JPH0198964A - Adsorptive carrier for chromatography - Google Patents

Abstract

PURPOSE:To obtain an adsorptive carrier which has a good sepn. characteristic and less nonspecific absorption by using a base material provided with all of the properties desired as the adsorptive carrier for affinity chromatography and covalent-bonding benzamidine via a suitable bond group thereto. CONSTITUTION:This adsorptive carrier is a hydroxyl group-modified gelatinous copolymer formed by bonding a compd. having a hydroxyl group to the epoxy group based on the component (A) of the gelatinous copolymer which consists essentially of (A) glycidyl monovinyl ester or glycidyl monovinyl ether and (B) alkylene glycol divinyl ester and in which the component (A) is crosslinked by the component (B). The carrier is further formed of the porous copolymer contg. the benzamidine bonded by the covalent bonding via the bond group to the hydroxyl group-modified gelatinous copolymer. This adsorptive carrier is not only usable as a packing material for the affinity chromatography but also sufficiently usable even under pressurization by packing the same into a pressure-resistant column in particular.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to an adsorption carrier for chromatography that can be used in various chromatography including affinity chromatography.

Affinity chromatography, one of the conventional chromatography techniques, uses the affinity of pairs of substances that specifically interact with each other to separate and purify biological substances. It is useful in identifying and purifying a property, ie, a specific chemical structure on a molecule.

An adsorption carrier for affinity chromatography (affinity gel) is, for example, an active support obtained by bonding a binding group (spacer) to an insoluble carrier (matrix), and a ligand is bonded to the spacer. An adsorption operation is performed by selecting a combination of substances that interact with each other.

As one of the ligands, benzamidine is a compound in which an amidino group is bonded to a benzene ring. Due to its similarity to arginine, affinity chromatography using benzamidine as a ligand is useful for trypsin group enzymes such as trypsin, plasmin, thrombin, kallikrein, etc. It is said to be useful for the separation, purification, analysis, and research of enzymes such as transferases.

For example, in separation, purification, and analysis by affinity chromatography using benzamidine as a ligand and a trypsin family enzyme as the adsorption target substance, the desired properties of the active support, which is the main component of the adsorption carrier for affinity chromatography, are as follows: Low non-specific adsorption, high porosity, easy binding of ligands and large immobilization capacity, chemical stability and sufficient stability under a wide range of pH ranges, salt concentrations, and temperatures. The characteristics include no volume change, sufficient mechanical strength and stability, good flow characteristics, and resistance to biological contamination.

Cellulose, dextran, polyacrylamide, agarose, and the like, which have been conventionally used as base materials for adsorption carriers for affinity chromatography, do not necessarily have these desired properties. In particular, since it is a so-called soft gel lacking in hardness, it has serious drawbacks such as poor flow properties and poor separation properties, and also has a short lifespan.

Furthermore, although the silica beads that have been used in recent years are satisfactory in terms of hardness, they cannot be used under alkaline conditions, which poses the problem of placing significant restrictions on the selection of separation conditions and elution/washing conditions. Ta.

Furthermore, adsorption carriers using styrene polymers or acrylate polymers as base materials have recently been developed, but these have not been satisfactory due to the problem of nonspecific adsorption.

Problems to be Solved by the Invention The object of the present invention is to overcome the drawbacks of the conventional adsorption carriers for chromatography and to provide a chromatography carrier that fully has the above-mentioned properties desired in an adsorption carrier for affinity chromatography. An object of the present invention is to provide an adsorption carrier for

Means for Solving the Problems The present invention provides an adsorption carrier for chromatography that can achieve the above objects.

That is, the present invention comprises (A) glycidyl ether ester or glycidyl monovinyl ether and (B) alkylene glycol divinyl ester as main components, and (A)
The component is a gel-like copolymer crosslinked with component (B) (hereinafter referred to as
A hydroxyl group-modified gel copolymer in which a compound having a hydroxyl group is bonded to an epoxy group based on the component (A) of the gel copolymer according to the present invention, further comprising: The present invention relates to an adsorption carrier for chromatography characterized by being made of a porous copolymer containing benzamidine covalently bonded via a bonding group.

