JPH059233A - Crosslinked copolymer particle and its production - Google Patents

Abstract

PURPOSE:To obtain the title particle which can selectively separate a polymeric substance such as a protein and is advantageous in that it has hydrophilicity equivalent to that of a natural separating agent though it is a synthetic separating agent. CONSTITUTION:A hydrophilic separating agent which is a crosslinked copolymer of glycidyl methacrylate with a crosslinking agent of general formula (I): CH2= CH(CH3)-COO-(R-O)n-OC-C(CH3)=CH2 (wherein R is 2-4 C alkylene group, and n is an integer of 1-4) or its hydrolyzate, has pendant glycerol groups and optionally further amino or sulfo groups in and has a specific volume after drying of 2.0-3.0ml/g and a specific volume in water of 6.0-13.0ml/g; a process for producing the same; and a process for separating a protein by using the same.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a crosslinked copolymer particle which swells remarkably in water and a method for producing the same.
It also relates to a hydrophilic separating agent derived therefrom, an ion exchange resin, a method for producing them, and a method for separating a protein using the ion exchange resin.

[0002]

2. Description of the Related Art A specific polymer substance is often recovered from an aqueous solution containing various polymer substances. One example is the recovery of proteins from aqueous solutions. For example, it is to recover an enzyme or a protein having physiological activity from a culture solution of a microorganism or a cell. In addition, a specific protein such as albumin is also collected from blood. Another example is the recovery of proteins from whey.

Several methods have been used for recovering such polymeric substances, such as proteins, from an aqueous solution. Among them, there are molecular sieves and adsorbents (hereinafter, these are collectively referred to as "separating agents"). There is a method of using. For example, a gel-like particle produced by reacting a natural polysaccharide such as cellulose or dextran with a cross-linking agent and swelling without being dissolved in water is packed in a column, and an aqueous solution containing a plurality of kinds of proteins is flowed through this column. , Specific proteins are separated and collected. This method is called gel filtration chromatography. This utilizes the phenomenon that the time required for the solute molecules to diffuse in the gel particles differs depending on the size of the solute molecules in the aqueous solution, that is, the molecular sieving effect. In gel filtration chromatography, a desired protein can be separated and recovered from an aqueous solution containing plural kinds of proteins by adjusting the size of the mesh of gel particles.

In the case of separating and recovering an ionic polymer substance, it has been practiced to use gel particles having an ion exchange group inside as a separating agent. For example 3 inside
When an alkaline aqueous solution containing a polymeric substance having a carboxyl group and a nonionic polymeric substance is flowed through a column packed with gelled particles having a primary amino group, the polymeric substance having a carboxyl group is To be trapped in the amino groups present there. On the other hand, even if the nonionic polymer substance diffuses into the gel-like particles, it is not captured there and escapes from the particles. When the eluent is passed through the column after the nonionic polymer substance flows out from the column, the polymer substance having a carboxyl group trapped in the gel particles comes out and is collected. For separation of such ionic polymer substances, gel-like particles obtained by reacting the aforementioned natural polysaccharide with a cross-linking agent, and introducing ion-exchange groups such as amino groups and sulfonic acid groups are used as separation agents. Used as.

However, such a gel-like particle made of a natural product generally has a small physical strength. Further, it has a drawback that it is easily decomposed by an acid or an alkali and easily decomposed by microorganisms. Instead of gel-like particles made of natural products, particles made of hydrophilic synthetic polymers have also been proposed. For example, in Japanese Examined Patent Publication No. 58-58026, a mixture of glycidyl methacrylate and ethylene glycol methacrylate is suspension polymerized in an aqueous medium to obtain a porous crosslinked copolymer particle having a glycidyl group, and then the glycidyl group is hydrolyzed. What was decomposed into hydrophilic porous crosslinked copolymer particles having a pendant glycerol group was used as a separating agent,
Performing chromatography is disclosed. This method is called gel permeation chromatography.
This is because, unlike the above-mentioned natural polymer-based separating agent, the porous cross-linked copolymer particles have innumerable pores inside, and the solute molecule is not inside the mesh of the particles. This is because they diffuse into the pores. Therefore, in general, a porous separating agent has been studied in the direction of increasing the volume of pores contributing to the diffusion of solute molecules as much as possible. Further, JP-A-53-90991 discloses that porous cross-linked copolymer particles having a glycidyl group are reacted with dimethylamine or the like to prepare a porous separating agent having an amino group.

The major difference between the porous separating agent composed of these synthetic polymers and the separating agent made of the above-mentioned natural product is that the former hardly swells in water but the latter does not swell in water. It is to swell significantly. In the dry state, the former particles are composed of a dense and solid skeleton part composed of a cross-linked copolymer and space parts or pores surrounded by this skeleton part, and have a remarkably large specific volume (ml / g).
have. When this is put in water, water penetrates into the space inside the particles. However, the rigid skeleton prevents the particles from swelling.

On the other hand, the latter hydrophilic separating agent made of a natural product has neither pores nor a solid skeleton in a dry state. When this is put in water, water penetrates into the meshes in the particles and expands the meshes, so that the particles swell.
In general, the lower the crosslink density, the better the swell.

