GB2068002A - Plasminogen preparations and their production - Google Patents

Abstract

A plasminogen preparation contains at least one of a physiologically acceptable inorganic salt, lysine, a salt thereof, phenylmethanesulfonyl fluoride, aprotinin, or soybean trypsin inhibitor in an amount effective for stabilizing plasminogen. The preparation may take the form of an aqueous solution or a dry powder. The preparation can be prepared by adding the abovementioned stabilizer or stabilizers to an aqueous solution containing plasminogen, if necessary subjecting the resulting solution to digestion at 4 DEG to 37 DEG C at pH 2 to 4 for 20 to 60 minutes, and then to heat-treatment at 60 DEG C for 10 hours, and lyophilizing the resulting solution.

Description

SPECIFICATION Plasminogen preparations and their production The present invention relates to a preparation of plasminogen originated from warmblooded animals including humankind and to a method for stabilization thereof. More particularly, it relates to a plasminogen preparation characterized by being admixed with an effective amount of a plasminogen stabilizer and to a method for stabilizing a plasminogen preparation characterized by adding thereto an effective amount of a plasminogen stabilizer.

Plasminogen is activated by urokinase or by streptokinase turning into plasmin, which in turn decomposes fibrin into soluble matter, i.e., causes fibrinolysis, so that plasminogen is noted together with urokinase and with streptokinase as a medicine widely available for clinical uses besides for the treatment of thrombosis. It is known, however, plasminogen is inactivated by treatment under severe conditions such as heat treatment or lyophilization or by a long-term storage. This a drawback to be improved in the production of a plasminogen preparation.

In view of the above, the present inventors made extensive studies, and as a result, have found that, when a particular compound, which was selected from a great number of compounds, is added to a plasminogen preparation in a sufficient amount and preferably within a range of specified proportions, plasminogen can be stabilized even under severe conditions such as those of heat treatment or of lyophilization, needless to say, under usual mild conditions. Thus, they have accomplished the present invention.

An object of this invention is to provide a method for stabilizing plasminogen which comprises adding an effective amount of plasminogen stabilizer to an aqueous solution containing plasminogen or to plasminogen dry powder.

Another object of this invention is to provide stabilized plasminogen preparations.

Other objects and advantages of this invention will be apparent from the following description.

The plasminogen-containing aqueous solution used in this invention is not particularly limited. It includes those obtainable by various known purification methods from the plasminogen-containing fractions, such as fraction Ill of Cohn's low temperature alcohol fractionation in the blood plasma protein fractionation generally applied to the production of important biological medicines such as blood serum, blood plasma and ascites of human and animals, and further, such as fibrinogen, y-globlin, and albumin in the blood plasma. The dry powder of plasminogen includes the dry powder from the above aqueous solutions containing plasminogen, particularly the lyophilized powder thereof.

As typical examples of methods for purifying crude plasminogen, there are methods by use of a fixed plasminogen stabilizer [Japanese Patent Application Kokai (Laid-open) No. 153,592/1980] and by use of lysine-sepharose [Science, 170, 1095(1970)].

The plasminogen stabilizer used in this invention include physiologically acceptable inorganic salts, lysine, salts thereof, phenylmethanesulfonyl fluoride, aprotinin, and soybean trypsin inhibitor.

These stabilizers can be used each alone or in combination thereof.

Any inorganic salts can be used in this invention so long as they are physiologically acceptable; for example, alkali metal salts (e.g., sodium salts and potassium salts), alkali earth metal salts (e.g., magnesium salts and calcium salts), and ammonium salts of mineral acids such as hydrochloric, sulfuric, boric acids (orthoboric acid, metaboric acid, and tetraboric acid), and phosphoric acids (pyrophosphoric acid, orthophosphoric acid, and metaphosphoric acid) may be used. Preferred examples of these salts are NaCl, KCl, MgCL2, (NH4)2SO4, Na2SO4, Na2B4O7, KH2HPO4, K2HPO4, NaH2PO4, Na2HPO4, etc.

Any salts of lysine can also be used in this invention so long ast they are physiologically acceptable; for example, alkali metal salts (e.g., sodium salts and potassium salts) and alkali earth metal salts (e.g., magnesium salts and calcium salts), and the like.

