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WO2018174022A1 - Matériau de séparation - Google Patents

Matériau de séparation Download PDF

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Publication number
WO2018174022A1
WO2018174022A1 PCT/JP2018/010880 JP2018010880W WO2018174022A1 WO 2018174022 A1 WO2018174022 A1 WO 2018174022A1 JP 2018010880 W JP2018010880 W JP 2018010880W WO 2018174022 A1 WO2018174022 A1 WO 2018174022A1
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WIPO (PCT)
Prior art keywords
separation material
porous polymer
group
hydroxyl group
polymer particles
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PCT/JP2018/010880
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English (en)
Japanese (ja)
Inventor
優 渡邊
史彦 河内
健 安江
後藤　泰史
道男 佛願
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日立化成株式会社
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Application filed by 日立化成株式会社 filed Critical 日立化成株式会社
Priority to JP2019507669A priority Critical patent/JPWO2018174022A1/ja
Publication of WO2018174022A1 publication Critical patent/WO2018174022A1/fr

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  • the present invention relates to a separating material.
  • ion exchangers based on porous synthetic polymers and ion exchanges based on cross-linked gels of hydrophilic natural polymers are generally used.
  • the body is used.
  • an ion exchanger based on a porous synthetic polymer the volume change due to the salt concentration is small. Therefore, when packed in a column and used for chromatography, the pressure resistance during liquid passage tends to be excellent.
  • this ion exchanger is used for separation of proteins, etc., nonspecific adsorption such as irreversible adsorption based on hydrophobic interaction occurs, resulting in peak asymmetry or ion exchange due to the hydrophobic interaction. There is a problem that the protein adsorbed on the body cannot be recovered while adsorbed.
  • an ion exchanger based on a cross-linked gel of a hydrophilic natural polymer typified by polysaccharides such as dextran and agarose has an advantage that there is almost no non-specific adsorption of protein.
  • this ion exchanger swells remarkably in an aqueous solution, and has the disadvantage that the volume change due to the ionic strength of the solution, the volume change between the free acid form and the loaded form is large, and the mechanical strength is not sufficient.
  • a cross-linked gel is used in chromatography, there is a disadvantage that the pressure loss during liquid passage is large and the gel is consolidated by liquid passage.
  • an object of the present invention is to provide a separation material that has a small amount of nonspecific adsorption of protein, is excellent in protein adsorption, and has excellent liquid permeability when used as a column.
  • the present invention provides the separating material described in [1] to [10] below.
  • a porous polymer particle containing a polymer containing a styrene monomer as a monomer unit, and a coating layer containing a polymer having a hydroxyl group and covering at least a part of the surface of the porous polymer particle A separation material having a change ratio of a specific surface area before and after that of 0.50 to 0.85.
  • the separation material according to [1] wherein the specific surface area is 30 m 2 / g or more.
  • the separation material according to [1] or [2], wherein the coefficient of variation of the particle size of the porous polymer particles is 5 to 15%.
  • the present invention it is possible to provide a separation material with less non-specific protein adsorption, excellent protein adsorption, and excellent liquid permeability when used as a column.
  • the separating material of the present embodiment includes a porous polymer particle containing a polymer containing a styrene monomer as a monomer unit, and a coating layer containing a polymer having a hydroxyl group that covers at least a part of the surface of the porous polymer particle.
  • the change ratio of the specific surface area before and after coating is 0.50 to 0.85.
  • Such a separation material has less non-specific protein adsorption, and is excellent in protein adsorption and liquid permeability when used as a column. The reason why the non-specific adsorption is small in the separation material of the present embodiment is considered to be that the hydrophobic interaction is small.
  • the separating material of the present embodiment is also excellent in alkali resistance, pressure resistance and durability.
  • the separation material of the present embodiment is excellent in dynamic adsorptivity when the liquid is passed at a high flow rate.
  • the separation material of the present embodiment is excellent in strength and can have a shape close to a true sphere.
  • Spherical separators are considered hydrodynamically advantageous when used in chromatography. Therefore, it is considered that such a separation material is easy to suppress pressure loss and perform a chromatography operation, for example.
  • the surface of the porous polymer particle includes not only the surface of the porous polymer particle but also the surface of the pores inside the porous polymer particle.
  • the porous polymer particle according to the present embodiment includes a polymer containing a styrene monomer as a monomer unit. Such porous polymer particles are considered to be excellent in durability and alkali resistance.
  • the porous polymer particles according to this embodiment are, for example, particles obtained by polymerizing a monomer containing a styrenic monomer in the presence of a porosifying agent.
  • the porous polymer particles can be synthesized by, for example, conventional suspension polymerization.
  • the styrene monomer means a monomer having a styrene skeleton. Examples of the styrenic monomer include the following polyfunctional monomers and monofunctional monomers.
