WO2016204644A1

WO2016204644A1 - Method of producing a drug based on a non-replicating viral vector expressing a human toll-like receptor gene and a modified flagellin gene

Info

Publication number: WO2016204644A1
Application number: PCT/RU2015/000379
Authority: WO
Inventors: Максим Михайлович ШМАРОВ; Ирина Алексеевна КАЛУГИНА; Александра Владимировна САМОРУКОВА; Наталья Сергеевна АЛФЕРОВА; Рустам Равшанович АТАУЛЛАХАНОВ; Наталья Вахитовна ЕРЕМИНА; Василий Игоревич КАЗЕЙ
Original assignee: Общество С Ограниченной Ответственностью "Панацела Лабс"
Priority date: 2015-06-19
Filing date: 2015-06-19
Publication date: 2016-12-22
Also published as: RU2590588C1

Abstract

The invention relates to biotechnology and concerns a method of producing the drug "Mobilan (M-VM3)". The invention improves the stages of production, increasing production efficiency and achieving an optimal ratio between production time, drug yield and the amount of resources required for production purposes. In particular, in the process of modernization, the following stable production parameters have been established: batch size - 1500 vials; production time - 18-22 days; total volume of culture medium required 44 l; volume of virus-containing M-VM3 suspension - 25 l; cell density at time of infection - from 0.8 х 10⁶ to 1 х 10⁶ cells/ml; time of cell harvest - on day three (i.e. after approximately 72 hours); temperature at time of infection - 36°С; multiplicity of infection - 5-10 PFU/cell; total concentration of viral construct at harvest - 8 х 10⁸ PFU/ml.

Description

A method of manufacturing a preparation based on a non-replicating viral vector expressing a human TOLL-like receptor gene and a modified flagellin protein gene.

The invention relates to biotechnology and relates to a method for the production of the drug "Mobilan (M-VM3)". The invention optimizes the stages of production, increasing its profitability, making the optimal ratio of production time / amount of drug received / amount of funds spent on production. In particular, during the modernization, the following stable parameters of the production process were developed: the volume of a batch of 1,500 bottles, the production time was 18-22 days, the total consumption of the nutrient medium was 44 l, the volume of the virus-containing M-VM3 suspension was 25 l, and the cell density was infection time - in the range from 0.8 x 10 ⁶ to 1 x 10 ⁶ cells / ml; cell collection time - on the third day (approximately 72 hours); the temperature at infection is 36 ° C, the multiplicity of infection is 5-10 PFU / cell, the final concentration of the viral construct when collecting 8 X 10 ⁸ PFU / ml.

Innovative drug Mobilan (M-VM3), a concentrate for the preparation of a solution for intratumoral administration, containing a replicable bicistronic human adenovirus vector expressing the human Toll-like receptor type 5 gene and a pharmacologically optimized gene for secreted protein fragment of Salmonella bacteria flagellin, 10 ¹² particles of the bicistronic human adenovirus vector unable to replicate in 1 ml of buffer solution.

The prior art it is known to obtain virus concentrates on an industrial scale, in particular, from RU2029561 from 02.27.1995. The closest solution found in the prior art is RU2465327 from 10.27.12, which describes the purification of recombinant human adenoviruses. The method includes preparing a virus-containing material in HEK293 cell culture, including: collecting these cells, preparatory cell lysis and release of adenovirus from cell debris, as well as directly cleaning the adenovirus on a carrier with monomeric avidin, including preliminary preparation of the carrier and planting on it an adenovirus, precipitation and washing of the carrier with adenovirus, elution of the adenovirus from the carrier and the final selection of the virus after centrifugation.

SUMMARY OF THE INVENTION

The viral construct M-VM3 (Mobilan) was produced on a semi-industrial scale using an optimized cell culture production process for this specific construct and the conditions for lysis and purification of human serotype 5 adenovirus, i.e. purification by anion exchange chromatography. Production was carried out in accordance with GLP rules, all analyzes were carried out using good documentation practice.

