FR2602681A1 - PROCESS FOR PREPARING A RECOMBINANT VACCINE AGAINST HEPATITIS B - Google Patents

Abstract

THE INVENTION CONCERNS THE FIELD OF BIOTECHNOLOGY. THE PROCESS FOR THE PREPARATION OF THE RECOMBINANT VACCINE AGAINST HEPATITIS B CONSISTS OF CULTIVATING A STRAIN OF YEAST PRODUCING THE HBSAG ANTIGEN SUCCESSIVELY IN A NUTRITIONAL ENVIRONMENT ENRICHED WITH A SOURCE OF PHOSPHORUS, THEN IN AN ENVIRONMENT CONTAINING NO PHOSPHORUS, SOURCE OF SOURCE WITH THE SUBSEQUENT DESTRUCTION OF THE YEAST CELLS SIMULTANEOUS ISOLATION AND PURIFICATION OF THE ANTIGEN BY ADSORPTION CHROMATOGRAPHY ON POROUS SILICATES, WITH SUBSEQUENT ULTRACENTRIFUGATION WITH A PRODUCT DENSITY GRADIENT OF 1.1 TO 1.3KGDM AND OBTAINING THE PRODUCT . THE VACCINE OBTAINED FOUND A MEDICAL APPLICATION FOR THE VACCINE PROPHYLAXIS OF MEN AGAINST HEPATITISB.

Description

The present invention relates to the field of biotechnology, and more

  specifically a method for preparing a recombinant vaccine against hepatitis B, finding an application in medicine for human prophylaxis with the vaccine.

  At the present time an effective means against hepatitis B mass infection is the vaccine.

  Various methods for the preparation of hepatitis B vaccines are known, consisting of culturing a yeast strain producing the HBsAg antigen on a nutrient medium with subsequent destruction of the yeast cells, isolation and purification of the antigen. . For example, there is known a method of preparing the hepatitis B vaccine (EP 0135435), consisting of the expression of the HBsAg antigen of the Saccharomyces culture.

  cerevisiae by cells of ATCC 20665 strain (strain) H52, for four days. The cells are incubated at a temperature of 28 ° C. in a shaker for 8 hours.

  The cell culture is then placed in a fermenter containing the YEPD nutrient medium. The fermentation process is conducted at a temperature of 28 ° C. with stirring for 14 - 40 hours (pH = 5.0). 40 hours later, the culture is added to 100 liters of YEPD medium and the process is carried out in the fermenter with stirring for 48 hours (pH = 5.0), at a dissolved O 2 concentration of 20%.

saturation.

  After the washing of the cell mass with a phosphate buffer solution and storage at 2-8 ° C for 3 days, 1% of

  PMSF to a final concentration of 2mM PMSF.

  Then the cells are destroyed in a homogenizer. The HBsAg antigen is obtained at a concentration of 0.9 mg / l, the total protein content being 1.523 g / l. The HBsAg antigen is then passed through the silicon oxide, washed with a phosphate buffer solution and incubated for 20 minutes at a temperature of 56 ° C. in a 5 mM borate buffer solution at pH = 9. 1 containing 0.25% by weight of deoxycholate. After that, it is purified by refined chromatography in a column containing antibodies against HBsAg and eluted with a 3M solution of ammonium thiocyanate at pH = 7.2. It is then dialyzed against the phosphate buffer solution. Aluminum hydroxide is added to the aqueous mass, the pH is brought to 6.7, stirred at room temperature, centrifuged and diluted with physiological solution (0.85% NaCl solution). . Another alternative preparation of the vaccine is that the cell extract obtained as described above is kneaded with silicon oxide, incubated for 2.5 hours at a temperature of 37.degree. C. The mixture is then washed with the phosphate buffer solution, the HBsAg antigen is eluted with 0.05M borate buffer solution at a pH = 8.9 - 9.2 containing 0.25% by weight of deoxycholate. The antigen obtained is centrifuged. The supernatant is coated on a cellulose column which has been previously treated with 5 mM NaCl solutions, 6.25 mM Tris, 0.5 mM MgCl 2. We realize

  the process at a temperature of 4 C and a pH of 7.5.

