WO2017003322A1 - Inactivated influenza split-virus vaccine and method of production thereof - Google Patents

Abstract

This invention relates to immunology, medicine, and pharmacology and concerns production of inactivated influenza vaccines which can be used in vaccination of the population of Russia against influenza. The method for producing a drug intended to treat influenza comprises inoculating embryonated chicken eggs with influenza virus, incubating the inoculated embryonated eggs, cooling the incubated embryonated eggs, harvesting viral allantoic fluid (VAF), processing the VAF for disaggregation and then inactivation thereof, subjecting the VAF to microfiltration on two cascade cartridges and then to ultrafiltration and purification in a sucrose density gradient, the purified inactivated viral concentrate is split using octyl-beta-glucoside detergent, virions that failed to split and ribonucleoprotein complexes with membrane protein are isolated by means of ultracentrifugation, the final purification of the isolated antigens is carried out using a chromatographic carrier, following which a stabilizing agent is added to the antigens produced. The method differs from the known one in that the inactivation of the viral activity is performed in the VAF processing step by using beta-propiolactone disinfectant, the virus disaggregation during the VAF processing is carried out using sodium chloride in a final concentration, and the final purification of the antigens is carried out using chromatographic carrier WorkBeads 40/100/sec (Bio-works, Sweden), followed by stabilizing with Triton X-100 detergent at a concentration of 100-300 µg/ml. A drug for treating influenza, which is produced by the above-mentioned method, is also proposed.

Description

 VACCINE INFLUENZA INACTIVATED CRACED AND

 METHOD FOR ITS PRODUCTION Inactivated split influenza vaccine and method for its preparation

(hereinafter referred to as the invention) relates to immunology, medicine, pharmacology, and relates to the production of inactivated influenza vaccines that can be used to vaccinate the Russian population against influenza.

 Currently, 2 types of vaccines are used for influenza vaccination: inactivated vaccines and live vaccines. In turn, inactivated vaccines are divided into whole-virion, split, subunit and virosomal. A significant part of the global market for inactivated vaccines is occupied by split vaccines. This is due to the fact that these vaccines have high immunogenicity, like whole-virion vaccines, but their reactogenicity is significantly lower than whole-virion vaccines. Subunit vaccines have a very low reactogenicity, however, their immunogenicity is inferior to the immunogenicity of split influenza vaccines.

 Currently, several agents are known for treating influenza and, accordingly, methods for producing these agents.

In the patent of the Russian Federation Ns2283139, publication date 10.09.2006, describes a tool and method for producing split antigens. In the description of the technological process, β-octylglucoside detergent is used to destroy the purified virions and the final cleaning of antigens is carried out by chromatography on a Sephadex-50 carrier from GE (Sweden). Inactivation of viral activity is carried out at the stage of dilution of the viral concentrate by ultraviolet irradiation. According to this method, virions are purified by a sequential combination of sorption-elution method on formalized chicken red blood cells and chromatographic purification method. However, the virion purification technology used does not allow reproducing the purification parameters due to the non-standard nature of chicken red blood cells, and the described chromatography method is not always effective for purification of virus-containing allantoic fluid (IMPORTANT) with a high content of destroyed cells. The Sephadex-50 carrier used at the stage of final antigen purification is not effective for the separation of influenza antigens and ovalbumin, due to the not optimal cutoff limit of the carrier itself. Inactivation with using UV irradiation is not ^'always allow to obtain reproducible results, since in case of highly aggregated virus concentrates are not all the viral particles are exposed to irradiation. Accordingly, the tool has an increased risk of reactogenicity in vaccinated people, as well as the danger of residual infectious ™ in the finished dosage form of the vaccine.

 RF patent N ° 2535153, publication date 12/10/2014, describes a tool and method for producing a highly purified viral concentrate using methods for treating virus-containing allantoic fluid (VAL) with PEG 6000 polyelectrolyte to reduce aggregation followed by microfiltration on 2 stages of cartridges (pore diameter 10 μm and 1 μm), by ultrafiltration on cassettes with a cut-off threshold of 300 KiloDalton and final purification of the virus in a sucrose density gradient. For the further production of inactivated influenza vaccines from concentrates obtained by this method, it is necessary to inactivate viral activity using the ultraviolet irradiation method, as in the previous method, which significantly reduces its attractiveness. As an electrolyte in this invention, a solution of polyethylene glycol-6000 in a sufficiently high concentration is used. This substance is harmless and is used in the medical industry, however, its cost is quite high, and taking into account the quantities that are used to process the IMPORTANT in this invention, the use of this reagent significantly increases the cost. In addition, based on this patent, you can get only purified viral concentrate, and not the dosage form of the flu vaccine. To obtain the dosage form of the vaccine, an additional block of technology for the destruction, isolation, and purification of viral antigens is required. Moreover, the resulting product has the risk of residual infectivity in the finished dosage form of the vaccine.

According to WHO recommendations, the inactivation of viral activity in the production of inactivated influenza vaccines is preferably carried out in the early stages of the technological process, in particular during the processing of IMPORTANT VALVES. For these purposes, various decontamination reagents are used, and, above all, a solution of β-propiolactone. According to the literature, treatment with this reagent does not violate the immunochemical structure of influenza antigens. Accordingly, during this treatment, the set of protective epitopes (immunocompetence) in composition of surface antigens (hemagglutinin, neurominidase). In addition, the functional properties of hemagglutinin are not impaired, namely, to carry out a fusion with the plasma membrane of the cell. In turn, the processing pathway of the hemagglutinin antigen in the antigen-presenting cell of the human immune system corresponds to that for the natural influenza virus, and thus allows inducing cellular immunity during vaccination with a preparation obtained using β-propiolactone as an inactivator. (A.Huckrade, 2012). Thus, the use of β-propiolactone to inactivate the IMPACT allows one to obtain deactivated material in the early stages of production, which drastically reduces the volume of liquid waste to be inactivated. In addition, the hemagglutinin antigen thus obtained has a higher protective potential than similar antigens obtained using formalin or ultraviolet radiation. It should also be noted that at the stage of processing the IMPORTANT, with the correct selection of components, β-propiolactone is guaranteed to inactivate viral activity for several hours, while the use of ultraviolet radiation at the stage of diluted HVA does not always lead to 100% inactivation. Currently, β-propiolactone is already used abroad in the production of influenza vaccines, however, in Russia there has not yet been a proven technology for the use of this reagent for the production of domestic anti-flu drugs.

