JP2005272403A - Method of treating plasminogen-containing solution - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of heat treating plasminogen which inhibits formation of impurities and reduces the ratio of inactivation. <P>SOLUTION: The method of heat treating plasminogen comprises heat treating a plasminogen-containing solution containing 0.25 M L-lysine hydrochloride in the presence of 10-20 w/v% glycine and 5-15 w/v% at least one kind selected from the group consisting of a monosaccharide, an oligosaccharide, and a sugar alcohol, for example, sorbitol and at least ≥5 v/v% glycine and/or polyethylene glycol at 50-70°C for 5-30 hours. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for treating a plasminogen-containing solution, specifically, prevention of a decrease in plasminogen activity associated with the heat treatment of the solution.

  The plasminogen preparation has been studied for clinical application as a thrombolytic agent and is one of the preparations attracting attention in recent years. Many of the plasminogens used in this preparation are derived from human plasma, and viruses such as hepatitis virus and AIDS virus may be mixed in the preparation. Therefore, in order to inactivate these viruses, the plasminogen-containing composition is heated in a solution state.

However, such heat treatment reduces the activity of plasminogen. For this purpose, for example, in Japanese Patent Application Laid-Open Nos. 55-145615 and 1-305036, sugar alcohols such as mannitol and sorbit and amino acids such as glycine and lysine are added as heat stabilizers. To suppress the decrease in activity. In JP-A-62-54286, a heat treatment is performed by adding an amino acid such as glycine, α- or β-alanine, hydroxyproline, or proline and a monosaccharide, sucrose, or sugar alcohol to reduce the activity. It is suppressed.
JP-A-55-145615 Japanese Patent Laid-Open No. 1-305036 JP-A-62-54286

  However, these methods cannot be said to be sufficient as a method for stabilizing plasminogen as described later, and a treatment method with less deactivation is required. In addition, if sugars (monosaccharides, sucroses, sugar alcohols) are added and heat-treated according to these methods, impurities may be produced depending on the addition concentration and heating conditions. As a result of causing deterioration of the properties of the solution, such as the occurrence of a problem, it may be considered that there was a problem in process control.

  The present invention has been made in view of such problems of the prior art, and the present inventors have made intensive efforts aimed at further stabilization. Not only the addition of amino acids and sugars, but also glycerin and It was found that the addition of polyethylene glycol further suppresses the decrease in activity, and the present invention has been completed.

  That is, the treatment method of the present invention comprises plasminogen in the presence of lysine and / or glycine, at least one selected from the group consisting of monosaccharides, sucrose, and sugar alcohols, and glycerin and / or polyethylene glycol. It is characterized by heat-treating the contained solution.

  At this time, it is preferable to heat-treat glycerin and polyethylene glycol in a plasminogen-containing solution at 5 v / v% or more, and it is preferable to use sorbitol as the sugar alcohol.

  According to the present invention, the decrease in activity is small compared to the conventional method in which amino acids and saccharides are coexisting and heated, and changes in the properties of the solution such as coloring and dusting after treatment can be prevented. Thus, this invention can suppress the fall of the activity in heat processing and can aim at the further effective utilization of the blood used as a raw material.

  The treatment method of the present invention is used as a method for inactivating a virus containing plasminogen, which is likely to be contaminated in the preparation. Therefore, although many plasminogens to which the present invention is applied are derived from human plasma, in addition to human plasma, animals derived from animals other than humans, such as those derived from porcine or bovine plasma, or so-called biotechnology techniques, It can also be applied to plasminogen obtained using bacteria.

  In the present invention, at least one of lysine or glycine, preferably both, is added as an essential component to the plasminogen solution to be heat-treated. In addition, at least one selected from the group consisting of saccharides used in conventional methods, specifically, monosaccharides, sucrose, or sugar alcohols, is added as an essential component. And as a component peculiar to the processing method of the present invention, at least one or both of glycerin and polyethylene glycol are added.

  The lysine or glycine used is an L-type amino acid and may be any salt such as a free amino acid or its hydrochloride. What is necessary is just to set the addition amount within the range of the method described in each said patent document, for example. Specifically, the free amino acid is 0.7M to 3.0M, preferably 1.8 to 2.3M. More specifically, it is as follows. Usually, a plasminogen-containing solution containing about 0.25 M of lysine hydrochloride is obtained in the purification step of plasminogen, which is the previous stage of the heat treatment. To this solution, glycine (as a free amino acid) is added in an amount of 5 w / v% or more, more preferably about 10 to 15 w / v%. Even if glycine exceeds 20 w / v%, the activity is not maintained.

  The monosaccharide may be a monosaccharide having any number of carbon atoms such as 5 monosaccharides and 6 monosaccharides, and examples thereof include glucose, mannose, galactose, and fructose. Among these, glucose is preferable. Further, as the sucrose, so-called oligosaccharides of about 2 saccharides, 3 saccharides, 4 to 6 saccharides are suitable, and examples thereof include sucrose, lactose, and maltose. Of these, sucrose is preferred. As the sugar alcohol, the reducing sugar of the monosaccharide is used. For example, sorbitol, mannitol, galactitol and the like can be mentioned, among which sorbitol is most preferable. Any one kind of these saccharides may be used, and two or more kinds may be used. What is necessary is just to set the addition amount of saccharide | sugar within the range of the method described in the said patent document. Specifically, it is added as a saccharide so as to be 3 w / v% to 15 w / v%, preferably 5 to 10 w / v% in the solution.

  In the present invention, glycerin and / or polyethylene glycol are further added. The amount of addition is 5 v / v% or more in the solution, preferably 8 v / v% or more, more desirably 10 v / v% or more and 30 v / v% or less. This is because no further stability can be expected even if it exceeds 30 v / v%.

  Next, the plasminogen-containing solution prepared by adding the amino acid, saccharide, glycerin and the like as described above is heat-treated. This heating condition is not different from the conventional condition, and may be within the range described in the patent document. Specifically, the heat treatment is performed at 50 to 70 ° C. for about 5 to 30 hours, generally at about 60 ° C. for about 10 hours. In the present invention, it is desirable to prepare the mixed solution so that the pH of the mixed solution is 6 to 8, preferably 6.5 to 7.5 immediately before the heat treatment. This is because heat treatment may cause a significant decrease in activity when it is deviated from the vicinity of neutral (pH 7.0).

  In this method, as will be described later, the solution after the heat treatment is not colored or dusty, and the generation of impurities that appear to be a degradation product of plasminogen is not observed. And the fall of activity is suppressed and the residual activity of plasminogen improves 1.1 to 1.2 times compared with the case where glycerin and polyethyleneglycol are not added.

  Hereinafter, although the present invention is further explained based on an example, it is needless to say that the present invention is not limited to the following example.

(Preparation of plasminogen)
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 resulting supernatant was loaded onto lysine sepharose 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 hydrochloric acid L-lysine gave a plasminogen-containing solution. Obtained.

[Inhibition of decrease in activity by addition of amino acid or saccharide (control example 1)]
Stabilizers such as glycine and glucose were added to 10 mL of the plasminogen-containing solution obtained above in the addition amounts shown in Table 1, filled in appropriate amounts in vials, and heat-treated at 60.5 ° C. for 16 hours. The residual activity [= (post-heating plasminogen activity / pre-heating plasminogen activity) × 100 (%)] was determined from the plasminogen activity before and after heating. The plasminogen activity was determined by test team PLG-2 kit using HD-valyl-L-leucyl-L-lysyl-p-nitroanilide / dihydrochloride synthetic substrate (Daiichi Chemical Co., Ltd.) It measured using. The results are shown in Table 1.

[Inhibition of decrease in activity by addition of amino acids and sugars (Control Example 2)]
Next, the decrease in activity was measured when amino acids and sugars were added. To 10 mL of the plasminogen-containing solution, glycine 15 w / v%, sucrose or sorbitol was added to a final concentration of 10 w / v%, followed by heat treatment in the same manner as described above, and plasminogen activity was measured. As a result, when sucrose was added, the residual activity was 76.1%, and when sorbitol was added, the residual activity was 74.3%.

[Inhibition of activity decrease by addition of glycerin (Example 1)]
Concentrated solution containing each of these components (20 mM phosphoric acid containing 0.25M lysine hydrochloride and 0.1M NaCl) so that the final concentrations of glycine, sorbitol and glycerin are as shown in Table 2 with respect to the plasminogen-containing solution. After adding a buffer solution (prepared with pH 6.9) and mixing, 20 mM phosphoric acid containing 0.25 M lysine hydrochloride and 0.1 M NaCl prepared at final concentrations of the respective components shown in Table 2. An acid buffer (pH 6.9) was added to adjust the absorbance at 280 nm to 0.3. This solution was heat-treated in the same manner as described above, and the decrease in plasminogen activity was measured. The results are shown in Table 2. The pH of the mixed solution immediately before heating was 7.0.

[Inhibition of contamination by adding glycerin]
In order to examine the inhibitory effect of the generation of impurities due to the addition of glycerin, the solution before and after the heat treatment was subjected to electrophoresis using SDS polyacrylamide gel under reducing conditions. Tests were performed on samples Nos. 1 to 5 and 11 among the samples shown in Table 2 above, and the appearance of a band considered to be a degradation product of plasminogen observed in the vicinity of a molecular weight of about 50 kD was observed (see FIG. 1). ).

[Inhibition of decrease in activity by addition of polyethylene glycol (Example 2)]
Concentrated solutions of these components (20 mM phosphoric acid containing 0.25M lysine hydrochloride and 0.1M NaCl) so that the final concentrations of glycine, sorbitol and polyethylene glycol 400 are as shown in Table 3 for the plasminogen-containing solution. After adding a buffer solution (prepared with pH 6.9) and mixing, 20 mM phosphoric acid containing 0.25 M lysine hydrochloride and 0.1 M NaCl prepared at final concentrations of the respective components shown in Table 3. An acid buffer (pH 6.9) was added to adjust the absorbance at 280 nm to 0.3. This solution was heat-treated in the same manner as described above, and the decrease in plasminogen activity was measured. The results are shown in Table 3. The pH of the heated solution was 7.0. Further, as a reference, a test using propylene glycol instead of polyethylene glycol was similarly tested and compared with the case of glycerin.

  The results of these tests are summarized as follows. That is, while only glycine alone had a residual activity of about 57.7% and glycine and saccharides added only about 76.1% (in the case of sucrose), in the treatment method of the present invention, glycerin was reduced to 5v. By adding about / v%, the residual activity became about 80%, and when it was favorable (for example, sample numbers 4 and 5), a residual activity of 85% or more could be obtained.

  In addition, it was confirmed that coloring of the solution due to heat treatment, dusting, and the like were suppressed, and at the same time, the band of plasminogen degradation product having a molecular weight of about 50 kD disappeared from electrophoresis. Therefore, it can be said that the addition of glycerin or polyethylene glycol suppresses the formation of decomposition products. Thus, the addition of glycerin or polyethylene glycol in addition to amino acids and sugars improves the stability during heating. However, the addition of propylene glycol did not improve the stability of plasminogen.

It is an expanded view at the time of carrying out electrophoresis of the plasminogen solution before and behind heat processing. The arrow shown in the figure indicates a band that seems to be a degradation product of plasminogen.

Claims (3)

  A plasminogen-containing solution is heat-treated in the presence of lysine and / or glycine, at least one selected from the group consisting of monosaccharides, sucrose and sugar alcohols, and glycerin and / or polyethylene glycol. A method for treating a plasminogen-containing solution.   The method for treating a plasminogen-containing solution according to claim 1, wherein the abundances of the glycerin and polyethylene glycol are 5 v / v% or more in the plasminogen-containing solution. The method for treating a plasminogen-containing solution according to claim 1, wherein sorbitol is used as the sugar alcohol.