  • the styrenic polyfunctional monomer examples include divinyl compounds having a styrene skeleton such as divinylbenzene, divinylbiphenyl, divinylnaphthalene, and divinylphenanthrene. These polyfunctional monomers can be used alone or in combination of two or more. Among these, divinylbenzene is preferably used from the viewpoint of further improving durability, acid resistance and alkali resistance. That is, the porous polymer particles may contain a polymer containing divinylbenzene as a monomer unit.
  • styrene monofunctional monomers examples include styrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, ⁇ -methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, 2 , 4-dimethylstyrene, pn-butylstyrene, pt-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, p- Examples thereof include styrene such as n-dodecylstyrene, p-methoxystyrene, p-phenylstyrene, p-chlorosty
  • Styrene derivatives having a functional group such as a carboxy group, an amino group, a hydroxyl group, and an aldehyde group can also be used. These monofunctional monomers can be used alone or in combination of two or more. Among these, styrene is preferably used from the viewpoint of further improving acid resistance and alkali resistance.
  • the porosifying agent examples include aliphatic or aromatic hydrocarbons, esters, ketones, ethers, alcohols, and the like, which are organic solvents that promote phase separation at the time of polymerization and promote pore formation of particles. It is done. Specific examples include toluene, xylene, diethylbenzene, cyclohexane, octane, butyl acetate, dibutyl phthalate, methyl ethyl ketone, dibutyl ether, 1-hexanol, 2-octanol, decanol, lauryl alcohol, cyclohexanol and the like. These porosifying agents can be used singly or in combination of two or more.
  • the above porous agent can be used, for example, in an amount of 0 to 200% by mass with respect to the total mass of the monomer.
  • the porosity of the porous polymer particles can be controlled by the amount of the porous agent. Further, the size and shape of the pores of the porous polymer particles can be controlled by the kind of the porous agent.
  • Water used as a solvent can be used as a porous agent.
  • the particles can be made porous by, for example, dissolving an oil-soluble surfactant in the monomer and absorbing water.
  • oil-soluble surfactant used for the porosification examples 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, such as sorbitan monolaurate, sorbitan Sorbitan monoesters derived from monooleate, sorbitan monomyristate or coconut fatty acid; diglycerol monoesters of branched C16-C24 fatty acids, chain unsaturated C16-C22 fatty acids or chain saturated C12-C14 fatty acids, for example di- Glycerol monooleate (for example, diglycerol monoester of C18: 1 (18 carbon atoms, 1 double bond) fatty acid), diglycerol monomyristate, diglycerol monoisostearate or diglycerol monoester of coconut fatty acid Ester; Branch C16 ⁇ 24 alcohol (e.g., Guerbet alcohols), linear unsaturated C16 ⁇ C24
  • sorbitan monolaurate eg, SPAN 20
  • Sorbitan monooleate e.g., SPAN 80
  • Diglycerol monooleate eg, diglycerol monooleate having a purity of preferably greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70%
  • diglycerol monoisostearate Form example, the purity is preferably greater than about 40%, more preferably about 50%.
  • diglycerol monoisostearate diglycerol monomyristate (eg, preferably greater than about 40%, more preferably greater than about 50%, even more preferably about 70% purity).
  • % Of sorbitan monomyristate a cocoyl (eg, lauryl and myristoyl group) ether of diglycerol; or a mixture thereof.
  • oil-soluble surfactants are preferably used in the range of 5 to 80% by mass relative to the total mass of the monomer.
  • content of the oil-soluble surfactant is 5% by mass or more, the stability of the water droplets is easily improved, so that a large single hole is easily formed.
  • content of the oil-soluble surfactant is 80% by mass or less, the porous polymer particles are more easily retained in shape after polymerization.
  • aqueous medium used for the polymerization reaction examples include water, a mixed medium of water and a water-soluble solvent (for example, lower alcohol), and the like.
  • the aqueous medium may contain a surfactant.
  • the surfactant any of anionic, cationic, nonionic and zwitterionic surfactants can be used.
  • anionic surfactant examples include fatty acid oils such as sodium oleate and castor oil potassium, alkyl sulfate salts such as sodium lauryl sulfate and ammonium lauryl sulfate, alkylbenzene sulfonates such as sodium dodecylbenzenesulfonate, and alkylnaphthalene sulfone.
  • Acid salts alkane sulfonates, dialkyl sulfosuccinates such as sodium dioctyl sulfosuccinate, alkenyl succinates (dipotassium salts), alkyl phosphate esters, naphthalene sulfonate formalin condensates, polyoxyethylene alkylphenyl ether sulfates Salts, polyoxyethylene alkyl ether sulfates such as sodium polyoxyethylene lauryl ether sulfate, and polyoxyethylene alkyl sulfate salts
  • cationic surfactant examples include alkylamine salts such as laurylamine acetate and stearylamine acetate, and quaternary ammonium salts such as lauryltrimethylammonium chloride.