 Closest to the proposed invention are the means and methods described in RF patents N22493872, date of publication 09/27/2013; and the Russian Federation jV-> 2523614 (Vaccine against influenza and its preparation), publication date 07/20/2014, which we consider to be the prototypes of the invention.

The first prototype (RF patent N ° 2493872) describes a means and method of purification of influenza viruses using microfiltration and ultrafiltration (without the use of formalized chicken red blood cells). For microfiltration, cartridges with a very small pore diameter (0.45 + 0.22 μm) are used. Ultrafiltration is carried out on cassettes with a cutoff limit of 300-500 KiloDalton. The virus purified in this way is then further purified by ultracentrifugation in a sucrose density gradient. This patent does not describe a method for inactivating the influenza virus, however, it follows from the text of the description of the invention that the inactivation process will be carried out after the stage of obtaining the purified viral concentrate. It should be noted that use in The process of microfiltration of cartridges with pore diameters of 0.45 + 0.22 μm, as a rule, leads to rapid clogging of these cartridges during the cleaning of the IMPORTANT, since as a result of the multiplication of viruses and destruction of chicken embryonic cells, a large number of debris cultures of various sizes are present in the IMPORTANT. Thus, due to the non-optimal pore size, the life of the above cartridges is very short. In addition, if the clogging of the cartridges occurs in the production process of cleaning the IMPORTANT, then replacing them leads to a violation of the sterility conditions of the process, and creates big problems with logistics. Therefore, in the world literature described cartridges with larger pore sizes for microfiltration of IMPORTANT. In particular, the patent of the Russian Federation N ° 2535153 describes the use of a cascade of cartridges of 10 μm and 1 μm, which allows you to clean from debris significant volumes of IMPORTANT.

The second prototype (RF patent N ° 2523614) describes a means and method of obtaining a split vaccine against influenza virus using methods of clarification (centrifugation), microfiltration, concentration, cleavage of the influenza virus concentrate, followed by ultrafiltration of the resulting viral lysate. A feature of this patent is the use of neutral or basic amino acids, as well as sodium chloride in a concentration of 0.3-2.0 mol as stabilizers. This patent does not indicate at what stage and in what way the inactivation of viral activity is performed. The description of this patent indicates that various detergents (cationic, anionic, and nonionic) are used to cleave the purified virions. However, far from all detergents make it possible to isolate hemagglutinin and neurominidase antigens with complete preservation of their immunocompetence. In particular, the most known and affordable anionic detergent Ν-lauryl sarcosinate Na in some serotypes of the influenza virus causes destruction of hemagglutinin and sharply reduces its antigenic and immunogenic activity. Another well-known non-ionic detergent Triton X-100 is a very mild reagent for the destruction of influenza viruses, but it is very poorly separated from the released antigens during their cleaning. Therefore, the selection of the optimal detergent for the destruction of the virus is the main part of the technology for the production of modern influenza inactivated vaccines. In addition, this patent describes methods for stabilizing isolated antigens using various amino acids in final concentrations up to 0.5 M, or with Tween 80 detergent to a final concentration up to 0.2%. However, it is known that some amino acids are biologically active substances and in high concentrations (up to 0.5 M) can be reactogenic with mass vaccination of people. In addition, these reagents, if used at different stages of the technological cycle, can significantly increase the cost of the technology. With respect to the tween-80 detergent, it should be noted that according to the literature, this detergent is used as a stabilizer only at a concentration of 1%, and there is no reason to believe that this substance at a concentration of 0.2% will be an effective stabilizer as in the process of splitting and purification of antigens, and as part of the final dosage form. Accordingly, the obtained drug has the danger of residual infectivity in the finished dosage form of the vaccine, as well as the danger of increased reactogenicity when vaccinating people.

 As a result of many years of experiments conducted by the authors of this invention, it was found that the most optimal approaches for obtaining an inactivated cleaved vaccine is the use of a β-propiolactone disinfectant to inactivate viral activity at the stage of treatment of IMPORTANT; to prevent aggregation of virions during the cleaning process, the most optimal is the use of sodium chloride in a final concentration of 0.22 M; for chromatographic purification of incense antigens, it is most effective to use the Bio-bad 40/100 / sec carrier, Bio works (Sweden) and stabilize the obtained monovaccines, as well as stabilize the trivalent dosage form of the vaccine using Triton X-100 detergent to a final concentration of 100- 300 mcg / ml. The above approaches formed the basis of this invention.

 The problem to which the present invention is directed is to develop a new technology for the production of Russian inactivated split vaccine, which will make it possible to produce a vaccine in our country with a world level of quality.

The technical result of the invention is to develop an industrial method of obtaining a split vaccine and funds based on it, which can be used for mass vaccination of the population of Russia. Therefore, the result obtained differs from the above prototypes in the degree of purification of the incorporated antigens, their higher level of immunocompetence, and also the higher efficiency of the presented technology The object of the invention is a method for producing a split vaccine and an agent based on it, which includes infection of chicken embryos with influenza virus, incubation of infected embryos, cooling of incubated embryos, collecting IMPORTANT, processing of IMPORTANT for the purpose of disaggregation and inactivation, microfilting of IMPORTANT on 2 stages of cartridges, ultrafiltration of IMPORTANT and purification of the viral concentrate in a sucrose density gradient. Cleavage of the purified inactivated viral concentrate is carried out with the beta-octyl glucoside detergent, the removal of the undeveloped virions and a significant part of the complexes of ribonucleoproteins with the membrane protein is carried out by ultracentrifugation, the final purification of the inserted antigens is carried out on a chromatographic carrier, and a stabilizer is added to the obtained monovaccine.

