CA1221307A

CA1221307A - Adsorbent and process for preparing the same

Info

Publication number: CA1221307A
Application number: CA000442312A
Authority: CA
Inventors: Nobutaka Tani; Tsuneo Hayashi
Original assignee: Kanegafuchi Chemical Industry Co Ltd
Current assignee: Kanegafuchi Chemical Industry Co Ltd
Priority date: 1982-12-02
Filing date: 1983-11-30
Publication date: 1987-05-05
Also published as: EP0110409B2; EP0464872B1; ATE195891T1; DE3382834D1; US4576928A; EP0225867B1; EP0225867A2; EP0464872A1; EP0110409B1; US4637994A; EP0225867A3; EP0110409A2; DE3382834T3; DE3379644D1; EP0110409A3; DE3382834T2; DE3382723T2; DE3382723D1; EP0464872B2

Abstract

Abstract of the Disclosure Adsorbent for removing a harmful substance to be removed from body fluid such as blood or plasma composed of a porous hard gel on which a ligand having an affinity for the harmful substance is immobilized. The adsorbent is suitable for selective removal of harmful substances such as VLDL, LDL, harmful proteins, viruses and harmful cells, in extracorporeal circulation treatment.

Description

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BACKGROUND OF THE INVENTION
The present invention relates to a novel adsorbent and a process for preparing the same, more particularly, to an adsorbent for removing harmful substances to be removed from body fluid such as blood or plasma in extra corporeal circulation treatment.
There has been required a means for selectively removing harmful substances which appease in body fluid and closely relate to a cause or a progress of a disease For example, it is known that plasma lipoprotein, especially very low density lipoprotein (hereinafter referred to as "VLDL") and/or low density lipoprotein (hereinafter referred to as "LDL") contain a large amount of cholesterol and cause arteriosclerosis. In hyperlipemia such as familial hyperlipemia or familial hypercholesterolemia, VLDL and/or LO show several times higher values than those in normal condition, and often cause arteriosclerosis such as coronary arteriosclerosis Although various types of treatments such as regimen and medications have been adopted, they have limitations in effect and a fear of unfavorable side effects. Particularly in familial hypercholesterolemia a plasma exchange therapy which is composed of plasma removal and compensatory supplement of exogenous human plasma protein solutions is probably the only treatment method hying effective nowadays. The plasma exchange therapy, however, has various defects such as (1) a need for using expensive fresh plasma or plasma fractions, (2) a fear of infection by hepatitis viruses and the like, and (3) loss of all plasma components containing not only harmful components but also useful ones, i.e. in case of lipoprotein, not only VLDL and/or LDL but also high density lipoprotein (hereinafter referred to as "HI." ) are lost. For the purpose of solving the above defects, a selective removal of harmful components by a membrane and the like has been adopted. These methods, however, are insufficient in selectivity and cause a large loss of useful components from body fluid. There has been also I

tried a selective removal of harmful components by means of adsorption. For example, a synthetic adsorbent such as active carbon or ~mberlite TAD (a registered trademark, commercially available from Room & Hess Co.) has been utilized for liver disease. Such an adsorbent however has many defects such as poor selectivity and disability for removing high molecular compounds. Furthermore, for the purpose of increasing selectivity, there has been adopted an adsorbent based on the principle of affinity chromatography composed of a carrier on which a material having an affinity for a substance to be specifically removed (such material is hereinafter referred to as "ligand") is immobilized. In that case, however, it is difficult to obtain a sufficient flow rate for an extra corporeal treatment because a carrier is a soft gel such as agrees. Accordingly, a particular modification in column shape is required in order to obtain a large flow rate and the risk of an occasional clogging still remains. Therefore, a stable extra corporeal circulation cannot be achieved by the above method.
An adsorbent of the present invention may be used for selectively removing not only the above-mentioned VLDL and/or LDL but also other harmful substances to be removed from body fluid.
It is an object of the present invention to provide an adsorbent for selectively removing harmful substances such as VLDL, LDL, virus and harmful cells from body fluid such as blood or plasma in extra corporeal circulation treatment of immune disease, metabolic disease, inflammatory disease such as hepatitis or nephritis, virus infection and the like.
A further object of the present invention is to provide a process for preparing the adsorbent.
These and other objects of the present invention will become apparent from the description hereinafter.

SUMMARY OF THE INVENTION

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In accordance with the present invention, there can be provided an adsorbent for removing a substance to be removed from body fluid in extra corporeal circulation treatment comprising a water-insoluble porous hard gel on which a ligand having an affinity for the substance is immobilized.

BRIEF DESCRIPTION F THE DRAYING
Figs. 1 and 2 are graphs, respectively, showing relations between flow rate and pressure-drop obtained in Reference Examples 1 and 2, and Fig 3 is a chart of polyacrylamide disc gel electrophoresis obtained in Example 41.

DETAILED DESCRIPTION OF THE INVENTION
It is suitable that carriers used in the present invention have the following properties:
(1) relatively high mechanical strength, I low pressure-drop and no column clogging in case of passing body fluid through a column packed with a carrier, (3) a large number of micro pores into which a substance to be removed permeates substantially, and (4) less change caused by a sterilizing procedure such as steam sterilization by autoclaving.
Therefore, the most suitable carrier used in the present invention is a water-insoluble porous polymer hard gel or a porous inorganic hard gel.
The porous hard gel used in the present invention is less swelled with a solvent and less deformed by pressure than a soft gel such as dextran, agrees or acrylamide.
The term "hard gel" and "soft gel" in the present invention is explained as follows A hard gel is distinguished from a soft gel by the following method described in Reference Examples 1 and 2. That is, when a relation between wow rate and pressure-drop is determined by passing water through a column uniformly packed with a gel, a hard gel shows a .,~

