RU2045073C1 - Method of preparing recombinant surface antigen of hepatitis b - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: recombinant surface antigen of hepatitis B is prepared by cultivation of yeast strain-producer on the nutrient medium consisting of pepton and yeast extract and containing 10-200 mg/l inorganic phosphate followed by incubation of antigen on the nutrient medium containing 5-50 mg/l inorganic phosphate. EFFECT: improved method of antigen preparing.

Description

 The invention relates to a method for producing hepatitis B surface antigen by microbiological synthesis, which can be used in the medical industry for the manufacture of hepatitis B vaccine.

 Hepatitis B vaccines based on HBs antigen from donor serum are known (J. med. Virol. 1982, 10, 65-74). However, such vaccines are unsafe for humans, as they may contain infectious particles of hepatitis B and can be contaminated with other viruses, for example, retroviruses contained in serum.

The method of obtaining vaccines from serum is multi-stage, contains 2-3 stages of inactivation, and the yield of surface antigen does not exceed 10%
A known method of producing HBs antigen from a yeast producer strain is to cultivate a producer strain in a phosphorus rich medium, transfer cells to a low phosphorus medium to reproduce the HBs antigen, followed by washing the cells in a centrifuge with a flowing rotor. Next, the cell paste freed from the medium is diluted to the original volume with phosphate buffer and the cells are destroyed using a high pressure homogenizer. The final product is obtained with a yield of 29% and with an impurity protein content of up to 20%, which makes its use as a vaccine impossible (PNAS, 1985, 82, 6830).

 Closest to the claimed combination of features is a method for producing a recombinant hepatitis B surface antigen, comprising culturing a yeast producer strain in a medium at the stage of the synthesis of НВs-antigen containing no phosphate, and isolating the antigen using adsorption chromatography on porous silicas, eluting with pH 9 bicarbonate buffer , 1-9.5, followed by centrifugation in a density gradient of 50% tartrate K (Na) 25% glycerol with a ratio of components 1: 1. The disadvantage of this method is the low yield of hepatitis B surface antigen after the fermentation and destruction step (200-300 μg of antigen with 1 liter of fermenter) (A. S. USSR N 1389060, class MKI A 61 K 39/29).

 The technical result of the invention is to increase the yield of hepatitis B surface antigen.

 A method for producing a recombinant hepatitis B surface antigen involves culturing and incubating a yeast producer strain on a nutrient medium, followed by destruction of the yeast cells and isolating the target HBsAg product, the difference of which is that the cultivation is carried out on a nutrient medium containing peptone and yeast extract with the final the total concentration of inorganic phosphorus 40-200 mg / l, and the incubation is carried out on a nutrient medium containing peptone and yeast extract with a final total concentration walkie inorganic phosphorus 5-50 mg / l.

 The use of a nutrient medium limited in inorganic phosphorus at the stage of yeast cultivation and biosynthesis of the antigen allows one to obtain the optimal yield of antigen regardless of the type of raw material.

 The use of a nutrient medium both at the stage of growing biomass and at the stage of antigen biosynthesis, with an inorganic phosphorus content in an amount that goes beyond the optimal values of the proposed method, does not lead to antigen production in good yield.

 During cultivation, a yeast strain is used containing a recombinant plasmid with the HBsAg gene and capable of producing immunogenic HBsAg. As the initial strain, any HBsAg producing yeast strain can be used, for example, the Saccharomyces cerevisiae DBY 746 / pNMVG-46 strain obtained by incorporating the transformed plasmid pNMVG-46 into the recipient strain DBY 746. The plasmid pNMVG-46 was obtained from a genomic engineering modification pADG47 (Mol. Genetics, Microbiology and Virology, 1986, N 8, N 12-19).

 The invention is illustrated by the following examples.

