RU2443779C2 - Method for making recombinant adenovirus preparation characterised by lower ratio of physical and infectious viral particles, and genetically therapeutic drug preparation produced by such method - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: all the stages of host cell culture cultivation are implemented in multilayered HYPERFlask culture vessels. Also, the present invention provides an recombinant adenovirus preparation produced by the method according to the present invention.

EFFECT: method provides intensifying production of the recombinant adenovirus drug preparation, considerably reducing time and space resources required for manufacturing and storage, as well as considerably lowering the ratio of physical and infectious viral particles that leads to higher quality of the drug preparation.

12 cl, 2 tbl, 4 ex

Description

FIELD OF THE INVENTION

The present invention relates to the field of biotechnology, virology and medicine, in particular gene therapy for cancer, and provides a method for producing a recombinant adenovirus that can be used to deliver the genes of target products with a therapeutic effect to the affected cells of a patient.

State of the art

Solid (solid) tumors make up almost 90% of all oncological diseases. One of the potentially promising methods of treating such tumors is gene therapy. The most effective methods of gene therapy are based on the delivery of therapeutic genes to the affected cells of the patient using Ad viruses called Ad virus vectors. Compared to other vector systems, recombinant replicatively defective adenoviruses are characterized by high transgene expression efficiency in various cell types, vector safety for humans and animals, and accumulation of recombinant viruses in producer cells in high titer (NEAltaras et al., Production and formylation of adenovirus vectors, Adv. Biochem Engin / biotecnology (2005), 99, 193-260), the large packing capacity of the vector (Black JL, Genome projects and gene therapy: gateways to next generation biological weapons // Milit. Med. - 2003. - Vol .108, No. 11. - P.864-871). To date, two adenovirus serotypes, 2 and 5, have been used as vectors for gene therapy. They are well suited for use in gene therapy in vivo, because they produce high titers of viral particles. Adenoviruses are able to replicate in both dividing and non-dividing cells. They do not integrate into the genome of host cells and remain epichromosomal. This reduces the risk of insertional mutagenesis (K. Roemer, T. Friedmann, Concepts and strategies for human gene therapy. Eur. J. Biochem., (1992) 268, 211-225; Mulligan RC The basic science of gene therapy. Science, ( 1993), 260, 926-931, Crystal RG, Transfer of Genes to Humans: Early Lessons and Obstacles to Success. Science, (1995), 270, 404-410) (see Table 1):

In addition, Ad viruses have a high degree of tropism in relation to tumor cells, which allows to selectively infect only cancer cells, reducing the toxic effect of gene therapy. Already received data on therapeutic drugs created on the basis of Ad viruses. Thus, the drug Gendicin, which carries the gene for the tumor suppressor protein protein (p53), is used to treat squamous epidermoid carcinoma and other solid tumors (Zhaohui Peng, Current Status of Gendicine in China: Recombinant Human Ad-p53 Agent for Treatment of Cancers, Human gene therapy 2005, 16, 1016-1027; M. Lusky, Human Gene Therapy, 2005, 16, 281-291).

Creating a genetic construct based on adenovirus serotype 5 (Ad5) has a number of practical advantages. Possessing the selective ability to infect malignant tumor cells, the Ad5 recombinant is not able to replicate in target cells, which allows prolonged expression of the gene insert of the target product with a therapeutic effect. In addition, the injection of the drug into the body does not pose a possible pathogenic threat to healthy cells, nor does it threaten the health of personnel involved in the manufacture of the pharmaceutical product.

Genetically modified (recombinant) adenoviruses can be used to efficiently transfer a therapeutic or marker-reporter transgene to cells of various types. The preparation and cultivation of replicatively defective Ad vectors is carried out in special genetically modified cell lines. Mammalian cells such as HeLa, CHO, W138, BHC, COS-7, HepG2, 3T3 RIN, MDCK, and A549 are used to build up the replicatively competent form of Ad, and after the corresponding gene modification to build up the replicatively defective form of Ad.

The most common cell line for building up a recombinant replicatively defective Ad is the HEK 293 line, obtained from human embryonic kidney cells, into whose genome an Ad5 DNA fragment capable of expressing E1a and E1b protein encoding viral replication was integrated (Graham, FL, Smiley, J ., Russel, WC, Nairn, R .; Characteristics of a human cell line transformed by DNA from human adenovirus type 5; J. Gen. Virol. (L977) 36: 59-74). PER.C6, 911, or IT293SF cell lines can also be used as host cells for replicatively defective adenoviruses.

