JPH0456807B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention is a bioactive substance that is stable, has little nonspecific adsorption, has a hydrophilic spacer, and has a high concentration of ligand (a substance that has affinity with the target substance). Regarding manufacturing methods. As a ligand, it is applicable to all physiologically active substances having an amino group, such as proteins, peptides, amino acids, and biogenic amines. [Prior Art] Conventionally, as a polymer carrier for immobilization used to produce an immobilized physiologically active substance, one that does not have non-specific adsorption and has a functional group that can be activated is considered suitable. . In particular, when used as an affinity adsorbent, the adsorption capacity can be increased due to the porosity, and the physical
A chemically stable, spherical polymeric carrier is preferred. Agarose gel, polyvinyl alcohol resin, and the like have been put into practical use as such polymer carriers. [Problems to be solved by the invention] There is a method of using cyanogen bromide as an immobilization method using covalent bonds [Nature, Vol. 215, 1491~
1492 pages (1967)]. This method is widely used for immobilizing amino groups and phosphate groups because the reaction steps are short and it can be easily prepared. In this case, the isourea bond forming the crosslinking part is unstable, and if the buffer used for affinity chromatography becomes alkaline, there is a risk that the immobilized ligand will leak out. is about 10, so it has a positive charge near neutrality, so it has the disadvantage of non-specific adsorption. Periodate oxidation of polysaccharides is a method for immobilizing substances with amino groups [Immunology, Vol. 20, pp. 1061-1065 (1971
Year)〕. This involves oxidizing the diol structure of the constituent sugar residues of polysaccharides with periodate, reacting the aldehyde group generated by oxidative cleavage with an amino group, and further reducing it with sodium borohydride to create an insoluble immobilized substance. It's a method. However, this method has decisive deficiencies as an affinity adsorbent in that a spacer (a molecule of arbitrary length to be introduced between the ligand and the carrier) cannot be introduced and the concentration of the ligand is low. [Means for Solving the Problems] The present invention significantly improves the drawbacks of the aforementioned cyanogen bromide method, polysaccharide periodate oxidation method, etc.
An insoluble polymer carrier having an epoxy group or a hydroxyl group introduced with a spacer having an epoxy group,
An insoluble polymer carrier having an amino group, a carboxyl group, or/and an amide group is reacted under alkaline conditions with a monosaccharide or disaccharide having a primary hydroxyl group and a diol group that can be cleaved by periodic acid oxidation. A physiologically active substance having an amino group is reacted with an aldehyde group generated by oxidative cleavage of a sugar-bonded insoluble polymer with periodic acid,
A Schiff base is formed between the two for immobilization, and if necessary, the bond is stabilized by reducing this reactant with commonly used sodium borohydride or sodium cyanoborohydride. It is. The insoluble polymer having an epoxy group used in the method of the present invention may be any water-insoluble and stable polymer that already has an epoxy group or into which an epoxy group has been introduced through a chemical reaction. Examples of insoluble polymer carriers having epoxy groups include polyglycidyl methacrylate or copolymers thereof, epoxy resins, etc. When these carriers are used, they are subjected to the reaction as they are. Examples of insoluble polymeric carriers having hydroxyl groups include agarose, polyvinyl alcohol resin, hydroxyethyl (or methyl) acrylate or its copolymer, hydroxyethyl (or methyl) methacrylate or its copolymer, N-methylol Examples of insoluble polymer carriers having amino groups include aminoethyl agarose, aminohexyl agarose, aminoethyl polyacrylamide, etc. Examples of insoluble polymer carriers having carboxyl groups include acrylamide or its copolymers, etc. There are polymers of maleic acid or copolymers thereof, polymers of acrylic acid or copolymers thereof, polymers of methacrylic acid or copolymers thereof, polymers of itaconic acid or copolymers thereof, etc., which are insoluble and have an amide group. Examples of polymeric carriers include polyacrylamide-based polymers, but when using these carriers, epoxy groups are introduced using epihalohydrin or bisoxirane compounds such as epichlorohydrin and epibromohydrin by known methods. After that, it is necessary to subject it to a reaction. To create a sugar-bonded insoluble polymer carrier, per 1 g of the carrier,
After adding 3 to 50 ml of an alkaline aqueous solution (PH 11 to 13), add a monosaccharide or disaccharide having a primary hydroxyl group and a diol group that can be cleaved by periodic acid oxidation,
Shake at 20°C to 40°C. The reaction time is preferably 5 to 30 hours. The monosaccharide or disaccharide to be added has an epoxy group [the amount of epoxy group is determined according to L. Sundberg et al. (J. Chromatogr., No. 90).
