WO1999032605A1

WO1999032605A1 - Method for producing heterologous proteins in eukaryotic cells on an industrial scale using nucleotide-manipulating agents

Info

Publication number: WO1999032605A1
Application number: PCT/DK1998/000561
Authority: WO
Inventors: Hendrik P. J. Bonarius
Original assignee: Novo Nordisk A/S
Priority date: 1997-12-19
Filing date: 1998-12-17
Publication date: 1999-07-01
Also published as: AU1663599A

Abstract

The invention relates to an improved method for expressing proteins through culture of transformed eukaryotic cells on an industrial scale. In particular, the present invention relates to increasing the yield of biological products produced by eukaryotic cells through arresting growth in order to devote more nutrients and energy to the synthesis of heterologous proteins, and less to the synthesis of biomass. The improved method comprises adding a (cyclic)-nucleotide-manipulating agent, e.g. a cAMP-elevating agent, to the production medium.

Description

TITLE

METHOD FOR PRODUCING HETEROLOGOUS PROTEINS IN EUKARYOTIC CELLS ON AN INDUSTRIAL SCALE USING NUCLEOTIDE-MANIPULATING AGENTS

NUCLEOTIDE-ANALOGUES

FIELD OF INVENTION

The present invention relates to an improved method for expressing proteins through culture of eukaryotic cells. In particular, the present invention relates to increasing the yield of biological products produced by transformed eukaryotic cells through arresting growth in order to devote more nutrients and energy to the synthesis of heterologous proteins, and less to the synthesis of biomass. The invention also relates to a method for blocking or inhibiting the proliferation of transformed eukaryotic cells producing heterologous proteins, and to the use of one or more nucleotide-manipulating (e.g. a cAMP-elevating agent) agent(s) for controlling cell proliferation, a medium for culturing eukaryotic cells comprising a nucleotide- manipulating agent, and to polypeptides produced by such methods.

BACKGROUND OF INVENTION

It is known that eukaryotic cells require relatively large amounts of nutrients and energy for the biosynthesis of biomass, whereas relatively small amounts are used for the synthesis of (heterologous) glycoproteins. It has therefore been suggested that a two-phase process, in which a production phase is separated from a growth phase will give significantly higher yields compared to processes in which growth and production processes occur simultaneously (Flickinger et al., 1992; 1993). Several research groups that work on optimisation of production processes based on cell culture have already attempted to control cell proliferation in mammalian-cell culture by genetic engineering (Hauser, 1995), by lowering the biore- actor temperature (Chuppa et al., 1997), addition of growth-inhibiting compounds (Suzuki et al., 1990; Takahashi et al. 1994), or by depletion of nutrients (Flickinger et al., 1992). By these techniques the overall cell metabolism (both proliferation and protein production) is discontinued or inhibited. For a two-phase process it is essential to inhibit growth specifically, without disturbing protein production. Some of these strategies have resulted in successful control of proliferation in large-scale industrial recombinant production of proteins (Chuppa et al. 1990; Hauser, 1995; Suzuki et al. 1990; Takahashi et al. 1994). To this date however, only a limited number of the mentioned references show that growth was inhibited while protein production is activated simultaneously. For example, Suzuki and OIlis (1990) showed that the addition of thymidine resulted in an increase of monoclonal-antibody product yields of 20 %, while growth was slowed down.

For decades, agents that block tumour growth have been both targets and tools in biochemical and biomedical research groups. Potent agents are known that selectively block the proliferation of mammalian cells (Seifert and Rudland, 1974; Langeveld et al., 1992; Hugo et al., 1992; Kimura and Ogihara, 1997) or yeast (Chung et al., 1992). No attempt or suggestion however, has been made to use these agents to enhance the production of heterologous proteins or peptides.

Prior art

• Hugo et al., 1992 (Journal of Cellular Physiology 153: 539-549), disclose the blocking of DNA synthesis by the addition of extracellular AMP in cancer cells. • Seifert W.E. and Rudland P.S. 1974. Possible involvement of cyclic GMP in growth control of cultured mouse cells. Nature 248: 138-140.

