JPS6040834B2 - Production method of urokinase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing urokinase, which comprises removing contaminating coloring components (pigments) from crude urokinase without reducing the titer of urokinase, and recovering urokinase.

Urokinase is an enzyme that is present in large amounts in human urine, and has the function of activating plasminogen contained in serum to produce blasmin, which has fibrinolytic ability.

Therefore, since urokinase is effective as an activator of the fibrinolytic system, it has been isolated and purified from human urine or tissue culture fluid, and is widely used clinically for the treatment of various thromboses. Methods for producing urokinase from an aqueous solution containing urokinase, such as urine, typically involve two main steps: concentration and purification and recovery.

Known methods for concentration include {1' cooling precipitation method, ■ adsorption method using barium sulfate, adsorption method using glue silica gel (Special Publication No. 7 of 1973), and '4} adsorption method using a cation exchanger (
Japanese Patent Publication No. 40-12783) and [5] Adsorption method using acrylonitrile (Japanese Patent Publication No. 48-10232) have been carried out. However, no matter which method is used, colored components in urine are adsorbed during concentration and are concentrated together with urokinase, resulting in the collection of inferior urokinase. Chromatography is generally used to further separate and purify the concentrated aqueous solution containing urokinase to recover urokinase, and chromatography is commonly used to recover urokinase. A/Gtite DEAE-Cellulose (Special Publication 1976-1980)
No. 64) etc. are used. By combining these methods, highly purified urokinase can be recovered in sufficient yield, and although these methods have been devised to increase specific activity and remove pyrogenic substances, It has not been sufficiently effective in removing pigments such as alkaloids. Literature has already been published examining methods for removing this pigment, for example, it has been reported that adsorption and removal using an anion exchange resin is effective to some extent (Kosho Hakama, 40).
No. 127830), but even this was not sufficient in terms of enhancing the purification effect of dependent processes. The present inventors conducted various studies on the selection and fractionation of carriers used in the manufacturing process for the purpose of effectively removing colored components such as pigments contained in crude urokinase.

As a result, we decided to incorporate an anion exchanger into the intermediate process between the concentration process and the recovery process, considering its relationship with the concentration process, which is currently being industrialized as a manufacturing method for urokinase, the purification and recovery process, and the effect of removing colored components. He discovered that a group of resins from among the hundreds of anionic exchange resins currently in use were extremely effective at adsorbing colored components in urine. . Next, as a result of examining the processing conditions of the selected anion exchange resin, it became possible to add it to the concentrated liquid before purification and recovery as an intermediate step.
Based on these new findings, we have completed the present invention, which aims to remove colored components. In the present invention, a strongly basic anion exchange resin having quaternary ammonium as an exchange group and a polystyrene/divinylbenzene copolymer as a base is equilibrated with a balanced solution of pH 7 to 9, and brought into contact with a crude urokinase aqueous solution. The aqueous solution containing unadsorbed urokinase is recovered, and the colored components in the crude urokinase aqueous solution are removed by adsorption onto the anion exchange resin.

The crude urokinase solution used in the present invention is not particularly limited, but is obtained using a known urokinase adsorbent such as silica gel, acrylonitrile, or barium sulfate, and has a specific activity of 100 to 10,001 U/. Aqueous solutions containing urokinase are suitable.

