JP2017125814A - Separation material and column - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a separation material capable of obtaining satisfactory dynamic attraction amount even when used in a chromatography filling in a column, and a column containing the separation material.SOLUTION: The separation material includes: a porous polymer particle; and a coating layer covering at least a part of the surface of the porous polymer particle. The coating layer includes a high polymer which has a hydroxyl group, and the high polymers are cross-linked. 5% weight loss temperature of the separating material is 200-350°C.SELECTED DRAWING: None

Description

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

  Conventionally, when separating and purifying biopolymers typified by proteins, generally, ion exchangers based on porous synthetic polymers and ion exchanges based on cross-linked gels of hydrophilic natural polymers are generally used. The body is used.

  Since an ion exchanger based on a porous synthetic polymer has a small volume change due to salt concentration, when packed in a column and used for chromatography, it tends to be excellent in pressure resistance during liquid passage. However, when this ion exchanger is used for separation of proteins, etc., nonspecific adsorption such as irreversible adsorption based on hydrophobic interaction occurs, peak asymmetry occurs, or nonspecifically adsorbed protein is adsorbed There is a problem that it cannot be recovered as it is.

  On the other hand, an ion exchanger based on a cross-linked gel of a hydrophilic natural polymer typified by polysaccharides such as dextran and agarose has an advantage that there is almost no non-specific adsorption of protein. However, this ion exchanger swells remarkably in an aqueous solution, and has a drawback that the volume change due to the ionic strength of the solution, the volume change between the free acid form and the loaded form is large, and the mechanical strength is not sufficient. In particular, when a cross-linked gel is used in chromatography, there is a drawback that the pressure loss during liquid passage is large and the gel is consolidated by liquid passage.

  In order to overcome the disadvantages of the hydrophilic natural polymer cross-linked gel, attempts have been made so far to combine it with a rigid substance that becomes a “skeleton” (for example, Patent Documents 1 to 7).

US Pat. No. 4,965,289 US Pat. No. 4,335,017 US Pat. No. 4,336,161 US Pat. No. 3,966,489 JP-A-1-254247 US Pat. No. 5,114,577 JP 2009-244067 A

  However, when the separation material as described above is packed in a column and used for chromatography, there is a problem that a sufficient amount of dynamic adsorption cannot be obtained, and it does not have sufficient properties as a separation material. .

  Accordingly, an object of the present invention is to provide a separation material that can obtain a sufficient amount of dynamic adsorption even when packed in a column and used in chromatography, and a column including the separation material.

The present invention provides the separating material described in [1] to [10] below.
[1] A separating material comprising porous polymer particles and a coating layer covering at least a part of the surface of the porous polymer particles, wherein the coating layer contains a polymer having a hydroxyl group, and the hydroxyl group A separation material in which a polymer having a cross-linkage is crosslinked and a 5% weight reduction temperature of the separation material is 200 to 350 ° C.
[2] The separation material according to [1], wherein the porous polymer particles contain a monomer unit derived from a styrene monomer.
[3] The separating material according to [1] or [2], wherein the polymer having a hydroxyl group is a polysaccharide or a modified product thereof.
[4] The separation material according to any one of [1] to [3], wherein the polymer having a hydroxyl group is agarose or a modified product thereof.
[5] The separation material according to any one of [1] to [4], wherein the porous polymer particles have an average particle size of 10 to 300 μm.
[6] The separation material according to any one of [1] to [5], wherein the mode diameter in the pore size distribution of the separation material is 0.05 to 0.5 μm.
[7] The separation material according to any one of [1] to [6], which is particles having a porosity of 30 to 70% by volume.
[8] The separating material according to any one of [1] to [7], which is a particle having a specific surface area of 30 m ² / g or more.
[9] The separation material according to any one of [1] to [8], wherein a coefficient of variation of the particle size of the porous polymer particles is 3 to 15%.
[10] The separating material according to any one of [1] to [9], comprising 30 to 500 mg of the coating layer per 1 g of the porous polymer particles.
[11] When water is passed through the column packed with the separation material so that the pressure in the column becomes 0.3 MPa, the linear flow rate of water is 800 cm / h or more. [1] -Separation material in any one of [10].
[12] A column comprising the separation material according to any one of [1] to [11].

  ADVANTAGE OF THE INVENTION According to this invention, even when it fills a column and uses it by chromatography, a separation material which can obtain sufficient dynamic adsorption amount, and a column provided with this separation material can be provided.

  Hereinafter, preferred embodiments of the present invention will be described, but the present invention is not limited to these embodiments.

  The separating material of the present embodiment includes porous polymer particles and a coating layer that covers at least a part of the surface of the porous polymer particles. The coating layer includes a polymer having a hydroxyl group, and the polymer having a hydroxyl group is crosslinked. The 5% weight loss temperature of the separating material is 200 to 350 ° C. In the present specification, 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 particle of this embodiment is a porous particle containing a polymer. The porous particles are obtained, for example, by polymerizing droplets containing a monomer and a porous agent dispersed in an aqueous medium. Examples of the polymerization method include conventional suspension polymerization and emulsion polymerization. Although it does not specifically limit as a monomer, From a viewpoint that it is excellent in durability and alkali resistance, a styrene-type monomer is preferable. That is, the porous polymer particles preferably contain a monomer unit derived from a styrene monomer. Specific examples of the monomer include the following polyfunctional monomers and monofunctional monomers.

  Examples of the polyfunctional monomer include divinyl compounds such as divinylbenzene, divinylbiphenyl, divinylnaphthalene and divinylphenanthrene. These polyfunctional monomers can be used alone or in combination. Among these, divinylbenzene is preferable from the viewpoint of more excellent durability, acid resistance, and alkali resistance.

