Title of Invention	METHOD OF PROTEIN PRODUCTION USING HIGH DENSITY CELL CULTURE
Abstract	The present invention relates to the field of protein expression in biotechnology. It more particularly relates to the use of aminoglycoside resistance gene product for achieving high-density growth of animal cells.

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The present invention relates to the field of protein expression in biotechnology. It specifically relates to the use of aminoglycoside resistance genes, in jpail^raar neomycin resistance £ene, for achieving high-density growth of animal cells, ajrespecjive method of protein production and a high-density cell culture. '*. % # rhe biotechnological production of therapeutic proteins by means ofj animal cell culture is a very laborious and costly endeveaour. The efficiency of productioii inducing downstream processing is mainly governed by the space-time yield cif the ii itial cell culture step. Both higji yields and concentration of product protein in| the cu ture broth are desired. In consequence, comparatively small increases in the maxintam eel density achieved before entering the still productive stationary phase of industrial s Strongtyjdepending on cell type and on the method of cultivation, conventional fed-batch culture systems optimized for high-density growth such as airlift reactors could not achieve growing serum-free cell cultures to a density of up to or even in excess of 1(7 cells/ml. Higher densities are potentially achievable in serum-supplemented feid-batcl culture or in the more modem perfusion reactor systems. However, both options entail se ious disadvantages. Fetal bovine serum-supplemented culture media which encoi ipass a whole range of natural growth-promoting substances are strongly dependent on the source of serum in quality and, most crucial, carry the permanent risk of unintentional y introducing animal viruses into cultures producing therapeutic proteins for medical applications. Hence for regulatory reasons, serum supplementation is to be avoided. Perfusion reactor systems however, are much more complex and demjaaiding to control during operation and are much more costly in operation than conventional fe i-batch culture systems. This due to the permanent infusion of fresh culture medium requiring state-of-the art microfiltration systems for parallel release of medium jfrom tl e reactor. In particular jamming of the filtration unit with cellular debris or protein! entails risk of premature shut-down of operation. In comparison, fed-batch culture has significant advantages in process economics and robustness. Further, the creation of stable recombinant cell lines producing a protein of interest requires the introduction of at least one usually constitutively expressed resistance marker gene. Expression of such marker gene constitutes an added metabolic load to the cell that may at best not impact growth behaviour or may quite adversely affect the growth rate and maximum viable cell density even in richly serum-supplemented cell culture medium (Gaigle et al., 1999, Aminoglycoside antibiotic phosphotransferases are also serine protein kinases, Chemistry and Biology 6,11-18 ; Maio et al, 1991, Gene activation mediated by protein kinase C in human macrophage and teratocarcinoma cells expressing aminoglycoside phosphotransferase activity, J. Cell. Physiology 149,548-559; Southern et al., 1982, Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter, J. Mol. Appl. Genet. 1,327-341). The present invention relates to a method of protein expression, comprising the step of growing an animal cell line to a viable cell density of at least or in excess of 10'cells/ml m a serum-free high-density growth culture medium in a fed-batch bioreactor which cell line is transfected with an expressable aminoglycoside resistance gene and is transfected with an expressable glutamine synthetase, and which cell line is further capable of expressing a product protein and further comprising the step of isolating the product protein from the culture broth. « It is the object of the present invention to avoid the disadvantages of the prior art and to provide a method for growing animal cells to high cell density in a serum-free culture system. This object is solved by expressing aminoglycoside resistance genes in animal cells, whereafter the cells are cultured in suitable media and in a suitable culture system according to the independent claims 1,2, 8. Surprisingly, depending on the culturing method and the culture medium as is customary in the art, cell lines treated according to the present invention can grow in serum-free cell culture medium to much higher cell densities than their non-treated parent cell line. In addition, some prolongation of stationary phase growth was observed. In this way, maximum viable cell densities are achievable in serum-free culture that could not have been realized prior to the invention. A possible embodiment of the invention is shown in the figure. What is shown is Fig. 1: Viable cell densities during growth of Neo-transformed NSO cell line in a 101 fed-batch airlift reactor system in comparison with a parent and a bcl-2 transformed cell line. Fig. 2: Productivity for secreted antibody expressed as cumulative cell time during growth of Neo-transformed NSO cell line in a 101 fed-batch