While the effective amount of the plasminogen stabilizer to be added depends upon the amount of plasm in in the plasminogen preparation and the kind of the stabilizers, the stabilizing effect of the stabilizer increases with amount thereof in the case of lysine. However, said amount should be adjusted according to the purpose of using the plasminogen preparation. For instance, for the purpose of studies on fibrinolysis, the concentration of the stabilizer is desirably less than the lowest value effecting fibrinolysis. Further, in case of the purpose of pharmaceutical applications where plasminogen preparations undergo a severe treatment, for example, heat treatment at 600C for 10 hours to inactivate viruses, a large amount of the stabilizer is added, and after a severe treatment of the preparation, it is removed by dialysis or some other suitable methods.In the cases of PMSF (phenylmethanesulfonyl fluoride; the same applies hereinafter), aprotinin, and soybean trypsin inhibitor, it is important to add an enough amount of stabilizer to inhibit contaminating plasmin completely.

Specific concentration of the stabilizers used in this invention are as follows: For aqueous solutions of plasminogen, a physiologically acceptable inorganic salt is used to give a final concentration of 0.002 to 0.4M, preferably 0.01 to 0.3M, and most preferably 0.05 to 0.2M, lysine 0.001 to 5W/V%, PMSF 0.01 to 100 mM, aprotinin 0.1 to 1,000 KlU/ml, and soybean trypsin inhibitor 0.1 to 1,000 BAEEU/ml. For dry powder of plasminogen, a physiologically acceptable inorganic salt is used to give a final concentration of 0.01 to 10 W/W%, lysin 0.01 to 10 W/W%, PMSF 0.01 to 10 W/W%, aprotinin 0.01 to 1,000 KlU/mg, and soybean trypsin inhibitor 0.1 to 1,000 BAEEU/mg.

Plasminogen is liable to be inactivated generaliy during the production process, particularly during heat treatment or lyophilization, as mentioned above. Consequently, the stabilization method of this invention is preferably applied not only to heat treatment of a plasminogen-containing aqueous solution or to lyophilization thereof, but also to all stages of the production process. In case of heat-treatment to inactivate viruses contained in a plasminogen-containing aqueous solution, e.g., heat-treatment at 600C for 10 hours, it is desirable to carry out the heat-treatment after adding the stabilizer and making the digestion at 40 to 370C at pH 2 to 4 for 20 to 60 minutes.

The stabilizers used in this invention may also be allowed to remain as it is in the preparations after the above-mentioned treatment, the remaining stabilizers also enhancing the stability of plasminogen on standing.

The method of this invention described hereinbefore improves the stability of plasminogen in its production stages including heat treatment, lyophilization, and other operations, thereby minimizing the loss of plasminogen in the production process, and the plasminogen preparations containing said stabilizer which are obtained by this method have high stability on standing: thus, the present invention provides a very favorable method as an industrial process for producing plasminogen preparations. It is a matter of course that the method of this invention can be utilized not only in the production of plasminogen preparations, but also widely in other cases where the stabilization of plasminogen is requested.

This invention will be illustrated in more detail by the following examples, but the invention is not limited thereto. In the Examples, as an activity of plasminogen is shown by the casein unit (CU unit) [Vox Sang., 5, 357-376 (1960)], an activity of Aprotinin is shown by the KIU [Freg. E.K. Kraut, H. Werle, E.

(1950) Kallikrein (Padutil) Enke Verlag Stuttgartl, and an activity of Soybean trypsin inhibitor is shown by the BAEEU [Laskowski, M. (1955) Method in Enzymology 2, 37 Ed. by Colowick, S.P. and Kaplan N.O. New York, Academic Press. Inc.] EXAMPLE 1 (Stabilizing Effect on Plasminogen Solution) Various kinds of inhibitors were each added to an aqueous solution of 100 CU/ml plasminogen in Tris-HCl buffer (pH 7.8). The solutions were allowed to stand in a thermostat of 370C to test the stabilities. The results are shown in Table 1.

TABLE 1

Remaining activity on Final standing at 37'C for Inhibitor concentration 24 hours (%) Lysine 0.1% 95 ,I 1.00/o 96 PMSF 0.1 mM 98 1 1 mM 98 Aprotinin 1 KIU/ml 89 5, 10 KlU/mi 95 Soybean trypsin inhibitor 10 BAEEU/ml 88 " 100 BAEEU/ml 92 No additive - 43 EXAMPLE 2 (Stabilizing Effect on Lyophilized Plasminogen) The solutions of Example 1, to which various kinds of inhibitors had been added, were lyophilized, and the resulting lycphilizates were allowed to stand for 1 month in a thermostat of 500C or for 3 months at room temperature to test the stabilities. The results are shown in Table 2.

TABLE 2

Remaining activity Remaining activity on standing at after 3-month 50'C for 1 month storage at room Inhibitor (%) temperature (%) Lysine 97 96 PMSF 94 93 Aprotinin 91 90 Soybean trypsin inhibitor 93 91 No additive 32 25 EXAMPLE 3 Fraction Ill obtained by the Cohn's cold ethanol fractionation method was suspended in an aqueous solution containing 1 w/v vÓ of sodium chloride, and after the suspension was stirred for a moment, the supernatant liquor was removed by centrifugation. According to the method of D.G.

