Method of producing continuous cell lines and use thereof. RU patent 2509803.

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and a method of producing continuous lines of living cells and use thereof. The disclosed method involves irradiating said living cells with a UV light dose of about 20 mJ/cm² to about 300 mJ/cm², at a wavelength between about 100 nm and about 400 nm, for about 30 s to 5 min and collecting cells that are capable of proliferation after at least 20 passages. The obtained cell lines are deposited in ECACC under numbers 08020602, 08020603 and 08020604 and can be used to produce viruses or gene products.

EFFECT: invention facilitates recombinant expression of biomolecules.

23 cl, 4 dwg, 1 tbl, 6 ex

The technical field to which the invention relates.

The invention relates to methods of obtaining cell lines.

The background to the invention

Cell lines have become a valuable tool in obtaining vaccines. Getting some important vaccines and viral vectors is still in the embryo chicken eggs or in primary chick embryo fibroblasts. Basically, fabric birds for viral replication get method SPF (free from specific pathogens). Get on the procedure SPF fabrics are expensive, and the quality of the supplied material is often difficult to control. In this regard, the mismatch between the requirements and the shortage of supply of fabric are the main disadvantage of the methods based on the use of eggs in the procedure of the SPF. The same is true for those strategies that use primary monolayer culture of fibroblasts. In order cell lines can be multiplied indefinitely, you must reach the immortalization of cells. Most currently used immortalized cell lines refers to the offspring of cancer cells or merged hybridoma cells. But the last of these technologies has limitations defined by the merger with myeloma cells. Accordingly, no basic technology that could be used to create immortalized cells of different types.

Short description of the invention

The aim of the present invention is to provide a continuously growing cell lines from cell material that does not belong to continuously growing. In particular, the aim of the invention is to create a continuous cell lines that have the potential to proliferation, but which are not entered alien viral genes.

In this regard, the present invention relates to a method for continuous cell lines, where this method includes obtaining of living cells in animals or humans, exposure of these cells to UV light, the achievement of the proliferation of these cells and the selection of cells can proliferate at least after 20 passages, as the cells that make up a continuous cell line.

This continuous cell line is a culture of cells that can reproduce and to use next to recombinant expression of biomolecules such as proteins, or for the production of viral products, such as viral antigens, or to create a viral populations, in particular, for the purposes of vaccination.

Accordingly, the present invention also relates to a method for viruses, where this method involves creating cells that make up a continuous cell line derived by the method of the present invention, infection of these cells specified by the virus reproduction of the given virus in these cells and collection of the given virus.

In another aspect, the present invention relates to a process for obtaining a product of recombinant gene, where this method involves creating cells that make up a continuous cell line that get in the way of the present invention, transfection these cells nucleic acid, coding specified gene product expression specified gene product and then optionally the selection of the specified gene product.

In yet another variant, the present invention relates to continuous cell line derived by the method that provides the presence of the living cells of an animal or person, with the subsequent exposure of these cells is an effective dose of UV light, the achievement of the proliferation of these cells and the selection of cells can proliferate after at least 20 passages, as the cells of the specified continuous cell lines.

Description of drawings

Figure 1 shows a schematic representation of procedure UV treatment.

Figure 2 illustrates the continuous cell line quail.

Figure 3 shows the phylogenetic tree and illustrated processing mode when receiving a continuous cell line quail.

Figure 4 illustrates the correlation UV dosage and time of exposure when using parameters suitable for the production of continuous cells.

Detailed description of the invention

The present invention relates to the production of continuous cell lines by UV-processing cells.

