JPH0553802B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an immobilized physiologically active substance, a method for producing the same, and a carrier used in the production method, and more particularly, an immobilized physiologically active substance that has a high ligand concentration and is stable, a method for producing the same, and a carrier for producing the same. This invention relates to a carrier used in the method. There are various immobilization methods for immobilizing physiologically active substances for industrial use, but considering the stability of the immobilized substance, non-specific reactivity between the immobilized substance and the reactant, etc. A method in which a ligand (physiologically active substance) is immobilized on an insoluble carrier by a covalent bond is desirable. Such methods include the cyanogen bromide method and the epoxy activation method [Organic Synthetic Chemistry, Vol. 38, 128-
138 (1980)], but the former has the disadvantage that nonspecific adsorption often occurs, and that once the immobilized ligand binds, the ligand often leaks out. However, the amount of ligand binding is also not satisfactory. On the other hand, specific adsorbents (immobilized physiologically active substances) prepared by the latter method have the advantages of excellent stability and low nonspecific adsorption, but when binding a ligand, , which must be treated under strong alkaline conditions at high temperatures, is unsuitable for immobilizing unstable physiologically active substances. In addition, the epoxy activated carrier is further treated with concentrated ammonia water,
Methods for binding various ligands after conversion to amino derivatives have also been invented, but they have the drawback of requiring a long time to react with many ligands, especially compounds with low reactivity carbonyl groups such as sugars. . There is also a method that utilizes a coupling reaction between an amino group and a carboxyl group using carbodiimide, but this method may cause side reactions such as the formation of isourea derivatives and Chapman rearrangement. Even at a certain PH4.5,
Furthermore, under conditions of high hydrogen ion concentration, side reactions in which the ligand polymerizes are likely to occur, and the yield of immobilized products is not satisfactory, so a carrier with more reactive functional groups is desired. . As a result of extensive research in order to solve the above-mentioned problems, the present inventors fixed a hydrazine derivative and/or dihydrazide derivative obtained by reacting a water-insoluble polymer having an epoxy group with hydrazine or a dihydrazide compound. By using it as a carrier for immobilization, physiologically active substances with low reactivity such as sugars can be immobilized under mild conditions, and the resulting immobilized substance has a high ligand concentration and is stable. This finding led to the completion of the present invention. That is, the immobilized physiologically active substance and insoluble polymer (immobilization carrier) of the present invention have the following formula (): [In the formula, M is a water-insoluble polymer residue; R ¹ , R ²
and ^R3 each represent a hydrogen atom or an alkyl group having 1 to 5 carbon atoms _; X is a hydrogen atom, a physiologically active substance residue having a carbonyl group or a _carboxyl group, _or In the formula, X ₁ is a physiologically active substance residue having an amino group; y is an integer from 2 to 10; Y is -(CH ₂ ) _o - (n is 0 ~8
), -CH=CH- or

[Formula]; x represents 0 or 1; ]. The insoluble polymer (immobilization carrier) of the present invention represented by the above formula () in which X is a hydrogen atom
can be obtained as follows. That is, a water-insoluble polymer is reacted with an epihalohydrin compound or a bisoxirane compound, and then this is reacted with NH ₂ NH ₂ and/or NH ₂ NH-CO-Y-.
