JPS59189906A - Hollow fiber membrane having physiologically active substance immobilized thereto and treatment of liquid using said membrane - Google Patents

Abstract

PURPOSE:To obtain an immobilized membrane excellent in the permeability of a macro-molecular substance with a M.W. of 10,000 or more and reduced in the effluence of a physiologically active substance, by using a hollow fiber membrane of which the transmittance of bovine serum albumin is 90% or more and to which a physiologically active substance is chemically bonded. CONSTITUTION:The pore size of max. probability in the pore size distribution curve of the thickness of a physiologically active substance immobilized fiber membrane is within a range of 0.035-15mum and the void ratio of the hollow fiber is 40% or more and the transmittance of bovine serum albumin thereof is 90% or more. This hollow fiber membrane comprises a substance with a M.W. of 10,000 or more or a substance with a M.W. of 10,000 or less and efficiently treats a liquid to be treated containing a substance converted to a substance with a M.W. of 10,000 or more by the treatment of said physiologically active substance. As the physiologically active substance to be immobilized, enzyme, a microbial cell, a cell, organera, an antigen, an antibody or medicine are represented.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a porous hollow fiber membrane to which a physiologically active substance is chemically bonded, and a method for treating a liquid using the membrane. In more detail, anti-inflammatory substances, hormones, enzymes, antigens, antibodies, cells, microbial boxes, organs, bacteria, nucleic acids, pharmaceuticals, etc. are chemically bonded to high molecular weight substances.
The present invention relates to a porous hollow fiber membrane that has excellent permeability to liquids, especially proteins and polysaccharides, and a method for treating liquids using the membrane.

Enzymes and microbial boxes, as well as animal and plant cells and organelles,
Materials in which antigens, antibodies, hormones, antibiotics, nuclear waves, pharmaceuticals, etc. are immobilized physically or chemically can be used as a means of synthesizing various useful substances, or for various sensors, medical purposes, and analytical purposes. Wide range of uses for 'c7pi'. Many of the conventionally known immobilization materials use granular materials such as polymer particles and activated carbon as carriers, and relatively few use hollow fiber type separation membranes as carriers; proposed a method for immobilizing enzymes on an asymmetric hollow fiber membrane made of polyacrylonitrile by chemical bonding. Ma/ζ Japanese Patent Publication No. 56-39788, No. 56-131387, No. 56
No. 16478-9 also discloses a method of physically and/or chemically immobilizing an enzyme on a hollow fiber type separation membrane having a dense layer on the surface. However, the enzyme-immobilized membranes obtained by these methods have an average pore size of 0.001μ to 0.01μ on the membrane surface to prevent the enzyme from leaking out.
It utilizes an ultra-permeable membrane with an enzyme-impermeable dense layer consisting of micro-sized pores, and has hollow membranes that are highly permeable to high molecular weight substances with a molecular weight of 10,000 or more, which is the object of the present invention. It is fundamentally different from the fiber membrane type fixation carrier.

The present inventors have earnestly attempted to immobilize a physiologically active substance on a hollow fiber membrane for precision filtration that does not have the above-mentioned permeable layer densa, and as a result, it is far more effective than conventional carriers. The FC membrane, in which physiologically active substances are chemically bonded to a hollow fiber membrane with large pores that allows over 90% of serum albumin to pass through, surprisingly has excellent practical performance with little outflow of active substances. He discovered something and came up with an invention. That is,
The present invention is a hollow fiber membrane fixed with a physiologically active substance as described below, and a method for treating a liquid using the membrane.

(1) The maximum probability (mode) pore size in the pore size distribution curve is in the range of 0.035 μ to 15 μ and the porosity is 4
0% or more, the permeability of bovine serum albumin is 90% or more,
and a physiologically active substance-fixed hollow fiber membrane characterized in that the physiologically active substance is chemically bonded.

(2) The pore diameter of the maximum probability (mode) in the pore size distribution curve is in the range of 0.035μ or more and 15μ or more, the porosity is 40 or more, the permeability of bovine serum albumin is 90% or more, and the bioactive substance is (a) A substance with a molecular weight of 10,000 or more to be treated with the physiologically active substance -! or (b) supplying and treating a liquid to be treated containing a substance with a molecular weight of 10,000 or less that is converted into a substance with a molecular weight of 10,000 or more when treated with the physiologically active substance;
A method for treating a liquid by using a physiologically active substance-fixed hollow fiber membrane, characterized in that a treatment membrane containing more than 10,000 substances is passed through the membrane wall of a hollow fiber.

