JPS6348452A - Adsorption carrier for chromatography - Google Patents

Abstract

PURPOSE:To obtain an adsorption carrier for chromatography which is hard and has good flowability and sepn. characteristic by using crosslinked or non- crosslinked chitosane porous beads as a base body and covalent-bonding lectin to such base body. CONSTITUTION:The chitin contained in the carapace of Crastaces and Insecta is subjected to a deacetylation treatment of obtain the chitosane porous beads consisting of polyglucosamine. The chitosane porous beads are otherwise subjected to a crosslinking treatment by using a dicarboxylic acid, dialdehyde, etc., to obtain the acid resistant chitosane porous beads. An alkane dicarboxylic anhydride is acted on the amino group contained in these porous beads to convert the same to an active supporting group having a carboxyl group. After the remaining amino group is acylated, the lectin is covalent-bonded to the carboxyl group.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an adsorption carrier for chromatography that can be used in affinity chromatography.

Affinity chromatography, a type of chromatography technology, uses the affinity of pairs of substances that specifically interact with each other to separate and purify biological substances. It is useful for identifying and purifying specific properties, that is, specific chemical structures on molecules.

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, lectin is a type of sugar-binding protein that has the property of specifically binding to sugars with a certain structure, but in recent years, glycoproteins or glycolipids on the cell surface have been used to recognize cells' self-other and cells. It is becoming clear that lectins play an important role in intercellular communication, and adsorption carriers with lectins as ligands are being actively used as one of the tools for these studies.

For example, in separation, purification, and analysis by affinity stomatography in which lectin is used as a ligand and sugar is used as 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, chemically stable and sufficient under a wide range of pH ranges, salt concentrations, and temperatures. Examples include stability and no volume change, sufficient mechanical strength and stability, good flow properties, and resistance to biological contamination.

Cellulose, dextran, polyacrylamide, agarose, etc., which have been conventionally used as base materials for adsorption carriers for affinity chromatography, do not necessarily have these desired properties, and in particular, are so-called soft gels lacking in hardness. Therefore, it has serious defects 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. was.

The object of the present invention is to overcome the drawbacks of the conventional adsorption carriers for chromatography and to develop a chromatography system that fully possesses 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 use in graphics.

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

That is, the present invention provides chitosan porous beads in which amino groups based on glucosamine constituting the chitosan or chitosan porous beads are crosslinked,
An adsorption for chromatography characterized by comprising porous chitosan beads containing a lectin covalently bonded via a bonding group to an amino group based on glucosamine and/or an amino group based on a crosslinking agent constituting the chitosan. Regarding the carrier.

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

The chitosan porous beads according to the present invention are made of polyglucosamine obtained by deacetylating chitin contained in the carapace of crustaceans and insects, and are made of polyglucosamine, which is obtained by deacetylating chitin contained in the carapace of crustaceans and insects. Products that are cross-linked using reagents such as dicarboxylic acids and their derivatives, dialdehydes, diisocyanates, etc. are also manufactured. Available as sex beads. These porous chitosan beads generally have amino groups based on glucosamine or amino groups based on crosslinking treatment agents, with a molar equivalent of several micrograms to several tens of grams. These amino groups can be converted into active supporting groups having carboxyl groups by the action of alkanedicarboxylic anhydrides.

Water is usually used as a solvent for the reaction, but other solvents such as ethers such as tetrahydrofuran and dioxane, carboxylic acids such as acetic acid, and pyridine are also used. Also,
In particular, a catalyst may not be used, but the pt+ of the reaction solution can also be adjusted by adding an acid such as hydrochloric acid or sulfuric acid, or an alkali such as sodium hydroxide or potassium carbonate.

Examples of the alkanedicarboxylic anhydride used in the reaction include succinic anhydride, glutaric anhydride, adipic anhydride, pimelic anhydride, speric acid, anhydride, azelaic anhydride, sebacic anhydride, 1. Examples include 10-decanedicarboxylic anhydride, 1,12-dodecanedicarboxylic anhydride, and 1,14-tetradecanedicarboxylic anhydride.

