JP2007068497A - Purification method of plasmin - Google Patents

Abstract

【Task】
Provided is a neutral plasmin composition that is constructed by plasma or genetic engineering techniques and is substantially free of plasminogen activator and has high purity and high specific activity.
[Solution]
After purifying plasminogen derived from plasma or a gene recombinant by a known method, it is contacted with a plasminogen activator to activate the plasminogen to plasmin, and then the plasmin-containing fraction is converted to omega-amino acid. The above-mentioned problems have been solved by subjecting it to affinity chromatography and gel filtration chromatography.
[Selection figure] None

Description

  The present invention relates to a method capable of highly purifying neutral plasmin. More specifically, the present invention relates to a method for producing neutral plasmin having a high specific activity that suppresses autolysis or inactivation of neutral plasmin and is substantially free of viruses, contaminants, degradation products, and plasminogen activator. .

Traditionally, a plasminogen activator is used to convert plasminogen, an inactive precursor of plasmin, to active plasmin, but the plasminogen activator is functional in plasmin, In other words, it may remain in such an amount that it exhibits its function to some extent, and therefore, preparation of pure plasmin substantially free of this is awaited.
That is, many of the plasmin preparations are slightly contaminated with plasminogen activators such as streptokinase or urokinase, and therefore the plasmin activity when administered in the body is rather that of plasminogen activators that are contaminated rather than plasmin itself. Affected and at risk of inducing bleeding. Plasmin preparations containing streptokinase can also cause immune reactions such as fever and anaphylactic shock.

  Another problem is that plasmin, which has a broad spectrum as a serine protease, tends to be highly self-degrading during its purification and lose its activity as a serious technical barrier limiting the clinical use of plasmin. It is. These conditions were factors that hindered the production of high-quality plasmin.

  It is known that the stability of plasmin is improved by addition of fibrinogen, ε-aminocaproic acid, high ionic strength, glycerol, and further addition of tranexamic acid, lysine and the like. However, these methods only stabilize for a very short time, and the activity of plasmin is significantly reduced during long-term storage. Moreover, since the essence of the plasmin stabilization effect by the addition of ε-aminocaproic acid, tranexamic acid, etc. is an activity inhibitor of plasmin, for example, the fibrinolytic activity such as fibrin clot dissolving activity is reduced. I could invite you. Therefore, it is preferable that the stabilizer of the plasmin preparation as a medical drug is reduced in concentration or not added as much as possible.

Furthermore, Dadd Christopher et al. Disclose plasmin compositions useful for thrombolysis therapy (Patent Documents 1 and 2). This composition is prepared and stored using a low pH-buffering agent to provide a substantially stable formulation. However, these formulations are stabilization of plasmin at a low pH, and since it is necessary to make the pH near neutral from the viewpoint of safety for administration as a pharmaceutical, no fundamental solution has been reached.
On the other hand, benzamidine has been disclosed as an affinity carrier that specifically adsorbs plasmin (Patent Document 1). However, benzamidine is extremely toxic and has a problem in safety as a carrier for producing a medical drug. Is not necessarily a suitable carrier.
Special table 2003-535574 gazette JP-T-2003-514789

  The present inventors consider that the pH of the plasmin-containing fraction should be near neutral from the viewpoint of safety as a pharmaceutical product, and stable plasmin that suppresses autolysis or inactivation under the neutral condition We have repeatedly studied to establish a purification method.

  The present inventors have performed relatively simple operations on highly purified neutral plasmin that has been inactivated / removed from virus and is substantially free of plasmin degradation products, contaminants, and plasminogen activator. As a result of intensive research on methods for obtaining high yields, plasminogen derived from plasma, induced plasma fractions or recombinants, for example, plasminogen-containing solutions purified by various methods are used as starting materials. A plasminogen activator, such as urokinase or tissue plasminogen activator, preferably immobilized on a carrier such as NHS-activated sepharose or cyanogen bromide-activated sepharose, and the plasminogen is The plasmin-containing fraction is activated by bringing it into contact with the carrier for immobilizing factor and activating it to plasmin. Moth - I learned that intended purpose can be achieved by subjecting the affinity chromatography and gel filtration chromatography comprises the amino acid.