The adsorption carrier for chromatography of the present invention will be explained below.

The gel-like copolymer according to the present invention is disclosed in JP-A-60-1042
As described in Publication No. 58, for example, (A) glycidyl monovinyl ester or glycidyl monovinyl ether and (B) alkylene glycol divinyl ester are subjected to aqueous suspension polymerization in the presence of a water-insoluble organic diluent. Although it can be prepared by obtaining a porous gel, aqueous suspension polymerization can be carried out using a simple oil-in-water suspension system. Copolymerization is carried out in the presence of an organic diluent, and due to the presence of this organic diluent, the alkylene glycol divinyl ester component (B) acts as the main crosslinking component, and the glycidyl monovinyl ester component (A) or Most of the epoxy groups in the glycidyl monovinyl ether component remain in the resulting copolymer without ring opening, and are also useful for adjusting the pore size of the resulting porous gel.

The component (A) is glycidyl ester of monovinylcarboxylic acid having 3 to 12 carbon atoms or 3 to 12 carbon atoms.
Glycidyl ether of monovinyl alcohol is used. Among these, those having a small number of carbon atoms are particularly preferably used, and examples thereof include glycidyl methacrylate, glycidyl acrylate, allyl glycidyl ether, and the like.

On the other hand, as the component (B) which acts as a crosslinking component, preferably used are alkylene glycols having 2 or 3 carbon atoms, hydroxyl-substituted alkylene glycols having 3 carbon atoms, or esters of polyalkylene glycols and acrylic acid or methacrylic acid. It will be done. Examples include alkylene glycol or hydroxyl substituted alkylene glycol divinyl esters such as ethylene glycol diacrylate, ethylene glycol dimethacrylate, propylene glycol diacrylate, propylene glycol dimethacrylate, glycerol diacrylate, glycerol dimethacrylate and polyethylene glycol dimethacrylate, polypropylene glycol dimethacrylate. Examples include polyalkylene glycol divinyl esters such as.

The ratio of component (A) to component (B) is 10 to 90 mol% for the former and 90 to 10 mol% for the latter. Preferably (A
) component 40 to 80 mol%, component (B) 60 to 20 mol%
It is.

Up to half of component (A) can be replaced by a comonomer, ie, a lower vinyl ester such as methyl methacrylate, methyl acrylate, or vinyl acetate.

The organic diluent used may be one that is inert to the polymerization reaction, insoluble or sparingly soluble in water, but capable of dissolving the raw material monomer. The amount used is at least 80% by volume based on the content of monomer components and organic diluent, preferably 40-8%.
0% by volume. Examples of organic diluents that can be suitably used in the present invention include cyclohexanone, chlorobenzene, benzene, toluene, n-propylacetate, n-
Examples include butyl acetate and n-octane.

The aqueous suspension polymerization can be carried out in the presence of a free-radical generating catalyst according to conventional methods known per se.

The amount of the aqueous phase is approximately the same volume or more based on the amount of the organic phase, and is not particularly limited, but may be used in an amount up to about 10 times the volume.

A compound having a hydroxyl group can be bonded to the gel copolymer according to the present invention by utilizing the reactivity of the epoxy group based on the component (A) of the gel copolymer according to the present invention. .

Examples of compounds having a hydroxyl group include polyols such as glycerin, erythritol, pentaerythritol, inositol, xylitol, annitol, and turbitol, epoxy compounds such as glycidol, glyceraldehyde, erythrose, xylose, arabinose,
Compounds with aldehyde groups such as ribose, glucose, galactose, annose, fructose, etc.
Examples include amino alcohols such as trimethylol aminomethane, glucamine, glucosamine, galactosamine, fucosamine, and the like.

