RU2710239C1 - Method of producing antigen or antigens for production of influenza vaccine and a vaccine based on it - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to immunology, medicine, pharmacology, and can be used for production of inactivated influenza vaccines. Proposed method for producing the influenza virus antigen or antigens includes production of chicken embryos (CE), their disinfection, pre-incubation, infection with a working solution of the seed virus, incubation of the infected CE, cooling after incubation, collecting a virus-containing allantoic liquid, disaggregating the recovered viruses by adding a NaCl solution to a final concentration of 0.23–0.30 M, inactivation by adding β-propiolactone to final concentration of 0.1 % and purification of virus-containing allantoic liquid by microfiltration using cascade of three filters with pore diameter of 10 mcm, 1 mcm, 0.6 mcm and ultrafiltration with cutoff limit of 300 kDa, then concentrated clarified virus-containing allantoic fluid is not more than 20 times, concentrate is purified by way of double ultracentrifugation in sucrose density gradient, after which inactivated viral concentrate is split using detergent n-octyl-β-D-glucopyranoside (octylglucoside) or tetradecyltrimethylammonium bromide (TDTAB), removal of undamaged virions and complexes of ribonucleoproteids with membrane protein by centrifugation, micro- and ultrafiltration, chromatographic purification and sterilizing filtration of the obtained antigen or antigens, and the antigen or antigen is stabilized using a Triton X-100 detergent in concentration of 100–200 mcg/ml. What is also presented is an influenza vaccine containing an antigen or antigens prepared by the above method.

EFFECT: development of industrial method for obtaining antigen and / or antigens of high-purity virus for production of inactivated vaccine, which can be used for mass vaccination of population.

3 cl, 9 dwg, 12 tbl, 10 ex

Description

The invention relates to immunology, medicine, pharmacology and can be used for the production of inactivated influenza vaccines, which can be used to vaccinate the 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.

Various methods are currently known for the production of inactivated influenza virus vaccines.

A known method for producing a virus-containing substance by culturing vaccine strains of the influenza virus in a culture of MDCK cells on a nutrient medium, purifying the viral substance from ballast impurities, introducing stabilizing additives into the purified substance and drying the finished vaccine form (patent RU 2330885, published on 08/10/2008). The disadvantage of this method is the inability to obtain antigen to create a vaccine on an industrial scale.

A known method of producing antigens for influenza vaccines by obtaining a crude virus concentrate, the virus concentrate is purified by gel filtration on a DIOL-500 carrier, then the viral concentrate is cleaved with β-octylglucoside detergent, and vaccine antigens are isolated by gel filtration on a Sephadex G- carrier 50 (patent RU 2283139, publ. 09/10/2006). The disadvantage of this method is the insufficient degree of purification of the antigen from ovalbumin.

The closest analogue of the claimed invention is a method for producing influenza antigens, including infection of chicken embryos with influenza virus, incubation of embryos, cooling of embryos, collection of virus-containing allantoic fluid (IMPORTANT), processing of IMPORTANT for the purpose of disaggregation and subsequent inactivation, microfiltration of IMPORTANT, ultrafiltration of IMPORTANT and purification of virus concentrate (patent RU 2584594, publ. 05.20.2016). The disadvantage of the prototype method is the insufficiently complete purification of the obtained antigen from egg albumin.

The problem to which the present invention is directed is to develop a new technology for the production of antigen and / or antigens of influenza virus for the production of Russian inactivated vaccines, which will allow us to produce in our country a vaccine with a world level of quality, the required degree of purification.

The technical result of the invention consists in the development of an industrial method for producing high purity antigen and / or antigens of influenza virus for the production of Russian inactivated vaccine, which can be used for mass vaccination of the population. This is achieved by the fact that in the proposed method, the disaggregation of viruses during the processing of IMPORTANT we carry out sodium chloride in a final concentration of 0.23-0.30 mol, inactivation of viral activity using β-propiolactone, purification of IMPORTANT by microfiltration using a cascade of three filters with pore diameters of 10 μm, 1 μm, 0.6 μm and ultrafiltration with a cutoff limit of 300 kDa, subsequent purification by double ultracentrifugation in a sucrose density gradient, the subsequent purification of antigen or antigens is carried out at x a carrier medium, and stabilization of the obtained antigen or antigens or the finished dosage form is carried out using the detergent Triton X-100 at a concentration of 100-200 μg / ml.

These conditions are defined as optimal as a result of many years of experiments conducted by the authors of this invention.

Thus, to solve this problem, we propose a method for producing an antigen or antigens of influenza virus, including obtaining chicken embryos (CE), disinfection, pre-incubation, infection with a working solution of seed virus, incubation of infected CE, cooling after incubation, collection of virus-containing allantoic fluid, disaggregation of the isolated viruses by adding a NaCl solution to a final concentration of 0.23-0.30 M, inactivating them by adding β-propiolactone to a final concentration of 0.1% and purification by IMPORTANT methods filtration using a cascade of three filters with a pore diameter of 10 μm, 1 μm, 0.6 μm and ultrafiltration with a cutoff limit of 300 kDa, then the clarified virus-containing allantoic fluid is concentrated no more than 20 times, the concentrate is purified by double ultracentrifugation in a density gradient sucrose, after which the inactivated viral concentrate is cleaved using the detergent n-octyl-β-D-glucopyranoside (octyl glucoside) or tetradecyltrimethylammonium bromide (TDTAB), removing edible destruction of virions and complexes of ribonucleoproteins with a membrane protein by centrifugation, micro- and ultrafiltration, chromatographic purification and sterilizing filtration of the resulting antigen or antigens, and antigen or antigens are stabilized using Triton X-100 detergent at a concentration of 100-200 μg / ml.

