AU5363699A

AU5363699A - Recombinant stable cell clone, its production and use thereof

Info

Publication number: AU5363699A
Application number: AU53636/99A
Authority: AU
Inventors: Friedrich Dorner; Wolfgang Mundt; Manfred Reiter
Original assignee: Baxalta GmbH; Baxalta Inc
Current assignee: Baxalta GmbH; Baxalta Inc
Priority date: 1999-08-05
Filing date: 1999-08-05
Publication date: 2001-03-05
Anticipated expiration: 2019-08-05
Also published as: AU780810B2; CA2381173A1; EP1200561A1; CY1106111T1; ES2267280T3; ATE330003T1; JP2003506077A; DE59913565D1; EP1200561B1; DK1200561T3; PT1200561E; WO2001011021A1

Abstract

The invention relates to a stabile recombinant cell clone which is stabile in a medium containing no serum and proteins for at least 40 generations, and to a biomass which is obtained by multiplying the stabile cell clone under cultivation conditions that do not involve the use of serum or proteins. The invention also relates to a method for producing recombinant proteins using the biomass, to a method for producing stabile recombinant cell clones, and to the production of a recombinant protein in a synthetic minimal medium that does contain serum or proteins.

Description

1 RECOMBINANT STABLE CELL CLONE, ITS PRODUCTION AND USE The present invention relates to a stable recombinant cell clone which is stable in a serum and protein-free medium for at least 40 generations, a biomass obtained by the propagation of the stable cell clone under serum- and protein-free culture conditions and a method for the preparation of recombinant proteins by means of the biomass. Moreover, the invention relates to a method for the preparation of stable recombinant cell clones. Furthermore, the invention relates to the preparation of a recombinant protein in a serum- and protein-free synthetic minimal medium. The preparation of recombinant proteins, in particular biomedical products, such as blood factors, is becoming increasingly important. To allow optimal growth of recombinant cells, serum is usually added to the medium. Because of the high cost of the serum and to prevent possible contaminations by viral or molecular pathogens through the serum in the culture medium, a number of serum-free media have been developed, which, in particular, should not contain any additives of bovine or human origin. The use of such media in the preparation process allows not only a low risk of contamination of the prepared products by viral and molecular pathogen, but also a more simple purification of the expressed proteins. Recombinant cells are mostly cultivated in the serum-containing medium until a high cell density, approximately equivalent to that of a "working cell bank," has been reached, and then, during the production phase, they are readapted to serum-free medium. Miyaji et al. (1990, Cytotechnology 3:133-140) selected a serum-independent cell clone in a serum-free medium which contained insulin and transferrin. However, it was shown that, after 16 days, the viable count and the expression rate continuously decreased. Miyaji et al. (1990, Cytotechnology 4:173-180) attempted to improve the expression rate and the productivity of the recombinant cells by coamplification with a marker gene. Yamauchi et al. (1992, Biosci. Biotechnol. Biochem. 56:600-604) established serum independent recombinant CHO subclones by culturing serum dependent cells on microtiter plate as a monolayer for 3-4 weeks in a serum-free medium which contained human serum albumin, insulin and transferrin. Approximately 0.1% of the cells were serum-independent. A portion of the subclones also grew in suspension culture in a serum-free medium, where the cells, however, formed aggregates and clumps. The doubling time of the cells was 1.5 days. However, no indications are provided on the stability of the serum-independent clones obtained or on the long term culturing of these clones under serum-free conditions. The media which allowed the maintenance of the metabolic activity and growth of cells during the cell free phase often contained additional substances, such as growth factors, insulin or transferrin, or adherence factors which replace the serum components.