When bonding these hydroxyl group-containing compounds to the gel copolymer according to the present invention, it can be carried out in an appropriate solvent using an appropriate catalyst or reaction reagent, if necessary. As the catalyst, acids such as hydrochloric acid and sulfuric acid; alkalis such as sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate are mainly used. especially,
For compounds having aldehyde groups, the epoxy groups of the gel-like copolymer according to the present invention are ring-opened using diamines such as ammonia or propanediamine, and then in the presence of a reducing agent such as sodium cyanoborohydride. Can be combined below. Further, compounds having an epoxy group can be ring-opened with water after bonding to form a hydroxyl group. Water, dimethylformamide, dioxane, etc. are usually used as a bonding solvent, but they can also be used as a buffer or inorganic salts can be added, if necessary.

The gel copolymer and the compound having a hydroxyl group according to the present invention have a weight ratio of, for example, 1:0.03 to 1, preferably 0.03.
05~0.5, reaction temperature 09C~150℃, preferably room temperature~80℃, reaction time 1~72 hours, preferably 2~
Binding can be carried out under conditions of 12 hours.

When a bonding group is further introduced into the gel copolymer of the present invention to which a compound having a hydroxyl group is bonded (hereinafter referred to as the hydroxyl group-modified gel copolymer of the present invention), the bonding group may be benzamidine or Although it is necessary to have a functional group capable of covalent bonding, there is no particular restriction on the structure of the bonding group other than having the above-mentioned functional group. Examples of the functional group capable of covalently bonding with the benzamidine include an epoxy group, a carboxyl group, and a formyl group. In this case, the bonding group can be easily introduced using a known method, and it does not necessarily have to be introduced in one reaction step, but can also be introduced in two or more reaction steps. .

A specific example of the introduction of a bonding group will be described below.

(1) Introduction of a bonding group having an epoxy group as a functional group For example, by bonding a compound having a glycidyl group to the hydroxyl group of the hydroxyl group-modified gel copolymer according to the present invention, a bonding group having an epoxy group based on a glycidyl group Introduce (
Hereinafter, it will be referred to as the epoxy-modified gel copolymer according to the present invention. ). Examples of compounds having a glycidyl group that are bonded to a hydroxyl group include epihalohydrins such as epichlorohydrin and epibromohydrin, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, and polyethylene glycol diglycidyl ether. diglycidyl ethers, alkyl diene diepoxides such as 1,3-butadiene diepoxide, and 1,7-octadiene diepoxide.

In order to act with a compound having a glycidyl group, for example, a hydroxyl group-modified gel copolymer is reacted with epichlorohydrin in an aqueous solution of an inorganic base such as sodium hydroxide or potassium carbonate, or a compound containing sodium borohydride is reacted with the hydroxyl group-modified gel copolymer. This is carried out by reacting with ethylene glycol, 1,4-butanediol diglycidyl ether, or polyethylene glycol diglycidyl ether in an aqueous solution of an inorganic base such as sodium hydroxide or potassium carbonate. The repeating unit -C2H40- of the polyethylene glycol diglycidyl ether used is 1
It is preferable to use 10 to 10.

(N) Introduction of a bonding group having a carboxyl group as a functional group For example, as a method for introducing a bonding group having a carboxyl group, the general Formula (I) H2NR' [wherein R' represents a hydrogen atom, an amino acid, an ω-aminoalkyl group or an ω-carboxyalkyl group. ] The above epoxy group is reacted with an amino compound represented by the above, or when an amino compound other than the ω-carboxyalkyl group is reacted with the above amino compound, an alkylenecarboxylic anhydride is further reacted with the epoxy group. General formula (
II) In the NHR C formula, R represents an ω-carboxyalkyl group, an ω-carboxyalkanoyl group, an ω-carboxyalkanoylamino group, or an ω-carboxyalkanoylaminoalkyl group. ] There is a method of introducing a bonding group represented by the following.

In this case, the reaction can be carried out according to the following reaction formula.

