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    • 专利摘要:
    improved cell culture medium. the present invention relates to an optimized medium for the growth of mammalian cells, as well as the production of polypeptides. the cell culture medium is distinguished for a sow ratio of sodium ions to potassium, and it further relates to the method for producing polypeptides using such cell culture media. in another aspect, the method for producing polypeptides using can also comprise a temperature shift and / or a pH shift to further optimize product growth and yield.
    公开号:BR112012027282B1
    申请号:R112012027282-9
    申请日:2011-04-25
    公开日:2020-09-29
    发明作者:Christian Leist；Petra Meissner；Jörg Schmidt
    申请人:Novartis Ag；
    IPC主号:
    专利说明:

    TECHNICAL FIELD OF THE INVENTION
    [001] The present invention relates to the generic field of biotechnology, particularly the cultivation of cells and their use for the production of polypeptides on an industrial scale.
    [002] The present invention provides cell culture media, which are suitable for the cultivation of cells with high cell viability, preferably mammalian cells such as CHO cells, and which are distinguished by their molar ratio of sodium ions to potassium. The cell culture media according to the present invention allows to obtain high polypeptide productivity when used for the production of a polypeptide, particularly by recombinant expression of polypeptides in mammalian cell culture systems, particularly on an industrial scale. TECHNICAL BACKGROUND OF THE INVENTION
    [003] The preparation of polypeptides using recombinant technology has developed in a standard procedure over the past two decades. Access to recombinant polypeptides by cloning the genes encoding the respective polypeptide, and then, by subsequent transformation of appropriate expression hosts with the gene to be expressed and final production and purification of the recombinant polypeptide product provided access to an entirely new class of products biologically designed and produced therapeutics.
    [004] Pharmaceutically active compounds have been prepared in increasing numbers in the pharmaceutical industry using recombinant DNA technology, and then by production processes developed in the field of bioengineering.
    [005] Such biological products include monoclonal antibodies, which have become important treatment options in several medical fields, including autoimmune diseases, inflammatory disorders, immunosuppression, oncology or similar fields.
    [006] The development of these therapeutic products of biological origin requires production on an industrial scale thus providing access to large quantities of recombinant polypeptides. Preferred expression systems are mammalian cell cultures that are better than most eukaryotic systems based on insect, yeast or similar cells, or even traditional prokaryotic expression systems.
    [007] However, the culture of mammalian cells includes tremendous challenges especially on an industrial scale. Production facilities for culturing mammalian cells require perfect optimization of many process conditions.
    [008] One of the most important parameters of the process to control the global production process is the medium in which cells are developed and polypeptide production takes place. The appropriate cell culture media should produce cell cultures with all the necessary nutrient substances, which is especially difficult if no components of animal origin, such as serum or proteins, for example, growth factors, are added to the media.
    [009] Consequently, a wide variety of cell culture media has been developed in some cases, with the focus being based on generic composition and media with a wide variety of different substances, has been proposed (documents n— US 5,122,469, EP 0 481 791, EP 0 283 942). In other cases, specific ingredients have been suggested to improve cell culture. The main goals were to improve cell growth or survival, or the quantity or quality of recombinantly expressed polypeptides.
    [0010] The aspects dealt with in the prior art documents are, among others, the contribution of trace ions (for example, documents n22 WO 02/066603, EP 0 872 487, EP 1160 314 A2), vitamins such as ascorbic acid (for example, US No. 6,838,284), carbohydrates (for example, EP No. 543,106) or the specific amino acid content in combination with additional characteristics (for example, EP22 0 0 501 435, US 5,830,761, US 7,294,484).
    [0011] The main ions and their concentrations in cell culture media are largely kept constant and unchanged. All classic types of media such as, for example, DMEM, DMEM / F12, BME or RPMI 1640 use relatively narrow and fixed ranges for major ion concentrations in general and monovalent cations Na + and K + in particular. This is in line with the fact that the ionic balance of major ions in general and monovalent cations Na + and K + in particular is a fairly universal property of almost all mammalian cells.
