MXPA00000677A - Identification of human cell lines for the production of human proteins by endogenous gene activation - Google Patents

Abstract

A process for selecting human cells for the production of human proteins by endogenous gene activation allows human proteins to be produced in economically feasible quantities and in a form suitable for producing a pharmaceutical composition. Also disclosed is a process for producing human proteins in a cell line identified in this manner.

Description

IDENTIFICATION OF HUMAN CELLULAR LINES FOR THE PRODUCTION OF HUMAN PROTEINS BY THE ACTIVATION OF THE GENE ENDOGENIC FIELD OF THE INVENTION This invention relates to methods for the selection of human cells for the production of human proteins by the activation of the endogenic gene as well as to produce human proteins in economic yields and in a form which is suitable for the production of a pharmaceutical preparation. This invention also relates to a method of preparing human proteins in a cell line identified in this way. The preparation of human proteins by the activation of the endogenic gene in a human cell line is known. For example, WO93 / 09222, O94 / 12650 and O95 / 31560 describe the production of erythropoietin and other human proteins in human cell lines by the activation of the endogenic gene. In the documents, however, it is not suggested that certain criteria be observed in the selection of the cell lines used for the preparation of human proteins. Therefore, it can not be guaranteed that the desired human protein in the selected cell line REF .: 32517 for its production can be obtained with the desired performance and shape, and free of contamination. Accordingly, only low yields of human proteins are achieved in the documents mentioned above in general.

DESCRIPTION OF THE INVENTION The problem of the present invention is to eliminate the disadvantages of the state of the art and especially to suggest the criteria for the selection of the human starting cell lines, which are suitable for endogenic activation of a target gene or predetermined target. This problem was solved by a method for the selection of human cell lines for the preparation of human proteins by the activation of a target gene endogenically present in the cell line which is characterized because: (a) a human cell line is tested for the presence of the following characteristics: (i) a target gene with the desired nucleic acid sequence, (ii) at least 5 duplications of populations with 14 days in a suspension culture, and (iii) at least 5 duplications of populations with 14 days in a serum-free culture medium, and (b) a cell line having the characteristics (i), (ii) and (iii) is used as the starting cell line for the endogenic activation of the gene as a target. If one approaches the task of activating a human gene as a target in a human cell line to target or target the gene and to obtain a cell which is qualified for the production of the protein as a target in a satisfactory yield and in the desired form according to the invention, one will test several cell lines for the presence of a number of characteristics that this cell line must have to be an appropriate candidate for the recent large-scale production of the target protein. Cell lines preferably immortalized, especially tumor cell lines will be tested as such, since they have important advantages over non-immortalized cells with respect to the culture. According to characteristic (i), the human cell line is tested to see if the target or target gene, i.e., the gene to be activated by endogenous gene activation, effectively has the desired nucleic acid sequence, which it is generally the nucleic acid sequence of the natural white gene. This is because tumorigenic cell lines and other cell lines in permanent culture frequently show a series of mutations in their genes. Therefore, an important aspect in the selection of a suitable cell line is whether the cells have the correct gene for the desired product. The determination of the sequence can be carried out by culturing the cells in a usual manner and sequencing the target gene. If desired, an amplification of the target gene can be carried out by PCR or other amplification methods prior to the formation of sequences. Another important characteristic for the selection of a cell line is the quality of cultivation in suspension. Cells in suspension are easier to ferment, and fermentation can be more easily adapted to large dimensions, for example, in a larger fermentor with a capacity of, for example, 10 I to 50,000 I. Therefore, the selected cells they should be cells in suspension or they should be easily adapted to a suspension culture. For this purpose the cells are cultured for 14 days with constant agitation. If within this period the cells show at least five duplications of population or colony of organisms, they are considered suitable for suspension culture. The multiplication number of the organism colony can be determined by periodically determining the cell count, for example, by counting the cells or by measuring the optical density of the cell suspension. Another important characteristic for the selection of human cells is cultivation in a serum-free medium. Since the protein purification of the serum free cell cultures is considerably easier and in serum-free culture there is no danger of contamination with animal pathogens, such as viruses, the selected cells could be able to grow in culture-free culture. serum. For this purpose the cells are cultured for 14 days at a density of 1 to 10 10 5 cells per ml in culture vessels with serum-free medium (eg, RPMI 1650 with ITS from Boehringer Mannheim). If the cells during this culture show at least 5 replications or duplications of population or colony of organisms, which can be determined by cell counting, they are considered suitable for the serum-free culture.

Another important feature, and a preferred one according to the invention, is the generation time (iv). The selected cells in medium such as 10% DMEM of total bovine serum or RPMI 1640 with 10% of total bovine serum should have a high proliferation, that is, within a week of culture they should have 16 to 256 duplications of population or colony of organisms, preferably 64 to 128 duplications of the colony of organisms. For this purpose the cells are seeded in culture dishes in a concentration of 0.1 to 10 • 105 cells per milliliter, preferably 0.5 to 2"105 cells per ml, and the cell count is taken by means of a cell chamber after or without The cells that have a sufficiently short generation period are especially suitable for the large-scale production of human proteins by activation of the endogenic gene.Another preferred feature is the absence of any detectable endogenous expression, ie, transcription and translation, of the target gene (v) Preferably, for the activation of the endogenic gene whose cell lines are selected which have essentially non-endogenic expression of the target gene.For this purpose the cells can be seeded for 24 hours at a cell density of 0.01 to 2 x 106 cells / ml, preferably 0.5 to 1 x 106 cells per milliliter of culture medium. After a predetermined period of time, for example 24 hours, the supernatant of cells is removed, the supernatant of cells is removed, the cells are discarded, and the content of the target protein in the cell supernatant is determined by the processes of known tests, for example, ELISA. In the case of EPO, the limit of detection is, for example, 10 pg / EPO / ml. Cells that, in a seeding of 106 cells / ml, synthesize less than 10 pg of protein are considered without production and are especially suitable. Yet another important and preferred feature is the polyiso of the target gene in the cell to be selected (vi). The presence of more than two chromosomal copies of the target gene in the cell significantly increases the yields in homologous recombination. For the production of EPO, whose gene is on chromosome 7, the cells, Namalwa (Nadkarni et al., Cancer 23 (1969), 64-79) or HeLa S3 (Puck et al., J. Exp. Med. 103_ (1956), 173-284) that have 7 chromosomes in triplicate have proved to be especially suitable. Additional examples of cell lines which contain 7 chromosomes in a high number of copies are the colon adenocarcinoma cell line S -480 (ATCC CCL-228; Leibovitz et al., Cancer Res. 36 (1976), 4562-4567), the malignant melanoma cell line SK-MEL-3 (ATCC HTB 69; Fogh and Tre p, in: Human Tumor Cells in vitro, pp. 115- 159, J. Fogh (ed.), Plenum Press, New York 1975), Colo-320 colon adenocarcinoma cell (ATCC CCL-220, Quinn et al., Cancer Res. 39 (1979), 4914-4924). ), the melanoma cell line MEL-HO (DSM ACC 62, Holz ann et al., Int. J. Cancer 41 (1988), 542-547) and the carcinoma cell line of the kidney A-498 (DSM ACC 55 Giard et al., J. Nati, Cancer Inst. 51 (1973), 1417-1423). Verification of the number of chromosomes in the genome of a cell line can be made by the use of DNA probes which are specific for the particular chromosome and / or the locus or target or target gene site. Yet another preferred feature of a starting cell line used for an activation of the endogenic gene is a correct glycosylation of the desired target protein (vii). A human cell line is preferably used, which synthesizes the target protein with a comparable glycosylation configuration, especially with respect to the number of sialic acid residues such as the target protein naturally present. The test for the presence of a correct glycosylation is preferably carried out by transfection of the cell temporarily with an extrachromosomal vector which contains the desired target gene under the control of a promoter activity in the cell. After the temporary expression of the target gene, the cell supernatant and / or the cell extract is analyzed by isoelectric focusing. With EPO as an example, the presence of correct glycosylation is very evident. Thus, non-glycosylated EPO, e.g., recombinant EPO from E. coli cells, has activity comparable to glycosylated EPO in in vitro experiments. In vivo experiments, however, non-glycosylated EPO is substantially less effective. To determine whether a starting cell line is capable of producing EPO with correct glycosylation, a comparison can be made with urinary EPO, but also with recombinant EPO of CHO cells of which it is known that they have a form of active glycosylation in and its glycosylation is largely identical with urinary EPO. The comparison of glycosylation is preferably carried out by isoelectric focusing. Yet another preferred feature for the selection of a human cell line is the ease with which the studied cell line suffers from infectious contamination (vii), for example, of infectious viral particles or mycoplasmas. The examination for the presence of viral contamination can be carried out by cell culture, in vivo analysis, and / or by detection of specific viral proteins. The invention also relates to a method for the preparation of human proteins by activation of the endogenous gene in a human cell line, which is characterized by the use of a cell line that satisfies the characteristics (i), (ii) and (iii) ) set forth above, as well as additionally at least one of features (iv), (v), (vi) and (vii) as given above. The method of the invention is used especially to prepare human factors, such as EPO, thrombopoietin (TPO), colony stimulation factors such as G-CSF or GM-CSF, proteins that affect blood coagulation such as tPA, interferons such as IFN-a, IFN-β or IFN- ?, interleukins such as IL-1 or IL-18, chemokines such as MIP, neurotrophic factors such as NGF or BDNF, proteins that affect bone growth such as IFG- BPs, hedgehog proteins or intractable persons, tumor growth factors such as TFG-β, growth hormones such as HGH, ACTH, encephaliñas, endorphins, receptors such as, interleukin receptors or insulin, expressed, in forms associated with the membrane and / or soluble and other proteins that bind or bind proteins. Special preference is given to the process for the preparation of EPO. Activation of the endogenous gene by itself is performed by known methods, and preferably involves the following steps: (a) Preparation of human starting cell lines, which contain at least one copy of an endogenic target gene with the nucleic acid sequence desired and which has been identified by examination of the selection criteria of the invention as is suitable for expression of the target gene. (b) Transfection of the cells with a DNA construct comprising: (i) two flanking DNA sequences which are homologous to areas of the target gene locus to allow homologous recombination, - (ii) a marker gene of positive selection, (iii) a marker gene of the negative selection if desired, (iv) an amplification gene if desired, and (v) a heterologous expression control sequence which is active in the human cell. (c) Culturing the transfected cells under conditions in which a selection is made in the presence of the positive selection marker gene and optionally in the absence of the negative marker gene. (d) Analysis of the cells that are selected according to step, (e) Identification of the cells that produce the desired target protein, and (f) amplification of the target gene in the selected cells, if desired. "The DNA construct used for the preparation of cells that produce the desired human protein contains two flanking DNA sequences homologous to areas of the target gene locus.The selection of suitable flanking sequences is made, for example, by methods described in WO90 / 11354 and O91 / 09955. Preferably the bleaching sequences each have a length of at least 150 bp. Especially, preferably the homologous DNA sequences are selected from the 5 'area of the target gene, for example, 5'-non-translated sequences, exons and introns that encode the signal sequence located in this region. The positive selection marker gene can be any suitable selection marker gene for eukaryotic cells, which in expression leads to a selection phenotype, e.g., antibiotic resistance, auxotrophy, etc. A marker gene of positive selection, especially preferred, is the neomycin phosphotransferase gene. Optionally a negative selection marker gene may be present, such as, expressed, the HSV thymidine kinase gene by those expression cells that are destroyed in the presence of a selection agent. The negative selection marker gene is located outside the two regions of flanking homologous sequence. If an amplification of the target gene endogenically activated in the human cell is desired, the DNA construct comprises an amplification gene. Examples of suitable amplification genes are the dihydrofolate reductase gene, the adenosine deaminase gene, and the ornithine decarboxylase gene, etc. An especially preferred amplification gene is the dihydrofolate reductase gene, especially a gene encoding a dehydrofolate arginine reductase mutant, which has a high sensitivity to the selective agent (methotrexate) as the wild-type or uncultivated gene ( Simonsen et al., Proc. Nati, Acad. Sci., USA 80 (1983), 2495). In addition, the DNA construct used for the activation of the endogenous gene contains a heterologous expression control sequence that is active in a human cell. The expression control sequence comprises a promoter and preferably additional expression enhancing sequences, for example, an enhancer. The promoter can be a controllable or constitutive promoter. Preferably the promoter is a strong viral promoter, for example, an SV40 promoter or a CMV promoter. Especially preferred is the CMV promoter / enhancer. The invention further relates to the use of the human cell line identified by the method described above after activation of a target gene present endogenically in the cell to obtain the polypeptide encoded by the activated target gene, preferably to obtain the polypeptide in a large-scale process that involves, for example, a large therminator. Still another object of the invention is a human cell which contains a copy of the endogenous gene in operative binding with a sequence controlling the active heterogenic expression in the human cell and is capable, without prior amplification of the gene, of the production of minus 200 ng of the polypeptide encoded by the endogenic gene / 1O6 cells / 24h. preferably, the human cell according to the invention is capable of producing 200 to 3000 ng of polypeptide / 10 6 cells / 24 h, and more preferably for the production of 1000 to 3000 ng of polypeptide / 10 6 cells / 24 h. Finally, a further object of the present invention is a human cell which is obtainable by the amplification of the gene from the aforementioned cell and contains several copies of an endogenic gene, each operably linked to an expression control sequence heterologous, active in the human cell and capable of the production of at least 1000 ng of the polypeptide encoded by the endogenic gene / 10 6 cells / 24h. Especially, preferably, the human cell line obtainable by amplification of the gene is capable of producing from 1000 to 25000 ng of polypeptide / 10 6 cells / 24h and more preferably for the production of from 5000 to 25,000 ng of polypeptide / 10 6 cells / 24h.

