JPH04193894A - Purification of human urinary trypsin inhibitor - Google Patents

Abstract

PURPOSE:To industrially advantageously produce human urinary trypsin inhibitor useful as an antiinflammatory agent by successively adsorbing condensed human urinary materials from human urine on an ion exchanger, a hydrophobic chromatographic carrier, etc., and subsequently eluting the adsorbed subject substance. CONSTITUTION:Human urinary materials obtained by concentrating human urine with an adsorbent followed by recovering is mixed with the salt of a metal such as Zn, Ca or Cu, and the precipitates are removed from the mixture. The remained solution is adsorbed on an anionic exchanger under an acidic condition, and the adsorbed substance is eluted with a highly concentrated salt solution. The eluent is adsorbed on a hydrophobic chromatographic carrier under a highly concentrated salt condition, and the adsorbed substance is eluted under a lowered concentration salt condition. The eluent is adsorbed on a reverse phase chromatographic carrier and eluted with a low polar solution to elute the objective trypsin inhibitor.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for purifying human urine trypsin inhibitor that can be carried out on an industrial scale.

[Conventional technology]

It is known that human urine contains trypsin inhibitors. Human urinary trypsin inhibitor (hereinafter referred to as
(abbreviated as UTI) has a molecular weight of 68,000 and an isoelectric point of pH 2~
It is a 3-glycoprotein and inhibits especially l-lipsin, chymotrypsin, plasmin, etc. (Sumi et al., Nippon Physiological Journal, 39.53 (1977)), so it has been clinically applied as an anti-inflammatory agent.

For example, purity 99% or more, specific activity 2500 units/Img
As mentioned above, UTI with an endotoxin concentration of 20 pg/l-lipsin inhibitor 1 mg or less is used as an anti-inflammatory agent.

By the way, in order to collect and purify UTI from a large amount of urine on an industrial scale, it is necessary to (I) obtain high-purity [JTr, (II) simple operation, (III) high efficiency, and (IV) reproducibility. (V) No decomposition or deactivation, etc.

The only known methods for purifying UTl are those that incorporate laboratory methods such as electrophoresis into the purification process, and these are only small-scale purification methods. Conventional methods include, for example, a method that combines methanol precipitation, immobilized trypsin affinity chromatography, and preparative electrophoresis (B. J., Bromke et al.
et, al.; Biochem, Med.

27.56 (1982)), a method combining perchloric acid treatment, immobilized trypsin affinity chromatography, isoelectric focusing, and gel filtration (Enzyme
:35, 225 (reported in 1986, etc.)

[Problem to be solved by the invention]

The present inventors have completed the present invention as a result of extensive research into a new method for easily purifying [JTI present in human urine] on an industrial scale to the level of purity for pharmaceutical use described above. .

[Means to solve the problem] That is, the gist of the present invention is as follows.

1. A method for purifying human urine trypsin inhibitor, which is characterized by comprising the following steps.

1st step: Concentrate urine or collect and concentrate urine with an adsorbent to obtain a urine stock solution. 2nd step: After adsorbing the urine stock solution obtained in the 1st step on an anion exchanger under acidic conditions, 3rd step: The solution obtained in the 2nd step is adsorbed onto a hydrophobic chromatographic carrier under conditions of high salt concentration, and then the salt concentration is lowered to elute the adsorbed material. 4th step: After the solution obtained in the 3rd step is adsorbed onto a reversed phase chromatography carrier, the polarity of the eluent is lowered to elute the adsorbed substance. Zinc, calcium,
1. The method for purifying human urine trypsin inhibitor according to item 1, which comprises adding at least one metal salt selected from metal salts of copper, barium, and aluminum, and then removing the resulting precipitate.

The present invention will be further explained in detail below.

First, to explain the invention of claim 1, this invention is capable of producing high purity U suitable for pharmaceuticals on an industrial scale from a large amount of urine.
This is a purification method for obtaining T[, and consists of the steps from the first step to the fourth step described above.

Each step will be explained below.

In this step, when UTI is extracted from a large amount of urine, it is concentrated or collected and concentrated using an adsorbent. That-
Examples of numerical means include ultrafiltration concentration and methods using adsorbents such as aluminum hydroxide gel, synthetic aluminum silicate, kaolin, silica gel, ion exchange resins, and chitosan. In the case of ultrafiltration concentration, the method etc. are not particularly limited.

