JP2020044496A - Separation material and filling column - Google Patents

Abstract

To provide a separation material having a coating layer containing a hydrophilic polymer having a hydroxy group, wherein a dynamic adsorption can be further improved.SOLUTION: Disclosed is a separation material having porous polymer particles, and a coating layer that coats at least part of the surface of the porous polymer particles. The coating layer has a hydrophilic polymer having a hydroxy group, and a graft chain bound to the hydrophilic polymer. The graft chain is a polymer containing a monomer unit having an ion exchange group.SELECTED DRAWING: None

Description

  The present invention relates to a separation material and a packed column.

  As a separation material for separating and purifying a biopolymer represented by a protein, generally, a porous polymer particle containing a synthetic polymer or a particle having a crosslinked gel of a hydrophilic natural polymer as a matrix is used. .

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 JP-A-1-254247 U.S. Pat. No. 5,114,577 JP 2009-244067 A

  A separation material having a coating layer containing a hydrophilic polymer having a hydroxyl group is excellent in that non-specific adsorption of proteins is small. However, this separation material has room for improvement in terms of dynamic adsorption.

  An object of one aspect of the present invention is to further improve the dynamic adsorption amount of a separation material having a coating layer containing a hydrophilic polymer having a hydroxyl group.

  One aspect of the present invention relates to a separation material including porous polymer particles and a coating layer that covers at least a part of the surface of the porous polymer particles. The coating layer includes a hydrophilic polymer having a hydroxyl group and a graft chain bonded to the hydrophilic polymer. The graft chain is a polymer containing a monomer unit having an ion exchange group.

  Another aspect of the present invention relates to a packed column, comprising: a column tube; and the separation material filled in the column tube.

  The separation material according to one aspect of the present invention can exhibit a sufficiently high dynamic adsorption amount in addition to good liquid permeability and reduced nonspecific adsorption. The separation material of the present invention can be used for separating and purifying biopolymers by, for example, electrostatic interaction or affinity purification.

  The separation material according to some embodiments can exhibit an excellent effect in operability that, when packed in a column tube, there is almost no volume change in the column tube regardless of the properties of the eluate.

  Hereinafter, some embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

  The separation material according to one embodiment is a particle including a porous polymer particle and a coating layer that covers at least a part of the surface of the porous polymer particle. The coating layer includes a hydrophilic polymer having a hydroxyl group and a graft chain bonded to the hydrophilic polymer. The graft chain is a polymer containing a monomer unit having an ion exchange group. In the present specification, the “surface of the porous polymer particles” also includes the surfaces of the pores formed inside the porous polymer particles.

(Porous polymer particles)
The porous polymer particles according to one embodiment can be particles containing a crosslinked polymer. The crosslinked polymer has, for example, a monomer unit derived from a crosslinkable monomer having an aromatic group and two or more vinyl groups bonded to the aromatic group. Since the porous polymer particles formed by the crosslinked polymer can have a high elastic modulus, they are not easily broken when packed in a column tube.

  The average particle size of the porous polymer particles may be 500 μm or less, 300 μm or less, or 100 μm or less. The average particle size of the porous polymer particles may be 10 μm or more, 30 μm or more, or 50 μm or more. When the average particle size becomes smaller, the pressure of the packed column filled with the separating material may increase.

  The coefficient of variation (CV) of the particle diameter of the porous polymer particles may be 5 to 15% or 5 to 10% from the viewpoint of liquid permeability. As a method of reducing the variation coefficient of the particle diameter, there is a method of monodispersing droplets containing a monomer using an emulsifying apparatus such as a microprocess server (manufactured by Hitachi, Ltd.).

The average particle size and the variation coefficient of the particle size of the porous polymer particles can be determined by the following measurement methods.
1) The particles are dispersed in water (including a dispersant such as a surfactant) to prepare a dispersion containing 1% by mass of the particles.
2) Using a particle size distribution meter (Sysmex Flow, manufactured by Sysmex), the average particle size and the coefficient of variation of the particle size are measured based on an image of about 10,000 particles in the dispersion.

  The ratio (porosity) of the pore volume to the total volume (including the pore volume) of the porous polymer particles or the separating material may be 30% or more and 70% or less. Most of the pores of the porous polymer may be macropores having a diameter of 0.1 μm or more and 0.5 μm or less. In other words, the mode diameter in the pore size distribution of the porous polymer particles may be 0.1 to 0.5 μm. The porosity may be 40% or more and 70% or less, and the mode diameter in the pore size distribution may be 0.05 μm or more and less than 0.3 μm. When the mode diameter in the pore size distribution is 0.1 μm or more, the substance tends to easily enter the pores. When the mode diameter in the pore diameter distribution is 0.5 μm or less, a sufficient specific surface area tends to be obtained. The porosity and the mode diameter can be adjusted by a porosity agent described later.

The specific surface area of the porous polymer particles may be 30 m ² / g or more, 35 m ² / g or more, or 40 m ² / g or more. When the specific surface area is small, the amount of adsorbed substances to be separated tends to be relatively small.

  The mode diameter (or average pore diameter), specific surface area, and porosity in the pore diameter distribution of the porous polymer particles are values measured by a mercury intrusion measuring device (Autopore: manufactured by Shimadzu Corporation). These can be measured as follows. A sample of about 0.05 g is taken in a standard 5 cc powder cell (stem volume 0.4 cc), and the pore distribution is measured under the conditions 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 a device default mercury contact angle of 130 degrees and a mercury surface tension of 485 dynes / cm. Each value is calculated limited to the range of the pore diameter of 0.05 to 5 μm.

