EA010735B1 - Medicament normalising reproductive male function and method for preparing thereof - Google Patents

Abstract

A number of inventions relates to a medicament and method for preparing thereof using animals' organs, in particular, to a medicament normalizing reproductive male functions, which is a medical preparation for parenteral administration as solution of a peptide complex, a low-molecular fraction content being from 70 to 90% with a molecular mass comprising the peptide components within 75 to 228 Da, peptide concentration being 2.5÷2.9 mg/ml and to a method for preparing thereof from testicles of sexually mature bull-calf aged less than 12 month or pigs by extracting acetic acid in the presence of zinc chloride; providing repeated deposition of formed homogenized deposition by twofold volumes of acetone at least two times and subsequent washing through a nutsch filter by twofold volumes of acetone; obtaining an aqueous solution of extract, polypeptide concentrate being 2.5÷2.9 mg/ml; centrifuging, filtering thereof with subsequent ultra-filtration purification at the apparatus with counter pressure not exceeding 1.0 kgs/cmusing materials with retention 15000 Da and adding glycocol in the ultrafiltrate to the final concentration 10÷20 mg/ml at pH=5.6÷6.6 mg/ml and subsequent sterile filtration, ampoulation and autoclaving for 8 minutes at 120°C and atmospheric pressure 1.1 kgs/cm, which provides stability of the medicament and preserving therapeutic effectiveness thereof.

Description

The group of inventions relates to a medicinal product and a method for its preparation from animal organs, in particular to the isolation of a biologically active substance from the testes and the preparation of a parenteral dosage form that can be used in medicine as a means of normalizing reproductive function in men.

Known methods for producing biologically active substances and medicines from animal raw materials (RF patent No. 944191, 1994; AS 8I No. 1112606, 1996; AS 8I No. 1218521, 1994; AS 8I No. 1227198, 1986; RF patent No. 1298879, 1993; RF patent No. 1522486, 1994; RF patent No. 2075944, 1997; RF patent No. 2104702, 1998; RF patent No. 2161501, 2001; I8 patent No. 4341765; CB patent No. 1161896, 1969; GB patent No. 2583982, 1987).

A substance is known that restores reproductive function and a method for producing it from testes of sexually mature cattle (RF patent No. 1448443, 1994, А61К 35/48), which is the closest analogue to the prototype for the proposed agent and method for its preparation.

According to the method described in the patent of the Russian Federation No. 1448443, the crushed testes of cattle are treated with acetic acid, precipitated with acetone. The raw material is treated with acetic acid at a pH of 3.5-4.0, for 12-24 hours at a temperature of 10-15 ° C with the addition of 0.5 g / l of zinc chloride, precipitation with acetone is carried out in three volumes at a temperature of minus 5 to minus 10 ° C, and pulped testis is added to the crushed testes after separation of a substance with hyaluronidase activity. Dissolve the precipitate in water, followed by filtration and drying of the target product.

The yield of active substance (i.e., a substance that restores reproductive function) is from 24 to 36 g per 1 kg of feedstock.

The disadvantages of this method include the extraction during the extraction from the testis tissue of a large number (more than 70%) of non-peptide substances, which, being impurities, do not determine the biological activity of the isolated active substance, as well as its low yield.

The present invention posed and solved the problem of developing a method for producing a means that normalizes reproductive function in men in the form of a parenteral dosage form isolated from the testes of sexually mature gobies no older than 12 months of age or pigs, the technical result of which is an optimal technology for isolating a peptide complex with content low molecular weight fraction from 70 to 90% with a molecular weight of its constituent peptide components ranging from 75 to 228 Da and obtaining aqueous extract creates a concentration of the polypeptides of 2.5-2.9 mg / ml, which allows not only to clean impurities from the resulting product, but also to increase its output.

Experimental verification showed that the proposed method provides pharmaceutical stability, maximum biological value and therapeutic efficacy of the product that normalizes reproductive function in men, made in the form of a parenteral dosage form, which is confirmed by the experimental results given in the examples illustrating the invention.

Another aspect of the invention is a means for normalizing reproductive function in men, in the form of a parenteral dosage form, which is a peptide complex with a low molecular weight fraction from 70 to 90% with a molecular weight of its constituent peptide components ranging from 75 to 228 Da, s the concentration of polypeptides 2.5-2.9 mg / ml, the technical result of which is that the isolated substance differs from known substances obtained previously from animal raw materials in molecular weight (from 500 d about 1,500 Da) of its peptide components (RF patents No. 1298879, 2104702, 2163129), as well as its non-toxicity and pyrogen-free nature due to complete purification from impurities.

