RU2614127C2 - Method for production of recombinant pseudoadenovirus particles concentrate, expressing hemagglutinin gene of influenza a/california/07/2009(h1n1) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for production of recombinant pseudoadenovirus particles (RPAN) concentrate, expressing the hemagglutinin gene of influenza a/california/07/2009(H1N1) is proposed. The method comprises: obtaining of a production cell culture; preparation of disaggregated starting RPAN culture samples; single-round infection of the production suspension cell culture, thereby obtaining a RPAN-containing crude suspension, with subsequent cell mass precipitation by centrifugation and spent culture medium diverting; precipitated cell mass resuspension in lysis buffer, then one-time freezing followed by suspension thawing the slurry and RPAN separation from the disrupted cells successively by centrifugation, ultrafiltration, anion exchange chromatography, size exclusion chromatography, sterile filtration to yield a sterile pharmaceutical grade RPAN concentrate with high level ofchromatographic purity.

EFFECT: method allows to obtain an increased yield of RPAN and a possibility to obtain a concentrate with high titers and chromatographic purity, that meets the quality of a pharmaceutical product suitable for industrial production of influenza vaccines.

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Description

Technical field

The invention relates to the field of biotechnology, in particular to the production of immunobiological preparations. The proposed method for producing recombinant pseudo-adenovirus particles expressing the A / California / 07/2009 (H1N1) or A / California / 07/2009 (H1N1) -like influenza hemagglutinin gene allows the component to be used as an active ingredient in highly immunogenic influenza vaccines.

State of the art

Every year in the world, seasonal increases in the incidence of influenza (epidemics) caused by type A and B viruses are recorded, leading to increased mortality and significant economic damage. As a rule, diseases are caused by influenza viruses of type A and B. In this regard, it is justified, guided by WHO recommendations, to use multivalent influenza vaccines that can prevent the disease by various seasonal subtypes of influenza virus. Therefore, of great interest is the large-scale production of highly immunogenic antigens of epidemiologically relevant strains for inclusion in such anti-influenza vaccines.

Among many subtypes of type A viruses, in recent years among people a significant share in the structure of seasonal incidence is occupied by subtypes of influenza A (H1N1) virus. In 2009, the World Health Organization (WHO) announced the onset of a human pandemic. The reason was the new virus A / California / 07/2009 (H1N1), the flu it caused was conditionally called "swine (California) flu." As a result of mutations, the swine flu virus acquired the ability to be transmitted from person to person, which became very dangerous and despite the abolition of the pandemic status in 2010, the new virus was included in seasonal vaccines as having epidemic significance.

The development of new vaccines is increasingly based on the use of not a classical antigen, but a vector based on recombinant viruses, which is able to express the target antigen gene as an active component. In particular, such drugs include influenza vaccines containing adenoviruses as a vector.

Recombinant pseudo-adenovirus nanoparticles (RPANs) are known and are a genetic construct based on human fifth adenovirus. The expression of hemagglutinin genes with RPANs occurs in the body after the introduction of the vaccine.

However, specialists in the production of vaccines containing the influenza virus hemagglutinin as an antigen have long faced the problem of aggregation of cultivated crops. In the case of recombinant pseudo-adenovirus nanoparticles, this problem is also present; after infection of the cell suspension culture with RPANs during production, hemagglutinins are produced with their localization on the cell membrane and further aggregation. This process negatively affects the yield of RPAN from the bioreactor and significantly increases the loss of RPAN during further purification of the drug. The reason for this is the natural purpose of the influenza virus hemagglutinin to bind to the virus-specific receptor of the host cell and adsorb its virions on the cell surface during the initial stage of infection. The composition of such a receptor includes sialic acid, which is important for the implementation of such binding. Due to the relatively low specificity of this interaction, influenza viruses can bind to cells of various types (Xiong X. Receptor binding properties of the influenza virus hemagglutinin as a determinant of host range - Receptor-binding properties of influenza hemagglutinin as a determinant of the host circle // Curr. Top. Microbiol. Immunol. 2014 - 385: 63-91).

In the case of influenza virus A / California / 07/2009 (H1N1), the process of growing it in a cell culture is even more complicated. We noted significant difficulties in the cultivation of cells in suspension culture by well-known methods of this strain in comparison with other strains of influenza A. It is known that in the process of obtaining on eukaryotic cells, RPANs expressing the hemagglutinins of strain A / California / 07/2009 (H1N1) form aggregates more strongly, give very weak accumulation and become less stable compared to RPANs carrying hemagglutinins of other seasonal strains. Perhaps this is due to mutational changes in the hemagglutinin gene of the “Californian” strain located at the receptor binding site, which increases their ability to bind to the cell receptor and enhances aggregation (Wang W. A mutation in the receptor binding site enhances infectivity of 2009 H1N1 influenza hemagglutinin pseudotypes without changing antigenicity - A mutation at the receptor binding site increases the infectivity of 2009 H1N1 influenza hemagglutinin - a pseudotype without changing antigenicity // Virology - 407 (2010) - 374-380).

Such features in the properties of hemagglutinin of the influenza A / California / 07/2009 (H1N1) virus do not allow the growth of RPANs expressing the gene of this hemagglutinin according to the standard procedure in large titers due to the concomitant strong aggregation of cell culture, which also increases the loss of RPAN during purification. This makes large-scale industrial production of influenza vaccine impossible and reduces its effectiveness.

