JPS61100261A - Sterilization of adsorbing body - 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 [Industrial Application Field] The present invention relates to a method for sterilizing an adsorbent. For more details,
The present invention relates to a method for sterilizing an adsorbent for extracorporeal circulation therapy, which is made by immobilizing a sulfated polysaccharide on a water-insoluble carrier.

[Prior art] Conventionally, for the purpose of selectively removing harmful components from body fluids,
Attempts have been made to use adsorbents in which substances (so-called ligands) that have a specific affinity for harmful components are immobilized on water-insoluble carriers for extracorporeal circulation therapy. Although sterilization is essential for use in treatment, 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 deactivation or desorption of the ligand occurs, 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.

[Problem that the invention seeks to solve]

When an adsorbent for extracorporeal circulation therapy 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 may hydrolyze and come off. A problem arises in that the ability to remove harmful components from the blood is not sufficient.

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 a method for steam sterilizing an adsorbent for extracorporeal circulation treatment, which is made by fixing a sulfated polysaccharide on a water-insoluble carrier.
The present invention relates to a method for sterilizing an adsorbent in which the W characteristic is sterilization in an aqueous solvent controlled at a temperature of 5 to 9.

[Example] 1 Examples of the sulfated polysaccharides used in the present invention include heparin, dextran sulfate, chondroitin sulfate, chondroitin poly-VA acid, heparanic acid, Kerata,
heparitin sulfate, xylan sulfate, caronine sulfate, cellulose sulfate, chitin sulfate, chitosan sulfate, pectin sulfate, inulin sulfate, alginate sulfate, glycogen sulfate, polylactose sulfate, carrageenan sulfate, starch sulfate, polyglucose sulfate, laminarin sulfate, Examples include galactan sulfate, levan sulfate, mebesulfate, etc., but are 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 polysaccharides,
Heparin, dextran sulfate, and chondroitin polysulfate are preferred.

The water-insoluble carrier used in the present invention includes, for example, agarose, which is a carrier usually used in affinity chromatography, a soft gel such as dextran and polyacrylamide, an inorganic porous material such as porous glass and porous silica, a polymer, etc. Examples include, but are not limited to, polymer gels made of adult bird molecules and porous cellulose gels. Among these, inorganic porous materials and hard gels such as polymer hard gels are preferred because they provide sufficient body fluid flow and are less likely to cause clogging.
In particular, porous cellulose gel has (1) II relatively high internal mechanical strength and is tough, so it is less likely to be destroyed or generate fine powder by operations such as stirring, and when packed in a column, it can hold body fluids at high levels. It does not become compacted or clogged even when flowing at a high flow rate, so it can be flowed at a high flow rate, and the pore structure is not easily changed by high-pressure steam sterilization. (2) The gel is composed of cellulose. Therefore, it is hydrophilic, has many hydroxyl groups that can be used to bind sulfated polysaccharides, and has low nonspecific adsorption. (3) Even with a large pore volume, it has relatively high strength, so it is inferior to soft gels. It has excellent features such as (4) higher safety than synthetic polymer gels, etc.; By retaining the I-oxidized polysaccharide, an adsorbent that can selectively adsorb and remove harmful components at a high flow rate can be used. Note that an adsorbent using porous cellulose gel is described in detail in Japanese Patent Application No. 1983-68116.

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.

Examples include physical methods, ionic bonding methods, and covalent bonding methods. 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. Also, if necessary, add a spacer to a water-insoluble carrier and III! i It may be introduced between the oxidized polysaccharide and the oxidized polysaccharide.

In the present invention, the adsorbent for extracorporeal circulation therapy produced in this manner is sterilized by steam sterilization in an aqueous solvent whose p is controlled to be 15 to 9, usually at 121°C for about 20 minutes. ,used.

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. Further, even if the pH exceeds 9, the same phenomenon as when the pH is less than 5 occurs. In addition, when the pH is less than 5, the adsorption activity of the adsorbent mainly occurs, and when the pH exceeds 9, desorption mainly occurs due to hydrolysis of the sulfated polysaccharide.
In either case, adsorbent performance deteriorates.

There are no particular limitations on the method of controlling the pH of the aqueous solvent used in steam sterilization, and pH changes may be automatically measured during steam sterilization to automatically adjust the pHJ. It may also be done using Adjusting the pH using a buffer eliminates the need for special equipment and stabilizes the pH.
It is possible to adjust the activity of the sterilized adsorbent for extracorporeal circulation treatment due to pH changes during storage,
This is preferable because the elimination of the sulfated polysaccharide which is the ligand 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 a mixture of two or more.