3~7 linear relationship while a soft gel shows a non-linear relationship. In case of a soft gel, a gel is deformed and consolidated over a certain pressure so that a flow rate does not increase further. In the present invention, a gel having the above linear relationship a-t least by 0.3 kg/cm is referred to as "hard gel".
A pore size of the porous hard gel it selected depending on molecular weight, shape, or size of a substance to be removed, and the most suitable pore size may be selected in each case. For measuring the pore size, there are various kinds of methods such as mercury porosimetry and observation by an electron microscope as a direct measuring method. With respect to water-containing particles, however, the above methods sometimes cannot be applied. In such a case, an exclusion limit may be adopted as a measure of pore size. The term "exclusion limit" in the present invention means the minimum molecular weight of a molecular which cannot permeate into a pore in a gel permeation chromatography (cf.
Heroic Hutton and Toshihiko lanai: Zikken Cossack Equity Chromatography (Experimental High-Pressure Liquid Cry-matography), published by Kabushiki Couch Kagaku Dojin).
Phenomenally, a molecule having a molecular weight of more than exclusion limit is eluded near the void volume.
Therefore, an exclusion limit can be determined by studying the relations between molecular weights and elusion volumes using substances of various molecular weights in a gel permeation chromatography. An exclusion limit varies with a kind of substances to be excluded.
In the present invention, an exclusion limit of the porous hard gel is measured by using globular proteins and/or viruses, and the preferable exclusion limit is 5 x 103 to 1 x 109~ When the exclusion limit is more than 1 x 109, the adsorbing amount of a substance to be removed decreases with a decrease of amount of immobilized ligand, and further a mechanical strength of gel is reduced.
Particularly, in case of removing VLDL and/or LDL being giant molecules having a molecular weight of more than 1 x 106, a porous hard gel having an exclusion limit of less than 1 x 106 is not practically available.
On the other hand, a porous hard gel having an exclusion limit of from 1 x 106 to several million which is near a molecular weight of VLDL or LDL per so may be practically available to a certain extent. A preferable exclusion limit for removal of VLDL and/or LDL is 1 x 106 to 1 x 109, more preferably 1 x 106 to 1 x 108.
With respect to a porous structure of the porous hard gel used in the present invention, a structure uniformly having pores at any part of the gel (hereinafter referred to as "uniform structure") is more preferable than a structure having pores only on the surface of the gel. It is preferred that a porosity of the gel is not less than 20 I. A shape of the carrier is selected depending on a kind of a substance to be removed. The carrier may be selected from suitable shapes such as particle, fiber, sheet and hollow fiber In case of using a carrier in the shape of particle, although a particle having a smaller size generally shows an excellent adsorbing capacity, the pressure-drop increases with an extremely small size. Therefore, a particle having a size of 1 em to 5000 em is preferred.
Furthermore, it is preferred that a carrier has functional groups to be utilized for the immobilization of ligand or groups to be easily activated. Examples of the group are, for instance, amino, carboxyl, hydroxyl, they'll, acid android, succinylimide, chlorine, alluded, amino, epoxy group, and the like.
Representative examples of the water-insoluble porous hard gel used in the present invention are, for instance, a porous hard gel of a synthetic polymer such as stylene-divinylbenzene copolymer, cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked vinyl ether-maleic android copolymer, cross-linked stylene-maleic android copolymer or cross-linked polyamide, a porous cellulose gel, an I

inorganic porous hard gel such as silica gel, porous glass, porous alumina, porous silica alumina, porous hydroxyapatite, porous calcium silicate, porous zircon or porous zealot, and the like. Of course it is to be understood that the porous hard gels used in the present invention are not limited to those set forth as above examples The surface of the above-mentioned porous hard gel may be coated with polysaccharides, synthetic polymers, and the like. These porous hard gels may be employed alone or in an admixture thereof.
In the above representative examples, some of the porous polymer hard gels composed of synthetic polymers have a fear of toxicity due to unrequited monomers and a less adsorbing capacity than that of a soft gel.
Therefore, in the above representative examples, a porous cellulose gel is one of the particularly preferable carriers for the present invention, and it satisfies the above all four points required for the carrier. In addi~ionr the porous cellulose gel has various excellent advantages such as hydrophilicity due to being composed of cellulose, a large number of hydroxyl group to be utilized for immobilization, less nonspecific adsorption, and sufficient adsorbing capacity not inferior to that of a soft gel due to its relatively high strength even with a large porosity. Therefore, the porous cellulose gel on which a ligand is immobilized provides a nearly ideal adsorbent.
As the porous cellulose gel used in the present invention, although cellulose per so is preferred, a cellulose derivative such as an esterified cellulose or an etherified cellulose, or a mixture of cellulose and the cellulose derivatives may be employed. Examples of the cellulose derivative are, for instance, acutely cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, and the like. It is preferred that the cellulose gel is in the spherical shape. The ~:Z~3~)~
- a -cellulose gel is prepared, for example, by dissolving or swelling cellulose and/or a cellulose derivatives with a solvent, dispersing the resulting mixture into another solvent being not admixed with the used solvent to make beads, and then regenerating the beads. The cellulose and/or cellulose derivatives may be cross-linked or not.
A porosity of a porous cellulose gel may be a measure of cellulose content. The cellulose content is expressed by the following formula:
W
Cellulose content (%) V x 100 wherein W is dry gel weight (g), Vet is a volume of column packed with Mel (ml) and Vow is a void volume (ml).
It is preferred that the cellulose content of the porous cellulose gel used in the present invention is