  • Nonionic surfactants include, for example, hydrocarbon nonionic surfactants such as polyethylene glycol alkyl ethers, polyethylene glycol alkyl aryl ethers, polyethylene glycol esters, polyethylene glycol sorbitan esters, polyalkylene glycol alkylamines, or amides. Agents, polyether-modified silicon nonionic surfactants such as silicon polyethylene oxide adducts and polypropylene oxide adducts, and fluorine nonionic surfactants such as perfluoroalkyl glycols.
  • hydrocarbon nonionic surfactants such as polyethylene glycol alkyl ethers, polyethylene glycol alkyl aryl ethers, polyethylene glycol esters, polyethylene glycol sorbitan esters, polyalkylene glycol alkylamines, or amides.
  • Agents polyether-modified silicon nonionic surfactants such as silicon polyethylene oxide adducts and polypropylene oxide adducts, and
  • zwitterionic surfactants include hydrocarbon surfactants such as lauryl dimethylamine oxide, phosphate ester surfactants, and phosphite ester surfactants.
  • Surfactant may be used alone or in combination of two or more.
  • anionic surfactants are preferable from the viewpoint of dispersion stability during monomer polymerization.
  • polymerization initiator examples include benzoyl peroxide, lauroyl peroxide, orthochlorobenzoyl peroxide, orthomethoxybenzoyl peroxide, 3,5,5-trimethylhexanoyl peroxide, tert-butyl peroxide.
  • Organic peroxides such as oxy-2-ethylhexanoate and di-tert-butyl peroxide; and 2,2′-azobisisobutyronitrile, 1,1′-azobiscyclohexanecarbonitrile, 2,2 And azo compounds such as' -azobis (2,4-dimethylvaleronitrile).
  • the polymerization initiator can be used, for example, in the range of 0.1 to 7.0 parts by mass with respect to 100 parts by mass of the monomer.
  • the polymerization temperature can be appropriately selected according to the type of monomer and polymerization initiator.
  • the polymerization temperature may be, for example, 25 to 110 ° C. or 50 to 100 ° C.
  • a polymer dispersion stabilizer may be added in order to improve the dispersion stability of the particles.
  • polymer dispersion stabilizer examples include polyvinyl alcohol, polycarboxylic acid, celluloses (hydroxyethyl cellulose, carboxymethyl cellulose, methyl cellulose, etc.), and polyvinyl pyrrolidone, and inorganic water-soluble polymer compounds such as sodium tripolyphosphate are also included. Can be used together. Of these, polyvinyl alcohol or polyvinyl pyrrolidone is preferred.
  • the amount of the polymer dispersion stabilizer added may be, for example, 1 to 10 parts by mass with respect to 100 parts by mass of the monomer.
  • a water-soluble polymerization inhibitor such as nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble vitamin Bs, citric acid, polyphenols and the like may be used.
  • the average particle diameter of the porous polymer particles may be, for example, 500 ⁇ m or less, 300 ⁇ m or less, or 200 ⁇ m or less.
  • the average particle diameter of the porous polymer particles may be, for example, 10 ⁇ m or more, 30 ⁇ m or more, or 50 ⁇ m or more from the viewpoint of easily suppressing an increase in column pressure after column filling. Good.
  • the coefficient of variation (CV) of the particle size of the porous polymer particles may be, for example, 5 to 15% or 5 to 13% from the viewpoint of improving liquid permeability. It may be up to 10%.
  • CV coefficient of variation
  • C. V. As a method for reducing the above, monodispersion by an emulsification apparatus such as a microprocess server (Hitachi Ltd.) can be mentioned.
  • C. of average particle size and particle size of porous polymer particles or separator V. can be determined by the following measurement method. 1) Disperse particles (porous polymer particles or separation material) in water (including a dispersant such as a surfactant) using an ultrasonic dispersion device to prepare a dispersion containing 1% by mass of particles. . 2) Using a particle size distribution meter (Sysmex Flow, manufactured by Sysmex Corporation), an average particle size and particle size of C.I. V. (Coefficient of variation) is measured.
  • the porosity (pore volume) of the porous polymer particles may be, for example, 30% to 70% by volume, and 40% to 70% by volume based on the total volume of the porous polymer particles. May be.
  • the porous polymer particles preferably have macropores (macropores).
  • the mode diameter in the pore size distribution of the porous polymer particles is preferably from 0.05 to 0.50 ⁇ m, more preferably from 0.10 to 0.50 ⁇ m, and from 0.10 ⁇ m to less than 0.50 ⁇ m. More preferably.
