JPS625599B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for preserving immobilized urokinase.

It is well known that immobilized urokinase has fibrinolytic ability and that filaments, tubes, etc. containing them are excellent antithrombotic materials [Sugitate et al., History of Medicine, Vol. 101]
Vol. 114 (1977) Oshiro et al., Artificial Organs, No. 6
Volume, 237 pages (1977)]. After producing immobilized urokinase, it is desired to store it stably and easily for a relatively long period of time until it is used as a medical material such as a vascular catheter, drainage tube, or suture thread. Immobilized enzymes are typically stored in water at low temperatures, but this storage format is not advantageous from the standpoint of packaging, transportation, storage, etc. For example, when immobilized urokinase is stored in water, special storage containers and low-temperature equipment are required, and there is a risk of water leakage during transportation and storage.

In view of the above-mentioned problems, the present inventors have investigated various ways to store immobilized urokinase in a simple and stable manner, and have surprisingly found that immobilized urokinase can be stored for a long period of time in a gas atmosphere or in a vacuum state. However, we have discovered that the decrease in activity is even smaller than when stored in water, and have arrived at the present invention.

That is, the present invention is a method for preserving an immobilized enzyme, which is characterized in that the immobilized urokinase is stored in a gas atmosphere or in a vacuum.

The immobilized urokinase in the present invention refers to urokinase immobilized on a carrier having a shape such as powder, beads, gel, tube, staple, filament, nonwoven fabric, sponge, woven fabric, knitted fabric, film, sheet, membrane, hollow fiber, etc. It refers to Urokinase contributes to fibrin dissolution by acting as a plasminogen activator. The materials that make up the carrier include:
For example, cellulose, starch, dextran,
Natural polymeric substances such as agarose, collagen, silk, and wool, polyester, polyamide, polyvinyl alcohol, polyamino acids, polyacrylamide, polystyrene, poly(meth)acrylic acid, poly(meth)acrylic acid ester, polyacrylonitrile, polyurethane silicone, Examples include synthetic polymer substances such as polyethyleneimine, polymaleic anhydride, polyethylene, polypropylene, and polyvinyl chloride, and inorganic substances such as activated carbon, glass alumina, silica gel, and kaolinite. Urokinase can be immobilized on these carriers by known methods such as covalent bonding, ionic bonding, and physical adsorption. In the case of covalent bonding, carboxyl groups, amino groups, chloroformyl groups, diazonium groups, azide groups, epoxy groups, formyl groups, bromoacetyl groups, isocyanate groups, carboxylic acid anhydride groups, imide groups, carbonate groups, etc. It is necessary to use a carrier that has a reactive functional group, and in the case of an ion bonding method, it is necessary to use a carrier that has an ion exchange group such as a carboxylate group, sulfonate group, ammonium group, sulfonium group, or phosphonium group. . A carrier having these reactive functional groups or ion exchange groups can be produced, for example, by the following method.

(1) A polymeric substance obtained by homopolymerizing or copolymerizing a monomer having a reactive functional group or an ion exchange group is molded into an appropriate shape.

(2) After introducing a reactive functional group or ion exchange group into a synthetic polymer material, natural polymer material, or inorganic material, the material is molded into an appropriate shape.

(3) Introducing reactive functional groups or ion exchange groups into the shaped carrier.

Immobilization of urokinase onto these carriers is carried out by bringing the carrier into contact with an aqueous urokinase solution. At this time, in order to renew the surface of the carrier as necessary, it is preferable to add operations such as stirring, shaking, and circulation. The obtained immobilized urokinase or immobilized streptokinase is washed by immersion in water or the like.

In the present invention, immobilized urokinase is stored in a gas atmosphere or in vacuum. When storing immobilized urokinase in a gas atmosphere or in vacuum, it is most convenient to remove the immobilized urokinase from water and store it in a storage container while still absorbing water. However, if necessary, the immobilized urokinase or streptokinase that has absorbed water may be air-dried, dried using a desiccant, dried under reduced pressure, or heated to remove water. It can also be stored.

Examples of the gas used in the present invention include inert gases such as nitrogen, helium, and argon, or air. The pressure of the gas is not particularly limited, but is preferably 2 atmospheres or less.