  The column is then washed with the same buffer solution and eluted with 0.2 M NaCl buffer solution. The unadsorbed fraction contains 7.95 mg of purified HBsAg antigen in 13 mg of total protein. The material is then placed on a Sepharose 6 B column and eluted with a

  phosphate buffer solution (60 ml / h). We get the fraction

  purified after dialysis of the antigen, purified on Sepharose 6B against a 3M solution of ammonium thiocyanate in phosphate buffer solution at pH 7.2 for 16 hours. Ammonium thiocyanate is removed by intense dialysis against the phosphate buffer solution. The purified product is diluted with the phosphate buffer solution and adsorbed on the hydroxide

  of aluminum and the process is conducted in a manner analogous to the previous variant.

  The indicated method provides for the use of affinity chromatography which decreases the antigenicity of HBsAg. In addition, during elution of the antigen, partial desorption of antibodies from the affinity sorbant is possible, which is undesirable in the case of preparation of a vaccine drug for humans ( the presence of foreign antibodies increases the reactogenicity of the drug). In addition to this, the use of toxic substances of PMSF and thiocyanate

  ammonium in said process is inadmissible for the preparation of the vaccine for humans.

  There is also known a method of preparing and purifying antigen against hepatitis B suitable for large scale production of hepatitis B vaccines (EP, B N 0155007). The process consists in culturing the Saccharomyces cerevisiae strain producing HBsAg antigen with subsequent adsorption of HBsAg by cell destruction and mixing with silicon oxide at dg 100C and with addition of phos phate buffer solution. The elution of HBsAg is then carried out with a mixture of a 0.05M solution of Na2CO3 or of NaHCO3 (pH = 5) and of urea at a temperature of 37 ° C. 10% acetic acid is added at room temperature. supernatant to a pH =

  7.2 and concentrated on an ultrafilter. Gel filtration was performed on Sepharose GL-6 B on the aqueous solution.

  phosphate and eluted with the same buffer solution.

  Purification of the antigen is then carried out on hydroxyapatite. The elution is carried out with a 0.05 M solution of KCl with the addition of a 0.5M solution of phosphate buffer K. The target product is obtained with a yield of 46% by weight. However, although the pure HBsAg antigen is obtained by the indicated method, its yield is not high enough. In the final step, the phosphate-K buffer solution is used, which is undesirable for the preparation of the vaccine intended for humans.

its toxicity.

  Also, there is known a method of preparing the recombinant hepatitis B vaccine from a yeast-producing strain of the HBsAg antigen (Waurpber D. et al., PNAS, v. 82, 1985, pages 6830 at 6834), culturing an antigen-producing strain in medium at a high phosphorus content, cells are transferred to medium at a low level.

  Phosphorus content for reproduction of HBsAg with subsequent washing of the cells in a centrifuge.

  Then the cell mass released from the medium is diluted to the starting volume with the phosphate buffer solution. The cells are destroyed with a high pressure homogenizer and the resulting extract containing 30-70 mg / ml protein is diluted 4-fold with phosphate buffer solution containing 0.1% Triton X-detergent. 100. After that the mixture is centrifuged, the supernatant is concentrated and the ladia-filtoetin is conducted. by two volumes of phosphate buffer solution. Triton X-100 detergent was removed using XAD-2 sorbent and centrifuged. Partial urification is then carried out using adsorption elution from silica oxide. The drug obtained is frozen at a temperature of 70 ° C. After thawing, the drug is cleaved by centrifugation. The product obtained is purified by chromatography on butylagarose or by affinity chromatography. The target product is obtained with the yield of 29% by weight and the content of 20% by weight of impure protein. The process indicated is characterized by a multiplicity of stages, the use of carriers containing cyanogen bromide hazardous to humans. The targeted product is obtained in a low yield (29% by weight) and an impure protein content of 20% by mass, which makes it impossible to use as a vaccine. Moreover, during the elution of HBsAg, solutions containing the rhodanates are used, which is also inadmissible for

  the preparation of the vaccine for humans.

  It has therefore been proposed, by modification of the technological operations, to develop a process which makes it possible to increase the yield and the purity of the product concerned. The solution consists in that, in the method of preparation of the recombinant vaccine against hepatitis B containing the culture of the yeast-producing strain of HBsAg antigen successively on a nutrient medium enriched with a source of phosphorus, then on a Nutrient medium ensuring the reproduction of the antigen, with subsequent destruction of the yeast cells, isolation, purification of the antigen and obtaining the target product, according to the invention, is used as a nutrient medium assuring the reproduction of the antigen, a nutrient medium containing no sources of phosphorus, and simultaneously carrying out the isolation and purification of the antigen by adsorption chromatography on porous silicas with subsequent ultracentrifugation with a gradient of density of 1.1

1.3 kg / dm3.

