AU2013206102A1 - Method for producing continuous cell lines - Google Patents

Abstract

A method for production of continuous cell lines comprises irradiating living cells of an animal or a human with an effective dose of UV light, and selecting cells capable to proliferate after at least 20 passages as cells of a continuous cell line.

Description

Regulation 3.2 Revised 2/98 AUSTRALIA Patents Act, 1990 ORIGINAL COMPLETE SPECIFICATION TO BE COMPLETED BY THE APPLICANT NAME OF APPLICANTS: Baxter International Inc, and Baxter Healthcare S.A. ACTUAL INVENTORS: REITER, Manfred MUNDT, Wolfgang FEIGL, Simone VON FIRCKS, Simone ADDRESS FOR SERVICE: Peter Maxwell and Associates Level 6 60 Pitt Street SYDNEY NSW 2000 INVENTION TITLE: METHOD FOR PRODUCING CONTINUOUS CELL LINES DETAILS OF ASSOCiATED APPLICATION NO(S): Divisional of Australia Patent Application No. 2009 244 776 filed on 20 February 2009 The following statement is a full description of this invention including the best method of performing it known to us: m:\docs\20091202\288583,doc Method for producing continuous ce1 lines Field of the invention The present invention relates to methods for producing cell lines. Background of the invention Cell lines have become a valuable tool for vaccine manufac turing. The production of some important vaccines and viral vec tors is still done in embryonated chicken eggs or primary chicken embryo fibroblasts. Primary avian tissue for virus rep lication is provided by SPF (specific pathogen free) production plants. SPF derived tissues are expensive and the quality of the supply material is often hard to control. Therefore, inconsis tency and shortage in supply are the most predominant disadvan tages of the technologies based on SPF eggs. The same is true for approaches where primary fibroblast monolayer cultures are used. To multiply cell lines indefinitely, the cells need to be immortalized. Most immortalized cell lines currently in use are descendants of cancer cells or of fused hybridoma cells. How ever, the later technology is limited to fusion with myeloma cells. No general technology exists that can generate immortal ized cells of different types. Summary of the invention It is an object of the present invention to produce a con tinuous cell from non-continuous cell material. In particular, the goal was to provide continuous cell lines that have the po tential to proliferate without the introduction of foreign viral genes. Therefore, the present invention provides a method for pro duction of continuous cell lines comprising providing living cells of an animal or a human, irradiating said cells with UV light, proliferating said cells and selecting cells capable to proliferate after at least 20 passages as cells of a continuous cell line. Such a continuous cell line is culture of cell that can be - 2 propagated and used for the recombinant expression of bio molecules such as proteins, or for the manufacture of viral products such as viral antigens or a whole virus population, in particular for vaccination purposes. Therefore, the present invention also provides a method of producing a virus comprising providing cells of a continuous cell line obtainable by the inventive method, infecting said cells with said virus, propagating said virus in said cells and collecting said virus. In another aspect the invention provides a method of produc ing a recombinant gene product comprising providing cells of a continuous cell line obtainable by the inventive method, trans fecting the cells with a nucleic acid encoding said gene prod uct, expressing said gene product and, optionally, collecting said gene product. In a further aspect the invention provides a continuous cell line obtainable by the method of providing living cells of an animal or a human, irradiating said cells with an effective dose of UV light, proliferating said cells and selecting cells capa ble of proliferating after at least 20 passages as cells of said continuous cell line. Brief description of the drawings Fig. 1 shows the scheme of the UV treatment procedure. Fig. 2 shows continuous quail cell cultures Fig. 3 shows the phylogenetic tree, and treatment route of producing a continuous quail cell line. Fig. 4 shows a correlation of the UV dosage to the irradia tion time with the set-up used to produce continuous cells. Detailed description of the invention The present invention provides the production of a continu ous cell line through UV treatment of cells. A cell line is a population of cells formed by one or more subcultures of a primary cell culture. Each round of subcultur ing is referred to as a passage. When cells are subcultured, they are referred to as having been passaged. A specific popula tion of cells, or a cell line, can be characterized by the number of times it has been passaged. The primary culture is the first culture following