Deutsch et al. [Science, 170, 1095 (1970)], the supernatant was poured into a lysine-Sepharose column to adsorb plasminogen, then impurity proteins were washed away with physiological sait-water, and the plasminogen adsorbed was eluted by use of an aqueous solution (pH 7.0) containing both 0.002M of E-aminocaproic acid and 1% of sodium chloride.

After dialyzed against distilled water, the purified plasminogen solution was divided into a large number of specimens, which were classified into four groups, and these groups were treated under the following different conditions, respectively: Condition D; various kinds of stabilizers were added to the specimens of this group respectively, and the resulting solutions were heat-treated at 6O0C for 10 hours.

Condition B; various kinds of stabilizers were addeci to the specimens of this group respectively, the resulting solutions were digested at a pH of 3.0 at room temperature for 30 minutes, and thereafter they were heat-treated at 600C for 10 hours.

Condition C; the specimens of this group were digested at a pH of 3.0 for 30 minutes, and after various kinds of stabilizers were added to the resulting specimens respectively, the resulting solutions were heat-treated at 600C for 10 hours.

Condition A; the specimens of this group were subjected to no treatment besides addition of various kinds of stabilizers.

Thereafter, the remaining activities of the followings samples were determined: The supernatants of the samples obtained under conditions D, B, C, and A after the pH values of these samples were corrected to 7.0 + 0.2.

The samples obtained by lyophilizing the solutions obtained under condition B (condition E).

The samples of condition E after standing at 40C for 6 months (condition F).

The activity determination were carried out by the casein decomposition method according to the procedure of Sgouris et al. [Vox Sang., 5,357 (1960)]. Table 3 shows the remaining activities of the various samples obtained by the treatments under the six conditions, expressed in percentages based on the activity of a never heated, no additive-containing plasminogen preparation assumed as 100%. TABLE 3 Remaining Activities (%) after Treatments under various Conditions

Condition Condition Condition Condition Condition Condition Additive (conc) A B C D E F No additive 100 27 27 20 -- - NaCl (0.05M) 100 72 25 23 -- - " (0.10M) 100 90 30 35 91 90 " (0.15M) 100 95 24 25 94 93 KCl (0.10M) 97 88 32 25 88 87 (NH4)2SO4 (0.02M) 97 80 33 35 81 80 " (0.05M) 95 95 25 27 93 94 " (0.10M) 100 95 25 20 94 93 Na2SO4 (0.02M) 98 77 17 23 76 77 " (0.05M) 100 85 17 26 84 83 " (0.20M) 100 90 20 30 91 90 Na2B4O7 (0.10M) 99 92 20 27 90 89 KHaPO4 (0.02M) 95 85 25 24 83 84 " (0.05M) 97 96 25 31 94 93 " (0.15M) 95 96 27 36 94 94 lysine (0.003M) 95 89 28 25 87 86 CH3COONa (0.10M) 97 40 12 30 -- -glucose (0.10M) 98 15 11 27 -- -glycine (0.10M) 95 17 20 25 -- - Mannitol (0.10M) 98 15 25 25 -- -sodium citrate (0.50M) -- 22 10 15 -- -sodium caprylate (0.10M) -- 17 10 20 -- -polyethylene glycol-4000 (2.0-%) 98 35 30 25 -- - The results shown in Table 3 are outlined as follows: Plasminogen solutions just after addition of sample stabilizers, exhibited activities of 95 to 100%, that is, practically no activity increase or no decrease due to sample stabilizers was observed (condition A).

The plasminogen solutions containing the stabilizers selected out in this invention, which were digested at a pH of 3.0 for 30 minutes and further heat-treated at 6O0C for 10 hours, retained activities substantially without being inactivated when pH values thereof were returned to neutral region. In contrast, the plasminogen solutions samely treated by containing no additive or containing acetic acid, glucose, glycine, sodium citrate, sodium caprylate, or polyethylene glycol-4000, when pH values thereof were returned to neutral region, yielded large amounts of insoluble matter and the activities of supernatants thereof were quite low (condition B).

The plasminogen solutions digested at a pH of 3.0 for 30 minutes before addition of stabilizer, even if admixed with the same amount of stabilizers as in condition B, yielded large amounts of insoluble matter after heated or when pH values thereof were returned to neutral region, and the remaining activities of the supernatants were low (condition C).

In case where the heat-treatment was carried out at 600C for 10 hours immediately after addition of stabilizer, a large amount of insoluble matters formed when pH values of the solution were returned to neutral region and the remaining activities of the supernatant were low (condition D).

From the above facts, it can be said that before making the heat-treatment at 600C for 1 0 hours it is necessary to carry out the digestion.

The remaining activities of the preparations obtained through lyophilization of the plasminogen solutions treated under condition B were almost in their integrity, that is, inactivation due to the lyophilization was scarcely observed (condition E). Further, said lyophilized preparations were stable during 6 months storage at 40C exhibiting no activity change (condition F).

It can be noted additionally that the lyophilized preparations exhibiting no decrease in activity were dissolved in injection-purpose distilled water and 1 ml of each solution was injected to a mouse weighing 20 + 2 g and observation was continued for 7 days, during which abnormal symptom was not observed at all.

Claims (14)

1. A plasminogen preparation containing at least one member selected from physiologically acceptable inorganic salts, lysine, salts thereof, phenylmethanesulfonyl fluoride, aprotinin, and soybean trypsin inhibitor in an effective amount for stabilizing plasminogen.

2. A plasminogen preparation according to Claim 1 , wherein said physiologically acceptable inorganic salt is an alkali metal salt, alkali earth metal salt, or ammonium salt of hydrochloric acid, sulfuric acid, boric acid, or of phosphoric acid.

3. A method according to Claim 2, wherein said physiologically acceptable inorganic salt is NaCI, KCI, MgCI2, (NH4)2SO4, Na2SO4, Na2B407, KH2PO4, K2HPO4, NaH2PO4, or Na2HPO4.

4. A plasminogen preparation according to Claim 1, wherein the salt of lysine is an alkali metal salt or an alkali earth metal salt.

5. A method according to Claim 4, wherein the alkali metal salt is a sodium or potassium salt and3 the alkali earth metal salt is a magnesium or calcium salt.

6. A plasminogen preparation according to Claim 1, wherein the preparation is in the form of an aqueous solution and contains at least one of the physiologically acceptable inorganic salts, lysine, saits thereof, phenylmethanesulfonyl fluoride, aprotinin, and soybean trypsin inhibitor in an amount, respectively, of 0.002 to OA M, 0.001 to 5 W/V%, 0.001 to 5 W/V%, 0.01 to 100 mM, 0.1 to 1,000 KlU/ml, and 1 to 1,000 BAEEU/ml.

7. A plasminogen preparation according to Claim 1, wherein the preparation is in the form of dry powder and contains at least one of inorganic salts, lysine, salts thereof, phenylmethanesulfonyl fluoride, aprotinin, and soybean trypsin inhibitor in an amount, respectively of 0.01 to 10 WtVV%, 0.01 to 10W/W%,0.01 to 10W/VV%,0.01 to 10W/W%,0.01 to 1,000 KU/mg, and 0.1 to 1,000 BAEEU/mg.

8. A method for producing a plasminogen preparation which comprises adding at least one member selected from physiologically acceptable inorganic salts, lysine, salts thereof, phenylmethanesulfonyl fluoride, aprotinin, and soybean trypsin inhibitor in an effective amount for stabilizing plasminogen, to an aqueous solution containing plasminogen solution to digestion at 40 to 370C at pH 2 to 4 for 20 to 60 minutes, and then to heat-treatment at 600C for 10 hours, and lyophilizing the resulting solution.

9. A method according to Claim 8, wherein said physiologically acceptable inorganic salt is an alkali metal salt, alkali earth metal salt, or ammonium salt of hydrochloric acid, sulfuric acid, boric acid, or of phosphoric acid.

1 0. A method according to Claim 9, wherein said physiologically acceptable inorganic salt is NaCI, KCI, MgCl2, (NH4)2SO4, Na2SO4, Na 213407, KH2PO4, K2HPO4, NaH2PO4, or Na2HPO4.

11. A method according to Claim 8, wherein said salt of lysine is an alkali metal salt or alkali earth metal salt.

12. A method according to Claim 1 wherein the alkali metal salt is sodium or potassium salt and the alkali earth metal salt is magnesium or calcium salt.

13. A method according to Claim 8, wherein said effective amounts of physiologically acceptable inorganic salts, lysine, salts thereof, phenylmethanesulfonyl fluoride, aprotinine, and soybean trypsin inhibitor are 0.002 to 0.4 M, 0.001 to 5 W/V%, 0.001 to 5 W/V%, 0.01 to 100 mM, 0.1 to 1,000 KlU/mI and 1 to 1,000 BAEEU/ml, respectively.

14. A plasminogen preparation according to any one of claims 1 to 7 substantially as herein described and exemplified.

1 5. A method according to any one of claims 8 to 13 for producing a plasminogen preparation, which method is substantially as herein described and exemplified.