JPH0718848B2 - Method for manufacturing carrier for affinity chromatography - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of use] The present invention relates to a method for producing a carrier suitable for carrying out affinity chromatography by immobilizing a substance having an amino group such as protein, peptide, amino acid, etc. as a ligand. It is a thing.

[Conventional technology]

It has been well known that a ligand having an amino group and a dialdehyde such as glutaraldehyde are used for affinity chromatography using chitosan. The use of a carbodiimide reagent as a peptide bond is also disclosed in Japanese Patent Publication No. 53-10150. The present inventors previously disclosed in Japanese Patent Application No. 62-92623 (Japanese Patent Laid-Open No. 63-258579) a method of treating a porous chitosan derivative having a carboxyl group with dicyclohexylcarbodiimide and N-hydroxysuccinimide to obtain a carrier. In addition, JP-A-62-315734 (JP-A-1-158000)
No.), there is proposed a method for obtaining a carrier for antibody immobilization by treating a partially acetylated granular porous chitosan with succinic anhydride and then with dicyclohexylcarbodiimide and N-hydroxysuccinimide.

[Problems to be Solved by the Invention]

The carrier previously filed by the present inventors is effective when affinity chromatography can be carried out because ligands such as antibodies and enzymes can be easily immobilized under mild conditions without nonspecific adsorption after immobilization. It was

However, there is no problem if the spacer is a short chain such as succinic acid, but if the steric hindrance of the ligand is expected, the spacer chain length must be increased. When an alkyl group is introduced as a spacer, it has the disadvantage that it becomes more hydrophobic and causes non-specific adsorption.

An object of the present invention is to solve the above-mentioned drawbacks, to enable the introduction of a spacer having an arbitrary chain length, and to reduce the hydrophobic interaction to obtain a carrier for affinity chromatography with small non-specific adsorption.

[Means for Solving the Problems]

In the present invention, granular porous chitosan is partially acetylated with acetic anhydride, treated with succinic anhydride, further treated with diamine and a condensing agent, and then treated with succinic anhydride, further dicyclohexylcarbodiimide and N-hydroxysuccinimide. To obtain a carrier for affinity chromatography.

The granular porous chitosan used in the present invention was previously disclosed in
It can be obtained by the method disclosed in 61-40337.
The chitosan used in the present invention is not particularly limited, but it is preferable to use low molecular weight chitosan having an average molecular weight of 10,000 to 230,000. Chitosan is dissolved in an aqueous solution of acetic acid, dichloroacetic acid, formic acid, etc. alone or in a mixture to obtain an acidic aqueous solution of chitosan. The chitosan acidic aqueous solution is dropped into a basic aqueous solution containing an alkaline substance such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, ammonia, and ethylenediamine under pressure from the discharge holes at a constant rate, and the particles are solidified and regenerated. Then, it is sufficiently washed with water until it becomes neutral to obtain granular porous chitosan. At this time, a polar solvent such as alcohol may be used in combination with the basic aqueous solution.

The granular porous chitosan obtained as described above is treated with acetic anhydride for partial acetylation. Thereafter, succinic anhydride, which is a dicarboxylic acid anhydride having a short chain length, is allowed to act to introduce a carboxyl group. Further treatment with diamine and condensing agent. At this time, it is important to react the diamine with a large excess of 3 times or more the molar amount of the carboxyl group in order to suppress the progress of the crosslinking reaction as much as possible. As the diamine to be used, ethylenediamine, tetramethylenediamine, hexamethylenediamine, octamethylenediamine or the like is used depending on the purpose, but if the chain length is longer than this, nonspecific adsorption occurs, which is not preferable. As the condensing agent, a carbodiimide type condensing agent such as dicyclohexylcarbodiimide is generally used, but any chemical substance capable of forming an amide bond between a carboxyl group and an amino group may be used. Then, succinic anhydride is reacted to obtain a granular porous chitosan derivative having an arbitrary carboxyl group. In the present invention, the step of introducing a carboxyl group and the subsequent step of forming an amide bond can be repeated a plurality of times. As described above, in the present invention, a long chain alkyl group compound is not used at one time, but a long chain alkyl group compound is used in order to lengthen a short chain chemical substance sequentially by an amide bond. You can weaken your sex. And the density of the carboxyl groups in this case can be controlled by the amount of acetic anhydride when the granular porous chitosan is first acetylated.