• Cristoffersen et al., 1994 (Cancer Research 54: 2245-2250), disclose the arrest of tumour cells in the G1-phase of the cell cycle when intracellular cAMP levels are increased.

• Cho-Chung, Y.S., 1988 (World Intellectual Property Organisation, WO 89/11487, discloses the use of derivatives of cyclic AMP as a treatment for cancer.

• Flickinger, M.C., and Rouse, M.P. ,1993 (Biotechnology Progress, 9: 555-572), investigate the possibility of sustaining protein synthesis by Escherichia coli during very slow growth, where the growth inhibition is induced by nutrient depletion.

• Flickinger, M.C., et al. 1992 (J. Biotechnol. 22: 201-226) investigate the possible stimulation of monoclonal antibody secretion during slow hybridoma growth by L- glutamine depletion.

• Hauser, H. 1995 (EU Biotechnology Framework III, PL 950291) suggests to apply genetic- engineering techniques for proliferation control of mammalian cell culture. • Renner, W.A., et al. 1995 (Biotechn. Bioeng. 47: 476-482) show that over-expression of cyclin-e stimulates the proliferation of chinese-hamster ovary (CHO) cells.

• Chuppa, S., et al. 1997. (Biotechnol. Bioeng. 55: 328-338) show that the temperature can be used to control the proliferation of mammalian cells in culture. • Markus, H. Z. and McAleer, W. J. 1983 (Methods of producing HBsAg. US Patent.

4416986) claim a method to produce hepatitis B surface antigen in hollow fiber reactors, in which a second (production) phase is introduced by lowering the temperature and by the addition of caffeine.

• Suzuki, E. and OIlis, D.F. (1990. Biotechnol. Progress. 6: 231-236) show that proliferation of monoclonal-antibody producing cells can be inhibited by the addition of DNA synthesis inhibitors.

• Kimura M., and Ogihara M .1997. Proliferation of adult rat hepatocytes in primary culture induced by insulin is potentiated by cAMP-elevating agents. Eur. J. Pharmacol. 327: 87- 95. • Poteat et al., 1996 (AIDS Research & Human Retroviruses 12(6): 527-533) describe a method for expressing human T-cell leukemia virus in lymphocytes.

It has now been found that certain (cyclic)-nucleotide-manipulating agents can block the growth of cells that produce a heterologous protein, and simultaneously stimulate heterolo- gous-protein production rates. Said cells and production rates are those associated with large-scale or industrial-scale recombinant production of polypeptides or proteins, such as continuous or batch or fed-batch processes. The addition of agents manipulating the level of intracellular cyclic nucleotides to the production medium in cell culture processes decrease the production of biomass and increase the production of heterologous protein. The agents used according to the present invention inhibit growth specifically without disturbing protein production and thus enhances protein production.

In most industrial cell-culture processes, cells are supplied with sufficient nutrients by the perfusion of medium through the bioreactor, while cells are retained using perfusion systems. Although this type of process ensures high cell densities, it is cost inefficient due to the need for large amounts of medium (for example 6 tank volumes per day, Chuppa et al., 1997), and due to the need for perfusion devices and medium tanks. When the proliferation of cells is blocked, perfusion rates can be lowered without loosing product, and process costs related to perfusion can significantly be reduced. Another object of the present invention is therefore to provide a more economical process for producing biological products in eukaryotic cells, the process being based on the addition of growth-blocking or inhibiting agents. Because requirements for nutrients are lower at low growth, medium addition rates, perfusion rates, and/or bleed rates can be reduced. Apart from cost reduction, process yields can be increased by the arrest of cells in a specific phase of the growth cycle. By specifically blocking cell growth in a protein-producing phase of the cell cycle, the fraction of protein- producing cells in a bioreactor will increase, and as a result the production both per cell and per reactor volume will be higher. Another object of the present invention is to provide a method of producing biological products in eukaryotic cells, in cell-culture or fermentation, which method results in higher product titres.

