The method of obtaining the immunoglobulin preparation

 

(57) Abstract:

The invention relates to medicine, in particular to immunology. The method of obtaining the immunoglobulin preparation is salt extraction alcohol precipitate of human serum, clearing the lipoproteins, followed by separation of the target product, adding a stabilizer, containing glucose, and sterilizing filtration, with a selection of immunoglobulins conduct alcohol precipitation, as stabilizer, a mixture of glycine and glucose at a ratio (1-1,5):1, a solution of immunoglobulins treated sorbent glass, at the stage of selection of the target product are conducting a flow-zonal centrifugation, and before sterilizing pass filtering the solution through a porous glass filter. Technical result: increasing output and lowering the cost of the drug.

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

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

Way to bookmark and purification of immunoglobulins by dissolving the precipitate in 0.05 M acetate buffer, pH 4.3 and treatment kaprilat sodium to a final concentration of 0.5%, pH of 4.8, the deposition of immunoglobulins alcohol, adding 0.15 M glycine and 2% sucrose, holding sterilizing filtration and the addition of 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 kaprilat sodium in the final product and the instability of the resulting solution immunoglobulin able to be kept after sterile filtration prior to bottling no more than 10 hours

Closest to the claimed technical essence and the achieved result is a method for immunoglobulin preparation (TRC) from sludge B (III fraction Kona) (patent RU 2060034, class a 61 K 39/395, published 06.09.91)

The method comprises the salt extraction of the alcohol precipitate B serum (III fraction Kona) in 0.9% sodium chloride solution. Clearing of lipoprotein extract sediment B are chloroform (10% by volume), followed by deposition of immunoglobulin fraction in the presence of 12% of polyethylene glycol. The precipitate immunoglobulins diluted in 0.9% sodium chloride solution, centrifuged to remove insoluble impurities. To the solution after centrifugation add Guetta, he provides a drug containing impurities of residual polyethylene glycol. In addition, the resulting solution of immunoglobulins contain significant amounts of fibrinogen and fibrin, shoesadidas with immunoglobulins. These impurities significantly complicate sterilizing filter and cause instability of the drug during storage in liquid form. The consequence of this is the need for immediate filling and freeze drying after filtration. Loss in the final release of the drug due to difficulties in filtration and the need to re-conduct this procedure reaches 30%.

The problem solved by the invention is obtaining immunoglobulin preparation that is free of impurities volatile components, complicating filtering.

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

This result is achieved in that in a method of producing immunoglobulin preparation from human blood by salt extraction of the alcohol precipitate serum, clearing the lipoproteins, followed by separation of zelenoklyniv conduct alcohol precipitation, as a stabilizer, a mixture of glycine and glucose at a ratio (1-1,5):1, a solution of immunoglobulins treated sorbents made of glass, at the stage of selection of the target product are conducting a flow-zonal centrifugation, and before sterilizing pass filtering the solution through a porous glass filter.

The method is as follows. Deposition of immunoglobulin fraction of the chloroform extract of the precipitate B is carried out with the use of ethyl alcohol. This avoids the presence in the final product of polyethylene glycol, which according to the method prototype must be removed by repeated washing. Alcoholic residue is dissolved in 0,55 - 0,65% solution of sodium chloride with the addition of glycine and glucose at a ratio (1-1,5):1 (i.e. 20.5% of glucose and 2-3% glycine), pH 6.5-7.5. These values of pH and salt are the best because they provide the most complete formation of a precipitate of insoluble fibrin, and do not require further correction. Reducing the salt concentration below 0,55% impedes the dissolution of the sediment, and increase more of 0.65% makes it difficult subsequently held the lyophilization. The increase in the ratio of the concentrations of glycine and glucose does not increase the yield of the final PR is of fibrin sediment spend processing solution using glass beads. The solution is incubated for about 30 minutes until the formation of fibrin, then subjected to flow-zonal centrifugation at 15000 rpm. /min. and the Supernatant passed through a filter of porous glass (filter Shota) in order to remove residual amounts of fibrinogen and fibrin, adsorbed on porous glass. The resulting solution was subjected to sterilizing filtration. This solution is stable and can be stored for up to filling in for 1 month without changing the physical-chemical properties (transparency, lack of deposited components).

Example 1. To 1 kg of sediment B add 10 l of 0.9% sodium chloride solution with stirring. The volume of extract 10 l protein 2,00,2%. To extract sediment B add 10% chloroform by volume. The mixture is slowly stirred for 2 hours the Precipitate is removed by centrifugation. Set the pH of the solution is equal to 6,90,5. Add ethanol to a final concentration of 26%. The precipitate was separated by centrifugation at 15000 rpm./minutes the precipitate immunoglobulins diluted in 0,60,05% solution of sodium chloride with the addition of 20.5% glucose and 3.0% glycine. To the solution add glass beads and incubated for about 30 minutes until the formation of fibrin. After processing, carried out the separation of the fibrin Ecay through the filter Shota. Then a solution of immunoglobulins is subjected to sterilizing filtration using a depth cellulose filters Cuno-60" and "Cuno-90", and lyophilized using the apparatus of TGS -15.

Specific activity (titer) of the obtained product is not different from the prototype (Shigella - 1:320, Salmonella 1:320 to 1:640). The content of immunologically inactive fractions, determined by electrophoresis on membranes from acetylcellulose, 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 raw material.

Example 2. Carried out analogously to example 1, but adding glycine to 2%. The final output of the product - 170-185 doses of 1 kg of raw materials, specific activity - titer antibodies to Shigella - 1:320, Salmonella - 1:1320 content immunologically inactive fractions, determined by electrophoresis on membranes from acetylcellulose, - 1%.

Use as precipitating reagent instead of glycol ethyl alcohol can improve the output of the immunoglobulin fraction by 15%, increase speed centrifugation also gives an increase in the yield of immunoglobulins by 15%. The removal of impurities fibrin leads to reduce the d drug is 170-185 doses of 1 kg of raw materials, while the method of the prototype allows you to receive no more than 100 doses of 1 kg of raw material. The total increase in the yield of the product is 70-85%. The cost of the drug compared with the prototype reduced by 50%.

The method of obtaining the immunoglobulin preparation from human blood by salt extraction of the alcohol precipitate serum, clearing the lipoproteins, followed by separation of the target product, adding a stabilizer, containing glucose, sterilizing filter, characterized in that the selection of immunoglobulins conduct alcohol precipitation, as stabilizer, a mixture of glycine and glucose in the ratio (1 to 1.5) : 1, treated with a solution of immunoglobulins sorbents made of glass, at the stage of selection of the target product are conducting a flow-zonal centrifugation, and before sterilizing filtration miss the immunoglobulin solution through a porous glass filter.

 

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