Then, wash thoroughly with water until neutral. The amount of the active group introduced is preferably 5 to 140 μmol / ml granular material. When the density of the active groups is 5 μmol / ml or less, the amount of the immobilized ligand is small and only the capacity of the used granule becomes large, which is not preferable, and when it is 140 μmol / ml or more, the immobilized ligand is sterically hindered. Occurs, and the activity of the ligand is not sufficiently effectively exhibited.

The obtained granular porous chitosan into which a carboxyl group has been introduced is suspended in a polar solvent such as dioxane, dimethylformamide, dimethylacetamide, dimethylsulfoxide, tetrahydrofuran, methyl alcohol, isopropyl alcohol, and then dicyclohexylcarbodiimide and N- Add hydroxysuccinimide and stir at room temperature for about 24 hours. After completion of the reaction, the carrier for affinity chromatography for immobilizing the ligand is obtained by sufficiently washing with the polar solvent used.

〔Example〕

The present invention is described below with reference to examples, but the present invention is not limited to the description of the examples. The active group density (CTV) and specific surface area were determined by the following methods.

(A) Active group density Approximately 50 ml of a sample is treated in 1,500 ml of 1 N-HCl for 1 hour with gentle stirring and thoroughly washed with deionized water until it becomes neutral to prevent absorption of carbon dioxide gas in the air. Carefully take 30 ml of dehydrated sample and take 1/50 N-NaOH 100m
Pour into the flask and leave for 5 hours with gentle stirring. 10 ml of this supernatant was collected and the methyl red solution was used as an indicator.
Neutralization titration is performed with 1/100 N-HCl and the value is calculated from the following formula.

a: 1/100 N-HCl volume required to neutralize 10 ml of test solution b: 1/50 N-HCl volume required to neutralize 10 ml of 1/50 N-NaOH before adding sample f : 1/100 N-HCl titer V: wet sample amount (30 ml) (B) Specific surface area Measured by BET method using a specific surface area measuring device.

Example 1 65 g of chitosan having a deacetylation degree of 77% and an average molecular weight of 42,000
Was dissolved in 635 g of 3.25% acetic acid aqueous solution, and the solution was dissolved in 7% NaO.
H, 30% ethyl alcohol, 63% water dropped into a mixed solution from a nozzle with a hole diameter of 0.15 mm at a constant pressure, coagulated and regenerated, and then washed with water until it became neutral. Got quality chitosan 0.9. Collect 50 ml (wet state) of each of the granular porous chitosan into 3 containers, and
The mixture was replaced with ethyl alcohol four times to completely remove water. 50 ml of ethyl alcohol was added to each, and acetic anhydride 5.6 g, 5.2 g,
7.8 g was added and reacted at 25 ° C. for 18 hours. Then 1N-NaOH
It was treated with water for 1 hour and washed with water until it became neutral. Then, each was replaced four times with ethyl alcohol, 50 ml of ethyl alcohol and 5 g of succinic anhydride were added, and the mixture was reacted at 25 ° C. for 18 hours. After treating with 1 N-NaOH for 1 hour, the mixture was replaced twice with 50 ml of dioxane, and hexamethylenediamine and dicyclohexylcarbodiimide were added to 50 ml of dioxane so that the concentration became 0.1 M, and the mixture was reacted at 25 ° C. for 18 hours with stirring. Dioxane,
50 ml each of methyl alcohol and dioxane in this order 2
Washed repeatedly. Then, 50 ml of dioxane and 5 g of succinic anhydride were added to each, and the mixture was reacted with stirring at 25 ° C for 18 hours.
After the treatment with NaOH, the mixture was washed until it became sufficiently neutral to obtain 42 ml each of three types of granular porous chitosan derivative having a carboxyl group before activation (sample numbers 1'-3 '). The ion exchange capacity of each was measured to be 59.02 μmol / ml granular material, 25.0
The particles were 2 μmol / ml granules and 18.76 μmol / ml granules. After substituting each with twice 50 ml of dioxane, dicyclohexylcarbodiimide and N-hydroxysuccinimide were added to 50 ml of dioxane so that the concentration became 0.1 M, and the mixture was stirred at 25 ° C. for 18 hours. Then, 50 ml each of dioxane, methyl alcohol, dioxane, and isopropyl alcohol were repeatedly washed twice to obtain 40 ml of affinity chromatography carriers (Samples 1 to 3).

