RU2545714C1 - Method of production of adjuvant for viral vaccines - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method comprises dissolving the mixture of birch bark triterpenoids in tetrahydrofuran to obtain the solution with a concentration of 5-10 g/l. Oleic acid is dissolved in an amount of 5-10% by weight of the birch bark triterpenoids. The sterilising filtration of the mixture is carried out. 25-fold excess of 0.01 M tris buffer is added, pH 9.0±0.2, when stirring. Sonication is carried out for 5-10 min. The organic solvent is removed using ultrafiltration on hollow membranes with exclusion threshold of 300 kDa at a rate of 1.0-1.2 L/min at a pressure of 0.6-0.8 atm. Cryoprotectant is added from the group of substances: mannitol, maltose, trehalose, mannose, sorbite, sucrose. The resulting concentrated mixture is frozen.

EFFECT: invention enhances the immunogenic activity of viral vaccines and provides their stability while storage.

2 cl, 2 dwg, 6 tbl, 7 ex

Description

The invention relates to biotechnology and immunology, in particular to the production of drugs specifically stimulating antibody formation, and can be used in medicine in the design and preparation of highly effective viral vaccines.

Vaccine prophylaxis is considered in modern conditions as one of the leading mass effective means of combating infections [Results and prospects of the development of vaccine prevention in the XXI century, I.V. Feldblyum, http://www.rusmedserv.com/epidinf/m-dokl/].

Currently, to increase the effectiveness of vaccines in experimental and clinical studies, adjuvants of various origins are used: mineral (aluminum hydroxide or phosphate, etc.); vegetable (saponins - QuilA, QS21); microbial (killed bacteria, lipopolysaccharide and its derivatives, DNA CpG motifs), etc.

Due to the toxicity or insufficient effectiveness of most adjuvants, only aluminum salts and the MF-59 water-in-oil emulsion are allowed for wide clinical use, and in some countries virus-like particles (VLP - virus-likeparticles) and immunostimulating complex (ISCOM - immunostimulatingcomplex). Foreign commercial influenza vaccines are produced with only one MF-59 adjuvant; such vaccines proved to be more immunogenic when vaccinating elderly people, however, they had increased reactogenicity.

The development of new classes of adjuvants is a promising area of modern immunobiology. Over the past decades, significant changes have occurred in the technology for the production of vaccines. The vaccines currently undergoing clinical trials are significantly different from traditional vaccines. First of all, these are recombinant vaccines based on purified proteins. The creation of new vaccines is, in turn, the reason for the search for new adjuvants. The use of adjuvants can reduce the dose of antigen in the vaccine, increase the immunogenicity of “weak” antigens, prevent competition of antigens in combination vaccines, increase the rate of development and duration of the immune response in vaccinated patients, induce protective properties of the mucous membranes, and also increase the strength of the immune response in children and elderly people age.

At present, adjuvants in the form of various forms are considered in world and domestic practice: liposomes, virosomes, immunostimulating complexes, nanoemulsions (MF59, SAF, Montanide), polymer nanospheres, virus-like particles, etc.

The most promising adjuvants, according to researchers, are nanoparticles (NPs). One of the reasons for using NPs as adjuvants is the well-known fact that they are effectively absorbed by antigen-presenting cells. Thus, if an antigen is associated with NPs, then it will be directionally absorbed by macrophages, which will lead to an increase in the immune response.

A promising area in this area are adjuvants based on birch bark triterpenoids.

When creating such adjuvants, there are difficulties associated with the extremely low solubility of triterpenoids in water. Thus, the solubility of betulin, as determined, is less than 1 μg / ml. Therefore, the creation of a water-soluble form is very important, as it should lead to an increase in the concentration of particles and an increase in the effectiveness of the adjuvant. The search for such forms led to the creation of spherical amorphous NPs based on birch bark.

Spherical amorphous nanoparticles (SANCH) based on the natural pentacyclic triterpene substance - betulin (betulenol, betulinol, lupendiol) are a composition of biologically active compounds. This compares them favorably with all known types of carriers. They, having a pronounced spectrum of biological activity (antimicrobial, antifungal, antiviral, hepatoprotective, anti-inflammatory, anti-cancer, anti-allergic, membrane-stabilizing, immunomodulating, antioxidant), have adjuvant properties.