the isolation of cells from tissue. Fol lowing the first subculture, the cells are described as a secon dary culture (one passage) . After the second subculture, the cells become a tertiary culture (passage 2), and so on. It will be understood by those of skill in the art that there may be many population doublings during the period of passaging; there fore, the number of population doublings of a culture is greater than the passage number. The expansion of cells (i.e., the num ber of population doublings) during the period between passaging depends on many factors, including but not limited to the seed ing density, substrate, medium, growth conditions, and time be tween passaging. Culturing can be performed by inoculation of a cell medium, letting the cells grow until a confluent cell cul ture or a continuous film is formed by the cells and inoculating a new cell medium with a portion of the confluent cells. Never theless, passaging is a tool to evaluate the capability to propagate. Normally, cells, including non-irradiated cells, iso lated from a tissue can be passaged about 10-20 times until they reach a state where no further propagation or cell doubling oc curs. The cells then enter a senescent state from which no fur ther subcultures can be obtained. Contrary thereto continuous cell lines are capable to propagate after more than 20 passages, such as after more then 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75 or 80 passages. It has now been found by the inventors that such a continuous cell line which can be passaged multiple times past the 2 0t passage, in particular immortalized cells, can be obtained through altera tion of cells by UV treatment, i.e. by irradiating these cells with an effective dose of UV light. The terms "effective dose of UV light" according to the present invention shall be the amount of irradiation needed for transforming the non-continuous cell lines into continuous cell lines. The effective dose of UV light ranges from the minimum dosage required for such transformations to the maximum dosage which is tolerated by these cells without lethal consequences for the cell culture as a whole. it is clear that above or under the effective dose limits continuous cell lines cannot be obtained. The skilled man in the art can easily determine optimum effective dosages for each cell line on the basis of the information and guidance contained herein withroutine optimization. The cells may be primary cells or cells capable of propagation after a few passages. Culturing of the cell lines can be performed with standard cell culture tech niques, such as in T-flask systems or roller bottle systems, or in stirred tank or other bioreactor formats. In several embodi ments of the invention, the culture is adapted to and held under serum-free conditions. In the present application the term "UV light" means ultra violet radiation having a wavelength of from 10 to 400nm, in particular 100 to 400nm. The UV light may be selected from the group consisting of UV C (100 to 280 nm), UV B (280 to 320 nm), and UV7 A (320 to 400 nrn) . In some embodiments of the invention, the wavelength is between 200 and 300nm. Photosensitizing agents such as those which intercalate into the DNA and which are acti vated by UV light may be used to enhance the altering effect of the UV radiation, although they are not necessary in all embodi ments of the invention. In one embodiment of the present inven tion the UV light is UV C having a wavelength of from about 100 to about 280nm. In anotherembodiment of the present invention the UV light has a wavelength of from about 240 to about 290nm. In another embodiment of the present invention about 85% or more of the T light has a wavelength of about 254nm. Without being bound by any theory it is believed that the UV light alters the genetic material of a cell, which introduces mutations. While such alterations can generally be repaired by the cell's repair mechanisms, some alterations might remain. These alterations can introduce lethal mutations and also al terations which result in cell immortalization. From UJV irradia tion experiments an optimal dosage can be selected which results in a significant portion of cells which are immortalized and can be cultured. After passaging, it is believed that only viable cells which are capable of multiplying are selected, which are expected to have only minor alterations with at least one al teration which results in immortalization. A significant portion of the irradiated cells will not be immortalized but gain dif ferent alterations, leading to apoptotic or necrotic cells. How ever, in principle, only one cell with the alteration inducing immortalization is sufficient to obtain a continuous cell cul ture, as this cell will continue to propagate and survive through the multiple rounds of passaging as described herein.

