JPS5838562A - Method and apparatus for regenerating protein adsorbent for body liquid treatment - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for regenerating an adsorbent on which proteins in body fluids are deposited, and an apparatus for the same.

In recent years, blood purification methods using activated charcoal have been used for patients with hepatic coma and drug addiction, and have been attracting attention because of their remarkable effects.

Furthermore, various adsorbents for adsorbing specific proteins in blood have been studied for the purpose of treating autoimmune diseases, rejection reactions after organ transplants, liver diseases, and kidney diseases. However, these protein adsorbents are generally more expensive than the previously mentioned activated carbon, and the amount of protein to be adsorbed may be large, making the cost per treatment expensive. There is.

In view of these circumstances, the present inventors have conducted intensive research and found that a porous material with silanol groups on its surface efficiently adsorbs proteins in body fluids. The present invention was completed by discovering that the ability to absorb Il can be easily recovered by using the method.

That is, the present invention provides a method for regenerating an adsorbent for body fluid treatment, in which an adsorbent adsorbing proteins in body fluids is treated with a washing liquid to desorb and remove the adsorbed proteins. This is a method for regenerating a protein adsorbent for treating body fluids, which is characterized in that a porous material having silanol groups of m'' is used, and an aqueous solution or a water-soluble solvent with a PR of 1 to 10 is used as a cleaning liquid. The body fluids mentioned include whole blood, plasma, serum, etc.

The adsorbent used in the present invention has at least α1 μmol/m” of silanol groups (38i01f
), and if the silanol group concentration is lower than this, the protein adsorption capacity will not be sufficient. The upper limit of the silanol group concentration is not particularly limited, but if it exceeds 100 μmol/m, the adsorption capacity tends to reach saturation, so it does not need to be higher than that. The concentration is a value measured by methyl red absorption method or titration method using methyl red as an indicator. Porous glass, porous silica, porous silica-alumina are preferably used as such adsorbents, but other It is also possible to use a porous body in which silanol groups are introduced onto a carrier by water glass treatment or coating with a silanol group-containing primer.The average pore diameter of these porous bodies is preferably in the range of 50 to 3000X, If the pore diameter is outside this range, the protein adsorption capacity will not be sufficient.Also, if the pore diameter is in the range of 18D to 1.2D and occupies more than 80% of the total pore volume (i.e. A material with a sharp pore size distribution is preferable for selectively adsorbing a specific protein.In other words, the average pore diameter of the porous material is appropriately selected depending on the molecular weight of the protein to be adsorbed, and the pore size distribution is By using sharp porous materials, target proteins can be selectively adsorbed.

Since these adsorbents are brought into contact with body fluids such as blood or plasma, the particle diameter is preferably within the range of 0.1 mm to 5 mm. Furthermore, in order to improve the affinity with blood, it is more preferable to be formed into a spherical shape or coated with a hydrophilic polymer.

A column filled with these adsorbents has a main body having an inlet and an outlet on both sides of the adsorbent packed bed, and an inlet and an outlet that are shaped to be easily connected to the blood circuit, and a main body that has an inlet and an outlet that are shaped to be easily connected to the blood circuit, and a main body that has an inlet and an outlet that are shaped to be easily connected to the blood circuit, and a main body that has an inlet and an outlet that are shaped to be easily connected to the blood circuit. 80 to 180, blood etc. pass through, but adsorbent does not pass through.
Although it is preferable to use a column equipped with a filter having a mesh for meshing, other shapes can be used for this purpose as long as the column has substantially the same function. The material of the column may be any physiologically inert material, such as glass, ethylene ethylene, polypropylene, gylycarbonate, polystyrene, polymethyl methacrylate, etc., but gylybrobylene, polycarbonate, etc., which can be sterilized by autoclaving, are particularly preferred. The filter may be made of any material that is physiologically inert and strong as a fiber, but one made of polyester is particularly preferably used.

Columns packed with adsorbents are usually sterilized before use, but
Autoclave sterilization and R-ray sterilization are preferred.

It is necessary that the cleaning liquid used in the present invention does not physically or chemically attack the adsorbent, column, etc., and it is preferable that the cleaning liquid has no or little toxicity to living organisms. Generally, silica-based adsorbents are attacked by strong alkaline aqueous solutions, so when using an aqueous solution as a cleaning solution, the PM must be within the range of 1 to 1°.
Also, those within this range are 15Il, which has a large # effect.

Although water-soluble solvents such as ethyl alcohol, propyl alcohol, and acetone can be used for the cleaning solution and shite, ethyl alcohol is preferable because it is less harmful to living organisms. If the bond between the adsorbed protein and the adsorbent is weak, the protein can be easily washed away with an aqueous solution such as distilled water or physiological saline.

