JPH055731A - Carrier for affinity chromatography - Google Patents

Abstract

(57) [Summary] [Constitution] A carrier particle for affinity chromatography, wherein a layer of a hydrophilic polymer having a thickness of 10 to 300 angstrom is formed on the surface of a hydrophobic cross-linked polymer particle. This hydrophilic polymer is a polymer having a functional group (for example, a hydroxyl group, a carbonyl group, an amino group, a glycidyl group, a cyano group) capable of immobilizing a ligand or a spacer in affinity chromatography. [Effect] A carrier having excellent pressure resistance, a low degree of swelling and contraction, and non-specific adsorption of proteins can be obtained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a carrier for affinity chromatography.

[0002]

2. Description of the Related Art What is affinity chromatography?
Chromatography using a column packed with a ligand-immobilized carrier obtained by immobilizing a substance (ligand) that specifically binds to the target substance for separation and purification on a carrier composed of insoluble natural or synthetic polymer And is particularly used for separation / purification of bio-related substances.

As a carrier for affinity chromatography which has been conventionally used, there is a porous silica gel particle as an inorganic carrier. Organic carriers include natural and synthetic polymer carriers, natural product-derived carriers include particles of polysaccharides such as agarose, dextran, and cellulose, and synthetic polymer carriers include polystyrene and polyacrylamide. There are particles such as.

The ideal conditions as a carrier are generally as follows: (1) The carrier itself does not nonspecifically adsorb to biological substances such as proteins in general; (2) the degree of porosity is high. (3) Having a functional group capable of immobilizing a ligand or a spacer under mild conditions, and having a large immobilization capacity; (4) Maintaining chemical and physical stability after immobilizing a ligand And (5) It has sufficient mechanical strength to perform column packing and chromatography operations. However, a carrier satisfying these conditions has not yet been developed (“Affinity Chromatography” Toshiaki Osawa, Yoshio Terao, co-edited, Protein Nucleic Acid Enzyme Separate Volume No. 22, 1980, pp5-10).

[0005] For example, among the above-mentioned conventional carriers, inorganic porous silica gel particles have strong physical strength and are good in separation, but they are actually used because they have a drawback of high nonspecific adsorption. There are very few examples. On the other hand, among organic carriers, as natural carriers, porous agarose particles (trade name: Sepharose) and dextran particles (trade name: Sephadex) into which various functional groups are introduced are commercially available. Is most commonly used. As a carrier, agarose gel is said to be suitable for handling high molecular weight biological substances because the limit molecular weight of impurities that can be eliminated is larger than that of dextran or acrylamide. However, a drawback common to these hydrophilic carriers is that they have low physical strength and thus lack pressure resistance,
High flow rate analysis, that is, it is difficult to perform analysis operation in a short time. JP-A-62-23437 discloses a carrier for chromatography in which the surface of core particles made of an inorganic or organic polymer is coated with agarose gel. The average particle size of the core particles of this carrier is 5 to 1000 μm, and the thickness of the agarose gel is 0.05 to 0.
It is 75 times. However, in this carrier, since the thickness of the agarose gel is relatively large, sufficient physical strength cannot be obtained.

In the synthetic polymer system, particles made of polyacrylamide gel (trade name: Bio-Gel P, Bio-Rad), polystyrene, ethylene-maleic anhydride copolymer, etc. have been developed. However, these polymers have a drawback that nonspecific adsorption of bio-related substances easily occurs due to their hydrophobicity. The purpose of developing the synthetic polymer carrier is to improve the physical strength while maintaining the performance of the natural polysaccharide carrier, and various improvements have been attempted in order to achieve such a purpose. However, in order to improve the physical strength, it is necessary to reduce the degree of porosity or increase the degree of crosslinking. These countermeasures are naturally limited because they reduce the specific surface area and the hydrophilicity (currently, "affinity chromatography" by Makoto Yamazaki, Shinichi Ishii and Koichi Iwai). , Kodansha, 1975, pp9-19).

[0007]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned conventional drawbacks, and an object thereof is to have a high pressure resistance and a small degree of swelling and contraction.
It is an object of the present invention to provide a carrier for affinity chromatography which is capable of high-speed separation, has sufficient hydrophilicity, and has extremely low nonspecific adsorption of proteins other than the substance to be separated.