The patent of the Russian Federation 2322091 “Composition of biologically active substances and a method for producing its nanodispersions”, A23L 1/30, A61K 36/00, publ. 04/20/2008 and the patent of the Russian Federation 2322998 “Carrier of medicines and diagnostic tools”, A61K 36/185, A61K 9/10, publ. 04/27/2008, concerning the preparation of nanodispersions of birch bark extract as a carrier of medicinal and diagnostic agents. The patents describe a method for producing nanodispersions, consisting in the fact that distilled water was added to the flask with birch bark extract in tetrahydrofuran (5 mg / ml) with vigorous stirring. The resulting dispersion was stirred, the solvent was evaporated on a rotary evaporator at a temperature not exceeding 40 ° C. Then the nanodispersion was processed in an ultrasonic bath and concentrated on a rotary evaporator to 1-1.5 mg / ml. The average size of the obtained nanoparticles was 187 nm.

The disadvantage of this method is unsuitability for industrial use - the difficulty of scaling the process, the limiting factor of which is the removal of the organic solvent on a rotary evaporator.

Known patent of the Russian Federation 2424516 “Method for isolating a mixture to obtain aqueous dispersions of spherical nanoparticles”, G01N 33/15, B82B 1/00, publ. 07/20/2011 on the allocation of a mixture to obtain aqueous dispersions of spherical nanoparticles from a mixture of poorly water-soluble birch bark triterpenoids, including the injection of excess water into a solution of birch bark triterpenoids in a water-miscible organic solvent (tetrahydrofuran) with the formation of a dispersion containing spherical nanoparticles and triterpeno crystals birch bark, The resulting dispersion is centrifuged, separating the fraction of spherical nanoparticles from the crystals, the separated nanoparticles are evaporated to obtain Doi triterpenoids mixture to form a morphologically homogeneous spherical nanoparticles by reinjection.

The disadvantage of this method of obtaining is the lack of a stage of purification from tetrahydrofuran. According to the classification of organic solvents, tetrahydrofuran belongs to the second class of non-genotoxic solvents and its content in drugs must be controlled.

Known patent of the Russian Federation 2355423 "Adjuvant", A61K 47/06, C08H 5/04, publ. 05/20/2009, which discloses a method for preparing betulin-based adjuvants in the form of nanoparticles. According to the known method, it is possible to obtain an adjuvant in the form of a 0.5% aqueous solution of betulin nanoparticles and in the form of a dried preparation of nanoparticles of birch bark extract. Adjuvants are prepared as follows. Dry birch bark extract (BES-65) is dissolved in an organic solvent at a concentration of 2.5-5 g / l; add a large amount of water to the specified solution (up to 25 volumes of water relative to the volume of the solution); remove the solvent and most of the water. A cryoprotectant (e.g., sorbitol) is added to the resulting aqueous solution of nanoparticles and lyophilization is carried out. Prior to immunization, the adjuvants are dissolved in phosphate buffer (pH 7.5), sonicated for 15 minutes and then cooled.

The disadvantage is the difficulty of scaling the process, the limiting factor of which is the removal of the organic solvent on a rotary evaporator.

As the closest analogue, a method for producing an adjuvant for vaccines in the form of spherical amorphous nanoparticles (SANCH) from a mixture of triterpenoids of birch bark (L. Gavrilova, NANOPARTICLES OF HYDROPHOBIC NATURAL COMPOUNDS AS ADJUVENTS, abstract of the dissertation, M., 2011) can be indicated as the closest analogue. An adjuvant is obtained by precipitating with excess water from water-miscible solvents such as tetrahydrofuran, adding miramistin or oleic acid to 2% by weight of triterpenoids, removing the organic solvent using a rotary evaporator, lyophilization in the presence of a cryoprotectant. Additionally, ultrafiltration can be used during evaporation. The sizes of spherical particles are 100-400 nm. However, although this method allows to increase the stability and effectiveness of the adjuvant, in its implementation there are losses of triterpeoids.

In addition, in the described method of obtaining there is no stage of sterilization, which is one of the main requirements for drugs administered parenterally. In accordance with the European Pharmacopoeia and with the regulatory documents in force in Russia, preparations for parenteral administration must be sterile, pyrogen-free, non-toxic.

The features of the prototype, which coincides with the features of the invention, are the presence of dissolution of a mixture of birch triterpenoids in tetrahydrofuran, addition of oleic acid, removal of tetrahydrofuran, addition of cryoprotectant and lyophilization.