CO-NHNH ₂ [Y is -(CH ₂ ) _o ^- (n is an integer from 0 to 8), -CH=CH- or

By reacting with [representing formula]], the following formula (): (In the formula, M, Y, R ¹ , R ² , R ³ and X have the same meanings as above.) An immobilization carrier can be obtained. Examples of water-insoluble polymers include insoluble polymers having hydroxyl groups such as agarose, cellulose, chitin, mannan, polyacrylamide resins, and polyvinyl alcohol resins, which are processed by known methods [Organic Synthetic Chemistry, Vol. 38] , pp. 128-138 (1980
2008)], epihalohydrins such as epichlorohydrin and epibromhydrin; or ethylene glycol diglycidyl ether, 1,3-propanediol diglycidyl ether, 1,4-butanediol diglycidyl ether, glycidyl ether, 1,6-
By treatment with bisoxirane compounds such as hexanediol diglycidyl ether;

[Formula] can be obtained. The insoluble polymer used as a carrier can be used in various forms depending on the purpose. For example, when used for affinity adsorbents or immobilized enzymes, in order to increase adsorption capacity or increase contact area,
A porous granular material is preferable, and a porous film-like material is preferable when used for treating a dilute physiologically active substance solution. As the hydrazines, various forms such as hydrazine hydrate, hydrazine dihydrochloride, and hydrazine sulfate are used. Examples of dihydrazide compounds include straight chain compounds having 2 to 12 carbon atoms such as oxalic acid dihydrazide, malonic acid dihydrazide, succinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, pimelic acid dihydrazide, suberic acid dihydrazide, azelaic acid dihydrazide, and sebacic acid dihydrazide. unsaturated dicarboxylic acid dihydrazides such as maleic acid dihydrazide and fumaric acid dihydrazide; and aromatic dicarboxylic acid dihydrazides such as phthalic acid dihydrazide and terephthalic acid dihydrazide. These hydrazines and dihydrazide compounds may be used alone or as a mixture of two or more. The amount of hydrazines and dihydrazide compounds to be used is 0.001 to 0.5 g for hydrazines and 0.001 to 0.5 g for dihydrazide compounds, depending on the type of dihydrazide compound, per 1 g (dry weight) of the water-insoluble polymer having the epoxy group or the epoxy-activated insoluble polymer. Although it varies, it is generally 0.005 to 1 g. As a solvent, water is generally used, and the amount thereof is 5 to 10 ml per 1 g (dry weight) of the insoluble polymer, and sodium carbonate, hydrochloric acid, etc. are added to the solvent as necessary. You may. Under the above conditions, at 0 to 100°C, preferably 4 to 50°C,
By reacting for 0.5 to 72 hours, preferably 1 to 48 hours, the immobilization carrier represented by the above formula () can be obtained. The method for immobilizing the physiologically active substance on the immobilization carrier thus obtained is appropriately selected depending on the type of the physiologically active substance. Physiologically active substances include sugars such as lactose, dextran sulfate, heparin, N-acetylglucosamine, N-acetylmuramic acid, tri-N-acetylchitotriose, chitin, glucose, and maltose; mucin, and acidic α ₁ -glycoprotein. Glycoproteins such as , prothrombin, haptoglobin, and gonadotropin; Glycolipids such as frenosin, kerasin, nervone, oxynervone, and ganglioside; Various glycosides such as glucoside, galactoside, mannoside, and fructoside; as well as carbonyl groups, aldehyde groups, and carboxyl groups. group, alkoxycarbonyl group,
In the case of a compound having a carbonyl group in a broad sense such as an aminocarbonyl group or a haloformyl group, it can be immobilized as follows. That is, by reacting the physiologically active substance with the immobilization carrier represented by the formula () in the presence of sodium cyanoborohydride,
The desired immobilized physiologically active substance can be obtained. The amount of sodium cyanoborohydride used is per 1 g (dry weight) of the immobilization carrier,
The amount may be 10 mg or more, but it is particularly preferably 100 to 500 mg. The amount of the physiologically active substance varies depending on its type, but is generally 50 to 500 mg per gram (dry weight) of the immobilization carrier. As the solvent, dipotassium hydrogen phosphate aqueous solution, phosphate buffer, acetate buffer, borate buffer, carbonate buffer, bicarbonate buffer, etc. are generally used, and the pH thereof should be 8 to 10. is preferable, and the amount is 3 to 20 per 1 g (dry weight) of the immobilization carrier.