  The porous polymer particles containing a crosslinked polymer are, for example, a monomer component containing a crosslinkable monomer and, if necessary, other monomers, a porogen, and a suspension component in a reaction solution containing an aqueous medium. Can be synthesized by a method including polymerization.

  The crosslinkable monomer may be, for example, a divinyl compound such as divinylbenzene, divinylbiphenyl, divinylnaphthalene, and divinylphenanthrene. These crosslinkable monomers may be used alone or in combination of two or more. In view of durability, acid resistance, and alkalinity, the crosslinkable monomer may be divinylbenzene.

  Monofunctional monomers may be polymerized with crosslinkable monomers. As the monofunctional monomer, for example, 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, and 3,4-dichlorostyrene, and derivatives thereof. These may be used alone or in combination of two or more. From the viewpoint of acid resistance and alkali resistance, the monofunctional monomer may be styrene. Styrene derivatives having a functional group such as a carboxyl group, an amino group, a hydroxyl group, and an aldehyde group can also be used.

  A porosifier is a component that promotes phase separation of the particles during polymerization, thereby forming porous polymer particles. One example of a porosifying agent is an organic solvent. Examples of organic solvents that can be used as a porosifying agent include aliphatic or aromatic hydrocarbons, esters, ketones, ethers, and alcohols. The porosifying agent is at least selected from the group consisting of, for example, toluene, xylene, cyclohexane, octane, butyl acetate, dibutyl phthalate, methyl ethyl ketone, dibutyl ether, 1-hexanol, 2-octanol, decanol, lauryl alcohol, and cyclohexanol. One can be included.

  The amount of the porogen may be 0 to 300% by mass based on the total amount of the crosslinkable monomer and other monomers. The porosity of the porous polymer particles can be controlled by the amount of the porogen. The size and shape of the pores of the porous polymer particles can be controlled by the type of the porosifying agent.

  The aqueous medium may include water. This water may function as a porogen. For example, when an oil-soluble surfactant is added to a reaction solution, particles containing a monomer and an oil-soluble surfactant are formed, and the particles can promote phase separation in the particles by absorbing water. By removing one phase from the phase-separated particles, the particles are made porous.

  Examples of oil-soluble surfactants include sorbitan monoesters formed from branched C16-C24 fatty acids, chain unsaturated C16-C22 fatty acids or chain saturated C12-C14 fatty acids and sorbitan, branched C16-C24 fatty acids, chains Diglycerol monoaliphatic ethers formed from linear unsaturated C16-C22 fatty acids or chain saturated C12-C14 fatty acids and diglycerol, and diglycerol monoaliphatic ethers. Examples of sorbitan monoesters include sorbitan monooleate, sorbitan monomyristate, and sorbitan monoester of coconut fatty acid. Examples of diglycerol monoester include diglycerol monooleate, diglycerol monomyristate, diglycerol monoisostearate, and diglycerol monoester of coconut fatty acid. Examples of diglycerol monoaliphatic ethers include branched C16-C24 alcohols (e.g., Guerbet alcohol), linear unsaturated C16-C22 alcohols or linear saturated C12-C14 alcohols (e.g., coconut fatty alcohol) and diglycerol. And monoaliphatic ethers formed from These can be used alone or in combination of two or more.

  The amount of oil-soluble surfactant may be from 5 to 80% by weight, based on the total weight of the monomers. As a result, droplets containing the monomer and the oil-soluble surfactant are easily formed stably.

  The aqueous medium contains water or a mixed solvent of water and a water-soluble solvent (eg, lower alcohol). The aqueous medium may include a surfactant. The surfactant may be an anionic, cationic, nonionic or zwitterionic surfactant.

  Examples of anionic surfactants include fatty acid oils such as sodium oleate and castor oil, alkyl sulfates such as sodium lauryl sulfate and ammonium lauryl sulfate, alkylbenzene sulfonates such as sodium dodecylbenzenesulfonate, and alkylnaphthalene sulfones. Acid, alkane sulfonate, dialkyl sulfosuccinate such as sodium dioctyl sulfosuccinate, alkenyl succinate (dipotassium salt), alkyl phosphate ester salt, naphthalene sulfonic acid formalin condensate, polyoxyethylene alkyl phenyl ether sulfate Salts, polyoxyethylene alkyl ether sulfates such as sodium polyoxyethylene lauryl ether sulfate, and polyoxyethylene alkyl sulfate salts.

  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 the nonionic surfactant include, for example, hydrocarbon nonionic surfactants such as polyethylene glycol alkyl ether, polyethylene glycol alkyl aryl ether, polyethylene glycol ester, polyethylene glycol sorbitan ester, polyalkylene glycol alkylamine or amide, and silicone polyethylene. Examples include polyether-modified silicone-based nonionic surfactants such as oxide adducts and polypropylene oxide adducts, and fluorine-based nonionic surfactants such as perfluoroalkyl glycols.

  Examples of the amphoteric surfactant include a hydrocarbon surfactant such as lauryl dimethylamine oxide, a phosphate ester surfactant, and a phosphite ester surfactant.

  The surfactants may be used alone or in combination of two or more. Among the above surfactants, anionic surfactants are preferable from the viewpoint of dispersion stability during polymerization of the monomer.

