The method of obtaining the surface antigen of hepatitis b virus from recombinant cells of the yeast hansenula polymorpha and vaccine for immunization against hepatitis b

 

The invention relates to medicine, namely to a method for the surface antigen of hepatitis b virus from recombinant cells of the yeast Hansenula polymorpha, and can be used in biotechnology for the preparation of a vaccine against hepatitis C. the Essence of this method is the cultivation of recombinant yeast cells, the destruction of cells in buffer containing chaotropic agent, adding a surfactant to the homogenate of the cells, centrifugation of the homogenate of cells, hydrophobic chromatography and ultracentrifugation in a density gradient of potassium bromide, the gel filtration fractions containing surface antigen, and hydrophobic chromatography is carried out in buffer containing 8-14% ethanol, the wash is carried out with the same buffer, with a temperature of 30oWith, the elution is conducted with the same buffer, with a temperature of 40-42oWith, and gel-filtration spend on copolymeres media. The preparation of the surface antigen rHBs obtained from recombinant cells of the yeast Hansenula polymorpha, is used for preparation of vaccines for immunization against hepatitis C. the Technical result is an improvement of the method of obtaining the surface antigen of hepatitis C. 2 C. p. F.-ly, 2 tab.

Naoussa hepatitis b from recombinant yeast cells.

Hepatitis b up to the present time is one of the most dangerous and widespread diseases, which are difficult to prevent due to the fact that the human body a long time can be hidden by the carrier of the virus of hepatitis C. hepatitis b often develops in cirrhosis and liver cell cancer, which can lead to death.

To combat this disease were directed studies virologists all over the world. In the hard years of labor were created vaccines available to combat hepatitis b and how to receive these vaccines.

The first generation of vaccines against hepatitis b was obtained by separation and purification of the surface antigen of hepatitis b virus (HBsAg) from the blood plasma of patients with hepatitis b (V. R. Hilleman, etc. Develop. Bio Stendart, 54, 3-12, 1983). Ways of receiving the vaccine from plasma costly and very time consuming. Blood is not an ideal source of raw material for production of vaccines, because it may contain pathogens and other diseases that may cause infection during the vaccination.

The next generation of vaccines against hepatitis b is obtained on the basis of recombinant producers (mainly bacteria and yeast fungi). The most famous in our country, the vaccine is for suspension which contains purified major surface antigen of hepatitis b virus, obtained using recombinant DNA technology and adsorbed on aluminum hydroxide. Vilenguela and others, have developed a method of obtaining HBsAg in yeast (Nature, 293,347-350,1982). Derived from yeast recombinant HBsAg (rHBsAg) consists predominantly of S protein (P25) having a 226 amino acids and when cleaning forming particles surface antigen identical to the HBsAg particles separated from the blood plasma.

The disadvantages of obtaining recombinant HBsAg from yeast Saccharomyces cerevisiae is the low productivity producers of this type and coupled with this the complexity and intensity of the treatment.

Currently, among the ways of getting vaccines best known methods described in Korean patent 065305 and USA 4742158.

In U.S. patent 4742158 (FROM 07 TO 3/20, 3/28, a 61 K 39/29, NCI 530-371) describes a method for antigen hepatitis b, including the preparation of yeast extract in the presence of various protease inhibitors, treatment of him surface antigen by a series of stages of the column-chromatographic separation using affinity columns, in which the polymer is albumin, human serum attached to the gel matrix and the hydrophobic column with elution surfactant. The method described in patent Equestrian currency. The disadvantages of the methods are: low yield of the target product, a low degree of purification of the target product, the possibility of damage to the native structure of the HBs antigen with significant pH shifts in the allocation process.

The closest known methods is the way (p. 2122430, Russia, MKI 6 a 61 K 39/29, 12 P 21/00, 21/02,Z. 08.12.95-prototype), including the incubation of recombinant yeast cells in buffer containing chaotropic agent, adding a surfactant to the homogenate of the cells, centrifugation of the homogenate of cells, hydrophobic chromatography, gel-filtration fractions containing surface antigen, followed by obtaining a fraction containing the target product.

The disadvantage of this method is that for the first purification method is used for adsorption/desorption on silicon-containing media, which leads to contamination of the intermediate denaturirovannyj fragments of the HBs antigen and impurities, and hydrophobic chromatography using toxic glycol.