In the following reaction formula, (base) CH-CH2 represents the epoxy-modified gel copolymer according to the present invention.
12 to represent epoxy groups. In the formula, m is 1 or 2, and n+ p+ q and r are each an integer of 1 or more (more preferably 1 to 12).

(The following is a blank space) -1= The reaction formula (D), general formula (Do), or general formula (F) is caused to act on the epoxy group of the epoxy-modified gel copolymer according to the present invention. The reaction can be carried out in the presence of a solvent, but if these compounds are liquid under the reaction conditions, a solvent may not be used. As a solvent,
Water is usually used, but other hydrocarbons such as n-hexane, benzene, and xylene, ethers such as tetrahydrofuran and dioxane, chlorinated hydrocarbons such as chloroform and chlorobenzene, alcohols such as ethanol and methyl cellosolve, and acetone In addition to ketones such as and methyl isobutyl ketone, dimethyl formamide and dimethyl sulfoxide are also used, and these may be used alone or in a mixed solvent system. Although no particular catalyst may be used, hydroxides or carbonates of alkali or alkaline earth metals such as sodium hydroxide and potassium carbonate may also be used.

Examples of the compound of general formula (Do) include ethylenediamine,
1,3-diaminopropane, 1,4-diaminobutane,
1,5-diaminopentane, 1,6-diaminohexane, ■,7-diaminohebutane, 1,8-diaminooctane, l,9-diaminononane, 1,10-diaminodecane, 1,12-diaminododecane, etc. Examples include diaminoalkanes.

Examples of the compound of general formula (P) include aminocarboxylic acids such as glycine, β-alanine, 4-aminobutyric acid, 6-aminocaproic acid, and 8-aminocaprylic acid.

Although there are no particular restrictions on the reaction conditions, it is generally preferable to select the following conditions to carry out the reaction.

Weight (a) of the epoxy-modified gel copolymer according to the present invention
and the weight (b) of the compound of formula (D), general formula (Do) or general formula (P): a:b=1:0.3-10 More preferably, a:b-1:0. 3-3 Reaction temperature 20-150°C, more preferably room temperature-100°C
C, reaction time = 1 to 60 hours, more preferably 1 to 30 hours, reaction pressure normal pressure to 10 atm, more preferably normal pressure.

There are no special requirements for post-treatment after the reaction, and it may be appropriately carried out by commonly used methods such as separate furnaces and washing.

The chromatography carrier according to the present invention obtained above can be prepared using the compound of formula (D) or general formula (Do). The terminal amino group can be converted into an active supporting group having a carboxyl group by reacting with an acid anhydride of general formula (E). Water is usually used as a solvent for the reaction, but ethers such as tetrahydrofuran and dioxane, carboxylic acids such as acetic acid, pyridine, and the like may also be used. Further, although no particular catalyst may be used, the pH of the reaction solution can be adjusted by adding an acid such as hydrochloric acid or sulfuric acid or an alkali such as sodium hydroxide or potassium carbonate.

In the reaction formula, the alkylenecarboxylic anhydride of general formula (E) or general formula (E') includes succinic anhydride, geltaric anhydride, adipic anhydride, pimelic anhydride, speric anhydride, and azelain. Examples include acid anhydrides and cepatic anhydride.

The reaction conditions are not necessarily limited, but the reaction can be carried out by selecting, for example, the same conditions as for the compounds of formula (D), general formula (Do) or general formula (P).

Another method for introducing a bonding group having a carboxyl group as a functional group is a method in which an aminocarboxylic acid having a hydroxyl group is made to act on the epoxy group of the epoxy-modified gel copolymer according to the present invention and bonded thereto.

Examples of aminocarboxylic acids having a hydroxyl group include serine, homoserine, threonine, 4-hydroxyproline, 4-
Examples include amino acid derivatives such as amino-3-hydroxybutyric acid, Nulith (hydroxymethyl), and methylglycine, and amino sugar derivatives such as glucosaminic acid.

