JPS62153755A - Adsorber carrier for chromatography - Google Patents

Abstract

PURPOSE:To obtain a desirable affinity gel, by constituting said gel of a copolymer based on glycidyl monovinyl ester A and alkylene glycol divinyl ester B and containing heparin bonded to the epoxy group of the A-component. CONSTITUTION:In a gel-like copolymer wherein (A) glycidyl monovinyl ester or glycidyl monovinyl ether and (B) alkylene glycol divinyl ester are main components and the (A)-component is crosslinked with the (B)-component, heparin is bonded to the epoxy group of the (A)-component through a covalent bond to obtain a heparin-containing porous copolymer used as the titled adsorbing carrier. This copolymer can be prepared by obtaining a porous gel through the aqueous suspension polymerization of the (A)-component and the (B)-component in the presence of a water-insoluble org. diluent. Because of the presence of said org. diluent, the (B)-component acts as a main crosslinking component and the greater part of the epoxy groups in the (A)-component is allowed to remain.

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, heparin is a mucopolysaccharide with anti-blood coagulation activity, but it has a strong affinity for adsorbing various factors of the blood coagulation system, enzymes such as lipoprotein lipase and DNA polymerase, and many other proteins. It has been widely used in recent years as a ligand for adsorption carriers for chromatography.

For example, in separation, purification, and analysis by affinity chromatography in which heparin is used as a ligand and enzyme or protein is used 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;
It has high porosity, easy binding of lycand and large immobilization capacity, chemical stability and pH range,
These include being sufficiently stable and not changing in volume under a wide range of salt concentration and temperature conditions, having sufficient mechanical strength and stability, and having good flow characteristics, and being resistant 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 characteristics and poor separation characteristics, 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.

SUMMARY OF THE INVENTION An object of the present invention is to overcome the drawbacks of the conventional adsorption carriers for chromatography, and to provide an adsorption carrier for chromatography that has the above-mentioned properties desired in an adsorption carrier for affinity chromatography. The purpose of the present invention is to provide an adsorption carrier.

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 monovinyl ester or glycidyl monovinyl ether and (B) alkylene glycol divinyl ester as main components;
The component is a gel-like copolymer crosslinked with component (B) (hereinafter referred to as
A chromatograph comprising a porous copolymer containing heparin covalently bonded to an epoxy group based on the component (A) of the gel-like copolymer according to the present invention via a bonding group. This invention relates to an adsorption carrier for graphics.

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. 56, 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. It can be prepared by obtaining a porous gel.

Since the components (A) and (B) are sparingly soluble in water, they can be copolymerized by an aqueous suspension polymerization method, and the aqueous suspension polymerization can be carried out by an oil-in-water suspension method. . 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, the (B) component that acts as a crosslinking component has 2 carbon atoms.
or 3 alkylene glycol or an ester of the polyalkylene glycol and acrylic acid or methacrylic acid is preferably used.

Examples include alkylene glycol divinyl esters such as ethylene glycol diacrylate, ethylene glycol dimethacrylate, propylene glycol diacrylate, propylene glycol dimethacrylate, and polyalkylene glycol divinyl esters such as polyethylene glycol dimethacrylate and polypropylene glycol dimethylate.

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%, (B) component 6
It is 0 to 20 mol%. (A) Less than half of the component is a comonomer, namely methyl methacrylate, methyl acrylate,
Lower vinyl esters such as vinyl acetate can be substituted. Of course, the greater the proportion of component (A), the greater the content of epoxy groups in the copolymer. The higher the proportion of component (B), the higher the degree of crosslinking of the copolymer, and therefore the denser the network structure, the lower the degree of swelling, and the harder the copolymer becomes. If the ratio of both components is outside the above range, a gel-like copolymer that fully satisfies the desired characteristics and properties cannot be obtained.