Cell line is a cell population, formed on the basis of one or several subcultures primary cell culture. Each round subculturing referred to as the passage. When cells are subculturing, this means that they have passed the passage. Specific populations of cells or a specific cell line can be characterized by the number of passages, which it passed. Primary culture is a culture, after the separation of the cells of the tissue. Retrieved from first subculture cells are described as secondary crops (one passage). After secondary subculture cells become tertiary culture (passage 2), etc. For professionals in this field it is clear that there can be many doubling populations during passage; in this connection, the number of doublings of populations of culture more than the number of passages. A multiplication of cells (i.e. the number of doublings of the population) within the period of time between passages depends on many factors, including, without limitation, the planting density, substrate used, environment, growth conditions and the length of time between passages. Cultivation can be done by inoculation of cells on Wednesday, while the cells to grow to merge cells in culture or before the formation of a continuous cell films and then inocularea in the new environment, the cells in the selection portion of fused cells. This passage is a tool to assess the ability of cells to reproduce. In normal cells, including non-irradiated cells from tissue can be 10-20 passages to achieve a state in which the reproduction or double cells is not already happening. The cells are then enter a phase of aging on the basis of which cannot be obtained by other subcultures. In contrast, continuous cell lines are able to multiply after more than 20 passages, for example, more than once 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75 or 80 passages. The authors of the present invention found that such continuous cell line, which can passerovat many times after 20 passages, in particular immortalized cells, can be obtained by changing cells by UV-processing, e.g. irradiation of these cells, the effective dose of UV light. The term "effective dose of UV light" according to the present invention is the amount of exposure to transform the cell lines that are not continuous, continuous cell lines. Effective dose of UV light varies from the minimum dose required for such transformations, to the maximum dose, which is tolerant to these cells, without causing fatal consequences for the whole cell culture in General. Obviously beyond the effective dose above or below a continuous cell line can not be obtained. Specialists in this field can easily determine the optimal efficient dosage for each cell line on the basis of information and practical guidelines described in this invention, without long-term approaches to optimization. Cells can be a primary cell or cells, capable of reproduction, obtained after a few passages. The cultivation of cell lines can be carried out by methods applied for a standard cell cultures, for example using the systems, including T-shaped bulb or Catalogne bulb, or cultivation stir in the tank or in other variant of the bioreactor. In some embodiments, the implementation of the present invention, culture adapted to the conditions of serum-free medium and kept in such conditions.

In the context of this discussion, the term "artificial light" refers to ultraviolet radiation with the wave length from 10 to 400 nm, in particular from 100 to 400 nm. UV-light can be selected from the group consisting of UV-C (100-280 nm), UV-B (280-320 nm) and UV-a (320-400 nm). In some embodiments, the implementation of the present invention, the wave length range from 200 to 300 nm. Photosensitizing agents, such as tools that interkaliruet DNA and which are activated by ultraviolet light, can be used to enhance the modifying effect of UV-light, although they are not binding in all variants of the implementation of the present invention. In one embodiment of the present invention, UV light is a UV-C With wave length from about 100 to about 280 nm. In another embodiment of the present invention, UV light has a wavelength range from about 240 to about 290 nm. In another embodiment of the present invention, approximately 85% or more of processing applied to UV light has a wavelength of 254 nm.

Emission of UV-light can be presented in the form of the continuous emission of UV light, for example, when using mercury lamps, or it can be pulsed UV light, for example, in the case of using the method of monochromatic laser. The desired intensity of the UV-irradiation can be achieved by combining two or more lamps. This can be combined with at least two procedures irradiation by introduction between them of a pause. The present invention relates to any effective dosage of UV light, that is, for such a dose of UV light, which changes the cell so that it starts continuously to proliferate. Effective dosage may depend on many factors, mainly well-known experts in this field, for example on the physical parameters of the cells for holding UV-learning, such as the size and diameter of the lamp and cameras, the distance between the medium containing the cells and the source of UV-light, Svetogorsky and reflective properties of the material of the camera. In particular variants of the implementation of the present invention, the cells irradiated in a monolayer when one cell layer is on the surface. Moreover, the wavelength and intensity of the UV-light and the time of contact of the exposed cells from UV light are also important parameters for determining effective dosage. In addition, the effective dosage is also influenced by the cell itself, the medium containing the virus and their sotouboua ability. In different variants of implementation of the present invention, the effective dosage is sufficient to change at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the cells contained in the sample, and in other variants of the implementation of the present invention, the effective dosage is sufficient to change the cells to a level where at least 10% of the cells changed so that achieved their continuous growth. However, 10%-90% of the cells may die under the action of irradiation. In some embodiments, the implementation of the present invention, sample containing cells exposed to an effective dose, which constitutes at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 MJ/cm 2 . In some embodiments, the implementation of the present invention, the effective dosage is approximately 500, 450, 400, 350, 300, 250, 200, 180, 150, 130 or 105 MJ/cm 2 . In particular variants of the implementation of the present invention, the specified UV dosage is approximately 70 to 105 MJ/cm 2 . In some embodiments, the implementation of the present invention, the dosage used in relation to UV-light. The term "approximately" has regard to the quality of conventional UV lamps that do not provide discrete UV light at a single wavelength (as in the case of lasers), and are characterized by a spectrum with the Gaussian distribution is emitted when the Ohr, the length of which is close to the specified wavelengths. In those versions of the implementation of the present invention that uses some of these lamps, the term "approximately" refers to the deviation from the specified value of the wavelength of 10%.

