polymer having a huge network structure is filled in the pores of an organic synthetic polymer substrate has been proposed (see, for example, Patent Documents 5 and 6).

U.S. Pat. No. 4,965,289 U.S. Pat. No. 4,335,017 U.S. Pat. No. 4,336,161 U.S. Pat. No. 3,966,489 Japanese Unexamined Patent Publication No. 1-254247 U.S. Pat. No. 5,114,577

However, conventional separating materials are required to reduce non-specific adsorption of proteins and to have excellent column properties such as liquid permeability when used as a column.

Therefore, an object of the present invention is to provide a separating material that reduces non-specific adsorption of proteins and has excellent column characteristics such as liquid permeability when used as a column, and a column using the separating material. And.

The present invention provides the separating material according to the following [1] to [7].
[1] A separating material comprising porous polymer particles and a coating layer containing a polymer having a sugar residue, which covers at least a part of the surface of the porous polymer particles.
[2] The separating material according to [1], wherein the sugar residue is a group based on at least one sugar selected from the group consisting of monosaccharides, disaccharides, trisaccharides, tetrasaccharides and oligosaccharides.
[3] The separating material according to [1] or [2], which has a specific surface area of 30 m ^{2 / g or more.}
[4] The separating material according to any one of [1] to [3], wherein the porous polymer particles contain a polymer having a structural unit derived from divinylbenzene.
[5] The separating material according to any one of [1] to [4], wherein the average particle size of the porous polymer particles is 10 to 300 μm, and the coefficient of variation of the particle size is 5 to 15%.
[6] The separating material according to any one of [1] to [5], which comprises a coating layer of 30 to 400 mg per 1 g of porous polymer particles.
[7] The separating material according to any one of [1] to [6], wherein when the column is filled, the liquid passing speed is 1500 cm / h or more when the column pressure is 0.3 MPa.
[8] A column comprising the separating material according to any one of [1] to [7].

According to the present invention, it is possible to provide a separating material which reduces non-specific adsorption of proteins and has excellent column characteristics such as liquid permeability when used as a column, and a column provided with the separating material.

Hereinafter, preferred embodiments of the present invention will be described, but the present invention is not limited to these embodiments.

<Separation material>
The separating material of the present embodiment includes porous polymer particles and a coating layer containing a polymer having a sugar residue, which covers at least a part of the surface of the porous polymer particles. In the present specification, the “surface of the porous polymer particles” includes not only the outer surface of the porous polymer particles but also the surface of the pores inside the porous polymer particles. Further, in the present specification, (meth) acrylic acid means acrylic acid or methacrylic acid, and the same applies to other similar expressions such as (meth) acrylate.

(Porous polymer particles)
The porous polymer particles according to the present embodiment are particles obtained by curing a monoma in the presence of a porosifying agent, and can be synthesized by, for example, conventional suspension polymerization, emulsion polymerization, or the like. The monoma is not particularly limited, but for example, a styrene-based monoma, a (meth) acrylic-based monoma, or the like can be used. Specific examples of the monoma include the following polyfunctional monomas and monofunctional monomas.

Examples of the polyfunctional monoma include divinyl compounds such as divinylbenzene, divinylbiphenyl, divinylnaphthalene, and divinylphenanthrene; (poly) ethylene glycol di (meth) acrylate, (poly) propylene glycol di (meth) acrylate, and (poly). (Poly) alkylene glycol-based di (meth) acrylates such as tetramethylene glycol di (meth) acrylates; trimethylolpropane tri (meth) acrylates, tetramethylolmethane tri (meth) acrylates, tetramethylolpropane tetra (meth) acrylates, penta. Trifunctional or higher functional (meth) acrylates such as erithitol tri (meth) acrylate, 1,1,1-trishydroxymethylethanetri (meth) acrylate, 1,1,1-trishydroxymethylpropanetriacrylate; ethoxylated bisphenol A Di (meth) acrylate, propoxylated bisphenol A di (meth) acrylate, tricyclodecanedimethanol di (meth) acrylate, 1,1,1-trishydroxymethylethanedi (meth) acrylate, cyclohexane ethoxylated Di (meth) acrylates such as dimethanol di (meth) acrylate and neopentyl glycol di (meth) acrylate; diallyl phthalate and its isomers; trimethylolisocianurate and its derivatives can be mentioned. Among these monomas, for example, NK ester (A-TMPT-6P0, A-TMPT-3E0, A-TMM-3LMN, A-GLY series, A-9300, AD-TMP, manufactured by Shin Nakamura Chemical Industry Co., Ltd. AD-TMP-4CL, ATM-4E, A-DPH) and the like are commercially available. These polyfunctional monomas can be used alone or in combination of two or more. Among the above, from the viewpoint of elastic modulus, it is preferable that the monoma contains divinylbenzene. That is, the porous polymer particles preferably contain a polymer having a structural unit derived from divinylbenzene.

When divinylbenzene is contained as a monoma unit, the porous polymer particles preferably contain divinylbenzene in an amount of 50% by mass or more, more preferably 60% by mass or more, and more preferably 70% by mass or more based on the total mass of monoma. More preferred. By containing divinylbenzene in an amount of 50% by mass or more based on the total mass of monoma, it tends to have excellent alkali resistance and pressure resistance.