2 In order to omit the addition of polypeptide factors, such as insulin or transferrin, and to allow protein-free culture conditions, various techniques have been developed. For example, specifically defined, complete protein-free media have been developed which allow cell growth even under protein-free conditions. WO 97/05240 describes the preparation of recombinant proteins under protein-free conditions where the cells coexpress a growth factor in addition to the desired protein. JP 2696001 describes the use of protein-free media for the preparation of factor VIII in CHO cells with the addition of a nonionic surfactant or cyclodextrin to improve the productivity of the host cells. To increase the effectiveness of these additives, it has been recommended, for example, to add butyrate and lithium. WO 96/26266 describes the culturing of cells in a medium which contains a glutamine-containing protein hydrolysate, whose free amino acid content is less than 15% of the total protein weight and whose peptide has a molecular weight of less than 44 kd. A synthetic minimal medium is used as base medium in the culturing media for cell cultures, to which base medium one also adds, besides protein hydrolysate, other additives, including fetal calf serum, gentamicin and mercaptoethanol. The use of this serum-containing medium for the recombinant preparation of blood factors is not mentioned. US 5,393,668 describes special synthetic surfaces which allow the growth of adherent cells under protein-free conditions. To stimulate the cell proliferation, CHO cells which overexpress human insulin were propagated on an artificial substrate to which the covalent insulin is bound (Ito et al., 1996, PNAS USA 93:3598-3601). Reiter et al. (1992, Cytotechnology 9:247-253) describe the immobilization of r-CHO cells which are cultured in the serum-containing medium at a high density on supports and the subsequent perfusion of the immobilized cells in the protein-free medium during the production phase, where a continuous release of protein into the cell supernatant was observed. However, the cells were perfused for less than 10 generations in the protein-free medium. The methods which are available to date for the successful preparation of an industrial "large-scale" cell culture under protein-free conditions have been described for continuous cell lines, particularly VERO cells (WO 96/15231). The cells were cultivated here under serum- and protein free conditions from the original ampule to the industrial scale of 1200 L. However, the cells used are not recombinant cells, but host cells which are used for the production of virus antigen in a lytic process. In contrast to adherent VERO cells, CHO cells, for example, are only dependent to a imited degree on adhesion. CHO cells which are cultured by conventional methods under serum-containing conditions are capable of binding both to smooth and porous microsupports (US 4,978,616, Reiter et 3 al., 1992, Cytotechnology 9:247-253). If CHO cells are grown under serum-free conditions, they lose this property and do not adhere to smooth supports, such as, for example, Cytodex 3, or they readily separate from them to the extent that no adherence-promoting additives, such as, for example, fibronectin, insulin or transferrin have been added to the medium. Because of the low adhesion of CHO cells to supports under serum-free conditions, the production of recombinant proteins is therefore usually carried out in suspension culture. The production process can here be run by a continuous or batch method. The recombinant cell culture is here cultivated in a bioreactor until an optimal cell density has been reached; the protein expression is optionally induced, and, for the harvest, the medium which contains the expressed proteins but also recombinant cells is drawn off at certain intervals from the reaction tank and thus removed from the production process. As a result of the continuous loss of biomass, the production efficiency in the bioreactor decreases, and it increases only after the slow addition of fresh medium, because the cells have to grow until the desired cell density is reached. Therefore, and in spite of the continuous process, there is always a delay phase in which the production rate decreases in this system. In addition, the capacities for growth and production are limited by the maximum achievable cell density in such a system. In the adaptation of cells cultured under serum-containing conditions on protein-free medium, it was consistently observed that the yield of expressed protein and the productivity of recombinant CHO cells strongly decrease after adaptation in a protein-free medium in comparison to serum-containing conditions (Paterson et al., 1994, Appl. Microbiol. Biotechnol. 40:691-658). This is explained by an instability or reduced growth of the recombinant clone as a result of changes in the culture conditions. Because of the changed fermentation conditions-and in spite of the use of a stable original clone-a large portion of the cells is always converted to cells with reduced expression or to nonproducing cells, which, during the production process, overgrow product-producing cells, resulting in a fermentation culture which, in the end, consists of a large portion of nonproducing cells or of cells with low expression. The result of this situation is that the maximum production capacity of the fermentation culture continuously decreases and the maximum product production is limited to a certain number of generations or cell passages. Therefore there is a need for a system which allows continuous production over as long a time period as possible, in particular in the industrial production of recombinant proteins under serum- and protein-free conditions. Moreover, it would be desirable to obtain recombinant cell clones which are stable over many generations in the production phase under protein-free conditions and which expresses a recombinant protein. Therefore the problem of the present invention is to provide an efficient method for the preparation of recombinant proteins under serum- and protein-free culture and production conditions.

