JPH084503B2 - Method for recovering urokinase and urokinase-containing fraction obtained thereby - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Application The present invention relates to a method for recovering urokinase and a urokinase-containing fraction obtained thereby.

B. BACKGROUND ART Urokinase is an enzyme that is present in tissue culture fluid of human urine and kidney cells, and has a function of activating plasminogen contained in human plasma to produce plasmin having fibrinolytic ability. Therefore, urokinase is collected and purified from human urine and the like, and is widely clinically used for the treatment of various thrombosis, myocardial infarction and the like. In addition, recently, it has been known that the combined use of anticancer agents improves the efficacy of the anticancer agents. Moreover, since urokinase is an extracted and purified product from human urine or tissue culture medium of kidney cells, it does not have antigenicity and therefore has no side effect due to antigen-antibody reaction and is widely used.

By the way, since human urine contains only a very small amount of urokinase, it is difficult to efficiently separate, concentrate and purify urokinase.

For example, in the method of adsorbing and eluting urokinase by contacting conventionally used minerals such as silicate and diatomaceous earth with urine itself, and recovering by various ion exchange chromatography, operability is complicated. However, since the amount of urine that can be treated at one time is limited, there is a problem in that it costs too much to collect a large amount of urokinase, which is a trace component.

C. Problems to be Solved by the Present Invention In concentrated recovery using a normal ultrafiltration membrane, a part of urokinase is incorporated into the membrane and the recovery rate is poor (40-50%).
No purification involved.

That is, the following problems are specifically mentioned.

(1) The membrane has a property of adsorbing a protein (urokinase), and if it is left as it is, the yield is significantly reduced. To increase the yield, pretreatment such as adjusting the pH to 10-11 or adding an adsorption inhibitor is necessary.

(2) As the protein is concentrated, it becomes turbid and causes troubles in the next step. Therefore, it is necessary to add a centrifugation or filtration step to filter the turbidity.

(3) When using raw urine with a lot of turbidity, pretreatment such as sodium hydroxide treatment must be performed to remove turbidity in advance.

(4) When several kinds of proteins are produced from urine, separation of protein molecules having similar molecular weights (such as urokinase and colony formation stimulating factor or kallikrein) is difficult in this step. It will be possible. However, it means that the protein is left in a liquid state for a long time, and it is considered that the protein is deactivated during that time.

Therefore, when a treatment method for efficiently collecting urokinase from urine was examined, it was found that the concentration and purification of urokinase from raw urine can be performed at once by using an alkali-treated polyacrylonitrile-based ultrafiltration membrane. Heading, completed the present invention.

D. Means for Solving Problems The urine (raw urine) as a raw material of the present invention contains urokinase in an amount of about 1 to 10 IU / ml.

The ultrafiltration membrane used as a carrier is a polyacrylonitrile-based
The molecular weight cutoff is preferably about 10,000 to 50,000. Specific examples of trade names that can be used in the present invention will be given below.

Daicel Chemical Industries, Ltd .: DUY-H (basic molecular weight cutoff 1
10,000), DUY-M (20,000 same), DUY-M (40,000 same), PLS-H
A1090 (10,000 same), PLS-MA (20,000 same), PLS-LA1090
(40,000) Millipore: PTGC (10,000), PTTK (30,000) Amicon: H1P10 (10,000), H1P30 (30,000), H1P50
(50,000 same), H1X50 (50,000 same), H5P10 (10,000 same), H5P3
0 (30,000 same), H5P50 (50,000 same), H10P10 (10,000 same), H1
0X50 (50,000 same) Romicon: PM10 (10,000 same), XM50 (50,000 same), etc.

In particular, it is preferable to use a polyacrylonitrile-based ultrafiltration membrane made of a copolymer of acrylonitrile and vinylporolidone.

 This has the following chemical structure.

The ultrafiltration membrane is treated with alkali. The alkali is preferably about 0.1 to 1 N hydroxide (pH about 10 to 13), more preferably sodium hydroxide.

The method of preparation is to filter the ultrafiltration membrane with an alkaline solution.
It is circulated for 10 to 60 minutes and then left to stand for 10 to 20 hours, then washed with cold purified water, and then circulated with raw urine. It is preferable to repeat this several times.

Raw urine is treated using the ultrafiltration membrane thus prepared.