Examples of the monofunctional monoma include styrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, α-methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, 2,4-. Dimethylstyrene, pn-butylstyrene, pt-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn-dodecyl Styrene such as styrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene, 3,4-dichlorostyrene and derivatives thereof; methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate, acrylic acid Isobutyl, hexyl acrylate, 2-ethylhexyl acrylate, n-octyl acrylate, dodecyl acrylate, lauryl acrylate, stearyl acrylate, 2-chloroethyl acrylate, phenyl acrylate, methyl α-chloroacrylate, methyl methacrylate , Ethyl methacrylate, propyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, hexyl methacrylate, 2-ethylhexyl methacrylate, n-octyl methacrylate, dodecyl methacrylate, lauryl methacrylate, stearyl methacrylate, etc. (meth) ) Acrylic acid ester; vinyl ester such as vinyl acetate, vinyl propionate, vinyl benzoate, vinyl butyrate; N-vinyl compound such as N-vinylpyrrole, N-vinylcarbazole, N-vinylindole, N-vinylpyrrolidone; Fluorinated monomas such as vinyl oxide, vinylidene fluoride, tetrafluoroethylene, hexafluoropropylene, trifluoroethyl acrylate, tetrafluoropropyl acrylate; and conjugated diene such as butadiene and isoprene. These monofunctional monomas may be used alone or in combination of two or more. Among the above, it is preferable to use styrene from the viewpoint of excellent acid resistance and alkali resistance. Further, a styrene derivative having a functional group such as a carboxyl group, an amino group, a hydroxyl group or an aldehyde group can also be used.

Examples of the porosifying agent include aliphatic or aromatic hydrocarbons, esters, ketones, ethers, alcohols, etc., which are organic solvents that promote phase separation during polymerization and promote porosification of particles. Be done. Specific examples of the porosifying agent include toluene, xylene, cyclohexane, octane, butyl acetate, dibutyl phthalate, methyl ethyl ketone, dibutyl ether, 1-hexanol, 2-octanol, decanol, lauryl alcohol, cyclohexanol and the like. .. These porosifying agents may be used alone or in combination of two or more.

The porosifying agent can be used in an amount of 0 to 200% by mass based on the total mass of the monoma. The porosity of the porous polymer particles can be controlled by the amount of the porosity agent. Furthermore, the size and shape of the pores of the porous polymer particles can be controlled by the type of the porosifying agent.

Water used as a solvent can also be used as a porosifying agent. When water is used as a porosifying agent, the particles can be made porous by dissolving an oil-soluble surfactant in a monoma and absorbing water.

Examples of the oil-soluble surfactant used for porosification include sorbitan monoesters of branched C16 to C24 fatty acids, chain unsaturated C16 to C22 fatty acids, and chain saturated C12 to C14 fatty acids, such as sorbitan monolaurate and sorbitan. Solbitan monoester derived from monooletate, sorbitan monomillistate or coconut fatty acid; diglycerol monoester of branched C16-C24 fatty acid, chain unsaturated C16-C22 fatty acid or chain saturated C12-C14 fatty acid, eg di Diglycerol monoester of glycerol monooleate (eg, C18: 1 (18 carbons, 1 double bond) fatty acid), diglycerol monomillistate, diglycerol monoisostearate or diglycerol mono of coconut fatty acid Esters; diglycerol monofatty acids of branched C16-C24 alcohols (eg, gelve alcohol), chain unsaturated C16-C22 alcohols or chain saturated C12-C14 alcohols (eg palm fatty alcohols); and mixtures thereof. Can be mentioned.

Of these, sorbitan monolaurate (eg, SPAN® 20, purity preferably greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70% sorbitan monolaurate). Rate), sorbitan monooleate (eg, SPAN® 80, purity preferably greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70% sorbitan monooleate. ), Diglycerol monooleate (eg, diglycerol monooleate having a purity of preferably greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70%), diglycerol monoisostearate. (For example, diglycerol monoisostearate having a purity of preferably greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70%), diglycerol monomillistate (purity preferably greater than about 40). %, More preferably greater than about 50%, even more preferably greater than about 70% sorbitan monomillistate), diglycerol cocoyl (eg, lauryl group, myristyl group, etc.) ether, or mixtures thereof. ..

These oil-soluble surfactants are preferably used in the range of 5 to 80% by mass with respect to the total mass of the monoma. When the content of the oil-soluble surfactant is 5% by mass or more, the stability of the water droplet is sufficient, so that a large single pore is easily formed. Further, when the content of the oil-soluble surfactant is 80% by mass or less, the porous polymer particles are more likely to retain their shape after polymerization.

Examples of the aqueous medium used in the polymerization reaction include water, a mixed medium of water and a water-soluble solvent (for example, a lower alcohol), and the like. The aqueous medium may contain a surfactant. As the surfactant, any of anionic, cationic, nonionic and zwitterionic surfactants can be used.

Examples of the anionic surfactant include fatty acid oils such as sodium oleate and potassium castor oil, alkyl sulfates such as sodium lauryl sulfate and ammonium lauryl sulfate, alkylbenzene sulfonates such as sodium dodecylbenzene sulfonate, and alkylnaphthalene sulfates. Dialkyl sulfosuccinates such as acid salts, alkane sulfonates, sodium dioctyl sulfosuccinate, alkernyl succinate (dipotassium salt), alkyl phosphate ester salts, naphthalene sulfonate formalin condensates, polyoxyethylene alkyl phenyl ether sulfate Examples thereof include salts, polyoxyethylene alkyl ether sulfates such as sodium polyoxyethylene lauryl ether sulfate, and polyoxyethylene alkyl sulfates.