ml is preferred. Under the above conditions, 0~
1 to 72 hours at 100°C, preferably 4 to 80°C,
The desired immobilized physiologically active substance can be obtained by reacting preferably for 2 to 48 hours. The immobilized physiologically active substance obtained in this way is
If necessary, unreacted sites may be acetylated by treatment with acetic anhydride. Next, the physiologically active substances are concanavalin A (hereinafter referred to as "ConA"), bovine serum albumin (hereinafter referred to as "BSA"), γ-globulin (antibody), enzymes, and phytohemagglutinin (PHA). , fibronectin, proteins such as protein A; kinin,
Peptides such as corticotropin-releasing factor (CRF), calcitonin, and glucagon; complex proteins such as nuclear proteins, pigment proteins, glycoproteins, and phosphoproteins; various amino acids; biogenic amines such as histamine, dopamine, glucosamine, and tryptamine; etc. In the case of a compound having an amino group of , it can be immobilized as follows. That is, in the presence of sodium cyanoborohydride, the immobilization carrier represented by the above formula () is treated with the following formula ():OHC-( _CH2 ) _y-2 -CHO() (wherein, y is 2 Representing an integer of ~10.) is reacted with a linear dialdehyde compound represented by the following formula (): (In the formula, M, Y, R ¹ , R ² , R ³ , x and y have the same meanings as above.) An aldehyde-activated insoluble polymer is used. The amount of sodium cyanoborohydride to be used varies depending on the water content of the carrier, but if it is 50 mg or more per 1 g (dry weight) of the immobilization carrier, the desired purpose can be achieved, but in particular, it should be 80 to 1000 mg. is preferred. The linear dialdehyde compounds generally include those having 2 to 10 carbon atoms, such as glyoxal, propane dial, butane dial, glutaraldehyde (pentan dial),
Decandial and the like can be used alone or as a mixture of two or more. Water is used as the solvent, and the amount is 1 g (dry weight) of the immobilization carrier.
It is preferably 5 to 20 ml per serving. The aldehyde-activated insoluble polymer can be obtained by reacting under the above conditions at 0 to 80°C, preferably 20 to 40°C, for 0.5 to 10 hours, preferably 1 to 4 hours. Next, the desired immobilized physiologically active substance can be obtained by reacting this aldehyde-activated insoluble polymer with the physiologically active substance in the presence of sodium cyanoborohydride. Examples of the solvent used at this time include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, etc., the pH of which is preferably 5 to 8, and the amount of aldehyde activated insoluble molecule 1g
(dry weight) is preferably 5 to 40 ml. The amount of sodium cyanoborohydride used may be 100 mg or more per 1 g (dry weight) of the aldehyde-activated insoluble polymer, but especially 200 mg or more.
~400mg is preferred. The amount of the physiologically active substance varies depending on its type, but is generally 100 to 1000 mg per gram (dry weight) of the aldehyde-activated insoluble polymer. Under the above conditions, 0~
The desired immobilized physiologically active substance can be obtained by reacting at 80°C, preferably 4 to 40°C, for 5 to 100 hours, preferably 12 to 72 hours. On the other hand, physiologically active substances include the aforementioned proteins, peptides, complex proteins, amino acids, heparin, N
-acetylmuramic acid; and fatty acids such as palmitic acid and oleic acid; and penicillin and prostaglandin; compounds having carboxyl groups can be immobilized as follows. That is, the desired immobilized physiologically active substance can be obtained by reacting the physiologically active substance with the immobilization carrier represented by the formula () in the presence of a carbodiimide compound. The carbodiimide compound used is 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter referred to as "EDC"), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide meso-p-toluenesulfonate, dicyclohexylcarbodiimide, etc. It can be used alone or as a mixture of two or more, and the amount is 10 to 10 per 1 g (dry weight) of the immobilization carrier.
Preferably it is 200 mg. The amount of physiologically active substance varies depending on its type, but is generally 30 to 100 per gram (dry weight) of the immobilization carrier.
mg. Generally, water is used as a solvent, and the amount thereof is 1 g (dry weight) of the immobilization carrier.