  Examples of the monofunctional monomer include styrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, α-methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, 2,4- Dimethyl styrene, pn-butyl styrene, pt-butyl styrene, pn-hexyl styrene, pn-octyl styrene, pn-nonyl styrene, pn-decyl styrene, pn-dodecyl Examples thereof include styrene such as styrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene and 3,4-dichlorostyrene and derivatives thereof. These monofunctional monomers can be used alone or in combination. Among these, styrene is preferable from the viewpoint of excellent acid resistance and alkali resistance. The monomer can be, for example, a styrene derivative having a functional group such as a carboxy group, an amino group, a hydroxyl group, and an aldehyde group.

  Examples of the aqueous medium include water and a mixed solvent of water and a water-soluble solvent (for example, a lower alcohol). The aqueous medium can contain a surfactant. Examples of the surfactant include anionic, cationic, nonionic and zwitterionic surfactants.

  Examples of anionic surfactants include fatty acid oils such as sodium oleate and castor oil potash, alkyl sulfate esters 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

  Examples of the cationic surfactant include alkylamine salts such as laurylamine acetate and stearylamine acetate, and quaternary ammonium salts such as lauryltrimethylammonium chloride.

  Nonionic surfactants include, for example, polyethylene glycol alkyl ethers, polyethylene glycol alkyl aryl ethers, polyethylene glycol esters, polyethylene glycol sorbitan esters, polyalkylene glycol alkylamines, amides and other hydrocarbon nonionic surfactants, silicon polyethylene Examples include polyether-modified silicon-based nonionic surfactants such as oxide adducts and polypropylene oxide adducts, and fluorine-based nonionic surfactants such as perfluoroalkyl glycol.

  Examples of zwitterionic surfactants include hydrocarbon surfactants such as lauryl dimethylamine oxide, phosphate ester surfactants, and phosphite ester surfactants.

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

  Examples of the porosifying agent include aliphatic or aromatic hydrocarbons, esters, ketones, ethers, and alcohols, which are organic solvents that promote phase separation during polymerization and promote pore formation of particles. Specific examples include toluene, xylene, diethylbenzene, cyclohexane, octane, butyl acetate, dibutyl phthalate, methyl ethyl ketone, dibutyl ether, 1-hexanol, 2-octanol, decanol, lauryl alcohol, and cyclohexanol. These porous materials can be used alone or in combination.

  The porosifying agent can be 0 to 200% by mass relative to the total mass of the monomer. The porosity of the porous polymer particles and the separating material particles can be controlled by the amount of the porous agent. The pore size and shape of the porous polymer particles and the separating material can be controlled depending on the kind of the porous agent.

  As the porosifying agent, water that is an aqueous medium can be used. When water is used as a porous agent, an oil-soluble surfactant can be added to water.

  Examples of the oil-soluble surfactant include branched fatty acids having 16 to 24 carbon atoms, chain unsaturated fatty acids having 16 to 22 carbon atoms, or sorbitan monoesters of chain saturated fatty acids having 12 to 14 carbon atoms (for example, sorbitan monoesters). Sorbitan monoesters derived from laurate, sorbitan monooleate, sorbitan monomyristate or coconut fatty acid); branched fatty acids having 16 to 24 carbon atoms, chain unsaturated fatty acids having 16 to 22 carbon atoms or 12 to 14 carbon atoms Diglycerol monoesters of chain saturated fatty acids (eg, diglycerol monooleate such as a diglycerol monoester of a fatty acid having 18 carbon atoms and 1 double bond); diglycerol monomyristate, diglycerol monoisostearate Diglycerol monoester of rate or coconut fatty acid; having 16 to 24 carbon atoms Diglycerol monoaliphatic ethers of dihydric alcohols (e.g. Gerve alcohol), chain unsaturated alcohols having 16 to 22 carbon atoms or chain saturated alcohols having 12 to 14 carbon atoms (e.g. coconut fatty alcohol); and mixtures thereof Is mentioned.

  Among these, sorbitan monolaurate (for example, SPAN (registered trademark) 20, preferably sorbitan monolaurate having a purity of 40% or more, more preferably 50% or more, and further preferably 70% or more); sorbitan Monooleate (eg, SPAN 80, preferably sorbitan monooleate having a purity of 40% or more, more preferably 50% or more, and even more preferably 70% or more); diglycerol monooleate ( For example, preferably a purity of 40% or more, more preferably a purity of 50% or more, more preferably a purity of 70% or more of diglycerol monooleate); a diglycerol monoisostearate (for example, preferably a purity of 40% or more, more preferably Is more than 50% purity, more preferably 70% purity Diglycerol monoisostearate above); diglycerol monomyristate (eg, preferably sorbitan monomyristate having a purity of 40% or more, more preferably 50% or more, and even more preferably 70% or more); Cocoyl (eg, lauryl and myristoyl groups) ethers; or mixtures thereof are preferred.

  These oil-soluble surfactants are preferably used in the range of 5 to 80% by mass with respect to the total mass of the monomer from the viewpoint of making the particles porous. When the content of the oil-soluble surfactant is 5% by mass or more, it is preferable because the particles are made porous. When the content of the oil-soluble surfactant is 80% by mass or less, the porous polymer particles are more easily retained in shape after polymerization.

  If necessary, a polymerization initiator can be further added to the aqueous medium. Examples of the polymerization initiator include benzoyl peroxide, lauroyl peroxide, orthochlorobenzoyl peroxide, orthomethoxybenzoyl peroxide, 3,5,5-trimethylhexanoyl peroxide, tert-butylperoxy-2-ethylhexano Organic peroxides such as ate and di-tert-butyl peroxide; and 2,2′-azobisisobutyronitrile, 1,1′-azobiscyclohexanecarbonitrile and 2,2′-azobis (2,4 An azo compound such as (dimethylvaleronitrile). The quantity of a polymerization initiator can be 0.1-7.0 mass parts with respect to 100 mass parts of monomers.