Examples of the cationic surfactant include alkylamine salts such as laurylamine acetate and stearylamine acetate, and quaternary ammonium salts such as lauryltrimethylammonium chloride.

Examples of nonionic surfactants include hydrocarbon-based nonionic surfactants such as polyethylene glycol alkyl ethers, polyethylene glycol alkylaryl ethers, polyethylene glycol esters, polyethylene glycol sorbitan esters, polyalkylene glycol alkylamines, and amides. Examples thereof include polyethylene oxide adducts of silicon, polyether-modified silicon nonionic surfactants such as polypropylene oxide adducts, and fluorine nonionic surfactants such as perfluoroalkyl glycols.

Examples of the amphoteric ion-based surfactant include hydrocarbon surfactants such as lauryldimethylamine oxide, phosphate ester-based surfactants, and phosphite ester-based surfactants.

As the surfactant, one type may be used alone or two or more types may be used in combination. Among the above-mentioned surfactants, anionic surfactants are preferable from the viewpoint of dispersion stability during monoma polymerization.

Examples of the polymerization initiator added as needed include benzoyl peroxide, lauroyl peroxide, orthochlorobenzoyl peroxide, benzoyl orthomethoxy peroxide, 3,5,5-trimethylhexanoyl peroxide, and tert-butylper. Organic peroxides such as oxy-2-ethylhexanoate, di-tert-butyl peroxide; 2,2'-azobisisobutyronitrile, 1,1'-azobiscyclohexanecarbonitrile, 2,2' Examples thereof include azo compounds such as −azobis (2,4-dimethylvaleronitrile). The polymerization initiator can be used in the range of 0.1 to 7.0 parts by mass with respect to 100 parts by mass of the monoma.

The polymerization temperature can be appropriately selected depending on the type of the monoma and the polymerization initiator. The polymerization temperature is preferably 25 to 110 ° C, more preferably 50 to 100 ° C.

In the synthesis of porous polymer particles, a polymer dispersion stabilizer may be added in order to improve the dispersion stability of the particles.

Examples of the polymer dispersion stabilizer include polyvinyl alcohol, polycarboxylic acid, celluloses (hydroxyethyl cellulose, carboxymethyl cellulose, methyl cellulose, etc.), polyvinylpyrrolidone, and an inorganic water-soluble polymer compound such as sodium tripolyphosphate is also used in combination. can do. Of these, polyvinyl alcohol or polyvinylpyrrolidone is preferable. The amount of the polymer dispersion stabilizer added is preferably 1 to 10 parts by mass with respect to 100 parts by mass of the monoma.

In order to prevent the monoma from polymerizing alone, water-soluble polymerization inhibitors such as nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble B vitamins, citric acid, and polyphenols may be used.

The average particle size of the porous polymer particles is preferably 300 μm or less, more preferably 150 μm or less, still more preferably 100 μm or less. The average particle size of the porous polymer particles is preferably 10 μm or more, more preferably 30 μm or more, still more preferably 50 μm or more, from the viewpoint of improving the liquid permeability.

The coefficient of variation (CV) of the particle size of the porous polymer particles is preferably 3 to 15%, more preferably 5 to 15%, and 5 to 5% from the viewpoint of improving the liquid permeability. It is more preferably 10%. C. V. As a method of reducing the amount of waste, a monodisperse can be mentioned by using an emulsifying device such as a microprocess server (Hitachi, Ltd.).

C. of the average particle size and particle size of the porous polymer particles or separating material. V. Can be obtained by the following measurement method.
1) The particles are dispersed in water (including a dispersant such as a surfactant) using an ultrasonic disperser to prepare a dispersion liquid containing 1% by mass of porous polymer particles.
2) Using a particle size distribution meter (Sysmex Flow, manufactured by Sysmex Corporation), an image of about 10,000 particles in the dispersion liquid shows the average particle size and particle size of C.I. V. To measure.

The pore volume of the porous polymer particles is preferably 30% by volume or more and 70% by volume or less, and more preferably 40% by volume or more and 70% by volume or less based on the total volume of the porous polymer particles. The porous polymer particles preferably have pores having a pore diameter of 0.05 μm or more and less than 0.6 μm, that is, macropores (macropores). The pore size of the porous polymer particles is more preferably 0.1 μm or more and less than 0.5 μm. When the pore diameter is 0.05 μm or more, the substance tends to easily enter the pores, and when the pore diameter is less than 0.6 μm, the specific surface area becomes sufficient. These can be adjusted by the above-mentioned porosifying agent.

The specific surface area of the porous polymer particles ^{is preferably 30 m 2} / g or more. From the viewpoint of higher practicality, the specific surface area is ^{more preferably 35 m 2} / g or more, and further preferably 40 m ² / g or more. When the specific surface area is 30 m ² / g or more, the amount of the substance to be separated tends to be large.

