JPH0585191B2 - - Google Patents

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to low-density lipoproteins, which are thought to be closely related to various diseases caused by increased plasma lipids, cancer, and immunoproliferative syndromes. , rheumatoid arthritis, systemic lupus erythematosus, allergies, diseases and phenomena related to the body's immune system such as organ transplant rejection, kidney diseases such as nephritis, liver diseases such as hepatitis, etc., and body fluids such as blood and plasma. The present invention relates to an adsorption column for body fluid purification that adsorbs and removes malignant substances that are expressed in body fluids and are thought to be closely related to the cause or progression of diseases from body fluids.

(Prior Art) The main adsorbents that have been used or studied for the purpose of purifying body fluids are:
Activated carbon or activated carbon coated with a hydrophilic polymer was used as an artificial liver for liver diseases, and heparin-immobilized agarose was used as a low-density lipoprotein adsorbent for familial hypercholesterolemia (Lupien, P-J, et.Al.：A new approach
to the management of familial
hypercholesterolemia.Removal of plasma−
cholesterol based on the principle of affinity
chromatography.Lancet, 2: 1261-1264,
1976.), glass powder, glass beads (Carlson, LA: Chromatographic separation
of serum lipoproteins on glass powder
columns.Description of the method and some
applications.Clin.Chim.Acta, 5:528-538,
1960.).

In addition, in recent years, special substances that the present inventors discovered as adsorbents for various autoantibodies and immune complexes have been chemically added to carriers that have biological and/or chemical selective interactions with the adsorbed substances. There are various adsorbents that are held together by bonding. (Unexamined Japanese Patent Publication No. 57-
77624, JP-A No. 57-77625, JP-A No. 57-122875, JP-A No. 57-134164, JP-A No. 57-156035) These adsorbents are usually packed in columns with fluid inlets and outlets. used.

(Problems to be Solved by the Invention) Since the conventional adsorbents are particulate adsorbents in the form of spheres or crushed bodies, it is difficult to pack them into a column closely, and the displacement capacity (the amount of adsorbent in the column) is difficult. external space), an increase in priming volume,
This led to a decrease in adsorption capacity. When columns are packed with conventional adsorbents, the rejected capacity is typically as much as 40% of the column capacity, and can exceed 50% if the adsorbent packing ratio is low.

Furthermore, among the various adsorbents mentioned above, there are still some that do not have sufficient adsorption capacity as adsorbents for body fluid purification.
When used as an extracorporeal circulation treatment device, sufficient clinical effects cannot be achieved unless a large amount of adsorbent is used, and if a large amount of adsorbent is used, the priming volume increases, which has an unfavorable effect on the patient. There were some things that had deteriorated, and improvements were desired.

(Means for Solving the Problems) The present inventors have solved the above-mentioned problems with the adsorbent, and further, in order to create an adsorption column with higher capacity, the present inventors have developed As a result of extensive research focusing on the method of filling the column,
An adsorption column in which the adsorbent is shaped into fibers or rods, bundled in large numbers in the length direction, placed in a container that is just large enough to hold the bundle of fibers or rods, and has fluid inlets and outlets on both ends of the bundle of fibers or rods. The present inventors have found that this material exhibits extremely high adsorption capacity, and have also confirmed that the resistance to liquid passage is relatively small, leading to the completion of the present invention.

That is, the present invention has a total pore volume of 0.5 cc per gram of dry porous material, which is made of non-activated carbon on which a ligand capable of binding an adsorbed substance is immobilized.
An adsorption column for body fluid purification, characterized in that a large number of the above fibrous or rod-shaped porous bodies are bundled and housed in a container, and each of the bundle of porous bodies has an inlet and an outlet for fluid at both end faces. It is. The porous material used in the present invention may be a porous material such as silica gel, glass, etc., which has an adsorbent property on its surface, on which a ligand capable of binding an adsorbed substance is immobilized, or it may not itself exhibit much adsorption ability. It may also be a porous carrier on which a ligand capable of binding to the substance to be adsorbed is immobilized. In any case, it is a porous body with a sponge-like mesh structure that extends to the inside, and is fibrous or rod-shaped with a ligand on its surface that can biochemically, physically, or scientifically bind to the adsorbed substance. means.