  The polymerization temperature can be appropriately selected according to the type of monomer and polymerization initiator. The polymerization temperature is preferably 25 to 110 ° C, more preferably 50 to 100 ° C.

  In order to improve the dispersion stability of the particles, a polymer dispersion stabilizer can be further added to the aqueous medium. Examples of the polymer dispersion stabilizer include polyvinyl alcohol, polycarboxylic acid, cellulose (for example, hydroxyethyl cellulose, carboxymethyl cellulose and methyl cellulose), and polyvinyl pyrrolidone. Of these, the polymer dispersion stabilizer is preferably polyvinyl alcohol or polyvinyl pyrrolidone. An inorganic water-soluble polymer compound such as sodium tripolyphosphate can also be used in combination. The addition amount of the polymer dispersion stabilizer is preferably 1 to 10 parts by mass with respect to 100 parts by mass of the monomer.

  In order to suppress the polymerization of the monomer alone, for example, a water-soluble polymerization inhibitor such as nitrite, sulfite, hydroquinone, ascorbic acid, water-soluble vitamin B, citric acid and polyphenol can be used.

  The average particle size of the porous polymer particles and the separating material is preferably 300 μm or less, more preferably 150 μm or less, and even more preferably 100 μm or less, from the viewpoint of improving separability. From the viewpoint of improving liquid permeability, the average particle size of the porous polymer particles and the separating material is preferably 10 μm or more, more preferably 30 μm or more, and further preferably 50 μm or more.

  The coefficient of variation (C.V.) of the particle diameter of the porous polymer particles and the separating material is preferably 3 to 15%, more preferably 5 to 15% from the viewpoint of improving liquid permeability. More preferably, it is 5 to 10%. Particle size C.I. V. As a method for reducing the above, for example, monodisperse droplets may be monodispersed by an emulsifying device such as a microprocess server (manufactured by Hitachi, Ltd.).

The average particle size and particle size of C.I. V. Can be determined by the following measurement method.
1) Disperse the particles in water (including a dispersing agent such as a surfactant) with an ultrasonic dispersing device to prepare a dispersion liquid containing 1% by mass of porous polymer particles.
2) Using a particle size distribution meter (Sysmex Flow, manufactured by Sysmex Corporation), C.I. V. Measure.

  The mode diameter (mode value of pore diameter distribution, maximum frequency diameter) in the pore diameter distribution of the separation material of the present embodiment is preferably 0.01 to 0.5 μm, and preferably 0.05 to 0.5 μm. It is more preferable. When the mode diameter in the pore size distribution is in this range, the liquid easily flows in the particles, and the amount of dynamic adsorption is likely to be increased.

  The pore volume of the porous polymer particles and the separation material particles is preferably 30 to 70% by volume, more preferably 40 to 70% by volume, based on the total volume of the porous polymer particles and the separation material, respectively. . The porous polymer particles and the separating material preferably have macropores (macropores). The average pore diameter of the macropore is preferably 0.1 to 0.5 μm, more preferably 0.2 to 0.5 μm. When the average pore diameter is 0.1 μm or more, a substance tends to easily enter the pores, and when the average pore diameter is 0.5 μm or less, the specific surface area of the particles becomes sufficient. The pore volume and average pore diameter of the porous polymer particles can be adjusted by the above-described porosifying agent.

The specific surface area of the porous polymer particles and the separation material particles is preferably 30 m ² / g or more from the viewpoint of improving the adsorption amount of the substance to be separated, and 35 m ² / g or more from the viewpoint of improving practicality. It is more preferable that it is 40 m ² / g or more.

  The porous polymer particles and the separating material preferably have a porosity of 30 to 70% by volume, more preferably 40 to 70% by volume, and still more preferably 50 to 70% by volume. When the porosity is within this numerical range, the adsorption amount of proteins and the like can be improved.

  The mode diameter, specific surface area and porosity in the pore size distribution of the porous polymer particles and the separation material particles can be measured as follows using a mercury intrusion measuring apparatus (Autopore: manufactured by Shimadzu Corporation). . About 0.05 g of a sample is added to a standard 5 mL powder cell (stem volume 0.4 mL), and the initial pressure is set to 21 kPa (about 3 psia, corresponding to a pore diameter of about 60 μm). The mercury parameters are a mercury contact angle of 130 degrees and a mercury surface tension of 485 dynes / cm. The mode diameter is limited to the range of 0.1 to 3 μm, and the mode diameter, specific surface area, and porosity in the pore diameter distribution of the porous polymer particles and the separation material particles are calculated. The mode diameter, specific surface area, and the like can be adjusted by appropriately selecting a raw material for porous polymer particles, a porosifying agent, a polymer having a hydroxyl group, and the like.

  The coating layer includes a crosslinked polymer having a hydroxyl group. By covering the porous polymer particles with a crosslinked polymer having a hydroxyl group, it is possible to suppress an increase in pressure inside the column (column pressure) when a liquid is passed through the column filled with the separation material. Moreover, while suppressing nonspecific adsorption | suction of the protein to a separation material, it exists in the tendency for the protein adsorption property of the outstanding separation material to be acquired.

  The polymer having a hydroxyl group preferably has two or more hydroxyl groups, and more preferably a hydrophilic polymer. Examples of the polymer having a hydroxyl group include polysaccharides and polyvinyl alcohol, and modified products thereof. Examples of the polysaccharide include agarose, dextran, cellulose, and chitosan.

