JPS59184134A - Production of carrier for immobilizing physiologically active substance - Google Patents

Abstract

PURPOSE:To obtain the titled carrier without deteriorating nor inactivating by wet heat sterilization, by immobilizing a physiologically active substance on a porous carrier having a defined linear expansion coefficient, Young's modulus and a constituent material within specific ranges, and sterilizing the resultant immobilized substance in wet heat. CONSTITUTION:A physiologically active substance is immobilized on a porous carrier having <=3X10<-5>deg<-1> at room temperature, >=1X10<11>dynes cm<-2> Young's modulus at room temperature and <=20% absorption ratio of the constituent material, and then sterilized in wet heat to give the aimed carrier. A metal, glass or alumina, etc. is used as the above-mentioned constituent material and has preferably >=1,000Angstrom average pore diameter and >=0.3mlg<-1> pore volume and chemically bondable active groups in >=1X10<-8>mol m<-2> concentration on the surface of the porous carrier. The physiologically active substance is immobilized through the above-mentioned active groups. Thus, the deformation of the carrier by the sterilization in wet heat is suppressed under the above-mentioned conditions.

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 refer to physiologically active substance immobilization carriers such as tissues, cells, intracellular tissues, enzyme antigens, antibodies, antigen-antibody complexes, complements, proteins, polysaccharides, nucleic acids, lipids, and complexes thereof. An active substance is immobilized on the surface of an insoluble carrier, and a mesovascular reaction specific to the physiologically active substance is caused to occur on the carrier.

Conventionally, agarose and various physiologically active substances immobilized on agarose 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. used>,6
, it is rarely applied for medical purposes that require sterilization or for the production of medical preparations.

The present inventors have conducted intensive research 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 physiologically active 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 has a linear expansion coefficient of 3 x 10 'deg'' or less at room temperature, and a Young's modulus of I x 1011 dyne at room temperature.
A method for producing a physiologically active substance-immobilized carrier, which comprises immobilizing a physiologically active substance on a porous carrier having a water absorption rate of s cm -2 or more and a constituent material having a water absorption rate of 20 cm or less, followed by heat sterilization. be.

Materials for forming the porous body that satisfies the above conditions used in the present invention include metals, glass, inorganic materials such as alumina, and silica;
and organisms having a glass transition temperature (hereinafter abbreviated as Tg) higher than the moist heat sterilization temperature, and complexes thereof. dyne whose coefficient of linear expansion exceeds 3 x 0-'deg or whose Young's modulus is IX I O'dyne
less than s cm-2, or the water absorption rate of the constituent materials is 20%
Under moist heat sterilization conditions, carriers that absorb water, swell and deform, are deformed by pressure, and are not suitable for this purpose. For example, agarose, which has traditionally been used for similar purposes, swells due to water absorption, and the immobilized physiologically active substances are deactivated because the agarose itself has a small Young's modulus and deforms under moist heat sterilization conditions. Generally, the physical properties of an organism greatly change 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, porous carriers can effectively immobilize physiologically active substances,
In addition, in order to facilitate contact between the immobilized physiologically active substance and the substance that reacts with a certain amount, the average pore diameter is 1.
00OX or more and the pore volume is 0.3 ml? −”
It is desirable that the porous body has the above-mentioned properties.

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. The shape of the physiologically active substance immobilized carrier is as follows:
It is preferable to use particles, fibers, sheets, tubular bodies, etc. depending on the type and purpose. In particular, those having a particle form of 0.05 to 5 m are preferred.

There are 1X10'mol of active groups on the surface of the carrier that can be chemically bonded.
Preferably, it is present at a concentration of em72 or higher.

If the active group concentration is less than 1 x 10' 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. Examples of surface treatment methods include a method of introducing a diamino group or a carboxyl group using a silane coupling agent such as agunoglobiltriethoxysilane, or the example disclosed in %Kokai No. 150433/1983. Examples of physiologically active substances include tissues, cells, intracellular tissues, antigens, antibodies, enzymes, antigen-antibody complexes, complements, etc. Includes proteins, polysaccharides, nucleic acids, lipids, and complexes thereof.

Methods for immobilizing a physiologically active substance on a carrier include a method in which an epoxy group or an aldehyde group on the carrier is directly bonded to an amino group of the physiologically active substance, or a method in which a carboxyl group or an amine group on the carrier and a physiologically active substance are bonded together. - A method of bonding with a dehydration condensation agent such as ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), a method in which the carboxyl group of the carrier is replaced with an active ester such as N-hydroxysuccinimide, and this has physiological activity. Examples include methods of substituting substances. Furthermore, the carrier and the physiologically active substance can be immobilized via a low molecular weight group such as t-aminocaproic acid, diaminoheptane, or glutaraldehyde.

