JPS60224618A - Carrier for immobilizing physiologically active substance - Google Patents

Abstract

PURPOSE:The titled carrier, obtained by treating a regenerated cellulose fiber aggregate with an acid anhydride based high polymer solution, easily handleable, and capable of immobilizing a physiologically active substance easily in a large amount. CONSTITUTION:A carrier for immobilizing a physiologically active substance, obtained by treating a regenerated cellulose fiber aggregate having 0.5-30 deniers, preferably 1-20 deniers fineness per fiber and >=1mm., preferably >=20mm. yarn length, e.g. staple fibers, spun yarns, knitted or woven fabric or nonwoven fabric, with a solution of an acid anhydride based polymer, e.g. polymaleic anhydride or polyacrylic acid anhydride, in 0.5-10wt% concentration, e.g. by dipping or coating, etc. to give 0.0002-20 milliequivalents, preferably 0.02-5 milliequivalents expressed in terms of carboxyl group content per g carrier, acid anhydride group content. The carrier retains 70-98% strength and elongation of the regenerated cellulose fibers used as the raw material even after immobilizing the physiologically active substance, and is capable of withstanding sufficiently use in various applications.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a carrier used for immobilizing physiologically active substances.

Enzymes, coenzymes, enzyme inhibitors, hormones, antibacterial agents.

Physiologically active substances such as antigens and antibodies are made of polyacrylamide.
Those immobilized on carriers such as cellulose, agarose, and glass are used as specific adsorbents for separation and purification of chemical reaction catalysts, materials for clinical tests, and medical materials.

For example, Weliky et al. (N, Weliky, F
, S. Brown.

and E, C, Dale+ Archives of
Biochemistry and Biophysics
s, 1311-8 (1969)) immobilize bar oxidase on fine powder of carboxymethylated cellulose using dicyclohexylcarbodiimide. Cellulose has carboxyl groups that can react with physiologically active substances such as enzymes under mild conditions.

Although it is a preferable carrier because it can introduce functional groups such as epoxy groups, finely powdered cellulose is difficult to handle and is not suitable for industrial use as a specific adsorbent for the separation and purification of chemical reaction catalysts. .

Further, JP-A-52-5393 discloses that carboxyalkylated molded articles made of cellulose are suitable for adsorbing physiologically active substances such as urokinase. However, even if this material is used as it is, it is not possible to immobilize physiologically active substances through covalent bonds, and the amount of immobilization itself cannot be expected to be large.

In view of these current circumstances, the present inventors have developed a carrier for immobilizing a physiologically active substance that is easy to handle and can easily immobilize a large amount of a physiologically active substance when used as a carrier for immobilizing a physiologically active substance. As a result of extensive research into carriers for immobilizing physiologically active substances made of cellulose, we have developed a single yarn fineness of 0.5 to 30 deniers and a yarn length of 1.
When an activated acidic cellulose fiber aggregate obtained by treating an oxidized cellulose fiber aggregate of 1 liter or more with an acid anhydride polymer solution is used as a carrier for immobilizing a physiologically active substance.

Like natural cellulose fiber, it has no uneven shape.

The present invention was achieved by discovering that it is easy to handle and can easily immobilize a large amount of physiologically active substances.

That is, in the present invention, the single yarn fineness is 0.5 to 30 deniers.

This is a carrier for immobilizing a physiologically active substance, which is made by treating a regenerated cellulose fiber aggregate with a thread length of 1 ms+ or more with an acid anhydride polymer solution.

In the present invention, cellulose means that glucose is β-1,
It refers to multiple Ws with 4-glucoside bonds. Regenerated cellulose is obtained by dissolving and regenerating pulp extracted from plant materials, and includes viscose rayon, copper ammonia rayon, saponified acetate, etc. These can be created by known methods.

Therefore, unlike natural cellulose, it is possible to obtain any single fiber denier and fiber length.1 For example, when used as a column packing material, it is possible to create and fill a column with a shape that prevents clogging. It is possible.