In the course of studies in animal experiments, it was found that the obtained aqueous solution of the agent in the concentration of polypeptides of 2.5-2.9 mg / ml is a therapeutically effective dose, which allows us to consider the use of the agent for various types of pathology of the reproductive system in men. This is confirmed by the following examples.

In the process of conducting research, the importance of the following stages of the method for producing a product that normalizes reproductive function in men was established in the form of a parenteral dosage form (hereinafter referred to as the drug):

repeated precipitation of the resulting homogenized precipitate with double volumes of acetone at least 2 times and subsequent washing on the suction filter with double volumes of acetone until a light gray precipitate is obtained, which indicates that the precipitate is completely purified from impurities such as lipid, protein, phospholipid, etc .;

obtaining an aqueous solution of the extract in a concentration of polypeptides of 2.5-2.9 mg / ml; centrifugation, filtration, followed by ultrafiltration cleaning at the installation with a backpressure of not more than 1.0 kgf / cm ² through materials with a retention capacity of 15,000 Da and the addition of glycocol to the ultrafiltrate to a final concentration of 10-20 mg / ml at pH 5.6-6, 6;

- 1 010735 sterilizing filtration, ampouling and autoclaving for 8 min at a temperature of 120 ° C and atmospheric pressure of 1.1 kgf / cm ² , which ensures the stability of the drug while maintaining therapeutic efficacy.

The mode and time of autoclaving were established experimentally.

The specified technical result is achieved in that the agent that normalizes the reproductive function in men is made in the form of a parenteral dosage form and is a peptide complex with a low molecular weight fraction from 70 to 90% with a molecular weight of the peptide components included in it ranging from 75 to 228 Yes, with a polypeptide concentration of 2.5-2.9 mg / ml and obtained from the testes of sexually mature gobies no older than 12 months of age or pigs by extraction with acetic acid in the presence of chloride Inca.

The indicated technical result is also achieved by the fact that the proposed method of obtaining a means normalizing reproductive function in men is characterized by the fact that the testes of sexually mature gobies not older than 12 months of age or pigs are frozen at a temperature of at least minus 40 ° C, kept at a temperature of minus (20 -22) ° C for at least two months, then crushed, add a 3% solution of acetic acid in a volume ratio of 1: 5 at a temperature of 20 ± 5 ° C, extraction is carried out with constant stirring, after receiving one To add foreign suspension, add a 1% solution of zinc chloride in a volume ratio of 50: 1, cool with constant stirring to a temperature of 7 ° -16 ° C, then mix for 1 hour after every 4 hours of sedimentation for 48 hours, the extract is separated from ballast substances separation, acetone is added to the extract in a volume ratio of 1: 5, kept at a temperature of 3-5 ° C for 4 hours, the resulting homogenized precipitate is re-precipitated with acetone at least 2 times, then the precipitate containing the active substance is washed twice with a suction filter volume we cooled acetone to a temperature of 7-16 ° C until a light gray precipitate was obtained, wiped through a metal sieve, dried, dissolved in distilled water at room temperature and constant stirring to a concentration of polypeptides of 2.5-2.9 mg / ml, solution centrifuged, filtered, subjected to ultrafiltration cleaning at the installation with a backpressure of not more than 1.0 kgf / cm ² through materials with a retention capacity of 15,000 Yes, glycol is added to the ultrafiltrate to a final concentration of 10-20 mg / ml at pH 5.6-6, 6, solution subjected to sterilizing filtration under a pressure of not more than 2.0 kgf / cm ² , poured into ampoules of 2 ml and autoclaved for 8 min at a temperature of 120 ° C and atmospheric pressure of 1.1 kgf / cm ² .

The invention is illustrated by tables and figure.

The figure shows the effect of the drug on the development of testis explants.

In the table. 1 shows the effect of the drug on the morphological and biochemical parameters of the peripheral blood of guinea pigs in the study of toxicity.

In the table. Figure 2 shows the effect of the drug on the content of sex hormones in peripheral blood in patients with male menopause.

The invention is illustrated by the following examples:

example 1 is a method of obtaining a drug;

example 2 - the study of the toxicity of the drug;

example 3, confirming the biological activity of the drug;

example 4, confirming the therapeutic effectiveness of the drug, made in the form of a dosage form for parenteral administration.

