JPH0516304B2 - - Google Patents

Description

[Detailed description of the invention]

[Object of the Invention] (Industrial Application Field) The present invention relates to an adsorbent for removing immunoglobulin from plasma. (Conventional techniques and their problems) Plasma exchange therapy is used to treat rheumatoid arthritis, myasthenia gravis, systemic lupus erythematosus (SLE), fulminant hepatitis, glomerulonephritis, and good-
It is becoming widely used as a means of treatment for intractable diseases such as (Pasture) syndrome and familial hypercholesterolemia. However, in plasma exchange therapy, 1
Two to four volumes of plasma are required for each plasmapheresis, and it is extremely difficult to obtain such amounts of plasma or albumin on a sustained basis. Against this background, methods have been studied and implemented in which plasma separated from a patient is purified and returned to the patient's body. One of the methods is to use hollow fibers of organic polymer compounds. However, this method sieves plasma components based only on molecular size, which has the disadvantage that useful components are also removed and the number of removed components fluctuates due to clogging. In addition, plasma purification methods using adsorbents have been investigated.
Applied clinically. Among adsorbents for removing unnecessary factors in plasma, examples of adsorbents that use physiologically active polymers such as antibodies and antigens as ligands include protein A-supported adsorbents, DNA
Examples include immobilized adsorbents, tryptophan-immobilized adsorbents, methylated albumin-immobilized adsorbents, and heparin-immobilized adsorbents. Although these have excellent adsorption specificity, the adsorption properties may be affected during sterilization, storage, and manufacturing. It has the disadvantage of causing a decrease in Furthermore, if a ligand such as an antigen or an antibody is detached from the adsorbent, it may form an immune complex and cause side effects. For these reasons, adsorbents that use bioactive polymers as ligands have many practical problems. In addition, as non-biological adsorbents, for example, polyethylene glycol (hereinafter referred to as "PEG") is made insolubilized with trifunctional isocyanate [Polymer Preprints,
Preprints, Japan) Volume 33, No. 7 (1984)]. However, this adsorbent has insufficient specificity for adsorbing plasma components, and when packed in a column, plasma cannot flow smoothly, and unreacted
There are problems such as complete removal of PEG is not easy. [Structure of the invention] (Means and effects for solving the problems) The adsorbent of the present invention contains agarose, cellulose,
The following formula (): (In the formula, R ₁ , R ₂ , R ₃ and R ₄ independently represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 10 to 200.) Adsorbent for removing immunoglobulins and their immune complexes from plasma to which compounds are bound The hydroxyl group-containing polymer compound used in the present invention may be any water-insoluble compound, such as agarose, cellulose, Examples include dextran and polyvinyl alcohol, and these may be used alone or as a mixture of two or more, but it is preferable to use a crosslinked one. The shape thereof is not particularly limited, such as particulate, fibrous, film, etc., but particulate and fibrous forms are preferred from the viewpoint of supporting reaction and ease of handling. In the case of particles, the spherical diameter is usually 20 to 5000μ, preferably 100 to 5000μ, in terms of clogging when packed in a column and adsorption rate of adsorbed materials.
1000μ, but is not limited to these. Two or more isocyanato groups (-NCO) in the molecule
Examples of compounds having the following formula (): OCN-R-NCO () (wherein R represents an aliphatic chain having 1 to 12 carbon atoms or an aromatic chain having 6 to 15 carbon atoms) In addition to the difunctional isocyanates shown, polyfunctional isocyanates of trifunctionality or higher are also included.
Examples of difunctional isocyanates include hexamethylene diisocyanate, tetramethylene diisocyanate, o-tolidine isocyanate, tolylene diisocyanate, naphthylene-1,5-
diisocyanate, 4,4'-diphenylmethane diisocyanate, and examples of polyfunctional isocyanates include triphenylmethane-4, 4,4'-diphenylmethane diisocyanate,
Examples of the compound represented by the above formula () include polyethylene glycol, polypropylene glycol, and poly-(1,2-butanediol). In the above formula (), n is 10-200
A compound having an average molecular weight of 500 to 50,000 corresponds to a compound having an average molecular weight of 500 to 50,000. Such compounds include the above formula ()
Particularly preferred are compounds in which n is 40 to 100 (corresponding to an average molecular weight of 2000 to 20000). The adsorbent of the present invention can be produced, for example, as follows. The water-insoluble polymer compound having a hydroxyl group is used in a dry state. When the polymer compound contains water, it is used after drying by conventional means or after repeated substitution treatment with an anhydrous organic solvent. The organic solvent used for substitution is usually the same as the solvent used for the reaction, and examples thereof include dimethyl sulfoxide, dimethyl acetamide, dimethyl formamide, tetrahydrofuran, acetone, methyl ethyl ketone, and methyl isobutyl ketone. The reaction is carried out by suspending a water-insoluble polymeric compound having a hydroxyl group in an organic solvent, adding a compound having two or more isocyanato groups in the molecule, and generally for 10 to 30 minutes.
