JP6897007B2 - 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 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 a drawback that it swells remarkably in an aqueous solution, the volume change due to the ionic strength of the solution and the volume change between the free acid type and the loaded type are 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 at the time of passing the liquid is large and the gel is consolidated by the passing of the liquid.

In order to overcome the drawbacks of crosslinked gels of hydrophilic natural polymers, for example, a complex in which a gel such as a natural polymer gel is held in the pores of a porous polymer is known in the field of peptide synthesis ( For example, see Patent Document 1). By using such a complex, the loading coefficient of the reactive substance is increased, and high-yield synthesis becomes possible. Further, since the gel is surrounded by a hard synthetic polymer material, there is an advantage that the volume does not change and the pressure of the flow-through passing through the column does not change even when used in the form of a column bed.

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). Diethylaminoethyl (DEAE) groups and the like are added to this gel in order to add sorption performance, and it is used for removing hemoglobin. Such a separating material has good liquid permeability in a column.

An ion exchanger of a hybrid copolymer in which the pores of a copolymer having a macronetwork structure are filled with a gel of a crosslinked copolymer synthesized from a monoma is known (see, for example, Patent Document 4). When the degree of cross-linking is low, the cross-linked copolymer gel has problems such as pressure loss and volume change, but by using a hybrid copolymer, the liquid passage characteristics are improved, the pressure loss is small, and the ion exchange capacity is improved. Leak behavior is improved.

Further, 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). Further, the synthesis of porous particles formed by copolymerization of glycidyl methacrylate and acrylic crosslinked monoma is known (see, for example, Patent Document 7).

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 Japanese Unexamined Patent Publication No. 2009-244067

However, the conventional separating material does not have a sufficient level of characteristics such as reduction of non-specific adsorption of protein and excellent column characteristics such as liquid permeability when used as a column. In addition, the liquid in which the protein is cultured may contain hydrophobic low molecular weight compounds such as fatty acids, and the hydrophobic low molecular weight compounds may be adsorbed on the column, thereby inhibiting the adsorption of the protein. ..

Therefore, the present invention is a separating material capable of reducing non-specific adsorption of proteins, excellent column properties such as liquid permeability when used as a column, and further suppressing adsorption of hydrophobic low molecular weight compounds, and the separation thereof. It is an object of the present invention to provide a column provided with a material.

The present invention provides the separating material described in the following [1] to [9] and the column described in the following [10].
[1] A separating material comprising: porous polymer particles and a coating layer containing a polysaccharide having a hydroxyl group and a water-soluble surfactant having a hydroxyl group, which coats at least a part of the surface of the porous polymer particles.
[2] The separating material according to [1], wherein the surfactant has a molecular weight of 20000 or less.
[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 mode diameter in the pore size distribution is 0.05 to 0.6 μm.
[5] The separating material according to any one of [1] to [4], which has an average particle size of 10 to 300 μm.
[6] The separating material according to any one of [1] to [5], wherein the polysaccharide is agarose or dextran.
[7] The separating material according to any one of [1] to [6], which comprises a coating layer of 30 to 450 mg per 1 g of porous polymer particles.
[8] The separating material according to any one of [1] to [7], wherein when the column is filled, the flow velocity of the liquid is 800 cm / h or more when the column pressure is 0.3 MPa.
[9] The separating material according to any one of [1] to [8], which has a cation exchange group or an anion exchange group.
[10] A column comprising the separating material according to any one of [1] to [9].

According to the present invention, it is possible to provide a separating material that reduces non-specific adsorption of proteins, has excellent column properties such as liquid permeability when used as a column, and can suppress adsorption of hydrophobic low molecular weight compounds. It will be possible.