That is, the present invention
(1)
Placing plasma-derived or genetically engineered plasminogen with a plasminogen activator to give plasmin, followed by an affinity chromatography step comprising omega-amino acids and a gel filtration chromatography step Purification method of sex plasmin,
(2)
The purification method according to (1), wherein the omega-amino acid is lysine, arginine, polylysine, ε-aminocaproic acid, tranexamic acid, β-alanine, γ-aminobutyric acid, σ-aminovaleric acid or a homologue thereof.
(3)
The purification method according to (1), wherein the omega-amino acid is lysine,
(4)
The purification method according to (1), wherein the plasminogen activator is an immobilized plasminogen activator,
(5)
The purification method according to (4), wherein the immobilized plasminogen activator is urokinase, tissue plasminogen activator, streptokinase or staphylokinase,
(6)
The purification method according to (1), wherein the gel filtration chromatography has a molecular weight cut-off of 10,000 to 1,500,000,
(7)
(1) In the plasmin purification step described above, glycerol is 1 to 30 v / v%, glycine is 1 to 30 w / v%, monoethanolamine is 0.1 to 5 v / v%, or glycylglycine is 0.5 to 15 w. The purification method according to (1), wherein a neutral buffer containing / v% is used,
(8)
In the affinity chromatography step comprising omega-amino acids, the purification method according to (1), wherein a neutral buffer containing 1 to 30 v / v% glycerol or 1 to 30 w / v% glycine is used,
(9)
In the gel filtration chromatography step, 1-30 v / v% glycerol, 0.1-5 v / v% monoethanolamine, 1-30 w / v% glycine or 0.5-15 w / v% glycylglycine The purification method according to (1), using a neutral buffer containing
(10)
The purification method according to any one of (7) to (9), wherein the neutral buffer has a pH of 6 to 8, and (11)
Neutral plasmin having an impurity content of less than 15% and a specific activity of 8 IU / A280 or more,
It is.

Any starting material plasminogen may be used as long as it has been purified by a conventionally known method. For example, it may be purified by affinity chromatography with lysine sepharose described by Deutsch & Mertz (1970) from plasma or from corn fraction II + III paste or the like obtained by further purifying plasma.
As another example, after dissolving and extracting the corn fraction II + III paste, adjusting the pH to 6.1 ± 0.1, collecting the precipitate by isoelectric precipitation, treating the precipitate solution with DEAE-Sephadex, It may be purified by Sephadex G-150 gel filtration chromatography (Methods in Enzymology, Vol. 19 p186-191).
The origin of plasminogen may be not only derived from human or mammalian plasma, but also derived from a recombinant such as a recombinant cell culture or a transgenic animal.

  Plasmin, the major fibrinolytic enzyme in mammals, is a serine proteolytic enzyme that is generated when plasminogen, an inactive precursor, is activated by a plasminogen activator at a catalytic concentration. Plasminogen, an inactive precursor, is a 791 amino acid single-chain glycoprotein that is activated by plasminogen activators such as urokinase, tissue plasminogen activator, streptokinase, and staphylokinase. When the Arg561-Val562 peptide bond undergoes limited degradation, it is cleaved into double-stranded plasmin. The two polypeptide chains of plasmin produced are retained by two internal chain disulfide bonds. The heavy chain on the N-terminal side of plasmin has five kringle structures that are triple loops with highly similar amino acid sequences. The kringle structure has a lysine binding site for interaction with α2-plasmin inhibitor, fibrin or other proteins. The light chain on the C-terminal side of plasmin has a catalytic center, and its enzymatic activity is homologous to trypsin and other serine proteases.

Plasmin not only degrades fibrin, blood coagulation factors V, VIII, fibrinogen, ACTH, etc., but also activates procollagenase and damages vascular wall endothelial cells. The activation reaction of plasminogen is skillfully controlled by an activation reaction inhibitor such as PAI-1 or the like, fibrin or an activator of plasminogen on the cell surface, and the produced plasmin is controlled by an α2-plasmin inhibitor. ing.
Examples of plasminogen activator include urokinase, tissue plasminogen activator, streptokinase, staphylokinase, etc., but other factors that can activate plasminogen are limited to this. There is no. A preferred plasminogen activator is urokinase. In these activation reactions, by adding a polyhydric alcohol such as glycerol, ethylene glycol, polyethylene glycol, or propylene glycol, the stability of plasmin itself can be improved and the yield can be enhanced.