 The difference of the proposed method from the prototype is that we carry out the inactivation of viral activity at the stage of processing the IMPORTANT using a betta-propiolactone disinfectant, disaggregate the viruses when processing the IMPORTANT, we carry out sodium chloride at a final concentration of 0.22 mol, the final cleaning of antigens is carried out on a WorkBeads chromatographic medium 40/100 / sec from BioWorks (Sweden), and we stabilize the monovaccine and the trivalent dosage form using the detergent Triton X-100 at a concentration of 100-300 μg / ml.

 The inventive method of obtaining consists of several technological steps: the 1st step is to obtain IMPORTANT, and consists of several steps: obtaining chicken embryos, disinfection, pre-incubation, infection with the working solution of seed virus, incubation of infected embryos, cooling after incubation, collecting IMP and cleaning IMPORTANT

 1.1. Obtaining chicken embryos (CE)

 The hatching egg enters the incubation site in accordance with the supply contract. After the input control, the fertilized egg is laid in an incubator, or preheated to 37.5 ° C. An incubation is carried out for 10-11 days at a temperature of (37.9 ± 0.2) ° C and relative humidity (54 ± 2)%.

On the 10th day, the obtained chicken embryos (CE) are unloaded from the incubator and the CE viability is monitored using a special ovoscope device. 36 pieces of chicken embryos in plastic trays are placed in carts transport shelving (TTS), load into the vehicle and transfer to stage 1.2.

 1.2 Disinfection of TBE

 The total time (from unloading the TTS to the aerosol chamber) is about 30 minutes. Before disinfection, an aerosol chamber is prepared: it is heated to a temperature of 32 ° C. At one time, no more than 18 FE carts are loaded into the camera. CE disinfection is carried out by spraying a disinfecting solution with a 1% Oxonium Active disinfectant solution, manufactured by Ecolab, Italy. Spraying is carried out using 2 “cold fog” generators. Disinfected TB cells are discharged into the thermal chamber for additional TB incubation.

 1.3. Pre-incubation

 The obtained CEs, from stage 1.2, are additionally incubated as follows: CEs are placed in a thermal chamber at a temperature of (37.5 ± 0.5) ° С and relative humidity (55 ± 5)%, and incubated for at least 12 hours

 1.4 TB infection

 The process of TBE infection is carried out under aseptic conditions. The container with the working solution of the seed virus is connected through the Medisart filter element to the infection line (inoculator), manufactured by Rame-Hart, USA.

70% ethanol is used for disinfection. This disinfectant treats the CE shell and needles during the infection process. Infection boards are installed on the inoculator (needles with punches sterilized in an autoclave at a temperature of (126 ± 2) ° С and steam pressure (1.5 ± 0.2) kgf / cm for (30 + 2) min.), The pressure is set compressed air (35 ± 5) kPa, after which the dose is checked / adjusted. The productivity of an automatic infection line is 30,000 CE per hour. Carts with FE from the thermal chamber are rolled out to the inoculator machine for the forthcoming infection. At the beginning of the work, plastic trays with FE are installed on the conveyor of the machine. Further, the plastic trays move along the conveyor, undergoing disinfection with 70% ethyl alcohol, and then infection occurs. In each TBE, (0.20 ± 0.02) ml of working dilution of the inoculum virus is administered. The control of the dose volume of the introduced virus is carried out every 30-40 minutes. The needles of the infection cards are automatically disinfected with 70% ethyl alcohol after each puncture of the CE shell. After the release of trays with infected CEs, their visual selective control is carried out. The controlled FEs are passed to stage 1.5. 1.5 Incubation of infected TBE

 Chicken embryos infected with the influenza virus are incubated in the technological mode (temperature, relative humidity) in accordance with the regimes indicated in the passports for the strain. For type A influenza viruses, the incubation time, as a rule, is from 36 to 46 hours, and for type B viruses, from 50 to 62 hours and at a relative humidity of 55 to 65%. After incubation, infected CEs are passed to step 1.6.

 1.6 Cooling CE after incubation

 CE is cooled in a refrigerating chamber for 12 to 20 hours at a temperature of (4 ± 2) ° С. After cooling, the CE collects the IMPORTANCE at stage 1.7.

 1.7 Collection of IMPORTANT

 The collection of VAZh is carried out using an automatic line for collecting allantoic fluid (harvester) manufactured by Rame-Hart, USA. On this line, CEs are automatically decapitated, their visual rejection (dead CEs, incubation defects, battle) and collection of IMPORTANT under aseptic conditions are performed. The collected IMPORTANT using Watson-Marlow 720 S peristaltic pumps is pumped into a 800-liter reactor. Before starting the collection of the IMPORTANT, the reactor is cooled using water and a cooling device to a final temperature in the reactor of (6 ± 2) ° С.

 1.8 IMPORTANCE

 At the end of the collection, the IMPORTANT turn on the stirrer at a speed of 40 rpm, using a Watson-Marlow model 720 S peristaltic pump, add a sterile solution of 2.5 M NaCl to a final concentration of 0.22 M. The contents of the reactor are altered for (30 ± 2 ) min at a temperature of (6 ± 2) ° С. Next, β-propiolactone is added to the same reactor to a final concentration of 0.1% to inactivate the virus, and the contents are again mixed for 1-2 hours at a temperature of (6 ± 2) ° С. After stirring, the mixer is turned off and the contents of the reactor are incubated for (16 ± 2) h at a temperature of (6 ± 2) ° С.

 Stage 2 consists in purifying the IMPORTANT by micro- and ultrafiltration methods and purifying the obtained IMPORTANT concentrate by ultracentrifugation in a sucrose density gradient.