(Coating layer)
The coating layer according to this embodiment contains a polymer having a sugar residue. By coating the porous polymer particles with a polymer having a sugar residue, it is possible to suppress the improvement of the column pressure, the non-specific adsorption of the protein, and the protein adsorption amount of the separating material. Can be equal to or higher than that in the case of using a natural polymer. The coating layer can be formed on the surface of the porous polymer particles, for example, by adsorbing a monoma having a sugar residue on the surface of the porous polymer particles and then polymerizing the monoma.

By coating the porous polymer particles with a polymer having a sugar residue, it is possible to reduce nonspecific adsorption of proteins and prepare a separating material having excellent column characteristics such as liquid permeability. The separating material of the present embodiment can achieve a high dynamic adsorption amount even when the column flow rate is high.

A monoma having a sugar residue is a vinyl or (meth) acrylic monoma having a structural unit based on a sugar. As the sugar, it is preferable to use at least one sugar selected from the group consisting of monosaccharides, disaccharides, trisaccharides, tetrasaccharides and oligosaccharides. In the case of a monoma having such a saccharide residue, since the steric hindrance does not become too large, the progress of polymerization is difficult to be suppressed, and the flexibility of the main chain of the polymer tends to be good.

As the monosaccharide, it is preferable to use hexose having 6 carbon atoms constituting the sugar. Examples of the hexose include allose, altrose, glucose, mannose, growth, idose, galactose, tarose, bushicose, fructose, sorbose and tagatose.

Examples of the disaccharide include sucrose, lactose, maltose, trehalose, turanose and cellobiose. Examples of trisaccharides include raffinose, melezitose and maltotriose. Examples of the tetrasaccharide include acarbose and stachyose.

An oligosaccharide is a saccharide in which a plurality of monosaccharides are bound by glycosidic bonds, but in the present specification, an oligosaccharide in which 5 to 20 monosaccharide molecules are bound is defined as an oligosaccharide. The molecular weight of the oligosaccharide is preferably 1000 to 3000. Examples of oligosaccharides include agarose and dextran.

The method for synthesizing the monoma having a sugar residue is not particularly limited, and for example, it can be synthesized as follows. When a sugar having a reducing end is used, an amino group is introduced into the reducing end with ammonium hydrogencarbonate or the like, and then the sugar residue is reacted with a vinyl monoma or a (meth) acrylic monoma having a functional group that reacts with the amino group. It is possible to synthesize a monoma having. In addition, a monoma having a sugar residue can be synthesized by reacting a vinyl monoma or (meth) acrylic monoma having an amino group or an azide group with a sugar having a reducing end. When a sugar having an amino group or a carboxyl group is used, a monoma having a sugar residue can be synthesized by reacting a vinyl monoma or a (meth) acrylic monoma having a functional group that reacts with the amino group or the carboxyl group. it can.

The method for forming a coating layer on the porous polymer particles using the monoma having a sugar residue is not particularly limited, and for example, it can be carried out as follows.

A coating layer can be formed by graft-polymerizing a monoma having a sugar residue on the surface of the porous polymer particles or inside the pores. Examples of the graft polymerization method include living radical polymerization methods such as atom transfer radical polymerization (ATRP) and reversible addition cleavage chain transfer polymerization (RAFT), radiation graft polymerization methods, and methods of introducing an initiator to carry out polymerization in a solution. Can be used. Above all, it is preferable to use living radical polymerization from the viewpoint of adjusting the coating amount and density.

When graft polymerization is carried out using a solvent, the concentration may be adjusted so that the amount of monoma having sugar residues is 5 to 500 mg with respect to 1 mL of the solvent. The solvent that can be used is not particularly limited as long as it does not inhibit graft polymerization and can uniformly dissolve monomas having sugar residues.

The amount of the coating layer is preferably 30 to 400 mg, more preferably 50 to 400 mg, and even more preferably 100 to 400 mg with respect to 1 g of the porous polymer particles. When the ratio of the coating layer is 400 mg or less with respect to 1 g of the porous polymer particles, the coating layer can be made into a thin film, and the liquid permeability when used as a column tends to be further improved. Further, when the ratio of the coating layer is 30 mg or more with respect to 1 g of the porous polymer particles, the amount of protein adsorbed tends to be further increased.

(Introduction of ion exchange group)
The separating material provided with the coating layer can be used for ion exchange purification, affinity purification, etc. by introducing an ion exchange group, a ligand (protein A), or the like via a hydroxyl group or the like on the surface. Examples of the method for introducing an ion exchange group include a method using an alkyl halide compound.

Examples of the alkyl halide compound include a monohalogenocarboxylic acid and its sodium salt, a primary or secondary or tertiary amine having at least one alkyl halide group, a hydrochloride thereof, and a quaternary having at least one alkyl halide group. Examples include ammonium salts. Examples of the monohalogenocarboxylic acid include monohalogenoacetic acid and monohalogenopropionic acid. Examples of the tertiary amine having at least one alkyl halide group include diethylaminoethyl chloride. These alkyl halide compounds are preferably bromides or chlorides. The amount of the alkyl halide compound used is preferably 0.2% by mass or more with respect to the total mass of the separating material to which the ion exchange group is imparted.

For the introduction of the ion exchange group, it is effective to use an organic solvent in order to promote the reaction. Examples of the organic solvent include alcohols such as ethanol, 1-propanol, 2-propanol, 1-butanol, isobutanol, 1-pentanol and isopentanol.