The organic diluent to be used may be one that is inert to the polymerization reaction, insoluble or sparingly soluble in water, but capable of dissolving the additives in the raw materials. The amount used is at least 30% by volume based on the total amount of monomer components and organic diluent, preferably 40-8% by volume.
0% by volume. The larger the amount used, the more suppressed is the ring opening of the epoxy group during the polymerization process. Generally speaking, use (A
) It is possible to easily make about 60 to 95% of the epoxy groups contained in the charged amount of the component remain in the produced polymer.

It is believed that the remainder is hydrolyzed during the polymerization process and a portion is used for crosslinking.

As mentioned above, the content of epoxy groups in the resulting gel can be adjusted by the amount of component (A) used, i.e. the ratio to component (B), or by partial substitution with a comonomer, and also by the amount of organic diluent. Preferably, the epoxy oxygen is present in the resulting polymer in an amount of 1% by weight or more, expressed as epoxy oxygen, up to about 10% by weight.

As mentioned above, the use of organic diluents during aqueous suspension polymerization also helps control the pore size of the resulting porous gel. When an organic diluent having a low swelling property is used for the resulting gel, phase separation occurs in the suspended particles during the polymerization process, and as a result, a gel with a large pore size, that is, a gel having a so-called giant pore structure can be obtained. On the other hand, if a highly swelling diluent is used, the polymerization will proceed in a swollen state during the polymerization process.
Phase separation is difficult to occur and a polymer with relatively small pores formed by swelling is obtained. The degree of swelling can be determined based on the solubility parameter of the organic diluent. Examples of organic diluents that can be suitably used in the present invention include cyclohexanone, chlorohenzene, benzene, toluene,
n-propyl acetate, n-butyl acetate, n-
Octane etc.

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.

The introduction of the bonding group into the gel-like copolymer according to the present invention includes:
This can be carried out by utilizing the reactivity of the epoxy group based on the component (A) of the gel-like copolymer according to the present invention, but in this case, the binding group has a functional group capable of covalently bonding with heparin. However, 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 heparin include an epoxy group, an amino group, a hydrazino group, and a carboxyl group. , formyl group, thiol group, etc. In this case, the bonding group can be easily introduced using a known method, and it is not necessarily necessary to introduce it in one reaction step;
It can also be introduced separately into more than one reaction step. A specific example of introducing a bonding group will be described below.

(1) Introduction of a bonding group having an epoxy group as a functional group For example, the epoxy group based on the component (A) of the gel copolymer according to the present invention is generated by ring-opening modification with a hydroxyl group-containing compound such as water. A compound having a glycidyl group is bonded to a hydroxyl group to introduce a bonding group having an epoxy group based on a glycidyl group. (Hereinafter, a polymer into which a glycidyl group has been introduced in this way will be referred to as a modified gel copolymer according to the present invention.) Ring-opening modification is performed in the presence of an acid or an alkali,
For example, when carrying out in the presence of an acid, these copolymers are heated at 70°C to 10°C in water using sulfuric acid or hydrochloric acid as a catalyst.
This is done by heating to 0° C. for about 2 hours. The concentration of acid is preferably 0.1N to 0.5N. Ring-opening modification is performed almost quantitatively.

Examples of compounds having a glycidyl group that are bonded to the hydroxyl group generated by ring-opening modification include shrimp halohydrins such as shrimp chlorohydrin and shrimp bromohydrin, ethylene glycol diglycidylethyl,
■, diglycidyl ethers such as 4-butanediol diglycidyl ether and polyethylene glycol diglycidyl ether, 1,3-butadiene dieboxide,
Examples include alkyl diene dieboxides such as 1,7-octazidine dieboxide.

In order to act with a compound having a glycidyl group, for example, a ring-opening modified gel copolymer is reacted with shrimp chlorohydrin in an aqueous solution of an inorganic base such as sodium hydroxide or potassium carbonate, or a hydrophilic copolymer is reacted with shrimp chlorohydrin. Reaction with ethylene glycol or 1,4-butanediol diglycidyl ether, or polyethylene glycol diglycidyl ether in an aqueous solution of an inorganic base such as sulfur hydroxide or potassium carbonate containing sodium borohydride in the polymer, etc. It is done as follows. Repeating unit of polyethylene glycol diglycidyl ether used -C2
It is preferable to use one having a repeating number of H3O- of 1 to 10.