  • the mode diameter in the pore size distribution is 0.05 ⁇ m or more, substances tend to easily enter the pores, and when the mode diameter in the pore diameter distribution is 0.50 ⁇ m or less, the specific surface area is sufficient. It tends to be.
  • the porosity and pore diameter of the porous polymer particles can be adjusted by, for example, the above-described 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 adsorption amount of the substance to be separated tends to increase.
  • the specific surface area of the porous polymer particles may be, for example, 500 m 2 / g or less, 200 m 2 / g or less, or 100 m 2 / g or less.
  • the mode diameter (mode value of pore diameter distribution, maximum frequency pore diameter), specific surface area and porosity in the pore diameter distribution of porous polymer particles or separation materials are measured with a mercury intrusion measuring device (Autopore: manufactured by Shimadzu Corporation). Measured in the following manner. About 0.05 g of a sample is added to a standard 5 mL powder cell (stem volume 0.4 mL), and measurement is performed under an initial pressure of 21 kPa (about 3 psia, corresponding to a pore diameter of about 60 ⁇ m). Mercury parameters are set to a device default mercury contact angle of 130 ° and a mercury surface tension of 485 dynes / cm. Each value is calculated by limiting the pore diameter to a range of 0 to 3 ⁇ m.
  • the coating layer according to the present embodiment includes a polymer having a hydroxyl group.
  • the coating layer contains a polymer having a hydroxyl group, it is easy to suppress nonspecific adsorption of protein and to easily improve the protein adsorption amount.
  • the polymer having a hydroxyl group may be cross-linked, for example, from the viewpoint of further suppressing the increase in the column pressure and from the viewpoint that the porous polymer particles and the coating layer are difficult to peel off.
  • the polymer having a hydroxyl group preferably has two or more hydroxyl groups in one molecule, and more preferably a hydrophilic polymer.
  • examples of the polymer having a hydroxyl group include polysaccharides and polyvinyl alcohol.
  • examples of the polysaccharide include agarose, dextran, cellulose, pullulan, and chitosan.
  • the weight average molecular weight of the polymer having a hydroxyl group may be, for example, 10,000 or more, 50000 or more, or 100,000 or more.
  • the weight average molecular weight of the polymer having a hydroxyl group may be, for example, 5000000 or less, 4500000 or less, or 4000000 or less.
  • the weight average molecular weight of the polymer having a hydroxyl group may be, for example, 10,000 to 5000000, 50000 to 450,000, or 100000 to 4000000.
  • the polymer having a hydroxyl group may be used singly or in combination of two or more.
  • the weight average molecular weight (Mw) refers to a value measured by the following method.
  • a solution obtained by dissolving 0.5% by mass of a sample in ultrapure water is measured using a gel permeation chromatography apparatus with 0.2M NaCl as an eluent.
  • a calibration curve is prepared by measuring pullulan and ethylene glycol as standard samples.
  • the polymer having a hydroxyl group may be a modified body (hydrophobic group-modified body) modified with a hydrophobic group from the viewpoint of improving the interfacial adsorption ability with the particles.
  • the hydrophobic group include an alkyl group having 1 to 6 carbon atoms and an aryl group having 6 to 10 carbon atoms.
  • 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 polymer having a hydroxyl group may be, for example, a polysaccharide or a modified product thereof from the viewpoint of alkali resistance.
  • modified polysaccharides include hydrophobic group-modified products.
  • the polymer having a hydroxyl group may be at least one selected from the group consisting of dextran, agarose, pullulan, and modified products thereof.
  • the coating layer according to the present embodiment can be formed by, for example, the following method.
  • a polymer solution 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 polymer 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 polymer solution is impregnated with the above solution.
  • porous polymer particles are added to a polymer solution having a hydroxyl group and left for a predetermined time. At this time, for example, for the purpose of dispersing the particles in the solution, stirring may be performed.
  • the impregnation time varies depending on the surface state of the porous polymer particles. However, when the impregnation is performed for 6 to 50 hours, the polymer concentration is in equilibrium with the external concentration inside the porous polymer particles. Then, it wash
  • Crosslinking treatment Next, a crosslinking agent is added to cause the polymer having a hydroxyl group adsorbed on the surface of the porous polymer particles to undergo a crosslinking reaction to form a crosslinked body.
  • an epihalohydrin such as epichlorohydrin
  • a dialdehyde compound such as glutaraldehyde
  • a diisocyanate compound such as methylene diisocyanate
  • a glycidyl compound such as ethylene glycol diglycidyl ether
  • two or more functional groups active on a hydroxyl group for example, an epihalohydrin such as epichlorohydrin, a dialdehyde compound such as glutaraldehyde, a diisocyanate compound such as methylene diisocyanate, a glycidyl compound such as ethylene glycol diglycidyl ether, and two or more functional groups active on a hydroxyl group.