airlift reactor system in comparison with a bcl-2 transfected cell line. Fig. 3: Plasmid map pEF-bcl-2 Fig.4: Plasmid map pEF-Neo Fig.5: Comparative growth experiment with NSO cell line adapted to non-lettjal doses of G418. Fig.6: Shake flask batch culture and productivity of Neo-transfoimed NSO cell line in comparison with a parent and a bcl-2 transformed cell line. According to the present invention, an aminoglycoside resistance gene pi.wu.tubt ID U.O^U. IUI achieving high-density growth of animal cells. The use according to the present invention comprises expressing the resistance gene product in the cells, selecting cells expressing such resistance gene product with an aminoglycoside antibiotic, preferably with the antibiotic Neomycin, which aminoglycoside is degraded by said corresponding resistance gene product, and finally cultivating such cells in a bioreactor, e.g. an airlift ot stirred bioreactor, in a suitable serum-free cell culture medium allowing for such higk-density growth, A resistance gene product according to the present invention is any aminoglycoside resistance marker such as known resistance genes to Gentamycin, Neomycin,Hygromycin and in particular to Neomycin, that can be used as a genetic marker for eukarjotic cells. Aminoglycoside resistance genes are commonly employed in the molecular biology of eukaryotic cells and are described in many standard textbooks and lab manuals (for description, cp. e.g. Shaw et aL, 1993, Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes, Microbiol. Rev. 57:138-163; WO 82/03087; Southern et aL, 1982, Transformation of matamalian cells to antibiotic resistance with a bacterial gene under control of the SV40 e irly region promoter, LMol. Appl. Genet 1,327-341). As can be inferred from the afore said, the aminoglycoside resistance gene product is said to be a functional gene product in view of its aminoglycoside-degrading activity according to the invention. It is therefore ^ . conceivable to employ genetically engineered, in the above sense functional \ ariants of known aminoglycoside resistance gene products in the present invention- Sue x variants can be generated e.g. by substitutions, deletions, insertions or truncations of the amino acid and its encoding DNA sequence, respectively. Methods for such are well known p the art and .1 -™w | usually comprise specific site directed mutagenesis or generation of diversity by random mutagensis of which is then followed by selecting desired variants by means of functional assays. Routine methods employed for mutagenesis maybe e.g. exposure to alkylating agents or UV irradiation, error-prone PCR or related gene shuffling PCR tec hniques and are usually performed in microorganisms ( Miller, J., Experiments in Molec liar Genetics, Cold Spring Harbor Laboratory 1972; Ling et aL, 1997, Approaches to DNA Mutagenesis, Analytical biochemistry 254,157-178; Cadwell et aL, 1992, Randomizatiomof genes by PCR mutagenesis in:PCR Methods, Cold Spring Harbor Laboratory Press 1192; Moore et aL, 1997, Strategies for the in vitro evolution of protein function, J. Mol. Bi Expediently, the resistance gene product is expressed from a DNA expression, construct suited for eukaryotic expression that has been transfected into the cells by kiown techniques; the resistance gene product may be constitutively expressed or can be inducibly expressed or repressed, ie. be expressable either way. At least during the periods of selection with an aminoglycoside antibiotic and during logarithmic upgrowth in a high density cell culture system for achieving maximum cell density, the resistance gene product should be expressed according to the present invention. Preferably, it is constitutively expressed e,g. from Thymidine Kinase (TK1) or Siamian Virus j(SV40) Late promoter, most preferably it is expressed from a strong viral enhancer-promoter constitutively active in eukaryotic cells such as e.g. Rous Sarcoma Virus(RSV)-Long Terminal Repeat (LTR)-promoter or Cytomegalovirus (CMV>promoter. It may also be possible to employ chimeric promoters constituted of the enhancer portion of a strong viral promoter and core promoter portion of another promoter, e.g. alpha-actin prcpnoter providing essential e.g. transcription start, TATA boxes and CAAT boxes. An aminoglycoside according to the present invention are the commonly known aminoglycoside antibiotics (Mingeot-Leclercq, M. et aL, Aminoglycosides: apitivity and resistance, 1999, Antimicrob..Agents Chemother. 43(4): 727-737) comprising at least one amino-pyranose or amino-furanose moiety linked via a glycosidic bond to thfe other half of the molecule. Their antibiotic effect is based on inhibition of protein synthesis. Examples are Kanamycin, Streptomycin, Gentamicin, Tobramycin, G418 (Geneticin), Neomycin B (Framycetin), Sisomicin, Amikacin, Isepamicin and the like. J In the context of the present invention, other compounds having antibiotic activity due to inhibition of protein synthesis in bacteria such as e.g. Spectinomycin, Anisoaaycin and in particular Puromycin, and which have chemical structures related to non-red icing amino-sugar moieties are considered being 'aminoglycoside antibiotics'according t According to the present invention, for selecting cells expressing the resistan ie gene product, resistance selection with an aminoglycoside antibiotic