WO1995004077A1

WO1995004077A1 - Method of purifying plasminogen

Info

Publication number: WO1995004077A1
Application number: PCT/JP1994/001124
Authority: WO
Inventors: Shinobu Mochizuki; Takashi Kobayashi
Original assignee: The Green Cross Corporation
Priority date: 1993-07-29
Filing date: 1994-07-08
Publication date: 1995-02-09
Also published as: JPH0739375A

Abstract

Plasminogen is purified by bringing a plasminogen-containing composition into contact with an anion exchanger and recovering the unadsorbed fraction. The contact is performed by adjusting the concentration of a salt such as sodium chloride to 0.1-2 w/v%, preferably to 0.4-0.8 w/v%, and, if desirable, adjusting the pH to 6 to 8. This method is useful to remove contaminants, in particular, a high-molecular substance having a plasminogen anti-genicity, from a plasminogen-containing composition. Further it permits plasminogen to be purified in a short time efficiently in a good yield at a low cost. Therefore this method is advantageous for preparing pure plasminogen from a large quantity of a plasminogen-containing composition and is extremely useful as a process for producing plasminogen on an industrial scale.

Description

Specification

Purification method of plasminogen

Technical field

The present invention relates to a method for purifying plasminogen, and more particularly to a method for producing high-purity plasminogen from which contaminants in a plasminogen-containing composition have been removed.

Background art

Plasminogen is activated by perokinase, streptokinase and the like to form plasmin, which breaks down fibrin and causes fibrinolysis. It is attracting attention as a drug that can be applied clinically. In particular, the usefulness of lysyl-type plasminogen whose N-terminal is a lysyl residue is expected.

Conventionally, plasminogen has been purified by isoelectric point-acid extraction, ammonium sulfate fractionation, polyethylene glycol fractionation, etc., to obtain crude plasminogen with a specific activity of about 0.1 to 0.1 C UZnig. Methods are known. As a purification method for further improving the specific activity of the crude plasminogen, an affinity chromatography method using lysine-agarose is known.

According to this lysine-agarose treatment, plasminogen is purified to a very high degree of purity, but still contains impurities. Some of these impurities have plasminogen antigenicity and cannot be removed by affinity chromatography.

Therefore, in order to remove such contaminants, as further purification treatments, a gel filtration method for fractionation based on a difference in molecular weight, and a method for separation and fractionation by adsorption to an ion exchanger have been performed.

However, in the gel filtration method, in order to obtain a high separation / purification ability, generally, 1) the added volume of the sample is reduced (usually, the added sample volume is about 1 to 5 vZv% of the column volume). Techniques such as increasing the length (increase the bet height) and (3) reducing the flow velocity are required. For this reason, according to such a gel filtration method, it is necessary to perform a concentration operation of the sample to be added to the column beforehand. There is a problem in industrial use in that a gel is required, and a long time is required for separation and purification.

In addition, the conventional ion exchange treatment is a method of once adsorbing and separating on an ion exchanger. According to this method, (1) it is necessary to optimize the conditions for adsorption and separation on the ion exchanger in detail. (2) Timely adjustment is necessary even after optimization. (3) A large amount of buffer is required for separation and elution. (4) A large amount of ion exchanger is required to adsorb the target substance. There is a problem.

As described above, the conventional method is time-consuming, labor-intensive, and involves problems that need to be solved in terms of cost, and is unsuitable for treating a large amount of a plasminogen-containing composition.

An object of the present invention is to provide a method for removing contaminants in a plasminogen-containing composition, in particular, a polymer substance having plasminogen antigenicity. Still another object of the present invention is to provide a method for purifying plasminogen in a short time, efficiently, with a high yield, and economically.

Disclosure of the invention

The present inventors have conducted various studies on an efficient method for purifying plasminogen in order to solve the problems in the conventional method, and as a result, have been able to remove contaminants from the plasminogen-containing composition in a short time to obtain a highly pure This has led to the establishment of a method that can obtain brassminogen in high yield and economically.

That is, the present invention provides a method for purifying plasminogen, which comprises contacting a plasminogen-containing composition with an anion exchanger and collecting a non-adsorbed fraction thereof.