The bonding state is such that the epoxy group of the epoxy-modified gel copolymer according to the present invention is bonded to the amino group of the aminocarboxylic acid having a hydroxyl group, and as a result, the epoxy group is ring-opened, and a hydroxyl group and a carboxyl residue having a hydroxyl group are formed. will be generated.

There are no particular restrictions on the solvent, catalyst, and reaction conditions in which the aminocarboxylic acid having a hydroxyl group is reacted; It can be made into a condition.

When a bonding group is introduced using an aminocarboxylic acid having such a hydroxyl group, nonspecific adsorption originating from hydrophobicity based on a portion other than the base material, that is, a portion of the bonding group can also be eliminated.

(Hi) Introduction of a bonding group having a formyl group as a functional group For example, the bonding group having an epoxy group as a functional group obtained in the above (1) can be easily hydrolyzed to form a
, 2-glycol, and this 1.2-
Glycol can be converted into formyl group by the action of periodic acid.

There are no particular restrictions on the reaction conditions, and the reaction can be carried out according to a conventional method using, for example, water, ethanol, acetic acid, or a mixture thereof as a solvent at room temperature to 50° C. by reacting periodic acid or an inorganic salt thereof. .

When covalently bonding benzamidine to the binding group,
For example, it is convenient to use p-aminobenzamidine. At this time, if necessary depending on the functional group possessed by the bonding group, the reaction can be carried out in an appropriate solvent using an appropriate catalyst or reaction reagent.

For example, as a catalyst, an acid such as hydrochloric acid, sodium carbonate or sodium bicarbonate, or an alkali is mainly used when the functional group is an epoxy group, and as a reaction reagent, for example, N-hydroxysuccinimide, dicyclohexylcarbodiimide or A condensing agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is used when the functional group is a carboxyl group, and a reducing agent such as sodium cyanoborohydride is used when the functional group is a formyl group. 1 - I can give examples such as being able to stay. Water is usually used as a solvent, but if necessary, it can also be used as a phosphoric acid or acetate buffer, or inorganic salts such as sodium chloride or sodium sulfate can be added thereto.

The conditions for the reaction with p-aminobenzamidine are not necessarily limited, but it is generally appropriate to carry out the reaction within the following range.

Weight ratio of hydroxyl-modified gel copolymer with bonding group to p-aminobenzamidine = 1 = 0.08 to 0.3,
Preferably 1:0.05-0.2; reaction temperature: 0°C
~100°C, preferably 4°C ~ 80°C; reaction time = 1~
72 hours, preferably 2-12 hours.

There are no special requirements for the post-treatment after the reaction, and it may be appropriately carried out by commonly used methods such as separate furnaces and washing.

Effects of the Invention The adsorption carrier for chromatography provided by the present invention uses a base having all the properties desired as an adsorption carrier for affinity chromatography as described above, and binds benzamidine to this base via an appropriate bonding group. By using the adsorption carrier of the present invention, it can not only be used as a packing material for affinity chromatography in the same way as adsorption carriers using conventional soft gels, but also can be used especially for packing in pressure columns. The fact that it can be used sufficiently even under pressure is the greatest advantage of significantly shortening the working time and therefore greatly improving the efficiency of separation and purification. Needless to say, it is essential for application to liquid chromatography and industrial separation and purification/equipment.

In particular, in contrast to gel-like copolymers which have many advantages as described above, adsorption carriers to which benzamidine is covalently bonded via a bonding group as described above, for example, Compared to adsorption carriers for affinity chromatography, etc., the ligand does not fall off during use, and the length of the binding group can be adjusted arbitrarily, so the adsorption ability of the ligand for the target substance is excellent. Furthermore, the adsorption carrier for chromatography provided by the present invention has great effects, such as very little nonspecific adsorption.

EXAMPLES Below, typical examples of the method for producing the adsorption carrier for chromatography of the present invention will be shown and explained in more detail.

However, these are merely examples for explanation, and it goes without saying that the present invention is not limited thereto.

