Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/005—Glycopeptides, glycoproteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/505—Erythropoietin [EPO]

Definitions

• the present invention relates to a process for the continuous fermentative production of erythropoietin (EPO).
• EPO erythropoietin
• the method is characterized in that it is carried out in a perfusion reactor with cell pressure maintenance and the fermentation process is controlled only by a few selected measurement and control parameters so that both the productivity of the selected host organism with respect to EPO and the product quality of EPO advantageous to be influenced.
• Erythropoietin is a glycoprotein that stimulates the formation of erythrocytes in the bone marrow.
• EPO is primarily produced in the kidneys and from there via the bloodstream to its destination. In kidney failure, the damaged kidneys produce too little or no EPO at all, with the result that too few erythrocytes emerge from the stem cells of the bone marrow. This so-called renal anemia can be treated by administering EPO in physiological amounts that stimulate the growth of erythrocytes in the bone marrow.
• EPO The therapeutic effect and application of EPO is described, for example, in Eckardt K.U., Macdougall I.C, Lancet 2006, 368, 947-953, Jelkmann W .; Physiol. Rev. 1992, 72, 449-489, Eschbach J.W. et al. , N. Engl. J. Med., 1987, 316, 73-78, EP-B 0 148 605, EP-B 0 209 539, EP-B 0 205 564, Huang SL, PNAS 1984, 2708-2712 , Lai, PH et al. , J. Biol. Chem. 1986, 261, 3116-3121, as well as in Dietzfelbinger H. et al. , Manual Supportive measures and symptom-oriented therapy, Tumor Center Munich, 2001, 70-77, described in detail.
• the EPO used for administration can either be obtained from human urine or prepared by genetic engineering methods. be put. Since EPO is present in the human body only in the smallest amounts, the isolation of EPO from the natural source for therapeutic applications is virtually impossible. Therefore, genetic engineering methods 5 offer the only economic possibility to produce this substance in larger quantities.
• the proteins with a higher degree of sialylation have a higher specific activity.
• Such proteins as EPO, t-PA (tissue plasminogen activator) or coagulation factor VIII, whose activity i.a. depends on their degree of sialylation
• the prior art has produced cultures of mammalian cells capable of such necessary posttranslational glycosylation or sialylation of the protein.
• the recombinant production of EPO is usually done in Chinese hamster ovary (CHO) -
• the culture medium has a significant influence on the growth rate, cell density, translation and transcription of the host cells and thus also on the glycosylation and sialylation pattern of the recombinantly produced protein.
• serum-free media are used, as are offered by various manufacturers, for example the medium MAM-PF2 (distributed by Bioconcept, Allschwil, Switzerland) or the media DMEM and DMENU12 (offered for example by Invitrogen / Gibco, Eggenstein, Germany ).
• Another disadvantage of the batch process is the unfavorable relationship between the time limited production time (typically in the range of 5 to 10 days) due to the limited supply of nutrients for the cell culture and the total cycle time, which additionally includes the time for assembly, cleaning and sterilization of the bioreactor (typically in the range of up to 4 days).
• the second known cultivation method is the continuous method in which fresh medium is constantly supplied and taken out to the same extent fermenter contents.
• This method can achieve higher cell densities and maintain them over a relatively long period of time.
• a special case of continuous process management is represented by so-called dialysis reactors in which high-molecular substances such as Proteins are retained in the fermenter, while low molecular weight substances such as substrates can be added or the main waste products ammonium and lactate can be removed from the system.
• the third possible method is the fed-batch fermentation, in which the culture is started in a fermenter filled only to a fraction with culture medium and, after a short growth phase, fresh medium is added little by little.
• This enables higher cell densities and longer process times than in the batch process.
• Another advantage of this method is that the metabolism of the cells can be influenced by the extent of the feed, which can lead to a lower production of waste substances.
• the product of the cells is accumulated here in the fermenter over a relatively long period of time and thus higher product concentrations are achieved, which facilitates the subsequent work-up.
• a major problem in the cultivation of mammalian cells is to provide the cells with sufficient nutrients without increasing the breakdown of the nutrients beyond a critical limit for cell physiology.
• the main energy sources of animal cells are glucose and glutamine, whose major degradation products, lactate or ammonium, in higher concentrations inhibit the growth and metabolism of cells and lead to cell death (Hasseil et al., Applied Biochemistry and Biotechnology 1991, 30, 29-41). Therefore, it is advantageous in the cultivation of animal cells to reduce the accumulation of lactate and ammonium with sufficient nutrient supply, thus achieving higher cell densities and a higher product yield.
• US Pat. No. 6,180,401 discloses a fed-batch cell culture method in which the glucose concentration is continuously measured and kept in the culture medium within a certain range by adapting the feed depending on the measured values. Also according to the teaching of US 2002/0099183, the feeding rate of glucose is determined via the glucose concentration, whereby the glucose concentration concentration in the culture medium in a certain range.