(11) Introduction of a bonding group having an amino group, hydrazino group, carboxyl group, etc. as a functional group For example, as a method for introducing a bonding group having an amino group, hydrazino group, carboxyl group, etc., the gel-like copolymer according to the present invention may be used. The epoxy group based on the component (A) or the epoxy group based on the glycidyl group of the modified gel copolymer according to the present invention is combined with the general formula (I) HzNR' (wherein R' is a hydrogen atom, an amino group, ω - represents an aminoalkyl group or ω-carboxyalkyl group. The epoxy group or glycidyl group is reacted with an acid anhydride to form a compound of the general formula (II) NHR (wherein R is a hydrogen atom, an amino group, an ω-aminoalkyl group, an ω-carboxyalkyl group, an ω-carboxyalkanoyl group). group, ω-carboxyalkanoylamino group or ω
- represents a carboxyalkanoylaminoalkyl group.

) There is a method of introducing a bonding group represented by

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

It represents one epoxy group or glycidyl group in the gel-like copolymer according to the invention or in these copolymers according to the invention. 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).

A compound of formula (D), general formula (D') or general formula (F) in the reaction formula is added to the epoxy group of the gel copolymer according to the present invention or the glycidyl group of the 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. Water is usually used as a solvent, but other solvents include hydrocarbons such as n-hexane, benzene, and xylene, ethers such as tetrahydrofuran and dioxane, chlorinated hydrocarbons such as chloroform and chlorobenzene, and ethanol and methyl cellosolve. In addition to alcohols and ketones such as acetone and methyl isobutyl ketone, dimethylformamide and dimethyl sulfoxide are also used, and these may be used alone or in a mixed solvent system, and there is no need to use a catalyst. ,
Alkali or alkaline earth metal hydroxides or carbonates such as sodium hydroxide and potassium carbonate can also be used.

Examples of the compound of general formula (D') include ethylenediamine,
1,3-diaminopropane, 1,4-diaminobutane,
1.5-diaminopentane, 1,6-diamithexane, 1.7-diaminohbutane, 1.8-diaminooctane, 1.9-diaminononane, 1.10-diaminodecane, 1.12-diaminododecane, etc. diaminoalkanes.

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

The reaction conditions are not necessarily limited, but it is generally preferable to select the following conditions to carry out the reaction.

Weight (a) and (D) of the gel-like copolymer according to the present invention or the modified gel-like copolymer according to the present invention, general formula (D')
Or the ratio of the weight (b) of the compound of general formula CF): a: b= 1: 0.3-10, more preferably a: b= l: 0.3-3, reaction temperature: O-150°C, more Preferably room temperature to 100
°C, reaction time = 1 to 60 hours, more preferably 1 to 30 hours, reaction pressure normal pressure to 10 ats, more preferably normal pressure.

There are no special requirements for post-treatment after the reaction, and conventional methods such as separation and washing may be carried out as appropriate.

The chromatography carrier according to the present invention obtained above can be prepared using an active support group as already shown in the general formula, if necessary, when the compound of formula (D) or general formula (D') is used. The terminal amino group of can be converted into an active supporting group having a carboxyl group by reacting with the 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 it is generally preferable to select the following conditions to carry out the reaction.

Weight (a') of the aminated product of the gel-like copolymer according to the present invention or the modified gel-like copolymer according to the present invention and the general formula (E)
Or the ratio of the weight (b') of the compound of general formula (E'): a':b'=l:o, 3-10, more preferably a':b'=1:0, 3-3 Reaction temperature :0~150℃, more preferably room temperature~100℃
°C, reaction time: 1 to 60 hours, more preferably 1 to 30 hours, reaction pressure normal pressure to 1 Qatm, more preferably normal pressure.