[0008]

The carrier for affinity chromatography of the present invention comprises a coating polymer in which a hydrophilic polymer layer is formed on the surface of hydrophobic cross-linked polymer particles, and the hydrophilic polymer The combined layer thickness is 10 to 300 angstroms, and the hydrophilic polymer is a polymer having a functional group capable of immobilizing a ligand or a spacer in affinity chromatography.

The carrier of the present invention is a two-layer structure polymer having hydrophobic cross-linked polymer particles as a skeleton and a hydrophilic polymer coated on the surface portion of the hydrophobic cross-linked polymer particles. The material for the hydrophobic crosslinked polymer particles is a (co) polymer obtained by (co) polymerizing a hydrophobic crosslinkable monomer, or a hydrophobic crosslinkable monomer and a hydrophobic noncrosslinkable monomer. And a copolymer thereof. These (co) polymers are homopolymers of hydrophobic crosslinkable monomers or copolymers of two or more crosslinkable monomers. Furthermore, if necessary, one or more hydrophobic non-crosslinking monomers can be added.

Examples of the hydrophobic crosslinkable monomer used in the present invention include ethylene glycol di (meth) acrylate, polyethylene glycol di (meth) acrylate, propylene glycol di (meth) acrylate and polypropylene glycol (meth). Di (meth) acrylic acid esters such as acrylates; poly (meth) acrylic acid esters of polyhydric alcohols such as tetramethylolmethane tri (meth) acrylate and tetramethylolmethane tetra (meth) acrylate; divinylbenzene, divinyltoluene, divinylxylene , And aromatic compounds having two or more vinyl groups such as divinylnaphthalene. Examples of hydrophobic non-crosslinkable monomers that can be added as necessary include, for example, methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth).
(Meth) acrylic acid ester such as acrylate, isopropyl (meth) acrylate, butyl (meth) acrylate, t-butyl (meth) acrylate; vinyl acetate,
Examples thereof include vinyl carboxylates such as vinyl propionate, vinyl butyrate and vinyl caproate; and styrene monomers such as styrene and methylstyrene. When the above crosslinkable and non-crosslinkable monomers are mixed and used, the amount of the crosslinkable monomer is 10 parts by weight or more, preferably 20 parts by weight or more based on 100 parts by weight of the total monomers. Used for. The particle size of the hydrophobic crosslinked polymer particles is usually 2 to 10
The thickness is 00 μm, preferably 5 to 500 μm.

The hydrophilic polymer for coating the hydrophobic cross-linked polymer particles used in the present invention is a hydrophilic polymer having a functional group that can be used for immobilizing a ligand (hereinafter referred to as a functional group for immobilization). It is obtained by polymerizing a monomer. Examples of the functional group for immobilization include the following groups. -OH group, -CO
OH group, -NH ₂ group, glycidyl group, -CN group. A monomer having two or more identical functional groups in the molecule and / or a monomer having two or more different functional groups can also be used. -OH
As the hydrophilic monomer having a group, 2-hydroxymethyl (meth) acrylate, 2-hydroxyethyl (meth) acrylate, glycerol mono (meth) acrylate,
Examples thereof include polyethylene glycol mono (meth) acrylate. Examples of the hydrophilic monomer having a -COOH group include (meth) acrylic acid, crotonic acid, maleic acid, phthalic acid and 2-carboxyethyl (meth) acrylate. As the hydrophilic monomer having an —NH ₂ group,
Examples thereof include (meth) acrylamide, maleic acid amide, fumaric acid amide, and acrylamine. As the hydrophilic monomer having a glycidyl group, glycidyl (meth)
Acrylate, oxiranyl methyl (meth) acrylate, oxiranyl nonyl (meth) acrylate, etc. are mentioned. Examples of the hydrophilic monomer having a -CN group include (meth) acrylonitrile and cyanoacrylate.

If desired, two or more kinds of the above hydrophilic monomers may be mixed and used. The amount of the hydrophilic monomer used varies depending on the kind of the monomer, but is 5 to 50 parts by weight with respect to 100 parts by weight of the hydrophobic crosslinked polymer particles to be coated.