In the method of the present invention, the purification is preferably carried out under the conditions of a salt concentration of 0.1 to 2 wZv%, more preferably a salt concentration of 0.4 to 0.8 wZv%, and more preferably a pH of 6 to 8 Under the conditions. That is, a preferred embodiment of the present invention is a method for purifying plasminogen, which comprises contacting a plasminogen-containing composition with an anion exchanger under the specific conditions and collecting a non-adsorbed fraction thereof.

The plasminogen which is the target product in the purification method of the present invention includes methionyl type (N-terminal amino acid is methionine), glutamyl type (N-terminal amino acid is Examples are glutamic acid) and lysyl type (the N-terminal amino acid is lysine). Further, a mixture thereof may be used. Preferably, it is lysyl-plus minogen.

The plasminogen-containing composition as a starting material in the present invention is subjected to the method of the present invention as an aqueous solution. Plasminogen is present in blood, body fluids, placenta and the like, and a plasminogen-containing composition can be prepared therefrom by a generally known method.

The degree of purification of the plasminogen-containing composition upon contact with the anion exchanger is not particularly limited, and the method of the present invention can be applied to any degree of purification. Therefore, contact with the anion exchanger can occur at any stage of plasminogen purification.

3 © used.

The plasminogen-containing composition as a starting material is not particularly limited as long as it contains plasminogen.For example, it is obtained from serum, plasma, ascites, placental extract, placental tissue extract, and plasma. Composition or gene set consisting of fractions n + m or II of the low-temperature alcohol fractionation method of corn and other fractions obtained by treating plasma or tissue extract by various fractionation methods A culture solution obtained by a recombinant host or tissue culture and a commercially available plasminogen preparation are exemplified.

Before applying the method of the present invention, the plasminogen-containing composition is subjected to fractionation operations such as ammonium sulfate fractionation, polyethylene glycol fractionation or dextran fractionation, lysine-affinity chromatography, gel Various separation operations by a filtration method or a combination thereof may be performed. Preferably, lysine affinity chromatography has been performed.

The plasminogen-containing composition thus obtained is subjected to the following anion exchange treatment.

The anion exchanger is obtained by binding a strong or weakly basic functional group to a suitable support such as cellulose, dextran, a hydrophilic polymer, silica gel synthetic resin or agarose.

A strongly basic functional group refers to a basic residue having an ion-exchange ability that is completely dissociated in a wide range of PH. Specifically, getyl (2-hydroxy) Examples thereof include quaternary aminoethylene (QAE-) such as cypropyl) aminoethyl and getylmethylaminoethyl, and quaternary aminomethyl (Q-) such as trimethylaminomethyl. The weakly basic functional group refers to a basic residue having an ion exchange ability whose degree of dissociation, ie, exchange capacity, is significantly changed by PH. Specifically, getylaminoethyl (DEAE ), Aminoethyl (AE), etc. _c The anion exchanger used in the present invention is not particularly limited, but is preferably an anion exchanger formed by bonding a strongly basic functional group. Are those having a trimethylaminomethyl, trimethylaminoethyl, or getyl- (2-hydroxypropyl) aminoethyl group as a functional group. In particular, QAE—Toyopearl 550 C (manufactured by Toso Corporation) and Q—Sepharose Fast Flow (manufactured by Pharmacia) are preferred. Preferred conditions for contacting the brassminogen-containing composition with the anion exchanger are as follows: the salt concentration in the contacting environment is 0.1 to 2%, particularly 0.4 to 0.8 wZv%, and the pH is 6 to 8 It is. The buffer used is not particularly limited as long as it has a buffering capacity in the above pH range. Preferred are a Tris-HCl buffer and a phosphate buffer. The concentration of the buffer used is usually 0.005 to 0.2M, preferably 0.01 to 0.05M. Further, lysine or the like of about 0.2 wv% may be blended.

Examples of the salt to be used include sodium chloride, potassium chloride, sodium citrate and the like, and preferably sodium chloride.

It is preferable that the anion exchanger be sufficiently equilibrated under the above conditions before use.

The plasminogen-containing composition to be brought into contact with the anion exchanger under the above conditions is preferably prepared under the same buffer conditions as the anion exchanger. Examples of the preparation method include a dialysis method using the same buffer as an external solution as the buffer used for equilibrating the anion exchanger.

