WO2020036151A1 - Matériau de séparation, procédé de fabrication de matériau de séparation, et colonne - Google Patents

Matériau de séparation, procédé de fabrication de matériau de séparation, et colonne Download PDF

Info

Publication number
WO2020036151A1
WO2020036151A1 PCT/JP2019/031740 JP2019031740W WO2020036151A1 WO 2020036151 A1 WO2020036151 A1 WO 2020036151A1 JP 2019031740 W JP2019031740 W JP 2019031740W WO 2020036151 A1 WO2020036151 A1 WO 2020036151A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
separation material
hydrophilic polymer
hydroxyl group
polymer particles
Prior art date
Application number
PCT/JP2019/031740
Other languages
English (en)
Japanese (ja)
Inventor
史彦 河内
真裕 青嶌
健 安江
後藤　泰史
Original Assignee
日立化成株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 日立化成株式会社 filed Critical 日立化成株式会社
Publication of WO2020036151A1 publication Critical patent/WO2020036151A1/fr

Links

Classifications

    • 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
    • B01J20/282—Porous sorbents
    • B01J20/285—Porous sorbents based on polymers
    • 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
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography

Definitions

  • the present invention relates to a separation material, a method for producing the separation material, and a column.
  • ion exchangers based on porous synthetic polymers and ion exchange based on crosslinked gels of hydrophilic natural polymers are generally used.
  • the body is used.
  • an ion exchanger having a porous synthetic polymer as a base the volume change due to the salt concentration is small. Therefore, when the ion exchanger is packed in a column and used for chromatography, it tends to be excellent in pressure resistance during liquid passage.
  • an ion exchanger whose base is a crosslinked gel of a hydrophilic natural polymer represented by a polysaccharide such as dextran or agarose has an advantage that there is almost no nonspecific adsorption of proteins.
  • this ion exchanger has the disadvantage that it swells remarkably in an aqueous solution, 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 crosslinked gel is used for chromatography, there is a disadvantage that the pressure loss at the time of passing the liquid is large and the gel is compacted by the passing of the liquid.
  • the conventional separation material has room for improvement in column characteristics such as liquid permeability when used as a column.
  • an object of the present invention is to provide a separation material having excellent liquid permeability when used as a column, a column including the separation material, and a method for producing the separation material.
  • the present invention provides a separation material, a method for producing a separation material, and a column according to the following [1] to [8].
  • R represents an alkylene group having 1 to 5 carbon atoms
  • L represents a carboxy group or a group represented by an alkali metal salt of a carboxy group.
  • Separation material [2] The separation material according to [1], wherein the content of the group represented by —ORL is 100 ⁇ mol or more per 1 mL of the separation material.
  • hydrophobic polymer particles are particles containing a polymer having a structural unit derived from a styrene-based monomer.
  • hydrophilic polymer contains a hydrophilic polymer derived from at least one selected from the group consisting of polysaccharides and modified products thereof.
  • hydrophilic polymer includes a hydrophilic polymer derived from at least one selected from the group consisting of agarose, dextran, cellulose, pullulan, chitosan, and modified products thereof.
  • the present invention it is possible to provide a separating material having excellent liquid permeability when used as a column, a column including the separating material, and a method for producing the separating material. Further, the present invention can also provide a separation material which is excellent in column characteristics such as protein dynamic adsorption and recovery while reducing non-specific adsorption of proteins.
  • the separation material according to the present embodiment includes hydrophobic polymer particles and a coating layer that covers at least a part of the surface of the hydrophobic polymer particles.
  • the coating layer includes a hydrophilic polymer, and has a high hydrophilicity.
  • -ORL O represents an oxygen atom derived from the hydroxyl group
  • R represents an alkylene group having 1 to 5 carbon atoms
  • L represents a carboxy group or an alkali metal salt of a carboxy group.
  • the separation material of the present embodiment has excellent liquid permeability when packed in a column.
  • the separation material of the present embodiment has excellent durability and alkali resistance, can reduce non-specific adsorption of protein, and has a sufficiently high dynamic adsorption amount and recovery rate of protein when packed in a column. It is considered high.
  • the separating material of the present embodiment has excellent strength and can have a shape close to a true sphere.
  • a truly spherical separating material is considered hydrodynamically advantageous when used in chromatography. Therefore, such a separation material is considered to be easy to suppress the pressure loss and to perform the chromatography operation, for example.
  • the “surface of the hydrophobic polymer particles” includes not only the outer surface of the hydrophobic polymer particles but also the surface of the pores inside the hydrophobic polymer particles.
  • the hydrophobic polymer particles according to the present embodiment are particles containing a polymer having hydrophobicity.
  • the method for producing the hydrophobic polymer particles is not particularly limited, and examples thereof include a method of polymerizing a monomer capable of forming a polymer having hydrophobicity.
  • the monomer is not particularly limited as long as it can form a polymer having hydrophobicity, and examples thereof include a styrene-based monomer. That is, the hydrophobic polymer particles may include, for example, a polymer having a structural unit derived from a styrene-based monomer. Such hydrophobic polymer particles are considered to be excellent in durability and alkali resistance.
  • the hydrophobic polymer particles according to the present embodiment may have a porous structure. That is, the hydrophobic polymer particles according to the present embodiment may be, for example, hydrophobic porous polymer particles.