The problem solved by the invention is to improve the quality of the adjuvant, allowing it to be obtained in accordance with the requirements for drugs administered parenterally: sterility, pyrogen-free, atoxicity.

The technical result that provides the solution to the mentioned problem is to eliminate the toxicity of the drug, reduce the residual concentration of THF, to ensure its sterility, pyrogen-free, ease of scaling and storage stability.

The technical result is achieved by the fact that in the method for producing an adjuvant, comprising dissolving a mixture of birch triterpenoids in tetrahydrofuran, adding oleic acid, removing tetrahydrofuran, adding cryoprotectant and lyophilization, according to the invention, a mixture of birch bark triterpenoids in tetrahydrofuran with a concentration of 5-10 g / l is obtained, followed by a solution of 5-10 g / l followed by a solution oleic acid in an amount of 5-10% by weight of birch triterpenoids, sterilize the mixture, sterilize the mixture, form a homogeneous dispersion of spherical amorphous nanoparticles by adding a 25-fold excess of 0.01 M Tris buffer, pH 9.0 ± 0.2, with stirring, followed by ultrasonic treatment for 5-10 minutes, remove tetrahydrofuran by ultrafiltration on hollow membranes with an exclusion threshold of 300 kDa at a speed of 1.0-1.2 l / min, at a pressure of 0.6-0.8 atm add a cryoprotectant from the group of substances: mannitol, maltose, trehalose, mannose, sorbitol, sucrose, freeze the resulting concentrated mixture containing a mixture of terpenoids 1 mg / ml below -35 ° C, incubated at this temperature for 4-6 hours lyophilized, lyophilization is carried out at a temperature of -35 ° C for 15 hours; followed by drying at 20-25 ° C for 15 hours.

Preferably, in the lyophilization process, sucrose is used as the cheapest carbohydrate.

The inclusion of the ultrasonic treatment step allowed the stabilizing of the adjuvant to a greater extent.

The determination of the eutectic temperature at which the aqueous solution passes into the solid phase is important for testing the lyophilization regime. The importance of this stage is determined by the fact that the presence of a liquid phase, even in externally frozen material, at the beginning of the sublimation phase can lead to irreversible phenomena, such as a violation of the integrity of the drug layer and its physical structure. The eutectic point for spherical amorphous nanoparticles was determined by analyzing the dependence of electrical resistance on temperature. Lyophilization of the samples was carried out on a TG-50 sublimation apparatus (Germany). According to the analysis of the definitions of the eutectic point, it was found that spherical amorphous nanoparticles with a cryoprotectant concentration of 1 / 10-1 / 20 by mass have a zone of eutectic temperatures of -15 ± 6 ° C. The most optimal stage of lyophilization is carried out at a temperature of -35 ° C, the duration of the stage is 15 hours; dry the adjuvant preparation at plus temperatures of 20-25 ° C for 15 hours.

The presence of oleic acid in the adjuvant in an amount of 5-10% by weight of birch bark triterpenoids ensures storage stability, the introduction of a sterilizing filtration stage ensured that the adjuvant is sterile and pyrogen-free, the use of ultrafiltration purification helped to eliminate toxicity and remove tetrahydrofuran, and reduce costs. The combination of distinctive features provides ease of scaling of the technological process from laboratory to industrial.

New features of the invention are that they obtain a mixture of birch bark triterpenoids in tetrahydrofuran with a concentration of 5-10 g / l, followed by dissolution of oleic acid in an amount of 5-10% by weight of birch bark triterpenoids, sterilize the mixture by sterilization, form a homogeneous dispersion of spherical amorphous nanoparticles by adding a 25-fold excess of 0.01 M Tris buffer, pH 9.0 ± 0.2, with stirring, followed by ultrasonic treatment for 5-10 minutes, tetrahydrofuran is removed by ultrafiltration on hollow membranes with an exclusion threshold of 300 kDa at a speed of 1.0-1.2 l / min, at a pressure of 0.6-0.8 atm, a cryoprotectant from the group of substances is added: mannitol, maltose, trehalose, mannose, sorbitol, sucrose, freeze the resulting concentrated a mixture containing a mixture of terpenoids 1 mg / ml below a temperature of -35 ° C, kept at this temperature for 4-6 hours and lyophilized, lyophilization carried out at a temperature of -35 ° C for 15 hours, followed by drying at 20-25 ° C within 15 hours.

