RU2741003C1 - Method for production of tetravalent subunit vaccine against influenza without adjuvants - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.SUBSTANCE: invention refers to medicine and pharmaceutics, namely to a method for producing a tetravalent subunit vaccine without adjuvants for influenza prevention, according to which a) obtaining four mono-vaccines containing one of serotypes of influenza viruses H1N1, H3N2, B (Victoria line) or B (Yamagata line), wherein to obtain monovaccine producing a virus-containing allantoic liquid, then inactivated with beta-propiolactone and purified and concentrated by microfiltration, ultrafiltration and two cycles of ultracentrifugation in a sucrose density gradient; destroying the virions in the obtained purified viral concentrate by successive treatment with Triton X-100 and TDTAB; sub-virion structures are removed by ultracentrifugation and microfiltration and detergents by ultrafiltration and concentration of the obtained purified supernatant with subsequent purification by hydrophobic chromatography using ExpandedBedAdsorption technology; diluting the concentrate with a phosphate buffer solution containing Triton X-100 to obtain a monovaccine; b) combining the four monovaccines into a single dosage form.EFFECT: technical result consists in improvement of purification and immunogenicity of tetravalent subunit vaccine without adjuvants during its production in accordance with the declared method.4 cl, 14 dwg, 2 tbl, 11 ex

Description

Technology area

The invention relates to biotechnology and relates to a method for the production of a tetravalent subunit vaccine for the prevention of influenza without adjuvants.

Prior art

According to the practice of world health care, the main means of combating the incidence of influenza is the immunization of the population with inactivated influenza vaccines. Inactivated influenza vaccines are categorized as whole virion, split and subunit. In the period from the 40s - 60s. XX century in the world for the vaccination of the population used mainly whole virion vaccines. These vaccines induced a stable protective immunity in vaccinated people, but they were highly reactogenic and therefore could not be used for vaccination of children. In the 60s and 70s, two new classes of vaccines appeared. The first to be developed was a split vaccine, which included both surface and internal antigens of influenza viruses, but which no longer contained virion and subvirionic structures. This class of vaccines was significantly less reactogenic than whole-virion vaccines, but these vaccines still had residual reactogenicity, presumably due to the presence of aggregated and partially denatured forms of the membrane protein (M1) in their composition. In the mid 70s. subunit vaccines began to appear, which mainly included surface antigens of influenza viruses (1). Due to the absence of various forms of membrane protein in their composition, as well as due to the higher quality of purification of virions, this class of vaccines is the least reactogenic, has sufficient immunogenicity and therefore can be used to vaccinate the population of all ages.

Over the past few years, WHO international centers have revealed that during the current epidemiological seasons, not three serotypes of influenza viruses (H3N2, H1N1, B) circulate in the human population, as it was before, but four. It was found that serotype B split into two independent lines, namely the Yamagato line and the Victoria line. Therefore, WHO recommends that all manufacturers of influenza vaccines switch to the production of tetravalent drugs, which more fully protect people from this disease.

Of great importance for the production of all types of inactivated vaccines is the quality of purification of virus-containing allantoic fluid (VAF) from debris structures that are formed during the reproduction of influenza viruses in chicken embryos (CE). In the Russian Federation, initially, the technology of sorption-elution on formalized chicken erythrocytes was used to remove these structures (4). This technology made it possible to sufficiently high purify IAF from debris structures, but did not allow achieving reproducible results due to the different ability of influenza virus strains to be sorbed on formalized chicken erythrocytes. Due to the non-reproducibility of the results at this technological stage, it was practically impossible to carry out the validation process in accordance with the rules of GMP ("good manufacturing practice"). The WHO guidelines for the production of drugs for human vaccination indicate that validation processes should be carried out in accordance with GMP rules for the main technological stages. In the process of the development of modern biotechnology in the Russian Federation, technological approaches have been developed that made it possible to purify VAZ without using erythrocytes. In one of these technologies, the separation methods using centrifugation, various types of microfiltration and ultrafiltration were used for the purification of VAS, and the method of ultracentrifugation in a sucrose density gradient was used to obtain HVA (5). The same authors further optimized their technology using neutral and basic amino acids, as well as detergents (6).

It should be noted that in both of these inventions for inactivation of the infectious activity of influenza viruses, the use of the ultraviolet irradiation method is indicated, which also does not allow obtaining reproducible results and, therefore, does not allow validating the entire production process in accordance with GMP requirements. Requirements for the production and quality of inactivated influenza vaccines are detailed in the EU regulatory document of 2014 (2) and the RF GF edition XIV (14). These documents indicate that in order to inactivate the infectious activity of influenza viruses in the process of obtaining vaccines, it is necessary to use disinfectant treatment: formalin or beta-propiolactone, since the use of these disinfectants ensures reliable and reproducible inactivation of influenza viruses. The use of beta-propiolactone to inactivate the infectious activity of influenza viruses as a part of VAV was first performed in 1991 (11). Later, it was shown that the use of this disinfectant in the process of inactivation of influenza viruses does not affect the antigenic structure of the HA antigen, in contrast to the use of the disinfectant formalin (10).

In the Russian Federation, a method for obtaining a split vaccine was developed (7), in which the disinfectant beta-propiolactone is used in the VAZ to inactivate influenza viruses, 2 cascades of depth filters with pore diameters of 5 μm and 1 μm are used for preliminary purification of the VAZ, the final purification of the virion concentrate is carried out by a single sucrose density gradient ultracentrifugation. To obtain a split vaccine, the destruction of OVK is carried out with a detergent Octylglucoside, antigens are purified from subvirionic structures by microfiltration through depth filters with a pore diameter of 10 microns and by ultracentrifugation, cleaning from detergent is carried out sequentially by diafiltration methods and gel chromatography using the WorkBeads 40/100 carrier "By Bio-Works, Sweden.

The disadvantages of this method are: 1) the method of preliminary cleaning of the VAZ using only two cascades of depth filters with pore diameters of 5 μm and 1 μm does not allow to completely clean the VAZ from debris structures; 2) the use of only one cycle of ultracentrifugation in a sucrose density gradient does not allow to completely purify the final HVAC preparation from ovalbumin impurities; 3) the use of the detergent Octylglucoside does not allow obtaining monovaccine preparations purified from internal antigens of influenza viruses; 4) the use of depth filters with a pore diameter of 10 μm at the stage of microfiltration of the split HVAC does not allow achieving adequate purification of the drug at this technological stage from subvirionic structures; 5) as a result of the combined action of the above factors, the obtained monovaccine preparations have a sufficiently high level of turbidity index.

Split and subunit influenza vaccines differ from each other, both in production technology and in composition (14). Split vaccines can contain both surface and internal antigens of influenza viruses, while subunit vaccines should contain mainly surface antigens, namely, hemagglutinin (HA) and neuraminedase (HA). In addition, according to the requirements of the GF RF XIV (14), these vaccines differ in the ratio of the total protein to the HA antigen, while in split vaccines this ratio can be (6.7: 1), while in subunit vaccines it should not be more than (3.7: 1).

RF patent No. 2446824 (3) describes a technology according to which a subunit vaccine with the adjuvant "Sovigripp" is produced in the amount of tens of millions of doses annually. This technology includes: the cultivation of influenza viruses in CE; receipt of VAZ and its preliminary cleaning; obtaining HVAC by ultracentrifugation in a sucrose density gradient; inactivation of viral material using the ultraviolet irradiation method; cleavage of HVAC with detergent TDTAB; purification of antigens from detergent by diafiltration.

The disadvantages of this method are: 1) the use of chicken erythrocytes for preliminary purification of VAZ, which does not allow the validation process of the entire production process in accordance with GMP requirements; 2) the use of ultraviolet radiation to inactivate viruses, which also does not allow the validation process to be carried out in accordance with GMP requirements; 3) the absence of a microfiltration stage at the stage of isolating monovaccines for additional purification of the drug from subvirionic structures, which does not allow obtaining monovaccines with a low level of turbidity; 4) the use of only one method of diafiltration at the stage of isolation of monovaccines, which is not enough for effective purification of surface antigens from the detergent TDTAB. In addition to the aforementioned shortcomings, in the production process of the Sovigripp vaccine, problems began to emerge related to the different efficiency of the production technology at the stage of HVAC cleavage for industrial strains of the influenza virus of serotypes H1N1 and H3N2c on the one hand, and industrial strains of influenza viruses of serotype B, on the other.