The physiologically active substance-immobilized carrier prepared in this way is generally heated at 121°C for 20 minutes as specified in the Japanese Pharmacopoeia, but in some cases it may be heated at 115°C for 30 minutes or even heated at 126°C for 15 minutes. Heating takes place for a minute. 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, the physiologically active substance may be selected appropriately depending on the disease to be treated.

For example, autoantibodies or immune complexes are the causative agents of autoimmune diseases such as autoimmune hemolytic anemia, glomerulonephritis, rheumatoid arthritis, and systemic lupus erythematosus.
These need to be removed from the blood. In order to remove these autoantibodies or immune complexes, 5 taphylococcus au
Protein A1 produced by certain strains of S. reus FC receptors, complement C1 components, anti-immunoglobulin antibodies, etc. present in the cell walls of lymphocytes and platelets are immobilized on a carrier and 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 that suppress the immune action against cancer cells are present in the blood, and these immunosuppressive factors are found in the antibody fraction and antigens in the 10,000 to 100,000 range. Antibodies against these antigens, protein A1 cell wall F
c receptor, anti-immunoglobulin antibodies are immobilized and used as a selective adsorbent for therapy. The second use is as an adsorbent for Affinichii Kumamatog 2 fee for the purpose of separating or synthesizing pharmaceuticals such as proteins, nucleic acids, polysaccharides, hormones, and tamins, or raw materials for medical preparations, or immobilization. Examples include enzymes. 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, the physiologically active substance to be immobilized is selected and used depending on the target substance to be separated and purified or synthesized.

The present invention will be explained in more detail below using Examples.

(Example) Porous glass with an average pore diameter of 1c + soL pore volume of 0.79jg-1 (average particle size of 0.15M, linear expansion coefficient of 2X
10'deg', -? / Doug rate 7 x 10” dyn
es Tsuba-2, water absorption rate 1% or less)? Amino groups were introduced by reacting with 3-7minoprobyltriethoxysilane in toluene (amino group concentration 8.4 x 10-"m).
Next, an anti-human IgG antibody (Miles M) was immobilized by dehydration condensation using KDC in a buffer solution, and then sterilized with moist heat at 121°C for 20 minutes to obtain the biological activity of the present invention. A substance-immobilized carrier (Example 1) was obtained.

Anti-human I
Comparative Example 1 was prepared by bonding glycine ether ester to the gG antibody.

Agarose activated with CNBr (manufactured by Pharmacia,
Product name CNBr-activated 5epbaro
Comparative Example 2 was prepared by binding the same anti-human IgG antibody as in Example 1 to se 4 B (water absorption rate of 100% or more).

Example 1 and Comparative Examples 1 and 2 before and after thermal sterilization (121°C, 20ml), 10d of human IgG solution (# degree I W mf'', 7.4 phosphate buffer for each 12 (dry weight)) ) was added and shaken at 37°C for 3 hours.

The concentration of human flies after 3 hours was calculated from the absorbance of 280 seaweed, and the amount of adsorption was calculated from the amount of decrease in concentration. The results are in Table-1
As shown in Example 1, the amount of adsorption hardly changed before and after moist heat sterilization, 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 antibody base on the same carrier as in Example 1, IgG
It was confirmed that the adsorption of Iρ in Example 1 was not non-specific but specific binding by the antibody. Furthermore, 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, so it did not adsorb human IgG at all after sterilization.

Table-1 Human fly adsorption: 1l (q/f carrier) Example 1 0.93 0.87 Comparative example】　　　　　　　- Comparative example 2 5.0 0

Claims (1)

[Claims] 1. The coefficient of linear expansion at room temperature is 3 x 10-'deg.
``Hereinafter, Young's modulus at room temperature is 1 x 1011d
ynesc*”-” or more, and the water absorption rate of the constituent material is 2
1. A method for producing a physiologically active substance-immobilized carrier, which comprises immobilizing a physiologically active substance on a 0- or less porous carrier and then sterilizing it with moist heat. 2. The constituent material of the porous carrier is metal, glass, alumina,
silica or glass transition temperature higher than the moist heat sterilization temperature (
The method for producing a physiologically active substance-immobilized carrier according to claim 1, which is made of an organism having Tg ) or a complex thereof. 3. The physiologically active substance according to claim 1 or 2, wherein the porous carrier has an average pore diameter of 1000λ or more and a pore volume of 0.3 mg or more. Method for producing immobilization carrier. 4. 1 x active group capable of chemical bonding on the surface of the porous carrier
4. A method for producing a physiologically active substance-immobilized carrier according to claims 1 to 3, characterized in that the physiologically active substance is transferred via an active group with an O concentration of 10-"molem-" or more. 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.