• EP-A-036 179 A need-based nutrient addition, depending on the glutamate concentration in the culture medium, is described in EP-A-036 179 based on a fed-batch process.
• WO 97/33973 discloses a culture method in which the production of an electrically charged metabolite is measured by the conductivity of the medium and the feed rate is adjusted accordingly.
• No. 5,912,113 describes a fermentation process for microorganisms in which feed is always carried out when the carbon source in the medium has been consumed and an increased pH or increased dissolved oxygen concentration in the medium is thereby measured.
• cell line-specific properties which can be expressed in different growth rates, production kinetics, cell vitality, post-translational processing for glycosylation and sialylation, play a central role in the product quality of the obtained EPO and overall productivity of the fermentation process.
• the cell metabolism has significant differences and thus in the present case EPO of different quantities and quality, in particular with respect to the Glycolization and sialylation can be formed.
• the technical problem addressed by the present invention was therefore to develop a process for the fermentative production of erythropoietin, which has advantages over both the simplicity of the process and the yield of high-quality erythropoietin over the processes of the prior art.
• the EPO obtained should meet all the requirements of the official EPO
• the technical problem is solved by a process for the continuous fermentative production of erythropoietin, in which eukaryotic EPO-producing cells are cultured in a perfusion reactor with retention of the cells, the glucose concentration in the reactor via the perfusion rate of the culture medium and the cell count in the reactor via the cell pressure retention rate be set within predetermined ranges.
• the perfusion rate of the culture medium (as a control parameter) as a function of the glucose concentration in the fermentation reactor (as a measurement parameter) and b) secondly, the cell pressure retention rate of the cell pressure maintenance device (as a control parameter) as a function of the cell density in the fermentation reactor (as measurement parameter)
• defined amounts of cell-containing culture medium can also be discharged from the bioreactor at intervals as required and in this way a specific cell density in the reactor can be achieved.
• the other relevant process parameters such as pH, temperature, oxygen partial pressure, Ruhr speed and the composition of the supplied medium are preferably kept constant over the entire period of the fermentation.
• the described method combines in a novel manner various measures to increase both the product yield and the product quality of erythropoietin:
• Perfusion ensures that both cell-toxic metabolic products are constantly removed and fresh nutrients are supplied, so that very high cell densities are achieved in the bioreactor and the cells are productive over a very long period of time.
• the perfusion rate is further selected so that the glucose content in the culture supernatant on the one hand on the other hand, however, is limited in such a way that in the cell metabolism of the "Metabolism Shift" occurs and the toxic metabolites lactate and ammonium are produced only in a reduced extent and thus in the perfusion with only smaller amounts of fresh medium must be discharged.
• the said measures setting a suitable Zellruckhalterate or regular discharging defined amounts of cell-containing culture medium while setting a suitable perfusion rate, act synergistically, so that in a very simple and technically easy Anlagenbaren process for a continuous process over a extremely Surprisingly, an erythropoietin can be obtained over a long period of time, which has an extremely high proportion of such EPO, which meets the requirements of pharmaceutical law, in particular with regard to its degree of glycosylation and sialylation and the distribution of isoforms.
• the measurement or monitoring of the glucose concentration and the cell number can take place continuously or at specific times.
• the adjustment of the glucose concentration and the cell number continuously.
• the adjustment of the glucose concentration is carried out via the perfusion rate, ie by adding fresh culture medium containing glucose as a function of the glucose concentration in the fermentation reactor.
• the glucose concentration in the culture supernatant within a range of 0.05 to 1.5 g / L and the cell count within a range of 0.5xl0 7 to 5, set OxIO 7 cells / mL.
• the eukaryotic erythropoietin-producing cells are mammalian cells, preferably human cells, and more preferably Chinese hamster ovary cells (CHO).
• the cells are retained using an ultrasound cell pressure retention system, which is preferably infinitely variable.
• process parameters pH value, temperature, oxygen partial pressure, rate of rotation and composition of the medium are kept constant over the entire period of the fermentation in the range of technical deviations.
• the productivity is at least 10, preferably at least 20, more preferably at least 25 and most preferably at least 30 mg erythropoietin / L fermentation supernatant.
• the mean productivity is at least 10, preferably at least 15 mg erythropoietin / L fermentation supernatant.
• the average specific productivity per cell and day amounts to at least 0.5 ⁇ g, more preferably at least 1.0 ⁇ g and more preferably at least 1.2 ⁇ g and very particularly preferably at least 1.4 ⁇ g of erythropoietin.
• the mean vitality of the cells is at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95%.
• the process according to the invention is preferably carried out at a perfusion rate of between 0.5 and 3 during the fermentation, preferably between 1 and 2.5 and particularly preferably between 1.5 and 2.0.
• the process according to the present invention is carried out over a period of at least 10, preferably of at least 20, more preferably of at least 30 days and most preferably of at least 40 days.