The raw urine is adjusted to pH 6-8, brought into contact with the ultrafiltration membrane, and concentrated by the ultrafiltration membrane. As the condition, it is preferable that the pleated module has an operating pressure of about 1 to 5 kg / cm ² . At this time, urokinase in the original urine is adsorbed.

The concentrated solution has a pH of 6 to 8 and low ionic strength (for example, 0.01 to 0.1).
It is also possible to recover urinary proteins other than UK (for example, colony-stimulating factor, kallikrein, etc.) by extruding under conditions of about M).

After removing the concentrated liquid (unadsorbed liquid), if necessary, the membrane is pH 6-8, low ionic strength (for example, 0.005-0.05M)
After washing under the conditions of pH6 ~ 8, ionic strength 0.05 ~ 0.3
Under conditions of about M, preferably about 0.2 to 0.3 M, while pushing out the urokinase adsorbed to the membrane at the same level as the operating pressure at the time of concentration described above, or to the permeate side by the drop pressure (that is, permeation through the membrane Elution) to obtain a urokinase-containing fraction free from endotoxin and fungi.

The thus-purified eluate, the endotoxin- and bacterium-free fraction containing urokinase, can be desalted, concentrated, and highly purified by a conventional method to give urokinase usable as a pharmaceutical.

E. Effect According to the method of the present invention, (1) no urine treatment is required for urokinase adsorption (2) it can be easily separated from other urinary proteins simply by pushing out the ultrafiltration membrane concentrate (3) the concentration and purification of urokinase It can be done at one time (4) The ultrafiltration membrane concentrate is small enough to be used for multiple purposes such as purification, etc. (5) Turbidity, endotoxin and fungi can be removed (6) As a result, urokinase yield The specific activity is improved and the manufacturing time can be shortened.

And so on.

Therefore, it is possible to purify a urokinase-containing fraction in which endotoxin and fungi have been removed from urine containing only a trace amount of urokinase and containing a large amount of impurities by a simple operation, with high efficiency, high purity, and low cost.

F. Experimental Examples / Examples Experimental examples and examples are given to describe the present invention in more detail, but the present invention is not limited thereto.

Experimental Example The viable cell count and the presence / absence of endotoxin in the urokinase fractions collected by the method of the present invention and the conventional method were compared (Table 1).

 The method of the present invention was carried out according to the examples.

In the conventional method, in Example, urokinase adsorbed on the membrane was eluted to the concentration side without performing an extrusion operation (that is, without permeating the membrane).

The viable cell count was determined by using the agar medium (made by Nissui or Eiken) and measuring the endotoxin by the Limulus reaction according to the JIS standard method.

Example 1 The urine was adjusted to pH 6.0, and the pleated polyacrylonitrile-based ultrafiltration membrane prepared according to the reference example (fraction molecular weight 2
After being concentrated using Daicel Co., Ltd., the concentrate was removed by extruding with 0.01M phosphate buffer (pH 7.0). After washing the membrane with 0.01 M phosphate buffer (pH 7.0), urokinase was eluted to the permeate side with 0.25 M ammonium sulfate at a head pressure. The values shown in Table 2 were obtained.

Reference Example 0.5N sodium hydroxide was circulated for 30 minutes in a pleated polyacrylonitrile-based ultrafiltration membrane (fraction molecular weight 20,000, manufactured by Daicel Corp.), then allowed to stand for 10 hours, washed with cold purified water, Circulate raw urine. This was repeated 5 times.

Claims (3)

[Claims] 1. An alkaline-treated polyacrylonitrile-based ultrafiltration membrane is brought into contact with raw urine and concentrated by ultrafiltration to adsorb urokinase in urine onto the membrane, and then the concentrated solution (unadsorbed solution) is removed. A method for recovering urokinase, which comprises removing and then eluting the urokinase adsorbed on the membrane through the membrane. 2. A polyacrylonitrile-based ultrafiltration membrane that has been treated with alkali is brought into contact with raw urine and concentrated by ultrafiltration to adsorb urokinase in urine onto the membrane, and then the concentrated solution (unadsorbed solution) is removed. A urokinase-containing fraction obtained by removing and then eluting the urokinase adsorbed on the membrane through the membrane. 3. The urokinase-containing fraction according to claim 2, from which endotoxin and fungi have been removed.