Vol. 87-98, 1974). In the produced monosaccharide- or disaccharide-bound insoluble polymer carrier, the monosaccharide or disaccharide is bound via the hydroxyl group of the primary alcohol, which is most reactive with the epoxy group. Monosaccharides having a primary hydroxyl group and a diol group cleavable by periodic acid oxidation used in the method of the present invention include aldohexoses such as glucose, mannose, galactose, and talose, N-
There are amino sugars such as acetylglucosamine and N-acetylgalactosamine, and disaccharides having a primary hydroxyl group and a diol group that can be cleaved by periodic acid oxidation include lactose, maltose,
There are sugar, etc. Oxidative cleavage of monosaccharide- or disaccharide-bound insoluble polymeric carriers is performed using periodic acid or its salts, preferably 0.04
Add 3 to 100 ml of ~0.2N sodium periodate aqueous solution per 1 g of the sugar- or disaccharide-bonded insoluble polymer carrier that has been suction-filtered, and react at 0°C to 20°C for 30 minutes to 5 hours. It is preferable to do so. When glucose is used as the monosaccharide having a primary hydroxyl group and a diol group that can be cleaved by periodic acid oxidation, a spacer of structural formula [1] is mainly introduced. (In the formula, G represents an insoluble polymer carrier, and ~ ~ represents a site that is oxidized and cleaved by sodium periodate. The same applies to the following formulas.) Other structural formulas [2] [3] [4] The spacer [5] is introduced, and all of the spacers have an aldehyde group and are effective when immobilizing a physiologically active substance. When lactose is used as a disaccharide having a primary hydroxyl group and a diol group that can be cleaved by periodic acid oxidation, spacers of structural formulas [6] and [7] are mainly introduced. In addition, as in the case of glucose, spacers with several types of aldehyde groups and spacers with sugars that are not oxidatively cleaved are introduced. A spacer having an aldehyde group is effective for immobilizing a physiologically active substance, and a spacer having a sugar is stable and does not interfere with affinity chromatography. Obtaining an immobilized physiologically active substance by reacting and immobilizing the aldehyde-activated insoluble polymer carrier obtained by the above reaction with a physiologically active substance having an amino group such as proteins, peptides, amino acids, and biogenic amines. I can do it. Examples of physiologically active substances having an amino group include:
Lectin, bovine serum albumin, γ-globulin (antibody), enzyme, fipronectin, protein A
complex proteins such as chromoproteins, glycoproteins, phosphoproteins, kinins, pupetides such as corticotropin-releasing factor (CRF), calcitonin, and glucagon, various amino acids, and biogenic amines such as histamine, dova, and tributamine. etc. The solvent used at this time is a buffer such as phosphate buffer or acetate buffer, and if the activity of the physiologically active substance to be immobilized is pH-dependent, the appropriate pH is used.
