JPH0547223B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for sterilizing an adsorbent. More specifically, the present invention relates to a method for sterilizing an adsorbent for extracorporeal circulation therapy, which is formed by immobilizing a sulfated polysaccharide on a water-insoluble carrier. [Prior art] For the purpose of selectively removing harmful components from body fluids, adsorbents in which substances with specific affinity for harmful components (so-called ligands) are immobilized on water-insoluble carriers have been used for extracorporeal circulation therapy. Attempts are being made to use it. Although sterilization is essential for therapeutic use, chemical sterilization is inappropriate due to safety issues and handling during use, and high-pressure steam sterilization is preferred. However, when these adsorbents are sterilized with high-pressure steam, the ligands are deactivated or desorbed, and in fact, no adsorbent for extracorporeal circulation treatment that has been sterilized with high-pressure steam has been realized. In particular, adsorbents using sulfated polysaccharides as ligands are known to be susceptible to decomposition such as desulfation during high-pressure steam sterilization. [Problems to be solved by the invention] When an adsorbent for extracorporeal circulation treatment in which sulfated polysaccharides are immobilized on a water-insoluble carrier is sterilized under strict conditions such as 121°C for 20 minutes, the immobilized sulfated polysaccharides are removed. This causes a problem in that the ability to remove harmful components from the blood is not sufficient because they may be hydrolyzed and removed. The present invention has been made in order to solve the problems that occur when steam sterilization is performed as described above. [Means for Solving the Problems] The present invention provides steam sterilization of an adsorbent for extracorporeal circulation treatment, which is made by immobilizing a sulfated polysaccharide on a water-insoluble carrier, in an aqueous solvent controlled at pH 5 to 9. The present invention relates to a method for sterilizing an adsorbent, which is characterized in that it is sterilized. [Example] Sulfated polysaccharides used in the present invention include, for example, heparin, dextran sulfate, chondroitin sulfate, chondroitin polysulfate, heparanic acid, keratan sulfate, heparitin sulfate, xylan sulfate, caronine sulfate, cellulose sulfate, chitin sulfate, and chitosan. Sulfuric acid, pectin sulfate, inulin sulfate, alginate sulfate, glycogen sulfate, polylactose sulfate, carrageenan sulfate, starch sulfate, polyglucose sulfate, laminarin sulfate, galactan sulfate, levanic acid, mebesulfate, etc.
The present invention is not limited to these, and any sulfated polysaccharide that is generally used in the production of adsorbents for extracorporeal circulation therapy may be used. Among the specific examples of the sulfated polysaccharide, heparin, dextran sulfate, and chondroitin polysulfate are preferred. Examples of water-insoluble carriers used in the present invention include agarose, which is a carrier commonly used in affinity chromatography, soft gels such as dextran and polyacrylamide, inorganic porous materials such as porous glass and porous silica, and synthetic polymers. Examples include, but are not limited to, polymer gel consisting of, porous cellulose gel, etc. Among these, inorganic porous materials and hard gels such as polymer hard gels are preferable because they provide sufficient body fluid flow and are less likely to cause clogging. In particular, porous cellulose gels are preferred because they (1) have relatively high mechanical strength; Because it is strong and strong, it is less likely to be destroyed or produce fine powder due to operations such as stirring, and when packed in a column, body fluids will not become compacted or clogged even when flowing at a high flow rate, so the flow rate is high. (2) Since the gel is composed of cellulose, it is hydrophilic and can be used to bind sulfated polysaccharides. (3) Even if the pore volume is increased, it has relatively high strength, so it can provide an adsorption capacity comparable to that of soft gels. (4) It has a high synthetic safety level. It has the advantage of being more expensive than molecular gels, etc., and by retaining sulfated polysaccharides in the porous cellulose gel, it can selectively adsorb and remove harmful components at high flow rates. I can get a body. Regarding the adsorbent using porous cellulose gel, please refer to the patent application No. 58-68116.
It is described in detail in the specification of the No. In the present invention, an adsorbent for extracorporeal circulation therapy is produced by immobilizing a sulfated polysaccharide on a water-insoluble carrier. Various known methods can be used to immobilize the sulfated polysaccharide on the water-insoluble carrier. That is, physical methods, ionic bonding methods, covalent bonding methods, etc. can be mentioned. Since it is important that the immobilized sulfated polysaccharide is difficult to desorb, a strong covalent bonding method is preferred, and even if other methods are used, it is preferable to prevent desorption.