  Here, the modified product refers to a product in which a hydrophobic group is introduced into the molecule. Examples of the hydrophobic group include an alkyl group having 1 to 6 carbon atoms and an aryl group having 6 to 10 carbon atoms. As a C1-C6 alkyl group, a methyl group, an ethyl group, and a propyl group are mentioned, for example. Examples of the aryl group having 6 to 10 carbon atoms include a phenyl group and a naphthyl group. Hydrophobic groups are 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.

  The polymer having a hydroxyl group is preferably a polysaccharide from the viewpoint of reducing non-specific adsorption of proteins, and is preferably a modified polysaccharide from the viewpoint of improving the interfacial adsorption ability. Of these, agarose or a modified product thereof is more preferable. The polymer having a hydroxyl group can have a weight average molecular weight of about 10,000 to 300,000.

  The content of the hydrophobic group of the polymer having a hydroxyl group is 5 to 30 from the viewpoint of balancing the maintenance of the hydrophobic interaction force for adsorption to the surface of the porous polymer particles and the suppression of nonspecific adsorption of the protein. % By mass is preferable, 10 to 20% by mass is more preferable, and 12 to 17% by mass is more preferable.

  The coating layer containing a polymer having a hydroxyl group can be formed by, for example, the following method. First, a porous polymer particle is impregnated with a solution containing a polymer having a hydroxyl group and a solvent. Thereafter, the particles are filtered and washed with a solvent such as water and alcohol to remove unadsorbed polymer.

  The solvent of the polymer solution having a hydroxyl group is not particularly limited as long as it can dissolve the polymer having a hydroxyl group, and may be water, for example. The concentration of the polymer having a hydroxyl group in the solution is preferably 5 to 20 mg / mL. The impregnation method is not particularly limited, and examples thereof include a method in which porous polymer particles are added to a polymer solution having a hydroxyl group and left for a predetermined time. Although the impregnation time depends on the surface state of the porous body, the polymer concentration is usually in equilibrium with the external concentration inside the porous body if it is impregnated overnight.

  Next, a crosslinking agent is added to the aqueous solution, and a polymer having a hydroxyl group adsorbed on the surface of the porous polymer particles is crosslinked to form a crosslinked body. The crosslinked body has a three-dimensional crosslinked network structure having a hydroxyl group.

  After completion of the crosslinking reaction, the produced porous polymer particles having a coating layer are filtered off, and then washed with a hydrophilic organic solvent such as methanol and ethanol, water or an aqueous solution, unreacted polymer and suspending medium, etc. By removing, a separation material in which at least a part of the surface of the porous polymer particles is coated with a coating layer containing a crosslinked polymer having a hydroxyl group is obtained.

  As the cross-linking agent, for example, epihalohydrin such as epichlorohydrin, dialdehyde compound such as glutaraldehyde, diisocyanate compound such as methylene diisocyanate, and glycidyl compound such as ethylene glycol diglycidyl ether, a functional group showing reactivity to hydroxyl groups. The compound which has 2 or more is mentioned. When a compound having an amino group such as chitosan is used as the polymer having a hydroxyl group, for example, a dihalide compound such as dichlorooctane can be used as a crosslinking agent.

  The crosslinking reaction can be performed by dispersing and suspending porous polymer particles impregnated with a polymer solution having a hydroxyl group in an appropriate medium and adding a crosslinking agent thereto. When the polysaccharide is used as the polymer having a hydroxyl group, the addition amount of the crosslinking agent may be selected within the range of 0.1 to 100 equivalents per 1 equivalent of monosaccharides according to the performance of the separating material. it can. When the addition amount of the crosslinking agent is not less than the above lower limit value, the coating layer tends to be favorably retained on the porous polymer particles. If the addition amount of the crosslinking agent is not more than the above upper limit, even if the reaction rate between the crosslinking agent and the polymer having a hydroxyl group is high, the properties of the polymer having a hydroxyl group are not easily impaired.

  A catalyst can be used for the crosslinking reaction. As a catalyst, a conventionally well-known thing can be used suitably according to the kind of crosslinking agent. For example, when the crosslinking agent is epichlorohydrin, an alkali such as sodium hydroxide is effective as a catalyst, and when the dialdehyde compound is used, a mineral acid such as hydrochloric acid is effective.

  The amount of the catalyst used depends on the type of the crosslinking agent. For example, when a polysaccharide is used as the polymer having a hydroxyl group, it is in the range of 0.01 to 10 equivalents with respect to 1 equivalent of the polysaccharide. It is preferable that it is in the range of 0.1 to 5 mole times.

  When the crosslinking reaction between the crosslinking agent and the polymer having a hydroxyl group can be controlled by adding a catalyst or the like, the porous polymer particle is impregnated with a polymer solution having a hydroxyl group mixed with a crosslinking agent in advance. Is dispersed and suspended in an appropriate medium, and a catalyst or the like is added thereto to cause a crosslinking reaction between the crosslinking agent and the polymer having a hydroxyl group. When a catalyst or the like is not used, the crosslinking reaction can be caused by changing the crosslinking reaction conditions such as temperature. For example, a porous polymer particle impregnated with a polymer having a hydroxyl group and a solution in which a crosslinking agent is dissolved is dispersed and suspended in an appropriate medium, and then a crosslinking reaction condition (for example, temperature) is a condition under which the reaction proceeds. By adjusting to, the crosslinking reaction between the crosslinking agent and the polymer having a hydroxyl group can be performed. When the crosslinking reaction condition is a temperature condition, the temperature of the reaction system is raised, and when the temperature reaches the reaction temperature, a crosslinking reaction occurs.