Before or after irradiation or passage, cell line may also be selected to carry out quality control, with into account criteria such as sterility, absence of pollution mycoplasmas, no contamination with pathogenic viruses, and/or to test the F-Pert with the purpose of determining whether the activity of reverse transcriptase inhibitors, and also to conduct other tests necessary for quality control, in accordance with the criteria applicable in this field in relation to the selection of cell lines for use in medical biotechnology. In this sense, the term "free/not contain" should be understood in such a way that pollution levels reduced to values below the detection limits, determined currently used by quality control procedures. Because the method of the present invention can be used for creating continuous cell lines without using viral vectors or without the introduction into the environment of retroviruses, consider cell lines often free from any retroviral activity that can be confirmed in a test on the activity of reverse transcriptase. However, this retroviral activity may be specially introduced in cell lines of the present invention by methods of molecular engineering, with the aim, for example, production of viruses or proteins in cell lines.

Irradiated cells can apply to any type of cells. In some embodiments, the implementation of the present invention, the specified fabric derived from embryos. In a number of options, use mixed culture more than one type of tissue that can be obtained in the tissue destruction or multiple fabrics. In other variants of the implementation of the present invention, these cells are cells of the umbilical cord of the embryo. Irradiated cells derived from one or more of fabrics, could constitute or may include, for example, endothelial cells, epithelial cells, pluripotent or totipotent stem cells, embryonic stem cells, neuronal cells, kidney cells, liver, muscle cells, intestinal cells, white blood cells, lung cells, the cells of the ovary, skin cells, cells of the spleen, stomach cells, the cells of the thyroid gland cells of blood vessels, cells of the pancreas and/or may present their precursor cells, as well as their combinations.

And many variations of the implementation of the present invention, the cells are fixed to the surface in the process of irradiation or during cultivation. Cultivation on the surface is particularly well suited for endothelial cells, since then these cells can be selected for monitoring quality according to current criteria, such as their ability to form monolayers, which may be degraded if UV dosage will introduce too many changes damaging nature. On the surface of cells can form monolayers. In particular, the cells are cultured or irradiated on microneedle. Alternatively, these cells or subjected to radiation exposure, either cultivated or both of these processes can be combined in suspension. Cells that were previously irradiated or cultivated on the surface, then we can be adapted for growth in suspension culture.

In another aspect, the present invention relates to a method for virus, where this method includes obtaining cells in continuous cell lines, the method of the present invention, then the infection of these cells specified by the virus reproduction of the given virus in these cells and collection of the given virus.

In accordance with the present invention produced described in the invention way viruses choose from DNA or RNA viruses, coated or not coated containing single-stranded or double-stranded (DNA) genomes, semantic or antisense chain, continuous or segmented. These viruses can be selected from the group consisting of baculoviruses, poxviruses, adenoviruses. Papovaviruses, parvovirus, hepadnaviruses, coronaviruses, flaviviruses, togaviruses, astroviruses, picornaviruses, retroviruses, orthomyxoviruses, filovirus, paramyxoviruses, rhabdoviruses, arenaviruses and bunyaviruses. In some embodiments, the implementation of the present invention, these viruses choose from a group enveloped viruses, including flavivirus, togavirus, retroviruses, coronaviruses, the filovirus, rhabdovirus, bunyaviruses, orthomyxovirus, paramyxoviruses, arenavirus, hepadnavirus, herpes viruses and poxviruses. In other variants of the implementation of the present invention, these viruses are enveloped viruses such as influenza, including influenza A, B or C, West Nile virus, vaccinia virus, a modified vaccinia virus or viruses river Ross. In other variants of the implementation of the present invention, these viruses are selected from the group enveloped RNA viruses, including flavivirus, togavirus, retroviruses, coronaviruses, the filovirus, rhabdovirus, bunyaviruses, orthomyxovirus, paramyxoviruses and arenavirus. In particular variants of the implementation of the present invention, the virus is a MVA (modified vaccinia virus Ankara (Ankara)), TBE virus (the virus of tick-borne encephalitis, yellow fever virus, West Nile virus, influenza virus New Caledonia or flu virus.