4 An additional goal is to provide a stable recombinant cell clone. According to the invention, the problem is solved by making available a recombinant cell clone which can be obtained from a cell culture, which cell clone is obtained after the culturing of the recombinant original cell clone on serum-containing medium and readaptation of the cells to a serum- and protein-free medium. Here, the cells are further cultured for at least 40 generations in a serum- and protein-free medium under conditions equivalent to the production conditions. The cell clone according to the invention therefore forms a population of cells which, in a predominant portion, can be cultured for at least 40 generations in a stable manner in the serum- and protein-free medium. Here, it is preferred that more than 80%, in particular more than 99%, of the cell population according to the invention or the cell clone according to the invention is stable for at least 40 generations. Here it is preferred for the culturing of the cells to be carried out without selection for the selection marker and/or amplification gene, for example, in the absence of MTX in the case of CHO-dhfr cells. In the context of the invention, the term original cell clone denotes a recombinant cell clone transfectant, which, after transfection of host cells with a recombinant nucleotide sequence expresses recombinant product in a stable manner under laboratory conditions. The original clone is cultured for growth optimization in the serum-containing medium. To increase the productivity, the original clone is optionally cultivated in the presence of a selection agent and with selection for the selection marker and/or amplification marker. For industrial production, the original cell clone is cultivated under serum-containing culturing conditions until a high cell density has been reached, and it is adapted to serum and/or protein-free medium shortly before the production phase. Here, the culturing is preferably carried out without selective pressure. It was found that under these conditions a large portion, more than 95%, of the cells in such a cell culture which has been readapted to serum- and protein-free medium is converted into non-product-producing cells. By means of immunofluorescence with product-specific antibodies, it was possible to show that, as a function of the generation time of the cells in serum- and protein-free medium, the number of non-producing cells in a culture increases and overgrows the product producing cells, resulting in a decrease in the production of the culture. The cell culture which is obtained after readaptation to serum- and protein-free medium is tested for the cell clone of the cell population which produces stable products under serum- and protein-free conditions, optionally in the absence of selective pressure. This can be achieved, for example, by immunofluorescence with specifically labeled antibodies made against the recombinant polypeptide or protein. The cells which have been identified as product-producing cells are isolated from the cell culture, and again propagated under serum- and protein-free conditions, which are preferably equivalent to the production conditions. The isolation of the cells can here be achieved by 5 isolation of the cells and testing for product-producing cells. Optionally, the cell culture which contains the stable cells is again tested for stable recombinant clones, which are then isolated from the cell culture and cloned. The stable recombinant cell clones obtained under serum- and protein free conditions are then further propagated under serum- and protein-free conditions. The recombinant cell clone according to the invention is characterized, in particular, in that it is stable in serum-free and protein-free medium for at least 40, preferably at least 50, and particularly advantageously more than 60, generations, and expresses a recombinant protein. Here, this stability appears without benefit of aids such as matrices or solid surfaces, for example, as supports. Furthermore, according to the invention it is not required to carry out the culturing using high cell densities. According to a special aspect of the invention, the stable recombinant cell clone is in an isolated form. Starting from the stable cell clones, a cell culture is obtained under serum- and protein-free conditions by propagation of the stable cells. The stable recombinant cell clone according to the invention is preferably derived from a recombinant mammalian cell. Here, the recombinant mammalian cells can be any cells which contain sequences coding for a recombinant polypeptide or protein. This definition comprises all continuously growing cells, both adherent and non-adherent. It is particularly preferred