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 comprises porous polymer particles and a coating layer containing a polysaccharide having a hydroxyl group and a water-soluble surfactant having a hydroxyl group, which coats at least a part of the surface of the porous polymer particles. To prepare. 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 which may contain soluble particles in the presence of a porosifying agent, and are obtained by, for example, conventional suspension polymerization, emulsion polymerization, or the like. Can be synthesized. The monoma is not particularly limited, but 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, and divinylnaphthalene; (poly) ethylene glycol di (meth) acrylate, (poly) propylene glycol di (meth) acrylate, and (poly) tetramethylene glycol. (Poly) alkylene glycol-based di (meth) acrylates such as di (meth) acrylates; trimethylolpropane tri (meth) acrylates, tetramethylolmethane tri (meth) acrylates, tetramethylolpropane tetra (meth) acrylates, pentaerythritol tris ( Trifunctional or higher functional (meth) acrylates such as meth) acrylates, 1,1,1-trishydroxymethylethanetri (meth) acrylates, 1,1,1-trishydroxymethylpropanetriacrylates; ethoxylated bisphenol A-based di (meth) acrylates. Meta) acrylate, propoxylated ethoxylated bisphenol A-based di (meth) acrylate, tricyclodecanedimethanol di (meth) acrylate, 1,1,1-trishydroxymethylethanedi (meth) acrylate, ethoxylated cyclohexanedimethanoldi. Examples thereof include di (meth) acrylates such as (meth) acrylate; diallyl phthalate and its isomers; trimethylolisocyanurate and its derivatives. Among these monomas, NK esters manufactured by Shin Nakamura Chemical Industry Co., Ltd. (A-TMPT-6P0, A-TMPT-3E0, A-TMM-3LMN, A-GLY series, A-9300, AD-TMP, AD-TMP -4CL, ATM-4E, A-DPH) and the like are commercially available. These polyfunctional monomas may be used alone or in combination of two or more. Among the above, it is preferable that the monoma contains divinylbenzene from the viewpoint of durability and swelling property. 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, the degree of swelling of the separating material can be lowered and the column pressure can be suppressed.

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-methyl 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 thereof include toluene, diethylbenzene, xylene, cyclohexane, octane, butyl acetate, dibutyl phthalate, methyl ethyl ketone, dibutyl ether, 1-hexanol, isoamyl alcohol, 2-octanol, decanol, lauryl alcohol and cyclohexanol. These porosifying agents may be used alone or in combination of two or more. When a porosifying agent having a property of easily taking in water is used, it tends to be easy to form large pores inside the particles.

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 or chain saturated C12 to C14 fatty acids, for example, sorbitan monooleate and sorbitan. Solbitan monoesters derived from monomillistate or coconut fatty acids; diglycerol monoesters of branched C16-C24 fatty acids, chain unsaturated C16-C22 fatty acids or chain saturated C12-C14 fatty acids, eg diglycerol monooleates ( For example, C18: 1 (18 carbons, 1 double bond) fatty acid diglycerol monoester), diglycerol monomillistate, diglycerol monoisostearate or coconut fatty acid diglycerol monoester; branched C16- Diglycerol monofatty ethers of C24 alcohols, chain unsaturated C16-C22 alcohols or chain saturated C12-C14 alcohols (eg, coconut fatty alcohols); and mixtures thereof.

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 with 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, diglyceride monoisostearate having a purity of preferably greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70%), diglyceride monomillistate (eg, preferably greater than purity). More than about 40%, more preferably more than about 50%, even more preferably more than about 70% sorbitan monomillistate), diglycerol cocoyl (eg, lauryl group, myristyl group, etc.) ether, or mixtures thereof. preferable.

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.

Soluble particles are particles that can be dissolved by, for example, an acid, an alkali, a solvent, or the like. Specifically, calcium carbonate, tricalcium phosphate, silica, polymer, metal colloid and the like can be used. From the viewpoint of ease of removal, it is preferable to use calcium carbonate or tricalcium phosphate. The particle size of the soluble particles is preferably 0.6 to 5 μm. It is preferably 1 to 5 μm from the viewpoint of improving the liquid permeability in the particles.

