JPS61152634A - Production of immobilized physiologically active substance - Google Patents

Abstract

PURPOSE:To produce the titled active substance, by reacting an insoluble polymer having epoxy group with a specific monosaccharide or disaccharide under alkaline condition, oxidizing and breaking the reaction product with periodic acid, and reacting the resultant aldehyde group with a physiologically active substance having amino group, thereby immobilizing the substance. CONSTITUTION:An insoluble polymer having epoxy group (e.g. polyglycidyl methacrylate) is made to react, under alkaline condition, with a monosaccharide (e.g. glucose) or a disaccharide (e.g. lactose) having primary hydroxyl group and a diol group incisable by oxidation with periodic acid. The resultant sugar- bonded insoluble polymer is incised by oxidizing with periodic acid, and obtained aldehyde group is reacted with a physiologically active substance (e.g. protein) having amino group. If necessary, the immobilized product is reduced with sodium borohydride or sodium cyanoborohydride to stabilize the bond to obtain the objective immobilized physiologically active substance. The product has low non-specific adsorption of spacer, contains the introduced spacer having high chemical stabilty, gives high ligand concentration and has extremely high reaction yield of ligand.

Description

Detailed Description of the Invention Field of Application in Ca Industry The present invention has a spacer that is stable, non-absorbent, has little heterosorption, is hydrophilic, and has a high ligand (having affinity with the target substance). Substance) Concerning a method for producing a physiologically active substance having a certain concentration.

Ligands include protein/bacterium, vegutide, amino acids,
and biogenic amines, etc., which have a 7ξ group in the pan
1 active substance.

[Conventional technology]

Conventionally, immobilization polymer carriers used for producing immobilized physiologically active substances have no non-transferential adsorption;
Those having a sensory fund that can be activated are preferred.

In particular, when used as an affinity adsorbent, a polymer carrier that is porous, can increase adsorption capacity, is physically and chemically stable, and is molded into a spherical shape is preferred. Agarase gel, polyvinyl alcohol resin, and the like have been put into practical use as such polymeric carriers.

[Problem that the invention seeks to solve]

As a covalent bond immobilization method, there is a method using 77 bromide (Nature, Vol. 215, 1491-
1492 Sada (1967)].

This method is widely used for immobilizing amim L phosphate groups because the reaction steps are short and #1 can be easily produced.
On the other hand, the cyanogen bromide used for activation is toxic.■
When coupled with an amino group, the inclair bond forming the crosslinking part is unstable, and if the buffer used for affinity chromatography becomes alkaline, there is a risk that the immobilized primary ligand will leak out. (2) Since the PKa of the izu do group is about 10, it has a positive charge near neutrality, so it has the disadvantage that nonspecific adsorption occurs.

As a method for immobilizing substances with amine groups, there is a periodate oxidation method for polysaccharides (Immunology,
Vol. 20, pp. 1061-1065 (1971)].

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, with this method, it is not possible to introduce subeners (molecules of arbitrary length that are introduced between the ligand and the carrier);
It has a decisive flaw as an affinity adsorbent in that the ligand concentration is low.

[Means for solving problems]

The present invention is based on the aforementioned cyanogen bromide method, polysaccharide periodic acid a!
This method has greatly improved the drawbacks of the folding method, etc., and has hydroxyl groups, amino groups, carboxyl groups, and Fi/$p and An insoluble polymer carrier having an amide group is reacted with a monosaccharide or 211 having a primary hydroxyl group and a diol group that can be cleaved by periodine afR reaction under alkaline conditions, and the resulting sugar-bonded insoluble polymer is 7(
) group to form a thick base between them to immobilize them, and if necessary, react this reaction product with commonly used sodium borohydride or sodium cyanoborohydride. There are nine ways to stabilize the bond by reducing it.

The epoxy group-containing non-habitat polymer conveniently used in the method of the present invention may be any water-insoluble and stable polymer in which an epoxy group has been introduced through a chemical reaction into a certain epoxy group. .

