JPS59193135A - Adsorbing body - Google Patents

Abstract

PURPOSE:To form an adsorbing body capable of removing harmful components by selectively adsorbing at high flow rate by supporting substance having affinity to the substance to be removed on porous cellulose gel. CONSTITUTION:A adsorbing body is formed for removing harmful components in the blood by supporting a substance (designated hereunder as ligand) having affinity to the substance to be removed on porous cellulose gel. For example, a complement component such as C1q, specific protein such as protein A, or anti- body for an immune complex may be used for the removal of an immune complex as the ligand. Further, a physical method, ionic bond method, or covalent bond method, etc. may be used for supporting a ligand on a carrier porous cellulose gel. When the adsorbing body is used for an external circulation therapy, the ligand is preferred to be supported by the covalent bond having higher bonding strength in order to prevent elimination of the ligand from the carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to an adsorbent. In more detail, blood, J
111 Harmful substances that appear in body fluids such as plasma, viruses,
This invention relates to an adsorbent that adsorbs and removes harmful cells.

Extracorporeal circulation therapy, which originated from artificial dialysis, has developed greatly in recent years, and the number of diseases that can be treated continues to increase. Along with this, there is a need for a means to more selectively remove harmful components that appear in body fluids and are thought to be closely related to the cause or progression of red skin. Separation methods using membranes have been studied for this purpose, but such methods have the disadvantage of insufficient selectivity and large losses of body fluid components other than the target substance. Attempts have been made to remove these components, and so-called synthetic adsorbents, typified by activated carbon and XAD, have been mainly used for liver diseases.

However, the synthetic adsorbents have many drawbacks, such as poor selectivity and inability to remove high molecular weight substances.

In order to further improve selectivity, attempts have been made to use so-called affinity adsorbents in which a water-insoluble carrier holds a substance that has an affinity for the substance to be removed. However, it has become clear that many of these attempts have been based on repurposed carriers that are normally used for affinity chromatography, and that they have various drawbacks that make them undesirable for use in extracorporeal circulation therapy. In other words, the carriers normally used for affinity chromatography are agarose, dextran, polyacrylamide, porous glass, porous silica, etc., but soft gels such as agarose, dextran, and polyacrylamide have (1) mechanical strength. (2) Due to its low pressure resistance, if body fluid is flowed at a high flow rate when packed in a column and used for extracorporeal circulation, it will cause compaction and the flow rate will become unstable. , it has drawbacks such as clogging.

On the other hand, although inorganic porous materials such as porous glass have high pressure resistance, (1) they are easily destroyed by operations such as stirring and produce fine powder, and (2) components other than the substance to be removed are adsorbed onto the surface of the carrier, so-called It has drawbacks such as non-specific adsorption which cannot be ignored.

Furthermore, polymer gels made from synthetic polymers have been proposed to overcome the drawbacks of paper, but these drawbacks cannot be said to be fully overcome, and there are concerns about toxicity due to unreacted monomers, and the adsorption capacity is also low. Inferior to soft gel.

The present inventors have conducted intensive research to overcome the drawbacks described in the paper, and as a result, we have found that by making porous cellulose gel hold a substance (hereinafter referred to as "ligand") that has an affinity for the target substance to be removed, it can be selectively removed at high flow rates. The present inventors have discovered an adsorbent that can adsorb and remove harmful components, and have completed the present invention.

In other words, the porous cellulose gel used in the present invention has (1) relatively high mechanical strength and toughness, so it is less likely to be destroyed or generate fine powder by operations such as stirring, and is easy to use when packed in a column. Even if body fluids flow at high flow rates, they will not become compacted or clogged, making it possible to flow at high flow rates, and the pore structure will not be easily changed by high-pressure steam sterilization. - It is hydrophilic because it is composed of carbon atoms, there are many hydroxyl groups that can be used for binding of ligands, and there is little non-specific adsorption. (3) Even if the pore volume is increased, the strength is relatively high) The porous cellulose gel has excellent features such as (4) higher adsorption capacity than soft gels, and (4) higher safety than synthetic polymer tar. Although cellulose itself is preferred as the porous cellulose gel used in the present invention, esterified or etherified cellulose derivatives, such as esterified or etherified cellulose derivatives, are a mixture of cellulose and said derivatives. Good too. Examples of such cellulose derivatives include acetylcellulose, methylcellulose, ethylcellulose, and carboxymethylcellulose. Moreover, the shape of the gel particles is preferably spherical.