The UV light emission may be a continuous form of UV light emission, e.g. mercury lamp technology, or pulsed UV light, e.g. monochromatic laser technology. The desired UV intensity may be generated by combining two or more lamps. At least two irradia tion procedures may be combined with a pause in between. The subject matter of the invention encompasses any effective dosage of UV light, i.e. any dosage of UV light which alters a cell to proliferate continuously. The effective dosage may depend on a variety of factors which are generally known in the field, e.g. the physical parameters of the UV irradiation chambers, such as size and diameter of the lamp and the chamber, distance between the cell containing medium and the UV light source, light ab sorption and reflection properties of the material of the cham ber. In particular embodiments of the invention, the cells are irradiated in a monolayer, one cellular layer on a surface. By the same token, the wavelength and intensity of the UV light as well as the contact time the cell is exposed to the UV light are also critical for the effective dosage. Furthermore, the effec tive dosage is also influenced by the cell itself, the medium containing the virus and their light absorption properties. In various embodiments of the invention, the effective dosage is sufficient to alter at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of cells contained in the sample, and in other em bodiments the effective dosage is sufficient to alter the cells to a level where at least 10% of the cells are either altered to grow continuously. 10% to 90% of the cells may be killed by the irradiation. In certain embodiments of the invention, a sample containing the cells is exposed to an effective dosage of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70mJ/cm 2 . In some embodiments the effective dosage is up to about 500, 450, 400, 350, 300, 250, 200, 180, 150, 130 or 105mJ/cm 2 . In particular embodiments of the invention, the UV dosage is be tween about 70 and 105mJ/cm 2 . In some embodiments, these dosages are employed by UV C light. The term "about" refers to the prop erty of common UV lamps which do not provide a discrete UV light at a single wavelength (as in lasers) but have a gauss shaped spectrum also emitting light in nearby wavelengths. In embodi ments utilizing some of these lamps, "about" refers to a devia tion of the wavelength value of 10%. Before or after irradiation or passaging, the cell line is can be further selected for fulfilling quality control criteria such as sterility, free of mycoplasma contamination, free of ad ventitious virus contamination, and/or passing the F-Pert test for the presence of reverse transcriptase activity, as well as other quality control criteria used in the art for selecting cell lines for medical biotechnology uses. In this sense "free of" is to be understood that contaminations are reduced to be below the detection limit of current quality test procedures. Since the present technology can generate continuous cell lines without the use of viral vectors or introduction of retrovi ruses, the inventive cell lines are often free of any retroviral activity, as can be tested by an assay for reverse transcriptase activity. However, such retroviral activity may be specifically introduced into the cell lines of the invention by molecular en gineering techniques for the purposes of, for example, produc tion of viruses or proteins in the cell lines. The cell line can be of any eukaryotic cell, particularly of a higher organism, such as in fish, avian, reptile, amphibious or mammal cells and even insect or plant cells. Some embodiments utilize mammal cells such as of hamster, mice, rat, dog, horse, cow, primate, or human; other embodiments utilize avian cells such as of chicken, duck, canary, parrot, quail, ostrich, emu, turkey or goose. In general, any bird species could be a source of avian cells for use in the invention. In some embodiments, it is advantageous to utilize a less frequently domesticated specie (such as quail or emu) to avoid potential contamination of stock tissues with viruses prevalent in more commonly domesticated species (such as chickens.) The irradiated cells can be of any type of tissue. In some embodiments the tissue is derived from an embryo. In many em bodiments, a mixed culture of more than one type of tissue is used, as can be obtained by disintegrate tissue or multiple tis sues. In further embodiments the cells are of the umbilical cord of an embryo. The irradiated cells can be or the tissue(s) can be of or include e.g. endothelial cells, epithelial cells, pluripotent or totipotent stem cells, embryonic stem cells, neu ronal cells, renal cells, liver cells, muscle cells, colon cells, leukocytes, lung cells, ovary cells, skin cells, spleen cells, stomach cells, thyroid cells, vascular cells, pancreatic cells, and/or precursor cells thereof and combinations thereof; In many embodiments the cells are attached to a surface dur ing irradiation or during culturing. Culturing on a surface is especially suitable for endothelial cells, whereby the cells can be further selected for fulfilling further quality criteria such as their capability to form monolayers, which can be hampered if the IV dosage introduces too much damaging alteration. On such a surface the cells may form monolayers. In particular the cells are cultured or irradiated on a microcarrier. Alternatively the cells may be either irradiated or cultured or both in suspen sion. Cells which are initially irradiated or cultured on a sur face may later be adapted to growth in suspension culture, In another aspect the present invention provides a method of producing a virus comprising providing cells of a continuous cell line obtainable by the inventive method, infecting said cells with said virus, propagating said virus in said cells and collecting said virus. In the present invention, the viruses to be produced are se lected from enveloped or unenveloped DNA or RNA viruses, with single or double (DNA) stranded genomes, sense or antisense, continuous or segmented. The viruses may be selected from the group consisting of baculoviruses, poxviruses, adenoviruses, pa povaviruses, parvoviruses, hepadnaviruses, coronaviruses, flaviviruses, togaviruses, astroviruses, picornaviruses, retro viruses, orthomyxoviruses, filoviruses, paramyxoviruses, rhab doviruses, arenaviruses, and bunyaviruses. In some embodiments of the invention, the viruses are selected from the group of en veloped viruses, including, flaviviruses, togaviruses, retrovi ruses, coronaviruses, filoviruses, rhabdoviruses, bunyaviruses, orthomyxoviruses, paramyxoviruses, arenaviruses, hepadnaviruses, herpesviruses, and poxviruses. In other embodiments, the viruses are enveloped viruses such as influenza, including influenza A, B or C, West Nile Virus, Vaccinia Virus, Modified Vaccinia Vi rus, or Ross River viruses. In other embodiments of the inven tion, the viruses are selected from the group of enveloped RNA viruses, including, flaviviruses, togaviruses, retroviruses, coronaviruses, filoviruses, rhabdoviruses, bunyaviruses, ortho myxoviruses, paramyxoviruses, and arenaviruses. In particular embodiments the virus is MVA (modified vaccinia virus Ankara) TBE (tick-borne encephalitis) virus, Yellow fever virus, West Nile virus, New Caledonia virus or an influenza virus.