The invention is illustrated by drawings, where in FIG. 1 shows the freezing mode of spherical amorphous nanoparticles; in FIG. 2 - sublimation mode of spherical amorphous nanoparticles.

The possibility of carrying out the invention can be demonstrated by the following examples.

Example 1. Obtaining adjuvant viral vaccines

In order to obtain an adjuvant that meets the requirements for preparations for parenteral administration, we have developed a technology in which all stock solutions must be sterilized at the initial stages of the production of adjuvant, and the entire further technological process must undergo sterile conditions. Therefore, at the first stage, a mixture of birch triterpenoids is dissolved, containing, wt.%: Betulin 65-71, luteol 12-16, 3-O-betulin 5-15 caffeate in tetrahydrofuran. To the resulting mixture add oleic acid in an amount of 5-10% by weight of birch bark triterpenoids. Sterilize the resulting mixture using a filter element with a modified surface charge that is resistant to organic solvents, such as a nylon membrane with a pore diameter of 0.22 μm brand NRG Pall N66 + c.

In order to obtain a homogeneous dispersion of spherical amorphous nanoparticles, a 25-fold volume of sterile buffer (pH 9.0 ± 0.2) is added to the sterile mixture using a peristaltic pump with constant stirring for 15 minutes with a propeller-type overhead stirrer. As a result of subsequent homogenization by ultrasound, spherical amorphous nanoparticles were formed for 5 min. In this case, the destruction of aggregated particles emerging on the surface was observed, and the dispersion became homogeneous.

In order to remove the organic solvent, hollow fiber ultrafiltration with a nominal cut-off molecular weight of 300 kDa is used. Purification from organic solvents is optimized by using a 0.01 M Tris buffer solution with a pH of 9.0 ± 0.2 for 2-3-fold dilution of the initial dispersion of spherical amorphous nanoparticles. In this case, the ultrafiltration rate was 1.0-1.2 l / min, the pressure was set within 0.6-0.8 atm. Nanodispersions are concentrated to a basic substance content of 1 mg / ml.

Lyophilization: the drug is frozen below -35 ° C and kept at this temperature for at least 4-6 hours; carrying out the stage of lyophilization at a temperature of the drug -35 ° C, the duration of the stage is 15 hours; dry the drug at positive temperatures of 20-25 ° C for 15 hours (figure 1, 2).

In appearance, the obtained lyophilized adjuvant was a porous mass of white or yellowish-white color, with a residual moisture content (3.5 ± 0.5)%.

The residual amount of THF in the finished product is determined by GLC. An analysis of the results showed that the proven ultrafiltration method provides effective removal of THF, its concentration in the preparation was 0.176 ± 0.009 mg / ml. The subsequent lyophilization step helped to reduce the residual concentration of THF to 0.122 ± 0.004 mg / ml, i.e. almost 30%. It should be noted that according to the GF of the XII edition, the maximum allowable amount of tetrahydrofuran taken as part of the daily dose in medicines should not exceed 7.2 mg / ml, which is 2360 times less than with the introduction of 25 μg SANCH (the estimated content in one dose of vaccine preparation).

The proposed method for producing spherical amorphous nanoparticles having a zeta potential of minus 44.3 mV and an average size of 160-180 nm provides conditions under which crystalline nanoparticles are not formed.

Example 2. Resuspension of freeze-dried adjuvant preparations

Freeze-dried preparation is resuspended with water for injection. Stir the resulting solution for 2-3 minutes, for example, using a shaker or mixer, avoiding foaming. Ultrasound treatment of the suspension for 3-5 minutes is also recommended.

Example 3. Test for pyrogenicity and sterility

To test for pyrogenicity, rehydrated adjuvant was administered to rabbits at 50 μg per kilogram of weight intravenously. In this case, the maximum sum of temperature changes in three rabbits was 0.9 ° C (Table 1), which indicates the pyrogen-free adjuvant.

Table 1 Adjuvant Pyrogenicity Test Results Rabbit number Weight of a rabbit, kg Temperature before administration, ° C Temperature in an hour, ° C Temperature after 2 hours, ° C Temperature after 3 hours, ° C Amount max temperature changes, ° C one 2.75 39,4 39.3 39.5 39.5 0.9 2 2.65 39.3 38.9 39.0 39.2 3 2,50 39.5 39.1 39.2 39.3

The sterility of the adjuvant was determined in accordance with the GF of the XII edition under two modes of 22.5 ± 2.5 ° C and 32.5 ± 2.5 ° C. The adjuvant was found to be sterile.

