JP2983955B2 - Device for removing glomerular basement membrane adhering protein - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a removing device for removing harmful components from a body fluid by adsorption. More specifically, removing the glomerular basement membrane adhering protein from the body fluid,
The present invention relates to a renal glomerular basement membrane adhering protein removing device for suppressing renal diseases such as nephritis.

[0002]

2. Description of the Related Art The kidney is an organ that tries to excrete waste products carried by blood from all parts of the human body as urine. That is, the kidney functions as a blood filter.
It is generally said that about 0.5 liter of blood flows per minute per kidney. Therefore, if the kidney does not work in the human body, waste products formed in the human body cannot be removed, and the waste products accumulate in the body, making it difficult to maintain life. Thus, the kidney plays an important role in maintaining life.

[0003] Among these kidneys are written books (body reading books,
As supervised by Shunji Ishiyama, Tatsuo Kobayashi and Tadao Takahashi, published by Living Book, 1971), there is a thread-like glomerulus with a diameter of about 0.2 mm. There are about 1.3 million glomeruli per kidney. The blood is then filtered through the glomeruli. This glomerulus has a membrane called a renal glomerular basement membrane on a surface that comes into contact with blood, and thereby recognizes the size and charge of blood components and sorts waste products and necessary products. Therefore, if a substance that adheres to the renal glomerular basement membrane enters or forms in the body, the renal glomerular basement membrane to which the substance adheres will not function, and the filtration function of the kidney will be lost, and life will not be maintained. Will lead to death.

[0004] Systemic lupus erythematosus (hereinafter referred to as SLE) is one of the typical renal diseases caused by such a mechanism. This disease is one of the representative examples of diseases of the immune system, and is a systemic disease characterized by symptoms such as nephritis and central nervous system disorders. In particular, the progression of nephritis (called lupus nephritis) in SLE patients
It has serious consequences for patients and is a guide for treatment.

[0005] In addition to normal immunoglobulins, SLE patients produce various and large amounts of abnormal antibodies known as autoantibodies, which are antibodies to their own components, and are present in the blood. SLE symptoms are caused by the deposition of these autoantibodies and immune complexes, which are the reaction products of autoantibodies and antigens, on tissues. SLE
In the case of lupus nephritis, which is the most severe of the symptoms, it is thought to be caused by the adhesion of autoantibodies to the most important part of renal function, that is, the glomerular basement membrane that performs blood component filtration. ing. This means that
It has been proved that immunoglobulins (hereinafter also referred to as anti-GBM antibodies) against renal glomerular basement membrane (hereinafter also referred to as GBM) are present in SLE patients (Shinichi Aozuka,
Rheumatism, 26, pp. 445-448 (1986)), and other reports (Faaber, P., et al., Journal of Clinical Investigation (J. Cli).
n. Invest.), Vol. 77, pp. 1824-1830 (1986)) that it has been demonstrated that immunoglobulins that react with heparan sulfate constituting the renal glomerular basement membrane are present in SLE patients. It is clear from

[0006] Thus, the importance of treatment of lupus nephritis in SLE, and G in general nephritis.
Because blood components are blocked in BM and renal function is inhibited,
For the treatment of LE patients, it is very important to remove proteins such as anti-GBM antibodies having the property of adhering to the renal glomerular basement membrane (hereinafter referred to as renal glomerular basement membrane adhering protein).

Hitherto, steroids, immunosuppressants, immunomodulators, anti-inflammatory agents and the like have been widely used for the treatment of lupus nephritis. Among them, steroids are the most commonly used, and a short-term ultra-high dose therapy of steroids called pulse therapy is also often performed. However, since steroids are liable to cause side effects even in small doses, it is obvious that short-term ultra-high dose steroid therapy tends to cause even greater side effects. In addition, these drugs are often used for a long period of time, and in such cases, side effects are more likely to occur, and in some cases, the dosage must be increased gradually due to drug resistance. There are many cases where it is impossible or does not exert a sufficient effect. In particular, the active phase of lupus nephritis is a time when it is necessary to suppress the production of glomerular basement membrane adhering protein, but for the above-mentioned reasons, there is a strong use of drugs such as pulse therapy and immunosuppressants. In many cases, no therapy is available. As a side effect, there is a problem that hospitalization is required because the protective ability for infection is significantly reduced during drug administration, and it takes several months to be discharged from hospital, delaying rehabilitation.