A monomer having a functional group which can be converted into the above-mentioned immobilizing functional group by a chemical reaction can also be used. These monomers include alkyl (meth) acrylates such as methyl (meth) acrylate, propyl (meth) acrylate and butyl (meth) acrylate; vinyl carboxylates such as vinyl acetate, vinyl propionate and vinyl butyrate. There is. These are -C by the hydrolysis reaction.
It can be converted to an OOH group or an -OH group. A monomer having a carbonyl group such as diacetone acrylamide carbonyl can be converted to an -OH group by reduction of the carbonyl group or a -COOH group having a terminal methoxy group by a haloform reaction. Allyl chloride reacts with alkali metal cyanide in hydrous ethanol to give -C
It can be converted to a nitrile cyanide having an N group.

When these monomers are used, it is necessary to carry out the above-mentioned chemical reaction under the condition that the above-mentioned chemical reaction acts only for converting to the functional group for immobilization and does not decompose the hydrophobic cross-linked polymer itself. For example, when methyl acrylate was polymerized in the presence of hydrophobic crosslinked polymer particles to form a polymer coating layer on the surface of the particles, it was formed by hydrolysis under alkaline conditions. Methyl acrylate polymers are easily converted to acrylic acid polymers. However, when there is an ester bond in the hydrophobic crosslinked polymer, there is a risk that even the ester bond may be cleaved, so as a material for the hydrophobic crosslinked polymer particles, non-hydrolyzable styrene,
It is preferable to select divinylbenzene or the like.

Next, a typical production method for preparing the carrier of the present invention will be described. However, the preparation of the carrier of the present invention is not limited to the following method.

To prepare the carrier for affinity chromatography of the present invention, hydrophobic crosslinked polymer particles are first prepared. First, the above-mentioned hydrophobic monomer (hydrophobic crosslinkable monomer and, if necessary, hydrophobic non-crosslinkable monomer) and a polymerization initiator are dissolved in a diluent. The polymerization initiator used in this case and the polymerization initiator (described later) to be impregnated in the obtained hydrophobic cross-linked polymer particles are catalysts that generate radicals and are not limited except that they are hydrophobic. For example, organic peroxides such as benzoyl peroxide and acetyl peroxide; azobisisobutyronitrile,
Any of the known radical generating catalysts, such as azo compounds such as azobisisobutyroamide; can be used. The diluent is added as a pore-forming agent, and any organic solvent that dissolves the monomer and does not dissolve the polymer can be used. For example, toluene,
Aromatic hydrocarbons such as xylene, diethylbenzene, dodecylbenzene; hexane, heptane, octane,
And saturated hydrocarbons such as decane; alcohols such as isoamyl alcohol, hexyl alcohol, and octyl alcohol; The amount used is not limited, but is 15 to 100 parts by weight of the above monomer mixture.
It is preferably 200 parts by weight.

The above-mentioned monomer mixture (monomer, polymerization initiator and diluent) is added to an aqueous phase in which a suspension stabilizer such as polyvinyl alcohol and calcium phosphate is dissolved, and the atmosphere is replaced with nitrogen, then the mixture is stirred at 40 to 40%. Suspension polymerization is carried out by heating to 100 ° C. When the polymerization is carried out by adding a diluent, the organic solvent that is a diluent is present in the obtained polymer particles in a dispersed state. Particles are obtained. By using various organic solvents having different compatibility with the above-mentioned hydrophobic monomer mixture as a diluent, it is possible to arbitrarily change the size of the pores of the porous polymer. Next, the obtained hydrophobic crosslinked polymer particles are impregnated with a polymerization initiator. In order to impregnate the polymerization initiator, the polymerization initiator is dissolved in a solvent having a low boiling point and a good affinity for the hydrophobic crosslinked polymer, and the above hydrophobic crosslinked polymer particles are immersed in the solvent. This causes the polymerization initiator to penetrate into the particles. If necessary, this is heated at a temperature not higher than the decomposition point of the polymerization initiator to remove the solvent, whereby hydrophobic crosslinked polymer particles containing the polymerization initiator therein are obtained. The polymerization initiator-containing particles are dispersed in an aqueous dispersion medium in which the hydrophilic monomer is dissolved, or the hydrophilic monomer is added to an aqueous dispersion medium in which the particles are dispersed and dissolved. Then, after substituting with nitrogen, polymerization is carried out by heating with stirring. By this polymerization, the hydrophilic monomer is polymerized on the surface of the hydrophobic crosslinked polymer particles to coat the particle surface. A dispersion stabilizer such as carboxymethyl cellulose or polyvinyl alcohol may be added to the above aqueous dispersion medium in order to stabilize the dispersibility of the hydrophobic crosslinked polymer. The temperature and time of polymerization are
Although it depends on the kind of the hydrophilic monomer to be reacted and the kind of the polymerization initiator, it is about 0.5 to 40 hours at 40 to 100 ° C. The polymer particles having the above-mentioned two-layer structure are prepared by the above method.