[0008]

DISCLOSURE OF THE INVENTION The present invention is intended to provide a separating agent composed of a synthetic polymer, which swells markedly in water, that is, similar to the separating agent based on the above-mentioned natural product. . One of the aims of the present invention is to provide a separating agent which has pendant glycerol groups and which swells markedly in water and is therefore suitable for gel filtration chromatography. Another object of the present invention is to have a sulfonic acid group or an amino group together with a pendant glycerol group,
The purpose of the present invention is to provide a separating agent which swells remarkably in water and is therefore suitable for adsorption separation of ionic polymer compounds.

Still another object of the present invention is to provide a method for separating a high molecular compound such as a protein by chromatography using these separating agents. Still another object of the present invention is to provide a method for adsorbing and separating a protein from an aqueous solution using the above separating agent having a sulfonic acid group or an amino group. Another object of the present invention is to provide a method for producing these separating agents. Still another object of the present invention is to provide a crosslinked copolymer having a glycidyl group, which is suitable for producing these separating agents, and a method for producing the same.

[0010]

The separating agent according to the present invention comprises:
Crosslinked polymer particles having pendant glycerol groups. This may further have either an amino group or a sulfonic acid group. This product has a relatively small specific volume of 2.0 to 3.0 ml / g in a dry state. However, it has a characteristic that it absorbs water in water and swells to a volume twice or more, and usually three times or more than that in a dry state.

To produce the separating agent of the present invention, first, glycidyl methacrylate and dimethacrylate as a cross-linking agent represented by the general formula (1) are copolymerized to form a cross-linking copolymer having a glycidyl group. _{CH 2 = C (CH 3)} -COO- (R-O) n -OC-
_{C (CH 3) = CH 2} (1) ( wherein, R represents an ethylene, n- propylene, i- propylene like alkylene group having 2 to 4 carbon atoms, n represents 1 to 4
Preferred as a cross-linking agent is ethylene glycol or an ethylene glycol oligomer dimethacrylate represented by the formula (2).

CH ₂ ═C (CH ₃ ) —COO— (CH ₂ —CH ₂ —
O) _n -OC-C (CH ₃ ) = CH ₂ (2) A separating agent derived from a copolymer of glycidyl methacrylate and a cross-linking agent represented by the formula (2) is generally swellable in water. Since it is rich and does not cause a specific interaction with a protein, it is suitable as a protein separating agent.

A particularly preferred cross-linking agent is diethylene glycol dimethacrylate. This is because this compound has a reactivity close to that of glycidyl methacrylate, so that only one monomer reacts preferentially during copolymerization, and the composition of the copolymer produced at the initial and final stages of the reaction is You can avoid the unwanted phenomenon of change.

The proportion of the cross-linking agent in the monomer mixture used in the copolymerization reaction is 3 to 15% by mol% of vinyl groups, but is generally 2.5 to 10% by weight. When the ratio of the cross-linking agent is large, a porous cross-linked copolymer is likely to be formed. Therefore, the swellability of the crosslinked copolymer produced in 1,4-dioxane and the swellability of the separating agent derived from this crosslinked copolymer in water tend to both decrease.
On the contrary, when the ratio of the cross-linking agent in the monomer mixture decreases, the swelling property of the product generally increases, but the physical strength of the finally obtained separating agent decreases. The preferred proportion of cross-linking agent in the monomer mixture is 3-10 wt%, especially 4-9 wt%.
%.

The copolymerization reaction can be carried out by various known methods. Usually, a method of suspension polymerization in an aqueous medium in which a dispersion stabilizer and inorganic salts are present is adopted according to a conventional method. As the dispersion stabilizer, gelatin, sodium polyacrylate, carboxymethyl cellulose, polyvinyl alcohol and the like are used. As the inorganic salts, sodium chloride, calcium chloride, sodium sulfate, etc. are used. Dissolving these inorganic salts in the aqueous medium helps prevent dissolution of the monomers in the aqueous medium.

For suspension polymerization, glycidyl methacrylate,
It is carried out by suspending an organic phase composed of a cross-linking agent, a polymerization initiator and an organic solvent which dissolves these components and does not dissolve in an aqueous medium in the above aqueous medium and reacting at 50 to 90 ° C. for 6 to 20 hours. Be done. Of course, if desired, the reaction can be carried out at a lower temperature for a longer time. Examples of the polymerization initiator include organic peroxides such as benzoyl peroxide and t-butyl hydroperoxide, and organic compounds such as azobisisobutyronitrile and 2,2′-azobis (2,4-dimethylvaleronitrile). A conventional one such as an azobis compound can be used. The polymerization initiator is generally used in an amount of 0.01 to 5 wt% based on the monomer mixture.

As the organic solvent, toluene, cyclohexanol, 1-octanol, dibutyl ether, n-butyl acetate, isooctane or the like is used. The amount of the organic solvent used is generally 0.5 to 1.5 times the volume ratio of the monomer mixture at room temperature. By dissolving the monomer mixture in an organic solvent and carrying out suspension polymerization, spherical crosslinked copolymer particles can be easily produced. In addition, when the amount ratio of the organic solvent to the monomer mixture increases, the specific volume of the cross-linked copolymer that is generally formed in the dry state increases. This tendency is remarkable especially in the case of a so-called poor solvent for a homopolymer of glycidyl methacrylate such as isooctane or 1-octanol. Further, when such a poor solvent is used, the crosslinked copolymer produced is likely to become porous. Therefore, when the ratio of the cross-linking agent in the monomer mixture is large, it is preferable to use toluene, cyclohexanol or the like in place of these poor solvents to avoid porosity of the resulting cross-linked copolymer.