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 sulfone. 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 polyether-modified silicon-based nonionic surfactants such as agents, polyethylene oxide adducts of silicon, polypropylene oxide adductants, and fluorine-based 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, a water-soluble polymerization inhibitor 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 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 micro process server (Hitachi, Ltd.).

C.I. 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), C.I. V. To measure.

The pore volume (porosity) of the porous polymer particles is preferably 30% by volume or more and 70% by volume or less based on the total volume of the porous polymer particles, and more preferably 50% by volume or more and 70% by volume or less. preferable. The porous polymer particles preferably have pores having a pore diameter of 0.01 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 0.6 μm. When the pore diameter is 0.01 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 adsorbed substances to be separated tends to increase.

[Coating layer]
The coating layer according to the present embodiment contains a polysaccharide having a hydroxyl group and a water-soluble surfactant having a hydroxyl group. By coating the porous polymer particles with a polysaccharide having a hydroxyl group and a water-soluble surfactant having a hydroxyl group, it is possible to suppress the improvement of the column pressure and also to suppress the non-specific adsorption of proteins. , It becomes possible to improve the amount of protein adsorbed on the separating material. Furthermore, when the polysaccharide having a hydroxyl group is crosslinked, it becomes possible to further suppress an increase in column pressure.

(Polysaccharide with hydroxyl group)
Examples of the polysaccharide having a hydroxyl group include agarose, dextran, cellulose, chitosan and the like. From the viewpoint of protein adsorption ability, the polysaccharide having a hydroxyl group is preferably agarose or dextran. As the polysaccharide having a hydroxyl group, a polysaccharide having a weight average molecular weight of about 10,000 to 200,000 can be used. These polysaccharides having a hydroxyl group may be used alone or in combination of two or more.

The polysaccharide having a hydroxyl group may be a modified product modified by a hydrophobic group from the viewpoint of improving the interfacial adsorption ability. Examples of the hydrophobic group include an alkyl group having 1 to 6 carbon atoms and an aryl group having 6 to 10 carbon atoms. Examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group and the like. Examples of the aryl group having 6 to 10 carbon atoms include a phenyl group and a naphthyl group. A hydrophobic group is introduced by reacting a functional group (for example, an epoxy group) that reacts with a hydroxyl group and a compound having a hydrophobic group (for example, glycidylphenyl ether) with a polysaccharide having a hydroxyl group by a conventionally known method. Can be done.

(Method of forming a coating layer containing a polysaccharide having a hydroxyl group)
The coating layer according to the present embodiment can be formed by, for example, the method shown below.

First, a solution of a polysaccharide having a hydroxyl group is adsorbed on the surface of the porous polymer particles. The solvent of the solution is not particularly limited as long as it can dissolve a polysaccharide having a hydroxyl group, but water is the most common. The concentration of the polymer dissolved in the solvent is preferably 5 to 20 (mg / mL).

The porous polymer particles are impregnated with this solution. The impregnation method is to add porous polymer particles to a solution of a polysaccharide having a hydroxyl group and leave it for a certain period of time. The impregnation time varies depending on the surface condition of the porous body, but usually, if impregnated all day and night, the polymer concentration becomes an equilibrium state with the external concentration inside the porous body. Then, it is washed with a solvent such as water and alcohol to remove the polysaccharide having an unadsorbed hydroxyl group.

(Crosslinking)
Next, a cross-linking agent is added to cause a cross-linking reaction of the polysaccharide having a hydroxyl group adsorbed on the surface of the porous polymer particles to form a cross-linked product. At this time, the crosslinked body has a three-dimensional crosslinked network structure having a hydroxyl group.

The cross-linking agent has two or more functional groups active on hydroxyl groups such as epihalohydrin such as epichlorohydrin, dialdehyde compound such as glutaraldehyde, diisocyanate compound such as methylene diisocyanate, and glycidyl compound such as ethylene glycol diglycidyl ether. Examples include compounds. When a compound having an amino group such as chitosan is used as the polysaccharide having a hydroxyl group, dihalide such as dichlorooctane can also be used as a cross-linking agent.