On the other hand, as an approach different from these drug therapies, attempts have been made to directly remove plasma containing renal glomerular basement membrane adhering protein in body fluids by extracorporeal circulation. The simplest method is the so-called plasma exchange therapy, which replaces the plasma of a patient with the plasma of a healthy person. According to this method, the concentration of renal glomerular basement membrane adhering protein in blood is significantly reduced, and symptoms have been improved. However, this method requires a large amount of healthy plasma and is not only expensive, but also has not been widely used due to the risk of infection such as serum hepatitis during the treatment.

[0009] In plasma exchange therapy, all components in the plasma are removed and replaced with healthy plasma. On the other hand, renal glomerular basement membrane adhering protein, which is a pathogenic substance, is selectively removed. For the purpose of removal, a plasma separation membrane method has been developed which separates pathogenic substances by molecular size. In this method, plasma is separated into a high molecular weight fraction and a low molecular weight fraction by a membrane, the high molecular weight fraction containing the pathogenic substance is discarded, and the low molecular weight fraction containing the main protein albumin is removed. Is returned to the patient. This method, as described in the book “Double Filtration Plasma Separation and Exchange Method, Tetsuzo Agashi, Medical Shoin (1984)”, is a rapidly progressive thread that is one of the renal lesions of systemic diseases such as SLE. Anti-GBM antibodies are observed in glomerulonephritis, and it is considered that anti-GBM antibodies contribute to the development of this nephritis. Therefore, they are used for rapidly progressive glomerulonephritis. However, since the anti-GBM antibody is composed of IgG (immunoglobulin G) having a molecular weight of about 160,000, its separation from normal IgG and other immunoglobulins is not always sufficient, and the renal glomerular basement membrane adhering protein cannot be separated. During the removal, immunoglobulins are also removed in a large amount, and furthermore, all proteins having a molecular weight equal to or higher than that of the anti-GBM antibody as a pathogenic substance are removed.

[0010] Therefore, the time until dialysis of currently serious patients can be extended by means other than drugs, and the rehabilitation of patients can be accelerated by using a minimal amount of drugs together with a new treatment system. There is a demand for a means for removing the glomerular basement membrane-adhering protein, which selectively removes the glomerular basement-membrane-adhering protein as an etiological agent and hardly loses useful components in body fluids. .

[0011]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies in view of such circumstances, and as a result, have found that an adsorbent capable of adsorbing a protein adhering to a renal glomerular basement membrane almost without losing an active ingredient in a body fluid. The present inventors have found a removal apparatus using the present invention, and have completed the present invention.

That is, according to the present invention, a container having a fluid inlet and a fluid outlet, a fluid and components contained in the fluid can pass through, but the water-insoluble porous carrier has a molecular weight of 1,000 or more.
A filter through which the adsorbent of the renal glomerular basement membrane-adhesive protein to which the sulfated polysaccharide is immobilized cannot pass, and the renal glomerulus comprising the adsorbent of the renal glomerular basement-membrane-adhesive protein filled in the container The present invention relates to an apparatus for removing a protein adhering to a basement membrane.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION In the present specification, a body fluid refers to blood, plasma, serum, ascites, lymph, intra-articular fluid and fraction components obtained therefrom, and other liquid components derived from living organisms.

In the present invention, the protein adhering to renal glomerular basement membrane refers to a bodily fluid having a property of adhering to an anionic functional group such as renal glomerular basement membrane or a component of renal glomerular basement membrane such as heparan sulfate or chondroitin sulfate. Refers to the protein inside.
Representative examples thereof include an anti-GBM antibody, an anti-heparan sulfate antibody, an anti-chondroitin sulfate antibody, and the like.
However, it is not limited to these.