By bringing the plasminogen-containing solution into contact with a plasminogen activator, the plasminogen-containing solution can be converted into an active form of plasmin. This contact is preferably carried out by immobilizing the plasminogen activator on a carrier and using it as an insoluble plasminogen activator, the released free plasminogen activator is mixed into the resulting solution. It can be prevented from coming.
The plasminogen activator can be immobilized by any method as long as it does not affect the activity of the activator and can be immobilized. For example, carrier binding methods include CNBr-activated Sepharose 4FF / 4B, NHS-activated Sepharose 4FF, ECH Sepharose 4B (Amersham Bioscience), Affi-Prep 10 (Bio-Rad), in which primary amino groups are covalently immobilized. ), Reacti-Gel (PIERCE), Epoxy-activated Sepharose 6B (Amersham Bioscience) for immobilizing primary amino groups, hydroxyl groups and thiol groups, EAH Sepharose 4B (Amersham bioscience) for selectively immobilizing carboxyl groups ), Thiopropyl Sepharose 6B that immobilizes substances containing thiol groups with disulfide (SS) bonds, Activated Thiol Sepharose (Amersham Biosciences), Affi- that immobilizes ligands covalently with oxidized aldehyde groups Prep Hz hydrazine carrier (Bio-Rad), TNB-Thiol for immobilizing ligands with sulfhydryl groups, Immobi lized p-Chloromercuribenzoate (PIERCE). In addition, a cross-linking method in which the ligand is immobilized by bridging between the ligands using a cross-linking reagent such as glutaraldehyde having two or more reactive groups, or the ligand is encapsulated in the lattice of the gel, or the ligand is formed by a semipermeable membrane. There is known a comprehensive method for immobilizing a ligand by coating with a microcapsule.

Further, as a carrier that can be used in the carrier binding method, insoluble substances derived from synthesis or nature are usually used, and for example, cellulose, agarose, crosslinked dextran, polyacrylamide, porous glass and the like are known.
It is preferable to use NHS activated sepharose (N-hydroxysuccinimide activated sepharose) for the activation of plasminogen to plasmin.

  The step of activating plasminogen to plasmin by the immobilized plasminogen activator is usually carried out by contacting the plasminogen-containing solution with the immobilized plasminogen activator at about 4 ° C. to about 37 ° C. It is typically completed by contact for about 15 minutes to 20 hours. The activated plasmin solution and the immobilized plasminogen activator can be separated by filtering the activated plasmin solution using a carrier-supporting mesh or filters.

  The plasmin activated by the plasminogen activator is then subjected to a step of binding to affinity chromatography comprising an omega-amino acid specific for the activated plasmin, resulting in a substantially high molecular weight mixture or heavy product. Remove the coalescence and also the inactive autolysis product of plasmin. The affinity chromatography step comprising this omega-amino acid makes it possible to concentrate plasmin and to use it in the next step of gel filtration chromatography.

Since plasmin has a lysine binding site, it can be specifically adsorbed by using an affinity adsorbent in which an omega-amino acid having a lysine-like structure is immobilized on a carrier.
An omega-amino acid having a lysine-like structure is a straight chain of 2 to 6, preferably 5 carbon atoms between a carboxyl group and an amino group (one of which is substituted with a nitrogen atom) Specifically, lysine, arginine, polylysine, ε-aminocaproic acid, tranexamic acid, β-alanine, γ-aminobutyric acid, σ-aminovaleric acid, and their homologues It is at least one selected from the group consisting of bodies. Particularly preferred is lysine. Homologues include those having a group that is not normally reactive as a side chain in the main chain of each compound, for example, a lower alkyl group such as a methyl group.
As the carrier, insoluble carriers such as cellulose, agarose, dextran, vinyl and polyacrylamide are used. Further, those having a hydrophilic spacer (long chain) between the carrier and the ligand can also be used.

  A neutral buffer is used for the affinity chromatography step comprising the omega-amino acid. The pH of the buffer is 6-8, preferably pH 6.5-7.5. For example, citric acid-disodium phosphate buffer, citric acid-sodium citrate buffer, β, β'-dimethylglutaric acid-sodium hydroxide buffer, sodium cacodylate-hydrochloric acid buffer, sodium maleate-hydroxylated Sodium buffer solution, phosphate buffer solution, imidazole-hydrochloric acid buffer solution, 2,4,6-trimethylpyridine-hydrochloric acid buffer solution and the like can be used. Preferred is a phosphate buffer. The concentration of the buffering agent in the buffer is usually 1 to 100 mM, preferably 10 to 75 mM, and more preferably 20 to 50 mM. This neutral buffer is preferably used as a buffer in all the steps of the present invention.

