RU2839314C2 - Method of producing an influenza virus concentrate for producing vaccine - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a method for producing an influenza virus concentrate, includes selecting a reassortant, selecting optimal conditions for incubation, in particular temperature and duration, infection 11 ± 1-day-old chicken embryos with a viral material containing hydrocortisone additives and an antibiotic, incubation, cooling of chicken embryos, collection of virus-containing allantoic fluid (VAF) with subsequent disaggregation with electrolytes, clarification of VAF, microfiltration of the obtained clarified VAF, purification of the obtained solution, inactivation of the purified viral concentrate or before the stage of clarification of the VAF and obtaining an influenza virus concentrate.

EFFECT: invention extends the range of methods for producing an influenza virus concentrate.

5 cl, 6 dwg, 6 tbl, 2 ex

Description

Field of technology

The claimed invention relates to the field of pharmaceutical preparations and can be used for the production of influenza vaccines.

Description of the Prior Art

Currently, two types of influenza vaccines are widely used: inactivated (split, subunit, virosomal) and live attenuated influenza vaccines. The production of seasonal influenza vaccines is based on the cultivation of the virus in chicken embryos or cell cultures. WHO annually updates the composition of influenza vaccines based on information provided by the Global Influenza Surveillance and Response System (GISRS). Since vaccine production takes about 6 months, the influenza vaccine is produced annually under great time pressure. In order to produce any version of influenza vaccines using chicken embryos, it is first necessary to obtain a sufficient amount of influenza virus concentrate. It should be noted that influenza virus reassortants prepared by WHO collaborating centers and used for influenza virus cultivation have different productivity, which affects the technology for obtaining the virus concentrate. In this regard, the problem that the present invention is aimed at solving is to develop a technology for obtaining influenza virus concentrates that will make it possible to produce a sufficient quantity of influenza virus concentrates of the appropriate quality for producing a vaccine in a short time.

Patent RU 2740751 dated 20.01.2021 describes a method for obtaining an influenza virus concentrate, which includes culturing influenza viruses in developing chicken embryos, purifying the collected virus-containing allantoic fluid (VAF) using microfiltration through a cascade of filter elements with pore sizes of 2.3; 1.6; 1.2; 0.8; 0.44 μm, respectively, concentrating on a tangential flow ultrafiltration unit with 4 cassette modules with a pore size of 300 kDa, purifying viral particles by density gradient ultracentrifugation using two sucrose solutions of 50% and 20% at a rotor speed of 30,000 rpm and gel chromatography on a polymer carrier. The proposed method cannot ensure high efficiency of the technology for obtaining a viral concentrate due to the loss of hemagglutinin due to sorption on the entire filtration area of debris cultures of various sizes, which are obtained as a result of the reproduction of the influenza virus and the destruction of chicken embryo cells and may contain hemagglutinin. Despite the fact that before the microfiltration process, it is proposed to add an amount of phosphate buffer solution equal to the volume of the VAG, which also increases the consumption of raw materials and the time of the VAG purification process, the proposed method does not include a diafiltration stage. At the same time, gel chromatography on a polymer carrier also reduces the yield of hemagglutinin in the viral concentrate. A similar purification of VAZ using microfiltration through cascades of filter elements with different pore sizes is described in patent RU 2741003 dated 22.01.2021, where preliminary purification of VAZ from debris structures is carried out by a three-stage microfiltration method with pore diameters of 10 μm, 1.2 μm and 0.6 μm, and the process of preliminary disaggregation and subsequent diafiltration is not described. Patent RU 2535153 dated 10.12.2014 discloses that microfiltration is carried out on 2 cascades of cartridges (pore diameter of 10 μm and 1 μm) without subsequent diafiltration, but preliminary purification is used using polyethylene glycol with a molecular weight of 6000 at a final concentration of 1.5%. However, the disadvantage of this method is an increase in the time of the technological process by 17 hours.