In suspension polymerization, the ratio of the organic phase to the aqueous medium phase (polymerization ratio) is preferably 1: 3 to 1:10. When the amount of the aqueous medium phase is too large, the amount of the monomer dissolved in the aqueous medium phase increases, and the yield of the crosslinked copolymer particles decreases. On the contrary, when the amount of the aqueous medium phase is too small, the suspension state of the organic phase becomes unstable, and it becomes difficult to obtain spherical crosslinked copolymer particles. The properties of the resulting crosslinked copolymer particles having a glycidyl group are preferably in the following range in order to induce a separating agent having a desired property.

Specific volume in dry state 1.8-3.0 ml / g, especially 2.0-3. 0 ml / g specific volume in water 1.8-3.0 ml / g, especially 2.0-3. Specific volume in 0 ml / g 1,4-dioxane 5.0-15.0 ml / g That is, the cross-linked copolymer particles having a glycidyl group are 1,
Those which remarkably swell in 4-dioxane are preferable.
The ratio of the specific volume in the dry state to the specific volume in 1,4-dioxane should be at least 2.5 or more.

In order to increase the dry volume and the specific volume in water, the ratio of the organic solvent to the monomer mixture in the suspension polymerization may be increased, or a poor solvent may be used as the organic solvent. To increase the specific volume in dioxane,
It suffices to reduce the proportion of the crosslinking agent in the monomer mixture.
The particle size of the cross-linked copolymer having a glycidyl group is generally 5μ.
m to 2000 μm, but it is specifically determined according to the particle size of the separating agent to be finally obtained. For example, when a separating agent having a pendant glycerol group used for gel filtration chromatography is to be obtained, a separating agent having a small particle size has better separation efficiency. Therefore, the particle size of the crosslinked copolymer having a glycidyl group is Generally 10 μm to 150 in dry state
μm, preferably 30 μm to 100 μm. Also,
In order to obtain a separating agent having an amino group or a sulfonic acid group in addition to the pendant glycerol group, a separating agent having a large particle size is easier to handle, and therefore the particle size of the crosslinked copolymer having a glycidyl group is 70μm-300μ in dry state
It is preferably m.

When the crosslinked copolymer having a glycidyl group is reacted with water to hydrolyze the glycidyl group, the separating agent having a pendant glycerol group according to the present invention can be obtained. This hydrolysis is preferably performed by swelling the above-mentioned crosslinked copolymer having a glycidyl group with a hydrophilic organic solvent, and adding water and an acid as a catalyst to the swollen crosslinked copolymer to cause a reaction. By doing so, a separating agent having a specific volume in the dry state of 2.0 to 3.0 ml / g and a specific volume in water of 6.0 to 13.0 ml / g, which remarkably swells in water, can be obtained. On the contrary, without swelling the above-mentioned cross-linked copolymer having a glycidyl group with an organic solvent, even if an acid aqueous solution is added thereto and heated, the glycidyl group is hydrolyzed to a glycerol group, which is thus obtained. The separating agent thus obtained generally has a poor swelling property in water.

As the organic solvent for swelling the cross-linked copolymer having a glycidyl group, 1,4-dioxane is generally used, but also tetrahydrofuran, N, N-
It is dissolved in water such as dimethylformamide and dimethylsulfoxide at an arbitrary ratio, and the volume of the crosslinked copolymer particles is adjusted to 2
A hydrophilic organic solvent that can swell more than twice can be used. The cross-linked copolymer having a glycidyl group is put in these organic solvents, and the mixture is heated at room temperature or under heating for 1-5.
When kept for a long time, the volume often swells more than double. Usually, it is preferable to swell sufficiently until no increase in volume occurs. Then, the swollen cross-linked copolymer was suspended in a large amount of an organic solvent, and a large excess of an aqueous acid solution was added to the glycidyl group, and the solution was added at 20-90 ° C. to 0.5-
When kept for 5 hours, the glycidyl group is hydrolyzed and converted into a glycerol group. The complete hydrolysis of the glycidyl group can be confirmed by the absence of absorption around 900 cm ⁻¹ corresponding to the absorption of the epoxy ring in the infrared absorption spectrum of the hydrolyzed particles.

The hydrolysis is preferably carried out under mild conditions so that the ester bond of the crosslinked copolymer is not cleaved as much as possible. The presence of a carboxyl group produced by cleavage of the ester bond in the produced separating agent has an unfavorable effect on the separation of the polymer compound having an ionic functional group such as protein. The amount of carboxyl groups in the separating agent having pendant glycerol groups produced by hydrolysis is 0.1 meq / m 2 when swollen in water.
It is preferably 1 or less, particularly preferably 0.05 meq / ml or less.

The water-swellable polymer particles having pendant glycerol groups thus obtained can be used as they are as a separating agent for gel filtration chromatography. In this case, the size of the particles, when swollen in water, is generally 20 to 300 μm, preferably 50 to 150 μm. The separating agent according to the present invention may have a tertiary amino group or a sulfonic acid group in addition to the pendant glycerol group. Such a water-swellable separating agent having an amino group or a sulfonic acid group is particularly effective for separating and recovering a polymer compound having an amino group or a carboxyl group such as a certain protein. The characteristics of these separating agents having amino groups or sulfonic acid groups also swell significantly in water, that is, the specific volume in the dry state is 2.0 to 3.0 ml / g, the specific volume in water is 6.0 to 13.0 ml. It is preferable to have / g.