Typically, the reduction of aggregation of hemagglutinins and cells in known similar methods is solved by optimizing the purification schemes (RF Patent No. 2535153). This method of obtaining highly purified virion concentrates is designed to reduce, as a result, the effect of particle aggregation on the quality of cleaning and to reduce the loss of antigen, but is not able to prevent the formation of aggregates at the growing stage. As a result, the cleaning process is multi-stage, costly in materials and time, but it still does not allow to minimize losses and improve the purity of the drug. Thus, it has still not been possible to reduce the effect of aggregation of cell cultures by hemagglutinins on the production process of influenza vaccines, which indicates the presence of a scientific and technical problem.

The closest solution known from the prior art and adopted as a prototype is a method for growing recombinant pseudo adenovirus particles (RPAN), based on the genome of the human adenovirus 5 serotype, strain A / California / 07/2009 (H1N1), expressing the genes for the hemagglutinins of influenza A / H1N1, proposed as a component of influenza vaccines (RF Patent No. 2507257).

In the present invention, recombinant pseudo-adenovirus particles are assembled in cells of the HEK293 line (human embryonic kidney - human embryonic kidney) (for example, No. 300192, CLS, Germany). The genome of this type of cell contains the E1 region, which allows recombinant pseudo-adenovirus particles with a similar region removed to collect and multiply in the cells of this line. The assembly of recombinant pseudo-adenovirus particles is accompanied by cell lysis, from the time of transduction to the time of cell lysis and the receipt of a new generation of recombinant pseudo-adenovirus particles with the insertion of the influenza virus hemagglutinin gene, 48 hours pass.

For the growth of HEK293 cells in an adhesion culture, it is necessary to use DMEM medium (for example, Invitrogen, No. 52100-047, USA) containing 25 mM glucose, 4 mM D-glutamine and 10% fetal bovine serum, a special incubator with a temperature of + 37 ° C and 5% CO ₂ .

All cultural cultivation methods used in the patent were well known.

Therefore, the prototype has significant disadvantages: the presented technology is laboratory and does not allow to obtain industrial quantities of the drug with pharmaceutical quality. The indicated activity of recombinant pseudo-adenovirus particles from 5 × 10 ⁷ to 5 × 10 ⁸ PFU / ml refers to a completely crude preparation and is indicated as a characteristic of the biological activity of the strain, since there is no description of the purification step. From the knowledge of a specialist it becomes obvious that the losses of RPAN after the stage of purification of such a drug will be significant, often reaching 70-80%. A big problem is also the process of aggregation when growing crops, which leads to poor accumulation and, consequently, to the impossibility of industrial use of this method.

Consequently, there is an unresolved scientific and technical problem in the process of obtaining RPAN concentrates with the hemagglutinin gene of influenza virus A / California / 07/2009 (H1N1) or A / California / 07/2009 (H1N1) -like in quantities sufficient for rapid industrial production of vaccines , which is especially important when pandemic situations occur.

Disclosure of invention

The technical task of the invention is directed to the development of a method for producing a concentrate of recombinant pseudo-adenovirus particles expressing the influenza virus hemagglutinin gene A / California / 07/2009 (H1N1) or A / California / 07/2009 (H1N1) -like, allowing to obtain a concentrated product with biological activity RPAN from × 10 ⁸ PFU / ml, pharmaceutical quality, with sufficient chromatographic purity and suitable for industrial production of influenza vaccines in high yield.

The technical problem is solved due to the fact that in the method for producing a concentrate of recombinant pseudo adenoviral particles expressing the A / California / 07/2009 (H1N1) or A / California / 07/2009 (H1N1) -like influenza hemagglutinin gene, the technological steps include the following sequences :

- receiving from the master bank of a production cell culture - a working bank of a production suspension cell culture with a concentration of 2.0 × 10 ⁶ cells / ml with an increase in cell yield of not less than 700 times, and receiving with an increase in active RPAN units of not less than 33 times from the RPAN master bank - the RPAN master bank with a titer of at least 2 × 10 ⁸ PFU / ml;

- preparation of samples of the disaggregated starting RPAN culture with activity of at least 2 × 10 ⁸ PFU / ml from the RPAN working bank obtained in the previous stage by purification from hemagglutinins expressed by RPAN during the growing of the RPAN working bank;

- conducting a one-round infection of a production suspension cell culture with a cell concentration of 1.0 × 10 ⁶ cells / ml in a bioreactor with a nutrient medium by introducing a disaggregated starting production culture of RPAN with a titer of at least 2 × 10 ⁸ PFU / ml, resulting in RPAN- containing suspension untreated with a titer of at least 10 ⁸ PFU / ml by cultivation for 24 hours with an increase in the yield of active RPAN units during the growth process by at least 10 times, with further sedimentation of the cell mass by centrifuge gyration and disposal of the spent nutrient medium;

- resuspension of the cell mass precipitated at the previous stage in a lysis buffer containing a non-ionic detergent Triton X-100 at a concentration of 1% for 3 hours, then a single freeze for 2 hours in a cryostorage followed by thawing of the suspension in a water bath at a temperature of 23-25 ° C, then treatment with benzonase, and separation of RPAN from the destroyed cells sequentially by centrifugation, ultrafiltration, anion exchange chromatography, size exclusion chromatography, sterilizing filtration to obtain sterile pharmaceutical grade concentrate Niemi RPAN with high chromatographic purity and with a titre of not less than 5 × ¹⁰ August pfu / ml.

FIG. one

The result of the analysis of RPAN concentrate after size exclusion chromatography.

Peak 1 - RPAN.

The analysis result showed that the purity of the obtained pseudo-adenovirus particles is 97%.