  It is advantageous to use as porous silicas silicas with a pore size of 1500.degree.

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  2000 to. It is better to conduct the desorption

  during chromatography with a bicarbonate buffer solution at a pH of 9.1-9.5. This makes it possible to increase the yield of HBsAg antigen by up to 90%.

  To increase the degree of purity of the antigen it is rational to carry out the centrifugation with a density gradient of a 50% solution of potassium or sodium tartrate and a 25% solution of glycerin, the ratio of indicated components being 1: I. The degree of purity then increases more than 2 times and

  the antigen activity yield of 10-20% (as compared with the known method).

  In order to increase the yield of the product aimed at the yeast cell destruction stage, it is advantageous to introduce a detergent, of which Tween-80 is preferably used. This makes it possible to increase the yield of HBsAg antigen by more than 2 times

compared to the known method.

  The claimed process makes it possible to obtain the targeted product with a yield of 60-67% by mass and with a purity

  high (protein impurity content being 10%).

  The claimed process is carried out as follows.

  Culture of a yeast strain carrying the recombinant plasmid with the HBsAg gene and capable of producing the immunogenic HBsAg is conducted. As a starting strain, any known yeast strain producing the HBsAg antigen can be used, for example the strain of Saccharomyces cerevisiae DBY.

  746 / pNKVG 46, obtained by inclusion of a transformed plasmid p4MVG 46 in the recipient strain DBY 746. The plasmid pNMVG 46 is obtained by modification by means of genetic engineering from the known plasmid of pADC-47 (Molekulyarnaya genetika, mikrobiologia i virusologia, 1986, No. 8, pages 12-19).

  The culture is carried out as follows. 3 - 5 large colonies of the yeast starter strain producing the HBsAG antigen are seeded onto a nutrient medium enriched with a phosphorus source. The culture is brought to a temperature of 30-32 ° C. and stirred for one day until a concentration of 2 - 6 × 10 7 cells / ml is obtained. After that, all the culture volume is transferred to a nutrient medium ensuring

the reproduction of the antigen.

  The culture is carried out in said medium at a temperature of 30 ° C., under aeration of air, for 24 hours up to a concentration of 1.8-2.10 × 10 7 / ml. The culture purity is checked microscopically. The cells are then harvested by centrifugation at a temperature of 4 ° C. The cells are then subjected to disintegration or freezing at a temperature of -40 ° C. Before the destruction, the cell mass is thawed, if frozen, and diluted in a buffer solution. The destruction can be carried out using, for example, 0.5 mm diameter glass beads, by triturating the yeast cells or in a high-pressure disintegrator, in which the cells are destroyed at a pressure of 500 - 1000. 25 atm. The disintegration is completed after the destruction of% of cells. It is advantageous, in order to increase the yield of product aimed at the destruction stage of the cells, to introduce a detergent, preferably such as

  the Tween-80. The HBsAg antigen yield then increases more than twice.

  The destroyed cells are frozen, decanted and then clarified by centrifugation at room temperature.

  C. The supernatant (clarified extract) containing recombinant HBsAg antigen was subjected to adsorption chromatography on porous silicas.

  For the adsorption chromatography, silicas (macroporous glasses) with pores having a size of 1500 to 2000 Å are preferably used. Such sorbents have the maximum capacity relative to recombinant HBsAg which accumulates in the form of particles with a size of 1822 nm and constitutes less than 1% in

  the clarified extract of yeast cells.

  After the adsorption of HBsAg on the sorbent, which goes from a few hours to a day under continuous stirring, the sorbent is precipitated, washed with a few volumes of phosphate buffer solution and transferred to the same buffer solution in the same manner. column, washed again with the phosphate buffer solution until the constant index of extinction at 280 nm. After washing, the HBsAg antigen is desorbed with the

buffer solution at pH = 9-10.