It is carried out at 200°C, preferably from 30 to 110°C, with stirring. In this case, there is no particular restriction on the ratio of the hydroxyl group (-OH) present in the water-insoluble polymer compound having a hydroxyl group to the isocyanato group (-NCO) present in the compound having two or more isocyanato groups in the molecule. , preferably -NCO/-OH>1. After stirring for a predetermined time and the hydroxyl groups and isocyanate groups react, the reaction product is repeatedly washed with a solvent in order to remove excess and unreacted isocyanate compounds and other by-products. After confirming that the cleaning solution does not contain isocyanate compounds,
The compound represented by the above formula () is added to a system consisting of a reaction product and a solvent, and the reaction is carried out with stirring at usually 10 to 200°C, preferably 50 to 110°C. All of the above reactions are carried out under conditions where no moisture enters the reaction system. After stirring for a predetermined period of time to carry out the reaction, water is added to the reaction system. After sucking off the reactants, the reaction product is added to a new reaction solvent and stirred for a while at room temperature to dissolve the remaining unreacted products in the solvent. The reaction product is aspirated, washed repeatedly with water, and stored wet, usually in physiological saline. (Examples) Hereinafter, the present invention will be explained in more detail with reference to Examples, but these Examples are not intended to limit the scope of the present invention in any way. Example 1 Cross-linked agarose (trade name: Sepharose CL-4B; manufactured by Pharmacia) was washed repeatedly with distilled water, and the water was thoroughly squeezed out by suction. 10.0 g of the thus obtained wet crosslinked agarose (0.77 g in absolute dry form) was added to 50 ml of dehydrated dimethyl sulfoxide (hereinafter referred to as "DMSO") and stirred at room temperature for 2 hours.
After removing DMSO in a water-free system, 30 ml of dehydrated DMSO was newly added and stirred at room temperature for 1 hour. The same procedure was repeated for 30 ml (12.5 hours) and 30 ml (3 hours), and finally 20 ml of DMSO was added. At this time, the water content in DMSO in the system was analyzed by the Karl Fischer method and was found to be less than 10 ppm. To this system, add 1.0 g of hexamethylene diisocyanate (hereinafter referred to as "HMDI") to the dehydrated DMSO 10
ml of the solution was charged and stirred at 100°C for 2 hours to react. After removing the reaction solvent, a new
25 ml of DMSO was added and the mixture was stirred at room temperature for 1.5 hours for washing. After that, 25ml (1.5 hours), 20ml
(1.5 hours), 20ml (1.5 hours), 20ml (11.5 hours),
Washing was carried out sequentially under conditions of 20 ml (2.5 hours). Titration analysis of isocyanate groups in 20 ml of the final washing solution revealed that no isocyanate groups were detected. continuation,
30 ml of dehydrated DMSO and 14.3 g of polyethylene glycol (hereinafter referred to as "PEG") having an average molecular weight of 6000 were charged, stirred at 100°C for 1.3 hours to react, and then 3 ml of water was added to stop the reaction. The reaction was sucked off and stirred in 20 ml of DMSO at room temperature overnight, then taken up again and washed repeatedly with large amounts of water. After suction, an adsorption test was performed using the adsorbent, which was immersed in physiological saline and water was squeezed out by suction. Adsorption test Add 0.5 ml of the above adsorbent to 1.5 ml of freeze-dried human plasma, shake at 37°C for 3 hours, and then mix at 3000 rpm.
Centrifugation was performed for 15 minutes and the supernatant was analyzed. An adsorption test was similarly conducted using fresh human plasma. Examples 2 to 5 and Comparative Example The same procedure as in Example 1 was carried out. The manufacturing method of the adsorbent and the results of the adsorption test are shown in Tables 1 and 2, respectively, along with the data of Example 1. After the adsorption test shown in Table 2, IgG, albumin (hereinafter referred to as "AL"), and total protein (hereinafter referred to as "TP") were measured in the first
It is shown in Fig. 2 and Fig. 2. The numbers in the figure indicate the example numbers. From FIG. 1 and FIG. 2, it can be seen that by using the adsorbent of the present invention, only IgG can be selectively removed. Example 6 Cross-linked agarose (trade name: Sepharose CL-4B; manufactured by Pharmacia) was repeatedly washed with distilled water and suctioned to thoroughly squeeze out the water. 153.0 g of wet cross-linked agarose (9.2 g in absolute dry form) thus obtained was dehydrated.
The mixture was added to 300 ml of DMSO and stirred at room temperature for 3 hours. After removing DMSO in a water-free system,
Another 200 ml of dehydrated DMSO was added, and the mixture was stirred at room temperature for 7 hours. Similarly below, 160ml (12 hours), 150ml
(12 hours), 140ml (12 hours), 160ml (12 hours),
Repeat the operation for 140ml (12 hours) and finally
250ml of DMSO was added. DMSO in the system at this time
When the moisture inside is analyzed using the Karl-Fitscher method,
It was below 10ppm. A solution of 11.4 g of HMDI added to 50 ml of dehydrated DMSO was added to this system, and the mixture was heated at 100℃ for 2 hours.
The reaction mixture was stirred for hours. After removing the reaction solvent by suction at room temperature under a dry atmosphere, add fresh DMSO.
