JPH04183392A - Production of membrane immobilizing physiologically active substance - Google Patents

Abstract

PURPOSE:To produce a membrane containing a physiologically active substance immobilized therein by reacting a cellulosic asymmetric membrane with a compound expressed by a specific formula, activating the aforementioned membrane and then immobilizing the physiologically active substance therein through chemical bonds. CONSTITUTION:A cellulosic asymmetric membrane is reacted with a compound expressed by formula I (R denotes formula II, III, TV or V) and activated and a physiologically active substance is then immobilized through chemical bonds to produce the objective membrane containing the physiologically active substance immobilized therein. The cellulosic membrane used in the aforementioned method is an asymmetric membrane in the form of a flat membrane or hollow fiber having a dense surface layer on one side or one membrane and a relatively coarse cellular layer on the surface on the opposite side. Fine agarose or cellulosic particles are reacted with 1,1'-carbonyldiimidazole, etc., in order to react and activate the cellulosic membrane with the compound expressed by formula I. The physiologically active substance to be immobilized is an enzyme, a coenzyme, an antibody, a hormone, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION (Technical Field of the Invention) The present invention j relates to a method for producing a membrane on which a physiologically active substance is immobilized.

(Prior art) Immobilized physiologically active substances obtained by immobilizing physiologically active substances such as enzymes, coenzymes, hormones, receptors, and inhibitors on insoluble carriers can be used as materials for bioreactors, biosensors, affinity chromatography, etc. chemicals·
It is widely used in food and pharmaceutical manufacturing, clinical diagnosis, treatment, etc. In these cases, the forms of the immobilized physiologically active substances used include particulates and membranes, both of which are being studied by various groups.

Immobilized bioactive substances using porous particulate carriers are the most well-studied, but the efficiency of immobilized bioactive substances is reduced due to diffusion limitations based on unstirred layers within and around the particles. It has been pointed out that in some cases, only a small portion of the latent activity is utilized (for example, Japanese Patent Publication No. 5G-3D6T3).

As a countermeasure to this problem, a reaction method has been developed in which, for example, an enzyme is immobilized on a microporous membrane and an aqueous substrate solution is permeated through the membrane using pressure.

This 1m bioactive substance is transferred to a bioreactor.
In order to be widely applied to biosensors, affinity separation materials, clinical diagnosis, etc., it is important that the sample carrier has as little nonspecific interaction with proteins as possible, and for this purpose, it must be highly hydrophilic. An uncharged polysaccharide such as cellulose is preferable as the material.

Several methods are already known for immobilizing enzymes and other physiologically active substances on porous cellulose membranes through chemical bonds. For example, JP-A No. 56-97235,
Japanese Patent Publication Nos. 58-44357 and 62-32919 disclose a method in which an oxidizing agent is applied to a cellulose membrane to activate it and then an enzyme is immobilized thereon.

Furthermore, British Patent No. 1.183.261) discloses a method in which a triazine derivative is applied to a membrane of cellulose or the like to activate it and then an enzyme is immobilized thereon.

These known methods make it possible to obtain cellulose membranes with immobilized biomaterials, but these methods still have some problems and are not practical. te;mana′,). In other words, in the activation method using an oxidizing agent, it is thought that the aldehyde groups generated on the carrier are immobilized by reacting mainly with the amide 7 group in the physiologically active substance to form a buffer base. However, since Nuffbase is unstable, it is easy to desorb the two-sided physiologically active substance O.18 Also, in order to prevent desorption, ＼a3H,
If you use a method of reducing constant NaBH mail, etc., the processing value will be lower than that of the second method. Physiology-Hundred substances! A method for immobilizing physiologically active substances using triazine derivatives was developed as a method that requires a second-step reaction such as reduction after settling, but with this method: Well! Because the rate of chemotactic reaction is slow, immobilization requires a relatively long period of time, and in the case of unstable physiologically active substances, deactivation of the chemotactic reaction may become a problem. Patent No. 1,183,260 shows that immersing a membrane (filter paper or Cobuton cloth) in an alkali upon activation of the membrane with a triazine derivative; When this method is applied to an asymmetric membrane consisting of a single thin fiber like a filter), the membrane is considerably deformed due to the hydrolysis of cellulose.
Furthermore, there is another problem when the strength deteriorates.

Therefore, it has been desired to develop an immobilization method that does not impair the physical properties of cellulose asymmetric membranes due to activation and that uses activation means that are highly reactive to proteins.

<Problems to be Solved by the Invention> The purpose of the present invention is to provide a method for immobilizing physiologically active substances through stable chemical bonds while maintaining physiological activity as much as possible on an asymmetric membrane made of cellulose that has excellent properties against fluids. Our goal is to provide the following.

(Means to solve the problem) (
The purpose of the present invention is to produce an asymmetric cellulose membrane with the following general formula (
This was achieved by reacting with the compound represented by I) to activate it, and then immobilizing the physiologically active substance through chemical bonding.

