JP6848203B2 - Separator and column - Google Patents

Description

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

Conventionally, when separating and purifying a biopolymer represented by a protein, porous particles based on a synthetic polymer and particles based on a crosslinked gel of a hydrophilic natural polymer are generally used. .. The above-mentioned ion exchanger based on a porous synthetic polymer has an advantage that the volume change due to salt concentration is small, and when it is packed in a column and used for chromatography, it has good pressure resistance during liquid passage. There is. However, when this ion exchanger is used for separating proteins and the like, non-specific adsorption such as irreversible adsorption based on hydrophobic interaction occurs, peak asymmetry occurs, or the hydrophobic interaction causes There is a problem that the protein adsorbed on the ion exchanger cannot be recovered while being adsorbed.

On the other hand, the above-mentioned ion exchanger based on a crosslinked gel of a hydrophilic natural polymer typified by a polysaccharide such as dextran or agarose has an advantage that there is almost no non-specific adsorption of proteins. However, this ion exchanger has drawbacks that it swells remarkably in an aqueous solution, the volume change due to the ionic strength of the solution, and the volume change between the free acid type and the loaded type are large, and the mechanical strength is not sufficient. In particular, when the crosslinked gel is used for chromatography, there is a drawback that the pressure loss at the time of passing the liquid is large and the gel is consolidated by the passing of the liquid.

In order to overcome the drawbacks of cross-linked gels made of hydrophilic natural polymers, attempts have been made to combine them with rigid substances that form a so-called “skeleton”.

For example, Patent Document 1 describes that a complex in which a gel such as a natural polymer gel is held in the pores of a porous polymer is used in the field of peptide synthesis, thereby increasing the load coefficient of a reactive substance. It is mentioned as an effect of the invention that it can be synthesized in a high yield. Moreover, in Patent Document 1, since the gel is surrounded by a hard synthetic polymer substance, there is an effect that the volume does not change and the flow-through pressure passing through the column does not change even when used in the form of a column bed. Has been done.

In Patent Documents 2 and 3, an inorganic porous material such as Celite retains a xerogel such as a polysaccharide such as dextran or cellulose, and a diethylaminomethyl (DEAE) group or the like is added to the gel in order to add sorption performance. It is used to remove hemoglobin. As an effect, good liquid permeability in the column is mentioned.

Patent Document 4 mentions an ion exchanger of a hybrid copolymer in which the pores of a copolymer having a so-called macro network structure are filled with a gel of a crosslinked copolymer synthesized from a monoma. In Patent Document 4, when the degree of cross-linking of the cross-linked copolymer gel is low, there are problems such as pressure loss and volume change. However, by using a hybrid copolymer, the liquid passage characteristics are improved and the pressure loss is reduced. It is also described that the ion exchange capacity is improved and the leak behavior is improved.

Further, a composite filler in which a crosslinked gel of a hydrophilic natural polymer having a huge network structure is filled in the pores of an organic synthetic polymer substrate has been proposed (see Patent Documents 5 and 6).

In Patent Document 7, porous particles composed of glycidyl methacrylate and acrylic crosslinked monoma are synthesized.

U.S. Pat. No. 4,965,289 U.S. Pat. No. 4,335,017 U.S. Pat. No. 4,336,161 U.S. Pat. No. 3,966,489 Japanese Unexamined Patent Publication No. 1-254247 U.S. Pat. No. 5,114,577 Japanese Unexamined Patent Publication No. 2009-244067

Conventional column fillers have sufficient problems such as liquid permeability and durability, which are problems of natural polymers, alkali resistance, pressure resistance, reduction of non-specific adsorption, and low protein adsorption amount, which are problems of polymer particles. It is difficult to solve at the level.

Therefore, an object of the present invention is to provide a separating material and a column capable of ensuring liquid permeability and durability, improving the amount of protein adsorbed, and reducing nonspecific adsorption.

The present invention provides the separating material described in the following [1] to [10] and the column described in the following [11].
[1] A coating layer containing water-soluble cellulose, which covers at least a part of the surface of the porous polymer particles, is provided, and the viscosity of a 2% by mass aqueous solution of water-soluble cellulose at 25 ° C. Separating material with 1 to 3000 mPa · s.
[2] The separating material according to [1], which has a porosity of 40 to 70% by volume.
[3] The separating material according to [1] or [2], wherein the porous polymer particles have an average particle size of 10 to 300 μm.
[4] The separating material according to any one of [1] to [3], wherein the mode diameter in the pore size distribution of the porous polymer particles is 0.1 to 0.5 μm.
[5] The separating material according to any one of [1] to [4], which has a hygroscopicity of 1 to 30% by mass.
[6] The separating material according to any one of [1] to [5], wherein the specific surface area of the porous polymer particles is 30 m ^{2 / g or more.}
[7] The separating material according to any one of [1] to [6], wherein the porous polymer particles contain divinylbenzene as a monoma unit in an amount of 50% by mass or more based on the total mass of the monoma unit.
[8] The separating material according to any one of [1] to [7], wherein the coefficient of variation of the particle size of the porous polymer particles is 3 to 15%.
[9] The separating material according to any one of [1] to [8], which comprises a coating layer of 30 to 500 mg per 1 g of porous polymer particles.
[10] The separating material according to any one of [1] to [9], wherein when the column is filled, the liquid passing speed is 800 cm / h or more when the column pressure is 0.3 MPa.
[11] A column comprising the separating material according to any one of [1] to [10].