If the adsorbed protein and adsorbent are strongly bound, acidic or alkaline buffer solution, surfactant aqueous solution,
It may be washed with a concentrated salt aqueous solution, ethyl alcohol, 8M ethyl alcohol water, or the like. The PM of the cleaning solution is appropriately selected depending on the protein to be adsorbed, but in general, an acidic cleaning solution is preferably used for cleaning neutral to basic proteins, and an alkaline cleaning solution is preferably used for acidic proteins. Moreover,
These cleaning solutions are preferably ones that do not easily remain on the adsorbent; for example, acetic acid buffers, citric acid buffer solutions, boric acid buffer solutions, carbonate buffers, nonionic surfactants, etc. are particularly preferably used. . For r-globulin, which is a typical protein, buffers of Pn1 to Pn9 are particularly preferably used. After washing, the adsorbent can be sufficiently washed with physiological saline and body fluids such as blood or plasma can be reintroduced to perform blood purification.

These cleaning solutions are usually used after being sterilized in an autoclave, but those whose components change due to autoclave sterilization are sterilized by R-ray sterilization or by using a sterilization filter.

In addition, the washing solution should be used at a temperature that does not denature the protein (i.e. 4
~50°C) is preferred. It is preferable to flow the cleaning liquid at least 10 times, more preferably at least 50 times, the apparent volume of the adsorbent, and it is also possible to use a combination of two or more cleaning liquids.

The apparatus for carrying out the method of the present invention includes an adsorbent for proteins in body fluids made of a porous material having at least 11 μmol/m'' of silanol groups on the surface, a means for supplying the body fluid to the adsorbent, and an adsorbent for the adsorption of proteins in body fluids. A body fluid treatment device mainly consisting of a container for storing a washing liquid for washing and removing proteins adsorbed on the adsorbent, a means for supplying the washing liquid to the adsorbent, and a means for switching the supply of the body fluid and the washing liquid to the adsorbent. As mentioned above, the adsorbent is used by being packed in a column, an example of which is shown in FIG.

The means for supplying body fluid to the adsorbent comprises a circuit and, if necessary, a pump. A commercially available blood pump is preferably used as the pump. The container for storing the cleaning liquid may be of any type as long as it can store the cleaning liquid in a sterile manner.
Plastic or glass bottles or plastic bags are preferably used. The means for supplying the cleaning liquid to the adsorbent comprises a circuit and, if necessary, a pump. In addition, as a means for switching the supply of body fluid and cleaning liquid to the adsorbent, a compression type opening/closing method using a cap, a drain, etc., or a three-way, four-way or six-way stopcock, etc. is preferably used. The above-mentioned device must have a structure that allows the entire interior to be kept sterile. Further, such an apparatus may be provided with a mechanism for adding heparin, protamine, etc., a mechanism for flowing physiological saline into an adsorbent, a mechanism for separating blood cells and plasma, etc., as necessary.

Next, an example of an apparatus for carrying out the method of the present invention will be explained using the drawings. FIG. 2 is a diagram showing an example of such a device, in which blood is extracted from a human body at a constant flow rate using a blood pump (6). First, in the adsorption operation, the flow path switching section (7)
is set to guide blood drawn out from the human body to the adsorbent-filled column (1), and the flow path switching section (in the flow path switching section) is set so that the blood that has passed through the adsorbent-filled column (1) returns to the human body. After performing the bite operation for a certain period of time, the cleaning operation begins. In the cleaning operation, the flow path switching section (7, 8
, 11), the blood is returned to the human body through the bypass circuit (13), the cleaning liquid is guided to the adsorbent-filled column (1) through the cleaning liquid pump (1), and the cleaning liquid that has passed through the adsorbent-filled column is sent to the waste liquid tank (12). is set to be sent to.

After washing for a certain period of time, the flow path switching section (8) is switched to introduce physiological saline into the adsorbent column. After passing through physiological saline fatigue for a certain period of time, return to the flow path switching section (7°8.11).
The blood is introduced into the adsorbent-filled column by operating the . If necessary, the adsorption and washing operations may be repeated. Operation of these pumps (6, 14), flow path switching section (7, 8, 11)
It is most preferable to use a device having a control unit that is automatically controlled by a preset program.

Figure 5 shows a centrifugal or membrane-based plasma separation device (
17) was used to demonstrate an embodiment of the present invention in which only plasma is introduced into an adsorbent-filled column. The method is exactly the same as the method shown in FIG. 2 except that plasma is separated. Alternatively, as shown in FIG. 4, the present invention can be carried out more efficiently by using two adsorbent-filled columns in parallel and alternately repeating adsorption and washing.

