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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0018—Culture media for cell or tissue culture
• • 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
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
• • 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
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/30—Organic components
  • C12N2500/34—Sugars

Definitions

• the invention relates to a method for the cultivation of cells for the production of substances according to the preamble of claim 1.
• Japanese Patent Application 001 101 882 A discloses a culture method for mammalian cells in which a minimum concentration of 0.2 mmol / l of glucose is maintained.
• US 544 39 68 discloses a cultivation method, in which a glucose limitation takes place. However, the method does not result in a higher specific production rate of the cells over the non-limiting feeding.
• the method should be particularly simple to carry out, with minimal measurement and control effort, and be particularly economical.
• the object is surprisingly achieved in that the cultivation of a substance-producing cell line is carried out under supply of a nutrient medium in such a way that adjusts a glucose limitation in the culture solution.
• the degree of glucose limitation can be defined as the ratio of the observed specific glucose consumption rate to the maximum specific glucose consumption rate known for these cells.
• DGL lies within the limits between DGL He haitung and 1 where DGL Erha ⁇ tU ng total
• Growth limitation means and 1 means no limitation or complete glucose excess. Together with the glucose limitation, there is a continuous decrease in the residual glucose concentration to a stationary concentration in the culture solution that is greater than 0 mmol / l, but less than 1 mmol / l, preferably less than 0.5 mmol / l. It can be observed that as the DGL sinks, a further increase in the living cell density in the culture vessel can occur. With increasing glucose limitation, the cell density then converges to a maximum value.
• the degree of glucose limitation converges to a minimum value, it is the DGL according to the invention is greater than or equal to the DGL, which leads to the preservation of the cell (maintenance metabolism)
• the growth medium must be such that glucose is limited at first.
• the method according to the invention increases space / time yield at a given cell density.
• the amount of glucose available per cell is reduced so that the glucose predominantly enters into the maintenance metabolism and connected therewith the product and less into cell growth.
• the method according to the invention does not require regulation of the glucose feed, so that the method is particularly simple, as it is possible to dispense with elaborate glucose control.
• a very high product concentration is achieved. This can lead to a reduction of the processing costs.
• the method according to the invention makes it possible to increase the production of proteins without having to additionally genetically modify a cell line for the implementation of the method according to the invention. Increasing the product titer allows the production of a desired amount of products in a smaller culture volume, resulting in lower investment costs.
• the process according to the invention can be carried out with the following process steps:
• the cells should preferably be in a continuous procedure with zeil Weghan, z. B. spin filter (perfusion culture) are cultivated. All common types of culture vessels, such as stirred tanks, and cell retention mechanisms such as spin filters, ultrasound or settlers are suitable.
• the culture system should allow high cell densities.
• a cell retention is realized, so that the cell density can not decrease when glucose limitation occurs.
• the DGL is further reduced.
• the high cell density allows the DGL to fall below a value of 0.4 at a set flow rate on the order of the maximum growth rate. For example, flow rates of 0.03-0.05 h -1 for the CHO MUC2-GFP-C-term cell used and the CH0 / MUCl-IgG2a PH3744 / 25 cell used can be used.
• the feeding strategy with glucose can therefore be as follows:
• the amount of glucose fed in is not increased with increasing living cell density in order to avoid glucose limitation. Rather, the amount of glucose added during the process is kept constant from the beginning.
• the amount of glucose fed in should be such that the DGL falls below the required values, namely DGL ⁇ 0.5, preferably ⁇ 0.4, more preferably ⁇ 0.3.
• the amount of glucose fed in is preferably not larger than 50%, more preferably not larger than 35% of that in the system Conventional, not glucose-limited process control expected maximum lifespan can consume.
• the amount of glucose fed in can be slowly increased, but should not allow for a DGL greater than 0.5, preferably greater than 0.4.
• the amount of added glucose can be influenced in a continuous process by the media feed rate and the glucose concentration in the feed medium.
• the decisive factor is that the mass flow of added glucose during the process is not increased or only to such an extent that the DGL reaches or falls below a value of less than 0.5, preferably less than 0.4 and this is then no longer exceeded.
• the figures show exemplary test results.
• Fig.l increase in the vital number of lines [ml "1 ] and representation of the medium flow rate [h " 1 ] against the process time [h] for the
• IgG2a [ ⁇ g / h * E9 cells]] and DGL versus the processing time in the perfusion reactor.
• Figure 3 Increase in the vital cell count [ml -1 ] and mM residual glucose, plotted against the processing time [h] for the production of MUCl-IgG2a from CHO MUCl / IgG2a PH3744 / 25 cells in the perfusion reactor.
• FIG. 4 Glucose and lactate concentration and concentration of glucose in the media feed [mmol / 1], plotted against the processing time [h] for the production of MUCl-IgG2a from CHO MUCl / IgG2a
• Fig. 5 Increase in the concentration of MUC1 - IgG2a [ ⁇ g / ml] and qMUCl - IgG2a [ ⁇ g / h * E9 cells)] versus time [h] for the production of MUCl-IgG2a from CHO MUCl / IgG2a PH3744 / 25 cells perfusion reactor.
• Fig.6 Increase in the vital cell count [ml "1 ]
• FIG. 8 Increase in the vital number of cells [ml.sup.- 1 ] and residual glucose [.mu.M], plotted against the processing time [h] for the production of MUC2-GFP-C-term from CHO MUC2-GFP-C-term cells in the perfusion reactor
• MUC2-GFP-C-term from CHO MUC2-GFP-C-term cells in the perfusion reactor
• Figure 10 Increase in concentration of MUC2-GFP-C-term [nM] and qMUC2 -GFP-C-term
• Table 1 shows the experimental data from the application of the method according to the invention with the CHO MUCl / IgG2a PH 3744/25 cell.
• Table 2 shows the experimental data from the use of the method according to the invention with the CHO MUC2 -GFP-C-term cell.
• the procedure according to the invention can be carried out with different production cell lines.
• the cell lines can be used as wild type or as genetically modified, recombinant cells.
• the genetic modification can be carried out, for example, by introducing additional genes of the same organism or another organism into the DNA, or a vector, or enhancing the activity or expression of a gene by introducing a more effective promoter, for example from CMV.
• the genes can code for various proteins, for example for proteins, such as fusion proteins or for antibodies.
• mammalian cells such as CHO cell lines, such as CHO-K1, BHK, such as BHK-21, hybridomas, NS / 0, others
• Myeloma cells and insect cells or other higher cells are particularly preferred. Particularly preferred is the use of cells that do not preferentially produce growth coupled.
• a recombinant CHO cell line whose productivity can be increased with the procedure according to the invention is the cell line CHO MUCl / IgG2a, PH 3744/25, with which the glycoprotein MUCl-IgG2a can be secreted.
• Another CHO cell line, CHO MUC2 -GFP-C-term is capable of secreting a fusion protein MUC2 -GFP-C-term when subjected to the procedure of this invention.
• a culture medium any glucose-containing medium can be used in principle, which is not limiting with respect to other components.
• ProCH04-CDM can be mentioned. It is also possible to use media based on known formulations, such as
• IMDM, DMEM or urinary F12 are used, which were optimized so that the procedure according to the invention that only glucose limitation occurs. This can be achieved, for example, by higher concentration of other components relative to glucose. In general, it is also possible to dose the glucose separately from the medium.
• the pH range is preferably between 6.7 and 7.7, more preferably between 7 and 7.3. However, other pH ranges are also conceivable.
• the temperature range is preferably between 35 ° C - 38.5 ° C, more preferably 37 ° C for CHO MUC1 IgG2a. But there are also other temperature ranges conceivable, such as ⁇ 35 ° C, in which there is no irreversible destruction of the product.
• FIG. 1 shows the course of the live cell density (cv) on CHO / MUCl-IgG2a cells and the medium flow rate (D) against the process time (h) in the perfusion reactor.
• cv live cell density
• D medium flow rate
• Figure 2 shows the specific productivity of MUCl-IgG2a (qMUCl-IgG2a) and DGL versus the processing time in the perfusion reactor. In it is:
• FIG. 3 shows a graph in which on the left side the vital cell count [ml -1 ] and on the right side the concentration of the residual glucose [mM] versus the processing time [h] for the production of MUCl-IgG2 in CHO MUC / IgG2a PH3744 / 25 is applied. In it is:
• Figure 5 plots the concentration of MUCl-IgG2a [ ⁇ g / ml] on the left and qMUCl-IgG2a [ ⁇ g / (h * E9 cells)] on the right side of the graph versus time [h]. In it are:
• FIG. 6 shows the course of the live cell density (cv) on CHO / MUC2-GFP cells and the medium flow rate (D) against the process time (h) in the perfusion reactor.
• D live cell density
• Process time in the perfusion reactor is:
• FIG. 8 shows a graph in which on the left side the vital cell count [ml "1 ] and on the right side the concentration of the residual glucose [mM] versus the processing time [h] for the production of MUC2 -GFP-C-term in FIG CHO MUC / IgG2a PH3744 / 25 is applied. In it is:
• FIG. 1 shows the procedure according to the invention by way of example with regard to the glucose feed.
• a constant amount of glucose is added.
• this is achieved by a constant media flow rate, the glucose concentration in the media feed constant is.
• the media flow rate is not increased with increasing living cell density. The process was started as a batch before the continuous procedure began.
• Figure 2 shows that in this procedure the DGL decreases in the course of the process and finally reaches a value below 0.4. While this happens, the specific productivity increases and eventually reaches a value 4 times higher than that before falling below the DGL value of 0.4.
• Table 1 shows data on the fermentation of MUCl-IgG2a.
• FIGS. 6 to 10 describe the results of using the method according to the invention with CHO MUC2-GFP-C-terminal cells.
• Table 2 shows data on the fermentation of MUC2 -GFP-C-term.
• the process according to the invention can also be operated as a fed-batch (feeding process), in addition to the perfusion process just described.
• the production culture is supplied once or recurrently, batchwise or continuously with glucose-containing medium or a separate glucose solution in a manner such that the DGL preferably has the value of 0.5, more preferably 0.4 and even better 0.3 below. Possible here is also a repetitive fed-batch.
• the process can be started in all well-known procedures.
• the culture may be batch, fed batch or continuous process. be operated with or without cell retention.

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• 2002
  • 2002-11-27 DE DE10255508A patent/DE10255508A1/de not_active Withdrawn
• 2003
  • 2003-11-07 HU HUE10152393A patent/HUE028862T2/en unknown
  • 2003-11-07 DK DK03767406.6T patent/DK1585810T3/da active
  • 2003-11-07 ES ES10152393.4T patent/ES2567161T3/es not_active Expired - Lifetime
  • 2003-11-07 ES ES10176622T patent/ES2749217T3/es not_active Expired - Lifetime
  • 2003-11-07 EP EP03767406A patent/EP1585810B1/de not_active Expired - Lifetime
  • 2003-11-07 DE DE50312554T patent/DE50312554D1/de not_active Expired - Lifetime
  • 2003-11-07 SI SI200332602T patent/SI2275530T1/sl unknown
  • 2003-11-07 EP EP10176622.8A patent/EP2275530B1/de not_active Expired - Lifetime
  • 2003-11-07 ES ES03767406T patent/ES2340377T3/es not_active Expired - Lifetime
  • 2003-11-07 EP EP10152393.4A patent/EP2226381B1/de not_active Expired - Lifetime
  • 2003-11-07 DK DK10152393.4T patent/DK2226381T3/en active
  • 2003-11-07 AT AT03767406T patent/ATE462013T1/de active
  • 2003-11-07 SI SI200331791T patent/SI1585810T1/sl unknown
  • 2003-11-07 PT PT03767406T patent/PT1585810E/pt unknown
  • 2003-11-07 US US10/535,581 patent/US20060127975A1/en not_active Abandoned
  • 2003-11-07 WO PCT/DE2003/003693 patent/WO2004048556A1/de not_active Ceased
  • 2003-11-07 JP JP2004554195A patent/JP4469283B2/ja not_active Expired - Lifetime
  • 2003-11-07 SI SI200332473A patent/SI2226381T1/sl unknown
• 2010
  • 2010-01-19 JP JP2010009094A patent/JP5221573B2/ja not_active Expired - Lifetime
  • 2010-06-11 CY CY20101100518T patent/CY1110267T1/el unknown
• 2014
  • 2014-01-21 US US14/160,336 patent/US20140329277A1/en not_active Abandoned
• 2016
  • 2016-05-13 CY CY20161100413T patent/CY1117558T1/el unknown

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