In a particular case, an influenza vaccine is proposed containing an antigen or antigens obtained by the above method, and it may contain 1, 3 or 4 antigens.

SUMMARY OF THE INVENTION

The inventive method of obtaining consists of several technological steps:

The first stage consists in obtaining IMPORTANT and consists of several stages: obtaining chicken embryos (CE), their disinfection, preliminary incubation, infection with a working solution of seed virus, incubation of infected CEs, cooling after incubation, collection of IMPORTANT and purification of IMPORTANT.

Hatching eggs are obtained only from clinically healthy poultry from epizootiologically safe poultry farms that are infectious and invasive diseases safe. The quality of the delivered eggs is confirmed by a veterinary certificate. After the input control, the fertilized egg is laid in an incubator for incubation. Disinfection of TBE is carried out by spraying a disinfectant solution of 0.5% P3-oxonium active 150 or another approved agent. The infection process of 9-11 daily TBE is carried out under aseptic conditions. The container with the working solution of the inoculum virus is connected to the inoculator, manufactured by Rame-Hart, USA or similar equipment. The processing of CE shells and needles during the infection process is carried out with ethyl alcohol. Further, the plastic trays move along the conveyor, undergoing disinfection with ethyl alcohol, and then infection occurs. In each TBE, (0.20 ± 0.02) ml of working dilution of the inoculum virus is administered. 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. Controlled CEs are transferred to the incubation stage of infected CEs. 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 or selected as a result of studies. For type A influenza viruses, the incubation time, as a rule, is from 35 to 50 hours, and for type B viruses, from 50 to 65 hours, and relative humidity from 55 to 65%. CE is cooled in the refrigerating chamber for 12 to 20 hours at a temperature of (5 ± 3) ° С. 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 is pumped to the reactor using pumps. 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. At the end of the collection of the IMPORTANT, a sterile solution of 2.5 M NaCl is added to the final concentration of 0.23-0.3 M using a pump to prevent aggregation and β-propiolactone to a final concentration of 0.1% to inactivate the virus. The contents are mixed for 1-2 hours. After stirring, the mixer is turned off and the contents of the reactor are defended for at least 6 hours. The selected time should ensure the inactivation of influenza virus and extraneous pathogenic agents for humans.

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

Purification and concentration of the IMPORTANT by micro- and ultrafiltration is carried out on a combined micro- and ultrafiltration unit in compliance with aseptic rules in the laminar flow zone. After processing and inactivation, the IMPORTANT from the reactor is pumped to a microfiltration unit using a pump and passed through a cascade of depth filters with a pore diameter of 10 μm, 1 μm, 0.6 μm into a sterile container. The clarified IMPORTANT obtained by microfiltration using a pump is fed to an ultrafiltration unit with Pellicon 2 Biomax-300 cartridge modules or similar with a cutoff limit of 300 kDa, for further purification and concentration of IMPORTANT. Concentration of clarified IMPORTANT is carried out no more than 20 times.

Purification and concentration of the influenza virus in a sucrose density gradient is carried out using a flow ultracentrifuge as follows: a sterile Tris buffer (pH = 7.4 ± 0.2) and a 60% sucrose solution at a temperature of (5 ± 3) ° C are loaded into the rotor. After acceleration to g = (85000 ± 5000) m / s2, the IMPORTANT concentrate is supplied to the rotor using a pump at a speed of (10 ± 1) l / h. At the end of the passage of the entire volume of concentrated IMPORTANT, a Tris buffer is fed into the centrifuge rotor and isoplastic centrifugation is carried out for (2.0 ± 0.5) hours. The solutions are used cooled to a temperature of (5 ± 3) ° С. After the end of isoplastic centrifugation, the ultracentrifuge is stopped and the obtained fractions of the inactivated virus concentrate (VK) are unloaded in a sterile box. 20 fractions of the sucrose density gradient contained in the rotor are collected: the first two fractions, 200 ml each, the next 100 ml each. A sample was taken from each vial to control hemagglutinating activity (HA titer) and% sucrose. After sampling, each vial 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, fractions of inactivated VC are used, having a GA titer of at least 1: 8000 and containing (28-51)% sucrose.

To improve the quality of cleaning, repeated centrifugation is carried out. For further processing, fractions of inactivated viral influenza (concentrated purified influenza virus, inactivated VK, secondary VK fractions) with a hemagglutinating activity titer of at least 1: 16000 and containing (19-50)% sucrose are used. 80 ml of glycerol are added to the selected VK fractions, the vials are mixed, closed with silicone stoppers and transferred to storage in the freezer at a temperature of minus 9 to minus 15 ° C for a period of not more than 6 months. It is allowed to store fractions at a temperature of (5 ± 3) ° С without adding glycerol for up to 3 days.