is applied at 1 ast for intially selecting transfectants after transfection of the resistance gene which tomrnonly is in the order of 48 hours up to several weeks. In consequence, the aminoglycoside resistance gene is stably transfected into the cell line. Stable transfectants commonly are genomic integrants of at least an expressed or expressable copy of the aminoglycoside resistance gene, giving rise to functional gene product More recent approaches in genetic engineering which might devise e.g. the creation of artifical, stable minichron osomes in a cells are likewise included in the present notion of 'stable transfectant\ It goe s without saying that, according to the well-known transfection protocols such as e.g. li] ►ofection, DEAE-Dextran, Ca-phosphate or electroporation all of which are possible transfection methods according to the present invention, freshly transfected cells are first c ultured in non-aminoglycoside supplemented medium before such supplement is added: or selection* This intervening period of non-selection is required for efficient expression oi the resistance marker gene product and is in the range of approximately 12 hours TO to 1-2 days, depending on cell type. Preferably, resistance selection is applied for at least 2 weeks post-transfection, more preferably for at least 5 weeks post-transfectioa most preferably for at least 8 weeks post-transfection. It is also possible to extend toe period of growth under selection pressure exerted by aminoglycoside antibiotic that has been added to the medium to further cultivation of the cells, e.g. during fermentation in the bioreactor. It is also possible to apply, after the initial selection of transfectants, selection Pressure in short interspersed intervals during further cultivation, by repeatedly adding sirajle doses of aminoglycoside with the culture medium which is then replenished by non- I aminoglycoside-supplemented medium. Preferably, during cultivation in a biareactor, the expressable or expressed resistance gene according to the present invention is stably integrated into the genome and cultivation is performed in the absence of sele Preferably, the aminoglycoside is employed in a concentration of at least 0,1 nlg/ml, preferably in a concentration of at least 1 mg/ml, most preferably in a concentration of at least 4 mg/ml. Usually, such amount of aminoglycoside according to the invention is added to the cell culture medium after transfection with the expression construct for t le corresponding resistance gene product, as is well-known in the art and is welln Lescribed in the standard lab manuals. In a further particularly preferred embodiment, amin >glycoside is employed in a concentration of 1 to 4 mg/ml for at least 2 weeks, more prefe ably for at least 5 weeks, most preferably for at least 8 weeks post-transfection during cocpnous cell cultivation. Preferably, the resistance gene product according to the present invention is a I eomycin-Phosphotransferase (the resistance gene usually being named Neo*) as describe I in WO82/03087. Various natural isofonns of such phosphotransferase enzymes axe known and are also comprised with the scope of the present invention. Selection with ©418 (Geneticine, as defined under Chemical abstracts Registry Number 49863-47-01 or Neomycin can be used to select for cells expressing the neomycin gene product! In a more preferred embodiment, G418 is used for selection of resistant cells. The animal cells or cell line according to the present invention may be any conventional cell line used in production of recombinant protein, such as e.g* Sf9 insect cells CHO . cells, Hela cells, COS-7 cells, VERO-96 cells, HepG2 cells, BHK cells, fibroblasts, hybridoma, EBV immortalized lymphoblasts or 'myeloma' cells such as e.g. thi NSO cell line. Myeloma cells such as NSO cells truly are B-lymphoid cell types althougjhjbeing routinely adressed in the art as 'myelomas' (Barnes et al., Cytotechnology 32:1*9-123, Preferably, the animal cells or cell line according to the present invention areianchorage-independent cells. Such cells do not rely on substrate contact for proper grow h and can grow being freely suspended in the culture medium. In a more preferred emb >diment, the producer cell line are lymphoid cell lines, e.g. hybridoma cells, EBV immorfc lized lymphoblasts or myeloma cells, most preferably the cell line are myeloma ce Is and in particular myeloma NSO cells such as e.g. cell line ECACC No. 85110503 ar I derivatives thereof, freely available from the European Collection of Cell Cultures (EGA X), Centre for Applied Microbiology & Research, Salisbury, Wiltshire SP4 OJG, United kingdom. NSO have been found to give potentially rise to extremely high product yields, in particular if used for production of recombinant antibodies. Most standard NSO cell line; are cholesterol-dependent, making cholesterol an obligate component of the cultijp medium. In a further preferred embodiment, the cell line according to the present invention that carries an aminoglycoside resistance gene is a cell line which is further capab e to express recombinant glutamine synthetase (GS), more preferably it is a NSO myelonu recombinant GS cell line. NSO cells are specifically of advantage if used with the Glutamine synthetase (GS) expression system (JBebbington et al, 1992, High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplififtble selctable marker, Bio/Technology 10:169-175; Cockett et al., 1990, High level expressi m of tissue inhibitor of metalloproteinases in Chinese Hamster Ovary (CHO) cells using recombinant antibodies using the glutamine synthetase (GS) system, Cytotechnilogy 9:231-236). NSO myeloma cells are phenotypically deficient in Glutamine-syntletase. Therefore the NSO cell line which was derived from a mouse tumour cell line (Clalfre, G. and Milstein,, C., Methods in EnzymoL 73,3-75,1981) is frequently the cell li* of choic In a further preferred embodiment, the cells according to the present invention employed for high-density cell culture are devoid of recombinantly expressed bcl-2 proteim bcl-xl protein or another functional, functional to be understood as apoptosis-preventink natural or genetically engineered variant of the apoptosis inhibiting-bcl-2 family (Pctros et aL, 2001, Solution structure of the antiapoptotic protein bcl-2, Proc. Natl. Acad. Scire mentioned preferred embodiment, namely the absence of selection pressure as ui derstooc as presence of aminoglycoside antibiotic during fermentation. Surprisingly, co-ekpressio of an aminoglycoside resistance marker and bcl-2 protein has been found to be refractory to the cell density promoting effect of aminoglycoside resistance according to thd present invention. Without being bound by theory, the growth promoting effect of the present invention therefore does not seem to be attributable to an anti-apoptotic effect or pcl-2 based effect, since co-expression e.g. bcl-2 and Neor should in that case be epistatic rathe than being refractory to high-density growth as has actually been observed in comoarative experiments. Suitable media and culture methods for mammalian cell lines are weU-laiowJa in the art, as described in US 5633162 for instance. Examples of standard cell culture me iia for laboratory flask or low density cell culture and being adapted to the needs o particular cell types are for instance: Roswell Park Memorial Institute (RPMI) 1640 mediu n (Moire, G., The Journal of the American Medical Association, 199, p.519 f. 1967), L-15 medium (Leibovitz, A. et al., Amer. J, of Hygiene, 78, lp.173 fi; 1963), Dulbecco's i lodified Eagle's medium (DMEM), Eagle's minimal essential medium (MEM), Ham's F12 medium (Ham, R. et al., Proc. Natl. Acad. Sc.53, p288 ff. 1965) or Iscoves' modified DMEM lacking albumin, transferrin and lecithin (Iscoves et al., J. Exp. medl 1, p. 923 ff., 1978). It is known that such culture media can be supplemented with fetal b During transfection and selection with aminoglycosides, any medium suited for sustaining growth of the cultured animal cells can be employed. For high-density grown of the animal cells in a fed-batch bioreactor according to the present invention, a high-density growth culture medium has to be employed According to the present invention, a cell culture medium will be a high-denlity growth culture medium by definition if the culture medium allows for growth of animal cells up to or in excess of a density of viable cells of 106 cells/ml in a conventional fed-jbatch bioreactor system. In the context of the present invention, such culture medium gives rise to even higher cell densites in combinantion with the afore described aminoglycoside selection. Usually, such a medium according to the present invention will co] aprise 1-10 g/1 Glucose or another source of energy, the concentration of glucose being c mtroled at this level during fed-batch cultivation. Preferably, the medium will comprise at least 2 g/1 Glucose, this concentration essentially being controled dining fed-batch fenr entation. The medium is isotonic, namely being in the range of 270-320 mOsm/kg, preferably at 280-300 mOsm/kg. Individual preferences of certain cell types, e.g. lymphoid cells, fcr certain media are well-known in the art, and are complexly correlated with the range! proportion and individual dosing of nutrients. Examples of a high-density growth media djuited e.g. for hybridoma cell lines as compared to the standard media mentioned above are even in GB2251 249 A; such high-density growth media can be usually supplemented with nutrients such as all amino acids, energy sources such as glucose in the range given above, inorganic salts, vitamins, trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), buffers, the four nucleosides br their corresponding nucleotides, antioxidants such as Glutathione (reduced), Vitamme C and other components such as important membrane lipids, e.g. cholesterol or I phosphatidylcholine or lipid precursors, e.g. choline or inositol. A high-density medium will be enriched in most or all of these compounds, and will, except for the inorganic salts based on which the osmolality of the essentially isotonic medium is regulated, [comprise them in higher amounts (fortified) than the afore mentioned standard media as pan be incurred from GB2251 249 in comparison with RPMI1640. Preferably, a toga-density culture medium according to the present invention is balancedly fortified in (hit all amino acids except for Tryptophane are in excess of 75 mg/1 culture medium. Preferably, in conjunction with the general amino acid requirement, Glutamine and/or Asparagine are jointly in excess of 1 g/1, more preferably of 2 g/1 of higji-density