JPS60118187A - Production of urokinase - Google Patents

Abstract

PURPOSE:To separate and purify urokinase useful as a remedy for various thromboses in high purity, by treating a solution containing crude urokinase with a water-insoluble carrier containing a cephem based compound as a ligand as an adsorbent. CONSTITUTION:A substance having the cephem skeleton of the formula, preferably cefalotin, cefotiam, cefotaxime, etc. is directly or through a spacer, e.g. L- lysine, is bonded to a water-insoluble carrier, e.g. cellulose. The resultant carrier as an adsorbent is brought into contact with a solution containing crude urokinase, e.g. urine, to separate and purify the urokinase.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a highly efficient method for producing urokinase from a crude urokinase-containing solution such as human urine. More specifically, the present invention relates to a method for efficiently separating and purifying highly pure urokinase from a crude urokinase-containing solution using a water-insoluble carrier having a substance having a cephem skeleton as a ligand.

Urokinase is a plasminogen activating enzyme present in large urine, and its importance is increasing year by year, as it is finding use not only in the treatment of thrombosis but also in combination therapy with anticancer drugs.

In order to separate and purify urokinase from human urine, etc., methods using silica gel ion exchangers, water-insoluble carriers with basic amino acids, etc. as ligands, etc. are well known.

The present inventors have conducted intensive research to search for separation and purification methods in order to obtain highly purified urokinase in higher yields. Substances with an unknown cephem skeleton (hereinafter referred to as cephem compounds)
We found that an adsorbent in which urokinase is bound to a water-insoluble carrier is extremely superior to previously known adsorbents for urokinase in terms of adsorption capacity, yield, degree of purification, etc. The present invention was completed based on the new findings.

The present invention relates to a method for producing highly pure urokinase from a crude urokinase-containing solution using a water-insoluble carrier having a cephem compound as a ligand as an adsorbent.

In the present invention, the crude urokinase-containing solution includes urine,
Examples include crude urokinase-containing aqueous solutions.

Adsorbents can be used for the separation and purification of urokinase, including those in which a cephem compound is directly bound to a water-insoluble tU body, as well as those in which a cephem antibiotic is bound to a water-insoluble carrier via a spacer. can.

Any cephem compound having the following formula as its basic skeleton can be used in the present invention. Purified ones are preferably used. Examples of -p-fem compounds include cephem antibiotics (eg, cephalothin, cefotiam, and cefoxitin).

The water-insoluble carrier is not particularly limited either, and any carrier can be selected and used as long as it can be used as a carrier for affinity chromatography. For example, polysaccharides such as cellulose and agarose, polyacrylamide gel, and anion exchangers such as aminoethyl cellulose and aminoethyl polyacrylamide gel (Bioland) can be used.

The spacer is not particularly limited either, and any spacer can be selected and used as long as it can be used as a spacer between the ligand for affinity chromatography and the carrier. For example, L-lysine, diaminoethane, 1,6-diamithexane, etc., with the general formula NH2-(CH2)n-NH2(
A diamino compound represented by the following formula, where n is an integer of 1 to 10, is conveniently used. In particular, when using a water-insoluble carrier that does not have an amino group, such as a polysaccharide or polyacrylamide gel, it is necessary to attach the spacer to introduce the amino group, and then attach the cephem compound.

In order to prepare the adsorbent used in the present invention, the cephem compound can be used as it is or through a spacer.
Any chemical method capable of binding to the water-insoluble carriers described above may be used, generally the amide formation reaction with water-soluble carbodiimides (Sh-eehan &
a (1957): J, Hachim, Cbcm, Soc.

79, 4528) is advantageously used.

When carrying out the separation and purification of urokinase using the adsorbent bound to the cephem-based compound according to the present invention, contacting the adsorbent with a urokinase-containing solution may be carried out in a batch manner or by a column method. Good too.

The outline of the method for separating and purifying urokinase of the present invention is as follows.

A crude urokinase-containing aqueous solution, such as human urine, or a crude urokinase-containing aqueous solution is brought into contact with a cephem compound-bound adsorbent. At this time, the pH of the crude urokinase solution is preferably 4 to 9. After adsorbing urokinase, unadsorbed substances are removed by washing.

Urokinase is mixed with an eluent (e.g., 1.0M sodium chloride solution, 0.1M arginine solution, 0.1M l'll
sodium chloride solution, etc.). In order to collect urokinase from the purified urokinase solution thus obtained, known methods such as ammonium sulfate salting out, ultrafiltration and freeze-drying are used.

The cephem-based compound-bonded adsorbent according to the present invention uses a new substance as a ligand, and can separate and purify highly pure urokinase in high yield through simple operations.

The method of the present invention will be illustrated in more detail by Examples below, but the present invention is not limited thereto. In addition,
The urokinase activity in the examples was determined using the method of Johnson et al.
ohnson+ A, J, et al. Thr-o
mb, Death, Haemorrh+ 21+2
59 (1969)), and the titer was expressed in international units (TU).

