SE459783B - STABLE PLASMINOGEN PREPARATION - 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

20 25 30 40 ' physiologically acceptable inorganic salt, 459 783 2 As typical examples of methods for purifying crude ' plasminogen, there are methods using a fixed plasminogen stabilizer [Japanese Laid-Open Patent Application 153 592 (1980)] and using lysine sepharose [Science, H 170, 1095 (1970)].

The stabilizing plasminogen preparation according to the present invention is characterized in that it contains at least one stabilizer selected from 0.0Û2~0.4 M of that consisting of Hgcl2, (NH4)2SÛ4, Naâ2SO4, Naâ2B4O7, KHZPO4, KZHPÛ4, NaH2PO4 and/or Na2HPO4, KCl, 0.01-100 mM phenylmethanesulfonyl fluoride and 1-1,000 BAEEU/ml soybean trypsin inhibitor, aqueous solution, in the form of one or which preparation contains at least that selected from 0.091-10 weight/weight X of the physiologically acceptable inorganic salt, KCl, a stabilizer consisting of MQCIZ, (NH4)2SÛ4, Naâ2SÜ4, Na2B4O7, KHZPÜQ, K2HPÛ4, NaH2PO4 and/or Na2HPO4, 0.01-10 wt/wt 1 phenylmethanesulfonyl fluoride and 0.1-1,000 BAEEU/ml soybean trypsin inhibitor, in the form of dry powder. The effective amount of the plasminogen stabilizer, is added, depends on the amount of plasmin in the plasminogen preparation and the type of stabilizer. However, the amount should be adjusted in accordance with the purpose of use of the plasminogen preparation. For example, for the purpose of studying fibrinolysis, the concentration of the stabilizer is desirably less than the lowest value that produces fibrinolysis. Furthermore, in cases of pharmaceutical applications where the plasminogen preparation undergoes a severe treatment, e.g. heat treatment at 80°C 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 method.

In the cases of PMSF (phenylmethanesulfonyl fluoride, the same is used hereinafter) and soybean trypsin inhibitor, it is important to add a sufficient amount of stabilizer to completely inhibit contamination of plasmin.

Specific concentrations of the stabilizers used in the present invention are as follows: For aqueous solutions of plasminogen, a physiologically acceptable inorganic salt was used to give a final concentration of 0.002 to 0.4 M, preferably 0.01 to 0.3 M, and most preferably 0.05 to 0.2 M, PMS 0.01 to 100 mM, and soybean trypsin inhibitor 1 to 1,000 BAEEU/ml. For dry powder of plasminogen, a physiologically acceptable inorganic salt was used to give a final concentration of 0.01 to 10 w/w 1, PMSF 0.01 to 10 w/w 2, and soybean trypsin inhibitor 0.1 to 1,000 BAEEU/mg.

Plasminogen is usually inactivated during the production process, especially during heat treatment or lyophilization, as mentioned above. Accordingly, the stabilization method of the present invention is suitably applied not only to heat treatment of a plasminogen-containing aqueous solution or to lyophilization thereof, but also to all steps of the production process. In the case of heat treatment to inactivate viruses, e.g., heat treatment at 60°C for 10 hours, it is desirable to carry out the heat treatment after adding the stabilizer and to perform the digestion at 4° to 37°C at pH 2 to 4 for 20 to 60 minutes.

Stabilizers used in the present invention may also be allowed to remain as they are in the preparations after the above treatment, whereby remaining stabilizers also increase the stability of plasminogen upon standing.

The addition of the stabilizer according to the present invention improves the stability of plasminogen in its production steps including heat treatment, lyophilization and other processes, thereby minimizing the loss of plasminogen in the production process, and the plasminogen preparation containing the stabilizer has high stability on standing; thus, the present invention provides a very favorable process such as an industrial process for producing plasminogen preparations. It goes without saying that the process according to the present invention can be utilized not only in the production of plasminogen preparations, but also generally in other cases where stabilization of plasminogen is required.

The present invention will be illustrated in more detail by the following examples, but the present invention is not limited thereto. In the examples, an action of plasminogen through the casein unit (CU unit) is shown [Vox Sang., 5, 357-376 (1969)] and an activity of soybean trypsin inhibitor is shown by BAEEU UI 10 15 20 25 30 459 783 4 ELaskowski, M. (1955) Method in Enzymology 2, Colowick S.P. and Kaplan, N.O., Inc.]. 37, published by New York, Academic Press, Exemgel 1 (Stabilizing action on plasminogen solution> Various kinds of inhibitors were each added to an aqueous solution of 100 CU/ml plasminogen in Tris (pH 7.8).

-HCl buffer The solutions were allowed to stand in a thermostat of 3?°C to test the stabilities. The results are shown in Table 1.

