Method for obtaining antithymocytic globulin for intravenous injection

FIELD: medicine.

SUBSTANCE: the present innovation deals with immunobiological medicinal preparations applied for intravenous injection at correcting immunodeficient states of different genesis. One should immunize animals with human thymus gland's cells, isolation of immune plasma, its depletion with plasmatic proteins AB (IV) human blood group followed by plasmatic fractioning with PEG 6000 and 20000, depleting semi-product with cellular components of the mixture of all four human blood groups and purification of the target product with PEG 6000. As a stabilizing agent on should apply glycine.

EFFECT: more simplified method.

4 cl, 3 ex

 

The invention relates to medicine, namely to obtain immunobiological drugs used for intravenous injection for the correction of immunodeficiency various origins.

A known method of producing immunoglobulin from biological fluids by adsorption on activated carbon [1].

The disadvantage of this method is the low capacity of coals in respect of immunoglobulins and low selective specified sorbent that provides for the allocation of not more than 1% of the immunoglobulins from the total amount of adsorbed protein.

There is also known a method for extracting antibodies from biological fluids by passage through sorbents, which are used macroporous copolymer of styrene and divinylbenzene containing groups of asymmetric dimethylation bis-ammonium bases [2].

The disadvantage of this method is its complexity and the low degree of purification that does not allow you to use the drug for intravenous administration because it does not meet modern requirements to such products.

A known method of producing immunoglobulins by fractionation of blood proteins with ethanol [3].

The disadvantage of this method is the necessity of using high end is crazy ethanol, creates the risk of denaturation of proteins, which makes it necessary to carry out the process at low temperatures.

Another disadvantage of this method is the necessity of using ion-exchange chromatography with subsequent ultrafiltration for additional purification of immunoglobulins from impurities ballast proteins and ethanol, which significantly complicates the process degrades the quality of the drug and increases its cost.

Closest to the claimed invention is a method for preparing antilimfocitarnyi globulin, based on obtaining the drug from the blood plasma of animals (such as rabbits, goats, horses)immunized with cells of the thymus gland of a human embryo, by fractionating it by rivanol for exemption from ballast proteins, followed by adsorption of heteroaggregation plasma to erythrocytes AB (IV) group donated human blood and purification of the intermediate product using polyethylene glycol (PEG) 6000 [4].

The main disadvantage of this method is the complexity of the process of fractionation by rivanol and the need for further purification of the intermediate product from this reagent.

In addition, currently, the production of rivanol in Russia ceased, and the cost of foreign reagent is very high, which makes it inaccessible for use in Shirakamut the service production of the drug antithymocyte globulin.

The objective of the invention is an improved method of obtaining high-purity reactogenic antithymocyte globulin (ATG) for intravenous administration to the extent necessary for purposes of health.

The technical result of the use of the invention is to develop a large-scale high-tech way of getting the APG, which fully meets the requirements of similar drugs for intravenous and used to prevent and relieve crises of transplants of organs, particularly the kidneys, liver, as well as for treatment of aplastic anemia, the control of inflammatory conditions, etc.

The essence of the proposed method to obtain the APG for intravenous injection is that animals, such as goats, rabbits, horses, subjected to immunization with cells of the thymus gland of a human embryo, isolated plasma of immunized animals, conduct its depletion of human serum AB (IV) group by adding the latest in a ratio of 20-25/1-1,5 (V/V), respectively. Then fractionary plasma proteins with PEG 6000, removed from the fractionated mixture of antibodies to non-lymphoid antigens sequential treatment with a mixture of washed donor erythrocyte and platelet concentrate four groups of human blood Then hold clearing the target product by precipitation with PEG 6000, adding it to the final concentration of 10-12% (W/V) and carrying out the subsequent centrifugation. The obtained residue protein consisting of immunoglobulins, diluted with saline, added as a stabilizer glycine, spend bleaching and sterilizing filtration, filling and lyophilization of the drug. This fractionation of proteins depleted plasma is produced in three stages: the first stage target product precipitated with PEG 6000, pH 7.0 to 7.2, with his final concentration of 10-12% weight/volume, in the second phase precipitated product is suspended in distilled water and remove high molecular weight proteins by precipitation of their PEG 20000 in his final concentration of 2-2,5% weight/volume, pH of 5.4, followed by centrifugation in a third stage, a re-deposition of the target product from the fractionating a mixture of PEG 6000 at a final concentration of 10-12% weight/volume, pH 7.0 to 7.2.

