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

As a typical example 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 (l970)].

The stabilizing plasminogen preparation according to the present invention is characterized in that it contains at least one stabilizer, which is selected from 0.0Û2 ~ 0.4 M of that consisting of Hgclz, (NH4) 2SÛ4, Nâ2SO4, Nâ2B407, KHZPO4, KZHPÛ4, NaH2PO4 and / or Na 2 HPO 4, KCl, 0.01-100 mM phenylmethanesulfonyl fluoride and 1-1000 BAEEU / ml soybean trypsin inhibitor, aqueous solution, in the form of one or more preparations selected from at least 0.091-10% w / w X of the physiologically acceptable inorganic salt, KCl, a stabilizer consisting of MQCl 2, (NH 4) 2 SO 4, Na 2 SO 4, Na 2 B 4 O 7, KH 2 P 1-1 000 BAEEU / ml soybean trypsin inhibitor, in the form of a dry powder. This depends on the amount of plasmin in the plasminogen preparation. The effective amount of the plasminogen stabilizer is added, added and the type of stabilizer. However, the amount should be adjusted according to the purpose of use of the plasminogen preparation. For example, for purposes of fibrinolysis studies, the stabilizer concentration is desirably less than the lowest value that fibrinolysis provides. Furthermore, in the case of pharmaceutical applications, where the plasminogen preparations undergo a rigorous treatment, eg heat treatment at 80 ° C for 10 hours to inactivate virus, a large amount of the stabilizer and after a strict treatment of the preparation it is removed by dialysis or any other suitable methods.

In the case 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 plasmin contamination.

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 .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 plasminogen dry powder, 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 Z, and soybean trypsin inhibitor 0.1 to 1,000 BAEEU / mg.

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

Stabilizers used in the present invention may also be allowed to remain as they are in the compositions after the above treatment, with remaining stabilizers also increasing the stability of plasminogen when standing.

The addition of 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. standing; thus, the present invention provides a very advantageous process, such as an industrial process for producing plasminogen preparations. It is obvious that the method according to the present invention can be used not only in the generation of plasminogen preparations, but also in general 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. The examples show an action of plasminogen through the casein unit (CU unit) [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 SP and Kaplan, N.O., Inc.]. 37, published by New York, Academic Press, Exemgel 1 (Stabilizing effect on plasminogen solution> Different types 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 3 ° C thermostat to test the stability. 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 BÅEEU / ml 92 No additive - 43 Example 2 (Stabilizing effect on lyophilized plasminogen) The solutions in Example 1 to which various kinds of inhibitors had been added, lyophilized and lyophilisates obtained were allowed to stand for 1 month in a thermostat at 50 ° C or for 3 months at room temperature to test the stability. 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 rumgtgmpi Z PMS? 94 93 Soybean Trypsin Inhibitor 93 91 No Additive 32 3 10 15 20 25 30 35 40 5 459 783 Example 3 Fraction III, obtained according to Cohn's method of fractionation with cold ethanol, was suspended in an aqueous solution. which contained 1% w / v sodium chloride and, after stirring the suspension for a moment, supernatant was removed by centrifugation. According to the procedure of oc. neueeen e: ei. (1095 0970)] handesaen supernatant the liquid in a lysine "Sepharose" column to absorb plasminogen, then crude proteins were washed away with physiological saline and adsorbed plasminogen was eluted using an aqueous solution (pH 7.0 ), which contained both 0.002 bl & 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: Ratio D: different kinds of stabilizers were added to the respective samples in this group and the resulting solutions were heated to 60 ° C for 10 hours.

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

Ratio C: the samples in this group were digested at a pH 3.0 for 30 minutes and then various kinds of stabilizers were added to the respective obtained samples, 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.

Thereafter, the remaining 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 f 0.2. Samples obtained by lyophilization of the solutions obtained under ratio B (ratio E). the tests in prehistoric E after: having a strain at 4 ° c for s months (ratio F).

The activity determinations were performed by the casein decomposition method according to the method of Sgouris et al. [Vox Sang., 459 783 6 å, 357 (1960l]. Table 3 shows residual activities of the various samples obtained by the treatments under the six conditions, expressed as a percentage, based on the activity of a never-heated plasminogen preparation, which does not contain any 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 SS _ ä ä 22:25 ßofïmz ä E o ... 3 å E:: Éïcv _. 2 å 3 2 2 E: šmoå: _. 2. i 2 i NI ä 22.8 åw fi å 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 Så - - 3 S. S 2: 3235 »5.3 .: 35 m m.. Q um <_ uwcwï fi kw .... .åâ fi å fifi pßm Bšïfëšm Bšqïæšë» »wšïmfwm åšëâgäë Üšå“ ÉWSC cowcn flfl qzpmw axw fi o hona: hmmc flfi scmzon »ouwo ^ wv» u ~ o fi> ~ uxc uw: c ~ æ> »=> zm ;; 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.