The aim of the present invention is to provide a method of producing the surface antigen of hepatitis b virus (HBsAg). This objective is achieved in that in the method of obtaining the HBs antigen from recombinant yeast cells, th agent, the solubilization of the target antigen by adding surfactants, centrifugation of the homogenate of cells, hydrophobic chromatography, gel-filtration fractions containing surface antigen, followed by obtaining a fraction containing the target product, before surgery, gel filtration spend hydrophobic chromatography and ultracentrifugation in a density gradient of potassium bromide, stage hydrophobic chromatography is carried out in buffer containing 8-14% ethanol, washing is conducted with the same buffer at a temperature of 30oWith, the elution is conducted with the same buffer at a temperature of 40-42oC, gel-filtration spend on copolymeres media, and as source material using recombinant cells of the yeast Hansenula polymorpha.

The method is as follows.

Example 1.

Stage 1. Cultivation of yeast cells.

Recombinant cells HP (strain DL-U/pH-HBs), which are able to Express the HBs-antigen, is cultivated at a temperature of 30oWith 7.5 liters of YEPD medium (1% yeast extract, 2% yeast peptone, and 1.6% glucose), enriched in methanol (1.5%).

Stage 2. The destruction of the yeast cells.

20 g obtained in stage 1 of the yeast cell mass is mixed with 250 ml of buffer for destruction (0.1 M sodium pratury +2oC in an ice bath and served with constant stirring with a rate of 10-15 ml/min in a homogenizer (APV Gaulin. The camera homogenizer is constantly cooled by the flow of water-methanol mixture with a temperature of -10oC.

Stage 3. Extraction and solubilization HBs antigen.

The cell homogenate obtained in stage 2, washed with distilled water using a double centrifugation. To the resulting washed precipitate add 0.2% (weight/volume) desoxycholate sodium; the mixture is stirred on ice for 1 hour. The mixture is then centrifuged for 45 minutes at 10,000 g and a temperature of 4oWith the removal of cellular residues. The total volume of the obtained supernatant is about 1000 ml.

Stage 4. Hydrophobic column chromatography.

The supernatant containing the solubilized antigen with stage 3, is applied to the chromatographic column section 50 mm, filled with 1500 ml of gel phenyl-agarose pre-equilibrated with buffer for hydrophobic chromatography (0.1 M sodium carbonate, pH of 9.2+6 M urea). The column was washed with 4500 ml of buffer for hydrophobic chromatography with the addition of 8-14% ethanol with a temperature of 20-25oC. Next, continue washing with the same buffer, heated to a temperature 30oC. Centrobanca) by the method of Bradford. Washing cease when: 1) the temperature of the solution in 50-ml fractions of the eluate is more than 28oOr 2) the optical density at a wavelength of 280 nm is less than 0,075 or 3) putative protein concentration in the determination by the Bradford method using human serum albumin as a calibrator is less than 50 µg/ml.

Next, the bound peroxidase on a column of material elute buffer for hydrophobic chromatography, preheated to a temperature of 40-42oC. the Entire volume of the eluate (about 350 ml) are collected and cooled in an ice bath; the resulting precipitate was separated by centrifugation and discarded.

Stage 5. The ultracentrifugation in a density gradient.

The density gradient of potassium bromide formed by making a solution in the density range from 1,025 to 1,200 in the zonal rotor Ti-45 ultracentrifuge Beckman Optima LC70K. The preparation of the eluate from the hydrophobic column, obtained in stage 4, translated by ultrafiltration in 0.1 M phosphate buffer with pH 8.0, containing 0.2 M sodium chloride, and concentrated until the concentration of protein is not more than 2500 μg/ml of the Concentrated eluate is applied to the gradient in the zonal rotor and centrifuged for 20 hours at 30,000 rpm at a temperature of 4oC. For konafa (HBs-AG ELISA Recombinant, ZAO Vector-best, p. Koltsovo, Novosibirsk region, Russia). The number of HBs antigen in the preparation according to the ELISA method is about 2500 mcg.

Example 2 (comparative example).

Repeat the same processes as in example 1, but in another modification stage 4. Instead hydrophobic column chromatography preparation of lysate from step 3 is transferred by ultrafiltration in 0.1 M phosphate buffer with pH 8.0, containing 0.2 M sodium chloride, and concentrated until the concentration of protein not less than 2500 μg/ml, the Number of HBs-antigen of hepatitis b vaccine in this preparation according to the ELISA method is about 2100 ug.

As this example shows, the inclusion of the hydrophobic phase chromatography allows to increase the output antigen of hepatitis b vaccine from recombinant yeast Hansenula polymorpha.