According to the present invention, it is possible to provide a separating material and a column capable of ensuring liquid permeability and durability, improving the amount of protein adsorbed, and reducing nonspecific adsorption. Further, the separating material and the column according to the present invention are also excellent in ion exchange capacity and alkali resistance.

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

<Separation material>
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. In the present specification, the "surface of the porous polymer particles" includes not only the outer surface of the porous polymer particles but also the surface of the pores inside the porous polymer particles.

(Porous polymer particles)
The porous polymer particles of the present embodiment can be obtained, for example, by polymerizing a monoma containing a styrene-based monoma, and may contain the styrene-based monoma as a monoma unit. Further, the porous polymer particles of the present embodiment can also be obtained by reacting a composition containing a styrene-based monoma and a porosifying agent. Porous polymer particles can be synthesized by, for example, conventional suspension polymerization, emulsion polymerization, or the like. As the monoma, a styrene-based monoma can be used. Specific examples of the styrene-based monoma include the following polyfunctional styrene-based monomas and monofunctional styrene-based monomas.

Examples of the polyfunctional styrene-based monoma include divinyl compounds such as divinylbenzene, divinylbiphenyl, divinylnaphthalene, and divinylphenanthrene. These polyfunctional styrene-based monomas can be used alone or in combination of two or more. Among the above, it is preferable to use divinylbenzene because it is more excellent in durability, acid resistance and alkali resistance.

When divinylbenzene is contained as a styrene-based monoma, the monoma preferably contains divinylbenzene in an amount of 50% by mass or more, more preferably 60% by mass or more, and more than 70% by mass based on the total mass of the monoma (unit). Is even more preferable. By containing divinylbenzene in an amount of 50% by mass or more based on the total mass of monoma (unit), the alkali resistance and pressure resistance tend to be further improved. That is, the porous polymer particles preferably contain divinylbenzene units in the above range with reference to all monoma units.

Examples of monofunctional styrene-based monomas include styrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, α-methylstyrene, o-ethylstyrene, m-ethylstyrene, p-ethylstyrene, 2, 4-Dimethylstyrene, pn-butylstyrene, pt-butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn Examples of styrene such as −dodecylstyrene, p-methoxystyrene, p-phenylstyrene, p-chlorostyrene, 3,4-dichlorostyrene and derivatives thereof. These monofunctional styrene-based monomas can be used alone or in combination of two or more. Of these, styrene is preferably used from the viewpoint of further excellent acid resistance and alkali resistance. Further, a styrene derivative having a functional group such as a carboxy group, an amino group, a hydroxyl group or an aldehyde group can also be used.

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

The porosifying agent can be used in an amount of 0 to 200% by mass based on the total mass of the monoma. The porosity of the porous polymer particles can be controlled by the amount of the porosity agent. Furthermore, the size and shape of the pores of the porous polymer particles can be controlled by the type of the porosifying agent.

Further, water used as a solvent can also be used as a porosifying agent. When water is used as a porosifying agent, it can be made porous by dissolving an oil-soluble surfactant in a monoma and absorbing water.

Examples of the oil-soluble surfactant used for porosification include sorbitan monoesters of branched C16 to C24 fatty acids, chain unsaturated C16 to C22 fatty acids or chain saturated C12 to C14 fatty acids, for example, sorbitan monolaurate and sorbitan. Solbitan monoesters derived from monooleates, sorbitan monomillistates 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 diglycerol monoesters. Diglycerol monoester of glycerol monooleate (eg, C18: 1 (18 carbons, 1 double bond) fatty acid), diglycerol monomillistate, diglycerol monoisostearate or diglycerol mono of coconut fatty acid Esters; diglycerol monofatty acids of branched C16-C24 alcohols (eg, gelve alcohol), chain unsaturated C16-C22 alcohols or chain saturated C12-C14 alcohols (eg palm fatty alcohols); and mixtures thereof. Can be mentioned.

Of these, sorbitan monolaurate (eg, SPAN® 20, preferably greater than about 40% pure, more preferably greater than about 50% pure, most preferably greater than about 70% pure sorbitan mono. Laurate); sorbitan monooleate (eg, SPAN® 80, preferably greater than about 40% pure, more preferably greater than about 50% pure, most preferably greater than about 70% pure sorbitan mono. Oleate); diglyceride monooleate (eg, preferably diglycerol monooleate with a purity greater than about 40%, more preferably greater than about 50% purity, most preferably greater than about 70% purity); diglycerol monooleate. Isostearate (eg, diglyceride monoisostearate, preferably greater than about 40% purity, more preferably greater than about 50% purity, most preferably greater than about 70% purity); diglycerol monomyristate (preferably greater than about 70% purity). Sorbitan monomillistates with a purity greater than about 40%, more preferably greater than about 50% purity, most preferably greater than about 70% purity; cocoyl (eg, lauryl group, myristyl group, etc.) ethers of diglycerol; and these. Is 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 monoma. When the content of the oil-soluble surfactant is 5% by mass or more, the stability of the water droplet is sufficient, so that a large single pore is easily formed. Further, when the content of the oil-soluble surfactant is 80% by mass or less, the porous polymer particles are more likely to retain their shape after polymerization.