When using a buffer solution 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 it can be easily washed before being used as an adsorbent for extracorporeal circulation therapy. 0.001 for reasons such as
~10% (weight%, the same applies hereinafter) is preferable, and 0.01~
2% is more preferred.

The adsorbent for extracorporeal circulation therapy manufactured in this manner and sterilized according to the present invention has, for example, an inlet and an outlet for body fluid acid.
Extracorporeal circulation treatment in which the adsorbent is packed into a column equipped with a filter, mesh, etc. that does not contain any adsorbent, the column is installed in an extracorporeal circulation circuit, and blood, plasma, etc. are passed through the column. It can be used for any purpose without limitation.

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 so, oo
o, ooo, particle size 50 to 100 mm, manufactured by Toyo Kaitakan) 1
0d, 6ml of saturated NaOH aqueous solution and 15ml of epichlorohydrin! was added and reacted at 50° C. for 2 hours with stirring to obtain an epoxidized gel. To the resulting gel was added 20 cm of concentrated ammonia water and stirred at 50°C for 2 hours to introduce amino groups.

Meanwhile, heparin 200119 was dissolved in 10 d of water and p
After adjusting to H4.5, the above gel 3d into which amine groups were introduced was added. Then, 200!I!g of 1-ethyl-3-(dimethylaminopropylde) was added while keeping the pH at 4.5.
Shake at 4°C for 24 hours. After the reaction was completed, the gel was washed with 2H saline solution, 0 and 5H saline solution, and water in this order to obtain a heparin-immobilized gel (hereinafter referred to as A-1).

Production Example 2 Toyopearl H75, a cross-linked polyacrylate gel
After adding saturated NaOH aqueous solution 6d and epichlorohydrin 15.d to 10d and reacting at 50° C. for 2 hours with stirring, the gel was washed with alcohol and water in this order to obtain an epoxidized gel.

The obtained gel 2d has an intrinsic viscosity of 0.055 dl/(],
Add 0.5 g of dextran sodium sulfate with an average degree of polymerization of 40 and a sulfur content of 19% and 2 m of water (the concentration of dextran sodium sulfate is approximately 13%).

The pH was then adjusted to 12, shaken at 40°C for 16 hours, and the gel was filtered off, followed by a 28% saline solution and 0.5% of the gel. 58 saline solution,
By washing in this order with water, a gel (hereinafter referred to as A-2) on which dextran sodium sulfate was fixed was obtained.

Production Example 3 Toyopearl 814 with heparin immobilized in the same manner as Production Example 2 except that dextran liQM was replaced with heparin.
75 (hereinafter referred to as A-3).

Production Example 4 Porous glass FPG2000 (average particle size: 1950 mm, specific surface area: 13 w), particle size: 80-12 G mesh, manufactured by Wako Saiyaku ■) was heated in dilute nitric acid for 3 hours, washed with water and dried, and then
After heating at 0° C. for 3 hours, the mixture was poured into a 10% toluene solution of γ-aminopropyltriethoxysilane, rapidly poured for 3 hours, and washed with methanol to obtain γ-7 minobrovir-treated glass.

On the other hand, dissolve heparin 200jlj in 10m of water,
'The pH was adjusted to 4.5. After adding 2 g of γ-aminopropyl treated glass to this, 1-ethyl-3,-(
dimethylaminepropyl) carbodiimide 200g II
was added while maintaining the pH at 4.5, and the mixture was shaken at 4°C for 24 hours. After completion of the reaction, add 2d saline solution, 0.5M□. l,
. □Eyu□, Yu1, and porous glass to which heparin was fixed (hereinafter referred to as To1) were obtained.

Production Example 5 Chondroitin polysulfate was immobilized in the same manner as Production Example 4 except that heparin was replaced with chondroitin polysulfate.
pc2ooo (hereinafter referred to as B-2) was obtained.

Production example 6 Dextran sulfur @ 800ay 0.25M Na1
It was dissolved in 10 d of Oa aqueous solution and stirred at room temperature for 4 hours, then 20011 g of ethylene glycol was added and stirred for 1 hour.