  The desorption of the antigen is preferably carried out with a bicarbonate buffer solution having a pH of 9.1-9.5. In this case, the maximum yield of HBsAg antigen (about 90%) was poor, which was not achieved by using other buffer solutions for adsorption. The eluate fractions containing the HBsAg antigen were pooled, concentrated if necessary by ultrafiltration and centrifuged with a density gradient of 1.1.3 kg / dm.sup.3. It is advantageous to carry out the ultracentrifugation in the density range of a 50% solution of potassium or sodium tartrate and a 25% glycerine solution, with a ratio of the indicated solutions of 1: 1 The use of the glycerine tartrate gradient makes it possible, compared to other gradients, to obtain the maximum purified drug while retaining its antigenic activity. The use of such a gradient increases the purification of the drug by more than 2 times and the antigen activity yield by -20%. The final yield of the target product increases respectively. After ultracentrifugation, the gradient was fractionated, the HBsAg titer was determined in the fractions, the fractions containing HBsAg were pooled and dialyzed against 0.8-0.9% NaCl solution in 0. buffer solution. 02 M phosphate at a pH of 7.4

- 7.6

  The dialyzed preparation was diluted to a concentration of 40 + 6 μg / ml HBsAg, sterilized by microfiltration, sorbed on aluminum hydroxide and packaged into ampoules. One dose of vaccine contains about 15 μg / ml of HBsAg and up to 10% of protein impurities sorbed on 1 mg of aluminum hydroxide. The vaccine obtained is sterile, non-toxic,

  contains no foreign viral antigens. The high immunogenicity of the vaccine obtained is confirmed in animal and human trials.

  In animal tests (guinea pigs) immunized with an experimental series of vaccines obtained by the claimed method, antibodies to HBsAg were detected in all animals,

senior titles (320 - 650 IU).

  Forty volunteers not containing antibodies to the HBsAg antigen were vaccinated with the vaccine obtained by the claimed method. The vaccination was carried out three times in the intervals between the first and the second vaccination - one month, between the second and third of six months. Volunteer blood was assayed for HBsAg antigen content each month. Low vaccine reactivity was noted compared to

known vaccines.

  One month after the second vaccination antibodies were detected in 30% of volunteers inoculated and one month after the third vaccination (8 months after the start of inoculations) the antibodies were found in 92% of vaccinated volunteers, the title of antibody was then 300 - 700 IU (while the

  protection of antibodies constitutes 10 IU).

  To better understand the present invention, the following are examples of concrete embodiments of the method.

Example 1

  Three colonies of Saccharo10 cerevisiae yeast cells (45 mm in diameter) of the strain DBY 746 / pNMVG 620, obtained from the strain DBY 746 by inclusion of the transformed plasmid pNMVG (Granovsky NN et al., Molekulyarnaya genetika, Mikrobiologia i virusologia, 1986, No. 8, pages 12-19) on the 2% agar are seeded in 200 ml of liquid nutrient medium of the following composition (by 1): KH 2 PO 4 0.9 - 1.1 NaCl 0.095 - 0.10 g glucose 19 - 21 g MgSO4 0.45 - 0.55 g CaCl2 0.095 0.105 g asparagine 2.1 - 2.2 g Vitamins: thiamine 190 - 210 μg riboflavin 190 - 210 μg pyridoxine 190 - 210 μg nicotinic acid 190 210 μg para-aminobenzoic acid 190 - 210 μg calcium pantothenate 190 - 210 μg inositol 9 - 11 mg biotin 1.9 - 2.1 μg Microelements: KJ 99 101 μg HBO3 9.9 - 10 , 1 μg MnSO 4 9.9 - 10.1 μg

2602681-

  (NH4) 2MoO4 9.9-10.1 μg FeSO4 49-51 μg The culture is incubated in a shaker at 300-350 rpm at a temperature of 30 ° C for one day. The cell concentration at the end of culture is 2.6 x 10 7 cells / ml. The sterile culture obtained is transferred into a nutrient medium of the following composition (by 1): KCl 0.9 - 1.1 g NaCl 0.095 - 0.10 g glucose 19 - 21 g MgSO4 0.45 0.55 g CaCl 2 0.095 0.105 g asparagine 2.1-2.2 g Vitamins: thiamine 190-210 μg riboflavin 190-210 μg pyridoxine 190-210 μg nicotinic acid 190 210 μg para-aminobenzoic acid 190-210 μg pantothenate calcium 190 - 210 μg inositol 9 - 11 mg biotin 1.9 - 2.1 μg Microelements: KJ 99 101 μg HBO3 9.9 - 10.1 μg MnSO4 9.9 - 10.1 μg (NH4) 214MoO4 9 , 9 - 10.1 μg FeSO4 49 - 51 μg

  The incubation is conducted for 26 hours.