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

Ratio between the weight of the chitosan porous beads (a) and the weight of the alkanedicarboxylic acid anhydride Cb); a:b=1:0.05~IO1 More preferably, a:b-1:0.1~3, Reaction temperature: 0 to 150°C, more preferably room temperature to 100°C
C. Reaction time: 1 to 60 hours, more preferably 1 to 30 hours.

The reaction pressure is normal pressure to lQatm, more preferably normal pressure.

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

The chitosan porous beads having carboxyl groups obtained above can then be treated with a monocarboxylic anhydride or an acyl halide to acylate the remaining amino groups.

The reaction conditions at this time can be the same as those for the alkane dicarboxylic anhydride described above, but when using an acyl halide as the acylating agent, it is better to use a solvent that is not near water. As a catalyst, an alkali is preferred.

Examples of the monocarboxylic anhydride used in the reaction include acetic anhydride, propionic anhydride, butyric anhydride, and examples of the acyl halide include acetyl chloride, acetyl bromide, propionyl chloride, butyryl chloride, etc. be able to.

Covalently bonding a lectin to a carboxyl group can be carried out in an appropriate solvent using an appropriate condensing agent or reaction reagent. Examples of the condensing agent and reaction reagent include N-hydroxysuccinimide, dicyclohexylcarbodiimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Water is usually used as a solvent, but it can also be used as a phosphoric acid or acetate buffer if necessary, or an inorganic salt such as sodium chloride can be added thereto.

There are no particular restrictions on the type of lectin that can be bound, but examples include concanavalin A, red bean lectin,
Plant lectins such as wheat germ lectin, castor bean lectin, soybean lectin, gorse lectin, kidney bean lectin, lotus lectin, vampiria lectin, lentil lectin, horse bean lectin, peanut lectin, horseshoe crab lectin, eel lectin, snail lectin, etc. Examples include animal lectins.

Although there are no particular restrictions on the conditions for the reaction with lectin, it is generally appropriate to carry out the reaction within the following range.

Weight ratio of chitosan porous beads according to the present invention to which carboxyl groups are bonded and retyl: 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 2 hours, preferably 2 to 12 hours.

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

Kyoto and KukuriThe adsorption carrier for chromatography provided by the present invention uses a base having the properties desired as the above-mentioned adsorption carrier for affinity chromatography, and a lectin is covalently attached to this base through 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 similar to the conventional adsorption carrier using foot gel, but also especially when packed in a pressure-resistant column. The fact that it can be used sufficiently even under pressure has the greatest advantage of greatly shortening the working time and therefore greatly improving the efficiency of separation and purification. Needless to say, it is essential for applications in chromatography and industrial separation and purification equipment.

In addition, the adsorption carrier for chromatography provided by the present invention completely acylates the amino groups of chitosan porous beads, thereby eliminating ionic nonspecific adsorption when coming into contact with proteins, etc. Therefore, the selectivity between the ligand and the target substance to be adsorbed can be further improved.

Furthermore, in particular, adsorption carriers in which lectins are bonded to chitosan porous beads through chemically neutral and stable covalent bonds can be used, for example, with affinity mediated by cyanogen bromide or glutaraldehyde, which are commonly used. Compared to adsorption carriers for chromatography, etc., the adhesive does not fall off during use, and the length of the bonding group can be adjusted arbitrarily, so it has excellent adsorption ability for the target substance of the ligand. Furthermore, the effect of the adsorption carrier for chromatography provided by the present invention is great in that it has less non-specific 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.