A distinctive feature of the M-VM3 construct from other recombinant adenoviral vectors is the presence in the genome of both the human Toll-like receptor type 5 gene, which is expressed on the membrane of infected cells, and the pharmacologically optimized secreted protein flagellin fragment of the Salmonella bacteria. When particles of the M-VM3 preparation (Mobilan) are grown in HEK293 cells, a Toll-like type 5 receptor is activated, which leads to the expression of the transcription factor NF-κΒ gene in the cells. The transcription factor NF-KB is a universal transcription factor that controls the expression of the immune response genes, it is also able to protect the cell from apoptosis and affect the cell cycle. It was not known from the prior art how the activation of the NF-KB factor in the cells in which the M-VM3 construct propagates will affect the effectiveness of the drug production and subsequent purification steps. As a result of the production of several series of the M-VM3 preparation (Mobilan), we developed the following method for the industrial production of the drug, which is conditionally divided into two stages: building up the M-VM3 virus construct in HE 293 cell culture and its multi-stage purification.

To obtain 25 L of the virus-containing cell suspension, 44 L of culture medium was expended. In this case, the cultivation is carried out in an inoculator with Cell spin magnetic stirrer, and when scaling, in a Biostat CultiBag wave bioreactor, using two disposable sterile plastic containers with a working volume of 10 l and one working volume of 25 l. During the growing process, the amount of cell suspension is scaled from a cell bank sample in the amount of 1 tube (1.5 ml) to 24.4 L. Moreover, in the process of scaling, the cell suspension is grown with a partial replacement of the spent nutrient medium with fresh in the ratio of 2/3 fresh to 1/3 old. The process of increasing the required volume of cell culture in the volume of 23.8 liters is carried out simultaneously with the cultivation of a viral flow of 1.2 liters for subsequent infection of this culture. The concentration of cells upon infection is 1.0 * 10 ⁶ cells / ml. Cell growth is carried out to a final virus concentration of 8> 10 ⁸ PFU / ml.

Next, the resulting suspension is transferred for purification. Initially, the suspension is centrifuged to drain the spent nutrient medium. It is further processed with lysis buffer, frozen and treated with nuclease for effective cell lysis with further successful release of the M-VM3 viral construct, taking into account the nuclear localization of the latter. The temperature and time conditions of gentle mixing were selected for efficient nuclease treatment. After lysis, repeated centrifugation is performed to release the viral runoff from cell debris. Then the virus-containing solution is transferred to ultrafiltration and chromatographic purification in accordance with the developed parameters for adenovirus. Multiple chromatographic purification procedures are performed with a filled column volume of ~ 174 ml using the AKTA Avant chromatographic system. Next, the drug is transferred to sterilizing filtration and bottling. After crimping the vials with aluminum caps, checking for tightness and labeling, the M-VM3 (Mobilan) preparation is frozen and stored at -70 ° C. Production output - 1,500 vials of the drug with a concentration of 10 ¹² particles of the bicistronic human adenovirus vector unable to replicate in 1 ml of buffer solution. Testing the final product according to the indicators description, pH, microbiological purity, the content of bacterial endotoxins (LAL test), infectious titer, concentration of viral particles and RCA (capable of replication adenovirus) showed that the product has the expected quality characteristics.

Examples

Example 1 Cultivation of Cell Suspension

Preparation and storage of culture medium

A sample of dry nutrient medium CD293AGT was prepared and dissolved in parts of purified water with stirring on a magnetic stirrer. After dissolution, a second part of purified water was added from the composition ratio. Then, sterilizing liquid nutrient media was carried out on a filtration system with Corning vacuum filters, pore diameter 0.22 μm. Glutamine was added to the sterile medium. The preparation of culture media was carried out in a room separate from the boxes intended for work with cell culture and M-VM3 culture. By optimizing the process (due to partial replacement of the nutrient medium during reseeding), the required volume of the nutrient medium is reduced. The total volume of 44 l (44000 ml). The finished medium was transported to:

a) cultural boxing

b) viral boxing

c) reactor

Obtaining cell suspension

Table 1

JVe Sequence of operations Displacement, ml

1 Cell culture in a volume of 1.5 ml with a concentration of 2 <10 ⁶ 6

cells / ml taken from the cryostorage were thawed at 37 ° C.