2 % to 60 I. In case of less than 2 %, the mechanical strength of gel is reduced, and in case of more than 60 %, the pore volume is reduced.
I Representative examples of the ligand used in the present invention are as follows:
Representative examples of the ligand using antigen-antibody reaction and the like are, far instance, a complement component such as Cluck, an anti-immune complex antibody, and the like for removal of immune complexes; an anti-nuclear antibody appeared in blood in general luaus erythematosus r and the like for removal of auto antibodies in auto immune diseases; a nucleic acid base, a nucleoside, a nucleated, a polynucleotide, DNA, RNA, and the like for removal of anti-DNA antibodies; an acetylcholine receptor fraction for removal of anti-acetylcholine receptor antibodies in myasthenia gravies; antibodies to various harmful components in blood such as an antibody to an antigen on a surface of virus for removal of hepatitis virus and an anti-DNA antibody for removal of DNA appeared in blood in general luaus erythemotosus; an anti-B cell antibody or anti-suppressor T cell antibody for removal of lvmpocytes in lymphocyte disorder, and the like. Furthermore, antigens to various auto antibodies may be used for removal of auto antibodies.
In addition to the above representative examples, representative examples of the ligand using a specific affinity are, for instance, a degenerated or agglutinated immuno~lobulin, globulin, or the fraction component thereof, an amino acid such as tryptophan, and the like for removal of rheumatoid factors in rheumatoid arthritic; a polyanion compound for removal of a lo lipoprotein such as VLDL or LDL; protein for removal of immunoglobulin; hemoglobin for removal of haptoglobin;
haptoglobin for removal of hemoglobin; Lawson for removal of plasminogen; immunoglobulin G jig G) for removal of Cluck; arginine for removal of precallicrein; transcortine for removal of courteously; hymen for removal of hemopexin;
polymyxin for removal of endotoxin, and the like.
Furthermore, pectin such as concanavalin Al conglutinin or phytohemagglutinin, nucleic acids, enzymes, substrates, consumes, and the like may be used. Of counsel it is to be understood that the ligands of the present invention are not limited to those set forth as above examples. These ligands may be used alone or in an admixture thereof.
As a substance to be removed, there may be included from a substance having a molecular weight of less than Lowe such as bilirubin to a substance having more than tens of millions of molecular weight such as viruses. The porous hard gel of the present invention is selected depending on molecular weight and molecular size of a substance to be removed, and also affected by various factors such as a kind of ligand and a shape of a substance to be removed. For example, it is suitable that the porous hard gels having from thousands to hundreds of thousands, tens of millions, and from tens of millions to hundreds of millions of exclusion limits are employed, respectively, to remove substances having hundreds, millions and tens of millions of molecular weights Al ~X~3q~7 When substances to be removed are VLDL and/or LDL containing a large amount of cholesterol and causing arteriosclerosis, polyanion compounds are preferred as a ligand. Examples of the polyanion compounds are, for instance, sulfated polysaccharides such as hep~rin, dextran sulfate, chondroitin sulfate, chondroitin polyp sulfate, heparan sulfate, Courtney sulfate, heparin sulfate, Dylan sulfate, caronin sulfate, cellulose sulfate, chutney sulfate, chitosan sulfate, pectin lo sulfate, insulin sulfate, arginine sulfate, glycogen sulfate, polylactose sulfate, carrageenan sulfate, starch sulfate, polyglucose sulfate, laminarin sulfate, galactan sulfate, lean sulfate and mepesulfate, phosphorus wolframic acid, polysulfated anthill, polyvinyl alcohol sulfate, polyphosphoric acid, and/or the salts thereof, and the like. Preferable examples of the above polyanion compounds are, for instance, heparin, dextran sulfate, chondroitin polysulfate, and/or the salts thereof, and particularly preferable examples are a dextran sulfate and/or the salt thereof. Examples of the salt of the above polyanion compound are, for instance, a water-soluble salt such as sodium salt or potassium salt, and the like.
Dextran sulfate and/or the salt thereof are explained in more detail hereinbelow.
Dextran sulfate and/or the salt thereof are sulfuric acid ester of dextran being a polysaccharide produced by Leuconostoc mesenteroides, etc., and/or the salt thereof. It has been known that dextran sulfate and/or the salt thereof form a precipitate with lipoproteins in the presence of a diva lent cation, and dextran sulfate and/or the salt thereof having a molecular weight of about 5 x 105 (intrinsic viscosity of about 0.20 dug are generally employed for this precipitation. However, as shown in the following Example 38 of Run Nos. I and (2), a porous hard Mel on which the above-mentioned dextran sulfate and/or the salt thereof are immobilized is sometimes poor in affinity -to Lo VLDL and/or LDL. As a result of extensive studies to solve the above problems, i-t has now been found that dextran sulfate having an intrinsic viscosity of not more than 0.12 dug preferably not more than 0.0~ dug and a sulfur content of not less than 15 % by weight has high affinity and selectivity to VLDL and/or LDL.
Furthermore, the adsorbent of the present invention employing such dextran sulfate and/or the salt thereof as a ligand has high affinity and selectivity even in the absence of a diva lent cation. Although a toxicity of dextran sulfate and/or the salt thereof is low, the toxicity increases with increasing of molecular weight From this point of view, the use of dextran sulfate and/or the salt thereof having an intrinsic viscosity of not more than 0.12 dug preferably not more than 0.08 dug can prevent a danger in case that the immobilized dextran sulfate and/or the salt thereof should be released prom a carrier In addition, dextran sulfate and/or the salt thereof are less changed by a sterilizing procedure such as steam sterilization by autoclaving, because they are linked mainly by 6)-glycosidic linkage. Although there are various methods for measuring a molecular weight of dextran sulfate and/or the salt thereof, a method by measuring viscosity is general. Dextran sulfate and/ox the salt thereof, however, show different viscosities depending on various conditions such as ion strength, pi value, and sulfur content (content of sulfonic acid group). The term "intrinsic viscosity" used in the present invention means a viscosity of sodium salt of dextran sulfate measured in a neutral 1 M Nail aqueous solution, at 25C. The dextran sulfate and/or the salt thereof used in the present invention may be in the form of straight-chain or branched-chain.
For coupling a ligand with a carrier, various methods such as physical adsorption methods, ionic coupling methods and covalent coupling methods may be employed. In order to use the adsorbent of the present ~2~3~3~