The amount is 2 to 10 ml per bottle, and sodium chloride, potassium chloride, etc. may be added to the solvent as necessary. The desired immobilized physiologically active substance can be obtained by reacting under the above conditions at 0 to 80°C, preferably 4 to 40°C, for 10 to 100 hours, preferably 24 to 72 hours. According to the production method of the present invention, under mild conditions and without adjusting the pH during the reaction,
The desired immobilized physiologically active substance can be obtained in a shorter reaction time than conventional methods. Furthermore, the obtained immobilized physiologically active substance has a higher concentration of ligand than conventional substances. Hereinafter, the present invention will be explained in more detail with reference to Examples. Example 1 (1) Preparation of hydrazine agarose Sepharose 4B manufactured by Pharmacia was used as agarose. After thoroughly washing with water on a glass filter, 20 g of the suctioned agarose was placed in a flask, and 30 ml of water, 13 ml of 2M NaOH aqueous solution, and 3 ml of epichlorohydrin were sequentially added. suspension
After shaking in an incubator at 40°C for 2 hours, the mixture was thoroughly washed with water on a glass filter to obtain epoxy-activated agarose. When the amount of epoxy groups introduced into this epoxy-activated agarose was measured by neutralization titration using Na ₂ S ₂ O ₃ , it was found that about 500 μmol of epoxy groups were introduced per 1 mg of dry weight. 1 volume (20g) of this epoxy activated agarose
1.5 volumes (30 ml) of 80% hydrazine hydrate aqueous solution was added, and the mixture was shaken for 1.5 hours in an incubator at 40°C. After the reaction, hydrazine agarose was obtained by thoroughly washing with water on a glass filter. (2) Preparation of immobilized lactose Add 520 mg of lactose and 256 mg of sodium cyanoborohydride to 2 g of hydrazine agarose obtained in (1) [dry weight (wet weight 20 g)] in a 0.2 MK ₂ HPO ₄ aqueous solution.
The solution was added to 15 ml and shaken at room temperature for 3 days. After the reaction, add 10ml of water and
It was washed sequentially with 10 ml of 0.2M CH ₃ COONa aqueous solution.
Next, 5 ml of acetic anhydride was added and the reaction was allowed to proceed at 0°C for 30 minutes, followed by further addition of 5 ml of acetic anhydride and the reaction was continued for 30 minutes at room temperature.
The mixture was treated for 1 minute to acetylate unreacted hydrazine residues (in which lactose was not immobilized). After the reaction, lactose-bound agarose was obtained by sequentially washing with water, 0.1M NaOH aqueous solution, water, and phosphate buffered saline (PBS). Example 2 (1) Preparation of adipic acid dihydrazide agarose Sepharose CL6B manufactured by Pharmacia was used as agarose. Epoxy-activated agarose 1 obtained by the same treatment as in Example 1 (1)
Add 115g of adipic acid dihydrazide to 0.1M
A solution dissolved in an aqueous Na ₂ CO ₃ solution and adjusted to pH 9 with hydrochloric acid was added, and the mixture was shaken at 40° C. overnight. After the reaction, the mixture was thoroughly washed with a 0.2M NaCl aqueous solution on a glass filter to obtain adipic acid dihydrazide agarose. (2) Preparation of immobilized dextran sulfate Adipic acid dihydrazide agarose obtained in (1)
A solution of 26 g of dextran sulfate (manufactured by Pharmacia) and 12.8 g of sodium cyanoborohydride dissolved in 750 ml of a 0.2M K ₂ HPO ₄ aqueous solution was added to 100 g [dry weight (wet volume 1)], and the mixture was shaken at room temperature for 3 days. . After the reaction, pour water on a glass filter,
Washed sequentially with 0.2M CH ₃ COONa aqueous solution. Next, 250 ml of acetic anhydride was added, and after shaking at 0°C for 30 minutes,
Further, 250 ml of acetic anhydride was added, and the mixture was shaken at room temperature for 30 minutes to acetylate unreacted adipic acid dihydrazide residues (in which dextran sulfate was not immobilized). After reaction, water, 0.1M NaOH aqueous solution,
Dextran sulfate-bound agarose was obtained by sequentially washing with water and PBS. Dextran sulfate in the 0.2M K ₂ HPO ₄ aqueous solution containing dextran sulfate before the reaction and in the supernatant after the reaction was measured according to the phenol sulfuric acid method.