The thus-prepared solution containing plasminogen is brought into contact with the equilibrated anion exchanger, and the treatment is carried out at a temperature of 0 to 60 '(: preferably 4 to 25 ° C).

The gel capacity of the anion exchanger used depends on the plasminogen-containing solution to be contacted. The total amount of protein contained in the solution is preferably not less than 1 Omg.

Under these conditions, impurities contaminating the brassminogen-containing composition are adsorbed to the anion exchanger, but the target plasminogen is not adsorbed. Therefore, according to the purification method of the present invention, plasminogen and impurities can be separated by one operation, and high-purity plasminogen can be obtained from the plasminogen-containing composition.

The contaminants are included in the plasminogen-containing composition before being subjected to the anion exchange treatment under the above-described conditions, and are a comprehensive concept representing substances other than the monomeric plasminogen. . Specific examples include high-molecular substances having plasminogen antigenicity (plasminogen dimers and polymers, riboprotein A, etc.), riboproteins, and the like. The method of the present invention is particularly effective for removing these substances. It is. The plasminogen thus obtained has a specific activity of 24 CUZmg and a high recovery, and the method of the present invention is particularly effective for removing these.

The plasminogen-containing composition to be subjected to the anion exchanger treatment may have been subjected to an SD (Solvent Detergent) treatment before that.

The SD treatment can be performed according to a known method (JP-A-60-511116, JP-A-3-218322). It is also preferable to carry out the reaction in the presence of a surfactant. More preferably, as a stabilizer during the SD treatment, benzamidines (such as benzamidine, i> -ryminobenzamidine) and basic amino acids (arginine, Lysine, etc.), abrotinin or ε-aminocaproic acid (EACA). As the surfactant, Tween 80, Teen 20, TritonXlO0 or the like is used. The amount of stabilizing agent added is about 0.0001 to 0.1 M for benzamidines, about 0.1 to 1 M for basic amino acids, and 10 to 100% for aprotinin, and 1 to 10% for KIZmL EACA ( w / v) degree.

The anion exchanger treatment may be performed by any method such as a batch method, a column method, and a membrane method. Preferably, the column method is used. According to this method, a series of operations such as equilibration of the anion exchanger (gel), purification of brassinogen, regeneration of the gel, etc. can be repeatedly and efficiently performed economically with the column packed. it can.

The anion exchanger (gel) can be regenerated under extremely mild conditions, preferably by increasing the salt concentration, and used repeatedly for the purification of plasminogen. Plasminogen obtained by anion exchanger treatment can be subjected to further purification treatment if necessary. For example, plasminogen which is more highly purified can be obtained by subjecting it to affinity chromatography again using lysine or aprotinin as a ligand, or by dialysis.

The plasminogen thus purified is used as a pharmaceutical. Therefore, the plasminogen is preferably subjected to a treatment for virus inactivation. Normal processing is applied as the virus inactivation processing. Specifically, for example, a method of heating a liquid composition of brassinogen at 50 to 100 ° C for 5 to 30 hours, and a method of heating a dried composition at 50 to 100 ° C for 10 to 1 hour. Examples include a method of heating for 50 hours, a method of contacting with a surfactant, and a combination of these treatments.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Reference example 1

To 100 g of the extraction residue in the corn I + BI fraction of plasma was added 0.9 wZ V% sodium chloride-added 5 OmM Tris buffer (pH 8.3) 50, and the mixture was stirred for 16 hours. 2.5 g of dextran sulfate sodium salt and 1.5 g of magnesium chloride were added, and the mixture was stirred for 3 hours. The mixture was centrifuged at 4,000 rpm for 30 minutes, and 5 Q Qm 5 of the supernatant fraction was collected. 121 g of ammonium sulfate was added, and the mixture was stirred for 2 hours. After centrifugation at 4,000 rpm for 30 minutes, 15 Og of the precipitated fraction was recovered. 0.9 wZv% sodium chloride added 0.9 wZv glycine solution (pH 7.4) 300 was added and dissolved. The mixture was applied to a lysin agarose (trade name: lysine sepharose 4B) column equilibrated with 0.9wZv 0.9wZv% glycine solution (pH 7.2) added with sodium chloride. After washing with 1 M sodium chloride-added 0.9% wZv glycine solution (pH 7.2), elution was carried out with 0.9 wZv% sodium chloride-added 0.25 M lysine solution (ρΗ7.2) to obtain eluate 35. Concentrate using an ultrafiltration membrane (trade name: Minitan, molecular weight cut off 30,000, manufactured by Millipore), and convert to 0.45 w / v% sodium chloride / 0.9 wZv% glycine solution (ρΗ7.2). PH 7.2.