The porous cellulose gel is produced by, for example, dissolving or swelling cellulose and/or cellulose derivatives using a solvent, and then dispersing the cellulose and/or cellulose derivatives in a separate solvent that is immiscible with the solvent to form spheres, and then gelling and regenerating the gel. It can be manufactured by the following method.

Cellulose and/or cellulose derivatives constituting the gel may be crosslinked. Typical examples of crosslinking agents include shrimp tetrahydrin, dichlorohydrin, jepoxy compounds, dialdehyde compounds, diisocyanate compounds, cyanuric chloride, dialkoxyalkylsilane compounds,
Examples include trialkoxyalkylsilane compounds, but are not limited to these crosslinking agents.

The pore diameter of the porous cellulose itself used in the present invention can be optimally selected depending on the molecular weight and size of the substance to be removed. There are various methods for measuring pore diameter, and direct measurement methods include mercury porosimetry and electron microscopic observation, but these methods may not be applicable to water-containing particles. In such cases, the exclusion limit molecular weight can be used as a guideline for the pore diameter. What is the exclusion limit molecular weight?
Written by Toshihiko Hana, Experimental High Performance Liquid Chromatograph, Chemistry Doujin)
As described in et al., it is the molecular weight of the smallest molecular weight among molecules that cannot enter (excluded) into the pores in gel permeation pomadography. Phenomenologically, molecules with molecular weights greater than the exclusion limit molecular weight are eluted near the volume of the mobile phase, so the exclusion limit molecular weight can be determined by examining the relationship with the elution volume using compounds of various molecular weights.

The exclusion limit molecular weight differs depending on the type of target compound, and the porous cellulose gel used in the present invention has an exclusion limit molecular weight (hereinafter referred to as exclusion limit molecular weight) of 5X103 to 1X1 when globular proteins and viruses are used.
The range is preferably 0.08. Exclusion limit molecular weight is 1
If it exceeds ×108, the amount of retained ligand decreases, the amount of adsorption of the substance to be removed decreases, and the strength of the gel decreases, which is not preferable.

The pore volume of the porous cellulose gel can be determined based on the cellulose content. The cellulose content is expressed by the formula shown below.

Cellulose content (%) =] τ, xio.

(In the formula, W is the gel weight (9), t is the body a (m7) of the column packed with gel, and Vo is a high molecular weight substance that cannot enter the pores of the gel, passed through the column packed with gel. (If so, it is the elution volume (mJ) until the high molecular weight substance is eluted from the column.) The porous cellulose gel used in the present invention preferably has a cellulose content of 2% to 60%; If it is less than 6%, the strength of the gel will decrease and
If it exceeds 0%, the pore volume becomes small, which is not preferable.

Generally, the smaller the particle size of the porous cellulose gel, the better from the viewpoint of adsorption capacity, but if the particle size becomes too small, the pressure loss will increase when packed in a column, etc., which is undesirable. Preferably, it is in the range of 5,000μ.

The following substances are representative examples of substances (ligands) that have an affinity for the substance to be removed used in the present invention.

To remove immune complexes, complement components such as C1q, specific proteins such as butetin A, antibodies against immune complexes, etc. can be used.

To remove autoantibodies that appear in autoimmune diseases, etc., for example, to remove anti-nuclear antibodies and anti-B cell antibodies that appear in the blood in systemic lupus erythematosus, use nucleobases, nucleosides, nucleotides, polynucleotides, and more. is DNA.

RNA etc. can be used, and an acetylcholine receptor fraction component can be used to remove anti-acetylcholine receptor antibodies that appear in myasthenia gravis.

In addition, antigens for each autoantibody can be used to remove autoantibodies.

Furthermore, antibodies against substances to be removed can be used to remove various harmful components that appear in the blood.

For example, antibodies against antigens on the surface of the virus can be used to remove hepatitis viruses, and anti-DNA antibodies can be used to remove DNA that appears in the blood in lupus erythematosus. In addition, for lymphocyte abnormalities, lymphocytes can be removed using anti-B cell antibodies, anti-pressor T cell bodies, and the like.