In general, it is preferable to carry out the reaction within the range of PH5 to PH10. The amount of the buffer solution in which the physiologically active substance is dissolved is preferably within the range of 2 ml to 10 ml per gram of the suction-filtered aldehyde-activated insoluble polymer carrier. The reaction temperature is set at a stable activity range of the physiologically active substance to be immobilized. Preferably it is carried out at 0°C to 40°C. The reaction time is determined by measuring the amount of physiologically active substance remaining during the immobilization reaction, and usually 5 to 72 hours is sufficient. After the above reaction, sodium cyanoborohydride or sodium borohydride is added to reduce and stabilize the generated Schiff base. In the case of physiologically active substances that are unstable to reducing agents, it is preferable to use sodium cyanoborohydride. It is sufficient that the amount of the reducing agent is 30 mg or more per gram of the suction filtration carrier, and it is sufficient that the reduction reaction is carried out at a temperature of 0°C to 40°C for 2 to 24 hours. Suction 1 g of sodium cyanoboride onto the obtained physiologically active substance-immobilized insoluble polymer carrier.
Add any amount of 0.5 to 2M Tris-HCl buffer (PH7.4) containing 30mg or more per bottle, and store at 0℃ to 40℃.
Leave it for 30 minutes to 2 hours. This allows the aldehyde groups remaining on the carrier to become inert groups. The above reaction makes it possible to introduce a high concentration of the ligand, and by changing the reaction conditions of periodic acid or by changing the amount of the physiologically active substance serving as the ligand, the concentration of the ligand can be adjusted freely. Can be changed. The immobilization yield is also excellent at approximately 90%. [Effects of the Invention] According to the method of the present invention, the drawbacks of conventional methods for immobilizing physiologically active substances can be significantly improved. That is, regarding the spacer, it has no electric charge, has little non-specific adsorption, is chemically stable, has a hydrophilic spacer that causes less leakage of the ligand seen in the cyanogen bromide method, It has the following features. Further, regarding the ligand, there are advantages such as a high concentration of the ligand can be obtained, the amount of the ligand can be changed freely, and the reaction yield of the ligand is very high. In addition, by introducing epoxy groups, crosslinks are formed and the carrier is strengthened. It can be stored for a long time with sugar bound to the carrier. Immediately before the immobilization reaction, periodic acid oxidation is performed to immobilize It also has the advantage of being able to be used for various purposes. [Example] Next, Examples of the present invention will be shown to further explain the present invention. Example 1 (1) Preparation of glucose-immobilized agarose Fumarmacia Sepharose 4B was used as the agarose gel. After thoroughly washing with water on a glass filter, epoxidation was performed by adding 50 g of suction-filtered agarose gel.
% dimethyl sulfoxide 45ml, 2M NaOH20ml,
4.5 ml of epichlorohydrin was added and the mixture was shaken at 40°C for 2 hours. After the reaction, transfer to a glass filter,
Epoxy-activated Cephalose by washing thoroughly with water.
Got 4B. The introduced epoxy group was measured by the method of L. Sundbeng et al. using Na ₂ S ₂ O ₃ and was found to be approximately
40 μmol of epoxy groups were introduced. To 20 g of this epoxy-activated agarose gel was added 80 ml of a 0.1N NaOH solution in which 3 g of glucose was dissolved, and the mixture was shaken at 37° C. for 20 hours. After the reaction, the mixture was thoroughly washed with water on a glass filter to obtain glucose-bonded agarose. (2) Immobilization of lectin Add 150 ml of 0.1N sodium periodate aqueous solution to 15 g of glucose-immobilized agarose, and
Shake for hours. After the reaction, the mixture was transferred to a glass filter, thoroughly washed with an acidic aqueous solution (PH3), and then thoroughly washed with water to obtain aldehyde-activated agarose. Concanavalin A (hereinafter referred to as "ConA"), 0.2M α-
0.1M vinegar buffer containing methylmannoside (PH6.0)
Added 10ml. ConA amount is 58mg, 110mg, 156
mg, 236 mg, and shaken at 4°C for 48 hours. After the reaction, 100 mg of sodium cyanoborohydride was added to each condition, and the mixture was further shaken at 4°C for 12 hours. The reaction mixture was transferred to a glass filter and thoroughly washed with 0.1M acetate buffer (PH6.0) to obtain ConA-agarose. 15 ml of 1M Tris-HCl buffer (PH7.4) containing 100 mg of sodium cyanoborohydride was added to 3 g of the obtained ConA-agarose, and after being left at room temperature for 1 hour, it was thoroughly washed with water on a glass filter. The amount of immobilization was determined from the difference between the absorbance of the original ConA at 280 nm and the absorbance of the remaining ConA at 280 nm. The results are shown in Table 1.