8 After the collecting step, Lhe virus can be inactivated by any known means for virus inactivation, e.g. as disclosed in the US publication number 2006/0270017 Al, which is incorporated herein by reference. In particular, inactivation can be per formed by formaldehyde treatment and/or UV irradiation, alone or in combination. In general, serum or serum-derived substances, such as, e.g., albumin, transferrin or insulin, may comprise unwanted agents that can contaminate the cell cultures and the biological products obtained thereof. Furthermore, human serum derived ad ditives have to be tested for all known viruses, including hepa titis viruses and HIV which can be transmitted via serum. There fore, according to some embodiments of the inventive method, the cells of the cell line are adapted for growth in serum free me dia, e.g. they are selected for their capability to grow in se rum free media. The media may be free of serum or serum frac tions, or also in general blood constituents. Media for these embodiments of the invention are selected from DMEM/HAM's F12, RPMI, MEM, BME, Waymouth's medium, in particular an oligopep dide- or protein-free medium as described in the US 2007/0212770 which is incorporated herein by reference in its entirety, or a combination thereof. Said oligopeptide free medium may be free of blood proteins or oligopeptides with a size of more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids but may com prise glutathione. The protein-free medium is substantially free of proteins but may contain proteins produced by the cell lines or proteases. In particular the medium may also comprise a poly amide as growth promoting agent and/or be a chemically defined medium as described in the US 2007/0212770. The term "chemically defined" means that the medium does not comprise any undefined supplements, such as, for example, extracts of animal compo nents, organs, glands, plants, or yeast. Accordingly, each com ponent of a chemically defined medium is accurately defined. The chemically defined media are substantially free of proteins, or cell hydrolysates but may contain proteins produced by the cell line or proteases. Examples of such media are given in "A guide to Serum-Free Cell Culture", GIBCO cell culture (2003) available the WWW at www.invitrogen.com/content/sfs/brochures/332 032442_SFMBrochure.pdf), These media, including the serum free medium, the oligopep- - 9 tide free medium or the chemically defined medium may also com prise glutathione and/or proteases, in particular trypsin such as porcine or recombinant trypsin prior or after virus inocula tion (Klenk et al. (1975) Virology, 68: 426-439) . Such proteases may also be required during culturing of the cell lines since cells attached to a surface by exhibit strong to very light ad herence. Strongly attached cells can be detached by proteases and/or chelating agents such as EDTA (Doyle et al. Chapter 4: Core Techniques, in: Cell & Tissue Culture: Laboratory Proce dures, ECACC, John Wiley & Sons, Chichester (1996)). Furthermore the medium, in particular the protein free medium, may comprise plant or yeast hydrolysates prior or after inoculation. of course the medium is also expected to comprise proteins or meta bolic products produced by the inventive cell lines. The cell lines obtainable by the inventive method are gener ally non-tumorigenic and/or non-carcinogenic. In some embodi ments the cells of the cell lines are tested and selected for to pass quality test such as the F-pert test. In a further aspect the invention provides a method of pro ducing a recombinant gene product comprising providing cells of a continuous cell line obtainable by the inventive method, transfecting the cells with a nucleic acid encoding said gene product, expressing said gene product and optionally collecting said gene product- The nucleic acid may be DNA, RNA or PNA. In addition to the gene, the nucleic acid may comprise promoters for expression in the cell, and selection markers, In a further aspect the invention provides a continuous cell line obtainable by the method of providing living cells of an animal or a human, irradiating said cells with an effective dose of UV light, proliferating said cells and selecting cells capa ble to proliferate after at least 20 passages as cells of said continuous cell line. The inventive cell lines also include the progeny of such produced cell lines. In particular the cell line is defined as being obtainable by the embodiments of the method described herein. The obtainable continuous cell lines may have characteristic features such as telomere activity of specific caryotypes associated with the UV irradiation necessary to pro duce the continuous cell line. In particular embodiments of the invention the cells of the cell line are non-tumorigenic and/or 10 non-carcinogenic and in particular also pass quality tests such as the F-pert test. In particular embodiments the cell line is a cell line de posited at the ECACC with the deposit accession number 08020602, 08020603 or 08020604 corresponding to filing references QOR/RE07-169, QORi CJO7 and CORECB/SFQB-06, respectively. Fur ther inventive cell lines have the characteristic features, like ability to propagate, cell cycle pattern, telomerase activity, caryotype, chromosome pattern or telomere length as said depos ited cell