The method of obtaining igm-containing concentrate of immunoglobulin

 

(57) Abstract:

The invention relates to medicine and for the production of therapeutic drugs from plasma or serum of donor blood, namely, the method of obtaining lgM-containing concentrate of immunoglobulin. The method consists in the fact that the precipitate II+III, obtained by ethanol fractionation of plasma (serum) blood is processed sequentially by the Aerosil-380 and sodium capricolum with intermediate zentrifugenbau removal of ballast proteins, subjected to ultrafiltration to remove low molecular weight impurities and concentration. After sterilizing filtration obtain the target product containing an average of 15% of total IgM immunoglobulin. The advantage of the invention consists in the enrichment of immunoglobulin IgM. table 1.

The invention relates to medicine and relates to a therapeutic drug from plasma donated blood

Raw material for the production of the drug is plasma donors tested for the absence of the surface antigen of hepatitis B virus (HBsAg), antibodies to human immunodeficiency virus (HIV) and hepatitis C virus

The prototype of the invention is a method of producing immunoglobulin is aceski immunoglobulin contains mainly immunoglobulin G class, which greatly limits its activity against gram-negative microorganisms.

The aim of the present invention to provide IgM-containing concentrate of immunoglobulin.

The method is as follows. Combined pool of plasma fractionary ethanol in the cold to the stage of precipitate II+III; crude residue is suspended in 0.9% sodium chloride in a weight ratio of 1:10 and treated with Aerosil-380, ballast sediment removed by centrifugation at 13,000 g; add sodium kapalbekuly to a final concentration of 0.4% at slightly acidic pH values; conduct maturation of the drug for the formation of a precipitate unstable proteins, which are removed by centrifugation; perform ultrafiltration to remove low molecular weight impurities and concentration and after sterilizing filtration obtain the target product.

Example. Immune plasma in the amount of 360 l obtained by the method of plasmapheresis. Spent the alcohol fractionation at low temperatures. Received 21000 g wet sediment fraction II+III. For the preparation of IgM-containing concentrate of immunoglobulin 260 g of sediment suspended under stirring in 2600 ml of 0.9% restr>C for 18 hours Aerosil-380. After 1 hour, Aerosil and ballast proteins were removed by centrifugation at 13,000 g. The volume of centrifugate was 2170 ml of 0.06 M acetate buffer brought the pH of centrifugate to 4.0, added kaprilat sodium to a final concentration of 0.4%. After maturation at a temperature of (203)oC for 18 hours spent centrifugation at 13,000 g. Then spent diafiltration of centrifugate 5-fold volume of sodium chloride 0.9 per cent, the ultrafiltration to a protein concentration of 5.3%, was added to the concentration of glucose (1,00,2)%, glycol to a concentration of (0,50,2)%. Spent sterilizing filtration of the solution. Received 200 ml of concentrate. The concentrate of immunoglobulin poured in bottles of 50 ml and 25 ml of the target product.

In laboratory preparations of blood of the Kirov research Institute of Hematology and blood transfusion of the developed technology made 7 series concentrate. The table below summarizes the results of the evaluation of physicochemical and biologic properties of the obtained series. The concentrate produced a clear, colorless or slightly opalescent solution, transparency averaged (0,0130,001), chromaticity (0,046 0,002) units). the density. During the observation period (up to 12 months.) education is Vila average (5,67 of 0.07)%.

Compared with the prototype concentrate has a low anticomplementary activity (AKA): in the experimental samples it ranged from 3.2 to 6,54 mg protein, not activating 2 CH50the complement. (Level AKA intramuscular immunoglobulin differs from that of figure 3 of the order and approximately 1,510-3mg).

Evaluation of the fractional composition of the samples obtained by the method of immunoelectrophoresis revealed the presence of 4 arcs of precipitation corresponding to IgG, IgA, IgM, and a small quantity of albumin. The percentage of IgG:IgA:IgM was equal to the average of 66.6:18,1:15,3%.

Molecular parameters of the samples was estimated by the method of gel chromatography of low pressure with an ACA-34. Elution was performed 0.05 M phosphate buffer. The chromatogram was allocated 3 peak corresponding to the fractions with molecular masses of more than 160 thousand D (IgM), 150-160 thousand D (total peak IgG and IgA), the third - 50-70 thousand Days Last indicate the presence of albumin. The absence in samples of polymers IgG was confirmed by the absence in the faction more than 160 thousand D immunoglobulin G, is proved by the method of radial immunodiffusion according to Mancini. The percentage fractions was as follows: the content of IgM ranged from 9 to onsite, aerogenes.

According to the TPHA concentrate has a high activity of antibodies to Pseudomonas aeruginosa: the average geometric return titer was 146,9. According to ELISA the presence of IgG antibodies to lipopolysaccharides of Pseudomonas aeruginosa ranged from 3,28 to 16,58 EDELISA/mg protein, IgM-antibodies - from 0.18 to 1.80 EDELISA/mg protein. The corresponding figures for E. coli averaged 8,31 of 0.85 and 1.12 of 0.25. EDELISA/mg protein. It must be emphasized that in the preparation of immunoglobulin produced by the method-prototype, reverse titer antiintegrin antibodies in TPHA hovers around 80, ELISA activity of IgG to LPS Escherichia coli is equal to the average of 1.60 0,27, IgM - 0,01 0,01; to E. coli - 10,46 0,87, IgM - 0,03 0,01 UELISA/mg protein.

Thus, the obtained concentrate immunologically active fraction of donor blood containing an average of 15% of total IgM immunoglobulin and highly active antibodies against gram-negative pathogens and low anticomplementary activity.

The proposed method of producing IgM-containing concentrate of immunoglobulin from the precipitate fraction II+III can be used when creating the production technology IgM-containing drug immunodeficiency is the conduct alcohol plasma fractionation at low temperatures, get the crude residue fraction II + III, characterized in that it is suspended in 0.9% sodium chloride in a weight ratio of 1 : 10 and treated with Aerosil-380, ballast sediment removed by centrifugation at 13000g, add sodium kapalbekuly to a final concentration of 0.4% at slightly acidic pH values, conduct maturation concentrate for the formation of a precipitate unstable proteins, which are removed by centrifugation, perform ultrafiltration to remove low molecular weight impurities and the concentration of the drug and after sterilizing filtration obtain the target product.

 

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