In addition to methods using antigen-antibody reactions as described above,
Substances that have a specific affinity for the adsorbed substance can be used. Typical examples include denatured or aggregated immunoglobulin to remove rheumatoid factors that appear in rheumatoid arthritis, and -globulin. or their fractionated components, amino acids such as tryptophan, sulfated polysaccharides such as heparin for the removal of riboproteins, protein A for the removal of immunotetrapurines.
1 haptoglobin, hemoglobin for bin removal, lysine for plasminogen removal, im/globulin G1 for C1q removal, arginine for prekallikrein removal, cortisol for transcortin removal, hemin for hemovexin removal, endotoxin Examples include polymyxin for removal. Furthermore, lectins such as concanavalin A1 funglutinin and phytohemagglutinin, nucleic acids, enzymes, substrates, coenzymes, etc. can also be used.

The substances to be removed and the ligands described above are merely representative examples, and the present invention is not limited to these. The substances to be removed include a variety of substances, from those with a molecular weight of less than 1,000, such as bilirubin urate, to those with a molecular weight of more than 1,000 million, such as viruses. In addition, the porous cellulose gel of the carrier is usually determined according to the molecular weight and molecular size of the substance to be removed, but it is also influenced by the type of ligand, the shape of the molecule of the substance to be removed, etc. hundreds,
For millions and tens of millions of molecules, it is appropriate to use molecular weights with exclusion limits of several thousand to hundreds of thousands, tens of millions, and tens of millions to 100 million, respectively. Further, the ligand may be used alone or in combination of two or more kinds. A known method can be used for retaining the ligand in the porous cellulose gel of the carrier. That is, physical methods, ionic bonding methods, covalent bonding methods, etc. can be mentioned. Unless the adsorbent of the present invention is used for extracorporeal circulation therapy, it is important that the Gantt does not desorb, so a strong covalent bonding method is preferred. It is necessary to find ways to prevent this. Furthermore, a spacer may be introduced between the carrier and the ligand if necessary.

There are various uses for the adsorbent of the present invention,
For example, an adsorbent is packed into a column equipped with a filter or mesh that allows body fluid components to pass through the inlet and outlet but not the adsorbent, and the column is installed in an extracorporeal circulation circuit, and blood, plasma, etc. are passed through the column. Although it is typically used for extracorporeal circulation therapy, it is not necessarily limited to such uses.

The adsorbent of the present invention can be used as long as the ligand is not significantly modified.
High-pressure steam sterilization is possible, and the sterilization process causes only slight changes in pores, particle shape, and volume.

Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Seven Rulofine A-3 (porous cellulose gel manufactured by Chisso ■, exclusion limit molecular weight 5X107, particle size 45-105
μm) 20% NaOH 4g, heptane 1 in 10mj
29, nonionic surfactant Twee 2Q (Twee
After adding one drop of m20) and stirring at 40°C for 2 hours, epichlorohydrin 59 was added and stirred for 2 hours. After standing still, the supernatant was discarded, and the gel was washed with water and filtered to obtain an epoxidized cellulose gel. 15 ml of concentrated ammonia water was added to the resulting gel, stirred at 40°C for 1.5 hours, and the contents were suction filtered and washed with water to obtain a cellulose gel into which 7 amino groups were introduced.

Next, 200 mp of heparin was dissolved in 10 ml of water, 6 ml of the gel containing amide 7 groups was added thereto, the pH was adjusted to 4.5, and 200 ml of 1-ethyl-6-(dimethylaminopropyl)-carbodiimide was adjusted to pH 4.5. The mixture was added while maintaining the temperature and shaken at 4°C for 24 hours.

After the reaction was completed, the gel was washed with a 2M saline solution, a 0.5M saline solution, and water to obtain a cellulose gel retaining heparin. The amount of heparin retained was 1.8m97ml. Furthermore, no breakage of the gel or generation of fine powder was observed during the operations up to this point.

The resulting adsorbent has an inner diameter of 9 mm and a length of 47 mm.