The physiologically active substances used in the present invention include, for example, enzymes,
Coenzymes, enzyme inhibitors, proenzymes, hormones, antibiotics,
Substances that have an important effect on the physiological functions of animals and plants, such as bactericidal agents, anticancer agents, and immunoreactive substances.

An example of an enzyme is alcohol dehydrogenase.

Lactate dehydrogenase, glucose-6-phosphate dehydrogenase,
glucose oxidase, luciferase.

Redox enzymes such as L-amino acid oxidase, catalase, tyrosinase, and peroxidase.

Transferases such as hexokinase, pyruvate dehydratase, carbamate kinase, acetokinase, ribonuclease, lipase, acetylcholinesterase, steroid esterase, amylase, cellulase, dextranase, inhertase, pepsin, renin, trypsin, chymotrypsin, papain, ficin , thrombin, kallikrein, streptokinase 2 urokinase, plasmin, brinolase, asparakinase, urease, penicillin amidase, hydrolytic enzymes such as avilase, lyases such as pyruvate decarboxylase, albaltase, threonine deaminase, isomerases such as glucose isomerase, tyrosyl- TRNA synthetase, acetyl-CoA
Examples include glue gauze such as synthetase.

Examples of the coenzyme include pyridoxal phosphate and nicotine adenine dinucleotide.

Enzyme inhibitors include, for example, the ohomucoid Kuni
tz soybean trypsin inhibitor, aprotinin, antitropin ■, alpha neemacroglobulin, alpha 11-antitrypsin, alpha 1-antiplasmin.

Examples include plasminogen antiactivator and heparin.

An example of a proenzyme is plasminogen.

Examples include fibrinogen, prothrombin, and blood coagulation factor X.

Examples of hormones include cortisone, testolone,
Examples include estrone, estradiol, progesterone, insulin, somustin, and gonadotropin.

An example of an antibiotic is cloxacillin.

Penicillins such as cycloxacillin, flucloxacillin, ampicillin, hetacillin, tarambicillin, cyclacillin, amoxicillin, bibmecillinum, piperagiline, cephalolidine, cephaloglycin, cephalexin, cefazolin.

Cephalosporins such as cefavirin, cefrazine, cefrazole, cefoxitin, and cefatridine; aminoglycosides such as streptomycin, kanamycin, fradiomycin, paromomycin, gentamicin, bekanamycin, ribostamycin, dibekacin, amikacin, tobramycin, and spectinomycin; oxytetracycline; , tetracyclines such as tetracycline, demethylchlortetracycline, methacycline, doxycycline, minocycline, erythromycin, kitasamycin, oleandomycin,
Macrolides such as spiramycin, josamycin, and midecamycin; lincomycins such as lincomycin and talindamycin; antigram-positive bacteria such as micamycin, gramicidin S, and gramicidin; and polymyxins such as colistin and polymyxin B.

Antimycobacteria such as biomycin, cabreomycin, enviomycin, and cycloserin, polyene macrolides such as amntotericin B and pimaricin, rifampicin, pyrrolnidoline, mitomycin C, actinomycin, pleomycin, daunorubicin, doxorubicin, Examples include neocarzinostatin.

Examples of fungicides include pigment preparations such as acrinol and acrylflavin, furan preparations such as nitrofladun, quaternary ammonium salts such as benzalkonium chloride and benzethonium chloride, guanidium salts such as chlorhexidine, and iodine complexes such as povidone-iodine. , amphoteric surfactants such as alkyldiaminoethylglycine hydrochloride, and the like.

Examples of anticancer drugs include nitrogen mustard, nitromine, chlorambucil, cyclophosphamide, melphalan, and uracil mustard.

Mannomustine, dopan, BCNυ, triethylenemelamine, thio-TEPA, Aza-TEP^, trenimon, inprocuone, busulfan, dimethylmyleran, vivosulfan, ethoglucide, epoxypropidine, epoxypiperazine, hexamethylmelamine, dibromomannitol, pivobroman, etc. Alkylating agents 9 folic acid, aminopritenmethotrexate, guanine, 8-azaganine.