Examples of insoluble polymeric carriers having epoxy groups include polyglycidyl methacrylate or copolymers thereof, and polyester resin (64). When these carriers are used, they are subjected to the reaction as they are. Examples of insoluble polymer carriers having hydroxyl groups include agarose, polyvinyl alcohol resin, hydroacetyl (or methyl) acrylate, copolymers thereof, hydroacetyl (t or methyl) methacrylate, or The combination, N-methylol acrylic eye F t 7t if
(-(') copolymers, etc.) Examples of insoluble polymeric carriers having amino groups include azunoethyl agarose, azinohenochyl agarose, ainoethyl polyacrylamide, etc. Examples of insoluble polymeric carriers having a polymer of Examples of insoluble polymer carriers having amide groups include polyacrylamide-based polyamides, etc. When using these carriers, shrimp bromohydrin, shrimp It is necessary to introduce an epoxy group using a bromohydrin such as bromohydrin or a bisoxirane compound, and then subject it to the reaction.

The sugar-bonded insoluble polymer carrier is prepared by adding 3 to 50 ml of an aqueous alkaline solution (PH 11 to 13) to the suction-filtered epoxy group-containing carrier, and then adding primary hydroxyl groups to the epoxy group-containing carrier. a A monosaccharide or disaccharide having a diol group that can be cleaved by oxidation is added, and the reaction time is preferably 5 to 30 hours while shaking at 20°C to 40°C.

The monosaccharide or disaccharide to be added has an epoxy group [the amount of epoxy group is determined according to Aundberg et al.
, Vol. 90, pp. 87-98, 1974).

In the produced monosaccharide or di-spun insoluble polymer carrier, the mono-m or Fi-disaccharide is the highly reactive monosaccharide or Fi disaccharide that is most reactive with the epoxy group.
It is bonded through the hydroxyl group of alcohol.

Examples of the monosaccharide having a primary hydroxyl group and a diol group which can be cleaved by periodic awakening to be used in the method of the present invention include aldohexyl saccharides such as glucose, manno-, ttyp-dose, and talose. North, N-acetylglucona (n,
Examples include mono-inosaccharides such as N-acetylgactone, etc., and lactose,! Lutose, Shiw@, etc. are included.

The oxidative cleavage of single IIL or 28m-bound insoluble polymeric carriers is carried out using a single IIL or a salt thereof, preferably α04-α
2Nm! The carrier 1 was added to the 11 or 2m bonded insoluble polymer carrier obtained by suction filtration of the sodium phosphate aqueous solution.
It is preferable to carry out the reaction by adding 100° C. and reacting at 0° C. to 20° C. for 30 minutes to 5 hours.

When glucose is used as the monosaccharide having a primary hydroxyl group and a diol group that can be cleaved by periodation, subener of structural formula [1] is mainly introduced.

(In the formula, G represents an insoluble polymer carrier, and %A/m represents the site of oxidative cleavage by sodium periodate.

The same applies to the following formulas. ) In addition, subenars of structural formulas (2) (5) (4) (5) are introduced, but all subenars have an aldehyde group and are effective for immobilizing physiologically active substances.

H ↓ HO When using lactose as 2111 having a primary hydroxyl group and a diol group that can be cleaved by converting it into a periodine unit, subates of structural formulas (6) and (7) are mainly introduced.

Others: (Similar to the case of glucose, Svener with several types of aldehyde groups and monosaccharides that cannot be oxidized are introduced. Svener with aldehyde groups is effective for immobilizing physiologically active substances. b, again,
Spacers containing sugar are stable and do not interfere with affinity chromatography.

When the aldehyde-activated insoluble polymer carrier obtained by the above reaction is reacted with a physiologically active substance having an agno group, such as protein, peptide, aξ) acid, bioactive amine, etc., and immobilized, An immobilized physiologically active substance can be obtained.

Examples of physiologically active substances having an amino group include lectins, comb serum albumin, r-globulin (antibodies), enzymes, fibronectin, proteins such as proteans, complex proteins such as chromoproteins, glycoproteins, and phosphoproteins, and kinins. , corticotropin-releasing factor (
ORF), peptides such as calcto-2/glucago-7, various 1ζ acids, and biogenic eins such as protein, dopa, and tryptamine.

