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WO2018181925A1 - Matériau de séparation pour filtration sur gel et procédé de purification d'une substance polymère soluble dans l'eau - Google Patents

Matériau de séparation pour filtration sur gel et procédé de purification d'une substance polymère soluble dans l'eau Download PDF

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Publication number
WO2018181925A1
WO2018181925A1 PCT/JP2018/013692 JP2018013692W WO2018181925A1 WO 2018181925 A1 WO2018181925 A1 WO 2018181925A1 JP 2018013692 W JP2018013692 W JP 2018013692W WO 2018181925 A1 WO2018181925 A1 WO 2018181925A1
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Prior art keywords
gel filtration
polymer
separation material
hydroxyl group
porous polymer
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PCT/JP2018/013692
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English (en)
Japanese (ja)
Inventor
史彦 河内
優 渡邊
亜季子 川口
笑 宮澤
健 安江
後藤　泰史
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日立化成株式会社
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Priority to JP2019510266A priority Critical patent/JPWO2018181925A1/ja
Publication of WO2018181925A1 publication Critical patent/WO2018181925A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/34—Size-selective separation, e.g. size-exclusion chromatography; Gel filtration; Permeation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/285—Porous sorbents based on polymers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

Definitions

  • the present invention relates to a separation material for gel filtration and a purification method of a water-soluble polymer substance.
  • the chromatographic method plays an extremely important role as a method for separating and purifying proteins
  • the gel filtration (gel chromatography) method is one of the most frequently used methods.
  • This method is generally a separation mode based on the molecular sieve effect based only on the molecular weight difference of globular proteins, and is performed under conditions that exclude influences such as ionic properties and hydrophobic properties. Therefore, it is a very versatile and sensitive separation technique in that it can be applied to many proteins and enzymes simultaneously.
  • the separation material for gel filtration carrier for gel chromatography
  • a synthetic polymer separation material and a natural polysaccharide separation material.
  • the synthetic polymer separator examples include a gel obtained by insolubilizing a water-soluble polymer such as polyvinyl alcohol or polyacrylamide by crosslinking. Synthetic polymer-based separators are generally considered to have higher strength than natural polysaccharide-based separators. On the other hand, the synthetic polymer-based separating material has a problem that the affinity with protein is low and the yield is likely to decrease due to denaturation or deactivation due to nonspecific adsorption such as hydrophobic adsorption.
  • natural polysaccharide separation material examples include microbial polysaccharides such as dextran gel, seaweeds such as agarose gel, celluloses, and those obtained by crosslinking these.
  • Natural polysaccharide separators are usually more expensive than synthetic polymer separators because they tend to be expensive to produce, such as extraction from natural products, purification, and gel preparation.
  • natural polysaccharide separation materials generally have a high swelling ratio and are weak, so that, for example, when an eluent is flowed at a high speed in a gel filtration column, the column pressure is likely to increase, and the particle strength is high. There is a problem that it is difficult to lengthen the column length because it is low.
  • an object of the present invention is to provide a separation material for gel filtration that has less nonspecific adsorption of proteins, is excellent in high molecular weight protein fractionation properties, and has excellent durability when used as a column.
  • Another object of the present invention is to provide a method for purifying a water-soluble polymer substance using the separation material for gel filtration.
  • the present invention provides a separation material for gel filtration described in [1] to [6] below and a method for purifying a water-soluble polymer substance described in [7] below.
  • a porous polymer particle 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, and a 5% compressive deformation modulus in a wet state is 100 MPa or more.
  • the gel filtration separation material according to [1], wherein the porous polymer particles include a polymer containing a styrene monomer as a monomer unit.
  • the present invention it is possible to provide a separation material for gel filtration that has less nonspecific adsorption of protein, is excellent in high molecular weight protein fractionation, and has excellent durability when used as a column.
  • the present invention can also provide a method for purifying a water-soluble polymer substance using the above-described separation material for gel filtration.
  • the separation material of this embodiment is a separation material for gel filtration.
  • the separation material of this embodiment includes porous polymer particles and a coating layer containing a polymer having a hydroxyl group that covers at least a part of the surface of the porous polymer particles, and is 5% compressive deformed in a wet state.
  • the elastic modulus is 100 MPa or more.