Next, the tube was centrifuged, the supernatant was drained

liquid and added a nutrient medium in a volume of 1 ml. AT

sterile culture box with laminar flow

class B seeded 1.5 ml of cell suspension in

plastic container with a surface area of 25 cm ² (containing 4.5 ml of culture medium). Cultivated in a CO2 incubator to a concentration of 2x10 ⁶ cells / ml (48 hours).

Concentration measurement.

The cell suspension was plated into a plastic container with 16 surface areas of 75 cm ² . The initial cell concentration is -0.8 10 ⁶ cells / ml. Cultivated in a C02 incubator to a concentration of 2.0 ^x 10 ⁶ cells / ml (24-48 hours).

Then a cell suspension of 16 ml volume was transferred to 40 Cellspin bioreactor with a volume of 100 ml, 24 ml of fresh nutrient medium (total volume 40 ml) was added.

Initial concentration = 0.8x 10 ⁶ cells / ml. Next, the bioreactor was placed in a CO2 incubator. Cultivated to a concentration of 2.0 to 10 ⁶ cells / ml (24-48 hours).

Upon reaching the indicated concentration, 60 ml of 100 nutrient medium (total volume of 100 ml) was added. Next, the bioreactor was placed in a CO2 incubator. Cell cultivation was performed until a concentration of 2.0 x 10 ⁶ cells / ml was reached.

Inoculated 100 ml of a cell suspension from a bioreactor 250 with a volume of 100 ml into a 250 ml Cellspin apparatus.

Added nutrient medium, total volume 250ml.

Cultivated in a CO2-incubator to the desired concentration concentration (24-48 hours).

Aseptically transferred 250 ml of a cell suspension from 650 bioreactor with a volume of 250 ml in Cellspin with a volume of 1000 ml.

Added 400 ml of culture medium. Initial concentration = 0.8x 10 ⁶ cells / ml. Upon reaching the required concentration, the cell suspension was defended. The supernatant was drained (2/3 of the total volume).

The remaining volume was brought up to 1600 ml. In reactor 1600 boxing with a Class C laminar flow into a Biostat CultiBag RM20 Optical wave fermenter mounted a sterile CultiBag RM 10 L bag. The cultivation mode was adjusted: temperature, platform angle, air flow, CO2 concentration, pH, p02. Aseptically transferred the resulting cell suspension. Cultivated to the required cell concentration. The supernatant was drained.

A suspension of up to 4000 volumes of 4000 ml was added with fresh nutrient medium. Upon reaching the required concentration, the cell suspension was defended and the supernatant was drained.

The remaining volume was brought up to 10200 ml. In a Class C laminar flow reactor box 10200, a sterile CultiBag RM 50 L bag was mounted in a Biostat CultiBag RM20 Optical wave fermenter. The resulting cell suspension was poured there. We made the adjustment of the cultivation mode, selected taking into account the optimization of the process of increasing cell culture:

temperature, angle of inclination of the platform, air flow, concentration of C0 ₂ , pH, p0 ₂ . Cultivated to a concentration of 2.0 x 10 ⁶ cells / ml (24-48 hours). Upon reaching the required concentration, a part of the cell suspension, in a volume of 600 ml, was transferred to the stage of “Production of culture

M-VM3. "

From the remaining cell suspension with a volume of 9600 ml, 12000 supernatants were drained (2/3 of the volume of ~ 6400 ml).