In addition, in the case of collection and concentration using adsorbents, the type is not particularly limited as long as UTI can be adsorbed, but the amount of each adsorbent added, addition conditions, elution conditions from the adsorbent, etc. should be carefully examined. There is a need.

For example, aluminum hydroxide gel (Kyoward 200
B: In the case of Kyowa Chemical Industry Co., Ltd., urine is adjusted to pH 3-5 with acetic acid, etc., and then added to a concentration of 0.25-2.0%, stirred and adsorbed for several hours, and then adjusted to pH 10-2.0%. It is possible to collect and concentrate UTI by elution with solution No. 12.

Furthermore, for the purpose of virus removal, urine undiluted solution was heated for 60~
It is preferably carried out for 10-15 hours at 65°C.

In this step, the urine stock solution obtained in the first step is dialyzed against a pH 3-5 buffer, adsorbed onto an anion exchange column equilibrated with the same buffer, and after thorough washing, UTI can be eluted by increasing the salt concentration. In this case, the anion exchange resin is diethylaminoethyl (DEAE
) groups and quaternized aminoethyl (QAE) groups as functional groups, but are not limited to these. The dialysis and column equilibration buffers contain 0.
About 0.05 to 0.15M sodium chloride may be added, and the salt concentration at which UTI elutes is preferably 0.2.
5-0, 35M sodium chloride.

In this step, a sample was prepared by adding ammonium sulfate to the eluate obtained in the second step to a concentration of 1.2 to 1.5 M, and the sample was equilibrated in advance with a buffer containing the same concentration of ammonium sulfate. hydrophobic chromatography column,
For example, a sample is fed to a column packed with a carrier having phenyl, octyl, or butyl functional groups to adsorb proteins, and the ammonium sulfate concentration is adjusted to 1.0 to 0.65.
The fraction eluted by lowering M was collected as a UTI active fraction and separated from other contaminant proteins.

The order of implementation of the second step and the third step may be arbitrarily selected without affecting the final purification result even if the order is reversed.

Process In this step, the solution obtained in the third step is used to chemically bond hydrophobic groups such as alkyl groups (C,, C,, C4, C,, C, 8), cyanopropyl groups, phenyl groups, etc. The silica or polystyrene resin used as a stationary phase is fed to a column (so-called reverse phase column), and after adsorption and thorough washing of the column, UTI is fractionated by lowering the polarity of the eluent (mobile phase). It is something to be collected. The reversed phase column is not particularly limited, and can be used with various functional groups as described above, various particle sizes (3 to 100 μm), and various pore sizes (100 to 100 μm).
However, when dealing with proteins, carriers with larger pores and relatively short alkyl chains are preferred. In addition, the particle size has a large effect on the separation ability, but carriers with small particle sizes are very expensive and are not suitable for use on an industrial scale. There is no problem at all as long as the particle size of the carrier used in the present invention is about 15 to 60 μm. As such a resin, bakebond WP-butyl (C4) (15 μm, 40 μm: J, T, Ba
(manufactured by KER) is exemplified, but is not limited thereto. Water-containing organic solvents are used as mobile phases as eluents to reduce polarity, and acetonitrile, 2-propanol, methanol, ethanol, etc., and combinations of these solvents are mainly used, but it is difficult to manufacture pharmaceuticals on an industrial scale. For this purpose, it is preferable to use ethanol.

The appropriate ethanol concentration is 20 to 50%, preferably 30 to 40%.

As described above, by carrying out the method for purifying UTI from the first step to the fourth step described above, it is possible to obtain highly pure UTI suitable for pharmaceuticals on an industrial scale from a large amount of urine.

Next, to explain the invention of claim 2, this invention produces high purity UT suitable for pharmaceuticals on an industrial scale from a large amount of urine.
This is a purification method for obtaining I, which is an improved invention of claim 1.

That is, after the first step according to claim I, a treatment step with a metal salt is carried out. This step removes contaminant proteins and mucopolysaccharides and other viscous substances in the urine stock without causing loss of UTI activity. As a result, from the second step onwards, (I) the load on the column can be reduced, and (II) sufficient separation can be performed.

This metal salt treatment step involves adding at least one metal salt selected from zinc, calcium, copper, barium, and aluminum metal salts to the urine stock solution obtained in the first step, and then removing the resulting precipitate. It is something to do. This precipitate contains viscous substances including contaminant proteins and mucopolysaccharides.

The metal salts to be added include zinc, calcium, copper, barium,
Aluminum nitrates, sulfates, acetates, chlorides, etc. can be used, and the added concentration is about 10mM to 5M.
Preferably it is 0.1-1.0M. The method of addition is not particularly limited, but it is preferable that the added metal salt is dissolved uniformly, and the addition is carried out under stirring and/or shaking in a temperature range of 4 to 80°C for 0.5 to 24 hours. It is preferable to leave it alone.

After this step, the second to fourth steps recited in claim 1 are similarly carried out.

As described above, by carrying out the method for purifying tJTl from the first step to the fifth step described above, high purity U suitable for pharmaceuticals can be obtained from a large amount of urine on an industrial scale and with good operability.
It is possible to get TI. Also, claims 1 and 2 of the present invention
By carrying out the purification method described in (C), it is also possible to remove so-called pyrogienes, which are substances that are mixed into pharmaceuticals and induce exothermic effects when administered into the body.

〔Example〕

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to the following Examples.

Example 1 First Step Hydrochloric acid was added to 1000 A of normal human urine to adjust the pH to 4.0 to obtain an acid-treated bed. 5 kg of aluminum hydroxide gel (Kyoward 200B; manufactured by Kyowa Chemical Industry Co., Ltd.) was added to this acid-treated bed, and the mixture was stirred at room temperature for 3 hours.

After standing still for 1 hour, the supernatant and adsorbent were separated by decantation, and the adsorbent was further washed with water by suction filtration. This washed adsorbent was stirred and eluted with 1% ammonia 50A for 2 hours, and an eluate was obtained by centrifugation (8000 rpm, 60 minutes).

Second Step The urine stock solution obtained in the first step was heat-treated for 10 hours with stirring in a constant temperature water bath at 65°C. The precipitate formed after treatment is
It was removed by centrifugation (8500 rpm, 60 minutes). This solution was diluted with pyrogen-free water to adjust the electrical conductivity to 15 mmho, and the pH was further adjusted to 4 with acetic acid. Anion exchange column (DEAB-Cellulofine A300, manufactured by Seikagaku Corporation) (5 cm in diameter, 30 cm in height) equilibrated in advance with 0 and 1 M acetate buffer (pH 4,0) containing 50 mM NaCl. and washed thoroughly with the same buffer.

0.1 M acetate buffer (p
Both fractions eluted by UT (H4,0) were separated as the active fraction. The activity recovery rate for both components was 86%, and the specific activity was 15270/mg.

3rd step Add 1.3 ammonium sulfate to the solution obtained in the 2nd step.
A phenyl sepharose column (manufactured by Pharmacia) (diameter 2.6 cm) was equilibrated with a buffer containing 1.3 M ammonium sulfate.
m', height 30 cm) and thoroughly washed with the same buffer. Both fractions eluted with a buffer containing 0.85M ammonium sulfate were separated as UTI active fractions.

The activity recovery rate for both parts was 91%, and the specific activity was 238.
It was 8U/mg.

4th step The solution obtained in the 3rd step was pre-equilibrated with a 2% ethanol solution to prepare baked carbon WP-butyl (C4).
(40 μm: J, T, Baker) packed column (7 cm in diameter, 11 cm in height), and after adsorption, it was washed with the same ethanol solution in an amount 10 times the column volume, and further washed with a 30% ethanol solution. The eluted fraction is U
It was separated as a TI active fraction.

The activity recovery rate for both parts was 89%, and the specific activity was 264.
It was 10/mg. In addition, the endotoxin concentration was 3,
It was 1 pg/mg-UTI. Purity was estimated to be greater than 99% by electrophoresis and HPLC methods.

Example 2 1st step normal human urine 1000I! Hydrochloric acid was added to the solution to adjust the pH to 4.0 to obtain an acid-treated bed. 5 kg of aluminum hydroxide gel (Kyoward 200B; manufactured by Kyowa Chemical Industry Co., Ltd.) was added to this acid-treated bed, and the mixture was stirred at room temperature for 3 hours.

After standing for 1 hour, the supernatant and adsorbent were separated by decantation, and the adsorbent was further washed with water by suction filtration. This washed adsorbent was stirred and eluted with 1% ammonia 50A for 2 hours, and an eluate was obtained by centrifugation (8000 rpm, 60 minutes).

2nd step After neutralizing the urine stock solution obtained in the 1st step with hydrochloric acid, add zinc chloride to 0.5M, leave it at room temperature for 3 hours, and remove the precipitate by centrifugation (8500 rpm, 60 minutes). did. Apply this zinc chloride treatment solution to a Pericon cassette (manufactured by Millibore).
It was concentrated to a concentration of 51% by ultrafiltration to obtain a zinc-treated stock solution.

Third step The urine stock solution obtained in the second step was heat-treated for 10 hours with stirring in a constant temperature water bath at 65°C. The precipitate that formed again after the treatment was removed by centrifugation (8500 rpm, 60 minutes). This solution was diluted with pyrogen-free water to adjust the electrical conductivity to 15 mmhO, and the pH was further adjusted to 4 with acetic acid. Anion exchange column (DEAE-Cellulofine A300) pre-equilibrated with 0, 1M acetate buffer (pH 4,0) containing 50mM NaCl.
: Manufactured by Seikagaku Corporation) (diameter 5cm, height 30cm)
) and washed thoroughly with the same buffer.

O, 1M acetate buffer containing 0.3M NaCl (
Both fractions eluted at pH 4, 0) were collected as tJTI active fractions. The activity recovery rate of this fraction was 88%, and the specific activity was 1829 U/mg.

4th step Add 1.3 ammonium sulfate to the solution obtained in the 3rd step.
A phenyl sepharose column (manufactured by Pharmacia) (diameter 2.6 cm) was equilibrated with a buffer containing 1.3 M ammonium sulfate.
m, height 30 cm) and thoroughly washed with the same buffer. Both fractions eluted with a buffer containing 0.85M ammonium sulfate were separated as UTI active fractions.

The activity recovery rate of this fraction was 93%, and the specific activity was 251
It was 6 U/mg.

5th step The solution obtained in the 4th step was equilibrated with a 2% ethanol solution in advance to prepare carbonated WP-butyl (C4).
(40 um: manufactured by J, T, Baker) was fed to a packed column (diameter 7 cm, height 11 cm), and after adsorption, it was washed with the same ethanol solution in an amount 10 times the column volume, and further washed with a 30% ethanol solution. The eluted fraction was collected as a UTI active fraction.

The activity recovery rate of this fraction was 93%, and the specific activity was 274.
It was 9 U/mg. Furthermore, the endotoxin concentration was below the level of distilled water for injection at a protein concentration of 5.3 mg/ml. Purity was estimated to be greater than 99% by electrophoresis and HPLC methods.

In addition, UTI activity, protein amount, endotoxin concentration, and purity in Example 1.2 were measured by the following methods.

1. UT [activity: method of Muramatsu et al. (
J, Biochemo, Niji, 402 (1965))
Measured using a casein solution in accordance with .

2. Protein: BCA method protein quantification kit (
(manufactured by Pierce Co.) was used.

3. Endotoxin concentration Nidoxy Color System LS
A colorimetric endotoxin measurement method was used using the -1 set and the Et-1 set (endodoxin standard solution) (manufactured by Seikagaku Corporation).

4. Purity assay electrophoresis method: 5DS-polyacrylamide electrophoresis (
After staining with Coomassie brilliant blue (CBB), quantitative analysis was performed using a densitometer.

HPLC method C4 column (AP-803-35
: manufactured by YMC) in the presence of 0.1% TFA with an acetonitrile elution system, and quantitative analysis of the peak area was performed.

〔Effect of the invention〕

By using the method for purifying UTI according to the present invention,
Easily and efficiently detect UTI from large amounts of urine on an industrial scale.
Not only can it be reproducibly recovered and purified, but it has also become possible to obtain highly pure UT that can be applied to pharmaceuticals.

Patent applicant: Denki Kagaku Kogyo Co., Ltd.

Claims (1)

[Scope of Claims] 1. A method for purifying human urine trypsin inhibitor, which comprises the following steps. 1st step: Concentrate urine or collect and concentrate urine with an adsorbent to obtain a urine stock solution. 2nd step: After adsorbing the urine stock solution obtained in the 1st step on an anion exchanger under acidic conditions, 3rd step: The solution obtained in the 2nd step is adsorbed onto a hydrophobic chromatographic carrier under conditions of high salt concentration, and then the salt concentration is lowered to elute the adsorbed material. 4th step: After the solution obtained in the 3rd step is adsorbed on a reversed phase chromatographic carrier, the polarity of the eluent is lowered to elute the adsorbed substance. The person according to claim 1, characterized in that the method comprises adding at least one metal salt selected from metal salts of zinc, calcium, copper, barium, and aluminum to the urine stock solution, and then removing the resulting precipitate. Method for purifying urinary trypsin inhibitor.