6-mercaptopurine, azathioprine, uracil, 5
- Synthesis of metabolic inhibitors such as fluorouracil, cytarabine, azaserine, diazomycin, antibiotics such as actinomycin D, sacromycin 3, mitomycin C, daunomycin, pleomycin, chromomycin 5 cardinophilin, 5-HP, IQ-1, etc. agent, thioteba, cyclophosphamide 9, plant ingredients such as doxorubicin, daunorubicin, neocartinosukun, Hg-hematoporphyrin, Co-protoporphyrin, stilbestrol, hydroxyurea, procarbazine, methylglyoxalubisuguanylhydrazone, L
- Examples include asparaginase.

Immunoreactive substances refer to substances capable of producing immunological binding, such as antigens and antibodies. An antigen is a substance that can induce an antigen-antibody reaction.

Common examples include peptides, proteins, polysaccharide triglucoproteins, and steroids. An antibody is a protein that is produced in a living body upon stimulation by an antigen and specifically binds to the antigen, and its chemical substance is an immunoglobulin. Such immunoreactive substances include, for example, microorganisms such as fungi, yeasts, protozoa, viruses, and their immunologically active components.

Examples include antibodies isolated from humans and animals, serum components, toxins, hormones, enzymes, alkaloids, extracts of cells and tissues, blood cells, lectins, etc.

A substance that can support the above-mentioned physiologically active substances through an immobilization process as described below is called a physiologically active substance immobilization carrier. It only becomes physiologically active when

The fiber aggregate in the present invention is, for example, stable,
Refers to an aggregate of short and long fibers such as spun yarn, nonwoven fabrics, and two-knit woven fabrics. The fineness of the single yarn forming the fiber aggregate is 0.5
It is necessary that the yarn length is 1+ms or more, preferably 5III11 or more, and particularly preferably 20 mm or more. Fineness is 0
．． If it is less than 5 denier, the strength per unit becomes small and it is not easy to manufacture. On the other hand, if it exceeds 30 deniers, the surface area per weight becomes small and only a small amount of physiologically active substance can be immobilized. If the yarn length is less than 1 ml, it becomes difficult to handle like a fine powder.

Furthermore, the regenerated cellulose fiber aggregate in the present invention must be treated with an acid anhydride polymer solution. Since cellulose has only a hydroxyl group, which is a low reactant, in its molecule, it goes without saying that it can immobilize physiologically active substances as it is.

In addition, reactant functional groups 2 other than acid anhydride groups, such as imidocarbonate group, isocyanate group, epoxy group, formyl group, acid chloride group, amino group, carboxyl group, carboxyalkyl group, diazonium group, azide group, etc. Cellulose treated with a compound contained in it is not as preferable to use as one treated with an acid anhydride-based polymer solution for the reasons listed below.In other words, it is not possible to obtain a sufficient amount of immobilization, and covalent bonds are not used. Other compounds such as dehydration condensation agents are required when immobilization is desired! It is. The treatment requires the use of reaction reagents that may have an adverse effect on biological physiology.1 Reaction reagents are difficult to handle, the treatment significantly reduces the mechanical strength of the fibers, and the physiologically active substances are deactivated. One or a combination of the following reasons.

In the present invention, the treatment of the regenerated cellulose fiber aggregate with the acid anhydride polymer solution is carried out as follows.

i.e. polymaleic anhydride.

Polyitaconic anhydride, polyacrylic anhydride.

Polycarboxylic anhydrides such as polymethacrylic anhydride and copolymers having these polycarboxylic anhydrides as constituent units, maleic anhydride/methyl vinyl ether copolymers, maleic anhydride/ethyl vinyl ether copolymers, maleic anhydride Copolymers of maleic anhydride and aliphatic vinyl ether such as acid/butanediol divinyl ether copolymers, maleic anhydride/ethylene copolymers, maleic anhydride/propylene copolymers, maleic anhydride/isobutylene copolymers copolymers of maleic anhydride and olefin monomers, copolymers of maleic anhydride and aromatic vinyl monomers such as maleic anhydride/styrene,
Copolymers of maleic anhydride and aliphatic vinyl esters such as maleic anhydride and vinyl acetate copolymers can be mixed with methanol, ethanol, propatool, isopropanol,
Butanol, tetrahydrofuran, ethyl acetate, methyl acetate, benzaldehyde, formaldehyde.

Acetone, cyclohexanone, methyl ethyl ketone, mesityl oxide, diacetone alcohol 22-pyrrolidone,
N-methyl-2-pyrrolidone, N-vinyl-2-pyrrolidone, butyrolactone, acetic acid.

Preferably about 0.1 to 30% by weight of dimethylformamide, pyridine, etc. or a mixed solvent thereof.

Particularly preferably, it is dissolved at a concentration of 0.5 to 10% by weight.

If necessary, sulfuric acid, hydrochloric acid, acetic acid, etc. are used as a catalyst, preferably about 0.01 to 10% by weight, particularly preferably 0.05% by weight.
~2% by weight is added, and the regenerated cellulose fiber aggregate is heated in the resulting solution at 0 to 150 t, preferably 0 to 100°C, particularly preferably 20 to 80 t, for about 10 minutes to 72 hours, preferably 30 minutes to The treatment is carried out for 36 hours, or for 10 minutes to 24 hours, preferably 30 minutes to 10 hours when treated at high temperature. As a treatment method, a known method such as a dipping method or a spray coating method can be employed.

Among the carriers for immobilizing physiologically active substances of the present invention prepared in this manner, the acid anhydride group content per 1 g of carrier is 0.0002 to 20 as carboxyl group content.
milliequivalents, even 0.002 to 1O milliequivalents, especially 0
．． 02 to 5 milliequivalents are preferred.

Acid anhydride group content is 0.000 as carboxyl group amount
If the amount is less than 2 milliequivalents, the ability to immobilize physiologically active substances tends to be poor, while if it exceeds 20 milliequivalents, the strength of the fiber tends to be weakened, or when used as a carrier, acid anhydride In the present invention, acid anhydride groups are hydrolyzed to form carboxyl groups, and then the amount of carboxyl groups is determined by neutralization titration.

In this way, the physiologically active substances can be covalently immobilized on the regenerated cellulose fiber aggregates treated with the acid anhydride-based polymer solution. Dioxane, dimethyl sulfoxide 5 It is dissolved or dispersed in a mixture of water and a water-miscible solvent such as dimethylformamide, and the regenerated cellulose fiber aggregate is heated in the resulting liquid preferably at -20 to 100°C.

Particularly preferably at 0 to 80°C, preferably for 5 minutes to 1
This can be carried out by treating for 00 hours, particularly preferably 109 to 80 hours. At that time, the pH is preferably 2 to 12. In order to particularly preferably adjust the temperature to 4 to 1o, a buffer such as phosphate or acetate may be used, or hydrochloric acid, sodium hydroxide, etc. may be added.

The regenerated cellulose fiber aggregate on which a physiologically active substance is immobilized in this manner can be used, for example, as a specific adsorbent for separation and purification of a chemical reaction catalyst, a material for clinical testing, a medical material, and the like.

That is, the regenerated cellulose fiber aggregate on which enzymes are immobilized as physiologically active substances can be used as a chemical reaction catalyst. For example, tyrosinase is immobilized for the production of L-DOPA, amylase or cellulase is immobilized for the production of glucose, and aspartase is immobilized for the production of L-aspartic acid.
Those with immobilized acetate kinase or carbamate kinase can be used for ATP regeneration, and those with immobilized glycose isomerase can be used for fructose production.

Furthermore, the regenerated cellulose fiber aggregate on which a physiologically active substance is immobilized can be used as a specific adsorbent for separation and purification. For example, immobilized enzymes include coenzymes,
For the separation and purification of enzyme inhibitors.

