Cell culture medium for culturing cells, cell culture method, and method for expressing at least one recombinant protein in cell culture

Abstract

A cell culture medium for culturing cells is provided, comprising an iron citrate-iron diphosphate complex as an iron source. Furthermore, a cell culture method is provided in which one or more cells are grown or maintained in the cell culture medium of the present invention. Furthermore, a method for expressing at least one recombinant protein in cell culture is presented, in which a nucleic acid that causes the production of at least one recombinant protein is introduced into cells grown or maintained in the cell culture medium of the present invention. The cell culture medium of the present invention has the advantage that the iron source contained therein is highly soluble and rapidly dissolved in aqueous solutions (e.g., cell culture medium, cell culture supplement, or water) and can be efficiently incorporated into the interior of cells, increasing the number of viable cells and increasing product titer (also called product concentration) in recombinant protein production, making it highly cost-effective.

Description

Technical Field

[0001] Provided is a cell culture medium for culturing cells, which contains an iron citrate diphosphate complex as an iron source. Further, provided is a cell culture method for growing or maintaining one or more cells in the cell culture medium of the present invention. Further, presented is a method for expressing at least one recombinant protein in cell culture, in which a nucleic acid that causes the production of at least one recombinant protein is introduced into cells grown or maintained in the cell culture medium according to the present invention. The cell culture medium according to the present invention has the advantage that the contained iron source dissolves very well and rapidly in an aqueous solution (for example, a cell culture medium, a cell culture supplement or water), can be efficiently taken up into the interior of cells, and causes an increase in the number of viable cells and an increase in the product titer (also called the product concentration) in the production of recombinant proteins, and is very cost-effective.

[0002] A cell culture medium for culturing living cells may contain an iron source, i.e., a source of iron ions that can be efficiently imported by living cells. In fact, the presence of an iron source in a cell culture medium is essential for culturing mammalian cells. The uptake of iron ions into living cells can be achieved, for example, by adding the protein transferrin to a cell culture medium containing an iron salt (for example, iron(III) nitrate, iron(II) sulfate and / or iron(III) chloride). This is because transferrin binds iron ions and makes them available in the cell culture medium in a way that is accessible (importable) to living cells.

[0003] Transferrin is usually obtained from plasma or produced recombinantly and is also commercially available in a dry form. The disadvantages of available transferrins are that they are expensive and thus uneconomical for cell culture on an industrial scale. Furthermore, since the available transferrins are already partially saturated with iron ions, the iron concentration in the cell culture medium cannot be adjusted in a precisely defined manner. Therefore, a cell culture medium without transferrin is required for culturing living cells. In such a cell culture medium, iron ions must be provided to the living cells in a different way so that they are effectively biologically accessible (i.e., can be taken up inside the cells).

[0004] In the prior art, it is known to make different forms of iron effectively accessible to the cell culture medium.

[0005] CA 2 756 247 C discloses that an iron salt or an iron complex can be used as an iron source in a serum-free medium. The iron source can be selected, for example, from the group consisting of iron(III) phosphate, iron(III) pyrophosphate, iron(III) nitrate, iron(II) sulfate, iron(III) chloride, iron(II) lactate, iron(III) citrate, ammonium ferrous citrate, iron dextran and sodium ferric EDTA. However, many of the listed iron sources are not suitable for making iron available to living cells in an easily accessible way, and thus it is difficult to take up important iron ions into the cells. As a result, when expressing a recombinant protein, the cells grow more slowly and show a low productivity titer. Iron(II) citrate is the most effective among the aforementioned iron sources, but it has the disadvantage that it dissolves very slowly when it is present in a dry cell culture medium and the culture medium is prepared with water for culturing the cells. The long dissolution period of iron(II) citrate is a time disadvantage in the industrial production of cell culture media for cell culture, and the use of iron(II) citrate is uneconomical.

[0006] WO 2016 / 156476 A1 discloses a cell culture medium without transferrin that enables iron to be accessible to living cells via an iron choline citrate complex. Since the iron choline citrate complex contributes to a significant increase in the productivity titer in cell culture, it is taught to be more advantageous than commonly used iron sources such as iron(II) phosphate, iron(III) pyrophosphate, and iron(III) citrate. However, using the iron choline citrate complex in the cell culture medium has the disadvantage that the manufacturing cost of the cell culture medium is very high due to the iron complex, and thus the cell culture medium is uneconomical, especially when a very large cell culture volume is required.

