JPH03164176A - Production of carrier for immobilizing biologically active substance - Google Patents

Abstract

PURPOSE:To obtain the title carrier free from absorption of excessive protein by non-specific absorption without lowering of activity by reacting a functional aldehyde compound with a formed article under condition of pH being in a specific range and subjecting the reaction product to diazotization treatment and then decomposing the diazotized product. CONSTITUTION:(A) A formed article composed of a polymer having a primary amino group at least surface layer part is reacted with (B) a bifunctional aldehyde compound and then when residual primary amino group is subjected to diazotization treatment and then decomposed to convert the amino group into OH group, the ingredient B is reacted therewith in pH 1.5-5 to provide the aimed carrier. Furthermore, the ingredient A is preferably polyphosphazene having primary amino group on the side chain and the ingredient B is preferably glutaric aldehyde.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention relates to a method for producing a carrier for immobilizing a biologically active substance.More specifically, at least the surface layer of the carrier for immobilization has a hydrophilic side chain. A method for producing a carrier for immobilizing a biologically active substance composed of a polymer is provided.

(Prior Art) In recent years, research and development of chemical industrial processes using biologically active substances, materials for clinical chemistry, etc. has been actively conducted.

Compared to organic chemical reactions, biochemical reactions controlled by enzymes, which are one of the biologically active substances that catalyze various reactions within living organisms, require less energy because they proceed at room temperature and pressure. Significant savings can be made.

(2) Since the reaction occurs specifically at a specific position of a specific structure, there are few by-products and the yield can be improved, and purification is easy.

(3) Since substrate specificity is strict, only a specific substance can be selectively changed even in the presence of a mixture of various compounds.

It has the following advantages.

However, enzymes, which are water-soluble globular proteins, are unstable to heat, organic solvents, acidic and alkaline solutions, and easily lose their activity. In addition, until now, enzymes have been used dissolved in aqueous solutions and it has been difficult to recover them.
This was extremely wasteful as it was discarded after each reaction.

Enzyme immobilization is a technique for utilizing the advantages of such enzymes while eliminating their disadvantages.

Enzyme immobilization methods include ({) carrier binding method, (2) crosslinking method, (3) entrapping method, and a composite method that combines these methods. Using these techniques, chemical industrial processes using enzymes are being developed. Furthermore, in recent years, not only enzymes but also NAD(P)/NAD(P)H and ATP
Techniques have been reported to immobilize and utilize cofactors such as , intracellular organelles, microbial cells, animal and plant cells, etc. In order to carry out biochemical reactions using enzymes and the like in these technologies, it is desirable to have a large number of enzymes and the like capable of expressing their functions.

Furthermore, in the field of clinical testing, biologically active substances such as antigens and antibodies are immobilized on solid carriers such as polystyrene latex and used for diagnosis. These immobilization methods can be divided into adsorption type and chemical bond type. Most of the products currently in practical use seem to be adsorption types, but these mainly use physical adsorption based on the hydrophobic interaction between biologically active substances (antibodies, proteins, etc.) and latex. There is a possibility that proteins that are undesirable for desorption of active substances and immunochemical reactions in diagnosis may be adsorbed.Therefore, it is possible that proteins with high reactivity and stable functional groups on the surface are present in a high concentration and quantitatively and are not suitable for aqueous systems. The above-mentioned disadvantages can be solved if there is a carrier that can easily immobilize proteins and the like through chemical bonding.

Furthermore, separation and purification techniques that utilize biological affinity, such as affinity chromatography, also require the immobilization of biologically active substances onto a carrier.

In this technique as well, it is desirable to immobilize a large number of biologically active substances that have biological affinity with the substance to be purified, and to ensure that the substance to be purified does not non-specifically adsorb to the carrier. Needed.

First, the present inventors treated the surface of a compound made of polyphosphazene to add functional groups (further reactive proposed a carrier for immobilizing biologically active substances (including those that can be converted into functional groups that can be immobilized by Substances other than substances may be adsorbed by hydrophobic bonds (referred to as non-specific adsorption), and when applied to affinity chromatography or diagnostic reagents,
There was a problem that separation accuracy or diagnostic accuracy decreased.

In order to solve this problem, the present inventors further developed a carrier that is more structured than polyphosphazene, in which the polyphosphazene in at least the surface layer of the carrier has a functional group whose side chain can bind a ligand. (Japanese Patent Application No. 1122/2003, 5'). In this product, nonspecific adsorption of substances (eg, proteins) that have no affinity with the ligand was extremely reduced.