culture mediim. It goes without saying that the latter preferred embodiment is less suitable in case of a recombinant cell line transfected with a Glutamine synthetase (GS) vector. In spch a ceU line, an excess of e.g. glutamine stemming both from exogenous and endogenous source would lead to production of ammonia which is to be avoided. Culture conditions for GS transfected cell lines are described in the examples. Expediently, the high-density cell culture medium according to the present invention is devoid of fetal calf serum (FCS or FBS), which is being termed *serum-free\ l| has not only been devised possible, according to the present invention, to achieve high-ldensity growth at a viable cell density of or in excess of 107cells/ml with serum-free medium, but it has surprisingly been found that supplementation with fetal serum is refractofy to high-density growth of at least some cell lines tested in the context of the present imiention, contrary to the usual expectations of the person skilled in the art. Cells in serum-free medium generally require insulin and transferrin in a serum-free medium for ontimal growth. Transferrin may at least partially be substituted by non-peptide siderophores such as tropolone as described in WO 94/02592. Most cell lines require one or more lof synthetic * growth factors (comprising recombinant polypeptides), including e.g. epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin like growth fectors I arid II (IGFI, IGFII), etc.. Other classes of factors which maybe necessary include: prostaglandins, transport and binding proteins (e.g. ceruloplasmin, high and low density lipoproteins, bovine serum albumin (BSA)), hormones, including steroid-hormones, and mtty acids. Polypeptide factor testing is best done in a stepwise ashion testing new polypeptide factors in the presence of those found to be growth stimulatory. There a several methodological approaches well-known in animal cell culture, an exemplary being described in the following. The initial step is to obtain conditions where the cells will survive and/or grow slowly for 3-6 days after transfer from serum-supplemented culture medium! In most cell types, this is at least in part a function of inoculum density. Once the optimal hormone/growth factor/polypeptide supplement is found, the inoculum density required for survival will decrease. In a more preferred embodiment, the cell culture medium is free of growth factors, meaning that it is free of fetal serum or of addition of essentially pure protein growth factors that are triggering signal transduction and cell cycle progression, respectively. Such medium may still comprise other proteins such as insulin or transferrin, useful for recruiting iron from the medium,or BSA required for delivery of lipids such Is cholesterol. More preferably, such medium is employed according to the present invention in conjunction with lymphoid cell lines, most preferably in conjunction with myeloma cell lines, in particular NSO cell lines. Suitable bioreactors according to the present invention are batch bioreactors is e.g. airlilt bioreactors or stirred bioreactors as routinely employed for high-density animal cell culture. Expediently, for high-density cell culture such bioreactor will be operated in a fed-batch mode. This definition includes continous feed operation as well. Preferably, fed-batch bioreactors according to the present invention have a volumetric oxygen mass transfer coeJEficient Kt,a (as defined in Bailey, J. et al., Biochemical Engineering Fundamentals, McGraw-Hill, N.Y. 1986) of at least 6 h"1, more preferably ofkt least 10 h"1. Most preferably, a fed-batch bioreactor having said preferred oxygen mass transfer properties according to the present invention is an airlift bioreactor. Airlift bioreactors are well-known to the skilled person and the crucial parameters for reactor desiga have been I t well described (for review, see e.g. Chisti, M. et al-, 1987, Airlift reactors, CheJn. Eng. Commun. 60,195-242; Koch, A, et aL, 1987, Measurement and modeling of mass transport in airlift-loop reactors in relation to the reactor design, Chem. Ing. Tefeh. 59,964-965). Though being self-evident, it is emphasized that fed-batch culture according to the present invention does not comprise perfusion culture systems. In the context of the present invention, high-density cell culture is defined as a Population of animal cells having a density of viable cells of at least or in excess of 106 cels/ml, preferably of at least or in excess of 107 cells/ml, more preferably in excess of 1.2 3 xlO7 cells/ml, most preferably in excess of 1.3 xlO7 cells/ml, and which population las been continously grown from a single cell or inoculum of lower viable cell density ii a cell culture medium in a constant or increasing culture volume. In a further preferred embodiment, the fed-batch culture is a culture system wherein at least Glutamin, optionally with one or several other amino acids, preferably gljicine, is fed to the cell culture as described in GB2251 249 for maintaing their concentration in the medium, apart from controlling Glucose concentration by spearate feed. More preferably, the feed of Glutamin and optionally one or several other amino acids is combined with feeding one or more energy sources such as glucose to the cell culture as described