  • a dihalide compound such as dichlorooctane can also be used as a crosslinking agent.
  • a catalyst is usually used for this crosslinking reaction.
  • the catalyst varies depending on the type of crosslinking agent. For example, when the crosslinking agent is epichlorohydrin or the like, 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. It is.
  • the crosslinking reaction with a crosslinking agent is usually performed by adding a crosslinking agent to a system in which porous polymer particles adsorbing a polymer having a hydroxyl group are dispersed and suspended in an appropriate medium.
  • the amount of the crosslinking agent added is within a range of 0.1 to 100 mol times with respect to 1 mol of one unit of the monosaccharide. It can be selected according to the performance of the target separation material.
  • the addition amount of the crosslinking agent is 0.1 mol times or more, the coating layer tends to hardly peel from the porous polymer particles.
  • the addition amount of the crosslinking agent is 100 mol times or less, even when the reaction rate with the polymer having a hydroxyl group is high, the characteristics of the polymer having a hydroxyl group tend to be easily maintained.
  • the amount of the catalyst used in the crosslinking reaction depends on the type of crosslinking agent, but when a polysaccharide is used as the polymer having a hydroxyl group, usually 1 mol of one unit of monosaccharide forming the polysaccharide is used. Then, it is preferably used in the range of 0.01 to 10 mole times, more preferably 0.1 to 5 mole times.
  • the cross-linking reaction condition is a temperature condition
  • the temperature of the reaction system is raised, and the cross-linking reaction occurs when the temperature reaches the reaction temperature.
  • the polymer, the cross-linking agent, etc. are not extracted from the adsorbed polymer solution and are not effective in the crosslinking reaction. It needs to be active. Specific examples thereof include water and alcohol.
  • the crosslinking reaction can be usually performed at a temperature in the range of 5 to 90 ° C. for 1 to 30 hours.
  • the crosslinking reaction may be performed, for example, at a temperature in the range of 5 to 90 ° C. for 1 to 10 hours.
  • the temperature of the crosslinking reaction is preferably 25 to 90 ° C.
  • the particles are filtered off and then washed with a hydrophilic organic solvent such as methanol or ethanol or water to remove unreacted polymer, suspending medium and the like.
  • a hydrophilic organic solvent such as methanol or ethanol or water to remove unreacted polymer, suspending medium and the like.
  • a separating material is obtained in which at least a part of the surface of the porous polymer particles is covered with a coating layer containing a polymer having a hydroxyl group, and the polymer having a hydroxyl group is crosslinked. If necessary, the above crosslinking treatment may be omitted.
  • the separating material of the present embodiment may include, for example, a coating layer of 30 mg or more per 1 g of porous polymer particles, may include a coating layer of 50 mg or more, or may include a coating layer of 100 mg or more. Good.
  • the separating material of the present embodiment may include, for example, a coating layer of 450 mg or less per 1 g of porous polymer particles, may include a coating layer of 400 mg or less, or may include a coating layer of 400 mg or less. Good.
  • the ratio of the coating layer is 450 mg or less with respect to 1 g of the porous polymer particles, the coating layer tends to be a thin film, and the column pressure tends to be suppressed when used as a column.
  • the separating material of the present embodiment preferably has a coating layer of 30 to 450 mg per 1 g of porous polymer particles, preferably has a coating layer of 50 to 400 mg, and preferably has a coating layer of 100 to 400 mg.
  • the amount of the coating layer can be measured by reducing the weight of pyrolysis.
  • the separation material of the present embodiment includes a coating layer of 30 to 450 mg per 1 g of the porous polymer particles from the viewpoint of further suppressing the increase in the column pressure and from the viewpoint of difficult separation of the porous polymer particles and the coating layer.
  • the polymer having a hydroxyl group is preferably crosslinked.
  • the separation material provided with the coating layer may have an ion exchange group, a ligand (protein A), and the like.
  • the separation material can be used for ion exchange purification, affinity purification, and the like by introducing these via a hydroxyl group on the surface.
  • Examples of the method for introducing an ion exchange group include a method using a halogenated alkyl compound.
  • halogenated alkyl compound examples include monohalogenocarboxylic acids such as monohalogenoacetic acid and monohalogenopropionic acid and sodium salts thereof, primary, secondary or tertiary amines having at least one halogenated alkyl group such as diethylaminoethyl chloride, halogen And quaternary ammonium hydrochloride having an alkyl group.
  • halogenated alkyl compounds are preferably bromides or chlorides.