lines and of course being a continuous cell line. The present invention is further illustrated by the follow ing examples without being limited thereto. Examples Example 1: T r d r radiation of Vero cells with UV light for producing mutants Materials: TC-Vero medium Nl-buffer Trypsin (1:10 dilution) Trypsin inhibitor 6-well plates, Corning Cat. No, 3516 25cm 2 T-Flask, Nunc Cat. No.: 163371 UV lamp, VL 50C, 240nm Grid-Tube, soW, company Vilber-Lourmet Procedure: Set-up is done in 6-well plates with 1x10 6 cells/well and 5ml of medium volume (in double set-up). A total of 7 plates (each time 2 wells/plate) is set up, After 24h there was a good monolayer culture. The 5ml of medium were drained off to iml, and the opened plates were irradiated with UV light (distance of the plates from the UV lamp = 9cm) plate A: 15min plate B: 30min plate C: 45min plate D: 60min plate E: 90min plate F: 120min plate G: control., no irradiation After irradiation, the cells of both wells are trypsinized (1ml Trypsin + 0.Sml trypsin inhibitor/well), wherein the cells of the 1 " well are used for determination of cell count (CC) and viability, and the cells of the 2 'a well are passaged in 25cm 2 roux with 1l of medium. Test no. Irradiation TCC/ B~rker- Thrk time well viab. [%] [x1Ol .1 A 15min 1.50 60.8 B 30min 1.25 27.9 C 45min. 1.15 5.6 D 60_min 0.95 23.6 E 90min 0.55 not determined F 120min 0.30 not determined G control 1.25 94.2 T-flask 25 content was trypsinized, the TCC and viability is de termined using Cedex: Test no. TCC/Roux Viab. Microscopic picture [x10 6 ] [%] A 0.80 23.2 spheroidal cells, no adherence B 0.60 18.8 cells in the supernatant, no adher ence C 0.60 34.4 individual cells in the super natant, no adherence D 0.50 25.0* only cell debris left E 0.50 22.7* only cell debris left F 0.40 11.1* only cell debris left G 1.80 196.6 good monolayer, 95-100% * the actual values are lower since the cell count in the Cedex is too low for correct cell-count determination! Example 2: UV irradiation of avi an cells The aim of this study was to investigate the potential use 12 of UV-light treatment as a tool for the generation of continuous cell lines suitable for vaccine production., Primary chicken and quail embryos were used as starting ma terial for production of initial primary monolayer cultures. Quality controlled cell cultures derived therefrom were used for derivation procedure based on the UV light exposure. Exposure of primary cells to UV light ( 2 54nm) . The continu ous cell line was developed from primary cells of bobwhite quail or chicken embryos by means of UV irradiation. The detailed course of development of the cell line derived from the primary cells of quail embryos up to the production of safety banks is illustrated in Fig. 3 in the form of a phyloge netic tree. As starting material for UV irradiation, in each case one ampoule of the first evaluation cell banks (chicken, Japanese quail and bobwhite quail) which originate from a cell prepara tion of the chicken embryos, embryos of the Japanese quail and of the bobwhite quail (mixed culture of disintegrated complete embryos) was thawed. The set-up for UV irradiation was done in 6-well plates with a cell seed of 1x16 cells/well and 5ml of medium volume. TBE me dium (FSME) with 5% of FBS and antibiotics (penicillin, strepto mycin and gentamycin) were used as medium. A total of 7 plates with 2 well/plate each was set up. After 24h, a uniform monolayer culture could be observed in the wells. For irradia tion of the cells, the 5ml of medium were drained off to lml and the opened plates were irradiated with UV light in the laminar flow bench as follows. The distance of the plates from the UV lamp was 9cm. A UV lamp of the company Vilber-Lourmet (VL 50C, 240nm Grid-Tube, 50W) was used as UV light source, plate A: 0.5min plate B: 1min plate C: 2min plate D: 3min plate E: 4min plate F: 5min plate G: control, no irradiation After irradiation, the cells were trypsinized in the wells (lml trypsin 1:10 diluted with N1 buffer), wherein 1ml of the cell suspension (a total of 6ml) was used for determination of - 13 CC and viability, and the remaining cells were passaged with Bmi of medium in 25cm 2 roux. The results are summed up in the table of this example During the first culturing period (about 25-35 days) there were only media exchanges, and morphology and adherence of the cells were optically assessed in the individual tests. Only af ter island formation of the adherently growing cells in the T-25 flasks had been observed, the cells of tests A-E were trypsinized and transferred to 6-well plates (smaller surface than T-25 flasks) in order to promote a homogeneous, adherent cell colonization. From this point in time, about K40-K50, the cells that had reached a confluence of 80-100% were further pas saged in T-25 and T-75 flasks every 6-9 days and set up in 1-2 safety ampoules which served as starting material for producing the evaluation cell banks (about 10 ampoules). Trypsinization and passaging of said cell populations is described in Example 3. Preparations used Medium: - TBE medium (PSME) + 5% FBS + mixture of antibiotics (penicillin/streptomycin 100mg/I and 50mg/i gentamycin) - TBE medium (FSME) + 10% FBS TC Vero medium + 10% FBS Ni buffer Gamma trypsin - DMSO company Sigma Abbreviations: CC.. .cell count, T-25/75/175. ..