[0007] Accordingly, an object of the present invention is to provide a cell culture medium without transferrin (or completely without serum) that contains an iron source capable of performing cell culture with a high productivity titer faster and more inexpensively (more economically). Furthermore, a corresponding method for culturing cells and a corresponding method for expressing at least one recombinant protein in cell culture should be provided.

[0008] The above object is achieved by a cell culture medium having the characteristics described in claim 1, a method for culturing cells having the characteristics described in claim 11, a method for expressing at least one recombinant protein in a cell culture having the characteristics described in claim 13, and the use having the characteristics described in claim 14. The dependent claims show advantageous developments.

[0009] According to the present invention, there is provided a cell culture medium characterized by containing an iron diphosphate citrate complex.

[0010] The term "cell culture medium" is understood to mean, in particular, a nutrient substrate suitable for the growth and maintenance of living cells (e.g., microorganisms, plants, human and / or animal cells), optionally also viruses. This understanding of the term "cell culture medium" is based on HS code 38210000 of the so-called "Harmonized Commodity Description and Coding Systems" (abbreviation: HS) defined by the "World Customs Organization" (abbreviation: WCO).

[0011] The cell culture medium can be based on Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / F12).

[0012] The cell culture medium can contain trace elements and salts, preferably the elements calcium, iron, cobalt, copper, potassium, magnesium, manganese, molybdenum, sodium, nickel, phosphate, selenium, silicon, zinc and / or tin (most preferably all of these elements).

[0013] Furthermore, the cell culture medium can contain essential and non-essential amino acids, preferably glycine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and / or L-valine (most preferably all of these).

[0014] The cell culture medium can contain at least one component (preferably all of these components) selected from the group consisting of biotin, choline, folic acid, glucose, Hepes buffer, hypoxanthine, linoleic acid, lipoic acid, myo-inositol, niacinamide, pantothenic acid, putrescine, pyridoxal, pyridoxine, riboflavin, thiamine, thymidine, pyruvic acid, and vitamin B12. Depending on the application, sodium bicarbonate and / or L-glutamine can be present in or added to the cell culture medium. For example, cell culture medium in powder form usually does not contain sodium bicarbonate. Sodium bicarbonate is often added only during the production of liquid cell culture medium. Since L-glutamine decomposes naturally in aqueous solution, liquid cell culture medium is often prepared without L-glutamine to increase its shelf life. In this case, L-glutamine is often added as a stock solution immediately before use.

[0015] The cell culture medium according to the present invention does not require transferrin from serum or recombinant transferrin as an iron source, and the diphosphate citrate complex present in the cell culture medium dissolves very rapidly and well in an aqueous solution (e.g., medium, supplement, or water), and is less expensive than the known use of the iron citrate choline complex in cell culture medium. Therefore, compared with comparable cell culture media in the prior art, it has the advantage of enabling cell culture with a high productivity titer in a faster and less expensive (more economical) way.

[0016] In a preferred embodiment, the cell culture medium is present in a non-dissolved form, preferably in powder or granule form. The advantage here is that the cell culture medium has a long shelf life and the transportation cost is lower than that of the dissolved cell culture medium. The cell culture medium preferably contains the iron diphosphate citrate complex in an amount of 0.16% to 12% by weight, preferably 0.22% to 6% by weight, particularly preferably 0.3% to 2% by weight, more particularly preferably 0.4% to 1% by weight, especially 0.5% to 0.7% by weight.

[0017] The cell culture medium can be present in a dissolved form, preferably in an aqueous solution form. The advantage here is that adding water eliminates the time required to dissolve the cell culture medium in water, allowing the cultivation of living cells to start immediately. The dissolved cell culture medium preferably contains an amount of ferric diphosphate citrate complex such that the iron concentration of the cell culture medium set via the (undissociated) ferric diphosphate citrate complex is in the range of 80 μM to 5800 μM, preferably 100 μM to 3000 μM, particularly preferably 150 μM to 1000 μM, more particularly preferably 200 μM to 5000 μM, and especially 250 μM to 350 μM.