The ion-exchange capacity of these separating agents is usually 0.05 to 0.5 meq / ml, preferably 0.4 meq / ml or less per 1 ml of resin when swollen in water. A protein generally has a very large amino group equivalent and carboxyl group equivalent (molecular weight per mol of amino group or carboxyl group). Therefore, unlike the case of capturing a low molecular weight ionic compound, in the case of capturing a protein, the separating agent may have a small ion exchange capacity per unit volume. Rather, the large ion exchange capacity is disadvantageous because a large amount of eluent must be used to elute the adsorbed protein.

Separation of proteins by ion exchange or ion exchange chromatography using the separating agent according to the present invention.
When recovering, the preferred ion exchange capacity of the separating agent is 0.1 per ml of resin when swollen in water.
It is 1 to 0.3 meq. When the ion exchange action of the separating agent is used, it is preferable that the separating agent has a relatively large particle size. Therefore, the separating agent of the present invention having an amino group or a sulfonic acid group is usually 1 when swollen in water.
It is preferably in the range of 00 to 500 μm.

To obtain a separating agent having a pendant glycerol group and an amino group and swelling remarkably in water,
An amino group may be introduced into the water-swellable polymer particles having the above-mentioned pendant glycerol group. The introduction of the amino group is carried out by introducing the above-mentioned water-swellable polymer particles having a pendant glycerol group into a mixed solution of an aqueous alkali hydroxide solution and a hydrophilic organic solvent, and allowing the mixed solution to penetrate the polymer particles. The swelled state in the mixed solution is activated, and the hydroxyl group of the glycerol group is activated by alkali hydroxide, and then an amine having a haloalkyl group or a salt thereof is added thereto to react the haloalkyl group with the hydroxyl group while maintaining the swollen state. It is preferable to carry out in two steps. By doing so, it is possible to obtain a basic anion exchange resin having a specific volume in a dry state which is substantially equal to that of the polymer particles having a pendant glycerol group as a raw material and swelling more than the raw material in water.

On the contrary, when the polymer particles having a pendant glycerol group are put into an alkali hydroxide aqueous solution and swollen with water, and then the amine having a haloalkyl group or a salt thereof is reacted with it, the reaction proceeds. Since the particles shrink, the basic anion exchange resin which is highly swellable in water cannot be obtained. The hydrophilic organic solvent is usually 1
A polyhydric or polyhydric alcohol is used. Especially carbon number 2
Alcohol 4 is preferable because it has good compatibility with the aqueous alkali hydroxide solution. Of these, alcohols having a secondary hydroxyl group, especially isopropanol, are considered to be effective in activating the hydroxyl group of the glycerol group, and are therefore preferable. It is preferable to use the hydrophilic organic solvent in an amount of 0.3 to 3 (volume) times the aqueous alkali hydroxide solution. It is considered that the hydrophilic organic solvent maintains the swollen state of the polymer particles during the reaction between the polymer particles and the haloalkylamine and promotes the uniform diffusion of the haloalkylamine into the polymer particles. As the alkali hydroxide, sodium hydroxide is usually used, but potassium hydroxide, lithium hydroxide and the like are also used. The concentration of alkaline hydroxide aqueous solution is 1 to 1
0 is appropriate. When the concentration of the aqueous solution is low, the action of activating the hydroxyl group of the glycerol group is weak. Conversely, if the concentration of the aqueous solution is too high, the ester bond of the polymer may be cleaved.

The mixed solution of the aqueous alkali hydroxide solution and the hydrophilic organic solvent is preferably used in a large excess with respect to the polymer particles so that the polymer particles can remain in a slurry state even after swelling. As the amine having a haloalkyl group, a tertiary amine represented by the formula (3) is used.

[0030]

[Chemical 2]

(Wherein X represents chlorine, bromine or iodine, R ₁ represents an alkylene group, and R ₂ and R ₃ represent an alkyl group or a hydroxyalkyl group). In formula (3), a haloalkyl group (X Preferred as -R _1- ) is a lower haloalkyl group having 2 to 4 carbon atoms such as 2-chloroethyl group, 2-bromoethyl group, 3-chloropropyl group and 4-chlorobutyl group. Further, as R ₂ and R ₃ , preferred are lower alkyl groups or carbon atoms having 1 to 4 carbon atoms such as methyl group, ethyl group, 2-hydroxyethyl group, n-propyl group, n-butyl group, i-butyl group. Number 2-4
Is a hydroxy lower alkyl group. Especially preferred are 1-halo-2 (N, N-diethylamino) ethane and 1-
Halo-2- (N, N-dimethylamino) ethane and the like. The amine is usually used in an amount of 0.1 to 3 times the molar amount of the glycerol group of the polymer particles. The amine may be used in a free form, or may be used in a salt form such as a hydrochloride, carbonate or bicarbonate.