The solvent used at this time is a buffer such as a phosphate buffer or an acetate buffer, and if the activity of the physiologically active substance to be immobilized is pH-dependent, the appropriate pH is 0, and in general, the pH is 1j.
A method in which the reaction is carried out within the range of PH5 to PH10 is preferred.
.

The amount of the buffer solution dissolved in the physiologically active substance tm is filtered by suction, and the 9-aldehyde-activated insoluble polymer carrier 1 is added to the 2- to 10-
It is preferable that it is within the range of .

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 residual physiologically active αIIJ mass during the immobilization reaction, but is usually 5 hours to 72 hours.
Time is enough.

After the above reaction, fA of sodium borohydride is added to the cyanoborated sodium borohydride to reduce and stabilize the generated thick 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 reducing agent is 30 m9 or more per 12 suction filtration carriers.
Reaction for ~24 hours is sufficient.

The obtained physiologically active substance-immobilized insoluble polymer carrier was suctioned with cyanofluorinated sodium filtration carrier (12 equivalents F) 30 m
? Any amount including above [L5~2M to! Add HCl buffer solution (pm 7.4) and incubate at 3°C within the range of 0°C to 40°C.
Leave it for 0 minutes to 2 hours. This allows aldehyde groups remaining on the carrier to be guided to inert groups.

As shown in the above reaction, it is possible to introduce high-concentration glue, and by changing the reaction conditions of persuccinic acid or by changing the amount of the physiologically active substance that serves as the ligand, the ligand concentration can be freely adjusted. can be changed. The immobilization yield is also excellent at about 90%.

〔Effect of the invention〕

According to the method of the present invention, the drawbacks of conventional immobilization methods for physiologically active substances can be significantly improved.

In other words, regarding subener, ■ it has no charge and less nonspecific IIk deposition, ■ it is chemically stable after introduction (b#), there is less leakage of the ligand given by the cyanogen bromide method, and ■ it is hydrophilic. It has characteristics such as having a soft and smooth texture.

Regarding the ligand, it has the following characteristics: (1) high ligation/performance ratio can be obtained, and the amount of the ligand can be changed freely; and (2) the reaction yield of the ligand is very high.

In addition, 1) the introduction of epoxy groups forms crosslinks and strengthens the carrier; 2) the carrier KJI can be bonded and stored for a long period of time in the following conditions;
It also has the -5 feature that it can be converted into m and used for immobilization.

〔Example〕

Next, examples of the present invention will be shown, and the misfire 1jII will be further explained.

Example t (Pharmacia Co., Ltd. 77 Allose 4B was used as a g4 agarose gel of recurse-immobilized agarose.

After washing thoroughly with water on a glass filter.

Epoxidation is suction filtered agarose gel 30? 50 to
% dimethylsulfonate 45++d, 2M NaO
H20d, shrimp chlorhito, phosphorus 4.5- added, 4
After the 0 reaction, which was shaken at 0°C for 2 hours, it was transferred to a glass filter, thoroughly washed with ice, and the epoxy activated 7 allose 4
I lost B.

Using the introduced epoxy group FiN&28203, steel was determined by the method of Sundbeng et al.
Approximately 40 μmol of epoxy groups were introduced per 1F of suction filtration Sepharose 4E.

Glue: l-
A 1N NaOH bath solution of 80-Q in which x5f was dissolved was added, and the mixture was shaken at 37°C for 20 hours. After the reaction, the mixture was washed thoroughly with water on a glass filter to obtain glucose-bound agate.

(2) Immobilization of lectin CLIN sodium periodate water bath solution 150- was added to glucose-immobilized agarose 15f, and the mixture was shaken at 4°C for 1 hour. After the reaction, transfer to a glass filter and add acidic water bath solution (
After thorough washing with pH 3) and further washing with water, aldehyde-activated agarose was obtained.