to use recombinant CHO cells or BHK cells. The recombinant polypeptides or proteins can be blood factors, growth factors and other biomedically relevant products. According to the present invention, it is preferred to use stable recombinant cell clones which contain the coding sequence for a recombinant blood factor such as factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, protein S, protein C, an activated form of one of these factors, or vWF, and which are capable of expressing these blood factors under stable conditions over several generations. Here, it is preferred to use CHO cells which express vWF or a polypeptide with vWF activity, factor VIII or a polypeptide with VIII activity, vWF and factor VIII, factor IX or factor II. The cell clone which was selected under serum- and protein-free conditions according to the invention is characterized, in particular, in that it is stable for at least 50 [sic; 40], preferably at least 50, generations, and particularly advantageously for more than 60 generations in serum- and protein free medium. In order to develop a master cell bank, 30 generations are needed. To carry out an average batch culture on the 1000 L scale, at least 40 generations are required. Thus, for the first time it is possible to prepare from an individual clone a "master cell bank" (MCB), a "working cell bank" (WCB) with approximately 8-10 generations and thus to prepare a cell culture on the production scale (production biomass) with up to 20-25 generations under these conditions, because the cell clones available to date became unstable after a few generations of growth on serum- or protein-free 6 medium, resulting in the inability to obtain a) a uniform cell culture with product-producing cells, and b) stable product productivity over a longer time period. The cell clone according to the invention is thus stable for at least 40 generations under production conditions in serum- and protein-free medium. The methods which have been described to date provided only a generation number of less than 10 generations with product productivity under protein-free conditions (Reiter et al., 1992, supra). As stability criterion, a minimal number of at least 40 generations, preferably more than 50, and particularly advantageously more than 60, generations are used in the production process, during which stable expression of the proteins occurs and the cell morphology and phenotype do not change and no tumorogenic characteristics are present. Unexpectedly, it was found that the cell clone according to the invention, under serum- and protein-free conditions, presents an increased productivity even in comparison to the original cell clone which was cultured in serum-containing medium. According to another aspect, the present invention makes available a cell culture which contains at least 90%, preferably more than 95%, and particularly more advantageously more than 98%, stable recombinant cells which, under serum- and protein-free conditions, are stable for at least 40 generations, in particular at least 50 generations, and express recombinant product. In the context of the present invention, a cell culture denotes a master cell bank (MCB), a working cell bank (WCB-working cell bank) or a production biomass in an industrial production bioreactor. According to the invention, the cell culture is obtained, in particular, by culturing a stable recombinant cell clone of the type mentioned above under serum- and protein-free conditions. The cell culture according to the invention can here be obtained by propagation of the isolated stable cell clone from the individual clone, that is the seed cells, to the MCB, the WCB or a biomass on the production scale in the bioreactor under serum- and protein-free conditions, preferably without selective pressure on the selection and/or marker gene. In particular, it has been shown that the recombinant cells in a cell culture obtained from the stable recombinant clone according to the invention are stable for at least 40 generations under serum- and protein-free conditions. The cell culture made available according to the present invention, which is prepared from a serum and protein-independent stable cell clone, under most protein-free culturing and production conditions presents at least 90%, preferably at least 95%, particularly advantageously at least 98%, stable recombinant cells. The term "stable recombinant cells" here denotes, in particular, recombinant mammalian cells which are derived from the stable cell clone. It is here preferred to use recombinant CHO cells, preferably CHO-dhfr- cells, CHO-KI cells, and BHK cells which express a blood factor, preferably recombinant vWF, factor VIII, factor VIII and vWF, factor IX or factor II.