As a method of proving the existence of renal glomerular basement membrane adhering protein, there are the following publications (Japanese clinical study, fall 1985, extensive blood, urine chemistry test, immunological test-how to read the values-Japan) There are various examples such as those described in Clinical Publishing (1985), etc., and any method may be used, but enzyme-linked immunosorbent assay (ELISA) is simple. That is, books (Spiro, R, G.), Journal of Biological Chemistry (J. Biol.
m.), Volume 242, pp. 1915-1922, (1967), etc., and then renal glomerular basement membrane is solubilized. After fixing the renal glomerular basement membrane to a microplate, the patient's body fluid is brought into contact. After removing the body fluid, it is reacted with a substance that binds to a body fluid component such as a fluorescent substance, immunoglobulin labeled with peroxidase, etc., and the amount of the labeled substance is measured by various measuring means, and the renal glomerular basement membrane adhesion is measured. Demonstrate the presence of sex proteins.

In the present invention, the water-insoluble porous carrier is
It refers to a substance having the property of being insoluble in water for fixing sulfated polysaccharides having a molecular weight of 1000 or more. The water-insoluble porous carrier used in the present invention preferably has large pores of continuous fine pores. That is, since the renal glomerular basement membrane adhering protein is a macromolecule having a molecular weight of 100,000 or more, including anti-GBM antibodies, the renal glomerular basement membrane adhering protein is required to be efficiently adsorbed. It must be able to easily penetrate into the porous body.

There are various methods for measuring the pore diameter, and the mercury intrusion method is most often used, but it is difficult to apply when measuring a hydrophilic porous body. The exclusion limit molecular weight is often used as a standard of the pore diameter instead, and can be applied to any of a hydrophilic porous body and a hydrophobic porous body. As described in a compendium (eg, Hiroyuki Hatano, Toshihiko Hanai, Experimental High Performance Liquid Chromatography, Chemical Doujinshi) etc., molecules that cannot enter (exclude) pores in gel permeation chromatography Of those having the smallest molecular weight.

It is known that the exclusion limit molecular weight varies depending on the compound of interest. Generally, globular proteins, dextran, polyethylene glycol and the like have been well examined, but in the case of the carrier used in the present invention, renal glomeruli It is appropriate to use the values obtained with globular proteins which appear to be most similar to basement membrane adhering proteins.

As a result of examination using various water-insoluble porous carriers having different exclusion limits, the range of pore diameters suitable for adsorbing proteins adhering to renal glomerular basement membrane is found to be 400,000 to 60,000,000 or less. It was revealed. That is, when a water-insoluble porous carrier having an exclusion limit molecular weight of less than 400,000 is used, the adsorbed amount of the protein adhering to the renal glomerular basement membrane tends to be too small for practical use. On the other hand, as the exclusion limit molecular weight increases, the amount of adsorbed renal glomerular basement membrane adhering protein increases, but eventually reaches a plateau, and when the exclusion limit molecular weight exceeds 60 million, the surface area is too small and the amount of adsorption decreases only noticeably. In addition, there is a tendency that the adsorption of proteins other than the target protein adhering to the glomerular basement membrane of the renal glomerulus, that is, non-specific adsorption, increases and the selectivity decreases significantly.

Therefore, the water-insoluble porous carrier used in the present invention has a preferable exclusion limit molecular weight of from 400,000 to 60,000,000, and more preferably from 600,000 to 20,000,000 in view of a larger selective adsorption capacity. Good.

Next, the porous structure of the water-insoluble porous carrier is preferably total porosity rather than surface porosity, and the pore volume is desirably 20% or more from the viewpoint of a large adsorption capacity. As the shape of the water-insoluble porous carrier, any shape such as a granular shape, a spherical shape, a fibrous shape, a membrane shape, and a hollow fiber shape can be selected.

When a granular water-insoluble porous carrier is used, the pressure loss is large when the particle size is less than 1 μm, and when the particle size exceeds 5000 μm, the adsorption capacity is small. Preferably it is.

The water-insoluble porous carrier used in the present invention may be either organic or inorganic, but adsorbs body fluid components other than the desired protein adhering to the glomerular basement membrane (so-called non-specific adsorption). Is preferred. Since hydrophilicity causes less nonspecific adsorption, the water-insoluble porous carrier is more preferably hydrophilic than hydrophobic.

