JPH0359080B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing an immobilized physiologically active substance, and more particularly, to a method for producing a stable immobilized physiologically active substance that has a high ligand concentration. There are various methods for immobilizing physiologically active substances having amino groups such as proteins, peptides, amino acids, and biogenic amines, and these are appropriately selected depending on the intended use of the immobilized material. The embedding method is often used when the purpose is to produce specific adsorbents, immobilized enzymes, specific membranes, etc. that have high utility value in the physiological and chemical industry. However, this encapsulation method requires (1) the molecular weight of the substance in the mobile phase that acts on the immobilized physiologically active substance to be low, and (2) when the substance to be immobilized is a polymer, it is particularly difficult to form a cage. Disadvantages include that the insoluble polymer tends to have a mechanically weak structure, and (3) mass production is difficult. A method that overcomes these drawbacks is to immobilize the ligand on an insoluble carrier by covalent bonding, and is particularly suitable for enzymes that act on polymeric substrates, immune proteins and hormones that bind to polymeric substances, or their It is said to be an excellent immobilization method for physiologically active substances such as receptors. The insoluble carrier used in this method is preferably a physically and chemically stable polymer that does not non-specifically adsorb and has a binding group and an activated functional group. Among these, affinity adsorbents used in batch methods or chromatography have a high flow rate, no clogging, and
It is preferable to use particles that can increase the adsorption capacity, and when used as an immobilized enzyme, particles of porous gel-like substances are preferable in order to increase the contact area with the substrate and improve the catalytic efficiency. As such, spherical agarose gel, cellulose gel, polyacrylamide gel, polyvinyl gel, etc. have been put into practical use. An example of a covalent bond immobilization method is a method using cyanogen bromide (hereinafter referred to as "CNBr"), in which the hydroxyl group on the carrier is converted (activated) into a cyanate ester or imidocarbonate, and an amide group is added to this. There is a method of immobilization by bonding the ligands and spacers (arms that separate the ligands from the carrier to facilitate adsorption by the ligands) through isourea crosslinking. This method is often used because immobilization can be performed relatively easily, but adsorption often occurs nonspecifically, and
There is a drawback that the ligand often leaks because the binding of the immobilized ligand is unstable. In other words, the imide group formed in the crosslinking part of this method has a pKa of 10.4 and is positively charged near neutrality, whereas normal proteins are negatively charged near neutrality, so they are not susceptible to static electricity. Adsorption occurs nonspecifically due to interaction. In addition, the isourea bond forming the crosslinked portion is easily decomposed, especially in alkaline conditions. Among the methods devised to eliminate these drawbacks, one particularly effective method is a method starting from an epoxy activated carrier (hereinafter referred to as the "epoxy activation method") [Organic Synthetic Chemistry, Vol. Volume 38,
128-138 (1980)]. This epoxy activation method has the following advantages. (1) There is no electrostatic non-specific adsorption as seen in the CNBr method. (2) Spacer will be inserted automatically. (3) The alkylamino bond or ether bond, which is a bond with a ligand having an amino group or a hydroxyl group, is stable, and there is little leakage as seen in the CNBr method. (4) Some crosslinking occurs between carrier polymers, strengthening the carrier. (5) The degree of activation can be measured by the sodium thiosulfate method. However, this epoxy activation method has the disadvantage that the reaction must be carried out under alkaline conditions at high temperatures for a long time when binding the ligand. As a method for immobilizing a physiologically active substance having an amino group, the diol structure of the constituent sugar residue of a polysaccharide is oxidized with periodic acid, and the resulting aldehyde group is immobilized with a physiologically active substance having an amino group in the presence of sodium borohydride. Methods for immobilizing active substances [Immunology, Vol. 20, pp. 1061-1065 (1971
year)] is known. However, this method has the following drawbacks: (1) no spacer is included, (2) the structure of the carrier becomes weak, and (3) the concentration of the ligand is low. The present inventor first made an epoxy activated carrier into an amino derivative with ammonia, and then treated it with succinic anhydride to make a succinylamino derivative, and then
A method for immobilizing a ligand by interacting carbodiimide with a ligand having an amino group [Organic Synthetic Chemistry, Vol. 38, pp. 128-138 (1980); J.