To this, tri-W-butyl phosphate (TNB P) and Tween 80 were added so that the final concentrations were 0.3% (w / v) and 1% (w / v), respectively, and 30 was added. The mixture was heated for a period of time to obtain a plasminogen-containing solution.

Example 1

The plasminogen-containing solution obtained in Reference Example 1 was added to each of the anion exchangers shown in Table 1 (ø5 mm x 5 cm; 1), and ion-exchange chromatography (flow rate: 0.5 ^ / min. Buffer: 0.9 wZv% glycine, 0.45 w / v% NaC £, pH 7.2) The unadsorbed fraction that passed through the column as it was was analyzed by gel filtration (hereinafter referred to as GPC). Each ion exchanger had been equilibrated with the above buffer in advance.

Recovery rate of plasminogen by each column treatment [Recovery rate of recovered protein mass (activity) when protein amount before treatment is 100, same hereafter. ] And (when the sample was G PC analysis, A _28. Impurities relative _nm A _28. _Nm ratio of the sample, the same. Or less) amount of impurities shown in Table 1.

table 1

Example 2

OS epharose Fast Fow (05 dragon x 5 cm; 1 m; Fuar Mashia) and QAE—To yopearl 55 0 C (ø5 mm x 5 cm; 1 nil; Toso One), Su per Q—To yopearl 65 0 M (ø 5 nun x 5 cm; \ nH Plasminogen obtained in Reference Example 1 was subjected to ion exchange chromatography (flow rate: 0.5 / min .; equilibration and washing buffer: 2 OmM Tris buffer). , 0.2 wZv% lysine, 0.45 wZv% NaC pH 7.2; Elution buffer: 2 OmM Tris buffer, 0.2 wZv% lysine, 1 M NaC pH 7.2). The unadsorbed fraction that passed through the column as it was and the adsorbed fraction eluted with the elution buffer were analyzed by GPC to determine the plasminogen recovery rate and the amount of impurities. Table 2 shows the results.

Table 2

Example 3

① Effect of salt concentration

Perform ion-exchange chromatography (flow rate: 0.5 Zmin.) On the plasminogen-containing solution obtained in Reference Example 1 using Q-Sepharose Fast Flow (ø5 mm X 5 cm; 1 τηβ; manufactured by Pharmacia). In this case, the salt concentration of the equilibration and washing buffer used (2 OmM Tris buffer, 0.2 w / v% U-zine, pH 7.2) was adjusted to 0, 0.1, 0.1 5, 0.3, 0.4, 0.4 5, 0.6 , 0.75, 0.8, 0.9 w / v% and the effect of salt concentration on plasminogen recovery and purification were investigated. Get Table 3 shows the results of measurement by GPC of the unadsorbed fraction and the adsorbed fraction eluted with the elution solution (2 OmM Tris buffer, 0.2 wZv% lysine, 1 MNa C £, PH 7.2). Show.

Table 3

② Influence of pH

The plasminogen-containing solution obtained in Reference Example 1 is subjected to ion-exchange chromatography (flow rate: Ο, δτ ^ ιΰη.) Using Q-Sepharose Fast Flow (ø5 mm x 5 cm; 1 τη &; manufactured by Pharmacia). At this time, change the ^ 1 of the equilibration buffer (2 OmM Tris buffer, 0.2 w / v% lysine, 0.45% 〇 3〇 ^) to 6.2, 6.7, 7.2, 7.7, 8.3, The effect of pH on gen recovery and purification was investigated. The results of GPC measurement of the unadsorbed fraction obtained and the adsorbed fraction eluted with the elution solution (2 OmM Tris buffer, 0.2 wZv% lysine, 0.1 M Na Ci, pH 7.2) are shown in Table 4. Show. Table 4

③Effect of lysine concentration and buffer concentration

Perform ion-exchange chromatography (flow rate: 0.5 Zmin .; pH 7.2) on the plasminogen-containing solution obtained in Reference Example 1 using Q-Sepharose Fast Flow (ø5 mm x 5 cm; 1 id Pharmacia). At this time, the lysine concentration of the equilibration and washing buffer (0.9 wZv% glycine, 0.45 w / v% NaC ^) and the concentration of Tris buffer were changed to improve the recovery and purification of plasminogen. Effects were investigated. Tables 5 and 6 show the results of measurement of the obtained unadsorbed fraction by GPC.