Thus, the developed technology allows to obtain adjuvants sterile and pyrogen-free, which meets the requirements for drugs for parenteral administration and allows them to be used as vaccine adjuvants.

Example 4. Evaluation of the sorption ability of the adjuvant on the example of diphtheria, tetanus toxoids and surface antigen of hepatitis B virus

One of the main mechanisms of action of adjuvants is the "depot" - an effect that is realized through the binding of antigen and adjuvant. In this case, the sorption ability of the adjuvant can be realized due to electrostatic interactions.

When testing the technology, the sorption ability of the adjuvant was determined using diphtheria, tetanus toxoids, and hepatitis B surface antigen as an example. To obtain vaccine compositions, the required amount of adjuvant with a pH of 9.00 was selected and the calculated volume of antigen was added, mixed and left for 30 min for sorption. Then brought 0.9% sodium chloride solution to obtain a vaccine dose. The prepared vaccine samples were centrifuged at 10,000 rpm, the supernatant was separated, which was analyzed by ELISA and the sorption ability of the adjuvant was determined by titration (Table 2).

table 2 Determination of sorption ability of adjuvant Adjuvant concentration, mcg / ml The efficiency of inclusion of antigens,% diphtheria toxoid tetanus toxoid HBsAg fifty 3,59 5.84 96.56 one hundred 1.24 1.34 97.85 200 9.91 3.11 98.92 400 13.56 14.73 99.47 500 29.35 20.98 99.39

As can be seen from the data in Table 2, an adjuvant at a concentration of 50 μg / ml effectively binds the surface antigen of hepatitis B. In turn, even with an adjuvant concentration of 500 μg / ml, the binding efficiency with respect to diphtheria and tetanus toxoid is less than 30%.

Example 5. The study of adjuvant properties on the example of a vaccine against hepatitis B

Adjuvant properties were studied on a model of hepatitis B vaccine in experiments on guinea pigs weighing 350-400 g. Animals were injected twice subcutaneously with an interval of 14 days with hepatitis B vaccines with various adjuvants in a volume of 0.5 ml (10 μg HBsAg). On the 14th and 28th day after immunization, antibodies to the hepatitis B virus surface antigen were determined in serum from guinea pigs by enzyme immunoassay.

Table 3 The results of a study of the immunogenic properties of the hepatitis B vaccine with adjuvants of various nature in experiments on guinea pigs Vaccine compositions (total dose per animal) Geometric mean titer (CGT), mIU / ml I immunization II immunization 20 mcg HBsAg + 50 mcg adjuvant 40.02 * 1276.63 ** [17.53-91.35] [830.24-1963.03] 20 mcg HBsAg + 500 mcg adjuvant 44.92 * 1192.26 ** [15,40-130,96] [681.91-2084.57] Control commercial hepatitis B vaccine (20 mcg HBsAg + 500 mcg Al (OH) ₃ ) 22.81 586.69 [10.94-47.52] [401.97-856.30] * Differences in average values are not significant compared with the control; ** Differences in average values are significant compared to control.

In our studies, it was found that the experimental adjuvant in concentrations significantly lower than the widely used adjuvant - aluminum hydroxide gel, provides a high immune response to the hepatitis B vaccine.

Example 6. The study of adjuvant properties on the example of a vaccine against influenza virus

White mice weighing 10–13 g were used in the experiment with influenza vaccines. Immunization was carried out as follows: 100 μl of freshly prepared solutions (with or without adjuvants) were injected intramuscularly at the rate of 1.5 μg of hemagglutinin in each of the hind legs of mice. ) per animal. Blood sampling was performed one month after immunization. The specific activity in mouse sera was determined by ELISA.

Table 4 Immunogenic properties of an influenza vaccine with experimental adjuvant Vaccine compositions The amount of hemagglutinin (HA) per animal, mcg The geometric mean optical density 1.5 mcg HA + 25 mcg adjuvant 1,5 1.015 [0.877-1.173] ** 1.5 mcg HA + 250 mcg adjuvant 1,5 0.791 [0.596-1.051] ** 1.5 mcg HA + 500 mcg adjuvant 1,5 0.676 [0.546-0.836] *** 1.5 mcg HA * 1,5 0.551 [0.429-0.708]  * Control (unsorbed hemagglutinin); ** Differences in average values are significant compared with the control; *** Differences in average values are not significant compared with the control.