SUMMARY OF THE INVENTION

The present invention relates to a method for proliferation control of genetically engineered eukaryotic cells cultured at industrial scale.

The present invention relates to the culturing of cells and the production of heterologous polypeptides or proteins in large-scale or industrial-scale recombinant production of polypep- tides or proteins, such as in continuous or batch or fed-batch processes

In one aspect the present invention relates to a method for producing a heterologous polypeptide in eukaryotic host cells transformed as to produce said polypeptide, comprising inhibiting the proliferation of said cells by adding at least one (cyclic)-nucleotide-manipulating agent to the medium.

In another aspect the invention relates to a method for producing a heterologous polypeptide in eukaryotic host cells transformed as to produce said polypeptide, comprising culturing said transformed host cells in a culture medium; adding at least one (cyclic)-nucleotide- manipulating agent to the medium; and isolating said polypeptide from the medium.

In another aspect the invention relates to a method for inhibiting proliferation of transformed eukaryotic cells, comprising culturing the transformed cells in a culture medium comprising at least one (cyclic)-nucleotide-manipulating agent. In still another aspect ,the invention relates to a medium for culturing transformed eukaryotic cells comprising at least one (cyclic)-nucleotide-manipulating agent.

In a further aspect, the invention relates to a protein produced by the process of the present invention.

In a further aspect, the invention relates to the use of at least one (cyclic)-nucleotide- manipulating agent for the inhibition of cell proliferation when producing a heterologous polypeptide in eukaryotic cells.

In different embodiments, the (cyclic)-nucleotide-manipulating agent is selected from a list of adenyl-cyclase-stimulating agents, cAMP-phosphodiesterase inhibitors, ribose-phosphate- pyrophosphokinase inhibitors, nucleotides, nucleosides, and cyclic nucleotide analogues, e.g. theophylline, rapamycin, dibutyryl adenosine cyclic monophosphate (DBcAMP), isobutyl- methylxanthine, forskolin, prostaglandins, metaproterenol, isoproterenol, adenosine, adenosine (5-)monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine, guanosine (5-) monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), 8-4-chlorophenylthio- adenosine 3':5'-cyclic monophosphate (8CP-AMP), 8-chloro-cAMP, 8-lodo-cAMP, N⁶-benzyl-cAMP, N⁶-benzoyl-cAMP, N⁶- phenylcarbomoyl-cAMP, and mixtures thereof.

In a preferred embodiment, the (cyclic)-nucleotide-manipulating agent(s) is/are agent(s) manipulating the level of cAMP or cGMP, preferably increasing the intracellular level of cAMP or cGMP.

In an preferred embodiment, at least one (cyclic)-nucleotide-manipulating agent is added to the medium when the cell density has reached a level of at least 10⁶ cells/ml in the case of mammalian or insect cells or when the optical density (OD₆₀₀) has reached a level of at least 5.0 in the case of yeast cells. In different embodiments, the cells are selected from a list consisting of yeast cells, mammalian cells or mammalian tissue, insect cells, fungal cells, or filamentous fungal cells.

In a preferred embodiment, the agent is AMP. In another preferred embodiment, the agents are AMP and one or more agents different from AMP, selected from the group of Ro-20- 1724, theophylline, rapamycin, N⁶,2-O-dibutyryladenosine 3:5-cyclic monophosphate (DBcAMP), forskolin, prostaglandins, metaproterenol, isoproterenol, adenosine diphosphate (ADP), adenosine, 8-4-chlorophenylthio- adenosine 3':5'-cyclic monophosphate (8CP- cAMP), 8-chloro-cAMP, 8-lodo-cAMP, N⁶-benzyl-cAMP, N⁶-benzoyl-cAMP, N⁶- phenylcarbomoyl-cAMP.

In a preferred embodiment, the cells are mammalian cells selected from a list of CHO cells, HeLa cells, BHK cells, or hybridomas, and the growth-inhibiting agent is AMP, 8-4- chlorophenylthio-adenosine 3':5'-cyclic monophosphate (8CP-cAMP), 3-lsobutyl-1- methylxanthine, or a combination of these agents.