There are no special requirements for post-treatment after the reaction, and conventional methods such as separation and washing may be carried out as appropriate.

Another method for introducing a bonding group having a carboxyl group as a functional group is to introduce an aminocarboxylic acid having a hydroxyl group into an epoxy group based on the component (A) of the gel copolymer according to the present invention or a modified method according to the present invention. There is a method of binding by acting on the glycidyl groups of a gel copolymer.

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, N-tris(hydroxymethyl), and methylglycine, and amino sugar derivatives such as glucosaminic acid.

The bonding state is as a result of the bonding between the epoxy group of the gel copolymer according to the present invention or the glycidyl group of the modified gel copolymer according to the present invention with the amino group of the aminocarboxylic acid containing a hydroxyl group. The glycidyl group opens to form a hydroxyl group and a carboxyl residue having a hydroxyl group.

The action of the aminocarboxylic acid having a hydroxyl group can be carried out in the presence of a solvent.

Water is usually used as a solvent, but other hydrocarbons such as n-hexane, benzene, and xylene, ethers such as tetrahydrofuran and dioxane, chlorinated hydrocarbons such as chloroform and chlorobenzene, ethanol and methyl cellosolve, etc. In addition to alcohols, ketones such as acetone and methyl isobutyl ketone, dimethylformamide 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.

There are no particular restrictions on the reaction conditions, and for example, the same conditions as in the case of reacting the compound of general formula (1) above can be used.

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.

(iii) 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 (i) can be easily hydrolyzed into 1
, 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. .

(iv) Introduction of a bonding group having a thiol group as a functional group For example, the bonding group having an epoxy group as a functional group obtained in (1) above is prepared by treating the functional group with sodium thiosulfate and then adding hydrochloric acid. A thiol group can be introduced by, for example, allowing the reaction to occur.

There are no particular restrictions on the reaction conditions, and the reaction can be easily carried out according to a conventional method, for example, at room temperature in an aqueous solvent.

When covalently bonding heparin to a binding group, it can be carried out in an appropriate solvent using an appropriate catalyst or reaction reagent, if necessary depending on the functional group possessed by the binding group.

For example, as catalysts, acids such as hydrochloric acid, sodium carbonate, or sodium bicarbonate, and alkalis are mainly used when the functional group is an epoxy group, and as reaction reagents, for example, N-hydroxysuccinimide and dicyclohexylcarbodiimide are used. When the functional group is a carboxyl group,
The condensing agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide has a carboxyl functional group,
In the case of an amino group or a hydrazino group, reducing agents such as sodium cyanoborohydride are used when the functional group is a formyl group, and so on. Water is usually used as a solvent, but if necessary, it can also be used as a phosphoric acid or acetate buffer, or an inorganic salt such as sodium chloride can be added thereto.

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

Weight ratio of the gel-like copolymer or modified gel-like copolymer according to the present invention to which a binding group is attached and heparin: 1:0.03~
0.3, preferably 1:0.05-0.2; Reaction temperature:
0°C to room temperature, preferably 4°C to room temperature; reaction time: 1 to 7
2 hours, preferably 2-12 hours.

There are no special requirements for post-treatment after the reaction, and conventional methods such as separation and washing may be carried out as appropriate.

3. The adsorption carrier for chromatography provided by the present invention uses a base having all the properties desired as the above-mentioned adsorption carrier for affinity chromatography, and binds it to the base through a suitable bonding group. Since heparin is commonly bonded, the adsorption carrier of the present invention can not only be used as a packing material for affinity chromatography similar to the conventional adsorption carrier using soft gel, but also particularly suitable for pressure-resistant columns. The biggest advantage is that it can be filled with water and used under pressure, which greatly reduces working time and therefore greatly improves the efficiency of separation and purification. Needless to say, this is indispensable for applications such as high performance liquid chromatography and industrial separation and purification equipment.

