DE1207550B - Process for obtaining plasminogen from animal blood serum or blood plasma, preferably from pig blood - Google Patents

Description

Process for obtaining plasminogen from animal blood serum or Blood plasma, preferably from pig blood It is known to obtain plasminogen from human Obtain blood plasma by excreting the fibrinogen content from the plasma and the so-called euglobulin content from the blood serum obtained in this way is precipitated and then from this euglobulin component through appropriate further treatment the plasminogen is obtained. Subsequently, the plasminogen can through a corresponding Treatment with enzymes such as streptokinase, trypsin or urokinase activated and converted into plasmin.

After the excretion of fibrinogen, some of the well-known Process for obtaining plasminogen from human blood first of all that Serum heavily diluted, then a portion of euglobulin at a certain pH value precipitated and this euglobulin content is then processed into plasminogen. That's the way it is for example, known to dilute human blood serum twenty-fold and adjusting the pH of the liquid thus obtained to 5.2 to 5.3 to obtain a To be able to precipitate euglobulin. Furthermore, it is also known to be tenfold Use dilution and then adjust the pH when working between 0 and 4 ° C 5.3 to be set.

The clinical use of plasmin obtained from animal blood on the other hand, there has always been a certain reluctance to oppose it, because here a protein is present that is foreign to the human body.

In addition, human and animal blood are also different on various other points. First of all, the quantitative composition gives way of human blood from that of animal blood, for example with regard to the ratio between the constituents which can be separated electrophoretically from one another Albumin and the a-, ß- and γ-globulins from each other. There are also some coming the proteins present in one of the blood types are not present in the other, or vice versa. Third, there is one and the same thing obtained from the blood of different animals Blood protein content often both in its chemical structure and in terms of its chemical properties often show certain differences. Among other things, conditionally In particular, the latter circumstance is the difference between the human Plasminogen and the animal plasminogen. For example, human Plasminogen can be activated by streptokinase, while ox plasminogen and Pig plasminogen are practically not activated by this enzyme.

In view of the differences described above, it is understandable that so far again and again at the risk of antigen formation when using animal Plasmin for clinical use has been advised. For example, it is a Known preparation that contains animal plasmin and is intended for wound treatment is. However, intravenous use of the preparation is prohibited.

It has now been found that it is possible to use an animal plasminogen to gain that, after its activation to plasmin, also to human Treatment can be used without creating the risk of serum sickness Anaphylaxis or other harmful effects is present.

The invention featuring a process for the extraction of plasminogen from animal blood serum or blood plasma, preferably from pig blood now the task is to obtain a euglobulin precipitate that is satisfactory Has plasminogen content, while at the same time the rest, through further treatment organic substances to be separated are reduced in terms of quantity.

The invention is based on the following experiments: In each case an amount of 50 ml of serum obtained from pig blood treated in the manner described below was diluted with different amounts of distilled water. The individual sera diluted fractions were each adjusted to a pH of 5.25 and allowed to stand at O 'C overnight. In this way a precipitate was obtained which was dissolved in 25 ml of dilute sulfuric acid having a pH of 2 to 3 after the liquid had been separated by a centrifugal method. The solutions thus obtained were then analyzed to determine their plasmin content (plasmin activity after activation of the plasminogen content), and the total content of organic substances was also determined.

After setting the plasmin activity to 100 and after detection the total amount of at a pH of 5.3, a temperature of 0 ° C and a The degree of dilution of 20 precipitated organic substances was determined by the The experiments described above determined the plasma content and the total amount organic substances in relation to these comparative figures converted in percent. With the "degree of dilution" is the ratio between the volume of the diluted Precipitation solution and the volume of the applied serum amount meant.

The results of these tests have been compiled accordingly and are shown graphically in the drawing. The degree of dilution is on the abscissa and on the ordinate are those for the plasmine content and that in the precipitates present total content of organic substances determined values in percent applied. The upper, full curve shows the relationship between the plasmin activity of the precipitate and the degree of dilution, while the lower curve drawn in dashed lines shows the total amount of precipitated organic Shows substances in terms of the degree of dilution.

It has also been found that the course of these curves does not change significantly when the precipitation is carried out at higher temperatures, for example at 25 ° C, although at a temperature of O 'C the yield of both precipitated substances and plasmin is higher than is the case at 25'C.

As can be seen from the two curves, the plasmin activity remains practically equal to 1000 /, the reference activity, even if the degree of dilution of 20 is decreased to 10 while the total yield of precipitated organics due to this reduction in the degree of dilution, it is also correspondingly uniform is decreased. If, however, the degree of dilution is now to a value below 10 brought a surprising result in the course of the curves to each other sudden change noticeable. This consists in the fact that the curves at a Degree of dilution of approximately 8 run practically parallel to each other, since both the curve illustrating the plasma content as well as the total amount of precipitated The curve showing organic substances suddenly drops downwards.

There is now the full curve with dilution values below 5 shows a drastically decreasing plasma level and after the two Curves quickly approach each other within this dilution range, it can be seen that that when using a relatively low degree of dilution, a plasma content is achieved, which is quite suitable for production on a technical basis, at the same time the amount of still to be eliminated in further processing organic substances is considerably reduced. The experiments described above Similar experiments have shown that it is not necessary to use the fibrinogen yet to be separated from the plasma before the precipitation of the plasminogen and that when it is no longer present of this step, d. H. when using animal blood plasma as the starting material, can also receive a plasminogen precipitate after activation has a satisfactory plasmin activity.

In view of the unexpected result of the above In the method according to the invention, the serum or the plasma are investigated Water diluted about five to eight times, the pH adjusted to 5 to 6 and the precipitate thus obtained is separated off.

This euglobulin precipitate can in a manner known per se in such a way be treated further that received plasminogen and then activated this to plasmin will.

In this context, both ordinary tap water and Distilled water can also be used as a diluent. The diluent may contain salts, but these may only be present in such small quantities that that they do not change the osmotic pressure significantly, since if one is present, too high osmotic pressure, the composition of the precipitated protein portion is disadvantageous is changed, The low degree of dilution used for the animal blood serum or plasma is particularly advantageous when plasmin is produced in large quantities should be because of the use of large amounts of liquid out of the way gone, which are processed further when using high degrees of dilution would have to.

Since, as can be seen from the curves explained above, with a Degree of dilution of approximately 8, on the one hand, a euglobulin precipitate with a high level Plasma content and, on the other hand, only a relatively small amount at the same time Occurring organic substances is obtained, it would obviously be obvious, preferably a degree of dilution in the upper half of the range between 5 and 8 to choose. On closer investigation, however, it turns out that the most economical Work possibilities with a degree of dilution between 5 and 6 are given. According to the invention preference is thus given to this degree of dilution. The basis for the Establishing and determining what is optimal from an economic point of view The degree of dilution lies in the fact that the plasmin obtained per unit is obtained Price can be calculated as the sum of two variables, the lowest of which Value of the one variable at degrees of dilution in the upper half of the for Use range is, while the lowest value of the other Variable in the lower half of the range.

With regard to the above-described differences between human and animal blood was not expected to be between about 5.2 and 5.3 lying pH value, which in the known processes for precipitating euglobulin from human blood serum was used, also for the precipitation according to the invention would represent an optimal value. According to the invention, however, it was found that it is also most expedient here to reduce the pH to 5.25 after dilution to adjust. The method according to the invention is below Explained in more detail with reference to individual examples. That in the examples and blood plasma used as the starting substance in the experiments described above and Blood serum was obtained in the following way: Immediately after the tapping, it is to the blood a solution containing an anticoagulant and optionally one or more antibiotics were also added and then cooled down afterwards. That treated blood is spun in a centrifuge and so are the blood cells removed therefrom, so that ultimately the blood plasma remains.

The serum is obtained in such a way that it is added to the plasma Calcium is incorporated by calcium chloride. Then the plasma is under stirred vigorously for a few hours so that the fibrin precipitates.

The latter is then separated and the remaining serum for the Experiments used in which the serum serves as the starting substance.

Obtaining plasminogen from serum obtained from pig blood Example 1200 cc of distilled water became 50 cc of serum obtained from pig blood added (degree of dilution f = 5). The pH is raised with the help of acetic acid 5.00 and the mixture was left to stand at a temperature of 0 ° C overnight. The mixture was then spun in a centrifuge. The thus obtained Euglobulin precipitate is separated and dissolved in 25 cc of dilute sulfuric acid, whereupon the solution to determine the total amount of organic substances present and analyzed the plasmin activity of the plasminogen portion after activation became; those with a corresponding euglobulin precipitate at a degree of dilution of 20, a pH of 5.3 and a temperature of 0 ° C were obtained set at 100 and then the experimentally determined amounts calculated in percent with reference to the comparative values.

In this way, a plasmin content of 71.5 % and a relative specific activity (the ratio between the plasmin content in percent and the amount of precipitated organic substances present, also stated in percent, after multiplication by a factor of 100) of 124 "were determined .

In a similar manner, the following results were obtained on the basis of combinations of temperature, pH and degree of dilution which differed from the above-mentioned example. Ver Tem- Plas- Relative At_ minogen-specific thinning temperature pH value game ad activity activity ° C 0/0 0/0 2 8 0 5.00 89.3 123 3 5 0 5.25 75.4 122 4 8 0 5.25 100.6 122 5 5 0 5.50 68.8 116 6 8 0 5.50 86.2 116 7 5 12.5 5.25 58.5 121 8 8 12.5 5.25 82.8 120 9 5 25 5.25 57.6 122 10 8 25 5.25 81.6 117 As can be seen from the table above, both a high plasminogen activity and a high relative specific activity are obtained within a relatively wide temperature range, in particular at a pH of 5.25, if the degree of dilution is selected to be 5 or 8.

Plasminogen production from serum obtained from bovine blood Examples 11 and 12 From serum obtained from bovine blood in portions of 50 ccm each, euglobulin precipitates were produced in the manner described in the above examples: Ver Tem- Plas- Or- Relative At- thinner- pera- pH- minogen- ganic specific play ture value activity s anzen activity degrees @C ° r '° ° i ° °% ° 11 8 0 5.3 101 79.5 127 12th 5 0 5.3 85.1 63.6 134 Obtaining Plasminogen from Plasma Obtained from Pig Blood Example 13 50 cc of plasma obtained from pig blood were diluted five-fold with 200 cc of distilled water. The pH is adjusted to 5.3 with acetic acid and the mixture is left to stand at a temperature of 0 ° C. overnight. The mixture is then spun in a centrifuge. The euglobulin precipitate thus obtained is separated from the mixture and dissolved in 25 cc of dilute sulfuric acid. The solution is then analyzed to determine its total content of organic substances and also to determine the plasmin activity of the precipitated plasminogen after activation; the values for the variables, which are determined with the aid of a euglobulin precipitate from a serum with a degree of dilution of 20, a pH value of 5.3 and a temperature of 0 ° C, are set at 100, and then the experimental ones amounts determined in relation to the comparative values, converted into percentages.

In this way, in the above-described example, a plasma activity of 81.8%, an inorganic substance content of 149% and a relative specific activity of established. In a similar way, the results summarized in the table below are obtained when using pig plasma and different combinations of degree of dilution, temperature and pH value: Ver Tem- Plas- Or Relative At- thinner- pera- pH- minogen- ganic specific activity play value activity punching degrees f oC ° % o ° / o ° / o 14 5 0 5.3 78.8 159 49.6 15 8 0 5.3 98.4 153 64.3 16 8 0 5.3 101 155 65.2 As can be seen from the examples described above, if the conditions which correspond to those for precipitation from serum are observed, it is possible to precipitate a considerable proportion of the plasmin activity directly from porcine plasma.

Claims (3)

Claims: 1. Process for obtaining plasminogen from blood serum or blood plasma, preferably from pig blood, the serum or plasma with Diluted water and adjusted to a pH of 5 to 6 and then the so obtained precipitate, which contains plasminogen, is separated, characterized in that, that animal serum or plasma, preferably from pig blood, with water to the Is diluted five to eight times. 2. The method according to claim 1, characterized in that that the serum is diluted five to six times. 3. The method according to claim 1 or 2, characterized in that the pH is adjusted to 5.25. In Publications considered: German Patent No. 884 854.