  The reaction solution for suspension polymerization may contain a polymerization initiator. Examples of the polymerization initiator include benzoyl peroxide, lauroyl peroxide, orthochlorobenzoyl peroxide, orthomethoxybenzoyl peroxide, 3,5,5-trimethylhexanoyl peroxide, tert-butylperoxy-2-ethylhexano Organic peroxides such as ethate and di-tert-butyl peroxide; 2,2′-azobisisobutyronitrile, 1,1′-azobiscyclohexanecarbonitrile, 2,2′-azobis (2,4- Azo compounds such as dimethylvaleronitrile). The amount of the polymerization initiator may be 0.1 to 7.0 parts by mass with respect to 100 parts by mass of the monomer.

  The reaction solution may contain a dispersion stabilizer in order to improve the dispersion stability of the particles containing the monomer. Examples of the dispersion stabilizer include polyvinyl alcohol, polycarboxylic acid, celluloses (such as hydroxyethylcellulose, carboxymethylcellulose, and methylcellulose), and polyvinylpyrrolidone. These may be used in combination with an inorganic water-soluble polymer compound such as sodium tripolyphosphate. The dispersion stabilizer may be polyvinyl alcohol or polyvinyl pyrrolidone. The amount of the dispersion stabilizer may be 1 to 10 parts by mass based on 100 parts by mass of the monomer.

  The reaction solution for suspension polymerization may contain a water-soluble polymerization inhibitor such as nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble vitamins B, citric acid, and polyphenols.

  The polymerization temperature for suspension polymerization can be appropriately selected according to the type of the monomer and the polymerization initiator. The polymerization temperature may be between 25 and 110C, or between 50 and 100C.

(Coating layer)
The coating layer covers at least a part of the surface of the porous polymer particles. The coating layer contains a hydrophilic polymer having a hydroxyl group. By providing the coating layer covering the surface of the porous polymer particles, it is possible to suppress an increase in column pressure. The coating layer containing a hydrophilic polymer having a hydroxyl group can suppress non-specific adsorption of proteins. The hydrophilic polymer may be cross-linked.

  The amount of the coating layer containing the hydrophilic polymer and the graft chain may be 30 to 750 mg per 1 g of the porous polymer particles.

  The 5% weight loss temperature of the separating material under a nitrogen atmosphere may be 200 ° C. or more. When the 5% weight loss temperature is 200 ° C. or higher, the dynamic adsorption amount tends to be particularly large. The upper limit of the 5% weight loss temperature is not particularly limited, but is usually about 300 ° C. The 5% weight loss temperature is the temperature at which the mass of the sample has decreased by 5% from the initial stage in thermogravimetric analysis in which the mass change of the sample is measured while increasing the temperature.

  The hydrophilic polymer having a hydroxyl group may have two or more hydroxyl groups. The hydrophilic polymer having a hydroxyl group may be, for example, a polysaccharide or a modified product thereof. Polysaccharides include agarose, dextran, cellulose, polyvinyl alcohol, and chitosan. A modified polysaccharide refers to a polysaccharide into which a hydrophobic group has been introduced. By introducing a hydrophobic group, the interfacial adsorption ability of the coating layer can be improved. 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, and a propyl group. Examples of the aryl group having 6 to 10 carbon atoms include a phenyl group and a naphthyl group. The hydrophobic group can be introduced by reacting a functional group that reacts with a hydroxyl group (for example, an epoxy group) and a compound having a hydrophobic group (for example, glycidyl phenyl ether) with a hydrophilic polymer having a hydroxyl group. The weight average molecular weight of the hydrophilic polymer having a hydroxyl group may be about 10,000 to 2,000,000.

  The coating layer containing a hydrophilic polymer having a hydroxyl group may be, for example, that a solution of a hydrophilic polymer having a hydroxyl group is adsorbed on the surface of the porous polymer particles, and that the hydrophilic layer adsorbed on the surface of the porous polymer particles has a high hydrophilicity. And crosslinking the molecules. Although the solvent of the solution of the hydrophilic polymer is not particularly limited, water is usually the most common. The concentration of the hydrophilic polymer in the solution may be 5 to 50 (mg / ml). The porous polymer particles are immersed in the solution of the hydrophilic polymer and left for a certain period of time in this state to impregnate the pores of the porous body with the solution of the hydrophilic polymer. The standing time for the impregnation varies depending on the surface condition of the porous polymer particles, but if left for one day, the concentration of the hydrophilic polymer is in equilibrium with the external concentration inside the porous polymer particles. Thereafter, washing with a solvent such as water or alcohol is performed to remove the unadsorbed hydrophilic polymer. At this stage, the solution of the hydrophilic polymer is usually held in the pores. Next, a crosslinking agent is added to crosslink the hydrophilic polymer to form a crosslinked gel of the hydrophilic polymer. The crosslinking reaction is performed in a reaction solution obtained by dispersing a porous polymer particle holding a solution of a hydrophilic polymer in pores in a medium and adding a crosslinking agent to the obtained dispersion. Can be. The reaction solution may be heated if necessary. The crosslinked gel has a three-dimensional crosslinked network structure.