The method for purifying VAV described in patent RU 2493872 dated 27.09.2013 provides for centrifugation (1000-1500 g) of VAV before the stage of filtration through polypropylene fiber. However, microfiltration of VAV is carried out on filter elements of 0.1-0.2 μm with subsequent diafiltration with a phosphate buffer solution containing 0.25-0.5 M sodium chloride, EDTA or sodium citrate, concentration of the influenza virus on membranes with a cutoff threshold of 300-500 kDa followed by diafiltration with a phosphate buffer solution containing 0.25-0.5 M sodium chloride, EDTA or sodium citrate, 0.02-0.002% anionic detergent. The disadvantages of this method are: there is no disaggregation stage and insufficient centrifugal force, which will not allow removing debris up to 1 µm in size, and, accordingly, sorption will occur on filter elements with a non-optimally small pore size of 0.1-0.2 µm.

The closest prototypes of the invention are the methods for producing influenza virus concentrates described in patent RU 2754398 dated 09/01/2021 and RU 2710239 dated 12/25/2019.

Patent RU 2754398 (OOO Fort) discloses a method for obtaining influenza virus antigen concentrates, which includes preparing seed material, culturing the influenza virus in developing 10-12-day chicken embryos until a given infectious activity is achieved, collecting the virus-containing fluid and inactivating it in the presence of betapropylactone at a final concentration of no more than 0.1% by volume, purifying the virus by micro- and ultrafiltration using the tangential method, and isolating the virus by ultracentrifugation in a sucrose gradient at 90,000-100,000 g.

The method does not describe the method for establishing optimal conditions for culturing the influenza virus, and there are no methods for increasing the productivity of the seed material when infecting chicken embryos. The cooling time of chicken embryos before collecting the influenza virus, which affects further losses of hemagglutinin during purification, is also not established. The disadvantages of the proposed method for purifying the influenza virus are the lack of a disaggregation stage, which is necessary to prevent the appearance of aggregates from debris that may contain influenza virus particles, which in turn may escape at the separation stage. There is no information on the pore sizes of microfiltration, where, with a non-optimal choice of pore sizes, hemagglutinin losses are possible.

In patent RU 2710239 (FSUE SPbNIIVS FMBA of Russia), before the stage of purification of VAZ, viruses are disaggregated with sodium chloride at a final concentration of 0.23-0.30 M, viral activity is inactivated using β-propiolactone, VAZ is purified by microfiltration methods 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, followed by purification by double ultracentrifugation in a sucrose density gradient. Just as in the previous prototype, there is no description of methods for increasing the productivity of the seed material when infecting chicken embryos. The disadvantages of the proposed method for purifying VAZ are the loss of hemagglutinin at the microfiltration stage due to the presence of a large filtration area, as well as the lack of a diafiltration stage. As a result of long-term experimental work carried out by the authors of this invention, it was established that complete separation of debris from influenza virions cannot be achieved with any combination of filters.

None of the documents under consideration described a method for obtaining influenza virus concentrate with optimal conditions for infection and incubation of chicken embryos and with increased productivity of seed materials when infected with the influenza virus. Also, the issue of maximum reduction of technological losses in obtaining inactivated virus concentrates for the production of influenza vaccines was not considered. Research on the development of a universal technology for obtaining virus concentrates was aimed at solving these problems, which would make it possible to produce a sufficient amount of virus concentrates of appropriate quality for vaccine production in a short time, with the highest yield of active and effective influenza virus concentrate.

Description of figures

Fig. 1 shows a graphical dependence of the concentration of hemagglutinin (HA) in the VAF (μg/ml) on the influence of the factors of time and incubation temperature.

Fig. 2 shows a graphical dependence of the concentration of GA in the VAF (μg/ml) on the influence of the factors of the amount of hydrocortisone and the incubation time.

Fig. 3 shows a graph of the dependence of the temperature of the VAZ in chicken embryos on the cooling time in a refrigeration chamber.

Fig. 4 shows the dependence of the change in the turbidity of the aqueous solution on the concentration of sodium chloride.

Fig. 5 shows the quality indicators of the fractions of the influenza B/Phuket/3073/2013 virus concentrate obtained according to Example 1.

