JPH06201671A - Adsorbent for chromatography - Google Patents

Abstract

PURPOSE:To promote the dispersion and the purification of a saccharide effectively by using a specific adsorbent for chromatography, in which a lectin has been bonded as a ligand. CONSTITUTION:An adsorbent uses a specific support (a cross-linked copolymer) out of all porous active carriers, and a lectin as a ligand is covalent-bonded to an alcoholic hydroxyl group for the support. That is to say, it is an adsorbent for chromatography, which is used to separate and purify a saccharide, and it is composed of a porous cross-linked copolymer containing 0.005 to 200mumol/g of a lectin covalent-bonded to a hydroxyl group via a bonding group of a cross- linked copolymer which is provided with 1.2 to 12.0mmol/g of an alcoholic hydroxyl group, with 0.5 to 3.0g/g of water and with 10 to 800m<2>/g of a specific surface area per adsorbent. The adsorbent satisfies the necessary performance such as a pH region, a temperature region, a mechanical strength, the capacity of capable of fixing the ligand and the like in a wide range.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an adsorption carrier for affinity chromatography such as high performance liquid affinity chromatography used for separation and purification of saccharides,
The present invention relates to an adsorption carrier using a lectin as a ligand.

[0002]

2. Description of the Related Art Affinity chromatography, which is a type of chromatography, is an affinity adsorption carrier having a biological affinity that is difficult to reach by the physical or chemical affinity of general chemical catalysts used in vivo. The biological substance can be separated and purified even in the presence of various compounds. In addition, high performance liquid affinity chromatography is one in which the above adsorption carrier is used as a packing material for high performance liquid chromatography.

The above-mentioned adsorption carrier for affinity chromatography, for example, binds / immobilizes a ligand having a biological affinity to an insoluble support through a binding group (spacer) without losing the properties of the ligand. It has been converted into a compound and has a property that only a specific compound can be selectively taken out.

For example, among the diseases that occur in the human body, DN
Some are caused by a defect in A, and some of the biomaterials of such patients are not found in healthy persons. As an example, there are innumerable sugar chains on the cell surface, and many proteins in the body exist as glycoproteins, but cancer patients have sugar chains that are not found in healthy people, and immune globin in chronic rheumatism patients. Is known to be very different from that of a healthy person.

As a method for clinical diagnosis of such patients,
The number of methods using high performance liquid affinity chromatography in which a lectin is immobilized as a ligand of an adsorption carrier (filler) has been increasing in recent years. Lectin is a generic name for a group of proteins known to have specific interactions with sugar chains, and high-performance liquid affinity chromatography using lectins as a diagnostic device as described above is extremely effective. It seems that it is. At this time,
The desired performance of the support, which is the main component of the packing material for chromatography, is that it is stable in a wide range of pH and temperature ranges, that it has high mechanical strength despite its sufficiently small particle size, and that it binds ligands. Is easy and has a large immobilizable capacity, and can withstand biological contamination.

[0006]

Conventionally, cellulose, agarose, etc. have been used as a support which is said to satisfy such performances. However, since these supports are particularly insufficient in mechanical strength, when the column is filled with the adsorption carrier by these supports, the mobile phase cannot be flowed at a high speed, and the separation takes a long time. However, there are drawbacks such as short life. In addition, the silica beads used recently have sufficient mechanical strength, but cannot be used under alkaline conditions.
Since the number of theoretical plates, which is a guideline for column performance, is low, there was a problem in that lectin or the like was selected as a ligand and separation / purification was severely limited.

The present invention overcomes the drawbacks of the above-mentioned conventional supports, and sufficiently provides the performance desired for a support for affinity chromatography used for separation and purification of sugars, and It is an object of the present invention to provide an adsorption carrier for chromatography, which can favorably separate and purify these sugars when a lectin is used as a ligand.