The hollow fibers used in the present invention have a pore diameter of 0.0 or less at a mode of 0.05 to 0.05.
035μ or more, 15μ or less, more preferable tL, <0.05
It must not exceed μ or more and must not exceed 10μ. In the present invention, the pore size M aEI can be measured using a mercury porosimeter, and the mode pore size and V'
J, the maximum size of the pores is 4, which corresponds to the total size of the pores in the membrane.

If the mode pore size is smaller than 0.03!5μ, the immobilization of enzymes, antigens, and antibodies will be severe and the activity will be low; if it is larger than 15μ, cells, organelles, A fixed crown is a bad virtue.

Mag ζ The empty space ratio of the hollow fibrous membrane used in the present invention is 4.
Must be 0% or more. It is preferably 50% or more, more preferably 60% or more.

When the porosity is less than 40, the surface area ratio per unit volume of the membrane is small, and the amount of immobilization is small, so the activity per membrane area is low. Note that the porosity (P) is a value determined by the following formula.

゛ p- (1-5) The most important requirement of a fiber membrane is its permeability to high molecular weight substances having a molecular weight of 10,000 or more, preferably 60,000 or more. Conventional hollow fiber fermentation membranes with an enzyme-impermeable layer and self-contained gel structure membranes have small pore diameters in their membrane walls, and cannot allow such high molecular weight substances to completely permeate. However, it was virtually impossible to process a substance with a molecular weight of 10,000 or more while thoroughly heating the membrane wall, and to obtain all of the permeate containing the substance with a molecular weight of 10,000 or more. In addition, for the same reason, in conventional solid-foot membranes, substances with a molecular weight of less than 10,000 are assembled within the membrane wall, or are combined with other substances, and substances with a molecular weight of more than 10,000 are deposited in the permeate. Even the awning φ did not come out, but those who failed to do so,
The transmittance of ten-plate groove albumin (Funa Senji 67,000) is 9.
(J% or more, preferably 100% globulin G (molecular weight 1
Using a porous hollow fiber membrane in which physiologically active substances are chemically immobilized on a membrane with a permeability of 56,000% or more, it is impossible to remove physiologically active substances containing substances with a molecular weight of 10,000 or more. Activity*
We have found that the permeate can be easily and efficiently obtained by the process. When the permeability to bovine serum albumin is 90%, the permeability to substances with a molecular weight of 10,000 or more is low, and the activity does not decrease over time.

The transmittance here is 37℃, pf (isotonic buffer bath of 7,4)
” From an accidental experience? The protein concentration Co at the entrance of the hollow fiber is Cin - the protein concentration Co at the entrance of the hollow fiber
The protein concentration at the outlet is CuF' = p The protein concentration at the outlet The concentration of the hollow fiber membrane used in the present invention is not particularly limited, and examples include glass, silica, ceramic, alumina,
Inorganic materials such as metal oxides and graphite, natural organic polymers such as Textran, Sephadex, cellulose, collagen, and chitin, Bouillon, styrene, polyacrylamide, polyvinyl alcohol, polyacrylonitrile, polymethacrylate, polymethylmethacrylate, and polyvinylpyrrolidone. There are synthetic organic polymer materials such as polyester, polyvinylchloride, polycarbonate, polyethylene, polypropylene, polybutadiene, polytetrafluoroethylene, polysulfone, polyetheretherketone, and polyamino acid. These materials may be used alone, or may be used as a copolymer or polymer blend.

In addition, materials that have been grafted or have functional groups introduced through light irradiation, radiation irradiation, chemical treatment, etc. may also be used.
Hydroxyl group, aldehyde, carboxyl group, amine 7'
Those having a group, an imide group, etc. are suitable because they require fewer steps for immobilization. Representative ones will be briefly explained below.