Normally, the ion exchange group is introduced into the hydroxyl group on the surface of the separating material. Therefore, the wet particles are drained by filtration or the like, immersed in an alkaline aqueous solution having a predetermined concentration, left for a certain period of time, and then water-organic. In a solvent mixing system, the above alkyl halide compound is added and reacted. This reaction is preferably carried out at a temperature of 40 to 90 ° C. under reflux for 0.5 to 12 hours. The type of alkyl halide used in the above reaction determines the applied ion exchange group.

As a method for introducing an amino group which is a weakly basic group as an ion exchange group, at least one of the above alkyl halide compounds is substituted with an alkyl halide group, mono-, di -Or a method of reacting tri-alkylamine, mono-alkyl-mono-alkanolamine, di-alkyl-mono-alkanolamine, mono-alkyl-di-alkanolamine, etc., or at least one of the alkanol groups is halogenated. Methods for reacting mono-, di- or tri-alkanolamines, mono-alkyl-mono-alkanolamines, di-alkyl-mono-alkanolamines, mono-alkyl-di-alkanolamines, etc. substituted with alkanol groups, etc. Can be mentioned. The amount of these alkyl halide compounds used is preferably 0.2% by mass or more with respect to the total mass of the separating material. The reaction conditions are preferably 40 to 90 ° C. and 0.5 to 12 hours.

As a method for introducing a quaternary ammonium group of a strongly basic group as an ion exchange group, first, a tertiary amino group is introduced, and the tertiary amino group is reacted with an alkyl halide group-containing compound such as epichlorohydrin, and 4 A method of converting to a quaternary ammonium group can be mentioned. Further, a quaternary ammonium halide such as quaternary ammonium chloride may be reacted with the separating material.

Examples of the method for introducing a carboxy group, which is a weakly acidic group, as an ion exchange group include a method in which a monohalogenocarboxylic acid such as monohalogenacetic acid or monohalogenopropionic acid or a sodium salt thereof is reacted as the alkyl halide compound. .. The amount of these alkyl halide compounds used is preferably 0.2% by mass or more with respect to the total mass of the separating material into which the ion exchange group is introduced.

As a method for introducing a sulfonic acid group, which is a strongly acidic group, as an ion exchange group, a glycidyl compound such as epichlorohydrin is reacted with a separating material, and a sulfite such as sodium sulfite or sodium bisulfite or a sulfite is obtained. A method of adding a separating material to the saturated aqueous solution of the above can be mentioned. The reaction conditions are preferably 30 to 90 ° C. for 1 to 10 hours.

On the other hand, as a method of introducing an ion exchange group, a method of reacting 1,3-propane sultone with a separating material in an alkaline atmosphere can also be mentioned. It is preferable to use 1,3-propane sultone in an amount of 0.4% by mass or more based on the total mass of the separating material. The reaction conditions are preferably 0 to 90 ° C. for 0.5 to 12 hours.

The average pore diameter, specific surface area, and porosity of the separating material or the porous polymer particles according to the present embodiment are values measured by a mercury intrusion measuring device (Autopore: manufactured by Shimadzu Corporation), and are as follows. Measure. Approximately 0.05 g of a sample is added to a standard 5 mL powder cell (stem volume 0.4 mL) and measured under the condition of an initial pressure of 21 kPa (approximately 3 psia, equivalent to a pore diameter of approximately 60 μm). The mercury parameters are set to the device default mercury contact angle of 130 ° and mercury surface tension of 485 days / cm. Further, each value is calculated by limiting the pore diameter to the range of 0.1 to 3 μm.

The separating material of the present embodiment is suitable for separating proteins by electrostatic interaction and for affinity purification. For example, the separating material of the present embodiment is added to a mixed solution containing a protein, and only the protein is adsorbed on the separating material by electrostatic interaction, and then the separating material is filtered off from the solution to obtain a salt concentration. When added to a high aqueous solution, the protein adsorbed on the separating material can be easily desorbed and recovered. In addition, the separating material of the present embodiment can also be used in column chromatography.

As the biopolymer that can be separated using the separating material of the present embodiment, a water-soluble substance is preferable. Specifically, it is a protein such as blood protein such as serum albumin and immunoglobulin, an enzyme existing in the living body, a protein physiologically active substance produced by biotechnology, a DNA, and a biopolymer such as a peptide having physiological activity. Yes, preferably the molecular weight is 2 million or less, more preferably 500,000 or less. In addition, it is necessary to select the properties, conditions, etc. of the separating material according to the isoelectric point, ionization state, etc. of the protein according to a known method. As a known method, for example, the method described in JP-A-60-169427 can be mentioned.

In the separating material of the present embodiment, by introducing an ion exchange group and protein A on the surface of the separating material, particles made of a natural polymer or particles made of a synthetic polymer can be separated in the separation of biopolymers such as proteins. Has advantages. In particular, since the porous polymer particles in the separating material of the present embodiment have a coating layer containing a polymer having sugar residues, the separating material of the present embodiment reduces nonspecific adsorption of proteins and reduces protein. Adsorption and desorption tend to occur easily. Further, the separating material of the present embodiment tends to have a large adsorption amount (dynamic adsorption amount) of proteins and the like under the same flow velocity.