Further, it is convenient that a functional group which can be used for a ligand immobilization reaction is present on the surface of the water-insoluble porous carrier. Representative examples of these functional groups include a hydroxyl group, an amino group, an aldehyde group, a carboxyl group, a thiol group, a silanol group, an amide group, an epoxy group, a halogen group, a succinylimide group, and an acid anhydride group. However, it is not limited to these.

The water-insoluble porous carrier is particularly preferable when it is made of a compound having a hydroxyl group among the above functional groups, since non-specific adsorption is small. It goes without saying that a water-insoluble porous carrier having these functional groups introduced as a spacer can also be used.

Representative examples of the water-insoluble porous carrier used in the present invention include soft porous materials such as agarose, dextran and polyacrylamide; inorganic porous materials such as porous glass and porous silica gel; polymethyl methacrylate; Examples include, but are not limited to, porous polymer hardgels made from synthetic polymers such as alcohols and styrene-divinylbenzene copolymers and / or natural polymers such as cellulose.

When the adsorbent used in the present invention is used for extracorporeal circulation treatment, it is necessary to flow a high-viscosity fluid such as blood or plasma at a high speed, so that a hard material having sufficient mechanical strength that does not cause consolidation is required. It is preferable to use a water-insoluble porous carrier. That is, the hard water-insoluble porous carrier is, as shown in Reference Examples described later, the water-insoluble porous carrier is uniformly packed in a cylindrical column, and the relationship between the pressure loss and the flow rate when the aqueous fluid flows is at least 0.3 kg. / Cm ^{2 in a} linear relationship.

Sulfuric acid having a molecular weight of 1,000 or more used in the present invention
Polysaccharides have multiple sulfate groups in one molecule
A polysaccharide having a molecular weight of 1,000 or more .

[0029] Representative examples of the sulfated polysaccharide used in the present invention, heparin, dextran sulfate, chondroitin sulfate.

The sulfated polysaccharide having a molecular weight of 1000 or more immobilized on the adsorbent used in the present invention may be one type or two or more types.

The adsorbent used in the present invention refers to a state in which a sulfated polysaccharide having a molecular weight of 1,000 or more is immobilized on a water-insoluble porous carrier. Such sulfur having a molecular weight of 1000 or more
There are various methods for introducing the oxidized polysaccharide into the water-insoluble porous carrier , and any method may be used .

Polyanion compound having a molecular weight of 1,000 or more
And other compounds with anionic functional groups are immobilized
As a typical method for introducing an anionic functional group into a carrier to obtain a state in which the anionic functional group is obtained , (1) an anionic functional group or a functional group which can be easily converted to an anionic functional group is contained. (2) a method of immobilizing a compound containing an anionic functional group on a water-insoluble porous carrier, (3) a method of forming an anionic functional group A method in which a compound having an anionic functional group is immobilized on the water-insoluble porous carrier by directly reacting the compound with the water-insoluble porous carrier is exemplified.

Representative examples of monomers or cross-linking agents containing an anionic functional group or a functional group which can be easily converted to an anionic functional group used in the method (1) include acrylic acid and its ester, methacrylic acid and its Examples include, but are not limited to, esters and styrene sulfonic acids.

The method of (2), that is, the method of immobilizing a compound containing an anionic functional group on a water-insoluble porous carrier includes a method by physical adsorption, a method by ionic bonding, a method by covalent bonding and the like. Yes, any method may be used, but it is important for the preservation and stability of the adsorbent that the anionic functional group-containing compound is not eliminated. desirable.

When the compound having an anionic functional group is fixed by a covalent bond, it is preferable that the compound having an anionic functional group has a functional group other than the anionic functional group that can be used for fixing.

Typical examples of the functional groups that can be used for immobilization include amino groups, amide groups, carboxyl groups, acid anhydride groups, succinyl imide groups, hydroxyl groups, thiol groups, aldehyde groups, halogen groups, epoxy groups, silanol groups and the like. But are not limited to these.