    [0012] In more detail, the transmembrane gradient of sodium and potassium ions is a basic property of mammalian cells with a high concentration of potassium ions inside the cell and a high concentration of sodium ions outside the cell. The sodium / potassium pump is one of the main ion pumps of the cell membrane, which is electrogenic and helps to establish and maintain the respective sodium and potassium ion gradient across the membrane (Kaplan, "Membrane cation transport and the control of proliferation of mammalian cells ", Annu. Rev. Physiol., 40: 19-41 (1978)). The pump uses about 30% of the cells' energy and is one of the main processes of energy consumption of the cells. Many basic biochemical processes are coupled with the electrochemical gradient of sodium ions, such as, for example, the Na7Ca + exchanger or the transport of amino acids into cells. The concentrations of sodium and potassium ions outside a cell are therefore parameters of paramount importance that influence the gradient of these ions across the membrane and the basic state of the cell.
    [0013] According to the typical concentration of sodium ions inside and outside a generic mammalian cell (Alberts et al., Molecular Biology of the Cell (1994)), most often sodium concentrations of about 145 mM they are chosen together with potassium ion concentrations of about 5 mM. For most types of media, this results in a ratio between sodium and potassium ions that is in the range of about 20-30 (see Table 1 below and, for example, document No. 5,135,866).
    [0014] Only a few documents from the prior art describe cell culture media suitable for culturing mammalian cells or the production of recombinant proteins mentioning specific ratios of sodium ions to potassium. These documents suggest media compositions with specifically high ratios in the high range of about 30.7 in US No. 5,232,848, or in the range of about 25 to 35, thus reaching even higher values (documents n— EP 0 283 942, EP 0 389 786). Other media such as HAM's-F12 or animal cell culture media, as proposed in US No. 2008/0261259, also specifically suggest higher values (for example, 27.9 to 57.5, in US No. 2008/0261259 ). Only very few documents describe media that have a sodium ions to potassium ratio below 20, such as 11.5-30 (document No. 2 7,294,484) or a ratio of about 15 (document No. 2, 6,180,401). These documents still use ratios higher than 10 and also do not designate a specific advantage for changing this parameter to the mentioned values.
    [0015] In addition to the effects related to the ionic balance between specific ions, the contribution of the main ions of the global osmolality of the environment must also be considered. Most conventional media such as, for example, DMEM, MEM alpha, or Fischer media are characterized by a high amount of sodium chloride.
    [0016] Document No. WO 02/101019 deals with the high glucose content in the medium in combination with the use of a higher osmolality. The high concentration of glucose between about 2-40 g / L has been achieved by reducing or even completely eliminating agents such as sodium chloride, thereby maintaining osmolality at a given level.
    [0017] Considering the above challenges and the existing disadvantages, there is a continuing need in the field of industrial biotechnology to obtain improved cell culture media that allow to produce recombinant polypeptides on an industrial scale. SUMMARY OF THE INVENTION
    [0018] The present invention provides cell culture media with a reduced Na7K + ratio, that is, a Na7K + ratio below a value of about 10. This is achieved by decreasing the number of total sodium ions and increased content of total potassium ions. This low ratio has been found to have several beneficial effects, particularly better mammalian cell viability, growth and productivity.
    [0019] Therefore, the present invention provides a cell culture medium optimized for the development of mammalian cells, as well as for the production of polypeptides, which is distinguished by a ratio of sodium ions to potassium, measured as molar content , between about 10 to 1 and about 1 to 1, alternatively between about 8 to 1 and about 6 to 1. The implementation of this feature may include concentrations of sodium ions in the range between 50 and about 90 mM and potassium ions between about 8 and about 12 mM.
    [0020] In another aspect, the optimization of the cell culture medium includes selecting a total amino acid content between about 40 mM and about 100 mM, alternatively between about 50 mM and about 100 mM. This characteristic can be combined with a low specific molar ratio between the total ion and total amino acid concentration of about 1.9 to about 4.
    [0021] In another aspect, the invention provides a process in which the cell culture medium according to the invention is used to grow mammalian cells for the production of a desired recombinant polypeptide. The process involves culturing mammalian cells in a medium according to the invention and expressing the recombinant polypeptide.
    [0022] Some implementations of the process include culture conditions in which the temperature and / or the pH of the medium are shifted at least once during the culture. As another option, feeding is carried out by a batch feeding process.
    [0023] The desired polypeptide products include glycosylated polypeptides and particularly antibodies and antibody fragments.
    [0024] The mammalian cells used in the process of the present invention are preferably selected from the group consisting of CHO cells, HEK cells and SP2 / 0 cells.