An example of a cell according to the invention is the production clone of EPO, "Aladin", which was deposited on July 15, 1997, in accordance with the provisions of the Budapest Agreement, with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Mascheroder eg Ib, 38124 Brunswick, under the DSM ACC 2320 file number. The invention is further explained by the following examples and figures and sequence listings.

Figure 1 shows a schematic representation of the amplification of homologous regions of the EPO gene from the area of the 5'-non-translated sequences, exon 1 and intron 1, Figure 2: a schematic representation of a plasmid which contains regions of homology from the area "of the 5 'sequences - not translated, exon 1 and intro 1, Figure 3: a schematic representation of an activation sequence of the gene containing the Rous sarcoma virus promoter (RSV), the neomycin phosphotransferase gene (NEO), the early SV40 polyadenylation region (SVI pA), the promoter of initial SV40 (SVI) of the dihydrofolate reductase gene (DHFR) and immediate initial promoter of cytomegalovirus and mej orator (MCMV), Figure 4a: production of the vector pl76 that converts the EPO gene into target, Figure 4b: production of the pl79 and pl87 vectors that target the EPO gene, Figure 4c: the production of the EPO gene blank vector pl89, Figure 4d: the production of the EP90 gene blank vector pl90, Figure 4e: the production of the pl92 vector that forms the target of the EPO gene, SEC ID NO. 1 and nucleotide sequences NO. 2 of the primers used to make the PCR product 1 (Figure 1), SEC. ID NO. 3 and sequences NO. 4 of the primers used to produce the PCR product 2 (Fig. 1).