2.1 Purification and concentration of the IMPORTANT by micro- and ultrafiltration The work is carried out on a combined micro and ultrafiltration unit in compliance with aseptic rules in the laminar flow zone. After processing and inactivation of the IMPORTANT from the reactor using a Watson-Marlow model 720S peristaltic pump, they are fed to a microfiltration unit and passed through 2 stages of depth filters with pore diameters of 5 μm and 1 μm, respectively, into a sterile container with a capacity of 16-20 l. The clarified IVA obtained by microfiltration using a Watson-Marlow model 720 S peristaltic pump is fed to an ultrafiltration unit with Pellicon 2 Biomax-300 cassette modules with a cutoff limit of 300 kDa to concentrate the IMPORTANT. Concentrate clarified IMPORTANT 10-15 times. The permissible pressure value during ultrafiltration should not exceed 0.5 bar. The microfiltration process and the ultrafiltration process are carried out at room temperature. The resulting concentrate in a volume of 20-40 liters is transferred to the next stage of purification.

 2.2 Purification and concentration of influenza virus in sucrose density gradient

In the rotor of the K2 ultracentrifuge manufactured by Alfa-Wasserman (USA), sterile phosphate buffer solution, pH = 7.2 (FBI) and a 60% sucrose solution are loaded at a temperature of (4 ± 2) ° С. After the rotor is accelerated to (34000 ± 1000) rpm, the WAG concentrate from stage 2.1 is supplied to the rotor using the Watson-Marlow model 520 S peristaltic pump at a speed of (10 ± 1) l / min. At the end of the passage of the entire volume of concentrated IMPORTANT, the FBI is fed into the centrifuge rotor and centrifuged for (2.0 ± 0.5) hours. The whole process is carried out at a rotor cooling temperature of (4 ± 2) ° С. After centrifugation is completed, the ultracentrifuge is stopped and the obtained fractions of the inactivated virus concentrate (VK) are unloaded in a sterile box. Then 20 fractions of the sucrose density gradient contained in the rotor are collected: the first two fractions, with a volume of 200 ml, the subsequent ones with 100 ml each. A 5 ml sample was taken from each vial to control hemagglutinating activity and% sucrose. After sampling, each bottle is closed with a silicone stopper and placed in the refrigerator for storage at a temperature of (5 + 3) ° С for up to 3 days.

For further processing, inactivated VK fractions are used that have a GA titer of at least 1: 8000 and contain (35-48)% sucrose, which are stored in a pharmaceutical freezer at a temperature of from -10 to -20 ° C for no more than 3 months. Stage 3 includes the cleavage of inactivated viral concentrates, micro- and ultrafiltration of the obtained lysate, precipitation of the undeveloped virions by ultracentrifugation, concentration of the supernatant, chromatographic purification and sterilizing filtration of monovaccine.

 3.1 Cleavage of inactivated viral concentrate

 Fractions of the inactivated viral concentrate from stage 2.2 in the required amount 15-18 hours before work are placed for thawing in a refrigerator at a temperature of (5 ± 3) ° С. The selected VK fractions are combined in a sterile bottle with a capacity of 16 l, in compliance with aseptic rules. The cleavage of inactivated VC is carried out using the octyl glucoside detergent. The amount of detergent added is calculated by the protein content of the viral concentrate.

 The determination of the optimal detergent / protein ratio is carried out for each serotype separately when changing the production strain or changing the lot of detergent. The determination is carried out as follows: in polypropylene tubes, a volume of 2 ml is placed 1 ml of the investigated combined inactivated VC, diluted to a protein concentration of (1 ± 0.2) mg / ml FBI.

 0.5 ml of a concentrated solution of octyl glucoside are added to 4 test tubes with a solution of diluted VK so that the mixture contains 5 mg of detergent, 7 mg of detergent, 10 mg of detergent and 15 mg of detergent (ratio 5: 1, 7: 1, 10: 1, 15: 1). The solutions are thoroughly mixed and incubated for 30 minutes at room temperature. Instead of the detergent, the FBI is added to the 5th test tube. After incubation in all samples (including control without detergent), the hemagglutinin content is determined. The minimum ratio is selected at which the amount of specific hemagglutinin is maximum.

 The calculated sample of detergent is dissolved in the FBI and the resulting β-octyl glucoside solution is added to a glass bottle with a capacity of 10-16 L with combined inactivated VC

 Then, an overhead electric mixer is inserted into the neck of the bottle and VK is split at a stirrer speed of (130 ± 10) rpm for (1.0 ± 0.2) hours. The resulting lysate is transferred to the next purification step.

3.2 Micro and ultrafiltration of the resulting lysate The lysate is filtered through a microfiltration unit with cartridges with a pore size of 10 μm. Residuals displace the FBI. The filtered lysate is fed to an ultrafiltration unit with cartridges with a cutoff limit of 30 kDa. Carry out the technological process (concentration of the material) at a pressure of (0.7 ± 0.1) MPa. The final volume of concentrated lysate is 5 l.

 3.3 Precipitation of undestructed virions

 Underdeveloped virions from the viral lysate are precipitated by ultracentrifugation on Tierto Fisher Scientific equipment) model Sorvall Lynx 6000.

 Using a Watson Marlow peristaltic pump, model 720 N, a 5 L FBI is pumped into the centrifuge rotor during fluid flow at a speed of (15 ± 1) l / h. Then, the concentrated lysate from step 3.2 is fed into the rotor by the same pump. and accelerate the rotor to a speed of 18000 rpm The speed of transmission of material through the rotor is (5 ± 1) l / h, after which the material is displaced with 5 liters of FBI. The supernatant and the FBI after displacing the material are combined in a 16 liter bottle and transferred to the next stage of purification.

 3.4 Ultrafiltration of the supernatant

 Ultrafiltration of the supernatant (concentration of the material) is carried out at the ultrafiltration unit, as described in paragraph 3.2. Concentration of the supernatant is carried out to a final volume of 2.0 L. The resulting product is transferred into a sterile glass container and transferred to stage 3.5 chromatographic purification.