  The medium for dispersing and suspending the porous polymer particles impregnated with the polymer solution having a hydroxyl group does not extract the polymer having a hydroxyl group impregnated into the porous polymer particles and the crosslinking agent, and the like. It can be inert to the crosslinking reaction. Examples of the medium include toluene, dichlorobenzene, and nitromethane.

  The crosslinking reaction can be carried out at 5 to 90 ° C. for 1 to 10 hours. The temperature for the crosslinking reaction is preferably 30 to 90 ° C.

  When adjusting the degree of crosslinking, the crosslinking reaction can be carried out stepwise. For example, the degree of progress of crosslinking can be adjusted by causing the crosslinking material once crosslinked to react again.

  The 5% weight loss temperature due to thermal decomposition of the separating material is measured, and from the value, the degree of crosslinking of the polymer having a hydroxyl group in the coating layer can be determined. When the degree of crosslinking is high, the weight reduction start temperature is high, and when the degree of crosslinking is low, the weight reduction start temperature is low. The 5% weight loss temperature is 200 to 350 ° C., preferably 220 to 330 ° C., more preferably 230 to 320 ° C., from the viewpoint of maintaining the characteristics of the polymer having a hydroxyl group. When the 5% weight loss temperature is not less than the lower limit of the above numerical range, the coating layer is satisfactorily retained on the porous polymer particles, and when the 5% weight reduction temperature is not more than the upper limit of the above numerical range, Since the porous polymer particles have sufficient swellability and functional group amount, when a solution such as a protein solution is passed through a column packed with a separating material, the amount of protein adsorbed at the same flow rate (dynamic adsorption amount) ) Is excellent.

  Here, the 5% weight loss temperature is the weight of the sample when the weight change of the sample is continuously measured while heating the sample at a heating rate of 10 ° C./min in a nitrogen atmosphere. It means the temperature at the time of 5% reduction with respect to 100%.

  The amount of the coating layer is preferably 30 to 500 mg, more preferably 50 to 500 mg, and further preferably 100 to 500 mg with respect to 1 g of the porous polymer particles. When the amount of the coating layer is not more than the above upper limit value, the coating layer can be made into a thin film, and the liquid permeability when used as a column tends to be further improved. When the amount of the coating layer is equal to or higher than the lower limit, the amount of adsorption of protein and the like and the amount of dynamic adsorption tend to be further increased. The amount of the coating layer can be measured, for example, by reducing the weight of pyrolysis of the separating material.

  The separating material may have an ion exchange group or a ligand (for example, protein A). An ion exchange group or a ligand can be introduced, for example, via a hydroxyl group on the surface of the separation material. When the separation material has an ion exchange group or a ligand, the separation material can be used for ion exchange purification and affinity purification. Examples of the method for introducing an ion exchange group include a method using an alkyl halide compound.

  Examples of the ion exchanger include an amino group that is a weakly basic group, a quaternary ammonium group that is a strongly basic group, a carboxy group that is a weakly acidic group, and a sulfonic acid group that is a strongly acidic group.

  Examples of the method using an alkyl halide compound include, for example, draining particles of a separating material in a wet state by filtration or the like, and then immersing it in an alkaline aqueous solution having a predetermined concentration for a certain period of time. And reacting them. This reaction is preferably carried out at 40 to 90 ° C. for 0.5 to 12 hours. The ion exchange group to be imparted is determined depending on the type of the halogenated alkyl compound.

  Examples of the halogenated alkyl compound include monohalogenocarboxylic acids such as monohalogenoacetic acid and monohalogenopropionic acid, and sodium salts thereof, primary, secondary or tertiary amines having at least one halogenated alkyl group such as diethylaminoethyl chloride; A quaternary ammonium hydrochloride having a halogenated alkyl group may be mentioned. These halogenated alkyl compounds are preferably bromides or chlorides. The addition amount of the halogenated alkyl compound is preferably 0.2% by mass or more with respect to the total mass of the separating material imparting ion exchange groups.

  When an amino group that is a weakly basic group is introduced as an ion exchange group, examples of the halogenated alkyl compound include mono- and di-, wherein at least one of the alkyl groups is substituted with a halogenated alkyl group. Alternatively, tri-alkylamines, mono-, di- or tri-alkanolamines, mono-alkyl-mono-alkanolamines, di-alkyl-mono-alkanolamines and mono-alkyl-di-alkanolamines can be used.

  As a method for introducing a strongly basic quaternary ammonium group as an ion exchange group, for example, first, a tertiary amino group is introduced, and a halogenated alkyl compound such as epichlorohydrin is reacted with the tertiary amino group, Examples thereof include a method of converting to a quaternary ammonium group and a method of reacting a quaternary ammonium hydrochloride or the like with a separating material.

  When a weakly acidic carboxy group is introduced as an ion exchange group, examples of the halogenated alkyl compound include monohalogenocarboxylic acids such as monohalogenoacetic acid and monohalogenopropionic acid, and sodium salts thereof.

  As a method for introducing a sulfonic acid group which is a strongly acidic group as an ion exchange group, for example, a glycidyl compound such as epichlorohydrin is reacted with a separating material, and a sulfite such as sodium sulfite or sodium bisulfite or A method of adding a separating material to a saturated aqueous solution of bisulfite is mentioned. The reaction conditions are preferably 30 to 90 ° C. and 1 to 10 hours.

  Examples of the method for introducing an ion exchange group include a method in which 1,3-propane sultone is reacted with a separation material in an alkaline atmosphere. The amount of 1,3-propane sultone is preferably 0.4% by mass or more based on the total mass of the separating material. The reaction conditions are preferably 0 to 90 ° C. and 0.5 to 12 hours.