In addition to the method of subjecting the crosslinked polymer particles impregnated with the above polymerization initiator to the polymerization reaction of the hydrophilic monomer, the hydrophilic monomer is reacted subsequent to the preparation of the hydrophobic crosslinked polymer particles. The above-mentioned two-layer structure polymer particles can also be prepared by a continuous method. In this method, first, a polymerization reaction for preparing the above hydrophobic crosslinked polymer particles is started.
The above-mentioned hydrophilic monomer is added to the reaction system when the polymerization proceeds to some extent and the unreacted polymerization initiator remains. In such a state, since the polymerization initiator is present in the organic phase in the system and inside the generated hydrophobic crosslinked polymer particles,
Subsequently, the hydrophilic monomer is polymerized, and a layer of the hydrophilic polymer is formed so as to cover the surface portion of the hydrophobic crosslinked polymer particles. Therefore, even with this continuous method,
Polymer particles similar to those described above are obtained.

In each of the above reactions, it is preferable to adjust the amount of the monomers, the reaction conditions, etc. so that the average thickness of the coating layer is 10 to 300 angstroms.
When the coating layer exceeds 300 angstroms, the swelling and shrinking effects of the coating layer become so large that they cannot be ignored.
Therefore, the resolution may decrease or the column pressure may increase. Further, this coating layer needs to be controlled so as to completely cover the surface of the hydrophobic crosslinked polymer. If there is a portion where the hydrophobic cross-linked polymer is exposed, non-specific adsorption may occur when the portion comes into contact with the substance to be separated (for example, protein) in affinity chromatography.

The polymer particles obtained by each of the above-mentioned methods are thoroughly washed with hot water and an organic solvent, and the suspension stabilizer, solvent and residual monomer contained in or attached to the particles. Etc. are removed. Furthermore, if necessary, the particles are classified to obtain a carrier for affinity chromatography.

The particle size of the carrier particles thus obtained,
Observation and measurement of the thickness and uniformity of the coating layer are carried out as follows. A section having a thickness of 1000 angstroms or less is prepared from a carrier particle as a sample by a microtome. The section is treated with a labeling agent or staining reagent specific to the functional group in the sample, and observed with a transmission electron microscope.

When the carrier of the present invention is used for affinity chromatography, the functional group for immobilization on the surface of the carrier is
The ligand is immobilized directly or via a spacer. As the ligand, any conventional spacer having a functional group capable of being covalently bound to the functional group on the surface of the carrier by a chemical reaction can be used. Examples include sugars, lipids, amino acids, peptides, polynucleotides, and various antigens and antibodies.

As a method for immobilizing these ligands on the carrier, known methods are used. For example, the CNBr method is often used when a protein is immobilized on a carrier having a —OH group on the surface. According to this method, the carrier is first treated with CNBr under alkaline conditions to activate the functional groups present in the carrier (imidocarbonate; C = NH is generated), and the resulting activated carrier is weakly alkaline. Act on the ligand below. If the ligand is a protein,
The activation site reacts with the amino group of the protein, and as a result, the protein is fixed by CN bond (partly C = N bond). Other methods include a method using a condensation reagent (carbodiimide) of —COOH group and —NH ₂ group, and a method of introducing a spacer having a functional group capable of reacting with a carrier and a ligand. Examples of the spacer include alkyldiamines such as ethylenediamine and hexyldiamine having amino groups at both ends of the molecule; ε-having an amino group at one end of the molecule and a carboxyl group at the other end.
Amino fatty acids such as aminocaproic acid;

The carrier whose ligand has been identified in this way is
It is used as a carrier for chromatography for separating and purifying a substance that specifically binds to the ligand.