A column having an internal volume of approximately 6 ml was packed, and 18 mJ of plasma from a hyperlipidemic patient was passed through the column at a rate of 0.6 ml/min. The pressure loss in the column was below 15+n+r+H9 throughout, and no clogging was observed.

By passing through the column, 65% of the total cholesterol in plasma was adsorbed, and total protein etc. decreased only slightly.

Example 2 The particle size of Cellulofine A-3 used in Example 1 was changed to 15
A heparin-retained cellulose gel was obtained in the same manner as in Example 1, except that the heparin content was changed to 0 to 200 μm.The amount of heparin retained at 0 μm was 1.5 m97 m! Met.

The gel on paper was packed into the same column as in Example 1, and 18 mj of plasma from a hyperlipidemic patient (added with 200 U of heparin) was added to 0.0 mj of plasma from a hyperlipidemic patient.
It was passed through the column at 2 m/min. The pressure drop in the column is 3
55% of the total cholesterol in plasma was adsorbed by passing through the 0 column, which had a value of 0 mmH9 or less and little change, and there was only a slight decrease in total protein and blood cells. Furthermore, there was little thrombus formation on the adsorbent surface.

Example 3 The heparin-retained gel prepared in Example 1 was sterilized using an autoclave at 120°C for 15 minutes, and filled into the same column as in Example 1, and the heparin-retained gel obtained in Example 1 was prepared using plasma 18+ from a hyperlipidemic patient.
nf! was passed through Kara A at 0.3 ml/min. Both the pressure drop in the column and the adsorption of total cholesterol were the same as in Example 1.

Example 4 An epoxidized cellulose gel was prepared in the same manner as in Example 1. Add 50% protein A to 10ml of the resulting gel.
After adding m9 and adjusting the pH to 9.5 and reacting at room temperature for 24 hours, the mixture was washed with a 2M* salt aqueous solution, a 0.5M salt aqueous solution, and water. Then add monoethanolamine and add 1
The mixture was reacted for 6 hours to seal unreacted epoxy groups, and then washed with water to obtain a cellulose gel in which protein A was retained.

The obtained adsorbent was packed into the same column as in Example 1, and 18 m7 of human plasma was passed through the column at O, Erm for 17 minutes, and about 35% of globulin was adsorbed.

Note that the decrease in albumin was slight.

Example 5 20 ml of Cellulofine A-3 (7) used in Example 1
was dispersed in water, and cyanogen bromide 69 was gradually added thereto while maintaining the pH at 11 to 12, followed by stirring for 10 minutes. The gel was separated and washed with cold water and 0.1M NaHOO3 aqueous solution to obtain an activated gel. Next, ho.

Remixin sulfur #jn 1.59 to 0.1 M N an
After dissolving in QOml of OO3 aqueous solution, adding the gel on paper and incubating at 4°C for 24 hours, excess active groups were sealed using a monoethanolamine solution to form a cellulose gel with polymyxin retained. I got it.

1 ml of the resulting gel was packed into an open column with an inner diameter of 7 mm, and when 5 ml of bovine plasma supplemented with endotoxin (approximately 100 μ7/ml) was passed through the column for 7 minutes, 60% of endotoxin was adsorbed. .

Example 6 Cellulofine A-3 was mixed with Cellulofine A-2 (porous cellulose gel manufactured by Chisso fII, exclusion limit molecular weight 7).
An epoxidized cellulose gel was produced in the same manner as in Example 1, except that the gel was changed to a particle size of 45 to 105 μm). Human immunoglobulin 0 was added to 1Qml of the resulting gel.
After adding 10 m9 of sodium chloride solution and adjusting the pH to 9.0 and reacting at room temperature for 24 hours, the gel was taken from house to house and treated with a saline solution for 21 days.
Washed with 5M saline Pa H and water. Then, unreacted epoxy groups are sealed using a monoethanolamine bath solution.
A cellulose gel in which immunoglobulin G was retained was obtained.

Next, the obtained gel was packed into the same column as in Example 1,
When 18 ml of human plasma was passed through the column at 0.2 mj/min, about 60% of C1q was adsorbed.

Claims (1)

[Claims] 1. An adsorbent in which a porous cellulose gel holds a substance that has an affinity for the substance to be removed.