OBJECT OF THE INVENTION The object of the present invention is, as described above, that the polymer of at least the surface layer of the carrier has a side chain having a functional group capable of binding to a ligand, is non-reactive with the ligand, and is hydrophilic. It is an object of the present invention to provide a novel method for producing a support for immobilizing a biologically active substance containing a side chain and having less non-specific adsorption.

(Construction of the Invention) As a result of intensive studies to achieve the above-mentioned object, the present inventors have discovered that, under specific conditions, a molded article composed of a polymer having at least a surface layer having a primary amino group has a The present invention was achieved by discovering that a method of reacting a functional aldehyde and then converting the remaining amide 7 groups into hydroxyl groups is extremely simple and causes less non-specific adsorption of the resulting carrier.

That is, the present invention involves reacting a bifunctional aldehyde compound with a molded article made of a polymer having at least a surface layer having primary amino groups, and then diazotizing the remaining primary amino groups and decomposing them to form hydroxyl groups. A method for producing a carrier for immobilizing a biologically active substance, which comprises reacting the bifunctional aldehyde compound at a pH of 1.5 to 5. It is.

The biologically active substance as used in the present invention refers to a substance that interacts with a substance in the living body, and may be a polymer such as an enzyme, an antibody, or a nucleic acid, or a low molecule such as an enzyme-capturing enzyme or a habten. It may be.

In the present invention, it is necessary to use a molded article in which at least the surface layer of the molded article is composed of a polymer having a primary amino group in its side chain. Examples of such a polymer include chitosan, t-class amino Examples include polyphosphazene having a group, polyaminostyrene obtained by reducing nitrated polystyrene, and polyamino obtained by partially or completely reducing polyacrylonitrile. Further, these may be partially crosslinked to make them insolubilized. Polyphosphazene polymers having primary amino groups in their side chains are suitable, and examples of such side chains include the following.

-OCH2CH2NH2 OH -OCH2CH20CH2CH2NH2-NH (CH2) NH2 -NHNHC○(CHz)a CONHNH2 Such a side chain is usually introduced as follows.

First, a polydialkoxyphosphazene is obtained by a known method, and then a suitable alcohol or phenol or a metal salt thereof is reacted to obtain a polydialkoxyphosphazene or a polyaryloxyphosphazene.

In this case, if an aminoalkoxide or aminoaryloxide is used, it can be selectively introduced as an aminoalkoxy group or aminoaryloxy group, and a product having the desired side chain can be obtained.

In addition, reactive functional groups such as alkoxy groups, hydroxyl groups, or amino groups can be added to polydichlorophosphazene, and reactive functional groups such as nitro groups, which are inert to polydichlorophosphazene but can be converted into amino groups by post-treatment, can be used. It can also be obtained by using an organic compound having a functional group, reacting polydichlorophosphazene with the organic compound, and then subjecting it to an appropriate post-treatment.

In the present invention, if at least the surface layer portion of the molded product is made of the polymer having the above-mentioned primary amino group,
Any shape may be used, and there is no need to specify the manufacturing method. That is, the shape may be particulate, fibrous, film, or any other shape depending on the final usage. The manufacturing method is also arbitrary, and it is possible to mold a polymer into which primary amino groups have been introduced in advance, or to process a polymer molded product so that at least the surface layer of the polymer has primary amino groups. It may also be one in which a side chain having the following is introduced. Furthermore, the surface of a molded article made of another type of polymer may be laminated and coated with a polymer having a primary amino group.

In the present invention, a molded article having at least a surface layer made of a polymer having these primary amino groups is reacted with a bifunctional aldehyde compound to introduce ligand fixing points. At this time, the pH of the treatment liquid is 1.5 to 5,
Preferably, it is necessary to use an aqueous solution controlled at 1.7 to 3.5. When the pH becomes less than 1.5,
The reactivity between the primary amino group and the aldehyde group becomes extremely low, making it impossible to introduce a desired ligand fixation point.

On the other hand, if the pH exceeds 5, the reactivity between the primary amino group and the aldehyde group becomes too high, which is undesirable because both aldehyde groups of the bifunctional aldehyde compound react and crosslinking occurs. .

The pH of the treatment liquid may be controlled with an aqueous acid solution, and as the acid, any of inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid, and organic acids such as acetic acid can be used. Among them, hydrochloric acid. Sulfuric acid is preferred. Note that an organic acid, salts, or the like may be mixed with the above-mentioned inorganic acid.