Examples of the aqueous medium used in the polymerization reaction include water, a mixed medium of water and a water-soluble solvent (for example, a lower alcohol), and the like. The aqueous medium may contain a surfactant. As the surfactant, any of anionic, cationic, nonionic and zwitterionic surfactants can be used.

Examples of the anionic surfactant include fatty acid oils such as sodium oleate and potassium castor oil, alkyl sulfates such as sodium lauryl sulfate and ammonium lauryl sulfate, alkylbenzene sulfonates such as sodium dodecylbenzene sulfonate, and alkylnaphthalene sulfone. Dialkyl sulfosuccinates such as acid salts, alkane sulfonates, sodium dioctyl sulfosuccinate, alkernyl succinate (dipotassium salt), alkyl phosphate ester salts, naphthalene sulfonate formalin condensates, polyoxyethylene alkyl phenyl ether sulfate Examples thereof include salts, polyoxyethylene alkyl ether sulfates such as sodium polyoxyethylene lauryl ether sulfate, and polyoxyethylene alkyl sulfates.

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

Examples of nonionic surfactants include hydrocarbon-based nonionic surfactants such as polyethylene glycol alkyl ethers, polyethylene glycol alkylaryl ethers, polyethylene glycol esters, polyethylene glycol sorbitan esters, polyalkylene glycol alkylamines, and amides. Examples thereof include polyethylene oxide adducts of silicon, polyether-modified silicon nonionic surfactants such as polypropylene oxide adducts, and fluorine nonionic surfactants such as perfluoroalkyl glycols.

Examples of the amphoteric ionic surfactant include hydrocarbon surfactants such as lauryldimethylamine oxide, phosphoric acid ester-based surfactants, and phosphite ester-based surfactants.

As the surfactant, one type may be used alone or two or more types may be used in combination. Among the above-mentioned surfactants, anionic surfactants are preferable from the viewpoint of dispersion stability during monoma polymerization.

Examples of the polymerization initiator added as needed include benzoyl peroxide, lauroyl peroxide, orthochlorobenzoyl peroxide, benzoyl orthomethoxy peroxide, 3,5,5-trimethylhexanoyl peroxide, and tert-butylper. Organic peroxides such as oxy-2-ethylhexanoate, di-tert-butyl peroxide; 2,2'-azobisisobutyronitrile, 1,1'-azobiscyclohexanecarbonitrile, 2,2' Examples thereof include 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 with respect to 100 parts by mass of the monoma.

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

In the synthesis of porous polymer particles, a polymer dispersion stabilizer may be used in order to improve the dispersion stability of the particles.

Examples of the polymer dispersion stabilizer include polyvinyl alcohol, polycarboxylic acid, celluloses (hydroxyethyl cellulose, carboxymethyl cellulose, methyl cellulose, etc.), 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 preferable. The amount of the polymer dispersion stabilizer added is preferably 1 to 10 parts by mass with respect to 100 parts by mass of the monoma.

In order to prevent the monoma from polymerizing alone, a water-soluble polymerization inhibitor such as nitrites, sulfites, hydroquinones, ascorbic acids, water-soluble B vitamins, citric acid, and polyphenols may be used.

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

The coefficient of variation (CV) of the particle size of the porous polymer particles is preferably 3 to 15%, more preferably 5 to 15%, from the viewpoint of further improving the liquid permeability. It is more preferably 5 to 10%. C. V. As a method of reducing the amount of the above, monodisperse can be mentioned by using an emulsifying device such as a micro process server (for example, manufactured by Hitachi, Ltd.).

C.I. V. The (coefficient of variation) can be obtained by the following measurement method.
1) Using an ultrasonic disperser, the particles are dispersed in water (including a dispersant such as a surfactant) 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 (coefficient of variation).

The porosity (pore volume) of the porous polymer particles is preferably 30% by volume or more and 70% by volume or less based on the total volume of the porous polymer particles, and more preferably 40% by volume or more and 70% by volume or less. preferable. The porous polymer particles preferably have pores having a mode diameter (mode, maximum frequency diameter of the pore diameter distribution) of 0.1 to 0.5 μm in the pore diameter distribution, that is, macropores (macropores). The mode diameter in the pore size distribution of the porous polymer particles is more preferably 0.2 to 0.5 μm. When the mode diameter in the pore diameter distribution is 0.1 μm or more, substances tend to enter into the pores easily, and when the mode diameter in the pore diameter distribution is 0.5 μm or less, the specific surface area becomes sufficient. Become. These can be adjusted by the above-mentioned porosifying agent.

The specific surface area of the porous polymer particles ^{is preferably 30 m 2} / g or more. From the viewpoint of higher practicality, the specific surface area is ^{more preferably 35 m 2} / g or more, and further preferably 40 m ² / g or more. When the specific surface area is 30 m ² / g or more, the amount of adsorbed substances to be separated tends to increase.