[0025]

As described above, the carrier for affinity chromatography of the present invention is a polymer particle having a two-layer structure in which the hydrophobic cross-linked polymer fine particles are the core particles and the surface of the particles is coated with the hydrophilic polymer. is there. Since a polymer having a high degree of cross-linking is used in the core part, a carrier for affinity chromatography having extremely high mechanical strength and excellent pressure resistance can be obtained. Since there is no hydrophilic group in the core of the carrier, the degree of swelling and shrinking is extremely small.
Since the surface is coated with a hydrophilic polymer, non-specific adsorption of proteins etc. does not occur. By selecting an appropriate hydrophilic monomer, a desired functional group can be imparted to the particle surface, so that a desired carrier can be obtained according to the type of ligand to be immobilized. This carrier can be used in a wide pH range. Further, as described above, the pressure resistance is large, and the degree of swelling and contraction is extremely low,
It is possible to reduce the particle size so that separation with high precision is possible and rapid analysis can be done. On the other hand, in the case of a carrier that generally has low pressure resistance and a large degree of swelling and contraction, if the particle size is reduced, the particles will be broken or the liquid will not flow.

[0026]

EXAMPLES The present invention will be described below with reference to examples.

The methods for measuring the physical properties and evaluating the performance of the carriers obtained in the following Examples and Comparative Examples are as follows.

(Method for measuring the thickness of the coating layer) After embedding carrier particles as a sample in an epoxy resin, a 900 Å section is prepared using a microtome ULTRACUTE manufactured by Reichert-Jung. Among the obtained slices, the slice of the carrier having a -COOH group is labeled with a silver nitrate solution (for volumetric analysis, manufactured by Wako Pure Chemical Industries, Ltd.). -NH ₂ group and-
The carrier section having an OH group is stained with an osmic acid solution (for electron microscope, manufactured by Wako Pure Chemical Industries, Ltd.), observed and photographed with a transmission electron microscope JEM100S manufactured by JEOL Ltd. The distribution of hydrophilic groups and the thickness of the coating layer are measured.

(Evaluation Method by Affinity Chromatography) Carrier particles to be used as a sample are packed in a stainless steel column having an inner diameter of 6 mm and a length of 75 mm, and the pressure resistance and the swelling property in water are examined. The pressure resistance is measured by passing purified water through the column at various flow rates and examining the relationship between the flow rate and the pressure loss. The swelling property is measured by examining the change in column pressure when liquids having different ionic strengths are passed. Affinity chromatography is performed using Sekisui Chemical Co., Ltd. liquid chromatography system SSLC-20.

Example 1 250 g of triethylene glycol dimethacrylate (hydrophobic crosslinkable monomer), 50 g of tetramethylolmethane tetraacrylate (hydrophobic crosslinkable monomer) and benzoyl peroxide (polymerization initiator) 1 g was dissolved in 200 g of toluene (diluent). This was added to 2.5 L of a 4% aqueous solution of polyvinyl alcohol, and the particles were sized with stirring, and then 80 under nitrogen substitution.
Suspension polymerization was carried out by heating to ℃. After polymerizing at 80 ° C. for 8 hours, the product was washed successively with hot water and acetone and dried to obtain fine hydrophobic crosslinked polymer particles. 200 g of the hydrophobic crosslinked polymer particles were immersed in 1 L of acetone in which 0.5 g of benzoyl peroxide (polymerization initiator) was dissolved to impregnate the polymerization initiator. Next, add 2 acetone
Evaporate under reduced pressure at 0 ° C. Disperse the above-mentioned impregnated hydrophobic cross-linked polymer in 2.5 L of 1% polyvinyl alcohol aqueous solution, add 50 g of methacrylic acid (hydrophilic monomer) with stirring, replace with nitrogen, and then perform polymerization reaction at 80 ° C. for 5 hours. I went. The product was washed successively with hot water and acetone and dried. The fine polymer particles obtained were classified by an air classifier ELBOW-JET LABO EJ-L-3 manufactured by Nittetsu Mining Co., Ltd. to collect particles having a particle size of 8 to 12 μm. In this way, a carrier for affinity chromatography having —COOH group on the particle surface was obtained.

The pressure resistance and swelling property of the above carrier were evaluated by the above-mentioned methods. In the evaluation of pressure resistance, 1
The pressure loss and the flow velocity were proportional to 20 kg / cm ² . In the evaluation of swelling property, when the eluent was changed from 40 mM phosphate buffer to 200 mM phosphate buffer, no increase in column pressure was observed. The thickness of the coating layer was measured according to the method described above and was about 50 angstroms.