After the collection phase, the virus can be inactivated by any known means used to inactivate viruses, for example, using the techniques described in the publication for U.S. patent no 2006/0270017 A1 included in this work of reference. In particular, inactivation can be carried out using a formaldehyde-treatment and/or UV-irradiation, separately or in combination.

Mainly serum or derived from the serum of a substance, such as, for example, albumin, transferrin, or insulin may include undesirable substances, which pollute cell culture and produced from these biological products. In addition, additives derived from human serum should be tested for the presence of all known viruses, including viruses of hepatitis and HIV, which can be transported through the serum. In this connection, according to some versions of the method of the present invention, the cells of the considered cell lines adapting for growth in serum-free medium, so that they can be selected on their ability to grow in serum-free media. These environments can be free from serum or fractions serum or major blood components. Environment suitable for use in such scenarios, the implementation of the present invention, are selected from DMEM/HAM's F12, RPMI, MEM, BME, environment Weymouth (Waymouth), in particular of the environment, not containing oligopeptides or proteins, as described in U.S. patent 2007/0212770, which is included in this discussion is completely link, or can be used a combination of both. Specified does not contain oligopeptides environment can be also free of protein or oligopeptides blood, which exceed 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4-amino acid(s), but may include glutathione. Protein-free environment in essence does not contain protein, but may include proteins produced cell lines, or protease. In particular, the environment may also include polyamide as the growth stimulating means, and/or it may be the environment, the chemical composition of which is specified as described, in particular, in the US patent US 2007/0212770. The term "chemically defined" means that the environment does not include any undefined additives, such as, for example, extracts components animals, organs, glands, plants or yeast. Accordingly, each component chemically specific environment carefully defined. Chemically defined environment is essentially not contain proteins or hydrolysates cells, but may contain proteins produced cell line, or protease. Examples of these environments are given in the "Guide for serum-free cell cultures" ("A guide to Serum-Free Cell Culture", GIBCO cell culture (2003)), which is available through the Internet on the page: www.invitrogen.com/content/sfs/brochures/332-032442_SFMBrochure.pdf).

The above environments, including serum-free medium, not containing oligopeptides, or chemically certain environment, may also include glutathione and/or protease, in particular trypsin, such as pork trypsin or recombinant trypsin, before or after inoculation of virus (Klenk et al. (1975) Virology, 68: 426-439). Such protease may also be required under cultivation cell lines, because the cells were fixed on the surface and the extent of such fixation could vary from severe to very weak. Strongly attached cells can be separated from the surface using protease and/or chelating agents such as EDTA (Doyle et al. Chapter 4: Core Techniques, in: Cell & Tissue Culture: Laboratory Procedures, ECACC, John Wiley & Sons, Chichester (1996)). In addition, this environment, in particular protein-free environment, may include hydrolysates of plants or yeast, before or after inoculation. Obviously, such an environment will also include proteins or metabolic products are produced in cell lines of the present invention.

Cell lines obtained using the method of the present invention, are mostly nepokorennye and/or non-cancer. In some embodiments, the implementation of the present invention, cells, cell lines tested and selected for testing the quality, such as F-pert test.

In another aspect, the present invention relates to a process for obtaining a product of recombinant gene, where this method includes obtaining cells in continuous cell lines created by the described in the present invention of the method, followed by transfection of cells of nucleic acid, which encodes the specified gene product, expression specified gene product and, optionally, the collection specified gene product. Nucleic acid can be a DNA, RNA or NCP. In addition to the gene nucleic acid may include promoters for expression in the cell and markers in plant breeding.

Below the present invention is illustrated by examples, which are in no way aimed at its limit.

Examples

Example 1: the Distinguished by the duration of irradiation of Vero cells UV light with the purpose of reception of the mutants

Materials:

Wednesday TC-Vero

N1-buffer

Trypsin (1:10 dilution)

Trypsin inhibitor

6-hole tablets, Corning Cat. No. 3516

The t-shaped flask on 25 cm 2 , Nunc Cat. No.: 163371

UV lamp, VL 50C, 240 nm Grid-Tube, 50 W, the Company Vilber-Lourmet

Procedure

Installs in a 6-hole tablets with a density of 1 x 10 6 cells/well and add 5 ml of the medium (in the double repetition). Just use 7 tablets (each measured in the experiment time: 2 wells/tablet).