After adjusting this solution to pH 8, γ-7 minoprovir-treated FPG 20G04d obtained in the same manner as in Production Example 4.
was added and shaken for 24 hours. After the reaction is complete, filter the gel.
This was washed with water, suspended in 10 d of 1% NaBHt aqueous solution, reduced for 15 minutes, filtered, and washed with water to obtain FPG200G (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.
! After heating and washing with water, the mixture was heated at 500°C for 3 hours. Add this to 10% of γ-glycidoxypropyltrimethoxysilane.
% toluene solution, followed by rapid flow for 3 hours and washing with methanol to obtain γ-glycidoxyprobil-treated glass.

On the other hand, dextran sulfur M2g was dissolved in 10H1 water,
I) After adjusting the temperature to 89.2, 2 g of the above-mentioned γ-glycidoxypyl-treated glass was added and reacted at 45° C. for 16 hours. After the reaction, 2d saline solution, 0.5M
It was washed with a saline solution and water in this order to obtain dextran sulfate-immobilized FPG2000 (hereinafter referred to as 8-4).

Production Example 8 Gel A-3 (exclusion limit molecule matrix so, ooo, ooo, particle size 45-105A5T) in C as a porous cellulose gel
(manufactured by Chisso ■) 10d with 14g of 20% NaOt, 12g of heptane, and nonionic surfactant TWEEN20.
Added 1 drop. After stirring at 40° C. for 2 hours, epichlorohydrin 59 was added and stirred for 2 hours, and the gel was washed with water and filtered to obtain an epoxidized cellulose gel. The introduced epoxy group has a column volume of 11+! i! It was 30 gM per 100 gM.

The resulting gel 2d had an intrinsic viscosity of 0.027 dl/CI,
Dextran sodium sulfate 0.1 with sulfur content 17.7%
27 and 2 m of water were added (the concentration of sodium dextran sulfate was approximately 2.5%), the pH was adjusted to 11, and the mixture was shaken at 45°C for 16 hours. Thereafter, the gel was filtered and washed with a 2d saline solution, a 0.5d 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 Gel A-3 was suction filtered into C, 10 g was taken, and 20
% NaOH49 and 12 g of heptane were added, and one drop of nonionic surfactant TWEEN20 was added to the colander and stirred to disperse the gel. After stirring at 40°C for 2 hours, 5 g of epichlorohydrin was added thereto, and the mixture was stirred at 40°C for 2 hours. The supernatant liquid from the stationary machine was discarded, and the gel was washed with water and filtered to obtain an epoxidized gel. Add 15 m of ammonia water to this and add 4
The mixture was stirred at 0°C for 1.5 hours, and the contents were suction filtered and washed with water to obtain a cellulose gel into which amine groups had been introduced.

On the other hand, heparin 200j19 is 107! dissolved in water,
Add the above amino group-introduced cellulose gel to this and adjust the pH to 4.
, 5. Thereafter, 200q of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide was added to pH 4,
The mixture was added while maintaining the temperature at 5°C, and the mixture was shaken at 4°C for 24 hours. After the reaction, the heparin-immobilized gel (hereinafter referred to as C-
2) was obtained.

Production Example 10 Gel A-3 (hereinafter referred to as C-3) was obtained in the same manner as Production Example 8 except that chondroitin polysulfate was used instead of dextran sulfate.

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 Gels (adsorbents) shown in Table 1 obtained in Production Examples 1 to 11
10 g (wet weight) was placed in a hard glass flask, 10 d of the aqueous solvent shown in Table 1 was added, and the mixture was autoclaved at 121° C. for 40 minutes.

For each adsorbent, the amount of pH1 ligand in the aqueous solvent was measured before and after sterilization. The results are shown in Table 1.

[Margin below]! □ [Effects of the Invention] According to the method of the present invention, even if an adsorbent for extracorporeal circulation therapy is steam sterilized at 121°C for 40 minutes, which is stricter than the usual conditions of 121°C and 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, pH 5 to 9 is required.
A method for sterilizing an adsorbent, the method comprising sterilizing the adsorbent in an aqueous solvent under controlled conditions. 2. The sterilization method according to claim 1, wherein the pH-controlled aqueous solvent is a buffer solution. 3. The sterilization method according to claim 2, wherein the buffer is a buffer containing at least one of citric acid, phosphoric acid, acetic acid, boric acid, tartaric acid, carbonic acid, maleic acid, glycine, and salts thereof.