  • the porous polymer particles are, for example, particles containing a polymer obtained by polymerizing a monomer in the presence of a porosifying agent, and can be synthesized by conventional suspension polymerization, emulsion polymerization, or the like.
  • Specific examples of the monomer include the following polyfunctional monomers and monofunctional monomers.
  • the polyfunctional monomer examples include divinyl compounds such as divinylbenzene, divinylbiphenyl, divinylnaphthalene, and divinylphenanthrene. These polyfunctional monomers can be used alone or in combination of two or more. Among them, the monomer preferably contains divinylbenzene from the viewpoint of durability, acid resistance and alkali resistance. That is, the hydrophobic polymer particles preferably include a polymer having a structural unit derived from divinylbenzene.
  • Examples of the monofunctional monomer 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, pn-dodecyl Styrene such as styrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene, and 3,4-dichlorosty
  • the monomer preferably contains styrene from the viewpoint of excellent acid resistance and alkali resistance.
  • a styrene derivative having a functional group such as a carboxy group, an amino group, a hydroxyl group, and an aldehyde group can also be used.
  • the porosifying agent examples include an organic solvent that promotes phase separation during polymerization and promotes porosity of particles, such as aliphatic or aromatic hydrocarbons, esters, ketones, ethers, and alcohols. Can be used.
  • the porosifying agent include diethylbenzene, toluene, xylene, cyclohexane, octane, butyl acetate, dibutyl phthalate, methyl ethyl ketone, dibutyl ether, 1-hexanol, 2-octanol, decanol, lauryl alcohol and cyclohexanol. These porosifying agents can be used alone or in combination of two or more.
  • the above-mentioned porous agent can be used in an amount of 0 to 200% by mass based on the total mass of the monomers.
  • the porosity of the hydrophobic polymer particles can be controlled by the amount of the porosifying agent.
  • the size and shape of the pores of the hydrophobic polymer particles can be controlled by the type of the porosifying agent.
  • Water used as a solvent can be used as a porosifying agent.
  • the oil-soluble surfactant is dissolved in the monomer, and the droplets of the monomer absorb the water, whereby the particles can be made porous.
  • oil-soluble surfactant used for making the porous material examples include a sorbitan monoester of a branched C16-C24 fatty acid, a chain unsaturated C16-C22 fatty acid or a chain saturated C12-C14 fatty acid, for example, sorbitan monolaurate, sorbitan Sorbitan monoesters derived from monooleate, sorbitan monomyristate or coconut fatty acids; diglycerol monoesters of branched C16-C24 fatty acids, chain unsaturated C16-C22 fatty acids or chain saturated C12-C14 fatty acids, such as Glycerol monooleate (for example, diglycerol monoester of C18: 1 fatty acid (18 carbon atoms, 1 double bond) fatty acid), diglycerol monomyristate, diglycerol monoisostearate or coconut fatty acid diglycerol monoester Ester; Branch C16 ⁇ 24 alcohol (e.g., Guerbet alcohols
  • sorbitan monolaurate e.g., SPAN® 20, having a purity preferably greater than about 40%, more preferably greater than about 50%, and even more preferably greater than about 70%
  • sorbitan monooleate e.g, SPAN® 80, preferably having a purity of greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70%
  • Diglycerol monooleate e.g, diglycerol monooleate having a purity preferably greater than about 40%, more preferably greater than about 50%, even more preferably greater than about 70%
  • diglycerol monoisostearate Eg, having a purity preferably greater than about 40%, more preferably about 50% More preferably more than about 70% diglycerol monoisostearate
  • diglycerol monomyristate purity preferably more than about 40%, more preferably more than about 50%, even more preferably more than about 70%.
  • Diglycerol monomyristate cocoyl (e.g., lauryl, myristo
  • oil-soluble surfactants are preferably used in the range of 5 to 80% by mass based on the total mass of the monomers.
  • the use amount of the oil-soluble surfactant is 5% by mass or more, the stability of water droplets becomes sufficient, so that it is difficult to form large single holes.
  • the amount of the oil-soluble surfactant used is 80% by mass or less, the shape of the hydrophobic polymer particles can be more easily maintained after the 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 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 salts alkane sulfonates, dialkyl sulfosuccinates such as sodium dioctyl sulfosuccinate, etc., alkenyl succinates (eg, dipotassium alkenyl succinate), alkyl phosphate salts, naphthalene sulfonic acid formalin condensates, polyoxyethylene Polyoxyethylene alkyl ether sulfates such as alkyl phenyl ether sulfate, sodium polyoxyethylene lauryl ether sulfate, and polyoxyethylene alkyl sulfate; Ester salts.
  • alkenyl succinates eg, dipotassium alkenyl succinate
  • alkyl phosphate salts alkyl phosphate salts
  • naphthalene sulfonic acid formalin condensates polyoxyethylene Polyoxyethylene alkyl ether sulfates such as alkyl phenyl ether sulf
  • cationic surfactant examples include alkylamine salts such as laurylamine acetate and stearylamine acetate, and quaternary ammonium salts such as lauryltrimethylammonium chloride.
  • nonionic surfactant examples 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, and amides.
  • hydrocarbon nonionic surfactants such as polyethylene glycol alkyl ethers, polyethylene glycol alkyl aryl ethers, polyethylene glycol esters, polyethylene glycol sorbitan esters, polyalkylene glycol alkylamines, and amides.
  • agents polyether-modified silicon-based nonionic surfactants such as polyethylene oxide adducts of silicon and polypropylene oxide adducts of silicon, and fluorine-based nonionic surfactants such as perfluoroalkyl glycols.
  • amphoteric surfactant examples include a hydrocarbon surfactant such as lauryl dimethylamine oxide, a phosphate ester surfactant, and a phosphite ester surfactant.
  • the surfactant may be used alone or in combination of two or more.
  • anionic surfactants are preferable from the viewpoint of dispersion stability during polymerization of monomers.