In the present invention, the fibrous or rod-like shape has a length that is sufficiently larger than the cross-sectional dimension. Although the cross-sectional shape is not particularly limited, a circular shape is particularly preferably used. The dimension of the cross section is preferably in the range of 0.01 mm to 10 mm, more preferably 0.02 mm to 5 mm, as a diameter when the cross sectional area is converted into a perfect circle. A range of 0.03 mm to 2 mm is desirable, and a range of 0.05 mm to 1 mm is particularly desirable. The smaller the diameter, the faster the rate of diffusion of the substance to be adsorbed into the pores, which is preferable, but the pressure drop increases. The cross-sectional shape and dimensions are preferably substantially uniform, and the length is usually 100 mm or more, preferably 200 mm or more.

The pore volume and pore diameter of a fibrous or rod-like porous body can be determined by, for example, the mercury intrusion method (Catalyst Engineering Course-4, Catalyst Measurement Method, edited by the Catalysis Society, Chijinshokan, pp. 69 to 73).
The total pore capacity is preferably 0.5 cc/g (dry adsorbent weight) or more, and more preferably 1.0 cc/g or more, as determined from the mercury intrusion curve obtained by (Page). It is preferably greater than 2.0 cc/g, and more preferably 3.0 cc/g or greater. If the adsorbent material is the same, the pore capacity is
The larger the value, the larger the internal space volume of the adsorbent per unit volume, and the adsorption capacity of the adsorbed substance can be increased accordingly.

The pore size and pore size distribution can be freely selected by taking into consideration the size, concentration, etc. of the adsorbed substance and the coexisting substance, but pore sizes ranging from tens of thousands to tens of thousands are possible, and the pore size distribution is also very sharp. It is possible to range from to broad.

The fibrous or rod-shaped porous adsorbent can be formed into a fibrous or rod-like shape by, for example, fusion, adhesion, etc. of particulate adsorbent having a surface capable of bonding with the substance to be adsorbed. Although it is possible to make a fibrous or rod-like adsorbent by molding resin into a fibrous or rod-like shape and then heat-treating it, the fibrous or rod-like adsorbent obtained by these methods
A rod-shaped adsorbent is not preferable because it is substantially the same as a particulate porous adsorbent, and the actual amount of adsorbent is small.

The fibrous or rod-shaped porous body referred to in the present invention preferably does not substantially have extremely large pores, and the entire fiber or rod has pores that participate in adsorption.

Such fibrous or rod-like porous bodies are
For example, by heating a glass fiber or glass rod made of a ternary system of Na ₂ O, B ₂ O ₃ -SiO ₃ to separate the phases, and extracting the phase rich in Na ₂ O and B ₂ O ₃ with an acid. can get. The obtained porous glass fibers or rods have adsorption properties for proteins as they are, but it is also possible to impart adsorption selectivity by partially blocking the surface silanol groups, or by using a silane coupling agent. Alternatively, a ligand capable of binding to the adsorbed substance can be immobilized on the surface.

Examples of ligands that can bind to adsorbed substances include heparin and anti-low-density lipoprotein antibodies for adsorption of low-density lipoproteins for treatment of familial hypercholesterolemia.

For the treatment of systemic lupus erythematosus, adenine,
Examples include homopolymers or copolymers of mono-, di-, and trinucleotides such as guanine, cytosine, uracil, and thymine, and naturally occurring nucleic acids such as DNA and RNA. In addition, DNA present in the blood,
Anti-single-stranded DNA antibody, anti-double-stranded DNA antibody, anti-RNA antibody, anti-ENA for adsorption and removal of RNA and ENA.
Examples include anti-nucleic acid antibodies such as antibodies, and basic compounds such as methylated albumin actinomycin D. In addition, for the adsorption and removal of immune complexes in blood, Ciq
Examples include antibodies against complement components such as, specific proteins such as protein A, and immune complexes such as anti-heavistein constant region second phase antibodies.

For the treatment of rheumatoid arthritis and malignant rheumatoid arthritis, chemical denaturation (agglomeration) methods such as urea, guanidine hydrochloride, mercaptoethanol, surfactants, and organic solvents, physical denaturation (agglomeration) methods such as heat, ultrasound, and gas bubbling are used. ) Modified γ modified by method
-Globulin, modified immunoglobulin, aggregated γ-
Examples include antigen-like substances against rheumatoid factors such as globulin, aggregated immunoglobulin, Fc region of immunoglobulin, heavy chain constant region 2 of immunoglobulin, and modified products thereof by the above-mentioned modification method, and anti-rheumatoid factor antibodies. In addition, for removing immune complexes from rheumatism, there may be mentioned complement components such as Clq, specific proteins such as protein A, and antibodies against immune complexes such as anti-heavichein constant region 2 phase antibody.