The liquid passing speed in the present specification represents the liquid passing speed when a stainless column having a diameter of 7.8 × 300 mm is filled with the separating material of the present embodiment and the liquid is passed through. When the separating material of the present embodiment is packed in a column, the liquid passing speed is preferably 800 cm / h or more, and more preferably 1500 cm / h or more when the column pressure is 0.3 MPa. When protein is separated by column chromatography, the flow rate of the protein solution or the like is generally in the range of 400 cm / h or less, but when the separating material of the present embodiment is used, it is used for normal protein separation. It can be used at a liquid passing speed of 800 cm / h or more, which is faster than that of the separating material.

The average particle size of the separating material of the present embodiment is preferably 10 to 300 μm, and for use in preparative or industrial chromatography, 10 to 100 μm in order to avoid an extreme increase in column internal pressure. Is more preferable, and 50 to 100 μm is further preferable.

When the separating material of the present embodiment is used as a column filler in column chromatography, it is excellent in operability because there is almost no volume change in the column regardless of the nature of the eluate used.

The 5% compressive deformation elastic modulus of the separating material of the present embodiment can be calculated as follows.
Using a micro-compression tester (manufactured by Fisher), the load and load when the particles are compressed to 50 mN by the smooth end face (50 μm × 50 μm) of a square column at a load load rate of 1 mN / sec under room temperature (25 ° C.) conditions. Measure the compressive displacement. From the obtained measured values, the compressive elastic modulus (5% K value) when the particles are compressionally deformed by 5% can be obtained by the following formula. Further, the load at the point where the displacement amount during the measurement changes most is defined as the fracture strength (mN).
5% K value (MPa) = (3/2 ^1/2 ) ・ F ・ S ^-3/2・ R- ^{1 / 2}
5% K value (MPa) = (3/2 ^1/2 ) ・ F ・ S ^-3/2・ R- ^{1 / 2}
F: Load (mN) when porous polymer particles are compressed and deformed by 5%
S: Compressive displacement (mm) when the porous polymer particles are compressed and deformed by 5%
R: Radius (mm) of porous polymer particles

The compressive elastic modulus (5% K value) when the separating material is compressionally deformed by 5% is preferably 100 to 1000 MPa, more preferably 150 to 1000 MPa, and even more preferably 170 to 1000 MPa.

If the compressive elastic modulus is too small, the flexibility of the porous polymer particles becomes high, the particles are easily deformed when the liquid is flowed in the column, and the column pressure is likely to be high.

The pore volume of the separating material is preferably 30% by volume or more and 70% by volume or less, and more preferably 40% by volume or more and 70% by volume or less based on the total volume of the separating material. The separating material has pores having an average pore diameter of 0.05 to 0.6 μm, that is, macropores (macropores). The average pore size is preferably 0.1 to 0.5 μm. When the pore diameter is 0.05 μm or more, the substance tends to easily enter the pores, and when the pore diameter is 0.6 μm or less, the specific surface area becomes sufficient.

The specific surface area of the separating material ^{is preferably 30 m 2} / g or more. From the viewpoint of higher practicality, the specific surface area is ^{more preferably 35 m 2} / g or more, and further preferably 40 m ² / g or more. When the specific surface area is 30 m ² / g or more, the amount of the substance to be separated tends to be large.

The compressive elastic modulus, average pore size, specific surface area, etc. of the separating material can be adjusted by appropriately selecting a raw material for porous polymer particles, a porosifying agent, a polymer having sugar residues, and the like.

The separating material of this embodiment can be used for a column. That is, the column of the present embodiment includes the above-mentioned separating material. In this embodiment, the separating material in the form of introducing an ion exchange group has been described, but it can be used as a separating material without introducing an ion exchange group. Such separators can be used, for example, in gel filtration chromatography.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to these Examples.

(Synthesis of monomas with sugar residues)
Put 10 g (55 mmol) of glucose (manufactured by Tokyo Chemical Industry Co., Ltd.) in a 300 mL beaker, add 30 g of an aqueous solution containing ammonium hydrogencarbonate 5 times every 24 hours to dissolve it, and then stir at 37 ° C for 96 hours while keeping it open. It was. Then, 200 mL of distilled water was added and the water was distilled off to 20 mL, and then 150 mL of water was added to concentrate the water to 10 mL. This operation was repeated until the ammonia odor disappeared, and after freeze-drying, glucose having an amino group introduced was obtained.

5.75 g of glucose into which an amino group has been introduced is dissolved in 50 mL of a 1 M aqueous solution of KOH, and 10.49 g (67.0 mmol) of 2-isocyanatoethyl methacrylate (Showa Denko KK, trade name "Karenzu MOI") is added. The mixture was vigorously stirred for 12 hours while being kept at 3 ° C. Then, the precipitated white solid was filtered and solid-liquid separated, and the solid phase was freeze-dried to obtain a white substance (a). Further, in order to remove unreacted 2-isocyanatoethyl methacrylate, the white substance (a) was washed 4 times with 20 mL of diethyl ether, and then a mixed solution of water and methanol (volume ratio 2: 1) l0 mL. Was dissolved in, and the mixture was added dropwise to a mixed solution of 80 mL of diethyl ether and 20 mL of acetone and cooled. The precipitated solid was filtered through a G3 glass filter to obtain a monosaccharide methacrylate having a glucose-based sugar residue.