  • 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; 2,2′-azobisisobutyronitrile, 1,1′-azobiscyclohexanecarbonitrile, 2,2 ′ Azo compounds such as -azobis (2,4-dimethylvaleronitrile);
  • the polymerization initiator can be used in the range of 0.1 to 7.0 parts by mass based on 100 parts by mass of the monomer.
  • the polymerization temperature can be appropriately selected according to the type of the monomer and the polymerization initiator.
  • the polymerization temperature is preferably from 25 to 110 ° C, more preferably from 50 to 100 ° C.
  • a polymer dispersion stabilizer may be used to improve the dispersion stability of the particles.
  • polymer dispersion stabilizer examples include polyvinyl alcohol, polycarboxylic acid, celluloses (such as hydroxyethylcellulose, carboxymethylcellulose, and methylcellulose) and polyvinylpyrrolidone, and an inorganic water-soluble polymer compound such as sodium tripolyphosphate is also used in combination. can do. Of these, polyvinyl alcohol or polyvinylpyrrolidone is preferred.
  • the addition amount of the polymer dispersion stabilizer is preferably 1 to 10 parts by mass based on 100 parts by mass of the monomer.
  • a water-soluble polymerization inhibitor such as nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble vitamins B, citric acid, and polyphenols may be used.
  • the average particle size of the hydrophobic polymer particles is preferably 500 ⁇ m or less, more preferably 150 ⁇ m or less, and further preferably 110 ⁇ m or less.
  • the average particle size of the hydrophobic polymer particles is preferably 10 ⁇ m or more, more preferably 30 ⁇ m or more, and further preferably 50 ⁇ m or more, from the viewpoint of improving liquid permeability.
  • the coefficient of variation (CV) of the particle diameter of the hydrophobic polymer particles is preferably from 3 to 15%, more preferably from 5 to 15%, from the viewpoint of improving liquid permeability. More preferably, it is 10%.
  • CV coefficient of variation
  • a method of monodispersing with an emulsifying apparatus such as a microprocess server (manufactured by Hitachi, Ltd.) can be mentioned.
  • the average particle diameter and the coefficient of variation of the particle diameter of the hydrophobic polymer particles or the separating material can be determined by the following measurement methods. 1) Disperse the hydrophobic polymer particles or the separating material in water (including a dispersant such as a surfactant) using an ultrasonic dispersing device and include 1% by mass of the hydrophobic polymer particles or the separating material. Prepare a dispersion. 2) Using a particle size distribution meter (Sysmex Flow, manufactured by Sysmex Corporation), the average particle size and the coefficient of variation of the particle size are measured from the image of the hydrophobic polymer particles or about 10,000 separating materials in the dispersion. .
  • a particle size distribution meter Sysmex Flow, manufactured by Sysmex Corporation
  • the pore volume (porosity) of the hydrophobic polymer particles is preferably 30% by volume to 70% by volume, and more preferably 40% by volume to 70% by volume based on the total volume of the hydrophobic polymer particles. Is more preferred.
  • the hydrophobic polymer particles preferably have pores having a pore diameter (mode diameter) of 0.05 ⁇ m or more and less than 0.6 ⁇ m, that is, macropores (macropores).
  • the pore diameter of the hydrophobic polymer particles is more preferably 0.2 ⁇ m or more and less than 0.5 ⁇ m. When the pore diameter is 0.05 ⁇ m or more, the substance tends to easily enter the pores, and when the pore diameter is less than 0.6 ⁇ m, the specific surface area tends to be sufficient.
  • the pore volume and the pore diameter can be adjusted by the above-mentioned porosifying agent.
  • the specific surface area of the hydrophobic polymer particles is preferably 20 m 2 / g or more. In light of higher practicality, the specific surface area is more preferably equal to or greater than 35 m 2 / g, and still more preferably equal to or greater than 40 m 2 / g. If the specific surface area is 20 m 2 / g or more, the amount of adsorption of the substance to be separated tends to increase.
  • the mode diameter (or average pore diameter), specific surface area, and porosity in the pore diameter distribution of the hydrophobic polymer particles and the separating material 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 mL powder cell (stem volume: 0.4 mL) and measured under conditions of an initial pressure of 21 kPa (about 3 psia, pore diameter of about 60 ⁇ m). 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 ⁇ m.
  • the coating layer according to this embodiment includes a hydroxyl group and —ORL (O represents an oxygen atom derived from the hydroxyl group, R represents an alkylene group having 1 to 5 carbon atoms, and L represents a carboxy group or a carboxy group. And a group represented by an alkali metal salt of a group).
  • the separation material according to the present embodiment is excellent in liquid permeability when used as a column, because the coating layer contains a hydrophilic polymer having a hydroxyl group and a group represented by —ORL.
  • the separation material according to the present embodiment tends to be excellent in characteristics such as dynamic adsorption property and recovery rate of protein while reducing non-specific adsorption of protein by providing the above-mentioned coating layer.
  • the alkylene group constituting R may be linear or branched, but is preferably linear from the viewpoint of better liquid permeability.
  • R is preferably an alkylene group having 1 to 4 carbon atoms, and more preferably an alkylene group having 1 to 3 carbon atoms, from the viewpoint of more excellent liquid permeability.
  • L preferably contains at least one selected from the group consisting of a carboxy group, a sodium salt of a carboxy group, and a potassium salt of a carboxy group, from the viewpoint of more excellent liquid permeability, and is composed of a sodium salt of a carboxy group and a carboxy group. More preferably, it contains at least one member selected from the group.
  • the hydrophilic polymer having a hydroxyl group and a group represented by —ORL may include a hydrophilic polymer derived from at least one selected from the group consisting of polysaccharides and modified products thereof.
  • the hydrophilic polymer may be a hydrophilic polymer derived from at least one selected from the group consisting of agarose, dextran, cellulose, pullulan, chitosan, and modified products thereof.
  • the hydrophilic polymer may be cross-linked.
  • the hydrophobic polymer particles By coating the hydrophobic polymer particles with a hydrophilic polymer having a hydroxyl group, non-specific adsorption of proteins tends to be easily suppressed. Further, the hydrophilic polymer having a hydroxyl group is preferably crosslinked. That is, the coating layer preferably contains a crosslinked body of a hydrophilic polymer having a hydroxyl group. Since the hydrophilic polymer having a hydroxyl group is crosslinked, an increase in column pressure tends to be suppressed.
  • the hydrophilic polymer having a hydroxyl group preferably has two or more hydroxyl groups in one molecule.
  • examples of the hydrophilic polymer having a hydroxyl group include a polysaccharide or a modified product thereof, and a polyvinyl alcohol or a modified product thereof.
  • the hydrophilic polymer having a hydroxyl group is preferably a polysaccharide or a modified product thereof.
  • the hydrophilic polymer having a hydroxyl group may include at least one selected from the group consisting of polysaccharides and modified products thereof. Polysaccharides include, for example, agarose, dextran, cellulose, pullulan and chitosan.
  • hydrophilic polymer having a hydroxyl group for example, a polymer having an average molecular weight of about 10,000 to 200,000 can be used.
  • the hydrophilic polymer having a hydroxyl group may include at least one selected from the group consisting of agarose, dextran, cellulose, pullulan, chitosan, and modified products thereof.
  • the hydrophilic polymer having a hydroxyl group is preferably a modified polymer having a hydrophobic group introduced from the viewpoint of improving the interfacial adsorption ability.
  • 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 hydrophobic group is obtained by reacting a functional group (for example, an epoxy group) that reacts with a hydroxyl group and a compound (for example, glycidyl phenyl ether) having a hydrophobic group with a hydrophilic polymer having a hydroxyl group by a conventionally known method. , Can be introduced.
  • a functional group for example, an epoxy group
  • a compound for example, glycidyl phenyl ether
  • the content of the hydrophobic group in the modified hydrophilic polymer in which the hydrophobic group is introduced is determined from the balance between the retention of the hydrophobic interaction force for adsorption on the particle surface and the suppression of non-specific adsorption of the protein. It is preferably from 5 to 30% by mass, more preferably from 10 to 20% by mass, even more preferably from 12 to 17% by mass.
  • the separating material of the present embodiment includes a step (1) of coating at least a part of the surface of the hydrophobic polymer particles with a hydrophilic polymer having a hydroxyl group, and a step of coating a hydrogen atom of the hydroxyl group of the coated hydrophilic polymer.
  • Step (2) of partially substituting a part of the group represented by -RL R represents an alkylene group having 1 to 5 carbon atoms, and L represents a carboxy group or an alkali metal salt of a carboxy group.
  • the coated hydrophilic polymer may be cross-linked before step (2).
  • the method for producing the separation material of the present embodiment includes, for example, a step of adsorbing a hydrophilic polymer having a hydroxyl group on the surface of the hydrophobic polymer particles, a step of cross-linking the adsorbed hydrophilic polymer, And introducing a group represented by —RL into the hydrophilic polymer via a hydroxyl group.
  • the coating layer according to the present embodiment can be formed, for example, by the following method.
  • a solution of a hydrophilic polymer having a hydroxyl group is adsorbed on the surface of the hydrophobic polymer particles.
  • the solvent for the solution of the hydrophilic polymer having a hydroxyl group is not particularly limited as long as it can dissolve the hydrophilic polymer having a hydroxyl group, but water is the most common.
  • the concentration of the hydrophilic polymer dissolved in the solvent is preferably 5 to 20 (mg / mL).
  • This solution is impregnated with hydrophobic polymer particles.
  • hydrophobic polymer particles are added to a solution of a hydrophilic polymer having a hydroxyl group, and the solution is left for a predetermined time.
  • the impregnation time varies depending on the surface state of the hydrophobic polymer particles. However, if the impregnation is carried out for 24 hours, the concentration of the hydrophilic polymer is in an equilibrium state between the inside and the outside of the hydrophobic polymer particles. Thereafter, washing with a solvent such as water or alcohol is performed to remove the hydrophilic polymer having a hydroxyl group that has not been adsorbed.
  • Crosslinking treatment Next, a crosslinking agent is added to cause a crosslinking reaction of the hydrophilic polymer having a hydroxyl group adsorbed on the surface of the hydrophobic polymer particles, thereby forming a crosslinked body. At this time, the crosslinked body has a three-dimensional crosslinked network structure having a hydroxyl group.
  • crosslinking agent examples include two or more functional groups active on hydroxyl groups such as epihalohydrin such as epichlorohydrin, dialdehyde compounds such as glutaraldehyde, diisocyanate compounds such as methylene diisocyanate, and glycidyl compounds such as ethylene glycol diglycidyl ether.
  • epihalohydrin such as epichlorohydrin
  • dialdehyde compounds such as glutaraldehyde
  • diisocyanate compounds such as methylene diisocyanate
  • glycidyl compounds such as ethylene glycol diglycidyl ether.
  • 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.
  • 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 a separating material is dispersed and suspended in an appropriate medium.
  • a crosslinking agent is in the range of 0.1 to 100 moles per mole of one unit of the monosaccharide forming the polysaccharide. Within, it can be selected according to the performance of the separating material. In general, when the amount of the cross-linking agent is reduced, the coating layer tends to be easily separated from the hydrophobic polymer particles. If the amount of the crosslinking agent added is excessive and the reaction rate with the hydroxyl group-containing hydrophilic polymer is high, the properties of the raw material hydroxyl group-containing hydrophilic polymer tend to be impaired.
  • the amount of the catalyst used depends on the type of the cross-linking agent. Usually, when a polysaccharide is used as the hydrophilic polymer having a hydroxyl group, when one unit of the monosaccharide forming the polysaccharide is 1 mol, Is used in a range of 0.01 to 10 mole times, preferably 0.1 to 5 mole times.
  • the cross-linking reaction conditions are temperature conditions
  • the temperature of the reaction system is increased, and when the temperature reaches the reaction temperature, a cross-linking reaction occurs.
  • Specific examples of the medium include water, alcohol, and the like.
  • the crosslinking reaction is usually performed at a temperature in the range of 5 to 90 ° C. for 1 to 24 hours.
  • the temperature is in the range of 30 to 90 ° C.
  • the crosslinking reaction may be performed stepwise.
  • the degree of crosslinking can also be adjusted by subjecting the particles that have undergone a crosslinking reaction to a crosslinking reaction again.
  • the degree of cross-linking is evaluated at the temperature at which 5% weight loss due to thermal decomposition occurs. When the degree of progress of crosslinking is high, the temperature at which weight loss starts is high, and when the degree of progress of crosslinking is low, the temperature at which weight loss starts is also low.
  • the temperature at the time of 5% thermal weight loss is preferably from 200 to 350 ° C, more preferably from 220 to 330 ° C, and more preferably from 230 to 320 ° C, from the viewpoint of maintaining the characteristics of the hydrophilic polymer having a hydroxyl group. Is more preferable. If the degree of cross-linking is too low, the hydrophilic polymer having a hydroxyl group tends to fall off the particles. If the degree of cross-linking is too high, the hydrophilic polymer having a hydroxyl group can be prevented from falling off the particles, but the hydrophilic polymer has Swelling or a decrease in the amount of functional groups tends to decrease the amount of dynamic adsorption.
  • the generated particles are separated by filtration, and then washed with a hydrophilic organic solvent such as methanol and ethanol to remove unreacted polymer and suspending medium. Particles at least part of the surface of which are coated with a coating layer containing a hydrophilic polymer having a hydroxyl group (hereinafter sometimes referred to as “coated particles”) are obtained.
  • a hydrophilic organic solvent such as methanol and ethanol
  • a carboxy group can be introduced into the coated particles via a hydroxyl group of the hydrophilic polymer. That is, in the coating layer according to the present embodiment, a group represented by —ORL, in which an alkylene group having a carboxy group is directly bonded to an oxygen atom derived from a hydroxyl group of the hydrophilic polymer, is introduced. ing.
  • a method for introducing a group represented by —ORL for example, a method using a halogenated alkyl compound can be mentioned.
  • the alkyl halide compound include monohalogenoacetic acid, monohalogenopropionic acid, monohalogenobutanoic acid, monohalogenopentanoic acid, monohalogenohexanoic acid, and alkali metal salts thereof.
  • the alkyl halide compound is a bromide or chloride.
  • the hydroxyl group of the hydrophilic polymer is reacted with a diglycidyl compound such as epichlorohydrin or ethylene glycol diglycidyl ether such as epichlorohydrin to introduce an epoxy group, and then the epoxy group is converted into a carboxylic acid thiol such as thioglycolic acid.
  • a carboxy group can be introduced.
  • the introduction of the epoxy group also causes the cross-linking reaction of the coating layer to the hydrophilic polymer to proceed in parallel, so that the swellability of the hydrophilic polymer such as a sugar chain is impaired, and the amount of dynamic adsorption of the protein decreases. There is a tendency.
  • the method for introducing a group represented by —ORL according to the present embodiment does not involve a step of introducing an epoxy group, and thus avoids the progress of the crosslinking reaction as described above. It is possible to improve the protein dynamic adsorption amount.
  • a separation material having excellent liquid permeability can be produced.
  • the content of the group represented by -ORL in the separation material is preferably 100 ⁇ mol or more, more preferably 110 ⁇ mol or more, and more preferably 120 ⁇ mol per 1 mL of the separation material. More preferably, it is more preferably at least 125 ⁇ mol.
  • the amount of the carboxy group is 100 ⁇ mol or more per 1 mL of the separation material, the liquid permeability of the separation material can be further improved, and the electrostatic force of the separation material increases. Can be increased.
  • the content of the group represented by —ORL in the separation material is too large, the protein is hardly diffused into the separation material from the viewpoint of difficulty in diffusing the protein into the separation material.
  • the amount is preferably 1000 ⁇ mol or less, more preferably 950 ⁇ mol or less per 1 mL of the separation material.
  • the separation material of the present embodiment has a coating layer of 30 to 500 mg per 1 g of the hydrophobic polymer particles.
  • the amount of the coating layer can be measured by a weight loss due to thermal decomposition or the like.
  • the liquid passing speed in the present specification indicates a liquid passing speed when a separating material of the present embodiment is packed in a stainless steel column of ⁇ 7.8 ⁇ 300 mm and the liquid is passed.
  • the flow rate of water is preferably 1300 cm / h or more when water is passed so that the pressure of the column becomes 0.3 MPa. It is more preferably at least 1500 cm / h, even more preferably at least 2000 cm / h.
  • the flow rate of a protein solution or the like is generally in the range of 400 cm / h or less.
  • a normal protein separation speed is used. It can be used at a flow rate of 1300 cm / h or more, which is faster than the separation material.