Example 1. The method of obtaining the drug.

The raw materials used are the seeds of mature bulls no older than 12 months of age or pigs that are frozen at a temperature of at least minus 40 ° C and kept at a temperature of minus (20-22) ° C for at least two months.

400 l of a 3% solution of acetic acid are pumped into the extraction reactor and cooled to a temperature of 20 ± 5 ° C, then 80 kg of crushed material are loaded into the reactor with constant stirring until a homogeneous mass of raw material is obtained. The extraction is carried out with constant stirring, after obtaining a uniform suspension, a 1% solution of zinc chloride in a volume ratio of 50: 1 is added to it, cooled with constant stirring to a temperature of 7-16 ° C, then it is stirred for 1 hour after every 4 hours of sedimentation for 48 hours. The extract is separated from the ballast substances by separation at 5000 ± 500 rpm for 1 hour. Acetone in a volume ratio of 1: 5 is added to the extract. It is kept at a temperature of 3-5 ° C for 4 hours. The precipitate formed is homogenized and reprecipitated with acetone at least 2 times. Then the precipitate containing the active substance is washed on a suction filter with twice volumes of acetone cooled to a temperature of 7-16 ° C until a light gray precipitate is obtained. The washed precipitate is wiped through a metal sieve, spread in a thin layer into enameled cuvettes, covered with a double layer of cotton cloth and dried with periodic stirring in a fume hood until the smell of acetone is completely removed.

The yield of active substance (powder of a biologically active peptide complex isolated from the testes) is 65 g per 1 kg of feedstock.

- 2 010735

The resulting powder is dissolved in distilled water with constant stirring at room temperature for 40 minutes to a concentration of peptides of 2.5-2.9 mg / ml. The resulting solution was centrifuged at 3000 ± 200 rpm for 20 ± 5 minutes. The centrifuge is filtered through an AP-15 filter or similar.

The filtrate is subjected to ultrafiltration purification in an ultrafiltration unit with a backpressure of not more than 1.0 kgf / cm ² through materials with a retention capacity of 15,000 Da.

In the ultrafiltrate add the calculated sample of glycol, to its final concentration of 10-20 mg / ml, mix until the glycol is completely dissolved while maintaining a pH of 5.6-6.6.

The solution is subjected to sterilizing filtration, which is carried out under a pressure not exceeding 2.0 kgf / cm ² .

The resulting solution is poured into 2.0 ml ampoules and autoclaved, and the ampoules with the preparation are sterilized for 8 min at a temperature of 120 ° C and atmospheric pressure of not more than 1.1 kgf / cm ² .

The preparation is a colorless, transparent solution and contains a peptide complex with a concentration of polypeptides of 2.5-2.9 mg / ml.

Testing for the absence of high molecular weight protein components is carried out by adding to the contents of 1 ampoule of the drug 1 ml of a 10% solution of trichloroacetic acid. The transparency of the solution indicates the absence of high molecular weight protein components.

To determine peptide bonds in a preparation, a biuret reagent is added to its solution. Staining the solution with violet color indicates the peptide bonds present in the preparation.

In order to determine the polypeptides and their fractions in the preparation, the methods of ultraviolet spectrophotometry, gel chromatography, mass spectrometry, high performance liquid chromatography and polyacrylamide gel electrophoresis are used.

The ultraviolet spectrum of the drug solution is recorded in the wavelength region from 250 to 350 nm, the absorption maximum is observed at a wavelength of 270 ± 5 nm.

The molecular weight of the polypeptides included in the preparation is determined by the following methods: gel chromatography on Sephadex 6-25 and 6-50 (Rääggääa, Sweden). To calibrate a 1.6x60 cm column, use Rörje Mo1essiag \ Uef111 Κίΐ Μδ III (“§egua”, Germany);

mass spectrometry method. The spectra are obtained on a Wouadeg ΌΕ Vyukresyosheyu time-of-flight mass spectrometer with laser desorption and ionization using a matrix (ΜΑΕΌΙ-ΤΟΕ) at a relative intensity of a nitrogen laser of 2300-2400, an accelerating voltage of 25000 V, a delay time of 90 ns, and a pressure in the vacuum chamber of 2.6 x 10 ⁶ torus;

by electrophoresis in a 15% polyacrylamide gel in comparison with a standard set of marker proteins.