LIST OF FIGURES

Fig. 1a and 1 b show the cell density and the polypeptide titers, respectively, of CHO cells cultured in t-flasks with and without the addition of different nucleotide-manipulating agents.

Fig. 2a and 2b show the cell density and the relative specific productivity (= amount of polypeptide produced per cell per day relative to the controls), respectively, of CHO cells cultured in t-flasks with the addition of different nucleotide-manipulating agents.

Fig. 3 shows the cell density and the polypeptide titers of CHO cells cultured in spinner flasks in a batch mode, with and without the addition AMP to the culture medium

Fig. 4a and 4b show the cell density and the polypeptide titers, respectively, of CHO cells cultured in spinner flasks in a fed-batch mode, with and without the addition AMP to the culture medium

Fig. 5 shows the cell density and the polypeptide titers of CHO cells cultured in a stirred tank reactor.

DETAILED DESCRIPTION OF THE INVENTION

The term "eukaryotic cells" is meant to include • yeast cells (f.e. Saccharomyces, Candida, Kluyveromyces, or Pichia) which include asco- sporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfect! (Blastomycetes);

• mammalian cells or mammalian tissue (e.g. Chinese hamster ovary cells (CHO), HeLa cells, baby hamster kidney (BHK) cells, or hybridomas) or any other immortalized cell line available at the ATCC (the American Type Culture Collection).

• insect cells (e.g. Lepidoptera cell line, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf. US 5,077,214)

• fungal cells or "fungi" which include the phyla Asco ycota, Basidiomycota, Chytridiomy- cota, and Zygomycota (as defined by Hawksworth et al., 1995) as well as the Oomycota

(as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra), or

• filamentous fungal cells, which include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra).

Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F A., Pass- more, S.M., and Davenport, R.R., eds, Soc. App. Bactehol. Symposium Series No. 9, 1980.)

Preferred are mammalian cells or mammalian tissue (e.g. Chinese hamster ovary cells

(CHO), HeLa cells, baby hamster kidney (BHK) cells, or hybridomas) or any other immortalized cell line available at the ATCC (the American Type Culture Collection), and insect cells (e.g. Lepidoptera cell line, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf. US 5,077,214). More preferred are mammalian cells or mammalian tissue (e.g. Chinese hamster ovary cells (CHO), HeLa cells, baby hamster kidney (BHK) cells, or hybridomas). Even more preferred are CHO cells, BHK cells and HeLa cells, most preferred are Cho cells and BHK cells.

In this context, the term "(cyclic)-nucleotide-manipulating agent" means any compound able to increase or decrease the intracellular level of cyclic nucleotides regardless of mechanism for changing said level. Cyclic nucleotides are nucleotides in which the phosphate group forms a ring. Such compounds may act for example through stimulating the enzyme adenyl cyclase or they may be inhibitors to cAMP-phosphodiesterase, or to ribose phosphate pyro- phosphokinase, such as nucleosides, nucleotides and cyclic nucleotide analogues. The compounds may also be cytostatics (i.e. agents that suppresses cell growth and multiplication of cancer cells). When controlling the proliferation of cells according to the present invention the agent(s) used may be a single compound or a combination of compounds. The agent may, for example, manipulate (e.g. increase) the intracellular level of cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP).

Examples of (cyclic)-nucleotide-manipulating agents are adenyl-cyclase-stimulating agents, e.g. forskolin, cAMP-phosphodiesterase inhibitors, e.g. Ro-20-1724 (Matsukawa et al., 1988), isobutyl-methylxanthine, theophylline, rapamycin, N⁶,2-O-dibutyryladenosine 3:5- cyclic monophosphate (DBcAMP), prostaglandins, metaproterenol, isoproterenol, nucleotides, e.g. adenosine (δ-)monophosphate (AMP), 8,4-chlorophenylthio-AMP, adenosine diphosphate (ADP)), nucleosides.e.g. adenosine, and cyclic nucleotide analogues, e.g. 8,4- chlorophenylthio-cAMP (8CP-cAMP), 8-chloro-cAMP, 8-lodo-cAMP, N⁶-benzyl-cAMP, N⁶- benzoyl-cAMP, N⁶-phenylcarbomoyl-cAMP (Cho-Chung, 1988, WO 89/11487).