There are many anionic functional group-containing compounds having these functional groups, and examples thereof include taurine, sulfanilic acid, glycine, and phosphorylethanolamine, which will be described later.

Typical examples of the compound containing a sulfate ester group among the compounds containing an anionic functional group include sulfate esters of hydroxyl group-containing compounds such as alcohols, saccharides, and glycols. Partial sulfate compounds of polyhydric alcohols, especially sulfated saccharides, contain both sulfate groups and functional groups required for immobilization, have high biocompatibility and high activity, and furthermore, sulfated polysaccharides are easily prepared. It is particularly preferable because it can be fixed to a water-insoluble porous carrier.

Next, the compound having an anionic functional group is immobilized on the water-insoluble porous carrier by the method (3), ie, by reacting the compound forming an anionic functional group with the water-insoluble porous carrier. As a typical example of the method of introducing an anionic functional group, a reaction of introducing a sulfate group into a hydroxyl group-containing porous carrier can be mentioned. In this case, a sulfate group can be directly introduced by reacting a hydroxyl group-containing water-insoluble porous carrier with a reagent such as chlorosulfonic acid or concentrated sulfuric acid.

The amount of the introduced anionic functional group is preferably 0.01 μmol or more and 10 mmol or less per 1 ml of the adsorbent. 0.0
If it is less than 1 μmol, the adsorption capacity is not sufficient, and if it exceeds 10 mmol, non-specific adsorption becomes too large to make it practically practical. A more preferred anionic functional group introduction amount is 1 μm.
It is preferably in the range of mol to 100 μmol.

There are various methods for using the adsorbent used in the present invention for treatment. The simplest method is to draw the patient's blood out of the body, store it in a blood bag, mix the adsorbent used in the present invention with the adsorbent used in the present invention to remove renal glomerular basement membrane adhering protein, and remove the adsorbent through a filter. There is a way to return blood to the patient. This method does not require a complicated device, but requires a small amount of processing at one time and requires a long time for treatment.
There is a disadvantage that the operation becomes complicated.

In the following method, the adsorbent is packed in a column, incorporated into an extracorporeal circuit, and adsorbed and removed on-line. That is, a container having an inlet and an outlet for a fluid, the fluid and components contained in the fluid can pass therethrough,
A filter in which a renal glomerular basement membrane-adhering protein adsorbent in which a sulfated polysaccharide having a molecular weight of 1000 or more is immobilized on a water-insoluble porous carrier cannot pass through, and the renal glomerular basement membrane adhering filled in the container A simple and preferable method is to pass a bodily fluid through a device for removing renal glomerular basement membrane adhering protein comprising an adsorbent of a soluble protein.

FIG. 1 is a schematic sectional view showing one embodiment of the apparatus for removing proteins adhering to the glomerular basement membrane of the present invention. In FIG.
(1) and (2) are the inlet and outlet of the respective fluid, (3) is the adsorbent, (4) and (5) are the fluid and the components contained in the fluid, but the adsorbent passes A filter or mesh that cannot be used, (6) is a column, and (7) is a container. Here, the filter (4) on the fluid inlet side may not be present.

Representative examples of nephritis to which the adsorbent used in the present invention can be applied include, in addition to the above-mentioned lupus nephritis, Goodpasture's syndrome, rapidly progressive glomerulonephritis, and the like, but are not limited thereto. Absent.

Hereinafter, the removing apparatus of the present invention will be described in more detail by way of examples, but the present invention is not limited to only such examples.

[0046]

EXAMPLES Reference Example Agarose gel (Biogel A5m, manufactured by Biorad) was applied to a glass cylindrical column (inner diameter 9 mm, column length 150 mm) equipped with a filter having a pore size of 15 μm at both ends.
l A5m), particle size 50-100 mesh), polymer hard gel (Toyopearl HW65 manufactured by Tosoh Corporation, particle size 50-100μ)
m, and Cellulofine GC-700m manufactured by Chisso Corporation,
(Particle size: 45-105 μm) were uniformly filled, water was circulated through the column by a peristaltic pump, and the relationship between the flow rate and the pressure loss ΔP was determined. The result is shown in FIG.