Currently, along with other viruses for gene therapy, recombinant adenoviruses of types 2 and 5 are also used (Ad2 and Ad5, respectively), which cause human respiratory diseases. Both Ad2 and Ad5 both belong to a subclass of adenoviruses that are not associated with malignancy of human tissues. It has been proven that the recently developed Ad5 / F35 hybrid adenoviral vector is very promising for the development of gene therapeutic drugs and related fields of science (P. Yotnda, N. Onishi, HE.Heslop, DM.Shayakhmetov, A.Lieber, MK. Brenner, and . A. Davis. Highly efficient transduction of primitive hematopoietic stem cells by modified Adenovirus. Hum Gene Ther. 2001 Apr 10; 12 (6): 659-70.)

Recombinant adenoviruses have the ability to provide an exceptionally high level of transgene delivery. The efficacy of delivering a therapeutic transgene with this system to restore disturbed genetic balance has been demonstrated in animal models of a variety of genetic disorders (Y. Watanabe, "Serial Inbreeding of Rabbits with Hereditary Hyperlipidemia (WHHL-Rabbit)", Atherosclerosis 36: 261-268 (1980); K. Tanzawa et al, "WHHL-Rabbit: A Low Density Lipoprotein Receptor-Deficient Animal Model for Familial Hypercholesterolemia", FEBS Letters, 118 (l): 81-84 (1980); J. Goldstein et al, "Defective Lipoprotein Receptors and Atherosclerosis-Lessons from an Animal Counterpart of Familial Hypercholesterolemia ", New Engl. J. Med., 309 (5): 288-296 (Aug. 4, 1983); S. Ishibashi et al," Massive Xanthomatosis and Atherosclerosis in Cholesterol -Fed Low Density Lipoprotein Receptor-negative Mice ", J Clin. Invest. 93: 1885-1893 (May, 1994). Indeed, a replication-defective adenovirus encoding c-DNA for the transmembrane cystic fibrosis regulator (CFTR) has recently been approved for use against cystic fibrosis as a result of at least two clinical test (J. Wilson, "Cystic Fibrosis-Vehicles for Gene Therapy", Nature, 365: 691-692 (Oct. 21, 1993). M. Horwitz, "Adenoviridae and Their Replication", 2d edition, ed. BNFields, Raven Press, Ltd., New York, Chapter 60, pp. 1669-1721 (1990) showed the therapeutic efficacy of adenoviral delivery-based gene therapy in a mouse cancer model (retinoblastoma). The need for clinical quality adenoviral vectors is increasing dramatically with the expansion of clinical trials. Estimation of annual demand for adenoviral vectors during clinical trials in 300 patients can reach approximately 6 × 10 ¹⁴ PFU (plaque-forming units).

In the production of a drug based on Ad5, considerable attention is paid to the quality and safety of drugs based on adenoviruses. One of the most important indicators of the drug is the ratio of the total number of viral particles (including) to infectious, plaque-forming units (PFU). As recommended by the U.S. FDA, this ratio should not be higher than 30 (Bauer et al., 2002, In Curiel DT, Douglas JT (eds) Adenoviral vector for gene therapy, Academic, New York, p.615). The presence of a high titer of the ballast population of defective r.h. leads to high immunogenicity of the drug, which leads to the rapid achievement of the maximum acceptable threshold of virus-neutralizing antibodies.

To date, the most common way to build recombinant adenovirus in the laboratory and at the pilot level in the production of pilot batches of the drug for preclinical and clinical trials has been the method using plastic culture mattresses and Petri dishes (Koneman's color atlas and textbook of diagnostic microbiology, Washington C. Winn, Elmer W. Koneman, 2006, 1377).

The method comprises the following steps:

C1. Cultivation of host cells for virus infection

For this, a suspension of host cells is plated in a mattress with an area of 162 cm ² with the addition of 25 ml of DMEM growth medium and 10% blood serum. The cell culture is incubated in a CO ₂ incubator for 48 hours until a cell monolayer with a confluency of 90-100% is formed. After incubation, the resulting cell suspension is poured into 3 culture mattresses with an area of 162 cm ² , 1 ml of cells in each mattress and 25 ml of DMEM culture medium. Cells are cultured in a CO ₂ incubator for 48 hours until a cell monolayer with a confluency of 90-100% is formed. The depleted medium is removed by vacuum. The resulting cell suspensions are seeded on 9 mattresses with an area of 162 cm ² . A cell culture in 9 mattresses is incubated in a CO ₂ incubator for 48 hours until a cell monolayer with a confluency of 90-100% is formed. The resulting cell suspensions are seeded on 9 mattresses and 18 mattresses and incubated 48 hours until a cell monolayer with confluency of 90-100% and 70-80%, respectively, is formed.