Since proteins in body fluids can be efficiently removed by the method and device of the present invention, various diseases can be treated. Main indications include systemic lupus erythematosus, rheumatoid arthritis, chronic glomerulonephritis, myasthenia gravis, multiple sclerosis, polymyositis, Bechit's disease, 8J5Gren syndrome, scleroderma, and soft tissue eosinophilia. Granulomatosis, Heerfordt
syndrome, severe Wegner granulation, bronchial asthma, hypersensitivity pneumonitis, eosinophilic pneumonitis, chronic active hepatitis, primary biliary cirrhosis, idiopathic inflammatory bowel disease, ankylosing spondylitis, diabetes,
Immune-related diseases such as polyarteritis, aplastic anemia, autoimmune hemolytic anemia, Fe1ty syndrome, idiopathic thrombocytopenic purpura, rejection after organ transplantation, jaundice, cancer diseases, familial high cholesterol Examples include blood pressure, liver disease, and kidney disease.

The present invention will be explained in more detail with reference to Examples below.
The present invention is not limited to such embodiments.

Example 1 Porous glass with average pore diameter 0 of 580X (surface ranol group concentration 0.67 μmol 7 m'' (measured by methyl red absorption method), pore volume proportion of pore diameter between α8D and 1.2D 86 %) was packed into a polypropylene column (equipped with a Bossester 180 mesh filter at both ends of the adsorbent layer, and a boat section outside the filter that could be connected to the blood circuit) to adsorb feces. Column (adsorption purpose IF! quality: r-globson)
And so. 10 m/m of this columnar human plasma was circulated for 2 hours at a flow rate of approximately 5 Inl/Ill I n. Afterwards, a wash solution of 200+n4 was circulated at room temperature at a flow rate of approximately 5 Inl/Ill I n.
Washing was carried out by flushing with water.

Furthermore, add 300 mj of physiological saline at a flow rate of 10 ml/'
After draining at mi 11°, add 110 ml of fresh human blood.
1 liter for 2 hours at 57'C with a flow rate of 5 mj/min
It was. Human plasma after the first and second adsorption was subjected to high performance liquid chromatography (Waters system).
soaiatem Inj, manufactured by Mu LC/GPG244 type, column manufactured by Toyo Soda Co., Ltd. G-5000sw (inner diameter 7.
5 mm, length 600 mu)', eluate 1/1
5M phosphate buffer (contains α15MNa01, PHHO2,
Analyze with a flow rate tomj/min and analyzer UV (280 m)), determine the removal rate from the reduction rate of r-globulin, and
The regeneration rate was calculated from the ratio of the removal rate of the first time and the second time after washing. The following four types of cleaning solutions were used. ■Citrate buffer (secondary sodium citrate [01, PHL?4), boric acid buffer wave (boric acid-[01-NaOH, P H9,
48), 0 Triton x-too (manufactured by Wako Pure Chemical Industries, Ltd., (L
5% aqueous solution, PK'sS 5 ), O ethyl alcohol (9t5%, special grade reagent manufactured by Wako Pure Chemical Industries). The results are summarized in Table 1.

Table I11 in the margin below Regeneration rate of adsorbent when using various cleaning solutions Example 2 Aqueous γ-globulin solution (2 g/dj, phosphate buffered saline, PHPH24) was placed in a column packed with the same porous glass as in Example 1. 10 at 57°C for 2 hours at a flow rate of approximately 5 m
j/min to adsorb γ-globulin onto the adsorbent. After that, 300mj of washing solution was filtrated at room temperature.
The column was washed with Dm17min. Furthermore, after flowing 100 mJ of physiological saline at a flow rate of 10 mJ/min, 10 m of r-globulin aqueous solution with the same concentration as the first time was added.
j was circulated under the same conditions and adsorption was performed for the second time. The concentration of the γ-globulin aqueous solution before and after the first and second adsorption was measured by the biuret method to determine the amount of adsorption. The regeneration rate was calculated from the ratio of the adsorption amount of 21 g of the first adsorption amount and the adsorption amount of the first adsorption amount. As a washing solution, ■P■1.88 buffer (secondary sodium citrate-Mo
l), ■ pH 585 buffer (II sodium citrate-flyCI), ■ pH 4-7 buffer (secondary sodium citrate-HCl), @ pH 7,14 buffer (KHl
PO4-NalHPO4), ■ pHa29 buffer (K
Physiological saline (pH&o) of pH 9,04 buffer (Hontsusuna-HCjl) was used. The results are summarized in Table 2.