3-Aminophenylboronic acid (hereinafter referred to as APB) as a ligand was immobilized on the obtained carrier.
The immobilization method is water-soluble carbodiimide [1-ethyl-3-
(3-Dimethylaminopropyl) carbodiimide hydrochloride; manufactured by Wako Pure Chemical Industries, Ltd.] was performed as follows (“Protein Chemistry 1” Shiro Akahori, Takeo Kaneko, Kozo Narita, Kyoritsu Shuppan, 1979, pp524 ~ 52
8). Approximately 5 g of carrier was added to 6 mM aqueous carbodiimide solution
It was dispersed in 0 mL and stirred at 4 ° C. for 30 minutes. afterwards,
A 6 mM APB solution was added and reacted at 4 ° C. for 5 hours.
The obtained ligand-immobilized carrier (hereinafter referred to as a filler) was packed in a stainless steel column having an inner diameter of 6 mm and a length of 75 mm. For filling, take 2 g of packing material in 30 mL of purified water and
After stirring for 0 minutes, a constant flow rate of 2.0 mL / min was used for filling.

Glycated hemoglobin was separated using the obtained column as follows. 2 mL of healthy human blood was collected using a heparin-containing vacuum blood collection tube (Sekisui Chemical Co., Ltd.). This blood was centrifuged at 4 ° C. to separate blood cells from plasma, and then red blood cells were washed 3 times with 10 mL of physiological saline. Then wash the red blood cells with 1.5 volumes of distilled water and 0.5
Toluene was added and shaken vigorously to hemolyze. This 4
After centrifugation at 3000 rpm for 15 minutes at 3000 rpm, the supernatant of the hemolyzed layer was incubated at 4 ° C for phosphate-cyanide buffer (NaH ₂ PO ₄ · H).
₂ O 4.59 g / L, Na ₂ HPO ₄ 1.19 g / L, KCN
It was dialyzed at 0.65 g / L for 24 hours (using a dialysis tube made by Nippon Ichika Plastic). 1 μL of this dialyzed hemolyzed blood was injected into a liquid chromatograph as a sample. As the adsorbent, 50 mM HEPES having a pH of 8.0, 10
50 mM as desorption solution using mM MgCl buffer
HEPES, 10 mM EDTA 2Na, 100 m
M sorbitol solution was used. After adsorbing the saccharified product with the adsorbent and eluting the non-saccharified hemoglobin, the adsorbed liquid was used to elute the adsorbed glycated component. Detection is 415nm
The measurement was carried out by measuring the absorbance of visible light. The result of analysis using the column obtained in this example is shown in FIG. 1,
In addition, in FIGS. 2 and 3 described later, peak 1 represents non-glycated hemoglobin and peak 2 represents glycated hemoglobin.

Example 2 150 g of styrene (hydrophobic non-crosslinkable monomer), divinylbenzene (hydrophobic crosslinkable monomer; 45% mixture of p- and m-forms and ethylvinylbenzene) 55% product) 150 g and benzoyl peroxide (polymerization initiator) 1 g toluene (diluent)
It was dissolved in 200 g. This was added to 2.5 L of a 4% aqueous polyvinyl alcohol solution, and the particles were sized with stirring, and then heated at 80 ° C. under nitrogen substitution to carry out suspension polymerization.
After polymerizing at 80 ° C. for 8 hours, the product was washed successively with hot water and acetone and dried to obtain fine hydrophobic cross-linked polymer particles. Using 200 g of the hydrophobic crosslinked polymer particles and 50 g of acrylic acid (hydrophilic monomer), a carrier was prepared according to Example 1 and evaluated.

In the evaluation of pressure resistance, 100 kg / c
The pressure loss and the flow velocity were proportional to m ² . In the swelling evaluation, the eluent was changed from 40 mM phosphate buffer to 200
No increase in column pressure was observed when switching to mM phosphate buffer. When the coating layer thickness was measured by treating with a silver nitrate solution, it was about 100 Å. As in Example 1, APB was immobilized by the carbodiimide method. The obtained filler was filled in the same manner as in Example 1 and analyzed for glycated hemoglobin. The obtained chromatogram was similar to that shown in FIG.