18 mattresses with cell confluence of 70-80% are used for viral infection. The remaining 9 mattresses with 100% confluency are left to maintain the culture with the aim of further building Ad5.

C2. Getting Ad5 viral seed

A cell culture of 18 mattresses with a confluency of 70-80% is infected with an initial viral seed from a master bank with a known viral titer. Culture mattresses with an infected cell culture are incubated in a CO ₂ incubator for 36-48 hours. The depleted culture medium of each mattress is used to suspend the Ad5 culture with a pipette.

C3 Cultivation of infected cell culture

Each of the 9 mattresses with an area of 162 cm ² obtained in stage 1 is sown in two Petri dishes with an area of 150 cm ² . 25 ml of DMEM growth medium containing 10% serum was added to each culture vessel and incubated in a CO ₂ incubator for 36-48 hours at 37 ° C. The obtained cell cultures on 18 Petri dishes with cell confluence of 70-80% are used for infection by virus seed (p.2), and the cell culture of 9 mattresses with 90-100% confluency is left to maintain the culture with the aim of further building Ad5.

Other methods for increasing adenovirus using bioreactors, roller systems, cell factories are also known. However, the use of these methods is not effective, because they are low-power and require considerable time (cups, mattresses) or are spatially and technologically cumbersome (bioreactors, cell factories) for the production of an average, pilot level of production of experimental batches of drugs.

The method of the present invention using multilevel Hyperflask culture mattresses (HyperFlask, Corning (USA, Cat. No. 10010)) can be considered the most optimal way of building Hell in the mid-level production environment. These mattresses were successfully used in the case of lentivirus (LV) building using direct transfection DNA of the LV vector, auxiliary packaging, and plasmid-cover DNA into cell culture 293T (CRL-11268), HOS (CRL-1543) and HeLa (CCL-2) (RHKutner et al., Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography, BM C Biotechnology 2009, 9:10). As a result of the implementation of the claimed method, a large volume (550 ml) of the supernatant with a high titer of the drug vector is obtained, which differs by an order of magnitude compared with the use of 150 cm ² plates. However, this work deals only with transfection viral units, and the ratio of infectious and non-infectious viral units, which is an important indicator of the quality of the drug, is not indicated.

Disclosure of invention

The present invention is based on the fact that the inventors discovered unexpectedly that the use of multilevel culture mattresses HYPERFlask for growing a host cell culture and subsequent cultivation of a recombinant adenovirus can not only intensify the production of an antitumor gene therapeutic drug based on a recombinant adenovirus, but also significantly reduce the time and spatial resources required for the production and storage of viral inoculum but. The time taken to build up the virus in HYPERFlask at the stages of spreading the cell culture for infection, obtaining virus seeds, infection with adenovirus and obtaining infected cell material is reduced by at least 5 times compared with building Ad5 on the equivalent area of culture plates and mattresses. Spatial resources thereby increase by 5 times.

In addition, it was unexpectedly found that the use of multilevel culture mattresses HYPERFlask leads to a decrease in the population of defective adenoviral particles, which is manifested in a decrease in the ratio of physical and infectious viral particles. Since this coefficient reflects the level of immunogenicity of the drug, its decrease leads to an increase in the quality of the drug and its effectiveness.

The proposed method for producing adenovirus is ideal at the stage of obtaining prototypes of drugs for preclinical and clinical trials.

In one of its aspects, the present invention relates to a method for increasing recombinant adenovirus, comprising the following steps:

a) growing a culture of host cells in a culture medium;

b) obtaining a working bank of seed for the recombinant adenovirus by infection of the host cell culture obtained in step a) with a recombinant adenovirus from the master bank, followed by cultivation of the infected host cell culture;

c) infection of the host cell culture grown as described in step a) with the virus seed obtained in step b);

d) culturing an infected culture of host cells;

e) collection of infected cells obtained in stage d), characterized in that all stages of cultivation of the host cell culture are carried out in multilevel culture mattresses HYPERFlask.