General formula (1) The process of the method of the present invention is carried out according to the following scheme in which cellulose> -[]H-R-c-R→(cellulose)- and -R
Mouth (cellulose> -0-mouth-R-112N - (physiologically active substance) □ (cellulose) -〇-lowNH- (physiologically active substance) Cellulose membrane used in the present invention; dense membrane on one S side of the membrane It is an asymmetric membrane in the shape of a flat membrane or hollow fiber, which has a surface layer and a relatively rough porous layer on the opposite surface.Porous membranes with pore diameters in the range of 0.1μ to 10μ
For example, a microfilter) or an extraocular filtration membrane having a smaller pore size and a molecular weight cut-off of 1.000 to 1,000.000 can be preferably used. Regarding the manufacturing method of this membrane, for example, Japanese Patent Publication No. 45-4633, Japanese Patent Publication No. 15642-1982, Japanese Patent Publication No. 34343-1983.
Disclosed in the issue. , pancreatic grass (thickness of the wall portion in the case of hollow fibers); tioμ or more can be preferably used, and 10μ to 300μ is more preferred.

Next, a method of activating a cellulose membrane by reacting it with a compound represented by the general formula (I>) will be described.

It is known that an activation reaction based on a similar @ structure is performed on particles (St, T, W, Heavn: !Je
thods inEnzymology vol, 1
35.102-11-. (1987)), and its contents: microscopic agarose and cellulose particles with 1,
It is activated by the action of 1'-carbonylimidasole, L+1'-carbonyl-1,2,4-1-lyasole, etc. Its standard activation method is 1 year old, micro-;
1 for 0.3 g of agarose or cellulose particles,
74 mmol of 1'-carbonylimidasole Q and 20 ml of dioxane were added and reacted at room temperature for about 15 minutes.

However, the activation method for membranes is unknown.
.

The present inventor attempted to activate the asymmetric cellulose membrane j= under the reaction conditions described in the above-mentioned document J, but A was not activated. As a result of searching for various reaction conditions, we found that cellulose membranes could be activated under the following conditions.

That is, 5 to 80 ITIITloβ,
Preferably 20-4QmmoA and solvent 10-100
-1 preferably 50 mI! Activation was performed by adding and reacting at room temperature for 5 to 15 minutes. Examples of the solvent used here include acetone, dioxane, pyridine, acetonitrile, toluene, ethyl acetate, and chloroform. Reaction method discovered at Uni: Well, one sentence of the compound represented by general formula (I) is extremely high.'')
This method is significantly different from known methods.

The amount of the active group introduced by the activation reaction is determined by the following scheme: As shown, a mino group is introduced by reacting with ethylenediamine, and the following formula is used:

1: Ta, Vol. 58, p. 248, 1972), it can be quantified by measuring the amount of ionic bond between picric acid and amine group.

(cell o-s)-0-[knee R=lh['LCLNL-
(Cell D-su) -0CNHCH2CH2NH2 Also, 0.0.
1-1Qmoβ%, more preferably 0.5-3.5mo
f% is preferably introduced for immobilization of physiologically active substances.

Next, we will describe a method for immobilizing physiologically active substances on activated cellulose membranes. First, the physiological activity (fi: dissolves the substance) to be defined in a buffer in the pH range of 7 to 9.5.
(MES>Buffer, one that does not have a primary amine group, such as human ethyl piperazine ethane sulponfer, is preferable.) Also, if necessary, a stabilizer for the physiologically active substance may be added to the container. Moyoshi 1. Pour the second solution into the cellulose membrane under pressure and let it circulate and react at 4 to 40°C for 1 to 12 hours. After the immobilization reaction, pass the buffer solution through the membrane and remove the membrane. Further, by passing an aqueous solution of an alkali or an amine such as ethanolamine, the active groups remaining on the membrane are reacted with and inactivated, and finally, the membrane is washed again with a buffer solution.

By the method described above, a physiologically active substance can be immobilized on an asymmetric cellulose membrane.

Physiologically active substances immobilized according to the present invention include enzymes, coenzymes, antibodies, hormones, receptors, lectins, inhibitors, and the like. Enzymes; for example, hydrolytic enzymes such as vitripsyne, chymotrybun, thermolysin, papain, asparaginase, reversese, amylase, cellulase, lysozyme, urease, alcohol dehydrogenase, lactate dehydrogenase, glucose dehydrogenase, aldehyde dehydrogenase, D-amino acid oxidoreductases such as ocintase, peroxidase, catalase, and super occhintodismutase; transferases such as hexokinase, taleatin kinase, and alanine transaminase; lyases such as fumarase, aspartate ammonia lyase, threonine aldolase, and pyruvate decarboxylase; Examples include isomerases such as glucose phosphate isomerase and methylmalonyl CO^mustase, glutamine ncetase, acetyl CoA carboxylase, and glue gauze such as D N and A IJ gauze. Other physiologically active substances include cofermented S derivatives such as Ng-(6-aminohexyl)-A~fp, immunoglobulins and their Fab fragments, lacteal Fc receptors such as protein A, and lectins such as concanavalin A and wheat germ agglutinin. , soybean tribone, inhibitors, amino acids such as phenylalanine and triadophane, and fibronectin and its partial sequences.