7 The cell culture according to the invention can contain the stable recombinant cells as suspension culture. The cells can also be immobilized on a support, in particular a microsupport, where porous microsupports are particularly preferred. It was found that porous supports such as, for example, Cytoline@ or Cytopore@, are particularly suitable. According to another aspect, the present invention represents a method for the industrial production of a recombinant product under serum- and protein-free conditions, using the stable cell clone made available according to the invention. The method here comprises the steps of preparation of an isolated, stable recombinant cell clone of the above described type for the preparation of a cell culture. Here the propagation of the isolated stable cell clone occurs under serum- and protein-free conditions from the stable individual cell clone to the cell culture. In particular, the subculturing of the stable cell clone also occurs under protein-free conditions, in particular without the addition of a protease such as, for example, trypsin. As a result, it is guaranteed that at no time during the preparation of a cell culture used in the production of a recombinant product that contamination occurs which may under certain circumstances be caused by the addition of serum and protein-containing additives of human or animal origin to the cell culture. Thus, for the first time a method is described which allows the preparation, starting from the initial clone and via the preparation of a working cell bank, a production biomass, and the subsequent production of recombinant protein under serum- and protein-free conditions. The preparation of the recombinant products with the cell culture according to the invention, which contains more than 90%, preferably more than 95%, and particularly advantageously more than 98%, of stable product-producing cells, can be carried out as suspension culture or with cells immobilized with a support. The process here can be carried out in batch or continuous mode, or by a perfusion technique with serum- and protein-free medium. The recombinant proteins expressed are then obtained from the cell culture supernatant, then purified with known methods of the state of the art and further processed. As serum- and protein-free medium one can use any known synthetic medium. Conventional synthetic minimal media can contain inorganic salts, amino acids, vitamins and a carbohydrate source and water. For example, it can be a DMEM/HAM F12 medium. The content of soy and yeast extract can be 0.1-100 g/L, particularly advantageously 1-5 g/L. In a particularly preferred embodiment one can use soy extract, for example, soy peptone. The molecular weight of the soy peptone is less than 50 kd, preferably less than 10 kd. It is particularly preferred to use a medium with the following composition: synthetic minimal medium (1-25 g/L), soy peptone (0.5-50 g/L), L-glutamine (0.05-1 g/L), NaHCO 3 (0.1-10 g/L), ascorbic acid (0.0005-0.05 g/L), ethanolamine (0.0005-0.05 g/L), Na selenite (0.0001-0.01 g/L). A nonionic surfactant such as, for example, polypropylene glycol (PLURONIC F-61, 8 PLURONIC F-68, SYNPERONIC F-68, PLURONIC F-71 or PLURONIC F108) as defoaming agent can optionally be added to the medium. This agent is generally used to protect the cells from the negative effects of aeration, because, without the addition of a surfactant, the ascending bursting air bubbles can lead to damage to those cells located on the surface of these air bubbles ("sparging") (Murhammer and Goochee, 1990, Biotechnol. Prog. 6:142-148). The quantity of nonionic surfactant can here be 0.05-10 g/L, however it is particularly preferred to use as small a quantity as possible of 0.1-5 g/L. Moreover, the medium can also contain cyclodextrin or a derivative thereof. The addition of the nonionic surfactant or of cyclodextrin is, however, not essential to the invention. It is preferred for the serum- and protein-free medium to contain a protease inhibitor, such as, for example, seine protease inhibitors which are suitable for use in tissue culture and of synthetic or plant origin. The parameters for the culturing of the cells, such as 02 concentration, rate of perfusion or change in medium, pH, temperature and culturing technique are here dependent on the individual cell types used and they can be determined in an easy manner by a person skilled in the art. For example, the culturing of CHO cells can be carried out in a stirred vessel and perfusion with protein free medium can occur at a perfusion rate of 1-10 volume changes/day, a pH of 7.0-7.8, preferably at pH 7.4, an 02 concentration of 40-60%, preferably 50%, and a temperature of 34-38'C, preferably 37 0 C. According to an additional aspect, the present invention makes available a method for the obtention of a stable recombinant cell clone, comprising the steps of - propagation of a recombinant original clone up to the cell culture in serum-containing medium, preferably without selective pressure, - culturing the cells under serum- and protein-free conditions which are preferably equivalent to production conditions, - testing the cell culture for product-producing cells under serum- and protein-free conditions, - cloning the stable recombinant cell clone under serum- and protein-free conditions, where the cloning can be carried out by generally known techniques such as isolation of the cells by dilution and growing of the individual clones, - propagation of the isolated cell clones under serum- and protein-free conditions, and, - optionally, testing the cell culture for product-producing-cells. Here only those recombinant cell clones should be considered stable which express stable recombinant protein in a protein-free medium for at least 10, preferably at least 20, and particularly advantageously at least 50 generations.