invention in extra corporeal circulation treatment, it is important that the ligand is not released. Therefore, a covalent coupling method having a strong bond between ligand and carrier is preferred. In case of employing other methods, a modification is necessary to prevent the release of ligand. If necessary, a spacer may be introduced between ligand and carrier.
It is preferred that a gel is activated by a reagent such as a cyanogen halide, epichlorohydrin, a polyoxirane compound such as bisepoxide or treason halide, and then reacted with a ligand to give the desired adsorbent. In that case, it is preferred that a gel having a group to be activated such as hydroxyl group is employed as a carrier In the above reagents, epichlorohydrin or a polyoxirane compound such as bisepoxide is more preferred, because a ligand is strongly immobilized on a carrier activated by using such a reagent and a release of a ligand is reduced.
Epichlorohydrin and a polyoxirane compound, however, show lower reactivity, particularly lower to dextran sulfate and/or the salt thereof, because dextran sulfate and/or the salt thereof have hydroxyl group alone as a functional group. Therefore, it is not easy to obtain a sufficient amount of immobilized ligand.
As a result of extensive studies, it has now been found that the following coupling method is preferred in case of using dextran sulfate and/or the salt thereof as a ligand. That is, a porous polymer hard gel is reacted with epichlorohydrin and/or a polyo~irane compound to introduce epoxy groups into the golf and then dextran sulfate and/or the salt thereof is reacted with the resulting epoxy-activated gel in a concentration of not less than 3 % based on the weight of the whole reaction system excluding the dry weight of the gel, more preferably not less than 10 %. This method gives a good immobilizing efficiency. In that case, a porous - cellulose gel is particularly suitable as a carrier.
On the other hand, when a porous inorganic hard 2~3~7 gel is employed as a carrier, it is preferred that the gel is activated with a reagent such as an epoxysilane, e.g. r-glycidoxypropyltrimethoxysilane or an aminosilane, e.g. Y-aminopropyltriethoxysilane, and then reacted with a ligand to give the desired adsorbent.
The amount of immobilized ligand varies depending on properties of the ligand used such as shape and activity. For sufficient removal of VLDL and/or LDL
by using a polyanion compound, for instance, it is preferred that the polyanion compound is immobilized in an amount of not less than 0.02 mg/ml of an apparent column volume occupied by an adsorbent (hereinafter referred to as "bed volume"), economically 100 my or less. The preferable range is 0.5 to 20 mg/ml of bed volume. Particularly, for removal of VLDL and/or LDL by using dextran sulfate and/or the salt thereof as a ligand, it is preferred that the amount of immobilized ligand is not less than 0.2 mg/ml of bed volume. After the coupling reaction, the unrequited polyanion compound may be recovered for reuse by purification, etc.
It is preferred that the remaining unrequited active groups are blocked by ethanol amine, and the like.
In accordance with the present invention, an adsorbent composed of porous cellulose gel having an exclusion limit of 106 to 10~ and a particle size of 30 to 200 em on which sodium salt of dextran sulfate having an intrinsic viscosity of not more than 0.12 dug and a sulfur content of not less than 15 % by weight is immobilized is particularly suitable for removal of VLDL
and/or LDL in extra corporeal circulation treatment of hypercholesterolemia.
The adsorbent of the present invention may be employed for various kinds of use. Representative example of the use is extra corporeal circulation treatment performed by incorporating a column into extra corporeal circulation circuit and passing body fluid such as blood or plasma through the column, the column being packed with the adsorbent of the present invention. The use of

3~7 the adsorbent it not necessarily limited to the above example.
The adsorbent of the present invention can be subjected to steam sterilization by autoclaving 50 long as the ligand it not largely degenerated, and this sterilization procedure does not affect on micro pore structure, particle shape and gel volume of the adsorbent.
The present invention it more specifically described and explained by means of the following Reference Examples and examples; and it is to be understood that the present invention is not limited to the Reference Examples and Examples.

Reference Example 1 Bejewel Arm (a commercially available agrees gel made by Byrd Co., particle size: 50 to 100 mesh as a soft gel and Toyopearl HOWE pa commercially available cross-linked polyacrylate gel made by Toy Soda Manufacturing Co., Lid particle size: 50 to 10P em) and Cellulo~ine ~C-700 (a commercially available porous cellulose gel made by Chihuahuas Corporation, particle size:
45 to 105 em) as a hard gel were uniformly packed, respectively, in a glass column (inner diameter: 9 mm, height: 150 mm) having filters (pore size: 15 em) at both top and bottom of the column. Water was passed through the thus obtained column, and a relation between flow rate and pressure-drop was determined. The results are shown in Fig. 1. As shown in Fig. 1, flow rate increased approximately in proportion to increase of pressure-drop in the porous polymer hard gels. On the other hand, the agrees gel was consolidated. As a result, increasing pressure did not make flow rate increase.
Reference Example 2 The procedures of Reference Example 1 were repeated except that FOG 2000 (a commercially available * Trade Mark - 15 3~7 porous glass made by Wake Pure Chemical Industry Ltd., particle size 80 to 120 mesh) instead ox porous polymer hard gets was employed as a porous inorganic hard gel The results are shown in Fig 2. As shown in. Fig. 2, slow rate increased approximately in proportion to increase of pressure-drop in the porous glass t while not in the agrees gel.

