JPH0549651B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a sterilized physiologically active substance-immobilized carrier that can be effectively used in the production of medical products or medical preparations. Physiologically active substance immobilized carriers are biologically active substances such as tissues, cells, intracellular tissues, enzymes, antigens, antibodies, antigen-antibody complexes, complements, proteins, polysaccharides, nucleic acids, lipids, and complexes thereof. A substance is immobilized on the surface of an insoluble carrier, and biological reactions specific to the physiologically active substance are caused to occur on the carrier. BACKGROUND ART Conventionally, agarose and agarose with various physiologically active substances immobilized thereon have been widely used for separation and analysis in order to carry out biological reactions specific to physiologically active substances. However, these methods are limited to cases where sterilization is not necessary because they are inactivated under general ethylene oxide gas sterilization and moist heat sterilization conditions, unless the immobilized physiologically active substance inherently has good heat resistance. It is used as
It is rarely applied for medical purposes that require sterilization or for the production of medical preparations. The present inventors have conducted extensive research in order to provide a method for producing a physiologically active substance-immobilized carrier that can be used for medical purposes or in the production of medical preparations. The present invention was achieved based on the discovery that in order to impart sterilization resistance to a substance, it is necessary that at least the carrier on which the physiologically active substance is immobilized does not deform under moist heat sterilization conditions. That is, the present invention
Linear expansion coefficient at room temperature is 3×10 ^-5 deg ^-1 or less,
A bioactive substance characterized by immobilizing a bioactive substance on a porous carrier having a Young's modulus of 1×10 ¹¹ dynescm ^-2 or more at room temperature and a constituent material having a water absorption rate of 20% or less, followed by moist heat sterilization. This is a method for producing a substance-immobilized carrier. The constituent materials of the porous body that satisfy the above conditions used in the present invention include metals, glass, inorganic substances such as alumina, and silica, and organic substances having a glass transition temperature (hereinafter abbreviated as Tg) higher than the moist heat sterilization temperature; Examples include these complexes. Linear expansion coefficient exceeds 3×10 ^-5 deg ^-1 or Young's modulus is 1
A carrier with a water absorption rate of less than ×10 ¹¹ dynescm ^-2 or a constituent material with a water absorption rate of more than 20% is not suitable for this purpose because it absorbs water and swells and deforms under moist heat sterilization conditions, or deforms due to temperature and pressure. For example, agarose, which has been conventionally used for similar purposes, swells when it absorbs water, and because agarose itself has a small Young's modulus and deforms under moist heat sterilization conditions, immobilized physiologically active substances are deactivated. Generally, the physical properties of organisms change significantly around the Tg, so even if the conditions of the present invention are satisfied at room temperature, the effects of the present invention will not be satisfactorily exhibited if the Tg is lower than the moist heat sterilization temperature. In addition, in order to effectively immobilize a physiologically active substance and facilitate contact between the immobilized physiologically active substance and a substance that reacts with the porous carrier,
The average pore diameter is 1000Å or more and the pore volume is
It is desirable that the porous material is 0.3 mlg ^-1 or more.
Porous materials that satisfy these conditions include porous glass, porous silica/alumina, porous alumina, porous metal, etc. Among these, porous glass has particularly good physical strength and pore size. It is most preferable in that it is easy to obtain a uniform product. Appropriate shapes of the physiologically active substance immobilized carrier include particles, fibers, sheets, tubular bodies, etc., and it is preferable to use the appropriate shape depending on the purpose. In particular, those having a particulate form of 0.05 to 5 mm are preferred. There are 1× active groups on the surface of the carrier that can be chemically bonded.
Preferably, it is present at a concentration of 10 ^-8 molem ^-2 or higher. If the active group concentration is less than 1×10 ⁻⁸ molem ⁻² , the physiologically active substance will not be effectively immobilized and sterilization resistance will deteriorate, which is not preferable. The active groups that can be chemically bonded here include amino groups, carboxyl groups, aldehyde groups, epoxy groups, etc., and are substituents that can form covalent bonds with physiologically active substances such as proteins, polysaccharides, and nucleic acids. be. These may be present naturally in the above-mentioned carriers or may be introduced onto the carrier surface by surface treatment. As a surface treatment method, a method of introducing an amino group or a carboxyl group using a silane coupling agent such as aminopropyltriethoxysilane, or a method of introducing an amino group or a carboxyl group using a silane coupling agent such as aminopropyltriethoxysilane, or
As typified by the example disclosed in No. 150433, there is a method of coating the surface with a polymer having a carboxyl group or an amino group. Physiologically active substances include tissues, cells, intracellular tissues, antigens, antibodies, enzymes, antigen-antibody complexes, proteins such as complements,
Examples include polysaccharides, nucleic acids, lipids, and complexes thereof. Methods for immobilizing a physiologically active substance on a carrier include a method in which the epoxy group or aldehyde group of the carrier and the amino group of the physiologically active substance are directly bonded;
Alternatively, a method in which the carboxyl group or amino group on the carrier and the physiologically active substance are bonded using a dehydration condensation agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC); Examples include a method in which an active ester such as -hydroxysuccinimide is substituted with a physiologically active substance. Furthermore, the carrier and the physiologically active substance can be immobilized via a low molecular weight group such as ε-aminocaproic acid, diaminoheptane, or glutaraldehyde. The physiologically active substance immobilized carrier prepared in this way is generally used as specified in the Japanese Pharmacopoeia.