  As the cross-linking agent, for example, epihalohydrin such as divinyl sulfone and epichlorohydrin, dialdehyde compounds such as glutaraldehyde, methylene diisocyanate, diisocyanate compounds, and hydroxyl-functional functional groups such as glycidyl compounds such as ethylene glycol diglycidyl ether and the like. Compounds having two or more are exemplified. When the hydrophilic polymer has an amino group like chitosan, a dihalide such as dichlorooctane can be used as a crosslinking agent. A catalyst is usually used for the crosslinking reaction. The catalyst varies depending on the type of the crosslinking agent. For example, when the crosslinking agent is epichlorohydrin, an alkali such as sodium hydroxide is effective, and when the crosslinking agent is a dialdehyde compound, a mineral acid such as hydrochloric acid is effective.

  When the hydrophilic polymer is a polysaccharide, the amount of the cross-linking agent may be in the range of 0.001 to 100 times as much as 1 mol of one unit of the monosaccharide. In general, when the amount of the crosslinking agent is small, the coating layer tends to be easily peeled off from the porous polymer particles. If the amount of the crosslinking agent is excessive, the properties of the hydrophilic polymer may be impaired. When the hydrophilic polymer is a polysaccharide, the amount of the catalyst at the time of the crosslinking reaction is 0.01 to 10 times, or 0.1 to 10 times, assuming that 1 unit of the monosaccharide forming the polysaccharide is 1 mol. The ratio may be 1 to 5 times.

  The dispersion medium for the cross-linking reaction must be difficult to extract the hydrophilic polymer and the cross-linking agent from the solution of the hydrophilic polymer, and must be inert to the cross-linking reaction. Specific examples of the medium include water and alcohol.

  The crosslinking reaction is usually performed at a temperature of 5 to 90 ° C. for 1 to 10 hours. The temperature of the crosslinking reaction may be from 30 to 90C. After the cross-linking reaction is completed, the particles taken out by filtration are washed with a hydrophilic solvent such as water, methanol, and ethanol to remove the unreacted hydrophilic polymer and the medium. Coated particles are obtained. The coating layer is usually also formed in the pores. The amount of the coating layer can be measured by a weight loss due to thermal decomposition, anthrone method or the like.

  The coating layer further includes a graft chain bonded to the hydrophilic polymer. This graft chain is a polymer containing a monomer unit having an ion exchange group. By introducing a graft chain having an ion exchange group into the coating layer, the amount of dynamic adsorption of proteins can be greatly improved. The monomer for deriving the monomer unit having an ion exchange group may be a compound that is soluble in a reaction solvent used for forming a graft chain from the viewpoint of preventing nonspecific adsorption.

The ion exchange group can be a cation exchange group or an anion exchange group. The cation exchange group may be at least one selected from a sulfo group (—SO ₃ H), a carboxyl group (—COOH), and a salt thereof. The anion exchange group may be at least one selected from a diethylaminoethyl group, a dimethylaminoethyl group, and a trimethylammonium group.

  Examples of monomers for deriving a monomer unit having a cation exchange group include acrylic acid, methacrylic acid, sodium methacrylate, trimethylsilyl methacrylate, potassium 3-sulfopropyl methacrylate, 2-acrylamide-2-methylpropanesulfonic acid, and vinyl. Sulfonic acid, sodium vinyl sulfonate, allyl sulfonic acid, sodium allyl sulfonate, sodium 2-methyl-2-propene-1-sulfonate, 2- (methacryloyloxy) ethyl phosphate, 2-methacryloyloxyethyl succinic acid, 2-metachloroxyethyl acid phosphate, and oxoethane acid.

  Examples of the monomer that derives a monomer unit having an anion exchange group include 2- (dimethylamino) ethyl methacrylate, 2- (diethylamino) ethyl methacrylate, (3-acrylamidopropyl) trimethylammonium chloride, and trimethyl-2-metachloro. Yloxyethylammonium chloride, 2- (methacryloyloxy) ethyl phosphate 2- (trimethylammonio) ethyl, 3- (methacrylamido) propyltrimethylammonium chloride, 2- (methacryloyloxy) ethyltrimethylammonium methyl sulfate, and 3 -(Methacrylamido) propyltrimethylammonium methyl sulfate.

  The graft chain may be a homopolymer of a monomer unit having an ion exchange group, or a copolymer further containing another monomer unit. The proportion of the monomer unit having an ion exchange group in the graft chain may be 50 to 100 mol% based on the amount of all the monomer units in the graft chain. Thereby, the effect of improving the amount of dynamic adsorption can be obtained particularly remarkably.

  The graft chain may further include a monomer unit derived from a monomer having a hydroxyl group. Examples of the monomer that derives a monomer unit having a hydroxyl group include 2-hydroxyethyl (meth) acrylate, 3-hydroxybutyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, and 2- (2-methacrylate). Hydroxyethoxy) ethyl, 2,3-dihydroxypropyl methacrylate, polyethylene glycol (meth) acrylate, and N- (2-hydroxyethyl) acrylamide.

  The weight average molecular weight of the graft chain having an ion exchange group may be 0.01000 to 500,000. In the present specification, the weight average molecular weight means a value in terms of standard polystyrene, which is determined by gel permeation chromatography.

  The graft chain can be formed by polymerizing a monomer component containing a monomer having an ion exchange group in the presence of the coated particles having a coating layer. For example, by graft polymerization of a monomer component in a reaction solution containing a coated particle, a monomer component, a radical polymerization initiator containing a peroxide, and a reaction solvent, a graft chain bonded to a hydrophilic polymer is formed. Is done. The introduction of the graft chain having an ion exchange group can be confirmed by, for example, XPS before and after the reaction, thermogravimetric analysis, quantification of the amount of the ion exchange group, and the like. When the hydrophilic polymer is a polysaccharide, usually, a hydrogen atom bonded to the carbon at the α-position of the primary alcohol of the sugar chain is extracted to generate a radical, and the graft chain grows from the radical.