  • Such a separation material has less non-specific protein adsorption, and is excellent in high molecular weight protein fractionation (separability) and durability when used as a column.
  • the separation material of this embodiment is also excellent in liquid permeability, pressure resistance and handling when used as a column.
  • 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.
  • the separation material of the present embodiment is excellent in the fractionation property between proteins having similar molecular weights and can stably separate the target substance over a long period of time
  • the “surface of the porous polymer particle” includes not only the outer surface of the porous polymer particle but also the surface of the pores inside the porous polymer particle.
  • the porous polymer particles according to this embodiment are, for example, particles obtained by polymerizing a monomer in the presence of a porosifying agent.
  • the porous polymer particles can be synthesized by, for example, conventional suspension polymerization and emulsion polymerization.
  • a styrene-type monomer can be used. That is, the porous polymer particles may be obtained by polymerizing a monomer containing a styrene monomer.
  • the styrene monomer means a monomer having a styrene skeleton.
  • the porous polymer particles according to this embodiment may contain a polymer containing a styrene monomer as a monomer unit.
  • the porous polymer particles according to the present embodiment may include a polymer having a structural unit derived from a styrene monomer.
  • styrene monomer examples 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.
  • 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, 300 ⁇ m or less, 150 ⁇ m or less, or 100 ⁇ m or less, from the viewpoint of further improving the separability. From the viewpoint of improving liquid permeability, the average particle diameter of the porous polymer particles may be, for example, 1 ⁇ m or more, 10 ⁇ m or more, 30 ⁇ m or more, or 50 ⁇ m or more. Also good. From these viewpoints, the average particle diameter of the porous polymer particles may be, for example, 1 to 300 ⁇ m, 10 to 150 ⁇ m, 30 to 100 ⁇ m, or 50 to 100 ⁇ m. Also good.
  • the coefficient of variation (CV) of the particle size of the porous polymer particles may be, for example, 1 to 50% or 3 to 15% from the viewpoint of easy improvement of liquid permeability. It may be 5 to 15% or 5 to 10%.
  • CV coefficient of variation
  • 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 coating layer according to the present embodiment includes a polymer having a hydroxyl group.
  • a polymer having a hydroxyl group By covering the porous polymer particles with a polymer having a hydroxyl group, it is easy to suppress an increase in column pressure and nonspecific adsorption of proteins.
  • 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, and chitosan.
  • the weight average molecular weight of the polymer having a hydroxyl group may be, for example, 10,000 or more.
  • the polymer having a hydroxyl group may be used singly or in combination of two or more.
  • 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.
  • a hydrophobic group is 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 polymer having a hydroxyl group by a conventionally known method. Can do.
  • a functional group that reacts with a hydroxyl group for example, an epoxy group
  • a compound having a hydrophobic group for example, glycidyl phenyl ether
  • the total molar amount of all structural units constituting the hydrophobic group-modified product (sum of the molar amounts of the structural unit containing a hydrophobic group and the structural unit not containing a hydrophobic group)
  • the content ratio of the structural unit containing a hydrophobic group (hereinafter also referred to as “hydrophobic group content”) is to maintain the hydrophobic interaction force for adsorbing to the particle surface and to suppress nonspecific adsorption of proteins. From the viewpoint of balance, for example, it may be 5 to 30%, 10 to 20%, or 12 to 17%.
  • the polymer having a hydroxyl group may be, for example, a polysaccharide or a modified product thereof from the viewpoint of easily improving the hydrophilicity of the separation material surface.
  • modified polysaccharides include hydrophobic group-modified products.
  • 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.
  • the impregnation time varies depending on the surface state of the porous polymer particles, the polymer concentration is in equilibrium with the external concentration inside the porous polymer particles if it is usually impregnated overnight. 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. At this time, the crosslinked body has a three-dimensional crosslinked network structure having a hydroxyl group.
  • 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.
  • a conventionally known catalyst can be appropriately used according to the type of the crosslinking agent.
  • the crosslinking agent is epichlorohydrin or the like
  • an alkali such as sodium hydroxide
  • the crosslinking agent is a dialdehyde compound.
  • a mineral acid such as hydrochloric acid is effective.
  • 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.
  • a crosslinking agent added 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 may be appropriately adjusted from the viewpoint of the molecular weight of the object to be purified by the separating material, for example.