Example 1 Toyopearl HOWE to commercially available cross linked polyacrylate gel made by Toy Soda Manufacturing Co., Ltd., exclusion limit- 7 x 105, particle size 50 to 10C ye) having a uniform structure was ~Iployed as a carrier.
To 10 ml of the gel were added 6 ml of saturated Noah aqueous solution and 15 ml of epichlorohydrin, and the reaction mixture was subjected to reaction with stirring at 50C for 2 hours. The gel was washed successively with alcohol and water to introduce epoxy groups into the gel. To the resulting epoxy-activated gel was added 20 ml of concentrated aqueous ammonia, and the reaction mixture was stirred at 50C for 2 hours to introduce amino groups into the gel.
Three ml portion of the thus obtained activated-gel containing amino groups was added to 10 ml of aqueous solution tpH 4.5) containing 200 my of heparin~ To the resulting reaction mixture was added 200 my of l-ethyl-3-(dimethylaminopropyl)-carbodiimide while maintaining the reaction mixture at pi 4.5, and than the up reaction mixture was shaken at 4C for 24 hours. After completion of the reaction, the resulting reaction mixture was washed successively with 2 M Nail aqueous solution, 0.5 M Nail aqueous solution and water to give the desired gel on which heparin was immobilized (hereinafter referred to as "heparin-gel"). The amount of immobilized heparin was 2.2 mg/ml of bed volume Examples 2 to 4 * Trade Mark The procedures of Example 1 were repeated except that Toyopearl HOWE (exclusion limit- 1 x 106d particle size; 50 to 100 em), Superalloy Ho 65 (exclusion limit:
5 x 10~, particle size; 50 to 100 em) and Toyopearl WOW
(exclusion limit: 5 x lD7, particle size 50 to 100 em) instead of Toyopearl HOWE were employed, respectively, to give each heparin-gel. Toyopearl WOW, Toyopearl WOW
and Toyopearl H~75 are all commercially available crcss-linked polyacrylate gels having a uniform structure made by Toy Soda Manufacturing Co., Ltd. The amounts of immobilized heparin were, respectively, 1~8 my, 1.4 my and 0.8 mg/ml of bed volume Example 5 Cellulofine GO 700 pa commercially available porous cellulose gel made by Chihuahuas Corporation, exclusion limit: 4 x lug, particle size: 45 to 105 ye) having a uniform structure was employed as a carrier.
The gel was filtered with suction and 4 g of 20 % Noah and 12 g of Hutton were added to 10 g of the suction-filtered gel. One drop of Tweet 20 (non ionic surfactant~ was further added to the reaction mixture which was stirred for dispersing the gel. After stirring at 40~C for 2 hours, 5 g of epichlorohydrin was added to the reaction mixture which was further stirred at 40C for 2 hours. After the reaction mixture was allowed to stand, the resulting supernatant was discarded, and the gel was washed with water to introduce epoxy groups into the gel. To the resulting epoxy-activated gel was added 15 ml of concentrated aqueous ammonia, and the reaction mixture was stirred at 40C for 1.5 hours, filtered with suction and washed with water to introduce amino groups into the gel.
Three ml portion of the thus obtained activated gel containing amino groups was added to I ml of aqueous solution (pi 4.5) containing 200 my of heparin~ To the resulting reaction mixture was added 200 my of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide while maintaining the * Trade Mark I

reaction mixture at pi OWE and then the reaction mixture was shaken at 4C for 24 hours. After completion of the reaction, the resulting reaction mixture was washed successively with 2 M Nikko aqueous solution, 0.5 M Nail aqueous solution and water to give the desired heparin-Cellulofine A-3. The amount of immobilized heparin was 2.5 mg/ml of bed volume.

Examples 6 to 7 The procedures of Example 5 were repeated except that Cellulofine A 2 (exclusion limit: 7 x 105, particle size: 45 to 105 em) and Cellulofine A-3 (exclusion limit:
5 x 107, particle size: 45 to 105 em) instead of Cellulofine GO 700 were employed, respectively, to give each heparin-gel. Both Cellulofine A-2 and Cellulofine A-3 are commercially available porous cellulose gels having a uniform structure made by Chihuahuas Corporation.
The amounts of immobilized heparin were, respectively, 2~2 my and 1.8 mg/ml of bed volume.
Example 8 The procedures of Example S were repeated except that Cellulofine A-3 having a particle size of 150 to 200 em instead of 45 to 105 em was employed. The amount of immobilized heparin was 1.5 mg/ml of bed volume.

Example 9 The procedures of Example 1 were repeated except that Toyopearl HOWE instead of Toyopearl HOWE and chondroitin polysulfate instead of heparin were employed, to give the desired chondroitin polysulfate-Toyopearl HESS. The amount of immobilized chondroitin polysulfate was 1.2 mg/ml of bed volume.

Example 10 To 4 ml of Cellulofine A-3 was added water to make the volume up to 10 my and then 0.5 mole of Noah was added. After stirring at a room temperature 301~

for one hour, the reaction mixture was washed with water by filtration to introduce alluded groups into the gel.
The thus obtained gel was suspended in 10 ml of phosphate buffer of pi 8 and stirred at a room temperature for 20 hours after addition of 50 my of ethylenediamine. The gel was filtered off and then suspended in 10 ml of 1 %
Nub solution. After reducing reaction for 15 minutes, the reaction mixture was filtered and washed with water to introduce amino groups into the gel.
In 10 ml of 0.25 M Noah solution was dissolved 300 my of sodium salt of dextran sulfate. After stirring at a room temperature for 4 hours, 200 my of ethylene glycol was added to the resulting solution and stirred for one hour. The resulting solution was adjusted to pi 8, and then the above gel containing amino groups was suspended in the solution and stirred for 24 hours.
After completion of the reaction, the gel was filtered washed with water, and then suspended in 10 ml of 1 %
Nub solution. The resulting suspension was subjected to reducing reaction for 15 minutes and washed with water by filtration to give the desired sodium salt of dextran sulfate-Cellulofine A-3. The amount of immobilized sodium salt of dextran sulfate was 0.5 mg/ml of bed volume.
Example 11 Cellulofine A-3 was treated in the same manner as in Example 5 to introduce epoxy groups into the gel.
Two ml of the thus obtained epoxy-activated gel was added to 2 ml of aqueous solution containing 0.5 g of sodium salt of dextran sulfate (intrinsic viscosity 0.055 dug average polymerization degree: 40, sulfur content:
19 % by weight), and the reaction mixture was adjusted to pi 12~ The concentration of sodium salt of dextran sulfate was about 10 by weight. The resulting reaction mixture was filtered and washed successively with 2 M
Nail aqueous solution, 0.5 M Nail aqueous solution and water to give the desired sodium salt of dextran sulfate 2~3~

CelluloEine A-3. The remaining unrequited epoxy groups were blocked with monoethanolamine. The amount of immobilized sodium salt of dextran sulfate was 1.5 mg/ml of bed volume.