[0018] In particular, the ferric diphosphate citrate complex exists in an undissociated form in the dissolved cell culture medium (see, for example, p. 1097, right column, first paragraph in Gupta et al., Physicochemical characterization of ferric pyrophosphate citrate, Biometals, 2018, vol. 31, pp. 1091 - 1099). Therefore, the iron concentration set via the ferric diphosphate citrate complex means the iron concentration resulting from the undissociated ferric diphosphate citrate complex. In other words, the iron concentration set via the ferric diphosphate citrate complex in the cell culture medium refers to the concentration of iron atoms that form complexes in the ferric diphosphate citrate complex, that is, the concentration of iron atoms that exist freely in the cell culture medium (e.g., free Fe 2+ ions and / or Fe 3+ ions). When the ferric diphosphate citrate complex has four iron atoms (see, for example, Figure 5b of Gupta et al.), the dissolved cell culture medium contains the ferric diphosphate citrate complex in an amount of preferably 20 μM to 1450 μM, preferably 25 μM to 750 μM, particularly preferably 37.5 μM to 250 μM, more particularly preferably 50 μM to 1250 μM, and especially 62.5 μM to 87.5 μM to set the iron concentration of the cell culture medium within the above range.

[0019] The iron diphosphate citrate complex can be selected from the group consisting of sodium iron diphosphate citrate complex, potassium iron diphosphate citrate complex, ammonium iron diphosphate citrate complex, and mixtures thereof. The iron diphosphate citrate complex is preferably the sodium iron diphosphate citrate complex, and particularly preferably the sodium iron diphosphate citrate complex having CAS No. 85338-24-5 and / or EC No. 286-697-4. The advantage of the sodium iron diphosphate citrate complex compared to other complexes is that sodium ions are tolerated at higher concentrations in the cell culture medium by many living cells, in contrast to other cations such as ammonium.

[0020] In a preferred embodiment, the cell culture medium does not contain at least one serum component and preferably does not contain transferrin and / or lactotransferrin.

[0021] The cell culture medium may contain a serum replacement, preferably Ultroser G serum replacement, in the composition available in August 2018. In this case, the cell culture medium particularly does not contain transferrin and / or lactotransferrin.

[0022] Furthermore, the cell culture medium can contain growth factors. In this case, the cell culture medium particularly does not contain transferrin and / or lactotransferrin.

[0023] In a preferred embodiment, the cell culture medium does not contain animal or human components.

[0024] The cell culture medium may not contain proteins.

[0025] Furthermore, the cell culture medium can be free of hydrolysates.

[0026] Furthermore, the cell culture medium can be chemically defined.

[0027] The cell culture medium contains at least one, preferably a plurality of amino acids, and the at least one, preferably plurality of amino acids are preferably selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and combinations and / or salts thereof, and the cell culture medium can be characterized in particular by containing all of the amino acids.

[0028] Furthermore, the cell culture medium contains at least one, preferably a plurality of lipid precursors, and the at least one, preferably plurality of lipid precursors are preferably selected from the group consisting of choline chloride, ethanolamine, glycerol, inositol, linolenic acid, fatty acids, phospholipids, cholesterol-related compounds, and combinations and salts thereof.

[0029] Furthermore, the cell culture medium contains at least one, preferably a plurality of carboxylic acids having at least 6 carbon atoms, and the at least one, preferably plurality of carboxylic acids are preferably selected from the group consisting of linoleic acid, linolenic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, arachidonic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, octanoic acid, and combinations and salts thereof, and the cell culture medium can be characterized in particular by containing all of the fatty acids and / or salts thereof.

[0030] The cell culture medium contains at least one, preferably a plurality of carboxylic acids having less than 6 carbon atoms, and the at least one carboxylic acid can be preferably butyric acid or a butyrate.

[0031] In a preferred embodiment, the cell culture medium contains at least one, preferably a plurality of nucleosides, and the at least one, preferably plurality of nucleosides are selected from the group consisting of adenosine, guanosine, cytidine, uridine, thymidine, hypoxanthine, and combinations and salts thereof, and in particular, the cell culture medium is characterized by containing all of the nucleosides and / or their salts.