24 hours a good monoclona culture.

Sucked off 5 ml environment to 1 ml and public tablets irradiated by ultraviolet light (distance tablets from UV lamp = 9 cm).

Tablet A: 15 minutes

Tablet B: 30 minutes

Tablet C: 45 minutes

Tablet D: 60 minutes

Tablet E: 90 minutes

Tablet F: 120 minutes

Tablet G: control, without radiation

After irradiation of the cells in both parallel holes treated with trypsin (1 ml trypsin + 0.5 ml inhibitor of tripsin /well), where the cells in the first hole is used for determine the number of cells (CC) and their viability, and the cells in the second hole passedout in the flask by 25 cm 2 with 10 ml of the medium.

The test room

Time exposure

TCC/well [x 10 6 ]

Vitality Barkero-Turku [%]

15 minutes

30 minutes

45 minutes

60 minutes

90 minutes

not determined

120 minutes

not determined

The content of T-shaped bulb 25 ml were treated with trypsin and defined indicators TCC and the viability of using Cedex:

The test room

TCC/Roux [x 10 6 ]

The viability [%]

The picture when using the microscope

spheroidal cells, not sticking

cells in the supernatant, not sticking

the individual cells in the supernatant, not sticking

only the remains of cells

only the remains of cells

only the remains of cells

good monolayer, 95-100%

* Actual value is lower because the counting of cells using Cedex gave too low results for correct number of cells.

Example 2: UV-irradiated cells birds

The purpose of this test was to explore the possibility of application processing UV light as a tool to create a continuous cell lines, suitable for the production of vaccines.

Primary chicken embryos, embryos quail used as source material for obtaining the original primary monolayer cultures. Derived cell culture with controlled as was used to develop a procedure, based on the effects of UV light.

The impact on primary cells of UV light at 254 nm). Continuous cell line was derived from primary cells of embryos virgin quail and chicken by UV exposure.

In detail the process of creating a cell line from primary cells of embryos quail to create a secure cell banks illustrated in figure 3 in the form of a phylogenetic tree.

As a material for UV-irradiation, in each case selected and thaw one ampoule of cell banks, taking into account the results of the first evaluation (cells chicken, Japanese quail and virgin quail), obtained on the basis of the cell preparation of chicken embryos, embryos Japanese quail and virgin quail (mixed culture full destroyed embryos).

Installation for UV-irradiation collected in the 6-hole tablets at sowing cells with a density of 1 x 10 6 cells/well and adding 5 ml of the medium. As a medium used TBE environment (FSME) with the addition of 5% of the FTS and antibiotics (penicillin, streptomycin and gentamicin). Only used 7 tablets with two holes on the tablet for each option. After 24 hours in the wells were observed formation of a uniform monolayer culture. For irradiation of the cells have been pumped out of 5 ml environment to rest in 1 ml and open the tablets were irradiated by ultraviolet light in laminar flow, as indicated below. Distance tablets from UV lamp was 9 see As the source of UV light is used in UV light from the company Vilber-Lourmet (VL 50C, 240 nm Grid-Tube, 50 W).

Tablet A: 0.5 minutes

Tablet B: l min

Tablet C: 2 minutes

Tablet D: 3 minutes

Tablet E: 4 minutes

Tablet F: 5 minutes

Tablet G: control, not irradiated

After irradiation, the cells were treated with trypsin in the wells (1 ml trypsin when breeding N1 buffer in the ratio 1:10), which used a 1 ml of cell suspension (6 ml) to determine the number of cells and their viability, and the remaining cells were passively 5 ml environment in the flask by 25 cm 2 . The results are shown in the table, attached to this example.