  For introducing the ion exchange group, an organic solvent can be used in order to promote the reaction. Examples of the organic solvent include alcohols such as ethanol, 1-propanol, 2-propanol, 1-butanol, isobutanol, 1-pentanol, and isopentanol.

  The separation material of this embodiment is suitable for separation by protein electrostatic interaction or affinity purification. For example, after adding the separation material of the present embodiment to a solution containing protein, adsorbing the protein to the separation material by electrostatic interaction, the separation material is filtered from the solution, and in an aqueous solution having a high salt concentration. Can be easily desorbed and recovered from the protein adsorbed on the separation material. The separation material of this embodiment can be used in column chromatography. That is, the column of this embodiment is provided with the separation material of this embodiment.

  When the separation material of this embodiment is packed in a column, it is preferable that the flow rate of water is 800 cm / h or more when water is passed so that the column pressure is 0.3 MPa. In this specification, the flow rate is determined by allowing water to flow through a stainless steel column having a size of φ7.8 × 300 mm filled with the separation material of the present embodiment so that the pressure in the column becomes a predetermined value. Represents the linear flow velocity of water. When separating proteins and the like by column chromatography using a separation material generally used in the past, the flow rate of the protein solution or the like is generally in the range of 400 cm / h or less. On the other hand, when the separation material of this embodiment is used, a high dynamic adsorption amount can be maintained even if the liquid flow rate is 800 cm / h or higher, which is faster than the conventional separation material for protein separation.

  The biopolymer separated using the separation material of this embodiment is preferably a water-soluble substance. Specific examples include blood proteins such as serum albumin and immunoglobulin, enzymes present in the living body, protein bioactive substances produced by biotechnology, DNA, and biopolymers such as peptides having physiological activity. . The molecular weight of the biopolymer is preferably 2 million or less, and more preferably 500,000 or less. The properties and conditions of the separating material can be selected according to a known method depending on the isoelectric point and ionization state of the protein. Examples of the known method include the method described in JP-A-60-169427.

  The separation material of this embodiment has the advantages of particles made of natural polymers and particles made of synthetic polymers in the separation of biopolymers such as proteins, and introduces ion exchange groups and ligands on the surface of the separation material. By doing so, a more remarkable effect can be obtained. The separating material of this embodiment has durability and alkali resistance. In addition, non-specific protein adsorption tends to be reduced and protein adsorption / desorption tends to occur. Furthermore, the separation material of this embodiment tends to have a large amount of dynamic adsorption.

  The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the above-described embodiments, and various modifications can be made. For example, a separation material that does not have an ion exchange group can be used for gel filtration chromatography.

EXAMPLES Hereinafter, although an invention is concretely demonstrated based on an Example, this invention is not limited to a following example.
(Example 1)
1-1. Synthesis of porous polymer particles In a 500 mL three-necked flask, 16 g of divinylbenzene having a purity of 96% (manufactured by Nippon Steel & Sumikin Co., Ltd., trade name: DVB960) as a monomer, 16 g of hexanol and 16 g of diethylbenzene as a porogen, and excess as a polymerization initiator A mixed solution was prepared by adding 0.64 g of benzoyl oxide to an aqueous solution of polyvinyl alcohol (0.5% by mass) containing a dispersant. This mixed solution was emulsified using a microprocess server, and then the emulsified solution was transferred to a flask and stirred for about 8 hours using a stirrer while heating in a water bath at 80 ° C. The produced particles were filtered and washed with acetone to obtain porous polymer particles 1. The particle size of the porous polymer particles 1 was measured with a flow-type particle size measuring device, and the average particle size and particle size C.I. V. Was calculated. The results are shown in Table 1.

1-2. Introduction of hydrophobic group into polymer having hydroxyl group To 480 mL of agarose aqueous solution (concentration 2% by mass), 0.98 g of sodium hydroxide and 4.90 g of glycidyl phenyl ether were added and reacted at 60 ° C. for 6 hours. Was introduced. The obtained modified agarose was reprecipitated with isopropyl alcohol and washed. The hydrophobic group content of the modified agarose was calculated by the following method and found to be 14.2%.

Dry powder agarose (non-denatured agarose) and denatured agarose whose volatile content was dried to less than 0.1% by mass were each dissolved in pure water at 70 ° C. to prepare a 0.05% by mass aqueous solution sample. . The absorbance at 269 nm of each aqueous solution was measured with a spectrophotometer to determine the concentration, and the hydrophobic group content was calculated from the following formula. In the following, an agarose constituent unit means a disaccharide unit of agarose, and a modified agarose constituent unit means one in which a phenyl group is introduced into at least one of the hydroxyl groups in the agarose disaccharide unit. To do.
Hydrophobic group content (%) = (C _AG / (C _HAG + C _AG )) × 100
C _AG : Density of denatured agarose constituent unit (mmol / L)
= A / ε _GPE × 1000
C _HAG : Concentration of unmodified agarose constituent unit (mmol / L)
= (Concentration of unmodified agarose constituent unit (g / L) / Agarose constituent unit (306 g / mol)) × 1000
A: True absorbance of denatured agarose = Absorbance of denatured agarose-Absorption of _unmodified agarose-ε _GPE : Absorption coefficient of glycidyl phenyl ether = 1372 (L / (mol · cm))
Absorption of undenatured agarose = absorbance of undenatured agarose × (sample concentration of denatured agarose (mmol / L) / sample concentration of unmodified agarose (mmol / L))
Non-denatured agarose constituent unit concentration (g / L) = denatured agarose sample concentration (mass%) × 10—denatured agarose constituent unit concentration (g / L)
Denatured agarose constituent unit concentration (g / L) = (C _AG × modified agarose constituent unit (456 g / mol)) / 1000

2-1. Formation of coating layer 1 g of porous polymer particles 1 was added to 70 mL of a 20 mg / mL modified agarose aqueous solution and stirred at 55 ° C. for 24 hours to adsorb the modified agarose to the porous polymer particles 1. After the adsorption, the porous polymer particles 1 on which the modified agarose was adsorbed were separated by filtration and washed with hot water.