In one embodiment, the method of the present invention further comprises isolating and purifying the virus from harvested infected cells. In one preferred embodiment, the purification is carried out by centrifugation in a density gradient of CsCl. In a further preferred embodiment, the purification is carried out by tangential ultrafiltration — anion exchange chromatography.

In yet another embodiment of the method of the present invention, the recombinant adenovirus is an adenovirus derived from serotype 5 adenovirus (Ad5).

In a further embodiment of the method of the present invention, the recombinant adenovirus expresses the thymidine kinase gene of herpes simplex virus type 1, p53, α-interferon, β-interferon, or green fluorescent protein (GFP), etc.

In one preferred embodiment, HEK293 cells are used as host cells. In a more preferred embodiment, the cultivation of the host cells in step a) comprises the following steps: growing the cells in a culture medium until a cell monolayer with a confluency of 100% is formed; cell suspension in the culture medium in the presence of trypsin-EDTA; inoculation of the fresh culture medium with the resulting suspension and cell growth to form a cell monolayer with 100% confluence; cell suspension in the culture medium in the presence of trypsin-EDTA; inoculation of fresh culture medium with the resulting suspension and cell growth until a cell monolayer with a confluency of 70% is formed.

In another more preferred embodiment, the preparation of the virus seed in step b) comprises the following steps: infection of cells obtained as described in claim 6 with recombinant Ad5 and culturing an infected cell culture; collection of infected cells with their subsequent lysis by freezing-thawing.

In a further aspect, the present invention relates to a recombinant adenovirus preparation obtained by the method of the present invention. In one preferred embodiment, the recombinant adenovirus is recombinant Ad5. In yet another preferred embodiment, the recombinant adenovirus is an adenovirus expressing the thymidine kinase gene of herpes simplex virus type 1, p53, α-interferon, β-interferon, or green fluorescent protein (GFP), etc.

The implementation of the invention

The claimed method is universal and can be used both for building replicatively competent and replicatively defective Hell. The growth of Hell can be carried out in any suitable cell line, allowing for the full replication of Hell. The specific serotype to which cultured adenovirus belongs is not significant, however, from the point of view of the practical use of the obtained adenovirus as a drug, the method of the present invention is preferably used for growing adenoviruses 2 and 5 of serotypes.

An example of a replicatively competent virus is a virus such as, for example, ONYX-015 (M. Sunamura et al, Restricted replication-competent adenovirus: a novel strategy for cancer gene therapy, International Journal of Clinical Oncology, 2000, 147-152) . The therapeutic effect of this recombinant virus is based on the fact that it has the unique ability to replicate only in cells with a deficiency in the expression of the gene encoding the onc suppressor p 53. The active protein p53 inhibits growth and induces cell apoptosis in response to cell DNA damage. Since ONYX-015 has a partial deletion in the region of the replicative E1B gene, the virus is also grown in the HEK293 cell line (Giuseppe Portella et al., ONYX-015, an E1B Gene-Defective Adenovirus, Induces Cell Death in Human Anaplastic Thyroid Carcinoma Cell Lines , The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 6 2525-2531).

Obtaining a culture of replicatively defective Ad5 or Ad2 virus with a high content of infectious viral particles can be carried out by infection of a cell culture, such as, for example, HEK-293, PER.C6, 911 and IT293SF.

The most preferred host cells for building up replicatively defective Ad5 are HEK-293 cells. This is due to the fact that the genome of the HEK-293 cell line contains a stable integrated copy of the E1 gene encoding two E1 proteins: Ela and Elb, encoding viral replication (Graham, FL, Smiley, J., Russell, WC and Nairn, R. (1977) J. Gen. Virol. 36: 59-74).