Below is Table 2 in the margin Regeneration rate of adsorbent when using cleaning solutions with various pH values 1゜1; 1:

[Brief explanation of the drawing]

FIG. 1 is a diagram showing an example of a column filled with an adsorbent, and FIGS. 2 to 4 are schematic diagrams showing examples of the body fluid treatment apparatus of the present invention. 1. Karago body 2 Body fluid introduction ROM Body fluid outlet Book Filter! LL dan arrival
Drug & Blood, Pump 1 Flow path switching section ■
a Flow path switching unit 9 Cleaning liquid storage capacity fi1α Physiological saline storage container 11, flow path switching unit■ 1L! 1
1gL tank, -433- L t<Ihas circuit 14 Washing liquid pump i Heparin 1 melon Protamine-plasma separation device
1a Plasma Pump Patent Applicant Kuraray Co., Ltd. Agent Patent Attorney Mizunen Kenai 1 Figure 3 Figure 3 Procedural Amendment (Voluntary) January 6, 1981 1, Case Indication Patent Application No. 157281 1981 Device for the purpose (108) RiRa Co., Ltd. Representative Director Tsugu Okabayashi 4, Agent ≦, Subject of amendment 6 The following is written between Table 2 on page 17 of the detailed statement of amendments and the column for a brief explanation of the drawings. Insert text. [Example 3 Porous glass with an average pore diameter of 0.420 mm (surface silanol group concentration 0.47 μmO) and a narrow pore diameter of 0.8 D to 1
．． 291 Example 1
It was packed into a column similar to the above and used as an immunoglobulin adsorbent. To this column, 12 ppd of a mixed solution of r-globulin and albumin (initial concentration of each 2.t/di, IJ acid buffered saline PII 7.4) was applied at a flow rate of 5.1 sl/m.
in for 50 minutes, then saline at a flow rate of 5.4
#! It was washed and regenerated by flushing at a rate of 4/min for 50 minutes. After that, the same protein mixed solution as the first time was circulated again for 30 minutes. Similarly, the physiological saline solution was poured off for 30 minutes to wash and regenerate. This operation was repeated a total of 6 times. The concentration of the protein mixture solution before and after circulation was measured by the bromocresol green method for albumin and the biuret method for total protein, and r-globulin was determined as the difference between the total protein concentration and albumin concentration. Table 5 summarizes the amount of each protein adsorbed and the integrated amount of protein removed for the first to sixth times. It is clear from the table that
The amount of r-globulin class deposited decreased to a target η after the second time, but
The total removal amount for 6 times is 2 per fI of adsorbent! i8 mg, which reached more than 5 times that of the single treatment. However, albumin is 2
No adsorption was observed after the first adsorption, and the total amount removed was 7q per gram of adsorbent.1 As described above, by using the regeneration method and apparatus of the present invention, the amount of adsorption and selectivity can be dramatically improved. did it. Table 5 Protein adsorption lid and above when reusing immunoglobulin adsorbent

Claims (1)

[Claims] (1) Proteins in body fluids! 12 In a method for regenerating an adsorbent for body fluid treatment in which adsorbed adsorbent is treated with a cleaning solution to desorb and remove adsorbed proteins, the adsorbent has a silanol group of at least α1 and amol 7m'' on its surface as an adsorbent. Using a porous material, the cleaning liquid has a P■ of 1 to 10.
A method for regenerating a protein adsorbent for treating body fluids, the method comprising using an aqueous solution or a water-soluble solvent, or a mixture thereof. (2) The cleaning liquid is a liquid selected from the group consisting of distilled water, physiological saline, d-infusion, surfactant aqueous solution, concentrated salt aqueous solution, ethyl alcohol aqueous solution, and ethyl alcohol. How to play. (5) The regeneration method according to claim 1 or 2, wherein the volume of the cleaning liquid used is 10 times or more the volume of the adsorbent. (4) The porous body has an average pore diameter of 50 to 5000.
Claims 1 to 3 are porous bodies in which the sum of the volumes of pores having pore diameters in the range of a8D to 1.2D accounts for 80% or more of the total pore volume. The reproduction method described in any of the sections. (5) The regeneration method according to any one of claims 1 to 4, wherein the porous body is any one of porous glass, porous silica, and porous silica-alumina. (6) The protein is r-globulin and the washing solution is Fli
Claims 1 and 5 to 9 are buffer solutions of 1 to 9.
5. The regeneration method described in any of paragraphs. (7) At least CL 1 pmo: / m” on the surface
An adsorbent for proteins in body fluids made of a porous material having silanol groups, a means for supplying body fluids to the adsorbent, a container for storing a cleaning solution for washing and removing proteins adsorbed on the adsorbent, and a container for storing the cleaning solution. A body fluid treatment device mainly comprising means for supplying an adsorbent and means for switching between supplying body fluid and cleaning fluid to the adsorbent. (8) The porous body has an average pore diameter in the range of 30 to 3000 μm, and the pore size is the sum of the volumes of pores in the range of α8D − L 2D, or 80% of the total pore volume. 8. The body fluid treatment device according to claim 7, wherein the body fluid treatment device is a porous body that accounts for at least % of the porous body. (9) The body fluid treatment device according to claim 7 or 8, wherein the porous body is porous glass, porous silica, or porous silica-alumina.