The remaining volume was brought up to 12000 ml (adding 8800 ml of culture medium). The cultivation mode was adjusted: temperature, angle of inclination of the platform, air flow, concentration of С0 ₂ , pH, р0 ₂ . Cultivation continued for 48-96 hours until reaching

cell concentrations 2.0> 10 ⁶ cells / ml.

1 1 Cell suspension was defended and the supernatant drained 23800

liquid. We brought the remaining volume to 23800 ml, so

thus, the cell concentration was 1.0 * 10 ⁶ cells / ml.

Next, this cell suspension was transferred to the stage

"Assembling M-VM3 in a cell suspension."

Thus, in order to obtain 600 ml of the cell suspension as a substrate for the viral seed of M-VM3 and 23800 ml of the cell suspension for the preparation of the M-VM3 virus construct, 43 liters of culture medium were used in the process of optimized scaling. In the standard method for culturing a eukaryotic cell culture of HEK293, which includes centrifuging the cell suspension at each stage and completely replacing the culture medium, the flow rate of the culture medium is at least 52.1 liters (Table 1).

Table 1.

Consumption Practical output

Nutrient Cell Culture Filter ion Volume Cell Culture Medium HEK293 Nutrient HEK293 CD293AG Vacuum

T dry system, medium, l Amount Volume

Corning, 0.22 cell count, μm, 1000 ml L cells

Weight, g Volume, Concentration Quantity,

ml tion, cells / ml pcs

Optimized 770 1.5 2.0 * 10 ⁶ 5 43 2.5 x 1 0 "24.4 production process

suspensions HEK. 293

Standard process 930 1.5 2.0 ^χ 10 ⁶ 6 52, 1 2.0 x 10 "24.4 receiving supensi

HEK293 Example 2. Obtaining the viral construct M-VM3 in cell suspension

To obtain a viral construct, it is necessary to obtain a virus “seed” using the cell suspension described in Example 1 in parallel with the cell suspension buildup. To do this, 600 ml of the cell suspension were sterilized from the fermenter and transferred to 1000 ml of Cellspin, sedimented and the supernatant was drained . The remaining volume was brought up to 1200 ml, thus, the cell concentration was 0.8-1, 0><10 ⁶ cells / ml. In a class B viral laminar box with a laminar flow, 70 ml of M-VM3 virus material was added from a working bank (with a titer of at least 10 PFU / ml) into the resulting cell suspension. Thus, the ratio of culture from the working bank (source) and the resulting cell suspension is 1/17 and cultured in it for 48-72 hours). Incubated in a CO2 incubator. The OCC controller performed a titration seed test. The titer must be at least 2 ^x 10 ⁸ PFU / ml.

At this stage, the following was consumed: a cell suspension of HEK293 0.6 L; nutrient medium 1, 0 l; viral material M-VM3 0,07l. Total: 1, 67l.

Obtained at the stage: viral "seed" M-VM3 1, 27 l; spent nutrient medium 0.4 l. A “seed” of 20 ml is selected for quality control.

Obtaining the virus construct M-VM3 volume of 25 l

In the viral laminar box with a class B laminar flow, the cultivation mode was adjusted: temperature, platform angle, air flow, CO2 concentration, pH, p02. Inoculated with “seed” M-VM3 (titer of at least 2 * 10 PFU / ml), 1250 ml bag, CultiBag RM 50 bag with 23750 ml cell suspension. Cultivation was continued until the concentration of M-VM3 was not less than 8 10 ⁸ PFU / ml for 48-72 hours.

Spent on stage: HEK293 cell suspension 23.75 L; M-VM3 virus "seed" of 1.25 liters. Total: 25 L

Obtained at the stage of 25 l of M-VM3 viral construct.

Consumption Practical output Cell culture Viral seed M- Time, hours M-VM3 viral HE 293 V 3 construct

Amount Volume, Active Volume, l

Art.