Example 1 Chitosan porous beads with a particle size of 0.1 tm (trade name Shodex Skillepearl, aromatic crosslinked product) were reacted with glutaric anhydride, and the remaining amino groups were acetylated to obtain beads (carboxyl groups). : 0.35 mmol per 1 g of dry gel) was thoroughly washed with anhydrous dioxane, then added to 4 ml of anhydrous dioxane, further added with 80 mg of N-hydroxysuccinimide and 144 s+g of dicyclohexylcarbodiimide, and shaken at room temperature for 2 hours. 0 then enka the beads and anhydrous dioxane 20 mj! , methanol 6
(2) Washed quickly in the following order: 11 with 3 IllV of cold water. The beads were mixed with 30 mg of concanavalin A and 0.1 mmol of calcium chloride, 0.1 mmol of methyl-α-mannopyranoside 40B.
, 1 mmol manganese chloride and 0.01 mol sodium bicarbonate in 2 yal of water, shaken at room temperature for 2 hours, and then left overnight at 4°C. After washing the beads with a 1M aqueous sodium chloride solution and water, they were added to 2Ill of a 1M Tris-HCl buffer (pH 8,0) at room temperature.
After shaking for an hour, the beads were washed with water again. It was confirmed from the amount of unreacted recovered concanavalin A that the adsorption carrier thus obtained supported 15 mg of concanavalin A per Ig of dry beads.

Example 2 In place of the concanavalin A30B solution in Example 1, 5.0 mg of wheat germ lectin and N-acetyl-
Using a solution of 0.05 molar potassium phosphate, 0.15 molar sodium chloride and 0.01 molar sodium bicarbonate containing 10 mg of α-glucosamine in 3 ml of water,
Otherwise, by performing the same operation, an adsorption carrier supporting 3.2 mg of wheat germ lectin per gram of dry beads could be obtained.

Test Example 1 The adsorption carrier obtained in Example 1 was packed into an 8φ x 50M stainless steel column, and p-nitrophenyl-α-D-galactopyranoside (compound 1), p-nitrophenyl-α-D-galactopyranoside (compound 1), When p-nitrophenyl-α-D-mannopyranoside (compound 2) was analyzed, good results were obtained as shown in the following table.

Eluent: 0.02 mol) IJS hydrochloric acid buffer, 0.02 mol)
5M sodium chloride, 0.5mM calcium chloride, 0.5mM manganese chloride (pH 7,4) Elution rate 1. Oml/min Detector Ultraviolet Spectrophotometer 260 nm Test Example 2 Ovalbumin was analyzed in the same manner as Test Example 1 using an adsorbent containing a non-acylated amino group.

Eluent ■: 0.01M sodium phosphate buffer pH 7
,0 ■:■+0.05 mol α-methylglucoside t? ! Departing speed: Omj2/min, detector: UV spectrophotometer: 280nr @ When using eluent ■, most of the ovalbumin is adsorbed
When the eluent was switched to ■, some of the adsorbed material began to elute, but a considerable portion still remained adsorbed (Figure 3).

Next, when the same ovalbumin was analyzed using the adsorbent obtained in Example 1, most of the ovalbumin adsorbed with eluent ■ was eluted with eluent ■ (Figure 4).
. When the same amount of ovalbumin was analyzed, there was a clear difference in the elution amount between FIGS. 3 and 4, which was thought to be due to the influence of non-arylated amino groups.

[Brief explanation of the drawing]

FIG. 1 and FIG. 2 both show concanavalin A according to the present invention.
p-nitrophenyl-α-D- using an adsorbent, respectively.
Galactopyranoside and p-nitoverophenyl α-D-? The elution of 'Nnobilacinod was investigated. Figures 3 and 4 are diagrams showing the patterns of adsorption and elution of ovalbumin when using the adsorbent containing non-acylated amino groups and the adsorbent of the present invention in Test Example 2. be. Figure 1 Figure 2 Figure 3 Figure 4

Claims (1)

[Claims] Porous chitosan beads, in which amino groups based on glucosamine constituting the chitosan are crosslinked, or porous chitosan beads are crosslinked, and amino groups based on glucosamine constituting the chitosan and/or a crosslinking treatment agent are used. An adsorption carrier for chromatography comprising porous chitosan beads containing a lectin covalently bonded to an amino group via a bonding group.