  • 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 network size of the three-dimensional network structure of the crosslinked body tends to be small even when the reaction rate with the polymer having a hydroxyl group is low.
  • the fractionation property of a substance having a low molecular weight is further improved.
  • 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 or crosslinking agent is not extracted from the impregnated polymer solution, and a crosslinking reaction is performed.
  • the crosslinking reaction can be usually performed at a temperature in the range of 5 to 90 ° C. for 1 to 10 hours.
  • the temperature of the crosslinking reaction is preferably 30 to 90 ° C.
  • the produced particles are filtered off and then washed with a hydrophilic organic solvent such as methanol or ethanol to remove unreacted polymer, suspending medium and the like.
  • a hydrophilic organic solvent such as methanol or ethanol 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 cross-linking treatment step may be omitted.
  • the 5% compression deformation elastic modulus of the separating material in a wet state is 100 MPa or more.
  • the 5% compressive deformation elastic modulus of the separating material in a wet state is less than 100 MPa, it is considered that the separating material is easily deformed by increasing the flexibility of the separating material, so that the column pressure is increased due to consolidation in the column.
  • the 5% compression deformation modulus of the separating material in a wet state is 100 MPa or more, it is considered that such an increase in column pressure is easily suppressed and nonspecific adsorption of proteins is easily reduced.
  • wet state refers to a state saturated with moisture. In order to maintain the wet state, it is preferable to use the separating material taken out of water immediately before the measurement. In the “wet state”, the particle surface and the pores in the particle usually contain water (pure water or the like).
  • the 5% compression deformation elastic modulus (for example, 5% compression deformation elastic modulus when the separation material is compressed at 50 mN) of the separation material of the present embodiment can be calculated as follows. Using a micro compression tester (manufactured by Fisher), the load when the separating material is compressed from 0 mN to 50 mN with a smooth end face (50 ⁇ m ⁇ 50 ⁇ m) of a square column at a load rate of 1 mN / sec at room temperature (25 ° C.). And measure the compression displacement. From the measured value obtained, the compression deformation elastic modulus (5% K value) when the separating material is 5% compressively deformed can be obtained by the following formula.
  • the 5% compressive deformation elastic modulus of the separating material in a wet state is, for example, 110 MPa or more from the viewpoint of further reducing nonspecific adsorption of proteins and from the viewpoint that particles are not easily deformed even if the column length is increased. It may be 120 MPa or more, or 130 MPa or more.
  • the 5% compressive deformation elastic modulus of the separating material in the wet state may be, for example, 1000 MPa or less, 950 MPa or less, 900 MPa or less, or 500 MPa or less.
  • the 5% compressive deformation modulus of the separating material can be adjusted by the type and amount of crosslinking agent used, the amount of coating layer, and the like. For example, as the amount of the crosslinking agent used or the amount of the coating layer increases, the 5% compressive deformation modulus tends to increase.
  • the average particle diameter of the separation material of the present embodiment may be, for example, 300 ⁇ m or less, 150 ⁇ m or less, or 100 ⁇ m or less from the viewpoint of further improving the separation performance.
  • the average particle diameter of the separating material may be, for example, 1 ⁇ m or more, 10 ⁇ m or more, 30 ⁇ m or more, or 50 ⁇ m or more.
  • the average particle diameter of the separating material may be, for example, 1 to 300 ⁇ m, 10 to 150 ⁇ m, 30 to 100 ⁇ m, or 50 to 100 ⁇ m. .
  • the variation coefficient (CV) of the particle size of the separation material of the present embodiment may be, for example, 1% or more, 3% or more, or 5% or more.
  • the variation coefficient may be, for example, 50% or less, 15% or less, or 10% or less.
  • the coefficient of variation (CV) of the particle size of the separating material of the present embodiment may be, for example, 1 to 50% or 3 to 15% from the viewpoint of easy improvement of liquid permeability. It may be 5 to 15% or 5 to 10%.
  • the separation material of the present embodiment preferably includes a coating layer of 30 to 500 mg per 1 g of porous polymer particles.
  • the amount of the coating layer can be measured by reducing the weight of pyrolysis.
  • the liquid passing speed is 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. It can be used at a liquid passing speed of 800 cm / h or more faster than the separating material.
  • liquid flow rate in this specification represents the liquid flow rate when the separation material of this embodiment is filled in a stainless steel column of ⁇ 7.8 ⁇ 300 mm and the liquid is passed.
  • the separation material of the present embodiment is provided with a coating layer containing a polymer having a hydroxyl group on porous polymer particles, whereby particles made of natural polymers or particles made of synthetic polymers in the separation of biopolymers such as proteins.
  • a coating layer containing a polymer having a hydroxyl group on porous polymer particles whereby particles made of natural polymers or particles made of synthetic polymers in the separation of biopolymers such as proteins.
  • the separation material of the present embodiment is suitable for use in separation by size exclusion purification of biopolymers, and the separation material of the present embodiment can be used in column chromatography.
  • the separation material of this embodiment is for liquid chromatography, for example.
  • the biopolymer that can be separated using the separation material of the present embodiment is preferably a water-soluble substance.
  • the purification method of the water-soluble polymer substance of the present embodiment performs gel filtration chromatography using the separation material for gel filtration of the present embodiment.
  • the water-soluble polymer substance to be purified include water-soluble biopolymers.
  • water-soluble biopolymers include proteins such as serum albumin and blood proteins such as immunoglobulins, enzymes present in living bodies, protein bioactive substances produced by biotechnology, DNA, and bioactivity. Peptides are mentioned.
  • the weight average molecular weight of the water-soluble polymer substance to be purified may be, for example, 2 million or less, or 500,000 or less.
  • the properties, conditions, etc. of the separation material may be selected according to the isoelectric point, ionization state, etc. of the protein. Examples of known methods include the methods described in JP-A-60-169427.
  • water-soluble polymer substances to be purified include thyroglobulin, gamma globulin, bovine serum albumin, myoglobin, and uracil.
  • the mass (kDa) of the water-soluble polymer substance may be, for example, 0.01 to 10,000 kDa, 0.05 to 5000 kDa, or 0.1 to 1000 kDa.
  • Example 1 Synthesis of porous polymer particles
  • 16 g of 96% pure divinylbenzene manufactured by Nippon Steel & Sumikin Chemical Co., Ltd., trade name: DVB960
  • 16 g of hexanol as a porous agent
  • 16 g of diethylbenzene and benzoyl peroxide as an initiator
  • 0.5 mass% polyvinyl alcohol aqueous solution was used as a continuous phase.
  • porous polymer particles 1 After emulsifying the continuous phase and the dispersed phase using a microprocess server, 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 and then washed with acetone to obtain porous polymer particles (hereinafter, the porous polymer particles obtained in this example are referred to as “porous polymer particles 1”). The particle size of the porous polymer particles 1 was measured with a flow type particle size measuring device, and the average particle size and particle size C.I. V. The value (coefficient of variation) was calculated. The results are shown in Table 1.
  • the hydrophobic group content of the modified agarose adsorbed on the particles was determined by treating 0.2 g of the particles in 10 mL of 1M sulfuric acid at 70 ° C. for 5 hours, and measuring the absorbance at 269 nm with a spectrophotometer. It can calculate similarly by calculating
  • Porous polymer particles 1 were added to a 20 mg / mL modified agarose aqueous solution at a concentration of 70 mL / particle g and stirred at 55 ° C. for 24 hours to adsorb the modified polymer particles 1 to the porous polymer particles 1.
  • the particles adsorbed with the modified agarose were filtered off and further washed with hot water.
  • the modified agarose was crosslinked as follows. 10 g of particles adsorbed with modified agarose were dispersed in a 0.4 M aqueous sodium hydroxide solution, a crosslinking agent (epichlorohydrin) was added to a concentration of 0.04 M, and the mixture was stirred at room temperature for 8 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 dried to obtain a separating material. The mass of the coating layer per 1 g of porous polymer particles (mg / particle g) was calculated by thermogravimetric analysis of the obtained separating material. Further, the particle size of the obtained separating material was measured with a flow type particle size measuring device, and the average particle size and particle size of C.I. V. The value (coefficient of variation) was calculated. These results are shown in Table 2.
  • Example 2 In the same manner as in Example 1, porous polymer particles were synthesized, and the obtained porous polymer particles were evaluated (hereinafter, the porous polymer particles obtained in this example are referred to as “porous polymer particles 2”). . The results are shown in Table 1.
  • a separating material was prepared and evaluated in the same manner as in Example 1 except that the amount of the crosslinking agent added was such that the concentration of the crosslinking agent (epichlorohydrin) was 0.4M. The evaluation results are shown in Tables 2 and 3.
  • agarose particles 1 Commercially available agarose particles (Sepharose 6 FF, GE Healthcare) were prepared (hereinafter referred to as “agarose particles 1”). In the same manner as the porous polymer particles 1 and 2, the average particle diameter and the C.I. V. The value (coefficient of variation) was calculated. The results are shown in Table 1. Agarose particles 1 were used as they were as a separating material and evaluated in the same manner as in Example 1. The results are shown in Tables 2 and 3.
  • the separation materials of Examples 1 and 2 have less protein non-specific adsorption, excellent fractionation of high molecular weight proteins, and excellent durability when used as a column. I understand that. Moreover, it turns out that a water-soluble polymer substance can be refine

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Abstract

La présente invention concerne un matériau de séparation pour la filtration sur gel, le matériau comprenant : des particules polymère poreuses; et une couche de recouvrement qui comprend un polymère ayant un groupe hydroxyle, et qui recouvre au moins une partie des surfaces de particules polymère poreuses. Pour le matériau de séparation, le module d'élasticité de déformation par compression à 5 % à l'état humide est de 100 MPa ou plus.
PCT/JP2018/013692 2017-03-30 2018-03-30 Matériau de séparation pour filtration sur gel et procédé de purification d'une substance polymère soluble dans l'eau WO2018181925A1 (fr)

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JP2023519448A (ja) * 2020-02-19 2023-05-11 バイオ－ラッド ラボラトリーズ インコーポレーティッド ポリマー充填クロマトグラフィー樹脂を調製する方法
WO2025169969A1 (fr) * 2024-02-09 2025-08-14 株式会社レゾナック Substrat pour charge, procédé de production de substrat pour charge, charge et procédé de production de protéine

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JP2012515640A (ja) * 2009-01-22 2012-07-12 フレセニウス メディカル ケア ドイチェランド ゲーエムベーハー タンパク質結合物質を除去するための着収剤
WO2016117574A1 (fr) * 2015-01-19 2016-07-28 日立化成株式会社 Matériau de séparation
US20160231208A1 (en) * 2013-10-03 2016-08-11 3M Innovative Properties Company Ligand-functionalized substrates with enhanced binding capacity

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JP2012515640A (ja) * 2009-01-22 2012-07-12 フレセニウス メディカル ケア ドイチェランド ゲーエムベーハー タンパク質結合物質を除去するための着収剤
US20160231208A1 (en) * 2013-10-03 2016-08-11 3M Innovative Properties Company Ligand-functionalized substrates with enhanced binding capacity
WO2016117574A1 (fr) * 2015-01-19 2016-07-28 日立化成株式会社 Matériau de séparation

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Publication number Priority date Publication date Assignee Title
JP2023519448A (ja) * 2020-02-19 2023-05-11 バイオ－ラッド ラボラトリーズ インコーポレーティッド ポリマー充填クロマトグラフィー樹脂を調製する方法
JP7577538B2 (ja) 2020-02-19 2024-11-05 バイオ－ラッド ラボラトリーズ インコーポレーティッド ポリマー充填クロマトグラフィー樹脂を調製する方法
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WO2025169969A1 (fr) * 2024-02-09 2025-08-14 株式会社レゾナック Substrat pour charge, procédé de production de substrat pour charge, charge et procédé de production de protéine

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