EXAMPLES Example 1 Selection Methods 1.1 Determination of Generation Time The tested cell lines were seeded at a concentration of 0.1-5 x 105 cells per ml, preferably 0.5-2x105 cells per ml in culture dishes with DMEM and 10% FCS or RPMI 1640 and 10% FCS respectively and cultured in a medium recommended for particular cells, for example, by ATCC and under appropriate conditions, and the cell count was likewise determined from two to three days with a counting chamber, for example, Neubauer, without and with trypsinization. The cells which within a week of culture had from 16 to 256 duplications of population or colony of organisms, preferably 64 to 128 population duplications, were evaluated as positive (+, ++ or +++). 1. 2 Cultivation in Suspension To determine the culture in suspension the cells were cultured for 14 days with constant agitation in medium (see 1.1 with and without the addition of serum, for example, fetal bovine serum) in a Spinner War with accessories corresponding to 37 ° C and 7% of C02. Cells which show at least 5 population doublings during this phase were evaluated as appropriate (+) for a suspension culture. 1. 3 Culture in serum-free medium To determine the cell culture in the serum-free medium, the cells were cultured for 14 days at a density of 1-10 x 10 5 cells / ml in culture vessels in the base medium (ie, without addition of serum) recommended by ATCC for particular cells and with ITS (Boehringer Mannheim), Cat. No. 1074547), under conditions as in 1.1. Cells which during this period show at least 5 population duplicates (determined by cell count) were evaluated as being suitable for the serum free culture. 1. 4 Determination of the Endogenic Expression of the White Gene To determine if the target protein is endogenously produced in the selected cell line, the cells were seeded at a cell density of 0.01 to 2 x 106 cells / ml, preferably 0.5 to 1 x 106 cells / ml of the culture medium, 24 hours. The last 24 hours, the supernatant of the cells was removed, the cells were discarded, and the content of the cellular protein in the cell supernatant was determined by known methods, for example, by a specific immunoassay for the particular protein. In the case of EPO, the content was determined by ELISA. For this purpose the streptavidin-coated microtiter plates were coated with biotinylated anti-EPO antibodies (Boehringer Mannheim) and incubated with a solution containing a protein (1% w / v) to block unspecified bonds. Then, 0.1 ml of the culture supernatant was added and incubated overnight. After washing, the monoclonal anti-EPO antibody conjugated with peroxidase (Boehringer Mannheim) was added for two hours. The reaction with peroxidase was carried out using ABTS® as substrate in a Perkin-Elmer photometer at 405 nm. The limit of detection of EPO in this test was 10 pg EPO / ml. The cells which with a seeding of 106 cells / ml produced less than 10 pg of EPO / ml, were evaluated without production and as was adequate (+). 1. 5 Determination of the Number of Copies of the White Gene To determine the number of copies of the target gene in the cell line, human genomic DNA was isolated from about 108 cells and quantified (Sambrook et al., Molecular Cloning, A Laboratory Manual (1989), Cold Spring Harbor Laboratory Press). After cleaving or splitting the DNA with restriction enzymes, for example Agel and AscI or BamHI, HindIII and SalI, respectively the DNA fragments were separated by agarose gel electrophoresis according to their size and finally they were transferred to a membrane of nylon and immobilized. The immobilized DNA was hybridized with a DNA probe labeled with digoxigenin which was specified for the locus of the target gene or the chromosome in which the target gene is localized, and washed under stringent conditions. Specific hybridization signals were detected by means of a chemiluminescence process using radiation sensitive films. - 1. 6 Determination of the Nucleic Acid Sequence of the White Gene ___ Genomic DNA was isolated from approximately 10 7 cells using a set of DNA isolation (e.g., QIAGEN Blood &Cell Culture DNA Kit). A pair of PCR primers was used for the amplification of the target gene. The sequences of the primers were complementary to the sequences which flank the coding region of the target gene. Thus, the entire coding region of the target gene was able to be amplified. The PCR product was directly subjected to sequence analysis or cloned into a vector and then formed in sequence. Sequence-forming primers were used whose sequences are complementary to sequences from the intron areas of the target gene, so that the sequence of the exon areas of the target gene can be obtained in full. Sequencing was performed in a PE / ABI sequencing machine using, for example, "Prism ™ Ready Reaction Dye Terminator Cycle Sequencing" sets (PE / ABI, Forest, City, USA), according to the information given by the manufacturer . 1. 7 Determination of the Glycosylation Configuration To determine the glycosylation configuration of EPO, the cell lines to be tested were transfected with the plasmid pEP0227 which contains a 4 kb HindIII / EcoRI fragment of the human EPO gene sequence under the control of the SV40 promoter (Jacobs et al. al., Nature 313 (1985), 806; Lee-Huang et al., Gene 128 (1993), 272). Cells were transfected in the presence of lipofectamine using a commercially available reagent set according to instructions of the producer or manufacturer. In the supernatant of cells obtained 2 to 5 days later, the EPO content was determined by ELISA. The cell supernatant was concentrated and compared with known EPO products by isoelectric focusing (Righetti, PG, in: Work, TS, Burdon, RH (ed.) Isoelectric Focusing: Theory, Methodology and Applications, Elsevier Bio edical Press, Amsterdam ( 1983) Human cells that provide a comparable glycosylation configuration for EPO products, such as urinary EPO, were evaluated as suitable (+). 1. 8 Determination of Viral Contamination 1. 8.1 Analysis by Cell Culture For the detection of possible infectious viral contamination of the human cell line to be tested, a lysate of the cells was incubated with a detector cell line to be able to detect cytopathic effects. In addition, hemaadsorption analysis was performed. To prepare the lysate, a suspension of 10d cells in 1 ml of buffer was treated by lysis by a rapid melting and freezing process. The cell debris was separated by centrifugation and the supernatant was provided to the detector cell lines. Detector cell lines used were HepG2 (ATCC HB-8065, Nature 282 (1979), 615-616), MRC-5 (ATCC-1587) and Vero (ATCC-CCL-171).; Jacobs, Nature 227 (1970), 168-170). Polio SV viruses and type of Influenza were used as positive controls. The negative controls were detector cell lines which have been cultured without lysate. To detect cytopathic effects, the detector cell lines were studied regularly in a period of at least 14 days. For the Vero hemadsorption analysis cells which have been incubated with the cell lysates and the controls, they were treated after 7 days with chicken, pig or man erythrocytes. The adhesion of the erythrocytes to the monolayer of the cultured cells indicates the viral contamination of the cultures. 1. 8.2 In vivo analysis The lysates of the cell lines to be studied were prepared as in 1.8.1 and injected into newborn mice intraperitoneally or intercerebrally, in an amount of 0.1 ml. During a period of 14 days the mice were observed for morbidity and mortality. 1. 8.3 Specific Detection of Viral Proteins The presence of specific viral proteins, such as Epstein-Barr virus proteins (nuclear protein or capsid antigen), was tested by providing immobilized cells of the cell line under the EBV positive band-positive human serum. The detection of the virus antigens was then carried out through the administration of complement and the fluorescein conjugate of the corresponding anti-human complement C3 (for the detection of the nuclear antigen) and through the fluorescein of anti-human globulin (for the detection of the antigen). detection of capsid antigen).