Biochem., Vol. 87, pp. 535-540 (1980)], and achieved some results, but coupling reactions using water-soluble carbodiimides may cause side effects such as Chapman rearrangement, and are not optimal. Even at an acidity of PH4.5, side reactions in which the ligand polymerizes are more likely to occur under conditions of even higher hydrogen ion concentrations, and the yield of the immobilized product was not satisfactory. Additionally, it is necessary to adjust the pH during the reaction, and the reaction time is quite long, usually taking 12 hours. The present invention aims to eliminate the drawbacks of the conventional immobilization methods described above, to provide a novel method for producing an immobilized physiologically active substance that can be immobilized under mild conditions and has a high ligand concentration. do. As a result of extensive research, the present inventor has discovered that a polysaccharide having an aminoalkyl group, an aminated epoxy-activated polysaccharide, or an aminated epoxy-activated polyvinyl alcohol resin is reacted with a dialdehyde compound. By reacting the obtained aldehyde-activated insoluble polymer with a physiologically active substance having an amino group in the presence of sodium cyanoborohydride, it can be immobilized under mild conditions and has a high ligand concentration. , discovered that a stable immobilized physiologically active substance could be obtained,
The present invention has now been completed. That is, the first aspect of the present invention is to convert a water-insoluble polysaccharide having an aminoalkyl group into the following formula (): OHC-(CH ₂ -) _o-2 CHO () (formula (in which n represents an integer of 2 to 10) is reacted with a linear dialdehyde compound represented by the following formula (): OHC-(CH ₂ -) _o-1 NH-M ₁ () (formula (wherein, M ₁ -NH- represents a water-insoluble polysaccharide residue having an aminoalkyl group, and n represents an integer of 2 to 10) to obtain an aldehyde-activated insoluble polymer,
This is then reacted with a physiologically active substance having an amino group in the presence of sodium cyanoborohydride to form the following formula (): X-NH-(CH ₂ -) _o NH-M ₁ () ( In the formula, X-NH- represents a physiologically active substance residue having an amino group, and M ₁ -NH- and n are the same as above. Furthermore, the second aspect of the present invention is the following formula () obtained by reacting a water-insoluble polysaccharide or polyvinyl alcohol resin having a hydroxyl group with epihalohydroline or bisoxirane, and then treating it _with ammonia: −NH ₂ () (where M ₂ is

[Formula] or , m represents a water-insoluble polysaccharide residue or polyvinyl alcohol resin residue having a hydroxyl group,
(P represents an integer of 2 to 8) in the presence of sodium cyanoborohydride, the following formula (): OHC-(CH ₂ -) _o-2 CHO () ( (wherein n represents an integer of 2 to 10) A linear dialdehyde compound represented by the following formula () is reacted to form the following formula (): OHC-(CH ₂ -) _o-1 NH-M ₂ () ( (wherein M ₂ -NH- represents the aminated insoluble polymer residue, n is the same as above) to obtain an aldehyde-activated insoluble polymer,
This is then reacted with a physiologically active substance having an amino group in the presence of sodium cyanoborohydride to form the following formula (): X-NH-(CH ₂ -) _o NH-M ₂ () ( In the formula, X-NH- represents a physiologically active substance residue having an amino group, and M ₂ -NH- and n are the same as above. The present invention will be explained in detail below. Physiologically active substances having amino groups used in the present invention include concanavalin A (hereinafter referred to as "ConA"), bovine serum albumin (hereinafter referred to as "BSA"), γ-globulin (antibody), enzymes, vegetable Proteins such as hemagglutinin (PHA), fibronectin, and protein A; peptides such as kinin, corticotropin-releasing factor (CRF), calcitonin, and glucagon; various amino acids; biogenic amines such as histamine, dopamine, glucosamine, and tryptamine; These include various hormones and their receptors. Examples of the water-insoluble polysaccharide having an aminoalkyl group include aminoalkyl agarose such as aminoethyl agarose and aminohexyl agarose; and aminoethyl cellulose. Examples of the water-insoluble polysaccharide having a hydroxyl group include agarose, cellulose, chitin, mannan, etc., and