As is clear from the figure, the agarose gel which is a soft gel consolidates at a certain flow rate or more, and the flow rate does not increase even when the pressure is increased, whereas the hard gels such as Toyopearl and Cellulofine are used. The flow velocity increases almost in proportion to the increase in pressure.

Production Example 1 Cellulose CK Gel A3, a porous cellulose gel (trade name, manufactured by Chisso Corporation, exclusion limit molecular weight of globular protein of 5000)
20 g (weight%, the same applies hereinafter) of 40 g of NaOH, 120 g of heptane and Tween 20 nonionic surfactant (trade name, manufactured by Kao Atlas Co., Ltd.) per 100 ml of 100,000 ml
Ten drops (0.5 ml) were added. After stirring at 40 ° C. for 2 hours, epichlorohydrin (50 g) was added, and the mixture was stirred for 2 hours. The gel was washed with water and filtered to obtain a cellulose gel having epoxy groups introduced therein (hereinafter referred to as epoxidized gel).

Comparative Production Example 1 Sulfanilic acid was added to 5 ml of the epoxidized gel obtained in Production Example 1.
A solution adjusted to pH 9.9 by dissolving 17 g in 10 ml of water was added and shaken at room temperature for 24 hours. The gel was then filtered off and
A 5% aqueous solution of monoethanolamine was added, and the mixture was shaken to seal unreacted epoxy groups to obtain a cellulose gel in which sulfanilic acid was fixed.

The amount of the anionic functional group introduced by the immobilized sulfanilic acid was 6.5 μmol / ml of the adsorbent.
Met.

Comparative Production Example 2 A solution adjusted to pH 9.6 by dissolving 0.1 g of phosphorylethanolamine in 10 ml of water was added to 5 ml of the epoxidized gel obtained in Production Example 1, and the mixture was shaken at 40 ° C. for 4 hours. Thereafter, the gel was separated by filtration, a 0.5% aqueous solution of monoethanolamine was added, and the mixture was shaken to seal unreacted epoxy groups to obtain a cellulose gel on which phosphorylethanolamine was fixed.

The amount of the anionic functional group introduced by the immobilized phosphorylethanolamine was 4 μmol / ml of the adsorbent.

Example 1 To 5 ml of the epoxidized gel obtained in Example 1 was added 4 g of sodium dextran sulfate having a molecular weight of about 5,000 and a sulfur content of 15% in water 5.
The solution was dissolved in ml and adjusted to pH 9, and shaken at 45 ° C for 16 hours. Thereafter, the gel was filtered off, and a 2M saline solution was added to the solution.
Wash with 5M saline solution and water in this order, 0.5%
An aqueous monoethanolamine solution was added and shaken to seal unreacted epoxy groups to obtain a cellulose gel on which sodium dextran sulfate was fixed.

The amount of the anionic functional group introduced by the immobilized dextran sulfate was 10 μmol / ml of the adsorbent.
Met.

Comparative Production Example 3 0.25 g of glycine was added to 5 ml of the epoxidized gel obtained in Production Example 1.
A solution dissolved in 10 ml of water and adjusted to pH 9.8 was added and shaken at room temperature for 24 hours. Thereafter, the gel was separated by filtration, a 0.5% aqueous solution of monoethanolamine was added, and the mixture was shaken to seal unreacted epoxy groups to obtain a cellulose gel on which glycine was fixed.

The amount of the anionic functional group introduced by the immobilized glycine was 9 μmol / ml of the adsorbent.

Comparative Production Example 4 0.35 g of taurine was added to 5 ml of the epoxidized gel obtained in Production Example 1.
A solution dissolved in 10 ml of water and adjusted to pH 9.0 was added, and the mixture was shaken at room temperature for 24 hours. Thereafter, the gel was separated by filtration, a 0.5% aqueous solution of monoethanolamine was added, and the mixture was shaken to seal unreacted epoxy groups to obtain a cellulose gel in which taurine was fixed.

The amount of anionic functional groups introduced by the immobilized taurine was 5 μmol / ml of adsorbent.