Furthermore, a spacer may be introduced between the water-insoluble carrier and the sulfated polysaccharide if necessary. In the present invention, the adsorbent for extracorporeal circulation therapy produced in this way is sterilized by steam sterilization in an aqueous solvent controlled at pH 5 to 9, usually at 121°C for about 20 minutes, and then used. be done. If the pH during steam sterilization is less than 5, the adsorption activity of the steam sterilized adsorbent will be significantly reduced, or the fixed sulfated polysaccharide will be significantly desorbed by hydrolysis. Also, even if the pH exceeds 9
A similar phenomenon occurs when the pH is less than 5. Note that when the pH is less than 5, the adsorption activity of the adsorbent mainly decreases, and when the pH exceeds 9, desorption due to hydrolysis of the sulfated polysaccharide mainly occurs, and in both cases, the performance of the adsorbent decreases. There are no particular limitations on the method of controlling the PH of the aqueous solvent used in steam sterilization, and while automatically measuring changes in PH during steam sterilization,
Adjustments may be made and buffers may be used. Adjusting the pH using a buffer solution allows for stable pH adjustment without the need for special equipment, and also prevents activity reduction due to pH changes during storage of sterilized extracorporeal circulation treatment adsorbents, and the ability to reduce the activity of sulfuric acid, which is a ligand. This is preferable because the elimination of polysaccharides is also suppressed. Buffers used in the preparation of buffer solutions include, for example, citric acid, phosphoric acid, acetic acid, boric acid, tartaric acid, carbonic acid, maleic acid, glycine, etc., or their sodium salts, potassium salts, calcium salts, etc. Safe ones are preferred, and these may be used alone or in combination of two or more. When a buffer solution is used to adjust the pH of an aqueous solvent, there is no particular limit to the concentration of the buffer solution, and it can be used as long as it functions as a buffer solution, but cleaning is easy before using it as an adsorbent for extracorporeal circulation therapy. For these reasons, it is preferably 0.001 to 10% (by weight, hereinafter the same), and more preferably 0.01 to 2%. The adsorbent for extracorporeal circulation therapy manufactured in this way and sterilized according to the present invention is packed with the adsorbent in a column equipped with a filter, mesh, etc. at the inlet and outlet that allow body fluid components to pass through but not the adsorbent. However, the column can be incorporated into an extracorporeal circulation circuit and used without limitation in applications such as extracorporeal circulation therapy in which blood, plasma, etc. are passed through the column. Next, the method of the present invention will be explained based on examples. Production example 1 Toyopearl HW75, a cross-linked polyacrylate gel (fully porous hard gel) (protein exclusion limit 50000000, particle size 50-100 μm, manufactured by Toyo Soda Co., Ltd.)
6 ml of a saturated NaOH aqueous solution and 15 ml of epichlorohydrin were added to 10 ml, and the mixture was reacted at 50° C. for 2 hours with stirring to obtain an epoxidized gel. 20 ml of concentrated ammonia water was added to the resulting gel and stirred at 50°C for 2 hours to introduce amino groups. Meanwhile, dissolve 200 mg of heparin in 10 ml of water and
After adjusting to 4.5, 30 ml of the above-mentioned amino group-introduced gel was added. Then, 200 mg of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide was added.
The mixture was added while maintaining the pH at 4.5, and the mixture was 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 in this order to obtain a heparin-immobilized gel (hereinafter referred to as A-1). Production example 2 TOYOPEARL, a cross-linked polyacrylate gel
Add 6 ml of saturated NaOH aqueous solution and 15 ml of epichlorohydrin to 10 ml of HW75, and while stirring,
After reacting for 2 hours at °C, the gel was washed with alcohol and water in this order, and the epoxidized gel was added. 2 ml of the resulting gel has an intrinsic viscosity of 0.055 dl/g,
0.5 g of dextran sodium sulfate having an average degree of polymerization of 40 and a sulfur content of 19% and 2 ml of water were added (the concentration of dextran sodium sulfate was approximately 13%). Then
The pH was adjusted to 12 and the gel was shaken at 40°C for 16 hours. The gel was filtered and washed with 2M saline solution, 0.5M saline solution, and water in this order to obtain a gel with dextran sodium sulfate fixed (hereinafter referred to as A -2). Production Example 3 Heparin-immobilized Toyopearl HW75 (hereinafter referred to as A-3) was obtained in the same manner as Production Example 2 except that heparin was used instead of dextran sulfate. Production example 4 Porous glass FPG2000 (average particle size 1950 Å, specific surface area 13 m ² /g, particle size 80-120 mesh, Wako Pure Chemical Industries, Ltd.)
Co., Ltd.) in dilute nitric acid for 3 hours, washed with water and dried.
After heating at 500°C for 3 hours, it was poured into a 10% toluene solution of γ-aminopropyltriethoxysilane, refluxed for 3 hours, washed with methanol, and the γ-
Obtained aminopropyl treated glass. Meanwhile, dissolve 200 mg of heparin in 10 ml of water and
Adjusted to 4.5. After adding 2 g of γ-aminopropyl treated glass to this, 1-ethyl-3-
(dimethylaminopropyl)carbodiimide 200mg
was added while maintaining the pH at 4.5, and the mixture was shaken at 4°C for 24 hours. After the reaction was completed, the glass was washed with a 2M saline solution, a 0.5M saline solution, and water in this order to obtain a porous glass on which heparin was fixed (hereinafter referred to as B-1). Production Example 5 FPG2000 (hereinafter referred to as B-2) on which chondroitin polysulfate was immobilized was obtained in the same manner as Production Example 4 except that heparin was replaced with chondroitin polysulfate. Production example 6 Dextran sulfate 800mg in 0.25M _NaIO4 aqueous solution
After dissolving in 10 ml and stirring at room temperature for 4 hours, 200 mg of ethylene glycol was added and stirred for 1 hour. After adjusting this solution to pH 8, 4 ml of γ-aminopropyl-treated FPG2000 obtained in the same manner as in Production Example 4 was added.