The method is as follows. Repeatedly subjected to immunization of animals such as goats, rabbits, horses, a suspension of cells of the thymus gland of a man, taken from the fruit of a person who dies as a result of asphyxia or birth trauma (first immunization using the full beta-blockers). Later, 2 weeks after the last immunization conduct blood sampling of animals-producers, followed by the separation of immune plasma. To the obtained plasma on billaut plasma donor human blood AB (IV) groups in a ratio of 20-25:1-1,5 respectively. Then spend the fractionation of proteins depleted plasma in three stages: the first stage target product precipitated with PEG 6000, pH 7.0 to 7.2 by adding it to the final concentration of 10-12% (W/V) and conduct low-speed centrifugation at 3000 rpm for 10 minutes in a centrifuge type HERMLE ZK 630. Then suspended in distilled water draught add PEG 20000, pH of 5.4, to a final concentration of 2-2,5% (wt/V), centrifuged and the separated precipitate high molecular weight proteins. In the third stage to the supernatant, the pH of which was adjusted to 7.0 to 7.2, add PEG-6000 to a final concentration of 10-12% (weight/about), carry out the centrifugation, remove the supernatant liquid, dissolve the precipitate immunoglobulins in physiological solution. Antibodies to non-lymphoid antigens drain successively with a mixture of erythrocytes and platelets four groups of human blood and centrifuged. The target product is precipitated with PEG 6000, adding it to the final concentration of 10-12% (weight/volume). After centrifugation at 3000-3500 rpm for 10-15 minutes to precipitate proteins, representing (at least 90%) of the immunoglobulin G and M, dissolved in physiological solution, added as a stabilizer glycine (15-25 g/l of dissolved protein), followed by bleaching and sterilizing filtration, bottling and subsequent lyophilization. Target product p is ed using dissolved in physiological solution. The activity of the drug in lymphocytotoxicity test (LCT) not less 1024-2048.

Example 1. Healthy goats of both sexes aged 1-3 years weighing 18-30 kg 6 times subjected to immunization suspension cells derived from the thymus glands of the fetus or corpses of infants who died as a result of asphyxia or birth trauma in the first days after birth. When the first immunization, the animals, the antigen is administered simultaneously with complete adjuvant's adjuvant. A mixture consisting of 2 ml of suspended cells of the thymus gland of a man at a concentration of 3×108and 2 ml of the adjuvant, injected animals intramuscularly 1 ml in four points. After two weeks, spend 5 subsequent immunization with an interval of 3-5 days. Each animal injected with 2 ml of antigen. Two weeks after the last immunization produce a preform of blood geoconservation that prevents hemolysis of red blood cells. Immune plasma is separated from blood cells by centrifugation. Then the plasma is treated with a plasma donor human blood AB (IV) groups in a ratio of 20:1. The mixture was thoroughly stirred for at least 30 minutes. Then spend the isolation and purification of the target product in three stages. At the first stage target product precipitated with PEG 6000, pH 7.0 to 7.2 by adding it to the final concentration of 10% (weight/volume) and conducting low-speed centrifugation at 3000 rpm for 10 minutes in a centrifuge type HEMLE ZK 630. At the second stage of the precipitated product, suspended in distilled water, add PEG 20000 to a final concentration of 2.5% (weight/volume), adjusted pH to 5.4, centrifuged under the same conditions and to remove high molecular weight proteins. In the third stage to the supernatant, the pH of which was adjusted to 7.0 to 7.2, add PEG-6000 to a final concentration of 10% (weight/volume), perform centrifugation at 3000 rpm for 10 minutes, remove the supernatant liquid, dissolve the precipitate immunoglobulins in physiological solution.

To release the fractionated mixture of antibodies to non-lymphoid antigens contribute consistently mixture was washed donor red cells and platelet concentrates four groups of human blood. A suspension of washed erythrocytes add in the ratio of 0.75:1 (on/about), several times to thoroughly mix. After 10 minutes of exposure to the mixture centrifuged for 10 min at 2500 rpm with a 10°and then add a human platelets from the calculation of 1.75 ml/l of source plasma, leave for 30-minute contact at room temperature with periodic mixing, and then centrifuged 20 min at 3900 rpm at a temperature of 10°C. the Supernatant sterile collect and conduct the selection of the target product using PEG 6000, which contribute to a final concentration of 11% (wt/V) under continuous re is eshiwani within 30 minutes Then the mixture is centrifuged for 10 min at 3000 rpm, the supernatant removed, and to the precipitate add protein physiological sodium chloride solution to dissolve the precipitate. The total protein content in the intermediate defined, for example, biuret method, is 10-15 mg/ml were Then added as a stabilizer glycine rate of 20 g/l of dissolved protein, adjusted pH to 7.3 and 7.5, and then spend the bleaching and sterilizing filtration of the drug through membrane filters, type "Millipore" with appropriate pore sizes, the filling of the drug in vials and lyophilization.

In one ampoule of the target product with a volume of 3-5 ml contains 50±10 mg of protein, which you control by electrophoresis on cellulose acetate films more than 90% consists of immunoglobulins. Such purification meets national and international requirements intravenous immunoglobulins. The product contains no preservatives and antibiotics, its activity in LCTT not less than 1:1024-2048.