(Coating layer)
The coating layer of this embodiment contains water-soluble cellulose. By coating the porous polymer particles with water-soluble cellulose having a hydroxyl group, it is possible to suppress an increase in column pressure and suppress non-specific adsorption of proteins. Furthermore, when the water-soluble cellulose is crosslinked, it becomes possible to further suppress an increase in column pressure.

(Water-soluble cellulose)
The water-soluble cellulose is not particularly limited, and examples thereof include hydroxyethyl cellulose, hydroxypropyl cellulose, cellulose acetate and carboxymethyl cellulose, and modified products thereof.

Here, the modified product refers to a molecule in which a hydrophobic group is introduced. The water-soluble cellulose is preferably a modified product from the viewpoint of improving the interfacial adsorption ability. Examples of the hydrophobic group include an alkyl group having 1 to 6 carbon atoms and an aryl group having 6 to 10 carbon atoms. Examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group and the like. Examples of the aryl group having 6 to 10 carbon atoms include a phenyl group and a naphthyl group. The hydrophobic group can be introduced by reacting a functional group (for example, an epoxy group) that reacts with a hydroxyl group and a compound having a hydrophobic group (for example, glycidyl phenyl ether) with water-soluble cellulose by a conventionally known method. ..

When the water-soluble cellulose is impregnated with the porous polymer particles, the water-soluble cellulose is a 2% by mass aqueous solution from the viewpoint of suppressing clogging of the water-soluble polymer particles inside the pores and forming a suitable coating layer. Is preferably a polymer having a viscosity of the aqueous solution at 25 ° C. of 1 to 3000 mPa · s. From the same viewpoint, the viscosity of a 2% by mass aqueous solution of water-soluble cellulose at 25 ° C. is preferably 1 to 1500 mPa · s, more preferably 1 to 1000 mPa · s. The 2% by mass aqueous solution of water-soluble cellulose here means an aqueous solution substantially composed of water and water-soluble cellulose.

The viscosity of a 2% by weight aqueous solution of water-soluble cellulose is measured according to the following procedure.
First, a 2% by mass aqueous solution is prepared by dissolving water-soluble cellulose in pure water. Next, the prepared aqueous solution is heated to 60 ° C., and the viscosity of the aqueous solution is measured with a viscometer. If the water-soluble cellulose is not dissolved at 60 ° C., the aqueous solution is further heated to dissolve the water-soluble cellulose, the temperature of the aqueous solution is lowered to 60 ° C., and then the viscosity of the aqueous solution is measured.

(Method of forming the coating layer)
The coating layer containing water-soluble cellulose can be formed, for example, by the method shown below.
First, the water-soluble cellulose solution is adsorbed on the surface of the porous polymer particles. The solvent for the solution of the water-soluble cellulose is not particularly limited as long as it can dissolve the water-soluble cellulose, but water is the most common. The concentration of the polymer dissolved in the solvent is preferably 5 to 20 (mg / mL).
The porous polymer particles are impregnated with this solution. The impregnation method is to add porous polymer particles to a water-soluble cellulose solution and leave it for a certain period of time. The impregnation time varies depending on the surface condition of the porous body, but usually, if impregnated all day and night, the polymer concentration becomes an equilibrium state with the external concentration inside the porous body. Then, it is washed with a solvent such as water and alcohol to remove the unadsorbed water-soluble cellulose.

(Crosslinking)
Next, a cross-linking agent is added to cause a cross-linking reaction of the water-soluble cellulose adsorbed on the surface of the porous polymer particles to form a cross-linked product. At this time, the crosslinked body has a three-dimensional crosslinked network structure having a hydroxyl group.

The cross-linking agent has two or more functional groups active on hydroxyl groups such as epihalohydrin such as epichlorohydrin, dialdehyde compound such as glutaraldehyde, diisocyanate compound such as methylene diisocyanate, and glycidyl compound such as ethylene glycol diglycidyl ether. Examples include compounds.

A catalyst is usually used for this cross-linking reaction. As the catalyst, conventionally known catalysts can be appropriately used according to the type of the cross-linking agent. For example, when the cross-linking agent is epichlorohydrin or the like, an alkali such as sodium hydroxide is effective, and when the cross-linking agent is a dialdehyde compound, it is effective. Mineral acids such as hydrochloric acid are effective.

The cross-linking reaction with a cross-linking agent is usually carried out by adding the cross-linking agent to a system in which the separating material is dispersed and suspended in a suitable medium. The amount of the cross-linking agent added can be selected according to the performance of the separating material within the range of 0.1 to 100 mol times, assuming that 1 unit of the monosaccharide in the water-soluble cellulose is 1 mol. .. In general, when the amount of the cross-linking agent added is small, the coating layer tends to be easily peeled off from the porous polymer particles. Further, when the amount of the cross-linking agent added is excessive and the reaction rate with the water-soluble cellulose is high, the characteristics of the water-soluble cellulose as a raw material tend to be impaired.

The amount of the catalyst used varies depending on the type of cross-linking agent, but usually, when 1 unit of the monosaccharide forming water-soluble cellulose is 1 mol, the range is 0.01 to 10 mol times that of 1 mol. More preferably, it is used in an amount of 0.1 to 5 mol times.