When polyvinyl alcohol is used as a hollow fiber membrane material for immobilizing physiologically active substances, it is necessary to first make the membrane water-insoluble and to maintain its porous structure. As a method for the insolubilization treatment, formalization using formalin, benzaldehyde, etc., crosslinking using a crosslinking agent such as glutaraldehyde, titanium hydroxide, etc., and crosslinking reaction using electron beams, gamma rays, etc. are used. Although some of the hydroxyl groups in the original polyvinyl alcohol are lost through these insolubilization treatments,
When the physiologically active substance (2) of the present invention is used as a regular carrier, the residual ratio of hydroxyl groups is preferably 20 molar or more, more preferably 35 molar or more.

-Also, when a vinyl alcohol copolymer is used as the membrane material, the content of vinyl alcohol residues in the copolymer is preferably at least 20 mol%, more preferably at least 35 mol%. If the residual rate is less than 20%, the amount of immobilization will be small only by the bonding method using hydroxyl groups, and the specific activity of the membrane will be low.

The porous hollow fiber membrane itself used in the present invention can be manufactured by a method known by Kojiro.

For example, a porous membrane of polyvinyl alcohol is
A porous membrane of ethylene vinyl alcohol copolymer was prepared by the method described in Japanese Patent Application Laid-open No. 51-1-21420.
It can be manufactured based on the method described in No. 45474. In addition, the profile of these porous hollow fiber membranes is usually such that the inner diameter is 50μ or more and 20,000μ or less, preferably 100μ or more and 5000μ or less, and more preferably 1
It is 75μ or more and 2000μ or less. If it is thinner than this, the hollow fiber membrane will be mechanically too deep, and when it is molded into a module, the pressure loss on the middle nitrogen flow path side will be too large. If it is thicker than this, the membrane area per unit volume will be too small.
・A sufficient amount of immobilization cannot be obtained, and the processing efficiency of the liquid to be processed is low.

Physiologically active substances to be immobilized include enzymes (including coenzymes);
It can open microbial capsules, cells, organelles, antigens, antibodies, hormones, antibiotics, nucleic acids, medicines, etc.

Examples of pharmaceuticals include immunosuppressants such as cyclophosphamide and azathioprine, immunomodulators such as levamisole, immunostimulants such as interferon and interphyleukin, phrostacindin, thromboxane, and prostacyclin. , leukotrienes, adrenal corticosteroids, etc. are identified.

Examples of antigens include thyroglobulin, microsome, thyroid stimulating hormone receptor, acetylcholine receptor,
Insulin receptors, autoimmune disease-related antigens such as cell nuclei, cancer-specific antigens such as α-fetoprotein, agglutinin, non-pathogenic Trebonema, or pseudo-substances thereof are immobilized on the 2'L. Examples of the antibodies that can be immobilized include anti-albumin antibodies, anti-immunoglobulin antibodies, hepatitis B antibodies, and human ciliated gonad antibodies.

Organelles such as Cf'i, such as mitochondria, nucleus,
Chromatophores, chloroplasts, peroxygens, etc. are immobilized.

Cells such as kidney cells, hepatocytes, islets of Langerhans cells, various lymphocytes, and hybridized cells are immobilized.

Examples of biological substances that can be immobilized include bacteria such as Escherichia coli, Bacillus subtilis, actinobacterium, phlegmon, methanol 1, yeast, and molds of various types.

In addition to isolated and purified enzymes, enzymes that exist inside cells, such as the imitative organism Kyohanaikokon, can also be used, and 11!
Enzymes extracted from I cells may also be used. Alternatively, instead of just a single enzyme, a plurality of enzymes may be immobilized, or they may be immobilized together with a complementary element, ATP, AIJP, or the like. Specific examples include amino acid oxidase, catalase, xanthine oxidase, glucose oxidase, glucose-6-phosphate tehydrogenase, glutamate dehydrogenase, cytochrome C oxidase, telosinase, lactate dehydrogenase, berogysidase, 6-phosphogluconate dehydrogenase, Oxidoreductases such as malate dehydrogenase, asbanate acetyltransferase, aspartate amine transferase, chrysin aminotransferase, glutamate-oxaloacetate amine transferase, glutamate-pyruvate aminotransferase, creatine phosphokinase, histamine methyltransferase, Transferases such as pyruvate kinase, fructokinase, hexokinase, 8-IJ resin cetyl transferase, leucine aminopeptidase, asnola kinase, acetylcholinesterase, aminoamylase, amylase, arginase, L-arginine deiminase, invertase, maltase, Lactase, urease, uricase, urokinase, esterase, β-galactolyse, kallikrein, chymotrypsin, trypsin, thrombin, pepsin, no()kuin, nokunureanan, naringinase, nucleotidase, 1f uronidase, plasmin, pectinase , hesperidinase, venicillinase, penicillin amitase, lipase, phosphorinase, bosphatase 11 nobonuclease, rennin, melibiase, aldolase, cellulase, an/tocyapase, nalnondinase, tannase, hydrolytic enzymes, aspartate decarboxylase, Lyases such as Asno<lutase, citrate lyase, glutamate decarboxylase, histidine ammonia lyase, phenylalanine ammonia lyase, fumarase, fumarate hydratase, malate synthetase, alanine racemase, curcose inomerase,
Isomerase enzymes such as glycose phosphate isomerase, glutamate racemase, lactate racemase, methionine racemase, glue gauzes such as asparagine synthase, daltathione synthase, biru and 6/acid synthase, IJNA ligase, EcoRI, Hindll, B
There are restriction enzymes such as am HI, Sat I, and Pst I.