Volume, l Active cell PFU / ml

BOO

Getting 2.5 ^χ | O ⁹ 24.4 at least 1.25 96- 144 25

viral 10 ⁸

construct M-VM3 PFU / ml

in HEK.293 cells

25 l

Obtaining other 2.0x 10 ⁹ 24.4 at least 1, 7 48-96 25 5 ^χ 10 ¹² viral vectors 10 ⁸

in HEK293 PFU / ml cells

25 l

Example 3 Purification of the M-VM3 Viral Construct

Centrifugation

The obtained and characterized M-VM3 viral construct from the bioreactor was sterilely poured into centrifuge tubes, balanced, and centrifuged at 6000 g for 15 minutes. The supernatant, the spent nutrient medium, metabolic products, and other physical particles were discharged, transferred to inactivation by autoclaving.

The precipitate from 25 l of the viral suspension was resuspended in the buffer (concentration coefficient x67), thus, the ratio of the volume of the precipitate to the volume of the buffer was 1-16.6, and mixed on a magnetic stirrer. The volume of the suspension was 380 ± 5 ml.

- freezing

M-VM3-containing suspension was poured into the required number of 50 ml vials (20 ml of suspension in each). It was then stored for 2 hours in the refrigerator.

- Cell destruction and nuclease treatment

Thawed the suspension in a water bath (23-25 ° C), not allowing to warm. The contents of all tubes were combined into one glass bottle. Visually inspected color and turbidity of the suspension. Treated with benzonase to a final concentration of 150 U / ml (-240 μl). Spent gentle stirring on a magnetic stirrer for 3 hours at room temperature (21-23 ° C).

Spent on stage: 0.38 L of M-VM3 containing suspension; benzonal 2.4x 10 ^"4. Obtained at the stage: M-VM3 containing suspension (lysate) 0.377 l;

Centrifugation

The dissolved M-VM3-containing suspension was poured into 5 centrifuge tubes of ~ 25 ml each. Centrifuged at 9000g for 10 min. The operation was repeated twice using the remaining lysate volume. 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 removed in the form of cell debris for inactivation for subsequent disposal.

Spent on stage: M-VM3 containing suspension (lysate) 0.377 l.

Obtained at the stage: M-VM3 containing suspension (clarified lysate) 0.370 l; cell debris of 0.007 liters.

- Ultrafiltration

The system was washed with purified water of 7 l volume 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 0.5 l of ultrafiltration buffer.

The suspension with a volume of ~ 400 ml was brought with ultrafiltration buffer to a volume of 1600 ml, ultrafiltered in an ultrafiltration unit. The retentate pathway was washed with 200 ml empty buffer, this wash was combined with the filtered retentate and 800 ml of empty buffer was added. The retentate volume was measured. 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 (should not exceed 1.2 atm).

Spent on stage: M-VM3 containing suspension (clarified lysate) 0.370 L; buffer for ultrafiltration 1, 7 l, "empty" buffer 1, 0 l, purified water 7 l. Obtained at the stage: M-VM3 retentate 1, 2 l;

Anion exchange chromatography The anion-exchange column was balanced with buffer A and the channels were filled: until a constant conductivity of -40-50 mS / cm was established (1.4CV = 500 ml). Buffer B was prepared and

they balanced the column to a conductivity of ~ 28-30 mS / cm (1.4СУ = 500ml). The pressure was monitored during chromatography (should not exceed 2 bar).

A solution containing M-VM3 was applied to the column, flow 193 ml / min (300 cm / h). Column volume 400 ml.

Washed the contents of the column with buffer A in a volume of 7CV (2800 ml) at a flow rate of 100 ml / min. Impurity Elution - 1.5 CV (600 ml) of buffer B at a flow rate of 100 ml / min; during elution, a peak (fraction) containing M-VM3 converges. The column was conditioned with buffer A - 1.5 CV (600 ml). The fractions of the desired peak were collected.