The strongly basic anion exchange resin used has quaternary ammonium as the exchange group and polystyrene/divinylbenzene copolymer as the base, and selected commercial products include Dowexll (manufactured by Dow Chemical Company) and Diaon SA.
The strongest basic anion exchange resin of 20 to 100 mesh of the Diaon PA series (manufactured by Mitsubishi Chemical Industries, Ltd.) is preferred. Equilibration for the assisted contact of the anionic resin with the aqueous solution containing urokinase is carried out by adjusting the pH using a suitable low-salt buffer (e.g. 0.1M phosphate buffer).
It is necessary to adjust the pH to 7 to 9, and the more preferable pH is 7.
．． It is 5 to 8.5. It is sufficient that the amount of anion exchange resin used is about 20 to 100 urokinase per 10 ratio c of an aqueous solution containing about 10,001 U' of urokinase. Both contacts may be simply mixed, or the urokinase fraction solution may be passed through a column packed with anionic exchanger 4-polar resin. Urokinase remains in the aqueous solution without being adsorbed by the anion exchange resin, and colored components such as dyes are adsorbed by the anion exchange resin, so the colored components are removed together with the resin. The contact conditions are not particularly limited, but the appropriate temperature is 3 to 20°C and the time is 500°C for about 1 hour. In order to sufficiently recover urokinase, the urokinase aqueous solution after contact is purified with the same buffer as the one used for the above-mentioned equilibration, using a buffer with an amount of about 1.5 to 3.3 times the amount of resin used for contact. The aqueous urokinase solution that has been treated to remove colored components in this manner is highly purified using a known cation exchanger or other adsorbent to produce a urokinase preparation that is safe for medical use. The effect of removing colored components in the present invention is extremely effective, as more than 80% of the colored components are removed when expressed as a ratio of absorbance at the absorption wavelength 40 rule m, as shown in the experimental example. The recovery rate is also about 93 to 100%, and since the process is simple and the processing time is short, it can be easily added as an intermediate process.

EXAMPLES The present invention will be specifically explained below with reference to Examples, and the effects of the present invention will be demonstrated through experimental examples, but the present invention is not limited to these Examples.

In addition, IU is Pharmaceutical Research Vol. 3}, 295-308 (
It is an international unit described in 1974). Example 1 Dowex-11100 (manufactured by Dow Chemical Company) was suspended in distilled water, fine particles were removed by a decant method, and the suspension was packed into a glass column with an inner diameter of 20 so as to have a height of 35 ribs.

Next, a caustic soda solution of IN was flowed down in an amount 5 times the amount of the resin,
The column was washed with 5 times or more of distilled water, followed by 5 times of IN hydrochloric acid solution, and in the same way with 5 times or more of distilled water. After repeating this operation three times, a phosphate buffer solution with a pH of 8.0 and 0.1 M was flowed down a single volume of the resin to equilibrate it. Crude urokinase aqueous solution collected and concentrated from urine (activity 405a)
U' skin (9) with a specific activity of 3721 U/ was passed through the column, and the above phosphate buffer solution was allowed to flow down twice as much as the resin.

Comparing the urokinase activity, protein content (absorbance at 28 m), and degree of coloration (absorbance at 40 m) of the passed-through liquid, we found that the residual titer was 100% and the impurity protein removal rate was 2.
0.4%, and the coloring component removal rate reached 82.1%. The specific activity at this time was 2 times higher than that of the solution before passage. Example 2 Diaon SA-11A5 manufactured by Mitsubishi Chemical Industries, Ltd.
After suspending the suspension in distilled water and removing fine particles by decantation, it was packed into a glass column with an inner diameter of 3 mm to a height of 25 mm, and the same treatment as in Example 1 was performed for equilibration.

Crude urokinase aqueous solution 150' (activity 145 aU/
', specific activity 6421 U/mg) was passed through the column,
The buffer solution was allowed to flow down in the same manner as in Example 1. Comparing before and after the passage, the residual titer rate was 100%, the impure protein removal rate was 16.9%, and the coloring component removal rate was 66.4%. Experimental Example An experiment was conducted to compare the removal rate of colored components using anion exchange resin.

Claims (1)

[Claims] 1 Using quaternary ammonium as an exchange group, polystyrene,
A strongly basic anion exchange resin based on a divinylbenzene copolymer is equilibrated with a buffer solution of pH 7 to 9, and an aqueous solution of crude urokinase is brought into contact with the resin to recover an aqueous solution containing unadsorbed urokinase. A method for producing urokinase, which comprises removing a colored component in an aqueous solution of urokinase by adsorbing it to the anion exchange resin.