Carrier binding methods are used to immobilize these physiologically active substances on porous hollow fiber membranes, but microbial cells,
When fixing cells, organelles, etc., an entrapment method may be used in combination. Among carrier bonding methods, covalent bonding methods and ionic bonding methods are preferred; examples of covalent bonding methods include diazo method,
Alkylation methods and peptide methods are preferred. 1/ko,
child! Stripes using chemicals such as numbing glue, light and shoulder green +11: <Using erection to joint shoulders, ion pressure is also applied to the coffin (2) can.

Hydroxyl groups in insolubilized polyvinyl alcohol, ethylene vinyl alcohol polymers, cellulose, etc.
It can be bonded to hydroxyl groups, -amino groups, carboxyl groups, etc. of enzymes, antigens, antibodies, cells, organelles, microbial cells, nucleic acids, urumones, antibiotics, I'/JJA1 pharmaceuticals, etc., using an appropriate reagent. Below, all the bond formation methods between the hydroxyl group of vinyl alcohol and the amino group of the ancient Japanese language ψ1 are listed. Here, the amino group of the immobilized product is
! :H te "'j-0H 1 α" Immobilization of a physiologically active substance onto a membrane made of an acrylic acid polymer can be carried out, for example, as follows. This represents an enzyme.

1- CO-NI(-NkLq +NaNO2-machi→membrane made of nylon-\) is fixed with an example of (
, 1st order. 6. Convex 〇 O crystal iron, R-NH2 destroys enzymes 〇1 By the physiologically active substance fixed inner wall fiber membrane of the present invention (a) Molecular weight of 10,000 or more substances (body fluids, plasma, etc.) - or molecular weight 1
Substances with a molecular weight of 10,000 or less that are converted into substances with a molecular weight of 10,000 or more when treated with the physiologically active substance (culture fluid containing amino acids, sugars, etc. that serve as nutritional sources for enzymes, cells, etc.; for example, ME
; M, NCTC, F-12°cMwh, -1415, etc., in which immune-related substances such as albumin and globulin with a molecular weight of 10,000 or more are biosynthesized) can be processed. The liquid to be treated is efficiently treated while passing through the membrane wall of the hollow fibers, which are porous. The entire amount of the liquid to be treated does not need to pass through the membrane wall, but if it is circulated inside or outside the hollow fiber, a portion of the liquid passes through the membrane wall and the useful substance is taken out through the membrane wall of the hollow fiber. good.

The treated solution fixes physiologically active substances/ζ membrane reaction yield,
Reaction speed, objective? ! Depending on the desired purity of the 7-quality βf, the treatment can be carried out by either a batch method or a continuous method. Also, from the same point of view, it is possible to select conditions for the presence or absence of circulation and the optimum ratio between the amount of circulation and the amount of membrane permeation. The flow path for the liquid to be treated may be in the hollow part of the hollow fiber membrane or on the outer surface side. Normally, in the circulation method, the entire amount is pushed through the hollow part (non-circulation focusing).
In terms of chemical engineering, it is more advantageous to flow the water on the outer surface side. In addition, when processing by the method described above, the hollow fiber membrane of the present invention can be used not only as a single fiber, but also by being bundled and formed into a known module form such as a double-ended round-one-end closed type. I can do things.