Reference Example 1 12 g of an epoxy group-containing gel copolymer obtained from glycidyl methacrylate and ethylene glycol dimethacrylate was added to 72 g of aqueous ammonia (25%),
The mixture was stirred for 12 hours while warming to ℃.

Next, when the gel was taken out and washed with water, dried gel 1
It was confirmed by neutralization titration that 1.4 mmol of amino groups were supported per g. This gel was added to 36 g of 0.2 molar dipotassium hydrogen phosphate aqueous solution, 7.92 g of glucose and 7.9 g of sodium cyanoborohydride.
2 g was added and stirred at 80°C for 10 hours. After the gel was taken out, washed with water and dried, it was added to 18 g of acetic anhydride, 18 g of sodium acetate was added, and the mixture was stirred at room temperature for 2 hours. Next, the gel was taken out and washed with water. The gel thus obtained (
Hydroxyl group-modified gel copolymer) was packed into a stainless steel column with an inner diameter of 4.6 m11% and a length of 75 m+, and the retention time ratio of butanol and ethylene glycol was determined using a high performance liquid chromatography device. The following results were obtained.

*Retention time ratio of butanol/ethylene glycol Reference example 2 When analysis was performed using α-chymotrypsinogen A and ovalbumin in place of butanol and ethylene glycol in Reference example 1, the following results were obtained.

Elution conditions Eluent ■: 0.1M sodium phosphate buffer (pH 1
7, (1) Eluent ■: Added 168 mol ammonium sulfate to eluent ■, linear gradient elution rate from eluent ■ to eluent ■ in 60 minutes: 1. Oml/min, Detector: Ultraviolet spectrophotometer (280 nm) Reference Example 3 Add 4 g of an epoxy group-containing gel copolymer obtained from glycidyl methacrylate and ethylene glycol dimethacrylate to 12 ml of dimethylformamide, and then add 15 ml of glycerol and Powdered potassium hydroxide 1.5g
was added and stirred at 60°C for 3 hours. Next, the gel was taken out, washed with water, and then packed into the same column as in Reference Example 1.

When this was used in place of (column A) in Reference Example 2 and the same analysis as in Reference Example 2 was conducted, α-chymotrypsinogen A and ovalbumin were not retained.

Example 1 An epoxy group-containing gel copolymer obtained from glycidyl methacrylate and ethylene glycol dimethacrylate was modified with hydroxyl groups using ammonia water and glucose in the same manner as in Reference Example 1, and further epoxy groups were introduced with shrimp chlorohydrin. gel (epoxy group: o, s per 1 g of dry gel)
To a suspension of 2 g of 0.1 molar sodium phosphate buffer (pH 7.5) in 6 ml was added 340 mg of p-aminobenzamidine dihydrochloride and stirred at 50°C.
Shake for hours. The gel was taken off and washed with water. It was confirmed by high-performance liquid chromatography analysis of unreacted p-aminobenzamidine dihydrochloride that the adsorption carrier thus obtained supported 0.115 mmol of benzamidine per gram of dry gel. .

Example 2 In Example 1, 1.4 was used instead of epichlorohydrin.
- 2.0 g of an epoxy group-introduced gel obtained using butanediol diglycidyl ether (epoxy group: 0.22 mmol per 1 g of dry gel) was mixed with acetic acid water containing 2.0 g of sodium periodate (9: 1) Added to 20ml of solution and shaken at room temperature for 2 hours. After removing the gel and washing with water, 0.
In addition to 6 ml of an aqueous solution containing 0.03 mol acetic acid and 0.06 mol sodium acetate, 0.34 g of p-aminobenzamidine dihydrochloride was added, and the mixture was shaken at 60°C for 8 hours. The gel was taken out and washed with a 1 molar aqueous sodium chloride solution and water, then added to a solution of 40 mg of sodium cyanoborohydride in 6 ml of a 0.2 molar dipotassium hydrogen phosphate solution, and after shaking at room temperature for 1 hour, the gel was washed again. It was taken out and washed with water and methanol. It was confirmed that the adsorption carrier thus obtained supported 0.012 mmol of benzamidine per 1 g of dry gel.