For the treatment of Hashimoto's disease, thyroglobulin, a thyroid microsomal fraction component, and for the treatment of myasthenia gravis, a neuromuscular acetylcholine receptor fraction component can be cited.

Glomerular basement membrane components are used to treat glomerulonephritis, platelet membrane components and platelet granule fraction components are used to treat idiopathic thrombocytopenic purpura, and transcortisone and anticortisone antibodies are used to treat Cushing's syndrome. It will be done.

For the prevention and treatment of hepatitis, hepatitis A virus,
Examples include antibodies against surface antigens of viruses such as hepatitis B virus, and anti-angiotensin antibodies for treating hypertension.

Anti-lymphocyte antibodies such as anti-B cell antibodies, anti-suppressor T antibodies, and anti-helper T antibodies are used to treat immune diseases based on lymphocyte abnormalities, and protein A and anti-immune antibodies are used to treat cancers such as breast cancer. Examples include globulin antibodies.

Ligands that can be used in the present invention are not limited to the above examples, but include lectins such as congnitinin, concanavalin A, and phytohemaagglutinin, nucleic acids, amino acids, lipids, protamines, antigens, antibodies, enzymes, substrates, Known substances that can bind to adsorbed substances such as coenzymes can be used.

Furthermore, it is also possible to use a carrier holding two or more types of ligands. Furthermore, two or more types of carriers holding ligands can be used in combination.

The carrier needs to be a fibrous or rod-like porous body, and it must contain an activated functional group (a nucleophilic reactive group having active hydrogen such as a hydroxyl group, an amino group, a carboxyl group, a thiol group, a silanol group, etc.) It is preferable to have this on the surface.

Methods of binding the ligand to the carrier include covalent bonding,
Any known method such as ionic bonding, physical adsorption, embedding, or precipitation and insolubilization on the polymer surface can be used, but in view of the elution properties of the bound substance, it is preferable to use covalent bonding for immobilization and insolubilization. Therefore, it is possible to use commonly known methods for activating immobilized enzymes, carriers used in affinity chromatography, and binding methods for ligands.

Examples of activation methods include cyanogen halide method, epichlorohydrin method, bisepoxide method,
Examples include the halogenated triazine method, the bromoacetyl bromide method, the ethyl chloroformate method, the 1,1'-carbonyldiimidazole method, and the silane coupling agent activation method. The activation method of the present invention is limited to the above examples as long as it can perform a substitution and/or addition reaction with a nucleophilic reactive group having active hydrogen such as an amino group, a hydroxyl group, a carboxyl group, a thiol group, or a silanol group of the ligand. It's not a thing.

In the case of an inorganic porous carrier, a silane coupling agent is preferably used.

The fibrous or rod-like porous material is filled into a container, for example, as shown in the drawings.

The drawing is a schematic cross-sectional view showing an example of an adsorption column for body fluid purification of the present invention.
A cap 5 having a body fluid inlet 4 is fitted with a screw through a packing 3 with a filter 2 stretched on the inside, and a filter 2' is inserted inside the opening at the other end of the cylinder 1.
A cap 7 having a body fluid outlet 6 is screwed into the cap 7 through the packing 3' which is stretched with a body fluid, thereby forming a container.
An adsorbent layer 8 is formed by filling and holding an adsorbent in the gap between the filters 2 and 2'.

Here, the adsorbent layer 8 includes a large number of fibrous or rod-shaped porous bodies made of non-activated carbon on which a ligand capable of binding to the adsorbed substance is immobilized, aligned in the length direction and bundled. It is housed in cylinder 1. If there is a gap between the wall surface of the cylinder 1 and the fibrous or rod-like porous adsorbent and there is a concern about one-sided flow of body fluids, it is preferable to put a filler or adhesive into this gap. Care must be taken not to crush the pores of the rod-shaped porous body. In the bundle of fibrous or rod-like porous materials, each fibrous or rod-like porous adsorbent may be independent, but it may be fused or adhered at multiple points to form a single bundle. may be easier to handle.