in EP-229 809-A. Feed is usually initiated at 25-60 hours after start of the culture; for instance, it is useful to start feed when cells have reached a density of about 106 cells/mL 1 he total glutamine and/or asparagine feed (for substitution of glutamine by asparagine, see Kurano, N. et al., 1990, J. Biotechnology 15,113-128) is usually in the range from 0.5 to 3 g per 1, preferably from 1 to 2 g per 1 culture volume; other amino acids that can be present in the feed are from 10 to 300 mg total feed per litre of culture, in particular glycine, lysine, arginine, valine, isoieucine and leucine are usually fed at higher amounts of at least 150 to 200 mg as compared to the other amino acids. The feed can be added as shot-addition or as contionusly pumped feed, preferably the feed is almost continously pumped intp the bioreactor. It goes without saying that the pH is carefully controlled during fedlbatch cultivation in a bioreactor at an approximately physiological pH optimal for a given cell line by addition of base or buffer. When glucose is used as an energy source th| total glucose feed is usually from 1 to 10, preferably from 3 to 6 grams per litre of tt|e culture. Apart from inclusion of amino acids, the feed preferably comprises a low amoijpt of choline in the range of 5 to 20 mg per litre of culture. Preferably, the animal cell or cell line according to the present invention is a producer cell line which produces a second product protein or is capable of expressing a seco id product protein, e.g. upon induction. According to the present invention, the second pro iuct protein is the protein that is sought to be produced and harvested in high amount It ma;' be any protein of interest, e.g. therapeutic proteins such as interleukins or enzymes, e\g| enzyme inhibitors or antibodies or fragments thereof (a fab fragment for instance). It cam be a recombinant protein, carried on a plasmid or another type of vector including ginetically engineered viruses, or it can be stably integrated in the genome of the cell by any technique. It can also be a naturally occuring expression construct such as an antibody secreted by plasma or hybridoma cells. The product protein may include a signal sequence allowing secretion of the polypeptide from the host producer cell. It may be corjfstitutively expressed or may be inducibly expressed. Preferably, the product protein is a recombinant protein, most preferably a recombinant protein expressed from a constitutive promoter. 'Recombinant' according to tti4 present 1 invention means a protein expressed from at least one exogenous copy of the f corresponding gene in a cell line that has originally been introduced into said cell line by any technique of genetic engineering, irrespective of whether the very protein i| occuring in the producer cell line in at least one naturally present copy. Such additional ciopies of a recombinant gene can e.g. be integrated in the genome or can be carried on an In a further preferred embodiment of the present invention, the product protein Is a i secreted protein. More preferably, the product protein is an antibody or engineered antibody or a fragment thereof most preferably it is an Immunoglobulin G (Ig(p) antibody. i 'b A respective method of protein expression and further a high density cell culture corresponding with what has been said in the preceding sections on use of aminoglycoside resistance gene product are further objects of the present invention. The description of possible and preferred embodiments in the foregoing apply likewise to these objects. w X I Examples 1. Generation of cell lines and plasmids I The NSO 6A1 (100)3 cell line (6A1 for short; Lonza Biologies pic, Slough/Ul.) secretes a a human-mouse chimeric IgG antibody (cB72.3) from a stably integrated recon binant glutamine synthetase (GS) expression construct. Recombinant cell lines carrying Neomycin resistance were obtained by transfection of the 6A1 cell line with pliismid vectors either dicistronically expressing bcl-2 and Neor or solely expressing Nep from a single plasmid construct. For generation of pEF- Neor, the MCI Neo poly A cassette from pMClneopA (Stratagene) was inserted into pEF BOS (Mizushima et al., 1990, bEF BOS, A powerful mammalian expression vector, Nucleic Acids Research 18,5322) which has the staffer, to make pEF MCI neo poly A (Visvader et el., Mol. Cell Biol. 199^ Feb;15(2):634-41). Fig. 4 shows the plasmid map. For generation of pEFbcl-2-Neor, the EcoRI/Taq I fragment of human bcl-2 cDNA (long anti-apoptotic transcript veasion, Cleary et al., Cell 47:19-28(1986)) was subcloned into pIC194 (Gene32:482f 1984) opened with EcoRJ/Clal and excised as Sail (blunt)/EcoRV fragment that was further cloned into the blunted Xbal site of the multiple cloning site (MCS) of pEF-MClneopA. bcl-2fis thus expressed from the Elongation Factor (EF) 1-a promoter (plasmid map, Fig* 3)J The Neor- expression cassette (MClneopA) is an integral part of the pEF-Vector, though in the pEF- Neo vector construct finally employed, the MCS beyond the EFl-a promoter wjas opened at the Xho I site, blunted and a random staffer 300 bp fragment from a non-coding sequence was inserted for proper control (plasmid map, Fig. 4). Lipofectin (Gittco, Paisely, Scotland)-mediated transfection was performed according to the manufacturers! protocol. Plasmid