The bifunctional aldehyde compound used in the present invention is preferably water-soluble, and glutaraldehyde is particularly preferred. In addition, there is no need to particularly limit the concentration of the treatment solution containing the bifunctional aldehyde compound, but if it is not crushed too much, a crosslinking reaction will easily occur, and if it is too thin, it will be difficult to introduce a sufficient amount of ligand fixation points. Usually about 0.01 to 15%, preferably 0.02 to 3%.
A concentration of about 100% is used. Note that the ratio of the bifunctional aldehyde compound to the polymer formed of a polymer having at least a surface layer portion having a primary amino group is arbitrary, but it is usually 0.1 to 0.1 to the weight of the molded product. 100 times the amount of bifunctional aldehyde compound is used.

In the present invention, the remaining primary amino group is then converted into a diazonium salt and then decomposed and converted into a hydroxyl group, but since the reaction proceeds under strongly acidic conditions,
The imine bonds produced by reacting the above aldehyde compounds tend to be hydrolyzed. Therefore, it is preferable to reduce the generated imine bond prior to the diazo reaction.

As the reducing agent, dimethylaminoborane, sodium cyanoborohydride, sodium borohydride, trimethylaminoborane, tetramethylammonium hydride, etc. are preferably used.

The compound into which the ligand fixation point has been introduced is usually washed with water and immersed in acid to protonate all remaining amino groups, and then reacted with an aqueous solution of sodium nitrite in an acidic reaction system. The diazonium salt is produced using a diazonium salt, and then a hydroxyl group is introduced by reacting it with water. At this time, the acid concentration is preferably as high as possible in order to speed up the protonation of amino groups inside the polymer. Usually, 1N hydrochloric acid is preferably used. The amount of acid used here depends on the concentration of the acid, but is usually 1 % of the weight of the molded product.
It is at least twice as large, preferably at least 20 times.

It is desirable that the concentration of sodium nitrite be as high as possible in consideration of its permeability into the interior of the polymer. Usually 0.1M or more and 2M or less, preferably 0.3M
A concentration of M or more and 1.5M or less is preferable.

The amount of sodium nitrite added needs to be equal to or greater than the number of moles of primary amino groups in the entire polymer.

In addition, during the diazotization reaction, the nitrous acid generated may exit the system without reacting with the primary amino group, and to what extent the initial crosslinking reaction or ligand fixation point introduction reaction Since it may be unclear whether there are any primary amino groups left, it is necessary to add sodium nitrite in an amount that is at least 10% more than the number of moles of primary amino groups in the polymer that has at least a portion of primary amino groups initially used. It is preferable to use If it is less than this, unreacted primary amino groups may remain, which is undesirable because it may cause non-specific adsorption.

The reaction of converting a primary amino group into a hydroxyl group proceeds extremely easily even at room temperature, and the reaction is almost complete once the generation of nitrogen has stopped. However, the reaction may be completed by subsequent treatment with an alkali or by heating.

This reaction of converting a primary amino group into a hydroxyl group is a reaction via a carbon cation, and therefore often involves a rearrangement reaction.

Rearrangement reactions occur to form more stable carbon cations, but are complex and the products difficult to identify. but,
Even if a rearrangement reaction occurs, a hydroxyl group is often introduced, so there is no particular problem as long as it is hydrophilic and no crosslinking reaction occurs. In addition, when the polymer having a primary amino group is polyphosphazene, rearrangement can be prevented by making the side chain contain the following structure.

-○CH2CH2NH2 -NHCH2CH2NH2 -SCH2CH2NH2 (Effect of the invention) The support for immobilizing biologically active substances obtained by the method detailed above does not adsorb excess protein due to non-specific adsorption, and the ligand is It does not cause any structural changes that reduce its activity.

Furthermore, the shapes of the precepts used in the manufacturing method of the present invention are arbitrary, and can be not only granular but also various textile products such as threads, braids, knitted fabrics, nonwoven fabrics, and paper, as well as films and sheets. A sheet-like molded product of can be used. Therefore, it is possible to provide a carrier for immobilizing a biologically active substance that can be used in a wide range of fields, such as a carrier for affinity chromatography, a carrier for immobilized enzymes, and a carrier for diagnostic agents.

(Example) The present invention will be explained in more detail with reference to the following example. Example 1 Sodium alcoholate is prepared from 105 g of 2-(2-aminoethoxy)ethanol and 2.0 g of sodium metal.

When polyphosphazene fibers (50 single fibers, 20 fibers) were run continuously and soaked at 80°C for 15 minutes, 76% of the side chains of polyphosphazene were 2-(2 -Aminoethoxy> It was found by gas chromatography to measure trifluoroethanol derived from trifluoroethoxide liberated into the reaction system by post-treatment that it was substituted with an ethoxy group.

When this fiber was observed with an optical microscope, it appeared that the substitution reaction had progressed up to about 273 points from the fiber surface, but the boundary was not clear.

In addition, absorption derived from primary amino groups was observed in the infrared absorption spectrum, indicating that primary amino groups were introduced into the side chains of polyphosphazene.