Using the methods listed above, it was found that the composition of the drug includes polypeptides with a molecular weight of 75 to 228 Da.

Using reverse-phase high-performance liquid chromatography in an acetonitrile gradient (Ysygokog C18 sorbent, 2x62 mm column), it was established that the composition of the preparation consists mainly of low molecular weight fractions - from 70 to 90%, and there are no high molecular weight components in the preparation.

Pyrogenicity of the drug is determined in rabbits by the generally accepted method (GF XI, issue 2, p. 183) with a test dose of 0.25 mg per 1 kg of animal weight in 1.0 ml of isotonic 0.9% sodium chloride solution for injection. It is shown that the drug is pyrogen-free.

Thus, in the above method, a preparation is obtained - a parenteral dosage form, which is a peptide complex with a low molecular weight fraction from 70 to 90%, with a molecular weight of its constituent peptide components ranging from 75 to 228 Da, with a concentration of 2.5 polypeptides -2.9 mg / ml.

Example 2. The study of the toxicity of the drug.

The general toxic effect of the drug was investigated in accordance with the requirements of the Guidelines for the experimental (preclinical) study of new pharmacological substances (2000): acute toxicity with a single administration of the drug, as well as subacute and chronic toxicity with prolonged administration of the drug.

A study of acute toxicity was conducted on 72 white mongrel male mice weighing 20-22 g. Animals were randomly divided into 6 equal groups. The drug was administered to animals once intramuscularly at doses of 35, 50, 100, 150, 200 mg / kg in 0.25 ml of a sterile 0.9% solution of IAl. Animals of the control group were injected with the same volume of 0.9% solution of IAl.

Studies on the study of subacute toxicity were carried out on 65 white mongrel rats with a body weight of 150-180 g. Daily, once a day, animals of experimental groups were injected intramuscularly with doses of 1, 10, 100 mg / kg in 0.5 ml of sterile 0.9 % solution of IAl. The animals of the control group were injected in the same volume with a sterile 0.9% solution of IAl. Before administration of the drug on the 30th, 60th and 90th day after the start of drug administration, the animals were examined morphologically

The composition and properties of peripheral blood. At the end of the experiment, biochemical and coagulological blood parameters were examined.

Studies of chronic toxicity were carried out for 6 months, based on the duration of the recommended clinical purpose of the drug, in 84 male guinea pigs weighing 250-280 g. Animals of the experimental groups received intramuscularly once daily drug for 6 months in doses of 1, 10, 100 mg / kg in 0.5 ml of sterile 0.9% solution No. 1C1. In the control group, animals were injected according to a similar scheme to a sterile 0.9% solution No. C1 in the same volume. In animals, the number of erythrocytes, hemoglobin, reticulocytes, platelets, leukocytes, leukocyte formula, erythrocyte sedimentation rate (ESR), and erythrocyte resistance were determined by the generally accepted methods in peripheral blood. Along with this, the content of total protein in blood serum was determined by the method of Lowry, potassium and sodium by plasma spectrophotometry. After the experiment was completed, a pathomorphological study of the brain and spinal cord, spinal ganglia, thyroid gland, parathyroid glands, adrenal glands, testes, pituitary gland, heart, lungs, aorta, liver, kidney, bladder, pancreas, stomach, small intestine, colon, thymus, spleen, lymph nodes, bone marrow.

When studying acute toxicity, it was found that a single administration of the studied drug to animals in a dose exceeding the therapeutic recommended for clinical use by more than 5000 times does not cause toxic reactions, which indicates a large therapeutic breadth of the drug.

The study of subacute and chronic toxicity of the drug indicates the absence of side effects with prolonged use of the drug in doses exceeding the therapeutic by 3003000 times. When studying the effect of the drug on the morphological composition and biochemical parameters of the peripheral blood of guinea pigs after 3 and 6 months after the start of its administration, no significant change in the indicators was revealed (Table 1).

When assessing the general condition of animals, morphological and biochemical parameters of peripheral blood, the morphological state of internal organs, the state of the cardiovascular and respiratory systems, and liver and kidney function, pathological changes in the body were not detected.

Therefore, the drug obtained by the proposed method, with long-term administration to animals does not have toxic properties that prevent its further use as a medicine, made in the form of a dosage form for parenteral administration.

Example 3. The effect of the drug on the development of testis explants.