  • the amount of the halogenated alkyl compound used is preferably, for example, 0.2% by mass or more with respect to the total mass of the separating material imparting ion exchange groups.
  • organic solvent examples include alcohols such as ethanol, 1-propanol, 2-propanol, 1-butanol, isobutanol, 1-pentanol, and isopentanol.
  • the ion exchange group is introduced into the hydroxyl group on the surface of the separation material, the wet particles are drained by filtration or the like, immersed in an alkaline aqueous solution of a predetermined concentration, left for a certain time, and then water-organic.
  • the halogenated alkyl compound is added and reacted in a solvent mixture system. This reaction is preferably performed at a temperature of 40 to 90 ° C. under reflux for 0.5 to 12 hours.
  • the ion exchange group to be provided is determined depending on the kind of the halogenated alkyl compound used in the above reaction.
  • alkaline aqueous solution sodium hydroxide aqueous solution is mentioned, for example.
  • the separation material of this embodiment may have a cation exchange group or an anion exchange group as an ion exchange group.
  • a separating material can be used for ion exchange. That is, the separation material of the present embodiment may have a cation exchange group or an anion exchange group and be used for ion exchange.
  • the cation exchange group include a carboxy group and a sulfonic acid group.
  • the anion exchange group include an amino group and a quaternary ammonium group.
  • a mono- having at least one alkyl group in which a part of hydrogen atoms is substituted with a chlorine atom Di- or tri-alkylamine, mono-, di- or tri-alkanolamine, monoalkyl-monoalkanolamine, dialkyl-monoalkanolamine, monoalkyl-dialkanolamine and the like.
  • the amount of the alkyl halide compound used is preferably 0.2% by mass or more based on the total mass of the separating material into which the ion exchange group is introduced.
  • the reaction conditions are preferably 40 to 90 ° C. and 0.5 to 12 hours.
  • a strongly basic quaternary ammonium group as an ion exchange group first, a tertiary amino group is introduced, and then the tertiary amino group is reacted with a halogenated alkyl compound such as epichlorohydrin. The method of converting into an ammonium group is mentioned. Further, quaternary ammonium hydrochloride or the like may be reacted with the separation material.
  • Examples of the method for introducing a carboxy group that is a weakly acidic group as an ion exchange group include a method in which a monohalogenocarboxylic acid such as monohalogenoacetic acid or monohalogenopropionic acid or a sodium salt thereof is reacted as the halogenated alkyl compound. .
  • the amount of these halogenated alkyl compounds used is preferably 0.2% by mass or more based on the total mass of the separating material into which ion exchange groups are introduced.
  • a glycidyl compound such as ebichlorohydrin is reacted with a separating material, and a sulfite or bisulfite such as sodium sulfite or sodium bisulfite.
  • a method of adding a separating material to the saturated aqueous solution are preferably 30 to 90 ° C. and 1 to 10 hours.
  • 1,3-propane sultone is reacted with a separating material in an alkaline cloud atmosphere.
  • 1,3-propane sultone is preferably used in an amount of 0.4% by mass or more based on the total mass of the separating material into which the ion exchange group is introduced.
  • the reaction conditions are preferably 0 to 90 ° C. and 0.5 to 12 hours.
  • the change ratio of the specific surface area before and after coating refers to a value calculated from the following formula using the specific surface area before and after coating the porous polymer particles with a polymer having a hydroxyl group.
  • the change ratio of the specific surface area is the ratio of the specific surface area of the separation material to the specific surface area of the porous polymer particles, and is expressed by (specific surface area of the separation material) / (specific surface area of the porous polymer particles). expressed.
  • the change ratio of the specific surface area before and after coating is 0.50 to 0.85.
  • the change ratio may be 0.55 or more, for example.
  • the change ratio may be, for example, 0.80 or less, 0.75 or less, or 0.70 or less.
  • the change ratio of the specific surface area before and after coating may be, for example, 0.50 to 0.80, 0.55 to 0.80, .55 to 0.75 or 0.55 to 0.70.
  • the change ratio of the specific surface area before and after coating is determined by appropriately selecting the type and weight average molecular weight of the polymer having a hydroxyl group, the raw material of the porous polymer particles, the mode diameter in the porosity and pore size distribution, the amount of the coating layer, and the like. Can be adjusted.
  • the liquid flow rate is preferably 800 cm / h or more when the column pressure is 0.3 MPa.
  • the flow rate of protein solution or the like is generally in the range of 400 cm / h or less.
  • the separation rate for normal protein separation is as follows. Even when used at a liquid flow rate of 800 cm / h or higher, which is faster than that of the ion exchanger, it is considered that a high adsorption amount can be maintained.
  • the liquid passing speed may be, for example, 4000 cm / h or less.