examples of the polyvinyl alcohol resin include Toyopearl (trade name of Tosoh Corporation). These insoluble polymers are used in various forms depending on the purpose. For example, when used in affinity adsorbents or immobilized enzymes, porous granules are preferable in order to increase adsorption capacity and contact area, and when used in the treatment of dilute physiologically active substance solutions, , porous membrane-like ones are preferred. The immobilized physiologically active substance according to the present invention can be produced as follows. It can be produced as a carrier. When using a water-insoluble polysaccharide having an aminoalkyl group as a carrier, it is first reacted with a linear dialdehyde compound ( ) in the presence of sodium cyanoborohydride to form an aldehyde-activated insoluble polymer. (). At this time, water is used as the solvent, and the amount thereof is preferably within the range of 10 to 100 ml per 1 g of insoluble polysaccharide. The amount of sodium cyanoborohydride used may be 100 mg or more per 1 g of insoluble polysaccharide, but it is particularly preferably within the range of 200 to 1000 mg. The linear dialdehyde compound () generally includes one having 2 to 10 carbon atoms, such as glyoxal, propane dial, butane dial, glutaraldehyde (pentan dial), decane dial, etc. alone or in combination of two or more. Used as a mixture. Under the above conditions, 1 to 100 hours at 4 to 100℃, preferably 37 to 80℃, preferably
By reacting for 12 to 48 hours, an aldehyde-activated insoluble polymer can be obtained. Next, the desired immobilized physiologically active substance () is obtained by reacting this aldehyde-activated insoluble polymer () with the physiologically active substance having an amino group in the presence of sodium cyanoborohydride. be able to. At this time, examples of the solvent used include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, etc., and the pH thereof is preferably within the range of 6 to 9, and the amount thereof is determined to have aldehyde activity. The amount is preferably within the range of 5 to 50 ml per gram of the insoluble polymer (). The amount of sodium cyanoborohydride used is the amount of aldehyde-activated insoluble polymer ()
100mg or more per 1g is fine, but especially 200~
Preferably, it is within the range of 500 mg. The amount of the physiologically active substance varies depending on its type, but is generally in the range of 200 to 2000 mg per gram of aldehyde-activated insoluble polymer (). Under the above conditions,
1-72 at 0-100℃, preferably 4-40℃
By reacting for a period of time, preferably 12 to 48 hours, the desired immobilized physiologically active substance () can be obtained. In addition, when using a water-insoluble polysaccharide or polyvinyl alcohol resin having a hydroxyl group as a carrier, a known method [Organic Synthetic Chemistry, Vol. 38, 128
138 (1980)], epihalohydrins such as epichlorohydrin and epibromohydrin;
Or bis such as ethylene glycol diglycidyl ether, 1,3-propanediol diglycidyl ether, 1,4-butanediol diglycidyl ether, 1,5-pentanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, etc. Using an oxirane compound;
After the water-insoluble polysaccharide or polyvinyl alcohol resin is activated with epoxy, it is treated with ammonia, and the resulting aminated insoluble polymer () is treated in the same manner as described above to obtain the desired immobilized physiologically active substance. can be obtained. According to the production method of the present invention, it is possible to obtain the desired immobilized physiologically active substance under mild conditions, without adjusting the pH during the reaction, and in a shorter reaction time than in conventional methods. can. Furthermore, the obtained immobilized physiologically active substance is 3 to
It has a 200x higher ligand concentration. Hereinafter, the present invention will be explained in more detail with reference to Examples. Example 1 (1) Preparation of aminated agarose Sepharose 6B and Sepharose 4B manufactured by Pharmacia were used as agarose. After thoroughly washing with water on a glass filter, put 20 g of the vacuumed agarose into a flask, add 30 ml of water,
13 ml of 2M NaOH aqueous solution and 3 ml of epichlorohydrin were added sequentially. The suspension was shaken in an incubator at 40°C for 2 hours, and then 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 a neutralization titration method using Na ₂ S ₂ O ₃ , it was found that cephalose