During the first period of cultivation (25-35 days) was conducted only by the replacement of the media, and the morphology and cell adhesiveness optical assessed in the individual tests. Only after the formation of the Islands adhesive growing cells in T-25 flasks, cells in variants a-E were treated with trypsin and taken to the 6-hole tablets (less surface area than in flasks T-25), so as to achieve homogeneous colonization of adherent cells. The cells, approximately at the level of K40-K50, who achieved Mergence 80-100%, then it was passively in T-25 and T-75 envelopes, each option for 6-9 days, and carried in 1-2 sealed ampoule, which served as the source material for the receipt and evaluation cell banks (about 10 vials). Tropinova processing and transfer of these cell populations described in example 3.

Drugs used

Environment: the environment TBE (FSME) + 5% FTS + mix antibiotics (penicillin/streptomycin, 100 mg/l + gentamicin, 50 mg/l)

TBE environment (FSME) + 10% FTS

Wednesday TC Vero + 10% FTS

Buffer N1

Gamma-trypsin

DMSO from Sigma

Abbreviations: CC - number of cells; T-25/75/175 25/75/175... cm 2 - T-bulb

The number of cells and their viability according to individual tests conducted after irradiation

The test room

Time exposure

CC/ml [x 10 6 ]

Vitality Barkero-Turku [%]

TCC/well [x 10 6 ]

30 seconds

1 minute

2 minutes

3 minutes

4 minutes

5 minutes

not determined

Because in variants A-G found similar values by the number of cells and their viability, the data of the individual tests, that, therefore, there were no significant differences according to the results of UV-irradiation cells depending on the duration of exposure. For this reason, morphology and adgezivnuu ability to compare cultures were evaluated almost daily to identify features.

Of all the investigated options A-F, cell population of variant E had the best properties as continuous, adhesive growing cell lines, such as the presence of a homogeneous cellular structures, the possibility of cultivation in various T-shaped tubes, constant growth of cells after a few passages, the ability to cryopreservation and suitability for the propagation of viruses (for example, MVA virus).

In the case of cells quail, cell population in option F unable to cultivate. Reduced growth of cells in the formation of inhomogeneous cell lawn (large associations) have been observed after more than 6 passages cells (test G), which are not irradiated by ultraviolet light. After 16 passage and next, the cells have lost the ability to share and could not continue to be cultivated. While similar results were obtained in tests with cells and quail and chicken.

Example 3: Tropinova processing and transfer cells

Tropinova processing and transfer of adhesive growing cells quail conducted under the scheme of passage, a similar scheme, which is typically used for Vero cells. After draining the culture medium was stage leaching N1 buffer, after that culture was layered one or more layers of an appropriate number of gamma-trypsin, diluted in the ratio 1:10, and then all were incubated at a temperature of (in the case of a 6-hole tablets and Kolb T-25 (T-25...25 cm 2 T-flask) was enough room temperature 37 C up to Department of cells from the vessel for cultivation (with a gentle scraping). Adding tropinovogo inhibitor to stop the actions of trypsin is optional, because the culture medium containing the FTS. The cells are then transferred to a new cultural environment and distributed to other vessels for cultivation, in accordance with certain tests, and then again raised.

The table below shows the amounts used in this tripsinei processing.

The vessel for cultivation

Buffer N1

Gamma-trypsin (diluted 1:10 N1 buffer)

6-hole tablet

T-bulb, 25 cm 2

T-bulb, 75 cm 2

T-bulb, 175 cm 2

Example 4: UV Dosimetry-for the immortalization of cells using UV lamps VL 50C

Defined the dose necessary to obtain continuous cell lines by UV exposure. Installation for dosimetry was similar to the device used for the processing of cells. UV irradiation With light causes the transformation of potassium iodide and Iodate potassium dissolved in buffer solution, accompanied by the formation of brown-yellow three-iodide. Tri-iodide has the maximum absorption at a wavelength of 352 nm, and this absorption can be quantitatively measured on a spectrophotometer. This principle allows to measure UV dose used for the influence on the cell monolayers, depending on the length of exposure. In this regard, on the basis of measurements made in a 6-hole tablets, it was shown that the duration of exposure is from 0.5 to 5 minutes corresponded to the UV doses from 20 to 120 MJ/cm 2 (figure 4).

Three model solution was prepared in sufficient quantities.

The composition of model solutions

Model solution 1

Model solution 2

Model solution 3

Boric acid

Dissolve 6,18 g/l of distilled water

NaOH pellets

Desirable the pH value 9,15; approximately 2 g/l

PVP R17 PF

2,414 g/l

Potassium iodide higher purity

of 1.41 g/l

2.57 m g/l

4,30 g/l

Potassium Iodate higher purity

0.55 g/l

0,92 g/l

Model solutions could be stored in a dark place before their use, at least for 47 days.