[0008]

[Means for Achieving the Object] As a result of repeated studies to achieve the above object, the present inventor has found that (1) JP-A-60-
A specific one of the all-porous activated carriers disclosed in Japanese Patent No. 257836 is effective as a support for lectin, and (2) the one in which lectin is immobilized on this specific carrier. The present invention has been developed based on the finding that the separation / purification of saccharides, particularly monosaccharides, can proceed extremely effectively by affinity chromatography using an adsorption carrier.

That is, the adsorption carrier of the present invention is an adsorption carrier for chromatography used for the separation and purification of sugars, and the alcoholic hydroxyl group is 1.2 to 1.2 per adsorption carrier.
12.0 mmol / g, water content 0.5-3.0 g / g,
A cross-linked copolymer having a specific surface area of 10 to 800 m ² / g, comprising a porous cross-linked copolymer containing 0.005 to 200 μmol / g of lectin covalently bonded to the hydroxyl group via a bonding group. Characterize.

The adsorbent carrier of the present invention uses a specific one of the above-mentioned all-porous activated carriers (cross-linked copolymer) as a support, and a lectin as a ligand is bound to the alcoholic hydroxyl group of this support. It is a covalent bond through a group.

In the adsorption carrier of the present invention having such a constitution, the alcoholic hydroxyl group is 1.2 to 12.0 m per adsorption carrier.
By including it in the range of mol / g, it has hydrophilicity and does not show hydrophobic adsorption and partitioning of most water-soluble substances in water when used as a filler. When the alcoholic hydroxyl group is less than 1.2 mmol / g, the adsorption selectivity of the substance is lowered, and when it is more than 12.0 mmol / g,
The adsorption selectivity of the substance decreases, and the mechanical strength also decreases. The amount of hydroxyl groups is more preferably 2.0-10.
Those in the range of 0 mmol / g are suitable. In addition,
The amount of the hydroxyl group can be determined by reacting the adsorption carrier with acetic anhydride in a pyridine solvent, and by consuming the acetic anhydride consumed by reacting with the hydroxyl group or by changing the weight of the adsorption carrier.
At this time, the amount of hydroxyl groups when 1 g of the adsorption carrier (dry weight) reacts with 1 mol of acetic anhydride is 1 mol / g.

The above-mentioned conventional adsorption carrier having cellulose or agarose as a support has a large amount of water retention and a small mechanical strength. This tendency was more remarkable as the pore size increased, but in the adsorption carrier of the present invention, sufficient adsorption performance can be obtained regardless of the pore size by setting the water content in the range of 0.5 to 3.0 g / g. It can have mechanical strength. If the water retention amount is less than 0.5 g / g, the adsorption performance will decrease, and if it exceeds 3.0 g / g, the mechanical strength will decrease. The water retention amount of the adsorption carrier is obtained by sufficiently swelling the adsorption carrier in water, centrifuging the adhering water on the surface with a centrifuge tube (sedimentation tube for centrifuge), and then drying, and determining the weight change before and after drying.

The specific surface area of the adsorption carrier is 10 to 800 m ^2.
/ G range. If it is less than 10 m ² / g, the adsorption performance is lowered, and if it exceeds 800 m ² / g, the mechanical strength is lowered. The specific surface area is measured by the BET method using nitrogen gas after the adsorption carrier is sufficiently dried.

The average particle size and shape of the adsorption carrier of the present invention are not particularly limited, but when used as a packing material for high performance liquid affinity chromatography, the average particle size is 3 to 30 μm, preferably 6 to 18 μm, The shape is preferably spherical. The average particle size can be measured by a commercially available particle size measuring device.

The lectin immobilized on the adsorption carrier of the present invention by a covalent bond via a binding group (spacer) has a content in the range of 0.005 to 200 μmol / g. If it is less than 0.005 μmol / g, it is difficult to exert an affinity, and if it exceeds 200 μmol / g, the adsorption ability is reduced due to steric hindrance between lectins.