(Example 3) In this example, a continuous method of reacting a hydrophilic monomer following the preparation of hydrophobic crosslinked polymer particles was adopted.

150 g of tetraethylene glycol dimethacrylate (hydrophobic crosslinkable monomer) and tetramethylolmethane triacrylate (hydrophobic crosslinkable monomer 15)
1. 1 g of benzoyl peroxide (polymerization initiator) was dissolved in 0 g of a mixed solution to prepare a 4% aqueous polybiyl alcohol solution.
Add to 5 L, adjust the particle size with stirring, and then under nitrogen replacement, 8
Polymerization was carried out by heating to 0 ° C. After polymerizing at 80 ° C. for 2 hours, 50 g of acrylic acid (hydrophilic monomer) was added and further polymerized at 80 ° C. for 2 hours. The obtained product was washed successively with hot water and acetone, dried and classified. The fine polymer particles obtained were evaluated in the same manner as in Example 1.

In the evaluation of pressure resistance, 350 kg / c
The pressure loss and the flow velocity were proportional to m ² or more. In the swelling evaluation, the eluent was changed from 40 mM phosphate buffer to 2
When the phosphate buffer was changed to 00 mM, the column pressure was not increased. When the coating layer was treated with a silver nitrate solution and the thickness of the coating layer was measured, it was about 80 Å. As in Example 1, APB was immobilized by the carbodiimide method. The obtained filler was filled in the same manner as in Example 1 to analyze glycated hemoglobin. The obtained chromatogram was similar to that shown in FIG.

Comparative Example 1 100 g of styrene (hydrophobic non-crosslinkable monomer), 200 g of divinylbenzene (hydrophobic crosslinkable monomer) and 100 g of acrylic acid (hydrophilic monomer).
g and 1 g of benzoyl peroxide (polymerization initiator) are dissolved in 270 g of toluene (diluent), added to 2.5 L of a 4% polyvinyl alcohol aqueous solution, and granulated with stirring, and then at 80 ° C. under nitrogen substitution. The polymerization reaction was carried out for 8 hours. After the completion of the polymerization, the product was classified and filled in the same manner as in Example 1 and evaluated.

In the evaluation of pressure resistance, 50 kg / cm
Up to ² , the pressure loss and flow velocity were proportional. In the evaluation of swelling property, the eluent was 200 m from 40 mM phosphate buffer.
When changing to M phosphate buffer, the column pressure is 20k.
It increased by g / cm ² . When the coating layer was treated with a silver nitrate solution to measure the thickness of the coating layer, the carboxyl groups were evenly distributed inside the carrier particles. As in Example 1, APB was immobilized by the carbodiimide method. The obtained filler was filled in the same manner as in Example 1 and analyzed for glycated hemoglobin. The obtained chromatogram is shown in FIG. From comparison with FIG. 1, it was found that the separation was clearly poor and the measurement time was long.

(Comparative Example 2) Using 250 g of triethylene glycol dimethacrylate (hydrophobic crosslinkable monomer) and 50 g of tetraethylene glycol triacrylate (hydrophobic crosslinkable monomer), the same procedure as in Example 1 was carried out. To prepare a hydrophobic crosslinked polymer. Further, when 300 g of acrylic acid (hydrophilic monomer) was used and polymerization was attempted in the same manner as in Example 1, agglomeration occurred during the reaction, and soft fine polymer particles were obtained.

When the polymer particles were classified and packed in the same manner as in Example 1, the particles could not be filled because the packing liquid did not flow because the particles were soft. When the coating layer thickness of the carrier particles was measured by treating with a silver nitrate solution, it was about 400 Å.

(Comparative Example 3) 250 g of triethylene glycol dimethacrylate (hydrophobic crosslinkable monomer) and 50 g of tetraethylene glycol triacrylate (hydrophobic crosslinkable monomer) were used in accordance with Example 1. To prepare a hydrophobic crosslinked polymer. Further, using 10 g of acrylic acid (hydrophilic monomer), a carrier was obtained in the same manner as in Example 1 and evaluated. In the evaluation of pressure resistance, 15
The pressure loss and the flow velocity were proportional to 0 kg / cm ² . In the evaluation of swelling property, when the eluent was changed from 40 mM phosphate buffer to 200 mM phosphate buffer, no increase in column pressure was observed. When the coating layer was treated with a silver nitrate solution and the coating layer thickness was measured, it was about 8 angstroms, and a part of the surface not coated was observed.