    [0025] In another aspect, the present invention relates to a process for the production of a cell culture medium according to the invention, where the different components are mixed with each other. In particular, the concentration of sodium chloride added to the composition of the medium can be in the range between about 7 and about 15 mM. The concentration of potassium chloride can be added to the composition of the medium to be in a range between about 8 and about 12 mM. BRIEF DESCRIPTION OF THE DRAWINGS
    [0026] The invention will be better understood with reference to the following examples and figures. The examples, however, are not intended to limit the scope of the invention.
    [0027] Figure 1 illustrates the viable cell density of a mAb1-producing CHO cell clone as a function of culture time in shaking culture flasks (see Example 1) using a medium with a reduced Na7K + ratio, as described for the invention. In addition, the effect of a constant temperature versus a temperature shift is represented.
    [0028] Figure 2 illustrates the viability of a clone of mAb1 producing CHO cells (see Example 1), using a medium with a reduced Na7K + ratio, and the effect of a constant temperature versus a temperature shift on Day 3.
    [0029] Figure 3 illustrates the product title as a function of cultivation time for flask cultures with shaking a clone of mAb1-producing CHO cells with and without a temperature shift (see Example 1). The cells were cultured in a medium with a reduced Na7K + ratio, according to the invention.
    [0030] Figure 4 illustrates the lactate concentration as a function of culture time in a mAb2 producing clone (see Example 2). The cells were cultured in a medium with a reduced Na7K + ratio, according to the invention.
    [0031] Figure 5 illustrates the density of viable cells as a function of culture time in a 300 L bioreactor with a CHO cell clone. Culture conditions included a temperature step (Day 5) and two pH shifts due to pH regulation with an adjustment and deadband (see also Example 2). The cells were cultured in a medium with a reduced Na7K + ratio, according to the invention.
    [0032] Figure 6 illustrates the product title as a function of culture time in a 300 L bioreactor with a CHO cell clone. The process combined a temperature with two pH shifts (see also figure 5 and Example 2). The cells were cultured in a medium with a reduced Na7K + ratio, according to the invention. DETAILED DESCRIPTION OF THE INVENTION
    [0033] The cell culture media according to the present invention are used to develop mammalian cells, preferably CHO cells, HEK cells and SP2 / 0 cells, and for the production of recombinant polypeptides, using such cells. CHO cells are especially preferred. The term "cell culture medium" refers to an aqueous solution of nutrients that can be used to develop cells over an extended period of time. Typically, cell culture media include the following components: an energy source that will usually be a carbohydrate compound, preferably glucose, amino acids, preferably the basic set of amino acids, including essential amino acids, vitamins and / or other organic compounds that they are needed in low concentrations, free fatty acids, and inorganic compounds, including microelements, inorganic salts, buffering compounds and nucleosides and bases.
    [0034] The cell culture medium according to the present invention can be used in various cell culture processes. Cell cultivation can be carried out in adherent culture, for example, in monolayer culture or, preferably, in suspension culture.
    [0035] The use of cell culture media in the field of the pharmaceutical industry, for example, for the production of recombinant therapeutically active polypeptides, does not generally allow the use of any material of biological origin due to safety and contamination problems. Therefore, the cell culture medium according to the invention is preferably a medium free of serum and / or proteins. The term "serum and / or protein-free medium" represents a completely chemically defined medium, containing no additives of animal origin such as tissue hydrolysates, for example, fetal bovine serum or the like. In addition, proteins, especially growth factors such as insulin, transferrin, or the like, preferably, are also not added to the cell culture according to the present invention. Preferably, the cell culture medium according to the present invention is also not supplemented with a hydrolyzed protein source such as soy, wheat or rice peptone or yeast hydrolyzate, or the like.
    [0036] The osmolality and pH of the media are adjusted to values that allow cell growth, for example, values between about pH 6.8 and about pH 7.2. The osmolality of the media at the beginning of the culture is typically between about 280 and about 365 mOsm, but it can also gradually increase during the culture, and the addition of feed solutions up to values less than about 600 mOsm / kg. Preferably, the media according to the present invention has an initial osmolality between about 285 and about 365 mOsm / kg.
    [0037] The temperature of the cell culture is selected in a range in which the cells are viable and grow. A typical temperature for cell culture is in the range of about 30 ° C to about 38 ° C. For example, cells are initially grown at temperatures from about 36 to about 37 ° C which is great for CHO cells. However, the exact temperature can be adapted to the needs of the cells and also changed during the culture to allow their viability, growth or optimal production.