The adsorption carrier of the present invention comprises a cross-linked copolymer, and the cross-linked structure is not particularly limited. Further, the ratio of the crosslinkable monomer unit to all the monomer units forming the adsorption carrier is preferably in the range represented by the formula (1).

[0017]

## EQU1 ## 0.25 ≦ na / (b + na) ≦ 0.4

In the formula (1), a is the mole fraction of the crosslinkable monomer unit, b is the mole fraction of the monomer unit other than the crosslinkable monomer in the adsorption carrier, and n is the crosslinkable unit amount. It represents the number of polymerizable functional groups contained in one molecule of the body.

Next, an example of a method for producing the adsorption carrier of the present invention having the above constitution will be described. For example, the ester group of a copolymer composed of a carboxylic acid vinyl ester and a crosslinkable monomer having a triazine ring is subjected to a saponification reaction or a transesterification reaction to convert about 15 to 75% of the ester group into a hydroxyl group. . A lectin such as concanavalin A may be covalently bonded to this hydroxyl group via a spacer molecule such as epichlorohydrin.

Here, the carboxylic acid vinyl ester is
It refers to a compound having a polymerizable carboxylic acid vinyl ester group, and in the present invention, one or more lower carboxylic acid vinyl ester having 4 to 7 carbon atoms can be used. Among these, those having a small number of carbon atoms are particularly preferably used, and examples thereof include vinyl acetate and vinyl propionate. On the other hand, as the crosslinkable monomer having a triazine ring, triallyl isocyanurate, triallyl cyanurate, or the like can be used.

The polymerization reaction for obtaining a copolymer from these monomers can be carried out by a usual polymerization method, but it is preferable to use suspension polymerization. The organic solvent used at this time is inert to the polymerization reaction, is a solvent insoluble or hardly soluble in water, as long as it can dissolve the monomer,
Specific examples include toluene, n-butyl acetate, butanol and the like. The amount used is preferably 20 to 70 wt% of the total amount of the monomer component and the organic solvent, and more preferably 25 to 50 wt%. The organic solvent also helps control the pore size of the crosslinked copolymer to be produced, and if an organic solvent having a small swelling property is used for the produced crosslinked copolymer, a crosslinked copolymer having a large pore size can be obtained and the swelling will be increased. When an organic solvent having a high property is used, a cross-linked copolymer having a small pore size can be obtained.

The polymerization initiator used in the above polymerization reaction is not particularly limited and may be any one, for example, a peroxide such as benzoyl peroxide, or 2,
Examples thereof include azo compounds having no coupling property such as 2'-azobisisobutyronitrile.

The saponification reaction and transesterification reaction for converting the ester group of the crosslinked copolymer obtained by the polymerization reaction into the hydroxyl group can be carried out by a usual method. For example, a solution obtained by dissolving the crosslinked copolymer in water, alcohol, a mixed solution of water and alcohol, or the like may be mixed with an acid or an alkali. The reaction rate at this time can be controlled by the reaction solvent, reaction time, temperature, etc. By selecting these conditions, the reaction rate is about 15 to 75% (that is, the ester group of the cross-linked copolymer). Is converted to hydroxyl group).

In order to covalently bond the ligand to the crosslinked copolymer via a spacer molecule, the hydroxyl group converted by the saponification reaction or transesterification reaction is reacted with the spacer molecule to covalently bond the hydroxyl group to the spacer molecule. The ligand may be immobilized, or the spacer molecule and the ligand may be reacted first, and then the spacer molecule and the hydroxyl group may be covalently bound and immobilized.

As the spacer molecule, those commonly used can be used, and examples thereof include epichlorohydrin, epoxysilane, and 1,4-butanediol diglycidyl ether. The length of the spacer molecule is not particularly limited, but it is equivalent to a methylene group and is C2 to C1.
It is preferably 0, and a spacer molecule having an appropriate length may be selected from this range depending on the kind of ligand to be immobilized.