Intracellular cAMP level may be assayed by the Biotrak cAMP assay system from Amersham (cf.: Internet address http://www.amersham.co.uk/life/lcat/biotrak/sig- trans/protocols/rpa556.htm).

Intracellular nucleotides may be quantified by the method described by Ryll and Wagner (J. of Chromatography 570: p.77-88, 1992)

In this context, the term "inhibiting cell proliferation" means any substantial reduction of cell proliferation as well as a total inhibition of proliferation.

The term "host cell" encompasses any progeny of a parent cell which is not identical to the parent cell due to mutations that occur during replication.

The term "transformed or transfected host cell" refers to cells that have been genetically engineered to produce a desired (glycosylated or non-glycosylated) polypeptide.

In this context, the term "basic medium" means the chosen medium for growing the eukaryotic cells without the addition of one or more (cyclic)-nucleotide-manipulating agent(s). In this context, the terms "continuous process" means a process in which the viable cell density is kept constant at a certain desired level by the continuous addition of fresh medium into the bioreactor or fermentation tank, while medium from the tank is harvested continuously.

In this context, a "(fed)-batch process" means a process in which the cells and product accumulate to a certain desired level, after which harvest is collected, and the process is either stopped ("batch") or restarted by the addition of fresh medium ("fed-batch").

An "industrial-scale" or "large-scale" process means a production in large, industrial scale fermentors, i.e. the eukaryotic cells which produces the polypeptide (for example, a thera- peutically active or pharmaceutically applicable polypeptide or a polypeptide suitable for diagnostic purposes) is grown under controlled conditions in a fermentor of 10-300 m³ (10.000- 300.000 I).

Methods of Production

For mammalian cell culture and insect cell culture:

The transformed or transfected eukaryotic host cells are cultured in a suitable nutrient medium (see below). Mammalian cells are cultured either in suspension or attached to a sur- face in a suitable bioreactor, which can be any system to cultivate cells, e.g. a stirred tank, an airlift reactor, a spinner flask, a roller bottle, a hollow-fibre bioreactor, a cell factory, or a cell cube. Insect cells are cultured and infected as described in Vlak et al., 1996 (Insect cell cultures. Fundamental and applied aspects. Kluwer Academic Publishers, Dordrecht, NL). The (cyclic)-nucleotide-manipulating agent(s) can be added any time to the culture medium. However, preferably the agent(s) is (are) added after the cell density has reached the desired level for the production of the protein. Methods for determining the desired level for the production of a protein, hereunder determining the minimum cell density before harvesting the produced protein, is well known to the skilled person (Chuppa, S. et al. 1997 Biotechnol. Bioeng. 55: 328, and references therein). The nucleotide-manipulating agents are added af- ter the cell density has reached a value of at least 1*10⁶ cells/ml, and preferably at higher cell densities (beyond 5^*10⁶ cells/ml). The agent(s) may be added pulse-wise or in one step. For yeast fermentation:

Yeast cells are cultured in a suitable fermenter as described by for example Rose, A. H., and Harrison, J. S (1993) in The Yeasts. (Vol. 1-5. Academic Press, London, UK). The (cyciic)- nucleotide-manipulating agent(s) can be added any time to the culture medium. However, preferably the agent(s) is (are) added after the cell density has reached the desired level for the production of the protein, which is known to the skilled person. For example, after the optical density (OD₆₀₀) has reached a value of 5.0 or any value higher than 5.0.

The (cyciic)-nucleotide-manipulating agent(s) may be added to the culture or production medium in a concentration of from 10^'9 Mol/litre to 10^"1 Mol/litre.