- Example 12 To 5 g of suction filtered Cellulofine A-3 were added 2.5 ml of 1,4-butanediol diglycidyl ether and 7.5 ml of 0.1 N Noah aqueous solution, and the reaction mixture was stirred at a room temperature for lo hours to introduce epoxy groups into the gel.
The thus obtained epoxy-activated gel was reacted with sodium salt of dextran sulfate in the same manner as in Example 11 to give the desired sodium salt of dextran sulfate-Cellulofine A-3. The amount of immobilized sodium salt of dextran sulfate was 1.8 mg/ml of bed volume.

Example 13 The procedures of Example 11 were repeated except that Cellulofine A-6 (a commercially available porous cellulose gel made by Chihuahuas Corporation, exclusion limit: 1 x 108, particle size: 45 to 105 em) having a uniform structure instead of Cellulofine A-3 was employed to give the desired sodium salt of dextran sulfate-Cellulofine A 6. The amount of immobilized sodium salt of dextran sulfate was 1.2 mg/ml of bed volume.

Example 14 Toyopearl HOWE was treated in the same manner as in Example 1 to introduce epoxy groups into the gel.
Two ml of the thus obtained epoxy activated gel was treated in the same manner as in Example 11 to give the desired sodium salt of dextran sulfate Toyopearl HOWE. The amount of immobilized sodium salt of dextran sulfate was 0.4 mg/ml of bed volume.

~2~3~

Example l_ Cellulofine A-3 was treated in the same manner as in Example 5 to introduce epoxy groups into the gel.
To 10 ml of the thus obtained epoxy-activated gel was added 50 my of protein A. The reaction mixture was adjusted to pi 9.5 and subjected to reaction at a room temperature for 24 hours. The resulting reaction mixture was washed successively with 2 M Nikko aqueous solution, 0.5 M Nail aqueous solution and water. The remaining unrequited epoxy groups were blocked by reacting with ethanol amine for 16 hours. The reaction mixture was then washed with water to give the desired protein A-Cellulofine A-3.

Example 16 The procedures of Example 15 were repeated except that Cellulofine A-7 (a commercially available porous cellulose gel made by Chihuahuas Corporation, exclusion limit: 5 x 106, particle size: 45 to 105 em) having a uniform structure instead of Cellulofine A-3 was employed and the coupling reaction was carried out a-t pi 8~5, to give the desired protein A-Cellulofine A 7.

Example 17 Toyopearl HOWE was treated in the same manner as in Example 1 to introduce epoxy groups into the gel. The thus obtained activated gel was reacted with protein A in the same manner as in Example 15 to give the desired protein A-Toyopearl HOWE.
sample 18 Twenty ml of Cellulofine A-3 was dispersed in water to which 6 g of cyanogen bromide was slowly added while maintaining the reaction mixture at pi 11 to 120 After stirring for 10 minutes, the gel was filtered off and washed with cold water and 0.1 M Nikko aqueous solution to give an activated gel. The thus obtained activated gel was added to 20 ml of 0.1 M Nikko aqueous ~2~3~
- I
solution containing 1.5 g of polymyxin B sulfate and shaken at 4C for 24 hours. The remaining unrequited active groups were blocked with monoethanolamine solution, and then the desired polymyxin B-Cellulofine A-3 was obtained.

Cellulofine A-3 was treated in the same manner as in Example 5 to introduce epoxy groups into the gel.
The thus obtained epoxy-activated gel was reacted with polymyxin B sulfate in the same manner as in Example 18 to give the desired polymyxin B-Cellulofine A.

Example 20 Cellulofine A-2 was treated in the same moaner as in Example 5 to introduce epoxy groups into the gel.
To 1 ml of the thus obtained epoxy-activated gel was added 10 my of Gig, and the reaction mixture was adjusted to pi 9 and subjected to reaction at a room temperature for 24 hours. The gel was filtered off and washed successively with 2 M Nail aqueous solution, 0.5 M Nail aqueous solution and water. After the remaining unrequited epoxy groups were blocked with monoethanolamine solution, the desired IgG-Cellulofine A-3 was obtained.

Cellulofine A was treated in the same manner as in Example 5 to introduce epoxy groups into the gel.
One ml of the thus obtained epoxy-activated gel was reacted with 10 my of heat denatured Gig in the same manner as in Example 20 at pi 8.5 to give the desired heat-denatured IgG-Cellulofine A-3.

Cellulofine A-7 was treated to the same manner as ill Example 5 to introduce epoxy groups into the gel.
One ml of the thus obtained epoxy-activated gel was reacted with 100 my of hemoglobin in the same manner as in Example 20 at pi 8.5 to give the desired hemoglobin-Cellulofine I

Example 23 Toyopearl WOW was treated in the same manner as in Example 1 to introduce epoxy groups and amino groups into the gel. The thus obtained activated gel was reacted with hemoglobin in the same manner as in Example 22 to give the desired hemoglobin-Toyopearl HOWE.
Example 24 Cellulofine A-7 was treated in the same manner as in Example 5 to introduce epoxy groups into the gel The thus obtained epoxy-activated gel was reacted with DNA in the same manner as in Example 20 to give the desired DNA-Cellulofine A-7.