  • the average particle size of the separating material is preferably from 10 to 500 ⁇ m, more preferably from 30 to 300 ⁇ m, further preferably from 50 to 150 ⁇ m, and more preferably from 50 to 110 ⁇ m, from the viewpoint of improving liquid permeability. Is particularly preferred.
  • the coefficient of variation (CV) of the particle size of the separating material is preferably 3 to 15%, more preferably 5 to 15%, and more preferably 5 to 10% from the viewpoint of improving liquid permeability. It is even more preferred.
  • the pore volume of the separating material is preferably 30% by volume or more and 70% by volume or less, more preferably 40% by volume or more and 70% by volume or less, and more preferably 50% by volume or more and 70% by volume based on the total volume of the separating material. % Is more preferable.
  • the separation material preferably has pores having a pore diameter of 0.05 ⁇ m or more and less than 0.6 ⁇ m, that is, macropores (macropores).
  • the pore size of the separation material is more preferably 0.1 ⁇ m or more and less than 0.5 ⁇ m. When the pore diameter is 0.05 ⁇ m or more, the substance tends to easily enter the pores, and when the pore diameter is less than 0.6 ⁇ m, the specific surface area tends to be sufficient.
  • the specific surface area of the separation material is preferably 20 m 2 / g or more. In light of higher practicality, the specific surface area is more preferably equal to or greater than 35 m 2 / g, and still more preferably equal to or greater than 40 m 2 / g. If the specific surface area is 20 m 2 / g or more, the amount of adsorption of the substance to be separated tends to increase.
  • the average pore diameter, specific surface area and the like of the separating material can be adjusted by appropriately selecting the raw material of the hydrophobic polymer particles, the porosifying agent, the hydrophilic polymer having a hydroxyl group and the like.
  • the 5% compressive deformation elastic modulus in water of the separating material may be, for example, 70 MPa or more, 100 MPa or more, 150 MPa or more, or 200 MPa or more.
  • the upper limit of the 5% compressive deformation elastic modulus is not particularly limited.
  • the 5% compressive deformation elastic modulus (5% K value) of the separating material in water can be calculated as follows. Using a micro-compression tester (manufactured by Fisher), the separation material previously immersed in water was applied at a load application speed of 1 mN / sec at room temperature (25 ° C.) and a smooth end surface (50 ⁇ m ⁇ 50 ⁇ m) of a square pillar. The load and the compression displacement when compressed to 50 mN are measured. From the obtained measured values, the compression elastic modulus (5% K value) when the separation material undergoes 5% compression deformation can be obtained by the following equation. The load at the point where the amount of displacement changes the most during the measurement is defined as the breaking strength (mN).
  • a ligand eg, protein A
  • an ion exchange group can be introduced into the separation material of the present embodiment via the carboxy group in the group represented by —ORL.
  • a ligand for example, protein A
  • an ion exchange group can be introduced into the separation material of the present embodiment in addition to the group represented by —ORL.
  • the separation material of the present embodiment can be suitably used for ion exchange purification, affinity purification, separation of biological macromolecules such as proteins by electrostatic interaction, and the like.
  • the separating material of the present embodiment is added to a mixed solution containing a protein, and only the protein is adsorbed to the separating material by electrostatic interaction.
  • the separation material of the present embodiment can be used in column chromatography.
  • a water-soluble substance is preferable.
  • proteins such as blood proteins such as serum albumin and immunoglobulin, enzymes present in living bodies, protein bioactive substances produced by biotechnology, DNA, and biopolymers such as bioactive peptides.
  • the molecular weight of the biopolymer is preferably 2,000,000 or less, more preferably 500,000 or less.
  • the properties of the separation material, separation conditions, and the like can be selected depending on the isoelectric point, ionization state, and the like of the protein.
  • Known methods include, for example, the method described in JP-A-60-169427.
  • the separation material of the present embodiment by introducing a carboxy group or an alkali metal salt of a carboxy group which serves as an ion exchange group into the coating layer, particles of natural polymer or synthetic polymer can be used for separating biopolymers such as proteins. Each particle has the advantages of molecules.
  • the separation material of the present embodiment has excellent liquid permeability, reduces nonspecific adsorption of proteins, and tends to easily cause protein adsorption and desorption. Further, the separation material of the present embodiment tends to have a large adsorption amount (dynamic adsorption amount) of proteins and the like under the same flow rate.
  • the column of the present embodiment includes the above-described separation material.
  • the column can be manufactured by packing the above-mentioned separating material in the column.
  • the method for filling the column with the separating material is not particularly limited, and for example, a known method can be employed.
  • the particle size of the hydrophobic polymer particles 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.
  • the average particle size of the hydrophobic polymer particles is 102 ⁇ m, and the C.I. V. Was 8%.
  • an agarose constituent unit means a disaccharide unit of agarose
  • a modified agarose constituent unit means one in which a phenyl group is introduced into at least one of the hydroxyl groups in the disaccharide unit of agarose. I do.
  • A: true absorbance of denatured agarose absorbance of denatured agarose-absorbance of undenatured agarose ⁇
  • -Concentration of undenatured agarose constituent unit (g / L) sample concentration of denatured agarose (% by mass) x 10-concentration of denatured agarose constituent unit (g / L) Absorption of undenatured aga
  • Example 1 ⁇ Formation of coating layer> Hydrophobic polymer particles are added to a 20 mg / mL modified agarose aqueous solution at a concentration of 70 mL / g of particles, and the mixture is stirred at 55 ° C. for 24 hours to adsorb the modified agarose to the hydrophobic polymer particles. And washed with hot water.