In the same manner, disaccharide methacrylate having a sugar residue based on cellobiose (manufactured by Tokyo Chemical Industry Co., Ltd.), tetrasaccharide methacrylate having a sugar residue based on cellotetraose (manufactured by Tokyo Chemical Industry Co., Ltd.), and agarooligosaccharide ( Oligosaccharide methacrylates having sugar residues based on Ina Foods, molecular weight 2300) were synthesized respectively.

(Synthesis of Porous Polymer Particle 1)
16 g of divinylbenzene (Nippon Steel & Sumikin Chemical Co., Ltd., trade name "DVB960") having a purity of 96%, 9.6 g of Span80, and 0.64 g of benzoyl peroxide were added to a 500 mL three-necked flask to prepare a dispersed phase. An aqueous polyvinyl alcohol solution (0.5% by mass) was used as the continuous phase. After emulsifying this aqueous solution using a microprocess server (25% by volume), the obtained emulsion was transferred to a flask and stirred using a stirrer for about 8 hours while heating in a water bath at 80 ° C. The obtained particles were filtered and then washed with acetone to obtain porous polymer particles 1. The particle size of the porous polymer particles 1 was measured with a flow-type particle size measuring device, and the average particle size and the particle size of C.I. V. The value was calculated. The results are shown in Table 1.

(Synthesis of Porous Polymer Particles 2)
Porous polymer particles 2 were synthesized in the same manner as in the synthesis of porous polymer particles 1 except that the amount of Span 80 used was changed to 8.0 g.

(Synthesis of Porous Polymer Particles 3)
Porous polymer particles 3 were synthesized in the same manner as in the synthesis of porous polymer particles 1 except that the amount of Span80 used was changed to 6.4 g.

(Synthesis of Porous Polymer Particles 4)
Porous polymer particles 4 were synthesized in the same manner as in the synthesis of porous polymer particles 1 except that the amount of Span 80 used was changed to 6.0 g.

(Synthesis of Porous Polymer Particles 5)
Porous polymer particles 5 were synthesized in the same manner as in the synthesis of porous polymer particles 1 except that 9.6 g of Span 80 and 2 g of octanol were used.

(Example 1)
<Formation of coating layer>
After stirring 0.2 g of dopamine hydrochloride, 0.49 g of 2-bromoisobutyl bromide and 0.21 g of triethylamine in a 10 mL solution for 3 hours, 1 g of porous polymer particles were added, and then 10 mM Tris-HCl buffer (pH 8). .5) was added in an amount of 90 mL, and the mixture was stirred at room temperature for 24 hours and then washed with water to obtain particles in which the living initiation group was introduced into the porous polymer particles 1.

1 g of the obtained particles, 1.3 g of monosaccharide methacrylate, 67 mg of copper (II) bromide, trisdimethylaminoethylamine and 80 g of water were mixed, nitrogen bubbling was performed, and then 20 g of water in which 119 mg of ascorbic acid was dissolved was added. Then, polymerization was carried out for 5 hours. The polymerized particles were filtered and washed to obtain polymer-coated particles having sugar residues. The coating amount of the polymer having a sugar residue was measured by thermogravimetric analysis of the obtained particles. The results are shown in Table 2.

<Evaluation of non-specific adsorption capacity of protein>
0.5 g of the obtained particles were put into 50 mL of phosphate buffer (pH 7.4) having a BSA (Bovine Serum Albumin) concentration of 20 mg / mL, stirred at room temperature for 24 hours, and then the supernatant was removed by centrifugation. The amount of BSA adsorbed on the particles was calculated from the BSA concentration of the filtrate with a spectrophotometer. The concentration of BSA was confirmed by a spectrophotometer from the absorbance at 280 nm. The results are shown in Table 2.

<Introduction of ion exchange group>
After removing water from 2 g of the obtained particles by centrifugation, the mixture was dispersed in 16 mL of a 3.5 M aqueous diethylaminoethyl chloride hydrochloride solution, the temperature was raised to 70 ° C., 16 mL of a 5 M aqueous NaOH solution was added, and the mixture was stirred. While reacting for 2 hours. After completion of the reaction, the mixture was filtered and washed with water to obtain a separating material having a diethylaminoethyl (DEAE) group as an ion exchange group. The average pore size and specific surface area of the obtained separating material were measured by the mercury intrusion method. The results are shown in Table 2.

<Column characterization>
The obtained separating material was filled as a slurry (solvent: methanol) having a concentration of 30% by mass with a stainless column having a diameter of 7.8 × 300 mm for 15 minutes. Then, water was passed through the column while changing the flow velocity, the relationship between the flow velocity and the column pressure was measured, and the liquid flow velocity (linear flow velocity) at 0.3 MPa was measured.
The amount of dynamic adsorption was measured as follows. A 20 mmol / L Tris-hydrochloric acid buffer (pH 8.0) was passed through the column by 10 column volumes. Then, a 20 mmol / L Tris-hydrochloric acid buffer having a BSA concentration of 2 mg / mL was passed through, and the BSA concentration at the column outlet was measured by UV measurement. The buffer was passed through the buffer until the BSA concentrations at the column inlet and outlet were matched, and the mixture was diluted with 1 M NaCl Tris-hydrochloric acid buffer in a volume of 5 columns. The amount of dynamic adsorption at 10% breakthrough was calculated using the following formula. The results are shown in Table 2.
q ₁₀ = c _f F (t ₁₀ −t ₀ ) / V _B
q ₁₀ : Dynamic adsorption amount at 10% breakthrough (mg / mL wet resin)
cf: Injecting BSA concentration F: Flow velocity (mL / min)
V _B : Bed volume (mL)
time at t ₁₀ : 10% breakthrough (min)
t ₀ : BSA injection start time (min)

<5% compressive deformation elastic modulus>
The 5% compressive deformation elastic modulus of the separating material was measured by the method described above. The results are shown in Table 2.