The experiments were carried out on 52 fragments of the testes of rats of the “XV Mag” rat with a body weight of 150-200 g. Nutrient medium for explant cultivation consisted of 35% Eagle's solution, 25% fetal calf serum, 35% Hanks' solution, 5% chicken embryonic extract, on Wednesday glucose (0.6%), insulin (0.5 units / ml), penicillin (100 units / ml), glutamine (2 mM) were added. Fragments of the testes were placed in this medium and cultured on a collagen substrate in Petri dishes in a thermostat at a temperature of 36.7 ° C for 2 days. The preparation was added to the experimental medium at concentrations of 1, 10, 50, 100, 200, and 400 ng / ml. The criterion of biological activity was the area index (PI) - the ratio of the area of the entire explant along with the growth zone to the outgoing area of the testis fragment. The IP values were expressed as a percentage, the control IP value was taken as 100%.

The figure shows the effect of the drug on the development of testis explants.

It was found that after 1 day of cultivation, the explants spread on a collagen substrate and the proliferation of proliferating and migrating cells began on the periphery of the explant. On the 3rd day of cultivation at a drug concentration of 100 ng / ml, a significant increase in the explant PI by 35% was observed compared to the control PI values. In the study of testis explants for longer periods of cultivation (7 days), a similar stimulating effect of the drug at the same concentration was revealed.

Thus, in relation to the testes, the drug had a tissue-specific effect, manifested in stimulating the growth of explants, which indicates the normalizing effect of the drug on the function of the testes cells.

Example 4. The effectiveness of the drug with male menopause.

Treatment with the drug was carried out in 19 patients of the main group aged 43 years to 61 years with negative manifestations of male menopause. Patients complained of rapid fatigue, decreased physical and mental performance, weakened memory, sudden hot flashes, increased sweating, irritability, but most men paid special attention to the appearance of sexual weakness. Patients have not previously sought medical help.

The drug was administered to patients of the main group at a dose of 5 mg in 2 ml of saline intramuscularly once daily for 10 days.

The control group included 17 patients who received injections of saline according to a similar pattern.

Patients' complaints were evaluated in dynamics, a general clinical study of blood and urine, a biochemical study of blood was performed. Using the radioimmunological method, the content was determined by

- 4 010735 blood hormones in the blood serum of patients. In addition, they performed palpation of the prostate gland, a laboratory study of its secretion and ejaculate.

The results of the studies showed that in the examined patients, the manifestations of male menopause are the result of mainly hormonal changes in the body, characteristic of this age group. Although it is possible that the severity of these manifestations in young people is also due to the influence of various adverse environmental and professional factors. In some patients, an increase in blood glucose was noted, which probably indicates a breakdown of the insulin regulation system. Characteristic was a change in the concentration of sex hormones in the blood (Table 2).

In almost all patients, edematous changes in the prostate were determined by palpation.

The use of the drug contributed to improving the overall well-being of patients, all patients noted an improvement in sexual function, a decrease in the phenomena of sexual weakness and an increase in libido.

The drug had a regulatory effect on the content of sex hormones in the blood (table. 2). Moreover, there was a significant increase in testosterone concentration, which correlated with improved sexual function and the results of microscopic examination of ejaculate.

Microscopic examination of the ejaculate showed an increase in the number of sperm and their motility, a decrease in the pathological forms of sperm, a decrease in the number of leukocytes, which confirms the normalizing effect of the drug on the function of the testes.

Thus, the results of the study indicate the therapeutic efficacy of the drug and the feasibility of its use in the complex treatment of male menopause to normalize the reproductive system in men at a therapeutic dose of 5 mg (2.5 mg / ml) intramuscularly once daily for 10 days.