Further, depending on the spacer molecule selected, the functional group is changed according to a conventional method before the immobilization reaction with the lectin. For example, when epichlorohydrin is used as the spacer molecule, epichlorohydrin and the cross-linking copolymer are immobilized in an alkaline aqueous solution such as sodium hydroxide or calcium hydroxide to be immobilized, and then the epoxy group of the immobilized epichlorohydrin is converted to a diol. Groups and aldehyde groups are converted to react these functional groups with lectins.

The reaction method with lectin is not particularly limited, and it can be immobilized by a conventional method. The conditions at this time are not particularly limited, either, depending on the type of lectin or spacer molecule to be immobilized, temperature, time,
The ratio between the lectin and the cross-linked copolymer can be arbitrarily selected.

The type of lectin is also not particularly limited, and examples thereof include plant lectins such as concanavalin A, chickpea lectin, soybean lectin and pea lectin, and animal lectins such as horseshoe crab lectin and snail lectin.

The adsorption carrier for chromatography of the present invention prepared as described above is used for separating and purifying monosaccharides such as mannose, glucose, galactose, fructose and rhamnose, and polysaccharides such as maltose, saccharose and lactose. However, since it can greatly improve the degree of separation, it is preferably used for the separation and purification of monosaccharides.

[0030]

【Example】

[Production Example of Adsorption Carrier] Example 1 100 g of vinyl acetate and triallyl isocyanurate 4
7.5 g, heptane 20 g, decalin 60 g, acetic acid n-
A homogeneous mixture of 40 g of butyl and 3.5 g of 2,2'-azobisisobutyronitrile as a polymerization initiator was dissolved in water 1 in which 1.5 wt% of polyvinyl alcohol was dissolved.
000 milliliters (hereinafter, the liter is referred to as "L",
Suspension polymerization was carried out by mixing milliliters with "mL") and holding at 60 ° C for 20 hours and then at 75 ° C for 4 hours with vigorous stirring. The obtained copolymer was filtered, washed with water, extracted with acetone, and then dissolved in methanol (60 g), methanol 20.
The saponification reaction was performed by mixing with 00 mL and maintaining at 20 ° C. for 24 hours while stirring. The obtained granular copolymer was filtered, washed with water, and dried.

2.5 g of the above-mentioned granular copolymer and 10 ml of epichlorohydrin, 6 m of 10N sodium hydroxide solution
L and 6 mL of water were mixed, and the mixture was stirred under ultrasonic irradiation for 3 minutes, and then stirred at 50 ° C. for 2 hours to bind epichlorohydrin, filtered, and washed with water. The obtained compound is mixed with 50 mL of 0.01N hydrochloric acid solution, and the mixture is heated to 60 ° C
After stirring for 1 hour, the epoxy group is ring-opened to give a diol, which is filtered and washed with water.
mL, 2 g of sodium metaperiodate are mixed, and the temperature is 25 ° C.
Stirring for 2 hours, filtering, washing with water, washing with acetone,
The diol was aldehyde based.

Next, 5 containing sodium 0.25M, manganese sulfate 0.5mM and calcium sulfate 0.5mM
1 L of 0 mM Tris-sulfate buffer (pH 7.6) was prepared (this is referred to as solution A), and 5 mL of this solution was mixed with an active carrier having an aldehyde group as an active group in the above reaction, and the mixture was irradiated with ultrasonic waves. Stir for 5 minutes. This solution and a solution of sodium cyanotrihydroborate in 1 mL of solution A,
3 mL of solution A containing 150 mg of concanavalin A was mixed, stirred at 4 ° C. for 48 hours, adjusted to pH 7 with 1M sulfuric acid solution, added with sodium borohydride 50 mg, and further heated at 4 ° C. Stir for 2 hours,
Concanavalin A was immobilized, filtered, washed with water, and dried.