(Example 2)
A separating material was prepared in the same manner as in Example 1 except that the monosaccharide methacrylate was changed to disaccharide methacrylate, and evaluated in the same manner as in Example 1.

(Example 3)
A separating material was prepared in the same manner as in Example 1 except that the monosaccharide methacrylate was changed to tetrasaccharide methacrylate, and evaluated in the same manner as in Example 1.

(Example 4)
A separating material was prepared in the same manner as in Example 1 except that the monosaccharide methacrylate was changed to oligosaccharide methacrylate, and evaluated in the same manner as in Example 1.

(Example 5)
A separating material was prepared in the same manner as in Example 1 except that the amount of monosaccharide methacrylate used was changed to 1.0 g, and evaluated in the same manner as in Example 1.

(Example 6)
A separating material was prepared in the same manner as in Example 1 except that the amount of monosaccharide methacrylate used was changed to 0.7 g, and evaluated in the same manner as in Example 1.

(Example 7)
A separating material was prepared in the same manner as in Example 1 except that the porous polymer particles 1 were changed to the porous polymer particles 2, and evaluated in the same manner as in Example 1.

(Example 8)
A separating material was prepared in the same manner as in Example 1 except that the porous polymer particles 1 were changed to the porous polymer particles 3, and evaluated in the same manner as in Example 1.

(Example 9)
A separating material was prepared in the same manner as in Example 1 except that the porous polymer particles 1 were changed to the porous polymer particles 4, and evaluated in the same manner as in Example 1.

(Comparative Example 1)
A separating material was prepared in the same manner as in Example 1 except that the monosaccharide methacrylate was changed to hydroxyethyl methacrylate, and evaluated in the same manner as in Example 1.

(Comparative Example 2)
A separating material was prepared in the same manner as in Example 1 except that the monosaccharide methacrylate was changed to glycerin monomethacrylate, and evaluated in the same manner as in Example 1.

(Comparative Example 3)
Porous polymer particles 1 are added to a 20 mg / mL polyvinyl alcohol (Nippon Synthetic Chemical Industry Co., Ltd., trade name "GH-17") aqueous solution at a concentration of 70 mL / particle g, stirred at 55 ° C. for 24 hours, and then filtered. The particles were recovered and washed with hot water to obtain particles adsorbed with polyvinyl alcohol.

Then, the polyvinyl alcohol adsorbed on the particles was crosslinked as follows. In an aqueous solution having a concentration of ethylene glycol diglycidyl ether of 0.64 M and a concentration of sodium hydroxide of 0.4 M, particles adsorbed with polyvinyl alcohol were added at a ratio of 1 g to 35 mL of the aqueous solution, and the mixture was brought to room temperature for 24 hours. Stirred. Then, it was washed with 2 mass% hot sodium dodecyl sulfate aqueous solution and then with pure water. Using the obtained particles, a separating material having a DEAE group as an ion exchange group was prepared by the same operation as in Example 1, and evaluated in the same manner as in Example 1.

(Comparative Example 4)
A separating material was prepared in the same manner as in Comparative Example 3 except that polyvinyl alcohol was changed to methylcellulose 50 (Wako Pure Chemical Industries, Ltd.), and evaluated in the same manner as in Example 1.

(Comparative Example 6)
Commercially available agarose particles (GE Healthcare Japan Co., Ltd., trade name "Capto DEAE") were used as they were as a separating material, and evaluated in the same manner as in Example 1.

It was found that the separating material provided with the coating layer containing the polymer having a sugar residue had a very high liquid passing rate at 0.3 MPa and maintained a high value even when the dynamic adsorption amount was 1500 cm / h or more. In addition, it was confirmed that non-specific adsorption can be reduced by coating with a polymer having a sugar residue.

Claims (5)

It comprises a porous polymer particle containing a polymer having a structural unit derived from divinylbenzene, and a coating layer containing a polymer having a sugar residue, which covers at least a part of the surface of the porous polymer particle.
Ri der specific surface area of ^30m 2 / g or more,
A separating material in which the polymer having the sugar residue is a polymer of vinyl or (meth) acrylic monoma having a structural unit based on the sugar. The separating material according to claim 1, wherein the sugar residue is a group based on at least one sugar selected from the group consisting of monosaccharides, disaccharides, trisaccharides, tetrasaccharides and oligosaccharides. The separating material according to claim 1 or 2, wherein the porous polymer particles have an average particle size of 10 to 300 μm and a coefficient of variation of the particle size of 5 to 15%. The separating material according to any one of claims 1 to 3, comprising the coating layer of 30 to 400 mg per 1 g of the porous polymer particles. A column comprising the separating material according to any one of claims 1 to 4.