Spent on stage: M-VM3-containing suspension (retentate + flush) 1, 2 l; buffer "A" 3.9 L (NaCl 0.5 M), buffer "B" 1.1 L (NaCl 0.27 M); sorbent Sepharose 4 Fast Flow 0.4 l.

Obtained at the stage:

M-VM3-containing solution (peak M-VM3) 0.4 l

- Size exclusion chromatography

The column was equilibrated with size exclusion chromatography buffer in a volume of 1.5CV (1.2L). The preparation was eluted from the anion exchange column in a volume of 130-140 ml, flow 100 ml / min (77 cm / h). Collected a slip. Elution 1.5CV (1.2L) size exclusion chromatography buffer, flow 100 ml / min (-12 min). They collected the peak and the next eluate. The operation was repeated twice with the remaining volume of the eluate.

Spent on stage: M-VM3-containing solution (peak M-VM3)

0.4 l; 4.8l exclusion chromatography buffer, Q SepharoseXL Virus sorbent

Licensed 0.8 L

Obtained at the stage: solution (M-VM3) 1.65 L, including M-VM3 1, 1x10 particles / ml.

- Sterilizing Filtration (M-VM3) RU2015 / 000379

12

Sterilized filtration was performed through the system using a filter cartridge with a pore size of 0.45 μm and a filter cartridge with a pore size of 0.22 μm under a pressure of (1, 5-2.0) atm.

Spent on stage: Intermediate solution (M-VM3) 1.65 L, including M-VM3

1 7

1.1x10 particles / ml.

Obtained at the stage: Solution (M-VM3) 1, 65 L, including M-VM3 1, 0x10 particles / ml.

Example 4. Filling and packaging of the drug Mobilan

- Bottling Mobilan solution (M-VM3) in bottles

On the dispenser, set the volume of the dosed solution - 1.0 ml with the help of the movable clamp of the eccentric, fixing it with a clamping screw. Turn on the machine and check the accuracy of the set dose. Fill the vibratory hopper with sterile plugs of the correct size. Fill the feeder table with sterile bottles of the required capacity. Carry out bottling. Periodically during the operation, every (50 - 100) vials (depending on the volume of the series), the technologist and the OK controller carry out a dose control of the solution in the bottle.

Spent on stage: solution (M-VM3) 1.65 L, including M-VM3 1, 0x1012 particles / ml, 1650 sterile bottles and rubber stoppers.

Obtained at the stage: Mobilan (M-VM3) in vials of 1610 pcs, including M-VM3 1.0x1012 particles / ml.

- Rolling bottles

Bottles corked with rubber stoppers were rolled with aluminum caps on a Cozzoli GM 200 bottle rolling machine. Next, a leak test was performed. Then labeling and packaging of the bottles.

Spent on stage: Mobilan (M-VM3) in bottles of 1610 pcs, including M-VM3

1.0x10 ¹² particles / ml. , 1650 sterile aluminum caps.

Obtained at the stage: Mobilan (M-VM3) in 1520 bottles, including M-VM3 Ι, Ο ^χ ΙΟ ¹² particles / ml.

- Freezing finished products

Finished products were put in containers, on which they hung a yellow label “In quarantine” with the name of the preparation, series number, production date product, date of delivery for analysis in the JCC and the number of packs in the container. The containers were sealed with a lid. Then the containers are placed in a pharmaceutical refrigerator at a temperature of -70 ° C for 24 hours. Next, the OCC controller selected the required number of packages and handed over at -70 ° C to conduct analysis for compliance with the FSP and arbitration storage. After receiving a positive opinion of the JCC, the containers were marked with a green label that indicated the series number, the number of packs in the series, the number of packs in each container and the date of delivery of the finished product to the warehouse. The packaged preparation was transferred to the finished goods warehouse under transportation conditions of -70 ° C.

Example 5. Quality control of the drug Mobilan

The innovative drug Mobilan (M-VM3) is transferred to quality control and must comply with the standards described in the pharmacopoeial article of the enterprise:

1) Description. The drug should be a colorless or bluish tinge with an opalescent solution.