  The intensity of aeration during the incubation (air: medium ratio) constitutes 1: 2.5. The concentration of cells in the medium at the end of incubation reaches 2.1 x

  10 cel / ml. The cells are precipitated by centrifugation

  at 10,000 x g for 20 minutes at a temperature of +4 C. The precipitated cells are resuspended in a 0.02 M sodium phosphate buffer solution, 0.5 M

  NaCl, pH 7.5, 100 ml volume and triturated with glass beads for 2 hours.

  The destroyed cells are precipitated by centrifugation at 10,000 × g at a temperature of 40 ° C. for minutes. We empty the supernatant, we add

  Again, 100 ml of buffer solution is added to the precipitate and the procedure is repeated twice more. The combined supernatant is then frozen at 40 ° C. for 14 hours.

  The thawed supernatant is again clarified by centrifugation under the same regime. We obtain

  280 ml clarified cell extract with HBsAg antigen content of 5 g / ml (based on antigenic activity).

  A macroporous glass with a pore diameter of 1050 X is added to the preparation obtained. The glass is regenerated beforehand and quenched in distilled water. The volume of macroporous glass constitutes cm3. The flask is kept with the suspension at +4 ° C. for 10 hours. After adsorption, the HBsAg antigen content of the supernatant corresponds to 0.04 μg / ml. The supernatant was purged, and the sorbent was washed twice with the phosphate buffer solution (ratio 1: 1), adding glass after the entire precipitation. The sorbent is then transferred to the column, the column is washed with phosphate buffer solution to remove the majority of impurities, after which a desorbent buffer solution HBsAg: 0.05 M tris-HCl is passed through the column. pH = 9. The eluate is pooled in the 4 ml volume fractions, in which the HBsAg antigen is determined. Fractions were pooled to give 64 ml of eluate containing 18 μg / ml HBsAg (

  HBsAg fractions is determined by the immunofermentary method. The yield of HBsAg constitutes 80% of the antigenic activity in the clarified supernatant.

  The eluate obtained is coated on the sucrose gradient (at 25 - 55%, on density 1.1 - 1.26 kg / dm 3) with 10 ml of eluate on a 24 ml gradient, prepared on the tris buffer solution. -HCl. Ultracentrifugation was carried out at 95000 x g and 4 C for one day, after which the gradients were fractionated and HBsAg was determined therein. The fractions containing the antigen are pooled and dialyzed against the phosphate buffer solution at pH = 7.6. 16 ml of drug containing 42 μg / ml HBsAg is definitely obtained. HBsAg yield

  is 58% of that coated on the gradient.

  The drug is sterilized, passed through the filters, diluted twice with aluminum hydroxide at a concentration of 2 mg / ml

  and poured in 1 ml one hour later in ampoules.

  Beforehand, before kneading with aluminum hydroxide, the impurities are determined by electrophoresis.

  Their content does not exceed 15% of the total protein content. Finally, a drug containing pg / ml HBsAg, 1 mg / ml aluminum hydroxide in phosphate buffer solution at pH 7.6 was obtained. The final yield of the product concerned (in relation to the extract

  of clarified cells) constitutes 47%.

Example 2

  The clarified cell extract of the HBsAg-producing DBY 746 / pNMVG yeast strain was obtained as in Example 1 except that the cells

  are destroyed by pressure in a special disintegrator. The final concentration of HBsAg antigen of the clarified extract (supernatant) constitutes 5 μg / ml. For the adsorption chromatography a macro glass is used.

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  porous with a pore diameter of 1900. To 70 ml of macroporous glass previously moistened with water is added to 280 ml of clarified extract of cells. After kneading for two hours, the sorbent is washed twice with the phosphate buffer solution, as in Example 1, and transferred to the column. The column is washed with the phosphate buffer solution until the constant extinction coefficient is obtained.

  280 nm, then desorbed HBsAg as in Example 1.

  30 ml of eluate containing 40 μg / ml of antigen are obtained.

  This corresponds to 86% of the starting activity in the clarified cell extract.

  The ultracentrifugation, dialysis, sterilization and packaging of the drug is carried out as in Example 1. The final antigen yield of the vaccine is 56% of the content in the clarified extract, the amount of impurities being 10%. % of the

total protein content.

Example 3

  The clarified extract of cells of

  the strain-producing HBsAg as in Example 1.