 3.5. Chromatographic purification of concentrated supernatant

 For purification of the concentrated supernatant, a WorkBeads 40/100 chromatographic carrier for exclusive chromatography (gel filtration) is used, manufactured by Bio-Works, Sweden (information on the carrier is available on the manufacturer’s website: www.bio-works.net, section: Life scienses).

Gel filtration is carried out on an axial compression glass production column (BPG 200/950, 30 L). A concentrated supernatant in a volume of 2 l is applied to a column with a volume of 15 l of the carrier using a peristaltic pump. Chromatography is carried out at a speed of 10 l / h with an FBI buffer and using an K-200 ultraviolet detector manufactured by Klaig, Germany. Registration of the detector data is carried out on a personal computer with the MultiChrom system. The peak of the purified material begins to come out after displacement free volume, while the process is accompanied by a sharp increase in optical density by 280 nm. Material collection is stopped after the optical density drops to 30-40 units and the chromatogram reaches the plateau phase. The volume of concentrate after passing through the column increases by 20-30%.

 The resulting material is passed to stage 3.6.

 3.6. Sterilizing Mono-Vaccine Filtration

 The material purified from the detergent from step 3.5 is transferred to sterilizing filtration.

 The purified supernatant is poured into a 50 L reactor prepared for operation, the FBI is added until the total protein content is 400-500 μg / ml; 1% solution of merthiolate in an amount providing its final concentration of 100 μg / ml. Then stirred at a speed of 200-220 rpm for 15-20 minutes at room temperature.

Next, the consolidated mono-vaccine is filtered through a 0.2 μm Sartobran type disposable filter element, 0.2 m ² filtration area, or through a capsule with similar technical characteristics into a disposable sterile plastic container of the Flexboy type (Sartorius, Germany) with a capacity of 20 l.

 The obtained sterile monovaccine is transported to pharmaceutical refrigerators and stored at a temperature of (5 ± 3) ° C for no more than 6 months. The vaccine obtained is monitored according to the following indicators: specific activity, protein content, authenticity, ovalbumin content, bacterial endotoxin content, sterility.

 Stage 4 - includes mixing the components of the vaccine, sterilizing the filtration of the vaccine, filling the drug into ampoules, labeling and packaging.

 4.1 Vaccine Component Mixing

The process involves mixing monovaccines of 3 types of influenza viruses Α (ΗιΝι), A (H ₃ N ₂ ) and B, with a 1% solution of the Triton X-100 detergent, 1% solution of merthiolate and the FBI in a 200-liter reactor.

Semi-finished products of a single vaccine of three different strains of the influenza virus: type Α (ΗιΝ, type A (H ₃ N ₂ ) and type B, having passed all the controls, from sterile disposable containers under aseptic conditions are loaded into the reactor.

Then, the calculated amounts of PBS, 1% solution of thiolate to a final concentration of (85-115) μg / ml, and 1% solution of Triton X-100 detergent to a final concentration of 100-300 μg / ml are added to the reactor. The contents of the reactor are mixed in for 15 minutes The process of mixing the dosage form is carried out at room temperature. Next, sterilized filtration of the mixed solution is carried out.

 4.2 Sterilizing filtration of the vaccine dosage form

A hose mounted on the outlet fitting of the reactor under aseptic conditions is connected to the fitting at the inlet of the filter element. The other sterile hose is connected at one end to the fitting at the outlet of the filter element, and the other is connected to the inlet port of a 200 liter sterile plastic bag. Filter through a disposable filter element of the type Sartobran 0.2 μm, the filtration area of 0.45 m ² . A sample is taken from the filled sterile bag under aseptic conditions for analysis of specific activity, protein content, and content of thiolate. Label with the name of the vaccine, N2 series, and date of filtration is glued onto the frame with the container containing the semi-finished vaccine. The container with the prefabricated tri-vaccine is transported to the finished dosage forms workshop. Storage of the container with the semi-finished product dosage form of the drug is carried out in a refrigerator at a temperature of 2 to 8 ° C for no more than 14 days. After passing the controls, the container with the sterile semi-finished product is transferred for filling into ampoules.

 4.3 Pouring the drug into ampoules

 The bottling of the drug is carried out on a machine for filling and sealing ampoules RSF manufactured by CO.RI.M.A., Italy. A disposable container with the drug under aseptic conditions is connected through the lower port to the dispenser system. Set the desired dose. The system is filled with a solution of the drug and pumped to remove air bubbles.

 The drug is poured into ampoules with a capacity of 1.0 ml of type IP-1 In 1 or similar. Sterility control of the bottling process is carried out at the beginning and at the end of the bottling.

 All ampoules with the drug are monitored for leaks and transmitted for viewing to a room that is protected from direct sunlight. Control of the vaccine for mechanical inclusion is carried out by an inspection machine A-30, manufactured by Brevetti, Italy.

 Then the drug is transferred to the quarantine storage warehouse of the packaging department and stored at a temperature of 2 to 8 ° C.

Ampoules with the finished dosage form of the drug are selected from the quarantine storage warehouse and quality control is carried out according to the documentation. The main controls include visual control, determination of protein content by the Lowry method, determination of specific activity by the ARID method, determination of the content of ovalbumin and endotoxins, determination of the content of Triton X-100, study of antigenic activity, sterility.

 4.4 Labeling and packaging

 A series of ampoules with a finished dosage form, having passed all the controls, comes from the quarantine storage warehouse to the packaging workshop. Before starting work, the marking machine is set up, a cliche is filled, which indicates: the brand name of the drug, batch number, release date, volume of the drug in ml, number of doses, shelf life.

 Marked ampoules are placed in boxes or packs of cardboard for consumer packaging (GOST 7933-89, or TU OP 5453-010-04766354-2005, or imported). A label is glued onto the box or bundle in accordance with regulatory documents.

 Group and transport packaging in accordance with GOST 17768-90.