(Effects of the invention) There is little deactivation during the immobilization of physiologically active substances on activated carriers, and the physiologically active substances do not desorb over time (): This is extremely important for immobilized physiologically active substances. be.

In this respect, the known methods for cellulose membranes, namely the activation method with oxidizing agents and the activation method with triazine derivatives, still have to be improved.
. The cellulose membrane activated according to the present invention has high reactivity with physiologically active substances, and also requires time for diffusion because a pump is used to send the physiologically active substances into the membrane.
,). As a result, in the case of many physiologically active substances, the
Immobilization is completed in a few hours, forming a stable chemical bond. This: In contrast to the above-mentioned known methods in which immobilization is carried out at 4°C for 12 hours or at 40°C for one day and night, it only takes a short time, so there is little decrease in physiological activity'-)C Since the bonding site with the cellulose membrane and physiologically active substances is phylic and nonionic, there is no non-specific adsorption of proteins, etc., making it suitable for applications in sensors and affinity separation materials 5).

Since cellulose membranes can withstand most solvents, the immobilized physiologically active substances of the present invention can also be used in organic solvent systems.

(Example) Next, the present invention will be explained in further detail by giving examples.
The main departure station is limited to this one: Yona-).

Example 1. Production of activated membrane 4 g (25 mmol) of 1゜1'-carbonyldiimidazole and 50 mmol of anhydrous dioxane were added to 0.3 g of cellulose microfilter FR-4o (made by x±photographic film ■, pore size 0.4 μ). . After activation by shaking for 15 minutes at room temperature, the membrane was thoroughly washed with methanol and acetone. This was dried to obtain an activated cellulose membrane into which 3.2 mol % of active groups were introduced.

Similar? ; Microfilter FR-40 (0,3g
) is activated at room temperature and the reaction conditions and activation amount are shown in Table 1. In addition, the standard for activating particles -
Table 1 shows comparative examples in which one activation method was applied to membranes.

Table 1 Example 5 Preparation of trypsin-immobilized membrane Trypsin was immobilized using the activation procedure shown in Example 1.

50 mg of trypsin, containing 50 m~ACaCR,
It was dissolved in HEPES Hasofa-50 at pH 7.3.

The trypsin solution was permeated through the activated cellulose membrane set in a filter holder for filtration at 4° C. using a pump, and the membrane was immobilized by circulation for 5 hours. Next, in order to remove active groups that did not react with trypsin, 0
．． An IM ethanolamine aqueous solution was stirred at 4° C. for 5 hours to react with ethanolamine. Finally, the membrane was thoroughly washed by passing the sofa solution. The amount of immobilized enzyme was determined by quantifying the amount of enzyme remaining in the solution by ninhydrin reaction.

The activity of this trypsin-immobilized membrane was measured.

As a substrate, L-benzoylarginine-paranitroanilide (L-BAPA) was used at a concentration of 10-'M.
A solution of pf 18.2 containing M. CaCR2 dissolved in 0.05 N. Trishampher was used. Set the trypsin-immobilized membrane in the 'a universal filter holder L2
The substrate solution was permeated at 5°C, and the product concentration in the permeate was determined. The results are shown in Table 2. Comparative Example 2. Structure of trypsin-immobilized membrane by activation method (using oxidizing agent) FR40 membrane was immersed in 0.5 M sodium metaperiodate and reacted at 40°C for 5 hours. After washing this membrane with pure water, it was immobilized by circulating the same trypsin solution as in Example 1 at 4° C. for 5 hours.

Next, borohydride (IJ) at a concentration of 1/ml was added to the enzyme solution and stirred at 4°C for 2 hours. after that,
Activity measurement was carried out in the same manner as in Example 1. The results are shown in Table 2.

m-ri

Claims (1)

[Claims] A physiologically active substance, characterized in that the asymmetric membrane made of cellulose is activated by reacting with a compound represented by the following general formula (I), and then the physiologically active substance is immobilized by chemical bonding. A method for producing a membrane with immobilized. General formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ In the formula, R is ▲There are mathematical formulas, chemical formulas, tables, etc.▼, ▲There are mathematical formulas, chemical formulas, tables, etc.▼, ▲There are mathematical formulas, chemical formulas, tables, etc. Represents ▼ or ▲There are mathematical formulas, chemical formulas, tables, etc.▼.