The implementation of the invention

The implementation of the invention is confirmed by examples. Examples of the invention implemented on the influenza virus A / California / 07/2009 (H1N1), but not limited to, but can be reproduced on any A / California / 07/2009 (H1N1) -like strain of influenza virus, as specialists under this the name implies the identity of constantly isolated strains in different places of the world with respect to the reference virus A / California / 07/2009 (H1N1).

Example 1

Preparatory work

Obtaining from the master bank of a production cell culture - a working bank of a production suspension cell culture with a concentration of 2.0 × 10 ⁶ cells / ml with an increase in cell yield of not less than 700 times and obtaining with an increase in active RPAN units of not less than 33 times from RPAN master bank - an RPAN master bank with a titer of at least 2 × 10 ⁸ PFU / ml;

The basic principle of obtaining RPANs is their cultivation in a specialized culture of eukaryotic cells, in which the propagation of RPANs is possible. The master cell bank and the RPAN master bank were obtained earlier, permanently stored in the cryo-vault and used as starting cultures for the full production cycle of the vaccine series.

Master Bank of cell production culture. A culture seed called a “master cell bank” is stored in a cryogenic storage with a concentration of 2 × 10 ⁶ cells / ml. The well-known specialized suspension cell culture HEK-293 (human embryonic kidney, human kidney embryonic cell line) was used as the production one. Master Bank RPAN. The RPAN culture seed is stored as a “master bank” with a titer of at least 10 ⁸ PFU / ml in a cryogenic storage (at liquid nitrogen temperature). The RPAN in this invention can be any human type 5 adenovirus-based adenovirus vectors expressing the H1N1 type A influenza virus hemagglutinin genes. The RPAN must be replicatively defective, thereby achieving drug safety. These designs can be obtained by well-known genetic engineering manipulations known from the state of the art (for example, as shown in RF patent No. 2507257).

Here and in the text, the activity (titer) of RPAN is measured in PFU (plaque-forming units). The number of PFU is determined by the well-known method - by the cytopathic effect of the sample on the layer of HEK293 cells. This indicator characterizes the number of active viral particles that retained not only physical integrity, but also physiological activity, i.e. the ability to penetrate the cells and activate the expression of their genetic material.

1) Preparation of culture medium

The process began with the preparation of a growth medium suitable for growing a HEK-293 cell culture.

A dry culture medium (for example, CD293AGT) suitable for growing a HEK-293 cell culture was taken in an amount of 118 g and dissolved in 5 L purified water (Milli-Q) with stirring on a magnetic stirrer. After dissolution, 1.65 L of purified water was added. Next, sterilization of the liquid nutrient medium was carried out on a filtration system with vacuum filters with a pore diameter of 0.22 μm. Glutamine was added to a sterile medium in an amount of 1.933 g (at a concentration of 2.92 × 10-3 g / l immediately before use of the growth medium). All bulk components were weighed on an electronic balance. The preparation of culture media was carried out in a room separate from the boxes intended for work with cell culture and RPAN culture. The media were stored in a refrigerator at a temperature of + 4 ° C.

The finished medium was transported and used for the implementation of further technological processes in the culture box, viral box and reactor.

The prepared and mixed nutrient medium was monitored for sterility. The pH was measured.

2) Cultivation and analysis of the working bank of cell suspension

To obtain a working bank of a production cell culture, a suspension culture of HEK293 cells from a master bank was used. The cell culture in a volume of 1.5 ml with a concentration of 2 × 10 ⁶ cells / ml, taken from the cryogenic storage, was thawed at 37 ° C. Next, the tube was centrifuged, the supernatant was poured off and culture medium was added in a volume of 1 ml In a sterile culture box with a class B laminar flow, 1.5 ml of cell suspension were seeded in a plastic container with a surface area of 25 cm ² (containing 3.5 ml of culture medium). Cultivated in a CO ₂ incubator to a concentration of 1 × 10 ⁶ cells / ml (48-72 hours). Next, the cell suspension was passaged into a plastic container with a surface area of 75 cm ² (containing 5 ml of culture medium, total volume of 10 ml). The initial cell concentration is 0.5 × 10 ⁶ cells / ml. Cultivated in a CO ₂ incubator to a concentration of 2 × 10 ⁶ cells / ml (24-48 hours). The cell suspension was transplanted into a plastic container with a surface area of 75 cm ² (containing 15 ml of culture medium, the total volume of 25 ml). The initial cell concentration was 0.8 × 10 ⁶ cells / ml. Cultivated in a CO ₂ incubator to a concentration of 2.0 × 10 ⁶ cells / ml (24-48 hours), which is ≈5.0 × 10 ⁷ cells. Next, the cell suspension was passaged into a laboratory bioreactor (with an agitator for culturing cells in vials) of 200 ml with 35 ml of culture medium (total volume of 60 ml). Next, the laboratory bioreactor was placed in a CO ₂ incubator. Cell cultivation was carried out until a concentration of 2.0 × 10 ⁶ cells / ml was reached. Inoculated 60 ml of a cell suspension from a laboratory bioreactor with a volume of 200 ml into a similar laboratory bioreactor, but with a volume of 500 ml (with 90 ml of culture medium, the resulting total volume of 150 ml). Cultivated in a CO ₂ incubator to a concentration of 2.0 × 10 ⁶ cells / ml (24-48 hours). As a result, 3 × 10 ⁸ cells were grown. Then 150 ml of a cell suspension were transferred from a laboratory bioreactor with a volume of 500 ml to a laboratory bioreactor with a volume of 2000 ml, adding 250 ml of culture medium, so that the total volume was 400 ml, the initial concentration of ≈0.8 × 10 ⁶ cells / ml. Cultivated in a CO ₂ incubator to a concentration of 2.0 × 10 ⁶ cells / ml (24-48 hours). Upon reaching the required concentration, 650 ml of culture medium was added (total volume 1050 ml). Initial concentration ≈0.8 × 10 ⁶ cells / ml. Cultivated in a CO ₂ incubator to a concentration of 2.0 × 10 ⁶ cells / ml (24-48 hours).