Fig. 6 shows the quality indicators of the fractions of the influenza B/Phuket/3073/2013 virus concentrate obtained according to Example 2.

Description of the invention

The present invention provides a method for producing an influenza virus concentrate, comprising:

- selection of a reassortant for the production of an influenza vaccine;

- selection of optimal conditions for incubation for each strain, such as temperature and duration, with the temperature selected within 34±2°C and the duration within 35-72 hours;

- infection of 11±1-day-old chicken embryos with viral material containing hydrocortisone additives at a concentration of 4-16 μg/EC or not containing this additive, and an antibiotic at a concentration of 40-60 μg/ml;

- incubation at a humidity of 60±5% under conditions selected for each strain;

- cooling of chicken embryos at a temperature of 5±3°C for at least 8 hours;

- collection of virus-containing allantoic fluid (VAF) with subsequent disaggregation with electrolytes;

- lightening is IMPORTANT;

- microfiltration of the obtained clarified VAZ;

- purification of the resulting solution;

- inactivation of the solution with β-propiolactone to a final concentration of 0.1% at the stage of concentrated VAF, purified viral concentrate or before the stage of VAF clarification;

- obtaining influenza virus concentrate for the production of influenza vaccine.

At the infection stage, various antibiotics can be used as antibiotics, for example, kanamycin, amikacin, amoxicillin, cefuroxime and others, preferably gentamicin is used.

The method also assumes that at the stage of collecting virus-containing allantoic fluid (VCF) with subsequent disaggregation with electrolytes, strong electrolytes are used as electrolytes, for example, sodium chloride at a final concentration of 0.2-0.3 M, potassium chloride, calcium chloride, and others.

In some embodiments of the method according to the invention, microfiltration is carried out on filters with a pore size of at least 0.6 μm, for example, with a pore size of 0.6 μm.

In the method according to the invention, it is possible to use influenza virus types A and B, for example, subtypes A (H1N1), A (H3N2), line B - B/Yamagata, B/Victoria. Also, after obtaining as described above, this obtained influenza virus concentrate, without preliminary splitting, can be used as an antigen of an influenza vaccine.

Also, according to the invention, an anti-flu vaccine is proposed, obtained using a flu virus concentrate obtained by the described method. The said vaccine can be obtained from one, three or four flu virus antigen concentrates, and can include flu virus antigen concentrates of types A or B.

It is also proposed to use the influenza virus concentrate obtained by the method according to the invention for the production of an influenza vaccine.

The technical result of the invention is a flu virus concentrate obtained by a method with optimal conditions and increased productivity, with less time expenditure, while the flu virus concentrate has high efficiency.

As a result of long-term experimental work carried out by the authors of this invention, it was established that it is the method according to the invention that allows obtaining a concentrate that is most effective for vaccine production. The technical result is achieved by implementing a method that includes the following stages:

- selection of a reassortant for the production of an influenza vaccine;

- selection of incubation conditions, such as temperature within 34±2°C, and duration within 35-72 hours;

- infection of 11±1-day-old chicken embryos with viral material containing hydrocortisone additives at a concentration of 4-16 μg/EC or not containing this additive, and an antibiotic at a concentration of 40-60 μg/ml;

- incubation at a humidity of 60±5% depending on the conditions selected for each strain;

- cooling of chicken embryos at a temperature of 5±3°C for at least 8 hours;

- collection of virus-containing allantoic fluid (VAF) with subsequent disaggregation with electrolytes;

- lightening is IMPORTANT;

- microfiltration of the obtained clarified VAZ;

- purification of the resulting solution;

- inactivation of the solution with β-propiolactone to a final concentration of 0.1% at the stage of concentrated VAF, purified viral concentrate or before the stage of VAF clarification;

- obtaining influenza virus concentrate for the production of influenza vaccine.

The authors of the present invention have established that the introduction of hydrocortisone and an antibiotic into the viral material during infection of 11±1 day old chicken embryos allows for a significant increase in the yield and purity of the resulting influenza virus concentrate. In particular, when studying possible concentrations that affect the production of an effective influenza virus concentrate, it was established that the highest concentration of the virus in the concentrate is achieved by introducing hydrocortisone at a concentration of 4-16 μg/EC and the antibiotic gentamicin at a concentration of 40-60 μg/ml per EC or per ml of viral material used during infection.

In addition, it was established that incubation at a temperature of 34±2°C for 35-72 hours and cooling of chicken embryos at a temperature of 5±3°C for at least 8 hours allows achieving the maximum density (concentration) of antigen in the VAZ.

The claimed method for obtaining influenza virus concentrate consists of several stages, the implementation of which allows for a significant reduction in process losses and an increase in the quality of the obtained influenza virus concentrate (increase in efficiency), which in turn will reduce the time required to produce influenza virus concentrates for the production of influenza vaccine.

The selection of the most productive reassortant involves infecting at least 36 10-12-day-old chicken embryos (CE) with each reassortant recommended by WHO, incubating at 34±2°C for 35-50 hours for type A and 50-72 hours for type B, then collecting the VAI and combining the samples into pools (at least 6 pools) and assessing the hemagglutination titer and hemagglutinin (HA) concentration in the resulting pools using the single radial immunodiffusion (SRID) method or RP-HPLC or the number of viral particles using quantitative PCR. The reassortant that gives the highest HA and HA titer values is selected. An example of the results obtained for selecting the most productive reassortant of the A/Victoria/2570/2019 (H1N1) strain is shown in Table 1.

The selection of more precise cultivation conditions is similar and consists of infecting at least 36 10-12-day-old chicken embryos (CE) with the influenza virus of the selected reassortant, incubating at different temperatures in the range of 34±2°C for different periods of time in the range from 35 to 72 h, then collecting the VAI and combining the samples into pools (at least 6 pools) and assessing the hemagglutination titer and hemagglutinin (HA) concentration in the resulting pools by the single radial immunodiffusion (SRID) method or the RP-HPLC method or the number of viral particles by the quantitative PCR method. An example of the results obtained for selecting the cultivation conditions for the influenza virus B/Phuket/3073/2013 is shown in Fig. 1. The presented dependence shows that the optimal point of hemagglutinin production is 33°C for 65 h.

Infection of 11±1 day old chicken embryos with viral material (0.2 ml) containing hydrocortisone additive at a concentration of 4-16 μg/EC increased hemagglutinin production to 70% under optimally selected cultivation conditions. The result of mathematical processing in the MINITAB software application based on regression and dispersion analysis showed the dependence of hemagglutinin yield on the concentration of hydrocortisone and incubation time using the example of influenza virus B/Phuket/3073/2013. This dependence is shown in Fig. 2, as can be seen, it is quite complex and nonlinear.

Cooling of chicken embryos in a refrigeration chamber at a temperature of 5±3°C for at least 8 hours is necessary to achieve a temperature of the IM in the egg of no more than 6°C (shown in Fig. 3), at which optimal collection of IM occurs with further minimal losses during cleaning. An example of reducing losses due to a decrease in the temperature of IM during collection is shown in Table 2.

Table 2 - Losses of hemagglutinin during the purification of VAZ depending on the cooling time of chicken embryos in the refrigerator before collection Cooling time of chicken embryos before collection of VAZ, h Average losses of hemagglutinin during the purification process of VAZ, % Influenza A virus Influenza B virus 2 29±6 39±6 4 27±6 33±5 6 18±3 21±3 8 15±2 17±3

As can be seen from the data, it is the holding for at least 8 hours in the refrigeration chamber that allows achieving the lowest losses of hemagglutinin during the purification of the VAF. The introduction of the disaggregation stage before the purification of the VAF is also necessary to reduce the loss of the influenza virus at subsequent stages of purification. Fig. 4 shows the dependence of the change in the turbidity of the VAF on the concentration of the added sodium chloride. The turbidity of the VAF was determined by the nephelometric method on a HACH 2100Q turbidimeter.