For example, when the cross-linking reaction condition is a temperature condition, the temperature of the reaction system is raised, and when the temperature reaches the reaction temperature, a cross-linking reaction occurs.

As a medium for dispersing and suspending porous polymer particles impregnated with a solution of water-soluble cellulose, etc., the polymer, cross-linking agent, etc. are not extracted from the impregnated polymer solution, and the cross-linking reaction is not possible. Must be active. Specific examples thereof include water and alcohol.

The cross-linking reaction is usually carried out at a temperature in the range of 5 to 90 ° C. over 1 to 10 hours. Preferably, the temperature is in the range of 30 to 90 ° C.

After the cross-linking reaction is completed, the generated particles are filtered off, and then washed with a hydrophilic organic solvent such as water, methanol, ethanol, etc. to remove unreacted polymers, suspension medium, etc., to obtain porous polymer particles. A separating material is obtained in which at least a part of the surface is coated with water-soluble cellulose. The separating material of the present embodiment includes a coating layer of preferably 30 to 500 mg, more preferably 30 to 400 mg per 1 g of the porous polymer particles. The amount of the coating layer can be measured by reducing the weight of thermal decomposition or the like.

(Introduction of ion exchange group)
The separating material provided with the coating layer can be used for ion exchange purification, affinity purification, etc. by introducing an ion exchange group, a ligand (protein A), or the like via a hydroxyl group or the like on the surface. Examples of the method for introducing an ion exchange group include a method using an alkyl halide compound.

Examples of the alkyl halide compound include monohalogenocarboxylic acids such as monohalogenoacetic acid and monohalogenopropionic acid, sodium salts thereof, and primary, secondary or tertiary amines and halogens having at least one alkyl halide group such as diethylaminoethyl chloride. Examples thereof include quaternary ammonium hydrochloride having an alkylated group. These alkyl halide compounds are preferably bromides or chlorides. The amount of the alkyl halide compound used is preferably 0.2% by mass or more with respect to the total mass of the separating material to which the ion exchange group is imparted.

For the introduction of the ion exchange group, it is effective to use an organic solvent 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.

Normally, the ion exchange group is introduced into the hydroxyl group on the surface of the separating material. Therefore, the wet particles are drained by filtration or the like, immersed in an alkaline aqueous solution having a predetermined concentration, left to stand for a certain period of time, and then water-organic. In a solvent mixing system, the above alkyl halide compound is added and reacted. This reaction is preferably carried out at a temperature of 40 to 90 ° C. for 0.5 to 12 hours. The type of alkyl halide used in the above reaction determines the ion exchange group to be imparted.

As a method for introducing an amino group which is a weakly basic group as an ion exchange group, a mono-alkanolamine compound having at least one alkyl group in which a part of hydrogen atoms is replaced with a chlorine atom. , Di- or tri-alkylamine, mono-, di- or tri-alkanolamine, mono-alkyl-mono-alkanolamine, di-alkyl-mono-alkanolamine, mono-alkyl-di-alkanolamine, etc. The method can be mentioned. The amount of these alkyl halide compounds used is preferably 0.2% by mass or more with respect to the total mass of the separating material. The reaction conditions are preferably 40 to 90 ° C. and 0.5 to 12 hours.

As a method for introducing a quaternary ammonium group of a strongly basic group as an ion exchange group, first, a tertiary amino group is introduced, and the tertiary amino group is reacted with an alkyl halide compound such as epichlorohydrin to be quaternary. A method of converting to an ammonium group can be mentioned. Further, a quaternary ammonium hydrochloride or the like may be reacted with the separating material.

Examples of the method for introducing a carboxy group, which is a weakly acidic group, as an ion exchange group include a method in which a monohalogenocarboxylic acid such as monohalogenacetic acid or monohalogenopropionic acid or a sodium salt thereof is reacted as the alkyl halide compound. .. The amount of these alkyl halide compounds used is preferably 0.2% by mass or more with respect to the total mass of the separating material into which the ion exchange group is introduced.

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

On the other hand, as a method of introducing an ion exchange group, a method of reacting 1,3-propane sultone with a separating material in an alkaline atmosphere can also be mentioned. It is preferable to use 1,3-propane sultone in an amount of 0.4% by mass or more based on the total mass of the separating material. The reaction conditions are preferably 0 to 90 ° C. for 0.5 to 12 hours.

The hygroscopicity of the separating material of the present embodiment is measured by the following method. After leaving 1 g of the dry separating material in a constant temperature and humidity test tank (temperature 60 ° C., humidity 90%) for 18 hours, the moisture absorption is calculated by the following formula by measuring the mass of the separating material again.
(Amount of separated material after moisture absorption-1) g / 1g x 100 = moisture absorption (mass%)

The hygroscopicity of the separating material of the present embodiment is preferably 1 to 30% by mass, more preferably 1 to 20% by mass, and further preferably 1 to 10% by mass. When the moisture absorption of the separating material is 30% by mass or less, it is possible to suppress a decrease in the liquid permeability of the separating material due to the thickness of the coating layer.