The volume of the adsorbent layer 8 is, when used for extracorporeal circulation,
Approximately 50 to 400 ml is appropriate. When using the adsorption column for body fluid purification of the present invention in extracorporeal circulation, there are roughly two methods as follows. One method is to separate blood taken from the body into blood cell components using a centrifuge or membrane plasma separator, and then pass the plasma component through the adsorption column to purify it. One method is to return the blood to the body together with blood cell components, and the other method is to directly pass the blood taken out from the body through the adsorption column for purification.

The method for passing body fluids may be either continuous or intermittent, depending on clinical needs or equipment conditions.

(Effects of the Invention) First, the adsorption column for body fluid purification of the present invention has a carrier made of porous thin rod-shaped or fibrous bodies bundled in an orderly manner in parallel. Compared to an adsorbent filled with a carrier, body fluids can easily flow into the porous parts.
Therefore, the porous portion that can contribute to adsorption can be utilized more effectively and in an amount closer to the theoretical value than in a particulate adsorbent.

In addition, in the adsorption column for body fluid purification of the present invention, the above-mentioned rod-shaped or fibrous bodies are firmly fixed to the inner walls at both ends of the column at both ends, so that when body fluid is introduced, the flow resistance is low and the pressure loss is also reduced. This can be kept lower than in columns packed with particulate carriers.

Furthermore, since the adsorption column for body fluid purification of the present invention has a ligand capable of binding to any adsorbed substance immobilized on the above-mentioned rod-like or fibrous body, body fluid can be purified by appropriately selecting the ligand. It can be used to remove various malignant substances inside.

The adsorption column for body fluid purification of the present invention has a much higher adsorption capacity per unit volume than conventional ones, so the priming volume of the adsorption column can be reduced, so blood can be drawn out of the body and delivered to the patient. This burden can be greatly reduced,
It also has excellent operability.

(Example) Example 1 Fibrous porous glass with a diameter of 0.2 mm and a length of 16 cm (average pore diameter measured using a mercury intrusion porosimeter manufactured by Carlo Erba: 498‖, pore volume 0.6 cc) /g) was immersed in 500 ml of 0.5N aqueous sodium hydroxide solution, gently shaken, and treated at room temperature for 8 hours. Wash thoroughly with water, then with acetone, and then immerse in 200 ml of 20 v/v% γ-glycidoxypropyltrimethoxysilane in acetone solution at 50°C with gentle shaking.
It was activated by reacting for 40 hours. The activated fibrous porous glass was washed sequentially with acetone, water, and 0.1 sodium carbonate buffer (PH9.8).

Contains 1w/v% polyacrylic acid (molecular weight approximately 13000) with an amino group at one end, synthesized using 2-aminoethanethiol as a chain transfer agent and α,α′-azobisisobutyronitrile as an initiator.
In 200ml of 0.1M sodium carbonate buffer (PH9.8),
The above activated fibrous porous glass was immersed and an immobilization reaction was carried out at 50° C. for 72 hours with gentle shaking.

The obtained fibrous porous adsorbent has an inner diameter of 2 cm and a length of
A 16 cm column with an internal volume of 50 ml was filled with 3,817 columns to form an adsorption column.

300 ml of familial hypercholesterolemia patient plasma kept at 37° C. was perfused into the adsorption column at a flow rate of 1 ml/min. When the low-density lipoprotein concentration before and after adsorption was measured by turbidimetry, it was 720 mg/dl before adsorption, whereas it was 115 mg/dl after adsorption, indicating high adsorption.

Example 2 Average pore size 0.2μm, pore volume 0.6ml/g, diameter 0.2
A porous glass fiber with a length of 16 cm and a length of 16 cm was fabricated.

10 g of this was immersed in a 25% solution of γ-glycidoxypropyltrimethoxysilane (acetone solution solvent) and reacted at 50° C. for 40 hours with shaking. After the reaction, wash with 500ml of acetone, then wash with water,
Place in 20 ml of 0.1M sodium carbonate buffer at 50°C.
Immobilization was performed with shaking for 40 hours, and then 60 mg/
Add 5 ml of tris(hydroxyethyl)aminomethane solution and conduct blocking reaction (to block remaining active groups) while stirring at 50°C for 5 hours.
I went there. Thereafter, the fibers were washed with water to obtain tryptophan-immobilized fibers. Immobilized tryptophan is
It was 55 μmol/(ml fiber).