Husband Once you measure these specific surface area people 65.3m ² /g,72.6m ² / g,
It was 80.5 m ² / g.

Application Example 1 1 ml each of the three types of carriers for affinity chromatography (Sample Nos. 1, 2, 3) obtained in Example 1 was collected and washed with water, and immediately thereafter, 0.1 M carbonate buffer solution (pH 8) containing 0.5 M NaCl was immediately added. .3)
2 ml of 25 mg / ml trypsin inhibitor solution dissolved in
Was added, and the mixture was gently stirred at 4 ° C. for 18 hours. After removing unreacted trypsin inhibitor solution, 0.1M Tris-HCl was added.
Add 0.1 ml of buffer solution (pH7.4) and react for 1 hour, then 0.1M
Washing with Tris-HCl buffer (immobilized trypsin inhibitor granules were obtained. This was packed in a column having a diameter of 1 cm and a length of 5 cm, washed thoroughly with 0.1 M Tris-HCl buffer, and then washed with 0.1 M tris-HCl buffer.
2 ml of 1% tribucin solution dissolved in M Tris-HCl buffer
At a flow rate of 2 ml / min. After the completion, the plate was washed with 0.1 M Tris-HCl buffer, and 10 ml of 0.01 N-NCl containing 0.5 M NaCl was flown to elute the adsorbed trypsin. Again 0.1M Tris-HCl
2 ml of bovine serum albumin (BSA) solution washed with a buffer solution and dissolved in 0.1 M Tris-HCl buffer was flowed at a flow rate of 2 ml / min for adsorption. The results are shown in Table 1.

The amount of protein adsorbed in the reaction solution before and after adsorption was determined by the BCA protein assay kit (US, Piercechemical companion
(made by y). The amount of adsorbed HCl indicates the amount of trypsin eluted by HCl.

As is clear from this result, trypsin having affinity for the trypsin inhibitor used as a ligand adsorbed 10 mg / ml granules or more in both sample numbers 1, 2, and 3, and by flowing 0.01 N-HCl. More than 80% is eluted. Therefore, trypsin affinity chromatography is a satisfactory result. And BSA, which has no affinity with the ligand, is hardly adsorbed. That is, since the non-specific adsorption hardly occurs, the carrier of the present invention is excellent as a carrier for affinity chromatography.

Example 2, Application Example 2 As to the granular porous chitosan having an average particle diameter of 0.3 mmφ obtained in the same manner as in Example 1, the same amount of acetic anhydride was used as in Example 1.
After fixing to 8 g and reacting with the same amount of ethyl alcohol succinic anhydride as in Example 1, the hexamethylenediamine of Example 1 was replaced with ethylenediamine and tetraethylenediamine and octamethylenediamine, and treated. The same amount of ethyl alcohol and succinic anhydride were treated to obtain 42 ml of three types of granular porous chitosan derivatives having spacers having different chain lengths (Sample Nos. 4'-6 '), respectively. These ion exchange capacities are 50.3 μmol / ml granules, 76.
There were 2 μmol / ml granules and 68.5 μmol / ml granules.
This sample was treated with dicyclohexylcarbodiimide and N-hydroxysuccinimide in the same manner as in Example 1 to obtain 40 ml of a carrier for affinity chromatography (sample numbers 4 to 6), respectively. These specific surface areas are 6 respectively
5.3m ² /g,76.2m ² / g, and 68.5m was ² / g. Affinity chromatography was performed on these three samples with trypsin inhibitor-trypsin in the same manner as in Example 1, and the results shown in Table 2 were obtained.