The mode diameter, specific surface area, and porosity in the pore size distribution of the separating material or the porous polymer particles of the present embodiment are values measured by a mercury intrusion measuring device (Autopore: manufactured by Shimadzu Corporation) and are as follows. And measure. About 0.05 g of a sample is added to a standard 5 mL powder cell (stem volume 0.4 mL) and measured under the condition of an initial pressure of 21 kPa (about 3 psia, equivalent to a pore diameter of about 60 μm). The mercury parameters are set to the device default mercury contact angle of 130 degrees and mercury surface tension of 485 days / cm. Further, the specific surface area and the porosity are calculated by limiting the pore diameter in the obtained pore distribution to a range of 0.1 to 3 μm.

The mode diameter in the pore size distribution of the separating material of the present embodiment is preferably 0.05 to 0.5 μm, more preferably 0.1 to 0.5 μm. When the mode diameter in the pore size distribution is in this range, the liquid easily flows into the particles, and the amount of dynamic adsorption can be increased.

The specific surface area of the separating material of the present embodiment ^{is preferably 30 m 2} / g or more. From the viewpoint of higher practicality, the specific surface area is ^{more preferably 35 m 2} / g or more, and further preferably 40 m ² / g or more. When the specific surface area is 30 m ² / g or more, the amount of adsorbed substances to be separated tends to increase. The upper limit of the specific surface area of the separating material is not particularly limited, but can be, for example, 300 m ² / g or less.

The porosity of the separating material of the present embodiment is preferably 40 to 70% by volume. When the porosity is in this range, the amount of protein adsorbed can be increased.

The separating material of the present embodiment is suitable for using proteins for separation by electrostatic interaction, ion exchange purification, and affinity purification. For example, the separating material of the present embodiment is added to a mixed solution containing a protein, and only the protein is adsorbed on the separating material by electrostatic interaction, and then the separating material is filtered off from the solution to obtain a salt concentration. When added to a high aqueous solution, the protein adsorbed on the separating material can be easily desorbed and recovered. The separating material of the present embodiment can also be used in column chromatography. That is, the column of the present embodiment includes the separating material of the present embodiment, and is filled with, for example, the separating material of the present embodiment.

As the biopolymer that can be separated using the separating material of the present embodiment, a water-soluble substance is preferable. Specifically, it is a protein such as blood protein such as serum albumin and immunoglobulin, an enzyme existing in the living body, a protein physiologically active substance produced by biotechnology, DNA, a biopolymer such as a peptide having physiological activity, and the like. Yes, preferably the molecular weight is 2 million or less, more preferably 500,000 or less. In addition, it is necessary to select the properties, conditions, etc. of the separating material according to the isoelectric point, ionization state, etc. of the protein according to a known method. As a known method, for example, the method described in JP-A-60-169427 can be mentioned.

The separating material of the present embodiment has a natural height in separating biopolymers such as proteins by introducing an ion exchange group and protein A into the surface of the separating material after cross-linking the coating layer on the porous polymer particles. It has the advantages of particles made of molecules or particles made of synthetic polymers. In particular, the porous polymer particles in the separating material of the present embodiment are obtained by the above-mentioned method, and therefore have durability and alkali resistance. In addition, the separating material of the present embodiment reduces non-specific adsorption of proteins and tends to cause protein adsorption / desorption. Further, the separating material of the present embodiment tends to have a large adsorption amount (dynamic adsorption amount) of proteins and the like under the same flow velocity.

The liquid passing speed in the present specification represents the liquid passing speed when a stainless steel column having a diameter of 7.8 × 300 mm is filled with the separating material of the present embodiment and the liquid is passed through. When the separating material of the present embodiment is packed in a column, it is preferable that the liquid passing speed is 800 cm / h or more when the column pressure is 0.3 MPa. When protein is separated by column chromatography, the flow rate of the protein solution or the like is generally in the range of 400 cm / h or less, but when the separating material of the present embodiment is used, it is used for normal protein separation. It can be used at a liquid passing speed of 800 cm / h or more, which is faster than that of the separating material.

The average particle size of the separating material of the present embodiment is preferably 10 to 300 μm. For use in preparative or industrial chromatography, it is more preferably 10 to 100 μm, even more preferably 50 to 100 μm, in order to avoid an extreme increase in column internal pressure.

When the separating material of the present embodiment is used as a column filler in column chromatography, it is excellent in operability because the volume change in the column is small regardless of the nature of the eluate used.

The mode diameter, specific surface area, etc. in the pore size distribution of the separating material can be adjusted by appropriately selecting the raw material of the porous polymer particles, the porosifying agent, the water-soluble cellulose, and the like.

In this embodiment, the separating material in which the ion exchange group is introduced has been described, but it can be used as the separating material without introducing the ion exchange group. Such a separating material can be used, for example, in gel filtration chromatography.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to these Examples.