For aldehyde-activated agarose 5tK, 11M6 acid buffer (PH6,0) containing Concanavalia A (hereinafter referred to as "0onAJ") and α2Mα-methylmannoside (PH6,0)
Add 11t and go north. The amount of ConA is 58m? , 110mF
, 156m? , 256m? .

and shaken at 4°C for 48 hours.

After the reaction, 100 mW of sodium cyanoborohydride was added to each reaction mixture, 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 Q, 1M acetate buffer (PH6,0) to obtain ConA-1 galose.

Add 1M Tris-hydrochloric acid laxative solution (P) 17.4) 15- containing 100 mft of sodium cyanoborohydride to the obtained ConA-agarose 3f, and after leaving it at room temperature for 1 hour, place it on a glass filter with ice. Washed.

The amount of immobilization was determined by the absorbance of the original ConA at 280 nm and the absorbance of the remaining ConA at 28 On! Based on the difference in absorbance of 11, determine the amount of immobilization and ratio. The results are shown in Table 1.

1!1 The amount of immobilization is indicated by 00nk, and as shown below, 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 immobilized para-aminophenylmannoside that interacts with 90onk.

Gel F1111It [Column (diameter J wm x length x
83) E was used, and para-aminophenyl galactoside, which has no interaction with 0orsk, was used as a control. The results are shown in Table 2.

Table 2 Furthermore, the amount of adsorption was assayed using a high-inose type soybean lectin (hereinafter referred to as "sB^") having 5ift. aon*-agarose 1 ml (18 mW
0 onk/ml gel) 34 my of j58BA was adsorbed.

Immobilized 0onA agarose has sufficient adsorption capacity,
Adsorption even when used repeatedly! a! No decrease was observed.

Example 2 (1) Lactose-immobilized agarose! 14 made A3
a-Sedge) Pharmacia Sepharose 4Bt as b
-Used.

After thoroughly washing with water on a glass filter, shrimp quaruhydrin 4.5- was added and shaken at 40C for 2 hours.
After the reaction, it was transferred to a glass filter and thoroughly washed with water to obtain epoxy-activated 7ya-x4Bk. Using the introduced epoxy group FiNa28205, Sundb
The ratio measured by the method of SNG et al. Approximately 40 μmol of epoxy groups were introduced into 7Aq-4B1f through suction filtration.

This epoxy-activated agarose sedge h20P<lactose 6ff: 80m cLIN NaOH solution t
-tI& was added and shaken at 37C for 20 hours. After the reaction, the mixture was thoroughly washed on a glass filter at 7 ml to obtain lactose-bonded agarose.

(2) Bovine serum albumen (hereinafter referred to as "88 people")
An aqueous solution of immobilized agarose 15PKGINi Miku Mokudou Sodium 150ml was added thereto, and the mixture was shaken at 4°C for 1 hour. After the reaction, transfer to a glass filter and add an acidic aqueous solution (p
After thorough washing with water (n3), aldehyde-activated agarose was obtained.

Aldehyde activated agarose 5fKBBA, 7-5. Q
my a1M phosphate buffer (PH7,2) 10
-t- and shaking at 25°C for 24 hours. Thereafter, 90 mft of sodium borohydride was added and the mixture was shaken for 1 hour. After the reaction was completed, the supernatant was thoroughly washed with a 1M acid buffer to obtain immobilized BAA agarose, with the same amount of immobilization as FiOonk. The difference in absorbance at 280 nm was determined.

The warping results are shown in Figure 5 together with the immobilized Con in Example 1.

For reference, the conventional method of directly oxidizing agarose with periodate (Methoda in Enzymo)
gy, Volume 38, Part B, Purchase 82, (1974)
) The amount of albumin immobilized is also shown.

Claims (1)

[Claims] (1) 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;
The aldehyde group produced by oxidative cleavage of the resulting sugar-bound insoluble polymer with periodic acid is reacted with a physiologically active substance having an amino group to immobilize it. A method for producing an immobilized physiologically active substance, which is characterized by stabilizing the bond by reduction with sodium or sodium cyanoborohydride.