Example 25 Cellulofine A-3 was treated in the same manner as in example 5 to introduce epoxy groups into the gel.
The thus obtained epoxy-activated gel was reacted with anti-DNA rabbit antibody in the same manner as in Example 20 at pi 8~5 to give the desired anti-DNA rabbit antibody-Cellulofine A-3.
-I- Example 26 -Cellulofine A-3 was treated in the same manner as in Example 5 to introduce epoxy groups into the gel.
The thus obtained epoxy-activated gel was reacted with an acetylcholine receptor fraction in the same manner as in Example 20 to give the desired acetylcholine receptor fraction-Cellulofine A-3.

Example 27 FOG 2000 (exclusion limit: 1 x 109, particle size 80 to 120 mesh, average pore size: 1950 A) was heated in diluted nitric acid for 3 hours. After washing and drying, the gel was heated at 500C for 3 hours and ~2~3~

then reflexed in 10 % Y-aminopropyltriethoxysilane solution in Tulane for 3 hours After washing with methanol, a Y-aminopropyl-activated glass was obtained.
Two g of the thus obtained activated glass we added to 10 ml of aqueous solution (pi 4.5) containing 200 my of heparin. The reaction mixture was treated in the same manner as in Example 1 to give the desired heparin-FPG 2000. The amount of immobilized heparin was I mg/ml of bed volume.
Examples 28 to 30 The procedures of Example 27 were repeated except that FOG 700 pa commercially available porous glass made by Wake Pure Chemical Industry Ltd., exclusion limit:
5 x 107, particle size: 80 to 120 mesh, average pore size: 70 A), FOG lock (a commercially available porous glass made by Waco Pure Chemical Industry Ltdo exclusion limit: 1 x 108, particle size: 80 to 120 mesh, average pore size 1091 A) and Lichrospher Sue (a commercially available porous silica gel made by Merck & Coy Inc., exclusion limit 1 x 109~ average particle size: 10 em, average pore size: 4000 A) instead of PUG 2000 were employed The amounts of immobilized heparin were, respectively, OWE my, 2.2 my and 0.5 Mel of bed volume.
Example 31 The procedures of Example 27 were repeated except that chondroitin polysulfate instead of heparin was employed to give the desired chondroitin posy sulfate-FPG 2000. The amount of immobilized chondroitin polysulfate was 1.0 mg/ml of bed volume.

Example 32 FOG 2000 was treated in the same manner as in Example 27 to introduce Y-aminopropyl groups into the gel.
The thus obtained activated gel was reacted with sodium salt ox dextran sulfate in the same manner as in Example 10 to give the desired sodium salt of dextran sulfate-FPG

* Trade Mark 2000. The amount of immobilized sodium salt of dextran sulfate was 0.5 mg/ml of bed volume.

Example 33 FOG 2000 was reflexed in 10 % solution of Y-glycidoxypropyltrimethoxysilane for 3 hours and then washed with methanol. The thus obtained activated gel was reacted with sodium salt of dextran sulfate in the same manner as in example 11 except that the reaction was carried out at pi 8.5 to 9 and at SKYE to give the desired sodium salt of dextran sulfate-FPG 2000.

Example 34 FOG 1000 was activated in the same manner as in Example 27. The thus obtained activated gel was reacted with protein A in the same manner as in Example 15 to give the desired protein A-FPG 1000.

Example 35 FOG 2000 was activated in the same manner as in Example 33. The thus obtained activated gel was reacted with polymyxin B sulfate in the same manner as in Example 18 to give the desired polymyxin B-FPG 2000.

Example 36 FOG 1000 was activated in the same manner as in Example 27. The thus obtained activated gel was reacted with heat-denatured Gig in the same manner as in Example 20 to give the desired heat-denatured IgG-FPG 1000.
Example 37 FOG 700 was activated in the same manner as in Example 33. The thus obtained activated gel was reacted with DNA in the same manner as in Example 20 to give the desired DNA-FPG 700.

Each adsorbent obtained in Examples 1 to 37 was I

uniformly packed in a column (internal volume: about 3 ml, inner diameter: 9 mm, height: 47 mm) and 18 ml of plasma containing 200 U of heparin was passed through the column at a flow rate of 0.3 ml/minute with varying the plasma origins depending on the kind of the desired substance to be removed. That is human plasma derived from familial hypercholesterolemia, normal human plasma, normal human plasma containing about lo gel of a commercially available endotoxin, human plasma derived from lo rheumatism, human plasma derived from systemic luaus erythematosus and human plasma derived -from myasthenia gravies were used, respectively, for the tests of removing VLDL and/or LDL; Gig, Cluck or haptoglobin; endotoxin;
rheumatoid factor; anti-DNA antibody or DNA; and anti-acetylcholine receptor antibody. The pressure-drop in the column was 15 mmHg or less throughout the test period and no cogging was observed. In each adsorbent, a substance to be removed in plasma which was passed through the column was determined to obtain a removal efficiency. The results are summarized in Table 1.

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Lo With respect to the coupling method in Table 1, Epichlorohydrin method, Epichlorohydrin-ammonia method, NaIO4-Diamine, Bisepo~ide method, CNBr method, Aminosilane method and Epoxysilane method are conducted, respectively, in the same manners as in Examples 1 and 5, 1 and 5, 10, 12, 18, 28 and 34.