2 5/75/175cm T flasks, Table: Cell counts and viabilities of the individual tests after irradiation Test no. Irradiation CC/ml Birker-Tirk TC/well t ime {x101] viab. fJ [x106] A 30sec 0.75 87.3 0.15 B 1min 0.75 79.5 0.15 C 2min 0 .80 84.1 0.16 D 3min 0.70 90.4 0.14 El 4min 0.80 80. 016 F 5min 0.70 014 14 GEcontrol 0.5 84.1 0.15 1 * not determined Due to the similar cell-count values and liabilities of the individual test set-ups A-G, no significant difference could be shown with respect to UV-irradiation time of the cells. This is the reason why morphology and adherence of the cultures compared were assessed nearly daily to recognize particularities. From all test set-ups A-F, the cell population from set-up E showed the best properties of a continuous, adherently growing cell line, such as homogeneous cell structure, culturing in dif ferent T-flasks, constant cell growth after several passages, capability for cryoconservation and suitability for virus propa gation (e.g. MVA virus). - In the case of quail cells, the cell population from set-up F could not be successfully cultured. Reduced cell growth with inhomogeneous cell-lawn formation (large wholes) could be ob served after more than 6 passages with the cells (test C) which had not been irradiated with UV light. From passage 16 on, the cells lost their division capability and could not be cultured any longer. All in all, similar results could be reached with quail and chicken cell tests. Example 3: Trypsinization and passaging of cells Trysinization and passaging of the adherently growing quail cells were done in a passaging scheme similar to that usually used for Vero cells. After pouring off the culture medium, a washing step is performed with N1 buffer, thereafter, the cul ture is covered with layer(s) of the corresponding amount of gamma trysin, diluted 1:10, and is incubated at a temperature (with 6-well plates and T-25 (T-25... 25cm 2 T-flasks) room tem perature is sufficient) of 37CO until the cells detach from the culturing vessel (by soft knocking) . Addition of the trypsin in hibitor to stop the effect of trypsin is not necessary due to the FBS contained in the culture medium. Subsequently, the cells are transferred to a new culture medium and are divided up into further culturing vessels in correspondence with the respective splits, and are, again, left to grow. The following table indicates the amounts used during trypsinization. culturing vessel N1 Gamma trypsin buffer (1:10 diluted with Ni buffer) 6-well plate 2ml iml 25cm 2 T-flask I 5ml iml 75cm T-flask 1Oml iml 175cm 2 T-flask 20ml 2ml Example 4: UV-C Dosimetry for Cell Immortalization with the UV lamp VL 50C The dosage to obtain continuous cell lines with UV irradia tion was measured. The dosimetry set up was similar to the set up for cell treatment. The radiation with UV-C light causes a transformation of potassium iodide and potassium iodate dis solved in buffer solution into brown-yellow tri-iodide. Tri iodide has its absorption maximum at 352nm and can be measured quantitatively in a spectral photometer. This principle allows to measure the UV dosage applied during cell monolayer exposure depending on exposure time. Therefore, based on measurements in 6-well plates, an exposure time of from 0.5 to 5 minutes corre sponds to an UV dosage of from 20 to 12OmJ/cm 2 (figure 4). Dosimetry is done as precisely as possible, as is the cell line test. In each case 1ml of the model solutions with absorp tion coefficients (367mn) of about 2.5/cm, 4.5/cm and 7.5/cm is irradiated in one well of the 6-well plate. Each model solution is irradiated 6 times. Irradiation times = 30sec, 1min, 2min, 3min, 4min und 5min. In order to find out the exact dosage for the respective irradiation time, the OD (253,7nm) of the medium used is determined. Materials used: portable UV lamp, VL50C, 254nm, SOW, company Vilber Lourmat Spectral photometer, company Therma, Device No.: PA5007 012MM - 6-well plate Boric acid 99 9%, company Riedel- de Haen, Lot No. :60460 NaOH pellets, company Baxter, Lot No.: 318608 16 - PVP K17 PF (polyvinyl-pyrrolidon Collidon K17), company Basf, Lot No.: 30408609TO - Potassium iodide, company Sigma Aldrich, Lot No.: P2963 500G Potassium iodat e, company Merck, Lot No. : K32577451622 TC VERO medium (VT), Charge: ORSFVTC0700401 - WFI water, company Baxter, PP2 Three model solutions are prepared