9 According to another aspect, the invention makes available a method for the obtention of a stable recombinant cell clone, comprising the steps of - propagation of a nonrecombinant initial cell or cell line under serum- and protein-free conditions and cloning a stable nonrecombinant cell clone under serum- and protein-free conditions, - transfecting the stable cell clone with a recombinant nucleic acid and isolation of stable recombinant cell clones, - culturing the stable cell clone transfectants in a serum- and protein-free medium under conditions which are optionally equivalent to production conditions, - testing the stable recombinant cells for production and product stability. The invention is described with reference to the following examples, without being limited to the examples. Brief description of the figures Figure 1: shows the microscopic view of a working cell bank of an original clone at the time of the readaptation from serum-containing medium to serum- and protein-free medium (A), after 10 generations in serum- and protein-free medium (B), and after 60 generations in serum- and protein free medium (C). Figure 2: shows the microscopic view of a cell culture starting from a stable recombinant cell clone under serum- and protein-free conditions at the stage of the working cell bank (A), after 10 generations (B) and after 60 generations (C). Figure 3: shows the results of culturing an rFVIII CHO cell clone in a 10 L perfusion bioreactor. a) FVIII activity (mU/mL) and perfusion rate (1-5/day) over a time period of 42 days. b) Volumetric productivity (units factor VIII/1/day) in the perfusion bioreactor. Examples: Example 1: Stability of rvWF CHO cells after readaptation from serum-containing medium to serum- and protein-free medium CHO-dhfr- cells cotransfected with plasmid phAct-rvWF and pSV-dhfr, and vWF expressing clones, as described in Fischer et al. (1994, FEBS Letters 351:345-348), are subcloned. From the subclones which expressed rvWF in a stable manner, a working cell bank (WCB) was prepared under serum-containing conditions but in the absence of MTX, and the cells were immobilized under serum-containing conditions on a porous microsupport (Cytopore@). After a cell density of 2 x 107 cells/mL support matrix was reached, the conversion of the cells to serum- and protein-free medium was carried out. The cells were further cultured for several generations under serum- and protein-free conditions. By means of immunofluorescence with labeled anti-vWF antibodies, the 10 cells were tested at various times in the serum- and protein-free medium. The evaluation of the stability of the cells was carried out on the working cell bank before the change of medium, after 10 and 60 generations in serum- and protein-free medium. While the working cell bank still presented 100% rvWF producing cells (Figure 1 A), the proportion of rvWF producing cells decreased to approximately 50% after 10 generations in serum- and protein-free medium (Figure 1 B). After 60 generations more than 95% of the cells were identified as nonproducing cells (Figure 1 C). Example 2: Cloning of stable recombinant CHO clones From the rVWF CHO cell-containing cell culture according to Example 1, which had been cultured for 60 generations in serum- and protein-free medium (Figure 1 C), a dilution was prepared, and in each case 0.1 cell/well was inoculated in a microtiter plate. The cells were cultured in DMEM/HAM F12 without serum or protein additives and without selective pressure for approximately 3 weeks, and the cells were tested by means of immunofluorescence with labeled anti-vWF antibodies. A cell clone which had been identified as positive was used as starting clone for the preparation of a seed cell bank. From the seed cell bank a master cell bank (MCB) was prepared in serum- and protein-free medium, and individual ampules were frozen and stored for the later preparation of a working cell bank. Starting from an individual ampule, a working cell bank was prepared in serum- and protein-free medium. The cells were immobilized on porous microsupports and continued to be cultured for several generations under serum- and protein-free conditions. By means of immunofluorescence with labeled anti-vWF antibodies, the cells were tested at different times in serum- and protein-free medium for productivity. The evaluation of the stability of the cells was carried out at the stage of the working cell bank and after 10 and 60 generations in serum- and protein-free medium. At the stage of the working cell bank (Figure 2A), and after 10 (Figure 2B) and 60 generations (Figure 2C), approximately 100% of the cells were identified as positive stable recombinant clones which express rvWF. Example 3: Cell specific productivity of the recombinant cell clones From the defined stage during the culturing of recombinant cells, a defined cell number was removed and incubated with fresh medium for 24 h. The rvWF:Risto-CoF [ristocetin cofactor] activity was determined in the cell culture supernatants. Table 1 shows that the cell-specific productivity in the stable recombinant cell clones according to the invention was stable even after 60 generations in serum- and protein-free medium, and it was elevated even in comparison with the original clone which was cultured in serum-containing medium.