Comparative Production Example 5 Cellulose CK gel A3,10 similar to that used in Production Example 1
After washing with water, suction filtration was performed, and dimethyl sulfoxide 6 was added thereto.
ml, 2N-NaOH 2.6 ml, and epichlorohydrin 1.5 ml.
Stirred at 0 ° C. for 2 hours. After the reaction, the gel was separated by filtration and washed with water to obtain a cellulose gel into which an epoxy group was introduced.

6 ml of concentrated aqueous ammonia was added thereto and reacted at 40 ° C. for 2 hours to obtain an aminated cellulose gel.

In 5 ml of this gel, 0.2 g of sodium polyacrylate having a molecular weight of 190,000 to 500,000 was dissolved in 10 ml of water to obtain a pH 4.5.
Was added, and 200 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide was further added while maintaining the pH at 4.5, followed by shaking at 4 ° C. for 24 hours. After the reaction, the gel was separated by filtration and washed with water to obtain a cellulose gel into which polyacrylic acid had been introduced.

The amount of anionic functional groups introduced by the immobilized polyacrylic acid was 14 μmol / ml of the adsorbent.

Example 2 A porous cellulose gel was used as a cellulose CK gel A22 (trade name, manufactured by Chisso Corp., exclusion limit molecular weight of globular protein of 2000).
10,000, particle size 45-105 μm), Cellulofine GCL-2000m (trade name, manufactured by Chisso Corp., exclusion limit molecular weight of globular protein 30)
100,000, particle size 45-105μm), Cellulofine GC-700m (trade name, manufactured by Chisso Corporation), exclusion limit molecular weight of globular protein 40
10,000, particle size 45-105 μm), Cellulofine GC-200m (trade name, manufactured by Chisso Co., Ltd., exclusion limit molecular weight of globular protein 12)
10,000, particle size 45-105 μm), Cellulofine GCL-90 (trade name, manufactured by Chisso Co., Ltd., exclusion limit molecular weight 3.5 of globular protein)
Cellulose gel in which dextran sodium sulfate was fixed was obtained in the same manner as in Production Example 1 and Example 1 except that the particle size was changed to 45-105 μm.

The amount of the anionic functional group introduced by the immobilized dextran sulfate was each
16, 18, 24, 30, and 37 μmol.

Production Example 3 Epoxy Activated Sepharose CL-6B (trade name, manufactured by Pharmacia Fine Chemicals, exclusion limit molecular weight of globular protein of 4,000,000, particle size of 45 to 165 μm), which is an epoxidized crosslinked agarose gel, was used. Otherwise, dextran sulfate sodium was immobilized in the same manner as in Example 1.

The amount of the anionic functional group introduced by the immobilized dextran sulfate was 20 μmol / ml of the adsorbent.
Met.

Example 4 A hydrophilic porous hard hydrogel FP-HG (trade name, Mitsubishi Kasei Co., Ltd.) containing polymethyl methacrylate as a main component
Made, globular protein exclusion limit molecular weight 4,000,000, particle size 120μ
Except for using m), a gel having sodium dextran sulfate immobilized thereon was obtained in the same manner as in Production Example 1 and Example 1.

The amount of the anionic functional group introduced by the immobilized dextran sulfate was 9 μmol / ml of the adsorbent.
Met.

Example 5 An aminated cellulose gel obtained in the same manner as in Comparative Example 5
To 2 g, the same molecular weight as that used in Production Example 1 of about 500
0, 4 g of dextran sulfate sodium with 15% sulfur content
A solution dissolved in 8 ml of 0.1 M phosphate buffer (pH 8.0) was added, and the mixture was shaken at room temperature for 16 hours. After the reaction, 20 mg of NaCNBH ₃ was added, and the mixture was stirred at room temperature for 30 minutes. After heating at 40 ° C. for 4 hours, the gel was separated by filtration and washed with water to obtain a cellulose gel on which dextran sulfate sodium was fixed.

The amount of the anionic functional group introduced by the immobilized dextran sulfate was 18 μmol / ml of the adsorbent.
Met.