was added and shaken for 24 hours. After the reaction was completed, the gel was filtered and washed with water, suspended in 10 ml of 1% HaBH ₄ aqueous solution and reduced for 15 minutes, filtered and washed with water to obtain FPG2000 (hereinafter referred to as B-3) on which dextran sulfate was fixed. . Production Example 7 Porous glass FPG2000 was heated in dilute nitric acid for 3 hours, washed with water, and then heated at 500°C for 3 hours. This is γ
-10 of glycidoxypropyltrimethoxysilane
% toluene solution, refluxed for 3 hours, and washed with methanol to obtain γ-glycidoxypropyl-treated glass. On the other hand, dissolve 2 g of dextran sulfate in 10 ml of water and adjust the pH to 9.2, then add 2 g of the above γ
−45℃ with addition of glycidoxypropyl treated glass
The reaction was carried out for 16 hours. After the reaction was completed, the product was washed with a 2M saline solution, a 0.5M saline solution, and water in this order to obtain dextran sulfate-immobilized FPG2000 (hereinafter referred to as B-4). Production example 8 To 10 ml of CK gel A-3 (exclusion limit molecular weight 50000000, particle size 45 to 105 μm, manufactured by Chitsuso Co., Ltd.) as a porous cellulose gel, add 4 g of 20% NaOH, 12 g of heptane, and 1 drop of nonionic surfactant TWEEN 20. Ta. After stirring at 40° C. for 2 hours, 5 g of epichlorohydrin was added and stirred for 2 hours, and the gel was washed with water and filtered to obtain an epoxidized cellulose gel. The amount of epoxy groups introduced was 30 μM per ml column volume. 2 ml of the resulting gel has an intrinsic viscosity of 0.027 dl/g,
Dextran sulfate sodium with 17.7% sulfur content
Add 0.12 g and 2 ml of water (concentration of sodium dextran sulfate is approximately 2.5%), adjust the pH to 11, and heat at 45°C.
The mixture was shaken for 16 hours. After that, filter the gel and
The gel was washed with a 2M saline solution, a 0.5M saline solution, and water in this order to obtain a cellulose gel (hereinafter referred to as C-1) on which dextran sodium sulfate was immobilized. Production Example 9 10 g of CK gel A-3 was collected by suction filtration, 4 g of 20% NaOH and 12 g of heptane were added thereto, and 1 drop of nonionic surfactant TWEEN 20 was added and stirred to disperse the gel. After stirring at 40℃ for 2 hours, 5g of epichlorohydrin was added to the mixture at 40℃.
Stirred at ℃ for 2 hours. After standing still, the supernatant was discarded, and the gel was washed with water and filtered to obtain an epoxidized gel. 15 for this
ml of concentrated ammonia water was added and 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 amino groups had been introduced. On the other hand, dissolve 200 mg of heparin in 10 ml of water, add the above amino group-introduced cellulose gel, and adjust the pH.
Adjusted to 4.5. Then, 200 mg of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide was added to the PH
The mixture was added while maintaining the temperature at 4.5°C, and the mixture was shaken at 4°C for 24 hours.
After the reaction is complete, add 2M saline solution, 0.5M saline solution,
The gel was washed with water in this order to obtain a heparin-immobilized gel (hereinafter referred to as C-2). Production Example 10 CK Gel A-3 (hereinafter referred to as
C-3) was obtained. Production Example 11 Heparin-immobilized CK gel A-3 (hereinafter referred to as C-4) was obtained in the same manner as Production Example 8 except that heparin was used instead of dextran sulfate. Examples 1 to 18 and Comparative Examples 1 to 11 10 g (wet weight) of the gel (adsorbent) shown in Table 1 obtained in Production Examples 1 to 11 was placed in a hard glass flask, and 10 ml of the aqueous solvent shown in Table 1 was added. and 121℃
High-pressure steam sterilization was performed for 40 minutes. of aqueous solvent before and after sterilization for each adsorbent.
PH and ligand amount were measured. The results are shown in Table 1.

【table】

[Table] [Effects of the Invention] According to the method of the present invention, an adsorbent for extracorporeal circulation therapy can be steam sterilized at 121°C for 40 minutes, which is stricter than the usual condition of 121°C for 20 minutes. , a good quality adsorbent for circulation therapy with less desorption of the ligand can be obtained.

Claims (1)

[Scope of Claims] 1. When steam sterilizing an adsorbent for extracorporeal circulation therapy consisting of a sulfated polysaccharide immobilized on a water-insoluble carrier, sterilization is performed in an aqueous solvent controlled at pH 5 to 9. How to sterilize adsorbents. 2. The sterilization method according to claim 1, wherein the PH-controlled aqueous solvent is a buffer solution. 3 The buffer solution is citric acid, phosphoric acid, acetic acid, boric acid,
The sterilization method according to claim 2, which is a buffer solution using at least one of tartaric acid, carbonic acid, maleic acid, glycine, and salts thereof.