Preferred compounds are Preferred concentration as single agents adenosine (5-)monophosphate (AMP), 0,5-10 gr/L adenosine diphosphate (ADP) 0,5-10 gr/L adenosine, 0,5-10 gr/L theophylline, lO lO^ M rapamycin, 10^"9-10^"5 M

N⁶,2-dibutyryladenosine-3:5-cyclic mono- 10^"8-10^"" M phosphate prostaglandins, 10^-IO^ M metaproterenol, lO^' O^ M isoproterenol 10^~8-10^"4 M

8-4-chlorophenylthio-adenosine-3:5-cyclic lO^' O^"4 M monophosphate isobutyl-methylxanthine 10^"7-10^"3 M

When combinations of nucleotide manipulating agents are used, lower concentrations than mentioned above may be preferable for each component, due to synergistic effects.

Media

The basic medium used to culture the cells, and to which the (cyclic)-nucleotide-manipulating agent(s) is/are to be added, may be any conventional medium suitable for growing the eukaryotic host cells, such as minimal or complex media containing appropriate supplements. The media may comprise serum or other proteins of animal origin, or it may be free of such proteins of animal origin (e.g. serum-free). Suitable basic media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The basic media are prepared using procedures known in the art (see, e.g., general references for bacteria and yeast; Bennett, J.W. and LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991).] Examples of suitable media are, e.g., DMEM, DMEM/F12, RPMI.

Isolation and purification

If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered di- rectly from the medium. If the polypeptide is not secreted, it is recovered from cell lysates. The polypeptide are recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chro- matography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the type of polypeptide in question.

The polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate.

The expressed polypeptides may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chro- matofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

Polypeptides that may be produced by the method according to the present invention includes human coagulation factors (e.g. FVII, FVIII, FIX, and FXIII) in inactive single-chain form (zymogen) or in activated, double-chain form, TPO, tPA, prethrombin, insulin, insulin variants and -analogues, monoclonal antibodies, PDGF.

The invention will be further illustrated by the following examples. EXAMPLES

Example 1. Growth-inhibition of CHO cells by the addition of various agents without influ- encing product titers.

Figure 1a and 1 b show cell counts and product titers, respectively, of CHO cells engineered to produce prethrombin (see US 5,476,777) cultured in Modified Eagle's Medium (MEM) (Life Technologies, Tastrup, DK) with 5 % fetal calf serum (FCS) (Hyclone, Logan, UT). Cells were cultured as monolayers in 75 cm² flasks in temperature- and gas-controlled incubators (37 °C and 5 % CO₂/95 % air).

At Day 1 , medium (controls) or a growth-inhibiting agent dissolved in medium was added to the culture. The concentrations of growth-inhibiting agents are shown in the legend of Figure 1 and given in molar of the final culture volume. In all cases, growth was inhibited without influencing the product titer (Figure 1 b). In particular, the combination of AMP (1 g/L) and

8CP-cAMP (0,1 μM) resulted in a strong growth inhibition: 36 % of the control. Surprisingly, despite cell densities were significantly lower, prethrombin titers were not affected under any of these conditions (Figure 1b).

Abbreviations in Figure 1 :

AMP = Adenosine 5' monophosphate

CHO cells = Chinese Hamster Ovary cells

DMEM/F12 medium = Dulbecco's Modified Eagle's Medium/F12 E-7 = 10^"7 M ; E-6 = 10^"6 M ; E-5 = 10^"5 M

FCS = Foetal Calf Serum

MEM = Modified Eagle's Medium

MetaP = Metaproterenol

8CP = 8-4-Chlorophenylthio- adenosine 3':5'-cyclic monophosphate (8CP-cAMP) IBMX = 3-lsobutyl-1 -methylxanthine Example 2. Growth-inhibition of CHO cells by the addition of various agents influences the specific productivity.

Figure 2a show cell counts of CHO cells engineered to produce prethrombin cultured in tis- sue flasks. The cell line, medium and culture conditions in this experiment are identical to those of Example 1 , except for the type of growth-inhibiting agents and the time point of addition of the agents, which are different. At Day 20, medium (controls) or a growth-inhibiting agent dissolved in medium was added to the culture. In Figure 2a it is shown that in all cases growth is inhibited. Similar to example 1 , growth was inhibited without influencing the product titer (data not shown).

