RU2590588C1 - Method for production of preparation based on non-replicating viral vector expressing gene of human toll-like receptor and gene of modified protein flagellin - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and concerns a method for production of "Mobilan (M-VM3)" preparation. During updating developed following stable parameters of the production process: batch volume is 1,500 flasks, time of production is 18-22 days, total consumption of the nutrient medium is 44 l, the volume of virus-containing suspension of M-VM3 is 25 l, density of cells during infection in range from 0.8×10⁶ to 1×10⁶ cl/ml; time for gathering cells is on third day (approximately 72 h); temperature during infection is 36 °C, multiplicity of infection 5-10 PFU/cl, total concentration of viral construct at collection is 8×10⁸ PFU/ml.

EFFECT: invention optimises production, increase its profitability, making optimum ratio of production time/amount of produced/amount spent on production.

1 cl, 2 tbl

Description

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: batch volume - 1,500 bottles, production time - 18-22 days, total consumption of nutrient medium - 44 l, volume of virus-containing suspension M-VM3 - 25 l, cell density infection time - in the range from 0.8 × 10 ⁶ to 1 × 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 during collection is 8 × 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 secreted gene fragment of the flagellin of Salmonella bacteria, at a concentration of 10 ¹² particles of replication-bicistronic human adenovirus vector in 1 ml of buffer solution.

The prior art it is known to obtain virus concentrates on an industrial scale, in particular, from RU 2029561 from 02.27.1995. The closest solution found in the prior art is RU 2465327 from 10.27.12, which describes the purification of recombinant human adenoviruses. The method includes preparing virus-containing material in a 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 landing of an adenovirus on it, precipitation and washing the carrier with adenovirus, eluting 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-κB 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-κ factor in 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 the HEK293 cell culture and its multi-stage purification.

To obtain 25 l of virus-containing cell suspension, 44 l of culture medium was expended. At the same time, cultivation is carried out in a cell spin magnetic inoculator 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 sample of the cell bank 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 a volume of 23.8 liters is carried out simultaneously with the cultivation of a 1.2 liter viral flow 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 efficient 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 ÄKTA Avant chromatographic system. Next, the drug is transferred to sterilizing filtration and bottling. After crimping the bottles with aluminum caps, checking for leaks and labeling, the M-VM3 preparation (Mobilan) 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 of the final product by indicators: description, pH, microbiological purity, bacterial endotoxin content (LAL test), infectious titer, concentration of viral particles and RCA (adenovirus capable of replication), 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 sterilized liquid nutrient media 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. Total volume 44 l (44000 ml).

The finished medium was transported to:

a) cultural boxing

b) viral boxing

c) reactor

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, including centrifuging the cell suspension at each stage and completely replacing the culture medium, the consumption of culture medium is at least 52.1 liters (Table 1).

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

In order 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 growth of the cell suspension. 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 to 1200 ml, thus, the cell concentration was 0.8-1.0 × 10 ⁶ cells / ml. In a Class B virus laminar box with a Class B laminar flow, 70 ml of M-VM3 virus material was added from a work bank (with a titer of at least 10 ⁸ PFU / ml) to the resulting cell suspension. Thus, the ratio of culture from the working bank (initial) and the resulting cell suspension is 1/17, and cultured in it for 48-72 hours. Incubated in a CO ₂ incubator. The controller of the JCC conducted an analysis of the "seed" titration. The titer should be at least 2 × 10 ⁸ PFU / ml.

At this stage, the following was consumed: HEK293 cell suspension 0.6 L; nutrient medium 1.0 l; viral material M-VM3 0.07 l. Total: 1.67 liters.

Obtained at the stage: virus "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, CO ₂ concentration, pH, pO ₂ . Inoculated with “seed” M-VM3 (titer of at least 2 × 10 ⁸ PFU / ml), 1250 ml bag of CultiBag RM 50 bag with 23750 ml cell suspension. Cultivation was continued until a concentration of M-VM3 of at least 8 × 10 ⁸ PFU / ml was reached 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.

Example 3. Purification of the viral construct M-VM3

- 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 of the spent nutrient medium, metabolic products, and other physical particles was removed, 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 sediment to the volume of the buffer is 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 conducted a control of 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 2.4 × 10 ^-4 . Obtained at the stage: M-VM3 containing suspension (lysate) of 0.377 liters.

- 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 volume of the lysate. 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 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; ultrafiltration buffer 1.7 l, “empty” buffer 1.0 l, purified water 7 l.

Obtained at the stage: M-VM3 - 1.2 L retentate.

- 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 (1.4CV = 500 ml) was established. Buffer B was prepared and the column was equilibrated to a conductivity of ~ 28-30 mS / cm (1.4CV = 500 ml). 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 (1.2 L). 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 (1.2 L) size exclusion chromatography buffer, flow 100 ml / min (~ 12 min). They collected the peak and the next eluate. Repeated operation twice with the remaining volume of the eluate.

Spent on stage: M-VM3-containing solution (peak M-VM3) 0.4 l; size exclusion chromatography buffer 4.8 l, sorbent Q SepharoseXL Virus Licensed 0.8 l.

Obtained at the stage: solution (M-VM3) 1.65 L, incl. M-VM3 1.1 × 10 ¹² particles / ml.

- Sterilizing Filtration (M-VM3)

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 product-solution (M-VM3) 1.65 l, incl. M-VM3 1.1 × 10 ¹² particles / ml.

Obtained at the stage: Solution (M-VM3) 1.65 L, incl. M-VM3 1.0 × 10 ¹² 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 a movable clutch 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) bottles (depending on the volume of the series), the technologist and the QC controller monitor the dose of the solution in the bottle.

Spent on stage: solution (M-VM3) 1.65 L, incl. M-VM3 1.0 × 1012 particles / ml, 1650 sterile vials and rubber stoppers.

Obtained at the stage: Mobilan (M-VM3) in bottles of 1610 pcs., Incl. M-VM3 1.0 × 10 ¹² 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., Incl. M-VM3 1.0 × 10 ¹² particles / ml., 1650 sterile aluminum caps.

Obtained at the stage: Mobilan (M-VM3) in bottles of 1520 pcs., Incl. M-VM3 1.0 × 10 ¹² particles / ml.

- Freezing finished products

The finished product was put in containers, on which they hung a yellow label “In quarantine” with the name of the preparation, batch number, date of production of the product, date of delivery for analysis in the OKC 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 transferred them at -70 ° C for analysis on the conformity of the FSP and arbitration storage. After receiving a positive opinion, the containers were marked with a green label on which the batch 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 were indicated. 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 should withstand comparison with reference No. IV (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 × 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) × 10 ¹² particles / ml.

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

The preparation, 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 ¹² particles / ml.

Claims (1)

A method for producing a preparation based on a non-replicating adenoviral 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 cell line, for this, HEK293 cell line is thawed, centrifuged, the supernatant is drained and culture medium is added and cultured to the required concentration of 2.0 × 10 ⁶ cells / ml, periodically replanting; the HEK293 cell suspension 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” of 1.0 × 10 ⁶ cells / ml, M- is added VM3 viral construct in a volume equal to approximately 1/17 of the total volume of the suspension and culture medium, and cultured to 2 × 10 ⁸ PFU / ml; the resulting “seed” infects the volume of cell suspension grown in the wave bioreactor to a final concentration of 8 × 10 ⁸ 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 units / ml, mix, centrifuged, the supernatant is subjected to ultrafiltration; sequentially anion exchange and size exclusion chromatography; carry out sterilizing filtration; poured into bottles; rolling the bottles and freezing the finished product.