Products with immobilized coenzymes or enzyme inhibitors are used for the separation and purification of enzymes, products with immobilized hormones are used for the separation and purification of hormone receptors, products with immobilized antigens are used for the separation and purification of antibodies, and products with immobilized antigens are used for the separation and purification of antibodies. The immobilized product can be used for separation and purification of antigens. In addition, these specific adsorbents are used as clinical test materials for enzyme immunoassays and radioimmunoassays such as thyroid stimulating hormone, thyroid hormone, insulin, steroid hormones, human placental gonadotropin, angiotensin, α-fetoprotein, ferritin, and HBs antigen. It can be used for quantitative determination of etc. As shown in Table 1, various diseases can be treated by removing various harmful substances from body fluids using these specific adsorbents.

In addition, the strength and elongation of the regenerated cellulose fibers treated with the acid anhydride polymer solution in which the physiologically active substances immobilized thus obtained retained 70 to 98% of the regenerated cellulose used as the raw material. I'm here.

It can be satisfactorily used as a fixation carrier for various purposes as mentioned above.

Table 1 Application of specific adsorbent to night carriers Table 1 (continued) The carrier obtained by the present invention is easy to handle.

It also has the feature of being able to impart high physiological activity, so it can be used as a specific adsorbent for separating and purifying the chemical reaction catalyst 1 as described above.
It has a wide range of uses including clinical testing materials and medical materials.

The present invention will be explained in more detail below by giving examples.

Example 1. Comparative Examples 1 and 2 9 g of viscose rayon yarn (120 denier 150 filament, yarn length 5 cm) was mixed with 4% by weight Ganty
ezAN169 (manufactured by GAF, maleic anhydride/methyl vinyl ether copolymer) acetone solution 100+++
] and treated at 40°C for 24 hours. After treatment, wash with acetone and hot water to remove the attached Gantre.
z AN] After thoroughly rinsing out 69, heat it at 110°C.
A carrier for immobilizing a physiologically active substance was obtained by vacuum drying for 0 hours.

The content of acid anhydride groups per gram of this carrier was determined to be about 0.5 milliequivalent as the amount of carboxyl groups by neutralization titration. After this, 1g of anti-hepatitis B human immunoglobulin (
HB3 antibody) in 1 wt% phosphate buffer (1/IOM,
After being immersed in 10 ml (pH 8,0) at 7° C. for 24 hours, it was thoroughly washed with a physiological saline solution. 20 g of the anti-hepatitis B HB3 antibody immobilized carrier obtained in this manner was added to 1 volume.
11m1! HB is packed in a column of
Plasma with s antigen titer 1:2s 2Illj! When filtered, the amount of plasma antigen after filtration was reduced to 1:22. When 2IIIl of plasma was subsequently filtered through this column at a ratio of 1:28 four times, the amount of plasma antigen after filtration was reduced to 1:22 in each case.

For comparison, cellulose powder was treated with Gantrezl solution in the same manner as viscose rayon filament yarn, and then HB and body were reacted.

HB, antibody-immobilized cellulose 200+wg in a volume of 1 r
When HB and 2 tal of plasma with an antigen titer of 1:2'' were filtered through this column, the amount of plasma antigen after filtration was reduced to 1:22.

Four consecutive times in this column (i.e. from the second to the fifth)
When 2 ml of HB blood with an antigen titer of 1:21' was filtered, the amount of antigen after filtration was 1=2'' for the second time.

The time for the 3rd to 5th times was 1:21.

Furthermore, for comparison, 9 g of viscose rayon yarn similar to that used in Example 1 was mixed with methanol (17.3% by weight) containing 2.7 g of sodium hydroxide - water (13.7% by weight).
-2-Mixed solvent 1 consisting of propatool (69% by weight)
After alkalization in 64 g at 20° C. for 35 minutes, 4.2 g of monochloroacetic acid was added to this reaction system, and the reaction system was treated at 70° C. for 3 hours to introduce carboxymethyl groups. After reaction.