Repeated centrifugation significantly increases the degree of purity of inactivated VC with minimal loss of hemagglutinin (GA). This is observed by a decrease in the ratio of total protein to GA and a decrease in the ratio of ovalbumin to GA (Table 1) and by chromatograms (Fig. 1 - Electrophoregram of samples of primary fractions obtained after concentration in sucrose density gradient by the example of strain A / Michigan / 45/2015 (H1N1), where 1 - molecular weight markers, 2 - combined fractions No. 3 + 4, 3 - combined fractions No. 5 + 6, 4 - combined fractions No. 7 + 8, 5 - volume dinene fractions No. 9 + 10, 6 - combined fractions No. 11 + 12, 7 - combined fractions No. 13 + 14, 8 - combined fractions No. 15 + 16, 9 - combined fractions No. 17 + 18, 10 - combined fractions No. 19 + 20. The selected area defines the boundaries of the selected fractions and Fig. 2 - Electrophoregram of samples of fractions obtained after concentration in the sucrose density gradient by repeated centrifugation using strain A / Michigan / 45/2015 (H1N1), where 1 is the combined fraction No. 3 + 4 , 2 - combined fractions No. 5 + 6, 3 - combined fractions No. 7 + 8, 4 - combined fractions No. 9 + 10, 5 - combined fractions nos. 11 + 12, 6 - combined fractions No. 13 + 14, 7 - combined fractions No. 15 + 16, 8 - combined fractions No. 17 + 18, 9 - combined fractions No. 19 + 20, 1 - molecular weight markers. The selected area defines the boundaries of the selected fractions).

Stage 3 includes the cleavage of inactivated viral particles from the obtained concentrates, the deposition of undestructed virions and complexes of ribonucleoproteins with a membrane protein by ultracentrifugation, micro and ultrafiltration, chromatographic purification and sterilizing filtering of antigens (monovaccines).

The cleavage of viral particles is carried out using an octyl glucoside detergent or TDTAB.

Detergents, by virtue of their mechanism of action, may well be a factor in the partial denaturation of conformational hemagglutinin epitopes. As a result of lengthy analyzes and technological experiments, it was concluded that for each influenza virus serotype there is its own optimal detergent, which allows one to isolate surface antigens from the virions of this serotype with the maximum preservation of their initial immunogenicity and protective activity.

The choice of detergent is carried out on the basis of the results of determining the specific activity in the studied samples. In the case when at the maximum protein / detergent ratios when using one of the detergents, a decrease in specific activity is observed, compared with lower ratios (the phenomenon of "burning" of antigen), then detergent is chosen as the only one for this serotype that does not produce such a phenomenon. In FIG. 3 - Morphological analysis of precipitation rings in the reaction of single radial immunodiffusion (ARID), samples of serotype B / Phuket / 3073/2013, obtained using various detergents, the results of determining the specific activity of samples of serotype B / Phuket / 3073/2013 are presented, from which it follows that the sample obtained using the octyl glucoside detergent forms well-colored rings of a clearly defined shape (rows No. 1 and No. 4), which in morphology are no different from the precipitation rings of the international standard antigen, produced NIBSC, UK (rows No. 3 and No. 6). A sample obtained using the TDTAB detergent forms faintly colored precipitation rings with fuzzy outlines (rows No. 2 and No. 5), which differ significantly in morphology from the precipitation rings of the international standard antigen, manufactured by NIBSC, United Kingdom (rows No. 3 and No. 6).

After choosing a detergent, a protein / detergent ratio is selected in which the yield of specific activity is within the maximum value, taking into account the results of the assessment of the polypeptide composition of the studied samples.

The determination of the optimal detergent and the protein / detergent ratio is carried out separately for each serotype when changing the production strain, since they may have sensitivity to a particular detergent or when changing the lot of detergent, because due to strain differences and fluctuations in the properties of the detergent from batch to batch, the optimal the protein / detergent ratio may vary.

The deposition of core structures (undestructed virions and complexes of ribonucleoproteins with a membrane protein) and aggregated virions from the resulting viral lysate is carried out by centrifugation (Fig. 4 - Electrophoregram of the precipitate from the centrifuge rotor and the virus concentrate using the example of strain A / Michigan / 45/2015 (H1N1) where 1 is a viral concentrate, 2 is a precipitate from a centrifuge rotor, 3 are molecular weight markers).

Using a pump, a Tris buffer is fed to the centrifuge rotor, then split VK at a flow rate of 4-6 l / h at a pump speed of 16,000 to 20,000 rpm (g = (13040-34774) m / s2) at a temperature of 2 up to 8 ° C. The split VK is centrifuged in a closed cycle. In FIG. Figure 4 shows the effectiveness of using centrifugation to remove non-protective flu virus proteins.

Then, the obtained influenza supernatant is passed through a peristaltic pump into a sterile glass container through depth filters whose pore diameters are 0.6 and 0.2 μm, respectively, at a pressure of not more than 0.5 bar. The filtered supernatant, using a peristaltic pump, is passed through an ultrafiltration unit with membrane cassettes with a cutoff limit of 30 kDa to concentrate the material.