As in Example 1, APB was immobilized by the carbodiimide method. The obtained filler was filled in the same manner as in Example 1 and analyzed for glycated hemoglobin. The obtained chromatogram is shown in FIG. From the comparison with Fig. 1,
It was found that the amount of hemoglobin eluted was small and the recovery rate was low. This is probably because proteins and the like in the sample were nonspecifically adsorbed to the non-coated portion of the carrier particles by the hydrophobic bond.

Example 4 According to Example 1, 250 g of triethylene glycol dimethacrylate (hydrophobic crosslinkable monomer) and 50 g of tetraethylene glycol triacrylate (hydrophobic crosslinkable monomer) were used. To prepare a hydrophobic crosslinked polymer. Further, using 50 g of octaethylene golycol monomethacrylate (hydrophilic monomer), a carrier having —OH groups only on the surface thereof was obtained in the same manner as in Example 1 and evaluated.

For the evaluation of pressure resistance, 150 kg / c
The pressure loss and the flow velocity were proportional to m ² . For swelling evaluation, eluent was changed from 40 mM phosphate buffer to 200
When the buffer was changed to mM phosphate buffer, no increase in column pressure was observed. It was about 80 Å when the coating layer thickness was measured by staining with osmic acid.

The obtained carrier particles were applied with AP by the CNBr method.
B was immobilized. First, the carrier was dispersed in a 2M Na ₂ CO ₃ solution, an acetonitrile solution of CNBr was added thereto, and the mixture was stirred at room temperature for 2 minutes. Then, the cells were quickly washed with a buffer solution.
Next, APB to be immobilized was added and reacted with stirring at 4 ° C. for 15 hours. Finally, residual functional groups were blocked with ethanolamine, and the APB-immobilized filler was filled in the same manner as in Example 1 to analyze glycated hemoglobin. The obtained chromatogram was similar to that shown in FIG.

Example 5 In this example, an example in which a spacer is introduced into the carrier obtained in Example 1 will be shown. First, the carrier obtained in Example 1 was activated by the carbodiimide method described above, a hexamethylenediamine solution (pH 10) was added to the activated carrier, and the mixture was stirred at 4 ° C. for 12 hours to prepare C ₆
A carrier having a —NH ₂ group at the end of the carbon chain was obtained. Next, this carrier was thoroughly washed with a buffer solution, and APB was immobilized by the CNBr method. When glycated hemoglobin was measured according to Example 1, the same chromatogram as in FIG. 1 was obtained.

Example 6 The carrier obtained in Example 5 was
Ethylenediamine diacetic acid was immobilized by the carbodiimide method under the same conditions as above. Ribonucleotides were separated using the obtained packing material. The result is shown in FIG. Peak 3 shows cytidine, peak 4 shows guanosine, and peak 5 shows adenosine (manufactured by Wako Pure Chemical Industries, Ltd.). Elution was performed with ethyl morpholine acetate, pH 5.6.

[0050]

As described above, according to the present invention, a carrier having excellent pressure resistance, a small degree of swelling and contraction, and non-specific adsorption of proteins does not occur. Such a carrier can be widely used as a carrier for affinity chromatography for separation and purification of various bio-related substances.

[Brief description of drawings]

FIG. 1 is a chromatogram obtained when a packing material was prepared using the carrier obtained in Example 1 and glycated hemoglobin was separated using this packing material.

FIG. 2 is a chromatogram obtained when a packing material was prepared using the carrier obtained in Comparative Example 1 and glycated hemoglobin was separated using this packing material.

FIG. 3 is a chromatogram obtained when a packing material was prepared using the carrier obtained in Comparative Example 3 and glycated hemoglobin was separated using this packing material.

FIG. 4 is a chromatogram obtained when a packing material was prepared using the carrier obtained in Example 6 and ribonucleotides were separated using this packing material.

Claims (1)

Claim: What is claimed is: 1. A coating polymer comprising a hydrophilic polymer layer formed on the surface of hydrophobic cross-linked polymer particles, wherein the hydrophilic polymer layer has a thickness of 10 To 300 angstroms, and the hydrophilic polymer is a polymer having a functional group capable of immobilizing a ligand or a spacer in affinity chromatography.