As a specific example of the use of the hollow fiber membrane of the present invention, for example, for medical purposes, blood is introduced under pressure into the hollow part of the hollow fiber membrane on which an antigen (antibody) is immobilized, and a portion of plasma λ1 is absorbed by the membrane.
(~, j) A method for selectively removing antibodies (antigens) while allowing them to pass through the membrane wall, obtaining normal serum that does not contain malignant substances, and returning it to the blood. In the same way, it can be used to remove anti-inflammatory complexes, cholesterol/sterol, fibrinogen, α,-talicoprotein, anti-inflammatory factor, and rheumatoid factor.

In the field of biochemistry, for example, breeding is done using recombinant DNA technology.
The simulated material was applied inside the membrane wall of the hollow fiber membrane and (also (Ci,
) Immobilized on the surface, 1 g of low molecular weight peptides, amino acids, sugars, ATP, m-type substances, and other substances are supplied from one side of the membrane along with the culture medium, and human hormones, polypeptides, and interferon are supplied from the other side. It can be used to obtain corner molecular weight products such as ferrons and vaccines.

Similarly, using a membrane immobilized with extracellular protein Namae bacteria,
It is also possible to produce protein for animal feed.

In the food industry, for example, cheese whey can be supplied from the outer surface of a hollow fiber membrane on which β-galactosigose is immobilized, and the lactose in the whey can be continuously hydrolyzed while passing through the membrane wall. According to the present invention, in addition to this, η-kuni proteins with a higher molecular weight can also pass through the membrane wall. concentration, gel layer generation,
The accompanying decrease in the rate of hydrolysis can be completely prevented.

In addition to the above-mentioned examples, liquids to be treated that are the object of the present invention include body fluids, plasma, culture fluids of cells and microorganisms, raw materials for the synthesis of polysaccharides, glycoproteins, proteins, peptides, nucleic acids, etc., and (or ) Product-containing liquids, as well as manufacturing process liquids and waste liquids in the food industry and pharmaceutical manufacturing.

The present invention will be explained in more detail with reference to Example 1. Example 1 Polyvinyl alcohol with an average degree of polymerization of 1700 8 kf,
A total of 50 tons of polyethylene glycol, 16 tons of boric acid, and 302 tons of acetic acid were dissolved in hot water.

Pour this solution through an annular nozzle and add caustic soda soy7.
t.

It was discharged into a coagulation bath of 230μ of Glauber's salt to obtain hollow fibers. Then, in a bath containing 0.5μ of glutaraldehyde and 3 parts of hydrochloric acid,
Crosslinking treatment was performed at 0°C for 2 hours, and then heat treatment was performed in hot water at 100°C for 1 hour, followed by washing with water. The polyvinyl alcohol hollow fiber thus obtained had an inner diameter of 700 μm and an outer diameter of 1,000 μm, and its membrane wall had a homogeneous porous structure. The mode pore size determined from the pore size distribution curve using a mercury porosimeter was 0.26 μm, and the porosity determined from the apparent specific gravity of the hollow fibers was 57%. Also, the transmittance of vertical τW Alhumin was 100%, and the transmittance of Gamma Globulin G was 97%.

This polyvinyl alcohol hollow fiber membrane was immersed in a solution consisting of 5% aminoacetaldehyde dimethyl acetal and 10% sulfuric acid at 80° C. for 5 hours to convert the hydroxyl groups into aminoacetals, and then washed with running water for 5 hours. Then 5
% glutaraldehyde aqueous solution at a constant temperature for 5 hours to completely form perilla base, and washed with running water for 5 hours. 100 hollow fiber membranes obtained in this way were placed in the prefecture and 10
α is molded into a module with both ends open, and inside the hollow fiber of the module are free dog hepatocytes i 5 ′? Earl's solution containing a concentration of /l was introduced, and after circulating for 1 hour while a part of the solution permeated through the membrane wall, 10 uALes of penicillin was introduced.
Earl1's containing 10μ of streptomycin
The hollow part and the inside of the membrane wall were washed with 2 tons of liquid to obtain a porous polyvinyl alcohol hollow fiber module in which free hepatocytes were fixed. The entire MEM culture solution (molecular weight 200 or less) was introduced into the hollow part of this module, and the entire amount was allowed to circulate for 4 hours while passing through the membrane wall, and the albumin produced by the immobilized hepatocytes was detected using an anti-albumin antibody. It was quantified by The increase in the amount of albumin produced over time shown in Figure 1 shows that the amino acid components in the culture solution are converted into albumin by the hepatocytes fixed on the membrane surface and/or within the membrane wall. It can be seen that the particles pass through the membrane wall and are extracted.

