JPS6348453A - Carrier for chromatography and its production - Google Patents

Abstract

PURPOSE:To improve flowability and sepn. characteristic by using crosslinked or uncrosslinked chitosane porous beads as a base body, bonding an omega-carboxyl alkanoyl group to the amino group of the base body and acylating the remaining amino group. CONSTITUTION:The chitin contained in the carapace of Crastacea and Insecta is subjected to a deacetylation treatment to 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. A monocarboxylic anhydride or halogenated acyl is acted on the remaining amino group to acylate the same.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a chromatography carrier that can be used in various chromatography including affinity chromatography, and a method for producing the chromatography carrier.

As a type of conventional chromatography technology, affinity chromatography is a method of separating and purifying biological materials by utilizing the affinity of pairs of substances that specifically interact with each other. It is useful for identifying and purifying specific properties, that is, specific chemical structures on molecules.

7. An adsorption carrier (affinity group) for affinity chromatography is, for example, one in which a ligand is bound to the spacer of an active support obtained by binding a spacer to an insoluble carrier (matrix). An adsorption operation is performed by selecting a combination of substances that interact with this ligand.

Examples of the combination of the ligand and the adsorption target substance include the following combinations.

Enzymes - substrates, products, inhibitors, coenzymes.

effector, antigen-antibody, receptor agonist, nucleobase pairs, Lectin - sugar (protein), metal chelate-protein, hydrophobic group - protein, Host - Dust.

In separation/purification and analysis by affinity chromatography, the desired properties of the active support for affinity chromatography include low nonspecific adsorption, high porosity, easy binding of ligands, and immobilization. It is chemically stable and stable enough under a wide range of vocal ranges, salt concentrations, and temperatures, has no volume change, has sufficient mechanical strength and stability, and has good flow characteristics. Examples include resistance to biological contamination.

Cellulose, dextran, polyacrylamide, agarose, etc., which have been conventionally used as base materials for adsorption carriers for cyaffinity chromatography, do not necessarily have these desired properties. In particular, since it is a so-called soft dull with insufficient 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.

Problems to be Solved by the Invention It is an object of the present invention to overcome the drawbacks of the conventional chromatography supports and to provide a chromatography support that has all of the above-mentioned properties desired as an active support for affinity chromatography. An object of the present invention is to provide a carrier and a method for producing the same.

Means for Solving the Problems The present invention provides a chromatography carrier and a method for producing the same 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,
A carrier for chromatography characterized in that an ω-carboxyalkanoyl group is bonded to an amino group based on glucosamine and/or an amino group based on a crosslinking agent constituting the chitosan, and the remaining amino groups are acylated. .

The present invention also provides chitosan porous beads, in which amino groups based on glucosamine constituting the chitosan or chitosan porous beads are cross-linked,
The amino groups based on glucosamine and/or the amino groups based on the crosslinking treatment agent constituting the chitosan are treated with alkafudic acid anhydride, and then the remaining amino groups are treated with monocarboxylic acid anhydride or acyl halide. The present invention relates to a method for producing a chromatography carrier characterized by:

Hereinafter, the chromatography carrier of the present invention and its manufacturing method will be explained.

The chitosan porous beads according to the present invention are composed of polyglucosamine obtained by deacetylating chitin contained in the carapace of crustaceans and insects, and are bifunctional in order to have acid resistance. Products that are crosslinked using a chemical reagent such as dicarboxylic acid and its derivatives, dialdehyde, diisocyanate, etc. are also manufactured, and have a particle size of 0.
It is available as porous beads with a diameter of 1 to 3.0 mm and a pore size of 500 to 3000X. These porous chitosan beads generally have an amino group based on glucosamine with a molar equivalent of several microns to several tens of 11, or amino groups, including those based on a crosslinking agent for those that have been crosslinked. C,
These amino groups can be converted into active supporting groups in which Cal has a xyl group by the action of an alkanedicarboxylic anhydride.

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 -ness 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 anhydride, azelaic anhydride, cepacic anhydride, 1,10 Examples include -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.

The ratio of the weight of chitosan porous beads (a) to the weight of alkanedicarboxylic acid anhydride (b): a: b = 1: 0.05-10, preferably a: b = 1: 0.1- 3. Reaction temperature 20
-150°C, preferably room temperature - 100°C, reaction time: 1-60 hours, preferably 1-30 hours, reaction pressure: normal pressure - 10 atm, preferably normal pressure.

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

The chitosan porous beads having VZ xyl 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 preferable to use a solvent other than water. is preferred, and an alkali is preferred as the catalyst.

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, glopionyl chloride, butyryl chloride, etc. be able to.

Effects of the Invention The chromatography carrier provided by the present invention is
It is possible to easily covalently bond an affinity chromatography ligand (ligand) to an already bound carbon via an active support group having a xyl group. It is very useful as an insoluble active support.