Example 2 Selection Results The human cell lines, HepG2, HT1080, Namalwa, HeLa and HeLaS3, were tested by the methods set forth in Point 1. The results are described in the following Table 1.

Table 1 From Table 1 it can be seen that the HT1080, Namalwa and HeLa S3 cell lines are to be considered as suitable for the activation of the endogenous EPO gene. Namalwa and HeLa S3 are especially suitable.

Example 3 Cloning of homologous regions of EPO The homologous regions of the EPO gene were amplified by PCR using a genomic placenta DNA (Boehringer Mannheim). From a 6.3 kb homologous region along the area of the 5'-untranslated sequences of the EPO gene, exon 1 and intron 1, two PCR products were prepared (see, Figure 1). The primers used in the production of the PCR product 1 had the following sequences: 5 '-CGC GGC GGA TCC CAG GGA GCT GGG TTG ACC GG-3' (SEQ ID NO: 1) and 5 '-GGC CGC GAA TTC TCC GCG CCT GGC CGG GGT CCC TCA GC-3 '(SEQ ID NO. ~ 2). The primers used to prepare the PCR product 2 had the following sequences: 5 '-CGC GGC GGA TCC TCT CCT CCC TCC CA CA GCT ATC-3' (SEQ ID NO 3) and 5 '-GGC CGC GAA TTC TAG AAC AGA TAG CGA GGC TGA GAG-3 '(SEQ ID NO 4). The desired segments were obtained from PCR products 1 and 2 by the restriction cleavage (PCR product 1: HindIII, PCR product 2: HindIII and Eco RV) and cloned into the pCRII vector (Invitrogen), which has been unfolded with HindIII and Eco RV. The recombinant vector obtained in this manner was referred to as 5epopcrl000 (see Figure 2).

Example 4 Construction of Target Formers EPO Gene 4. 1 An activation sequence of the gene, which contains the NEO gene, the DHFR gene and a CMV promoter / enhancer (cj Figure 3) was inserted into the Agel site of the 5epocrl000 plasmid containing the homologous EPO region, where plasmid pl76 was obtained (cj Fig. 4a). To bind the CMV promoter as close as possible to the translation initiation site of the EPO gene, a 963 bp long segment was deleted between the Ascl and Agen restriction sites (partial cleavage), where the plasmid pl79 was obtained (Figure 4b). 4.2 To achieve the optimization of expression, the nucleosides in exon 1, which code for the start of the EPO reading sequence Met-Gly-Val-His, were replaced by the synthetic sequence Met-Ser-Ala-His. This sequence was obtained by the amplification of a genomic EPO DNA sequence, for example from the plasmid pEP0148, which contained a 3.5 kb fragment of BstEII / EcoRI (including exons 1-5) of the human EPO gene sequence under control of the SV40 promoter (Jacobs et al., Nature 313 (1985), 806, and Lee-Huang et al., Gene 128 (1993), 227), when calibrated with the corresponding primers. Thus plasmid pl87 was obtained (Fig. 4b). Plasmid pl89 was prepared from plasmid pl87 by insertion of the thymidine kinase gene of the simple herpes virus (HSV-TK), which originates from Psvtk-1 (PvuII / Narl fragment) (Fig. 4c). The HSV-TK gene is under the control of the SV40 promoter at the 3 'end of intron 1 (EcoRV / CIal) in opposite orientation relative to the CMV promoter and should serve for negative selection for homologous recombination. For the construction of plasmid pl90, a Sfil / BglII fragment of pHEAVY, a plasmid which contains the cDNA of an arginine mutant of DHFR described in Simonsen et al. (Proc. Nati, Acad. Sci. USA 80 (1983) 2495), was subcloned into the plasmid p-Genak-1 cut with Sfil and BglII, which contains the NEO gene under the control of the RSV promoter and the latter SV40 polyadenylation site as a terminator, the murine DHFR gene under control of the initial SV40 promoter and the initial SV40 polyadenylation site as a terminator (Kaufmann et al., Mol.Cell Biol. 2 (1982), 1304; Okayama et al., Mol. Cell.