Selected 60 ml each model solutions 1, 2 and 3 to construct the calibration curve. In protected from light form, these samples were sent for analysis and construct a calibration curve in the unit IBC. 100 ml each of the model solutions 1, 2 and 3 moved in flasks of SCHOTT and closed from the incident light. Portable UV light VL 50C put on the net. The distance between the base of the tablet, and the bottom side portable UV lamp was 9 see Portable UV lamp was adjusted so that the filter was sent on the basis of the tablet (i.e. the bottom).

Portable UV light has included 10 minutes before use.

Has prepared 3 bulb Shota with 100 ml of model solutions 1, 2 and 3, as well as the pipette, holders for pipettes, an empty flask of SCHOTT and three 6-hole tablet. 1 ml model solution 1 suffered a pipette in the upper left a hole 6-hole tablet. This well placed below portable UV lamp without coverage, so it was located in the center bottom of the filter. After a 30-second exposure, the hole was quickly removed from its provisions below portable UV lamp. 370 MKL from 1 ml of irradiated solution was transferred into a thin-walled quartz cuvette and within 5 minutes measured OP 367 nm. The same measurement is repeated 3 times, and recorded the results of measurements. Determined the average value of these 3 results. If the measured value was above the site of calibration for a photometer, respectively, used a cell with another wall thickness. Supernatant from the hole was sucked out and threw it away.

These stages repeated for all variants of irradiation of different duration. Based on the obtained curve and OP (253.7 nm) VT environment, calculated appropriate dosage (MJ/cm 2 ) in relation to the period of time from 30 seconds to 5 minutes. The results obtained are presented in the table below.

UV dosage, calculated on the basis functions of the corresponding curve

The exposure time: 30 seconds

The function of the potential curve

y=21,767x -0,1945

Environment And over 253.7 VT (=x)

UV dosage (MJ/cm 2 )

Time exposure: 1 minute

The function of the potential curve

y=56,953x -0,4709

Environment And over 253.7 VT (=x)

UV dosage (MJ/cm 2 )

to 29.61

The exposure time: 2 minutes

The function of the potential curve

y=98,154x -0,4322

Environment And over 253.7 VT (=x)

UV dosage (MJ/cm 2 )

Time exposure: 3 minutes

The function of the potential curve

y=147,31x -0,5363

Environment And over 253.7 VT (=x)

UV dosage (MJ/cm 2 )

The exposure time: 4 minutes

The function of the potential curve

y=212,7x -0,5159

Environment And over 253.7 VT (=x)

UV dosage (MJ/cm 2 )

The exposure time: 5 minutes

The function of the potential curve

y=264,53x -0,5377

Environment And over 253.7 VT (=x)

UV dosage (MJ/cm 2 )

As can be seen from the above table, the curve of the function of the dose is y=24,h+4,3125. X denotes exposure time is in minutes, and y represents the dose in MJ/cm 2 . (Figure 4).

Example 5: getting the virus in continuously growing cells

MVA, r-MVA, TBE influenza virus was replicated in ever-growing culture of cells quail. Cell culture quail in katalozni flasks were set according to the procedure as described above. Culture infected with the use of MVA, TroVax, TBE virus and influenza (according to the Protocol GMP). The multiplicity of infection (MOI) was selected in accordance with currently accepted way of getting MVA. Viral products collected within 3-4 days. As a culture medium used the environment TC-Vero with 10% of the FTS and were incubated at a temperature of 32 C.

The infection was performed using 10 ml after incubation for 1 hour at the end of 60 ml.

TBE: 50 ul of virus

MVA: 250 cells / mm virus

The influenza virus "New Caledonia" (NC): 50 ml

Abbreviation: KXX... - day incubation culture XX

Sampling: 3 x 1 ml of sample NOVA, NaBr - for NC, HA, obtaining microphotographs

Glutamine

The titer of virus

TBE-Elisa

lg PFU/ml

the negative.