TABLE 1 Final Residual Activity Inhibitor Concentration at 37°C for 24 h (Z) PMSF 0.1 mM 98 - - 1 mM 98 Soybean Trypsin Inhibitor 10 BAEEU/ml 88 - - 100 BAEEU/ml 92 No addition - 43 Example 2 (Stabilizing effect on lyophilized plasminogen) The solutions in Example 1 to which different types of inhibitors had been added were lyophilized and the resulting lyophilizates were allowed to stand for 1 month in a thermostat of 50°C or for 3 months at room temperature to test the stabilities. are shown in Table 2.

Results TABLE 2 Residual activity Residual activity when standing at 50°C after 3 months storage Inhibitor for 1 month 1 rino at room temperature Z PMS? 94 93 Soybean trypsin- * inhibitor 93 91 No addition 32 25 10 15 20 25 30 35 40 5 459 783 Example 3 Fraction III, which was obtained according to Cohn's fractionation method with cold ethanol, was suspended in an aqueous solution containing 1% w/v sodium chloride and after the suspension was stirred for a moment, the supernatant liquid was removed by centrifugation. According to the procedure according to oc. neueen e: ei. _fseienee,' 1095 0970)] handesaen overlaid the liquid on a lysine-"Sepharose" column to absorb plasminogen, then impure proteins were washed away with physiological saline and adsorbed plasminogen was eluted using an aqueous solution (pH 7.0) containing both 0.002 bI&aminocaproic acid and 1% sodium chloride.

After dialyzing against distilled water, the purified plasminogen solution was divided into a large number of samples, which were classified into four groups, and these groups were treated under the following different conditions respectively: Condition D: different kinds of stabilizers were added to the respective samples in this group and the obtained solutions were heated to 60°C for 10 hours.

Condition B: Different types of stabilizers were added to respective samples in this group; the resulting solutions were dissolved at a pH of 3.0 at room temperature for 30 minutes and then heat-treated at 60°C for 10 hours.

Condition C: The samples in this group were dissolved at a pH of 3.0 for 30 minutes and then different types of stabilizers were added to the respective obtained samples, the obtained solutions were heat-treated at 60°C for 10 hours.

Condition A: The samples in this group were not subjected to any treatment other than the addition of various types of stabilizers.

Then, the residual activities of the following 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 lyophilization of the solutions obtained under condition B (condition E). The samples in condition E after: having been stored at 4°C for 6 months (condition F).

The activity determinations were carried out by the casein degradation method according to the procedure of Sgouris et al. [Vox Sang., 459 783 6 å, 357 (1960l]. Table 3 shows the residual activities of the various samples obtained by the treatments under the six conditions, expressed in percent, based on the activity of a never heated plasminogen preparation, containing no additive, assumed as 100%. 459 785 .wuHOW å å 3 _ 2 å ä 32.8 _. 2 å S .WN å .å šmoáv _ _. .å 2 i 3 2 ä 242.8 vomß: 2 ä S S _ ä ä 22:25 ßofïmz ä E o... 3 å E: :Éïcv _. 2 å 3 2 2 E: šmoå: _. 2. i 2 i N I ä 22.8 åwfiå 2 å S ä. ä E: 29:: _. ä ä S 2. ä ä E22: _. 3 E m» 2 3 E šäx: Jwmtfz, 2 ä 2 S ä E cáñm: Sv. 2 å 2 å ä E: šmíâ ._ ä å mm S ä 2: 9.2.8 ._ - - 2 2 2 E: 52.3 So - - 3 S. S 2: 3235» 5.3.: 35 m m. . Q u m < _ uwcwïfikw... .åâfiåfifipßm Bšïfëšm Bšqïæšë» »wšïmfwm åšëâgäë Üšå "ÉWSC cowcnflflqzpmw axwfio hona: hmmcflfiscmzon »ouwo ^wv»u~ofi>~uxc uw:c~æ>»=>z m ;;mm

Claims (1)

10 15 11 459 785 Patent claims Stable plasminogen preparation, characterized in that it contains at least one stabilizer, selected from 0.002-0.4 M of the physiologically acceptable inorganic salt, which consists of KCl, MgCl 2, (NH 4) 2 SO 4, Na 2 SO 4, Na 2 B 4 O 7, KHZPO4, KZHPO4, NaH2PO4 and / or Na2HPO4, 0.01 ~ 100 mM phenylmethanesulfonyl fluoride and 1-1000 BAEEU / ml soybean trypsin inhibitors, in the form of an aqueous solution, or which preparation contains at least one stabilizer, which is selected from 0.01-10% w / w of the physiologically acceptable inorganic salt consisting of KCl, MgCl 2, (NH 4) 2 SO 4, Na 2 SO 4, Na 2 B 4 O 7, KHZPO 4, KZHPO 4, HaH 2 PO 4 and / or Na 10 w / w Z phenylmethanesulfonyl fluoride and 0.1-1,000 BAEEU / ml soybean trypsin inhibitor, in dry powder form.