Protein quantification was performed by the method of Lowry et al.

Example 1 5 g of aminoethyl cellulose (manufactured by Hanui Chemicals) was swollen in 500 ml of distilled water. Swollen cellulose 065
After purifying 1.000 ml of M sodium chloride solution, it was suspended in 200 ml of distilled water. The pH of this suspension
was adjusted to 4.5 with IN-hydrochloric acid. After pHfli adjustment, 1
1! ! To the suspension were added 2 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Peptide Research Institute, Protein Research Association III) and 1 g of cephalothin, and the mixture was stirred at pH 4, 5, and 4°C for 24 hours. .0.5
M sodium chloride solution2. By washing with IIooml, an adsorbent with cephalothin bound at a rate of 150 ma/g cellulose was obtained. 1g of adsorbent bound to cephalothin
was packed into a column (φ2.0×10 cm), and the amount of urokinase adsorbed was examined at 4°C. That is, 0.5
0.1 M phosphate buffer containing M sodium chloride (11
117,4) 1.0 (l) After flowing 1 ml of C into the column and equilibrating the adsorbent, the crude urokinase solution (50
,000 Iυ/ml, specific activity 5.011010/mg protein). After washing the column with 300 ml of the above buffer, the adsorbed urokinase was eluted with 0.1 M acetate buffer (p115.5) containing 0.5 M sodium chloride. The amount of urokinase adsorbed by the adsorbent at this time was 48
0.000 +1 J/mg adsorbent. The specific activity of the obtained urokinase was 130,0001 [1/mg protein, and the yield was 92%.

Example 2 1.1 was added to 50 ml of AH-Sepharose (manufactured by Pharmacia) into which 6-diamithexane was introduced as a spacer.
-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride 50011g Cefazolin 0.5g
was added and reacted at pH 4.5 for 24 hours at 4°C to obtain cefazolin-bound Sepharose. After equilibrating 50 ml of cefazolin-bound Sepharose with 0.1 M phosphate buffer (pH 7.4) containing 0.5 M sodium chloride, the entire volume was mixed with fresh human urine IQl (I
III/m+) to adsorb urokinase in a batch manner. After washing the urokinase-adsorbed and cefazolin-bound Sepharose in the column with the above buffer, urokinase was eluted with a 0.1M phosphate buffer (pH 7.0) containing 0.1M arginine. The specific activity of the obtained urokinase was 80,00010/mg protein, and the yield was 7.
It was 5%.

Example 3 3 g of cephalothin-bound cellulose obtained in Example 1 was added to 50 j' (810/m+) of fresh human urine and stirred at 4°C for 5 hours. After separating the urokinase-adsorbed and cephalothin-bound cellulose by centrifugation, it was washed with 0.1M phosphate buffer (p117.4) containing 0.5M sodium chloride, and the urokinase was separated from the urokinase in 0.1M containing 0.1M arginine.
Batchwise elution was performed with phosphate buffer (pH 7, A) IR. The specific activity of urokinase in the eluate is 75,00
010/mg protein, yield was 85%. This eluate was concentrated by ultrafiltration, and 0.05M phosphate buffer (
The product was dialyzed against pH 7.2) and freeze-dried to obtain a urokinase sample. As a result of conducting a pyrogenic substance test on this preparation according to the method described in the Japanese Pharmacopoeia Law (9th revision),
This specimen was negative for pyrogenic substances.

Comparative Example 1 The cephalothin-bound cellulose of Example 1 and the aminohenzamidine-bound cellulose conventionally used for purifying urokinase were compared in terms of urokilyase adsorption capacity, specific activity of the resulting urokinase, and yield of urokinase. As shown in Table 1, the cephalothin-conjugated cellulose showed approximately 14 h410 adsorption capacity, purity, and yield for aminohesobumami panosho.

Table 1 Procedural amendments (sealed) January 11, 1982 1. Indication of the case 1988 Patent Application No. 225168 2. Name of the invention Process for producing urokinase 3. Person making the amendment Relationship to the case Patent applicant Name: Mitori Juji Co., Ltd. 4, Agent ■541 Address: New Life Hirano-cho 4-53-3, Higashi-ku, Osaka 406 Telephone: (06) 227-1156 6. Number of inventions increased by amendment 7 , "Column 8 of Detailed Description of the Invention j, Contents of Amendment (1) "Silica gel ion" in the first line of page 2 of the specification is corrected to "Silica gel, ion" in the specification to be amended.

(2) "Adjustment" on page 4, line 18 of the same book is corrected to "Made by RIM."

(3) “Over-freezing” in the second line of page 6 of the same book is “over-freezing”
Correct.

that's all

Claims (1)

[Claims] A method for producing highly pure urokinase from a crude urokinase-containing solution using a water-insoluble carrier having a cephem skeleton-containing substance as a ligand as an adsorbent.