PRI me R 1. In a bioreactor with a working volume of 10 l, containing 9 l of sterile medium composition, wt. peptone from casein acid hydrolyzate (domestic) with an initial inorganic phosphorus content of 0.5-0.6 mg / g 3.0; yeast extract (domestic) with an initial inorganic phosphorus content of 2.0-2.5 mg / g 1.0; glucose 2.0, containing inorganic phosphorus 40-42 mg / l, at pH 5.0-5.5, a yeast culture of Sc DBY 746 / pNMVG-46 was added in an amount of 1.0 l grown on minimal medium (see Appendix 1 ) and having a cell titer of 4.1 × 10 ⁷ cm ^{3 of} culture fluid. Cultivation is carried out at a temperature of 29 + 1 ^about C for 20 hours. An accumulative culture is obtained with a cell titer of 2.0 × 10 ⁸ cells / cm ^{3 of} culture fluid.

The cell culture in an amount of 10 l is transferred to a bioreactor with a working volume of 100 l, containing 90 l of sterile nutrient medium composition, wt. peptone from casein acid hydrolyzate (domestic) with an initial inorganic phosphorus content of 0.5-0.6 mg / g 1.5; yeast extract (domestic) with an initial inorganic phosphorus content of 2.0-2.5 mg / g; glucose 2.0, salts, trace elements, vitamins (see Appendix 2), containing inorganic phosphorus 5.0-6.0 mg / l at pH 5.5-6.0. Cultivation is carried out at a temperature of 30 ^about C for 22 hours

A culture fluid with a cell content of 1.4 × 10 ⁸ cells / cm ^{3 is} fed to a Westfalia type flow centrifuge with a flow rate of 500 l / h. The cell paste is freed from the medium by washing with 0.05 M carbonate buffer with a pH of 9.0 by microfiltration using membrane filters with a pore diameter of 0.2-0.45 μm or washing three times in a buffer of the same composition, followed by separation of the biomass in a flow type centrifuge Westfalia at a flow rate of 500 l / h. Obtain 1.2 kg of biomass, which is suspended in 10 l of carbonate buffer composition: 0.05 M Na ₂ CO ₃ NaHCO ₃ , 0.15 M NaCl, 10 mM EDTA, 0.2 mm phenylmethylsulfonyl fluoride and 0.3% Tween-20 . Destroy using a homogenizer type Gaulin APV at a pressure of 600-700 atm in 3 cycles. 15 l of clarified homogenizate are obtained, in which the antigen content is determined. The antigen titer is 20 μg / ml clarified homogenizate. The antigen yield of 3.0 mg per liter of culture fluid. The content of HBsAg is determined by enzyme immunoassay using Abbott Auszyme II kits. Antigenic activity is measured in μg / ml using the industry standard HBsAg activity standard (OSO 42-28-153-88), manufactured by GISK im. L.A. Tarasevich. On electrophoregrams in polyacrylamide gel under reducing conditions, the recombinant antigen is presented as a 24 kD monomer band. The immunospecificity of this band is shown by immunoblotting using commercial anti-HBs labeled with iodine-125.

PRI me R 2. In a bioreactor with 9 l of a sterile nutrient medium composition, wt. peptone (DIFCO company) with an initial inorganic phosphorus content of 4.5-5.0 mg / g 2.0; yeast extract (DIFCO company with an initial inorganic phosphorus content of 10-11 mg / g 1.0; glucose 2.0 containing inorganic phosphorus 195-200 mg / l, at pH 5.0-5.5, yeast culture is introduced in an amount of 1 l grown on minimal medium (see para. Annex 1) and having a titer 4,4˙10 ⁷ cells / cm ³ of the culture medium. Cultivation is carried out at a temperature of 29 + 1 ^{° C} for 24 hours. a batch culture with a titer of 2.8 ˙10 ⁸ kg / cm ^{3 of} culture fluid.

The cell culture in an amount of 10 l is transferred to a bioreactor containing 90 l of a sterile nutrient medium composition, wt. peptone (DIFCO company) with an initial inorganic phosphorus content of 4.5-5.0 mg / g 1.0; yeast extract (DIFCO company) with an initial inorganic phosphorus content of 10-11 mg / g 0.1; glucose 2.0; salts, trace elements, vitamins (see Appendix 2), containing inorganic phosphorus 50 mg / l, at pH 5.5-6.0. Cultivation is carried out at a temperature of 30 ^about C for 24 hours

The culture fluid containing 2.0 × 10 ⁸ cells / cm ³ is subjected to processing as described in example 1.