Example 38 [Effects of intrinsic viscosity and sulfur content of dextran sulfate and/or the salt thereof]
Cellulofine A-3 was treated in the same manner as in Example 5 to introduce epoxy groups into the gel.
The thus obtained epoxy-activated gel was reacted with each sodium salt of dextran sulfate having the intrinsic viscosity and sulfur content shown in the following Table 2 (Run Nos. S13 to (7)) in the same manner as in Example 11 .
One ml portion of the resulting each adsorbent was packed in a column, and then 6 ml of human plasma containing 300 mg/dl of total cholesterol derived from a familial hypercholesterolemia patient was passed through the column at a flow rate of 0.3 ml/minute. The removal efficiency for LDL was determined from the amount of adsorbed LDL measured by using the total amount of cholesterol as an indication. That is, the amount of cholesterol in the human plasma used was mostly derived from LDL. The results are shown in Table 2.

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Example 39 effect of amount of epoxy group introduced]
Toyopearl 65 was treated in the same manner as in Example 1 to introduce epoxy groups into the gel and SCHICK (a commercially available porous cellulose gel made by Chihuahuas Corporation, exclusion limit. 5 x 107, particle size: 45 to 105 em) having a uniform structure was treated in the same manner as in Example 5 to introduce epoxy groups into the gel. The amounts of epoxy groups introduced were, respectively, 250 moles and 30 ~moles/ml of bed volume.
Each gel was reacted with sodium salt of dextran sulfate (intrinsic viscosity: 0.027 dug sulfur content: 17.7 % by weight) in the same manner as in Example 11 except that the concentration of sodium salt of dextran sulfate based on the weight of the whole reaction system excluding the dry weight of the gel was charged.
The thus obtained adsorbent was subjected to the determination of removal efficiency for LDL in the same manner as in Example 38. The results are summarized in Table 3.

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En Example 40 One ml portion of the adsorbent obtained in Example 38 of Run No. I was uniformly packed in a column having an internal volume of 1 ml, and 6 ml of normal human plasma containing LDL and HAL cholesterol in the ratio of approximately 1 : 1 was passed through the column. LDL in the plasma passed through the column was greatly reduced, while HAL was scarcely reduced.

Example 41 One ml portion of the adsorbent obtained in Example 38 of Run No. (3) was uniformly packed in a column having an internal volume of 1 ml, and 6 ml of normal rabbit plasma containing lipoproteins of VLDL, LDL
and HAL was passed through the column. The plasma obtained before and after the column treatment were, respectively, examined by polyacrylamide disc gel electrophoresis. The results are shown in Fig. 3. In Fig. 3, curves A and B show, respectively, the results obtained before and after the column treatment. The axis of ordinates indicates the ahsorbance at 570 no and the axis of abscissas indicates the-migration positions at which bands of VLDL/ LDL and HAL were, respectively appeared.
As shown in Fig. 3, VLDL and LDL were significantly adsorbed, while DO was not.

Example 42 The adsorbent obtained in Examples 1 to 7 and 11 to 14 were sterilized in an autoclave at 120C for 15 minutes. Each resulting sterilized adsorbent was subjected to the determination of removal efficiency for LDL in the same manner as in Test Example 1. As a result, the removal efficiencies were not inferior to those obtained without sterilizing by autoclaving. In addition, pressure-drop was not changed.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are de-fined as follows:

1. An adsorbent for removing a substance to be removed from body fluid in extracorporeal cir-culation treatment comprising a porous cellulose gel on which a ligand having an affinity for the substance is immobilized by a covalent linkage by reacting the porous cellulose gel with epichlorohydrin or a poly-oxirane compound to introduce epoxy groups into the gel and reacting the resulting epoxy-activated gel with the ligand.

2. The adsorbent of claim 1, wherein said ligand is a polyanion compound.

3. The adsorbent of claim 2, wherein said polyanion compound is a sulfated polysaccharide.

4. The adsorbent of claim 3, wherein said sulfated polysaccharide is a member selected from the group consisting of heparin, dextran sulfate, chon-droitin sulfate, and their salts.

5. The adsorbent of claim 1, wherein said substance to be removed is a lipoprotein.

6. The adsorbent of claim 5, wherein an exclusion limit of the porous cellulose gel is from 106 to 109 daltons.

7. The adsorbent of claim 6, wherein said exclusion limit is 106 to 108 daltons.

8. The adsorbent of claim 5, wherein said lipoprotein is very low density lipoprotein, low den-sity lipoprotein or an admixture thereof.

9. The adsorbent of claim 1, comprising a porous cellulose gel having an exclusion limit of 106 to 108 daltons on which a polyanion compound having an affinity for a lipoprotein is immobilized.

10. The adsorbent of claim 1, wherein said polyanion compound is immobilized in an amount of 0.02 mg to 100 mg/ml of bed volume.

11. The adsorbent of claim 4, wherein said dextran sulfate, a salt thereof or a mixture of the dextran sulfate and the salt has an intrinsic viscos-ity of not more than 0.12 dl/g and a sulfur content of not less than 15% by weight.

12. The adsorbent of claim 4, wherein said dextran sulfate, a salt thereof or a mixture of the dextran sulfate and the salt is immobilized in an am-ount of not less than 0.2 mg/ml of bed volume.

13. The adsorbent of claim 1, wherein a dex-tran sulfate, a salt thereof or a mixture of the dex-tran sulfate and the salt is immobilized on a porous cellulose gel by the covalent linkage.

14. A process of preparing an adsorbent for removing a substance to be removed from body fluid in extracorporeal circulation treatment which comprises immobilizing a ligand having an affinity for the sub-stance on a porous cellulose gel by reacting the por-ous cellulose gel with epichlorohydrin of a polyoxi-rane compound to introduce epoxy groups into the gel and reacting the resulting epoxy-activated gel with the ligand.

15. The process of claim 14, wherein said ligand is dextran sulfate, a salt thereof or a mix-ture of the dextran sulfate and the salt, said dex-tran sulfate, the salt thereof or the mixture of the dextran sulfate and the salt being reacted with said epoxy-activated gel in a concentration of not less than 3% based on the weight of the whole reaction system excluding the dry weight of the porous hard gel.