transfectants were selected with 1 mg/ml G418 in GMEM medium (Gfbco) supplemented with 5% fetal calf serum and further supplements as described id Tey et al. (Tey, B. et al., Bcl-2 mediated suppression of apoptosis in myeloma NSO cultures, J. Biotechnology, 79(2000), 147-159). Stable transfectants were then adapted to |erum-free if high-density growth medium EX-CELL 302. t 2. High density fed-batch cell culture For high density cell culture, the NSO 6A1 parent cell line, 6A1 Neo-cell line aid the 6A1 bcl-2/Neo control cell line were adapted to serum-free medium EX-CELL 302 IjRH Biosciences Inc., KS/U.S.A.) supplemented with lml/50 ml GSEM (GS expreslion medium supplement, product number G9785, Sigma, Poole, UK). Whilst passaging, 25 \iM MSX and 0.7 g/1 G418 were supplemented to the medium but were omitted from inoculum and bioreactor culture medium- Density of viable cells during bioreador cell culture is shown in Fig. 1 for culture of individual cell lines. Fed batch culture far each cell line was set up as follows: | The myeloma cell line was grown in a 101 airlift fermenter in serum-free EX-CELL 302 medium. The starting cell population density was approximately 105 cells/ml frdm fresh lij mid-exponential culture. After continous growth to a density of about 106 cells/ml, shot additions of supplements were made and the pumped supplement iniated (Tablell). Feed 1 was continous over 100 h. pH was controlled at about pH 7. Culture density wd determined approx. every 25 hours by counting and cell viability was determinep by Trypan blue exclusion. For total and viable cell concentration, an appropriately liluted sample was diluted with Trypan Blue (Sigma, Poole, UK) followed by a microsiopic examination using a modified Fuchs-Rosental haemocytometer. For counterchecpng, a CASY counter was used to measure the total and viable cell concentrations, basld on cell size. 1 Whereas the bcl-2/Neo recombinant cell line grew to a maximum density of liable cells of only about 107 cells/ml, in contrast to Tey et aL who described having obtained maximum cell densities of Space time yield of recombinant antibody was in favor of the recombinant jleo cell line (Fig.2). This is shown by graphically displaying product titer in the bioreactor versus cumulative cell time (CCT; unit: 109 cell h / L). CCT was calculated by integration of the cell growth curve, essentially as described in Renard et al. (1988, Biotechnology Letters 10: 91-96). As can be inferred from Fig. 2, mean single cell productivity of the bcl-2/Neo „ recombinant cell line was 0.260 pg protein/celLh, whereas the Neo cell line amounted to 0.210 pg protein/cell. The slight difference in single cell productivity (qp) w^s clearly outnumbered at the level of total yield by the difference in viable cell densities. 3. Gene copy number determination Hi Gene copy number of the Neomycin resistance gene was determined for the 6A|-Neo cell line as deposited by means of quantitative PCR assay, employing the 6A1 parfent as a comparative standard. An gene copy number of 3.0 copies (±0.4) was detenrnoM. Given concatemeric integration of stably transfected genes, this means that single integration of the recombinant neoymcin resistance marker took place in the 6Al-Neo cell lin| without further amplification. This in concert with the low MSX concentrations of about 25 pM MSX; for amplification of a GS marker gene to occur 10-20x fold higher concentrations of the selection agent need to be applied. The assay was carried out by a specializld contract laboratory (Lark Technologies Inc., Houston, Texas). Apart from testing for thelaeomycin resistance gene from Transposon Tn5, a negative control was establised by testing likewise for the mouse glyceraldehyde 3-phosphate dehydrogenase [GAPDH] gene. A climed plasmid standard was used to calibrate the assay plates.Copy numbers were calculated as Si the number of targets (NEO) per cell (MUSGAPDH), assuming that the DNA n|ass of the standard diploid mammalian cell is about 6.6 pg and that GAPDH is present in two copies per cell. Genomic DNA extracted from cells was quantitated spectiophotometridally by measuring optical densities at 260 and 280 mn as is routine in the art For assaying neomycin resistance gene copy number, a QPCR standard curve was generated ising eight dilutions of the pMCl neopA plasmid provided by Lonza Biologies. The dilution series represented the range from 5xl06 copies to 49 copies of plasmid DNA. Each standard was diluted into GS-NSO control genomic DNA (equivalent to 1000 cells) in order bf mimic the matrix effect of the extracted cell line DNA Negative control reactions containing no template or containing only the control plasmid (5000 to 50 000 copies) or the QNA I equivalent of 1000 GS-NSO cells (parent cell line, about 6.6 ng) were assembled! Copy number was determined with 95% confidence interval by determination of threshold cycle (Ct). The Ct is the fractional number at which the reporter fluorescence produced by probe cleavage crosses a fixed threshold set above the baseline. Each increase in|the numerical value of Ct represents a 2x increase in target, assuming 100%PCR eff|ciency. - i ~ Six independent dilutions of each test article DNA were prepared and analyzed! in duplicate. Each QPCR reaction contained about 6.6 ng of test sample DNA. THe QPCR reactions were assembled according to the TaqMan™ Universal PCR Master Mix protocol (Applied Biosystems, Foster City, CA). The reactions were thermal cycled andldata was collected by the ABI Prism 7700 Sequence Detection System version 1.6.3 (Applied Biosystems). 