FI62624B - FOERFARANDE FOER FRAMSTAELLNING AV FOER INTRAVENOEST BRUK LAEMPLIGT GAMMAGLOBULIN SOM INTE INNEHAOLLER ANTIKOMPLEMENTAERT GAMMAGLOBULIN UR ORENT GAMMAGLOBULIN - Google Patents

Description

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Patent · <Xh r «glsterstyr« lMn · AmMcan uttagd och utl.tkdfMn published 29.10.82 (32) (33) (31) Pyydetty «moMmm — Β« | Μ priority «c 07.02.76

Federal Republic of Germany-Förbundsrepubliken lyskland (DE) P 260 ^ 759.6 Proven-Styrkt (71) Armor Pharmaceutical Company, 6991 East Camelback Road, Scottsdale,

Arizona, USA (72) Waldemar Schneider, Hagen, Dietrich Wolter, Hagen, Germany Federal Republic of Germany (DE) (7J0 Leitzinger Oy (510 Method for the preparation of anti-complement gamma globulin for intravenous administration of gamma globulin, in which case gamma globulin,

The invention relates to a process for the preparation of gamma globulin free of anticomplementary gamma globulin suitable for intravenous use from impure gamma globulin obtained, for example, by cryoethanol fractionation, in which a gamma globulin crude precipitate is dissolved in is 1000 to 900,000 and finally the anticomplementary gamma globulin moiety is compounded by the addition of aqueous polyethylene glycol.

British Patent Publication No. 1,372,953 discloses a process for the purification of gamma globulin in which polyethylene glycol is used to precipitate the purified globulin. Instead of said glycol, water-soluble linear polymers such as dextran, polyvinyl alcohol, polyvinylpyrrolidone and polyalkylene glycol can also be used as filler in equivalent amounts. However, the purified gamma globulin thus fractionated still contains intravenous anti-complementary gamma globulin, which according to the known method is sought to be removed by post-treatment with saline, for example aluminum saline. Such post-treatment is cumbersome and reduces the yield.

U.S. Pat. No. 3,449,313 and French Pat. No. 2,677,115 describe methods for purifying globulin using bentonite for precipitation. The product prepared by these methods also still contains anticomplementary gamma globulin, which is harmful for intravenous administration.

Since the gamma globulin solution contains both damaged, i.e. anti-complementary and undamaged gamma globulin molecules, the aim is to bind the damaged molecules so that they can be chemically separated from the physiologically sound molecules. Damaged molecules are capable of forming hydrogen bond bonds. They therefore bind strongly inside the silicate layers, while the intact gamma globulin molecules remain in solution. Damaged gamma globulin molecules bound to the layers of layered silicates can then be easily removed, for example by centrifugation.

The object of the present invention is to eliminate the above-mentioned disadvantages by using layered silicate together with starch.

This gives a method which can be carried out simply and which gives a product suitable for intravenous administration.

These objects are achieved according to the invention by adding bentonite or vermiculite 0.2 to 10% by weight to the crude gamma globulin solution in addition to Hvd-roxyethyl starch and after a sufficient time, preferably 3-10 hours, polyethylene glycol is added, followed by an anticomplementary portion of gamma globulin or bentonite is separated from the solution from which pure gamma globulin is separated by precipitation.

In addition, the combination of the hydroxyethyl starch and the bentonite or vermiculite according to the invention gives a very pure gamma globulin in a simple manner. This advantageous effect achieved is probably due to the swelling effect of hydroxyethyl starch on gamma-globulin molecules as well as the selective filtration effect of layered silicate.

Based on the experiments, the reaction takes place in the temperature range of 4 to 20 ° C, and the process operates at pH values of 4.0 to 7.6. The reaction time should be 3 to 10 hours. In this case, it is best to work overnight because the reaction is relatively slow, so a longer interval improves the results. Separation of the silicate is best performed by centrifugation. However, other methods generally known in the art may be used, for example, flotation filtration.

In the experiments carried out, it has been found that the gamma globulin prepared according to the invention is purer than the products prepared by known methods. We refer to the attached drawing showing the gel filtration of five products (Sephadex G 200). In the Annex, Experiment A represents Cohn standard gamma globulin, B represents proteolytically treated gamma globulin, C represents beta-propiolactone-treated gamma globulin, D represents pH-4 modified gamma globulin, and E represents gamma globulin prepared according to Example 1 of this application. As shown in results A and B, the products contain both high molecular weight proteins (aggregates) and small molecule moieties (degradation products). Experiments C and D contain only minor amounts of high molecular weight proteins and hardly any degradation products at all. Product E, on the other hand, is practically only the desired gamma globulin.

The following examples illustrate the invention:

Example 1 a) Separation of gamma globulin: Starting from pooled plasma to which 8% ethanol is added and precipitated at pH 7.2 and -3 ° C. Fraction I is then separated. 19% 4,62624 ethanol is then added to the resulting liquid at -5 ° C and pH 5.8. In this case, the fraction II-lii, which consists of gamma globulin, is separated. The precipitate is redissolved and remixed at pH 5.8 with 19% ethanol. The remaining mother liquor is then reprecipitated at pH 7.2 with 25% ethanol. The precipitate thus obtained (= fraction II) consists of at least 90% of gamma globulins.

b) Reduction of the anti-complement effect:

The gamma globulin precipitate is dissolved to a concentration of about 6% in a buffered, aqueous solution having a pH of 7.0, to which about 10% of hydroxyethyl starch is added to the aqueous solution. Aqueous bentonite SF slurry (main component: montmorillonite; grain size less than 80 μm; manufactured by SERVA Feinbiochemika, Heidelberg) is further added to the solution. The solution then contains 2.5% by weight of bentonite and is mixed well. The solution is then allowed to stand for 6 hours at 15 ° C ± 2 ° C. 10% polyethylene glycol is added to the solution and the bentonite and undesired materials are removed by centrifugation.

(c) Conversion to physiological saline:

The supernatant obtained by centrifugation contains the desired gamma globulin moieties in solution. The upper layer is adjusted with 0.1 NaOF. to pH 7.2 and 20% by weight of polyethylene glycol is added. In this case, the pure gamma globulin precipitate differs. The precipitate is collected by centrifugation and added to physiological saline to give a protein concentration of 5.2%, after which the solution is sterile-filtered. It is then ready for therapeutic use.

Example 2

Steps a) and c) are the same as in Example 1. Step b) is amended as follows:

The gamma globulin precipitate is dissolved as a solution of about 4% by weight in a buffered aqueous solution having a pH of 7.4, to which about 8% of hydroxyethyl starch is added to the aqueous solution. An aqueous slurry of finely ground (grain size less than RO pm) vermiku connector is further added to the solution up to 5% by weight. The whole mixture is stirred vigorously. The solution is then allowed to stand for 8 hours at 15 ° C ± 2 ° C. About 10% by weight of PEG is then added to the solution, after which the vermiculite together with the undesired substances is removed by centrifugation.

Step c) continues from here.

Summary;

From the immunoelectrophoresis diagrams, it can be calculated and measured that the yield of pure gamma globulin becomes higher when the method according to the invention is used. The resulting gamma globulin is not modified or chemically altered. It is very suitable for intravenous use and has virtually no anti-complementary properties. Besides, it is very permanent which has been shown by storage experiments.

Claims (2)

62624 6 A process for the preparation of an anti-complement gamma globulin-free gamma globulin suitable for intravenous use from impure gamma globulin obtained, for example, by cryoethanol fractionation, in which a gamma-globulin crude precipitate is dissolved in an optionally buffered 1% aqueous solution, , having a molecular weight of 1,000 to 900,000 and finally precipitating the anticomplementary gamma globulin moiety by adding aqueous polyethylene glycol, characterized in that in addition to hydroxyethyl starch, bentonite or vermiculite is added to the crude gamma globulin solution after 0.2 to 10% by weight and preferably for 3 hours, preferably polyethylene glycol is added, after which the anticomplementary gamma globulin moiety together with the bentonite or vermiculite is separated from the solution from which the pure gamma globulin is separated by precipitation. Process according to Claim 1, characterized in that the treatment takes place at a temperature of +4 to 20 ° C. Process according to Claim 1 or 2, characterized in that the treatment with bentonite or vermiculite takes place at a pH of the solution of 4.0 to 7.6.