Furthermore, regarding the properties desired in the above-mentioned active support for affinity chromatography, the hydrophilic properties provided by the chitosan porous beads according to the present invention suppress hydrophobic non-specific adsorption when coming into contact with proteins, etc. It is also clear that the porous structure has an advantageous effect on the interaction between the ligand and the adsorbed target substance.

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

Furthermore, the chromatography carrier provided by the present invention is capable of bonding a siligant with a chemically neutral and stable covalent bond, compared to the commonly available carriers using cyanogen bromide or glutaraldehyde. As an adsorption carrier for affinity chromatography, it has a great advantage that it is completely unaffected by the conditions of separation, elution, and washing of the target substance. These properties are indispensable especially as a carrier for high-speed affinity chromatography and a carrier for industrial separation and purification processes.
The effects of the chromatography carrier provided by the present invention are very large.

Examples The chromatographic carrier provided by the present invention is
As mentioned above, it is useful as an active support for affinity chromatography, and below, representative examples of the method for producing the chromatography support of the present invention will be shown and more specifically explained. 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 061 m (trade name Shodex Chitonosele, aromatic crosslinked product) 1. A solution of 1.0 g of glutaric anhydride in water was added to OI, and after adjusting the - to 6 by adding 4N aqueous sodium hydroxide solution, it was shaken at room temperature for one day. The beads were separated and washed in the order of 0.1N aqueous sodium bicarbonate solution, 0.1N hydrochloric acid, and water. Next, a 0.2 molar aqueous sodium acetate solution 5- was added to the beads, and 0.5 g of acetic anhydride was added thereto, followed by shaking at room temperature for 2 hours. The beads were washed in large amounts of water and dried.

Conobeads were confirmed to be amino-free using a colorimetric method using sodium trinitrobenzenesulfonate. The quantitative value of carboxylic acid determined by neutralization titration was 0.35 mmol per gram of dry beads.

Example 2 Chitosan porous beads with a particle size of 0.3 mm (trade name Shodex Chitonosol, aliphatic crosslinked product) 1. A solution of 0.11 succinic anhydride in 5 parts of dioxane was added to the OJ, followed by 0.1 g of pyridine and shaken at room temperature for 6 hours. After washing the beads several times with dioxane, a solution of 0.5 F of acetyl chloride in 5 Tnt of dioxane was added, 0.5 g of triethylamino was added, and the mixture was shaken for 30 minutes at room temperature. The beads were taken out and washed with dioxane, then washed with a large amount of water and dried. The beads were confirmed to be amino-free using a colorimetric method using sodium trinitrobenzenesulfonate. What is the quantitative value of carboxylic acid by neutralization titration per 1g of dry peas? It was 0.80 m', 1 mole.

Reference Example 1 Beads obtained according to Example 1 1. After thoroughly washing the OF with anhydrous dioxane, it was added to 4 ml of anhydrous dioxane, 801M9 of N-hydroxysuccinimide and 144 ml of dicyclohexylcarbodiimide were added, and the mixture was shaken at room temperature for 2 hours. The beads were then washed quickly in the following order: 20 ml of anhydrous dioxane, 6 ml of methanol, and 3 ml of cold water. The beads were added to a solution of concanavalin A 307n9 and methyl-α-mannopyranoside 401n9 in 0.1 mmol calcium chloride, 0.1 mmol manganese chloride and o, oi moles sodium bicarbonate in water for 2 hours at room temperature. After shaking, it was left at 4°C overnight. After taking the beads and washing them with a 1M aqueous sodium chloride solution and water, they were added to a 1M Tris-HCl buffer (pH 8,0) 2-
After shaking at room temperature for 1 hour, the beads were washed again with water. It was confirmed from the amount of unreacted concanavalin A that the adsorption carrier thus obtained supported 15 m9 of concanavalin A per gram of dry ffl beads.

Reference Example 2 Regarding the beads obtained in Example 2, protein A10 was added instead of the concanavalin A30 solution of Reference Example 1.
m9 0.01 mol sodium bicarbonate water 2rnlK
By using the solution in 6.5 hours and performing the same procedure as above, 1 g of dried peas white cypronaine A was added. Orn9
It was possible to obtain an adsorption carrier on which the particles were supported.

Patent issued: Showa Denko Co., Ltd.

Claims (2)

[Claims] (1) Chitosan porous beads, in which the amino groups based on glucosamine constituting the chitosan are cross-linked, or chitosan porous beads are cross-linked, and the amino groups based on glucosamine constituting the chitosan and/or A chromatography carrier characterized in that an ω-carboxyalkanoyl group is bonded to an amino group based on a crosslinking agent, and the remaining amino group is acylated. (2) Chitosan porous beads, in which amino groups based on glucosamine constituting the chitosan are cross-linked; A method for producing a chromatography carrier, which comprises reacting an alkanedicarboxylic anhydride with an amino group based on a crosslinking agent, and then reacting a monocarboxylic anhydride or an acyl halide with the remaining amino group.