Example 3.

Stage 1. Cultivation of yeast cells.

Recombinant cells of Hansenula polymorpha (strain DL-U/pH-HBs), which are able to Express the HBs-antigen, is cultivated at a temperature of 30oWith 6.0 l of YEPD medium (1% yeast extract, 2% yeast peptone, and 1.6% glucose), enriched in methanol (1.5%).

Stage 2. The destruction of the yeast cells.

50 g obtained in stage 1 of the yeast mass resuspended in distilled evina + 0.2 M NaCl + 20 mm ethylenediaminetetraacetic acid). The mixture is cooled to a temperature of 2oC in an ice bath and served with constant stirring with a rate of 10-15 ml/min in a homogenizer (APV Gaulin. The camera homogenizer is constantly cooled by the flow of water-methanol mixture with a temperature of -10oC.

Stage 3. Extraction and solubilization of the HBs antigen.

The cell homogenate obtained in stage 2, washed with distilled water using a double centrifugation. To the resulting washed precipitate add 0.1% (weight/volume) desoxycholate sodium and 10% of ammonium sulfate; the mixture is stirred on ice for 1 hour. Adding ammonium sulfate leads to the deposition of ions thiocynate. The mixture is then centrifuged at a temperature of 4oFor sediment removal. The total volume of the obtained supernatant is about 700 ml.

Stage 4. Hydrophobic column chromatography.

The supernatant containing the solubilized antigen with stage 3, is applied to the chromatographic column section 50 mm, filled with 1500 ml of gel phenyl-agarose pre-equilibrated with buffer for hydrophobic chromatography (0.2 M sodium carbonate, pH of 9.2 + 6 M urea). The column was washed with 4500 ml of buffer for hydrophobic chromatography with the addition of 8-14% ethanol with temperatureand components (protein) by the method of Bradford. Washing stops when 1) the temperature of the solution in selected fractions of the eluate is more than 28oOr 2) the optical density at a wavelength of 280 nm is less than 0.07 or 3) putative protein concentration in the determination by the method of Bradford using human serum albumin as a calibrator is less than 50 µg/ml.

Associated with the column material elute buffer for hydrophobic chromatography, preheated to a temperature of 40-42oC. the Entire volume of the eluate (about 350 ml) are collected and cooled in an ice bath; the resulting precipitate was separated by centrifugation and discarded.

Stage 5. The ultracentrifugation in a density gradient.

The density gradient of potassium bromide formed by making a solution in the density range from 1,025 to 1,200 in the zonal rotor Ti-45 ultracentrifuge Beckman Optima LC70K. The preparation of the eluate from the hydrophobic column, obtained in stage 4, translated by ultrafiltration in 0.1 M phosphate buffer with pH 8.0, containing 0.2 M sodium chloride, and concentrated until the concentration of protein is not more than 2500 µg/ml.

The concentrated eluate is applied to the gradient in the zonal rotor and centrifuged for 20 hours at 30,000 rpm at a temperature of 4oC. On the Windows of the ELISA (HBs-AG ELISA, ImBio, Nizhny Novgorod, Russia). The fractions containing less than 100 µg/ml of immunoreactive antigen, are combined and translated by ultrafiltration in 0.1 M phosphate buffer, pH 8.0, containing 0.2 M sodium chloride. The average amount received drug is 400 ml.

Stage 6. Gel filtration on a column with copolymeres media Toyopearl HW65F.

5 liters of pre-washed copolymeres carrier for gel filtration Toyopearl HW65F contribute to column cross-section 100 mm, and balance its phosphate buffer (pH 8.5) containing 0.3 M sodium chloride. The product obtained at stage 5, making the column and elute with the same buffer with control fractions of 50 ml volume of the content and electrophoretic purity of the HBs antigen. The HBsAg content determined by ELISA (HBs-AG ELISA, ImBio, Nizhny Novgorod, Russia). The fractions containing HBs antigen at a concentration of not less than 100 µg/ml and the protein purity of at least 95%, unite, filtered through a sterilizing filter and stored at a temperature of 4oC.

Example 4 (comparative example).

Repeat the same processes as in example 3, but in another modification stage. Instead copolymeres media Toyopearl HW65F use the column with 5 l of agarose media Sephacryl CL-4B-section of 100 mm, the balanced f is irout the same buffer with control of the volume fractions of 50 ml. the content and electrophoretic purity of the HBs antigen. The HBsAg content determined by ELISA (HBs-AG ELISA, ImBio, Nizhny Novgorod, Russia). The fractions containing HBs antigen at a concentration of not less than 100 µg/ml and the protein purity of at least 95%, unite, filtered through a sterilizing filter and stored at a temperature of 4o(See tab. 1).