Figure 2b shows the specific productivity at day 4 (amount produced per cell per day), shown as relative values to the controls. In particular when AMP was used in combination with 8-CP or IBMX, the productivity per cell increases significantly.

Example 3 . Simultaneous growth-inhibition and product-titer increase by the addition of AMP in a batch spinner-flask culture.

In addition to the experiments with surface-dependent growth in serum-containing medium, the effect of AMP was investigated for suspended cells in serum-free medium. Figure 3 shows the cell density and product titers of prethrombin-producing CHO cells cultured in 100 ml spinner flasks in DMEM/F12 medium. In contrast to the examples described above, the medium was serum free. Instead of serum the medium contained additional insulin (5 mg/L) (Novo Nordisk, Bagsvaerd, DK), iron(iii)citrate (50 μM / 1 mM), L-proline (134 mg/L), sodium pyruvate (110 mg/L), sodium selenite (0,036 μM), ethanolamine (1 ,22 mg/L), and pluronic (1 g/L). The spinner flasks (Bellco, Vineland , NJ) were stirred at approximately 60 rpm, the temperate was kept at 37 °C, spinners were aerated with a gas mixture of 5 % CO₂/95 % air.

At day 1 , medium without (circles) or with (triangles) AMP was added. In the latter case, AMP was added to a final concentration of 5 g/l. Similar to the experiments described above, growth is significantly inhibited by AMP. Surprisingly, in contrast to the experiments with anchorage-dependent cultures, in which prethrombin titers were not affected by the addition of growth inhibitors, the product titer increased with 71 % compared to the control culture. Example 4 . Simultaneous growth-inhibition and product-titer increase by the addition of AMP in a fed-batch spinner-flask culture.

To investigate the long-term effect of the growth-inhibiting agent AMP, a similar spinner-flask experiment as described in example 3 was carried out in a fed-batch mode. Everyday, beginning with day 3, 15 ml of the culture broth was removed before adding 15 ml of medium ("draw and fill"). At day 6, 30 ml was drawn and refilled. At day 1 , 4, and 8 medium without (circles) or with (triangles) AMP was added. In the latter case, AMP was added to a final concentration of 5 g/l.

Figure 4a shows that the cell density decreased when AMP is added to the culture. This is in agreement with the other examples shown above. Prethrombin titers doubled under the high levels of AMP stress. These experiment indicate that growth inhibition by the addition of AMP is feasible for long-term, serum free, suspension cell culture. A large-scale bioreactor study, in which not only temperature, but also dissolved oxygen, pH, and medium addition is measured and controlled at a constant level is shown in Example 5.

Example 5. Simultaneous growth-inhibition and product-titre increase in bioreactors by the addition of AMP

CHO THR 101 cells were cultivated in a 5-liter-scale bioreactor (Biolafitte, St. Germain, F) in serum-free DMEM/F12 medium. The cell line and culture medium are described in example 1 and 3 respectively. Temperature, pH, and dissolved oxygen were controlled at 37 °C, 7,2, and 50 % of air saturation, respectively. The bioreactor was perfused at 0,9 tank volume per day and a cell bleed of 0,1 tank volume per day. Cells were retained in the bioreactor by a Centritech Lab centrifuge (Sorvall, Newton, CT). Figure 5 shows cell density (circles) and prethrombin titers (triangles). At day 34, AMP was added to the bioreactor (to a final concentration of 5 g/L). In addition, AMP was added to the medium tank (5 g/L). Immediately after AMP addition the cell density dropped (See Figure 5). Because proliferation is blocked by AMP stress, the cell bleed caused a rapid decrease in cell number. To prevent that the cell density dropped further, the cell bleed was stopped at day 35.

Even at lower cell densities, the product titers doubled in 5 days from -25 to -50 mg/L.