2-2. Evaluation of adsorption amount of polymer having hydroxyl group The adsorption amount of the modified agarose to the porous polymer particles 1 was calculated from the concentration of the modified agarose in the filtrate. Adsorption of denatured agarose when the adsorbed amount of denatured agarose per 1 g of the porous polymer particles is 50 mg or more is “◯”, the case of 30 mg or more and less than 50 mg is “Δ”, and the case of less than 30 mg is “x”. The amount was evaluated.

2-3. Crosslinking of the coating layer The modified agarose adsorbed on the surface of the porous polymer particles 1 was crosslinked as follows. 10 g of particles adsorbed with modified agarose were dispersed in a 0.4 M aqueous sodium hydroxide solution, 0.02 M epichlorohydrin was added, and the mixture was stirred for 10 hours at room temperature. Then, after washing | cleaning with 2 mass% hot sodium dodecyl sulfate aqueous solution, it wash | cleaned with the pure water and obtained the separating material by making it dry with an 80 degreeC dryer for 1 hour.

2-4.5% weight loss temperature measurement The obtained separation material (using a material whose weight change is within 5% after drying for 1 hour in a dryer at 80 ° C.) Using EXTRA 6300 (manufactured by Hitachi High-Tech Science Co., Ltd.), a 5% weight loss temperature was measured at a temperature rising rate of 10 ° C./min in a nitrogen atmosphere to evaluate the degree of crosslinking. The results are shown in Table 2.

2-5. Evaluation of non-specific adsorption ability of protein 0.2 g of the separating material was added to 20 mL of Tris-hydrochloric acid buffer (pH 8.0) having a BSA (Bovine Serum Albumin) concentration of 24 mg / mL, and stirred at room temperature for 24 hours. Thereafter, the amount of BSA adsorbed on the separation material was calculated from the BSA concentration of the supernatant obtained by taking the supernatant by centrifugation and measuring the absorbance at 280 nm of the supernatant with a spectrophotometer. The amount of BSA adsorbed per 1 mL of the separation material was evaluated as “◯” when the amount was less than 1 mg, “Δ” when the amount was 1 mg or more and less than 10 mg, and “x” when the amount was 10 mg or more. . The results are shown in Table 3.

3-1. Introduction of ion exchange group The separating material (dry mass 20 g) was added to 100 mL of 5 M aqueous sodium hydroxide solution and allowed to stand at room temperature for 10 minutes. Thereafter, 100 mL of an aqueous solution in which 20 g of diethylaminoethyl chloride hydrochloride was dissolved was added and stirred at 70 ° C. for 2 hours. After completion of the reaction, the reaction solution was filtered, and the separation material was washed three times with water / ethanol (volume ratio 5/1) to obtain a DEAE-modified separation material having diethylaminoethyl (DEAE) groups as ion exchange groups. In the following evaluation, a DEAE-modified separation material was used.

3-2. Measurement of Mode Diameter, Specific Surface Area and Porosity After drying the DEAE-modified separation material, the mode diameter, specific surface area and porosity in the pore diameter distribution were measured by mercury porosimetry. The results are shown in Table 1.

3-3. Evaluation of ion exchange capacity 5 mL of DEAE-modified separator was immersed in 20 mL of 0.1N sodium hydroxide and stirred at room temperature for 1 hour. Thereafter, suction filtration was performed using a filter, and the particles on the filter were washed until the washing liquid became neutral. The washed particles were immersed in 10 mL of 0.1N hydrochloric acid aqueous solution and stirred at room temperature for 1 hour. Thereafter, suction filtration was performed using a filter, and the particles on the filter were washed until the washing liquid became neutral. About this washing | cleaning liquid, the ion exchange capacity (mmol / mL) of the separation material was calculated | required by performing titration with 0.1-N sodium hydroxide aqueous solution using an automatic potentiometric titrator. The results are shown in Table 3.

3-4. Evaluation of column characteristics The DEAE-modified separation material was mixed with methanol to obtain a slurry having a concentration of 30% by mass. This slurry was packed in a φ7.8 mm × 300 mm stainless steel column over 15 minutes and used for the following evaluation.

3-5. Evaluation of liquid permeability Water was passed through the column packed with the DEAE-modified separation material while changing the liquid flow speed, and the relationship between the liquid flow speed and the column pressure was measured. The liquid flow rate (linear flow rate) when the column pressure was 0.3 MPa was determined. The results are shown in Table 2.

3-6. Evaluation of dynamic adsorption amount 10 column volumes of 20 mmol / L Tris-hydrochloric acid buffer (pH 8.0) were passed through a column packed with DEAE-modified separation material. Thereafter, the BSA concentration in the eluate at the column outlet was measured by UV absorbance measurement while flowing a 20 mmol / L Tris-HCl buffer solution having a BSA concentration of 2 mg / mL. The liquid passage speed was the same as the speed when the column pressure was 0.3 MPa in the liquid permeability evaluation. After flowing the buffer until the BSA concentrations at the column inlet and outlet coincided with each other, 5 column volumes of 1 M NaCl Tris-HCl buffer were flowed. The amount of dynamic adsorption at 10% breakthrough was calculated using the following formula. The results are shown in Table 2. The dynamic adsorption amount at 10% breakthrough refers to the adsorption amount at the time when 10% of BSA is eluted with respect to the initial concentration of the passed BSA solution.
_{· Q 10 = c f F (} t 10 -t 0) / V B
Q ₁₀ : Dynamic adsorption amount in 10% breakthrough (mg / separation material mL)
C _f : BSA concentration of infusion (mg / mL)
F: Flow rate (mL / min)
・ V _B : Bed volume (mL)
T ₁₀ : Time at 10% breakthrough (min)
T ₀ : BSA injection start time (min)