    [0038] The first aspect of the invention is related to the ionic balance between sodium and potassium ions in cell culture. The present invention describes a molar ratio of sodium ions to potassium that is between about 10 to 1 and about del to 1. In other implementations of the invention the ratio is selected between about 9 to 1 and about 5 to 1. Alternatively , the ratio is between about 8 to 1 and about 6 to 1.
    [0039] The concentration of sodium and potassium ions and the respective ratio is defined here through their molar content. The concentration of sodium and potassium ions is determined by calculating the total number of these ions in the growth medium, after the respective salts have been added and dissolved in the medium solution.
    [0040] To reach the required sodium concentration usually different salts are added to the medium. Sodium salts, for example, NaCI, sodium monobasic or dibasic phosphate salts, sodium carbonate, sodium citrate, trace ions such as, for example, sodium selenite are commonly used, but are not limited to these examples. In addition, the sodium hydroxide base (NaOH) that can be added to the pH adjustment media contributes to the total sodium ion content. The term "ion" in this regard refers to the dissociated state. Calculating the molar content of ions thus means taking into account the valence of ions. The sodium chloride (NaCI) 1 mM added a medium would therefore contribute with 1 mM sodium ions, while sodium dibasic phosphate (Na2HPO4) 1 mM would consequently contribute 2 mM sodium ions. According to the invention, the sodium concentration used in the medium is selected between about 50 and 90 mM. Alternatively, the sodium ion concentration is selected to be about 65 to about 85 mM.
    [0041] The potassium salt that is used for the medium is typically KCI, but it also includes, for example, K2SO4 or diacid potassium phosphate (KH2PO4). The potassium salt is not limited to these specific examples. Alternatively, the concentration of potassium used in the media is between about 8 and about 12 mM, or about 10.7 mM.
    [0042] Table 1 indicates examples of media that are traditionally used for the growth of mammalian cells. These classic media such as DMEM, DMEM / F12, BGJ and others have a particularly high ratio of Na / K ions. The means according to the present invention are characterized by a particularly low Na / K ratio of less than about 10 to 1 (see Table 2). The means according to the present invention are suitable for cultivating CHO cells and other mammalian cells and show better cell growth and / or allow better production of polypeptides. / two examples of these media are shown in Table 3, which represents the composition of two media examples and how a low Na / K ratio can be achieved. The synergistic effects between low sodium ions to potassium ratio and the other characteristics of the media even have an advantageous effect on cell growth and production of recombinant proteins.
    [0043] In addition to the specific concentration of sodium and potassium ions and their specific ratio, the present media are also distinguished by a particularly low concentration of sodium chloride (NaCI) which is added to the mixture of components of the medium. Preferably, concentrations of about 7 to about 15 mM are used. This low amount of sodium chloride is uncommon. In some implementations of the present invention, the concentration (in mM) of sodium chloride is even lower than the concentration (in mM) of the respective potassium salt that is added. In addition, the media according to the present invention often have a low total chloride ion content. As illustrated by the examples in Table 2, this results in initial chloride concentrations between about 36 and 46 mM. Most of the time, inorganic salts such as NaCl or CaCh contribute to this value, but also components of the medium such as sodium chloride, or amino acids such as, for example, L-histidine hydrochloride or L-lysine hydrochloride can add the total concentration.
    [0044] Another advantage of the media compositions according to the present invention is the combination of a low molar ratio of Na / K with an initial concentration of amino acids in the range between about 40 mM, alternatively about 50 mM, and about 100 mM. Classical media use comparatively low concentrations of amino acids and / or high Na / K ratios. The combination of both characteristics can provide additional effects that are advantageous for cell growth and polypeptide production.
    [0045] Table 2 indicates different implementations of these parameters according to the invention. In addition to their total amino acid content, the appropriate media according to the present invention, optimized for cell growth, preferably contain initial concentrations of amino acids according to the following ranges.


    [0046] The means of the present invention further specified by amino acids as defined in the table above can be used favorably in the improved cell culture processes according to the present invention.
    [0047] In a particularly preferred embodiment, the media according to the present invention are optimized for production and, preferably, contain initial concentrations of amino acids according to the following ranges.


    [0048] The means of the present invention that contain amino acids as defined in the table above can be used favorably in the improved cell culture processes according to the present invention.