<Example 1>
(Synthesis of Porous Polymer Particle 1)
In a 500 mL three-necked flask, 14 g of 90% pure divinylbenzene (DVB960, Nippon Steel & Sumikin Chemical Co., Ltd.), 2 g of octanol, and 0.64 g of benzoyl peroxide were added to an aqueous polyvinyl alcohol solution (0.5% by mass), and microprocessed. Emulsified using a server. The obtained emulsion was transferred to a flask and stirred with a stirrer for about 8 hours while heating in a water bath at 80 ° C. to obtain particles. The obtained particles were filtered and then washed with acetone to obtain porous polymer particles 1. The particle size of the obtained porous polymer particles 1 was measured with a flow-type particle size measuring device, and the average particle size and the particle size of C.I. V. The value was calculated. In addition, the mode diameter, specific surface area, and porosity in the pore size distribution of the porous polymer particles 1 were measured by the mercury intrusion method. The results are shown in Table 1.

(Formation of coating layer)
Add 4 g of sodium hydroxide and 0.4 g of glycidylphenyl ether to 100 mL of an aqueous solution of hydroxypropyl cellulose (hydroxypropyl cellulose 150-400 cP, Wako Pure Chemical Industries, Ltd.) (2% by mass, viscosity at 25 ° C.: 18 mPa · s). The reaction was carried out at 70 ° C. for 12 hours, reprecipitated with 2 L of isopropyl alcohol, and suction filtered to obtain modified hydroxypropyl cellulose. The obtained modified hydroxypropyl cellulose was dissolved again in 100 mL of pure water at 70 ° C., reprecipitated with 2 L of isopropyl alcohol, and purified by repeating the steps of suction filtration twice.

10 g of the porous polymer particles 1 was added to 700 mL (concentration 20 mg / mL) of the purified modified hydroxypropyl cellulose aqueous solution, and the mixture was stirred at 55 ° C. for 8 hours to form a coating layer on the surface of the porous polymer particles 1. The particles were then filtered and washed with hot water. The amount of modified hydroxypropyl cellulose adsorbed (the amount of the coating layer) on 1 g of the porous polymer particles was calculated by measuring the thermogravimetric reduction of the dried particles. The results are shown in Table 2.

The modified hydroxypropyl cellulose adsorbed on the particles was crosslinked as follows. 39 g of ethylene glycol diglycidyl ether was added to a 0.4 M aqueous sodium hydroxide solution in which 10 g of particles were dispersed, and the mixture was stirred at room temperature for 10 hours. Then, the particles were washed with heated 2 mass% sodium dodecyl sulfate aqueous solution, washed with pure water, and the particles were stored in water.

(Introduction of ion exchange group)
Water was removed from the dispersion of the separating material by centrifugation, and then 20 g of the separating material was dispersed in 100 mL of an aqueous solution in which a predetermined amount of diethylaminoethyl chloride hydrochloride was dissolved, and the mixture was stirred at 70 ° C. for 10 minutes. Then, 100 mL of a 5 M NaOH aqueous solution heated to 70 ° C. was added, and the mixture was reacted for 1 hour. After completion of the reaction, the mixture was filtered and washed twice with water / ethanol (volume ratio 8/2) to obtain a DEAE-modified separator in which a diethylaminoethyl (DEAE) group was introduced as an ion exchange group. The mode diameter, specific surface area, and porosity of the obtained DEAE-modified separator in the pore size distribution were measured by a mercury intrusion method. The results are shown in Table 2.

(Evaluation of ion exchange capacity)
The ion exchange capacity of the DEAE modified separator was measured as follows. A 5 mL volume of separator was immersed in 20 mL of 0.1 N aqueous sodium hydroxide solution for 1 hour and stirred at room temperature. Then, washing was performed until the pH of the water used as the washing liquid became 7 or less. The washed separator was immersed in 20 mL of 0.1 N hydrochloric acid and stirred for 1 hour. After removing the separating material by filtration, the ion exchange capacity of the separating material was measured by neutralizing and titrating the aqueous hydrochloric acid solution of the filtrate. The results are shown in Table 3.

(Column characteristic evaluation)
The separating material was filled in a stainless steel column having a diameter of 7.8 × 300 mm at a slurry (solvent: methanol) concentration of 30% by mass over 15 minutes. Then, water was flowed through the column at different flow velocities, the relationship between the flow velocities and the column pressure was measured, and the linear flow velocity at a column pressure of 0.3 MPa was calculated. The results are shown in Table 3.
In addition, the amount of dynamic adsorption was measured as follows. A 20 mmol / L Tris-hydrochloric acid buffer (pH 8.0) was flowed through the column by 10 column volumes. Then, a 20 mmol / L Tris-hydrochloric acid buffer having a BSA concentration of 2 mg / mL was flowed, and the BSA concentration at the column outlet was measured by UV. The solution was run until the BSA concentrations at the column inlet and outlet were the same, and diluted with Tris-hydrochloric acid buffer of 1 M NaCl in 5 column volumes. The amount of dynamic adsorption at 10% breakthrough was calculated using the following formula. The results are shown in Table 3.
q ₁₀ = c _f F (t ₁₀ −t ₀ ) / V _B
q ₁₀ : Dynamic adsorption amount at 10% breakthrough (mg / mL wet resin)
c _f : Injecting BSA concentration F: Flow velocity (mL / min)
V _B : Bed volume (mL)
t ₁₀ : Time at 10% breakthrough t ₀ : BSA injection start time

(Evaluation of non-specific adsorption capacity of protein)
0.5 g of the obtained particles (separator) was put into 50 mL of a phosphate buffer solution (pH 7.4) having a BSA concentration of 20 mg / mL, and the mixture was stirred at room temperature for 24 hours. Then, after the supernatant was obtained by centrifugation, the amount of BSA adsorbed on the particles was calculated by calculating the BSA concentration of the filtrate with a spectrophotometer. The concentration of BSA was calculated from the absorbance at 280 nm by a spectrophotometer. The case where the non-specific adsorption amount was 1 mg / mL particles or less was evaluated as ◯, the case where the non-specific adsorption amount exceeded 1 mg / mL particles and 10 mg / mL particles or less was evaluated as Δ, and the case where the non-specific adsorption amount exceeded 10 mg / mL particles was evaluated as ×. The results are shown in Table 3.