Table 5: Effect of lysine concentration and tris concentration (impurity amount:%)

Example 4 (Effect of removing high molecular substances)

Q- S epharose Fast? 1

Pharmacia) and the plasminogen-containing solution obtained in Reference Example 1 was applied in different amounts (1.0, 0.5, 0.), and ion exchange chromatography was performed (flow rate: 0.5 mS). min.; Equilibration buffer: 0.9 w / v% glycine, 0.45 w / v% sodium chloride, ρΗ7.2, Elution buffer: 0.9 wZv% glycine, 1 M sodium chloride, pH 7.2) The unadsorbed fraction was analyzed by GPC and the results are shown in Table 7. Table 7: GPC analysis of unadsorbed fractions (polymer content:

Figure 1 shows the results of analysis of plasminogen treated with ion-exchange chromatography on gel filtration chromatography. In the figure, 1 shows the peak of the polymer substance. In the starting sample containing the molecular substance, the addition of the gel (imO 1Z5 amount (0.2 to 1Z10 amount (0.1) removed the high molecular substance completely).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing the results of analysis of plasminogen treated with ion exchange chromatography in Example 4 by gel filtration chromatography.

In the figure, “untreated” indicates a gel filtration chromatogram of a plasminogen-containing solution that has not been subjected to ion exchange chromatography. The amounts shown at the upper left of each chromatogram (1.0 m £, 0.5 md, 0.5 li, 0.1 indicate the amount of the plasminogen-containing solution added to the ion exchanger.

Industrial applicability

According to the purification method of the present invention, it is possible to easily remove substances contaminating the plasminogen-containing composition, especially high molecular substances having plasminogen antigenicity. A high-purity plasminogen can be obtained from a gen-containing composition in a short time, efficiently, with good yield, and economically. Therefore, it is particularly advantageous in purification from a large amount of a plasminogen-containing composition, and is extremely useful as a process for producing plasminogen on an industrial scale.

Claims

The scope of the claims

1. A method for purifying plasminogen, comprising contacting a plasminogen-containing composition with an anion exchanger and collecting a non-adsorbed fraction thereof.

2. The plasminol according to claim 1, wherein the plasminogen-containing composition is brought into contact with the anion exchanger under a salt concentration of 0.1 to 2%, preferably 0.4 to 0.8%. Gen purification method.

3. The method for purifying plasminogen according to claim 1, wherein the plasminogen-containing composition is brought into contact with an anion exchanger under conditions of pH 6 to 8.

4. The method for purifying brassinogen according to claim 1, wherein the brassinogen is lysyl-brasminogen.

5. The method for purifying plasminogen according to claim 1, wherein the plasminogen-containing composition has been subjected to lysine affinity chromatography.

6. Purification of plasminogen according to claim 1, wherein the anion exchanger is formed by bonding a trimethylaminomethyl, trimethylaminoethyl, or getyl- (2-hydroxypropyl) aminoethyl group. Method.

7. The method for purifying plasminogen according to claim 2, wherein the salt is sodium chloride.

8. Plasminogen one plasminogen-containing composition, the method of purifying brass plasminogen according to claim 1, wherein the characterized by being prepared in the anion exchanger and the same buffer conditions _c

9. The method for purifying plasminogen according to claim 1, wherein the plasminogen-containing composition is brought into contact with an anion exchanger in a Tris-HCl buffer or a phosphate buffer.

10. The method for purifying plasminogen according to claim 1, wherein the gel capacity of the anion exchanger is not less than 1 Omg of total protein contained in the plasminogen-containing solution.

11. The method for purifying plasminogen according to claim 1, wherein the anion exchanger treatment is a column method.