    [0049] In another aspect of the invention, in addition to the specific ratio between sodium and potassium ions, also the overall balance between total ionic concentrations (contributing to the overall ionic intensity of the medium), and the amino acids in the medium is important. The ratio between the total ion concentration and the amino acids in the nutrient growth medium is mainly dominated by the major inorganic salts such as, for example, sodium chloride, potassium chloride, sodium bicarbonate, and others, which are important ingredients of the majority types of cell culture media for animal cells. In addition, trace ion salts, amino acids or vitamins contribute to this value (for example, cupric sulfate, L-arginine hydrochloride, L-histidine hydrochloride, choline chloride, calcium D-pantothenate and others). It can be advantageous for cells to also adjust the concentrations of these ions. Therefore, the total ion concentration is defined here as the sum of all the main organic and inorganic salts added to the medium, which are ionizable in an aqueous solution of the medium plus the NaOH base and the HCI acid. Microelements are not included. Therefore, 1 mM NaCI, NaOH or organic salts such as Lysine-HCI or choline chloride would contribute each 2 mM ions. 1 mM MgCh would consequently add 3 mM ions, while 1 mM of the organic trisodium citrate salt contributes 4 mM.
    [0050] According to the present invention, the molar ratio between ions and amino acids is thus selected to be between about 1.9 and 4. In some implementations the ratio is selected to be in the range between about 2.0 and 3 , 9. These low specific ratios are not only achieved by a relatively high content of amino acids, but also by a relatively low content of ions in the medium. The ion content in the medium is generally less than 250 mM. For example, the values are selected to be between about 150 and 220 mM, or alternatively, between about 170 and 200 mM.
    [0051] In short, the specific characteristics of the described media have important effects on cell metabolism and physiology, while, at the same time, also affecting generic parameters such as osmolality or, for example, the availability of nutritional components. The balance of the different characteristics of the medium thus leads to unique properties that lead to unexpected synergistic effects for the cells.
    [0052] Low Na / K ratio media are generally suitable for the growth of different mammalian cells and the manufacture of recombinant polypeptides / proteins in large-scale production. Polypeptides and proteins, as used herein, refer to recombinant polypeptides that are expressed by the respective mammalian cell after transfection of the cells with the DNA construct or constructs encoding the product of interest. Any polypeptide that can be expressed in a host cell can be produced in accordance with the present invention. After the polypeptides have been produced by the process of the present invention, they are either secreted extracellularly, bound to cells or remain in cells, depending on the specific product and the cell line used. The polypeptide product can be recovered from the culture supernatant directly or after lysis of the cells by usual procedures. In addition, additional isolation and purification are done by usual techniques known to those skilled in the art. The polypeptide of the invention can be included in a pharmaceutical composition.
    [0053] Another aspect of the invention relates to a process for the production of a recombinant polypeptide, comprising culturing mammalian cells in a medium according to the present invention, where the culture conditions comprise at least one temperature shift and at least minus a pH shift.
    Consequently, in another aspect of the invention, it may be advantageous to change the temperature during the course of the culture and include one or more temperature shifts that are initiated at certain points in time. A change / shift in temperature does not refer to spontaneous fluctuations in temperature, but changes in temperature of at least 1 ° C, or alternatively, at least 2 ° C that are intentional, and where the second temperature is maintained for at least one day . A shift / shift can be implemented by changing the crop setpoint. The timing is dependent on the growth status of the culture, a predetermined number of days after the start of the culture or the metabolic needs of the cells. Therefore, the temperature can be shifted in a period of about 1 to 10 days after the start of the culture, / preferably, a temperature shift is made during the cell growth phase or towards the end of this phase. Depending on the volume of the culture vessel, the change may occur quickly or more slowly and lasts several hours. In one example, such a shift in temperature is implemented during the growth phase of the crop when the density is between about 40 and about 90% of the maximum density. In one example, the first temperature is between about 33 and about 38 ° C. The second temperature is between about 30 and about 37 ° C, or alternatively, between about 32 and about 34 ° C.
    [0055] In another aspect of the present invention, it may be advantageous to change the pH during the course of the culture, including one or more pH shifts. In other aspects of the invention, shifts in temperature can also be combined with one or more shifts in pH. Although pH (for example, pH 7.0) is chosen to be favorable for the rapid expansion of cells, it is advantageous to modify the pH of the culture after a certain cell density is reached. This change or shift in pH is accompanied by changing the pH set point of the bioreactor / culture vessel or by defining a pH set point in combination with a dead band. A change in pH does not refer to small fluctuations in pH, it refers instead to an intended change. The second pH value (for example, 6.8) is selected to reduce cell death and to allow high rates of specific production by polypeptide cells of adequate quality. The second pH can be maintained until the end of the culture or until additional pH shifts have been introduced. In an implementation, it may be useful to change the pH by at least 0.2. In one embodiment, the first pH is selected to be in the range between pH 6.8 and 7.5. In another modality, the first pH is selected to be in the range between pH 6.8 and 7.2. The second pH value that is reached after a shift in pH can be in the range between pH 6.0 and pH 7.5, or alternatively, between 6.5 and 6.8.