  In an affinity chromatography step comprising an omega-amino acid, neutral buffer is used as a stabilizer for plasmin, for example, polyhydric alcohols such as glycerol, ethylene glycol, polyethylene glycol, propylene glycol, glycine, alanine, asparagine, Oligopeptides consisting of amino acids such as methionine, and amino acids selected from alanine, asparagine, glycine, methionine and the like can be added alone or in combination. Preferable are glycerol and glycine, and glycylglycine may be further added to these. More specifically, glycerol is 1-30 v / v%, preferably 5-15 v / v%, glycine is 1-30 w / v%, preferably 5-15 v / v%, glycylglycine is 0.5 It is used at a concentration of ˜15 w / v%, preferably 7 to 12 w / v%.

  Plasmin bound to an affinity carrier comprising omega-amino acids is lysine, arginine, polylysine, ε-aminocaproic acid, tranexamic acid, β-alanine, γ-aminobutyric acid, σ-aminovaleric acid or their homologues, etc. It can be eluted with a buffer containing omega-amino acids, preferably a buffer containing lysine. Suitably, lysine can be eluted with a neutral buffer containing 10 to 500 mM, preferably 150 to 350 mM, pH 6 to 8, more preferably 6.5 to 7.5. The final eluted plasmin solution is neutral and substantially free of high molecular weight contaminants or polymers, as well as inactive autolysates of plasmin.

The plasmin eluted by the affinity chromatography step is then subjected to gel filtration chromatography for further purification and exchange for a buffer containing pharmaceutically and pharmaceutically acceptable additives as the final plasmin formulation. Attached to the process.
Gel filtration chromatography can use a molecular weight cut off of 1,000 to 8,000,000, and more preferably a separation carrier that can be separated in the range of 10,000 to 1,500,000. Examples thereof include insoluble carriers such as cellulose, agarose, dextran, vinyl and polyacrylamide. Specifically, Superdex, Superose, Sephacryl (all of which are manufactured by Amersham Biosciences), Bio-Gel ( Bio-Rad), Toyopearl (Tosoh), Cellulofine (Biochemical Industrial), and the like.

  In gel filtration chromatography, neutral buffer, plasmin stabilizer, polyhydric alcohols such as glycerol, ethylene glycol, polyethylene glycol, propylene glycol, primary amines such as methylamine hydrochloride and monoethanolamine Oligopeptides consisting of amino acids such as glycine, alanine, asparagine, methionine, etc., amino acids selected from alanine, asparagine, glycine, methionine, etc. Can be separated and purified. Preferably, glycylglycine, glycerol, glycine, and monoethanolamine can be used, and among them, glycylglycine is preferable. More specifically, glycerol is 1-30 v / v%, preferably 5-15 v / v%, glycine is 1-30 w / v%, preferably 1-5 v / v%, glycylglycine is 0.5 It is used at a concentration of ˜15 w / v%, preferably 3 to 12 w / v%, monoethanolamine 0.1 to 5 v / v%, preferably 0.3 to 1 v / v%.

  On the other hand, as a method for desalting and molecular weight fractionation, an ultrafiltration method is generally known. For example, Biomax, Ultracel (Millipore), CENTRASETTE, CENTRAMATE, MAXIMATE, MAXISETTE (Paul), Zaltcon, Hydrosart (Sartorius), etc. are commercially available and are industrially used for protein desalting, concentration and purification. However, in these methods, when desalinating, concentrating, and purifying an activated substance typified by plasmin, significant deactivation occurs due to physical abuse or the like, so it may not always be suitable for separation of the target substance. .

The finally obtained plasmin solution may be used as a liquid, or may be lyophilized and dissolved at the time of use.
For a more stable preparation, pharmaceutically acceptable additives such as sugar alcohols such as mannitol and sorbitol, or disaccharides such as sucrose, lactose and maltose, or glucose as protective substances that prevent enzyme denaturation , Saccharides such as galactose, mannose, fructose, or amino acids such as lysine, arginine, glycine, histidine, isoleucine, glutamic acid, aspartic acid, alanine, valine, leucine, asparagine, glutamine, or lysine, arginine, glycine, histidine, isoleucine , Oligopeptides consisting of amino acids selected from glutamic acid, aspartic acid, alanine, valine, leucine, asparagine, glutamine, or glycerol, ethylene glycol, polyethylene glycol Le, polyhydric alcohols alone or can be used in combination thereof (freeze-drying and protective substance Nei outer Yoshio Hen 1972 Foundation University of Tokyo Press), such as propylene glycol.