Biol. 3 (1983), 280, and Schimke, J. Biol. Chem. 263 (1988), 5989) and the CMV promoter (Boshart et al., Cell 41 (1995), 521). Then a fragment of Hpal containing the cDNA coding for the arginine mutant of DHFR was ligated to plasmid pl89 cut with Hpal, where plasmid pl90 was obtained (Fig. 4d). 4.5 To obtain a transfection vector without the HSV-TK gene, an Ascl / Nhel fragment of the pl90 plasmid that contained the gene activation sequence was ligated into an Ascl / Nhel fragment of plasmid pl87 containing exon 1. The plasmid The resultant is named pl92 (Fig. 4e).

Example 5 Transfection of Cells Several cell lines were selected for the production of EPO and transfected with target-forming vectors. . 1 Namalwa cells Cells were grown in T150 tissue culture canisters and transfected by electroporation (1 x 10 7 cells / 800 μl electroporation buffer 20 mM Hepes, 138 mM NaCl, 5 mM KCl, 0.7 mM Na2HP04, 6 mM of D-glucose monohydrate pH 7.0, 10 μg linearized DNA, 960 μF, 260 V BioRad Gene Pulser). After electroporation, the cells were cultured in RPMI 1640, 10% (v / v) fetal bovine serum (for its acronym in English, FCS), 2 mM L-glutamine, 1 mM sodium pyruvate in forty plates of 96 cavities. After two days the cells were cultured for 10 to 20 days in 1 mg / ml medium containing G-418. The supernatant was tested in a solid phase ELISA for the production of EPO (see Example 1.4). The clones that produce EPO expanded into 24-well plates and T-25 tissue culture bottles. The aliquots were frozen and the cells were subcloned by FACS (Ventage, Becton Dickinson). The subclones were repeatedly tested for EPO production. . 2 HT 1080 cells Conditions were as described for Namalwa cells, except that HT 1080 cells were cultured in DMEM, 10% (v / v) FCS, 2 mM L-glutamine, 1 mM sodium pyruvate. For transfection by electroporation, the cells were released from the walls of the culture vessels by trypsinization. After electroporation, 1 x 107 cells were cultured in DMEM, 10% (v / v) FCS, 2 mM L-glutamine, 1 mM sodium pyruvate in five 96-well plates. . 3 HeLa S3 cells Conditions were as described for Namalwa cells, except that HeLaS3 cells were cultured in RPMI 1640, 10% (v / v) FCS, 2 mM L-gluta ina, 1% (v / v) non-essential amino acids of NEM (Sigma), and 1 mM of sodium pyruvate. For transfection by electroporation the cells were released from the walls of the culture vessels by trypsinization. The conditions for electroporation were 960 μF / 250 V. After electroporation the cells were cultured in RPMI1640, 10% (v / v) FCS, 2 mM L-glutamine, 1% (v / v) NEM , 1 mM sodium pyruvate, in tissue culture flasks of T75. 24 hours after electroporation, the cells were trypsinized and cultured for 10 to 15 days in a medium containing 600 μg / ml of G-418 in ten 96-well plates.

Example 6 Amplification of the EPO Gene To increase EPO expression, the EPO producing clones were cultured in the presence of increasing concentrations (100 pM - 1000 nM) of methotrexate (MTX). At each MTX concentration the clones were tested by ELISA (see Example 1.4) for the production of EPO. Strong producers were subcloned by limited dilution.

Example 7 Characterization of EPO Production Line Cell Lines -_ Three different cell lines (Namalwa, HeLa S3 and HT 1080) were chosen for the activation of the EPO gene.

The EPO-producing clones were obtained by transfection with plasmids pl79, 0187, pl89, pl90 or pl92 (cj, Examples 2 and 3). Approximately 160,000 clones resistant to NEO were tested for the production of EPO, of which 12 to 15 EPO secreted repeatedly in significant field in the cell supernatant. Of these a total of 7 EPO clones could be surprisingly identified without amplification of the gene by MTX, and EPO was produced in sufficient quantities for the large industrial production. The EPO production of these clones varies from 200 ng / ml to more than 1000 ng / ml / 106 cells / 24h. After amplification of the 500 nM MTX gene, the EPO production of the identified EPO clones was increased to more than 3000 ng / ml / 10 6 cells / 24 h. A further increase in the MTX concentration to 1000 nM results in a production of up to more than 7000 ng / ml / 106 cells / 24h. The obtained clones shown in EPO production also under serum free culture conditions and in the absence of MTX.