[0032] Furthermore, the cell culture medium contains at least one, preferably a plurality of carbohydrates, and the at least one, preferably plurality of carbohydrates are preferably selected from the group consisting of glucose, galactose, glucosamine, fructose, mannose, ribose, sucrose, and combinations thereof.

[0033] The cell culture medium contains at least one, preferably a plurality of buffering substances, and the at least one, preferably plurality of buffering substances are preferably selected from the group consisting of ACES, HEPES, MES, MOPS, NaHCO3, PIPES, phosphate buffer, TRIS, and combinations and / or salts thereof.

[0034] Furthermore, the cell culture medium contains at least one, preferably a plurality of trace elements, and optionally, further contains a chelating agent, preferably EDTA, and the at least one, preferably plurality of trace elements are preferably selected from the group consisting of calcium, cobalt, copper, potassium, magnesium, manganese, molybdenum, sodium, nickel, phosphate, selenium, vanadium, zinc, tin, and combinations thereof, and the trace elements can optionally be present in salt form, and the cell culture medium can in particular be characterized by containing all of the trace elements.

[0035] Furthermore, the cell culture medium contains at least one, preferably a plurality of vitamins, and the at least one, preferably the plurality of vitamins, are preferably selected from the group consisting of biotin, choline, folic acid, myo-inositol, niacinamide (B3), pantothenic acid, pyridoxal, pyridoxine, riboflavin, thiamine, vitamin B12, and combinations thereof and salts thereof.

[0036] In a preferred embodiment, the cell culture medium particularly preferably does not contain iron salts in a non-dissolved form.

[0037] Furthermore, the cell culture medium particularly preferably does not contain iron citrate in a non-dissolved form, and preferably does not contain citrate and / or citric acid.

[0038] Also, the cell culture medium particularly preferably does not contain ferric diphosphate in a non-dissolved form, and preferably does not contain diphosphate and / or diphosphoric acid.

[0039] The cell culture medium includes a cell culture medium selected from the group consisting of DMEM, DMEM / F12 medium (Media), Ham F-10 medium, Ham F-12 medium, Medium 199, MEM, RPMI 1640 medium, ISF-1, Octomed, Ames medium, BGJb medium (optionally in the Fitton-Jackson modification), Click medium, CMRL-1066 medium, Fischer medium, Glascow Minimum Essential medium (GMEM), Iscove's Modified Dulbecco's medium (IMDM), L-15 medium (Leibovitz), McCoy 5A modified medium, NCTC medium, Swim S-77 medium, Waymouth medium, William's medium E, and combinations thereof, and each of the cell culture media means those in the compositions available in August 2018.

[0040] In a preferred embodiment, the cell culture medium comprises a cell culture medium selected from the group consisting of DMEM, DMEM / F12 medium, Ham F-10 medium, Ham F-12 medium, Medium 199, MEM, RPMI 1640 medium, and combinations thereof, optionally modified versions thereof, and each cell culture medium means the one in the composition available in August 2018.

[0041] According to the present invention, there is also provided a method for culturing cells, comprising the following steps. a) providing a cell culture medium according to any one of the preceding claims in an aqueous solution; and b) growing or maintaining at least one cell in the aqueous solution of the cell culture medium.

[0042] The cells in this case can be cells of a primary cell line or a continuous cell line, preferably can be selected from the group consisting of mammalian cells, avian cells, and insect cells, the cells are particularly preferably mammalian cells, and particularly preferably are mammalian cells selected from the group consisting of CHO, NS0, SP2 / 0, hybridoma, HEK293, PERC-6, BHK-21, and Vero-76, and more particularly preferably are CHO cells, and in particular are CHO cells selected from the group consisting of CHO-DG44, CHO-DUKX, CHO-S, and CHO-K1.

[0043] Furthermore, according to the present invention, there is provided a method for expressing at least one recombinant protein in a cell culture, and in this case, the method of the present invention for culturing cells further comprises introducing a nucleic acid into at least one cell, and this nucleic acid causes constitutive or inducible production of at least one recombinant protein, preferably further causes secretion of the protein produced in the aqueous solution of the cell culture medium, and this recombinant protein is particularly preferably selected from the group consisting of therapeutic proteins, antibodies, fusion proteins, enzymes, vaccines, biosimilars, and combinations thereof.

[0044] According to the present invention, the use of the iron diphosphate citrate complex (preferably the sodium iron diphosphate citrate complex, particularly CAS No. 85338-24-5 and / or EC No. 286-697-4) in a cell culture medium is proposed, preferably as a component in a cell culture medium for culturing at least one cell in the cell culture medium and / or as an additive in the cell culture medium during culturing of at least one cell in the cell culture medium. The iron diphosphate citrate complex can be used such that the cell culture medium has at least one of the above characteristics. For example, the iron diphosphate citrate complex can be used at one of the above concentrations in the cell culture medium. Further, the iron diphosphate citrate complex can be used as a component and / or additive in a cell culture medium for culturing at least one of the above cells.

Brief Description of the Drawings

[0045] The problems of the present invention will be described in more detail based on the following examples and figures without being limited to the specific embodiments shown herein.

[0046]

Figure 1

[0047]

Figure 2

[0048]

Figure 3

[0049]

Figure 4

[0050]

Figure 5

[0051] Example - Living cells that can be cultured in a cell culture medium according to the present invention [Table 1]

[0052] Example 2 - Determination of the optimal concentration of iron citrate diphosphate complex in a cell culture medium To determine the optimal concentration range of the iron citrate diphosphate complex in a cell culture medium, the iron citrate diphosphate sodium complex was tested at various concentrations in a cell culture medium without transferrin.

[0053] The cell culture medium used was based on Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / F12 medium). The medium contained salts of trace elements and the elements calcium, iron, cobalt, copper, potassium, magnesium, manganese, molybdenum, sodium, nickel, phosphate, selenium, silicon, zinc and tin. The iron concentration in the cell culture medium used, without the addition of the ferric diphosphate citrate complex, was less than 0.8 μM. Furthermore, the cell culture medium used contained glycine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine. In addition, the cell culture medium used contained biotin, choline, folic acid, glucose, HEPES buffer, hypoxanthine, linoleic acid, lipoic acid, myo-inositol, niacinamide, pantothenic acid, putrescine, pyridoxal, pyridoxine, riboflavin, thiamine, thymidine, pyruvic acid and vitamin B12.

[0054] The cell growth of cells genetically engineered to produce a specific protein was investigated as a function of iron concentration, which was set via the sodium ferric diphosphate citrate complex in a liquid cell culture medium based on the above DMEM / F12 medium and dissolved in water. Furthermore, the production of the desired protein product was investigated as a function of the iron concentration in the liquid medium set via the sodium ferric diphosphate citrate complex.

[0055] The following table shows the respective weights of the ferric diphosphate citrate complex, expressed in mg per liter of liquid medium (concentration of ferric diphosphate citrate complex in mg / l), used to set each desired iron concentration in the liquid medium.

Table 2

[0056] The results of cell proliferation and protein production as a function of iron concentration set via the sodium iron diphosphate citrate complex are shown in FIGS. 1A and 1B. The final iron concentration in the liquid medium in the range of about 80 μM to 5800 μM was found to be optimal for cell proliferation and protein production. Since the iron concentration in the cell culture medium used was less than 0.8 μM without adding the sodium iron diphosphate citrate complex, the final iron concentration in the range of about 80 μM to 5800 μM was achieved using a practically equivalent molar amount of the sodium iron diphosphate citrate complex (i.e., about 80 μM to 5800 μM of the sodium iron diphosphate citrate complex).

[0057] Example 3 - Effect of sodium iron diphosphate citrate complex on cell proliferation and protein production compared with other iron sources Separate from the iron sources used, a total of four different media corresponding to the transferrin-free medium described in Example 2 and thus having the same composition except for the iron sources used were tested. The iron sources used were added to all the media in an amount such that the final iron concentration reached 300 μM in the liquid medium.

[0058] The iron sources used were as follows: Medium 1: Sodium iron diphosphate citrate complex (iron concentration 300 μM); Medium 2: Iron(II) sulfate heptahydrate (iron concentration 300 μM); Medium 3: Iron(III) citrate anhydrous (iron concentration 300 μM); Medium 4: Iron(III) citrate (iron concentration 300 μM).