 Packaged vaccine is placed in the refrigerator compartment of the finished product warehouse and stored at a temperature of 2 to 8 ° C.

 The feasibility of the described invention can be demonstrated by the following examples:

 Example 1. Obtaining monovaccine serotype H3N2.

For infection with TBE, a 10-pass level of a reassortant virus A / Switzerland / 9715293/2013 / H3N2 containing an infectious titer of 10 'ID in 0.2 ml was selected as the working solution. A reassortant strain for the production of inactivated influenza vaccines was obtained from WHO. CE after 11 days of incubation in the amount of 56366 pcs. After disinfection and preliminary incubation were infected with the above dose of the working solution of seed virus. Incubation after infection was carried out at a temperature of 35 ° C and relative humidity of 60% for 43-46 hours. Cooling after incubation was carried out in a cold chamber at a temperature of (4 ± 1) ° C for 12-18 hours. After cooling for 6 hours, an IMPORTANT volume of 370 liters was collected. 37 L of a 2.5 M NaCl solution was added to the reactor with the collected IMPORTANT, followed by stirring, after which 0.37 L of β-propiolactone was added. Inactivation took place at a temperature of (4 ± 2) ° C for 14 hours. In the next stage of purification and concentration, 40 L of IMPORTANT concentrate were obtained, which were transferred for purification in a density gradient sucrose by ultracentrifugation. 600 ml (6 fractions) with the optimal sucrose concentration and RGA titer were selected from the gradient. This volume of the combined viral concentrate (HVAC) contained 14.21 g of protein (as determined by the Bradford method) and 6.12 g of GA (determined by the ORID method). The entire amount of HVAC (600 ml) was used to prepare one batch of monovaccine. To carry out the destruction of this HVAC, 200 g of octyl glucoside detergent, manufactured by Ferae Berlin, Germany, were used. In the lysate, 6.12 g of HA were determined (by the ORID method). The volume of the lysate after microfiltration was 20 liters. Then the filtered lysate was concentrated to a volume of 5 l using the ultrafiltration method using cassettes with a cutoff limit of 30 kDa. Further, this volume was purified from undeveloped virions by ultracentrifugation for 120 minutes at a flow rate of 5 l / h at a temperature of +8 ° C. The supernatant was concentrated using those cassettes to a volume of 2 L; chromatography was performed at room temperature using a column with 16 L of support. The peak density in the “free” zone was collected by optical density, the protein was determined in it according to the Bradford method, after which the material was diluted with FBI buffer to a final protein concentration of up to 400 μg / ml, Triton X-100 detergent was added to a final concentration of 200 μg / ml, the mixture was thoroughly mixed for 20 minutes, and filtered through 0.22 μm sterile cartridges into a sterile plastic container. The resulting monovaccine is transferred to a pharmaceutical refrigerator and stored at a temperature of (5 ± 3) ° C for no more than 3 months. Samples are taken from a container with a sterile mono-vaccine for technological controls. It was determined that out of 6.12 g of hemagglutinin found in HVAC, 5.41 g of hemagglutinin is determined in the resulting monovaccine. Thus, the efficiency of the technology for the release of hemagglutinin is 88.8%.

 A comparative analysis of the polypeptide composition of the HVAC serotype H3N2 and the resulting monovaccine was performed. The results are presented in Appendix 1.

It follows from the presented results that hemogglutinin trimers, undigested hemagglutinin, hemagglutinin heavy chain, trace amounts of nucleocapsid protein and hemagglutinin light chain are present in the monovaccine preparation. In the original viral concentrate from which this monovaccine was produced, Ml polypeptide (membrane protein) and essential the amount of protein nucleocapsid. This indicates that the resulting monovaccine preparation is a split preparation and does not contain viral particles.

 Example 2. Obtaining monotype vaccine serotype B.

 To obtain HVAC, the same procedures were carried out as in Example 1. The differences were as follows: for infection, the 9th passage level of the production strain of the B / Phuket / 3073/2013 virus was used; 56774 pcs were used for infection. CE at a temperature of 33 ° C and a relative humidity of 60% for 60-63 hours. After incubation and cooling, the same parameters were collected 440 l of IMPORTANT, which were concentrated 20 l in 2 bottles. After ultracentrifugation in a sucrose density gradient, 600 ml of the preparation containing 11.36 g of total protein (determined by the Bradford method) and 3.35 g of HA (determined by the ORID method) were selected. After degradation, ultrafiltration, ultracentrifugation and chromatography, and sterilizing filtration were carried out, as described in Example 1, 14.6 L of a single vaccine was obtained. This volume contained 5.76 g of total protein and 1.96 GA (by the ARID method). Thus, the efficiency of the technology for GA output was 58.3%.

 A comparative analysis of polypeptides in monovaccines obtained in 2014 and 2015 was carried out. and HVAC. The results are presented in the app>.

 It was shown that the polypeptide composition of a monovaccine is significantly different from the polypeptide composition of the original viral concentrate from which the presented monovaccine was produced. It seems obvious that the resulting mono-vaccine is a split-type vaccine against influenza serotype B.

 Example 3. Obtaining monovaccine serotype H1N1.

 All technological stages corresponded to those in examples N ° l and 2.

However, there were differences: 52571 pieces were taken for infection. CE, for their infection, the inoculum virus A / California 07/2009 / HlNl with a passage level of 12 was used. After infection, CE were incubated at 35 ° C and 60% relative humidity for 43-46 hours. 436 L of VAC was collected from this amount of CE After purification and concentration, 500 ml of HAC were obtained. This HVAC volume contained 6.75 g of total protein (determined by the Lowry method) and 2.587 g of HA (determined by the ORID method). From this series of HVAC, a series of monovaccines was obtained in a volume of 11.6 L, which contained 4 g of total protein (determined by the Lowry method) and 2.55 g of HA (determined by the ORID method). Thus, the GA output for this technology amounted to 98.6%. The polypeptide composition of the obtained monovaccine is presented in Appendix N ° 3.

