RU2041716C1 - Method for obtaining myelopid - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves culturing pig spinal cord cells in nutrient medium, separating supernatant by centrifuging, exposing it to ultrafiltration on membranes having 10 000 Dalton permeability threshold at a temperature from 4 C to the room temperature, concentrating by means of lyophilization, performing gel filtration chromatography with G-25 Sephadex. The separated fractions of 600-3000 Dalton molecular mass are repeatedly concentrated by means of lyophilization. EFFECT: higher immunostimulating activity; lowered therapeutic doses. 2 tbl

Description

 The invention relates to medicine, namely to immunology and can be used in the production of an immunologically active drug myelopid.

 A method for producing myelopid, formerly known as a stimulator of antibody producers, is described by isolating cells from a pig’s bone marrow, culturing them in a nutrient medium, separating the supernatant by centrifugation, concentrating and isolating the target product by gel chromatography on a column filled with Sephadex G-50. Fractions eluted in the exit region of cytochrome G with a molecular weight of about 13,000 Da are concentrated by lyophilization.

 There is also known a method for producing myelopid by culturing bone marrow cells in a nutrient medium, separating the supernatant by centrifugation, concentration, gel filtration of the concentrated supernatant on a Sephadex G-25 column, selecting fractions with a molar mass of 1000-2000 and desalting on a Sephadex C-10 column. The finished product is dried by lyophilization.

A disadvantage of the known methods is the small yield of the drug (from 1 kg of uncleaned bones, 1.0 ^. 10 ¹⁰ bone marrow cells receive 25-30 doses of the drug) and its low specific activity. Myelopid was standardized so that 1 dose of the drug contained 3 mg of protein determined by the Lowry method. The stimulation index was at least 1.6 at a drug concentration of 50 μg / ml.

 The purpose of the invention is an increase in the immunostimulating activity of myelopid.

The goal is achieved in that the bone marrow cells of pigs cultured in a culture medium, the supernatant was separated by centrifugation and subjected to an ultrafiltration on membranes with 10.000 Da threshold permeability at a temperature from ^about 4 C to room temperature, then concentrated by lyophilization, is performed by gel chromatography on Sephadex G- 25, selected fractions with a molecular weight of 600-3000 Da are re-concentrated by lyophilization.

Prirealom distinguishing method is that the isolation of the desired product is carried out by ultrafiltration on membranes with 10.000 Da threshold permeability at a temperature from ^about 4 C to room temperature.

 In more detail, the method is carried out in the following way.

Bone marrow from swine rib bones after they are washed grinding shaking thermostatic shaker at ³⁷ ° C for 1 h in serum free medium 199. Next, the cells were washed twice by centrifugation and placed in a culture medium. The culture medium is prepared based on 199 medium. Cultivation was carried out in a thermostated shaker or spinner apparatus at 37 ^{° C} for 20 hours. The supernatant was separated from the cell mass by centrifugation at + 4 ^{° C} at a speed of 8000 rev / min for 20 min.

The resultant culture supernatant was subjected to bone marrow cells ultrafiltration cassettes company "Millipore" with a threshold of 10,000 Da permeability at a temperature from ^about 4 C to room temperature and excess pressure system at the inlet 0.5 atm. The sterile ultrafiltrate obtained at the outlet is lyophilized at a temperature of (-30) -0 ^о С. The dry lyophilized product is dissolved in sterile pyrogen-free water, centrifuged, the clarified concentrate is subjected to microfiltration, and then gel chromatography on a column filled with Sephadex G-25, selected fractions with molecular weight 600-3000 Da re-subjected to lyophilization. From 1 kg of raw materials (1.0x10 ¹⁰ cells) receive 250-300 doses of the drug.

 Checking the claimed technical solution for compliance with its criterion of "Inventive step" showed that neither in the patent nor in the scientific and technical literature found the totality of the features given in the claims.

 The following are specific examples of the method and analysis of the biological properties of myelopid.

PRI me R 1. 25 kg of cleaned rib bones of pigs are crushed under sterile conditions. 3 liters of sterile 199 medium are added to the homogenizate. The suspension was shaken on a shaker at ³⁷ ° C for 1 hour to wash out bone marrow cells. 3 l of cell-containing supernatant was separated by filtration. Cells were washed twice with medium 199 by centrifugation at + 4 ° ^C. The cells obtained 50 ml ^(15-25. October ¹⁰⁾ is poured 25 liters of medium and 199, bringing the cell concentration to 10 ^10-cell / liter and incubated for 20 hours at 37 ^о С. Cells are separated by centrifugation at 5000 rpm at a temperature of + 4 ^о С. The supernatant of 25 l is ultrafiltered on Millipor membranes with a permeability of 10,000 daltons at a pressure of not more than 0.5 atm and a temperature of + 4 ^о С. volume of 23 l freeze-dried at (-30 ° ^C) -0 ° ^C. The dry product is stretched oryayut at 4 ^{° C} in 350 ml of pyrogen-free sterile water for 30 min with vigorous stirring. The insoluble precipitate is separated by centrifugation at 8000 rev / min at 4 ^{° C} for 20 min. The supernatant of 300 ml was sterile filtered on a filter with a pore diameter of 0.2 microns, and applied to a column of Sephadex G-25 size 10h100 cm. Gel chromatography is carried out under sterile conditions at 4 ^{° C,} using as an eluate of 10 mM phosphate buffer pH 7.00, selecting a fraction with a mol. mass corresponding to 2000 Da. The concentration of myelopid in the resulting eluate was adjusted to 0.1-0.2 mg / ml by adding sterile 10 mM phosphate buffer pH 7.0. The myelopid solution is sterile filtered through a filter with a pore diameter of 0.2 μm, poured into 3 ml vials and freeze-dried at a temperature of (-30) -0 ^о С.

 The product yield is 300 doses of the drug with a Lowry protein content of 0.3-0.6 mg / dose. The stimulation coefficient is 1.9.

 PRI me R 2. The method is carried out similarly to that described in example 1, but carry out ultrafiltration on membranes with a permeability threshold of 10,000 Da at room temperature.

The output of myelopid is 350 doses (from 10 ¹⁰ bone marrow cells) with a stimulation coefficient of 1.8.

 PRI me R 3. The effect of stimulation of antibody formation is assessed at the peak of the immune response to ram red blood cells in vitro, adding test material to the cell culture of lymph nodes obtained from mice on the fourth day after immunization.

 The stimulation coefficient is calculated as the ratio of the number of antibody-forming cells (AOK) in the culture with the test sample to the number of AOK in the control culture.

 The anti-stimulating effect of myelopid obtained by known and proposed methods is shown in table 1.

 From the data of table 1 it follows that the myelopid obtained by the proposed method has a 100 times greater specific activity.

 The anti-stimulating effect of myelopid at different temperatures of the ultrafiltration process is given in Table 2.

Conducting ultrafiltration process at a temperature above room temperature or below 4 ^{° C} leads to reduction in yield and loss of activity of the drug.

 Myelopid obtained by the proposed method has a 100 times greater specific immunostimulating activity, which allows to reduce the dose of the drug administered during treatment and to receive 10 times more doses from the same number of bone marrow cells.

Claims (1)

METHOD FOR PRODUCING MYELOPID by preparing a suspension of pig bone marrow cells, incubating them in a nutrient medium, separating the supernatant with subsequent isolation of the target product, characterized in that the target product is further purified by ultrafiltration on membranes with a permeability threshold of 10,000 D and at a temperature of +4 ^o С to room temperature and by gel filtration on Sephadex G 25, and a fraction with a molecular weight of 600 3,000 Da was taken.

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