[Table] As shown in ConA, by changing the amount of lectin used in the reaction, the amount of immobilization can be changed accordingly. The adsorption capacity of the obtained immobilized ConA-agarose was tested by front-end analysis using para-aminophenyl mannoside that interacts with immobilized ConA. 1 ml of gel [column (diameter 4 mm x length 8 cm)]
was used, and para-aminophenyl galactoside, which has no interaction with ConA, was used as a control.
The results are shown in Table 2.

[Table] Furthermore, the amount of adsorption was assayed using soybean lectin (hereinafter referred to as "SBA") having a high mannose type sugar chain. ConA-Agarose 1 ml (18
34mg of SBA was adsorbed per mgConA/ml gel). The immobilized ConA agarose had sufficient adsorption capacity, and no decrease in adsorption capacity was observed even after repeated use. Example 2 (1) Preparation of lactose-immobilized agarose Sepharose 4B manufactured by Pharmacia Co., Ltd. was used as the agarose gel. After thoroughly washing with water on a glass filter, epoxidation was performed by adding 50 g of suction-filtered agarose gel.
% dimethyl sulfoxide 45ml, 2M NaOH20ml,
4.5 ml of epichlorohydrin was added and the mixture was shaken at 40°C for 2 hours. After the reaction, transfer to a glass filter,
Epoxy-activated Cephalose by washing thoroughly with water.
Got 4B. The introduced epoxy group was measured by the method of L. Sundbeng et al. using Na ₂ S ₂ O ₃ .
Approximately 40 μmol of epoxy groups were introduced per gram of suction-filtered Sepharose 4B. 80 ml of a 0.1N NaOH solution containing 6 g of lactose was added to 20 g of this epoxy-activated agarose gel, and the mixture was shaken at 37° C. for 20 hours. After the reaction, the mixture was thoroughly washed with water on a glass filter to obtain lactose-bonded agarose. (2) Immobilization of bovine serum albumin (hereinafter referred to as "BSA") Add 150 ml of 0.1N sodium periodate aqueous solution to 15 g of lactose-immobilized agarose, and
Shake for an hour. After the reaction, the mixture was transferred to a glass filter, thoroughly washed with an acidic aqueous solution (PH3), and then thoroughly washed with water to obtain aldehyde-activated agarose. 3g of aldehyde-activated agarose and 250mg of BSA
10 ml of 0.1M phosphate buffer (PH7.2) in which 20% was dissolved was added, and the mixture was shaken at 25°C for 24 hours. Thereafter, 90 mg of sodium borohydride was added and the mixture was shaken for 1 hour. After the reaction was completed, the supernatant was removed and thoroughly washed with 0.1M phosphate buffer to obtain immobilized BSA agarose. The amount of immobilization was determined from the difference in absorbance at 280 nm, similar to ConA. The results are shown in Table 3 together with the immobilized ConA of Example 1. For reference, the conventional method of directly oxidizing agarose with periodate [Methods in Enzymology,
38, Part B, p. 82, 1974)] is also shown.

【table】

Claims (1)

[Claims] 1. A sugar-bonded insoluble polymer obtained by reacting an insoluble polymer having an epoxy group with a monosaccharide or disaccharide having a primary hydroxyl group and a diol group that can be cleaved by periodic acid oxidation under alkaline conditions. A physiologically active substance having an amino group is reacted with the aldehyde group produced by the oxidative cleavage of the compound with periodic acid to immobilize it, and if necessary, this reactant is treated with sodium borohydride or sodium cyanoborohydride. A method for producing an immobilized physiologically active substance, characterized by stabilizing the bond by reduction.