The reaction system was cooled, treated with hydrochloric acid, and then the carboxymethyl group-introduced rayon yarn was washed repeatedly with water until the pH reached 3 or more, and then dried to obtain a carboxymethylated carrier for immobilizing a physiologically active substance. 200mg later
ll111 column and pass H through this column.
B. When plasma 21 with an antigen titer of 1 F 2'' was filtered, the amount of plasma antigen after filtration was reduced to 1:24.

Using this column, HB was performed 4 times, antigen titer l: 28.
When 2 ml of plasma was filtered, the amount of antigen after filtration was 1:2' for the second and third times, and 1:25 for the fourth and fifth times.
Met.

The measurements of HB and antigen titers were carried out in accordance with Kanai and Kanai (eds.), "Clinical Test Overview," Revised and Expanded 28th Edition (Kanehara Publishing) XX-40.

Example 2 Use cyclohexanone instead of acetone.

A carrier for immobilizing a physiologically active substance was obtained by treating with a cyclohexanone solution of Gantrez AN169 in the same manner as in Example 1, except that the treatment temperature was changed from 40° C. to 100° C. The #I anhydride group content per gram of the other carrier was approximately 2.4 milliequivalents as carboxyl group content.

Example 3 10g copper ammonia rayon yarn (100 denier/1
00 filament) to 10% by weight of 5MA3000 (
It was immersed in a methyl ethyl ketone solution 1001 of maleic anhydride/styrene copolymer (manufactured by ARCO/Che+mlcal Company) and treated at 60° C. for 36 hours.

After treatment, methyl ethyl ketone cleaning and acetone cleaning were performed to thoroughly wash away the attached 5MA3000, and then
Vacuum drying was performed at 10° C. for 10 hours to obtain a carrier for immobilizing a physiologically active substance. The acid anhydride group content of the carrier was approximately 0.04 milliequivalent per 1 g of carrier as the amount of carboxyl groups. Example 4 5MA3000 (ARCO/Chemical Co
A carrier for immobilizing a physiologically active substance was prepared in the same manner as in Example 3, except that a methyl ethyl ketone solution of maleic anhydride/ethylene copolymer was used instead of a methyl ethyl ketone solution of maleic anhydride/methyl vinyl ether copolymer (manufactured by Mpany Co., Ltd.). Obtained. The content of acid anhydride groups in this carrier was approximately 0.03 milliequivalent as carboxyl per gram of carrier.

Example 5 Viscose rayon long fiber fabric (120 denier 15
0 filament, single yarn fineness 2.4 denier filament, 10cm x 10cm) with 2% Ga by weight
n trezAN169 (manufactured by GAP, maleic anhydride/methyl vinyl ether copolymer) acetone solution 10
After being immersed in 0 ml of water and treated at 50°C for 24 hours, it was washed with acetone and hot water, and vacuum-dried at 110°C for 8 hours to obtain a carrier for immobilizing a physiologically active substance. The acid anhydride group content of this carrier was 0.4 milliequivalent as carboxyl group per gram of carrier. This was mixed with urokinase phosphate buffer (pH 7,0,1/IOM, 2000 units/m
1) After immersing in 100+nl for 24 hours, it was thoroughly washed with physiological saline solution. Measurement of fibrinolytic activity is based on the revised and expanded 28th edition of "Clinical Test Methods Overview" edited by Kanai and Kanai (Kanehara Publishing)
A fibrin plate was prepared according to Vl-105, a piece of fabric (circular with a diameter of 1 cI11) was placed on it, and the dissolution of fibrin was observed. As a result, after 24 hours, it was observed that fibrin had dissolved over a 3.5 cm area around the fabric piece on which urokinase was immobilized.

Patent applicant: Uni-Tei Riki Co., Ltd. Continuation of page 1

Claims (1)

[Claims] +11 Single yarn fineness 0.5-30 denier, yarn length 1-+11
A carrier for immobilizing a physiologically active substance, which is obtained by treating the above regenerated cellulose fiber aggregate with an acid anhydride polymer solution.