Example 2. Received the proposed method, the drug APG (commercial name of Antilepton) was used to treat the patient Peninsula 34 years old (case history No. 748). Diagnosis: acute hemorrhagic pancreatic necrosis, extensive retroperitoneal abscess, peritonitis. Comorbidities: l postoronni hydrothorax, hydroperiod.

Despite all accepted methods of treatment, modern complex therapy, General condition of the patient remains very serious. The immunological witnessed a sharp decline of the immune status of the patient. On the 2nd day after the introduction of Antilymphoma significantly increased the phagocytic activity of neutrophils (FAN). On the 7th day after drug use has increased significantly (2-3 times) the number of immune cells was observed further increase the FAN; there was an increase in the number of immunoglobulins G and M. the Use of Antilymphoma had immunostimulating effect and contributed to a significant improvement of patient's condition, which was soon transferred from the ICU to the ward General surgery with subsequent discharge to home..

Example 3. Patient K., 23 years old (case history No. 4819) was admitted to the hospital with complaints of General weakness, shortness of breath during physical activity, nasal and gingival bleeding. Production had contact with benzene compounds. Blood test on admission: Hb - 47 g/l, erythrocytes-1,5·1012/l, leukocytes 2,6·109/l, segmented - 12% (0,312·10 /l), lymphocytes 87% (of 2.26·109/l), platelets 6·109/liter punctate bone marrow: myelokaryocytes 9,0·109/l, immature granulocytes of 7.5%, Mature - 17,5%, limfotsity,5%, retronasally of 4.5%. In drepanosiphidae - aplasia of the blood. Revealed only small accumulations of eritrotsitov, lymphocytes and isolated siderophore. In the immunological: CD3+CD8+- 47% (0,9·109/l), CD3+CD4+- 40,7% (0,8·109/l), CD20+2,5% (0,05·109/l), CD3+CD16+to 18.6% (0,4·109/l). MDK in the bone marrow: erythroid series - 20%, granulocyte - 40%, lymphoid series is 40%. MDK in the peripheral blood: erythrocytes - 36%, granulocytes - 15%, lymphocytes 30%. Within 5 days the patient received Antilepton 15 mg/kg of body weight. The total dose amounted to 4.62 g of protein. On the third day of injection temperature rose to 38°appeared cutaneous petechiae, increased hemorrhagic manifestations. Was also conducted antihistamine therapy. Subsequently was added transfusion, hemostatic, antibacterial therapy in connection with the emerging infectious complications (right-sided pneumonia, acute pyelonephritis). 1.5 months after treatment was achieved clinical remission and the patient was discharged home. A blood test at discharge: Hb - 86 g/l, erythrocytes - 2,7·1012/l, leucocytes - 3,0·109/l, segmented - 31%, lymphocytes - 65%, platelet - 7,0·109/HP GE is graycheeked there are no symptoms. Dynamics of peripheral blood and bone marrow was positive. During the period of observation is maintained clinical and hematological remission. The patient learns and works.

LITERATURE

1. Lopukhin, Y.M., Molodenkov mathematical SCIENCES. Hemosorption. - M - Medicine. - 1978. - Pp.31-34; 139-141.

2. USSR author's certificate No. 1121619 from 29.03.83.

3. USSR author's certificate No. 1403414 from 15.02.88.

4. RF patent №2178309 from 12.01.2000.

1. The method of receiving antithymocyte globulin for intravenous administration, comprising immunizing animals with cells of the thymus gland of a man, depletion obtained plasma processing her human serum AB (IV) group, the fractionation of plasma proteins, the removal of the fractionated mixture of antibodies to non-lymphoid antigens sequential treatment with a mixture of washed donor erythrocyte and platelet concentrate of the four blood groups in man, purification of the target product by precipitation with PEG 6000, dissolving the obtained antibodies in the physiological solution, the addition of glycine and bleaching and sterilizing filtration, characterized in that the fractionation of proteins depleted plasma is produced in three stages: the first stage target product precipitated with PEG 6000, pH 7,0-7,2, suspended precipitated product in distilled water and remove impurities low, zentrifugenbau is m, in the second stage of the precipitated product to remove high molecular weight proteins by precipitation of their PEG 20000, pH 5.4 and at the third stage, a re-deposition of the target product from the fractionating a mixture of PEG 6000, pH 7.0 to 7.2.

2. The method according to claim 1, characterized in that the deposition of the target product from a depleted plasma in the first stage fractionation spend PEG 6000 at a final concentration of 10-12% weight/volume.

3. The method according to claim 1, characterized in that the high molecular weight proteins in the second phase precipitated PEG 20000, pH of 5.4 with a final concentration of 2-2,5% weight/volume.

4. The method according to claim 1, characterized in that the re-deposition of the target product from the fractionating the mixture in the third stage is carried out at a final concentration of PEG 6000 10-12% weight/volume.

5. The method according to claim 1, characterized in that after sterilizing and clarifying filtration of the received antithymocyte globulin is subjected to lyophilization.



 

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