11 Table 1 Cell clone Cell specific Cell specific Cell specific productivity of the productivity after 10 productivity after 60 working cells, mU generations, mU generations, mU rvWF/1 06 cells/day rvWF/1 06 cells/day rvWF/1 06 cells/day rvWf-CHO *808.68 55 30 <10 original cell clone r-vWF-CHO F7 *) 62 65 60 stable clone *) deposited in accordance with the Budapest Treatise of January 22, 1998 (ECAC (European Collection of Cell Cultures, Salisbury, Wiltshire SP4 OJG, UK), deposit No. 98012206) Example 4: Culturing of rFVIII CHO cells in protein and serum-free minimal medium An rFVIII CHO cell-containing cell culture was cultured in a 10 L stirred tank and perfused. Thereby a serum- and protein-free medium was used. The cells were here immobilized on a porous microsupport (Cytopore@, Pharmacia) and cultured for at least 6 weeks. The perfusion rate was 4 volume changes/day, the pH was 6.9-7.2, the 02 concentration approximately 20-50%, and the temperature 37*C. Figure 3 shows the results of culturing an rFVIII CHO cell clone in a 10 L perfusion bioreactor. a) FVIII activity (mU/mL) and perfusion rate (1-5/day) over a time period of 42 days. b) Volumetric productivity (units factor VIII/L/day) in the perfusion bioreactor. Table 2 Culturing days Cell specific productivity Immunofluorescence (mU/106 cells/day) (% FVIII positive cells) 15 702 n.a. 21 1125 n.a. 28 951 >95% 35 691 >95% 42 970 n.a. Table 2 shows the stability and specific productivity of the rFVIII expressing cells. For these results, samples were removed after 15, 21, 28, 35 and 42 days, centrifuged at 300 G and 12 resuspended in fresh serum- and protein-free medium. After an additional 24 h, the factor VIII concentration in the cell culture supernatants and the cell count were determined. From these data, the specific FVIII productivity was calculated. A stable average productivity of 888 mU/106 cells/day was reached. This stable productivity was also confirmed by immunofluorescence with labeled anti-FVIII antibodies after 15, 21, 28, 35 and 42 days in serum- and protein-free medium.