The literature describes the patent of the company "Novartis" (8). This patent recommends, in order to increase the efficiency of the technology, to add two detergents sequentially in the process of HVAC destruction. In this case, the nonionic detergent (TWIN-80) was added first to carry out the disaggregation process, and then the second cationic detergent (CTAB, TDTAB, etc.) was added to conduct the actual cleavage process. It was through the use of both detergents that it was possible to achieve an increase in the percentage of degradation compared to using only one cationic detergent. However, the above patent does not indicate the technological parameters of the degradation process using two detergents. In addition, the detergent TWIN-80 is determined in the composition of monovaccines with great difficulties due to its chemical structure. Accordingly, when using this detergent in industrial production, serious problems may arise with the method of its detection in the composition of the obtained monovaccines and the finished dosage form, since the detergents included in the HVAC destruction system must be determined using production control methods that have passed the validation procedure (2, 14). This means that to carry out such controls, equipment that is sufficiently available to the relevant production units must be used. In particular, in order to detect traces of the TWIN-80 detergent in monovaccines, it is necessary to use a mass spectrometric detector, which, due to its high cost, is not available as a means of production control in the Russian Federation.

Disclosure of invention

The problem to which the present invention is directed is to create a new highly efficient method for the production of a tetravalent subunit influenza vaccine.

The technical results of the proposed invention are as follows:

1. Creation of a new technology for preliminary treatment of VAZ with the maximum reduced content of debris structures in the resulting filtrate.

2. Creation of a new technology for HVAC purification with the maximum reduced content of ovalbumin and other EC proteins.

3. Creation of a new technology for HVAC cleavage with detergents with increased technology efficiency at this stage for all serotypes of influenza viruses.

4. Creation of a new technology for the purification of a split HVAC preparation from subvirionic structures with a maximum decrease in turbidity indicators in monovaccine preparations.

5. Creation of a new highly effective technology for cleaning surface antigens from detergents with a maximum reduction in the content of detergents in the preparations of monovaccines obtained.

6. Creation of a new technology for preparing a dosage form of a tetravalent subunit vaccine without adjuvants.

The product obtained by the claimed method is a highly purified and highly immunogenic subunit vaccine with low rates of rectogenicity for vaccinated people.

The essence of the invention lies in the creation of a new method of production, which allows not only to obtain highly purified preparations of a four-valent subunit vaccine, but also allows to achieve a high indicator of the efficiency of the technology of the entire production process. The method includes obtaining vaccinated allantoic fluid (VAF), inactivation of VFA, preliminary purification of VFA from debris structures by three-stage microfiltration, concentration of VFA by ultrafiltration and final purification of virions by the method of sequential double ultracentrifugation in a sucrose density gradient. Then the purified viral concentrate (VVC) obtained in this way is sequentially treated with detergents Triton X-100 and TDTAB for digestion, subvirionic structures are removed from the suspension by ultracentrifugation and two-stage microfiltration. Removal of detergents from the resulting preparation is carried out using the methods of tangential ultrafiltration and industrial chromatography. Chromatography is carried out using a hydrophobic carrier "SDRHyperD", firm Pall (USA) using a special technology for the chromatographic process. The resulting purified preparation is diluted with a buffer containing detergent Triton X-100 as a means of disaggregation, after which a sterilizing filtration step is carried out using cartridges with a pore diameter of 220 nm. The resulting 4 monovalent of 4 different serotypes are combined into a single dosage form so that the final concentration of each monovalent is in the range from 25.6 to 34.4 μg / ml for the hemagglutinin protein. The detergent Triton X-100 is added to the resulting solution as a disaggregating additive, the solution is thoroughly mixed, subjected to sterilizing filtration, and then poured into sterile vials or special syringes. The use of the method according to the present invention ensures the production of a highly purified and highly immunogenic subunit vaccine, and also allows to increase the efficiency of the technology at various stages of the production process in comparison with prototypes.

The technical result is achieved by the fact that the cultivation of influenza viruses in EC, the selection of VAZ, inactivation of VAZ with a disinfectant beta-propiolactone is carried out. Preliminary purification of VFA is carried out by microfiltration using three cascades of folded filters, and HVAC production is carried out using two successive cycles of ultracentrifugation in a sucrose density gradient. The HVAC is cleaved with a mixture of Triton X-100 and TDTAB detergents in certain ratios. Removal of subvirionic structures after cleavage is carried out sequentially by ultracentrifugation and microfiltration using two cascades of folded filters. Purification of surface antigens from detergents is carried out sequentially by diafiltration and hydrophobic chromatography using an industrial chromatographic carrier. To preserve the specific activity during storage of the vaccine dosage form, a disaggregating detergent additive Triton X-100 is used.

Brief Description of the Drawing Figures

FIG. 1.presents an analysis of the polypeptide composition of serotype B / Phuket / 3073/2013 antigen samples obtained using the detergents Octylglucoside and TDTAB by electrophoresis under non-reducing conditions.

The positions in FIG. one:

1 - standard of molecular weights of the company "Biorad";

2 - antigen sample obtained using detergent TDTAB in the ratio of protein: detergent (1: 1). Specific activity by ORID was not revealed;

3 - an antigen sample obtained using the detergent Octylglucoside protein: detergent in the ratio (1:15). Specific activity - 432 μg / ml;

4 - an antigen sample obtained using the detergent Octylglucoside protein: detergent in the ratio (1:10). Specific activity - 451 μg / ml;

5 - a sample of the original purified viral concentrate (VVC) without detergent treatment. Specific activity according to ORID is 514 μg / ml.

FIG. 2 shows the analysis of the polypeptide composition of samples of serotype B / Brisbane / 60/2008 antigens obtained using the detergents Octylglucoside and TDTAB by electrophoresis in non-reducing conditions.

The positions in FIG. 2:

1 - standard of molecular weights from Bio-Rad;

2 - antigen sample obtained using detergent TDTAB in the ratio of protein: detergent (1: 1). No specific activity was found.

3 - sample of antigen obtained using detergent TDTAB in the ratio of protein: detergent (1: 0.8). Specific activity - 223 μg / ml (pale rings);

4 - sample of antigen obtained using the detergent Octylglucoside in the ratio of protein: detergent (1:15). Specific activity - 317 μg / ml;

5 - sample of antigen obtained using the detergent Octylglucoside in the ratio of protein: detergent (1:10). Specific activity - 286 μg / ml;

6 - the original sample of HVAC without the addition of detergents. The specific activity is 362 μg / ml.

FIG. 3 shows the analysis of the polypeptide composition of antigen samples of serotype B / Maryland / 15/2016 using detergents TDTAB and Octylglucoside, by electrophoresis in non-reducing conditions.

The positions in FIG. 3:

1 - standard of molecular weights from Bio-Rad;

2 - antigen sample obtained using detergent TDTAB in the ratio of protein: detergent (1: 0.4). Specific activity - 191 μg / ml;

3 - sample of antigen obtained using detergent TDTAB in the ratio of protein: detergent in the ratio (1: 0.5). Specific activity - 273 μg / ml;

4 - sample of antigen obtained using detergent TDTAB in the ratio of protein: detergent (1: 0.6). Specific activity - 224 μg / ml;

5 - antigen sample obtained using detergent TDTAB in the ratio of protein: detergent (1: 0.8). No specific activity was found;

6 - an antigen sample obtained using the detergent Octylglucoside in the ratio of protein: detergent (1:10). Specific activity - 354 μg / ml;

7 - a sample of antigen obtained using the detergent Octylglucoside in the ratio of protein: detergent (1:12). Specific activity - 348 μg / ml;

8 - antigen sample obtained using the detergent Octylglucoside in the ratio of protein: detergent (1:15). Specific activity - 338 μg / ml;

9 - A sample of the original HVAC without treatment with detergents. The specific activity is 368 μg / ml.

FIG. 4 shows the analysis of the precipitation ring morphology in the ORID reaction of serotype B / Brisbane / 60/2008 antigen samples obtained using the detergents Octylglucoside, TDTAB and TDTAB Plus.

The positions in FIG. 4:

1 - an antigen sample obtained using a mixture of detergents in the ratio of protein: TDTAB Plus (1: 1);

2 - international standard ORID;

3 - sample of antigen obtained using a mixture of detergents in the ratio of protein: TDTAB Plus (1: 0.8);

4 - a sample of antigen obtained using the detergent Octylglucoside in the ratio of protein: detergent (1:10);

5 - sample of antigen obtained using a mixture of detergents in the ratio of protein: DTAB Plus (1: 1);

6 - international standard ORID;

7 - sample of antigen obtained using a mixture of detergents in the ratio of protein: TDTAB Plus (1: 0.8);

8 - a sample of antigen obtained using the detergent Octylglucoside in the ratio of protein: detergent (1:10).

FIG. 5 shows an analysis of the polypeptide composition of antigens of serotype A / Michigan / 45/2105 (H1N1) obtained using detergents TBTAB Plus, TDTAB and Octylglucoside.

The positions in FIG. five:

1 - standard of molecular weights from Bio-Rad;

2 - sample of antigen obtained using detergent TDTAB Plus in the ratio of protein: detergent (1: 1). Specific activity - 503 μg / ml;

3 - sample of antigen obtained using detergent TDTAB Plus in a protein: detergent ratio (1: 0.8). Specific activity - 528 μg / ml;

4 - sample of antigen obtained using detergent TDTAB in the ratio of protein: detergent (1: 1). Specific activity - 490 μg / ml;

5 - an antigen sample obtained using the detergent Octylglucoside in the ratio of protein: detergent (1:15). Specific activity - 496 μg / ml;

6 - an antigen sample obtained using the detergent Octylglucoside in the ratio of protein: detergent (1:10). Specific activity - 480 μg / ml;

7 - sample of the original HVAC without the addition of detergents. The specific activity is 587 μg / ml.

FIG. 6 shows an analysis of the polypeptide composition of antigen samples of the A / HongKong / 4801/2014 (H3N2) certificate obtained using the detergents TDTAB, Octylglucoside and TDTAB Plus.

The positions in FIG. 6:

1 - standard of molecular weights from Bio-Rad;

2 - antigen sample obtained using detergent TDTAB in the ratio of protein: detergent (1: 0.8). Specific activity - 489 μg / ml;

3 - an antigen sample obtained using the detergent Octylglucoside in the ratio of protein: detergent (1:15). Specific activity - 637 μg / ml;

4 - antigen sample obtained using detergent TDTAB Plus in the ratio of protein: detergent (1: 0.8). Specific activity - 456 μg / ml;

5 - sample of antigen obtained using detergent TDTAB Plus in the ratio of protein: detergent (1: 1). Specific activity - 394 μg / ml;

6 - sample of the original HVAC without the addition of detergents. The specific activity is 663 μg / ml.

FIG. 7 shows the analysis of the polypeptide composition of serotype B / Brisbane / 60/2008 antigen samples obtained using the detergents Octylclucoside, TDTAB and TDTAB Plus by electrophoresis in non-reducing conditions.

The positions in FIG. 7:

1 - standard of molecular weights from Bio-Rad;

2 - antigen sample obtained using detergent TDTAB Plus in a protein: detergent ratio (1: 1). Specific activity - 316 μg / ml;

3 - sample of antigen obtained using detergent TDTAB Plus in a protein: detergent ratio (1: 0.8). Specific activity - 340 μg / ml;

4 - antigen sample obtained using detergent TDTAB (1: 1). No precipitation rings were found;

5 - antigen sample obtained using detergent TDTAB in the ratio of protein: detergent (1: 0.8). Specific activity - 255 μg / ml (pale rings);

6 - An antigen sample obtained using the detergent Octylglucoside in the ratio of protein: detergent (1:15). Specific activity - 336 μg / ml;

7 - an antigen sample obtained using the detergent Octylglucoside in the ratio of protein: detergent (1:10). Specific activity - 291 μg / ml;

8 - the original sample of HVAC without the addition of detergents; The specific activity is 384 μg / ml.

FIG. 8 shows an analysis of the polypeptide composition of serotype B / Phuket / 3073/2013 antigen samples obtained using the detergents Octylclucoside, TDTAB and TDTAB Plus by electrophoresis under non-reducing conditions.

The positions in FIG. 8:

1 - standard of molecular weights from Bio-Rad;

2 - sample of antigen obtained using detergent TDTAB Plus in the ratio of protein: detergent (1: 1). Specific activity - 481 μg / ml;

3 - sample of antigen obtained using detergent TDTAB Plus in a protein: detergent ratio (1: 0.8). Specific activity - 507 μg / ml;

4 - sample of antigen obtained using detergent TDTAB in the ratio of protein: detergent (1: 1). No precipitation rings were found;

5 - an antigen sample obtained using the detergent Octylglucoside in the ratio of protein: detergent (1:15). Specific activity - 428 μg / ml;

7 - an antigen sample obtained using the detergent Octylglucoside in the ratio of protein: detergent (1:10). Specific activity - 437 μg / ml;

8 - antigen sample without added detergents; The specific activity is 544 μg / ml.

FIG. 9 shows the analysis of the polypeptide composition of OVK samples of serotype B / Maryland / 15/2016 treated with detergents TDTAB and TDTAB PLUS by electrophoresis in non-reducing conditions.

The positions in FIG. nine:

1 - standard of molecular weights from Bio-Rad;

2 - antigen sample obtained using detergent TDTAB in the ratio of protein: detergent (1: 0.4). Specific activity - 252 μg / ml;

3 - antigen sample obtained using detergent TDTAB in the ratio of protein: detergent (1: 0.5). Specific activity - 280 μg / ml;

4 - sample of antigen obtained using detergent TDTAB in the ratio of protein: detergent (1: 0.6). Specific activity - 242 μg / ml;

5 - antigen sample obtained using detergent TDTAB in the ratio of protein: detergent (1: 0.8). No precipitation rings were identified;

6 - sample of antigen obtained using detergent TDTAB plus in the ratio of protein: detergent (1: 1); Specific activity - 359 μg / ml;

7 - sample of antigen obtained using detergent TDTAB plus in the ratio of protein: detergent (1: 0.8); Specific activity - 365 μg / ml;

8 - antigen sample obtained using detergent TDTAB plus in the ratio of protein: detergent (1: 0.7); Specific activity - 354 μg / ml;

9 - sample of HVAC serotype B / Maryland / 15/2016 without detergent treatment. The specific activity is 368 μg / ml.

FIG. 10 shows the analysis of the morphology of the antigen HA serotype B / Brisbane / 60/2008 by the METHOD of electron microscopy, the size of the comparison ruler -100 mn.

FIG. 11 - analysis of the morphology of the monovaccine serotype A / HongKong / 4801/2014 (H3N2) by electron microscopy, the size of the comparison ruler is 100 nm.

FIG. 12 shows the analysis of the polypeptide composition of the experimental monovaccine A / HongKong / 4801/2014 (H3N2) by electrophoresis in non-reducing conditions.

The positions in FIG. 12:

1 - HVAC sample from which the experimental monovaccine was obtained;

2 - a sample of an experimental monovaccine obtained using the detergent TDTAB Plus;

3 - standard of molecular weights from BioRad.

FIG. 13 shows the analysis of the polypeptide composition of the experimental monovaccine B / Brisbane / 60/2008 by electrophoresis in non-reducing conditions.

The positions in FIG. 13:

1 - standard of molecular weights from BioRad;

2 - a sample of an experimental monovaccine obtained using the detergent TDTAB Plus;

3 - HVAC sample from which the experimental monovaccine was obtained.

FIG. 14 shows polypeptide composition analysis of experimental monovaccine A / Michigan / 45/2015 (H1N1) by electrophoresis under non-reducing conditions.

The positions in FIG. 14:

1 - standard of molecular weights from BioRad;

2 - a sample of an experimental monovaccine obtained using the detergent TDTAB Plus;

3 - HVAC sample from which the experimental monovaccine was obtained.

Examples of implementation of the invention

Example 1 discloses the optimization of the processes of preliminary purification of VFA and purification of HVA in the density gradient of sucrose and shows the achievement of the technical result - the creation of a new technology for preliminary purification of VFA with the most reduced content of debris structures in the resulting filtrate and the creation of a new technology for purification of HVA with the most reduced content of ovalbumin and other proteins CE.

On the basis of the search for technological approaches, it was concluded that in order to increase the degree of purification of VAZ from debris structures during microfiltration, it is necessary to change both the type of filters themselves and the pore size of these filters. The fact is that the depth filters, which were used at the stage of microfiltration of VAZ in the source (7), have a high dirt holding capacity, but they have a very wide scatter of pore diameters (relative transmission rating). For this reason, these depth filters cannot be selected in such a way that they retain debris structures as much as possible, but at the same time do not delay the passage of virion particles. Therefore, to optimize the VAZ microfiltration process, it was decided to use a fundamentally different type of filters, namely folded filters.

Example 1.

The use of three cascades of folded filters with different pore diameters made it possible to carry out the process of microfiltration of VFA in such a way that practically no debris structures remained in the final filtrate of VAZ, however, the retention of virion particles during microfiltration did not occur.

To increase the degree of purification of OVK from ovalbumin and other EC proteins, the use of the technology of double sequential ultracentrifugation in a sucrose density gradient turned out to be the most optimal. It should be noted that, within the framework of this technology, in order to reduce the level of aggregation of virion particles during the first cycle of ultracentrifugation, it turned out to be optimal to introduce disaggregating additives into the first sucrose gradient, namely sodium citrate. During the second cycle of ultracentrifugation, this reagent was separated from the virion particles due to its chemical structure.

Two technological experiments demonstrate the effectiveness of the above new approaches. For these experiments, 1010 liters of A / Michigan / 45/2015 (H1N1) VAF were obtained using 108 thousand EC. This VAZ volume was divided into two equal parts.

The first part of the VAZ (510 liters) was used to conduct experiment No. 1, in which the first stage of obtaining HVAC was carried out according to the method indicated in the source (7). In this experiment No. 1, microfiltration of VAS was carried out through two cascades of depth filters with pore diameters of 5 μm and 1 μm, the process of ultracentrifugation in a sucrose density gradient was performed once without adding sodium citrate.

In experiment # 2, another 510 liters of VAH were used. In this experiment, three successive cascades of folded filters with pore diameters of 10 μm, 1.2 μm and 0.6 μm were used for the microfiltration process. The process of ultracentrifugation in a sucrose density gradient was carried out twice, while the composition of the first gradient included a sodium citrate solution to a final concentration of 0.2 M. Samples of two HVAC obtained as a result of the above experiments were analyzed for total protein content. Further, two independent experiments were carried out to obtain subunit monovaccines using the detergent TDTAB according to the method indicated in another source (3). The resulting experimental monovaccines were analyzed for total protein content, specific activity and ovalbumin content. Based on the results obtained, such indicators were calculated as the efficiency of the technology at the stage of obtaining monovaccines (the amount of GA obtained from 1 EC); the relative content of ovalbumin per 100 μg of HA antigen in each of the monovaccines and the ratio of the total protein content to the HA content. The data are presented in Table 1.

From the presented data, it follows that the use of new technological approaches at the first stage in the process of obtaining HVAC made it possible to significantly improve the quality of monovaccines that were obtained from these HVACs in the course of the further technological process. It is important that the use of these new technological approaches practically did not lead to a decrease in such an important indicator of the technological process as the efficiency of the technology, but at the same time such important indicators of the purification of monovaccines as a decrease in the content of ovalbumin and a decrease in the relative load of total protein improved significantly.

In the world literature, the quality of purification of inactivated influenza vaccines is compared precisely according to these indicators to assess the potential reactogenicity. In addition, the ratio of the total protein content to the HA content is of additional importance due to the fact that it is for this indicator that vaccines are divided into split and subunit (2, 14). Thus, the proposed new approaches made it possible to significantly optimize the first stage of the technology (HVAC production) and, as a result, to obtain monovaccine preparations with higher quality indicators.

Example 2.

Effect of detergent TDTAB on GA antigens of influenza viruses of various serotypes

This example shows the achievement of such a technical result as the creation of a new technology for the cleavage of HVAC with detergents with an increased efficiency of the technology at this stage for all serotypes of influenza viruses.

Further, studies were carried out to study the process of cleavage of the OVK of various serotypes of influenza viruses using the detergent TDTAB. To carry out these studies, HVAC preparations were used, obtained according to the new technological approaches described in example 1. The degradation process was carried out according to the method indicated in the source (3). The method of studying this effect was expanded in comparison with the method described in the source. We studied not only the efficiency of cleavage, but also studied the polypeptide composition of the samples after appropriate treatment with OVK and removal of subvirionic structures. The study of the polypeptide composition of HA samples makes it possible to conclude that the conformational structure of this antigen is preserved during the action of the detergent. The objects of study were taken samples of OVK, which were obtained using industrial strains of the last few years of all three serotypes (H1N1, H3N2 and B). To make sure that the results obtained are related specifically to the effect of the detergent TDTAB, in a number of experiments the detergent Octylglucoside was used as a comparison detergent, which is used to isolate HA in the production technology of the Ultrix split vaccine.

At the first stage of the research, it was revealed that the detergent TDTAB did not have any destructive effect on HA antigens of serotypes H1N1 and H3N2. However, in the course of experiments with HVAC obtained with the use of influenza serotype B virus, facts of a decrease in the indicator of the effectiveness of the technology at the stage of obtaining monovaccines were revealed. It was found that in the ORID reaction, serotype B HA antigens formed pale colored precipitation rings with blurred edges, in contrast to precipitation rings, which were formed in the same reaction with samples of the original OVA or an international antigen standard (produced by NIBCS). As indicated above, in these experiments, the polypeptide composition of HA samples after treatment with TDTAB was also studied. This allowed us to make a conclusion about the state of the conformational structure of the HA antigen in the sample. It is known from the literature that if an HO polypeptide in a preparation is spontaneously cleaved into HA1 (heavy chain) and HA2 (light chain) polypeptides, then the conformational structure of this antigen no longer corresponds to that which should be in an intact virion preparation. A change in the native conformational structure of the HA antigen indicates that the immunochemical structure of this antigen no longer corresponds to that which this antigen had in the composition of the virion preparation before treatment with detergents (9). The results are presented in Figures 1-3.

From the presented results, it can be concluded that samples of HA antigens obtained using three industrial strains of serotype B did indeed have a sensitivity to the denaturing effect of the detergent TDTAB. In experiments where the ratio of total RVA protein to TDTAB detergent was 1: 1, precipitation rings were not detected in the ORID reaction when analyzing GA antigens of all three serotypes. In experiments in which samples of OVK serotype B / Brisbane / 60/2008 were treated with detergent TDTAB in the ratio (1: 0.8), the specific activity was determined, however, significantly less compared to experiments using the detergent Octyl glucoside. When processing HVAC samples obtained using two other industrial strains of serotype B, treatment with detergent TDTAB in the ratio (1: 0.8) led to a complete loss of specific activity of the HA antigen in these samples. For serotype B HVA, treatment with TDTAB detergent in the ratios (1: 0.4), (1: 0.5), (1: 0.6) and (1: 0.7) allowed the production of HA antigens in which it was possible determine the specific activity. But in the reaction of ORID with these antigens, the precipitation rings were pale and with blurred edges, which made it difficult to determine the specific activity of the HA antigen in these samples.

Since the detergent used in these experiments was the detergent Octylglucoside, which did not have a denaturing effect on these samples, it can be argued that the denaturation of HA antigens during the treatment of serotype B OVA occurred precisely from the action of the detergent TDTAB. As follows from this study, at any ratios, the efficiency of degradation using the detergent TDTAB was significantly lower than the efficiency of degradation of the same HVA samples using the detergent Octyl glucoside.

Analysis of the polypeptide composition of antigen samples obtained using the detergent TDTAB revealed the phenomenon of spontaneous cleavage, in which the HA0 molecule is cleaved into HA1 and HA2 polypeptides without any outside influence. When Octylglucoside was used for cleavage, the phenomenon of spontaneous cleavage was not observed. Since this cleavage indicates a change in the conformational structure of HA, then, according to the literature data, it can be concluded that the immunochemical structure of the HA antigen also changed (9). Thus, the use of the detergent TDTAB for the production of monovaccines of serotype B is not optimal due to the low efficiency of the technology, and also because of the risk of the appearance of HA antigens with altered immunochemical structure.

It should be noted that those industrial strains of serotype B, which were used in this study to obtain HVAC samples, were the strains that were included in the inactivated influenza vaccines in recent years according to WHO recommendations. This means that the recently recommended WHO production strains of serotype B were susceptible to the denaturing effect of the detergent TDTAB.

Example 3.

Development of new technological approaches to optimization of the HVAC splitting stage

This example shows the achievement of such a technical result as the creation of a new optimal technology for the cleavage of the OVK preparation with various detergents to achieve maximum preservation of the immunochemical activity of the obtained surface antigens and increase the efficiency of the production process at this stage.

As a result of the experiments, it was revealed that the optimal technology for the stage of HVAC cleavage is the sequential use of non-ionic detergent Triton X-100 and detergent TDTAB. It was found that changes in the ratios of total protein to these detergents did not affect the immunochemical nativeness of the obtained HA antigens (Fig. 4).

It has also been shown that the sequence of addition of detergents and the incubation time are very important. First, the detergent Triton X-100 should be added and reacted with the HVAC for at least 20 minutes, and then the detergent TDTAB should be added. The second incubation should take place within 40 minutes. The system of detergents thus developed was named TDTAB Plus.

Example 4.

Effect of the TDTAB Plus detergent system on HA antigens of influenza viruses of serotypes H1N1, H3N2 and B

The analysis of samples of surface antigens obtained using the detergent system TDTABPlus was carried out by the same methods as in example 2. For greater illustration of the material, samples of HA antigens obtained using detergents TDTAB Plus, TDTAB and Octylglucoside were simultaneously studied. This allowed us to evaluate the comparative efficiency of the degradation of OVK using three different detergents (see Figures 5-9).

As a result of the study of the comparative effect of three different detergents on the HA antigens of different serotypes of influenza viruses, data were obtained, based on which the following conclusions can be drawn:

1. Detergents TDTAB Plus, TDTAB and Octylglucoside act differently on HA serotypes H1N1 and H3N2hB antigens. This difference is manifested in the percentage of cleavage, the morphology of the precipitation rings in the ORID reaction, and in the polypeptide composition, which was studied by electrophoresis under non-reducing conditions.

2. Detergents TDTAB Plus and Octylglucoside affected the GA antigens of all three serotypes in such a way that the native conformational (immunochemical) structure of these antigens did not change. This was expressed in the fact that the obtained HA antigens in the ORID reaction formed precipitation rings, the morphology of which did not differ from the morphology of the precipitation rings with samples of international standard antigens, as well as samples of the original OVK. Analysis of the polypeptide composition of these antigens did not reveal the phenomenon of spontaneous cleavage of HA0 molecules.

3. The detergent TDTAB had a similar effect on the HA serotypes H1N1 and H3N2 antigens, as did the other two detergents. However, the study of the effect of the detergent TDTAB on serotype B antigens revealed the fact that this detergent, in the process of cleavage, has a destructive effect on the HA antigens of four industrial strains of serotype B influenza viruses. This was expressed in the fact that at low ratios of protein: detergent (1: 0 , 4), (1: 0.5) and (1: 0.6), the obtained antigen samples in the ORID reaction formed pale colored precipitation rings with blurred edges. At higher ratios of protein: detergent (1: 1), the thus obtained HA antigens did not form precipitation rings in the ORID reaction. When studying the polypeptide composition of HA antigen samples obtained using the detergent TDTAB in various ratios, the phenomenon of spontaneous cleavage was observed. This indicates that some of the HA antigen molecules have passed from the so-called "prefusion" form to the "postfusion" form with corresponding large changes in the conformational and immunochemical structure (9).

4. As a result of the studies carried out, it can be concluded that with viruses of the H1N1 serotype, detergent TDTAB Plus gives the maximum percentage of degradation (90%) compared to the other two detergents.

5. With viruses of the H3N2 serotype, the detergent Octylglucoside showed the maximum percentage of cleavage (96%).

6.With viruses of serotype B (B / Brisbane / 60/2008, B / Phuket / 3073/2013 and B / Maryland / 15/2016), the action of the TDTAB Plus detergent system allowed to achieve higher percentages of degradation (93%, 93% and 99 %) compared to the action of the detergent Octylglucoside (87%, 80%).

7. Despite the fact that the detergent TDTAB had a destructive effect on the HA antigens of all studied industrial strains of serotype B in the process of cleavage, the degree of its destructive effect depended on the specific production strain and the ratio of protein to detergent. In particular, at a protein: detergent ratio (1: 0.8), the isolated antigens of serotype B / Brisbane / 60/2008 in the ORID reaction formed precipitation rings, and GA antigens of other studied B serotypes isolated using the detergent TDTAB in the protein-detergent ratio (1 : 0.8), no precipitation rings were formed.

8. Based on the above conclusions, we can conclude that detergents TDTAB Plus and Octylglucoside from the point of view of the process of obtaining monovaccines are universal for the serotypes of influenza viruses H1N1, H3N2 and B (Victoria and Yamagato lines), which are included in the "seasonal" vaccines.

At the same time, the use of detergent TDTAB Plus makes it possible to obtain a higher technology efficiency for 3 out of 4 serotypes included in tetravalent vaccines as compared to the use of Octylglucoside detergent.

The use of the detergent TDTAB for the production of monovaccines of serotypes Outside is optimal, since its use leads to an underestimation of the technology efficiency at the stage of cleavage of OVA, and can also lead to a change in the immunochemical structure of isolated HA antigens.

9. Thus, for the production of tetravalent subunit vaccines, the most optimal is the use of detergent TDTAB Plus.

Example 5.

Development of technology for purification of viral antigens from detergent TDTAB Plus and subvirionic structures.

This example demonstrates the achievement of technical results - the creation of new optimal technologies for cleaning surface antigens from the detergent TDTAB Plus and subvirionic structures.

The source (3) described the technology for cleavage of OVK using a single detergent TDTAB and the subsequent technology of the antigen purification scheme after cleavage. In this method, subvirionic structures are removed from the suspension only by ultracentrifugation. Then the supernatant is concentrated by tangential ultrafiltration using membranes with a pore diameter of 30 KD, after which the concentrated supernatant is subjected to a long-term diafiltration process with a large volume of buffer for purification from TDTAB. After the concentration of the detergent TDTAB in the solution is lower than 10 μg per 50 μg of HA antigen, the diafiltration process is stopped, the resulting suspension is diluted with a buffer without any additives and subjected to a sterilizing filtration process to obtain a monovaccine.

When using the detergent system TDTAB Plus to obtain monovaccines according to example 4, the above technology for the purification of surface antigens (3) did not give adequate results. The fact is that in aqueous solutions the detergent TDTAB forms micelles of small size, which can be separated from surface antigens by diafiltration using a membrane with a pore diameter of 30 KD. However, the detergent Triton X-100, which is part of the TDTAB Plus detergent system, forms very large micelles in aqueous solutions, which do not pass through a membrane with a pore diameter of 30 KD. This means that it is not possible to separate Triton X-100 micelles from the HA antigen using only one diafiltration method. It should be noted that as a result of complete removal of detergents, HA antigens are collected in supramolecular structures that are highly immunogenic (13). Thus, in order to obtain vaccines with a high protective potential, it is necessary that in the process of isolating monovaccines the process of self-assembly of GA into these supramolecular structures occurs. If, in the process of obtaining a monovaccine, a large amount of detergent remains in the suspension, then the self-assembly process will be blocked.

In order to ensure the production of highly purified HA antigens with high immunogenicity, it was necessary to develop effective technologies for the purification of surface antigens both from subvirionic structures and from a mixture of detergents Triton X-100 and TDTAB ..

After performing a series of experiments, a complex technology for obtaining monovaccines was developed, using which the self-assembly process was efficient. This technology consisted of the following stages:

1. Flow-through ultracentrifugation at a speed of 20 thousand rpm for three hours.

2. Sequential microfiltration of the obtained supernatant through two cascades of folded filters with pore diameters of 0.6 µm and 0.2 µm.

3. Ultrafiltration using membranes with a cut-off size of 30 KD and concentration of the resulting purified supernatant to a volume of 3-4 liters.

4. Chromatography of the resulting concentrate solution using a hydrophobic carrier "SDRHyperD" manufactured by Pall (USA) and a column "Streamline 200" manufactured by GE-Healthcare (USA) by the "Expandedbedadsorption" method. The use of this carrier, as well as this chromatography method, made it possible to dramatically increase the efficiency of the detergent removal process and obtain monovaccine preparations in which the content of the detergent TDTAB did not exceed 5 μg / ml, and the content of the detergent Triton X-100 was below the limit of the method. This is due to the fact that when using chromatography according to the "Expandedbedadsorption" method, the flow of material goes from bottom to top, and the chromatographic carrier passes into a suspension state, which makes it possible to increase the contact time of the liquid with the sorbent surface.

5. After chromatography and evaluation of the protein content in the obtained preparation, the preparation was diluted with a buffer containing detergent Triton X-100 as a disaggregating additive in concentrations from 200 to 300 μg / ml.

6. The solution thus diluted was subjected to sterilizing filtration through a membrane with a pore diameter of 220 nm.

Compared to the diafiltration process indicated in the source (3), the combined purification process by microfiltration, concentration and chromatography took half the time.

As an illustration of the fact that when using the proposed purification technology, HA antigens are actually collected in supramolecular structures, the results of the analysis of morphology by electron microscopy of samples of monovaccines of serotypes B and H3N2 are presented.

The analysis was carried out using the negative contrast technique using reagentauranyl acetate and using a Jeol-100B electron microscope. The results were obtained using a magnification of 50 thousand times.

In the process of studying each of the preparations, at least two hundred fields of view were examined and the most typical morphological structures were selected. The 100 nanometer bar at the bottom of each figure is shown as a comparative size.

The presented Figures 10 and 11 show that typical morphological structures of the studied subunit monovaccines are specific aggregates of HA molecules, which have an irregular spherical shape and a size of 25 to 40 nm. In the structure of these aggregates, separate morphological structures of GA trimers, the so-called "spines", are revealed.

Thus, it follows from the presented results that the proposed purification technology really allows the removal of detergents from the drug solution in such a way that the self-assembly of HA antigens occurs completely. It should be noted that after the self-assembly of HA antigens has already occurred, the addition of the detergent Triton X-100 at concentrations of 200-300 μg / ml is already unable to destroy the formed supramolecular structures. According to the literature (12), the detergent Triton X-100 in such concentrations acts only as a disaggregating additive and does not affect the morphological structure of the HA antigen in virions or other subviral structures.

Analysis of the commercial split vaccine Fluzone, manufactured by SANOFI-PASTER, revealed similar morphological forms of organization of HA molecules (13). It should be noted that the authors of this publication had a higher image clarity in the figures presented by them, since they used a more modern technique of cryogenic electron microscopy for analysis. This information confirms our results in the part that in the composition of commercial vaccines the HA antigen forms aggregates of approximately the same size and morphology as in the figures presented by us.

According to the literature, the vaccine "Fluzone" contains the detergent Triton X-100 at a concentration of 200 μg / ml as a disaggregating additive. This confirms our observation that the detergent Triton X-100 in this concentration as a disaggregating additive does not affect the morphology of the supramolecular HA structures after their self-assembly.

Example 6.

Obtaining monovaccine serotype H3N2 according to the claimed method

Examples 1, 3, 5 describe new technological approaches at the stages of preliminary purification of VFA, obtaining HVA, cleavage of HVA by the TDTAB Plus detergent system, purification of the cleaved HVA from subvirionic structures and purification of isolated surface antigens from the TDBAB Plus detergent system. Based on these new technological approaches, a new method for producing subunit monovaccines has been developed. To illustrate the suitability and high efficiency of the claimed method, the isolation of monovaccines of serotype H3N2 (example 6), serotype B (example 7) and serotype H1N1 (example 8) was carried out.

EC at the age of 10 days in the amount of 45 thousand pieces were obtained from a poultry farm and subjected to visual control for the purpose of rejection. Then they were disinfected upon entering the shop in two batches of disinfectant with a 2% solution of "Oxonium Active" for 30 minutes at a temperature (+34 C °). Next, the disinfected CEs were transported to the pre-incubation chamber. Incubation took place in the temperature range (from +36 to +37 C °), relative humidity 55-65% for 16 hours. After that, TBE was infected with the seventh passage level of the reassortant strain of serotype A / HongKong / 4801/2014 (H3N2). Infection was carried out using an automatic infection line manufactured by Technolabo (Italy). The infection process with 45 thousand CE took place within 2 hours. After infection, CEs were moved to a thermal incubation chamber with a temperature (+ 36.5 C °) and a relative humidity of 65%. The incubation lasted for 44 hours, after which the incubated CEs were transported to a special refrigerating chamber with a temperature (from +3 to +5 C °). The cooling of the FE was carried out for 16 hours, after which the cooled FE was fed to an automatic line for the selection of VAZ manufactured by Technolabo (Italy). After appropriate rejection, 42 thousand FE were used for the selection of VAZ. The total volume of the collected VAZ was 420 liters. The collection of VAS took place in a reactor with a capacity of 630 liters. Before the start of the collection of VAZ, this reactor was cooled to a temperature (from +5 to +8 C °). The total collection time for the VAZ was 4 hours. After the end of the collection of VAS, 40 liters of a sterile solution of 2 molar NaCl were added to the reactor and the contents were stirred for 15 minutes. Next, disinfectant beta-propiolactone was added to the reactor to a final concentration in the reactor of 0.1% and the contents were stirred for another 30 minutes. Then the stirrers were turned off, and the contents of the reactor were incubated at a temperature (from +5 to +8 C °) for 16-20 hours. After treatment with beta-propiolactone, subsequent operations with the obtained VAZ were carried out in the so-called "clean zone". Preliminary purification of the VAZ was carried out using three cascades of folded filters with pore sizes of 10 μm, 1.0 μm, and 0.6 μm manufactured by Domnic Hunter (USA). In parallel with the microfiltration process, the concentration process was carried out using the method of tangential ultrafiltration on membranes with a pore diameter of 300 KD. This entire stage was carried out at room temperature. The processes of preliminary purification and concentration of 440 liters of VAZ were carried out within 3 hours. The total volume of the obtained VAZ concentrate was 30 liters.

Further, the VAZ concentrate was purified in a sucrose density gradient in two stages. For this purpose, we used industrial centrifuges, model EK2, manufactured by Alfa Wassermann (USA). At the first stage, sucrose density gradients were used, which included sodium citrate at a final concentration of 0.2 M. The selection of fractions after the ultracentrifugation process was carried out in the range of sucrose concentrations from 32 to 50% and with a HA titer in the fraction of 1: 16000 and more. The fractions collected after the first gradients were diluted with phosphate buffered saline (PBS) and the purification on a sucrose gradient was repeated once more. The second sucrose gradient was prepared using PBS without any other additives. The whole process of centrifugation and selection of fractions, both in the first and in the second case, took place within 7 hours, the speed of centrifugation was 32-34 thousand rpm with cooling (from +3 to +5 C °). After the second centrifugation, the selection of fractions was carried out in a more stringent range from 34 to 48% sucrose concentration and a HA titer of at least 1: 64000. From a volume of 30 liters of concentrated VAF, 500 ml of HVA in sucrose was obtained after the second cycle of ultracentrifugation. Aliquots of the obtained fractions of the second sucrose gradient were pooled and their protein content was determined according to Lowry. The total protein content in 500 ml of HVAC was 16 grams. The HVAC fractions were stored at a temperature (from +3 to +6 C °) for several days for the subsequent receipt of a monovaccine.

To isolate the monovaccine, these fractions were diluted with PBS, mixed thoroughly, and a solution of the Triton X-100 detergent was added to its total amount in the reaction of 24 grams (the ratio of total protein to detergent was 1: 1.5). After that, the resulting solution was thoroughly mixed for 20 minutes, after which the detergent TDTAB was added to it so that its total amount in the final solution was 16 grams (the ratio of total protein to detergent TDTAB was 1: 1). After adding the second detergent, the solution was thoroughly mixed for another 40 minutes, after which it was transferred to the ultracentrifugation stage. The total volume of the solution was 12 liters. Ultracentrifugation was carried out in an EK2 centrifuge manufactured by AlfaWassermann (USA). Ultracentrifugation took place on a flow-through rotor at 20,000 rpm. within 3 hours at a liquid flow rate through the rotor of 10 l / h. After the end of centrifugation, the resulting supernatant was transferred to the next purification step. This stage of purification was carried out by the method of two-stage microfiltration using folded filters with pore diameters of 0.6 µm and 0.2 µm manufactured by DomnicHunter (USA). After the end of microfiltration, the cartridges were washed with buffer, after which the wash solution and filtrate were combined and concentrated by tangential ultrafiltration with membranes with a pore diameter of 30 KD. In this experiment, the volume of the supernatant, as well as the volume of solutions after washing the rotor and cartridges, was 16 liters; after concentration, this volume was 4 liters. The final purification of surface antigens was performed by industrial chromatography using a hydrophobic SDRHyperD carrier manufactured by Pall (USA). This carrier works on the principle of hydrophobic interactions and is able to remove practically all detergents with hydrophobic chains from aqueous solutions. To reduce the time of chromatography, a special technological approach “ExpandedBedAdsorbtion” was used, the essence of which is that the preparation for purification is supplied from the bottom of the column, and the upper layer of the hydrophobic carrier is not fixed. To implement this approach, a special column "StreamLine 200" manufactured by GE-Healthcare (USA) was used. The volume of the media in this column was 3.5 liters. This column was loaded with 4 liters of concentrated supernatant and circulated the solution five times through this volume of carrier at a rate of 10 liters / hour. After the fifth cycle, a sample was taken and it was verified that the content of the detergent TDTAB was 11 μg / ml. The entire process of purification of surface antigens, from microfiltration to chromatographic purification, took place at room temperature and took 3 hours. The result was a concentrate with a total protein content of 9.0 grams in a volume of 5 liters (with wash buffer). This volume of concentrate was diluted to 25 liters with PBS buffer containing Triton X-100 detergent to a final concentration of 300 μg / ml. The resulting 25 liters of solution was thoroughly mixed in a sterile reactor with a stirrer, after which sterilizing filtration was carried out through a sterile cartridge with a pore size of 0.22 μm from Millipore (USA). Filtration was carried out in a sterile disposable bag manufactured by HyClone (USA). The volume of the filtered liquid was 23 liters. Samples were taken from the obtained solution of the monovaccine for control according to the specification specified in the RF State Pharmacopoeia XIV. According to the results obtained, the total protein content in the monovaccine was 7.5 grams, and the total HA antigen content was 2.7 grams. The concentration of the detergent TDTAB in the resulting monovaccine solution was determined at 2.5 μg / ml. The level of technology efficiency at the monovaccine stage in this experiment was 64 μg of GA with 1 EC. The polypeptide composition of the sample of the obtained experimental monovaccine is shown in FIG. 12.

From the presented data, it follows that the main polypeptide in the experimental monovaccine is the HA0 polypeptide (an uncleaved form of the HA antigen). The nucleocapsid polypeptide (NP) is present in trace amounts compared to the relative content of the nucleocapsid polypeptide in the OVAC preparation from which the experimental monovaccine was obtained. The membrane protein polypeptide (M1) was not detected in the monovaccine. The presence of HA1 (HA antigen heavy chain) and HA2 (HA antigen light chain) polypeptides was practically not detected, which indicates the absence of the phenomenon of spontaneous cleavage during the isolation of the monovaccine.

Example 7.

Obtaining monovaccine serotype B (Victoria line) according to the claimed method

To obtain a monovaccine of serotype B (Victoria line), 44 thousand pcs were used. CE. Inoculum virus of the 8th passage level of serotype B / Brisbane / 60/2008 was used to infect TBE. The procedures for processing embryos, pre-incubation and infection with CE virus were carried out as in example 6. However, the incubation of CE after infection was carried out at a temperature (+32, 5 C °) for 68 hours. After the incubation and cooling step, 410 liters of VAF were collected, which were obtained in a volume of 30 liters after appropriate processing, purification and concentration. As a result, after two cycles of sucrose density gradient ultracentrifugation, 6 gradient fractions with a total protein content of 14 grams were collected. Isolation of monovaccines was carried out as described in example 6, however, the ratio of total protein: detergent Triton X-100: detergent TDTAB was chosen as (1: 1.2: 0.8). The volume of destruction was 14 liters. After the ultracentrifugation and microfiltration step, together with the wash buffer, the volume was 22 liters, which were concentrated to a volume of 5 liters. To purify the resulting concentrate, four circulation cycles were carried out at a rate of 10 l / h, after which the content of the detergent TDTAB reached a level of 4 μg / ml. The total protein content was 7 grams. This solution was diluted to 20 liters with PBS buffer containing Triton X-100 detergent. As a result, 19 liters of monovaccine were obtained. The total protein content in the monovaccine was 7.2 grams, the total HA content was 2.5 grams. The efficiency of the technology in this experiment was 56 μg of HA per 1 pc. CE. The detergent content TDTAB was 2 μg / ml. The polypeptide composition of a sample of the obtained experimental monovaccine is shown in FIG. 13.

From the presented data, it follows that the main polypeptide in the experimental monovaccine is the HAO polypeptide (an uncleaved form of the HA antigen). The nucleocapsid polypeptide (NP) is also found in the monovaccine, but its comparative content with respect to the HAO polypeptide is less than in the OVA preparation from which the experimental monovaccine was obtained. The membrane protein polypeptide (M1) was not detected in the monovaccine. The presence of HA1 (HA antigen heavy chain) and HA2 (HA antigen light chain) polypeptides was practically not detected, which indicates the absence of the phenomenon of spontaneous cleavage during the isolation of the monovaccine.

Example 8.

Obtaining monovaccine serotype B (Yamagato line) according to the claimed method

To obtain monovaccines of serotype B (Yamagato line), 46 thousand pcs were used. CE. To infect TBE, a serotype B / Phuket / 3073/2013 virus at the 10th passage level was used as an inoculum. The procedures for processing embryos, pre-incubation and the process of infecting the CE with the virus were carried out as in examples 6 and 7. However, the incubation of CE after infection was carried out at a temperature (+33.0 C °) for 64 hours. After the stage of incubation and cooling, 480 liters of IAF were collected, which after appropriate processing, purification and concentration were obtained in a volume of 35 liters. After two cycles of sucrose density gradient ultracentrifugation, 7 gradient fractions with a total protein content of 16 grams were collected. Isolation of the monovaccine was carried out as described in example 7.

The ratio Total protein: detergent Triton X-100: detergent TDTAB was chosen (1: 1.2: 0.8), as in example 7. The volume of destruction was 13 liters. After the ultracentrifugation and microfiltration step, together with the wash buffer, the volume was 21 liters, which were concentrated to a volume of 4 liters. To purify the resulting concentrate, five circulation cycles were carried out at a rate of 10 l / h, after which the content of the detergent TDTAB in the solution reached a level of 5 μg / ml. The total protein content was 7.9 grams. This solution was diluted to a volume of 22 liters with PBS buffer containing Triton X-100 detergent. As a result, 21 liters of monovaccine were obtained. The total protein content in the monovaccine was 7.7 grams, the total HA content was 2.8 grams. The efficiency of the technology in this experiment was 68 μg of HA per 1 pc. CE. The detergent content of TDTAB in the monovaccine solution was 2 μg / ml. The polypeptide composition of the sample of the obtained experimental monovaccine was similar to that shown in FIG. 13 (example 7).

Example 9.

Obtaining monovaccine serotype H1N1 according to the claimed method

To obtain a monovaccine of serotype H1N1, 48 thousand pcs were used. TBE and seed virus of the 6th passage level serotype A / Michigan / 45/2105 (H1N1). After infection with EC, incubation took place at a temperature (+ 35.5 C °) for 48 hours. From the received amount of EC, 470 liters of VAZ were collected. All parameters of further purification also corresponded to the parameters indicated in example 6. The concentrate after microfiltration was collected in a volume of 30 liters. After the second gradient, 5 fractions were collected, which contained 9 grams of total protein. The destruction of the HVAC was carried out with the ratios total

protein: detergent Triton X-100: detergent TDTAB as 1: 1.5: 1. After removal of the detergents, the total amount of protein was 4 grams, which were diluted to 12 liters with PBS buffer. In the resulting monovaccine, the total protein content was 3.5 grams, the total HA content was 1.9 grams. The concentration of the detergent TDTAB was 3.2 μg / ml. The efficiency of the technology in this experiment was 39.6 μg of GA per 1 pc. CE. The polypeptide composition of a sample of the obtained experimental monovaccine is shown in Figure 14.

As a result of the analysis, it was shown that the experimental monovaccine contains only the HA antigen in the form of two forms: the HAO polypeptide (non-cleaved form) and the HAO dimer polypeptide (a specific HA antigen aggregate). No nucleocapsid polypeptides (NP), membrane protein (M1) polypeptides, or spontaneous cleavage were detected.

Example 10.

Preparation of a dosage form of a tetravalent subunit vaccine without adjuvants.

For the preparation of the dosage form of the vaccine, preparations of monovaccines were used, the preparation process of which was described in examples No. 6, 7, 8, 9.

According to WHO recommendations, tetravalent inactivated influenza vaccines without adjuvants should contain HA antigens of each of the 4 recommended serotypes in the concentration range from 25.6 μg / ml to 34.4 μg / ml for each serotype. There are also recommendations that no bacterial growth preservatives should be added to prepare sterile single-dose vaccine packages.

From the literature data, it follows that foreign manufacturers of inactivated influenza vaccines to preserve the specific activity of the dosage form during its storage use various disaggregating additives, including the detergent Triton X-100.

Based on this information, an experimental batch of a quadrivalent vaccine in a volume of 10 liters was mixed. The calculation of this information was carried out in such a way that in the final vaccine solution the concentration of GA antigens of each of the four serotypes was in the above range, and the concentration of the detergent Triton X-100 was from 150 μg / ml to 200 μg / ml. From this volume of information, a 10 ml sample was taken, in which the above listed parameters were studied. After obtaining positive results, the entire volume of information was sterilized using sterile disposable MilliPak-60 capsules from Millipore (USA) in accordance with GMP rules. Then the sterile vaccine solution was poured into sterile 1 ml vials. The filling volume in each bottle was 55 ml (one dose). After filling, several vials were taken for analysis.

Based on the results of these analyzes, it was found that the content of HA serotype H1N1 in one dose of the vaccine is 16.2 μg, HA serotype H3N2 is 16.4 μg, HA serotype B (Victoria line) is 15.9 μg, HA serotype B (Yamagato line ) is 16.7 μg. The concentration of the detergent Triton X-100 was 170 μg / ml and the concentration of the detergent TDTAB was 2 μg / ml. The performed sterility control showed that the obtained dosage form is sterile and can be used for further research.

Example 11

Comparative study of the antigenic activity of the vaccine obtained by the proposed method

In order to confirm that the experimental vaccine obtained by the proposed method has high antigenic activity, a comparative study of this vaccine and the split vaccine "Vaxigripp" produced by SANOFI-Pasteur, (France) was carried out. Vaccine "Vaxigripp" was taken as a comparison vaccine, since, according to the world literature, it has high antigenic activity and, accordingly, high immunogenicity when vaccinating people. In order to obtain more adequate results, 40 outbred mice were taken into the study to study each of the two drugs. The control group consisted of 20 laboratory animals. Each animal was injected with 0.2 ml of the test drug intraperitoneally, the vaccination was carried out twice with an interval of 21 days. On the 21st day after the first vaccination and on the 14th day after the second vaccination, blood serum was collected from each animal for study in the reaction of inhibition of hemaglutinating activity (RTGA). The RTGA analysis was carried out according to the procedure specified in the State Pharmaceutical Foundation of the Russian Federation (14). In addition, an additional study of antibody titers in the microneutralization reaction (PMN) was carried out. RMN, according to the data of the world literature, significantly more adequately reveals the protective ability of the HA antigen as compared with RTGA. The titers were assessed both by the change in antibody titers after the first and second immunizations, and by the corresponding levels of seroprotection after each immunization. The data are presented in Table 2.

According to the data presented, both studied drugs showed high antigenic activity. After the first immunization, the level of specific antibodies in the RTGA reaction reached a protective level (not less than a titer of 1:40), while in the group of mice immunized with the proposed vaccine, this level was significantly higher for all serotypes compared with the Vaxigripp vaccine. The level of seroprotection in the RTGA reaction for all serotypes after the first immunization also reached the level specified in the relevant WHO recommendations (at least 70%). But in terms of seroprotection in the PMN reaction for serotype B / Brisbane / 60/2008, a difference was revealed between the proposed vaccine and the Vaxigripp vaccine, which in this experiment did not gain a level of seroprotection of 70% after the first immunization.

With regard to antibody titers and seroprotection levels after the second immunization, no significant differences were found. Thus, the proposed subunit vaccine showed antigenic activity, which is not inferior to that of the vaccine "Vaxigripp", which is considered the "gold" standard of immunogenicity in the world vaccination practice. Thus, the vaccine obtained by the proposed method has a high antigenic activity.

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Claims (13)

1. A method for the production of a quadrivalent subunit vaccine without adjuvants for the prevention of influenza, according to which a) receive four monovaccines containing one of the serotypes of influenza viruses H1N1, H3N2, B (Victoria line) or B (Yamagata line), and to obtain monovaccines 1) get vaccinated allantoic fluid (VAF); 2) inactivate VAZ with a disinfectant beta-propiolactone; 3) purify and concentrate VAS using microfiltration methods using three successive cascades of folded filters with pore diameters of 10 μm, 1.0 μm and 0.6 μm, ultrafiltration and two successive cycles of ultracentrifugation in a sucrose density gradient to obtain a purified viral concentrate (VVC); 4) destroy the virions in the obtained OVK by sequential treatment with detergents Triton X-100 and TDTAB; 5) remove subvirionic structures from the solution obtained at stage 4) by ultracentrifugation and microfiltration through folded filters with pore diameters of 0.6 μm and 0.2 μm; 6) remove detergents from the resulting solution by ultrafiltration methods using membranes with a cut-off size of 30 KD and concentrating the obtained purified supernatant, followed by purification of the resulting concentrate solution by hydrophobic chromatography using the Expanded Bed Addsorption technology, which consists in the fact that the preparation for purification is fed through the bottom of the column, and the top of the chromatographic support is not fixed; 7) dilute the concentrate with phosphate buffer solution containing the detergent Triton X-100 to obtain a monovaccine; b) combine four monovaccines containing the serotypes of influenza viruses H1N1, H3N2, B (Victoria line) and B (Yamagata line) into a single dosage form with the addition of the detergent Triton X-100 as a disaggregating additive, followed by filter sterilization and filling the resulting dosage form tetravalent vaccine in sterile containers. 2. The method according to claim 1, wherein the “SDRHyperD” support, manufactured by Pall (USA), is used for hydrophobic chromatography. 3. A method according to claim 1, in which the interaction of Triton X-100 with purified virions is carried out for 20 minutes, and the interaction of TDTAB with purified virions for 40 minutes. 4. The method according to claim. 1, in which the filling of the dosage form is carried out in sterile vials, ampoules and special syringes for pre-filling the vaccine.

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