 From the presented data it follows that in the initial viral concentrate, the main polypeptides are the NAO dimer, undigested hemagglutinin (NAO), the aggregate of neurominidase and protein nucleocapsid (NA + NP); nucleocapsid protein (NP), hemagglutinin heavy chain (HA1) and membrane protein (Ml). The same polypeptides are present in the preparation after detergent destruction, and in the monovaccine preparation, only the NAO dimer, the NAO itself, the aggregate (NA + NP), the hemagglutinin light chain (HA1) and trace amounts of the nucleocapsid protein remain. Based on these results, we believe that this mono-vaccine preparation is a split-type vaccine against influenza serotype H1N1.

 Example 4. Preparation of a vaccine dosage form.

For the preparation of a series of tri-vaccines (dosage form) from the monovalent obtained serotypes H1N1; H3N2 and B, we selected sterile solutions of monovaccines in such a way that each sample of the monovaccine contained an amount of hemagglutinin (determined by the ARID method) equal to 1.87 g. Then, under aseptic conditions, three samples of monovaccines were added to the reactor; FBI solution; 1% merthiolate solution (preservative), 1% Triton X-100 detergent solution, so that the final solution had a final concentration of all serotypes equal to (15 ± 2.2) μg / dose, the final concentration of merthiolate should be within 85- 115 μg / ml, and Triton X-100 - to a final concentration of 200-300 μg / ml (taking into account the concentration of Triton X-100 in monovaccines). The volume of liquid in the reaction after adding all the components reached 68 l. Next, the contents of the reactor are mixed for 15-20 minutes at a speed of 400 rpm and filtered through a disposable sterile filter capsule with a pore diameter of 0.22 μm into a sterile plastic container. We get the volume of sterile vaccine 67 l. Within 10 days, satisfactory results were obtained on the specific activity, protein content, bacterial endotoxins and ovalbumin, after which the container with the tri-vaccine was transferred for filling into ampoules. The drug was poured into ampoules with a capacity of 1 ml of 0.6 ml of trivacine in each ampoule. After bottling, 110,000 ampoules were obtained, which were checked for mechanical impurities. As a result, 1,100 ampoules were rejected for this indicator. The remaining ampoules were sent to a temporary storage warehouse with a temperature (6 ± 2) ° С. A part of the ampoules was selected for control according to the regulatory documentation.

 Antigenic activity, specific safety, toxicity, and sterility were in accordance with WHO requirements.

 Example 5. The study of the effect of additives stabilizer detergent Triton X-100 on

% yield of hemagglutinin (GA) in monovaccine, from hemagglutinin contained in HVAC.

 Preserving the stability of the specific activity of hemagglutinin (determined by the ARID method) both in the process of isolating a single vaccine and in the process of its subsequent storage is an extremely important technological task. As a result of lengthy experiments, from a number of stabilizing additives, for the purpose of stabilizing the vaccine presented, an additive of Triton X-100 detergent was added to a final concentration of 200 μg / ml. Using this additive, we obtained several series of monovaccines of different serotypes, which were investigated in order to determine the stability of preservation of specific activity (HA content). The results are presented in table N ° l.

 From the data presented it follows that with the use of a stabilizing additive it is possible to significantly increase the effectiveness of the technology at the final stage, namely, with the addition of Triton X-100 before sterilizing filtration of a monovaccine. The data obtained suggest that the stabilizing additive of this detergent is effective not only at the stage of obtaining a monovaccine, but also at the stage of storage of the trivalent dosage form, poured into ampoules.

 Example 6. Comparison of biochemical parameters of the proposed vaccine and vaccine "Vaksigripp"

 The obtained stabilized dosage form of the proposed vaccine was analyzed in international trials for some biochemical parameters in comparison with the split vaccine “Vaxigripp”, which is produced under license at the Butantan Institute, Brazil. As the main indicators of comparison were selected criteria for protein content; titer of hemagglutination, specific activity, ovalbumin content and bacterial endotoxin content. The data are presented in table Ns 2.

From the presented data it can be seen that according to the indicators “Ovalbumin content” and “bacterial endotoxin content” in the presented vaccine, they significantly differ from the “Vaxigripp” vaccine produced under license Sanofi, at the Butantan Institute, Brazil. Obviously, the significantly reduced amounts of reactogenic impurities in the presented vaccine are due to the fact that industrial gel chromatography was used in the process of purifying monovaccines. It should be noted that the content of ovalbumin and bacterial endotoxins are the main indicators for assessing the potential reactogenicity of the vaccine. According to WHO requirements, these indicators are regulated. In particular, in modern vaccines intended for immunization of children, the content of ovalbumin should not exceed 0.1 mg / ml.

 Example 7. The study of the antigenic activity of the obtained tri-vaccine.

 In the experiment, female BALB / c mice were used,

6-8 weeks, weighing 18-20 g. 3 groups of animals were formed (30 animals per group): 2 experimental groups and 1 control.

 On day 0 of the experiment, total blood sampling was performed in non-immune animals (10 animals from each group, including the control) in order to assess the presence of antibodies to vaccine strains in the blood serum of experimental animals. After that, the primary administration, VI (priming) of trivalent influenza vaccines was carried out to experimental animals (the vaccine we received and the vaccine “Vaksigripp”) intramuscularly, at a dose of 0.5 ml of the drug per animal. The control group of animals was injected with 0.9% sodium chloride in a volume of 0.5 ml per animal.

 On the 21st day of the experiment, total blood sampling was performed in animals (10 animals from each group, including control) in order to assess the level of specific antibodies to vaccine strains after the initial injection of vaccines. Since it is believed that mice are not susceptible to influenza infection and are “naive” for influenza antigens, a single injection of influenza vaccine is not enough to produce a full-fledged immune response. As a result, the vaccines were re-introduced, V2 (immunization proper) to the experimental animals according to the same scheme as for the initial vaccine administration. The control group of animals was injected with 0.9% sodium chloride in a volume of 0.5 ml per animal.

