Title of Invention	A PROCESS FOR THE PREPARATION OF A PROTIEN BY CULTIVATION OF EUKARYOTIC CELLS
Abstract	Cultivation is effected in the presence of a repressive substrate as carbon source which is controlled. Oxygen supply is maintained continuously during the cultivation. Protein produced is recovered by known means from the culture medium.

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Methylotrophic yeasts have been investigated during the last years as suitable expression systems for heterologous proteins. Particularly Hansenula polymorpha and Pichia pastoris have been described as easy to handle host organisms for a wide variety of foreign genes (for reviews see Gelissen & Melber 1996 and Gregg & Madden 1988). Since the promoters for the enzymes involved in methanol metabolism were found to be very strong, they are generally used to control the heterologous expression of proteins (see EP 0173 378, EP 0299 108, EP 0183 071). It is known that the carbon sources used for cultivations of these organisms have an enormous influence on promoter regulation. Gelissen et al. (1994) describe that the key enzjrmes of the methanol metabolism in Hansenula polymorpha are present in large amounts during growth on methanol. It was also found out that significant levels of the enzymes methanol- oxidase (MOX) and formate dehydrogenase (FMDH) can be detected in glycerol grown cells, but are absent if glucose is used as carbon source (see e.g. EP 0299 108). Based on this data, it was concluded that these enzymes are induced by methanol. Expression of MOX and FMDH during-grqwth on glycerol is explained with derepression of the promoters. In glucose limited chemostat cultures of Hansenula polymorpha and Kloeckera sp. 2201, FMDH is only produced below growth rates of 0.1 h"'^ in very small amounts (Egli et al. 1980). Therefore, so far non repressive carbon sources have been used for the heterologous expression of proteins with methylotrophic yeasts. Hansenula polymorpha is cultivated for the production of a wide, variety of pharmaceutical proteins .in a * fed batch process either in the single carbon source mode with glycerol or in the two carbon source mode with glycerol and additional methanol (Gelissen & Melber 1996). The common process strategy is described in more details by Weydemann et al. (1995) for the production of Hirudin. A process for the production of Thrombomodulin with a Pichia pastoris strain using also glycerol as a non repressive carbon source is described'by Chen et al. (1996). However, due to the high price of glycerol this process is until now not suitable for the production of low cost proteins like feed or industrial enzymes. Therefore it is an object of the present invention to overcome such obstacles and to provide a process for the preparation of an endogenous or heterologous protein by cultivation of a transformed or non-transformed eukaryotic cell which process is characterized therein that a repressive substrate is used as a carbon source, the carbon source is limiting during the feeding phase and there is no continuous oxygen limitation during the whole cultivation; or such a process wherein the protein is an enzyme, especially a feed enz3ane, e.g. phytase, cellulase, xylanase or an industrial enzyme, e.g. amylase, protease, invertase, lipase, catalase, cellulase, glucose oxidase, alcohol oxidase, pectinase, naraginase, collagenase, peroxidase or pullalanase; or such a process wherein the cell is methylotrophic and/or is transformed with a DNA sequence comprising a promoter for enzymes involved in the methanol metabolism, e.g. the formate dehydrogenase (FMD or FMDH) promoter, the methanol oxidase (MOX) promoter or the dihydroxyacetone sjmthase (DAS or DHAS) promoter; or such a process wherein the cell is a methylotrophic yeast, preferably Hansenula, Pichia, Candida or Torulopsis, e.g. Hansenula pol5anorpha or Pichia pastoris; or such a process wherein the repressive substrate counts for 1-100%, preferably 40-100% or more preferably 90-100% of the carbon source or is the sole carbon soure; or such a process wherein the substrate is a sugar like mono-, di-, oligo- or poly-saccharinde, e.g. glucose, fructose, sucrose, maltose, starch, glycogen, cellulose or dextrose or a sugar containing compound, e.g. molasses, glucose syrups or fructose S5T-ups; or such a process which is effected in form of repeated fed-batches. Furthermore such process is characterized therein that the feeding rate range is limited by the metabolic characteristics of the microorganisms and the mass transfer performance of the bioreactor, in particular for oxygen. Feeding rates are preferentially maintained between the minimum to allow production and the maximum to avoid oxygen limitation in the culture. In the case of a process for the production of a heterologeous protein, the eukaryotic cell has been transformed by a DNA sequence or a vector comprising a DNA sequence encoding such heterologeous protein, e.g. in the examples of the present case the phytase. A man skilled in the art of molecular biology is familiar with the methods used to prepare such transformed eukaryotic cells, see e.g. for the specific examples of the present case EP 684 313 or European Patent Application No.97810175.6. "Cultivation" in the context of the present invention has its usual meaning a man skilled in the art is femihar with. The specific cultivation conditions will depend on the cells used and the proteins to be produced and their expression systems. A man skilled in the art of fermentative production of proteins is also &miliar with such conditions. Furthermore it is understood that for flie practice of the present invention during the short batch phase, namely the growth phase of the cells, a repressive or a non-repressive carbon source, e.g. glucose or glycerol respectively, can be used. During the fed batch phase, which is the production phase of the desired protein, the cultivation process is conducted in the manner as provided by the present invention. In addition, methylotrophic yeasts of interest for the practice of the present invention can be taken e.g. fi-om EP173 378, pages 37 and 38. Accordingly the present invention provides a process for the preparation of a protein by cultivation of eukaryotic cells wherein a known repressive substrate such as herein described is used as a carbon source, controlling said carbon source during the feeding phase in a known manner maintaining oxygen saturation continuously through out said cultivation step, and recovering said protein fixjm the culture medium by known methods. Examples Example 1 Production of A. Fumisatus phvtase with Hansenula polymorvha using glycerol or glucose as main carbon source To compare the production of ph3i;ase using glycerol or glucose as feeding substrates, two fed bat'ch experiments were carried out in 15 1 reactors with a Hansenula polymorpha strain containing about 80 copies of the wild-type phytase gene from Aspergillus fumigatus (Pasamontes et al., 1997) under the control of the FMD promoter. Shake flask cultures were started-by inoculating 1 ml of. a glycerol stock cell suspension maintained at -70°C in 50 ml medium with the following composition: 30.0 g/1 glycerol; 2.5 g/1 KH2PO4; 5 g/1 NH4H2PO4; 2.25 g/1 MgS04-7H20; 2.5 g/1 (NH4)2S04; 1.15 g/1 KCl; 0.25 g/1 NaCl; 0.375 g/1 -CaCl2-2H20; 0.25 mg/1 H3BO3; 0.05 g/1 (NH4)2Fe(S04)2-6H20; 4 mg/1 CuS04-5H20; 15 mg/1 ZnS04-7H20; 20 mg/1 MnS04-H20; 0.05 g/1 Na-EDTA; 0.5 mg/1 NiS04-6H20; 0.5 mg/1 CoCl2-6H20; 0.5 mg/1 Na2Mo04-2H20; 0.5 mg/1 KI; 0.05 g/1 thiamin-HCl; 0.15 mg/1 biotin. They were cultivated at 30°C and 200 rpm for 24 h, 30 ml were transferred to a second shake flask stage with 300 ml of the same medium, cultivated at 30°C and 200 rpm for further 24h. 300 ml of this seed culture were used as inoculum for a 15 1 fermenter with 5 1 initial medium composed as follows: 10.0 g/1 glycerol; 5.0 g/1 KH2PO4; 10 g/1 NH4H2PO4; 4.5 g/1 MgS04-7H20; 5.0 g/1 (NH4)2S04; 2.3 g/1 KCl; 0.5 g/1 NaCl; 0.2 ml/1 antifoam; 0.75 g/1 CaCl2-2H20; 0.5 mg/1 H3BO3; 0.1 g/1 (NH4)2Fe(S04)2-6H20; 8 mg/1 CuS04-5H20; 30 mg/1 ZnS04-7H20; 40 mg/1 MnS04-H20; 0.1 g/1 Na-EDTA; 1.0 mg/1 NiS04-6H20; 1.0 mg/1 CoCl2-6H20; 1.0 mg/1 Na2Mo04-2H20; 1.0 mg/1 KI; 0.1 g/1 thiamin-HCl; 0.3 mg/1 biotin. During the whole process temperature, pH, and aeration were kept constant at 30°C, pH 4.6 and 5 1/h, respectively. The pH was controlled by addition of NH3 25%. Oxygen saturation was maintained at a minimum value of 20% saturation in the reactor by controlling the stirrer speed and the pressure (0-0.5 bar). The complete consumption of the initial glycerol in the medium led to a rapid increase in the p02 value and indicated the end of the batch phase. At this point, the feeding with 700 g/1 solutions of either glycerol or glucose was started. An initial feeding rate of 25 g solution per hour was used for the first 18 h feeding and then the rate was increased to 50 g/h. The culture volume increased due to the feeding of about 6 liters of carbon source solution up to about 11 liters at the end of the cultivation. In the case of the cultivation fed with glucose, 98% of the whole carbon source metabolized during the process is glucose, and the remaining 2% correspond to glycerol used during the batch phase. During the whole process, no glucose could be quantified in the supernatant. After 165 h process time, 5.86 g/1 phytase were quantified in samples taken fi-om the cultivation with glycerol by using the Bradford method for protein determination. From the cultivation with glucose, samples after 160 h process time contained 7.12 g/1 phytase determined by the same method. These results confirm that the repressive effect of glucose can be avoided with a feeding strategy ensuring a strict carbon limitation during the feeding phase. Fig. 1 shows the comparative time course for the cultivations done with glycerol and with glucose as main carbon source. Example 2 Production of A. Fumisatus phvtase with Hansenula polymorphn usingsucrose as main carbon source In this example, a cultivation was conducted in a 15 1 bioreactor using the same strain as that from Example 1. The seed culture and main cultivation conditions were exactly the same as for Example 1, except for the feeding solution, which contained 700 g/\ sucrose. During the process, sucrose concentrations in supernatant samples were never higher than 1 g/1, whereas neither glucose nor fructose could be detected in the same samples. After 167 h cultivation time, 5.27 g/1 phytase could be measured in the medium by the Bradford method for protein determination. Example 3 Production of A. Fumisatus phvtase with Hansenula volymorpha using glucose as the sole carbon source In this example, a cultivation was conducted with the same strain of Examples 1 and 2 in a 15 1 bioreactor. The seed culture was obtained under the same conditions as Examples 1 and 2. The main cultivation was done starting with 5 1 initial medium in the bioreactor with the same composition described in Example 1, except that the initial 10 g/1 glycerol were substituted by 10 g/1 glucose. The feeding solution was composed by 700 g/1 glucose, and was added to the cultivation under exactly the same conditions described in Example 1. After 160 h process time, 7.21 g/1 phytase could be detected in the medium, measured by the Bradford method. Example 4 Production of A. Fumisatus phvtase with Hansenula polvmorDha in a repeated fed batch mode In this example, repeated fed-batch cultivations in a 15 1 reactor with the same Hansenula polymorpha strain used in Examples 1, 2 and 3 were conducted. For the first cultivation, a seed culture was obtained by inoculating 1 ml of a glycerol stock cell suspension maintained at -70°C into 300 ml medium with the following composition: 30.0 g/1 glycerol; 13.3 g/1 NH4H2PO4; 3.0 g/1 MgS04-7H20; 6.7 g/1 (NH4)2S04; 3.3 g/1 KCl; 0.33 g/1 NaCl; 1.0 g/1 CaCl2-2H20; 0.67 mg/1 H3BO3; 0.067 g/1 (NH4)2Fe(S04)2-6H20; 5.3 mg/1 CuS04-5H20; 20 mg/1 ZnS04-7H20; 26 mg/1 MnS04-H20; 0.067 g/1 Na-EDTA; 0.67 mg/1 NiS04-6H20; 0.67 mg/1 CoCl2-6H20; 0.67 mg/1 Na2Mo04-2H20; 0.67 mg/1 KI; 0.13 g/1 thiamin*HCl; 0.4 mg/1 biotin. The shake flask was cultivated at 30°C and 200 rpm for 30 h, 300 ml were used as inoculum for the main culture in a 15 1 bioreactor containing: 150.0 g glycerol; 100.0 g NH4H2PO4; 22.5 g MgS04-7H20; 50.0 g (NH4)2S04; 25 g KCl; 2.5 g NaCl; 7.5 g CaCl2-2H20; 5 mg H3BO3; 0.5 g (NH4)2Fe(S04)2-6H20; 40 mg CuS04-5H20; 150 mg ZnS04-7H20; 40 mg MnS04-H02; 0.5 g Na-EDTA; 5.0 mg NiS04-6H20; 5.0 mg CoCl2-6H20; 5.0 mg Na2Mo04-2H20; 5.0 mg KI; 1 g thiamin-HCl; 3 mg biotin, for an initial volume of 7.5 1 medium. During the whole process temperature, pH and aeration were kept constant at 30°C, 5.0 and 9.0 1/min, respectively. The pH was controlled by addition of NH3 25%. Oxygen saturation was maintained at a minimum value of 20% saturation in the reactor by controlling the stirrer speed and the pressure (0-0.5 bar). The complete consumption of the initial glycerol in the medium led to a rapid increase in the p02 value and indicated the end of the batch phase. At this point, feeding was started with a solution containing 40% glucose and 60% glycerol to a total carbon source concentration of 700 g/1. The feeding rate was controlled at 45 g/h during the whole feeding phase. For the following repeated fed-batch cultivations, 0.5 to 1 1 of culture broth from the previous run were maintained in the bioreactor at the process end as inoculum for the next run. New sterile medium containing the same total amoimt of salts described above but no carbon source was added to the bioreactor up to a defined initial volume. Therefore, there was no batch phase and the feeding was started immediately as described above. Feeding solutions contained a total of 700 g/1 carbon source. The different proportions of glycerol and glucose as well as the initial volume were used as given below: 2°*^ cycle - 66% glucose, 34% glycerol, 7.51 initial volume 3"^ cycle - 90% glucose, 10% glycerol, 7.5 initial volume 4^^ and 5**^ cycles -100% glucose, 5 1 initial volume The concentrations of phytase attained at the end of each cycle were 4.7 g/1, 3.9 g/1, 4.3 g/1, 6.0 g/1 and 4.4 g/1, respectively for the 1'* to 5* cycles, as shown in Fig. 2. In the 4*^ cycle, a slower, extended feeding mode at the end of the growth phase led to a higher product concentration. Example 5 Production of consensus phvtase with Hansenula polymorpha In this example, a strain of Hansenula polymorpha containing about 40 copies of a consensus phytase gene (European Patent Application No. 97810175.6) under the control of the FMD promotor was cultivated in a 15 1 bioreactor with the same medium and under the same conditions described in Example 1, using a feeding solution containing 700 g/1 glucose for the fed-batch phase. After 165h cultivation time, the phytase concentration in the medium was 13.1 g/1, as determined by the Bradford method. Literature J. M. Cregg and K. R. Madden (1988): Development of methylotrophic yeast, Pichia pastoris, as a host system for the production of foreign proteins, Developments in Industrial Microbiology 29, 33-41 GeHssen and K. Melber (1996): Methylotrophic yeast Hansenula polymorpha as production organism for recombinat pharmaceuticals. Drug Res. 46, 943-948 G. Gelissen, C. P. HoUenberger, Z. A. Janowicz (1994): Gene expression in methylotrohic yeasts. In: Smith A. (ed): Gene expression in recombinant microorganisms, Dekker, New York, 195-239 Weydemann, P. Keup, M. Piontek, A. W. M. Strasser, J. Schweden, G. Gelissen, Z. A. Janowicz (1995): High-level secretion of hirudin by Hansenula polj^morpha, Appl Microbiol Biotechnol 44, 377-385 Th. Egli, J. P. Van Dijken, M. Veenhuis, W. Harder, A. Fiechter (1980): Methanol metabolism in yeasts: Regulation of the s5Tithesis of catabolic enz5anes, Arch. Microbiol. 124, 115-121 Y. Chen, J. Krol, and D. Freedman (1996): Continuous production of Thrombomodulin from a Pichia pastoris fermentation, J. Chem. Tech. Biotechnol. 67,143-148 L. Pasamontes, M. Haiker, M. Wyss, M. Tessier, A. P. G. M. Van Loon (1997): Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus, Appl. Environ. Microbiol. 63, 1696-1700 WE CLAIM: 1. A process for the preparation of a protein by cultivation of eukaiyotic cells wherein a known repressive substrate such as herein described is used as a carbon source, controlling said carbon source during the feedmg phase in a known manner maintaining oxygen saturation continuously through out said cultivation step, and recovering said protein from the culture medium by known methods. 2. The process as claimed in claim 1, wherein the protein is an enzyme, especially a feed enzyme such as phytase, cellulase, xylanase or an industrial enzyme such as amylase, protease, invertase, Upase, catalase, cellulase, glucose oxidase, alcohol oxidase, pectinase, naraginase, collagenase, peroxidase or puUulanase. 3. The process as claimed in claim 1 or 2, wherein the cell is methylotrophic and/or transformed in a known manner with a DNA sequence comprising a promoter for enzymes involved in the methanol metabolism, such as the formate dehydrogenase (FMD or FMDH> promoter, the methanol oxidase (MOX) promotor or the dihydroxyacetone synthase (DAS or DHAS) promotor. 4. The process as claimed in any one of claims 1 to 3 wherein the cell is a methylotrophic yeast, preferably Hansenula, Hansenula polymorpha, Pichia, Pichia pastories, Candida or Torulopsis. 5. The process as claimed in any one of claims 1 to 4, wherein the repressive substrate accounts for 1-100%, preferably 40-100 or more preferably 90-100% of the carbon source or is the only carbon source. 6. The process as claimed in any one of claims 1 to 5, wherein the repressive substrate is a sugar such as mono-, di-, oligo- or polysaccharide, e.g. glucose, fructose, sucrose, maltose, starch, glycogen, cellulose or destrose or a sugar containing substance such as molasses, glucose syrups or fructose syrups. 7. The process as claimed in any one of claims 1 to 6, wherein such process is carried out batch wise. 8. A process for the preparation of a protein by cultivation of eukaryotic cells substantially as herein described with reference to the accompnying drawings.

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