  • the modified agarose adsorbed on the hydrophobic polymer particles was crosslinked as follows. 10 g of the particles onto which the modified agarose had been adsorbed were dispersed in a 0.4 M aqueous sodium hydroxide solution, and 0.02 M epichlorohydrin was added thereto, followed by stirring at room temperature for 24 hours. Thereafter, the resultant was washed with a 2% by mass aqueous solution of hot sodium dodecyl sulfate, and then washed with pure water to obtain coated particles having a crosslinked product of modified agarose as a coating layer. The amount of the coating layer was measured by weight loss due to thermal decomposition, and the coating amount (mg / g) per 1 g of the hydrophobic polymer particles was calculated. Table 1 shows the results.
  • Non-specific adsorption amount of protein 0.2 g of the obtained coated particles was added to 20 mL of a Tris-hydrochloride buffer (pH 8.0) having a BSA (Bovine Serum Albumin) concentration of 24 mg / mL, and the mixture was stirred at room temperature for 24 hours. Thereafter, the supernatant was collected by centrifugation. Based on the BSA concentration of the supernatant, the amount of BSA adsorbed on the particles was calculated. The BSA concentration was confirmed from the absorbance at 280 nm of the supernatant with a spectrophotometer.
  • BSA Bovine Serum Albumin
  • Non-specific adsorption was evaluated as “A” when the amount of BSA adsorbed per 1 mL of the separating material was less than 1 mg, “B” when it was 1 to 10 mg, and “C” when it exceeded 10 mg. Table 1 shows the results. In Comparative Example 1, nonspecific adsorption could not be evaluated.
  • the particle size of the separation material was measured with a flow type particle size measuring device, and the average particle size and the coefficient of variation (CV) of the particle size were calculated.
  • the mode pore diameter, specific surface area, and porosity of the separation material were measured with a mercury intrusion measuring device. Table 1 shows the results.
  • the buffer was flowed through the packed column for 10 column volumes. Thereafter, a buffer containing a protein at a concentration of 2 mg / mL was passed through the packed column with a residence time of 2 minutes, and the protein concentration at the column outlet was measured by UV measurement. The solution was allowed to flow until the protein concentration at the inlet and the column of the column coincided with each other. Thereafter, a solution obtained by adding a 1 M NaCl solution to the buffer was passed through 10 column volumes as a desorption solution, and the adsorbed protein was recovered.
  • the dynamic adsorption amount (10% Dynamic Binding Capacity: hereinafter, 10% DBC) at 10% breakthrough was calculated using the following equation.
  • the amount of protein in the recovered solution was calculated, and the recovery rate R (%) of the protein relative to the amount of dynamic adsorption was calculated from the following equation.
  • a buffer a 20 mmol / L sodium acetate buffer (pH 5.0) was used.
  • the protein used was IgG (immunoglobulin G). Table 2 shows the results.
  • Example 2 A separation material having a group in which —CH 2 COOH or —CH 2 COONa is bonded to an oxygen atom derived from a hydroxyl group of modified agarose was prepared in the same manner as in Example 1 except that monochloroacetic acid 40 g was changed to monochloroacetic acid 20 g. Evaluation was performed in the same manner as in Example 1.
  • Example 3 Performed in the same manner as in Example 1 except that 40 g of monochloroacetic acid was changed to 40 g of 3-chloropropionic acid, and then 40 g of DMF (N, N-dimethylformamide) was added after adding 3-chloropropionic acid.
  • a separation material having a group in which — (CH 2 ) 2 COOH or — (CH 2 ) 2 COONa was bonded to the oxygen atom of was prepared, and evaluated in the same manner as in Example 1.
  • Example 4 Monochloroacetic acid 40g 4-chlorobutane was changed to acid 40g, 4-chlorobutane except that after the acid addition further DMF was added 40g is as in Example 1 and the oxygen atom derived from the hydroxyl group of the denaturing agarose - (CH 2) 3 A separating material having a group to which COOH or — (CH 2 ) 3 COONa was bonded was prepared and evaluated in the same manner as in Example 1.
  • Example 5 The same procedure as in Example 1 was carried out except that 40 g of monochloroacetic acid was changed to 40 g of 5-chloropentanoic acid, and 40 g of DMF was further added after the addition of 5-chloropentanoic acid, and the oxygen atom derived from the hydroxyl group of the modified agarose was replaced with-(CH 2 ) 4 COOH, or - (CH 2) 4 COONa to form a separation member having the bonded groups, was evaluated in the same manner as in example 1.
  • Example 6 The same procedure as in Example 1 was carried out except that 40 g of monochloroacetic acid was changed to 40 g of 6-chlorohexanoic acid, and 40 g of DMF was further added after the addition of 6-chlorohexanoic acid.
  • the oxygen atom derived from the hydroxyl group of the modified agarose was replaced with-(CH 2 ) 5 COOH or - (CH 2) 5 COONa is prepared separation material having attached groups, was evaluated in the same manner as in example 1.
  • Example 1 Evaluation was performed in the same manner as in Example 1 using a commercially available ion exchange chromatography carrier “CM Sepharose Fast Flow” (manufactured by GE Healthcare Japan) as a separating material.
  • CM Sepharose Fast Flow commercially available ion exchange chromatography carrier “CM Sepharose Fast Flow” (manufactured by GE Healthcare Japan) as a separating material.
  • the separation materials of Examples 1 to 6 were excellent in liquid permeability when packed in a column, and high in the amount of dynamic adsorption of protein and the recovery rate. In addition, it was also confirmed that the separation materials of Examples 1 to 6 can suppress nonspecific adsorption of proteins and have a high 5% compressive deformation elastic modulus.
  • the present invention it is possible to provide a separation material having excellent liquid permeability when used as a column, a column including the separation material, and a method for producing the separation material.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)