As this example shows, copolymeric carrier for gel filtration Toyopearl is preferred for fine purification stage of the drug rHBs-antigen from recombinant cells of Hansenula polymorpha strain DL-U/pH-HBs Example 5.

Stage 1. The formulation of the vaccine.

850 microliters (200 micrograms) drug HBs antigen obtained according to the method described in example 3, was dissolved in 9500 μl of 0.1 M phosphate-saline buffer (PBS) with a pH of 7.2, containing 0.15 M sodium chloride, and intensively mixed on a vortex mixer for 5 minutes. Suspension adjuvant Alhydrogel 3% (Brenntag Nordic, Denmark) are intensively mixed on a vortex mixer for 5 minutes. To the obtained mixture under stirring was added dropwise to 500 microlitres suspension adjuvant Alhydrogel 3% (Brenntag Nordic, Denmark), previously intensively mixed on a vortex mixer for 5 minutes. The mixture is then incubated in t is concentratie 50 μg/ml Formulated vaccine was stored at a temperature of 4-8oTo use (no more than 6 months).

Stage 2. Preparation of dilutions of the vaccine and the comparison drug.

Drug comparison served as the vaccine Engerix B (Glaxo Smith Kline), adult dosage, 20 mg/ml suspension. To obtain a dilution liquid to 10 ml of FBS was added 500 microliters suspension adjuvant Alhydrogel 3% (Brenntag Nordic, Denmark). The reference product and the product prepared in stage 1, was diluted with dilution liquid in 10, 50, 250 and 1250 times. The obtained diluted vaccine drugs and dilution liquid (negative control) the temperature of 4-8oTo use (no more than 2 weeks).

Stage 3. Immunization of mice.

The control was carried out on 9 groups of mice Balb/c mice (females, aged 10-12 weeks), 10 animals in the group. Each group immunization was performed with one of the 4 dilutions of the test product or the reference product, as well as a negative control. Animals once were injected intraperitoneally at 1.0 ml of the diluted vaccine. At 28 days after immunization, animals were bled. The obtained serum (separately from each animal) were stored at -20oTo control (no more than 5 days).

Stage 4. Fermentas test systems manufactured by Vector-best (Russia) according to the manufacturer's instructions. Take into account two indicators: 4.1) a qualitative result (positive or negative serum according to the manufacturer's instructions); in each group was calculated the percentage of seroconversion (percentage of animals that gave a positive reaction); 4.2) quantitative results of the optical density (OD) in the hole corresponding to the given serum); in each group was calculated the average OD and standard deviation. The results are shown in table 2.

From the table it follows that the immunogenicity of the preparation of the HBs antigen is almost identical to the immunogenicity of the drug comparisons and by the percentage of seroconversion in all dilutions, and the relative intensity of the immune response.

Example 6.

Vaccine for immunization against hepatitis b includes:
surface antigen preparation s - 202 µg/ml,
adjuvant - 1,50.1 ág/ml.

From the above materials, it is evident that the proposed method has several advantages:
- no use of toxic component, ethylene glycol, and ethanol is used, which is less toxic;
- the use of ultracentrifugation reduces the risk of undesired impurities in drugs,
- reduced pipe receiving surface antigen of hepatitis b virus from recombinant yeast cells, comprising culturing the recombinant yeast cells, the destruction of cells in buffer containing chaotropic agent, adding a surfactant to the homogenate of the cells, centrifugation of the homogenate of cells, hydrophobic chromatography, gel-filtration fractions containing surface antigen, followed by obtaining a fraction containing the target product, characterized in that the quality of raw materials using recombinant cells Hansemula polymorpha (strain DL-U/pH-HBs), before surgery, gel filtration spend hydrophobic chromatography and ultracentrifugation in a density gradient of potassium bromide, while hydrophobic chromatography carry out the washing with buffer containing 8-14% ethanol, an additional washing is conducted with the same buffer, with a temperature of 40-42oWith, and gel-filtration spend on copolymeres media Toyopearl HN65F.

2. Vaccine for immunization against hepatitis b, including surface antigen of hepatitis b virus produced by recombinantly cell yeast and adjuvant, characterized in that as the antigen of the hepatitis b virus using surface antigen obtained under item 1, in an effective amount.

 

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