Vacuum as used in the present invention refers to a state in which gas is substantially excluded. The simplest method for storing immobilized urokinase in a vacuum is to use a plastic film bag or the like and seal the immobilized urokinase under vacuum. Examples of containers used to store immobilized urokinase include plastic/film bags made of cellophane, polyethylene, polypropylene, nylon, and polyester, paper bags,
Examples include metal containers such as glass containers and cans. The storage temperature of immobilized urokinase is -20 to 60℃.
℃, particularly preferably in the range of 0 to 40℃. In particular, the preservation method of the present invention is extremely advantageous in that immobilized urokinase can be preserved at temperatures around room temperature.

When immobilized urokinase is stored for a long period of time according to the method of the present invention, it has the advantage that the decrease in activity is extremely small compared to the conventional storage method of immersing it in water. Furthermore, the preservation method of the present invention includes:
Special equipment is used for packaging, transportation, storage, etc.
It is economically advantageous in that it does not require complicated operations.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples.

In addition, the activity measurement of immobilized urokinase in the example is based on the 27th revised edition of "Clinical Test Methods Summary" edited by Kanai and Kanai.
The test was carried out in the following manner using a fibrin plate prepared by adding a physiological saline solution of thrombin to an aqueous fibrinogen solution, with reference to version M-100 (Kanehara Publishing). That is, a monofilament with immobilized urokinase is placed on a fibrin plate,
After being left at 37°C for 24 hours, the area of the dissolved fibrin membrane was taken as the activity titer. Then, the activity titer of the immobilized urokinase after storage for a predetermined period was divided by the activity titer before storage, and the value was calculated and expressed as an activity residual rate.

Example 1, Comparative Example 1 Nylon 6 tubes having an inner diameter of 3 mm and an outer diameter of 5 mm were cut into rounds of 2 mm thick and processed as follows.

(1) Immersed in 3N-HCl aqueous solution, stirred well at 30℃, and washed with water.

(2) Then, 2WT% polyethyleneimine and
It was immersed in a methanol solution containing 2WT% dicyclohexylcarbodiimide, stirred well at room temperature, washed with methanol, and dried.

(3) Then, immersed in an acetone solution containing 4WT% maleic anhydride-methyl vinyl ether copolymer and 0.8WT% nitrophthalic anhydride,
After stirring well at room temperature, the mixture was washed with acetone and dried.

(4) Next, a physiological saline solution of urokinase (600
Units/ml) and left at 7°C for 24 hours, followed by washing with physiological saline.

After wiping off the water adhering to the obtained immobilized urokinase (a ring section of nylon 6 on which urokinase was immobilized by a covalent bond) with a filter paper,
Immobilized urokinase was placed in glass reagent bottles and stored separately at 7°C and 25°C.
The activity retention rate after 3 months is 103% at 7℃,
The rate was 94% at 25°C.

For comparison, when stored in physiological saline at 7°C and 25°C for 3 months, the activity retention rates were 72% and 48%, respectively.

Example 2 The immobilized urokinase obtained in Example 1 was dried under reduced pressure at room temperature for 24 hours, and then stored separately at 7°C and 25°C. The activity retention rate after 3 months was 98% at 7°C and 97% at 25°C.

Example 3 Urokinase was immobilized in the same manner as in Example 1, except that a nylon 6 monofilament (diameter 0.2 mm) was used instead of the nylon 6 ring section.

The obtained immobilized urokinase (nylon 6 monofilament on which urokinase was covalently immobilized) was placed in a bag made of nylon 6/polyethylene laminate film and sealed under vacuum using a vacuum packaging machine. When vacuum-packaged immobilized urokinase was stored at 25°C for 3 months, its activity retention rate was 95%.

Comparative Example 2 A ring section of nylon 6 treated in steps (1) to (3) in the same manner as in Example 1 was immersed in a physiological saline solution of streptokinase (600 units/ml) for 24 hours.
After being left at ℃, it was washed with physiological saline. After wiping off the water adhering to the obtained immobilized streptokinase (a ring section of nylon 6 on which streptokinase has been covalently immobilized) with a filter paper, the immobilized streptokinase was placed in a glass reagent bottle. Stored separately at 7°C and 25°C. The activity retention rate after 3 months was 99% at 7°C and 93% at 25°C. Furthermore, when the same immobilized streptokinase was stored in physiological saline at 7°C and 25°C for 3 months, the activity retention rates were 80% and 62%, respectively.

Claims (1)

[Claims] 1. A method for preserving an immobilized enzyme, which comprises storing the immobilized urokinase in a gas atmosphere or in a vacuum.