SEQUENCE LIST (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Boehringer Mannheim GmbH (B) ADDRESS: Sandhofer Str. 112-132 (C) STATUS: Mannheim (E) COUNTRY: Germany (F) POSTAL GUIDE NUMBER: 68305 (ii) TITLE OF THE INVENTION: identification of human cell lines for the production of human proteins by the activation of the endogenic gene. (iii) NUMBER OF SEQUENCES: 4 ' (iv) COMPUTER LEGIBLE FORM: "- (A) TYPE OF MEDIUM: Diskette (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patent In Relay # 1.0, Version # 1.30 (EPA) (2) INFORMATION FOR SEQ ID NO. 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 32 base pairs (B) TYPE: nucleotide (C) TYPE OF HEBRA: single strand (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 1: CGCGGCGGAT CCCAGGGAGC TGGGTTGACC GG 32 (2) INFORMATION FOR SEQ ID NO. 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 38 base pairs (B) TYPE: nucleotide (C) TYPE OF HEBRA: single strand (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 2 GGCCGCGAAT TCTCCGCGCC TGGCCGGGGT CCCTCAGC 38 (2) INFORMATION FOR SEQ ID NO. 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleotide (C) TYPE OF HEBRA: single strand (D) TOPOLOGY: Linear [xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 3: CGCGGCGGAT CCTCTCCTCC CTCCCAAGCT GCAATC 36 (2) INFORMATION FOR SEQ ID NO. 4 (i) CHARACTERISTICS OF THE SEQUENCE: (C) LENGTH: 36 base pairs (D) TYPE: nucleotide (C) TYPE OF HEBRA: single strand (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 4 GGCCGCGAAT TCTAGAACAG ATAGCCAGGC TGAGAG 32 It is noted that in relation to this date the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property

Claims (14)

1. A method for the selection of human cell lines for the preparation of human proteins by endogenic activation of a target or target gene endogenously present in the cell, characterized in that (a) a human cell line is tested for the presence of the following characteristics: (i) a target gene with the desired nucleic acid sequence, (ii) at least 5 duplications of the population or colony of organisms within 14 days in a culture in suspension, and (iii) at least 5 doublings of the population or colony of organisms within 14 days in a serum-free culture medium, and (b) a cell line that satisfies characteristics (i), (ii) and (iii) is used as a starting cell line for endogenic activation of target or target gene.

2. A method according to claim 1, characterized in that a human cell line is used, which (iv) has a generation time of 16 to 256 population doubles within a week in a culture medium.

3. The method according to claim 2, characterized in that a human cell line is used which has 64 to 128 population doublings within a week in a culture medium.

4'. The method according to any of the preceding claims, characterized in that a human cell line is used which (v) shows non-substantial endogenous expression of target or target gene.

5. The method according to any of the preceding claims, characterized in that a human cell line is used which (vi) contains more than 2 chromosome copies of the target or target gene.

6. The method according to any of the preceding claims, characterized in that a human cell line is used which (vii) synthesizes the cellular protein with a glycosylation configuration comparable to the target protein that is naturally present.

7. The method according to any of the preceding claims, characterized in that a human cell line is used which (viii) is free of detectable infectious contamination.

8. The method for the preparation of human proteins by activation of the endogenous gene in a human cell line, characterized in that a human cell line is used which has been selected by the method according to any of claims 1 to 7, and which fulfills characteristics (i), (ii) and (iii) as defined in claim 1.

9. The method according to claim 8, characterized in that a cell line is used which also fulfills at least one of the characteristics (iv), (v), (vi), (vii) and (viii) as defined in a of claims 2, 4, 5, 6 and 7.

10. The method according to claim 8 or 9, characterized in that it is for the preparation of a human factor selected from EPO, TPO, colony stimulation factors, proteins which affect the blood coagulation, interferons, interleukins, chemokines, factors neurotrophs, proteins which affect the growth of bones, proteins for intractable people, growth factors of tumors, growth hormones, ACTH, encephaliñas f endorphins, receptors and other protein binding or protein binding.

11. The method according to claim 10, characterized in that it is for the preparation of EPO.

12. The use of human cell lines identified according to any of claims 1 to 7 after activation of a target gene endogenously present in the cell to obtain the polypeptide encoded by the active target gene.

13. The use in accordance with the claim 12 to obtain the polypeptide in a large thermenter.

14. The human cell line, characterized in that it contains a copy of an endogenous gene in operative connection with an active heterologous promoter in the human cell and is capable of producing at least 200 ng of the polypeptide / 10 6 cells / 24 h encoded by the endogenous gene , which can be obtained by the selection method according to any of claims 1 to 7. •