The authors of the present invention have demonstrated that the growth of AB in HEK 293 cell culture using multilevel cell factories (HyperFlask, # 10010, Corning) can significantly increase the productivity of obtaining AB. The authors of the present invention showed that if HEK 293 host cells are grown and sown in multilevel culture mattresses under infection with AB, the drug yield per unit time can be increased by at least 5 times. The intensification of AV growth was achieved by reducing the time and spatial resources at the stages of dissemination of cell culture for infection, infection of AB and at the stage of obtaining cellular material infected with the virus. It was also revealed a 10-fold improvement in quality and a 2-fold increase in the amount of viral seed material obtained by cultivating AB in multilevel culture mattresses, compared with traditional methods for building AB in laboratory conditions. This has significantly reduced the time and spatial resources for the production and storage of viral inoculum. In addition, a decrease in the population of defective AV particles was detected, which was reflected in a decrease in the ratio of physical and infectious viral particles. Since this coefficient reflects the level of immunogenicity of the drug, its decrease leads to an increase in the quality of the drug and its effectiveness. The specified method makes it possible to intensify the process of production of cellular material infected with recombinant AB, which in purified form and being enclosed in a pharmaceutical composition can be used in gene therapy to transform mammalian hyperproliferative cells, treat cancer, inhibit tumor proliferation in animals and humans, to reduce proliferation tumor cells. The claimed method is characterized in that the infection of the adhesive cell culture of HEK 293 with recombinant replicatively defective Ad5 is carried out using multilevel culture mattresses HYPERFlask (Corning). The total area of HYPERFlask is 1700 cm ² , which is equivalent to eleven culture dishes with an area of 150 cm ² . HYPERFlask consists of ten interconnected growth membranes, suitably treated to improve cell adhesion. All ten membranes have properties that allow oxygen to enter HYPERFlask and remove carbon dioxide in such a way that all growth surfaces inside HYPERFlask have effective access to gas exchange.

The finished pAD5 preparation can be prepared using a variety of purification methods that are well known to those skilled in the art. As an example, the finished product can be obtained by centrifugation in a CsCl gradient followed by dialysis against a buffer suitable for subsequent storage and injection (e.g. 10 mM Tris, pH 8.0-8.5, 10% glycerol or phosphate buffered saline (PBS) with glycerol), or using ultrafiltration, ion exchange chromatography and dialysis.

The following examples disclose the most preferred embodiments of the present invention and are provided solely for the purpose of better clarifying its essence. One skilled in the art will recognize that many modifications can be made both to the means and materials used, and to the methods used, without departing from the scope of the invention.

Example 1. Growing a culture of recombinant Ad5

1.560 ml of DMEM growth medium containing 7% serum, 146 mg of glutamine, 25,000 units. penicillin, 25,000 units. streptomycin, 25 ml of 7.5% sodium bicarbonate and 4 × 10 ⁷ HEK293 cells, are placed in a HYPERFlask (Cat. # 10010, Corning, USA) and cultured 48 hours until a cell monolayer with 100% confluence is formed.

2. A cell suspension is obtained by washing the cell monolayer with 100 ml of trypsin-EDTA solution and 50 ml of DMEM growth medium prepared as described above in claim 1.

3. To 510 ml of DMEM growth medium prepared as described in claim 1, add 50 ml of a cell suspension obtained as described above in claim 2 containing 4 × 10 ⁷ cells. The resulting cell suspension was placed in HYPERFlask and cultured for 48 hours until a cell monolayer with a confluency of 100% was formed.

4. To 535 ml of DMEM growth medium prepared as described in claim 1, add 25 ml of a cell suspension (claim 2) containing 3 × 10 ⁷ cells. The resulting cell suspension was placed in HYPERFlask and cultured for 48 hours at 37 ° C until a cell monolayer with a confluency of 70% was formed. This operation is carried out using 4 parallel culture devices.

5. Obtained, as described above in paragraph 4, cell monolayers are infected by adding virus seeds (5 × 10 ⁸ -10 ⁹ PFU) from the master bank of recombinant Ad5. For this, the nutrient medium in which the cells grew is drained from HYPERFlask (p. 4) and mixed with a virus seed. A final volume of medium and virus seed of 560 ml was placed back into the HYPERFlask containing the cell monolayer.

6. AB cultivation in HYPERFlask is carried out for 48-64 hours at a temperature of 37 ° C. At this stage, the morphology of infected cells is evaluated visually.

7. Infected cells are poured into sterile containers, lysed by freezing-thawing, stored at -70 ° C and subsequently used as a working bank of virus seed for large-scale growth of recombinant AB.

8. The titer of the obtained seed (p. 7) is 5 × 10 ⁸ -10 ⁹ PFU / ml, which is an order of magnitude higher than the titer of the virus seed obtained on cultural mattresses with an area

162 cm ² . In addition, the volume of the seed obtained (item 7) is 560 ml, which is two times more than the volume of the seed with 10 culture mattresses with an area of 162 cm ² .