25% were found to be combined. Example 3 (1) Preparation of aldehyde-activated agarose 1 ml of a 25% glutaraldehyde aqueous solution and 82 mg of sodium cyanoborohydride were added to adipic acid dihydrazide agarose obtained by the same treatment as in Example 2 (1). After shaking in an incubator at 40°C for 2 hours, the mixture was thoroughly washed with water on a glass filter to obtain aldehyde-activated agarose. (2) Preparation of immobilized ConA Add 1 g of aldehyde-activated agarose obtained in (1) to
ConA 100 mg and methyl-α-D-mannoside 110
After adding 2 ml of 0.2 M phosphate buffer (PH7.0) in which 1.0 mg of sodium cyanoborohydride had been dissolved, 24 mg of sodium cyanoborohydride was added, and the mixture was shaken in an incubator at 4°C for 2 days. After the reaction, the supernatant was removed and thoroughly washed with 0.2M phosphate buffer to obtain ConA-bound agarose. (3) Measurement of ligand concentration The amount of unbound remaining ConA in the supernatant and washing solution was
The amount of ConA bound to the agarose was calculated from the measured value of absorption at 280 nm. As a control, an enzyme bound to agarose by the CNBr method [Ichiro Chibata et al., "Affinity Chromatography", p. 40, Tokyo, Japan;
Kodansha (1976)] (Control 1), BSA bound to various polysaccharides by periodate oxidation method
[Immunology, Vol. 20, pp. 1061-1065 (1971
)] (Control 2) and BSA bound to a hydrazide derivative [Biochemistry, Vol. 8, pp. 4074-4082 (1969)] (Control 3) were used. The results are shown in Table 1.

[Table] From the table, it can be seen that the immobilized physiologically active substance of the present invention has a higher ligand concentration than the conventional one. Example 4 Preparation of immobilized BSA 15 ml of 80% hydrazine hydrate aqueous solution was added to 10 g of epoxy-activated agarose obtained by the same treatment as in Example 1 (1), and the mixture was incubated in an incubator at 40°C for 1.5 hours. Shake. After the reaction, it was thoroughly washed with water on a glass filter, and it was confirmed that no color was formed due to phenolphthalein. Next, 2 ml of 0.5 M NaCl aqueous solution in which 50 mg of BSA was dissolved.
was added, and then 33 mg of EDC was added. After adjusting the pH to 5, the mixture was shaken at room temperature overnight, and then 33 mg of EDC was added and shaken overnight. After the reaction, the mixture was washed on a glass filter with 8 ml of 0.5M NaCl aqueous solution and 100 ml of water in order to obtain BSA-bonded agarose. By calculating the amount of unbound residual BSA in the washing solution from its absorbance measurement at 280 nm, the resulting BSA-bound agarose has a
It was found that 47mg of BSA was bound. Example 5 (1) Preparation of adipic acid dihydrazide agarose 2.3 g of adipic acid dihydrazide was added to 20 g (wet weight) of epoxy activated agarose obtained by the same treatment as in Example 1 (1) with 0.1M Na ₂ CO ₃ A solution dissolved in an aqueous solution and adjusted to pH 9 with hydrochloric acid was added, and the mixture was shaken at room temperature overnight. After the reaction, add 0.2M on a glass filter until the washing solution shows no reaction with trinitrobenzenesulfonic acid (TNBS).