The adsorbent carrier of the present invention obtained as described above has an average particle size of 9.0 μm, a hydroxyl group density of 5 mmol / g, a water retention amount of 2.0 g / g and a specific surface area per adsorbent carrier. It was 38 m ² / g. In addition, it was confirmed from the amount of unreacted recovered concanavalin A that 25 mg / g of concanavalin A was immobilized.

Comparative Example 1 Vinyl acetate 100 g, triallyl isocyanurate 10
g, 120 g of heptane, 120 g of ethyl acetate, and 3.5 g of 2,2'-azobisisobutyronitrile as a polymerization initiator were mixed in the same manner as in Example 1 by suspension polymerization and saponification reaction. Then, the spacer and concanavalin A were immobilized in the same manner as in Example 1.

The comparative adsorption carrier obtained as described above has an average particle size of 80 μm, a hydroxyl group density of 10 mmol / g, a water retention amount of 4.0 g / g, and a specific surface area of 5 m ² / g per adsorption carrier. It was g. In addition, it was confirmed from the amount of unreacted recovered concanavalin A that 8 mg / g of concanavalin A was immobilized.

[Separation of mannose or galactose
Purification Example] Example 1 The adsorption carrier of the present invention and the comparative adsorption carrier obtained in Example 1 and Comparative Example 1, respectively, had an inner diameter of 4.6 mm and a length of 1
It was packed in a 50 mm stainless steel column, and UDP-mannose and UDP were used at room temperature and a flow rate of 0.5 mL / min, using 50 mM Tris sulfate buffer (pH 7.0) as a mobile phase.
-Galactose and 25 μg each were injected. SPD-6AV (Shimadzu Corporation) was used as a detector, and UDP-mannose and U were used at a detection wavelength of 261 nm.
The number of theoretical plates for DP-galactose was measured respectively. The results are shown in Table 1. In addition, the results obtained by using the columns packed with the respective adsorption carriers described above are shown in FIG.
And shown in FIG. FIG. 1 shows the results obtained by the column packed with the adsorption carrier of the present invention obtained in Example 1, and FIG. 2 shows the results obtained by the column packed with the comparative adsorption carrier obtained in Comparative Example 1.

From Table 1, as a result of comparing the adsorption carrier of the present invention with the adsorption carrier of the comparison, a difference of 2 to 3 times in theoretical plate number was observed. Considering this together with FIGS. It is clear that the column performance is excellent with the adsorbent carrier of.

[0038]

[Table 1]

[0039]

As described in detail above, the adsorption carrier of the present invention is used as a packing material for high performance liquid affinity chromatography, for example, UDP-mannose or UDP.
-When separating and purifying galactose, the theoretical fraction is extremely high under the same conditions as compared with the conventional adsorption carrier, and the degree of separation can be significantly improved, and more accurate separation and purification of the target compound is performed. be able to. Moreover, according to the adsorbent of the present invention, it is stable in a wide range of pH and temperature regions required for this type of adsorbent,
It is possible to sufficiently satisfy all that the mechanical strength is high even though the particle size is sufficiently small, the binding of the ligand is easy and the immobilization capacity is large, and it can withstand biological contamination.

[Brief description of drawings]

FIG. 1 shows the results of a column packed with the adsorption carrier of the present invention.

FIG. 2 shows the results of a column packed with a comparative adsorption carrier.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoichi Ono 1134-2, Gongendo, Satte City, Saitama Cosmo Research Institute Co., Ltd.

Claims (1)

[Claims] 1. An adsorption carrier for chromatography used for the separation or purification of sugars, wherein the alcoholic hydroxyl group is 1.2 to 12.0 per adsorption carrier.
0.005 to 200 μmol of a lectin covalently bonded to the hydroxyl group of the crosslinked copolymer having a mmol / g, a water content of 0.5 to 3.0 g / g and a specific surface area of 10 to 800 m ² / g. An adsorption carrier for chromatography, which is composed of a porous cross-linked copolymer containing / g.