9. The cell culture obtained in claim 3 is seeded, as described in claim 2, 3, 4, and infected with a virus seed (claim 7), as described in claim 5.

10. The cultivation of Ad5 in HYPERFlask is carried out for 48 hours at a temperature of 37 ° C.

11. The infected cells are collected by centrifugation, suspended in 10 ml of a buffer suitable for further purification, and stored at -70 ° C.

Example 2. Purification of recombinant Ad5 virus

Recombinant Ad5 is purified by concentrating virions by depositing them on the surface of a CsCl solution with a density of 1.365 g / cm ³ , as described in the monograph Virology. Methods ", B. Meikhi, 1988, p. 263, as well as using tangential ultrafiltration and anion exchange chromatography, as described in" Methods in molecular biology ", M. Le Doux, vol. 434, volume 2, Design and characterization of gene transfer vectors, p.13-23. The number of physical Ad5 particles is estimated by the optical density of the Ad5 solution when measuring absorbance at 260 nm, assuming that 1 OE corresponds to 10 ¹² viral particles per 1 ml of solution with an optical path length of 1 cm (Maizel JVJ, et al., 1968, Virology, 36-115). The yield of purified Ad5 is 2 × 10 ¹² -5 × 10 ¹² physical particles from one HYPERFlask culture device.

Example 3. Analysis of the quality of the obtained drug virus Ad5

The number of infectious viral particles was estimated using titration of the virus and calculation of the logarithm of TCD ₅₀ , as described in the monograph “Virology. Methods ", B. Meikhi, 1988, p. 267, and according to the plaque formation method, as described in the monograph" Virology. Methods ”, B. Meikhi, 1988, p. 265. A decrease in the population of defective Ad5 particles was found, which was reflected in a decrease in the ratio of physical and infectious viral particles from 260 to 54-14 compared with the build-up of Ad5 on 150 cm ² culture dishes. The absence of a population of defective low molecular weight Ad5 particles was also observed visually by centrifugation in a CsCl gradient.

Example 4. Indicators of efficiency and reproducibility of the method of the present invention

The results of several representative experiments in which the adenovirus buildup in multilevel HyperFlask culture mattresses was carried out in accordance with the method of the present invention are presented in Table 2.

table 2 The effectiveness of adenovirus build-up in multilevel HyperFlask culture mattresses Final cleaning Sample Name lgTCD ₅₀ Way of building Number of pfu in ml ** Qty * in ml h / pfu CsCl Gradient Centrifugation 04/17/09 8 Hyperflask ** 7 × 10 ¹² fourteen CsCl Gradient Centrifugation 04/24/09 8 Hyperflask ** 1 × 10 ¹³ twenty Chromatography 05/20/09 7 Hyperflask ** 2.7 × 10 ¹² 54 CsCl Gradient Centrifugation 10/29/08 6 Cups 150 cm ² ** 1.4 × 10 ¹² 280 Chromatography The control **
01/14/09 5 Cups 150 cm ² 5 × 10 ⁸ 1.3 × 10 ¹¹ 260 Chromatography *** (04/09/08) 04/09/08 four Cups 150 cm ² 4 × 10 ⁷ - - Cell lysate (seed) **** 05/20/09 6 Hyperflask ** - - Cell lysate (seed) 06/03/09 5 Hyperflask ** - - Cell lysate (seed) 06/03/09 5 Hyperflask ** - - Cell lysate (seed) 04/18/08 four Cups 150 cm ² ** - - Cell lysate (seed) 08/08/08 four Cups 150 cm ² ** - - Cell lysate (seed) 08/08/08 four Cups 150 cm ² ** - - Cellular **** Lysate 06/26/09 7 Hyperflask ** - - Cellular
lysate 07/01/09 7 Hyperflask ** - - Cellular
lysate 07/08/09 7 Hyperflask ** - - Cell lysate 10/29/08 6 Cups 150 cm ² 5 × 10 ⁹ - - Cell lysate 10/29/08 6 Cups 150 cm ² 2 × 10 ⁹ - - Cell lysate 10/29/08 5 Cups 150 cm ² 5 × 10 ⁸ - - * - The total number of viral particles (including) was determined in a purified preparation (centrifugation in a CsCl gradient or liquid chromatography) extinction coefficient - 1.0 OE _260nm = 1.2 × 10 ¹² hours. per 1 ml with a length of 1 cm through the optical path. ** - To control the number of infectious viral particles in the case of determining the biological activity of a viral sample by counting lgTCD ₅₀ , a standard sample with a known number of plaque forming units (PFU) was used. *** - Partially purified preparation. **** - Cell lysate (seed) for infection of cells was obtained by suspending infected cells in a depleted growth medium (25 ml from 1 mattress with an area of 162 cm ² and 50 ml from one surface of 170 cm ² HyperFlask), cell lysate for virus purification obtained by suspending one surface of 162-170 cm ² in 1 ml of PBS in the case of centrifugation in a CsCl gradient or in 1 ml of resuspension buffer in the case of chromatography and ultrafiltration (5 mM Tris-HCl, 75 mM NaCl, 1 mM MgCl ₂ , 5 % sucrose, 1% PS-80, pH 8.0).