  The hydrophilic polymer to which the graft chain is bonded is represented, for example, by the formula: R-CHX-OH. In the formula, X represents a graft chain, and R represents a hydrophilic polymer. R may include a graft chain other than X in the formula. When a graft chain is introduced into a polysaccharide, a structure represented by this formula is often formed.

  Examples of peroxides as radical polymerization initiators include sodium persulfate, potassium persulfate, ammonium persulfate, benzoyl peroxide, and hydrogen peroxide. From the viewpoint of the density of the graft chain and the molecular weight of the graft chain, the concentration of the peroxide may be 0.01 to 30% by weight based on the mass of the reaction solution.

  The reaction solution may contain a reducing agent to promote the polymerization. Examples of reducing agents include sodium hydrogen carbonate, sodium bisulfite, and sodium sulfite. The pH of the reaction solution can be adjusted by adding a reducing agent. From the viewpoint of inhibiting hydrolysis of the hydrophilic polymer, the pH of the reaction solution may be 4 to 9. From the viewpoint of the density of the graft chain and the length of the graft chain, the concentration of the reducing agent may be 0.01 to 30% by weight based on the mass of the reaction solution.

  The reaction solvent for forming the graft chain may be a solvent that dissolves the monomer component. The reaction solvent may be, for example, water, alcohol, or a mixed solvent of water and an organic solvent.

  The reaction for forming a graft chain is performed, for example, under the conditions of a temperature of 10 to 90 ° C and a reaction time of 0.5 to 12 hours.

  The coating layer may further have an additional ion-exchange group bonded to the hydrophilic polymer in a form other than the graft chain, in addition to the ion-exchange group introduced into the graft chain. This additional ion exchange group is introduced, for example, by reacting a compound having an ion exchange group and a halogenated alkyl group with a hydroxyl group of a hydrophilic polymer. Examples of the compound having an ion exchange group and a halogenated alkyl group include monohalogenocarboxylic acids such as monohalogenoacetic acid and monohalogenopropionic acid, and sodium salts thereof, and salts having at least one halogenated alkyl group such as diethylaminoethyl chloride. Primary, secondary and tertiary amines, and quaternary ammonium hydrochlorides having alkyl halide groups are mentioned. The halogenated alkyl group-containing compound may be bromide or chloride. The amount of the compound having an ion exchange group and a halogenated alkyl group may be 0.2% by mass or more based on the total mass of the separation material before the additional ion exchange group is introduced.

  Organic solvents may be used to promote the reaction to introduce additional ion exchange groups. The organic solvent may be an alcohol such as ethanol, 1-propanol, 2-propanol, 1-butanol, isobutanol, 1-pentanol, and isopentanol. For example, the separating material into which the graft chains have been introduced is drained by filtration or the like in a wet state, then immersed in an alkaline aqueous solution having a predetermined concentration, and left for a predetermined time. Thereto, a solution containing a mixed solvent of water-organic solvent and a compound having an ion exchange group and a halogenated alkyl group is added and reacted. This reaction is performed, for example, under the conditions of a temperature of 40 to 90 ° C and a reaction time of 0.5 to 12 hours.

  When the additional ion exchange group is an amino group, for example, mono-, di- or tri-alkylamino chloride, mono-, di- or tri-alkanol amino chloride, mono- (or di-) alkyl-mono (or di- -) Secondary or tertiary aminohalogenides such as alkanolamino chloride (however, 0 is not simultaneously shown) are used.

  When the additional ion exchange group is a quaternary ammonium group, a tertiary amino group is introduced, and then the tertiary amino group is reacted with a compound containing a halogenated alkyl group of epichlorohydrin to convert it into a quaternary ammonium group. Is also good. A quaternary amino halide such as quaternary ammonium chloride may be reacted with a hydroxyl group of the hydrophilic polymer. An alkyl halide compound having a quaternary ammonium group may be reacted under alkaline conditions.

  When the additional ion exchange group is a carboxyl group, for example, a monohalogenocarboxylic acid such as monohalogenoacetic acid or monohalogenopropionic acid or a sodium salt thereof is used. The amount of the carboxylic acid or its sodium salt may be 0.2% by mass or more based on the mass of the separating material before the additional ion exchange group is introduced.

  When the additional ion exchange group is a sulfo group, a glycidyl group such as shrimp chlorohydrin is reacted with the separating material to introduce a glycidyl group, and then a sulfite or bisulfite such as sodium sulfite or sodium bisulfite is used. The sulfo group can be introduced in a saturated aqueous solution of the salt. This reaction is carried out, for example, at a temperature of 30 to 90 ° C. and a reaction time of 1 to 10 hours.

  As another method for introducing an additional ion exchange group, for example, a method of reacting 1,3-propane sultone with a separating material in an alkaline atmosphere can also be mentioned. The amount of 1.3-propane sultone may be 0.4% by mass or more based on the total mass of the separation material. The reaction conditions may be, for example, 0 to 90 ° C. for 0.5 to 12 hours. A method in which an alkyl halide having a sulfonic acid or a sulfonic acid salt is reacted with a separating material in an alkaline atmosphere is also used. The amount of the alkyl halide having a sulfonic acid or a sulfonic acid salt may be 0.4% by mass or more based on the total mass of the separating material.