  The adsorption of the antigen is carried out on the macroporous glasses in a manner analogous to that described in Example 2. For the desorption of the HBsAg antigen, a 0.05 M buffer solution of bicarbonate is used and pH = 9.1. The eluate is collected in the 4 ml volume fractions and the antigenic activity of HBsAg is determined therein. 28 ml of 45 μg / ml antigenic activity eluate is obtained, corresponding to 90% of the starting activity of HBsAg. After ultracentrifugation, sterilization and conditioning the

  The antigen yield of the vaccine constituted 63% (of the clarified cell extract content), the protein impurity content did not exceed 10% of the total protein content.

Example 4

  It is realized obtaining the clarified extract

  yeast cells of the strain-producing HBsAg and adsorption chromatography in a manner analogous to that described in Example 3.

  Ultracentrifugation of the resulting eluate containing the HBsAg antigen in the density gradient was carried out using a 50% solution of potassium sodium tartrate as a heavy gradient, and in a light gradient grade of 25% solution. of glycerin in the phosphate buffer solution at pH = 7.8. Ultracentrifugation is carried out for 18 hours at 90000 xg and at a temperature of 4 C. Gradient fractionation, determination of HBsAg antigen and dialysis are carried out as in Example I. 12 ml of medicament are obtained. antigen at the activity of 82 μg / ml, which corresponds to 76% of the antigenic activity applied to the gradient. The final HBsAg antigen yield of the vaccine is 68% relative to the clarified extract, and the protein impurity content constitutes 5% of the

total protein.

Example 5

  The clarified extract of yeast cells of the producing strain is obtained in a manner analogous to that described in Example 1, except that in the phosphate buffer solution with which the cells are diluted before destruction, we

  Triton X-100 is added to the final concentration of 0.2% by weight. After the clarification, 250 ml of extract containing 8 μg / ml of HBsAg antigen is obtained.

  The clarified extract obtained was purified as in Example 4. 16 ml of antigen activity drug of 82 μg / ml was obtained. Dilution, sterilization and packaging of the vaccine are carried out as

2602681-

in example 1.

Example 6

  The clarified extract of yeast cells is obtained in a manner analogous to that described in Example 1, except that the Tween-80 detergent is added to the phosphate buffer solution to the concentration of 1. % by mass. After clarification, 280 ml of cell extract at the antigen concentration of 10 μg / ml are obtained. The resulting extract is purified by adsorption chromatography and ultracetrifugation as in Example 4. 18 ml of drug at the antigenic concentration of 104 μg / ml is obtained. Dilution, sterilization and packaging of the drug are performed

as in Example 1.

Example 7

  For the Iptipt of the recombinant vaccine, recombinant yeast strain-DBY 746 / pNiMVG 46 producing the HBsAg antigen (NN Granovsky et al., Molekulyarnaya genetika, mikrobiologia i virusologiea, 1986, N 8, pages 12-19) is used. . We conduct the process of obtaining the clarified extract

  in a manner analogous to that described in Example 1.

  250 ml of clarified extract with a content of

  HBsAg antigen of 30 μg / ml. The procedure is then carried out as in Example 4. 6 ml of drug and the activity of the HBsAg antigen of 760 μg / ml are obtained.

  The final antigen yield of the vaccine constitutes 61% (of the antigen content of the clarified cell extract). 17.

Claims (6)

  1. A method for preparing a recombinant vaccine against hepatitis B, comprising culturing a yeast strain producing HBsAg antigen successively in a nutrient medium enriched with a phosphorus source, and then in a medium ensuring the reproduction of the antigen, with the subsequent destruction of the yeast cells, with isolation, purification of the antigen and obtaining of the targeted product, characterized in that as a nutrient medium for the reproduction of the antigen, one uses a nutrient medium which does not contain phosphorus sources, and the isolation and purification of the antigen is simultaneously carried out by adsorption chromatography on porous silicas with subsequent ultracentrifugation with a gradient density of 1.1 - 1.3 kg / dm3.   2. Method according to claim 1, characterized in that as porous silicas is used   silicas having a pore size of 1500 to 2000.   3. Process according to claims 1 or 2,   characterized in that the desorption is carried out during chromatography with a bicarbonate buffer solution at a pH of 9.1-9.5.   4. Process according to one of Claims 1 to 3,   characterized in that the ultracentrifugation is carried out with a density gradient of a 50% solution of potassium or sodium tartrate and a solution   25% glycerin in a ratio of the indicated components of 1: 1.   5. Method according to one of claims 1 to   4, characterized in that, to increase the yield of product aimed at the destruction stage of the cells   yeasts, a detergent is introduced.   6. Process according to claim 5, characterized in that Tween-80 is used as a detergent.