Heating is carried out at 121℃ for 20 minutes, but in some cases
Heating is performed at 115°C for 30 minutes or at 126°C for 15 minutes. The above-mentioned physiologically active substance immobilized carrier does not become deactivated even in the above-mentioned high-temperature, high-pressure steam atmosphere. The first use of the moist heat sterilized physiologically active substance immobilized carrier is as a therapeutic selective adsorbent. In this case, an appropriate physiologically active substance may be selected depending on the disease to be treated. For example, autoimmune hemolytic anemia, glomerulonephritis,
In autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, autoantibodies or immune complexes are the causative agents of the disease, and it is therefore necessary to remove them from the blood. In order to remove these autoantibodies or immune complexes, protein A produced by certain strains of Staphylococcus aureus that specifically bind to them, Fc receptors present in the cell walls of lymphocytes and platelets, and complement are used. A _C1 component, anti-immunoglobulin antibody, etc. immobilized on a carrier is used as a selective adsorbent for treatment. Furthermore, in patients with renal failure, urea in the blood accumulates without being metabolized, so a carrier on which urease, an enzyme that decomposes urea, is immobilized can also be used as a therapeutic immobilized enzyme. Furthermore, in cancer patients, it has been proven that several factors exist in the blood that suppress the immune action against cancer cells. Antibodies against these antigens, protein A, cell wall Fc receptors, and anti-immunoglobulin antibodies are immobilized and used as therapeutic selective adsorbents. The second use is as an adsorbent for affinity chromatography or immobilized enzymes for the purpose of separating and purifying or synthesizing pharmaceuticals such as proteins, nucleic acids, polysaccharides, hormones, and vitamins, or raw materials for medical preparations. It will be done. Since pharmaceuticals or medical preparations must be kept in a sterile state, the physiologically active substance-immobilized carrier of the present invention is sterile and can be effectively used for this purpose. In this application as well, separation and purification,
Alternatively, a physiologically active substance to be immobilized is selected and used depending on the target substance to be synthesized. The present invention will be explained in more detail with reference to Examples below. (Example) Porous glass with an average pore diameter of 1950 Å and a pore volume of 0.79 mlg ^-1 (average particle diameter of 0.15 mm, linear expansion coefficient of approximately 2 ×
10 ^-4 deg ^-1 , Young's modulus of about 7 x 10 ¹¹ dynescm ^-2 , water absorption of 1% or less) was reacted with 3-aminopropyltriethoxysilane in toluene to introduce amino groups (amino group concentration 8.4 x 10 ^-8 molem ^-2 ). Next, anti-human IgG antibody (manufactured by Miles) was added to EDC in a buffer solution.
After dehydration condensation and immobilization using
A physiologically active substance immobilized carrier of the present invention (Example 1) was obtained by performing moist heat sterilization for 20 minutes. In a similar manner to the same porous glass as in Example 1,
Comparative Example 1 was prepared by binding glycine ethyl ester instead of the anti-human IgG antibody. Agarose activated with CNBr (manufactured by Pharmacia, trade name CNBr-activated Sepbarose 4B,
(water absorption rate of 100% or more) and the same anti-human IgG as in Example 1.
Comparative Example 2 was obtained by binding the antibody. Example 1 before and after moist heat sterilization (121℃, 20min),
Comparative Examples 1 and 2, each for 1 g (dry weight)
10 ml of human IgG solution (concentration 1 mg ml ^-1 , PH7.4 phosphate buffer) was added, and the mixture was shaken at 37°C for 3 hours. Determine the human IgG concentration after 3 hours from the absorbance at 280 mm,
The amount of adsorption was calculated from the amount of decrease in concentration. The results are in the table-
As shown in Example 1, the amount of adsorption hardly changed before and after moist heat sterilization in Example 1, and it is clear that the physiologically active substance-immobilized carrier of the present invention is effective in a sterilized state. On the other hand, in Comparative Example 1, in which glycine ethyl ester was immobilized on the same carrier as in Example 1 instead of the antibody, human IgG was not adsorbed at all.
It was confirmed that the adsorption of human IgG in Example 1 was not nonspecific but specific binding by the antibody. In Comparative Example 2, the carrier did not satisfy the conditions of the present invention, and the immobilized antibody was denatured and inactivated by moist heat sterilization.
It did not adsorb at all. 【table】

Claims (1)

[Claims] 1. The linear expansion coefficient at room temperature is 3×10 ^-5 deg ^-1 or less, and the Young's modulus at room temperature is 1×10 ¹¹ dynescm ^-2
A method for producing a physiologically active substance-immobilized carrier, which is characterized in that the physiologically active substance is immobilized on a porous carrier having the above structure and whose constituent materials have a water absorption rate of 20% or less, and then sterilized with moist heat. 2. The physiological material according to claim 1, wherein the porous carrier is made of metal, glass, alumina, silica, an organism having a glass transition temperature (Tg) higher than the moist heat sterilization temperature, or a composite thereof. A method for producing an active substance immobilized carrier. 3 The porous carrier has an average pore diameter of 1000 Å or more,
The method for producing a physiologically active substance-immobilized carrier according to claim 1 or 2, which is a porous carrier having a pore volume of 0.3 mlg ^-1 or more. 4 1 active group capable of chemical bonding on the surface of the porous carrier
4. The method for producing a physiologically active substance-immobilized carrier according to claims 1 to 3, wherein the carrier has a concentration of ×10 ^-8 molem ^-2 or more, and the physiologically active substance is immobilized via the active group. 5. The method for producing a physiologically active substance-immobilized carrier according to claim 4, wherein the active group capable of chemical bonding is one or more selected from the group consisting of an amino group, a carboxyl group, an aldehyde group, and an epoxy group.