  • the liquid passing speed represents a liquid passing speed when the separation material of the present embodiment is filled in a glass column of ⁇ 5.0 ⁇ 200 mm and the liquid is passed.
  • the average particle diameter of the separation material of the present embodiment may be, for example, 500 ⁇ m or less, 300 ⁇ m or less, or 200 ⁇ m or less. Further, the average particle diameter of the separation material may be, for example, 10 ⁇ m or more, 30 ⁇ m or more, or 50 ⁇ m or more from the viewpoint of easily suppressing an increase in the column pressure after column filling. . From the above viewpoint, the average particle diameter of the separating material may be, for example, 10 to 500 ⁇ m, 10 to 300 ⁇ m, 30 to 300 ⁇ m, or 50 to 200 ⁇ m. When used in preparative or industrial chromatography, the average particle size of the separating material is preferably, for example, 50 to 200 ⁇ m from the viewpoint of easily avoiding an extreme increase in the internal pressure of the column.
  • the mode diameter in the pore size distribution of the separating material may be, for example, 0.05 ⁇ m or more, 0.075 ⁇ m or more, or 0.10 ⁇ m or more.
  • the mode diameter may be, for example, 0.50 ⁇ m or less, or less than 0.50 ⁇ m.
  • the mode diameter in the pore size distribution of the separating material is preferably 0.05 to 0.50 ⁇ m, more preferably 0.075 to 0.50, and further preferably 0.10 to 0.50 ⁇ m. It is particularly preferably 0.10 ⁇ m or more and less than 0.50 ⁇ m.
  • the mode diameter in the pore size distribution is 0.05 ⁇ m or more, substances tend to easily enter the pores, and when the mode diameter in the pore diameter distribution is 0.50 ⁇ m or less, the specific surface area is sufficient. It tends to be.
  • the mode diameter may be, for example, 0.50 to 0.75.
  • the specific surface area of the separating material is preferably 30 m 2 / g or more from the viewpoint of easy adsorption of the substance to be separated and higher practicality.
  • the specific surface area of the separating material may be, for example, 500 m 2 / g or less, 200 m 2 / g or less, or 100 m 2 / g or less.
  • the separation material of this embodiment has the respective advantages of particles made of natural polymers or particles made of synthetic polymers in the separation of biopolymers such as proteins. Moreover, the separation material of the present embodiment tends to reduce non-specific adsorption and easily cause protein adsorption / desorption. Furthermore, the separation material according to the present embodiment tends to have a large adsorption amount (dynamic adsorption amount) of protein or the like under the same flow rate.
  • the separation material of this embodiment When the separation material of this embodiment is used as a column packing material in column chromatography, it has excellent operability because there is almost no volume change in the column regardless of the properties of the eluate used.
  • the separation material of the present embodiment may be used for liquid chromatography, for example. That is, the separation material of this embodiment may be, for example, a liquid chromatography column packing material.
  • the separation material of this embodiment is suitable for use in separation of proteins by electrostatic interaction and affinity purification.
  • a separation material having an ion exchange group hereinafter also referred to as “ion exchanger”
  • ion exchanger a separation material having an ion exchange group
  • the ion exchanger is filtered from the solution and added to an aqueous solution having a high salt concentration, the protein adsorbed on the ion exchanger can be easily desorbed and recovered.
  • the ion exchanger can also be used in column chromatography.
  • a water-soluble substance is preferable.
  • proteins such as serum albumin and blood proteins such as immunoglobulins, enzymes present in the living body, protein bioactive substances produced by biotechnology, DNA, and peptides having bioactivity.
  • the molecular weight of these substances may be, for example, 2 million or less, or 500,000 or less.
  • the nature and conditions of the ion exchanger may be selected according to the isoelectric point, ionization state, etc. of the protein. Examples of known methods include the method described in JP-A-60-169427.
  • Example 1 Synthesis of porous polymer particles
  • 12 g of 96% pure divinylbenzene manufactured by NS was added to obtain a dispersed phase.
  • 0.5 mass% polyvinyl alcohol aqueous solution was used as a continuous phase.
  • the obtained emulsion was transferred to a flask and stirred for about 8 hours using a stirrer while heating in a water bath at 80 ° C.
  • the obtained particles were filtered off and then washed with acetone to obtain porous polymer particles.
  • the particle size of the porous polymer particles was measured with a flow-type particle size measuring device, and the average particle size and particle size C.I. V. The value (coefficient of variation) was calculated. The results are shown in Table 1.
  • the obtained modified dextran was reprecipitated three times with isopropyl alcohol and washed.
  • porous polymer particles 10 g were put into 700 mL of a 20 mg / mL modified dextran aqueous solution and stirred at 55 ° C. for 24 hours to adsorb the modified dextran to the porous polymer particles.