A catalyst is usually used for this cross-linking reaction. As the catalyst, conventionally known catalysts can be appropriately used according to the type of the cross-linking agent. For example, when the cross-linking agent is epichlorohydrin or the like, an alkali such as sodium hydroxide is effective, and when the cross-linking agent is a dialdehyde compound, it is effective. Mineral acids such as hydrochloric acid are effective.

The cross-linking reaction with a cross-linking agent is usually carried out by adding the cross-linking agent to a system in which the separating material is dispersed and suspended in a suitable medium. The amount of the cross-linking agent added should be selected according to the performance of the separating material within the range of 0.1 to 100 mol times that of 1 mol of the monosaccharide in the polysaccharide having a hydroxyl group. Can be done. In general, when the amount of the cross-linking agent added is small, the coating layer tends to be easily peeled off from the porous polymer particles. Further, when the amount of the cross-linking agent added is excessive and the reaction rate with the polysaccharide having a hydroxyl group is high, the characteristics of the polysaccharide having a hydroxyl group as a raw material tend to be impaired.

The amount of the catalyst used varies depending on the type of cross-linking agent, but usually, in the polysaccharide having a hydroxyl group, if one unit of the monosaccharide forming the polysaccharide is 1 mol, 0.01 to 0.01 to this. It is used in the range of 10 mol times, more preferably 0.1 to 5 mol times.

For example, when the cross-linking reaction condition is a temperature condition, the temperature of the reaction system is raised, and when the temperature reaches the reaction temperature, a cross-linking reaction occurs.

As a medium for dispersing and suspending porous polymer particles impregnated with a solution of a polysaccharide having a hydroxyl group, a cross-linking reaction can be performed without extracting a polymer, a cross-linking agent, etc. from the impregnated polymer solution. Must be inactive. Specific examples thereof include water and alcohol.

The cross-linking reaction can usually be carried out at a temperature of 5 to 90 ° C. over 1 to 10 hours. The temperature of the cross-linking reaction is preferably 30 to 90 ° C.

After the cross-linking reaction is completed, the generated particles are filtered off, and then washed with a hydrophilic organic solvent such as water, methanol, ethanol, etc. to remove unreacted polymers, suspension medium, etc., to obtain porous polymer particles. A separating material is obtained in which at least a part of the surface is coated with a coating layer containing a polysaccharide having a hydroxyl group.

The separating material of the present embodiment preferably includes a coating layer of 30 to 450 mg per 1 g of the porous polymer particles, more preferably 50 to 400 mg of the coating layer, and further preferably 100 to 400 mg of the coating layer. preferable. When the ratio of the coating layer is 450 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. The amount of the coating layer can be measured by reducing the weight of thermal decomposition or the like.

The content of the polysaccharide having a hydroxyl group in the coating layer is preferably 80 to 99% by mass based on the total mass of the coating layer.

(Introduction of ion exchange group)
The separating material provided with the coating layer may have an ion exchange group, a ligand (protein A) and the like. The separating material can be used for ion exchange purification, affinity purification, etc. by introducing these through hydroxyl groups 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 monohalogenocarboxylic acids such as monohalogenoacetic acid and monohalogenopropionic acid, sodium salts thereof, and primary, secondary or tertiary amines and halogens having at least one alkyl halide group such as diethylaminoethyl chloride. Examples thereof include quaternary ammonium hydrochloride having an alkylated group. 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 to stand 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. for 0.5 to 12 hours. The type of alkyl halide used in the above reaction determines the ion exchange group to be imparted.

The separating material of the present embodiment may have a cation exchange group or an anion exchange group as the ion exchange group. Examples of the cation exchange group include a carboxy group and a sulfonic acid group. Examples of the anion exchange group include an amino group and a quaternary ammonium group.