• the glucose concentration in the culture supernatant is preferably set within a range of from 0.25 to 1.25 g / L and particularly preferably from 0.5 to 1.0 g / L.
• the cell number in the bioreactor is preferably set within a range of 1, 0x10 7 to 4.0 x 10 7 cells / ml and particularly preferably in a range of 1.5xl0 7 to 3.0 x 10 7 cells / ml of fermentation medium.
• the present invention relates to a process for the continuous fermentative production of erythropoietin, wherein eukaryotic EPO-producing cells are cultured in a perfusion reactor with retention of the cells, wherein the glucose concentration in the culture supernatant via the perfusion rate and the cell count on the Zellruckhalte- rate of a cell pressure maintenance device and / or regular discharge of defined amounts of cell-containing culture medium can be set within predetermined ranges.
• a glucose content in the culture supernatant of 0.05 to 1.5 g / L, preferably from 0.25 to 1.25 g / L and particularly preferably from 0.5 to 1, has been determined for the present CHO cell line. 0 g / L proved to be advantageous.
• the control of the perfusion rate is carried out according to the glucose content measurements in the reactor.
• the cell density in the reactor is maintained in the range from 0.5 ⁇ 10 7 to 5.0 ⁇ 10 7 cells / mL by appropriate adjustment of the ultrasonic cell pressure maintenance or regular discharge of defined amounts of cell-containing culture medium.
• the control of this parameter setting is accomplished by cell density measurements in the reactor.
• Cultivation preferably takes place in serum- and protein-free medium.
• the ingredients of such serum and protein-free culture media are known to those skilled in the art. They consist of a mixture of amino acids, fatty acids, vitamins, inorganic salts and hormones in various concentrations, as shown for example in EP-Bl 481 791 and WO88 / 00967 Al.
• the culture medium has a decisive influence on the growth rate, cell density, translation and transcription of the host cells 5 and thus also on the glycosylation and sialylation pattern of the recombinantly produced protein.
• serum-free media were used, as offered by various manufacturers, for example, the medium MAM-PF2 (distributed by Bioconcept, lo Allschwil, Switzerland), the media DMEM and DMENU12 (offered for example by Invitrogen / Gibco, Eggenstein, Germany) or the medium HyQPF CHO Liquid Soy (sold by HyCione / Perbio, Bonn, Germany, among others).
• the EPO prepared according to the invention is preferably recombinant human erythropoietin produced in eukaryotic cells.
• the recombinant EPO is produced in mammalian cells, more preferably in human cells and most preferably in CHO cells, e.g. generally described in EP-A-0 205 564 and EP-A-0 148 20 605.
• EPO erythropoietin
• the EPO may be the wild-type human erythropoietin or a variant thereof with one or more amino acids.
• this variant is only in 1 to 20, preferably in only 1 to 15, more preferably in only 1 to 10, and most preferably in only 1 to 5 amino acid positions of human wild-type erythropoietin by amino acid substitutions, deletions or additions.
• a CHO cell culture solution of 0.44 ⁇ 10 6 cells / mL was inoculated into a 10 L perfusion reactor (Applikon) equipped with a Biosep 50 (Applikon) in a volume of 10 L and kept for 3 days while maintaining the cultivation parameters held. On the 4th day, a 0.25-fold perfusion was started. The perfusion rate was successively in each 0.25 steps to max. increased to 2.5 times and then adjusted according to the glucose concentrations measured in the target range of glucose concentration (0.5 - 1.2 g / L). The target range for the cell number was adjusted by adjusting the cell retention rate of the ultrasound device and by discharging appropriate amounts of cell-containing culture medium.
• the other fermentation conditions were:
• Inoculum 0.44 x 10 6 cells / mL
• HyQPF CHO Liquid Soy from Hyclone / Perbio
• the base culture medium used was enriched with protein hydrolysates (Yeastolate from Becton Dickinson, HyPEP SR3 from Kerry Bio Science) and trace elements (CHO 4A TE Sock from Lonza).
• the crops were filtered cell-free and subjected to a known to the expert processing and purification, consisting of 3 to 4 chromatography steps subjected.
• FIG. 1 shows the time course of the glucose concentration or the EPO productivity ( ⁇ g EPO / ml) in the culture supernatant obtained by the process according to the invention.
• FIG. 2 shows the time course of the vital cell number (vit. ZZ) and the EPO productivity in the culture supernatant as well as the perfusion according to the method according to the invention.
• FIG. 3 shows the time profile of the percentage of vital cells in the totality of the cells in the culture supernatant and of the percentage of cell pressure retention according to the method according to the invention.
• FIG. 4 shows the time course of the lactate or glutamate concentration in the culture supernatant, which is obtained by the method according to the invention.

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• 2008
  • 2008-06-04 DE DE102008002210A patent/DE102008002210A1/de not_active Withdrawn
• 2009
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