All patents, publications, scientific articles, and other documents and materials cited or referenced herein are hereby incorporated by reference to the extent that each of these documents was individually incorporated by reference or is presented in its entirety.

The specific therapeutic genes, vectors, cell cultures, materials used and methods described herein refer to preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. In the study of this description, other objects, aspects and embodiments will come to mind to specialists in this field and they are covered by the scope of this invention. Specialists in this field will be clear that various replacements and modifications can be made in relation to the invention described here without deviating from its scope and scope, which are determined by the attached claims.

Claims (12)

1. A method of producing a recombinant adenovirus preparation characterized by a reduced ratio of the ratio of physical and infectious viral particles by reducing the population of defective viral particles, including:
but. growing a culture of host cells in a culture medium;
b. obtaining viral seed — a working bank of a recombinant adenovirus by infection of the host cell culture obtained in step a) with a recombinant adenovirus from the master bank, followed by cultivation of the infected host cell culture;
from. infection of the host cell culture grown as described in step a) with the viral seed obtained in step b);
d. culturing an infected host cell culture;
e. collection of infected cells obtained in stage d);
characterized in that all stages of cultivation of the host cell culture are carried out in multilevel culture mattresses HYPERFlask. 2. The method according to claim 1, further comprising isolating and purifying the virus from the collected infected cells. 3. The method according to claim 2, in which the purification is carried out by centrifugation in a density gradient of CsCl. 4. The method according to claim 2, in which the purification is carried out by tangential ultrafiltration - anion exchange chromatography. 5. The method according to claim 1, in which the recombinant adenovirus is an adenovirus derived from adenovirus serotype 5 (Ad5). 6. The method according to claim 1, in which the recombinant adenovirus expresses a gene selected from the group consisting of genes encoding herpes simplex thymidine kinase type 1, transcription factor p53, α-interferon, β-interferon and green fluorescent protein (GFP). 7. The method according to any one of claims 5 and 6, wherein HEK293 cells are used as host cells. 8. The method according to claim 7, in which the cultivation of host cells in stage a) includes the following stages: growing cells in a culture medium until a cell monolayer is formed with 100% confluence; cell suspension in the culture medium in the presence of trypsin-EDTA; inoculation of the fresh culture medium with the resulting suspension and cell growth to form a cell monolayer with 100% confluence; cell suspension in the culture medium in the presence of trypsin-EDTA; inoculation of fresh culture medium with the resulting suspension and cell growth until a cell monolayer with a confluency of 70% is formed. 9. The method according to claim 7, wherein obtaining the virus seed in step b) comprises the following steps: infection of cells obtained as described in claim 6 with recombinant Ad5 and culturing an infected cell culture; collection of infected cells with their subsequent lysis by freezing-thawing. 10. The gene therapeutic drug of the recombinant adenovirus, delivering the genes of the target products with the therapeutic effect to the diseased cells of the patient, while the drug obtained by the method according to any one of claims 1 to 9 is characterized by a reduced ratio of the ratio of physical and infectious viral particles due to reducing the population of defective viral particles. 11. The drug of claim 10, which is an aqueous solution for injection containing 2.5 · 10 ¹¹ viral particles of recombinant Ad5, 10 mm Tris, pH 8.0-8.5, 10% glycerol. 12. The drug of claim 10, in which the recombinant adenovirus expresses a gene selected from the group consisting of genes encoding herpes simplex thymidine kinase type 1, transcription factor p53, α-interferon, β-interferon and green fluorescent protein (GFP).