As a method for introducing an amino group which is a weakly basic group as an ion exchange group, a mono-alkanolamine compound having at least one alkyl group in which a part of hydrogen atoms is replaced with a chlorine atom. , Di- or tri-alkylamine, mono-, di- or tri-alkanolamine, monoalkyl-monoalkanolamine, dialkyl-monoalkanolamine, monoalkyl-dialkanolamine and the like 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 such as epichlorohydrin to be quaternary. A method of converting to an ammonium group can be mentioned. Further, a quaternary ammonium hydrochloride or the like 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 salt is obtained. A method of adding a separating material to the saturated aqueous solution of Sulfite can be mentioned. The reaction conditions are preferably 30 to 90 ° C. for 1 to 10 hours.

On the other hand, as a method for introducing a sulfonic acid 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-propanesulton 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.

(Water-soluble surfactant having a hydroxyl group)
By adding a water-soluble surfactant having a hydroxyl group to the porous polymer particles having a hydroxyl group at the time of adsorption, after adsorption, or after a cross-linking treatment, the surface of the porous polymer particles is coated with the polysaccharide. The portion that is not covered can be covered, and the non-specific adsorption of the separating material of the present embodiment can be further suppressed.

The water-soluble surfactant of the present embodiment is not particularly limited as long as it has one or more hydroxyl groups and is water-soluble. In the present embodiment, the fact that the surfactant is water-soluble means that when the surfactant is dissolved in water at 25 ° C., the aqueous solution does not become cloudy and the surfactant is dispersed. Examples of such a surfactant include ester compounds such as lauric acid, myristic acid, palmitic acid, stearic acid and oleic acid of sucrose or polyglycerin, and polyethylene glycol chains such as polyvinyl alcohol and poly (oxyethylene) octylphenyl ether. Examples thereof include compounds having. These surfactants may be used alone or in combination of two or more.

The molecular weight of the water-soluble surfactant is preferably 20000 or less, more preferably 500 to 20000, and even more preferably 600 to 19000. When the molecular weight of the water-soluble surfactant is 20000 or less, it tends to be easily adsorbed on the portion not coated with the polysaccharide.

The molecular weight of the water-soluble surfactant can be measured by gel permeation chromatography (GPC), mass spectrometer or the like.

The content of the water-soluble surfactant in the coating layer is preferably 1 to 20% by mass based on the total mass of the coating layer.

(Method of forming a coating layer containing a water-soluble surfactant)
When such a water-soluble surfactant is adsorbed (coated) at the same time as the polysaccharide having a hydroxyl group, it is preferable to adjust the content so as to be 10% by mass or less based on the total mass of the coated polysaccharide. When it is 10% by mass or less, the previously coated polysaccharide can be adsorbed without peeling. Even when the water-soluble surfactant is adsorbed after the polysaccharide is adsorbed, it is preferable to adjust the content so that it is 10% by mass or less based on the total mass of the coated polysaccharide. When the surfactant is adsorbed after the cross-linking treatment of the polysaccharide, it is unlikely that the polysaccharide will be cross-linked and peeled off. Good. The adsorbed water-soluble surfactant may be further crosslinked. The cross-linking treatment can be carried out in the same manner as the above-mentioned polysaccharide having a hydroxyl group.

The mode diameter (mode of the pore diameter distribution, the most frequent pore diameter) and the specific surface area in the pore diameter distribution of the separating material or the porous polymer particles of the present embodiment are determined by a mercury intrusion measuring device (Autopore: manufactured by Shimadzu Corporation). It is a value measured in the above, and is measured as follows. About 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 (about 3 psia, equivalent to a pore diameter of about 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 0.05 to 5 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. 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.