    [0056] The cell culture medium according to the present invention can be used in various cell culture processes. Cell cultivation can be carried out in adherent culture, for example, in monolayer culture or, preferably, in suspension culture.
    [0057] Large-scale cell cultivation can be used, for example, by the various fermentation processes established in industrial biotechnology. Continuous and batch cell culture processes can be used using the cell culture media according to the present invention. Other known reactor technologies, for example, perfusion or similar technologies can also be used. Batch processes are a preferred modality.
    [0058] Batch cell culture includes fed batch culture or simple batch culture. The term "fed batch cell culture" refers to cell culture in which mammalian cells and cell culture medium are supplied to the culture vessel initially and additional culture nutrients are fed continuously or in separate increments for culture during the culture process with or without periodic harvesting of cells and / or product before the end of culture. The term "single batch culture" refers to a procedure in which all components for cell culture, including mammalian cells and the cell culture medium are supplied to the culture vessel at the beginning of the culture process .
    [0059] According to a preferred embodiment of the present invention, the cells are fed in a batch-fed process. Such feeding is beneficial for cells to replace components and nutrients in the medium that are depleted in the medium during the culture process. Typically, feed solutions comprise amino acids, at least one carbohydrate as a source of energy, microelements, vitamins or specific ions. Feed solutions are added depending on the needs of the cells, which are based on a predetermined program that has been determined for the specific cell line or cell clone and product or measures during the culture process. It is particularly advantageous to use concentrated feed solutions to avoid a large increase in volume and dilution of the medium. In some preferred embodiments, it may also be useful to have at least two different feed solutions. This allows the independent dosing of two or more groups of nutrients and components for the cells, and therefore, a better adjustment of the feeding conditions regarding the optimal supply of certain nutrients.
    [0060] In another embodiment of the invention, one of the two feed solutions added to the cell culture medium is a feed comprising the cysteine dipeptide and the amino acid tyrosine. Preferably, the feed contains the dipeptide and the amino acid tyrosine in respective concentrations in the range of about 6.5 g / L and about 8.0 g / L and in the range of about 9 g / L and about 11 g / L in an aqueous solution at a basic pH greater than 10. In a specific embodiment, the concentrated feed comprises the dipeptide cysteine and the amino acid tyrosine in respective concentrations of 10.06 g / L of L-tyrosine and 7.25 g / L of cysteine at a pH above 10.
    [0061] Feed medium comprising cysteine and tyrosine, as described above, can be added based on the measured consumption of the respective amino acids or according to a fixed program, for example, at about 0.2 to about 0.8 % by weight of the initial weight of the cell culture medium per day, preferably about 0.4% by weight of the initial weight of the cell culture medium per day.
    [0062] In some examples, the other feed solution contains all the other amino acids that are also present in the basic medium except tyrosine and cysteine. In some instances, this additional feed solution may consist of specific selected components such as, for example, amino acids or carbohydrates. In another preferred embodiment of the invention, this concentrated feed medium preferably contains amino acids selected according to the following concentration ranges.

    [0063] Preferably, also carbohydrates, such as glucose, are added to this concentrated feed medium, with preferred concentrations between about 1,200 and about 1,400 mmol / L, or alternatively, between about 1,300 and about 1,395 mmol / L.
    [0064] The feed medium as just above, preferably including a carbohydrate, such as glucose, can be added based on the measured consumption of the respective amino acids or according to a fixed program, for example, at about 4% by weight of the initial weight of the cell culture medium per day, preferably about 2% by weight of the initial weight of the cell culture medium per day.
    [0065] Cells cultured in the cell culture medium according to the present invention include mammalian and non-mammalian cells. Non-mammalian cells include insect cells or the like. However, mammalian cells are preferred. The terms "cell", "cell line" and "cell culture" can be used interchangeably here.