When clarifying VAZ using microfiltration methods, high losses of the influenza virus are possible, which occur due to sorption on filters with a huge total filtration area. Moreover, to reduce these losses, the principle of a filter cascade is often used, which further increases the total filtration area, thereby even for optimally selected filter materials, the total losses of the influenza virus are noticeable. An alternative purification method that provides fewer losses while maintaining the same degree of purification is separation. The separator allows purifying VAZ from large precipitates of debris proteins and erythrocytes (with a size of more than 1 μm), which reduces the loss of hemagglutinin, which occur due to sorption in the process of microfiltration. The results of comparing the VAZ purification technology using a cascade of deep filters with a pore diameter of 10 μm, 1 μm (filter material - polypropylene) and using the Clara - 20 separator are presented in Table 3.

Table 3 - Evaluation of the efficiency of the technology when using a separator Influenza virus strain Cleaning of VAZ using a separator Cleaning of VAZ using depth filters
(10 µm and 1 µm) Average losses of GA, % Median GA loss Average losses of GA, % Median GA loss B/Washington/
02/2019 16.5 19.0 39.8 38.8 A/HongKong/
2671/2019(H3N2) 18.5 18.3 21.0 18.9 A/Guangdong-Maonan/
SWL1536/2019 (H1N1) 20.0 20.0 28.7 28.7

As follows from the data in Table 3, the use of separation allows to reduce hemagglutinin losses by an average of 10-20% depending on the strain.

However, when using a separator, there is a risk of foaming of the material, which can affect the increase of losses at the following technological stages. Therefore, the proposed method for obtaining influenza virus antigen concentrates includes a degassing stage, which can be carried out using a flow-through ultrasonic reactor or with subsequent foam removal by pumping and settling the VAG in an intermediate container. The results of the comparative tests are presented in Table 4.

Table 4 - Evaluation of the efficiency of the technology for obtaining influenza virus concentrates using the degassing stage after the separation stage and the diafiltration stage after the concentration stage Influenza virus strain Average losses of GA, % Cleaning of VAZ using a separator Purification of VAZ using a separator + ultrasonic degassing + diafiltration B/Washington/
02/2019 24±5 9±3 A/Victoria/2570/2019 (H1N1) 24±5 13±3 A/Tasmania/503/2020 (H3N2) 32±6 23±4

To reduce process losses at the stage of concentration by ultrafiltration (cutoff limit of not less than 300 kDa, not more than 1 MDa), the proposed method uses diafiltration with a buffer solution in an amount of not less than five volumes of concentrate. The results of the comparative tests are presented in Table 4.

As follows from the data in Table 4, the use of separation in combination with degassing allows for a reduction in hemagglutinin losses by an average of 10%, depending on the strain.

Examples of the invention implementation are given below. The examples are intended as illustrative and are not intended to limit the present invention in any way.

Examples

Example 1

Obtaining a viral concentrate of the influenza virus

To obtain the influenza B/Phuket/3073/2013 virus concentrate, the most productive reassortant was selected. For this purpose, 36 eleven-day-old chicken embryos (CE) were infected with the wild type B/Phuket/3073/2013 (NIBSC, UK), 36 with the reassortant BVR 1B (VIDRL, Australia), and 36 with the reassortant NYMC BX-59B B/California/12/2015 (NIBSC, UK). Next, incubation was carried out at 33°C for 63 h, after which the VAG was collected and the samples were combined into pools of 6 CE. The hemagglutination titer and hemagglutinin (HA) concentration were assessed using the single radial immunodiffusion (SRID) method. Table 5. - Selection of the most productive strain/reassortant B/Phuket/3073/2013.

The results are presented in Table 5.

Table 5 - Selection of the most productive strain/reassortant B/Phuket/3073/2013 Strain Reassortant Incubation temperature, °C Incubation time, h RGA titer GA concentration, μgGA/ml Average GA value (confidence interval for p=0.95) B/Phuket/3073/2013 Wild Strain 33.0 63 1:320 16.6 16.5 ± 0.8 1:320 15.8 1:640 17.5 1:640 15.8 1:640 16.0 1:640 17.2 B/Phuket/3073/2013 BVR-1B 33.0 63 1:640 15.1 16.9 ± 1.2 1:640 16.6 1:640 17.1 1:640 17.0 1:640 16.8 1:640 18.8 B/California/12/2015 NYMC BX-59B 33.0 63 1:320 13.9 14.9 ± 0.6 1:320 15.1 1:320 15.6 1:320 14.9 1:320 14.8 1:320 15.3

Next, cultivation conditions were selected for the wild type B/Phuket/3073/2013 (NIBSC, UK), for which eleven day-old chicken embryos were infected and incubated according to the scheme presented in Table 6.

Table 6 - Scheme for assessing optimal incubation conditions after infection of chicken embryos with influenza virus B/Phuket/3073/2013 Sample No. for titer and GA content control Number of CEs included in the sample/Number of samples Incubation time, h Incubation temperature, °C 1.1-1.6 6/6 55 32 2.1-2.6 6/6 65 3.1-3.6 6/6 72 4.1-4.6 6/6 55 33 5.1-5.6 6/6 65 6.1-6.6 6/6 72 7.1-7.6 6/6 55 34 8.1-8.6 6/6 65 9.1-9.6 6/6 72

The results of the assessment of the hemagglutination titer and HA concentration using the ORID method are presented in Fig. 2.

Thus, the optimal conditions for wild type B/Phuket/3073/2013 (NIBSC, UK) are an incubation temperature of 33°C and a time of 65 h.

To obtain WAG, 70,000 11-day-old EC were infected with a dose of the working solution of the wild-type seed virus B/Phuket/3073/2013 (NIBSC, UK) having an infectious titer of 10 ^3.33 EID ₅₀ /0.2 ml and containing the addition of hydrocortisone at a concentration of 50 μg / ml. Incubation after infection was carried out at a temperature of 33 ° C for 61-65 h. Cooling of EC after incubation was carried out in a cold chamber at a temperature of 5 ± 3 ° C for 10-14 h. After cooling, WAG was collected in a reactor in a volume of 728 l. Then 74 l of 2.5 M NaCl solution were added to the reactor with the collected WAG for disaggregation, followed by mixing. The yield of hemagglutinin at this stage of the technological process was 247 μg HA / EC.

After processing, the VAI was fed from the reactor to an Alfa Laval Clara-20 separator, rotor speed 9200 rpm (11000 g), liquid flow rate from 5 to 6 l/min at a back pressure of 2.0±0.2 atm. Then the VAI was fed through an ultrasonic flow reactor at the separator flow rate for microfiltration with a pore size of 0.6 μm (filter material - polypropylene, filtration area - 23.7 m ² ). Then the VAI was fed to an ultrafiltration unit with Pellicon 2 Biomax-300 cassette modules with a cutoff limit of 300 kDa (material) for further purification and concentration. The resulting volume of clarified concentrated VAI was 40 l. Next, the resulting concentrate was subjected to diafiltration with 400 l of phosphate buffer solution (pH=7.4±0.2) and re-concentrated to 40 l.

The yield of hemagglutinin at this stage of the technological process was 210 μg HA/EC.

Next, the influenza virus was inactivated for 12 hours using a chemical method by adding 40 ml of β-propiolactone.

Then, the influenza virus antigen was purified and concentrated in a sucrose density gradient. For this purpose, a tris buffer (pH = 7.4 ± 0.2) and a 60% sucrose solution were loaded into the ultracentrifuge rotor. The solutions were used cooled to a temperature of 5 ± 3 ° C. After acceleration to g = (85,000 ± 5,000) m / s ² , clarified concentrated VAG was fed into the centrifuge rotor using a peristaltic pump. After the entire volume of concentrated VAG had passed through, a tris buffer was fed into the ultracentrifuge rotor and isodensity centrifugation was performed.

After completion of isodensity centrifugation, the obtained fractions of the viral concentrate (VC) were unloaded. 20 fractions of the sucrose density gradient contained in the rotor were collected: the first two fractions, 200 ml each, the next ones, 100 ml each. 800 ml (8 fractions) with an optimal sucrose concentration of no more than 50% and an HA titer of at least 1:16000 were selected from the gradient for further work. The quality of the obtained influenza virus concentrate is shown in Fig. 5.

The yield of hemagglutinin at this stage of the technological process was 197 μg HA/CE. The total loss of hemagglutinin during the production of the influenza virus concentrate was no more than 20.2%.

This volume of viral concentrate contained 39.8 g of protein (determined using the Bradford method) and 13.8 g of GA (determined using the ORID method).

Example 2

Obtaining a viral concentrate according to the prior art method

The method described in patent RU 2710239 was adopted as a prototype.

To obtain the VAG, 70,000 11-day-old ECs were infected with a dose of the working solution of the NYMC BX-59B B/California/12/2015 seed virus, which has an infectious titer of 10 ^3.66 EID ₅₀ /0.2 ml. Incubation after infection was carried out at a temperature of 35 ° C for 48-52 h. Cooling of the EC after incubation was carried out in a cold chamber at a temperature of 5 ± 3 ° C for 4-8 h. After cooling, the VAG was collected in a reactor in a volume of 687 l. Then, 70 l of a 2.5 M NaCl solution were added to the reactor with the collected VAG for disaggregation, followed by mixing, after which 0.68 l of β-propiolactone was added. Inactivation was carried out at a temperature of 6±2°C for 10 hours. The yield of hemagglutinin at this stage of the technological process was 171 μg HA/EC.

Further, after purification and concentration of the VAZ using micro- and ultrafiltration, 40 liters of VAZ concentrate were obtained. The yield of hemagglutinin at this stage of the technological process was 104 μg HA/CE.

Then, the influenza virus antigen was purified and concentrated in a sucrose density gradient. After isodensity centrifugation was completed, the obtained fractions of the viral concentrate (VC) were unloaded. 20 fractions of the sucrose density gradient contained in the rotor were collected: the first two fractions, 200 ml each, the next ones, 100 ml each. 800 ml (8 fractions) with an optimal sucrose concentration of no more than 50% and an HA titer of at least 1:16000 were selected from the gradient for further work. The quality of the obtained influenza virus concentrate is shown in Fig. 6.

The yield of hemagglutinin at this stage of the technological process was 99 μg HA/CE. The total loss of hemagglutinin during the production of the influenza virus concentrate was about 42.1%.

This volume of influenza virus concentrate contained 21.2 g of protein (determined using the Bradford method) and 6.9 g of HA (determined using the ORID method).

As can be seen from the data presented in Figs. 1 and 2, the method according to the invention makes it possible to obtain influenza virus concentrates with increased yield and purity.

Claims (16)

1. A method for obtaining an influenza virus concentrate, comprising: - selection of a reassortant for the production of an influenza vaccine; - selection of incubation conditions, such as temperature within 34±2°C and duration within 35-72 hours; - infection of 11±1-day-old chicken embryos with viral material containing an antibiotic at a concentration of 40-60 μg/ml; - incubation at a humidity of 60±5% depending on the conditions selected for each strain; - cooling of chicken embryos at a temperature of 5±3°C for at least 8 hours; - collection of virus-containing allantoic fluid (VAF) with subsequent disaggregation with electrolytes; - clarification of VAZ using a separator with subsequent degassing; - microfiltration of the obtained clarified VAZ; - purification of the resulting solution; - inactivation of the solution with β-propiolactone to a final concentration of 0.1% at the stage of concentrated VAF, purified viral concentrate or before the stage of VAF clarification; - obtaining influenza virus concentrate for the production of influenza vaccine. 2. The method according to paragraph 1, characterized in that at the infection stage, gentamicin is used as an antibiotic. 3. The method according to item 1, wherein at the stage of infection of the embryos, an additional hydrocortisone additive is added at a concentration of 4-16 μg/EC. 4. The method according to item 1, characterized in that at the stage of collecting virus-containing allantoic fluid (VCF) with subsequent disaggregation with electrolytes, sodium chloride is used as the electrolyte at a final concentration of 0.2-0.3 M. 5. The method according to paragraph 1, characterized in that microfiltration is carried out on filters with a pore size of 0.6 µm.