As a result, the adsorption and desorption of trypsin having affinity for the trypsin inhibitor, which is a ligand, was satisfied as in Example 1, and BSA was hardly adsorbed, which is an excellent carrier for affinity chromatography.

Comparative Example 50 ml of each granular porous chitosan obtained by granulating in the same manner as in Example 1 was replaced with 50 ml of ethyl acol 4 times to remove water. Add 50 ml of ethyl alcohol to each and add acetic anhydride 7.
8 g each was added and reacted at 25 ° C. for 18 hours. The reaction solution was filtered off, 1N-NaOH (50 ml) was added, and the mixture was stirred for 1 hour. After thoroughly washing with water to neutrality, each was replaced four times with 50 ml of dioxane, one sample was added with adipic acid dichloride, and the other sample was added with a 0.1 M dioxane solution of sebacic acid dichloride, and then the hydrochloric acid generated in this reaction system was added. In order to remove the above, triethylamine was further added so that the concentration of each was 0.2 M, and the mixture was reacted at 25 ° C. for 1 hour. This was washed with water until it became neutral to obtain a carrier of adipic acid derivative and sebacic acid derivative before activation. The ion exchange capacity of each carrier was 20.0 μmol / ml granules and 21.5 μmol / ml granules.

After substituting 50 ml of dioxane for this carrier twice in the same manner as in Example 1, dicyclohexylcarbodiimide and N-hydroxysuccinimide were added to 50 ml of dioxane so that the concentration became 0.1 M, and the mixture was stirred at 25 ° C. for 18 hours to obtain dioxane, methyl alcohol, 50 ml each of dioxane and isopropyl alcohol were washed twice in this order to obtain 40 ml each of the carriers for affinity chromatography (sample numbers 7 and 8).

This carrier was used in the same manner as in Application Example 1 to give a trypsin inhibitor-
When affinity chromatography was performed with trypsin, the results shown in Table 3 were obtained.

As a result, trypsin was successfully adsorbed, but the hydrophobicity of the carrier was so strong that it could not be eluted with 0.01N-HCl, and the target product, trypsin, was not adsorbed to the ligand and was nonspecifically adsorbed on the carrier. It is considered that it was adsorbed. or,
The amount of BSA adsorbed is also large, and it is clear that nonspecific adsorption is increased due to the increase in hydrophobicity.

〔The invention's effect〕

The carrier for affinity chromatography for immobilizing a ligand according to the present invention is a granular porous chitosan partially acetylated with acetic anhydride, then treated with succinic anhydride, and further treated with a diamine and a condensing agent to reduce hydrophobic interaction. After introducing a spacer having an arbitrary chain length, it is treated with succinic anhydride, and further treated with dicyclohexylcarbodiimide and N-hydroxysuccinimide.

The carrier of the present invention can increase the adsorption rate of a physiologically active substance having an affinity for a ligand even when steric hindrance is expected, as is clear from the description of application examples, and further has an affinity for the ligand. Almost no physiologically active substance is adsorbed, and nonspecific adsorption does not occur.
Therefore, the carrier of the present invention is extremely preferable as a carrier for affinity chromatography.

Claims (1)

[Claims] 1. Granular porous chitosan is partially acetylated with acetic anhydride, then treated with succinic anhydride, further treated with diamine and a condensing agent, and then treated with succinic anhydride, further dicyclohexylcarbodiimide and N-dihydroxysuccinimide. A method for producing a carrier for affinity chromatography, which comprises: