RU2535153C1 - Method for preparing high-purity virion concentrate - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: chicken embryos are infected; a virus-containing allantoic fluid (VAF) is collected, and the VAF is purified by microfiltration. The VAF is purified with the use of polyethylene glycol of the molecular weight of 6000 in the concentration of 1.5% and microfiltered on depth filters having pore diameters of 1.0 mcm and 10.0 mcm. Further, the purified VAF is concentrated by ultrafiltration on membranes with the molecular weight cut 100 kD, and the prepared concentrate is purified in a sucrose gradient by ultracentrifugation.

EFFECT: using the method enables the higher quality of virion purification, as well as the higher number of viral antigens recovered from one chicken embryo.

1 tbl, 1 dwg, 6 ex

Description

A method of obtaining highly purified varion concentrates (hereinafter referred to as the Invention) relates to biotechnology, immunology and relates to virion concentrates, which can be used for the production of various inactivated influenza vaccines. Currently, there are several types of inactivated influenza vaccines: whole-virion, split, subunit, adjuvant vaccines and virosomal vaccines. Whole-virion vaccines have high immunogenicity, but they also have high reactogenicity, so they are not used to vaccinate children. Split vaccines, which are second-generation vaccines, have less reactogenicity and a fairly high immunogenicity. Therefore, they are currently the most widely used type of vaccine in the world. However, mainly subunit vaccines are used to vaccinate children, which have very low reactogenicity. At the same time, subunit vaccines in real practice show a relatively low rate of protective protection for vaccinated people, therefore, they have recently preferred vaccines with various adjuvants, in particular with MF-59 adjuvant. The widespread use of the Novartis pandemic vaccine against swine flu, which contained this adjuvant, showed that the reactogenicity of the vaccine due to the presence of adjuvant sharply increased. Therefore, the use of seasonal influenza vaccines containing this adjuvant has been suspended. In Russia, for the past 16 years, the polymer subunit vaccine Grippol, which includes the Polyoxidonium polymer, has been used. However, a study of the epidemiological efficacy of this vaccine, which was conducted in the early 2000s, showed that in this indicator it is much inferior to foreign subunit and split vaccines.

In connection with the foregoing, of greatest interest to modern health care are virosomal vaccines, which contain only surface antigens and phospholipids of the viral membrane. Such structures are called “pseudoviral,” because they look like the preparation of purified virions. However, they lack a membrane protein and a nucleocapsid of the influenza virus, which makes these structures practically unreactogenic. It should be noted that virosomal vaccines are able to induce not only humoral, but also cellular immunity, which distinguishes them from all other types of inactivated influenza vaccines.

In order to produce any variant of an inactivated influenza vaccine with the required level of quality, it is necessary to carry out a special purification of influenza virus virions from virus-containing allantoic fluid. This purification should separate virion particles from the lipotropic membranes of the destroyed cells (debris). The problem is that hemagglutinin, which is the main antigen of the influenza virus, is usually presented on the part of the debris.

The world literature describes various methods for cleaning the IMPORTANT. US Pat. No. 3,989,818 describes the use of polyethylene glycol (PEG) 6000 for the isolation of purified virions. The disadvantage of this method is that at low virus concentrations, PEG 6000 precipitation does not occur quantitatively, and this significantly reduces the effectiveness of the technology.

The use of depth filters with different pore diameters is described in several patents. In particular, US 8,247,207 describes the use of depth filters from 1.5 to 0.2 microns. However, different vaccine strains of the influenza virus are characterized by polymorphism. Therefore, viral particles with a size of more than 0.2 microns are retained on such filters, which reduces the effectiveness of this technology.

EP 0 870 508 describes the use of only one depth filter with a pore diameter of 0.5 μm and the use for ultrafiltration of membranes with a molecular weight cut-off of 300 KD. The disadvantage of this method is that in the case of strong heterogeneity of the IMPORTANT one filter with a pore diameter of 0.5 μm will not be enough and the number of liters of IMPORTANT that can be passed through this filter will be insignificant. In order for the technology to be effective according to the theory of microfiltration, you need to install a cascade of 2 or more cartridges. Only in this case the technology will be effective.

In Russia, the technology of purification of virions from IMPORTANT using formalinized chicken red blood cells is traditionally used. A similar method of cleaning the IMPORTANT is disclosed in patent RU 2446824, which is taken as a prototype for this invention. According to a known solution, in order to obtain a purified concentrate of influenza antigens, the split virus concentrate is centrifuged at 16,000 rpm for 2 hours. Then, ultrafiltration is performed in a tangential flow on a cassette with a membrane of 30,000 daltons until the concentration of detergent in the filtrate decreases to 10 μg / ml The resulting concentrate of purified influenza antigens is considered as a monovaccine. Monovaccine is filtered through two series-connected filters with pore sizes of 0.45 and 0.22 μm.

However, when using this technology, not only virions are adsorbed on red blood cells, but also part of the debris containing hemagglutinin. In the framework of this technology, elution of viruses from red blood cells is performed with a 10-fold decrease in volume compared with the initial IMPORTANT. Thus, not only a cleaning effect is achieved, but also a concentration effect. The problem is that debris is concentrated with virions. And therefore, in the process of elution, aggregation of virions and debris occurs. The resulting aggregates are very difficult to separate in the subsequent stages of purification, and the hemagglutinin that they contain is usually lost. This leads to a decrease in the effectiveness of the technology. In addition, the quality of virion purification using this technology is significantly worse than similar world samples obtained without the use of red blood cells. For the further isolation of hemagglutinin and the creation of various types of inactivated vaccines, the quality of virion purification and viral aggregation are of great importance, because the antigen preparation is not very stable from a poorly purified virus.

As a result of lengthy experimental work carried out by the authors of this invention, it was found that the complete separation of debris from influenza virions cannot be achieved with any combination of filters. It was also found that the addition of various electrolytes, in particular PEG 6000, leads to strong aggregation and subsequent deposition of debris structures, influenza virus virions at certain concentrations of PEG 6000 continue to remain in solution (unpublished data). These observations formed the basis of this invention.

The problem to which the present invention is directed is to develop a new technology for the purification of virions, which will allow the production of Russian inactivated vaccines with a world level of quality.

The technical result of the invention is to develop an industrial method for producing highly purified virion concentrate, which could be tested for the production of inactivated influenza vaccines.

The essence of the invention is to obtain highly purified virion concentrates using membrane technology and special additives.

The object of the invention is a method for producing highly purified virion concentrates for the production of an inactivated influenza vaccine, including infection of chicken embryos, collection of virus-containing allantoic fluid (IMPORTANT), purification of IMPORTANT by microfiltration, concentration by ultrafiltration and purification of the obtained concentrates by ultracentrifugation in a sucrose density gradient, characterized in that the cleaning of the IMPORTANT is carried out using polyethylene glycol with a molecular weight of 6000 in the final con a concentration of 1.5% followed by microfiltration on depth filters with pore diameters of 1.0 μm and 10.0 μm and concentration of the purified IMPORTANT on membranes with a molecular weight cut-off of 100 kilodaltons.

The difference of the proposed method from the prototype is that formalinized chicken red blood cells are not used to clean the IMPORTANT, and modern technologies and electrolytes approved for medical use are used. Therefore, the result obtained differs from the prototype in the degree of purification of the viral concentrate by the higher efficiency of the technology used.

A summary of the method of obtaining

To prepare the vaccine, use influenza virus strains that are consistent with WHO recommendations.

The chicken embryos infected with the influenza virus in the optimal dose are incubated at a temperature of 33-37 ° C (according to the passport attached to the strain of the virus) and humidity (65 ± 5)% in thermal rooms.

After ovoscopy, the remaining eggs are cooled in the refrigerator for (18 ± 2) h at a temperature of (4 ± 2) ° C.

The collection of virus-containing allantoic fluid (IMPORTANT) is carried out on a special machine for collecting allantoic fluid (Tekhno, Italy).

The selected allantoic fluid is collected by vacuum in 2 special containers of 250 l. After collection, polyethylene glycol (PEG-6000) is added to the IMPORTANT to a final concentration of 1.5%. The exposure time of the IMPORTANT with PEG is 17 hours

The collected IMPORTANT is passed through two series-connected Betapur cartridges (3M, USA) for microfiltration with pore sizes of 10 μm and 1 μm, respectively.

Ultrafiltration is carried out on an ASF-020 ultrafiltration unit (Vladisart, Russia) in a tangential flow with 10 cartridge modules. For concentration use modules (firms Vladisart, Russia) with a pore diameter of 100 kD.

The installation and piping system is sterilized with a 3% formalin solution with an exposure of at least 10 hours. Then, the unit is washed from formalin with sterile water for injection in a volume of 40 l, followed by verification and restoration of pH.

Purified IMPORTANT is concentrated 20 times. Concentration pressure should not exceed 0.8 bar.

After work, cassette membrane modules are regenerated with a 1 M sodium hydroxide solution, and then washed with purified water. After regeneration, the installation is sterilized using a 3% formalin solution.

The filtered liquid (filtrate) is disinfected in an autoclave.

Purification by concentrated IMPORTANT is carried out by ultracentrifugation in a sucrose density gradient.

A solution of 60% sucrose was prepared in 0.05 M Tris buffer, which was autoclaved at 121 ° C for 20 minutes.

In a flowing rotor with a volume of 3.2 liters of a 520N peristaltic pump (Watson-Marlow, Great Britain), 2 liters of a 60% sucrose solution are introduced and the rotor is started in standard mode (30 thousand rpm, temperature + 4 degrees). Then, with the same pump, a sterile phosphate-buffer solution is launched into the rotor at a rate of 10 l / h. After passing 5 liters of buffer into the rotor, it is fed in the same way purified and concentrated IMPORTANCE in portions of 10 liters (1 container of 200 liters - 10 liters of concentrate, 2 containers - 20 liters of concentrate).

After stopping the rotor, the sucrose gradient is dug in a laminar cabinet in fractions of 100 ml each (20 fractions are obtained). In fractions by method of viscometry determine the content of sucrose, as well as determine the protein content and authenticity. Fractions containing more than 4000 PIECES of hemagglutinin are separated from the rest (usually 5-6 fractions) in one pool, an equal volume of pharmacopeia glycerol (Panreac, Spain) is added to them, the liquid in the vials is thoroughly mixed and stored at minus 10 ° C no more than 2 months.

The possibility of carrying out the invention can be demonstrated by the following examples.

Example 1. Chicken embryos at 11 days of age in the amount of 46,000 pieces were infected with the optimal dose of seed virus. After incubation for 48 hours at 34 ° C, the embryos are transferred to a refrigerator and cooled for 17 hours. The embryos are then treated with a 0.2% alcohol solution of iodine. Next, the embryos are transferred to an automatic machine for collecting the IMPORTANT (company Techno, Italy). Allantoic fluid is collected in 2 containers of 250 liters. The volume of the IMPORTANT was 290 liters. After collecting the IMPORTANT, PEG 6000 is added to the container at a concentration of 1.0%. Incubated at plus 4 ° C for 17 hours. Microfiltration is then carried out through 2 Betapur cartridges in series (3M, USA) with pore diameters of 10 μm and 1 μm, respectively. After that, the obtained filtrate was concentrated 20 times on membranes with molecular cutoff of 100 KD on an ASF 0.20 installation (Vladisart, Russia). The resulting concentrate is applied to a sucrose density gradient at a flow rate of 10 l / h and 30,000 rpm. The supernatant is disinfected and removed into the sewer, and the sucrose gradient is dug up by fractions and hemagglutinin is determined in them. Fractions with high hemagglutinating activity (more than 4000 units of hemagglutinin) are combined into one pool and the protein content and specific antigen-hemagglutinin content in ORID are determined in this pool. The results are presented in table 1.

Example 2. Chicken embryos at 11 days of age in the amount of 47124 pieces were infected with the optimal dose of seed virus. After incubation for 48 hours at 34 ° C, the embryos are transferred to a refrigerator and cooled for 17 hours. The embryos are then treated with a 0.2% alcohol solution of iodine. Next, the embryos are transferred to an automatic machine for collecting the IMPORTANT (company Techno, Italy). Allantoic fluid is collected in 2 containers of 250 liters. The volume of the IMPORTANT was 315 liters. After collecting the IMPORTANT, PEG 6000 at a concentration of 0.5% is added to the container. Incubated at plus 4 ° C for 17 hours. Microfiltration is then carried out through 2 Betapur cartridges in series (3M, USA) with pore diameters of 10 μm and 1 μm, respectively. After that, the obtained filtrate was concentrated 20 times on membranes with molecular cutoff of 100 KD on an ASF 0.20 installation (Vladisart, Russia). The resulting concentrate is applied to a sucrose density gradient at a flow rate of 10 l / h and 30,000 rpm. The supernatant is disinfected and removed into the sewer, and the sucrose gradient is dug up by fractions and hemagglutinin is determined in them. Fractions with high hemagglutinating activity (more than 4000 units of hemagglutinin) are combined into one pool and the protein content and specific antigen-hemagglutinin content in ORID are determined in this pool. The results are presented in table 1.

Example 3. Chicken embryos at 11 days of age in the amount of 54174 pieces were infected with the optimal dose of seed virus. After incubation for 48 hours at 34 ° C, the embryos are transferred to a refrigerator and cooled for 17 hours. The embryos are then treated with a 0.2% alcohol solution of iodine. Next, the embryos are transferred to an automatic machine for collecting the IMPORTANT (company Techno, Italy). Allantoic fluid is collected in 2 containers of 250 liters. The volume of the IMPORTANT was 385 liters. After collecting the IMPORTANT, PEG 6000 was not added to the containers. They were incubated at plus 4 ° C for 17 hours. Microfiltration was then performed through 2 Betapur cartridges (3M, USA) in series with pore diameters of 10 μm and 1 μm, respectively. After that, the obtained filtrate was concentrated 20 times on membranes with molecular cutoff of 100 KD on an ASF 0.20 installation (Vladisart, Russia). The resulting concentrate is applied to a sucrose density gradient at a flow rate of 10 l / h and 30,000 rpm. The supernatant is disinfected and removed into the sewer, and the sucrose gradient is dug up by fractions and hemagglutinin is determined in them. Fractions with high hemagglutinating activity (more than 4000 units of hemagglutinin) are combined into one pool and the protein content and specific antigen-hemagglutinin content in ORID are determined in this pool. The results are presented in table 1.

Example 4. Chicken embryos at 11 days of age in the amount of 41112 pieces were infected with the optimal dose of seed virus. After incubation for 48 hours at 34 ° C, the embryos are transferred to a refrigerator and cooled for 17 hours. The embryos are then treated with a 0.2% alcohol solution of iodine. Next, the embryos are transferred to an automatic machine for collecting the IMPORTANT (company Techno, Italy). Allantoic fluid is collected in 2 containers of 250 liters. The volume of the IMPORTANT was 300 liters. After collecting the IMPORTANT, PEG 6000 was not added to the containers. They were incubated at plus 4 ° C for 17 hours. Microfiltration was then performed through 2 Betapur cartridges (3M, USA) in series with pore diameters of 10 μm and 1 μm, respectively. After that, the obtained filtrate was concentrated 20 times on membranes with molecular cutoff of 100 KD on an ASF 0.20 installation (Vladisart, Russia). The resulting concentrate is applied to a sucrose density gradient at a flow rate of 10 l / h and 30,000 rpm. The supernatant is disinfected and removed into the sewer, and the sucrose gradient is dug up by fractions and hemagglutinin is determined in them. Fractions with high hemagglutinating activity (more than 4000 units of hemagglutinin) are combined into one pool and the protein content and specific antigen-hemagglutinin content in ORID are determined in this pool. The results are presented in table 1.

Example 5. Chicken embryos at 11 days of age in the amount of 30542 pieces were infected with the optimal dose of seed virus. After incubation for 48 hours at 34 ° C, the embryos are transferred to a refrigerator and cooled for 17 hours. The embryos are then treated with a 0.2% alcohol solution of iodine. Next, the embryos are transferred to an automatic machine for collecting the IMPORTANT (company Techno, Italy). Allantoic fluid is collected in 2 containers of 250 liters. The volume of the IMPORTANT was 265 liters. After collecting the IMPORTANT, PEG 6000 at a concentration of 1.5% is added to the container. Incubated at plus 4 ° C for 17 hours. Microfiltration is then carried out through 2 Betapur cartridges in series (3M, USA) with pore diameters of 10 μm and 1 μm, respectively. After that, the obtained filtrate was concentrated 20 times on membranes with molecular cutoff of 100 KD on an ASF 0.20 installation (Vladisart, Russia). The resulting concentrate is applied to a sucrose density gradient at a flow rate of 10 l / h and 30,000 rpm. The supernatant is disinfected and removed into the sewer, and the sucrose gradient is dug up by fractions and hemagglutinin is determined in them. Fractions with high hemagglutinating activity (more than 4000 units of hemagglutinin) are combined into one pool and the protein content and specific antigen-hemagglutinin content in ORID are determined in this pool. The results are presented in table 1.

Example 6. Chicken embryos at 11 days of age in the amount of 29349 pieces were infected with the optimal dose of seed virus. After incubation for 48 hours at 34 ° C, the embryos are transferred to a refrigerator and cooled for 17 hours. The embryos are then treated with a 0.2% alcohol solution of iodine. Next, the embryos are transferred to an automatic machine for collecting the IMPORTANT (company Techno, Italy). Allantoic fluid is collected in 2 containers of 250 liters. The volume of the IMPORTANT was 230 liters. After collecting the IMPORTANT, PEG 6000 at a concentration of 1.5% is added to the container. Incubated at plus 4 ° C for 17 hours. Microfiltration is then carried out through 2 Betapur cartridges in series (3M, USA) with pore diameters of 10 μm and 1 μm, respectively. After that, the obtained filtrate was concentrated 20 times on membranes with molecular cutoff of 100 KD on an ASF 0.20 installation (Vladisart, Russia). The resulting concentrate is applied to a sucrose density gradient at a flow rate of 10 l / h and 30,000 rpm. The supernatant is disinfected and removed into the sewer, and the sucrose gradient is dug up by fractions and hemagglutinin is determined in them. Fractions with high hemagglutinating activity (more than 4000 units of hemagglutinin) are combined into one pool and the protein content and specific antigen-hemagglutinin content in ORID are determined in this pool. The results are presented in table 1.

Table 1 Example No. Number of embryos Volume IMPORTANT, l Content General ORID ORID / protein Antigen yield PC. total protein, l hemagglutinin per 1 embryo, mcg / embryo. one 2 3 four 5 6 7 one 46800 290.0 4.92 1.47 3.35 31,4 2 47124 315.0 4.31 1,2 3.6 25,4 3 54174 385.0 2.43 0.72 3.38 13.3 four 41112 300,0 2.85 0.6 4.42 14.6 5 30542 265.0 3.44 1.26 2.73 41.2 6 29349 230,0 3.42 1,08 3.17 36.8

Output

From the results presented in examples 1-6, it follows that without the addition of PEG 6000, the effectiveness of the technology is extremely low. In Example 3, the antigen yield per embryo was 13.3 μg hemagglutinin per 1 embryo. In example 4, this yield was 14.6 μg of hemagglutinin per 1 embryo. This is due to the fact that debris structures were aggregated together with virions and for the most part were retained on membrane cartridges. When PEG 6000 was added to a concentration of 0.5% (Example 2), the output of hemagglutinin per 1 embryo was 25.4 μg / embryo. When PEG 6000 was added to a concentration of 1.0% (Example 1), this yield was 31.4 μg / embryo. When PEG 6000 was added to a concentration of 1.5%, the yield was 41.2 μg / embryo. (example 5) or 36.8 μg / embryo. (example 6). Thus, it was proved that the greatest efficiency of this technology is achieved by adding PEG 6000 at a concentration of 1.5%.

For the first time in world practice, a double approach has been taken to purify the IMPORTANT: 1) separation of virions and debris due to PEG 6000 polyelectrolyte; 2) the complete separation of virions and debris is carried out using depth filters. The combination of these two approaches will maximize the purification of the debris from the debris structure, so that at the next stages of purification, namely concentration by ultrafiltration and ultracentrifugation in the sucrose gradient, it is possible to obtain a highly purified virion concentrate of influenza virus.

Figure 1 presents the results of the analysis of the polypeptide composition of purified virions obtained according to example 3 (line 1) and example 5 (line 2). The arrows indicate the location of the main polypeptides of the influenza virus serotype H ₁ N ₁ (HA0, NP, M1). Obviously, according to example 5, a more purified preparation of virions is obtained, which practically does not contain extraneous polypeptides.

Bibliography

1. RF patent 2446824, publ. 04/10/2012.

2. Patent US 3989818, publ. 11/02/1976.

3. Patent EP 8247207, publ. 08/12/2012

4. Patent EP 0870508, publ. 11/08/2000

Claims (1)

A method of obtaining highly purified virion concentrates for the production of an inactivated influenza vaccine, including infection of chicken embryos, collection of virus-containing allantoic fluid (IMPORTANT), purification of IMPORTANT by microfiltration, concentration by ultrafiltration and purification of the obtained concentrates by ultracentrifugation in a sucrose density gradient, characterized in that purification by IMPORTANT carried out using polyethylene glycol with a molecular weight of 6000 at a final concentration of 1.5% followed by mic by depth filtration with depth filters with a pore diameter of 1.0 μm and 10.0 μm and concentration of the purified IMPORTANT on membranes with a molecular weight cut-off of 100 kilodaltons.

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