In 6B, about 700 μmol of epoxy groups were introduced per 1 g of dry weight, and in Sepharose 4B, about 500 μmol of epoxy groups were introduced. 1.5 volumes of concentrated ammonia water was added to 1 volume of this epoxy-activated agarose, and the mixture was shaken for 1.5 hours in an incubator at 40°C. After the reaction, the mixture was thoroughly washed with water on a glass filter to obtain aminated agarose. This aminated agarose is
After treatment with 4,6-trinitrobenzenesulfonic acid, the amount of introduced amino groups was calculated by solubilizing with 50% acetic acid and measuring the absorption of trinitrophenylamine at 340 nm. , dry weight 1
Approximately 600 μmol per g, for Sepharose 4B,
Approximately 400 μmol of amino groups were introduced. (2) Preparation of immobilized ConA Aminated agarose (Sepharose) obtained in (1)
4B) 1g of 25% glutaraldehyde aqueous solution 1
ml and 82 mg of sodium cyanoborohydride were added thereto, and 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. To 1 g of this aldehyde-activated agarose,
ConA 100mg and methyl-α-D-mannoside
0.2M phosphate buffer (PH7.0) containing 110mg
After adding 2 ml, 24 mg of sodium cyanoborohydride was added, and the mixture was shaken for 2 days in an incubator at 4°C. After the reaction, remove the supernatant and
ConA-bound agarose was obtained by thorough washing with 0.2M phosphate buffer. (3) Measurement of ligand concentration The amount of unbound ConA in the supernatant and washing solution was calculated from the measured value of its absorption at 28 nm.
The amount of ConA was calculated. As a comparison,
Enzyme bound to agarose by the CNBr method [Ichiro Chibata et al., “Affinity Chromatography”, p. 40, Tokyo, Kodansha (1976)]
(Control 1), BSA bound to various polysaccharides by periodate oxidation method [Imuunology Vol. 20, 1061-
1065 (1971)] (Control 2) and BSA bound to a hydrazide derivative [Bio-chemistry, Vol.
Vol., pp. 4074-4082 (1969)] (Control 3) was 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 2 (1) Preparation of immobilized BSA 1 g of aminated agarose (Sepharose 4B) obtained by the same treatment as in Example 1 (1)
(wet weight) to 1 part of 25% glutaraldehyde aqueous solution
ml and 82 mg of sodium cyanoborohydride were added, and 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. To 0.5g of this aldehyde-activated agarose,
0.2M phosphate buffer (PH
7.0) After adding 1 ml, 12 mg of sodium cyanoborohydride was added, and the mixture was shaken overnight in an incubator at 37°C. After the reaction is complete, remove the supernatant, wash thoroughly with 0.2M phosphate buffer, and add BSA.
Bound agarose was obtained. (2) Measurement of ligand concentration The amount of unbound residual BSA in the supernatant and washing solution was
BSA bound to agarose was determined by calculating its absorption at 280 nm.
The amount of In addition, as a control, Example 1
Controls 1 to 3 similar to (3) were used. The results are shown in Table 2.

[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 3 (1) Preparation of aminated polyvinyl alcohol resin Toyopearl HW65F (manufactured by Tosoh Corporation, polyvinyl alcohol resin) 1 was washed with water, suspended in 1.5 of distilled water, and 2M NaOH aqueous solution was added to this.
650 ml was added and stirred, and then epichlorohydrin was added and reacted at 37 to 40°C for 2 hours with stirring to obtain epoxy-activated Toyopearl. Next, after washing the epoxy activated Toyo Pearl with water,
The suspension was suspended in 1.5 g of concentrated aqueous ammonia and reacted by shaking and stirring at 40° C. for 1 hour, and then the reaction product was washed with water to obtain aminated Toyopearl. (2) Preparation of immobilized wheat germ lectin (WGA) Add 100 ml of 25% glutaraldehyde aqueous solution and 8.2 g of sodium cyanoborohydride to 100 g of aminated Toyopearl obtained in (1). 2 at 40℃
The reaction was carried out for a period of time to obtain (glutar)aldehyde Toyopearl. To 100 ml of this aldehyde Toyopearl, 10 g of wheat germ lectin (WGA), 0.2 M phosphate buffer and 2.4 g of sodium cyanoborohydride were added, and after shaking and stirring at 30°C for 1 day, the supernatant was removed and 0.05 M WGA-bound Toyopearl was obtained by washing with Tris buffer. This WGA-conjugated Toyopearl is prepared in phosphate buffered saline (PBS).