Depending on the site and affected area where the drug is administered, the pH and osmotic pressure ratio of the drug are very important factors. The plasmin preparation obtained by the present invention has a pH of 6 to 8, preferably 6.5 to 7.5 and is isotonic with respect to the osmotic pressure ratio, and is more preferable from the viewpoint of safety of pharmaceuticals. is there.
Furthermore, the plasmin obtained by the present invention has a very low impurity content of less than about 15% because the high-molecular-weight mixture, the polymer, and the inactive autolysis product of plasmin are sufficiently removed. Conventionally, human serum albumin has been added as a stabilizer because plasmin is poorly stable near neutral, but neutral plasmin obtained in the present invention is a proteinaceous stabilizer such as human serum albumin. The specific activity is high and can be 8 IU / A280 or more, preferably 10 IU / A280 or more, more preferably 12 IU / A280 or more.
Therefore, when a drug is administered into the eye in anticipation of a direct pharmacological effect such as thrombolysis or vitreous dissolution, the dose of the drug solution is limited to a very small amount of 0.1 mL or less. Neutral plasmin with high specific activity obtained in (1) exhibits its effect sufficiently in that amount. In addition, since it is not necessary to administer unnecessary protein components into the body to treat an original disease such as adding human serum albumin, there is less risk of complications. The resulting neutral plasmin is very good.

  EXAMPLES The present invention will be described more specifically with reference to the following examples, but it goes without saying that the present invention is not limited thereto.

  1 L of 20 mM L-lysine hydrochloride and 100 mM NaCl-containing Tris buffer (pH 9) was added to 100 g of the precipitated fraction III obtained from normal human plasma by the low temperature ethanol fractionation method, and the mixture was allowed to stand. The obtained supernatant was loaded onto ECH lysine sepharose 4FF (Amersham Biosciences) equilibrated with 50 mM phosphate buffer (pH 7.5). After washing with 50 mM phosphate buffer (pH 7.5) containing 500 mM NaCl, elution with 50 mM phosphate buffer (pH 7.4) containing 100 mM NaCl and 0.25 M L-lysine hydrochloride, the plasminogen-containing solution was obtained. Obtained.

  0.4 mL of urokinase immobilized on NHS-activated Sepharose carrier is added to 10 mL of the concentrated plasminogen fraction containing 10 v / v% glycerol obtained above, and activated to plasmin while slowly mixing for 2 hours at room temperature. Went. Thereafter, the urokinase-immobilized carrier is removed from the drug solution using a nylon mesh capable of holding the carrier, and the obtained plasmin fraction containing 10 v / v% glycerol is added with 10 mM w / v% glycine and 20 mM phosphoric acid containing 10 v / v% glycerol. ECH lysine sepharose 4FF (Amersham Bioscience) equilibrated with buffer (pH 7) was loaded. After washing with the above buffer solution, the purified plasmin-containing solution was eluted with 50 mM phosphate buffer (pH 7) containing 100 mM NaCl, 10 w / v% glycine, 10 v / v% glycerol, 0.25 M L-lysine hydrochloride. Obtained.

  The purified plasmin-containing solution obtained above was loaded on a Sephacryl S-300 (Amersham Biosciences) gel filtration carrier equilibrated with 20 mM neutral phosphate buffer (pH 7), and after removing the polymer peak, the main peak was A final plasmin solution was collected.

The purified plasmin-containing solution was loaded on a Sephacryl S-300 (Amersham Bioscience) gel filtration carrier equilibrated with 20 mM neutral phosphate buffer (pH 7) containing 10 w / v% glycylglycine, and a polymer peak was obtained. After removal, the main peak was collected and used as the final plasmin solution.
[Experimental Example 1]

In the purification method described above, Example 1 in which no stabilizer was added to neutral buffer, Example 2 in which stabilizer was added to neutral buffer, Comparative Example 1 in which lysine sepharose was not used, and gel filtration instead The activity yield and the specific activity were compared for the solutions obtained in Comparative Example 2 using ultrafiltration membranes. The results are summarized in Table 1.
Plasmin activity (IU / mL) was tested using HD-valyl-L-leucyl-L-lysyl-p-nitroanilide / dihydrochloride (S-2251) synthetic substrate with plasmin international standard as standard. Measured with Team PLG-2 kit (Daiichi Chemical Co., Ltd.). The activity yield (%) was calculated by the following formula.
Activity yield (%) = [{Plasmin activity after purification (IU / mL) × Liquid volume (mL)} / {Plasmin activity before purification (IU / mL) × Liquid volume (mL)} × 100]
Specific activity (IU / A280) was calculated by dividing plasmin activity (IU / mL) by the absorption value at A280 nm. The absorption value at A280 nm was measured at a measurement wavelength of 280 nm by placing a sample in a 10 mm cell with a UV-1600GLP type double beam spectrophotometer (manufactured by Shimadzu Corporation).
Purity (%) was measured by high performance liquid chromatography analysis. The column was TSK-GEL (G3000SWXL 7.8 cm × 30 cm, manufactured by Tosoh Corporation), the sample was developed at a flow rate of 0.4 mL / min, and the elution pattern was detected at 280 nm.