Claims

1. Method for the preparation of a stable recombinant cell clone, characterized by the following steps: - preparation of a recombinant original cell clone, - culturing of the recombinant original cell clone on serum-containing medium, - readaptation of the cells to serum- and protein-free medium, - testing of the cell culture for stable product-producing cells, and - cloning of a stable product-producing cell clone under serum- and protein-free conditions.

2. Method according to Claim 1, characterized in that, after cloning, the stable cell clone is obtained in isolated form.

3. Method according to Claim 1 or 2, characterized in that a recombinant mammalian cell is prepared as the original clone.

4. Method according to Claim 3, characterized in that the mammalian cell is a recombinant CHO cell or a BHK cell.

5. Method according to one of Claims 1-4, characterized in that the recombinant cell clone contains the sequences coding for a recombinant polypeptide or protein.

6. Method according to Claim 5, characterized in that the recombinant protein is a blood factor chosen from the group consisting of factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, protein S, protein C, or an activated form of one of the factors, or vWF.

7. Method according to one of Claims 1-6, characterized in that, as the original clone, a recombinant CHO cell which expresses von Willebrand's factor is prepared.

8. Method according to one of Claims 1-6, characterized in that, as the original clone, a recombinant CHO cell which expresses factor VIII is prepared.

9. Method according to one of Claims 1-6, characterized in that, as the original clone, a recombinant CHO cell with coexpresses factor VIII and vWF is prepared.

10. Method according to one of Claims 1-6, characterized in that, as the original clone, a recombinant CHO cell which expresses factor IX is prepared.

11. Method according to one of Claims 1-6, characterized in that, as the original clone, a recombinant CHO cell which expresses factor II is prepared.

12. Recombinant cell clone, characterized in that it can be prepared by a method according to one of Claims 1-11.

13. Cell culture, characterized in that it can be obtained by the following steps: - propagation of a recombinant original clone in serum-containing medium, - culturing of the cell under serum- and protein-free conditions up to the cell culture, - testing of the cell culture under serum- and protein-free conditions for product-producing cells, 14 - cloning of stable cell clones under serum- and protein-free conditions, - propagation of stable cell clones under serum- and protein-free conditions.

14. Cell culture, characterized in that it can be obtained by culturing a stable recombinant cell clone according to Claim 12.

15. Cell culture according to Claim 13 or 14, characterized in that it can be induced to express recombinant product under serum- and protein-free conditions.

16. Cell culture according to one of Claims 13-15, characterized in that the stable recombinant cell clones are mammalian cells.

17. Cell culture according to Claim 16, characterized in that the mammalian cells are CHO cells, preferably CHO-DHFR~ cells, CHO-K1 cells or BHK cells.

18. Cell culture according to one of Claims 13-17, characterized in that the stable recombinant cells contain a sequence coding for a recombinant polypeptide or protein.

19. Cell culture according to one of Claims 13-18, characterized in that the cells are immobilized on a microsupport.

20. Method for the industrial production of a recombinant product under serum- and protein free conditions, characterized in that it comprises the following steps: - preparation of an isolated, stable recombinant cell clones according to Claim 12, - propagation of the stable cell clone in serum- and protein-free medium from the initial clone up to the cell culture, - preparation of the stable cell-containing cell culture in the bioreactor, and - harvesting of the proteins from the culture supernatant.

21. Method according to Claim 20, characterized in that the serum- and protein-free medium is a synthetic minimal medium containing a yeast or soy extract.

22. Method according to Claims 20 or 21, characterized in that the medium contains cyclodextrin or a derivative thereof.

23. Method according to Claim 20-22, characterized in that the serum- and protein-free medium contains a protease inhibitor.

24. Method for the obtention of a stable recombinant cell clone, characterized in that it comprises the following steps: - propagation of a recombinant original clone up to the cell culture in serum-containing medium, - culturing of the cells under serum- and protein-free conditions which are equivalent to production conditions, - testing of the cell culture for product-producing cells under serum- and protein-free conditions, 15 - cloning of the recombinant cell clones which are stable under serum- and protein-free conditions, and - propagation of the stable cell clone under serum- and protein-free conditions.

25. Method for the obtention of a stable recombinant cell clone, characterized in that it comprises the following steps: - propagation of a nonrecombinant starting cell under serum- and protein-free conditions, - cloning of a stable nonrecombinant cell clone under serum- and protein-free conditions, - transfection of the stable cell clone with a recombinant nucleic acid and isolation of stable transfectants, culturing of the transfectants in serum- and protein-free medium under conditions which are equivalent to production conditions, and testing of the cells for production stability.