Polymer particles having a glycerol group were added to a mixed solution of the above-mentioned aqueous solution of alkali hydroxide and a hydrophilic organic solvent, and the mixture was kept in a slurry state at 20 to 90 ° C. for 1 to 5 hours to remove the hydroxyl group of the glycerol group. After activating and swelling so as to have a volume more than twice as large as that at the time of drying, the above-mentioned tertiary amine is added to this to further swell 20 to 90
When kept at 1 ° C. for 1 to 10 hours, the haloalkylamine reacts with the glycerol group to form the basic anion exchange resin according to the present invention.

The thus obtained separating agent having anion exchange capacity has a specific volume in a dry state which is almost the same as that of the polymer particles having pendant glycerol groups used as a raw material and is equal to or superior to the raw material. It has a specific volume in water. In order to obtain a separating agent having a pendant glycerol group and a sulfonic acid group and swelling remarkably in water, a swellable crosslinked copolymer having the above-mentioned glycidyl group is reacted with sulfite or bisulfite. , The polymer particles are swollen with a hydrophilic solvent, and then an aqueous acid solution is added thereto to hydrolyze the glycidyl group.

As the sulfite or bisulfite, sodium sulfite or sodium bisulfite is usually used. The polymer is added to 0.1 mol / liter or a saturated aqueous solution of these salts and reacted at 30 to 90 ° C. for 1 to 10 hours. As a result, the swellable polymer having a glycidyl group becomes an acidic cation exchange resin having a glycidyl group and a sulfonic acid group. Next, the unreacted glycidyl group is hydrolyzed and converted into a pendant glycerol group. The hydrolysis of the glycidyl group of the sulfonated polymer needs to be carried out in the state where the polymer is swollen with a hydrophilic organic solvent, similarly to the hydrolysis of the glycidyl group of the crosslinked copolymer. If the polymer is simply put in the aqueous solution of mineral acid to hydrolyze the glycidyl group, a product having a high swelling property in water cannot be obtained.

Hydrolysis of the glycidyl group of the sulfonated polymer is carried out using 1,4-dioxane, tetrahydrofuran,
The polymer is placed in a hydrophilic organic solvent such as N, N-dimethylformamide, dimethyl sulfoxide, etc., which dissolves in water at an arbitrary ratio, and the polymer particles are kept at room temperature for about 1 to 5 hours to have twice the volume of the dried particles. Swell to above. Then, an aqueous acid solution is added to this and held at 20 to 90 ° C. for 0.5 to 5 hours to hydrolyze the glycidyl group to form a water-swellable acidic cation exchange resin having a pendant glycerol group and a sulfonic acid group. .

The separating agent according to the present invention is useful as a packing material for a chromatography column. For example, to a column filled with the separating agent according to the present invention and filled with water, a sample aqueous solution containing various proteins having different molecular weights is supplied from the upper part to flow down the column, and then water is supplied from the upper part of the column. Then, when the sample aqueous solution is extruded, various proteins in the sample aqueous solution are separated from each other and flow out from the bottom of the column. Since the separating agent according to the present invention has a higher mechanical strength than a natural material, the packing height of the column can be increased. For example, the separating agent according to the present invention can be packed in a column at a height of 50 cm or more and subjected to chromatography.

Among the separating agents according to the present invention, those having an amino group or a sulfonic acid group are useful as an ion exchange resin. For example, a sample aqueous solution containing various proteins is supplied to the upper part of the column packed with the separating agent having an amino group according to the present invention to flow down the column. At this time, the pH of the sample aqueous solution is adjusted to a pH higher than the isoelectric point of the protein to be captured. Monitor the effluent from the bottom of the column, and if the protein to be captured leaks more than the allowable amount (leak), stop supplying the sample aqueous solution to the column and supply water instead. The sample solution remaining in the column is extruded. Next, when a low-concentration aqueous acid solution, for example, a 0.1 mol / liter aqueous hydrochloric acid solution is supplied to the column, the protein adsorbed on the separating agent is eluted. Instead of the acid aqueous solution, a sodium chloride aqueous solution of about 0.5 to 1 mol / liter can be used.

As another method, the separating agent having the ion-exchange ability according to the present invention is put into a sample aqueous solution to adsorb a target component. Then, the separating agent may be recovered by filtration or centrifugation, and the separated agent may be put into an aqueous acid solution or an aqueous sodium chloride solution to elute the adsorbed components. For example, the separating agent having a sulfonic acid group according to the present invention is added to whey having a pH adjusted to about 3 to adsorb lactalbumin and lactoglobulin in the whey. Next, the separating agent is an alkaline aqueous solution having a pH of about 9 or 0.5 to
When it is put into an aqueous solution of sodium chloride of about 1 mol / liter, the adsorbed protein is eluted. Since the separating agent according to the present invention swells remarkably in water and allows the protein to easily diffuse into the inside of the separating agent, the adsorption capacity for the protein is remarkably large. For example, according to the invention, 30 g per liter of separating agent packed in the column
Above, preferably 50 g or more of protein can be adsorbed.

[0039]

The present invention will be described in detail with reference to the following examples, but the present invention is not limited to these examples. <Measurement Method of Specific Volume of Water-Swellable Polymer Particles Having Glycerol Group> About 10 ml of polymer particles swollen in water is put into a 10 ml graduated cylinder together with water,
The volume (V ₁ ml) is measured. After the polymer particles were transferred to a beaker and tilted to remove water, 100 ml of acetone was added and held for 10 minutes, and the operation of tilting to remove acetone was repeated twice. Next, 100 ml of toluene is added and held for 10 minutes, the operation of removing the toluene by tilting is repeated twice, and the same operation is repeated twice by replacing n-heptane with toluene. The polymer particles are collected by filtration and dried under reduced pressure to evaporate n-heptane. The weight (Wg) of the dried resin is measured. 10m of dried resin
It is put in a graduated cylinder of 1 and its volume (V ₂ ml) is measured.