The separating material of the present embodiment has a natural height in separating biopolymers such as proteins by introducing an ion exchange group and protein A into the surface of the separating material after cross-linking the coating layer on the porous polymer particles. It has the advantages of particles made of molecules or particles made of synthetic polymers. In particular, the porous polymer particles in the separating material of the present embodiment are obtained by the above-mentioned method, and therefore have durability and alkali resistance. In addition, the separating material of the present embodiment reduces non-specific adsorption of proteins and tends to cause protein adsorption / desorption. 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 flow velocity in the present specification represents the liquid flow rate when a stainless steel 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, it is preferable that the flow velocity of the liquid passing through the column is 800 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 flow velocity 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. For preparative or industrial chromatography use, it is preferably 10-100 μm to avoid extreme increases in column pressure.

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 mode diameter in the pore size distribution of the separating material is preferably 0.05 to 0.6 μm, more preferably 0.05 to 0.5 μm, and further preferably 0.1 to 0.5 μm. preferable. When the mode diameter in the pore diameter distribution is 0.05 μm or more, substances tend to enter into the pores easily, and when the mode diameter in the pore diameter distribution is 0.6 μm or less, the specific surface area becomes sufficient. Become.

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 adsorbed substances to be separated tends to increase. The upper limit of the specific surface area of the separating material ^{can be 500 m 2} / g or less.

The mode diameter, specific surface area, etc. in the pore size distribution of the separating material are adjusted by appropriately selecting a raw material for porous polymer particles, a porosifying agent, a polysaccharide having a hydroxyl group, a water-soluble surfactant having a hydroxyl group, and the like. can do.

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 which the ion exchange group is introduced has been described, but it can be used as the separating material without introducing the ion exchange group. Such a separating material 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 Porous Polymer Particle 1)
In a 500 mL three-necked flask, 16 g of divinylbenzene (manufactured by Nippon Steel & Sumitomo Metal Chemical Corporation, trade name "DVB960") having a purity of 96% as a monoma, 6 g of span 80 as a porosifying agent, and benzoyl peroxide as an initiator are 0. 64 g was added to prepare a dispersed phase. Further, an aqueous solution containing 0.5% by mass of polyvinyl alcohol was used as the continuous phase. After emulsifying these dispersions using a microprocess server (monoma concentration 25% by volume), the obtained emulsions are transferred to a flask and heated in a water bath at 80 ° C., and about 8 using a stirrer. Stirred for hours. 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 (FPIA-3000, manufactured by Sysmex Co., Ltd.), and the average particle size and particle size of C.I. V. The value was calculated. The results are shown in Table 1.

(Synthesis of Porous Polymer Particles 2)
The porous polymer particles 2 were synthesized in the same manner as in the synthesis of the porous polymer particles 1 except that the amount of the span 80 used was changed to 7 g.

(Synthesis of Porous Polymer Particles 3)
The porous polymer particles 3 were synthesized in the same manner as in the synthesis of the porous polymer particles 1 except that the amount of the span 80 used was changed to 8 g.

(Porous polymer particles 4)
Commercially available agarose particles (GE Healthcare Japan Co., Ltd., trade name "Capto DEAE") were used.

(Synthesis of Porous Polymer Particles 5)
Porous in the same manner as in the synthesis of porous polymer particles 1, except that 11.2 g of 2,3-dihydroxypropyl methacrylate was used as a monoma, 4.8 g of ethylene glycol dimethacrylate was used, and 5 g of span 80 was used as a porosifying agent. Quality polymer particles 5 were synthesized.

[Example 1]
<Adsorption and cross-linking of polysaccharides with hydroxyl groups>
4 g of sodium hydroxide and 0.14 g of glycidyl phenyl ether were added to 100 mL of an aqueous agarose solution (2% by mass) and reacted at 70 ° C. for 12 hours to introduce a phenyl group into the agarose. The obtained modified agarose was reprecipitated with isopropyl alcohol and washed. Next, the porous polymer particles 1 were added to a 20 mg / mL modified agarose aqueous solution at a ratio of 1 g to 70 mL of the aqueous solution, and the mixture was stirred at 55 ° C. for 24 hours to make the porous polymer particles 1 The modified agarose was adsorbed on the particles. After adsorption, it was filtered and washed with hot water. The amount of agarose coated per 1 g of the porous polymer particles was calculated from the concentration of modified agarose in the filtrate. The results are shown in Table 2.