4. Comparative example: Culture of 6A1 parent cell line in the absence of Ne|)-resistance gene but in presence of sublethal amounts of G418 In order to exclude any growth effect being brought forward by exposure to G418 rather ftian to the presence of an expressed Neomycin resistance gene, the parent cell line 6Al was adapted to G418-containing cell culture medium. Amount of G418 could Be gradually increased up to a concentration of 360 mg/ml G418 (Sigma, Poole, UK; note mat lot quality of G418 may vary considerably depending on source) without abolishing cell growth. Growth medium was Lonza's optimized proprietary NSO cell culture medium PM1 supplemented with 6mM glutamine in the absence of fetal serum. For 6A|l-Neo cell line, this serum-free medium reproducibly allowed of achieving the high cell densities of the present invention (>2xl07 cells/ml) as compared to bcl-2 transfectant or no|i-transfected parent cell line 6A1. Therefore the medium was suited to monitor potential eflfct of G418 adaption in the absence of neomycin marker gene. As compared to non-adapted parent cell line grown in PM1 + 6mM glutamine jthe adapted cells grown in G418(-) media were mildly more resistant to apoptotic and necrotic cell death (data not shown) but had lower growth rate and lower maximum cell density as compared to parenf cell line (Fig. 5A: Viable cell density in batch culture of tfsO 6A1 cell line/ non-adapted 6A1: closed circles, 6A1 adapted to 360mg/ml G418 grown m the presence (open ciicles) and absence (closed triangles) of G418). By substracong viable cell count from total cell count, number of dead or necrotic cells was determined and is expressed in Fig. 5B as percentage of total cell count. The experiment allowedjto conclude that exposure to 9418 in the absence of neomycin resistance marker thereforejmay account only for pptential effect of apoptosis resistance but may not account far effect of enhanced high density growth behaviour. Interestingly, bioreactor culture of adapted cell line in the presence of 360 mg/1 G418 even further minimized both growth ratefand maximum cell density of the adapted cell line in serum-free medium PML 5. qpin batch culture of 6AL 6Al-Neo and 6Al-bcl-2 Cells lines were already described in examples 1 and 2; cell culture was carriedlout as described in example 2, except instead of fed-batch culture in a airlift fermentel, simple batch culture was carried out in a shake flask. Single cell productivity was dete mined by assaying secretion of cB72.3 in an Elisa test format from culture aliquots. Aliqi ots were counted for viable cell number and resuspended in fresh medium in a 96 well p ate for a pre-set period of time. Culture samples were taken at about 80 hours and at harvest (about 240 hours). Results are shown in Fig. 6 B. Growth curve based on viable cell density as judged by Trypan blue exclusion is shown in Fig- 6 A (open circles: 6Al-Neo, closed triangles: 6Al-bcl2, closed circles: 6A1 -(100)3). The 6Al-Neo cell line consistlngly showed higher single cell productivity % than the 6Al-bcl-2 cell line. The parent 6A1 cell line has an ever higher qp that is negatively balanced by its much lower growth potential. [!; ^1 \i *> We Claim: 1. Method of protein expression, comprising the step of a. growing an animal cell line to a viable cell density of at least or in excess of 10' cells/ml in a serum-free high-density growth culture medium in a fed-batch bioreactor which cell line is transfected with an expressable aminoglycoside resistance gene and is transfected with an expressable glutamine synthetase, and which cell line is further capable of expressing a product protein, and further comprising the step of b. isolating the product protein from the culture broth. 2. Method according to claim 1, wherein the aminoglycoside resistance gene product is employed to produce high-density growth of animal cell line, characterized in that said resistance gene product is the neomycin resistance gene product and said cells are transfected with DNA carrying an expressable neomycin resistance cassette and are subsequently subjected to selection of cells having a neomycin resistance phenotype by incubation with neomycin or, preferably, G418. 3. Method according to claim 2, characterized in that the animal cells are lymphoid cells, more preferably myeloma cells, most preferably NSO cells. 4. Method according to claim 2, characterized in that the high-density growth is a viable cell density in excess of 10' cells/ml. 5. Method according to claim 2, wherein the incubation is carried at a concentration of at least 1 mg/ml in cell culture medium. 6. Method according to claim 5, characterized in that after selection of neomycin resistants, the cells are further cultured to high-density in a fed-batch bioreactor system. 7. Method according to claim 1, wherein the high-density growth of animal cell line is achieved by employing antibiotic aminoglycosides. 8. A method of protein expression substantially as herein described with reference to the accompanying drawings.

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