Abstract

Le but de la présente invention est de fournir un matériau de séparation qui présente une excellente perméabilité aux liquides lorsqu'il est utilisé en tant que colonne. Ce matériau de séparation comprend un polymère hydrophile qui comprend des particules polymère hydrophobe et une couche d'enrobage qui recouvre au moins une partie de la surface des particules polymère hydrophobe. La couche d'enrobage comprend un groupe hydroxyle et un groupe représenté par-O-R-L (O est un atome d'oxygène dérivé d'un groupe hydroxyle, R est un groupe alkylène ayant 1 à 5 atomes de carbone, et L est un groupe carboxyle ou un sel de métal alcalin d'un groupe carboxyle).
PCT/JP2019/031740 2018-08-14 2019-08-09 Matériau de séparation, procédé de fabrication de matériau de séparation, et colonne WO2020036151A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2018-152608 2018-08-14
JP2018152608A JP2021181037A (ja) 2018-08-14 2018-08-14 分離材、分離材の製造方法及びカラム

Publications (1)

Publication Number Publication Date
WO2020036151A1 true WO2020036151A1 (fr) 2020-02-20

Family

ID=69524735

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2019/031740 WO2020036151A1 (fr) 2018-08-14 2019-08-09 Matériau de séparation, procédé de fabrication de matériau de séparation, et colonne

Country Status (2)

Country Link
JP (1) JP2021181037A (fr)
WO (1) WO2020036151A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022257560A1 (fr) * 2021-06-09 2022-12-15 深圳普门科技股份有限公司 Microsphère non poreuse de copolymère d'agent de réticulation polyvinylique-monomère vinylique, son procédé de préparation, et son application

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016117574A1 (fr) * 2015-01-19 2016-07-28 日立化成株式会社 Matériau de séparation
JP2017196544A (ja) * 2016-04-25 2017-11-02 日立化成株式会社 分離材及びカラム
WO2018181738A1 (fr) * 2017-03-30 2018-10-04 日立化成株式会社 Matériau de séparation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016117574A1 (fr) * 2015-01-19 2016-07-28 日立化成株式会社 Matériau de séparation
JP2017196544A (ja) * 2016-04-25 2017-11-02 日立化成株式会社 分離材及びカラム
WO2018181738A1 (fr) * 2017-03-30 2018-10-04 日立化成株式会社 Matériau de séparation

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022257560A1 (fr) * 2021-06-09 2022-12-15 深圳普门科技股份有限公司 Microsphère non poreuse de copolymère d'agent de réticulation polyvinylique-monomère vinylique, son procédé de préparation, et son application

Also Published As

Publication number Publication date
JP2021181037A (ja) 2021-11-25

Similar Documents

Publication Publication Date Title
JP6778382B2 (ja) 分離材
JP6790834B2 (ja) 分離材
JP2017192897A (ja) 分離材及びカラム
JPWO2018181738A1 (ja) 分離材
JP6648565B2 (ja) 分離材
WO2020036151A1 (fr) Matériau de séparation, procédé de fabrication de matériau de séparation, et colonne
JP6972606B2 (ja) 分離材、カラム、及び分離材の製造方法
JP2021007915A (ja) 被覆粒子の製造方法及びカラムの製造方法
JP6627514B2 (ja) 分離材、カラム、及び分離材の製造方法
WO2016117567A1 (fr) Matériau de séparation
JP2017125815A (ja) 分離材及びカラム
JP6759679B2 (ja) 分離材及びカラム
JP6746919B2 (ja) 分離材及びカラム
JP6939021B2 (ja) 分離材及びカラム
JP6676976B2 (ja) 分離材及びカラム
JP6676975B2 (ja) 分離材及びカラム
JP6926573B2 (ja) 分離材及びカラム
JP6852259B2 (ja) 分離材及びカラム
JP6874276B2 (ja) 分離材及びカラム
JP2020018989A (ja) 分離材
JPWO2018174022A1 (ja) 分離材
JP6746915B2 (ja) 分離材及びカラム
JP6778378B2 (ja) 分離材及びカラム
WO2018181925A1 (fr) Matériau de séparation pour filtration sur gel et procédé de purification d'une substance polymère soluble dans l'eau
JP2020044496A (ja) 分離材及び充填カラム

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19849686

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19849686

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP