JPS615020A - Method for purifying urokinase converted protease and method for measuring amount of urokinase converted protease antigen - Google Patents

Abstract

PURPOSE:To purify economically the titled substance useful as an effect enhancer, e.g. urokinase pharmaceutical, by removing proteins, e.g. plasmin, from blood plasma and urine, and subjecting the resultant material to the steps through affinity chromatography, ion exchange and adsorption chromatography. CONSTITUTION:For example, blood plasma is passed through lysinesepharose gel, and plasmin, plasminogen, etc. are removed therefrom. The resultant liquid after passing through the gel is then subjected to affinity chromatography, and the adsorbed proteins are then eluted with a buffer solution and then dialyzed. The eluate after the dialysis is then subjected to ion exchange chromatography and gel filtration to purify stably urokinase converted protease in high purity and yield. The resultant purified protease can be supplied as an effect enhancer, e.g. urokinase pharmaceutical, tissue plasminogen activator pharmaceutical, etc. The method is useful for purifying a high polymer urokinase of high quality obtained by removing the titled substance utilizing the above-mentioned method.

Description

DETAILED DESCRIPTION OF THE INVENTION Industrial Applications and Related Applications The present invention provides a method for separating and purifying urokinase converting protease from plasma and urine, and a method for separating and purifying urokinase converting protease from plasma and urine, and the use of the purified urokinase converting protease mixed in advance in an adjuvant in animals. The present invention relates to a method for measuring the amount of urokinase-converted proteinase antigen, which measures the amount of antigen using an antibody obtained by immunization with urokinase. The present invention also enables the purification of highly pure and stable urokinase, particularly advantageously polymeric urokinase; in other words, it makes it possible to purify the polymeric urokinase. It is something.

DISCLOSURE OF TECHNICAL IDEA To briefly describe the content described below so that it can be easily understood and accurately grasped, the method for purifying urokinase converted protease of the present invention uses a predetermined adsorption gel in the purification process. After the removal of proteins such as plasmin, plasminogen, etc. from urine, it involves either affinity chromatography, or ion exchange and adsorption chromatography to achieve high purity. Moreover, it is possible to stably purify urokinase-converted protease with high yield, and in addition, it is possible to purify stable polymer urokinase. In addition, the method for measuring the amount of urokinase converted protease antigen contained in blood, urine, and tissues uses antibodies obtained by immunizing animals with the purified urokinase converted protease. This makes it possible to elucidate the relationship between various pathological conditions and diseases.

BACKGROUND ART Today, urokinase is widely used as a thrombolytic agent when treating patients with thrombosis, but there are two types of urokinase: high-molecular-weight urokinase and low-molecular-weight urokinase. The polymer urokinase has been used.

However, in the process of using high-molecular-weight four-kinases, it has been frequently experienced that they are not always sufficiently effective as thrombolytic agents. A similar experience would also be expected when tissue plasminogen activators and tissue culture urokinase are used as thrombolytic agents. Therefore, by using urokinase converting protease, which has the effect of enhancing urokinase and tissue activators, in combination with polymeric urokinase, tissue activators, and tissue culture urokinase, the effects of these thrombolytic agents can be enhanced and the desired effects can be fully obtained. It is conceivable that

As such, this invention originates from the physical research of polymer urokinase from a medical standpoint.According to our research, we have discovered that polymer urokinase can be degraded and
We were able to identify that the face that is converted into low-molecular-weight urokinase is a four-kinase converting protease.

Soneurokinase converting protease is an enzyme with a molecular weight of 120,000 to 140,000 that degrades proteins, and has excellent properties in medicine and for the purification of plasma, urine-derived urokinase, tissue culture urokinase, and tissue plasminogen actita. It is equipped with

OBJECTS AND PROBLEMS OF THE INVENTION An object of the present invention is to provide a method for purifying urokinase-converted protease, which enables stable purification of urokinase-converted protease with high purity and yield from plasma and urine.

Another object of the present invention is to provide urine-derived urokinase, tissue culture urokinase, which is prevented from being degraded and converted, and which is qualitatively stable, in particular, which maintains a polymeric state. and a method for purifying urokinase converting protease that can be used to purify tissue plasminogen activator.

Still another object of the present invention is to provide a method for measuring the amount of urokinase converted protease antigen using an antibody obtained by immunizing an animal with the purified urokinase converted protease.

Contents of the invention claimed in connection with solving the problems In relation to those problems, Party B's invention first involves a removal step in which plasma is passed through a lysine-Sepharose gel to remove plasmin, plasminogen, etc. , an adsorption step in which the pass-through from the removal step is subjected to affinity chromatography; a washing step in which the adsorbed solution from the adsorption step is washed away with a buffer to wash away unadsorbed proteins; A first separation step of eluting adsorbed proteins from the washed solution from the washing step, dialyzing the eluate from the first separation step using a buffer, and using an eluent and an ion exchanger. This method purifies urokinase-converted protease from plasma, including a second separation step in which the dialyzed eluate is subjected to ion-exchange pomagography, and a filtration step in which the eluate from the second separation step is gel-filtered. Yes, this invention involves bringing urine diluted in advance with distilled water into contact with a siliceous gel, allowing the proteins in the urine to be adsorbed to the siliceous gel, and using a buffer solution to absorb the proteins in the urine. a first washing step in which unadsorbed proteins are washed; a first separation step in which the proteins adsorbed to the silica gel from the first washing step are eluted using a buffer solution; a second washing step in which the eluted protein is subjected to affinity chromatography and washed with a buffer; a second separation step in which the washed protein from the second washing step is passed through the buffer; and from the second separation step. The present invention also includes a filtration step in which eluted protein is gel-filtered to purify urinary protein kinase converting protease.Furthermore, this invention involves passing plasma through a lysine-Sepharose gel to remove plasmin, plasminogen, etc. A second removal step in which the flow-through from the first removal step is salted out and passed through a lysine-Sepharose gel; A first separation step in which the pass-through from the second removal step is subjected to ion exchange and quadrupography; an intermediate filtration step in which the eluate from the first separation step is gel-filtered; and an intermediate filtration step in which an adsorbent and a buffer are used to a second separation step of adsorption chromatography of the filtrate from the filtration step, using an eluent and an ion exchanger, and within a predetermined range so as to obtain an elution curve showing two peaks;
and a third separation step of controlling the acidity (pH) of the eluent and subjecting the active eluate from the second separation step to hydrophobic ion exchange chromatography to purify urokinase-converted protease from plasma.

Furthermore, the present invention measures the antigen amount of urokinase conversion protease using an antibody obtained by immunizing an animal with purified urokinase conversion protease mixed with an adjuvant.

Industrial conveniences and benefits derived from the content of the invention based on the technical idea The conveniences and benefits obtained by the above-mentioned method for purifying urokinase converted protease of the present invention are based on the above-mentioned method for purifying urokinase converted protease. After removing proteins such as plasmin and plasminogen from urine, it is subjected to affinity chromatography or ion exchange and adsorption chromatography, so that urokinase converting protease can be produced with high purity and high yield. The urokinase-converted protease is purified from plasma and urine, and the urokinase-converted protease is separated, making it possible to purify stable polymeric urokinase.Furthermore, in the method of measuring the antigen amount of the urokinase-converted protease of this invention, Because it uses highly purified urokinase converting protease, plasma,
The amount of urokinase converted protease antigen in urine and tissues can be measured precisely and accurately.

Therefore, in the purification method of the present invention, urokinase converting protease is purified with high purity and high yield, so high quality urokinase converting protease can be supplied economically and in large quantities. Due to the properties of urokinase-converted protease, it can be supplied as an effect enhancer for urokinase preparations, kidney tissue culture ruminal kinase preparations, tissue plasminogen activator preparations, etc. In the process of this purification method, Urokinase conversion protease can be separated from urokinase converting protease by using the method, and it is possible to purify urokinase, especially stable polymer urokinase. In other words, it makes it possible to remove the urokinase-converting protease, making it possible to purify high-quality polymeric urokinase.

Furthermore, in the method for measuring the amount of antigen of this invention, an antiserum against urokinase converted protease developed by the inventor is used. The purification method and antigen amount measurement method of the present invention can be used to elucidate the relationship between various pathological conditions and diseases, and provide more effective treatment.
It dramatically improves treatment methods in the medical field and is very useful and beneficial to the medical field.

Further, the properties of the urokinase converted protease purified by the purification method of the present invention and the relationship between the antigen amount measurement method and the therapeutic method of the present invention will be described in more detail as follows.

U-kinase conversion protease is essentially the same whether derived from plasma or urine, and is degraded during the purification process, with a molecular weight of 128°000±s, ooo
and acetic acid with a molecular weight of 64,000±4,000, and the former molecular weight indicates that it exists as a dimer, and the latter molecular weight indicates that it exists as a simple substance, but the antigenicity is the same in both cases.

In addition, the urokinase conversion protease is a toxinbin-like protease that cleaves the algine binding site of the synthetic substrate, and degrades casein.
-2288 (registered trademark HKabi: Sweden)
Disassemble.

However, this protease does not contain fibrin (Five!
Although it does not degrade S-2251 (Kabi, Sweden) or convert plasminogen to plasmin, it enhances plasminogen activation by urokinase and also enhances plasminogen activation by tissue activators. do.

On the other hand, the protease does not enhance plasminogen activation by streptokinase.

In this way, the phenomenon of enhancement of urokinase activity by urokinase converting protease is caused by abrotiin (apro
tinin), epsilon amino carbtetraphosphate, soybean trypsin inhibitor, tranexamic acid, etc., inhibit the effect by about 10 to 20%, and penzole arginine ethyl ester inhibits it 100%. Therefore, the penzole arginine ethyl ester is recognized as a competitive inhibitor of urokinase converting protease, and the inhibition constant of the penzole arginine ethyl ester is 1.85m.

In addition, this urokinase converting protease is a commonly purified polymeric gastric kinase (molecular weight: 53,000-
55. OOO) is a low molecular weight urokinase (molecular weight?
33,000), and is further reduced to a lower molecular weight. However, the low-molecular-weight urokinase produced in this way was found to have different properties from the low-molecular-weight urokinase produced by degradation with plasmin.

Due to such properties, this urokinase converting protease can be used as an effect enhancer of conventionally purified urokinase, renal tissue culture urokinase, tissue plasminogen activator, etc., and can also be used as an effect enhancer of urokinase converting protease derived from urokinase converting protease. Commonly purified polymeric urokinase, which makes it available as a thrombolytic agent and is currently being widely used as a thrombolytic agent in treating patients with thrombosis, contains trace amounts of urokinase converting protease. Therefore, by removing the urokinase-converting protease from the high-molecular-weight urokinase, a stable t-co-high-molecular-weight urokinase whose conversion to low-molecular-weight urokinase is prevented even when left untreated can be obtained, which is extremely useful clinically.

In addition, it has become possible to measure the amount of urokinase converting protease antigen in blood, urine, and tissues using antiserum against urokinase converting protease, which has made it possible to elucidate the relationship between various pathological conditions and diseases. It also leads to the detection of protease deficiencies and abnormalities, and also allows more effective treatments to be implemented by monitoring the movement of urokinase-converting proteases during urokinase treatment and tissue plasminogen activator treatment.

Due to the properties described in detail and in relation to therapeutic methods, the purification method and antigen amount measurement method of the present invention are useful for the treatment of various diseases, and are highly accurate and safe. It will be understood that this is extremely useful for improving the law.

Description of Specific Examples Preferred specific examples of the method for purifying urokinase-converted protease and the method for measuring the amount of urokinase-converted protease antigen according to the invention will be described below.

Specific Example 1 This purification method consists of a removal step, an adsorption step, a washing step, a first separation step, a second separation step, and a filtration step, resulting in 400 ml of plasma! The urokinase converting protease was purified batchwise at low temperature, i.e. at a temperature of 4°C.

First, transfer 400 m of plasma to a lysine-Sepharose gel.
1 to remove plasmin, plasminogen, etc., and the flow-through from the removal step was then affinity chromatographed using Penzole Arginine Ethyl Ester-Sephalyse Gel.

Subsequently, the adsorbed liquid from the adsorption step was thoroughly washed with a buffer solution of acidity (pH) 7.6 containing 0.1 M phosphate and 0.5 M NaCl to wash away unadsorbed proteins. Ta.

Subsequently, the adsorbed proteins in the washed solution were eluted using a pH 7.6 buffer containing O, 1M phosphate, and 0.2M arginine.

Subsequently, pH containing 0.05M 1-lithic hydrochloric acid
The eluate was dialyzed using 8,3 buffer and 0.05M) as the eluent! JS hydrochloric acid buffer (pH 8,3)
The dialyzed solution was subjected to ion exchange chromatography using Sephadex ion exchanger A-50 as an ion exchanger, and 0.05M) lithium-hydrochloric acid,
and eluted with a pH 8.3 buffer containing 0°2M sodium chloride.

Thereafter, the eluate was gel-filtered using Sephadex G-100 to remove approximately 0% of the urokinase-converted protease.
．． 4+++g was obtained.

As a result of analysis, the purified urokinase converting protease had a molecular weight in the range of 128,000±6,000.

Specific Example 2 This purification method consists of a first washing step, a first separation step, a second washing step, a second separation step, and a filtration step.
! Urokinase converting protease was purified batchwise at low temperature.

Urine 41 was diluted three times with distilled water in advance, and the diluted urine was passed through a siliceous gel to adsorb the protein in the urine to the siliceous gel, which contained 0.1M phosphate. pH7,6
Wash thoroughly with buffer.

Remove unadsorbed protein from the urine.

Then pH 7 containing 0.1M phosphate and 1M NaCl
, 6 was used to elute the protein adsorbed on the siliceous gel.

Subsequently, the elution fraction, ie, the eluted protein, was treated with benzoyl arginine and ethyl ester.

After adsorption to ester-Sepharose, the mixture was thoroughly washed with a pH 7.6 buffer containing 0.1 M phosphate and 0.5 M sodium chloride.

Subsequently, the washed protein was eluted using a pH 7.6 buffer containing 0.1 M phosphate and 0.2 M arginine.

Thereafter, the eluted fraction, ie, the eluted protein, was gel-filtered using Sephadex G-100, a gel filtration carrier, to obtain urokinase-converted protease.

As a result of analysis, the thus purified urokinase converting protease has a molecular weight of 128,000±6,000.
It was within the range of

Specific Example 3 This purification method consists of a first removal step, a second removal step, a first separation step, an intermediate filtration step, a second separation step, and a third separation step. It was purified at low temperature.

200 mj of plasma was passed through a lysine-Sepharose gel to remove plasmin, plasminogen, and the like.

The flowthrough was then salted out with 50% saturated ammonium sulfate solution, the precipitate dissolved and passed through the lysine-Sepharose gel again.

The flowthrough was subsequently ion-exchange chromatographed using eluent and Sephadex ion exchanger A-50 and eluted with 0.15M NaCl.

Subsequently, the eluate was subjected to gel filtration using Sephadex G-75 for gel filtration carrier. In this case, Sephadex G-75 was used as the gel filtration medium, but it is of course possible to use Sephadex G-100 for gel filtration carrier instead.

Subsequently, the filtrate was subjected to adsorption chromatography using hydroxyapatite using an adsorbent and a phosphate buffer, and the active sites were collected by elution with a pH 7.0 buffer containing 0.2M phosphate. , p containing 0.2M acetic acid
Dialyzed against H4,0 buffer.

Then, using an eluent and Amberlite ion exchange resin (Amberlite CG-50), and adjusting the acidity of the eluent so that two peaks appear in the elution curve (
The dialyzed active eluate was hydrophobically ion-exchange chromatographed, controlling the pH to rise from 4.0 to 6.5, with the two peaks containing:
Approximately 0.2 μ of urokinase converted protease was obtained from the rear peak eluted.

The purified urokinase converting protease was analyzed to have a molecular weight in the range of 128,000±6,000.

Specific Example 4 This purification method was improved in order to simplify the steps of the purification method in Specific Example 3 described above and improve the yield.
Consisting of a removal step, a second removal step, a first separation step, a washing step, a second separation step, and a filtration step, 400 m of plasma
Urokinase-converted protease was purified batchwise at low temperature from L.

First, 400 m of plasma was added to a lysine-sepharose gel.
1 to remove plasmin, plasminogen, etc.

The flow-through fraction was then salted out with 50% saturated ammonium sulfate solution,
Dialysis was performed with a pH 8.3 buffer containing 0.05M) lithium-hydrochloric acid.

Subsequently, 0.05M) Lis-HCl buffer and DEA
E-Sephadex-Ion Exchanger A-50 (DEAE
The dialysed solution was subjected to ion exchange chromatography using 0.5ephedex A-50).
Elution was performed with 2M sodium chloride.

The eluted fraction was then passed directly through benzoyl arginine ethyl ester-Sepharose 4B.

Subsequently, unadsorbed proteins in the elution fraction were thoroughly washed with a pH 7.6 buffer containing 0.1M phosphate and 0.5M sodium chloride.

Subsequently, the adsorbed proteins in the washed solution were eluted with a pH 7.6 buffer containing 0.1 M phosphate and 6 M urea.

Thereafter, the elution fraction was concentrated and gel-filtered using Sephadex G-100 for gel filtration carriers to elute urokinase-converted protease.

As a result of analysis, the thus purified urokinase converting protease has a molecular weight of 128,000±6,000.
It was within the range of

Specific Example 5 This is an attempt to easily separate urokinase converting protease from urine, and this method is similar to the method described in Specific Example 2.
This purification method is basically the same as the one described above, and its distinctive feature is that affinity chromatography is performed using a substance with a strong affinity for the four-kinase conversion proteinase as a ligand.

That is, if urine is added to benzoyl arginine ethyl ester-Sepharose equilibrated with a pH 7.6 buffer containing 0.1 M phosphate and eluted as it is, urokinase conversion will occur during affinity chromatography. Protease is adsorbed to the column and is easily separated from urine.

A specific example is the purification of urokinase converting protease such that the urokinase converting protease stably maintains a predetermined clinical effect despite the separation and removal of the urokinase converting protease from the normally purified urokinase. This method is related to the above-mentioned urokinase preparations, and targets urine-derived tsushikinase and kidney cell culture urokinase, but it can also target tissue activator preparations, and in principle, the above-mentioned specific This is the same method as Example 5.

The feature of this method is that it incorporates an affinity chromatography step using benzoyl arginine ethyl ester-Sepharose during the current purification process of urokinase preparations, renal cell culture urokinase preparations, and tissue activator preparations. By this,
Urokinase converting protease is removed from urokinase.

Therefore, even if urokinase preparations, kidney cell culture urokinase preparations, and tissue activator preparations that do not contain urokinase converting protease are left untreated,
Conversion from a high molecular state to a low molecular state is prevented,
The clinical efficacy of these formulations is stabilized.

In addition, in order to mass-produce U-kinase converting protease from Specific Examples 1 to 4 mentioned above with high yield, a continuous purification method is required, but even in that case, the affinity for urokinase-converting protease is This is possible by affinity chromatography using strong benzoyl arginine ethyl ester-Sepharose or by using Sepharose, an antibody against urokinase converting protease.

Specific Example 7 This is a method for measuring the amount of antigen using the urokinase conversion protease purified in the above-mentioned Examples 4 to 4. It is based on a protease antiserum production method.

First, the purified urokinase converting protease is thoroughly mixed with Freund's complete adjuvant, and the mixture is injected into the sole and back of the rabbit's feet.

One month later, the rabbit is boosted with the mixture as a booster.

Two weeks after the booster immunization, blood is collected to obtain antiserum.

Once a specific antiserum against urokinase converting protease is obtained in this way, the antiserum can be used to
Urokinase converted protease antigen levels were measured. The specific measurement was carried out by the usual glue method.

Of course, the amount of urokinase converted protease antigen can also be measured using the antiserum by the commonly used radioimmunoassay method, enzyme immunoassay method, latex agglutination method, etc.

Specific Example 8 This is a method for measuring urokinase converted protease activity developed by the inventors for the convenience of using purified urokinase converted protease in therapy.

This method of measuring urokinase converting protease activity calculates the activity of urokinase converting protease by subtracting the activity of the four kinases from the activities of both urokinase converting protease and urokinase. ) synthesis substrate: Test Team S-2444 (registered trademark) was used as follows.

First, urokinase (600I u/mj) 10μ4
100 μm of the test solution was mixed with the mixture and incubated at a temperature of 37° C. for 5 minutes.

Next, 0.8 mJ of a buffer solution containing 0.05 M) Lis-HCl and 0.38 M NaCl was added to the test team S-2.
100μ4' of 444/3mM was added and incubated again for 15 minutes at a temperature of 37°C.

Once so incubated, then 40
Measure absorbance at 5 nm.

According to the measurement results, the absorbance was 0.120.

On the other hand, as a control, 100 m of water was added instead of the test solution.
j In addition, perform the above-mentioned operation in the same manner and measure the absorbance at 405 nm.

The absorbance in this case was 0.042.

Therefore, the activity of the urokinase converting protease is
It is given by the difference in absorbance between the two, and the latter is subtracted from the former (and becomes 0.078).

Although the method for measuring urokinase converting protease activity has been described using a synthetic substrate, the activity of urokinase converting protease can be measured without using the synthetic substrate.

In this case, first, urokinase and a test solution are mixed in fixed amounts and incubated at a temperature of 37° C. for 5 minutes.

Next, 5 μl of the incubated solution was added to a plasminogen-containing fibrin plate, incubated at a temperature of 37° C. for a fixed period of 12 hours, and the dissolved area was measured.

According to the measurement results, the melting area was 44 mm+F.

In addition, as a control, the same operation as described above was performed in a system without the test liquid, and the dissolution area in this case was 24 mm''.

Therefore, the activity of urokinase converting protease in this case is given by the difference in lysis area between the two, and subtracting the latter from the former yields 20 mrrl''.

Industrial convenience and benefits derived from the content of the invention based on specific examples As described above, the method for purifying the urokinase converted protease of the present invention includes pretreatment followed by affinity chromatography, ion exchange and adsorption. Since it includes a chromatography process, urokinase converting protease can be purified in large quantities from plasma and urine with high purity and yield, making it possible to supply large quantities of urokinase converting protease, which is useful for elucidating the relationship between various pathological conditions and diseases. As a result, more effective treatment, more accurate and safer treatment will be carried out. In other words, treatment methods will be dramatically improved, and this purification method will be improved. Utilizing this method makes it possible to separate the urokinase-converting protease and purify the removed urokinase, which is particularly advantageous for purifying stable polymeric urokinase.
Urokinase converting protease is separated and removed from urokinase purified by a conventional method, resulting in high quality, so-called
It becomes possible to supply a high-molecular-weight p-kinase that is not converted into a low-molecular-weight compound even if left untreated, which is extremely beneficial and useful medically.

In addition, the method for measuring the antigen amount of urokinase converted protease of this invention is the urokinase converted protease discovered by Kan, the inventor of this invention. Since the method is carried out based on the antiserum, the amount of p-kinase converting protease antigen in plasma, urine, and tissues can be measured, and the reliability of the measurement value is high. The relationship between pathological conditions and diseases can be clarified, and more effective treatments can be provided.
It is extremely beneficial and useful in terms of therapy.

As for the relationship between the invention and the specific examples, from the specific examples of the present invention described with reference to the drawings, various design modifications will be apparent to those who have ordinary knowledge in the technical field to which this invention pertains. Modifications and modifications may be easily made, and furthermore, the content of this invention may be essentially the same as that invention, which satisfies essentially the same problem and achieves the same effect as that invention. It could easily be replaced with the same version.

Claims (4)

[Claims] (1) Pass plasma through lysine-Sepharose gel,
A removal step to remove plasmin, plasminogen, etc., an adsorption step in which the flow-through from the removal step is subjected to affinity chromatography, and a buffer solution is used to wash the adsorbed solution from the adsorption step to remove unadsorbed proteins. a first separation step of eluting adsorbed proteins from the washed solution using a buffer; and dialysis of the eluate from the first separation step using a buffer. and a second separation step of performing ion-exchange chromatography on the dialyzed eluate using an eluent and an ion exchanger, and a filtration step of gel-filtering the eluate from the second separation step. Method for purifying converted protease. (2) A step in which urine diluted in advance with distilled water is brought into contact with a siliceous gel, the proteins in the urine are adsorbed on the siliceous gel, and unadsorbed proteins in the urine are washed away using a buffer solution. a first washing step, a first separation step in which the proteins adsorbed to the silica gel from the first washing step are eluted using a buffer solution, and the eluted proteins from the first separation step are subjected to affinity chromatography. , and a second washing step of washing with a buffer solution, a second separation step of passing the washed protein from the second washing step through the buffer solution, and gel filtration of the eluted protein from the second separation step. A method for purifying urokinase converted protease, comprising a filtration step. (3) passing plasma through a lysine-Sepharose gel;
a first removal step for removing plasmin, plasminogen, etc.; a second removal step for salting out the pass-through from the first removal step and passing it through a lysine-Sepharose gel; and an eluent and ion exchange. a first separation step in which the pass-through from the second removal step is subjected to ion-exchange chromatography using a ion-exchange chromatograph; an intermediate filtration step in which the eluate from the first separation step is gel-filtered; and an adsorbent and a buffer. a second separation step in which the filtrate from the intermediate filtration step is subjected to adsorption chromatography using an eluent and an ion exchanger, and within a predetermined range so as to obtain an elution curve exhibiting two peaks;
a third separation step of controlling the acidity (pH) of the eluent and subjecting the active eluate from the second separation step to hydrophobic ion exchange chromatography. (4) A method for measuring the amount of urokinase converted protease antigen, which measures the amount of antigen using an antibody obtained by immunizing an animal with purified urokinase converted protease mixed with an adjuvant.