Adipic acid dihydrazide agarose was obtained by washing with about 500 ml of NaCl aqueous solution. (2) Preparation of immobilized BSA Adipic acid dihydrazide agarose 1 obtained in (1)
0.5M NaCl aqueous solution 2 in which 50mg of BSA was dissolved in
ml and then 33 mg of EDC. After adjusting the pH to 5 and shaking at room temperature overnight, add 33 mg of EDC.
was added and shaken overnight. After the reaction, the mixture was washed on a glass filter with 8 ml of 0.5M NaCl aqueous solution and 100 ml of water in order to obtain BSA-bonded agarose. By calculating the amount of unbound residual BSA in the washing solution from the measured absorption at 280 nm, it was found that 38.5 mg of BSA was bound per gram of wet weight in the obtained BSA-bound agarose. found.

Claims (1)

[Claims] Primary formula: [In the formula, M represents a water-insoluble polymer residue; R ¹ , R ₂
and R ³ are each a hydrogen atom or a carbon number of 1 to 5
an alkyl group; _X is a hydrogen atom _{, a} physiologically active substance residue having a carbonyl group or a carboxyl group, _or the following formula: group; y is an integer of 2 to 10; each represents a residue; Y is -(CH ₂ ) _o - (n is 0 to 8
(an integer of ), -CH=CH- or [Formula]; x represents 0 or 1, respectively] An immobilized physiologically active substance and a water-insoluble polymer. 2 A water-insoluble polymer is reacted with an epihalohydrin compound or a bisoxirane compound, and then this is reacted with NH ₂ NH ₂ or NH ₂ NH-CO-Y-CO-
NHNH ₂ [Y is -(CH ₂ ) _o - (n is an integer from 0 to 8),
−CH=CH− or [representing the formula]], the following formula: (In the formula, M is a water-insoluble polymer residue; R ₁ , R ₂
and R ₃ are each a hydrogen atom or an alkyl group having 1 to 5 carbon atoms; Y is the same as above; x is 0 or 1
Represents each. ) Production of an immobilized physiologically active substance, characterized in that the hydrazine derivative and/or dihydrazide derivative shown in Law. 3. React a water-insoluble polymer with an epihalohydrin compound or a bisoxirane compound, and then react this with NH ₂ NH ₂ or NH ₂ NH-CO-Y-CO-
NHNH ₂ [Y is -(CH ₂ ) _o - (n is an integer from 0 to 8),
−CH=CH− or [representing the formula]], the following formula: (In the formula, M is a water-insoluble polymer residue; R ¹ , R ²
and R ³ are each a hydrogen atom or an alkyl group having 1 to 5 carbon atoms; Y is the same as above; x is 0 or 1
respectively) to obtain a hydrazine derivative and/or dihydrazide derivative represented by the following formula: OHC-( _CH2 ) _y-2 -CHO (y is 2 to An integer of 10) is reacted with a linear dialdehyde compound represented by the following formula: (In the formula, M, Y, R ¹ , R ² , R ³ , x and y are the same as defined above) Below, a method for producing an immobilized physiologically active substance, which comprises reacting with a physiologically active substance having an amino group. 4. React a water-insoluble polymer with an epihalohydrin compound or a bisoxirane compound, and then react this with NH ₂ NH ₂ or NH ₂ NH-CO-Y-CO-
NHNH ₂ [Y is -(CH ₂ ) _o - (n is an integer from 0 to 8),
-CH=CH- or [representing the formula]], the following formula: (In the formula, M is a water-insoluble polymer residue; R ₁ , R ²
and R ³ are each a hydrogen atom or an alkyl group having 1 to 5 carbon atoms; Y is the same as above; x is 0 or 1
Production of an immobilized physiologically active substance, which is characterized by obtaining a hydrazine derivative and/or dihydrazide derivative represented by: Law.