In particular, while the gel-like copolymer has many advantages as described above, the adsorption carrier to which heparin is covalently bonded via the bonding group as described above, for example, can absorb the commonly used cyanogen bromide. Compared to adsorption carriers for affinity chromatography, etc., the ligand does not fall off during use, and the length of the bonding group can be adjusted arbitrarily, so the adsorption ability of the ligand for the target substance is excellent. In addition, the adsorption carrier for chromatography provided by the present invention has great effects, such as low specific adsorption.

Big Arashi ■ Below, typical examples of the method for producing the adsorption carrier for chromatography of the present invention from known substances will be described in more detail. However, these are merely examples for explanation, and it goes without saying that the present invention is not limited thereto.

Example 1 A copolymer obtained from glycidyl methacrylate and ethylene glycol dimethacrylate was subjected to ring-opening modification of the epoxy groups with water, further introducing glycidyl groups with ethylene glycol diglycidyl ether, and then reacting with ammonia to obtain ( Amino group: 0.16 mmol per 1 g of dry gel) Add 0.6 g of gel to 30 μg of heparin sodium.
．． 2M dipotassium hydrogen phosphate water soluble? ff11.3mx
Then about 10 μm of sodium cyanoborohydride was added, heated to 60° C., and reacted for 15 hours with shaking. The gel was then collected, washed with a 1 molar aqueous sodium chloride solution and water, and then treated with 0.2 molar acetic acid (l-).
In addition to 1 mil of IJum aqueous solution, add 0.5 acetic anhydride.
After adding mj2 and shaking for 30 minutes at room temperature, the gel was renewed and added with water and 0. Washed with IN aqueous sodium hydroxide solution and again with water. It was confirmed by elemental sulfur analysis that the gel thus obtained supported about 8 mg of heparin per gram of dry gel.

Example 2 An epoxy group-containing gel copolymer obtained from glycidyl methacrylate and ethylene glycol dimethacrylate was ring-opened and modified with water, and further glycidyl groups were introduced with 1,4-butanediol diglycidyl ether. , and then the gel obtained by reacting with 4-aminobutyric acid 1.
After thoroughly washing 0 g with anhydrous dioxane, it was added to 4 ml of anhydrous dioxane, and then 8°■ of N-hydroxysuccinimide and 144°■ of dicyclohexylcarbodiimide were added.
After shaking at room temperature for 2 hours, the gel was renewed and added with 20 mβ of anhydrous dioxane, 6 ml of methanol, and 3 m of cold water.
Wash quickly in this order. This gel was mixed with 100 g of heparin sodium in a 0.1 molar aqueous solution of sodium bicarbonate 2
After adding to the solution in m12 and shaking at room temperature for 2 hours, it was left at 4°C overnight. After the gel was renewed and washed with 1 molar aqueous sodium chloride solution and water, it was washed with 1 molar Tris-HCl buffer (p
After adding 2 ml of H8,0) and shaking at room temperature for 1 hour, the gel was renewed and washed with water. It was confirmed that the gel thus obtained supported about 25 mg of heparin per gram of dry gel.

Test Example The adsorption carrier obtained in Example 1 had a diameter of 4.6 mm and a length of 75 mm.
When a thrombin adsorption test was performed using a high-speed chromatography device packed in a stainless steel column (No. 1),
0.0 with 0.15M sodium chloride as eluent
When 1M Tris-HCl buffer (pH 7.5) was used, the adsorption was good, and when the eluent was changed to 0.01M Tris-HCl buffer (pH 7.5) containing 1.25M sodium chloride, it eluted immediately.

Claims (1)

[Claims] Epoxy based on component (A) of a gel-like copolymer containing (A) glycidyl monovinyl ester or glycidyl monovinyl ether and (B) alkylene glycol divinyl ester as main components, and component (A) crosslinked with component (B). 1. An adsorption carrier for chromatography comprising a porous copolymer containing heparin covalently bonded to a group via a bonding group.