(Separation material)
The particle size of the separating material composed of the porous polymer particles and the coating layer may be, for example, 10 to 300 μm. When used as a packing material for preparative or industrial chromatography, the particle size of the separating material may be 50 to 100 μm in order to avoid an extreme increase in the internal pressure of the column.

  A separation material having a graft chain having an ion exchange group is used, for example, for separation of proteins by electrostatic interaction or affinity purification. For example, a separating material is added to a mixed solution containing proteins, only proteins are adsorbed to the separating material by electrostatic interaction, and then the separating material is separated from the solution and added to an aqueous solution having a high salt concentration. Then, the protein adsorbed on the separation material can be easily desorbed and recovered. The separation material is also useful as a column filler in column chromatography. A packed column used for column chromatography can include a column tube and a separation material (column packing material) filled in the column tube. A separation material into which a cation exchange group or an anion exchange group has been introduced can also be used as a carrier for ion exchange.

  As the biopolymer that can be separated using the separation material according to one embodiment, a water-soluble substance is preferable. The biopolymer may be a protein such as blood protein such as serum albumin or immunoglobulin, an enzyme present in a living body, a protein bioactive substance produced by biotechnology, or DNA. The molecular weight of the biopolymer may be 2,000,000 or less, or 500,000 or less. The properties and conditions of the ion exchange group can be selected depending on the isoelectric point, ionization state, and the like of the protein. In this regard, for example, JP-A-60-169427 can be referred to.

  When a protein is separated by a column, the flow rate of a protein solution or the like passed through the column is generally 400 cm / h or less. On the other hand, the separation material according to one embodiment can be used with a high adsorption capacity even at a liquid passing speed of 800 cm / h or more. The liquid passing speed as used herein means a liquid passing speed when a filler is filled in a stainless steel column tube having a diameter of 7.8 x 300 mm and the liquid is allowed to flow.

  The surface of the porous polymer particles is coated with a crosslinked polymer having a hydroxyl group having a hydroxyl group by crosslinking, and ion exchange is performed by introducing a graft chain having a polymer derived from a monomer having a cation exchange group or an anion exchange group. By introducing a group or the like, in the separation of biopolymers such as proteins, the respective advantages of the graft chain having a polymer derived from a monomer having an ion exchange group, the polymer having a hydroxyl group, and the porous polymer particles can be obtained. The combined properties can be shown. The porous polymer particles according to the above-described embodiment have high durability and alkali resistance. By forming the coating layer with a crosslinked polymer of a hydroxyl group-containing polymer, nonspecific adsorption hardly occurs. Further, by introducing a graft chain having a polymer derived from a monomer having an ion exchange group, desorption of a protein is facilitated. The separation material having an ion-exchange group has preferable properties as compared with a conventional ion-exchange resin also in that it has a large adsorption capacity (dynamic adsorption capacity) for proteins and the like under the same flow rate.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

1. Porous polymer particles In a 500 mL three-necked flask, 16 g of 96% pure divinylbenzene (trade name: DVB960, manufactured by Nippon Steel & Sumikin Chemical Co., Ltd.), 6 g of emulsifier (sorbitan monooleate, trade name: Span 80), and 6 g of benzoyl peroxide 0.64 g was added to an aqueous polyvinyl alcohol solution (concentration: 0.5% by mass). The resulting mixture was emulsified using a microprocess server to form an emulsion containing a polyvinyl alcohol aqueous solution as a continuous phase and a monomer phase dispersed in the continuous phase. The monomer phase mainly contains divinylbenzene, an emulsifier and benzoyl peroxide. The obtained emulsion was transferred to a flask. The emulsion was stirred for about 8 hours using a stirrer while heating the emulsion in a water bath at 80 ° C., thereby allowing the polymerization of divinylbenzene to proceed. Particles formed of a polymer of divinylbenzene (DVB) were taken out by filtration and washed with acetone to obtain porous polymer particles. The particle size of the obtained porous polymer particles was measured by a flow type particle size measuring device. The average particle size was 99 μm, and the coefficient of variation (CV value) of the particle size was 8%.

2. Example 1 of separation material
(Coating layer formation with polymer having hydroxyl group)
4 g of sodium hydroxide and 0.4 g of glycidyl phenyl ether were added to 100 mL of an agarose aqueous solution (concentration: 2% by mass), and these were reacted at 70 ° C. for 12 hours to introduce a phenyl group into the agarose. The phenyl group-introduced agarose (denatured agarose) was washed by reprecipitation three times with isopropyl alcohol.
A modified agarose aqueous solution (700 mL) having a concentration of 20 mg / mL was prepared, and 10 g of porous polymer particles were dispersed therein. By stirring the dispersion at 55 ° C. for 24 hours, the modified agarose was adsorbed on the surface including the inside of the pores of the porous polymer particles, thereby forming coated particles having a coating layer containing the modified agarose. The coated particles were removed by filtration and washed with hot water. The amount of the modified agarose adsorbed on the porous polymer particles was calculated from the concentration of the modified agarose in the filtrate. The calculated amount of adsorption was 300 mg per 1 g of the porous polymer particles.
10 g of the coated particles were dispersed in 350 g of a 0.4 M aqueous sodium hydroxide solution, and 10 g of ethylene glycol diglycidyl ether was added to the resulting dispersion. Then, the modified agarose in the coating layer was cross-linked by ethylene glycol diglycidyl ether by stirring the dispersion for 12 hours at room temperature. The coated particles taken out by filtration were washed with pure water, a heated 2% by mass aqueous solution of sodium dodecyl sulfate, and water in this order to obtain coated particles having a coating layer containing crosslinked modified agarose.