    [0066] Examples of mammalian cells include human retinoblasts; human cervical carcinoma cells, human embryonic kidney lineage, human lung cells, human liver cells, PER.C6 cells (a cell line derived from human retinoblasts), human hepatoma lineage and human cell lines such as AGE1.HN; monkey renal CV1 strain transformed by SV40; monkey kidney cells, African green monkey kidney cells, Chinese hamster ovary cells / -DHFR, baby hamster kidney cells; mouse Sertoli cells; mouse mammary tumor cells, canine kidney cells; Buffalo rat liver cells; TRI cells; MRC 5 cells; FS4 cells; CHO cells are a preferred cell line for practicing the invention.
    [0067] In a preferred embodiment of the invention, these cells can be strains other than CHO cells, such as wild type CHO K1, CHO dhfr (Dux1) or CHO dhfr (DG44), but also HEK cells, Sp2 / 0 cells. These cells are typically transfected with one or more DNA constructs that encode the polypeptide (s) of interest. Any polypeptide that can be expressed in these host cells can be produced according to the present invention.
    [0068] Another class of cells that can be used with the cell culture media according to the present invention includes hybridoma cells that are commonly used for the production of monoclonal or polyclonal antibodies.
    [0069] Polypeptides that can be produced from cell cultures and the cell culture media according to the present invention are not limited. Polypeptides can be recombinant or non-recombinant. The term "polypeptide", as used herein, encompasses molecules consisting of a chain of more than two amino acids joined by peptide bonds; molecules that contain two or more of these chains; molecules comprising one or more of these chains being further modified, for example, by glycosylation. The term "polypeptide" is intended to encompass proteins.
    [0070] The preferred class of polypeptides produced by cell cultures and the cell culture media according to the present invention are recombinant antibodies.
    [0071] The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including monoclonal antibodies in full length), polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies), nanoantibodies, modified antibodies, antibody subunits, antibody derivatives, artificial antibodies, combinations of antibodies with proteins and antibody fragments long enough to exhibit the desired biological activity. Monoclonal antibodies, as used herein, can be human antibodies.
    [0072] However, polypeptides other than antibodies can also be produced using cell cultures and cell culture media according to the present invention, for example, polypeptides such as transmembrane proteins, receptors, hormones, growth factors, proteases, coagulating and anticoagulating proteins, inhibitory proteins, interleukins, transport factors, fusion proteins and the like.
    [0073] The products obtained from these cell culture processes can be used for the preparation of pharmaceutical preparations. The term "pharmaceutical preparation" indicates a composition suitable or adapted for administration to a mammal, especially a human. In addition, the proteins according to the invention can be administered together with other components of biologically active agents such as surfactants, receptors, cartridges, diluents and pharmaceutically acceptable vehicles.
    [0074] Table 1 summarizes compositions of commercially available media and additional cell culture media from the prior art: values are based on published values; additions such as NaOH are therefore not included in the values. The sodium concentration or sodium to potassium ratio is thus quite underestimated and, furthermore, apart from the values of the present invention. Table 1

    [0075] Table 2 describes formulations of cell culture media according to the present invention distinguished by their low ratio of sodium to potassium ions. Table 2

    [0076] Table 3 describes the example compositions for chemically defined cell culture media according to the present invention. The individual components of these cell culture media are available from usual commercial suppliers. Table 3



    EXAMPLES
    [0077] In the examples described below, cell culture media 1 and 2 chemically defined are used, having the composition detailed in Table 3 above. The individual components of these cell culture media are available from common commercial sources.
    [0078] Table 4 below indicates the composition of a concentrated feed medium containing L-tyrosine and cysteine. The feed medium can be added based on the measured consumption of the respective amino acids, or according to a fixed program, for example, at 0.4% by weight per day. Table 4

    [0079] Table 5 below indicates the composition of an exemplary concentrated feed medium. The feed medium can be added based on the measured consumption of the respective amino acids, or according to a fixed program, for example, at 2% by weight per day.


    [0080] For the experiments in the examples, a parental CHO cell line that is derived from the CHO-K1 dhfr (+) cell line, ATCC CCL-61 (Kao et al., Genetics55: 513-524 (1967); Kao et al., PNAS 60: 1275-1281 (1968); Puck et al., J. Exp. Med. 108: 945-959 (1958)) for adaptation to protein-free and serum-free medium conditions. Two fractions of this parental cell line are transfected to express two different monoclonal antibodies, mAb1 and mAb2, respectively. Example 1
    [0081] In Example 1, two shaken flask cultures containing medium 1 are inoculated in parallel with a mAb1 producing CHO clone. Shaken flask cultures are incubated in a carbon dioxide incubator at 37 ° C. On day 3, a shaker flask is transferred to a carbon dioxide incubator at 33 ° C. Both shaking bottles are similarly fed with two feeding solutions. Feeding was supplemented according to a fixed schedule, with the addition of 0.4% by weight of the first feeding solution (Table 2) and 2% of the second feeding (Table 3) per day starting on Day 5 and lasting until the end of culture.
    [0082] The temperature shift to 33 ° C allows longer maintenance of viable cell density and culture viability over time (figures 1 and 2), and the achievement of a higher product titer (figure 3) in comparison with the culture that is kept at 37 ° C for the total duration of the experiment. This example illustrates the benefit of implementing a temperature shift to 33 ° C during a cell culture production process based on a CHO cell line. Example 2
    [0083] In this example, a 300 L bioreactor containing medium 2 is inoculated with a mAb2 producing CHO clone. On Day 5, the temperature of the bioreactor is shifted from 36.5 ° C to 33 ° C. The pH set point is 6.90 and the deadband is 0.10. As a result, the culture starts at pH 7.00, the pH moves to 6.80 between Day 2 and Day 4, and then progressively returns to 7.00 due to the consumption of lactic acid by the cells (figure 4). The shift to pH 6.80 allows to reduce the base addition compared to a scenario with a constant pH of 7.00. The return to pH 7.00 allows to reduce the concentration of CO2 in the medium compared to a scenario in which the pH is left at 6.80 after the first displacement. In this process that combines temperature and pH shifts, a high density of viable cells is achieved and the decrease in the density of viable cells over time is minimized (figure 5), making it possible to reach a high titer on day 14 (figure 6). product with the proper quality. The feed is applied similarly to the feed in Example 1.
    权利要求:
    Claims (15)
    [0001]
    1. Cell culture medium, serum-free, for developing mammalian cells, characterized by a molar ratio of sodium to potassium ions between 10 to 1 and 1 to 1.
    [0002]
    2. Cell culture medium according to claim 1, characterized by the fact that the medium is a protein-free medium.
    [0003]
    3. Cell culture medium according to claim 1 or 2, characterized by the fact that the molar ratio of sodium ions to potassium is between 8 to 1 and 6 to 1.
    [0004]
    4. Culture medium according to any of the preceding claims, characterized by the fact that the concentration of sodium ions is between 50 and 90 mM.
    [0005]
    5. Culture medium according to any of the preceding claims, characterized by the fact that the concentration of potassium ions is between 8 and 12 mM.
    [0006]
    6. Culture medium according to any of the preceding claims, characterized by the fact that the total concentration of amino acids is between 40 and 100 mM.
    [0007]
    7. Culture medium according to any one of the preceding claims, characterized by the fact that the molar ratio of the total ion concentration to the total amino acid concentration is between 1.9 and 4.
    [0008]
    8. Process for the production of a recombinant polypeptide, characterized in that it comprises culturing mammalian cells in a medium as defined in any one of claims 1 to 7 and expressing the recombinant polypeptide.
    [0009]
    9. Process, according to claim 8, characterized by the fact that the culture is made by a fed batch process.
    [0010]
    Process according to either of Claims 8 and 9, characterized in that the polypeptides produced are glycosylated.
    [0011]
    Process according to any one of claims 8 to 10, characterized in that the polypeptide is an antibody or antibody fragment.
    [0012]
    Process according to any one of claims 8 to 10, characterized in that the mammalian cells are selected from the group consisting of CHO cells, HEK cells and SP2 / 0 cells.
    [0013]
    13. Process for the production of a cell culture medium according to claim 1, characterized in that the different components are mixed with each other.
    [0014]
    14. Process according to claim 13, characterized by the fact that sodium chloride is added in a concentration between 7 and 15 mM.
    [0015]
    15. Process according to claim 13 or 14, characterized by the fact that potassium chloride is added in a concentration between 8 and 12 mM.
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    法律状态:
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-07-16| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|
    2020-02-11| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2020-06-16| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2020-09-29| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 25/04/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US32783610P| true| 2010-04-26|2010-04-26|
    US61/327,836|2010-04-26|
    PCT/EP2011/056508|WO2011134920A1|2010-04-26|2011-04-25|Improved cell culture medium|
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