  • the particles adsorbed with the modified dextran were separated by filtration and further washed with hot water.
  • the coating amount (adsorption amount) of the modified dextran per 1 g of the porous polymer particles was calculated from the concentration of the modified dextran in the filtrate. The results are shown in Table 1.
  • the modified dextran was crosslinked as follows. 10 g of particles adsorbed with modified dextran were dispersed in a 0.4 M aqueous sodium hydroxide solution, 39 g of ethylene glycol diglycidyl ether was added, and the mixture was stirred at room temperature for 24 hours. Then, after washing with 2% by mass of hot sodium dodecyl sulfate aqueous solution, it was washed with pure water. The obtained particles were stored as a dispersion in water.
  • the dynamic adsorption amount (dynamic binding capacity) was measured as follows. 10 column volumes of 40 mmol / L Tris-HCl buffer (pH 8.0) were passed through the column. Thereafter, a 40 mmol / L Tris-HCl buffer solution having a BSA concentration of 0.5 mg / mL was passed at 800 cm / h, and the BSA concentration at the column outlet was measured by UV measurement. The solution was passed through until the BSA concentration at the column inlet and outlet coincided, and diluted with 1 M NaCl Tris-HCl buffer solution for 5 column volumes. The dynamic binding capacity at 10% breakthrough was calculated using the following equation. The results are shown in Table 2.
  • the dynamic adsorption amount (dynamic adsorption amount / static adsorption amount) relative to the static adsorption amount was calculated. The results are shown in Table 2. The larger the dynamic adsorption amount relative to the static adsorption amount, the better.
  • Example 2 In the synthesis of the porous polymer particles, a separating material was produced and evaluated in the same manner as in Example 1 except that the amount of the porosifying agent was changed to 8 g of diethylbenzene and 16 g of hexanol.
  • Example 3 In the synthesis of the porous polymer particles, a separation material was prepared and evaluated in the same manner as in Example 1 except that the amount of the porosifying agent was changed to 4 g of diethylbenzene and 20 g of hexanol.
  • Example 4 A separation material was prepared and evaluated in the same manner as in Example 1 except that the Mw of dextran used was changed to 1 million.
  • Example 5 A separation material was produced and evaluated in the same manner as in Example 1 except that Mw of dextran used was changed to 500,000.
  • Example 1 A separation material was prepared and evaluated in the same manner as in Example 1 except that Mw of dextran used was changed to 40,000.
  • Example 2 The porous polymer particles synthesized in Example 1 were used as they were as a separating material and evaluated in the same manner as in Example 1.
  • the separation materials of the examples have less protein non-specific adsorption, excellent protein adsorption, and excellent liquid permeability when used as a column.

Landscapes

  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

La présente invention concerne un matériau de séparation qui comporte : des particules polymère poreuses qui contiennent un polymère qui contient un monomère de styrène en tant qu'unité monomère; et une couche de revêtement qui recouvre au moins une partie de la surface de chaque particule polymère poreuse, tout en contenant un polymère qui a un groupe hydroxyle. Le rapport de changement de la surface spécifique du matériau de séparation avant et après le revêtement est de 0,50 à 0,85.
PCT/JP2018/010880 2017-03-21 2018-03-19 Matériau de séparation WO2018174022A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
JPS6223437A (ja) * 1985-07-24 1987-01-31 Japan Synthetic Rubber Co Ltd 粒子状担体
JP2003093801A (ja) * 2001-06-08 2003-04-02 Mitsubishi Chemicals Corp 多孔性重合体粒子及び分離剤並びにそれを用いた分離方法
WO2016117574A1 (fr) * 2015-01-19 2016-07-28 日立化成株式会社 Matériau de séparation
JP2017125799A (ja) * 2016-01-15 2017-07-20 日立化成株式会社 分離材及びカラム

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6223437A (ja) * 1985-07-24 1987-01-31 Japan Synthetic Rubber Co Ltd 粒子状担体
JP2003093801A (ja) * 2001-06-08 2003-04-02 Mitsubishi Chemicals Corp 多孔性重合体粒子及び分離剤並びにそれを用いた分離方法
WO2016117574A1 (fr) * 2015-01-19 2016-07-28 日立化成株式会社 Matériau de séparation
JP2017125799A (ja) * 2016-01-15 2017-07-20 日立化成株式会社 分離材及びカラム

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Title
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QU JIAN-BO ET AL.: "An Effective Way To Hydrophilize Gigaporous Polystyrene Microspheres as Rapid Chromatographic Separation Media for Proteins", LANGMUIR, vol. 24, no. 23, 2008, pages 13646 - 13652, XP055465735, ISSN: 0743-7463, DOI: doi:10.1021/la801486t *

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