(Non-specific adsorption ability of protein)
0.5 g of the coated particles having a coating layer containing cross-linked denatured agarose was put into 50 mL of a phosphate buffer (pH 7.4) containing bovine serum albumin (BSA) at a concentration of 20 mg / mL. The dispersion was stirred at room temperature for 24 hours. Thereafter, the supernatant obtained by centrifugation was filtered, and the BSA concentration of the filtrate was measured with a spectrophotometer. From the obtained BSA concentration, the amount of BSA adsorbed on the coated particles was calculated. The concentration of BSA was confirmed from the absorbance at 280 nm using a spectrophotometer. The amount of adsorption was 5 mg or less, and nonspecific adsorption of protein was sufficiently suppressed.

(Introduction of graft chain)
A 500 mL three-necked flask was charged with 10 g of coated particles having a coating layer containing crosslinked modified agarose, 100 mL of aqueous solution of potassium 3-sulfopropyl methacrylate (SPMA) (concentration: 10% by mass), and 1 g of potassium persulfate. Nitrogen gas was blown into the reaction solution in the flask at a rate of 200 mL / min for 20 minutes. Subsequently, the reaction solution was stirred for 8 hours while heating in a water bath at 70 ° C. while maintaining the blowing of nitrogen gas, thereby forming a graft chain bonded to the modified agarose in the coating layer. The coated particles taken out of the reaction solution were washed with pure water to obtain particles of a separation material having a coating layer into which a graft chain having an ion exchange group was introduced. The ratio of the mass of the graft chain to 1 g of the porous polymer particles (mg / g of the particle, the amount of the graft) was determined by thermogravimetry / differential heat measurement.

Example 2
In the same manner as in Example 1 except that the concentration of potassium 3-sulfopropyl methacrylate was changed to 8% by mass and 2-hydroxyethyl methacrylate (HEMA) was further added to the reaction solution at a concentration of 2% by mass. Separation material particles having a coating layer into which graft chains having exchange groups were introduced were obtained. The graft amount of the obtained separation material was measured in the same manner as in Example 1.

Example 3
In the same manner as in Example 1, particles of a separation material having a coating layer into which a graft chain having an ion exchange group was introduced were obtained. 20 g of the obtained separating material was dispersed in 100 mL of an aqueous solution containing 20 g of 2-chloroethanesulfonic acid sodium salt and 20 g of sodium hydroxide. The dispersion was stirred for 8 hours while heating to 70 ° C. Subsequently, 100 mL of a 5M aqueous solution of NaOH heated to 70 ° C. was added, and the dispersion was stirred for 1 hour while heating to 70 ° C. After the completion of the reaction, the separating material was removed by filtration, washed twice with a mixed solution of water / ethanol (volume ratio: 8/2), and further ion-exchange groups bonded to the modified agarose via ethylene groups were further introduced. A separation material having a covering layer was obtained. The graft amount of the obtained separation material was measured in the same manner as in Example 1.

Example 4
In the same manner as in Example 1, coated particles before graft chains were introduced were obtained. Separation having a coating layer into which a graft chain having an ion exchange group was introduced in the same manner as in Example 1 except that nitrogen gas was blown into the reaction solution, and then 1 g of sodium hydrogen carbonate was added as a reducing agent to the reaction solution. Wood particles were obtained. The graft amount of the obtained separation material was measured in the same manner as in Example 1.

Example 5
In the same manner as in Example 1, coated particles before graft chains were introduced were obtained. The same procedure as in Example 1 was carried out except that the aqueous solution of potassium 3-sulfopropyl methacrylate was changed to an aqueous solution of sodium methacrylate (MA) (concentration: 10% by mass). Particles of a separating material having the same were obtained. The graft amount of the obtained separation material was measured in the same manner as in Example 1.

Example 6
In the same manner as in Example 1, coated particles before graft chains were introduced were obtained. A graft chain having an ion exchange group was obtained in the same manner as in Example 1 except that the aqueous solution of potassium 3-sulfopropyl methacrylate was changed to an aqueous solution of 2- (diethylamino) ethyl methacrylate (DEAE-MA) (10% by mass). Separation material particles having the introduced coating layer were obtained. The graft amount of the obtained separation material was measured in the same manner as in Example 1.

Example 7
In the same manner as in Example 1, coated particles before graft chains were introduced were obtained. Graft having an ion exchange group in the same manner as in Example 1 except that the aqueous solution of potassium 3-sulfopropyl methacrylate was changed to an aqueous solution of trimethyl-2-methacryloyloxyethylammonium chloride (Q-MA) (10% by mass). Separation material particles having a coating layer into which chains were introduced were obtained. The graft amount of the obtained separation material was measured in the same manner as in Example 1.

Comparative Example 1
In the same manner as in Example 1, coated particles before graft chains were introduced were obtained. Without introducing a graft chain, ion-exchange groups bonded to the modified agarose were introduced in the same manner as in Example 3 to obtain particles of a separating material.