 The weight of raw biomass is 1.5 kg; the volume of the clarified homogenizate is 15 l, the antigen titer is 22 μg / ml of the clarified homogenizate. The output of antigen from one liter of culture fluid is 3.3 mg.

 PRI me R 3. The process is carried out analogously to example 2; at the stage of growing biomass, a nutrient medium with an inorganic phosphorus content of 300 mg / l is used. The medium is prepared from 2.0 wt. peptone with an initial inorganic phosphorus content of 8.0 mg / g and 1.0 wt. yeast extract with an initial inorganic phosphorus content of 15.0 mg / g. At the stage of antigen biosynthesis, a nutrient medium with an inorganic phosphorus content of 100 mg / L, composed of 1.0 wt. peptone and 0.1 wt. yeast extract with the above content of the original inorganic phosphorus. 1.8 kg of crude biomass are obtained; antigen biosynthesis is absent.

PRI me R 4. In a bioreactor with 9 l of a sterile culture medium composition, wt. casein sulfuric acid hydrolyzate (domestic) with an initial inorganic phosphorus content of 2.0-2.5 mg / g 2.0; yeast extract (domestic) with an initial inorganic phosphorus content of 5.0-6.0 mg / g 1.0; glucose 2.0, containing inorganic phosphorus 100 mg / l, at pH 5.0-5.5, a yeast culture is introduced in an amount of 1 l grown on minimal medium (see Appendix 1) and having a titer of 4.3 × 10 ⁷ cells / cm ³ culture fluid. Cultivation is carried out at a temperature of 29 + 1 ^about C for 20 hours. An accumulative culture is obtained with a titer of 2.2 × 10 ⁸ cells / cm ^{3 of} culture fluid.

The cell culture in an amount of 10 l is transferred to a bioreactor containing 90 l of a sterile nutrient medium composition, wt. casein sulfuric acid hydrolyzate (domestic) with an initial inorganic phosphorus content of 2.0-2.5 mg / g 1.0; yeast extract (domestic) with an initial inorganic phosphorus content of 5.0-6.0 mg / g 0.1; glucose 2.0; salts, trace elements, vitamins (see Appendix 2), containing inorganic phosphorus 25-30 mg / l, at pH 5.5-6.0. Cultivation is carried out at a temperature of 30 ^about C for 24 hours

Get a culture fluid with a cell titer of 1.7 × 10 ⁸ cells / cm ³ . Separation, washing of biomass from residues of the nutrient medium, destruction of cell biomass and clarification of the homogenizate is carried out similarly to that described in example 1.

 The weight of raw biomass is 1.35 kg. 15 l of clarified homogenisate are obtained with an antigen titer of 21 μg / ml. The output of antigen from one liter of culture fluid is 3.15 mg.

PRI me R 5. In a bioreactor containing 9 l of a sterile culture medium composition, wt. peptone (domestic) with an initial inorganic phosphorus content of 0.05 mg / g 3.0; yeast extract (domestic) with an initial inorganic phosphorus content of 4.0-5.0 mg / g 1.0; glucose 2.0, containing 40 mg / l of inorganic phosphorus at pH 5.0-5.5, introduce a yeast culture and cultivate as described in Example 1. The cell titer at the biomass growth stage is 1.8˙10 ⁸ cells / cm ³ culture fluid.

A culture of 10 l is transferred to a bioreactor with a working volume of 100 l, containing 90 l of a sterile nutrient medium composition, wt. peptone (domestic) with an initial inorganic phosphorus content of 0.05 mg / g 3.0; yeast extract (domestic) with an initial inorganic phosphorus content of 4.0-5.0 mg / g 0.1; glucose 2.0, salts, trace elements, vitamins (see Appendix 2), containing inorganic phosphorus 4-5 mg / l at pH 5.5-6.0. Culturing was carried out at ³⁰ ° C for 24 h fermentation broth with a titer of cells 1,4˙10 ⁸ cells / cm ³ is worked up as described in Example 1. 1.15 kg wet biomass.; 15 l of clarified homogenizate with an antigen titer of 18 μg / ml. The output of antigen from one liter of culture fluid is 2.7 mg.