The modified agarose was crosslinked as follows. First, 1 g of porous polymer particles 1 adsorbed with modified agarose was added to 35 mL of an aqueous solution having ethylene glycol diglycidyl ether and sodium hydroxide concentrations of 0.64 M and 0.4 M, respectively, at room temperature for 24 hours. Was stirred. Then, it was washed with 2 mass% hot sodium dodecyl sulfate aqueous solution and then with pure water.

<Adsorption of water-soluble surfactant>
The porous polymer particles crosslinked in 100 g of an aqueous solution of decaglyceriooleic acid ester (0.1% by mass), which is a water-soluble surfactant, were stirred for 3 hours to adsorb the water-soluble surfactant. After washing the obtained particles with water, the porous polymer particles in which the water-soluble surfactant was adsorbed were added to 35 mL of an aqueous solution in which the concentrations of ethylene glycol diglycidyl ether and sodium hydroxide were 0.64 M and 0.4 M, respectively. It was added at a ratio of 1 g and stirred at room temperature for 6 hours. Then, it was washed with hot water of 2 mass% sodium dodecyl sulfate aqueous solution, and then with pure water.

(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, and the mixture was stirred at room temperature for 24 hours. Then, centrifugation was performed to remove the supernatant. The amount of BSA adsorbed on the particles was calculated from the BSA concentration in the supernatant obtained by measuring the absorbance at 280 nm of the supernatant with a spectrophotometer. The results are shown in Tables 3 and 4.

(Evaluation of adsorption amount of hydrophobic low molecular weight compounds)
Using 0.5 g of the obtained particles as a model compound for a hydrophobic low molecular weight compound, the concentration of octylphenol ethoxylate having a hydrophobic group (average chain length 9.5 units, Wako Pure Chemical Industries, Ltd., trade name "Triton-X100") The mixture was added to 50 mL at 50 mg / mL and stirred at room temperature for 3 hours. Then, the supernatant was taken, and the amount of octylphenol ethoxylate adsorbed on the particles was calculated from the absorbance of the filtrate with a spectrophotometer. The concentration of octylphenol ethoxylate was confirmed by a spectrophotometer from the absorbance of the phenyl group near 269 nm. The results are shown in Tables 3 and 4.

<Introduction of ion exchange group>
Particles were filtered off from an aqueous suspension containing particles crosslinked with modified agarose and a water-soluble surfactant. The obtained particles (dry weight 20 g) were put into 200 mL of a 5 M aqueous sodium hydroxide solution and left at room temperature for 1 hour. Separately, a predetermined amount (60 g) of diethylaminoethyl chloride hydrochloride was added to 200 mL of water to prepare an aqueous solution of diethylaminoethyl chloride hydrochloride. This aqueous solution was added to the above sodium hydroxide aqueous solution, the temperature was raised to 70 ° C., and the reaction was carried out for 2 hours with stirring. After completion of the reaction, the product was filtered off and washed with water to obtain a (DEAE-modified) separating material having a diethylaminoethyl (DEAE) group. The mode diameter and specific surface area in the pore size distribution of the obtained separating material were measured by the mercury intrusion method. Further, after the obtained separating material was dried, the mass (coating amount) of the coating layer was measured by thermogravimetric analysis. The results are shown in Table 2.

(Column characterization)
The 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 at different flow velocities, 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 results are shown in Tables 3 and 4.