Comparative Example 2
In the same manner as in Example 1, coated particles before graft chains were introduced were obtained. An ion-exchange group bonded to the modified agarose was introduced in the same manner as in Example 3 except that 20 g of 2-chloroethanesulfonic acid sodium salt was changed to 20 g of monochloroacetic acid without introducing a graft chain, thereby introducing a separation material. Particles were obtained.

Reference Example For comparison with a general separation material, a commercially available ion-exchange chromatography support (Capto S (manufactured by GE Healthcare)) was directly evaluated as a separation material of a reference example. Was measured by a flow-type particle size measuring device, and the average particle size was 98 μm and the coefficient of variation (CV value) of the particle size was 38%.

3. Coefficient of variation (CV) of mode diameter, specific surface area, porosity, and particle size in pore size distribution of evaluation of separation material
The mode diameter, specific surface area, and porosity in the pore size distribution of the separation material were measured by mercury intrusion measurement. The particle size of the separation material was measured by a flow type particle size measuring device, and the average particle size and the coefficient of variation (CV) of the particle size were calculated from the measurement results. Table 2 shows the results.

5% Weight Loss Temperature A 5% weight loss temperature of the separation material was measured using a thermogravimetric analyzer. If the 5% weight loss temperature is lower than 200 ° C., the coating layer may be split or detached by hydrolysis during the reaction for introducing the graft chain. When the coating layer is split or detached by hydrolysis, the swelling property and the amount of ion exchange groups of the coating layer tend to decrease, and as a result, the dynamic adsorption amount tends to decrease.

4. Preparation of Packed Column Packed Column Particles of the separation material were dispersed in ethanol to prepare a slurry having a particle concentration of 30% by mass. This slurry was packed in a φ7.8 × 300 mm stainless column tube over 15 minutes. Thus, a packed column having the separation material packed in the column tube was obtained.

Water Permeability Water was passed through the packed column while changing the flow rate, and the relationship between the linear flow rate and the column pressure at that time was recorded. From the measurement results, the linear flow rate at a column pressure of 0.3 MPa was determined. Based on the linear flow velocity, the liquid permeability of the separation material was evaluated according to the following criteria.
A: Linear flow velocity of 1500 cm / h or more B: Linear flow velocity of 1000 cm / h or more and 1500 cm / h or less C: Linear flow velocity of less than 1000 cm / h

Dynamic adsorption amount 10 column volumes of buffer solution was passed through the packed column. Then, while flowing a buffer containing protein at a concentration of 2 mg / mL through the packed column, the protein concentration at the column outlet was measured based on ultraviolet absorption. Buffer containing protein was passed through the packed column until the protein concentration at the inlet and outlet of the column matched. Subsequently, a solution in which 1 M NaCl was added to a buffer solution was prepared, and this solution was passed through a packed column for 10 column volumes, whereby the protein adsorbed on the separation material was recovered. In the evaluation of each packed column, the following proteins and buffers were used.
Examples 1 to 5 and Comparative Examples 1 and 2
Buffer: 20 mmol / L sodium acetate buffer (pH 5.0)
Protein: IgG (immunoglobulin G)
Examples 6 and 7 and Comparative Examples 3 and 4
Buffer: 20 mmol / L Tris-HCl buffer (pH 8.0)
Protein: BSA (bovine serum albumin)

The dynamic adsorption amount at 10% breakthrough was calculated by the following equation.
_{q 10 = c f F (t} 10 -t 0) / V B
q ₁₀ : Dynamic adsorption amount (mg / mL wet resin) at 10% breakthrough
cf: Injected protein concentration F: Flow rate (mL / min)
V _B : bed volume (mL)
t ₁₀ : time (min) at 10% breakthrough
t ₀ : protein injection start time (min)

  Table 1 shows the configuration of each separation material, and Table 2 shows the evaluation results. The separation material of the example having the coating layer in which the graft chain having a cation exchange group or an anion exchange group was introduced was compared with the separation material of the comparative example in which the graft chain was not introduced, and the commercially available separation material. , Showed significantly improved dynamic adsorption. Further, the separation materials of the examples also showed good liquid permeability.

Claims (7)

Porous polymer particles, comprising a coating layer that covers at least a part of the surface of the porous polymer particles,
The coating layer includes a hydrophilic polymer having a hydroxyl group and a graft chain bonded to the hydrophilic polymer, and the graft chain is a polymer including a monomer unit having an ion exchange group.
Separation material.   The ion exchange group is at least one cation exchange group selected from a sulfo group, a carboxyl group, and a salt thereof, or at least one cation exchange group selected from a diethylaminoethyl group, a dimethylaminoethyl group, and a trimethylammonium group. The separation material according to claim 1, which is an anion exchange group.   The separation material according to claim 1 or 2, wherein the hydrophilic polymer is a polysaccharide or a modified product thereof.   4. The method according to claim 1, wherein a ratio of the monomer unit having the ion exchange group in the graft chain is 50 to 100 mol% based on an amount of all monomer units in the graft chain. 5. Separation material.   The separation material according to any one of claims 1 to 4, wherein a 5% weight loss temperature of the separation material under a nitrogen atmosphere is 200 ° C or more.   The separation material according to any one of claims 1 to 5, which is a carrier for ion exchange.   A packed column, comprising: a column tube; and the separation material according to claim 1 filled in the column tube.