Specific Volume of Polymer Particles in Dry State ... V ₂ / W (ml / g) Specific Volume of Polymer Particles in Swelling State in Water ... V ₁ / W (ml / g) Amino Group or Sulfonic Acid Group In the case of the polymer particles having the above, the above operation is performed after the amino group or the sulfonic acid group is released.

<Measurement Method of Ion Exchange Capacity of Polymer Particles Having Glycerol Group and Amino Group> The polymer particles are treated with an aqueous 0.1N-NaOH solution to give amino groups in a free form, and then washed thoroughly with water. About 5 ml of this polymer particle
Was sampled in a 10 ml graduated cylinder and its volume (Vm
l) is measured. After removing water by centrifugation, the mixture is transferred to a flask, 100 ml of 0.1N HCl is added, and the mixture is kept for 12 hours. Take 10 ml of the supernatant and add 0.1N-NaO.
Titrate with aqueous H solution. The ion exchange capacity of the polymer particles is given by (10-M) / Vmeq / ml, where Mml is the consumption amount of the 0.1N-NaOH aqueous solution.

<Method for Measuring Ion Exchange Capacity of Polymer Particles Having Glycerol Group or Glycerol Group and Sulfonic Acid Group> Polymer particles are treated with 0.1N HCl aqueous solution to release carboxyl group or carboxyl group and sulfonic acid group. After shaping, wash with water. About 5m of this polymer particle
l was sampled in a 10 ml graduated cylinder and its volume (V
ml) is measured. After removing water by centrifugation, transfer to a flask and add 100 ml of 0.1N-NaOH to 1
Hold for 2 hours. Take 10 ml of the supernatant and add 0.1N-H
Titrate with Cl. Consume the consumption amount of 0.1N-HCl aqueous solution as M
If the volume is ml, the ion exchange capacity of the polymer particles is (10
-M) / Vmeq / ml.

<Measurement Method of Sulfonic Acid Group of Polymer Particles Having Glycerol Group and Sulfonic Acid Group> The polymer particles are treated with a 0.1N-HCl aqueous solution to give sulfonic acid groups in a free form, and then washed thoroughly with water. About 5m of this polymer particle
l was sampled in a 10 ml graduated cylinder and its volume (Vm
After measuring l), it is dried in a vacuum dryer at 100 ° C. for 24 hours. The dried polymer particles are subjected to elemental analysis by a conventional method to measure the sulfur content (Wmg). The amount of sulfonic acid groups of the polymer particles is given by (W / 32) / Vmeq / ml.

<Measurement method of exclusion limit molecular weight> Inner diameter 8.2 m
A glass column of m is packed with polymer particles having pendant glycerol groups (packing height 47.5 cm). Dextran is injected into this column while continuously flowing demineralized water at 0.5 ml / min, and the time until dextran appears in the effluent from the column is measured. The above-mentioned measurement is performed for dextran having various molecular weights, and the exclusion limit molecular weight of dextran is determined from the measurement results by a conventional method.

<Method for measuring albumin adsorption capacity> Polymer particles were buffered [in the case of polymer particles having an amino group, Tris-HCl buffer (pH 7.5), in the case of polymer particles having a sulfonic acid group, an acetic acid buffer ( pH 4.5)]]
And pH with sodium hydroxide or sodium acetate
Adjust. Collect 5 ml of particles with polymer and centrifuge to remove buffer. This is put into 200 ml of a 4 g / liter bovine serum albumin solution at 10 ° C., and the mixture is maintained at 10 ° C. for 6 hours while being gently stirred. 280 of supernatant
absorbance at 280 nm and the original albumin solution at 280 nm
The amount of albumin adsorbed on the polymer particles is calculated from the difference in absorbance at.

Example 1 Glycidyl Methacrylate 9
5.7 g, diethylene glycol dimethacrylate 4.
3 g and 1.0 g of 2,2-azobis (2,4-dimethylvaleronitrile) were dissolved in 87 g of toluene. This monomer solution was added to demineralized water to obtain 120 g of calcium chloride, 6 g of polyvinyl alcohol and 0.003 of sodium nitrite.
6 g was added to 600 g of the dissolved aqueous solution, and the mixture was stirred to disperse the monomer solution in the aqueous solution. In this state, suspension polymerization was carried out at 70 ° C. for 6 hours.
Cross-linked copolymer particles of 0 to 300 μm were obtained (yield 9
1%). The specific volume of this product in the dry state is 2.7 ml /
The specific volume in the swollen state in 1,4-dioxane was 10.8 ml / g. Further, even if the crosslinked copolymer particles were dried and then added to water, the volume thereof was hardly changed.

After adding 300 ml of 1,4-dioxane to 30 g of the crosslinked copolymer particles having a glycidyl group and keeping the mixture at room temperature for 3 hours for swelling with dioxane, 150 ml of 1,4-dioxane and 50 g / liter were further added. 150 ml of a sulfuric acid aqueous solution was added, and the mixture was heated to 50 ° C. and kept for 3 hours to hydrolyze the glycidyl group into a glycerol group.
The polymer particles having pendant glycerol groups thus obtained had a specific volume of 2.6 ml / g in a dry state and a specific volume of 8.5 m when swollen in water.
It was 1 / g. Estimated cation exchange capacity is about 0.05
It was meq / liter. Exclusion limit molecular weight is about 100
It was good.

Example 2 A copolymerization reaction and a hydrolysis reaction were conducted in the same manner as in Example 1 except that the amount of glycidyl methacrylate was 91.3 g and the amount of diethylene glycol dimethacrylate was 8.7 g. Was done. The specific volume of the cross-linked copolymer having an epoxy group is
It was 7.1 ml / g when swollen in 1.9 ml / g 1,4-dioxane in a dry state. The specific volume of the polymer particles having pendant glycerol groups was 7.5 ml / g when swollen in water. The exclusion limit molecular weight was about 500,000.

Example 3 Polymer particles 10 in swollen state having pendant glycerol groups obtained in Example 1
After dehydrating 0 ml by centrifugation, this was 5N-N
The mixture was placed in a mixed solution of 24 ml of an aOH aqueous solution and 40 ml of isopropanol and kept at 50 ° C. for 1 hour while gently stirring. 2-Chloroethyldiethylamine hydrochloride (Cl-C) was added to the slurry containing the swollen polymer particles.
_{H 2 -CH 2 -N- (CH 2} CH 3) 2 -HCl) 31
A solution of 10 g of water in 10 g of water was added, and the mixture was kept at 50 ° C. for 5 hours. The white spherical anion exchange resin thus obtained had a specific volume of about 2.0 ml / g in a dry state and about 8.5 ml / g in a state of being swollen in water. The ion exchange capacity was 0.3 meq / ml. The albumin adsorption amount was 58 g / liter.

Example 4 About 10 g of the glycidyl group-containing cross-linked copolymer obtained in Example 1 was placed in 100 ml of a 1 mol / liter sodium sulfite aqueous solution, and the mixture was kept at 50 ° C. for 3 hours while stirring. The polymer particles were recovered by filtration, and then this was added to 100 ml of tetrahydrofuran.
It was placed inside and kept at 25 ° C. for 3 hours to swell the polymer particles. Further add 50 ml of tetrahydrofuran and 50
150 ml of a g / liter sulfuric acid aqueous solution was added, and the mixture was maintained at 50 ° C. for 3 hours while stirring to hydrolyze the glycidyl group into a glycerol group. The obtained white spherical particles had a specific volume in a dried state of 2.5 ml / g and a specific volume in a swollen state in water of about 10 ml / g. The ion exchange capacity was 0.15 meq / ml. The amount of adsorbed albumin was 85 g / liter.

Example 5 Suspension polymerization was carried out in the same manner as in Example 1 except that the amount of glycidyl methacrylate used was 93.1 g and the amount of diethylene glycol dimethacrylate used was 6.9 g. I did.
Dry state and 1,4 of the produced white spherical cross-linked copolymer
-The specific volumes swollen with dioxane were 2.1 ml / g and 8.3 ml / g, respectively. The particle size of the dried polymer was about 30 to 250 μm. Tetrahydrofuran 300 is added to 30 g of the crosslinked copolymer particles.
After adding ml, the mixture was kept at room temperature for 3 hours to swell the particles, 150 ml of tetrahydrofuran and 150 ml of a 50 g / l sulfuric acid aqueous solution were further added, and the mixture was kept at 50 ° C. for 3 hours. The produced polymer particles having pendant glycerol groups had a specific volume in a dry state and a specific volume in a state swollen with water of 2.4 ml / g and 7.7 ml / g, respectively. The exclusion limit molecular weight was about 1 million.

Example 6 About 1 polymer particle in swollen state having pendant glycerol groups obtained in Example 1
After dehydrating 100 ml by centrifugation,
24 ml of N-NaOH aqueous solution and 40 m of isopropanol
1 l of mixed aqueous solution, with gentle stirring at 50 ° C for 1
Held for hours. Next, 62 g of a 50% aqueous solution of 2-chloroethyl-diethylamine hydrochloride was added to this slurry, and the mixture was kept at 50 ° C for 5 hours. The white spherical anion exchange resin thus obtained has a specific volume of 11 ml / g and an anion exchange capacity of 0.24 meq / in a state swollen in water.
It was ml.

Example 7 100 ml of swollen polymer particles having a pendant glycerol group obtained in exactly the same manner as in Example 1 was replaced with 24 ml of a 5N-NaOH aqueous solution.
It was put in and kept at 50 ° C. for 1 hour with gentle stirring. A solution of 31 g of 2-chloroethyl-diethylamine hydrochloride dissolved in 10 g of water was added to this slurry to give 50
Hold at 5 ° C for 5 hours. The thus-obtained colored spherical anion exchange resin has a specific volume of about 2 ml / g in a dry state and about 5 ml / g in a state of being swollen in water, which is inferior to that of Example 3 in swelling property. . The ion exchange capacity of this product was 0.3 meq / ml. The amount of adsorbed albumin was 5 g / liter.

[Example 8] 100 g / l% was added to 30 g of the crosslinked copolymer particles obtained in exactly the same manner as in Example 1.
150 ml of a sulfuric acid aqueous solution was added, and the mixture was heated to 50 ° C. and kept for 5 hours to hydrolyze the glycidyl group. The white polymer particles having pendant glycerol groups thus obtained had a specific volume of 3.2 ml when swollen in water.
/ G, which was significantly inferior to that of Example 1 in swelling property.

[0055]

EFFECTS OF THE INVENTION According to the present invention, it is possible to provide a hydrophilic synthetic separating agent which can remarkably swell in water. Therefore, the advantages of both natural and synthetic separating agents can be obtained. It is extremely useful because it can selectively separate high molecular substances such as proteins.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location // B01D 15/08 8014-4D G01N 30/48 P 8105-2J

Claims (9)

[Claims] 1. A cross-linking agent CH ₂ ═C (CH ₃ ) —COO— (RO) _n —OC— represented by the following general formula (1) with glycidyl methacrylate.
C (CH ₃ ) = CH ₂ (1) (wherein R represents an alkylene group having 2 to 4 carbon atoms, and n represents an integer of 1 to 4) and is a crosslinked copolymer in a dry state. And the specific volume in water is 1.8 to 3.0 ml /
and a specific volume in a state swollen with 1,4-dioxane is 5.0 to 15.0 ml / g. 2. A cross-linking agent CH ₂ ═C (CH ₃ ) —COO— (R—O) _n —OC— represented by glycidyl methacrylate and the following general formula (1).
Compatible with a monomer mixture consisting of C (CH ₃ ) = CH ₂ (1) (wherein R represents an alkylene group having 2 to 4 carbon atoms, and n represents an integer of 1 to 4), and the monomer mixture. In a method of suspension-polymerizing a polymerizable mixture consisting of a certain organic solvent and a polymerization initiator in an aqueous medium, the weight ratio of glycidyl methacrylate to the crosslinking agent in the monomer mixture is 97.5: 2.5-9.
By selecting the volume ratio of the monomer mixture and the organic solvent from the range of 0:10 to the range of 1: 0.5 to 1: 1.5, respectively, the specific volume in the dry state and in water is 1.8 to 3. 0.0 ml / g, and the specific volume in a state swollen with 1,4-dioxane is 5.0 to 15.0 ml.
/ G of cross-linked copolymer is produced 3. Glycidyl methacrylate and the following (1)
Crosslinking agents represented by the formula _{CH 2 = C (CH 3)} -COO- (R-O) n -OC-
C (CH ₃ ) = CH ₂ (1) (wherein R represents an alkylene group having 2 to 4 carbon atoms, and n represents an integer of 1 to 4), and a hydrolyzate of crosslinked copolymer particles And the specific volume in the dry state is 2.0 to
Hydrophilic separating agent having a specific volume of 3.0 ml / g and a specific volume of 6.0 to 13.0 ml / g when swollen with water. 4. Hydrophilic cross-linked copolymer particles having pendant glycerol groups, having a specific volume of 2.0 to 3.0 ml / g in a dry state and a specific volume of 6. when swollen with water. 0 to 13.0 ml / g and a cation exchange capacity of 0.05 meq / ml or less. 5. The crosslinked copolymer according to claim 1 is swollen with a hydrophilic organic solvent, and then the swollen crosslinked copolymer is reacted with water to hydrolyze a glycidyl group. Method for producing hydrophilic crosslinked copolymer particles that swell in water to a volume that is at least twice the volume when dried 6. A hydrophilic ion exchange resin having a pendant glycerol group and an amino group or a sulfonic acid group, which has a specific volume in a dry state of 2.0 to 3.0 ml /
g, specific volume in the swollen state with water is 6.0 to 1
What is characterized by being 3.0 ml / g. 7. Having a pendant glycerol group, a specific volume in a dry state is 2.0 to 3.0 ml / g, and a specific volume in a state swollen with water is 6.0 to 13.0 ml / g.
g of the hydrophilic cross-linked copolymer particles,
The mixture is treated with a mixture of water and a hydrophilic organic solvent to form particles swollen by permeating the mixture into haloalkylamine represented by the following general formula (2). (Wherein X represents a halogen atom, R ₁ represents an alkylene group, R ₂ and R ₃ represent an alkyl group or a hydroxyalkyl group) or a salt thereof, and when dried in water A method for producing a basic anion exchange resin which swells to twice or more its volume. 8. The crosslinked copolymer particles according to claim 1,
An aqueous solution containing at least one selected from the group consisting of sulfites and bisulfites is reacted, then treated with a hydrophilic organic solvent, the organic solvent penetrates into the interior, and is twice the volume when dried. Production of a water-swellable acidic cation exchange resin having both pendant glycerol groups and sulfonic acid groups, which is characterized in that it is swollen in the above volume and water is reacted with it to hydrolyze the glycidyl ring. Law 9. A hydrophilic ion exchange resin having a pendant glycerol group and an amino group or a sulfonic acid group, which has a specific volume in a dry state of 1.8 to 3.0 ml /
g, specific volume in the swollen state with water is 6.0 to 1
3.0 ml / g, ion exchange capacity 0.4 meq / ml
An aqueous solution containing a protein is passed through a column packed with the following to adsorb 30 g or more of protein per liter of resin, and then an eluent is passed through to elute the adsorbed protein. And a method for recovering the protein.