300 ml was charged and washed by stirring at room temperature for 2 hours. Thereafter, washing was performed in the same manner four times in sequence under the condition of 300 ml (2 hours). Titration analysis of isocyanate groups in 300 ml of the final washing solution revealed that no isocyanate groups were detected. Next, add 300ml of dehydrated DMSO and 60g of PEG with an average molecular weight of 6000, and heat to 90% at 100℃.
After stirring for several minutes to react, the temperature was lowered to room temperature and 100 ml of water was added. Aspirate the reaction and add DMSO
After stirring overnight at room temperature in 300 ml, remove again.
Washed repeatedly with large amounts of water. After squeezing out the water by suction, an adsorption test was conducted using the adsorbent, which was immersed in physiological saline and then squeezed out by suction. The generated adsorbent is 460g in wet form (absolutely dry).
13.5g). Adsorption test The inner diameter of the column packed with adsorbent was 10 mm.
The plasma used was separated from patients through plasma exchange after kidney transplantation. The total amount of plasma flowing through the adsorption test device was 28ml, and the plasma circulation rate was 0.3ml/
The temperature of the column was 37°C. TP, AL, IgG, anti-T cell antibodies, and anti-B cell antibodies in plasma were analyzed after conducting a circulation test by varying the adsorbent amount and circulation time. TP is the Biuret method, AL is the BCG method, IgG is the single radial immunodiffusion method, and anti-T cell antibodies and anti-B cell antibodies are CDC (complement-dependent cytotoxicity).
Measured by test. The test conditions and results are shown in Table 3. From Table 3, in terms of antibodies against donor lymphocytes, T-warm's score went from 8 to 1,
It can be seen that anti-T-warm antibodies that appeared during kidney transplantation were adsorbed. Example 7 Cross-linked agarose (trade name: Sepharose CL-4B; manufactured by Pharmacia) was repeatedly washed with distilled water and suctioned to thoroughly squeeze out the water. 102.6 g of the wet cross-linked agarose thus obtained was freeze-dried (weight after freeze-drying: 5.81 g. The freeze-dried agarose was placed in dehydrated methanol, and the water content in the methanol was analyzed by the Karl-Fisscher method. Based on the analysis values, the water content in agarose after freeze-drying is calculated.
It was 0.13%. ). 4.0g of freeze-dried agarose was dehydrated.
Add to 150ml of DMSO and add HMDI to this system.
Prepare a solution of 5.3g added to 30ml of dehydrated DMSO,
The reaction was stirred at 100°C for 2 hours. After the reaction solvent was removed by suction at room temperature under a dry atmosphere, 200 ml of fresh DMSO was added, and the mixture was stirred at room temperature for 2 hours to perform washing. After that, 200ml (2 hours)
It was washed 6 times under the following conditions. 200ml final cleaning solution
Titration analysis of the isocyanato groups in the solution revealed that no isocyanato groups were detected. Continue to dehydrate
200 ml of DMSO and 40 g of PEG with an average molecular weight of 6000 were charged, and the mixture was stirred and reacted at 90°C for 3 hours, and then the temperature was lowered to room temperature and 100 ml of water was added. The reaction was sucked off and stirred in 200 ml of DMSO at room temperature overnight, then taken up again and washed repeatedly with large amounts of water. After squeezing out the water by suction, an adsorption test was conducted using the adsorbent, which was immersed in physiological saline and then squeezed out by suction. Adsorption test Plasma circulation rate 0.25 ml/min, column temperature 30
The test was conducted in the same manner as in Example 6, except that the temperature was changed to
Analysis was performed in a similar manner. The test conditions and results are shown in Table 4. Table 4 shows that T-warm's score for antibodies against donor lymphocytes went from 8 to 2 to 1, indicating that it adsorbs anti-T-warm antibodies that appeared during kidney transplantation. [Effects of the Invention] By using the adsorbent of the present invention, only immunoglobulins and their immune complexes in plasma can be selectively removed.

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【table】 [Brief explanation of the drawing]

FIG. 1 and FIG. 2 are diagrams showing the results of Examples of the present invention.

Claims (1)

[Claims] 1. A water-insoluble polymer compound having a hydroxyl group selected from agarose, cellulose, dextran, and polyvinyl alcohol, via a compound having two or more isocyanato groups in the molecule, by the following formula (): (In the formula, R ₁ , R ₂ , R ₃ and R ₄ independently represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and n represents an integer of 10 to 200.) An adsorbent for removing immunoglobulins and their immune complexes from plasma to which compounds are bound. 2 A compound having two or more isocyanato groups in the molecule has the following formula (): OCN-R-NCO () (wherein R is an aliphatic chain having 1 to 12 carbon atoms or an aromatic chain having 6 to 15 carbon atoms) The adsorbent according to claim 1, which is a compound represented by the following. 3. The adsorbent according to claim 1, wherein the compound represented by formula () is polyethylene glycol. 4. The adsorbent according to claim 1, wherein the compound having two or more isocyanate groups in the molecule is hexamethylene diisocyanate. 5. The adsorbent according to claim 1, wherein the immunoglobulin is an anti-T cell antibody.