2) Authenticity. The gene for the bicistronic bicistronic human adenovirus vector and the genes for the human Toll-like type 5 receptor and a pharmacologically optimized secreted fragment of the flagellin protein of bacteria of the genus Salmonella must be present. There should be no nonspecific fragments.

3) Transparency. A 10% solution of the drug in a 5% aqueous glucose solution must withstand comparison with the Ns IV standard (according to GF XII).

4) Color. The drug should be colorless or the color intensity of the solution should be no more than the color of the standard Y6.

5) Mechanical inclusion. Visible and visible particles. The drug must withstand the requirements according to RD 42-501-98.

6) pH. From 7.0 to 9.0.

7) Recoverable volume. Not less than nominal (according to GF XII).

8) Sterility. It must be sterile (according to GF XII, the method of membrane filtration or direct seeding). 9) Viral safety. The drug should contain less than 7 ^x 103 replicatively competent adenoviruses per ml. Analysis of the cytopathic effect on cell culture (A549).

10) Abnormal toxicity. The drug should be non-toxic (according to GF XII).

11) Pyrogenicity. The drug should be pyrogen-free (according to GF XII).

12) Foreign matter. Unidentified impurities no more than 5%.

13) Quantification. The drug should contain (1.0 ± 0.2) x10

particles / ml

14) Biological activity. At least 1x10 ¹⁰ plaque forming units / ml.

The drug, produced in the amount of 1,500 bottles according to the optimized technology (Table 2), complies with the FSP standards for Mobilan (M-VM3), concentrate for the preparation of a solution for intratumoral administration, 10 particles / ml.

Table 2. Process parameters for the production of Mobilan (integrated data, 25 l).

Growing Obtained Obtaining a purified preparation of cell culture and three viral viruses, construct М-VM VM3

Consumption Volume Volume Multiple General Biologists Part Companion cultivator obtained quantitatively, twice as dilute active flasks of the medium, cells, constructions, physical therapy, and lentic particles of particles of particles yam nd (F

Joint venture)

Optimized 43 24.4 25 1: 4.125 1.5x10 ^,:> xlO '1500 Soya technology tv tst vu t

Time-consuming, 12-14 4-6 2 14 days

Claims

Formula

A method for producing a preparation based on a non-replicating adenovirus vector expressing a human type 5 TOLL-like receptor gene and flagelin protein fragment gene, comprising: preparing a nutrient medium from CD293AGT dry medium, glutamine and water; obtaining a cell suspension from HEK293 cells; for this, HEK293 cells are thawed, centrifuged, the supernatant is drained and culture medium is added and cultured to the required concentration of 2.0 x 10 ⁶ cells / ml, periodically reseeding; a cell suspension of the HEK293 line is placed in the fermenter and sedimented for 30 minutes, the supernatant liquid is drained - about 2/3 of the volume and the prepared nutrient medium is added to the fermenter to the cell concentration “under infection” 1, 0><10 ⁶ cells / ml, add M -VM3 viral construct in a volume equal to approximately 1/17 of the total volume of the suspension and culture medium and cultured up to 2 10 PFU / ml; the obtained “seed” infects the volume of cell suspension increased in the wave bioreactor to a final concentration of 8 ^x 10 PFU / ml; centrifugation and removal of the supernatant are carried out, the precipitate is resuspended in lysis buffer at a ratio of about 1 / 16.6, poured into vials and frozen for 2 hours; thawed in a water bath 23-25 ° C, not allowing to warm up, combine the contents of all the bottles and treat with benzonase to a final concentration of 150 U / ml, mix, centrifuge, the supernatant is subjected to ultrafiltration; sequentially conduct anion exchange and size exclusion chromatography; carry out sterilizing filtration; poured into bottles; rolling the bottles and freezing the finished product.