The presented ELISA results showed that the experimental adjuvant showed pronounced adjuvanting properties. The maximum level of antibodies in animals was provided by the vaccine composition with a low dose of experimental adjuvant.

Example 7. The study of the toxic properties of the experimental adjuvant

The toxic properties of the drug were evaluated in experiments to determine acute and chronic toxicity. To study acute toxicity, white mice were injected intraperitoneally once at a dose of 0.5 ml (25 μg / dose) per mouse (corresponding to 2994 human doses), guinea pigs (weighing 300-350 g) subcutaneously at a dose of 5.0 ml ( 250 mcg / dose) per guinea pig (corresponding to 1851 human dose). When studying chronic toxicity, adjuvant was administered to white mice at a concentration of 50 μg / ml in a dose of 0.5 ml (25 μg) three times intraperitoneally (1, 4, 9 days) in a total dose of 1.5 ml per mouse (which corresponds to 8982 human doses ) The animals of the comparison group were given 0.9% sodium chloride solution according to similar schemes.

The results of the observation showed that a single and multiple administration of the drug did not cause the death of animals, did not lead to a decrease in body weight (Tables 5, 6), changes in the hairline, and necrobiotic changes at the injection site.

Table 5 The dynamics of changes in the mass of animals after a single injection Test drug Animal species The initial mass of animals, g The mass of animals for 1 day, g The mass of animals for 8 days, g Experimental adjuvant White mice 18.24 ± 0.14 18.58 ± 0.184 * 21.48 ± 1.24 * Guinea pigs 317.50 ± 2.10 320.50 ± 1.90 * 367.00 ± 3.50 * NaCl 0.9% (control) White mice 18.09 ± 0.16 18.30 ± 0.14 22.20 ± 0.98 Guinea pigs 324.10 ± 2.39 327.50 ± 2.03 372.10 ± 4.15  * p> 0.05, the difference is insignificant compared with the control. Table 6 The dynamics of changes in the mass of white mice after repeated administration of the drug Test drug The initial mass of animals, g The mass of animals for 1 day, g The mass of animals on 22 days, g Experimental adjuvant 18.46 ± 0.18 18.78 ± 0.60 * 27.80 ± 2.89 * NaCl 0.9% (control) 18.60 ± 0.46 18.68 ± 0.62 27.63 ± 2.80 * p> 0.05, the difference is insignificant compared with the control.

24 hours and 13 days after the last injection, when assessing chronic toxicity in the comparison group and the experimental group, the internal organs of animals had all the characteristic signs, location, and structure. The membranes lining the internal cavity are moist, grayish-pink in color, with no signs of inflammation.

Thus, when studying acute and chronic toxicity, it was found that the experimental adjuvant does not cause intoxication symptoms, does not have a toxic effect on the systems and organs of laboratory animals, and does not contribute to the development of pathological, including inflammatory, dystrophic, and necrotic changes.

Claims (2)

1. A method of obtaining an adjuvant for vaccines, comprising dissolving a mixture of birch triterpenoids in tetrahydrofuran, adding oleic acid, removing tetrahydrofuran, adding cryoprotectant and lyophilization, characterized in that a mixture of birch triterpenoids in tetrahydrofuran with a concentration of 5-10 g / l, followed by dissolution oleic acid in an amount of 5-10% by weight of birch triterpenoids, sterilizing the mixture is carried out, form a homogeneous dispersion of spherical amorphous nanoparticles by adding 25 cr a clear excess of 0.01 M Tris buffer, pH 9.0 ± 0.2, with stirring, followed by ultrasonic treatment for 5-10 minutes, tetrahydrofuran is removed by ultrafiltration on hollow membranes with an exclusion threshold of 300 kDa at a speed of 1, 0-1.2 l / min, at a pressure of 0.6-0.8 atm, a cryoprotector from the group of substances is added: mannitol, maltose, trehalose, mannose, sorbitol, sucrose, the resulting concentrated mixture is frozen with a mixture of terpenoids 1 mg / ml below -35 ° C, maintained at this temperature for 4-6 hours and lyophilized, lyophilization pr lead at a temperature of -35 ° C for 15 hours followed by subsequent drying at 20-25 ° C for 15 hours. 2. The method according to claim 1, characterized in that sucrose is used as a cryoprotectant.

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