表１から明らかなように、実施例１、２の最終プラスミン溶液は比較例１、２の最終プラスミン溶液に比して有意に活性収率、比活性が高かった。
〔実験例２〕

As is clear from Table 1, the final plasmin solutions of Examples 1 and 2 had significantly higher activity yield and specific activity than the final plasmin solutions of Comparative Examples 1 and 2.
[Experimental example 2]

Using a commercially available plasmin solution (commercially available reagents from Company A, Company B and Company C) and the final plasmin solution of Example 2 purified by this method, high-performance liquid chromatography analysis was performed to measure purity.
As shown in Table 2, it was proved that the product of this example had significantly less impurities and a higher specific activity than the commercially available product.

Plasmin obtained by the present invention is a thromboembolism, myocardial infarction with thrombosis due to arterial blood coagulation, cerebral infarction, pulmonary thrombosis, pulmonary infarction with thrombosis due to venous blood coagulation, obstructive stroke, deep vein thrombosis , Peripheral arteriovenous disease or myocardial infarction, cerebral infarction, mesenteric artery thrombosis, ischemic colitis, portal vein / mesenteric venous thrombosis, transplantation vascular occlusion, deep venous thrombosis, etc. It is useful as a preventive and therapeutic drug for Furthermore, plasmin obtained by the present invention has low byproduct contamination and high specific activity, so retinal detachment, endophthalmitis, vitreous hemorrhage, vitreous opacification, proliferative vitreoretinopathy, diabetic retinopathy, proliferation Diabetic retinopathy such as diabetic retinopathy, diabetic macular disease, macular diseases such as macular hole, premacular membrane, cystoid macular edema, central retinal vein occlusion, branch retinal vein occlusion Even if it is used for vitrectomy for eye diseases accompanied by body peeling (PVD), the effect is sufficiently exerted, so that it is particularly useful.

Claims (11)

  Placing plasma-derived or genetically engineered plasminogen with a plasminogen activator to give plasmin, followed by affinity chromatography step comprising omega-amino acid and gel filtration chromatography step Purification method of sex plasmin.   The purification method according to claim 1, wherein the omega-amino acid is lysine, arginine, polylysine, ε-aminocaproic acid, tranexamic acid, β-alanine, γ-aminobutyric acid, σ-aminovaleric acid or a homologue thereof.   The purification method according to claim 1, wherein the omega-amino acid is lysine.   The purification method according to claim 1, wherein the plasminogen activator is an immobilized plasminogen activator.   The purification method according to claim 4, wherein the immobilized plasminogen activator is urokinase, tissue plasminogen activator, streptokinase or staphylokinase.   The purification method according to claim 1, wherein the gel filtration chromatography has a molecular weight cut-off of 10,000 to 1,500,000.   The plasmin purification step according to claim 1, wherein glycerol is 1 to 30 v / v%, glycine is 1 to 30 w / v%, monoethanolamine is 0.1 to 5 v / v%, or glycylglycine is 0.5 to 0.5%. The purification method according to claim 1, wherein a neutral buffer containing 15 w / v% is used.   The purification method according to claim 1, wherein a neutral buffer containing 1 to 30 v / v% glycerol or 1 to 30 w / v% glycine is used in the affinity chromatography step comprising an omega-amino acid.   In the gel filtration chromatography step, 1-30 v / v% glycerol, 0.1-5 v / v% monoethanolamine, 1-30 w / v% glycine or 0.5-15 w / v% glycylglycine The purification method according to claim 1, wherein the neutral buffer is used.   The purification method according to any one of claims 7 to 9, wherein the neutral buffer has a pH of 6-8. Neutral plasmin having an impurity content of less than 15% and a specific activity of 8 IU / A280 or more.