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 at 800 cm / h, and the BSA concentration at the column outlet was measured by UV measurement. The buffer was passed through until the BSA concentrations at the column inlet and outlet were the same, and diluted with 1 M NaCl Tris-hydrochloric acid buffer in 5 column volumes. The dynamic binding capacity at 10% breakthrough was calculated using the following equation. The results are shown in Tables 3 and 4.
q ₁₀ = c _f F (t ₁₀ −t ₀ ) / V _B
q ₁₀ : Dynamic binding volume at 10% breakthrough (mg / mL wet resin)
cf: Injecting BSA concentration F: Flow velocity (mL / min)
V _B : Bed volume (mL)
t ₁₀ : Time at 10% breakthrough t ₀ : BSA injection start time

[Example 2]
A separating material was prepared in the same manner as in Example 1 except that the sucrose oleic acid ester was used as the surfactant, 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 Triton-X100 was used as the water-soluble surfactant, 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 lauryl alcohol (EO) 15 glycidyl ether (Nagasechemtex Co., Ltd., molecular weight 971) was used as the water-soluble surfactant. Evaluated in the same way.

[Example 5]
A separating material was prepared in the same manner as in Example 1 except that polyvinyl alcohol (molecular weight 19000) was used as the water-soluble surfactant, 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 porous polymer particles 2 were used, 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 3 were used, 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 surfactant was not adsorbed, 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 polysaccharide was not adsorbed, and evaluated in the same manner as in Example 1.

[Comparative Example 3]
The porous polymer particles 4 were used as they were as a separating material and evaluated in the same manner as in Example 1.

[Comparative Example 4]
4 g of porous polymer particles 5 are added to 6 g of an aqueous solution prepared by dissolving 1 g of dextran (weight average molecular weight 150,000), 0.6 g of sodium hydroxide, and 0.15 g of sodium borohydride in distilled water, and the porous polymer particles are added. The pores of the particles were impregnated. The obtained dextran-impregnated porous polymer particles were added to 1 L of a 1 mass% toluene solution of ethyl cellulose and stirred to obtain a suspension. To the obtained suspension, 5 mL of epichlorohydrin was added, the temperature was raised to 50 ° C., and the mixture was stirred at this temperature for 6 hours to crosslink the dextran in the porous polymer particles. After the reaction, the suspension was collected by filtration to separate the gel, which was washed successively with toluene, ethanol and distilled water. A diethylaminoethyl group was introduced in the same manner as in Example 1 to prepare a separating material, which was evaluated in the same manner as in Example 1.

As shown in Tables 3 and 4, Examples 1 to 7 of the present application reduce non-specific adsorption of proteins, have excellent durability, and are hydrophobic low molecular weight compounds (for example, as compared with Comparative Examples 1 to 4). It was found that the adsorption of hydrophobic compounds having a molecular weight of 1000 or less) can be suppressed, and the column properties such as liquid permeability when used as a column are excellent.

Claims (9)

Porous polymer particles and
A coating layer containing a polysaccharide having a hydroxyl group and a water-soluble surfactant having a hydroxyl group, which covers at least a part of the surface of the porous polymer particles.
Equipped with a,
A separating material in which the water-soluble surfactant is an ester compound of sucrose or polyglycerin or a compound having a polyethylene glycol chain. The separating material according to claim 1, wherein the water-soluble surfactant has a molecular weight of 600 to 19000. The separating material according to claim 1 or 2 , wherein the specific surface area is 30 m ² / g or more. The separating material according to any one of claims 1 to 3 , which has an average particle size of 10 to 300 μm. The separating material according to any one of claims 1 to 4 , wherein the polysaccharide is agarose or dextran. The separating material according to any one of claims 1 to 5 , comprising the coating layer of 30 to 450 mg per 1 g of the porous polymer particles. The separating material according to any one of claims 1 to 6 , wherein when the column is filled, the flow velocity of the liquid is 800 cm / h or more when the column pressure is 0.3 MPa. The separating material according to any one of claims 1 to 7 , which has a cation exchange group or an anion exchange group. A column comprising the separating material according to any one of claims 1 to 8.