in sufficient amounts_ Table 1: Composition of the model solutions Reagent Model solution 1 Model solution 2 Model solution 3 Boric acid 6.16g/l in Aqua dest. dissolve NaOH pellets desired value pH: 9.15; about 2g/1 PVP K17 PF 2.4149/1 Potassium 1.41g/l 2.57g/l 4.3Og/1 iodide purest Potassium 0.3g/l 0.559/1 0.92g/1 iodate purest The model solutions can be stored in a dark place until they are used but at least up to 47 days. 60ml each are taken from model solutions 1, 2 and 3 to produce a calibration curve. Protected from incident light, these samples are sent to IBC which establishes the calibration curve100ml each are transferred from model solutions 1, 2 und 3 into Schott flasks and are protected from incident light. The portable UV lamp VL 50C ist placed on a framework. The distance between the table plate and the bottom side of the portable UV lamp is 9cm. The portable UV lamp is adjusted such that the filter points to the table plate (i.e. downwards), The portable UV lamp is turned on 30 minutes before it is used. The 3 Schott flasks with the 100m] of model solutions 1, 2 and 3, pipettes, pipettboy, an empty Schott flask and three 6 well plates are prepared. lml of model solution 1 is pipetted into the left upper well of a 6-well plate. This well is placed below the portable UV lamp without a cover such that it is posi- : 17 tioned centrally below the filter. After 30sec of irradiation, the well is quickly removed from its position below the portable UV lamp. 370pl of the irradiated 1-ml solution are transferred to a thin-layered silica cuvette and the OD 3 6 7 nm is determined within £ minutes. The same is measured three times and recorded. The mean value of these 3 values is determined. If a value meas ured is beyond the calibration region of the photometer, corre spondingly, a cuvette with a different layer thickness will be used. The supernatant in the well is sucked off and discarded. These steps are repeated for all irradiation times. Based on the obtained curve functions and OD (253.7nm) of the VT medium, the respective UV dosage [mJ/cm] is calculated far from 30 seconds to 5 minutes. The results are presentend in the following table. Table: UV dosage calculated based on the respective curve functions: irradiaton time irradiatiorime 30 seconds 3 mites potential curve function potential curve function y = 21.767x ' y = 147.31x03 A 253.7 VT medium (=x) A 253.7 VT medium (=x) 4.01 4.01 UV dosage [mJ/cmi UV dosage [ma/cmi 16.61 69.95 irradiation time irradiation time minute 4 minutes potential curve function potential curve function y = 56.953x 1 709 y = 212.7x- 5 159 A 253.7 VT medium (=x) A 253.7 VT medium (=x) 4.01 4.01 UV dosage [mJ/cm9) UV dosage [mJ/cmi 29.61 103.90 irradiation time irradiation tine 2 minutes 5 minutes potential curve function potential cucve function y = 98.154x' 4322 y = 264.53x' 5 3 7 1B A 253.7 VT medium (=x) A 253.7 VT medium (=x) 4.01 4.01 UV dosage [mJ/cm") UV dosage [mJlcmn 53.86 125,36 As can be seen from this table, the curve function of the dosage is y = 24.09x- 4,3125. X is the irradiation time in min utes and y is the dosage in mJ/cm2 (Fig. 4). Example e 5: Virus production in continuous cells MVA, r-MVA, TBE and Influenza were propagated in continuous quail cells. Roller bottle cultures of quail cells were estab lished as described above. Cultures were infected with (GMP) MVA, TroVax, TBE and Influenza virus. A MOI was chosen according to the current MVA production process. Viral products were har vested after 3 to 4 days. AS culture medium TC-Vero 10%FBS was used during incubation at incubation 32 0 C. Infect.: carried out with 10ml after 1 h at final volume (60ml) TBE: 50p1 virus MVA: 2 5 0 pl virus New Caledonia (NC) : 50gl Abbreviation: KXX, . .day of culture XX Taking of samples: 3xlml sample, NOVA, NaBr- with NC, HA, micro photography TBE day i ucosa ( ulamine Ilaciate " i NH H 0 ss tt - iesa lEE-K&H x x x x x 2.83 0.31 33 21 3 56 8 ni To ( 2.71 027 04 26 732 8 3 00 _ 16 MVA ay glucse glutamiie lactate NH4 pH C02 | C [3ill - ( mg [%iter % 22 01 0.4 2 7 4 x 0 3N2w 027 0 2 731 61 x x C New Caledonia 19 day ucose glutaine acetate NH-4 pH Cr2 2.76 3 7--2T1 T 2 x 3=HAU 70 3 2.2 0.26 03 2 73 57 5=32HAU 100 4_2 .. 19 0.48 45 74 5 550E+0 5 32HAU 100U* Control day g ucosne ta I actate NH4 pH 0 Nar - r a 1g mg % %1 1 x x x x x x x x 2 1.78 0.16 1.24 31 70 63 x C T h__ r