This surface-treated fiber was slowly run in water for about 10 minutes to remove unreacted sodium alcoholate, and then the fiber was wound up in water as it was. This fiber 0.3
g was taken out of water and immersed in 100 ml of glutaraldehyde (25% aqueous solution)/0.01N hydrochloric acid (mixture solution (pH 1.9, volume ratio 1/1001) for 1 hour at 0°C. This fiber was immersed in 100 ml of water. After washing three times with
It was immersed in 50ml of N-hydrochloric acid for 3 hours at O℃, and further soaked in O
0 at °C. 25 ml of 5M sodium nitrite aqueous solution was gradually added dropwise. After nitrogen has been released, leave it at room temperature for 3 hours, and add 100 ml of water 3 times and 100 ml of methanol 3 times.
0.1M phosphate buffer (pH 7.6) 100m
The fibers were washed three times with l.

The carrier fiber thus prepared was mixed with 20 mg of bovine serum albumin in 20 mM phosphate buffer (pH 7.6).
ml of the solution, and the amount of bovine serum albumin immobilized on the fiber was determined from the decrease in absorbance of the supernatant of the solution at 280 mm after 1 hour. It was 5 mg.

To this, add 90mM monoethanolamine in 0.1M phosphate buffer (adjusted to pH 7.6) to 100ml of solution.
The remaining aldehyde was sequestered by soaking for a period of time, and the recovered imine bonds were reduced with sodium borohydride cyanide.

50 ml of rabbit serum immunized with BSA was loaded onto the above carrier and subjected to a series of adsorptions using conventional affinity chromatography. Washing. Elution and regeneration operations were performed.

The anti-BSA antibody eluted at this time was 34. It was 1 mg, and it was found to be single by electrophoresis.

Example 2 0.2% dimethylformamide solution, 10 g of polyacrylonitrile with a logarithmic viscosity of 10.4 measured at 30°C was dissolved in 150 ml of dimethylformamide,
Extruded into a thread in a water bath containing 0% dimethylformamide,
A filamentary polyacrylonitrile having a porous structure was obtained.

2.5 g of lithium aluminum hydride was added to dry diethyl ether and stirred, followed by adding 1.2 g of the filamentous polyacrylonitrile obtained above and heating under reflux for 16 hours. After the reaction, water was added dropwise while cooling with water. to decompose unreacted lithium aluminum hydride, and further INHCI
was added dropwise to dissolve the decomposed product, and then the aminated polyacrylonitrile threads were recovered.

This filamentous material was mixed with glutaraldehyde (25% aqueous solution).
/0. Mixture of 0IN hydrochloric acid (volume ratio 1/100)<p
}l1.9) After immersing in 100 ml, the following reduction reaction, diazotization reaction, etc. were performed in the same manner as in Example 1.

The carrier thus obtained was immersed in a solution of 20 mg of bovine serum albumin in 20 ml of 20 mM phosphate buffer (pH 7.6), and the amount of bovine serum albumin immobilized on the carrier was determined. It was 6 mg.

To this, add 90mM monoethanolamine in 0.1M phosphate buffer (adjusted to pH 7.6) to 100ml of solution.
The remaining aldehyde was sequestered by soaking for a period of time, and the formed imine bond was reduced with sodium borohydride cyanide.

50 ml of rabbit serum immunized with BSA was loaded onto the carrier and subjected to a series of conventional affinity chromatography operations including adsorption, washing, elution, and regeneration.

The anti-BSA antibody eluted at this time was 32. It was 9 mg and was found to be single by electrophoresis.

Examples 3 and 4, Comparative Examples 1 and 2 A carrier on which bovine serum albumin was immobilized was obtained in the same manner as in Example 1, except that the conditions for reacting glutaraldehyde were changed. The amount of fixed bovine serum albumin is shown in the table below.

Claims (3)

[Claims] (1) A molded article made of a polymer having at least a surface layer having primary amino groups is reacted with a bifunctional aldehyde compound, and then the remaining primary amino groups are diazotized and then decomposed and converted to hydroxyl groups. A method for producing a carrier for immobilizing a biologically active substance, which comprises reacting the bifunctional aldehyde compound at a pH of 1.5 to 5 when producing the carrier for immobilizing a biologically active substance. (2) A polymer having a primary amino group has a primary amino group in its side chain.
Claim (1) wherein the polyphosphazene is a polyphosphazene having a class amino group.
) A method for producing a carrier for immobilizing a biologically active substance. (3) The method for producing a carrier for immobilizing a biologically active substance according to claim (1), wherein the bifunctional aldehyde is glutaraldehyde.