Table 1

Index The introduction of the drug (10 mg / kg) 3 months 6 months Control (n = 24) The drug (n = 24) Control (n = 24) The drug (n = 24) Red blood cells, x10 ¹² / l 5.3 ± 0.7 5.3 ± 0.7 5.2 ± 0.1 5.1 ± 0.3 Hemoglobin, g / l 14.3 ± 1.8 14.1 + 1.5 14.2 + 1.4 14.1 ± 1.2 Reticulocytes,% 1.1 ± 0.02 1.2 + 0.03 1.1 ± 0.05 1.3 ± 0.08 Platelets x10 ⁹ / L 142.2 ± 5.8 143.2 + 5.3 144.3 ± 7.2 144.1 ± 7.3 White blood cells, x10 ⁹ / l 9.6 ± 0.1 9.4 ± 0.7 9.7 ± 0.2 9.6 ± 0.4 Band neutrons,% 0.32 ± 0.01 0.31 ± 0.05 0.30 ± 0.03 0.32 ± 0.07 Segmented neutrophils,% 43.2 ± 1.6 44.6 ± 2.1 43.8 + 1.7 44.3 ± 2.6 Eosinophils,% 0.71 ± 0.05 0.70 + 0.06 0.72 ± 0.07 0.70 ± 0.01 Basophils,% 0.67 + 0.01 0.69 ± 0.04 0.71 + 0.04 0.60 ± 0.01 Monocytes,% 2.5 + 0.03 2.2 ± 0.05 2.4 ± 0.03 2.4 ± 0.04 Lymphocytes,% 48.5 + 1.9 50.1 ± 2.2 52.7 ± 2.7 47.8 ± 2.1 ESR, mm / h 1.82 ± 0.02 1.78 ± 0.04 1.91 ± 0.06 1.79 ± 0.03 Erythrocyte resistance,% No. C1 - maximum - minimum 0.41 + 0.04 0.33 ± 0.05 0.40 ± 0.03 0.31 ± 0.04 0.40 ± 0.04 0.32 + 0.02 0.43 ± 0.06 0.31 ± 0.05 Total protein in blood serum, g / l 72.3 ± 2.2 73.7 ± 2.2 71.0 ± 2.7 73.1 ± 3.6 Sodium in blood serum, mmol / l 153.7 ± 7.1 152.7 ± 5.6 153.3 ± 6.2 151.9 ± 7.1 Serum potassium, mmol / l 5.3 ± 1.7 5.2 ± 2.8 5.2 ± 1.4 5.3 ± 2.1

table 2

Index Before treatment After treatment with the drug LH, ng / ml 3.64 ± 0.17 2.95 ± 0.12 FSH, ng / ml 2.86 ± 0.09 2.34 ± 0.11 Testosterone ng / ml 5.7 ± 0.6 7.2 ± 0.1 ‘

* P <0.05 compared with the indicator before using the drug.

Claims (2)

1. A tool that normalizes reproductive function in men, which is a parenteral dosage form in the form of a solution of the peptide complex with a low molecular weight fraction of 70 to 90% with a molecular weight of its constituent peptide components ranging from 75 to 228 Da, with a concentration of polypeptides 2.5-2.9 mg / ml and obtained from the testes of sexually mature calves of calves no older than 12 months of age or pigs by extraction with acetic acid in the presence of zinc chloride 2. The method of obtaining funds according to claim 1, characterized in that the testes of sexually mature gobies not older than 12 months of age or pigs are frozen at a temperature of at least minus 40 ° C, kept at a temperature of minus (20-22) ° C for at least two months, then crushed, add a 3% solution of acetic acid in a volume ratio of 1: 5 at a temperature of 20 ± 5 ° C, the extraction is carried out with constant stirring, after obtaining a homogeneous suspension, add a 1% solution of zinc chloride in a volume ratio of 50 volumes of homogeneous suspend to 1 we take a 1% solution of zinc chloride, it is cooled with constant stirring to a temperature of 7-16 ° C, then it is stirred for 1 hour after every 4 hours of settling for 48 hours, the extract is separated from the ballast substances by separation, acetone is added to the extract in a volume ratio of raw materials: acetone, equal to 1: 5, is kept at a temperature of 3-5 ° C for 4 h, the resulting homogenized precipitate is re-precipitated with acetone at least 2 times, then the precipitate containing the active substance is washed on a nutfilter with two volumes of cooled to a temperature uri 7-16 ° C of acetone until a light gray precipitate is obtained, wiped through a metal sieve, dried, dissolved in distilled water at room temperature and constant stirring to a concentration of polypeptides of 2.5-2.9 mg / ml, the solution is centrifuged, filtered subjected to ultrafiltration cleaning at the installation with a backpressure of not more than 1.0 kgf / cm ² through materials with a retention capacity of 15,000 Yes, glycol is added to the ultrafiltrate to a final concentration of 10-20 mg / ml at pH 5.6-6.6, solution subjected to sterilizing fil tration under a pressure of not more than 2.0 kgf / cm ^2, filled into vials of 2 ml and autoclaved for 8 minutes at 120 ° C and atmospheric pressure of 1.1 kgf / cm ^2.