[0059] The effects on cell proliferation and protein production of cultured cells genetically modified to produce a specific protein were investigated 10 days and 13 days after the start of the cell culture experiment (fed-batch). The results are shown in FIGS. 2A and 2B. The iron source (sodium iron diphosphate citrate complex) present in Medium 1 was shown to have a beneficial effect on the viable cell count and product concentration, i.e., the amount of product, on the 10th and 13th days from the start of the experiment compared to the iron sources present in Media 2 - 4.

[0060] Solubility in water of a dry medium having the iron source sodium ferric citrate diphosphate complex compared to dry media having other iron sources Water was added to the medium of Example 3 in dry powder form, and the time until each of the dry powder media was completely dissolved was measured. The results are shown in Figure 3. The dry powder medium 1 having the sodium ferric citrate diphosphate complex as the iron source dissolved in water significantly faster than media 2, 3, and 4 having different iron sources, and it can be seen that it took more than 360 minutes to achieve complete dissolution of the originally dried powder media 2, 3, and 4.

[0061] Example 5 - Influence of sodium ferric citrate diphosphate complex compared to other iron sources, further having the individual components of sodium ferric citrate diphosphate complex on cell growth and protein production Excluding the iron source used, it corresponded to the medium without transferrin described in Example 2, and a total of five different media having the same composition were tested, excluding the specific individual components of the iron source and the ferric citrate - diphosphate - sodium complex used. The iron source used was added to all media in an amount such that the final iron concentration reached 300 μM in the liquid medium. Further individual components of the ferric citrate - diphosphate - sodium complex from medium 1, which allows complex formation in the dissolved medium, were added to the comparison media 5, 6, 7, and 8 in equimolar amounts as much as possible. A ratio of 4 to 3 to 3 of iron to citrate to pyrophosphate was used to calculate equimolar amounts of pyrophosphate and citrate as determined by the X - ray absorption spectroscopy of Gupta et al. (Physicochemical characterization of ferric pyrophosphate citrate, Biometals, 2018, vol. 31, pp. 1091 - 1099). In other words, the amounts of pyrophosphate and citrate in media 5 - 8 were maintained to be comparable to the concentrations of pyrophosphate and citrate in medium 1 provided by the sodium ferric citrate diphosphate complex. The purpose of this study was to investigate whether the individual components of the ferric citrate - diphosphate - sodium complex have an effect comparable to the complex or whether the complex forms spontaneously from the individual components in an aqueous solution.

[0062] The five tested media were composed as iron source, pyrophosphate source and citrate source: Medium 1: Sodium ferric diphosphate citrate complex (iron concentration 300 μM); Medium 5: Iron(III) citrate (iron concentration 300 μM) Sodium pyrophosphate tetrabasic (pyrophosphate concentration 225 μM); Medium 6: Iron(II) sulfate heptahydrate (iron concentration 300 μM) Sodium pyrophosphate tetrabasic (pyrophosphate concentration 225 μM) Sodium citrate tribasic dihydrate (citrate concentration 225 μM) Medium 7: Iron(III) nitrate nonahydrate (iron concentration 300 μM) Sodium pyrophosphate tetrabasic (pyrophosphate concentration 225 μM) Sodium citrate tribasic dihydrate (citrate concentration 225 μM) Medium 8: Iron(III) pyrophosphate (iron concentration 300 μM) Sodium citrate tribasic dihydrate (citrate concentration 225 μM)

[0063] The effects on cell growth and protein production of cultured cells genetically modified to produce a specific protein were investigated 10 days and 13 days after the start of the cell culture experiment (fed batch). The results are shown in Figures 4A and 4B. The iron source (sodium ferric diphosphate citrate complex) present in Medium 1 was found to have a beneficial effect on the number of live cells at 10 days and 13 days after the start of the experiment compared to the iron sources present in Media 6 - 8. Furthermore, it can be seen that the iron source (sodium ferric diphosphate citrate complex) present in Medium 1 has a beneficial effect on the product concentration, i.e., the amount of product, at 10 days and 13 days after the start of the experiment compared to the iron sources present in Media 5 - 8.

[0064] The experiment demonstrated that the sodium ferric diphosphate citrate complex is not formed in the liquid medium from its individual components, otherwise the results of Media 5 - 8 would have to be the same as those of Medium 1. However, this was not the case.

[0065] The data further show that sodium iron diphosphate citrate complex (medium 1) is beneficial with respect to the amount of product obtained, as compared to a mixture of iron(III) citrate and tetrabasic sodium pyrophosphate (medium 5). (Please compare medium 1 and 5 in Figure 4B). Since medium 5 contains pyrophosphate in its free anionic form rather than in complexed form (as in the sodium iron diphosphate citrate complex), this means that the pyrophosphate bound to cells in the sodium iron diphosphate citrate complex is more accessible, i.e., can be taken up more readily than free anionic pyrophosphate, and thus the intracellular pyrophosphate concentration that can be achieved via the iron-citrate-diphosphate-sodium complex may mean that it is higher than that possible with free pyrophosphate in the medium.

[0066] Media 5 - 8 were prepared to show that no iron complex forms spontaneously and there are performance differences.

[0067] Solubility in water of a dry medium having a sodium iron diphosphate citrate complex compared to dry media having other individual components of the complex (i.e., iron, citrate, pyrophosphate as individual components) Water was added to the media from Example 5 in dry powder form, and the time until each of the dry powder media was completely dissolved was measured. The results are shown in Figure 5. Dry powder medium 1 having a sodium iron diphosphate citrate complex dissolved significantly faster in water compared to media 5, 6, 7, and 8 having the individual components of the iron diphosphate citrate complex (i.e., salts to provide iron ions, citrate ions, pyrophosphate ions, sodium ions), and it was clear that it took more than 360 minutes for the originally dry powder media 5, 6, and 7 to completely dissolve, and medium 8 did not completely dissolve.

Claims

1. The cell culture medium exists in a non-dissolved form, the cell culture medium contains 0.16% to 12% by weight of an iron diphosphate citrate complex, and the iron diphosphate citrate complex is selected from the group consisting of a sodium iron diphosphate citrate complex, a potassium iron diphosphate citrate complex, an ammonium iron diphosphate citrate complex, and mixtures thereof. The cell culture medium further contains a cell culture medium selected from the group consisting of DMEM, DMEM / F12 medium, Ham F-10 medium, Ham F-12 medium, Medium 199, MEM, RPMI1640 medium, ISF-1, Octomed, Ames medium, BGJb medium (which may be Fitton-Jackson modified), Click medium, CMRL-1066 medium, Fischer medium, Glasgow Minimum Essential medium (GMEM), Iscove's Modified Dulbecco medium (IMDM), L-15 medium (Leibovitz), McCoy's 5A modified medium, NCTC medium, Swim's S-77 medium, Waymouth medium, William's medium E, and combinations thereof. A cell culture medium characterized by this.

2. The cell culture medium exists in a dissolved form, and the dissolved cell culture medium contains the iron diphosphate citrate complex in an amount such that the iron concentration of the cell culture medium set through the iron diphosphate citrate complex is in the range of 80 μM to 5800 μM, and the iron diphosphate citrate complex is selected from the group consisting of a sodium iron diphosphate citrate complex, a potassium iron diphosphate citrate complex, an ammonium iron diphosphate citrate complex, and mixtures thereof. The cell culture medium further comprises a cell culture medium selected from the group consisting of DMEM, DMEM / F12 medium, Ham F-10 medium, Ham F-12 medium, Medium 199, MEM, RPMI1640 medium, ISF-1, Octomed, Ames medium, BGJb medium (which may be modified by Fitton-Jackson), Click medium, CMRL-1066 medium, Fischer medium, Glasgow Minimum Essential medium (GMEM), Iscove's Modified Dulbecco medium (IMDM), L-15 medium (Leibovitz), McCoy's 5A modified medium, NCTC medium, Swim's S-77 medium, Waymouth medium, William's medium E, and combinations thereof. A cell culture medium characterized by this is provided.

3. The cell culture medium according to claim 1 or 2, wherein the iron diphosphate citrate complex is a sodium iron diphosphate citrate complex or the sodium iron diphosphate citrate complex of CAS No. 85338-24-5.

4. The cell culture medium according to any one of claims 1 to 3, characterized in that the cell culture medium has at least one of the following characteristics: i) It does not contain at least one serum component; ii) It contains a serum substitute; iii) It contains a growth factor; iv) It does not contain animal or human components; v) It does not contain proteins; vi) It does not contain hydrolysates.

5. The cell culture medium according to any one of claims 1 to 4, characterized in that the cell culture medium has at least one of the following characteristics: i) It contains at least one amino acid; ii) It contains at least one lipid precursor; iii) It contains at least one carboxylic acid having at least 6 carbon atoms; iv) It contains at least one carboxylic acid having less than 6 carbon atoms; v) It contains at least one nucleoside; vi) It contains at least one carbohydrate.

6. The cell culture medium according to any one of claims 1 to 5, characterized in that the cell culture medium has at least one of the following characteristics: i) It contains at least one buffer; ii) It contains at least one trace element; iii) It contains at least one vitamin.

7. The cell culture medium according to any one of claims 1 to 6, characterized in that the cell culture medium has at least one of the following characteristics: i) does not contain iron salts; ii) does not contain iron citrate; iii) does not contain iron diphosphate.

8. The cell culture medium according to any one of claims 1 to 7, characterized in that the cell culture medium contains a cell culture medium selected from the group consisting of DMEM, DMEM / F12 medium, Ham F-10 medium, Ham F-12 medium, Medium 199, MEM, RPMI 1640 medium, and combinations thereof. The cell culture medium according to any one of claims 1 to 7, characterized in that the cell culture medium contains a cell culture medium selected from the group consisting of DMEM, DMEM / F12 medium, Ham F-10 medium, Ham F-12 medium, Medium 199, MEM, RPMI 1640 medium, and combinations thereof.

9. A method for culturing cells, comprising the following steps: a) preparing a cell culture medium in an aqueous solution, wherein the dissolved cell culture medium contains an iron diphosphate citrate complex in an amount such that the iron concentration of the cell culture medium set through the iron diphosphate citrate complex is in the range of 80 μM to 5800 μM, and the iron diphosphate citrate complex is selected from the group consisting of sodium iron diphosphate citrate complex, potassium iron diphosphate citrate complex, ammonium iron diphosphate citrate complex, and mixtures thereof, and the cell culture medium further contains a cell culture medium selected from the group consisting of DMEM, DMEM / F12 medium, Ham F-10 medium, Ham F-12 medium, Medium 199, MEM, RPMI 1640 medium, ISF-1, Octomed, Ames medium, BGJb medium (which may be Fitton-Jackson modified), Click medium, CMRL-1066 medium, Fischer medium, Glasgow Minimum Essential medium (GMEM), Iscove's Modified Dulbecco medium (IMDM), L-15 medium (Leibovitz), McCoy's 5A modified medium, NCTC medium, Swim's S-77 medium, Waymouth medium, William's medium E, and combinations thereof; and b) growing or maintaining at least one cell in the aqueous solution of the cell culture medium.

10. The method according to claim 9, wherein the cell is a cell of a primary cell line or a continuous cell line.

11. A method for producing at least one recombinant protein in a cell culture, The method according to claim 9 or 10, further comprising introducing a nucleic acid into at least one cell, wherein the nucleic acid causes constitutive or inducible production of at least one recombinant protein.

12. Use of an iron diphosphate citrate complex in a cell culture medium, wherein the cell culture medium is in a dissolved form and contains the iron diphosphate citrate complex in an amount such that the iron concentration of the cell culture medium set through the iron diphosphate citrate complex is in the range of 80 μM to 5800 μM, and the iron diphosphate citrate complex is selected from the group consisting of a sodium iron diphosphate citrate complex, a potassium iron diphosphate citrate complex, an ammonium iron diphosphate citrate complex, and mixtures thereof, and the cell culture medium further comprises a cell culture medium selected from the group consisting of DMEM, DMEM / F12 medium, Ham F-10 medium, Ham F-12 medium, Medium 199, MEM, RPMI1640 medium, ISF-1, Octomed, Ames medium, BGJb medium (which may be Fitton-Jackson modified), Click medium, CMRL-1066 medium, Fischer medium, Glasgow Minimum Essential medium (GMEM), Iscove modified Dulbecco medium (IMDM), L-15 medium (Leibovitz), McCoy's 5A modified medium, NCTC medium, Swim's S-77 medium, Waymouth medium, William's medium E, and combinations thereof.

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