Upon reaching the required concentration, part of the obtained cell suspension, in a volume of 120 ml, was transferred to begin the stage of “Production of RPAN culture” (Example 3).

3) Obtaining a working bank of RPAN culture expressing the influenza virus hemagglutinin gene

Used cell suspension from the previous stage. 120 ml of the cell suspension was sterile taken from a 2000 ml laboratory bioreactor and transferred to a 500 ml laboratory bioreactor, and settled for 30 minutes. The supernatant was drained (2/3 of the volume ≈80 ml). The remaining volume was adjusted to 240 ml (200 ml of culture medium was added), thus, the cell concentration was 1.0 × 10 ⁶ cells / ml.

In a Class B laminar flow box with a laminar flow, 15 ml of RPAN culture from the master bank (with a titer of at least 10 ⁸ PFU / ml) were introduced into a laboratory bioreactor containing the resulting cell suspension of 240 ml (total volume 255 ml) and cultured it has 36-48 hours. Incubated in a CO ₂ incubator. The suspension was analyzed by titration, the titer was not less than 2 × 10 ⁸ PFU / ml.

The samples were analyzed for the detection of extraneous viruses and RCA, for the detection of Mycoplasma hominis DNA, sterility. For this, the analyzed samples of a working bank with a total volume of 5 ml were centrifuged for the purpose of concentration. No extraneous viruses, RCA, Mycoplasma hominis DNA were detected, the samples were sterile.

Thus, at this stage, from 15 ml of RPAN culture (with a titer of at least 10 ⁸ PFU / ml), 250 ml of RPAN culture with a titer of at least 2 × 10 ⁸ PFU / ml was obtained.

Example 2

Preparation of samples of a disaggregated starting RPAN culture with an activity of at least 2 × 10 ⁸ PFU / ml from the RPAN working bank obtained in the previous stage by purification of hemagglutinins expressed by RPAN during the growing of the RPAN working bank

In standard cultivation schemes, the RPAN culture obtained at the previous stage from the working bank immediately goes to the inoculation of the bioreactor without any additional manipulations. As a result: firstly, if the RPAN strain has a low level of accumulation, it is difficult to obtain a culture for sowing with the required activity titers; secondly, when such a culture is introduced into the bioreactor, a large amount of hemagglutinins expressed at the previous stage gets into the bioreactor, which contribute to the aggregation of the suspension culture in the bioreactor and reduce the number of infected RPAN cells. Both reasons will cause a sharp decline in the yield of RPAN. The additional stage introduced by the authors in the production process consists in a certain preparation of the starting culture of the RPAN.

The preparation is carried out by cleaning the working bank of RPAN, obtained at the previous stage, from hemagglutinins expressed by RPAN in the process of growing, aggregating a production cell culture.

The negative effect of cell aggregation of the culture is expressed in a significant decrease in the efficiency of infection of cell culture by RPANs in the subsequent main production production cycle. When such an unrefined working bank of RPAN is introduced into the cell culture, hemagglutinins are immediately adsorbed on the receptors of the culture cells and aggregate them, which does not allow RPANs to infect a significant part of the cells and further sharply reduces the yield of RPAN. In addition, the degree of aggregation of the culture during the main growing cycle is reduced, since the total amount of hemagglutinins will be less and consisting only of those expressed in this production cycle. A decrease in aggregation will affect an increase in RPAN yield at subsequent stages of drug purification.

During the experiments, it was found that the most effective method is the introduction of an additional stage of purification of the starting RPAN culture before introducing it for inoculation of the bioreactor.

To disaggregate the starting production culture of RPAN obtained at the previous stage with a volume of 250 ml with a titer of at least 2 × 10 ⁸ PFU / ml, it is subjected to purification.

Cleaning can be carried out by all appropriate methods to remove hemagglutinins from the culture, while maintaining the biological activity of RPAN.

The authors used a purification technique that completely repeats the process that contains all the steps developed by the authors and described below in Example 4 for the main cycle.

As a result, after purification, 5 ml of starting disaggregated RPAN culture with a chromatographic purity of 97% was obtained. Storage was carried out at + 4 ° C. Immediately before inoculation of the bioreactor, the volume of the starter culture was adjusted to 250 ml with culture medium.

Example 3

Getting culture RPAN

The RPAN culture was obtained by a one-round infection of a production suspension cell culture (RPAN is assembled in a cell suspension).

The one-round infection technique developed by the authors at this stage allowed a significant increase in RPAN yield due to a decrease in aggregation.

Experimentally, when selecting the cultivation conditions, it was found that harvesting after 24 hours provides a single infection of the HEK293 suspension culture cells with a disaggregated starting RPAN culture, although this slightly decreases the RPAN yield at this stage, it significantly increases the RPAN yield in the future after purification due to almost complete absence of aggregation of culture cells. This method was called a one-round infection. It is based on the fact that the assembly and release of RPAN particles takes about 24 hours. During a standard growth cycle for 48-96 hours, the collected RPANs repeatedly accumulate and re-accumulate suspension culture cells (i.e., a multi-round infection), which is simultaneously accompanied by an increase in hemagglutinin accumulation each time with a further increase in aggregation.

1) Cultivation of cell suspension

In a Class C laminar flow reactor box, a 10 L sterile bag was mounted in a wave bioreactor. The cell suspension remaining from the previous stage “Cultivation and analysis of the working bank of the cell suspension” (see Example 1) was poured there and the nutrient medium was brought to a final volume of 2380 ml (by adding 1450 ml of the nutrient medium). Cultivation was continued for 48-96 hours until a cell concentration of 2.0 × 10 ⁶ cells / ml was reached. To control the procedure, every 24 hours, the concentration of cells in the Goryaev chamber was counted. The result was 2380 ml of a suspension containing ≈4.76 × 10 ⁹ cells. The cell suspension was defended for 30 minutes. The supernatant was drained (2/3 of the volume ≈1590 ml). The remaining volume was adjusted to 4750 ml (3960 ml of culture medium was added), thus, the cell concentration was 1.0 × 10 ⁶ cells / ml.

Next, the grown cell suspension was transferred to the next stage "Assembly of RPAN in cell suspension" for infection with RPAN culture.

2) Assembly of RPAN in cell suspension

Direct assembly of RPAN in cell suspensions was performed in a bioreactor. For this, they further seeded with a disaggregated starting production culture RPAN with a titer of at least 2 × 10 ⁸ PFU / ml, a volume of 250 ml, a bioreactor with a cell suspension of 4750 ml. Cultivation was continued until an RPAN concentration of at least 10 ⁸ PFU / ml was reached for 24 hours.

RPAN-containing cell suspension was checked for the following indicators: determination of sterility and determination of titer.

Thus, at this stage, as a intermediate product from 250 ml of RPAN culture with a titer of at least 2 × 10 ⁸ PFU / ml, 5 L of RPAN-containing suspension of crude with a titer of at least 10 ⁸ PFU / ml was obtained (i.e., not all less than 5 × 10 ¹¹ PFU RAN) 24 hours after inoculation of the bioreactor.

3) Precipitation of cell mass by centrifugation

The characterized RPAN-containing suspension, crude from the bioreactor, was sterilely poured into 6 centrifuge tubes of 830 ml each, balanced. Centrifuged at 6000 g for 15 minutes. The assigned supernatant in the form of a liquid part (spent nutrient medium, metabolic products, and other physical particles) was inactivated by autoclaving.

A solid precipitate is the desired cell mass containing RPAN.

Example 4

Purification of RPAN culture to obtain pharmaceutical grade concentrate.

The target substance - recombinant pseudo-adenovirus particles - has an intracellular localization, so first you need to destroy the cells and then clean. The process is complex and carried out in stages. A problem is also the aggregation of culture cells with hemagglutinins, albeit in the smallest amount, but expressed during a one-round infection.

The authors experimentally optimized the purification scheme of the drug. The commonly used four-fold thawing-freezing step to destroy the cells of the culture in order to extract RPAN has been replaced by chemical lysis. The cleaning scheme is multi-stage and consists of the following steps.

a) Resuspension in lysis buffer

To perform the actual purification of the RPAN culture, the culture cells were destroyed by chemical lysis.

For this, the precipitate obtained in the previous step from 5 l of RPAN-containing suspension of the crude was resuspended in lysis buffer.

The composition of this lysis buffer was determined experimentally, in comparison with the standard method of repeated freezing, the use of chemical lysis is more technologically advanced - it reduces the number of manipulations with tubes, reduces their number and significantly reduces the stage time (it takes only 8 hours to freeze), and there are opportunities for scaling production.

The lysis buffer contained the following components of the following composition: Tris (hydroxymethyl) aminomethane 5 mM, 1% Triton X-100; magnesium chloride hexahydrate 1 mM, sucrose 5.0%; sodium chloride 0.075 M, purified water to 60 ml, pH 8.0. The cell suspension was treated with lysis buffer for 3 hours. Concentration coefficient × 67. The volume of RPAN-containing suspension of the crude (total lysate) was 80 ± 5 ml.

b) Extraction of RPAN from RPAN-containing cell suspension

Further, for more effective destruction of cells, the RPAN-containing suspension was once frozen in four 50 ml vials (20 ml of suspension in each) for 2 hours in a cryogenic storage.

Thawed the suspension in a water bath (23-25 ° C), not allowing to warm. The contents of all tubes were combined into one glass vial. Visually carried out the control of color, turbidity of the suspension.

To remove genomic cell DNA, sodium chloride up to 1 M (20 ml sodium chloride 5 M) was added to 80 ml of RPAN-containing suspension. Was treated with benzonase to a final concentration of 500 U / ml (≈160 μl).

Gentle stirring was carried out for 1.5 hours in a water bath (33-35 ° C).

At this stage, from 0.08 L of RPAN-containing suspension (total lysate), 0.1 L of RPAN-containing suspension (lysate) was obtained.

c) Separation of RPAN from destroyed cells by centrifugation

Separation of RPAN from the destroyed cells was carried out by centrifugation. The supernatant containing RPAN was selected.

The dissolved RPAN-containing suspension was poured into 4 centrifuge tubes (≈25 ml each), balanced. Centrifuged at 9000 g for 10 min. The supernatant was transferred to clean tubes, repeated twice.

The supernatant was transferred to a clean container, closed with a multi-port lid and placed on a magnetic stirrer for ultrafiltration.

The sediment was discharged in the form of cell debris for inactivation for subsequent disposal.

From a RPAN-containing suspension (lysate) with a volume of 0.1 L, a RPAN-containing suspension (clarified lysate) of 0.095 L was obtained.

A technologist selected a supernatant in a volume of 7 × 0.1 ml and analyzed for titer in PFU.

d) Ultrafiltration

Most impurities, such as DNA fragments, host cell proteins and other impurities associated with the process, are smaller than the physical size of the RPAN (about 70 nm). Therefore, we used tangential filtration, during which the fluid flow moves tangentially (i.e. tangentially) to the surface of the filter, usually in a closed loop. The part of the fluid flow that passes through the filter is called the filtrate or permeate, and the remaining part of the fluid that does not pass through the filter and is recycled is retentate. If a membrane is used in the process of tangential filtration, the pore size of which is from 0.1 μm to several nanometers, then this process is called ultrafiltration.

UV cleaning solutions are attractive due to their high performance, low cost and easy scalability. Using tangential filtration, it is also possible to concentrate the product or replace the buffer. Our task was to determine the conditions of the ultrafiltration process, which allows us to get rid of impurities that are smaller than the size of the RPAN, while maintaining a high yield of active pseudo-adenovirus particles. When developing the ultrafiltration process, a number of process parameters were determined:

I) a membrane (membrane material, pore size);

Ii) the load on the membrane, i.e. the concentration of the target substance in the sample for application;

Iii) recirculation rate, process temperature, time, degree of buffer replacement, transmembrane pressure.

During the ultrafiltration, the concentration of pseudo-adenovirus particles was monitored, since more than 50% of the material could be lost at this stage due to aggregation. It is believed that aggregation can cause a high local concentration of viral particles near the surface of the membrane. Thus, the optimization of this step was critical for increasing the product yield.

When working out the ultrafiltration process conditions, two types of membranes were analyzed: polyethersulfone membranes with pore sizes of 300 and 500 kDa, as well as regenerated cellulose membranes with pore sizes of 300 and 1000 kDa. Both materials are hydrophilic; they are characterized by low sorption of protein molecules. The results indicated a greater throughput of polyethersulfone membranes compared to a regenerated cellulose membrane and their advantage in terms of reducing the time of the ultrafiltration process. In parallel, retention of the target component in retentate was evaluated. Data on the effect of membrane pore size on the yield of active RPANs during ultrafiltration are shown in Table 1. The amount of viral particles was analyzed by the plaque formation method.

The results obtained indicate that when using membranes with a pore size of more than 300 kDa, part of the recombinant pseudo-adenovirus particles passes through the membrane, thereby reducing the yield of the product. However, the time of the ultrafiltration process when using a membrane with a pore size of 300 kDa increases from 1.5 to 3 times.

Cell biomass parameters may vary significantly for each cultivation. It was found that the smallest losses at the ultrafiltration stage correspond to the conditions when the amount of viral particles in the sample entering the ultrafiltration stage is equivalent to about 50-100 l of cultivation volume per square meter of membrane area.

As a result, the following ultrafiltration scheme was developed.

The clarified cell lysate entered the ultrafiltration stage to get rid of impurities smaller than the size of the RPAN. Ultrafiltration was carried out on a pre-prepared membrane of polyethersulfone 0.015 m ² with a pore size of 300 kDa. The solution was diluted 4 times with ultrafiltration buffer, then concentrated to the original volume. In the process of ultrafiltration, the transmembrane pressure in the system was controlled (0.6-1.0 bar).

Thus, the developed ultrafiltration stage is presented below.

First prepared the device for ultrafiltration. An ultrafiltration buffer of the following composition was prepared: Tris (hydroxymethyl) aminomethane 50 mM, magnesium chloride hexahydrate 2 mM, sucrose 5.0%, sodium chloride 1 M, water for injection - up to 1.62 L, pH 7.5.

The system was washed with 3 L of purified water until the preservation solution of 0.1 M NaOH was completely washed out (pH control - 6.0-7.0). Pumped the system with air, balanced 0.3 l of ultrafiltration buffer.

They carried out the ultrafiltration process itself.

For this, a suspension of 95 ml was made up to 400 ml with ultrafiltration buffer and ultrafiltered. Ultrafiltration was performed using 1 L of ultrafiltration buffer. The volume of retentate after ultrafiltration was ≈100 ml. Diluted the drug 4 times with “empty” buffer (Tris 40 mM). The resulting volume is 400 ml.

At the end, the volume of the buffer (permeate) pumped through the membrane was measured.

We measured the pressure on the manometer every 5 minutes (it should not exceed 1.2 atm).

At this stage, 0.095 L of RPAN-containing suspension (clarified lysate) was consumed and 0.4 L of RPAN-containing suspension (retentate) was obtained.

5) Anion exchange chromatography

Further purification was carried out by anion exchange chromatography.

The process began with the preparation of the column.

Buffer A was prepared containing the following components: Tris (hydroxymethyl) aminomethane 40 mM, magnesium chloride hexahydrate 2 mM, sucrose 5.0%, sodium chloride 0.5 M, water for injection up to 2100 ml, pH 7.5.

Buffer B was prepared containing the following components: Tris (hydroxymethyl) aminomethane 40 mM, magnesium chloride hexahydrate 2 mM, sucrose 5.0%, sodium chloride 0.27 M, polysorbate-80 0.1%, water for injection - up to 1300 ml pH 7.5.

The column was conditioned with buffer (phase) B - about 500 ml until the conductivity index reached the plateau (42-44 μS). Next, conditioning the column volume with buffer (phase) A is about 500 ml until the conductivity index reaches a plateau (25.6 μS).

A suspension containing RPAN was applied to a 70/300 column, flow rate 15 ml / min. Conductivity of the applied sample: 25.4-26 μS. Column volume 400 ml.

The pressure was monitored during chromatography (should not exceed 1 bar).

Sorption of fractions containing RPAN was carried out. For this, after application of the drug, impurities were eluted — 3.0 CV (1200 ml) of buffer A. Then, the viral peak (fraction) containing RPAN was removed using buffer B — 2.0 CV (800 ml). The column was conditioned with buffer A - 1.0 CV (400 ml). The desired peak fractions were collected — 400 ml eluate volume. The pressure was monitored during chromatography (should not exceed 1 bar).

The OCC controller selected 7 × 0.1 ml of eluate and the titer was monitored.

6) Size exclusion chromatography Started with column preparation

An exclusion chromatography buffer was prepared containing the following components: Tris (hydroxymethyl) aminomethane 10 mM, magnesium chloride hexahydrate 1 mM, sucrose 5.0%, sodium chloride 75 mM, polysorbate-80 0.05%, water for injection - up to 6, 5 l, pH 7.5.

The column was conditioned with size exclusion chromatography buffer in a volume of 700 ml - 5 times.

To sorb fractions containing RPAN, the preparation was eluted from the anion exchange column in a volume of 120-130 ml. Collected a breakthrough. An elution chromatography buffer was carried out in a volume of 1000 ml. The peak and the next eluate were collected. Several rounds of chromatography were performed (the amount depended on the volume of the eluate on AOX). The volume of combined viral peaks in size exclusion chromatography is 350 ml (350-500 ml).

OCC technologist selected 7 × 0.1 ml of eluate and the following parameters were monitored:

- Definition of titer in PFU;

- The amount of protein in the eluate according to the method of Bradford.

After collecting the peak of RPAN, EDTA and ethanol were added to the preparation to the following final concentrations: EDTA disodium salt - 100 μM, Ethanol, 96% concentration 0.5%, volume 0.3 ml.

A suspension of RPAN in a volume of 350 ml was concentrated in an ultrafiltration unit. Inlet pressure to the cartridge did not exceed 1.1 Bar.

The purity of the RPAN after the step was monitored using an analytical HPLC technique. In fig. 1 presents the result of the analysis.

The result of the analysis of RPAN concentrate after size exclusion chromatography. Peak 1 - RPAN.

The result of the analysis showed that the purity of the pseudo-adenovirus particles obtained at the stage of 97%.

Thus, at this stage, from 0 .6 ml of RPAN-containing suspension of under-purified (viral peak), 0.06 L of concentrate of suspension of RPAN not sterile was obtained.

7) Sterilizing filtration of RPAN concentrate

Poured from a glass bottle into a filtration system (0.22 μm filter from PES material (polyethersulfone)) RPAN concentrate is not sterile. The vacuum pump was connected. The load is 2.1 o / cm ² . Before filtering, the membrane was moistened with 15 ml of formulation buffer.

Filtered for 120 minutes. Store RPAN_ no more than three weeks in the refrigerator at a temperature of no more than + 4 ° C.

The OCC controller took a sample in a volume of 1 ml and analyzed for the following indicators and their compliance with the requirements:

- Residual DNA cell culture - not more than 150 ng / ml, corresponds;

- Residual cell culture protein - not more than 0.01% of the total protein, corresponds;

- Total cell culture protein - 22.5 mg / ml, consistent;

- Sterility - consistent;

- Specific activity - RPAN 2.02 × 10 ⁹ PFU / ml, consistent;

- Replicatively competent adenoviruses - less than 7 × 10 ³ - not found.

From 0.06 L of non-sterile RPAN concentrate (including RPAN 2.12 × 10 ⁹ PFU / ml, a total of 1.275 × 10 ¹¹ PFU), 0.059 L of sterile RPAN concentrate (including RPAN 2, 02 × 10 ⁹ PFU / ml, total 1.19 × 10 ¹¹ PFU).

The product yield minus losses at the stage is - 93.7%

The obtained sterile concentrate of RPAN expressing the A / California / 07/2009 (H1N1) influenza hemagglutinin gene with chromatographic purity of 97% fully complies with pharmaceutical quality requirements.

Example 5

Preparation of the finished form of influenza vaccine.

The RPAN concentrate obtained according to the invention can be used to prepare the finished form of the influenza vaccine. For this purpose, RPAN concentrates carrying the hemagglutinin genes of the three seasonal strains (one of which was the concentrate of the influenza virus strain of the invention) in sterile pharmaceutically acceptable buffer solution by stirring for 20 min were dissolved sterilely on a magnetic stirrer, to obtain an RPAN solution. After the mixing time, the pH of the solution was determined, it should be from 6.0 to 8.0 (corresponded). Next, sterilizing filtration of the RPAN solution was carried out. The RPAN solutions of the three strains carrying different hemagglutinins were mixed until an effective amount of RPAN was obtained:

A / H1 / N1 - at least 1 × 10 ⁸ PFU / dose

The second seasonal strain of influenza virus - at least 1 × 10 ⁸ PFU / dose

The third seasonal strain of influenza virus is at least 1 × 10 ⁸ PFU / dose in 0.5 ml of a pharmaceutically acceptable buffer solution.

The resulting influenza vaccine for intramuscular administration in vials, dose / 0.5 ml, after passing through quality control, met the requirements of the international and Russian pharmacopoeia according to the following indicators:

1. Authenticity (positive PCR reaction to the presence of the fifth genotype of the human adenovirus and the influenza virus hemagglutinin gene) - Corresponds;

2. Transparency (the drug must withstand comparison with standard IV) - Corresponds to standard IV;

3. Color (the drug should be colorless or the intensity of the color of the solution should be no more than the color of the standard Y6) - The drug is colorless;

4. Mechanical inclusion (the drug must withstand the requirements (RD 42-501-98) - Compliant;

5. The pH of the solution (from 6.0 to 8.0) - 7.0;

6. Recoverable volume (not less than nominal) - Corresponds;

7. Sterility (must be sterile) - Sterile;

8. Specific safety:

A. (the drug should not contain adeno-associated viruses) - Not detected;

B. (the drug should contain less than 7 × 10 ³ replicatively competent adenoviruses per dose) - Compliant;

9. Abnormal toxicity (should be non-toxic) - Non-toxic;

10. Pyrogenicity - (the drug must be pyrogen-free) - Pyrogen-free;

11. Specific activity, the drug should contain:

A / H1 / N1 - at least 1 × 10 ⁸ act. unit / dose;

The second seasonal strain of influenza virus is not less than 1 × 10 ⁸ act. unit / dose;

The third seasonal strain of influenza virus is not less than 1 × 10 ⁸ act. unit / dose;

- Conform

12. Residual DNA cell culture (the drug should contain no more than 10 ng / dose) - None.

13. Total protein - not more than 1.5 mg / dose - Compliant;

14. Residual cell culture protein - not more than 0.01% of the total protein - Compliant.

The intramuscular influenza vaccine has been shown to be highly effective and safe after administration to laboratory animals during preclinical studies.

It was concluded that from a sterile RPAN concentrate with a titer of 2.02 × 10 ⁹ PFU / ml obtained by the method of the invention, it is possible to produce an influenza vaccine that meets the quality of a pharmaceutical product with a total quantity of at least 900 doses.

Thus, all the above examples confirm the fulfillment of the technical task set in the invention.

Industrial applicability

All of the above examples also confirm the industrial applicability of the claimed invention.

The essence of the invention is to reduce the effect of hemagglutinins expressed by RPANs during the cultivation process on the main cycle of concentrate production. This is achieved by introducing an additional stage in the preparation of the starting RPAN culture, which consists in carrying out its disaggregation by purification from hemagglutinins. An additional effect of reducing aggregation in the main growth cycle is obtained by reducing the time of RPAN cultivation in the bioreactor to 24 hours in order to conduct only a one-round infection of the cell culture, which can dramatically reduce the number of hemagglutinins expressed by RPANs and, therefore, significantly reduce the aggregation of cell culture. At the same time, the selected process conditions make it possible to level out small losses in the RPAN crop due to shortening the cultivation time by 2–3 times in comparison with standard methods. This is due to an increase in the efficiency of infection of the production suspension culture with RPANs at the stage of introducing the starting disaggregated culture of RPANs, since there is no aggregation of the cell culture with hemagglutinins using the standard method introduced with the starting culture, therefore, according to the method specified in the invention, a larger amount of the production cell culture will be infected. The decrease in aggregation, together with the proposed conditions for multi-stage purification, will affect the increase in RPAN yield and the possibility of obtaining a concentrate with high titers, chromatographic purity, corresponding to the quality of a pharmaceutical product suitable for the industrial production of influenza vaccines.

List of abbreviations

RPAN - Recombinant Pseudo Adenoviral Particles

PFU - plaque-forming units

Claims (7)

A method for producing a concentrate of recombinant pseudo-adenovirus particles expressing the influenza A / California / 07/2009 (H1N1) hemagglutinin gene characterized in that technological stages include the following sequences: - receiving from the master bank of a production cell culture - a working bank of a production suspension cell culture with a concentration of 2.0 × 10 ⁶ cells / ml with an increase in cell yield of not less than 700 times, and receiving with an increase in active RPAN units of not less than 33 times from the RPAN master bank - the RPAN master bank with a titer of at least 2 × 10 ⁸ PFU / ml; - preparation of samples of the disaggregated starting RPAN culture with activity of at least 2 × 10 ⁸ PFU / ml from the RPAN working bank obtained in the previous stage by purification from hemagglutinins expressed by RPAN during the growing of the RPAN working bank; - conducting a one-round infection of a production suspension cell culture with a cell concentration of 1.0 × 10 ⁶ cells / ml in a bioreactor with a nutrient medium by introducing a disaggregated starting production culture of RPAN with a titer of at least 2 × 10 ⁸ PFU / ml, resulting in RPAN- containing suspension untreated with a titer of at least 10 ⁸ PFU / ml by cultivation for 24 hours with an increase in the yield of active RPAN units during the growth process by at least 10 times, with further sedimentation of the cell mass by centrifuge gyration and disposal of the spent nutrient medium; - resuspension of the cell mass precipitated at the previous stage in a lysis buffer containing a non-ionic detergent Triton X-100 at a concentration of 1% for 3 hours, then a single freeze for 2 hours in a cryostorage followed by thawing of the suspension in a water bath at a temperature of 23-25 ° C, then treatment with benzonase, and separation of RPAN from the destroyed cells sequentially by centrifugation, ultrafiltration, anion exchange chromatography, size exclusion chromatography, sterilization filtration to obtain the use of sterile RPAN concentrate of pharmaceutical quality with a high level of chromatographic purity and with a titer of at least 5 × 10 ⁸ PFU / ml.

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