For purification of the concentrated supernatant, a Work Beads 40/100 / sec chromatographic carrier for gel filtration was used by Bio Works Company Ltd. (Sweden) or similar. Gel filtration is carried out on a borosilicate glass production column. A concentrated supernatant in a volume of 2.0-2.5 l is applied to a column with a volume of 16-19 l of the carrier using a peristaltic pump. Chromatography is carried out at a flow rate of not more than 300 ml / min and at a pressure of not more than 1.0. The collection of material is carried out according to the indicators of the UV detector 200, KNAUER, Germany or the like. Data is recorded on a personal computer with the MultiChrom system. After the value at the detector reaches 10 mV, they begin to collect material. Material collection is stopped after a drop in optical density from 10 to 20% of the peak value. In FIG. Figure 5 shows a typical chromatogram for removing residual impurities from a process using chromatographic purification, where 1 is Antigen, 2 is Ovalbumin, 3 is Sucrose, 4 is Octylglucoside.

After chromatographic purification, the material is transferred to sterilizing filtration. The purified supernatant is poured into the reactor prepared for operation, the FBI is added to a hemagglutinin concentration of at least 100 μg / ml, Triton X-100 can also be added to a final concentration of 100-200 μg / ml and stirred at a speed of 200-220 rpm 15-20 minutes at room temperature.

Next, the resulting antigen (monovaccine) of the influenza virus is filtered using a sterile sterilizing capsule with a pore size of 0.22 μm (or less), the membrane material is polyethersulfone in a sterile disposable polymer container. The obtained sterile monovaccine is transported to pharmaceutical refrigerators and stored at a temperature of (5 ± 3) ° C for no more than a year.

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

The process of obtaining a trivalent influenza vaccine involves the reduction of antigens (monovaccines) of 3 types of influenza virus A (H1N1), A (H3N2) and B, with a 1% solution of detergent Triton X-100, 1% solution of thiomersal (for preparations containing preservative) and the FBI in the reactor.

The process of obtaining a tetravalent influenza vaccine involves the mixing of antigens (monovaccines) of 4 types of influenza virus A (H1N1), A (H3N2), B / Yamagata lineage and B / Victoria lineage, with a 1% solution of detergent Triton X-100, 1 % thiomersal solution (for preparations containing a preservative) and FBI in a reactor with a capacity of 200 l.

Semi-finished antigens (monovaccines) of the influenza virus, having passed all the controls, from sterile disposable containers under aseptic conditions are loaded into the reactor. Then, the calculated amounts of PBS, 1% thiomersal solution to a final concentration of (85-115) μg / ml (for a preparation containing a preservative) and 1% Triton X-100 detergent solution to a final concentration of 100-200 μg / ml are added to the reactor. The contents of the reactor are stirred at a stirrer speed of 400 rpm for 15 minutes. Next, turn off the stirrer and defend the cake mix for 10 minutes. After settling, a sample is taken to control the semi-finished vaccine. Next, sterilized filtration of the mixed solution is carried out through a 0.22 μm Sartobran disposable filter element, the filtration area is 0.45 m2. A sample is taken from the filled sterile bag under aseptic conditions to control the intermediate of the influenza vaccine in accordance with the requirements of the State Pharmacopoeia of the Russian Federation.

Bottling of the drug in ampoules or vials is carried out under aseptic conditions. The sterility of the process and the recoverable volume (filling dose) of the drug are monitored.

Further, all ampoules or vials with the drug are monitored for tightness and mechanical inclusion.

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

Ampoules or vials with the finished dosage form of the drug are selected from the quarantine storage warehouse and monitored in accordance with the requirements of the State Pharmacopoeia of the Russian Federation for the following quality indicators of influenza vaccines: description, authenticity, pH, total protein, sterility, bacterial endotoxins, abnormal toxicity, specific safety, specific activity of hemagglutinin, thiomersal (for a preparation containing a preservative), ovalbumin, Triton X-100, immunogenicity and reactogenicity.

The implementation of the invention

Example 1. Obtaining antigen (monovaccine) influenza virus serotype H3N2

For infection with TBE, a 5-pass level of the reassortant virus A / Singapore / INFIMH-16-0019 / 2016-like / (IVR-186) / H3N2 was used as the working solution. A reassortant strain for the production of inactivated influenza vaccines was obtained from WHO. 60061 pieces of eleven day old TBEs were infected with a dose of the inoculum virus solution having an infectious titer of 103.66 EID50 in 0.2 ml. Incubation after infection was carried out at a temperature of 35 ° C and relative humidity of 60% for 45-48 hours. Cooling after incubation was carried out in a cold chamber at a temperature of (5 ± 3) ° C for 12-15 hours. After cooling, the VAZH was collected in a reactor in a volume of 550 l. Next, 50-70 L of a 2.5 M NaCl solution was added to the reactor with the collected IMPORTANT, followed by stirring, after which 0.55 L of β-propiolactone was added. Inactivation took place at a temperature of (6 ± 2) ° C for 10 hours. Then, after cleaning and concentrating the IMPORTANT by micro- and ultrafiltration, 40 L of IMPORTANT concentrate were obtained, which were transferred for purification in the sucrose density gradient by ultracentrifugation. 700 ml (7 fractions) with the optimum sucrose concentration and hemagglutinin titer (HA) were selected from the gradient. This volume of viral concentrate (VK) contained 13.2 g of protein (determination was carried out by the Bradford method) and 6.0 g of HA (determination was carried out by the ARID method). The entire amount of the combined VK (700 ml) was used to prepare one batch of monovaccine. To carry out the destruction, 198 g of octyl glucoside detergent, manufactured by Carbosynh Limited, United Kingdom, were used. The volume of the lysate after cleavage was 12 liters. Next, the core structures were cleaned by ultracentrifugation at a flow rate of 4-6 l / h at a pump rotation speed of 16,000 to 20,000 rpm (g = (13040-34774) m / s2) at a temperature of 2 to 8 ° С . Next, the rotor of 5 l of Tris buffer was washed with a fluid flow rate of 14 to 16 l / h. The total volume of material was 17 liters. After which the material was filtered on a microfiltration unit. The supernatant was concentrated using cassettes with a cut-off limit of 30 kDa to 2.0 L, chromatography was performed at room temperature. According to the optical density, a peak of 2.9 L was collected; protein was determined in it by the Bradford method. The total amount of protein at the peak was 7.0 g. Next, the material was diluted to a final protein concentration of up to 300 μg / ml with FBI buffer. The resulting mixture was thoroughly mixed for 15 minutes. The semifinished product was filtered through a 0.22 μm disposable filter element. The resulting antigen (monovaccine) with a volume of 23.0 l is transferred to a pharmaceutical refrigerator and stored at a temperature of (5 ± 3) ° C for no more than 12 months. In the obtained antigen (monovaccine) of the influenza virus, 4.05 g of hemagglutinin was determined, while in the combined VK there was 6.00 g of hemagglutinin. Thus, the efficiency of the technology for the release of hemagglutinin is 67.5%.