Claims

1. A method for producing a heterologous polypeptide on an industrial scale in eukaryotic host cells transformed as to produce said polypeptide, comprising inhibiting the proliferation of said cells by adding at least one (cyclic)-nucleotide-manipulating agent to the medium.

2. A method for producing a heterologous polypeptide in eukaryotic host cells transformed as to produce said polypeptide, comprising

1) culturing said transformed host cells on an industrial scale in a culture medium; 2) adding at least one (cyclic)-nucleotide-manipulating agent to the medium,

3) and isolating said polypeptide from the medium.

3. A method for inhibiting proliferation of transformed eukaryotic cells on an industrial scale, comprising culturing the transformed cells in a culture medium comprising at least one (cyclic)-nucleotide-manipulating agent.

4. A method according to any of claims 1-3 wherein at least one (cyclic)-nucleotide- manipulating agent is added to the medium when the cell density has reached a density of at least 10⁶ cells/ml in the case of mammalian or insect cells or when the optical density (OD₆₀₀) has reached a level of at least 5.0 in the case of yeast cells;

5. A method according to any of claims 1-4, wherein the (cyclic)-nucleotide-manipulating agent(s) is/are manipulating the level of cAMP or cGMP.

6. A method according to claim 5, wherein the (cyclic)-nucleotide-manipulating agent(s) is/are increasing the intracellular level of cAMP or cGMP.

7. A method according to any of claims 1-4 wherein the agent(s) is/are selected from the group of adenyl-cyclase-stimulating agents, cAMP-phosphodiesterase inhibitors, nucleo- tides, nucleosides, and cyclic nucleotide analogues.

8. A method according to claim 7, wherein the agent(s) is/are selected from the group of Ro- 20-1724, theophylline, rapamycin, DBcAMP, forskolin, prostaglandins, metaproterenol, isoproterenol, N⁶,2-O-dibutyryladenosine 3:5-cyclic monophosphate (DBcAMP), 3-lsobutyl-1- methylxanthine (IBMX), adenosine (5-)monophosphate (AMP), adenosine diphosphate (ADP), adenosine, 8-4-chlorophenylthio- adenosine 3':5'-cyclic monophosphate (8CP- cAMP), 8-chloro-cAMP, 8-lodo-cAMP, N⁶-benzyl-cAMP, N⁶-benzoyl-cAMP, N⁶- phenylcarbomoyl-cAMP.

9. A method according to claim 8, wherein the agent is AMP, 8-4-chlorophenylthio- adenosine 3':5'-cyclic monophosphate (8CP-cAMP), 3-lsobutyl-1 -methylxanthine, or a combination of these agents.

10. A method according to claim 7 and claim 8, wherein one agent is AMP and one or more agents different from AMP are selected from the group of Ro-20-1724, theophylline, rapamycin, forskolin, prostaglandins, metaproterenol, isoproterenol, N⁶,2-O-dibutyryladenosine 3:5-cyclic monophosphate (DBcAMP), adenosine (╬┤-)monophosphate (AMP), adenosine diphosphate (ADP), adenosine, 3-lsobutyl-1 -methylxanthine (IBMX), 8-4-chlorophenylthio- adenosine 3':5'-cyclic monophosphate (8CP-cAMP), 8-chloro-cAMP, 8-lodo-cAMP, N⁶- benzyl-cAMP, N⁶-benzoyl-cAMP, N⁶-phenylcarbomoyl-cAMP.

11. A method according to any of claims 1-10, wherein the cells are selected from yeast cells, mammalian cells or mammalian tissue, insect cells, fungal cells, or filamentous fungal cells.

12. A method according to claim 11 , wherein the cells are mammalian ceils and are selected from CHO cells, HeLa cells, BHK cells, or hybridomas.

13. Use of at least one (cyclic)-nucleotide-manipulating agents for the inhibition of cell proliferation when producing a heterologous polypeptide in eukaryotic cells on an industrial scale.

14. A medium for culturing transformed eukaryotic cells on an industrial scale, comprising at least one (cyclic-) nucleotide-manipulating agent.

15. Protein produced by the method of claims 1-12.