RU2158137C1 - Method of immunoglobulin preparation producing - Google Patents

Abstract

FIELD: medicine, immunology. SUBSTANCE: method of preparing immunoglobulin preparation involves salt extraction of human blood serum alcoholic extract, its purification to remove lipoproteins followed by isolation of the end product, addition of glucose-containing stabilizing agent and sterilizing filtration. Immunoglobulins are isolated by precipitation with alcohol. Mixture of glycine and glucose taken in the ratio (1-1.5):1 is used as a stabilizing agent. Immunoglobulin solution is treated with glass sorbent and at the stage of isolation of the end product flowing-zonal centrifugation is carried out. Before sterilizing filtration solution is passed through the porous glass filter. EFFECT: increased yield, decreased cost of preparation. 2 ex

Description

 The invention relates to medicine, in particular to immunology, and can be used for immunotherapy and immunoprophylaxis of bacterial and viral diseases.

 A known method of obtaining an immunoglobulin preparation, protected by patent SU 1767742, class. A 61 K 37/04, published 11.06.90.

 The method consists in obtaining a preparation by treating precipitate B with a buffer solution, separating the precipitate formed and purifying immunoglobulins by dissolving the precipitate in 0.05 M acetate buffer, pH 4.3 and treating with sodium caprylate to a final concentration of 0.5%, pH 4.8, and precipitation immunoglobulins with alcohol, adding 0.15 M glycine and 2% sucrose, sterilizing filtration and adding pectin at a concentration of 0.3-0.5 g per 1 g of immunoglobulin.

 The disadvantage of this method is the presence of impurities of sodium caprylate in the final product and the instability of the resulting immunoglobulin solution that can be stored after sterile filtration until bottling for no more than 10 hours

Closest to the claimed technical essence and the achieved result is a method for producing an immunoglobulin preparation (CIP) from sediment B (III Cohn fraction) (patent RU 2060034, class A 61 K 39/395, published on 09/06/91)
The method involves salt extraction of an alcoholic precipitate B of blood serum (III Cohn fraction) in a 0.9% sodium chloride solution. Purification of the lipoproteins of sediment B extract is carried out with chloroform (10% by volume), followed by precipitation of the immunoglobulin fraction in the presence of 12% polyethylene glycol. The resulting precipitate of immunoglobulins is diluted in 0.9% sodium chloride solution, centrifuged to remove insoluble impurities. After centrifugation, glucose is added to the solution to a final concentration of 2% and sterilized by filtration.

 The disadvantage of this method is that it provides a preparation containing undesirable impurities of residual polyethylene glycol. In addition, the resulting immunoglobulin solution contains significant amounts of fibrinogen and fibrin, coprecipitating with immunoglobulins. These impurities significantly complicate the sterilizing filtration and cause instability of the drug when stored in liquid form. The consequence of this is the need for immediate bottling and freeze drying after filtration. Losses in the final yield of the drug due to difficult filtration and the need to repeat this procedure reach 30%.

 The problem solved by the invention is the preparation of an immunoglobulin preparation free of impurities of unstable components that impede filtration.

 The technical result from the use of the invention is to increase the yield of the target product and reduce its cost.

 The specified result is achieved by the fact that in the method for producing an immunoglobulin preparation from human blood by salt extraction of an alcoholic precipitate of blood serum, purifying it from lipoproteins, followed by isolation of the target product, adding a stabilizer containing glucose, sterilizing filtration, the isolation of immunoglobulins is carried out by alcohol deposition, using a stabilizer as alcohol a mixture of glycine and glucose in the ratio (1-1.5): 1, the solution of immunoglobulins is treated with glass sorbents, at the stage of isolation Spruce product is carried out in fluid-zonal centrifugation, and before the sterilizing filtration, the solution was passed through a porous glass filter.

 The method is as follows. Precipitation of the immunoglobulin fraction from the chloroform extract of precipitate B is carried out using ethanol. This avoids the presence of polyethylene glycol in the final product, which, according to the method of the prototype, has to be removed by repeated washing. The alcohol precipitate is dissolved in a 0.55 - 0.65% sodium chloride solution with the addition of glycine and glucose in the ratio (1-1.5): 1 (i.e. 2 ± 0.5% glucose and 2-3% glycine) pH 6.5-7.5. These pH and salt values are optimal because they provide the most complete formation of a precipitate of insoluble fibrin, and do not require further correction. A decrease in salt concentration below 0.55% complicates the dissolution of the precipitate, and an increase of more than 0.65% complicates the subsequent lyophilization. An increase in the ratio of glycine to glucose concentrations does not increase the yield of the final product, and its decrease does not provide sufficient dissolution of immunoglobulins. To accelerate the formation of fibrin precipitate, the solution is processed using glass beads. The solution is incubated for about 30 minutes until fibrin is formed, then subjected to flow-zone centrifugation at 15,000 vol. / min The supernatant is passed through a fine-porous glass filter (Shot filter) to remove residual amounts of fibrinogen and fibrin adsorbed on the porous glass. The resulting solution is subjected to sterilizing filtration. This solution is stable and can be stored until bottling for 1 month without changing the physico-chemical properties (transparency, lack of precipitating components).

 Example 1. To 1 kg of sediment B add 10 l of a 0.9% solution of sodium chloride with stirring. The volume of the extract is 10 l, the protein content is 2.0 ± 0.2%. Sediment extract B was added with 10% by volume chloroform. The mixture was stirred slowly for 2 hours. The precipitate was removed by centrifugation. Set the pH of the solution to 6.9 ± 0.5. Ethyl alcohol was added to a final concentration of 26%. The precipitate was separated by centrifugation at 15,000 rpm. The resulting precipitate of immunoglobulins is diluted in 0.6 ± 0.05% sodium chloride solution with the addition of 2 ± 0.5% glucose and 3.0% glycine. Glass beads are added to the solution and incubated for about 30 minutes until fibrin forms. After treatment, fibrin is separated by flow-zone centrifugation at 15,000 rpm on an OTP-101 centrifuge. The supernatant is passed through a Shot filter. Then the immunoglobulin solution is subjected to sterilizing filtration using Cuno-60 and Cuno-90 deep cellulose filters, and freeze-drying using a TGS-15 apparatus.

 The specific activity (antibody titer) of the obtained preparation does not differ from the prototype (for Shigella - 1: 320, for Salmonella 1: 320 - 1: 640). The content of immunologically inactive fractions, determined by electrophoresis on cellulose acetate membranes, also does not differ from the prototype and is 0-1%. The output of the immunoglobulin preparation is 170-185 doses of 1 kg of feedstock.

 Example 2. Carried out analogously to example 1, but with the addition of glycine to 2%. The final yield of the drug is 170-185 doses from 1 kg of raw materials, specific activity is the titer of antibodies to shigella - 1: 320, to salmonella - 1: 1320, the content of immunologically inactive fractions, determined by electrophoresis on cellulose acetate membranes, is 1%.

 The use of ethanol instead of polyethylene glycol as a precipitating reagent makes it possible to increase the yield of the immunoglobulin fraction by 15%, an increase in the centrifugation rate also gives an increase in the yield of immunoglobulins by 15%. Removal of fibrin impurities leads to a reduction in filtration losses by 40-55%, which, in turn, also improves economic performance. The output of the drug is 170-185 doses of 1 kg of feedstock, while the prototype technique allows you to get no more than 100 doses of 1 kg of raw material. The total increase in the yield of the drug is 70-85%. The cost of the drug compared with the prototype is reduced by 50%.

Claims (1)

 A method of obtaining an immunoglobulin preparation from human blood by salt extraction of an alcoholic precipitate of blood serum, purifying it from lipoproteins, followed by isolation of the target product, adding a stabilizer containing glucose, sterilizing filtration, characterized in that the isolation of immunoglobulins is carried out by alcohol precipitation, a glycine mixture is used as stabilizer and glucose in the ratio (1 - 1.5): 1, the solution of immunoglobulins is treated with sorbents from glass, at the stage of isolation of the target product oestriasis flow-zonal centrifugation, and before the sterilizing filtration, the immunoglobulin solution is passed through a porous glass filter.