PRI me R 6. In a bioreactor containing 9 l of a sterile culture medium composition, wt. peptone (domestic) with an initial inorganic phosphorus content of 0.05 mg / g 0.5; yeast extract with an initial inorganic phosphorus content of 2.0-2.5 mg / g 0.1; glucose 2.0, containing 2.0 mg / l of inorganic phosphorus at pH 5.0-5.5, introduce a yeast culture and cultivate as described in Example 1. The cell titer at the stage of biomass growth is 5.0 × 10 ⁷ cells / cm ³ culture fluid.

Culture in an amount of 10 l is transferred to a bioreactor with a working volume of 100 l, containing 90 l of sterile medium composition, wt. peptone (domestic) with an initial inorganic phosphorus content of 0.05 mg / g 0.1; yeast extract 2.0-2.5 mg / g 0.1; glucose 2.0, salts, trace elements, vitamins (see Appendix 2), containing inorganic phosphorus 2.0 mg / l at pH 5.5-6.0. Culturing was carried out at ³⁰ ° C for 24 hours. The culture broth with a titer in cells 1,0˙10 ⁷ cm ³ is worked up analogously to Example 1. 0.110 kg of raw biomass, 15 liters of a clarified homogenate antigen titer with 1.5 ug / ml The output of antigen from one liter of culture fluid 0.225 mg.

 Examples 3 and 6 show that the use of a nutrient medium at the stage of growing biomass and biosynthesis of antigen with inorganic phosphorus in an amount that goes beyond the optimal values does not allow to obtain an antigen in good yield.

 Thus, the proposed method allows to obtain a recombinant surface antigen of hepatitis B in good yield when using raw materials of various origins.

APPENDIX 1 Composition, wt. KH ₂ PO ₄ 0,100 NaCl 0,010 CaCl ₂ anhydrous 0,020 MgSO ₄ ˙ 7H ₂ O 0,102 (NH ₄ ) ₂ SO ₄ 0,500 Glucose 2.00 Micronutrient solution 0.50 ml / l Vitamin solution 1.00 ml / l Distil. water Up to 100.0
Microelement solution Composition, mg% KJ 20.0 H ₃ BO ₃ 2.0 MnSO ₄ 2.0 MoO ₄ (NH ₄ ) ₂ 2.0 FeSO ₄ 10.0 Dist. water Up to 100 ml
Vitamin solution Composition, mg% Thiamine chloride 20.00 Pyridoxine 20.00 Riboflavin (mononucleotide) 20.00 Pantothenate calcium 20.00 Nicotinic acid 20.00 p-Aminobenzoic acid 20.00 Biotin 0.20 Inositol 1000.0 Dist. water Up to 100 ml
APPENDIX 2 Composition, wt. KCl 0,100 NaCl 0,010 CaCl ₂ anhydrous 0,020 MgSO ₄ ˙ 7H ₂ O 0,102 (NH ₄ ) ₂ SO ₄ 0,500 Micronutrient solution 0.50 ml / l (see Appendix 1) Vitamin solution 1.00 ml / l ( cm.

 Annex 1)

Claims (1)

 METHOD FOR PRODUCING A RECOMBINANT SURFACE HEPATITIS B ANTIGEN, comprising culturing and incubating a yeast producer strain on a nutrient medium, followed by destruction of the yeast cells and isolating the target product, characterized in that the cultivation is carried out on a nutrient medium containing peptone and yeast extract with a non-final concentration phosphorus in a nutrient medium of 40-300 mg / l, and incubation is carried out in a nutrient medium containing peptone and yeast extract, with a final total concentration atsiey inorganic phosphorus in the nutrient medium 5-50 mg / l.