DE1148037B - Process for the production of a disaggregated gamma globulin which does not affect the complement system - Google Patents

Description

Process for producing a disaggregated, the complement system non-influencing gamma globulin The invention relates to a method for the production of a disaggregated gammaglobuhn preparation, which in contrast to the gamma globulin fraction previously isolated from human blood plasma forms the complement system unable to inactivate. The gamma globulin preparation obtained according to the claimed process can be used intravenously.

Antibody-containing gamma globulin preparations from human plasma are previously injected intramuscularly. The poor tolerance of the current, Preparations manufactured by various methods are available for intravenous use opposite. With intravenous administration of such preparations become the heaviest Side effects such as drop in blood pressure, rise in temperature, circulatory disorders, etc. like observed. On the other hand, the intravenous use of gamma globulin is aimed at, because they accelerate the onset of action of those contained in gamma globulin Antibodies, a better utilization of the administered gamma globulin and a more rational one Dosage of the preparation guaranteed.

There has now been a process for the production of a disaggregated, The complement system found not affecting gamma globulin that thereby is characterized in that the known manner, for. B. by ammonium sulfate precipitation or alcohol precipitation obtained gamma globulin fraction of the serum at a pH value from 1.5 to 5.5 and a temperature of 0 to 50 ° C for 2 hours to 2 days with Pepsin, e.g. B. with 25,000 to 200,000 units of pepsin per 100 g of protein to be broken down, under constant control of the anti-complementary effect and until elimination the complement inactivating effect treated, the thus obtained disaggregated Gamma globulin in a known manner, e.g. B. by fractional precipitation with neutral salts or organic solvents or by ultrafiltration, of low molecular weight Separate cleavage products, sterile-filtered and optionally freeze-dried.

The disaggregated gamma globulin produced by the present process can be used intravenously without side reactions and offers the above listed advantages. The good intravenous tolerance of the novel according to the invention The gamma globulin preparation obtained is based on its tolerance to the complement system.

It is known that gamma-globulins from different origins complement, but particularly the complement factor C 'l of human and guinea pig serum inactivate. This property already has the gentle way, z. B. by preparative electrophoresis or by DEAE cellulose chromatography obtainable main fraction of the antibody-containing gamma globulin with the sedimentation constant S = 7. These properties are even more pronounced by those gamma globulin preparations that have been heated to 40 to 50 ° C and their property of inactivating complement , with the aggregation detected in the ultracentrifuge after heating the gamma globulins. When gamma globulins are processed or stored, aggregation products are also formed, the sedimentation constant of which in the ultracentrifuge is S = 9. The complement is most strongly inactivated by the gamma-macroglobulin belonging to the gamma globulin component system with a sedimentation constant of S = 19 and higher than 19.

It is now generally accepted that the complement system is the cause immunopathological secondary reactions of an antigen-antibody binding reaction. In the process, enzymatic substances are exposed that lead to anaphylactic reactions to lead. It was therefore to be assumed that antibodies which do not influence the complement system do not lead to the side reactions mentioned. Indeed, this is true for that produced by the present process, disaggregated, antibody-containing Gamma globulin too. As already mentioned, it can be done safely without disruptive side reactions administered intravenously. The difference in effect different Gamma globulin preparations and the disaggregated gamma globulin produced according to the invention can already be proven by general complementary provisions, but it becomes clearer recognizable when considering individual complement factors, especially the complement factor C'1 after that of Fritzsche, Fischer, Schwick and Schultze in Klinische Wochenschrift, 36, p. 100 (1958), specified method. Appropriately, the procedure is as follows: that 1 ml guinea pig serum (complement) with a certain amount of gamma globulin, z. B. 5 mg is incubated at 37 ° C in a water bath and from this test batch after 1 hour, 3 hour and 5 hour samples for determination of complement factor activity can be removed.

Example 1 501 human serum are diluted to 851 with distilled water, adjusted to pH 7.0 and mixed with saturated ammonium sulfate solution up to 451110 saturation. The precipitate, ie the crude gamma globulin fraction, is isolated by centrifugation, dissolved in distilled water and made up to 701. After the complement-inactivating effect has been determined (see Table 1), the solution is adjusted to pH 4.0 with acetic acid. Then there are 90,000 units per 100 g of protein to be broken down. Pepsin is added and the solution is subjected to its proteolytic action at 40 ° C for 24 hours. A sample of the reaction mixture is filtered, the filtrate is adjusted to pH 7.0 and tested again for complement-inactivating effect (see Table 1). The majority of the reaction mixture is now worked up like the sample and freed from low molecular weight cleavage products by ultrafiltration. After a sterile filtration, the protein content is adjusted to 5.0%.

The yield is 41% of a 5% solution of disaggregated Gamma globulin, d. H. about 7511 / o of those present in the serum, now the complement system non-influencing gamma globulins. As can be seen from the following table, the deaggregated gamma globulin does not inactivate the complement system.

Explanation of Table 1 The test results listed in Table 1 show that the tested gamma globulin crude fraction inactivated complement to a large extent. The inactivation of the complement depends on the incubation time. The gamma globulin crude fraction has the greatest influence on the complement factors C 1 and C'4. The table also shows that the disaggregated gamma globulin practically no longer has any complement-inactivating effect and that the test results for the sample taken from the test batch and the processed main amount agree well. Table 1 °% complement inactivation (37 ° C) * C, 1 C2 C'3 C 4 lh 3h 5h Ih 1 3h 1 5h Ih [ 3h 1 5h Ih 3h 5h i 1 ml guinea pig serum (Complement) -f- 0.1 ml = 5 mg gammaglo- bulin crude fraction ....... 17 65 80 15 30 38 10 25 36 20 37 60 -I- 0.1 ml = 5 mg disaggregating- tes gamma globulin (sample) 0 0 7 0 0 0 0 0 0 0 3 5 0.1 ml = 5 mg disaggregating tes gamma globulin (main quantity) .................. 0 0 4 0 0 0 0 0 0 0 0 0 * C 1, C'2, C 3 and C '4 are different complement factors of the guinea pig serum. Ih, 3h and 5h are exposure times in hours (h). Example 2 101 of a 1611% solution of by the method of J. Horejsi and R. Smetana (cf. Act. Med. Scand., 155, p. 65 [1956]) using 2-ethoxy-6,9-diamono- acridine-derived gamma globulin. diluted to 100 liters with physiological saline solution, adjusted to pH 5.0 with acetic acid, added 160,000 units of Pepsind per 100 g of protein and subjected to its proteolytic action for 36 hours at 40 ° C. The further work-up takes place as in Example 1.

The yield is 191% of a 5% solution of disaggregated gamma globulin (tests in Table 2), corresponding to about 6011% of the gamma globulin in the starting material. Table 2 ° / o Complement inactivation after 3 hours (37 ° C) C'1 C'2 C'3 I C'4 1 ml guinea pig serum -f- 0.1 ml = 5 mg gamma globulin starting material ... 80 42 40 65 -I- 0.1 ml = 5 mg disaggregated gamma globulin ..... 0 0 0 0 Example 3 101 of a 16% solution of gamma globulin prepared by the method of HE Schultze, M. Schönenberger and HD Matheka, Behringwerke-Mitteilungen, 26, p.21 (1952), are worked up as in Example 2. The yield is 19.51 of a 5% solution of disaggregated gamma globulin (test in Table 3), corresponding to about 60% of the gamma globulin in the starting material. Table 3 % Complement inactivation after 3 hours (37 ° C) C'1 I C'2! C'3 C'4 1 ml guinea pig serum 0.1 ml = 5 mg gamma globulin starting material ... 65 38 35 50 - 0.1 ml = 5 m desa re 'ertes gamma obulin ..... 0 0 0 I 0 Example 4 101 of a 16% solution of gamma globulin obtained by fractionation with alcohol by the method of EJ Cohn and coworkers (J. Amer. Chem. Soc., 68, p.459 [1946]) are diluted with physiological saline solution to 701 diluted, adjusted to pH 4.5 with acetic acid, treated with 200,000 units of pepsin per 100 g of protein and subjected to its proteolytic action for 24 hours at 40 ° C. The further work-up was carried out as in Example 2.

The yield is 20.51 of a 5% solution of disaggregated gamma globulin (test, Table 4), corresponding to about 64% of the gamma globulin in the starting material. Table 4 ° / o Complement inactivation after 3 hours (37 ° C) C'1 I C'2 I C'3 C'4 1 ml guinea pig serum + 0.1 ml = 5 mg gamma globulin starting material ... 75 i 42 39 67 - 0.1 ml = 5 mg disaggregated gamma globulin ..... 2 (0 0 1 Example 5 101 16 '° / o, by the method of HE Schultze, M. Schonberger and HD Mat heka, Behring work messages, 26, p 21 (1952), prepared Gammaglobulinlösungwerden with physiologische.r saline 80 1 diluted with Acetic acid adjusted to pH 4.5, mixed with 120,000 units of pepsin per 100 g of protein and reacted at 40 ° C. for 28 hours. The reaction mixture is filtered, adjusted to pH 7.0 and freed from low molecular weight components by ultrafiltration. After sterile filtration, the gamma globulin solution (521 2 n / o solution) is lyophilized in the freeze-drying apparatus.

Yield 1.48 kg dry product with 70.3% disaggregated gamma globulin (remainder NaCl), thus 65% yield of gamma globulin. Check for influence on complement in Table 5. Table 5 % Complement inactivation after 3 hours (37 ° C) C'1 I C'2 I C'3 I C'4 1 ml guinea pig serum 5 mg gamma globulin starting material. . . . . . . . . . :. . . 65 38 35 50 5 mg disaggregated gamma globulin before lyophilization 0 0 0 0 5 mg disaggregated gamma globulin after lyophilization tion ......................................... 0 0 0 0

Claims (1)

PATENT CLAIM: Process for the production of a disaggregated, the Complement system not influencing gamma globulin, characterized in that one in a known manner, for. B. obtained by ammonium sulfate precipitation or alcohol precipitation Gamma globulin fraction of the serum at pH 1.5 to 5.5 and a temperature from 0 to 50 ° C for 2 hours to 2 days with pepsin, e.g. With 25,000 to 200,000 units Pepsin per 100 g of protein to be broken down, with ongoing monitoring of the anti-complementary Effect and treated until the elimination of the complement-inactivating effect, the disaggregated gamma globulin thus obtained in a known manner, e.g. B. by fractional Precipitation with neutral salts or organic solvents. or by ultrafiltration, separated from low molecular weight cleavage products, sterile-filtered and optionally freeze dried.