3-7. Evaluation of alkali resistance DEAE-modified separation material was stirred in a 0.5 M aqueous sodium hydroxide solution for 24 hours, then washed with a phosphate buffer, and packed in a column in the same manner as in the above column characteristic evaluation. The amount of dynamic adsorption in 10% breakthrough of BSA was measured. Compared with the amount of dynamic adsorption after 10% breakthrough before alkali treatment, the rate of decrease in the amount of dynamic adsorption after alkali treatment is less than 3%. The case of “Δ” and 20% or more was evaluated as “x”, and the alkali resistance was evaluated. The results are shown in Table 3.

3-8. Durability evaluation Water was passed through the column at a flow rate of 800 cm / h, and after measuring the column pressure, the flow rate was increased to 3000 cm / h and allowed to flow for 1 hour. Again, the flow rate is lowered to 800 cm / h, and the column pressure is increased by 10% or more from the initial value (before increasing the flow rate to 3000 cm / h). As a result, durability was evaluated. The results are shown in Table 3.

(Example 2)
Porous polymer particles 2 were synthesized in the same manner as porous polymer particles 1 except that 0.04M epichlorohydrin was used as the cross-linking material. The obtained porous polymer particles 2 were treated in the same manner as in Example 1 to obtain a separating material. Evaluation similar to Example 1 was performed about the porous polymer particle 2 and the separating material.

(Example 3)
Porous polymer particles 3 were synthesized in the same manner as the porous polymer particles 1 except that 0.08M epichlorohydrin was used as the cross-linking material. The obtained porous polymer particles 3 were treated in the same manner as in Example 1 to obtain a separating material. Evaluation similar to Example 1 was performed about the porous polymer particle 3 and the separating material.
Example 4
Porous polymer particles 4 were synthesized in the same manner as the porous polymer particles 1. About the obtained porous polymer particles 4, a coating layer was formed in the same manner as in Example 1, and the particles after the crosslinking treatment were dispersed again in a 0.4M aqueous sodium hydroxide solution, and 0.02M epichlorohydrin was added. For 24 hours at room temperature. Thereafter, it was washed with 2% by mass of hot sodium dodecyl sulfate aqueous solution, washed with pure water, and dried. Evaluation similar to Example 1 was performed about the porous polymer particle 4 and the separating material.

(Example 5)
Porous polymer particles 5 were synthesized in the same manner as the porous polymer particles 1 except that 0.04 M ethylene glycol diglycidyl ether was used as the cross-linking material. The obtained porous polymer particles 5 were treated in the same manner as in Example 1 to obtain a separating material. Evaluation similar to Example 1 was performed about the porous polymer particle 5 and the separating material.
(Comparative Example 1)
Porous polymer particles 6 were synthesized in the same manner as the porous polymer particles 1 except that 0.001 M epichlorohydrin was used as the cross-linking material. The obtained porous polymer particles 6 were processed in the same manner as in Example 1 to obtain a separating material. Evaluation similar to Example 1 was performed about the porous polymer particle 6 and the separating material.

(Comparative Example 2)
Porous polymer particles 7 were synthesized in the same manner as the porous polymer particles 1 except that 1M epichlorohydrin was used as the cross-linking material. The obtained porous polymer particles 7 were processed in the same manner as in Example 1 to obtain a separating material. Evaluation similar to Example 1 was performed about the porous polymer particle 7 and the separating material.

  As shown in Table 3, the separating materials of Examples 1 to 5 had a good dynamic adsorption amount. As shown in Tables 2 and 3, it was found that the separation materials of Examples 1 to 5 have sufficient linear flow velocity, nonspecific adsorption amount, and ion exchange capacity while maintaining alkali resistance and durability. .

Claims (12)

Porous polymer particles;
A separating layer comprising a coating layer covering at least a part of the surface of the porous polymer particle,
The coating layer includes a polymer having a hydroxyl group,
The polymer having a hydroxyl group is crosslinked;
5% weight reduction temperature of the separation material is 200 to 350 ° C.
Separation material.   The separation material according to claim 1, wherein the porous polymer particles contain a monomer unit derived from a styrenic monomer.   The separation material according to claim 1 or 2, wherein the polymer having a hydroxyl group is a polysaccharide or a modified product thereof.   The separation material according to any one of claims 1 to 3, wherein the polymer having a hydroxyl group is agarose or a modified product thereof.   The separation material according to any one of claims 1 to 4, wherein an average particle diameter of the porous polymer particles is 10 to 300 µm.   The separation material according to any one of claims 1 to 5, wherein a mode diameter in the pore size distribution of the separation material is 0.05 to 0.5 µm.   The separation material according to any one of claims 1 to 6, which is a particle having a porosity of 30 to 70% by volume. The separating material according to any one of claims 1 to 7, which is a particle having a specific surface area of 30 m ² / g or more.   The separation material according to any one of claims 1 to 8, wherein a coefficient of variation in particle diameter of the porous polymer particles is 3 to 15%.   The separation material according to any one of claims 1 to 9, comprising 30 to 500 mg of the coating layer per 1 g of the porous polymer particles.   The linear flow rate of water is 800 cm / h or more when water is passed through the column filled with the separation material so that the pressure in the column becomes 0.3 MPa. The separation material according to any one of the above.   A column comprising the separation material according to claim 1.