Glutamine

7,02e8V/ml

The influenza virus "New Caledonia"

Glutamine

was 2.76

0,00E+00

Glutamine

Viral titres obtained for MVA and r-MVA growing in katalozni asks, in the experiment are:

TCID 50 /ml

8 x 10 8

r-MVA (TroVax)

9 x 10 8

Viral titres obtained for TBE virus and influenza growing in katalozni asks, in the experiment are:

Title (log PFU/ml)

HA (HAU/50/ml)

The influenza virus "New Caledonia"

not determ.

Example 6: an Analysis of different cell cultures in the procedure of the F-test Pert

Testing procedure F-Pert allows you to determine the activity of reverse transcriptase PCR method and need to confirm security. Different cultures (Vero (negative control)), primary cells of chicken (positive control), continuous cell cultures quail and continuous cell cultures chicken received using the same procedure. Cultural supernatant collected and processed for quality assurance in the procedure of the test the F-Pert.

The results of the testing procedure F-test Pert

Cell culture

The result of the F-test Pert

Vero (control)

negative

CEC (primary cells of chicken)

positive

Cells quail (4 different culture)

negative

Cells of chicken (2 different culture)

negative

1. Way access to continuous cell lines animal or person, where these cell lines derived from living cells that are not embryonic human cells, including irradiation of these living cells dose of UV light from about 20 MJ/cm 2 to 300 MJ/cm2 at a wavelength of between about 100 nm and 400 nm within about 30 seconds to 5 minutes and selection of cells capable of proliferation after at least 20 passages, as the cells of continuous cell lines.

2. The method according to claim 1, characterized in that the effective dose of UV light has a wavelength of between 200 nm and 300 nm.

3. The method according to claim 1, wherein the dosage of UV light is at least 50 MJ/cm2 .

4. The method according to claim 1, characterized in that the specified dosage of UV light is at least 300 MJ/cm2 .

5. The method according to claim 1, characterized in that the cells are cells of birds or mammalian cells.

6. The method according to claim 1, characterized in that stage of the selection process includes the selection of these cells after at least 40 passages.

7. The method according to claim 1, characterized in that the cells are fixed on the surface, or they are in suspension.

8. The method according to claim 5, wherein these cells are embryonic cells.

9. The method according to claim 1, characterized in that the cells are found in the mixed culture that includes more than one type of tissue.

10. The method according to claim 1, characterized in that the cells are endothelial cells.

11. The method according to claim 1, characterized in that these cells form the monolayer.

12. The method according to claim 1, characterized in that the cells of cell lines is chosen so that they were nepokorennye and/or non-cancer.

13. The way to obtain virus, including: (i) infection of cells continuous cell lines obtained by the method according to claim 1, the virus in conditions that can achieve the proliferation of the virus; and (ii) collection of the given virus.

14. The method indicated in paragraph 13 notable that the virus choose from a baculovirus, poxvirus, adenovirus, papovavirus, parvovirus, hepadnavirus, coronavirus, flavivirus, togavirus, Astrovirus, picornavirus, retrovirus, orthomyxovirus, filovirus, paramyxovirus, rhabdovirus, arenavirus and bunyavirus, MVA, TBE virus, yellow fever virus, virus fever West Nile virus flu "New Caledonia" and influenza.

15. The method of obtaining of recombinant gene product, including: (i) transfection of cells continuous cell lines obtained by the method according to claim 1, nucleic acid, coding recombinant gene product, in conditions which allow to achieve the production of the specified gene product, and (ii) optional, collection specified gene product.

16. The method according to claim 1, characterized in that the cells in the specified cell lines adapted for growth in serum-free media.

17. The method according to article 16, wherein the specified environment selected from DMEM/HAM F12, RPMI, MEM, BME, environment Weymouth (Waymouth), environment, not containing oligopeptides, chemically specific environment or their combinations.

18. Cell line for the production of vaccines received by the method according to claim 1, deposited in European collection of cell cultures (ESAS) with access number 08020602.

19. Cell line for the production of vaccines received by the method according to claim 1, deposited in European collection of cell cultures (ESAS) with access number 08020603.

20. Cell line for the production of vaccines received by the method according to claim 1, deposited in European collection of cell cultures (ESAS) with access number 08020604.

21. Cell line on any of PP-20, wherein the cells of the specified cell lines are nepokorennye and/or non-cancer.

23. Cell line on any of PP-20, wherein the cells of the specified cell lines capable of cultivation in serum-free media.