It can be used by suspending it in (3) Measurement of Ligand Concentration As in Example 1, the amount of WGA bound to Toyopearl was calculated from the measured value of absorption at 280 nm to be 72 mg/ml. Example 4 (1) Preparation of aminated cellulose After washing commercially available cellulose powder (manufactured by Adavantek Co., Ltd., filter paper powder 100-200 mesh) with water, 100 g of wet cellulose obtained by degassing was added to 100 g of wet cellulose obtained by the following formula: 100 ml of 1,4-butanediol diglycidyl ether shown by was added and stirred. To this were added 0.2 g of sodium borohydride and 100 ml of a 2M aqueous NaOH solution, and the mixture was shaken and stirred to react at 25°C for 8 hours to obtain epoxy-activated cellulose. Next, epoxy-activated cellulose was suspended in 150 ml of concentrated ammonia water and reacted by shaking and stirring at 40°C for 1 hour, and then the reaction product was washed with water to obtain aminated cellulose. (2) Preparation of immobilized amylase To 100 g of aminated cellulose obtained in (1), 100 ml of 25% glutaraldehyde aqueous solution and 8.2 g of sodium cyanoborohydride were added, and the mixture was heated at 40°C for 2 hours.
The reaction was carried out for a period of time to obtain (glutar)aldehyde cellulose. 10g (wet weight) of this aldehyde cellulose
, glucamylase (manufactured by Seikagaku Corporation,
20 ml of a solution prepared by dissolving 1 g of Rhizpus nireus Glcoamylase) and 1 g of maltose in 0.2 M phosphate buffer (PH7.0) and 0.24 g of sodium cyanoborohydride were added, and the mixture was shaken and stirred at 4°C for 2 days. Remove the supernatant and add 0.1M Tris-HCl buffer (PH
8) and further washed with phosphate buffer to obtain immobilized glucamylase. (3) Measurement of Ligand Concentration As in Example 1, the amount of glucamylase bound to cellulose was calculated from the measured value of absorption at 280 nm to be 64 mg/ml.

Claims (1)

[Claims] 1. A water-insoluble polysaccharide having an aminoalkyl group is prepared by the following formula (): OHC-(CH ₂ -) _o-2 CHO () in the presence of sodium cyanoborohydride (wherein, n represents an integer of 2 to 10) to react with a linear dialdehyde compound represented by the following formula (): OHC-(CH ₂ -) _o-1 NH-M ₁ () (in the formula, M ₁ -NH- represents a water-insoluble polysaccharide residue having an aminoalkyl group, n represents an integer from 2 to 10) to obtain an aldehyde-activated insoluble polymer,
This is then reacted with a physiologically active substance having an amino group in the presence of sodium cyanoborohydride to form the following formula (): X-NH-(CH ₂ -) _o NH-M ₁ () ( (wherein, X-NH- represents a physiologically active substance residue having an amino group, and M ₁ -NH- and n are the same as above) A method for producing an immobilized physiologically active substance. 2 A water-insoluble polysaccharide or polyvinyl alcohol resin having a hydroxyl group is reacted with epihalohydrin or bisoxirane, and then treated with ammonia to obtain the following formula (): M ₂ −NH ₂ () (wherein, M ₂ is , [expression] or , m represents a water-insoluble polysaccharide residue or polyvinyl alcohol resin residue having a hydroxyl group,
P represents an integer of 2 to 8) In the presence of sodium cyanoborohydride, the aminated insoluble polymer represented by the following formula (): OHC-(CH ₂ -) _o-2 CHO () ( (wherein n represents an integer of 2 to 10) is reacted with a linear dialdehyde compound represented by the following formula (): OHC-(CH ₂ -) _o-1 NH-M ₂ () ( (wherein M ₂ -NH- represents the aminated insoluble polymer residue, n is the same as above) to obtain an aldehyde-activated insoluble polymer,
This is then reacted with a physiologically active substance having an amino group in the presence of sodium cyanoborohydride to form the following formula (): X-NH-(CH ₂ -) _o NH-M ₂ () ( (In the formula, X-NH- represents a physiologically active substance residue having an amino group, and M ₂ -NH- and n are the same as above.) A method for producing an immobilized physiologically active substance.