Example 2 An ethylene vinyl alcohol copolymer with an ethylene content of 32 moles was dissolved in total dimethyl sulfoxide, and the polymer @
A 20% solution was prepared. This was discharged from a TL-annular nozzle into a 20-layer dimethyl sulfoxide aqueous solution at 25°C, then stretched, heat treated, and dried to obtain a hollow fiber type porous membrane. The inner diameter of this hollow fiber membrane is 220μ and the outer diameter is 330μ.
So, the membrane structure is the outer surface! - It was a porous structure in which the pore diameter was slightly smaller than the pore diameter inside the membrane wall. The mode pore diameter determined from the pore diameter distribution curve using a mercury porosimeter is 0.56.
μ, and the porosity was less than 68. In addition, the permeability to bovine serum albumin is 96%, bovine gamma clopurin G.
The transmittance was 91%.

This ethylene vinyl alcohol copolymer hollow fiber membrane was
Example 1 by focusing 00 lines. It was shaped into a similar module. Next, all the pumps in the module were contacted with kF, L, the reaction liquid was circulated, and the washing liquid was passed in a single pass, and the entire treatment was carried out in the same manner as in Example 1, to obtain a porous membrane module into which aldehyde groups were introduced.

In this way, the hollow part of the kg module is filled with 01 pieces of jumen 3.
Giglopuri/G (IgG)! J Nsan Slow @Solution (1) H
7,4) Introduce 200 ml of water, circulate for 1 hour while passing through the membrane wall, then introduce 3 t of physiological saline into the hollow part, wash the 1NS while allowing it to pass through the membrane wall, and wash the IgG while passing through the membrane wall. An immobilized ethylene vinyl alcohol based porous hollow fiber module was obtained.

131-labeled anti-cattle i
Antibiotic IgG antibodies containing IgG antibodies were added to 200 iJ of 0.1% bovine serum albumin Linsan buffer solution (pH 7,4).
One part of the solution was introduced into the hollow part and circulated for 4 hours while passing through the membrane wall.The alhumin permeability and count change rate of each module after 4 hours were as shown in Table 1. 11 The count change rate referred to here is the value calculated by the following equation.

Count value before treatment From this result, it was found that in the IgG delimitation module, the serum albumin coexisting in the liquid to be treated passes through the entire membrane wall, but the antibiotic εG antibody is adsorbed and removed by the immobilized FGG. It's obvious.

Table 1

[Brief explanation of drawings]

FIG. 1 is a diagram showing the relationship between culture time and serum albumin profile when MEM culture solution was treated using the hepatocyte-like hollow fiber membrane obtained in Example 1. Patent applicant: RiRa Co., Ltd. Representative patent attorney: Ken Honda

Claims (2)

[Claims] (1) The pore size of the maximum probability (mode) in the pore size distribution curve is in the range of 0.035μ to 15μ, the porosity is 40% or more, the permeability of ten-plate albumin is 90% or more, and A physiologically active substance-fixed hollow fiber membrane characterized in that active VJ substances are chemically bonded. (2) The pore size of the maximum probability (mode) in the pore size distribution curve is in the range of 0.035 μ to 15 μ, the porosity is 40% or more, the permeability of serum albumin is 90% or more, and the bioactive substance is A substance with a molecular weight of 10,000 or less (a substance with a molecular weight of 10,000 or more that is treated with the biologically active substance) that is chemically bonded to the hollow fiber membrane and that is treated with the biologically active substance. Physiology characterized by supplying and processing a liquid to be treated containing a substance that is converted into a substance with a molecular weight of 10,000 or more, and taking out the treatment liquid containing a substance with a molecular weight of 10,000 or more through the entire membrane wall of the hollow fiber. A method for treating liquids by using active substance-immobilized hollow fiber membranes.