Approximately 5 g of 930 pieces of the fibrous adsorbent thus obtained were packed into a column with an inner diameter of 9 mm and a length of 16 cm, and 12 ml of rheumatism patient plasma was passed through the column at a rate of 0.4 ml/min. The concentration of immune complexes after perfusion was less than 10.0 μg/ml, and the reduction rate of immune complexes due to adsorption was more than 58%.

Example 3 High-density polyethylene (density 0.968, MI value 5.5, trade name Hi-Zex 2208J) was spun using a circular spindle with a diameter of 35 mm, spindle temperature 150°C, polyethylene output rate 16 g/min, spinning distance 5 m, and spinning cooling. Melt spinning was carried out at a temperature of 20° C., a winding tension of 10 gf, and a winding speed of 400 ml/min.

The obtained polyethylene thread was heated at room temperature (22℃).
Cold stretching was performed at a primary roller speed of 3.75 m/min and a secondary roller speed of 5 m/min. Next, following this cold stretching, at three temperatures of 108°C, 119°C, and 122°C,
Each roller speed 15m/min, 19.5m/min,
The pores were opened by hot stretching at a stretching speed of 21.8 m/min to obtain a fibrous porous polyethylene adsorption carrier. The total stretching ratio was 5.8 times.

The obtained fiber has a thread diameter of 176 μm, a winding length of 1 km, an average pore diameter of 0.2 μm by mercury intrusion method, a total pore volume of 3.6 ml/g, a porosity of 77.8%, and a total surface area of 71.03.
The volume of pores with m ² /g and pore diameter of 0.01 μm or more and 10 μm or less was 84% of the total pore volume.

This porous support (length 12 cm, 1000 pieces) was placed in chloroform containing 5% glycidyl methacrylate and irradiated with 25 kGy of γ-rays under a nitrogen atmosphere. After irradiation, solvent washing was performed, and the grafting rate determined from the weight change was 15.6%, indicating that a large amount of epoxy groups could be introduced.

An adsorbent containing phenylalanine in a 0.1N aqueous sodium hydroxide solution was obtained on this epoxy group-introduced porous support. The fixed amount of phenylalanine is
The amount was 73 μequivalent (including internal pores) per ml of adsorbent.

1000 pieces of this adsorbent were packed into a column with an inner diameter of 10 mm and a length of 12 cm, and 0.3
Perfusion was performed at ml/min. The concentration of immune complexes in plasma before perfusion was 28.6 μg/ml, whereas the concentration of immune complexes after perfusion was less than 10.0 μg/ml, and the rate of decrease of immune complexes due to adsorption was It was below 65%.

Comparative Example 1 Using the polyethylene of Example 3, the same operation as above was performed except that cold stretching was not performed, and the total stretching ratio was
A 5.8x non-porous polyethylene fiber was obtained.

Glycidyl methacrylate was grafted onto this fiber in the same manner as in Example 3. The grafting rate determined by weight change was 5.7%. This epoxy group-introduced fiber was reacted with phenylalanine in the same manner as above, and the amount of phenylalanine fixed was 30 μ equivalent per ml of adsorbent.

Using this phenylalanine-immobilized fiber and the same plasma, the concentration of the immune complex was measured by the method described above. The concentration of immune complexes after perfusion was 19.1 μg/ml.
The reduction rate of immune complexes due to adsorption is 33
% or more.

[Brief explanation of drawings]

The drawing is a schematic cross-sectional view showing an example of the adsorption column for body fluid purification of the present invention. 1...Cylinder, 2, 2'...Filter, 3,
3'...Packing, 4...Body fluid inlet, 5...
Cap, 6... Body fluid outlet, 7... Cap,
8...Adsorbent layer.

Claims (1)

[Claims] 1 1g of dry porous material made of non-activated carbon with a ligand capable of binding to the adsorbed substance immobilized on its surface
A large number of fibrous or rod-shaped porous bodies each having a total pore capacity of 0.5 cc or more are bundled together and stored in a container, and each end face of the bundle of porous bodies has an inlet and an outlet for fluid. An adsorption column for body fluid purification characterized by: