RU2073525C1 - Method to obtain immunoglobulin product - Google Patents

Abstract

FIELD: medicine, immunology, immune therapy and immune prophylaxis of bacterial and viral diseases. SUBSTANCE: alcohol residue is extracted at 10-fold volume of sodium chloride solution to isolate immunoglobulin fraction followed by polyethylene glycol treatment at 10-15% against total protein. Residue is dissolved in distilled water, centrifuged and supernatant is used as IgA immunoglobulin product. IgA immunoglobulin content is increased by 30-40%, technological cycle is shortened due to elimination of the most time-consuming stage. EFFECT: higher IgA immunoglobulin content, more simplified production technology. 1 tbl

Description

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

 Modern commercial gammaglobulin preparations contain mainly IgG immunoglobulins (85-93%) and only 1-3% of other classes of immunoglobulins.

 Currently, in a number of countries, preparations enriched with IgM and IgA for both intramuscular and intravenous administration are produced and successfully used in the clinic. The company "Behring-werke" Germany produces gamma-M and gamma-A-concentrates (80% and 20% IgM and IgA). In France, the drug Ig GAM (40% IgG, 20% IgA and 25% IgM) is available. Similar drugs are available in Italy, Hungary and other countries.

 To date, convincing data on the effectiveness of the introduction of immunoglobulin preparations by the enteral route for the prevention and treatment of intestinal diseases in children are tilted.

 In Japan, Switzerland, the USA, and England, immunoglobulin preparations for oral administration have been developed. In the United States, a drug containing up to 70% IgG and 20% filler carbohydrates is used orally for the prevention and treatment of intestinal infections in therapeutic practice. In France, England, Switzerland, and the USA, oral preparations containing a complex of colostrum immunoglobulins and serum immunoglobulins have been developed. According to Losonsky (1978), rotavirus infection is cured after oral administration of immunoglobulin, and rotavirus + immunoglobulin immune complexes, up to 50% of the administered drug, are found in feces.

 The company "Immuno" (Austria) produces IgAbulin for oral use for the prevention and treatment of necrotizing enterocolitis.

A known method for producing a complex immunoglobulin preparation (CIP) from sediment B (Cohn III fraction), which is the ballast fraction in the alcohol fractionation of serum (Experimental production regulation N 338-91 human Immunoglobulin, dry, for enteral use Approved on September 6, 1991). In CIP, elevated titers of antibodies against a number of enterobacteria (Shigella, Escherichia, Salmonella), as well as pseudomonads, meningococci, as compared with commercial gammaglobulin preparations, are noted. If normal immunoglobulin contains IgM and IgA in amounts not exceeding 4, then in the complex preparation their content is increased to 15-25%
The method for obtaining instrumentation involves the treatment of extract of sediment B in order to purify lipoproteins from chloroform and dextransulfate and subsequent precipitation of the immunoglobulin fraction with polyethylene glycol (Scheme 1).

 The disadvantage of this method is that the final product contains IgA - immunoglobulin in quantities insufficient to achieve therapeutic efficacy in a number of diseases (diseases of the gastrointestinal tract, chronic infections of the respiratory tract, skin, etc.) (≈ 15%), which limits the spectrum of its application, in addition, the technological process of obtaining the drug is quite time-consuming.

 IgA immunoglobulin selectively passes through glandular organs and, connecting with the so-called secretory component, is located in the form of a dimer on the surface of mucous membrane cells. When secreted by only a significant amount of IgA-IG, it can provide surface protection against a number of bacteria and viruses.

 The aim of the present invention is a method for producing an immunoglobulin preparation with a high content of IgA and simplification of the production technology of the drug. The goal is achieved by the following methods: in the known method for obtaining instrumentation after processing the extract of sediment B with polyethylene glycol (10-15% of the total volume), the precipitate containing immunoglobulins and lipoproteins is dissolved in distilled water, centrifuged and the supernatant is used as an IgA preparation. The precipitate containing euglobulins (IgG, part of IgM, etc.) and lipoproteins are removed, thereby eliminating one of the most time-consuming steps in the known method - treatment with chloroform a highly toxic reagent and dextransulfate, an expensive import reagent (Scheme 2). An increase in the content of IgA-IG in the preparation expands the spectrum of specific therapeutic efficacy against bacterial and viral infections.

 The drug is released in dry form for per os use and in liquid form for i / m and iv administration.

 The lyophilized preparation is intended for oral use for the treatment of a number of viral and bacterial infections of the gastrointestinal tract, especially with necrotizing enterocolitis in young children. In the form of a solution, the drug can be administered iv / iv or in the following indications.

 1. Respiratory tract diseases (relative immunological insufficiency, increased need for IgA), recurrent respiratory infections, recurrent tonsillitis, otitis, sinusitis, asthmatoid bronchitis.

 2. Diseases of the gastrointestinal tract (secondary failure), protein loss and malabsorption syndrome, steatorrhea.

 3. Skin diseases that are difficult to treat: pyoderma, eczema.

 4. Antibody deficiency syndrome (K.-D. Timpner. F. Neuhaus, 1979).

 Example. 1 kg of sediment B is extracted in a tenfold volume of a 0.9% sodium chloride solution, centrifuged for 1 hour at 3000 rpm in a centrifuge with cooling. The precipitate is removed. The volume of extract obtained is 10 liters.

A 50% solution of polyethylene glycol (mm 4-6 thousand) is added to 10 L of sediment B extract to a final concentration of 10-15%. The precipitate containing lipoproteins and immunoglobulins is separated by centrifugation and dissolved in five times the volume of distilled water (200 g sediment in 1000 ml of water). After stirring, the resulting suspension is left for 18 hours at 4-6 ^° C. The precipitate of euglobulins is separated by centrifugation and discarded. The supernatant is the final product in a volume of 1 liter with a protein content of 3-4%. Glucose is added to the finished product to a concentration of 2%. After clarifying and sterilizing the filter, the drug is lyophilized.

 The quantitative ratio of immunoglobulins in the preparation: IgG-55-65% IgA-30-40% IgM-5% Antibody titers are presented in table 1.

 Scheme 1.

 Sediment B.

 Extraction in 0.15 M NaCl, pH 5.5, 1 kg of sediment B, 10 l of 9% NaCl solution.

 Centrifuge

Chloroform 10% pH 5.5. Dextran sulfate 0.1%
Centrifuge

Polyethylene glycol m.m. 6000. Saturation 12%
Sediment.

 Rinsing with distilled water, pH 5.2. Dissolution in 0.15 M NaCl.

 Centrifuge

 Glucose 2% pH 7.2. Brightening and sterilizing filtration.

 Instrumentation.

IgG -65-70%
IgM -15-20%
IgA -15%
Scheme 2.

 Sediment B.

 Extraction in a 0.9% sodium chloride solution.

 Centrifuge

Polyethylene glycol m.m. 4-6 thousand Saturation 10-15%
The precipitate is removed.

 Centrifuge

 Glucose 2% pH 7.2. Brightening and sterilizing filtration.

 IgA immunoglobulin.

IgG -55-65%
IgA -30-40%
IgM 5%

Claims (1)

 A method for producing an immunoglobulin preparation from human blood by salt extraction of an alcoholic precipitate of human blood serum, characterized in that the separation of the immunoglobulin fraction is carried out in the presence of 10 15% polyethylene glycol, followed by dissolution of the immunoglobulin precipitate in distilled water, separation of the final product in the form of a supernatant by centrifugation.

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