(Evaluation of alkali resistance)
The separating material was stirred in 0.5 M aqueous sodium hydroxide solution for 24 hours, washed with phosphate buffer, and then filled under the same conditions as for column characteristic evaluation. The 10% breakthrough dynamic adsorption amount of BSA was measured and compared with the dynamic adsorption amount before the alkali treatment. The case where the reduction rate of the dynamic adsorption amount was 3% or less was evaluated as ◯, the case where it exceeded 3% and 20% or less was evaluated as Δ, and the case where it exceeded 20% was evaluated as ×. The results are shown in Table 3.

(Durability evaluation)
Water was flowed through the column at a flow rate of 800 cm / h, the column pressure was measured, the flow rate was increased to 3000 cm / h, and the solution was passed for 1 hour. When the column pressure was lowered to 800 cm / h again, the case where the column pressure increased by 10% or more from the initial value (before increasing the flow velocity to 3000 cm / h) was evaluated as ×, and the case where the increase was less than 10% was evaluated as ○. .. The results are shown in Table 3.

<Example 2>
Porous polymer particles 2 were synthesized in the same manner as the porous polymer particles 1. In the formation of the coating layer, a separating material was prepared and evaluated in the same manner as in Example 1 except that hydroxyethyl cellulose (HEC Daicel SP500, Daicel FineChem Co., Ltd.) was used instead of hydroxypropyl cellulose. The viscosity of a 2% by mass aqueous solution of hydroxyethyl cellulose at 25 ° C. was 340 mPa · s.

<Example 3>
Porous polymer particles 3 were synthesized in the same manner as the porous polymer particles 1. In the formation of the coating layer, a separating material was prepared and evaluated in the same manner as in Example 1 except that hydroxyethyl cellulose (HEC Daicel SE550, Daicel FineChem Co., Ltd.) was used instead of hydroxypropyl cellulose. The viscosity of a 2% by mass aqueous solution of hydroxyethyl cellulose at 25 ° C. was 1370 mPa · s.

<Comparative example 1>
Porous polymer particles 4 were synthesized in the same manner as the porous polymer particles 1. In the formation of the coating layer, a separating material was prepared and evaluated in the same manner as in Example 1 except that hydroxyethyl cellulose (HEC Daicel SP600, Daicel FineChem Co., Ltd.) was used instead of hydroxypropyl cellulose. The viscosity of a 2% by mass aqueous solution of hydroxyethyl cellulose at 25 ° C. was 4720 mPa · s.

<Comparative example 2>
Commercially available agarose particles (Capto DEAE, GE Healthcare) were used as the porous polymer particles 5 and evaluated in the same manner as in Example 1.

From the results in Tables 2 and 3, when water-soluble cellulose having a viscosity of 1 to 3000 mPa · s in a 2% by mass aqueous solution was used for the coating layer, the amount adsorbed on the particles (the amount of the coating layer) was improved, and nonspecific adsorption was achieved. It can be seen that there is almost no liquid, and that it is also excellent in liquid permeability, durability, and protein adsorption amount (dynamic adsorption amount).

Claims (9)

Porous polymer particles containing divinylbenzene as a monoma unit,
A coating layer containing water-soluble cellulose, which covers at least a part of the surface of the porous polymer particles, is provided.
Wherein the water-soluble cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose, and at least one to the parallel beauty selected from the group consisting of modified product,
A separating material having a viscosity of a 2% by mass aqueous solution of the water-soluble cellulose at 25 ° C. of 1 to 1500 mPa · s. The separating material according to claim 1, wherein the porous polymer particles have a porosity of 40 to 70% by volume. The separating material according to claim 1 or 2, wherein the porous polymer particles have an average particle size of 10 to 300 μm. The separating material according to any one of claims 1 to 3, wherein the mode diameter in the pore size distribution of the porous polymer particles is 0.1 to 0.5 μm. The separating material according to any one of claims 1 to 4, wherein the specific surface area of the porous polymer particles is 30 m ^{2 / g or more.} The separating material according to any one of claims 1 to 5, wherein the porous polymer particles contain divinylbenzene as a monoma unit in an amount of 50% by mass or more based on the total mass of the monoma unit. The separating material according to any one of claims 1 to 6, wherein the coefficient of variation of the particle size of the porous polymer particles is 3 to 15%. The separating material according to any one of claims 1 to 7, comprising the coating layer of 30 to 500 mg per 1 g of the porous polymer particles. A column comprising the separating material according to any one of claims 1 to 8.