-

m 3 1.24 0.12 142 4 6.87 9 x x 1.52 - 0 1 1.611 5 6.9 4.9 x Virus titer achieved for MVA and r-MVA grown in roller bottle experiments: Virus

TCID

5 0 /ml MVA 8x1O r-MVA (TroVax) 9x10 8 Virus titer achieved for TBE and Influenza grown in roller bottle experiments: Virus Titer (log pfu/ml) HA (HAU/50pl) CPE (%) TBE 69 64 100 New Caledonia not determinated 32 100 Example 6: F-Pert assay of different cell cultures The F-Pert assay allows to detect reverse transcriptase activity by PCR and is necessary for safety validation. Different cultures (Vero (neg. control), primary chicken (pos. control), continuous quail and continuous chicken cells) were prepared according to the same procedure. Culture supernatants were harvested and processed for F-Pert quality control testing Results F-Pert testing Cel Culture F-Pert Vero (control) negative CEC (primary chicken cells) positive quail cells (4 different cultures) negative chicken cells (2 different cultures) negative

Claims (26)

1. A method for production of continuous cell lines comprising irradiating living cells of an animal or a human with an effective dose of UV light, and selecting cells capable to proliferate after at least 20 passages as cells of a continuous cell line.

2. The method of claim 1 comprising irradiating said cells with UV ight vth a wavelength of between 10 nm and 400 nm,

3. The method of claim 2 wherein the wavelength is between 200 nm and 300 nm.

4. The method of claim 1 wherein the UV light dosage is at least 50 mJ/cm 2 .

5. The method of claim 1 wherein the UV light dosage is up to 300 mJ/cm 2 .

6. The method of claim 1 wherein the cells are avian or mammal cells.

7. The method of claim 1 wherein selecting said ceis comprises at least 40 passages.

8. The method of claim 1 wherein the cells are attached to a surface or are m suspension. 21

9. The method of claim 1 wherein the cells are cells of an embryo.

10. The method of claim I wherein the cells are a mixed culture of more than one type of tissue.

11. The method of claim I wherein the cells are endothelial cells.

12. The method of claim 1 wherein he cells are in a monolayer.

13. A method of producing a virus comprising infecting said cells of claim 1 with said virus under conditions which permit virus proliferation and collecting said virus,

14. The method of daim 13 wherein the virus is selected from baculovirus, poxvirus, adenovirus, papovavirus, parvovirus, hepadnavirus, coronavirus, flavivirus, togavirus, astrovirus, picornavirus, retrovirus, orthomyxovirus, filovirus, paramyxovirus, rhabdovirus, arenavirus, and bunyavirus,

15. The method of claim 13 wherein the virus is selected from MVA, TBE virus, Yellow fever virus, West Nile virus, New Caledonia virus or an influenza virus.

16. A method of producing a recombinant gene product comprising transfecting the cells of claim 1 with a nucleic acid encoding said gene product under conditions which permit production of said gene product and, optionally, collecting said gene product. 22

17. The method of claim 1 wherein the cells of the cell line are adapted for growth m serum free media.

18. The method of claim 17 wherein said medium is selected from DMEM/HAM's F12, RPMI, MEM, BME, WaymoutW' s medium, an oligopeptie free medium, a chemically defined medium or a combination thereof.

19. The method of claim 1 wherein said cels of the cell lines are selected for being non-tumorigenic and/or non-carcinogenic.

20. A continuous cell line obtainable by the method of providing living cells of an animal or a human, irradiating said cells with an effective dose of UV light, proliferating said cells and selecting cells capable to proliferate after at least 20 passages as cells of said continuous cell line,

21. The cell line of claim 20, wherein the dose of UV lght is at least 50 mJ/cm 2

22. The cell line of claim 20, wherein the dose of UIV light is up to 300 mj/cm.

23. A cell line deposited at the ECACC with the deposit accession number 08020602, 08020603 or 08020604.

24. A cell line according to claim 20, wherein cells of the cell line are non tumorigenic and/or non-carcinogenic. 23

25. A cell line according to claim 20, wherein cells of the cell line can be cultured on solid surfaces or m suspension.

26. A cell |ine according to claim 20, wherein cells of the cell line are capable for culturing in serum free media. Dated this 31t day of May 2013 Baxter international Inc. and Baxter Healthcare S.A. Patent Attorneys for the Applicant PETER MAXWELL AND ASSOCIATES