Example 3 An epoxy group-containing gel copolymer obtained from glycidyl methacrylate and ethylene glycol dimethacrylate was modified with hydroxyl groups using ammonia water and glucose in the same manner as in Reference Example 1, and then the epoxy groups were modified with ethylene glycol diglycidyl ether. After the introduction, 2 g of the gel into which carboxyl groups were introduced with 4-aminobutyric acid was thoroughly washed with anhydrous dioxane, and then added to 6 ml of anhydrous dioxane, and further 230 mg of N-hydroxysuccinimide and 412 mg of dicyclohexylcarbodiimide were added. Te5
After shaking at ℃ for 4 hours, the gel was taken out and dioxane 2
0m1. It was quickly washed with 6 ml of methanol and 3 ml of cold water. This gel was added to a solution of 340 mg of p-aminobenzamidine dihydrochloride in 6 ml of 0.1 molar sodium phosphate buffer (pH 6) and shaken overnight at room temperature. The gel was then taken out and washed with water.

It was confirmed that the thus obtained adsorption carrier supported 0.025 mmol of benzamidine per 1 g of dry gel.

Example 4 The epoxy group-introduced gel obtained in Example 2 was treated with 4-
Add 2 g of the gel obtained by reacting with aminobutyric acid (carboxyl group: 0.14 mmol per 1 g of dry gel) to 20 ml of 0.01 M sodium phosphate buffer (pH 5),
p-Aminobenzamidine dihydrochloride 34 while keeping at ℃
0 mg and 575 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added in three portions and shaken for 3 hours. Then, after leaving it at 4℃ overnight,
The gel was taken out and washed with 0.1N sodium chloride. It was confirmed that the adsorption carrier thus obtained supported 18 mmol of benzamidine at 0°O per gram of dry gel.

Example 5 The epoxy group-introduced gel obtained in Example 1 was
2 g of gel into which carboxyl groups were introduced by reacting with 3-diaminopropane and then with succinic anhydride (carboxyl group: 0.22 mmol per 1 g of dry gel) was added to 6 ml of tetrahydrofuran, and p-aminobenzamidine dihydrochloride was added. 340 ml and 412 mg of dicyclohexylcarbodiimide were added and stirred at 5°C for 4 hours. The gel was taken out and washed with methanol and water to obtain an adsorption carrier supporting 0.035 mmol of benzamidine per gram of dry gel.

Test Example The adsorption carrier obtained in Example 3 had an inner diameter of 4. [i+on+,
When trypsin (Sigma, Type I) was analyzed using a high-performance liquid chromatography device packed in a stainless steel column with a length of 75 mm, the results shown in the figure were obtained.

Elution conditions Eluent ■: 0.1M sodium acetate buffer (pH 7,
0) with 0.5 M sodium chloride added Eluent ■: 0.1 M sodium acetate buffer (pH 3,
0) with 0.5M sodium chloride added Linear gradient from eluent ■ to eluent ■ in 30 minutes Elution rate: 1.0 m 17 minutes Detector: Ultraviolet spectrophotometer (280 nm) The diagonally shaded fraction in the diagram is enzyme Fractions with confirmed activity are shown.

[Brief explanation of the drawing]

The figure is a chromatogram diagram showing a separation and elution pattern using a high performance liquid chromatography column using the chromatography adsorption carrier according to the present invention.

Claims (1)

[Claims] Based on the above (A) component of a gel-like copolymer containing (A) glycidyl monovinyl ester or glycidyl monovinyl ether and (B) alkylene glycol divinyl ester as the main components, and the above (A) component being crosslinked with the (B) component. A porous copolymer comprising a hydroxyl-modified gel copolymer in which a compound having a hydroxyl group is bonded to an epoxy group, and further contains benzamidine covalently bonded to the hydroxyl-modified gel copolymer via a bonding group. An adsorption carrier for chromatography, characterized in that it consists of a coalesce.