[0071] washing the CK gel A3 carriers used in Experimental Example 1 embodiment Preparation Example 1 Contact and 5, and Comparative Production Example 1 to 5 in Production Example 1 with adsorber and for comparative purposes, which is caught in saline Afterwards, each adsorbent 1.0m
l each to a polypropylene micro tube (7m capacity)
In l), 4.0 ml of serum containing the protein adhering to the glomerular basement membrane was added thereto, and the mixture was shaken at 37 ° C. for 2 hours. After completion of the adsorption operation, the gel was sedimented by centrifugation, and the concentration of the protein adhering to the glomerular basement membrane in the collected supernatant was measured by an enzyme-linked immunosorbent assay (ELISA). In other words, the diluted specimen was added to a plate coated with glomerular basement membrane extracted from human kidney, and the reaction between the glomerular basement membrane and the renal glomerular basement membrane-adhering protein in serum was performed, and the peroxidase-labeled anti-human An immunoglobulin antibody is dropped, and the enzyme color reaction is performed.
It was measured at a measurement wavelength of 486 nm with SLT-210 (trade name, manufactured by Labo Science Co., Ltd.). Table 1 shows the names of compounds having an anionic functional group immobilized on each adsorbent, and the renal glomerular basement membrane in the supernatant after adsorption by each adsorbent with respect to the concentration of the adhering protein to the glomerular basement membrane of the original serum. The adherent protein concentration is indicated as a percentage of the glomerular basement membrane adhering protein concentration in the supernatant.

Table 1 shows that the water-insoluble porous carrier had a molecular weight of 1000
The adsorbent in which the above polyanion compound is fixed,
It can be seen that the protein adhering to the renal glomerular basement membrane is adsorbed. Further, it can be seen that the adsorbent to which dextran sulfate is immobilized has particularly excellent ability to adsorb proteins adhering to renal glomerular basement membrane.

[0073]

[Table 1]

Experimental Example 2 The renal glomerular basement membrane adhering protein concentration in the supernatant was determined in the same manner as in Experimental Example 1 except that the adsorbents obtained in Examples 1 to 4 were used. Table 2 shows the names of various water-insoluble porous carriers using the obtained results.

From Table 2, it can be seen that the Cellulofine of Example 2 is a water-insoluble porous carrier having an exclusion limit molecular weight of 400,000 or less.
It can be seen that the ability of GC-700m, Cellulofine GC-200m and Cellulofine GCL-90 to adsorb proteins adhering to renal glomerular basement membrane is low. Conversely, even if the exclusion limit molecular weight is too large, 50,000,000, the results of the cellulose CK gel A3 of Example 1 show that the ability to adsorb the protein adhering to the glomerular basement membrane tends to decrease.

[0076]

[Table 2]

[0077]

The removal device of the present invention has the effect of removing renal glomerular basement membrane adhering proteins from body fluids.

[Brief description of the drawings]

FIG. 1 is a schematic cross-sectional view of one embodiment of the apparatus for removing proteins adhering to glomerular basement membrane of the present invention.

FIG. 2 is a graph showing the results of examining the relationship between flow rate and pressure loss using three types of gels.

[Explanation of symbols]

 1: Inlet 2: Outlet 3: Adsorbent 4, 5: Filter 7: Container

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁶ , DB name) B01J 20/26 A61M 1/36

Claims (3)

(57) [Claims] 1. A container having an inlet and an outlet for a fluid, the fluid and components contained in the fluid can pass therethrough,
A filter in which a renal glomerular basement membrane-adhering protein adsorbent in which a sulfated polysaccharide having a molecular weight of 1000 or more is immobilized on a water-insoluble porous carrier cannot pass through, and the renal glomerular basement membrane adhering filled in the container An apparatus for removing renal glomerular basement membrane adhering protein, which comprises an adsorbent of a soluble protein. 2. The adsorbent of renal glomerular basement membrane-adhering protein is a water-insoluble porous carrier having a globular protein having an exclusion limit molecular weight of 40 or less.
The removal apparatus according to claim 1, wherein the number is not less than 10,000 and not more than 60 million. 3. The removal device according to claim 2, wherein the water-insoluble porous carrier is made of a compound having a hydroxyl group.