A comparative analysis of the polypeptide composition, the combined VK and the antigen (monovaccine) of the influenza virus A / Singapore / INFIMH-16-0019 / 2016-like / (IVR-186) / H3N2 was performed by electrophoresis in SDS page under non-reducing conditions. The results are presented in FIG. 6 - Electrophoregram of the antigen (monovaccine) of the influenza virus A / Singapore / INFIMH-16-0019 / 2016-like / H3N2, where 1 is the molecular weight marker, 2 is the antigen (monovaccine) of the influenza A / Singapore / INFIMH-16-0019 / 2016-like / H3N2,3 - combined viral concentrate (VK).

From the presented results it follows that the obtained antigen (monovaccine) contains hemagglutinin trimers, undigested hemagglutinin (HA0), a heavy chain of hemagglutinin (HA1) and a certain amount of nucleocapsid (NP) protein and neuraminidase (NA). In the original pooled VC from which this antigen (monovaccine) of the influenza virus was produced, the M1 polypeptide (membrane protein) and significant amounts of NP protein are also present. This indicates that the resulting preparation is highly purified and does not contain unsplit viral particles.

Example 2. Obtaining antigen (monovaccine) of influenza virus type B / Yamagata lineage

To obtain the combined VK, the same procedures were carried out as in Example 1. The differences were as follows: for infection, a 3-pass level of the reassortant virus B / California / 2/2015 / BX-69B-like was used (B / Phuket / 3073/2013 ) 70,704 eleven-day TBEs were infected with a dose of the inoculum virus working solution having an infectious titer of 103.66 EID50 in 0.2 ml. Incubation after infection was carried out at a temperature of 32.5 ° C and a relative humidity of 60% for 60-64 hours. 720 L of IMPORTANT VALVES were collected, which concentrated to 40 L. After the second ultracentrifugation, 700 ml (7 fractions) with the optimal sucrose concentration and HA titer were selected from the gradient. This volume of VK contained 21.0 g of protein and 7.9 g of HA. After the destruction, ultracentrifugation, micro- and ultrafiltration, chromatography and sterilizing filtration, as described in example 1, was obtained 25.0 l of a single vaccine. This volume contained 12.63 g of total protein and 5.43 g of HA. Thus, the efficiency of the technology for GA output was 68.7%.

A comparative analysis of the polypeptide composition, the combined VK and the antigen (monovaccine) of the influenza virus B / California / 2/2015-like obtained by electrophoresis in PAAG under non-reducing conditions was carried out. The results are presented in FIG. 7 Electrophoregram of the antigen (mono-vaccine) of the influenza virus B / California / 2/2015-like, where 1 is the combined BK, 2 is the antigen (mono-vaccine) of the influenza virus B / California / 2/2015-like, 3 are molecular weight markers.

From the presented results it follows that the obtained antigen (monovaccine) of the influenza virus contains trimers and dimers of hemagglutinin, undigested hemagglutinin (HA0), the heavy and light chain of hemagglutinin (HA1 and HA2), and a certain amount of nucleocapsid protein (NP) and neuraminidase (NA). In the original pooled BK from which this antigen (monovaccine) of the influenza virus was produced, there are also the Ml polypeptide (membrane protein) and significant amounts of NP protein. This indicates that the resulting preparation is highly purified and does not contain unsplit viral particles.

Example 3. Obtaining antigen (monovaccine) of influenza virus type B / Victoria lineage

To obtain a combined VK, the same procedures were performed as in example No. 1 and No. 2. The differences were as follows: for infection, the 5th passive level of the production strain of the B / Maryland / 15/2016-like / BX-69A virus (B / Colorado / 06/2017-like virus) was used. 101101 eleven-day CEs were infected with a dose of the inoculum virus working solution having an infectious titer of 104.0 ID in 0.2 ml. Incubation after infection was carried out at a temperature of 32.5 ° C and a relative humidity of 60% for 60-64 hours. It was collected in 2 reactors 965 liters of IMPORTANT, which concentrated to 70 liters. After the second ultracentrifugation in a sucrose density gradient, 800 ml (8 fractions) were selected with the optimal sucrose concentration and HA titer. This volume of VK contained 23.6 g of protein and 7.4 g of HA. After carrying out the destruction, ultracentrifugation, micro- and ultrafiltration, chromatography and sterilizing filtration, as described in example 1, as indicated in example 1, was obtained 34.0 l of a single vaccine. This volume contained 15.27 g of total protein and 4.49 g. Thus, the efficiency of the technology for GA yield was 60.7%.

A comparative analysis of the polypeptide composition, the combined VK and the antigen (monovaccine) of the influenza virus B / Maryland / 15/2016-like obtained by electrophoresis in SDS page under non-reducing conditions was performed. The results are presented in FIG. 8 - Electrophoregram of the antigen (mono-vaccine) of the influenza virus B / Magu land / 15/2016-like, where 1 is the combined BK, 2 - the antigen (mono-vaccine) of the influenza virus B / Maryland / 15/2016-like, 3 - molecular weight markers.

From the presented results it follows that the obtained antigen (monovaccine) of the influenza virus contains trimers and dimers of hemagglutinin, undigested hemagglutinin (HA0), the heavy and light chain of hemagglutinin (HA1 and HA2), and a certain amount of nucleocapsid protein (NP) and neuraminidase (NA). In the original pooled VC from which this antigen (monovaccine) of the influenza virus was produced, the M1 polypeptide (membrane protein) and significant amounts of NP protein are also present. This indicates that the resulting preparation is highly purified and does not contain unsplit viral particles.

Example 4. Obtaining antigen (monovaccine) of influenza virus serotype H1N1

All technological stages corresponded to those in examples No. 1, No. 2 and No. 3. The differences were as follows: for infection, the 7th passage level of virus A / Michigan / 45/2015 / NYMC-275A / H1N1 was used. 106,749 eleven-day TBEs were infected with a dose of the inoculum virus working solution having an infectious titer of 103.66 ID in 0.2 ml. After infection, incubation was carried out at a temperature of 35 ° C and a relative humidity of 60% for 44-48 hours. It was collected in two reactors 1085 liters of IMPORTANT, which concentrated to 70 liters. After the second ultracentrifugation in a sucrose density gradient, 700 ml (7 fractions) were selected with the optimal sucrose concentration and HA titer. This volume of VK contained 16.8 g of protein and 6.8 g of HA. After degradation, ultracentrifugation, micro- and ultrafiltration, chromatography and sterilizing filtration, as described in Example 1, as described in Example 1, 20.0 L mono-vaccine was obtained. This volume contained 7.66 g of total protein and 4.32 g of HA. Thus, the efficiency of the technology for HA output was 63.9%.

A comparative analysis of the polypeptide composition, the combined VK and the antigen (monovaccine) of the influenza virus A / Michigan / 45/2015 / H1N1 obtained by electrophoresis in PAAG under non-reducing conditions was carried out. The results are presented in FIG. 9 - Electrophoregram of the antigen (monovaccine) of the influenza A / Michigan / 45/2015 / H1N1 virus, where 1 is the combined BK, 2 is the antigen (monovaccine) of the influenza A / Michigan / 45/2015 / H1N1 virus, 3 are molecular weight markers.

From the presented results it follows that the obtained antigen (monovaccine) of the influenza virus contains hemagglutinin trimers, undigested hemagglutinin (HA0), the heavy chain of hemagglutinin (HA1) and a certain amount of nucleocapsid protein (NP) and neuraminidase (NA). In the original pooled VC from which this antigen (monovaccine) of the influenza virus was produced, the M1 polypeptide (membrane protein) and significant amounts of NP protein are also present. This indicates that the resulting preparation is highly purified and does not contain unsplit viral particles.

Example 5. The preparation of the dosage form of the trivalent vaccine

To prepare a series of trivalent vaccines, sterile solutions of the obtained antigens (monovaccines) of serotypes A (H1N1), A (H3N2) and B were selected and under aseptic conditions they were added to the reactor in the amount of 7.4 l, 9.1 l, 7.4 l, respectively . Next, they were added FBI M in the amount of 25.2 l and 0.9 l of a 1% solution of Triton X-100 so that in the final solution there was a concentration of HA for all serotypes equal to 16 μg / dose. Next, the contents of the reactor were mixed for 15 min at a speed of 400 rpm, the stirrer was turned off, the semi-finished product was defended for 10 min, and filtered through a disposable sterile filter capsule with a pore diameter of 0.22 μm into a sterile plastic container. The volume of the semi-finished product after adding all the components corresponded to 50 l.

After sterilizing filtration, a sterile trivalent vaccine volume of 49 l was obtained. After obtaining satisfactory results for all necessary indicators of the quality of influenza vaccines, the container with the vaccine was transferred for filling into ampoules. The drug was poured into ampoules with a capacity of 1 ml of 0.6 ml of the trivalent vaccine in each ampoule. After bottling, 80,000 ampoules were obtained, which were checked for mechanical impurities. As a result, 800 ampoules were rejected for this indicator. The remaining ampoules were sent to a temporary storage warehouse with a temperature of (6 ± 2) ° С. Part of the ampoules was selected for the controls according to regulatory documentation.

Example 6. Preparation of a tetravalent vaccine dosage form

To prepare a series of tetravalent vaccines, sterile solutions of the obtained antigens (monovaccines) of serotypes A (H1N1), A (H3N2), B / Yamagata lineage and B / Victoria lineage were selected and they were added to the reactor in aseptic conditions in the amount of 7.4 l, 9 1 liter, 12.1 liters or 7.4 liters, respectively. Then, FBI M was added to them in an amount of 13.1 L and 0.9 L of a 1% Triton X-100 solution so that in the final solution there was a concentration of HA for all serotypes equal to 16 μg / dose. Next, the contents of the reactor were mixed for 15 min at a speed of 400 rpm, the mixer was turned off, the semi-finished product was defended for 10 min and filtered through a disposable sterile filter capsule with a pore diameter of 0.22 μm into a sterile plastic container. The volume of the semi-finished product after adding all the components corresponded to 50 l.

After sterilizing filtration, a sterile four-valent vaccine volume of 49 l was obtained. After obtaining satisfactory results on all necessary indicators of the quality of influenza vaccines, the container with the vaccine was transferred for filling into ampoules. The drug was poured into ampoules with a capacity of 1 ml of 0.6 ml of tetravalent vaccine in each ampoule. After bottling, 80,000 ampoules were obtained, which were checked for mechanical impurities. As a result, 800 ampoules were rejected for this indicator. The remaining ampoules were sent to a temporary storage warehouse with a temperature of (6 ± 2) ° С. Part of the ampoules was selected for the controls according to regulatory documentation.

Example 7. Comparison of the results of quality indicators of the proposed vaccine and vaccines of other manufacturers

The obtained stabilized dosage forms of the proposed vaccines (trivalent and tetravalent) were analyzed by some biochemical indicators of the quality of influenza vaccines in comparison with the vaccine Vaxigripp® (inactivated split vaccine for the prevention of influenza - Sanofi Pasteur S.A., France), Ultrix® (influenza vaccine inactivated cleaved - FORT LLC, Russia), Grippol® quadrivalent (tetravalent influenza vaccine; inactivated subunit adjuvant - NPO Petrovaks Pharm LLC, Russia). The results are presented in table No. 2.

Based on the presented results, it can be concluded that the obtained stabilized dosage forms of the proposed vaccines (trivalent and tetravalent) are not inferior in quality to other inactivated influenza vaccines registered in Russia. In some quality indicators, such as the content of ovalbumin or the ratio of total protein to hemagglutinin, they even surpass other influenza vaccines, both Russian and foreign.

The marker of the presence of the components of the producer organism in antigens (monovaccines) of the influenza virus is the ovalbumin protein contained in the CE allantoic fluid. This impurity is an important indicator of the quality of any vaccine whose antigen was obtained by culturing the vaccine strain in CE. In addition to the fact that the content of this protein can be interpreted as an integral indicator of the ability of a technology to purify a drug from high molecular weight impurities, ovalbumin requires special control due to its reactogenicity. Thus, influenza virus antigens, which may potentially contain ovalbumin and other proteins of the chicken embryo, are strictly contraindicated in persons with confirmed anaphylactic hypersensitivity, and vaccination of persons with a history of allergic reactions to chicken proteins should be carried out with extreme caution. Since vaccines with different ovalbumin content standards are present among the influenza drugs on the market, it is recommended for people with chicken protein allergy to select drugs whose specification allows the smallest threshold for the permissible concentration of ovalbumin.

Example 8. The study of the antigenic activity of the obtained trivalent and tetravalent vaccines

The study of the immunogenicity of the proposed tetravalent vaccine was carried out with a double intramuscular injection of the vaccine to Balb / c mice in females (6-8 weeks old, weighing (17.08 ± 0.03) g) in doses corresponding to 1 (group 1) and 1/2 vaccination dose for a person (group 2). The third group of mice was administered the comparison vaccine 1 Grippol® Quadrivalent twice intramuscularly in 1 dose for humans. Comparison vaccine 2 - the trivalent vaccine of the proposed vaccine was administered to group 4 mice twice intramuscularly in 1 dose for humans. The control group of mice (group 5) was injected intramuscularly with 0.5 ml of 0.9% sodium chloride solution. The number of mice in each group was 10. Blood from mice of the experimental and control groups was taken on the 14th and 28th day of the study.

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. Therefore, to develop a full-fledged immune response, the studied drug and comparison drugs were administered intramuscularly to Balb / c mice (the pathway that is intended for clinical use of the studied drug) twice with an interval of 14 days.

Evaluation of antigen-specific humoral immunity was carried out in the hemagglutination inhibition reaction (RTGA) and microneutralization reaction (PMN) in an MDCK cell culture with influenza A virus strains (H1N1); A (H3N2); B (Victoria lineage) and B (Yamagata lineage).

The reactions were carried out according to the methods recommended by WHO.

The results are presented in tables 3-6.

The results of a preclinical study of the immunogenicity of the proposed vaccines indicate that the proposed vaccines have a pronounced specific activity in relation to all strains of the influenza virus that are included in its composition.

Example 8. The study of the comparative immunogenicity of the obtained trivalent vaccine and vaccine Vaksigripp® in a clinical trial on volunteers

A double-blind, comparative, randomized, multicenter study of the proposed vaccine was conducted. The inactivated vaccine for influenza Waxigripp®, manufactured by Sanofi Pasteur, France, was used as a comparison drug. The vaccines were administered once intramuscularly to 400 volunteers aged 18 to 60 years. The study included 222 women (55.5%) and 178 men (44.8%), the average age of the volunteers was 33.6 ± 0.6 years. The vaccine of the proposed vaccine was given to 200 volunteers, the vaccine Vaksigripp® - 200 volunteers.

Evaluation of 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 multiplicity of the increase in antibody titer after vaccination:> 2.5 in people aged 18 to 60 years;

2. The level of seroconversion (an increase in the titer of antibodies to GA not less than 4 times):> 40% for persons aged 18 to 60 years;

3. The level of seroprotection (the number of persons with a protective titer ≥40):> 70% for persons aged 18 to 60 years.

The research results (Table 7-9) were statistically processed using Student's t-test and, in the absence of signs of a normal distribution, using nonparametric criteria (Wilcoxon and Mana-Whitney). Differences were considered significant at p <0.05.

Evaluation of the immunogenicity of the proposed vaccine in comparison with the vaccine Vaksigripp® with a single administration to volunteers aged 18-60 years revealed a high immunogenic activity to all strains of influenza virus (A (H1N1), A (H3N2) and B).

Significant differences in terms of “level of seroprotection”, “level of seroconversion” and “factor of seroconversion” with the introduction of the proposed vaccine and vaccine Vaksigripp® were not detected (p> 0.05).

Example 9. The safety study of the obtained trivalent and tetravalent vaccines

Safety studies of trivalent and tetravalent vaccines included the study of acute and subchronic, reproductive toxicity, assessment of pharmacological safety, local irritant effect, immunotoxicity, allergenicity, pyrogenicity and were carried out according to the design presented in table 10.

In the course of studies it was found that the proposed trivalent and tetravalent vaccines are safe and are characterized by good tolerance. Against the background of the use of vaccines, the death of animals was not observed, the deterioration of their condition, weight loss, fever were not installed, a negative effect on the reproductive, immune system was not noted. It is worth noting that the proposed vaccines caused some allergic reaction when administered to guinea pigs, however, these effects were observed with the introduction of a 10-fold dose and, in addition, allergic reactions are acceptable for immunobiological preparations.

Example 10. The safety study of the obtained trivalent vaccine in a clinical trial on volunteers

As part of Phase I, a simple, blind, placebo-controlled, one-center, prospective study was conducted, in which 45 volunteers, 33.3% men, 66.7% women, and an average age of 31.4 years took part. 15 volunteers received a trivalent vaccine with a preservative, 15 - without a preservative, 15 - a placebo. Control points of the study: screening visit, 3, 7, 21 days after vaccination. Examinations carried out: physical (temperature. Blood pressure, heart rate, NPV), examination by a general practitioner, collection of data on concomitant therapy, monitoring of adverse events and serious adverse events, blood and urine were also taken from volunteers. The results of evaluating the frequency of post-vaccination reactions among volunteers are presented in table 11.

For the entire period of post-vaccination observation, subjectively from 7 to 21 days, local and systemic reactions were not detected and registered.

Any deviations from normal values in terms of recorded indicators (temperature, blood pressure, heart rate, NPV, etc.) were detected and not registered.

The allergic properties of the trivalent vaccine were evaluated by total IgE and specific ovalbumin IgE (table 12).

It was found that the allergic properties of the trivalent vaccine are absent.

Thus, according to the results of the studies it can be concluded that the trivalent vaccine is characterized by weak reactogenicity and a high safety profile.

During phase II (see example 8), in addition to immunogenicity, the reactogenicity and safety of the trivalent vaccine were evaluated in 200 volunteers. The results obtained led to the conclusion that the trivalent vaccine is characterized by low reactogenicity and a high safety profile.

Claims (3)

1. The method of producing antigen or antigens of influenza virus, including obtaining chicken embryos (CE), disinfection, pre-incubation, infection with a working solution of seed virus, incubation of infected CE, cooling after incubation, collection of virus-containing allantoic fluid, disaggregation of the isolated viruses by adding NaCl solution to a final concentration of 0.23-0.30 M, inactivation by adding β-propiolactone to a final concentration of 0.1% and purification of the virus-containing allantoic fluid by microfiltration using cascade of three filters with a pore diameter of 10 μm, 1 μm, 0.6 μm and ultrafiltration with a cutoff limit of 300 kDa, then the clarified virus-containing allantoic fluid is concentrated no more than 20 times, the concentrate is purified by double ultracentrifugation in a sucrose density gradient, after which the inactivated viral concentrate is cleaved using the detergent n-octyl-β-D-glucopyranoside (octyl glucoside) or tetradecyl trimethylammonium bromide (TDTAB), and the removal of the intact virion and complexes with ribonucleoproteins membrane protein by centrifugation, micro-and ultrafiltration, chromatographic purification and sterilizing filtration is obtained an antigen or antigens, and stabilization of the antigen or antigens is carried out by using X-100 Triton detergent at a concentration of 100-200 ug / ml. 2. An influenza vaccine containing an antigen or antigens obtained by the method of claim 1. 3. The vaccine according to claim 2, characterized in that it contains 1, 3 or 4 antigens obtained by the method according to claim 1.

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