 On the 42nd day of the experiment (21st day after V2), total blood sampling was performed in all animals.

Evaluation of antigen-specific humoral immunity was carried out in the inhibition reaction of hemagglutination (RTGA) and the microneutralization reaction (RMN) in MDC cell culture with influenza virus strains. The reactions were carried out according to the methods recommended by WHO. Serums having an antibody titer of 1: 40 and higher were considered positive.

 The results are presented in tables 3 to 5.

 From the data we can conclude that the antigenic activity of the presented vaccine exceeds that of the vaccine "Vaksigripp" (manufactured by Sanofi, France), in particular by titers in the Russian Medical Library after primary immunization with serotype B and serotype H1N1. After secondary immunization, the indicator of antigenic activity is leveled between the two studied drugs, since the doses intended for human vaccination were used to immunize mice. It should be emphasized that the difference in the percentage of seropositive animals after primary vaccination indicates that the vaccine presented better induces an immune response in “naive” animals. It is likely that this is due to the fact that the presented vaccine contains virosomal components that were detected during morphological analysis by electron microscopy.

 Example 8. The study of the comparative immunogenicity of the presented vaccines and vaccines "Vaksigripp" in a clinical trial on volunteers.

The study was conducted as open in two parallel groups of participants. Vaccines for influenza prevention were used: vaccine presented and Vaxigripp ^® , France. According to the study protocol, vaccines were administered once. Vaccination was carried out to 120 volunteers aged 18 to 60 years. The study included 65 women (54%) and 55 men (46%), whose average age was 32.9 ± 1.5 and 30.5 ± 1.6 years, respectively. Vaccination with the presented vaccine was carried out to 61 volunteers, Vaxigripp vaccine - to 59 people, of which 58 and 59 participants respectively were examined on the 21-28th day of observation.

 Evaluation of the immunogenicity indicators of the studied vaccines was carried out according to the criteria for the effectiveness of the development of the WHO immune response:

 1. the rate of increase in antibody titer after vaccination:> 2.5 in people aged 18 to 60 years (> 2 in people over the age of 60 years);

2. seroconversion level (increase in titer of antibodies to GA not less than 4 times):> 40% for people aged 18 to 60 years (> 30% for people over 60 years old); 3. level of seroprotection (the number of people with a protective titer> = 40):> 70% for people aged 18 to 60 years (> 60% for people over the age of 60).

 The research results were statistically processed using the Student t-test and, in the absence of signs of a normal distribution, using nonparametric criteria (Wilcoxon and Mann-Whitney). The differences were considered significant at p <0.05

 The immune response in all vaccines who participated in the study are presented in table 6.

It was shown that in both groups the seroconversion coefficient, as well as the indicators of seroconversion and seroprotection to influenza viruses H1N1, H3N2 and B, met the WHO criteria for the development of the immune response. There were no statistically significant differences between the groups.

Bibliography

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Bryzgalova Svetlana Ivanovna,

Melnikov Sergey Yakovlevich,

Gusarova Natalia Anatolyevna,

Yaroslavtsev Ivan Vasilievich.

 2. RF patent JV "2,493,872" Method for the purification of influenza virus. "

Date of beginning of validity: 08.08. 2012 year

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Kataeva Valentina Vasilievna.

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Authors: Zagidulin Nail Vilenovich,

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27. doi: 10.1371 / journal.pone.0030898 Table 1 - Study of the effect of the stabilizer Triton X-100 on% yield of hemagglutinin antigen in the process of obtaining monovaccines from HVAC of various serotypes.

Table 2 - Comparison of biochemical parameters of the analysis of the split

 vaccine "Vaksigripp" and the proposed vaccine.

 Named Content Title Specific Content Content of a sample of RGA protein ovalbumin activity, bacterial by the ARID method, μg / dose μg / dose

 Bradford (0.5 ml) (0.5 ml) endotoxino

, mcg / dose H1N1 H3N2 B in, EE / dose

(0.5 ml) (0.5 ml)

Presents 50 640 14 169 15 0.005 0.24 to vaccine 0.48

Split 185 640 21 19 24 0.127 More than 48 vaccine

Waxigrip Table 3 - Geometric mean antibody titers (GMT) in the blood serum of mice with H1N1 serotype antigen and the proportion of seropositive animals in

 each r ppe

Table 4 - Geometric mean antibody titers (GMT) in the blood sera of mice with the H3N2 serotype antigen and the proportion of seropositive animals in

Table 5 - Geometric mean antibody titers (GMT) in the blood serum of mice with serotype B antigen and the proportion of seropositive animals in each

 r ppe

Table 6

 * p = 0.0064

5

Claims

29 SUMMARY OF THE INVENTION

1. The method of obtaining funds intended for the treatment of influenza, including infection of chicken embryos with influenza virus, incubation of infected embryos, cooling of incubated embryos, collection of virus-containing allantoic fluid (IMPORTANT), processing of IMPORTANT for the purpose of disaggregation and subsequent inactivation, microfiltration of IMPORTANT on 2 stages of cartridges, ultrafiltration of the IMPORTANT and purification of the viral concentrate in a sucrose density gradient, cleavage of the purified inactivated viral concentrate is carried out with beta-octylglyu detergent ozide, the removal of the undestructed virions and complexes of ribonucleoproteins with a membrane protein is carried out by ultracentrifugation, the final purification of the isolated antigens is carried out on a chromatographic carrier, after which a stabilizer is added to the resulting antigens, characterized in that the inactivation of viral activity is carried out at the stage of VIA processing using a betta-propiolactone disinfectant , the disaggregation of viruses during the processing of VALUE is carried out with sodium chloride in a final concentration of 0.22 mol, and the end The antigens are completely purified on a WorkBeads chromatographic medium, 40/100 / sec from Bio-works (Sweden) and stabilized with the Triton X-100 detergent at a concentration of 100-300 μg / ml.

2. The tool intended for the treatment of influenza, which is obtained by the method according to claim 1